Post on 04-Oct-2020
ROLE OF AUTOCHTHONOUS FUNGI IN
PHYTOEXTRACTION OF HEAVY METALS
FROM TOXIC TANNERY SOLID WASTE
AISHA NAZIR
DEPARTMENT OF BOTANY
UNIVERSITY OF THE PUNJAB
LAHORE, PAKISTAN.
ROLE OF AUTOCHTHONOUS FUNGI IN
PHYTOEXTRACTION OF HEAVY METALS
FROM TOXIC TANNERY SOLID WASTE
A Thesis Submitted to the University of the Punjab in
Partial Fulfillment to the Requirements for the Degree
of Doctor of Philosophy in Botany
By
AISHA NAZIR
DEPARTMENT OF BOTANY
UNIVERSITY OF THE PUNJAB
LAHORE, PAKISTAN.
August, 2013
Dedicated to my supervisor
Prof. Dr. Firdaus-e-Bareen
DECLARATION CERTIFICATE
I, Ms. AISHA NAZIR, under the supervision of DR. FIRDAUS E BAREEN
Professor, College of Earth and Environmental Science, University of the
Punjab, Lahore, admit that the data presented in the PhD dissertation entitled
“ROLE OF AUTOCHTHONOUS FUNGI IN PHYTOEXTRACTION OF HEAVY
METALS FROM TOXIC TANNERY SOLID WASTE” is original. All the
sections, sub-sections and formatting are performed by me. This work has not
been used in part or full in a manuscript already submitted or in the process of
submission in partial or complete fulfilment of the award of any other degree
from any institution.
(AISHA NAZIR)
PhD Scholar
Department of Botany,
University of the Punjab,
Lahore.
Dated:
ABSTRACT
The TSW generated by the Leather industry has been a hazardous
entity for agricultural soils in the vicinity of KTWMA landfill site, Kasur,
Pakistan. The presence of enormous amounts of toxic metals like Cr, Cd, Cu,
Fe, Na, K in the TSW has been a major hindrance in converting its organic
and combustible components into products like manure, compost and
fertilizer, etc. Finding solution for the decontamination of heavy metals present
in TSW has been one the primary concerns of environmental biotechnologists
in Pakistan. The current study is focused at phytoextraction of heavy metals
from TSW through phytoextraction bioreinforced with autochthanous saprobic
fungi isolated from TSW.
After repetitive analyses, the TSW was observed to have high pH (8.9),
ECe (2.89 dS cm-1), NaCl (421 %), bicarbonates and chlorides (359.9 and
3118 mgL-1 respectively), considerable amount (4.5 %) of organic matter and
very low bulk density (0.66 g cm-3). The multi-metal contaminated TSW had
high levels of both essential and trace metals. The total metal fraction of
Category-I and II metals was much higher than the in the upper part of
permissible limits of USEPA (1999) with concentrations of Ca, Mg and Na
(6320, 4210 and 9440 mg kg-1 respectively) as well as Cd, Cr, Cu, Fe, Mg, Ni,
Pb and Zn (10097, 25534, 10554, 2250, 3840, 590, BDL and 7590 mg kg-1
respectively).
Screening of hyperaccumulator fungi isolated from TSW on different
fungal nutrient media and selection of ornamental plants for phytoextraction
on the basis of germination response (%) on TSW-Soil mixtures short listed
the upper level of TSW (%) in TSW-Soil mixtures on the basis of toxicity
contribution in soil. The total thirteen autochthonous fungal species were
isolated from TSW and four of them viz. Alternaria alternata, Aspergillus niger,
Fusarium sp. and Trichoderma pseudokoningii were shortlisted for in vitro
mutual growth interaction studies. The four shortlisted fungi were also tested
for their in situ mutual interaction studies in soil by applying their inoculations
in different combinations to marigold (Tagetes patula). On the basis of plant
vegetative biomass production incurred by the fungal inoculations, the isolates
of Trichoderma pseudokoningii and Aspergillus niger were ultimately selected
for actual phytoextraction trials on marigold (Tagetes patula) and sunflower
(Helianthus annuus) in greenhouse and field conditions. The TSW-Soil
mixtures for these trials were 0 (only soil), 5, 10 and 20 % (TSW-Soil w:w).
The plants cultivated on 20% TSW-soil mixture showed less significant
growth as compared to 5 and 10 % lower TSW-soil Mixtures, with lower
values of all biochemical parameters in terms of chlorophyll content, total
protein, SOD and CAT activity. The metal extraction efficiency was found to
be the highest in F1 + F2 treatment. The metal extraction efficiency from
higher to lower order was in the order: F1 + F2 > F2 > F1 > C.
Both the tested plants were found to be effective accumulators of metals.
The plants given inoculation of both fungi (F1 + F2) showed a significantly
higher growth in all types of soil. Plants given only fungus (F1 or F2) also
showed significant growth rate as compared with control treatment. The
statistical analyses of the results showed increase in all growth parameters in
lower TSW-soil mixtures at all exposures followed by a decrease at the
highest TSW-soil ratio i.e. 20%.
According to Tolerance Index (TI) and translocation Index, H. annuus and
T. patula proved to be the suitable for phytoextraction of multimetal
contaminated TSW and showed the ability to serve as phytostabilizing plants
for metals in the phytoremediation process. Tolerance Index (TI) values more
than 1.0 for Cr and Zn suggested the hyperaccumulative potential of both
plants for these metals. Greater SEY (%) values also suggested the efficiency
of both these plants to remove metals from TSW.
Keeping in view the growth parameters and metal accumulation in the
plant, it was observed that lower percentage (5 and 10%) of tannery solid
waste was suitable for the phytoremediation of most of the studied metals.
The better growth, elevated levels of antioxidants (SOD and CAT), high
accumulation of metals and significant statistical data showed that there is
synergistic effect of both fungal inocula (F1 + F2). Thus autochthonous fungi
along with tolerant plants can be exploited for phytoremediation of tannery
waste by products.
Acknowledgements
Without encouragement and support of many people, this dissertation
would never have been accomplished. My personal very deep appreciation
goes to each of the contributors, right from my very induction into PhD till
submission of the final manuscript.
Firstly and fore mostly, I would like to express my deep gratitude to
learned and dignified teacher and supervisor, Prof. Dr. Firdaus-e-Bareen,
Principal College of Earth and Environmental Science, University of the
Punjab, Lahore, for her keen interest in supervision, valuable guidance
sympathetic attitude and stimulating ideas, which were real source of
inspiration for me throughout the course of this research work. She has the
attitude and the substance of a genius; she continually and convincingly
conveyed a spirit of adventure in regard to research. Without her persistent
help and guidance this synergistic product would not have been possible.
I am particularly indebted to Dr. Janice E. Thies, International Professor
of Soil Ecology at Cornell University, for always extending her expert, sincere,
valuable guidance and encouragement to me. Thanks Dr. J for being so kind
and helpful during my stay in your laboratory and giving us opportunity to
learn very advanced techniques in such a simple way.
Very special thanks to Prof. Dr. Khan Raas Masood, Chairman of
Department of Botany, for allowing me to conduct this research work and for
providing me all the necessary facilities for the accomplishment of the present
work.
I gratefully acknowledge the Project Director of KTWMA, who always kept
the door of sampling site open for me. Special thanks to Higher Education
Commission (HEC) for the financial support.
I feel a deep sense of gratitude for my father and late mother who formed
part of my vision and taught me the good things that really matter in life. The
happy memory of my mother still provides a persistent inspiration for my
journey in this life. I am grateful to my sisters who gave me love, cared for me
and encouraged me to pursue my education.
I cannot find words to express my gratitude to my brother Syed Abid
Shah, who took me on the process of learning, taught me the importance of
hard work and instilled in me the perseverance and strength to overcome all
hardships and made himself always available to complete this work, even
though it was an irrelevant subject for him. Thank you doesn’t seem sufficient
but it is said with appreciation and respect.
I owe my deepest gratitude to my dear husband Mr. Muhammad Shafiq,
for supporting me with his constant love, concern, interest, excellent
suggestions, and creative ideas and for helping to create an environment that
gave the peace of mind to enable me to really focus on research work.
Without his practical and emotional support this dissertation might not have
been accomplished.
I owe my deepest gratitude to my friends and colleagues. I share the credit
of my work with all my laboratory members.
Whenever my steps tried to lose balance and my perseverance during
the laborious journey of PhD become fainted, a glimpse of my wonderful son,
Muhammad Asad Shafiq, always made me forget the worries and instilled
new energy, passion and determination to complete this uphill task as at the
earliest possible. He is who proved to be the light of my life and the hope of
my heart.
I also place on record my deep sense of gratitude to one and all who,
directly or indirectly, have lent their helping hand in this venture from the
people who first persuaded and got me interested into the study of Botany to
those who with the gift of their company made my days more enjoyable and
worth living.
I would like to say more; however, “word are but empty thanks.”
(Aisha Nazir)
LIST OF ABBREVIATIONS
AAS Atomic Absroption Spectrophotometer AM Arbuscular Mycorrhizal AN Aspergillus niger AS Autoclaved Soil BDL Below Detection Limit Ca Calcium CAT Catalase Cd Cadmium CFUs Colony Forming Units Cr Chromium Cu Copper DMRT Duncan’s Multiple Range Test DTPA Diethylene Triamine Pentaacetic Acid ECe Electrical Conductivity of extract EPA Environment Protection Agency Fe Iron HEC Higher Education Commission HM Heavy Metals K Potassium KTWMA Kasur Tannery Waste Management Agency LSD Least Significant Difference MEA Malt Extract Agar Mg Magnesium MMN Modified Melin Norken Na Sodium NAS Non Autoclaved Soil Ni Nickel PDA Potato Dextrose Agar RCBD Randomized Complete Block Design ROS Reactive Oxygen Species SEY (%) Specific Extraction Yield percentage SOD Superoxide Dismutase SOM Soil Organic Matter SPAD Soil-Plant Analysis Development TH Trichoderma harzianum TI Tolerance Index TS Trichoderma pseudokoningii TSW Tannery Solid Waste Zn Zinc
TABLE OF CONTENTS
Sr. No. Title Page No.
Abstract
Acknowledgements
List of Abbreviations
List of Tables ............................................................................................................ i
List of Figures ........................................................................................................... xi
1. INTRODUCTION ...................................................................................................... 1
2. MATERIAL AND METHODS ................................................................................... 12
2.1 Sampling site, surveys and sampling of tannery solid waste ..................... 12
2.2 TSW sample processing for spiking garden soil ......................................... 15
2.3 Screening of fungi and plants tolerant for TSW based toxic metals ........... 19
2.4 Pot trial phytoextraction studies based on ultimate screened fungi and plants 26
2.5 Evaluation of pot-trial findings at field level ................................................. 31
2.6 Phytoextraction studies assisted with Trichoderma harzianum (TH) and TS in
a pot trial at Cornell University, New York USA .......................................... 35
2.7 Statistical analyses ................................................................... 35
3. RESULTS ....................................................................................................... 36
3.1 Physico-chemical properties of TSW and garden soil ................................ 36
3.2 Isolation and identification of TSW representative fungi ............................. 41
3.3 Screening and selection of heavy metal resistant autochthonous fungi ..... 43
3.4 In vitro fungal mutual growth interaction studies for Category-II metal
tolerance............................................. ........................................................ 44
3.5 In situ mutual growth interaction studies for screened Category-II metal tolerant fungal isolates with Tagetes patula in soil ..................................... 49
3.6 Screening of heavy metal tolerant ornamental plant species for phytoextraction of TSW-Soil mixtures ......................................................... 51
3.7 Pot experiments with Marigold on autoclaved (AS) and non-autoclaved TSW-
Soil mixtures (NAS) to verify bio-reinforcing role of fungi ........................... 53
3.8 Experiment with saprobic and AM fungi ..................................................... 78
3.9 Experiments with saprobic fungi ................................................................. 99
3.9A Experiments with Tagetes patula inoculated with saprobic fungi ............... 99
3.9B Experiments with Helianthus annuus inoculated with saprobic fungi ......... 121
3.10 Experiment with French marigold ............................................................... 141
3.11 Field experiments ................................................................... 161
3.11A Experiment with Helianthus annuus .............................................. 161
3.11B Experiment with Tagetes patula .................................................... 184
4. DISCUSSION ....................................................................................................... 209
4.1 Physico-chemical properties of TSW and garden soil ................................ 209
4.2 Isolation and identification of TSW representative fungi ............................. 215
4.3 Screening and selection of heavy metal resistant autochthonous saprobic
fungi........................................ ................................................................... 216
4.4 In vitro fungal mutual interaction studies for Category-II metal tolerance... 218
4.5 In situ mutual growth interaction studies for screened Category-II metals tolerant fungal isolates with Tagetes patula in soil ..................................... 219
4.6 Screening of heavy metal tolerant ornamental plant species for phytoextraction of TSW-Soil mixtures ........................................................ 220
4.7 Experiments with marigold cultivated on autoclaved and non autoclaved
TSW-Soil mixtures and inoculated with selective autochthonous saprobic
fungi..................................................................................................... ....... 221
4.8 Experiments with marigold cultivated on TSW-Soil mixtures and inoculated with selective autochthonous saprobic fungi and AM fungi under greenhouse conditions....................................................... ............................................. 227
4.9 Experiments with marigold and sunflower inoculated with selective autochthonous saprobic fungi under greenhouse and field conditions....... 229
5. REFERENCES ....................................................................................................... 239
6. PUBLISHED RESEARCH PAPERS....................................................................... 264
********************
i
LIST OF TABLES
Table
No. Title
Page
No.
2.1.1
Layout for pot experiments showing selected mixtures of tannery solid
waste with soil (TSW-Soil) either autoclaved (AS) or non-autoclaved
(NAS) and fungi (F1: Aspergillus niger and F2: Trichoderma
pseudokoningii) used for inoculation in soil
25
3.1.1 The physico-chemical properties of TSW, garden soil and their various
(% w:w TSW-Soil) mixtures 37
3.1.2
The total, water soluble and DTPA-extractable fraction of Category-I
metals (mgkg-1
) in TSW, garden soil and their various (% w:w TSW-
Soil) mixtures
38
3.1.3
The total, water soluble and DTPA-extractable fraction of Category-II
metals (mgkg-1
) in TSW, garden soil and their various (% w:w TSW-
Soil) mixtures
39
3.3.1
The tolerance index (TI) of various fungi cultivated on 2 % MEA
prepared in autoclaved extract of TSW along with control of each of
the fungus cultivated on 2 % MEA prepared in distilled autoclaved
water.
43
3.4.1
Comparison of six pairs of mutually interacting fungi for growth
competition on the basis of various morphological parameters
observed after 10 days of fungal inoculation.
47
3.5.1
Morphological and biochemical parameters for 50-days old pot
cultivated plants of Tagetes patula cultivated in soil and applied with
individual and combined fungal inoculations. The mean values ± SD
with different letters are significantly different according to Duncan’s
multiple range test (n = 3; P = 0.05).
49
3.6.1
Screening of plants for their phytoextraction potential on the basis of
percentage germination observed in different TSW-Soil (% w:w)
mixtures. The values ± S.D. are mean of three replicates.
51
3.7.1
The biochemical parameters observed in 50-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
55
3.7.2
Various morphological parameters observed in 50-days old Tagetes
patula cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
58
3.7.3 The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 50-days old Tagetes patula cultivated on TSW-Soil mixtures. The 60
ii
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
3.7.4
The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
50-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
61
3.7.5
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 50-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
65
3.7.6
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
50-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
68
3.7.7
The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 50-days old
Tagetes patula cultivated on TSW-Soil mixtures. The mean values
S.D. with common letters (small along the row & capital within a
column) are not significantly different according to Duncan’s multiple
range test (P = 0.05; n = 3).
70
3.7.8 The Category-I metals translocation index (%) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 71
3.7.9 The Category-II metals translocation index (%) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 73
3.7.10 The tolerance index (TI) analyzed in shoot and root of 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 74
3.7.11 The Category-I metals specific extraction yield (SEY %) analyzed in
50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 74
3.7.12 The Category-II metals tolerance index (TI) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 76
3.8.1
The biochemical parameters observed in 50-days old Tagetes patula
cultivated on TSW-Soil mixtures applied with different fungi. The mean
values S.D. with common letters (small along the row & capital within
a column) are not significantly different according to Duncan’s multiple
range test (P = 0.05; n = 3).
78
3.8.2
Various morphological parameters observed in 50-days old Tagetes
patula cultivated on TSW-Soil mixtures applied with different fungi. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
82
iii
3.8.3
The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied
with different fungi. The mean values S.D. with common letters
(small along the row & capital within a column) are not significantly
different according to Duncan’s multiple range test (P = 0.05; n = 3).
83
3.8.4
The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
50-days old Tagetes patula cultivated on TSW-Soil mixtures and
applied with different fungi. The mean values S.D. with common
letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
85
3.8.5
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied
with different fungi. The mean values S.D. with common letters
(small along the row & capital within a column) are not significantly
different according to Duncan’s multiple range test (P = 0.05; n = 3).
87
3.8.6
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
50-days old Tagetes patula cultivated on TSW-Soil mixtures applied
with different fungi. The mean values S.D. with common letters
(small along the row & capital within a column) are not significantly
different according to Duncan’s multiple range test (P = 0.05; n = 3).
90
3.8.7
The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 50-days old
Tagetes patula cultivated on TSW-Soil mixtures applied with different
fungi. The mean values S.D. with common letters (small along the
row & capital within a column) are not significantly different according
to Duncan’s multiple range test (P = 0.05; n = 3).
93
3.8.8 The Category-I metals translocation index (%) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 94
3.8.9 The Category-II metals translocation index (%) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 95
3.8.10 The root and shoot tolerance index (TI) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 96
3.8.11 The Category-I metals specific extraction yield (SEY %) analyzed in
50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 96
3.8.12 The Category-II metals specific extraction yield (SEY %) analyzed in
50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 98
3.9A.1
The biochemical parameters observed in 55-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
100
3.9A.2 Various morphological parameters observed in 55-days old Tagetes
patula cultivated on TSW-Soil mixtures. The mean values S.D. with
103
iv
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
3.9A.3
The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 55-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
105
3.9A.4
The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
55-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
107
3.9A.5
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 55-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
108
3.9A.6
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
55-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
112
3.9A.7
The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 55-days old
Tagetes patula cultivated on TSW-Soil mixtures. The mean values
S.D. with common letters (small along the row & capital within a
column) are not significantly different according to Duncan’s multiple
range test (P = 0.05; n = 3).
115
3.9A.8 The Category-I metals translocation index (%) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 115
3.9A.9 The Category-II metals translocation index (%) analyzed in 50-days old
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 116
3.9A.10 The tolerance index (TI) analyzed in 50-days old Tagetes patula
cultivated on TSW-Soil (% w:w) mixtures. 117
3.9A.11 The Category-I specific extraction yield (SEY %) analyzed in 50-days
old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 118
3.9A.12 The Category-II metals specific extraction yield (SEY %) analyzed in
50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 119
3.9B.1
The biochemical parameters observed in 52-days old Helianthus
annuus cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
122
v
3.9B.2
Various morphological parameters observed in 52-days old Helianthus
annuus cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 3).
125
3.9B.3
The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 52-days old Helianthus annuus cultivated on TSW-Soil mixtures.
The mean values S.D. with common letters (small along the row &
capital within a column) are not significantly different according to
Duncan’s multiple range test (P = 0.05; n = 3).
127
3.9B.4
The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
52-days old Helianthus annuus cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
128
3.9B.5
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 55-days old Helianthus annuus cultivated on TSW-Soil mixtures.
The mean values S.D. with common letters (small along the row &
capital within a column) are not significantly different according to
Duncan’s multiple range test (P = 0.05; n = 3).
130
3.9B.6
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
52-days old Helianthus annuus cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 3).
133
3.9B.7
The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 52-days old
Helianthus annuus cultivated on TSW-Soil mixtures. The mean values
S.D. with common letters (small along the row & capital within a
column) are not significantly different according to Duncan’s multiple
range test (P = 0.05; n = 3).
135
3.9B.8 The Category-I metals translocation index (%) analyzed in 52-days old
Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 136
3.9B.9 The Category-II metals translocation index (%) analyzed in 52-days old
Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 137
3.9B.10 The tolerance index (TI) analyzed in 52-days old Helianthus annuus
cultivated on TSW-Soil (% w:w) mixtures. 138
3.9B.11 The Category-I specific extraction yield (SEY %) analyzed in 52-days
old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 138
3.9B.12 The Category-II specific extraction yield (SEY %) analyzed in 52-days
old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 139
3.10.1
The physico-chemical properties, Category-I and Category-II metals
determined in TSW, Caldwell field soil and their various (% w:w TSW-
Soil) mixtures; The mean values with common letters (small along the
row & capital within a column) are not significantly different according
141
vi
to Duncan’s multiple range test (P = 0.05; n = 3).
3.10.2
The biochemical parameters observed in 45-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 4).
142
3.10.3
Various morphological parameters observed in 45-days old Tagetes
patula cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 4).
145
3.10.4
The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 45-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 4).
147
3.10.5
The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
45-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 4).
148
3.10.6
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 45-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 4).
150
3.10.7
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
45-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 4).
154
3.10.8
The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 45-days old
Tagetes patula cultivated on TSW-Soil mixtures. The mean values
S.D. with common letters (small along the row & capital within a
column) are not significantly different according to Duncan’s multiple
range test (P = 0.05; n = 4).
156
3.10.9
The Category-I metals translocation index (%) analyzed in 45-days old
Tagetes patula cultivated on Caldwell field mixed with different
percentages of TSW (% w:w).
157
3.10.10
The Category-II metals translocation index (%) analyzed in 45-days old
Tagetes patula cultivated on Caldwell field mixed with different
percentages of TSW (% w:w).
157
3.10.11 The tolerance index (TI) analyzed in 45-days old Tagetes patula
cultivated on Caldwell field mixed with different percentages of TSW 158
vii
(% w:w).
3.10.12
The Category-I metals specific extraction yield (SEY %) analyzed in
45-days old Tagetes patula cultivated on Caldwell field mixed with
different percentages of TSW (% w:w).
159
3.10.13
The Category-II metals specific extraction yield (SEY %) analyzed in
45-days old Tagetes patula cultivated on Caldwell field mixed with
different percentages of TSW (% w:w).
159
3.11A.1
The biochemical parameters observed in 78-days old Helianthus
annuus cultivated on field soil mixed with different percentages of
tannery solid waste (TSW-Soil mixtures). The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 6).
162
3.11A.2
Various morphological parameters observed in 78-days old Helianthus
annuus cultivated on field soil mixed with different percentages of
tannery solid waste (TSW-Soil mixtures). The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 6).
164
3.11A.3
The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 78-days old Helianthus annuus cultivated on field soil mixed with
different percentages of tannery solid waste (TSW-Soil mixtures). The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
167
3.11A.4
The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
78-days old Helianthus annuus cultivated on field soil mixed with
different percentages of tannery solid waste (TSW-Soil mixtures). The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
169
3.11A.5
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 78-days old Helianthus annuus cultivated on field soil mixed with
different percentages of tannery solid waste (TSW-Soil mixtures). The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
171
3.11A.6
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
78-days old Helianthus annuus cultivated on field soil mixed with
different percentages of tannery solid waste (TSW-Soil mixtures). The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
175
3.11A.7 The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of soil used for the
cultivation of 78-days old sunflower (Helianthus annuus) on field soil mixed
178
viii
with different percentages of tannery solid waste (TSW-Soil mixtures). The
mean values S.D. with common letters (small along the row & capital within a
column) are not significantly different according to Duncan’s multiple range test
(P = 0.05; n = 6).
3.11A.8
Meta-analytical phytoextraction indices of sunflower: the Category-I
translocation index (%) analyzed for sunflower cultivated on field soil mixed
with different percentages of tanner solid waste (TSW) and inoculated with
different fungi.
179
3.11A.9
Meta-analytical phytoextraction indices of sunflower: the Category-II
translocation index (%) analyzed for sunflower cultivated on field soil mixed
with different percentages of tanner solid waste (TSW) and inoculated with
different fungi.
180
3.11A.10
Meta-analytical phytoextraction indices of sunflower: the tolerance index (TI)
analyzed for shoot and root of sunflower cultivated on field soil mixed with
different percentages of tanner solid waste (TSW) and inoculated with different
fungi.
181
3.11A.11
Meta-analytical phytoextraction indices of sunflower: the specific extraction
yield (SEY %) for Category-I metals in sunflower cultivated on field soil mixed
with different percentages of tanner solid waste (TSW) and inoculated with
different fungi.
181
3.11A.12
Meta-analytical phytoextraction indices of sunflower: the specific extraction
yield (SEY %) for Category-II metals in sunflower cultivated on field soil mixed
with different percentages of tanner solid waste (TSW) and inoculated with
different fungi.
182
3.11B.1
The concentration of category-I category-II metals (mgkg-1
) observed
in soil amended with different concentration of tannery solid waste
(TSW-Soil % w:w) determined after the harvesting sunflower
(Helianthus annuus) and prior the sowing French marigold (Tagetes
patula). The mean values S.D. with common letters are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 6).
185
3.11B.2
The biochemical parameters observed in 82-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with
common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P =
0.05; n = 6).
187
3.11B.3
Various morphological parameters observed in 82-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters
(small along the row & capital within a column) are not significantly different
according to Duncan’s multiple range test (P = 0.05; n = 6).
189
3.11B.4
The concentration of Category-I Metals (mgkg-1
) observed in SHOOT
of 82-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
193
3.11B.5 The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
82-days old Tagetes patula cultivated on TSW-Soil mixtures. The 195
ix
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
3.11B.6
The concentration of Category-II Metals (mgkg-1
) observed in SHOOT
of 82-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
197
3.11B.7
The concentration of Category-II Metals (mgkg-1
) observed in ROOT of
82-days old Tagetes patula cultivated on TSW-Soil mixtures. The
mean values S.D. with common letters (small along the row & capital
within a column) are not significantly different according to Duncan’s
multiple range test (P = 0.05; n = 6).
200
3.11B.8
The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 82-days old
Tagetes patula cultivated on TSW-Soil mixtures. The mean values
S.D. with common letters (small along the row & capital within a
column) are not significantly different according to Duncan’s multiple
range test (P = 0.05; n = 6).
203
3.11B.9 The Category-I metals translocation index (%) analyzed in Tagetes
patula cultivated on TSW-Soil (% w:w) mixtures. 204
3.11B.10 The Category-II metals translocation index (%) analyzed in Tagetes
patula cultivated on TSW-Soil (% w:w) mixtures 205
3.11B.11 The translocation index (%) analyzed in Tagetes patula cultivated on
TSW-Soil (% w:w) mixtures. 206
3.11B.12 The Category-I metals specific extraction yield (SEY %) analyzed in
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 206
3.11B.13 The Category-II metals specific extraction yield (SEY %) analyzed in
Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 207
4.7.1 Pearson correlation among selected biochemical properties, dry
weight, Cr and Na uptake observed in Tagetes patula cultivated on
non-autoclaved soil (NAS) mixed with tannery solid waste (TSW).
223
4.7.2 Pearson correlation among selected biochemical properties, dry
weight, Cr and Na uptake in Tagetes patula cultivated in autoclaved
soil (AS) mixed with tannery solid waste (TSW)
225
4.8.1 Pearson correlation among selected biochemical properties and
metals for Tagetes patula cultivated in soil mixed with tannery solid
waste (TSW) and inoculated with AM (arbuscular mycorrhizal) and
saprobic fungi.
228
4.9.1 Pearson correlation among selected biochemical properties, CAT,
SOD, dry weight production, Cr and Na uptake in Tagetes patula
cultivated on TSW:Soil taken in pots and inoculated with saprobic
fungi.
230
4.9.2 Pearson correlation among selected biochemical properties and 232
x
metals for Tagetes patula cultivated in Caldwell field soil mixed with
tannery solid waste (TSW) and inoculated with saprobic fungi (incl.
Trichoderma harzianum).
4.9.3 Pearson correlation among selected biochemical properties and
metals for Helianthus annuus cultivated in soil mixed with tannery solid
waste (TSW) and inoculated with saprobic fungi (FIELD TRIAL).
234
xi
LIST OF FIGURES
Fig. No. Title Page No.
2.1.1 Map showing location of KTWMA plant in Kasur city of Pakistan (upper)
and detailed layout of the effluent treatment plant and landfill of KTWMA
(lower) from where TSW was sampled for the study.
13
2.1.2 View of the KTWMA landfill with open dumping of tannery solid waste
(TSW) selected as sampling site for TSW. (upper) The agricultural fields
can be seen in the vicinity of the landfill with no protective linings; (lower)
the diversity of the components of TSW can be observed.
14
2.2.1 Procurement steps of tannery solid waste (TSW) from landfill to the mixing
in soil at experimental station: (A) scrapping top 15 cm layer of TSW
before sampling; (B) filling sack with TSW dug with hoe; (C) TSW sample
filled sacks; (D) carriage of samples to the experimental station; (E) TSW
spread on polythene sheet for drying; (F) air dried TSW.
16
2.3.1 A view of the experimental station where pot trials were carried out; the
partially controlled conditions can be noted from the available type of wire
house applied with glass roof and airy fairy sides being open to natural
ambient temperature and moisture regimes.
23
2.5.1 The statistical layout of the experimental units showing distribution of field
plots lined with polythene sheet. The vertical grey lines show the
separation between experimental plots (25 × 3 feet each) arranged in split
plot design with no replication having soil mixed with tannery solid waste
(TSW-Soil % w:w) while the horizontal dark black lines show division of
each strip plot into sub-plots (5 × 3 feet each) applied with fungal
inoculations in a randomized complete block design.
32
2.5.2 Preparation of field plots in order to apply pot trial findings at field level: (A)
excavation of soil from strip plots (25 × 3 feet each) up to 2.5 feet depth
and lining with polythene sheet; (B) mixing of soil with 2 mm sieved
tannery solid waste (TSW); (C) refilling of excavated strip plots with soil
mixed with different percentages of TSW; (D) a strip plot refilled with TSW
mixed soil; (E) the vertical orientation of 5 strip plots; (F) the sub-plotting of
each of the strip plot into four experimental units each for fungal
inoculations in a randomized complete block design.
33
3.2.1 Alternaria alternata on Czapek’s medium at 250C isolated from TSW. 41
3.2.2 Aspergillus flavipus on 2% MEA medium at 250C. 41
3.2.3 Aspergillus fumigatus on Czapek’s medium at 250C 41
3.2.4 Aspergillus parasiticus on Czapek’s medium at 250C 41
3.2.5 Aspergillus terreus on MMN medium at 250C isolated from TSW 41
3.2.6 Fusarium sp. on Czapek’s medium at 250C isolated from TSW 41
3.2.7 Rhizopus arrhizus on 2% MEA basal medium at 250C isolated from TSW 42
3.2.8 Trichoderma pseudokoningii on Czapek’s medium at 250C isolated from
TSW
42
3.2.9 Aspergillus flavus on Czapek’s medium at 250C isolated from TSW. 42
3.2.10 Aspergillus japonicus on 2% MEA basal medium at 250C isolated from 42
xii
TSW.
3.2.11 Aspergillus niger on MMN medium at 250C isolated from TSW. 42
3.2.12 Aspergillus penicilloides on Czapek’s medium at 250C isolated from TSW 42
3.2.13 Aspergillus versicolor on 2% MEA medium at 250C isolated from TSW 42
3.4.1 In vitro mutual growth interaction (center) between screened heavy metal
tolerant Aspergillus niger (right) vs. Trichoderma pseudokoningii (left) 44
3.4.2 In vitro mutual growth interaction (center) between screened heavy metal
tolerant Alternaria alternata (left) vs. Trichoderma pseudokoningii (right)
45
3.4.3 In vitro mutual growth interaction (center) between screened heavy metal
tolerant Fusarium sp. (left) vs. Alternaria alternata (right)
45
3.4.4 In vitro mutual growth interaction (center) between screened heavy metal
tolerant Aspergillus niger (left) vs. Alternaria alternata (right)
46
3.4.5 In vitro mutual growth interaction (center) between screened heavy metal
tolerant Fusarium sp. (left) vs. Trichoderma pseudokoningii (right)
46
3.4.6 In vitro mutual growth interaction (center) between screened heavy metal
tolerant Fusarium sp. (left) vs. Aspergillus niger (right)
47
3.5.1 Pot cultivated 50-days old plants of Tagetes patula with vegetative growth
variation in response to individual and combined fungal inoculations in soil.
50
3.6.1 Screening of plants for their phytoextraction potential on the basis of
percentage germination on different TSW-Soil (% w:w) mixtures. A)
Tagetes patula B) Patunia xybrid C) Dahlia coccinea D) Zinnia elegans
52
3.7.1 The vegetative growth variation caN be observed in Marigold (Tagetes
patula) in response to autoclaved soil (AS on right) and non-autoclaved
soil (NAS on left) mixed with different percentages of TSW (% w:w)
ranging from the maximum in plants from 5 % (TSW:Soil) NAS inoculated
with F1 + F2 to the minimum in plants from 20 % (TSW:Soil) AS inoculated
with C i.e. no fungi.
57
3.8.1 The vegetative growth variation in Marigold (Tagetes patula) in response
to soil mixed with different percentages of TSW (% w:w) and inoculated
with different fungi.
81
3.9A.1 The vegetative growth variation in Marigold (Tagetes patula) in response
to soil mixed with different percentages of TSW (% w:w) and inoculated
with different fungi.
104
3.9B.1 The vegetative growth variation in sunflower (Helianthus annuus) in
response to soil mixed with different percentages of TSW (% w:w) and
inoculated with different fungi.
124
3.10.1 The vegetative growth variation in marigold (Tagetes patula) in response
to Caldwell field soil mixed with different percentages of TSW (% w:w) and
inoculated with different fungi; (upper) the representative pots of each of
the treatments with best growth of marigold; (lower) all of the experimental
units with replicates of all the treatments.
146
3.11A.1 Phytoextraction field trials with sunflower (Helianthus annuus) cultivated
on soil amended with different levels of tannery solid waste (TSW:Soil
w:w); 0 % the only treatment without geothermal membrane allowing
165
xiii
leaching (A), 0 % with geothermal membrane to avoid leaching (B), 5 %
(C), 10 % (D) and 20 % (E). The white lines across the strip plots (25 × 3
ft) indicate soil barriers (1.25 ft) subdividing each strip plot into four
subplots (5 × 3 ft each) for fungal inoculations viz. C: No fungal inoculum;
F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger +
T. pseudokoningii, applied in a randomized complete block design.
3.11A.2 The sunflower stem girth variation in response to soil amended with
different levels of tannery solid waste (% w:w) inoculated with fungi;
(clockwise from upper left) 10 % with F2, 20 % with F1 + F2; 5 % with F1 +
F2, 10 % with F1 + F2, 20 % with C, 5 % with F1 + F2.
166
3.11B.1 Phytoextraction field trials with French Marigold (Tagetes patula)
cultivated on soil amended with different levels of tannery solid
waste (TSW-Soil % w:w); upper: (A) 0* % the only treatment
without geothermal membrane allowing leaching, (B) 0 % with
geothermal membrane to avoid leaching, (C) 5 %, (D) 10 % and (E)
20 %. The white lines across the strip plots separated by bricked
walk ways (horizontal in above and vertical in lower; 25 × 3 ft) in
upper and the white arrows in the lower picture indicate soil
separations (1.25 ft) subdividing each strip plot into four subplots (5
× 3 ft each) for fungal inoculations viz. C: No fungal inoculum; F1:
Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A.
niger + T. pseudokoningii, applied in a randomized complete block
design.
190
3.11B.2 The Tagetes patula stem girth variation in response to soil mixed
with different levels of tannery solid waste (TSW : soil w:w)
inoculated with fungi (A- 0*% with F1+F2; B- 0% with F1+F2; C- 5%
with F2; D- 5% with F1+F2 ; E- 5% with C ;F-10% with F1+F2; G-
10% with F2; H- 20% with F2; I- 20% with F1+F2.
191
4.1.1 Simple non-linear regression between bulk density (gcm-3
) of TSW-soil
mixture and different fractions of Category-I metals (mgkg-1
): (A) between
bulk density and water-soluble fraction, (B) between bulk density and
DTPA-extractable fraction.
211
4.1.2 Simple linear regression between bulk density (gcm-3
) of TSW-soil mixture
and different fractions of Category-II metals (mgkg-1
): (A) between bulk
density and water-soluble fraction, (B) between bulk density and DTPA-
extractable fraction.
212
4.1.3 Correlation between TSW (% w/w) of TSW-soil mixture and different
fractions of Category-II metals (mgkg-1
): (A) between bulk density and
water-soluble fraction, (B) between bulk density and DTPA-extractable
fraction.
213
4.5.1 The growth variation observed in Tagetes patula in response to different
fungal inoculations in soil
219
Chapter 1
Introduction
1
CHAPTER 1
INTRODUCTION
Land and water are valuable natural resources that sustain agriculture
and ultimately civilization of mankind. For the last couple of decades they
have not only been subjected to the maximum exploitation and severe
degradation but also been polluted due to anthropogenic activities. The
escalating rate of human activities in the form of industrialization has been
posing unprecedented threats towards the biosphere. Both the number and
diversity of pollution sources have been increasing day by day. Burning of
fossil fuels, mining, smelting of metalliferous ores, electroplating, finishing of
metals and plastics, municipal wastes, fertilizers, pesticides, the use of
pigments, chemical works, wood preservation, vehicle service stations, metal
fabrication shops, paper mills, textile plants, waste disposal sites, and above
all tanning of leather industry have been particularly guilty of polluting the
environment (Wong, 2003; Freitas et al., 2004). Each of the pollution sources
have been variably contaminating the environment and exerting damaging
effects on plants, animals and ultimately human health by adding heavy
metals to the soils and waters. Such a scenario has been a serious concern
due to the persistence of heavy metals in the environment and potent
carcinogenicity to human beings. The contamination of the environment with
heavy metals has become a worldwide problem that affects crop yields, soil
biomass and fertility, and leads to the bioaccumulation of metals in the food
chain (Gratao et al., 2005; Rajkumar et al., 2009).
A heavy metal is a member of an ill-defined subset of elements that
exhibits metallic properties. They mainly include the transition metals, some
metalloids, lanthanides and actinides (Babula et al., 2008). These are metallic
chemical elements that have a relatively high density and are toxic even at
low concentrations. They cannot be destroyed biologically but are only
transformed from one oxidation state or organic complex to another (Garbisu
and Alkorta, 2001; Gisbert et al., 2003). Therefore, heavy metal pollution
poses a great potential threat to the environment and human health.
The leather processing has been one of the top most culpable
industries that cause heavy metal pollution. Among all the industrial wastes
2
tannery effluents have been ranked the greatest pollutants. Industrialized
countries have regulated the emission of toxic substances, but in developing
countries like Pakistan, rapid industrial development and population
explosion, coupled with lack of pollution control has caused an enormous
increase in heavy metal contamination of agricultural soils (Ji et al., 2000).
Because of the relatively inexpensive cost of labor and materials, over half of
the world’s tanning activities have been occurring in low and middle income
countries as in the case of Pakistan. Between 1970 and 1995, the percentage
of low to middle income countries contributing to the global production of light
leather increased from 35 % to 56 % and from 26 % to 56 % for the
production of heavy leather materials (Jenkins et al., 2004). According to a
study conducted by the Blacksmith Institute (2010), about 75% of Cr sites are
located in South Asia and of these, nearly a third have been associated with
tannery operations, with mining and metallurgy sites also contributing
significantly. The high concentration of chromium sites in South Asia has been
primarily due to the abundance of tanneries in the region. Majority of the
tanneries have poor environmental controls (Azom et al., 2012).
The process of turning raw leather into manufactured hide uses a lot of
chemicals, auxiliary materials and water. Various chemicals are used during
the soaking, tanning and post tanning processing of hides and skins (Inamul
Haq, 1998). The main chemicals used include sodium sulphite and basic
chromium sulphate including non-ionic wetting agents, bactericides, soda ash,
CaO, ammonium sulphide, ammonium chloride and enzymes. Others include
sodium bisulphate, sodium chlorite, NaCl, H2SO4, formic acid, sodium
formate, sodium bicarbonate, vegetable tannins, syntans, resins,
polyurethane, dyes, fat emulsions, pigments, binders, waxes, lacquers and
formaldehyde. Various types of processes and finishing solvents and
auxiliaries are also used, as well. It has been reported that only about 20 % of
the large number of chemicals used in the tanning process are absorbed by
leather while the rest are released as waste (Azom et al., 2012).
It has been reported that 90 % of all global production of tanned
leathers is tanned using chromium sulfates (Aslan et al., 2007). The
remainders are tanned using other metal sulfates, mostly aluminum,
vegetable tannins or a combination of both (Aslan, 2009). In the event of
3
leather being released to the environment as waste, the heavy metals within
the leathers might harm the ecosystem and threaten human health by
transferring directly or indirectly into the food chain (Aslan et al., 2006; Aslan,
2009). Chromium salts have been used commonly as tanning agents due to
their excellent tanning property, low cost, easy application and wide supply.
As a result, a world total of 540.000 tons of chrome tanned solid waste is
being produced each year (Afsar et al., 2011). A significant amount of
chromium (60 %) applied in tanning is taken up by the leather and the
remaining is discharged in the effluent (Feed International, 2003).
The major environmental issues of tanneries have been solid wastes,
sludge and wastewater. While processing of animal hides into leather, about
20 % of the materials have been produced as solid wastes, consisting of
leather scraps, hair, soluble proteins, curing salts and fleshing (animal fats,
collagen fibers, meat etc). The collagen based waste has been consisting of
4.5 % three valance chrome on an average. During the preservation of animal
hides, huge loads of pollution have been discharged as total dissolved solids
(TDS) and chlorides as a result of use of lime, sodium sulphide, ammonium
salts, sulphuric acid and chromium salts (Kanagaraj et al., 2006). The toxic
metallic compounds, chemicals, biologically oxidizable materials and large
quantities of putrefying suspended matter solid wastes of tanneries are
usually dumped improperly inside and around the factory area (Khuwaja et al.,
1995). Such solid waste has been observed to augment obnoxious smell
because of the degradation of proteins and fats in the skin and generation of
compounds such as NH3, H2S and CO2. Interest on this waste has increased
in recent years due to its volume and oxidation risk of Cr (III) to Cr (VI) which
is carcinogenic for human and more toxic for environment. Discharging of the
solid waste to restricted areas has been a problem for tanners due to the high
cost.
Tanning industry in Pakistan has not been a new industry like other
South Asian countries. It has existed in its indigenous form since long. It had
started in Pakistan about 100 years ago but received a big boost in 1971. The
province of the Punjab has been playing a major role in the development of
tanning industry in Pakistan. Presently, most of the cities where the industry
has been installed in the Province are located in Sialkot, Lahore, Multan,
4
Gujranwala and Kasur. There have been as many as 600 tanneries in the
formal sector of the country and 50% of these have been located in Kasur, a
district of Punjab province, with a long standing tradition of leather tanning
(Augustus, 1996).
The leather sector has been representing one of the most important
industrial sectors in Pakistan in terms of significant contribution to the national
economy. According to a review on Pakistan exports by Trade Development
Authority of Pakistan (TDAP, 2007-08), leather and its products showed an
increase of 21% with export reaching upto US$ 1220.12 Million from US$
1088.20 Million. However, concerns over the expansion of the leather sector
have been resulting in considerable pressures on the environment in the form
of pollution and occupational hazards. Leather industry has been one of the
key culprits of such pollution in Pakistan (Khan, 2001).
By far, proper solid waste disposal systems have been completely
lacking in the design of tanneries of Pakistan. Even in case of the treatment
plant constructed by Kasur Tannery Waste Management Agency (KTWMA) in
association with United Nations Organization (UNO) at Kasur, Pakistan; the
solid waste produced by the cluster of tanneries has been simply thrown away
at the designated spot with extreme violation of standard recommendations.
The solid waste from tanneries is being removed and disposed off in the solid
waste landfill site indiscriminately without any environmental consideration.
Such approach of solid waste disposal has so far been considered a low-cost-
solution although its actual cost may be many folds especially for small and
medium business enterprises. The presence of toxic compounds, especially
compounds of Cr, has been representing a real danger to the health of the
nearby population (TOOL, 2003). Improper and unguarded use of chemicals
has exposed the workers and residents of the adjoining areas to health
hazards of varied types. Respiratory disorders, lung infection and related
diseases, diarrhea/dysentery and typhoid have been common among the
community population (EMRO, 2003).
Conventional methods for removing metals from aqueous solutions or
extracts taken from solid wastes include chemical and physical methods
(chemical precipitation, chemical oxidation or reduction, ion exchange,
filtration, electrochemical treatment, reverse osmosis, membrane
5
technologies, evaporation recovery, etc.) and activated sludge biological
treatment (Ahluwalia and Goyal, 2007). These processes have been generally
efficient in removing the bulk of metals from solution at high or moderate
concentrations. These techniques might have been ineffective or extremely
expensive especially when the metals in solution are at low concentration i.e.
in the range of 1–100 mg L-1 (Ahluwalia and Goyal, 2007). As a consequence,
their limits (high cost, high reagent requirements, etc.) become more
pronounced when voluminous effluents containing complex organic matter
and low metal contamination have to be treated. Various expensive cleaner
technologies cannot eliminate Cr completely from tannery waste, because
some Cr remains in sludge and solid waste derived from tanning (IULTCS,
2008).
In such a scenario, technologies are badly needed that should not only
be able to remove small to large fractions of heavy metals in the polluted
entities like soil and water but also be sustainable in terms of cost and
efficiency. With regard to this, phytotechnologies involving the use of plants
for pollutant removal has gained importance during the last two decades
(Dhir, 2010). Such technologies have been very commonly used because of
their cost effectiveness. However, one of the major drawbacks is their
tremendously slow efficiency. In order to cover efficiency cons, such
technologies are incorporating various chemical, biological and/or
combination of both as phytoextraction enhancers. For example, application
of crude and commercial plant growth regulators (PGRs) along with the
saprobic fungus Trichoderma pseudokoningii have been found to enhance
both multi-metal accumulation and biomass production in Pennisetum
glaucum cultivated in tannery solid waste amended soils (Bareen et al., 2012).
Application of PGRs along with saprobic fungi help the plants to mitigate toxic
effects of HM by secretion of acids, proteins, phytoantibiotics and other
chemicals (Denton, 2007). Other microorganisms such as, bacteria,
filamentous fungi and yeasts have also been found to be capable of efficiently
accumulating heavy metals (Ahluwalia and Goyal, 2007; Mungasavalli et al.,
2007). When efficiency of plants used for phytoremediation is accelerated by
applying either synthetic chemicals or living microbes in the soil, it is termed
as bioaugmentation-assisted phytoextraction (Lebeau et al., 2008) or simply
6
assisted phytoremediation. The reinforced application of plants and
associated microbes to remove HM from the contaminated bodies has offered
an effective, low cost and sustainable means to achieve sustainable cleaning
(Terry and LeDuc, 2005). Researchers have found that microorganisms
associated with plants are able to degrade a number of contaminants (Suresh
and Ravishankar, 2004; Macek et al., 2004). Such microorganisms increase
the efficiency of plants to decontaminate the toxic environment.
Biotechnological approaches consisting of combined application of plants and
microbes could succeed in such areas (Malik, 2004). Therefore, the use of
biological resources such as those of autochthonous fungi has been explored
worldwide for treatment of toxic wastes and accepted owing to several
advantages including environmental safety and cost effectiveness (Dhir et al.,
2010). El-Kassas and El-Taher (2009) isolated Trichoderma viride from heavy
metal polluted area found to be a strong potent for removal of Cr (VI).
Whereas Ezzouhri et al. (2009) isolated highly tolerant fungi and yeasts from
multi-metal contaminated sites. There has also been work on extracellular
enzymatic activities in the rhizosphere of stress tolerant plants that could
potentially contribute to phytoremediation of HM (Reboreda and Cacador,
2008). Besides autochthonous fungi, people have been advocating the
application of allochthonous fungi to increase microbial diversity in the
historically contaminated soils that could cause fungal augmentation leading
to qualitative differences in the soil (Federici et al., 2007). The expanded and
multi-variate tendency of mycelial fungi could be attributed to their more
varied array of strategies for reducing HM toxicity (Baldrian, 2010). There
have been suggestions about applicability of mycelial fungi in both biosorption
(Prigione et al., 2009) as well as bioremediation (Hatvani and Mecs, 2003)
technologies to treat complex wastes contaminated with HM. One of the key
advances offered in the current research is expanding list of stress tolerant
autochthonous filamentous fungi isolated from the contaminated sites like that
of tannery solid waste dumping sites and putting them back in the
phytoextraction process as enhancers of heavy metal tolerance and uptake.
The introduction of heavy metal compounds into the environment
generally induces morphological and physiological changes in the microbial
communities (Vadkertiova and Slavikova, 2006), hence exerting a selective
7
pressure on the microbiota (Verma et al., 2001). Generally, the contaminated
sites are the sources of metal resistant micro-organisms (Gadd, 1993).
Therefore, it is important to explore autochthonous micro-organisms from
such contaminated niches for the bioremediation of heavy metals.
Besides autochthonous filamentous fungi, there has been extensive
work on isolation from polluted soils, application of arbuscular mycorrhizal
(AM) fungi in enhancing biomass as well as heavy metal uptake of plants
growing on heavy metal contaminated soils. The AM fungi have been
reported in tannery pollutants and affected soils (Raman and Sambandan,
1998). They also provide an attractive system to advance plant-based
environmental clean-up (Gohre and Paszkowski, 2006). The AM fungi have
acquired salient functions that make them thrive in stressed soils, enhance
plant growth and mobilize HM from soil. Such functions comprise nutrient
acquisition, cell elongation, metal detoxification, and alleviation of biotic /
abiotic stress (Rajkumar et al., 2012). It has been suggested that isolation of
the indigenous heavy metal stress-adapted AM fungi could be the potential
biotechnological tool for inoculation of plants for successful restoration of
degraded ecosystems (Gaur and Adholeya, 2004). Vivas et al. (2003) isolated
AM fungus namely G. mosseae from cadmium polluted soil and found its
mycorrhizal colonization potential on Trifolium to yield high growth and multi-
metal tolerance. Audet and Charest (2007) have presented models that
illustrate important compromise between plant growth, plant HM uptake and
HM tolerance, and importance of AM fungi in buffering the soil environment
for plants under stressed plant cultivation conditions. Similarly, the incidence
of AM fungi in HM polluted sites, their role in imparting HM tolerance in plants,
factors affecting AM fungi in HM polluted sites and their mechanism of HM
tolerance have been extensively reviewed by Khade and Adholeya (2009). It
has been reported that mycorrhizal associations increase the absorptive
surface area of the plants and the protection provided to roots and enhanced
capability for greater uptake of minerals results in greater biomass production,
a prerequisite for successful remediation (Khan 2001, 2005). The chemical,
biochemical, structural and genomic aspect of AM fungi colonized on roots of
plants in heavy metal soils have been extensively elaborated as a matter of
support for repeated application of AM fungi to alleviate heavy metal stress of
8
plants (Hildebrandt et al., 2007). A multi-scale application comprising pot
lysimeter and field plot experimental approach for studying the
biogeochemical role of AM fungi has been developed (Neagoe et al., 2013).
To a wider perspective, the AM fungi application in phytoextraction has
resulted in greater effectiveness with respect to increasing the tolerance of
lettuce because of induction of antioxidant enzymes (Kohler et al., 2009;
Azcon et al., 2009). Combined application of AM and filamentous fungi
isolated from multi-contaminated soil resulted in enhanced bioaccumulation of
HM (Azcon et al., 2010). The application of AM fungi in phytoremediation with
plants that cannot translocate HM from root to shoot could be of great interest
since such plants produce high biomass but show less bioaccumulation index
(Citterio et al., 2005).
Several heavy metal-tolerant autochthonous fungi have been isolated
from polluted soils, which can be useful for reclamation of degraded soils as
they are found to be associated with a large number of plant species in heavy
metal-polluted soil. Korda et al. (2004) reported that reintroduction of
indigenous microorganisms isolated from the contaminated sites after
culturing provide a highly effective bioremediation tool. Fungi employed in this
effort include many species that have been commonly found in soil, such as
species of Aspergillus, Fusarium, Rhizopus, Mucor and Penicillium (Ammarati
and Michelle, 2005).
Saprobic fungi are important and a common component of rhizosphere
soil, where they obtain enhanced nutritional benefit from organic and
inorganic compounds released from living roots, together with sloughed cells
(Alexander, 1977; Dix and Webster, 1995). Their importance lies in the large
microbial biomass they supply to the soil and in their capacity to degrade toxic
substances (Madrid et al., 1996). Some experimental results confirmed the
existence of synergism in the saprobic fungi, Fusarium concolor and
Trichoderma koningii, on plant roots colonized by AMF (Garcia-Romera et al.,
1998, Arriagada et al., 2005; Arriagada et al., 2007). Similar synergistic
interaction has been observed in Trichoderma pseudokoningii and the AM
fungus Glomus fasciculatum (Bareen and Nazir, 2010). So the cultivation of
heavily mycorrhizal trees that produce large amounts of biomass on
contaminated soil are recommended for phytoremediation practice to prevent
9
food chain contamination (Arriagada et al., 2009). The current work does
comprise isolation of AM fungi from stressed soils and their application in HM
alleviation endeavors with selected ornamental plants.
Increase in the level of heavy metals poses a pervasive threat to the
natural ecosystem. Although many heavy metals when in trace amounts are
essential for various metabolic processes in organisms, they create
physiological stress leading to generation of free radicals when in high
concentration. Stress in turn induces the production of reactive oxygen
species (ROS). Therefore, a mechanism to interrupt such an autocatalytic
process is required. Under normal circumstances, concentration of oxygen
radicals remains low because of the activity of protective enzymes, including
superoxide dismutase (SOD), catalase, and ascorbate peroxidase (Asada,
1984). In resistant forms, stress condition may enhance protective processes
such as accumulation of compatible solutes and increase in the activities of
detoxifying enzymes. Malondialdehyde (MDA) is a cytotoxic product of lipid
peroxidation and an indicator of free radical production and consequent tissue
damage (Ohkawa et al., 1979). Superoxide dismutase (SOD) is a
metalloenzyme that catalyzes dismutation of superoxide anion into oxygen
and hydrogen peroxide. Such enzymes provide a defense system for the
survival of aerobic organisms (Beyer et al., 1991). Proline accumulates
heavily in several plants under stress, providing the plants protection against
damage by ROS. Proline also plays important roles in osmoregulation
(Ahmad and Hellebust, 1988; Laliberte and Hellebust, 1989), protection of
enzymes (Nikolopoulos and Manetas, 1991; Laliberte and Hellebust, 1989;
Paleg et al., 1984), stabilization of the machinery of protein synthesis (Kadpal
and Rao, 1985), regulation of cytosolic acidity (Venekemp, 1989), and
scavenging of free radicals (Smirnoff and Cumbes, 1989). It also acts as an
effective singlet oxygen quencher (Alia et al., 2001).
Wise selection of hyperaccumulators has played a key role in
designing and successful implementation of an assisted phytoextraction plan.
The key factors involved in making such plant selection include HM tolerance
index of plant, continuous rotational frequency with respect to seasonality and
climatology, to let phytoextraction enhancers effectively integrate into the
rhizosphere of the plant, and be able to propagate under multi-metal
10
contaminated conditions. No plant has yet been found to fulfill all of the
desired pre-requisites of an ideal hyperaccumulator, however, some plants
show many of them. Sunflower (Helianthus annuus L.) has been found to
produce sufficient plant biomass and metal shoot translocation for efficient
phytoextraction (Nehnevajova et al., 2009). It (sunflower) has also been able
to show increased concentration of HM from tannery sludge amended soils
under synergistic effects of AM fungi and Trichoderma pseudokoningii (Nazir
and Bareen, 2011). As a matter of plant rotational frequency in the HM
contaminated site, it has been found to be suitable with its other floriculture
members such as, Marigold, Tagetes patula and T. erecta, while propagating
in soils ameliorated with tannery effluents (Chatterjee et al., 2010). Since the
integration of the phytoextraction enhancers is necessary for the ideal
hyperaccumulator, the Marigold (Tagetes patula) has been found to produce
maximum biomass and HM uptake when accompanied with combined
application of saprobic and mycorrhizal fungi while being cultivated in tannery
solid waste amended multi-metal contaminated soil (Bareen and Nazir, 2010).
Such studies with T. patula on tannery waste amended soils have been
repeated by other workers in India (Singh et al., 2011); with T. erecta in China
(Miao and Yan, 2013) and India (Sivasankar et al., 2012). There have also
been findings about bioremediation of sludge through soil amendments using
hyperaccumulator plant species (Singh and Sinha, 2004). On the basis of
their excellent qualities, Sunflower (H. annuus L.) and Marigold (T. patula)
have been selected for current research work.
Heavy metal exposure stimulates formation of toxic oxygen species
(TOS) or reactive oxygen species (ROS) in plants such as O2− and H2O2
(Halliwell and Gutteridge, 1984). Such studies in Sunflower have also been
reported (Singh et al., 2004). Toxic oxygen species are highly reactive and
damage lipids, proteins and nucleic acids (Foyer et al., 1994). Plants possess
certain defensive mechanisms that either prevent the formation of TOS/ROS
or scavenge free radicals. These include antioxidant enzymes and specific
compounds, the production of which is triggered during stress (Devi and
Prasad, 1998). The synchronous action of various antioxidant enzymes, viz.,
catalase (CAT) and ascorbate peroxidase (APX) with the thiol-regulated
enzymes dehydroascorbate reductase (DHAR), Monodehydroascorbate
11
reductase (MDHAR) and glutathione reductase (GR) of the ascorbate
glutathione (GSH) pathway is a predominant mechanism of ROS quenching
under heavy metal stress (Weckx and Clijsters, 1996; Hall, 2002). Apart from
these enzymes low molecular weight antioxidant metabolites like ascorbic
acid (Asc) and glutathione play an important role in protecting plants from
oxidative stress damage (Alscher, 1989; Dhir et. al., 2010). The assessment
of enzymology associated with plants in their defense mechanism while being
cultivated on tannery solid waste multi-metal contaminated soils have been
other key advances of the current research work.
Generation of free radicals under heavy metal stresses and induction
of a defense mechanism in plants inoculated with autochthonous fungi
(saprobic and/or mycorrhizal) have not been studied thoroughly so far. This
situation prompted to investigate whether free radicals are generated under
the stress imposed by tannery solid waste containing heavy metals such as
Pb, Cu, Cr and Zn. The other key purpose of the current work is to determine
the response of selected hyperaccumulator plants to heavy metals in terms of
growth and tolerance reflected by the parameters of plant defense mechanism
based on SOD, CAT, and proline under the influence of autochthonous fungal
inoculated in soils with different toxicity levels of tannery solid waste.
In the light of the given text, the current research work is targeted at
finding phytoextraction potential of sunflower and marigold under the influence
of AM and filamentous fungi isolated from the tannery solid waste in terms of
HM tolerance index and biomass production of selected plants, assessment of
biochemical plant enzymes involved in providing hyperaccumulation and
shoot translocation tendencies, as well as overall cleaning efficiency of the
proposed technology for targeted tannery solid waste (collected from dumping
site of Kasur Tannery Waste Management Agency (KTWMA) located at
Kasur, Pakistan) amended multi-metal contaminated soil both in pilot scale
greenhouse experiment and in the field.
Chapter 2
Materials and
Methods
12
CHAPTER 2
MATERIALS AND METHODS
2.1 Sampling site, surveys and sampling of tannery solid waste
2.1.1 Sampling site
The tannery solid waste (TSW) landfill site constructed and managed
by Kasur Tannery Waste Management Agency (KTWMA) is located at
Depalpur Road, Kasur (31005’16.29’N, 74028’36.16’E), Pakistan (Fig. 2.1.1).
The landfill is surrounded with agricultural fields from three sides while
on the fourth side (towards north) effluent treatment plant of KTWMA is
located (Fig. 2.1.1). The landfill has been receiving TSW from about more
than one hundred local tanneries of Kasur since its establishment in 1994.
The landfill has been ill-managed, lacking of regular leveling of TSW and
application of clay over improperly pressed TSW. As a result, localized heaps
of TSW with variable sizes are randomly distributed in the landfill area. Few of
the TSW heaps are high enough so that during rainy (monsoon) season in the
area, the runoff from TSW slopes flows down the adjacent agricultural fields,
severely affecting the cultivated crops.
The TSW comprises about more than thirty different kinds of
components, mainly being chrome shavings, pieces of partially or completely
tanned leather, wet shavings, bulk amount of salts, hairs, etc. Most of the
precipitation water is retained in the TSW igniting the putrification process of
TSW. As a result, obnoxious odors are produced that contaminate the air as
well.
The KTWMA landfill makes the Kasur city an acute example of
environmental degradation, not only surface water, fertile land and ground
water, but also a direct health hazard to the people living and working in the
affected area (Fig. 2.1.2). The putrification of TSW involving degradation of
proteins and fats from the leather further makes composition of TSW more
complex.
13
Figure 2.1.1. Map showing location of KTWMA plant in Kasur city of Pakistan (upper) and detailed layout of the effluent treatment plant and landfill of KTWMA (lower) from where TSW was sampled for the study.
14
Figure 2.1.2. View of the KTWMA landfill with open dumping of tannery solid waste (TSW) selected as sampling site for TSW. (upper) The agricultural fields can be seen in the vicinity of the landfill with no protective linings; (lower) the diversity of the components of TSW can be observed.
2.1.2 Surveys and sampling
The sampling site was extensively surveyed in order to assess the
variation of TSW and collection of representative sample of the whole lot.
N
15
During the course of study, the TSW landfill was surveyed on: May 05, 2007;
Jul 19, 2008; Sep 20, 2008; Dec 20, 2009; Feb 16, 2009; Apr 15, 2010; Nov
10, 2010; Feb 15, 2011; and Sep 20, 2011.
For sampling of TSW, a completely randomized design was followed.
After scrapping off top 15 cm layer of the surface debris at 1 m2 area, the top
10-inches of TSW was thoroughly mixed with the help of a shovel and placed
approximately 25 kg of TSW in a polypropylene sac. There were as many as
50 random spots from where TSW bulk samples were collected and carried to
the experimental site at Quid-e-Azam Campus, University of the Punjab,
Lahore, Pakistan.
2.2 TSW sample processing for spiking garden soil
A uniform layer of TSW sample from each sack was spread on
polythene sheet in sunlight for drying (Fig. 2.2.1) and turned every other day
for two weeks. Any aggregates were hammered before sieving through a
mesh size of 2.5 mm2. In addition, field soil from Botanical Garden near the
experimental site was collected, dried and sieved through 2.5 mm2 mesh size.
The sieved TSW and soil were further dried to bring them at the same
moisture content prior to mixing. To obtain 5 % TSW-Soil (w : w) mixture, 250
g of TSW and 4650 g of soil were taken in a tumbler. The resulting 5000 g of
mixture were homogenized for 2 minutes by rotating the tumbler and then
1500 g of the mixture were placed in each of the three regular 1.5 liter plastic
pots with no bottom holes taken as replicates while the remaining replicative
500 g of the mixture were placed in labeled zip-loc bag for physic-chemical
analysis in the laboratory. Similarly, 10, 20, 30 through 100 % TSW: Soil
mixtures were procured with three replicates each and replicative 500 g of
each of the mixture ratios in zip-loc bags for pre-sowing analysis in the
laboratory.
The tumbler was cleaned between mixing every other type of TSW-Soil
mixture ratio. The mixtures were allowed to stay by leaving pots in the
greenhouse for 30 days.
16
Figure 2.2.1. Procurement steps of tannery solid waste (TSW) from landfill to the mixing in soil at experimental station: (A) scrapping top 15 cm layer of TSW before sampling; (B) filling sack with TSW dug with hoe; (C) TSW sample filled sacks; (D) carriage of samples to the experimental station; (E) TSW spread on polythene sheet for drying; (F) air dried TSW.
2.2.1 Preparation of soil saturation extract
According to the method given by Rhoads (1982), 500 g of soil sample
were taken and mixed with adequate water to form a supersaturated paste
and left overnight until it glistened under the light and the spatula did not stick
with the paste. A soil saturation extract was obtained under suction pressure
exerted from a vacuum pump.
A B
C D
E F
17
2.2.2 Determination of pH
The soil saturation extract obtained as given in 2.2.1 was used to
determine the pH of the soil samples with the help of a bench type pH meter
(Model Inolab pH 720/Set WTW Germany).
2.2.3 Determination of Electrical Conductivity (ECe) and Sodium
Chloride (%)
The soil saturation extract was used to determine the ECe and NaCl
content of the soil sample using auto ranging portable water proof
microprocessor EC/ TDS/NaCl/oC meter (Model, Hanna 9835).
2.2.4 Determination of carbonates and bicarbonates
For the estimation of carbonates and bicarbonates, the soil saturation
extract was titrated against a standard solution of sulfuric acid using
phenolphthalein as an indicator for carbonates and methyl orange as an
indicator for bicarbonates (Saeed, 1980).
2.2.5 Determination of soil organic matter (SOM %)
The SOM in TSW-Soil mixtures was assessed by loss-on-ignition
method given by Wright et al. (2008).
2.2.6 Digestion of Soil-TSW mixtures
The TSW-Soil mixtures with three replicates each were digested on a
heating digester (Model DK 6, TMD 6 Velp, Italy) and mineralized in a
microwave oven with aqua regia (ISO 11466, 1995) to bring their volume up
to 50 ml final volume of soil saturation extract.
2.2.7 Determination of water soluble metals
The soil saturation extract obtained from the extraction process was
used to determine the water soluble fraction of metals. On the basis of their
method of detection, the metals were grouped into two categories i.e. the
Category-I and Category-II metals.
The Category-I metals were detected by using flame photometer
(Model: PFP7&PFP7/C JENWAY) such as, Ca, K, and Na. In order to
determine the water soluble fraction of Category-I metals, the soil saturation
extracts obtained were run on flame photometer. The quantification of metals
18
was carried out by plotting the absorption values of unknown samples against
the standards solutions of each of the Category-I metal.
The Category-II metals were detected by using flame atomic
absorption spectrophotometer (AAS Model: GBC SAVANT AA, Australia)
such as, Cd, Cr, Cu, Fe, Mg, Ni, Pb and Zn. The water-soluble fraction of
Category-II metals was determined from the soil saturation extracts of
unknown samples by running them on AAS after running the blank and
standard solution for each of the metal and then blank again. After running
every ten samples, the process of AAS calibration with respective standards
was repeated for each of the metal, following the recommendations given in
user manual by the manufacturer.
2.2.8 Determination of DTPA extractable metals
In order to determine diethylene triamine pentaacetic acid or DTPA-
extractable fraction of Category-I as well as Category-II metals, the TSW-Soil
mixtures, 10 g of the TSW-Soil mixtures were solubilized in 20 ml of
extracting solution (0.005M DTPA, 0.01M CaCl2 and 0.1M EDTEA adjusted
to pH 7.3) as given by Lindsay and Norvell (1978). The extracts thus obtained
were run on flame photometer and AAS, as illustrated in 2.2.7.
19
2.3 Screening of fungi and plants tolerant for TSW based toxic
metals
2.3.1 Isolation and screening of TSW-representative autochthonous
fungi
2.3.1.1 Preparation of culture media
The isolation of fungi from TSW was performed on three types of
fungal nutrient media namely, Malt Extract Agar (MEA), Czapek’s medium
and Modified Melin Norken (MMN) Medium.
The 2% MEA medium was prepared by solubilizing 20 gm each of Malt
Extract and Agar in one liter of distilled water as given by Tuite (1969).
For Czapek’s medium (Dox, 1910), 3 gm sodium nitrate, 1gm
potassium hydrogen phosphate, 0.5gm magnesium sulphate, 0.5gm
potassium chloride, 0.01gm ferrous sulphate, 30gm sucrose and 20gm agar
were taken per liter of distilled water.
In order to prepare MMN Medium (Morx, 1969), 3 gm malt extract, 10
gm d-glucose, 0.25 gm (NH4)2HPO4, 0.50 gm MgSO4.7H2O, 0.15 gm
CaCl2.2H2O 0.05 gm, 0.02/1.2ml (1% solution) NaCl, 100 µg thiamin HCl and
12 gm agar were taken per liter of distilled water.
For each of the fungal nutrient medium, their respective ingredients
were thoroughly mixed using a magnetic stirrer in individual volumetric flasks
and autoclaved at 1210C temperature and 15 lb/inch2 pressure for 15 minutes.
After autoclaving, the flasks were allowed to cool down to 650C. The nutrient
medium from flasks was poured into the petri plates with a uniform layer after
adding the antibacterial powder (Chloromycetin chloramphenicol) under
aseptic conditions maintained in a laminar air flow cabinet.
2.3.1.2 Isolating TSW-representative fungi by direct and spread plate
The TSW powder was sprinkled on the fungal nutrient medium taken in
petri plates by the direct and spread plate methods.
20
In the direct plate method, the sieved powder of TSW was sprinkled
directly on top of the medium under aseptic conditions. The petri plates were
sealed with parafilm and incubated at room temperature for 3 days.
In the spread plate method, 1gm powdered TSW was dissolved in
100ml distilled sterilized water by agitating for 20 minutes at room
temperature and then diluting (10 - 10,000 folds) in large beakers. Aliquots of
100 µl of different dilutions were plated onto all of the three fungal nutrient
media with three replicates each to ensure the growth of fungi present in
samples. The petri plates were sealed with parafilm and incubated.
After at least 3 days of incubation at 25°C, the developed fungal
colonies were randomly picked for isolation from MEA and sub-cultured on
petri plates contained with new flush of same nutrient medium. Similarly, the
picking and sub-culturing of fungal colonies was carried out for both Czapek’s
and MMN fungal nutrient media. The process of sub-culturing was kept
continuous until pure isolates for all of the TSW representative fungal colonies
on three fungal nutrient media were obtained.
2.3.1.3 Identification of the TSW-representative fungal species
The pure isolates from TSW were characterized to the genus level on the
basis of macroscopic characteristics (colonial morphology i.e. color,
appearance and shape) and microscopic characteristics (septation of
mycelium, shape, diameter and texture of conidia). The identification at
species level was authenticated at “The First Fungal Bank of Pakistan”,
University of the Punjab, Lahore Pakistan. The pure fungal isolates comprised
of 13 fungal strains.
2.3.1.4 Screening of TSW-representative autochthonous fungi for HM
tolerance
The purified isolates of TSW-representative fungi resulting from 2.3.1.3
were counter-screened for HM tolerance. For this purpose, 2% MEA medium
was prepared by solubilizing 20 gm each of malt extract and agar per liter of
pre-autoclaved extract taken from TSW with distilled autoclaved water. For
each of the pure isolates from 2.3.3.1, the picking and sub-culturing on 2%
MEA meant for counter-screening was performed as described earlier in
21
2.3.1.2. The inoculated plates were incubated at 25°C for at least 7 days. As a
matter of HM tolerance, the growth of the sub-cultured fungal isolates was
estimated by measuring the radius of the colony extension (mm) against the
control (2% MEA medium prepared in distilled and autoclaved water). While
the tolerance index (TI) of TSW representative fungal isolates was determined
by obtaining the ratio of the extension radius of the treated colony
(supplemented with TSW extract) to that of the untreated colony
(supplemented with distilled autoclaved water). The fungal isolates resulting
from the counter-screening process were termed ‘autochthonous saprobic
fungi’ and selected for further experimentation. Four of the 13 pure fungal
isolates were screened for in vitro and in situ mutual interaction studies on the
basis of HM tolerance during the screening process.
2.3.2 In vitro and in situ mutual interaction studies for the
autochthonous saprobic fungi
2.3.2.1 In vitro mutual interaction studies for the autochthonous
saprobic fungi
The four selected isolates of autochthonous saprobic fungi isolated
from TSW were studied for their mutual competitive growth response in petri
plates using 2% MEA medium. For interactions study, petri plates were
labeled as control and experimental on either side of the vertical line crossing
at the center of the petri dish. Two different fungi were introduced opposite to
the vertical line crossing at the center of petri plate. They were incubated at
room temperature and observed every day until the experiment was over. The
combination of fungi competing for mutual growth was as under:
Trichoderma pseudokoningii vs Aspergillus niger (TS vs AN)
Aspergillus niger vs Fusarium sp. (AN vs F)
Aspergillus niger vs Alternaria alternata (AN vs AA)
Trichoderma pseudokoningii vs Fusaium sp. (TS vs F)
Trichoderma pseudokoningii vs Alternaria alternata (TS vs AA)
Fusarium sp. vs Alternaria alternata (F vs AA)
22
2.3.2.2 In situ mutual growth competition studies for autochthonous
saprobic fungi
Parallel to in vitro, the in situ study of the aforementioned combinations
of the autochthonous saprobic fungi was performed for their mutual interaction
with respect to influence on biomass production tendency of Tagetes patula L.
(a reported hyperaccumulator of HM) cultivated on garden soil (mentioned as
control in 2.2) in a pot trial.
2.3.2.2.1 Preparation of Fungal inoculum
The autochthonous saprobic fungal strains namely, Trichoderma
pseudokoningii Persoon ex Gray, Aspergillus niger Van Tieghem, Fusarium
sp. Link ex Gray, and Alternaria alternata Nees ex Wallroth isolated from the
tannery solid waste were used as inoculum in pot experiments. The
autochthonous saprobic fungal strains were cultured on 2% MEA medium.
Then a suspension solution of each fungus was prepared by taking 2-3 loops
full of actively growing young fungal colonies were taken in autoclaved
distilled water under aseptic conditions. After vigorous shaking at least for 15
minutes the inoculum was given through injection to the roots of plants.
Approximately 1.5ml (having colony forming units (CFU) 95,000/ml of
Trichoderma pseudokoningii, 75,000/ml of Aspergillus niger, 10,000/ml of
Fusarium sp and 26,000/ml of Alternaria alternata) was given to each
replicate of each treatment 3 times a week, after germination.
2.3.2.2.2 Pot trial set up
The experiment was set in a wire house having a transparent glass
roof (Fig. 2.3.1) in the Department of Botany, University of the Punjab, Lahore
Pakistan, in a “Completely Randomized Design”.
The experiment was set in medium regular 1.5 liter plastic pots each
filled with 1.5kg soil. The experiment comprised of 12 treatments of fungi with
four replicates each. The pots were watered regularly after every 2 days and
maintained at pot capacity. The combinations of different fungal isolates were
as under:
C = Control (without any inoculum)
23
F1 = Aspergillus niger
F2 = Trichoderma pseudokoningii
F3 = Fusarium sp.
F4 = Alternaria alternata
F1+F2 = Trichoderma pseudokoningii + Aspergillus niger
F1+F3 = Trichoderma pseudokoningii + Fusarium sp.
F1+F4 = Trichoderma pseudokoningii + Alternaria alternata
F2+F3 = Aspergillus niger + Fusarium sp.
F2+F4= Aspergillus niger + Alternaria alternata
F3+F4 = Fusarium sp. + Alternaria alternata
F1+F2+F3+F4 = Trichoderma pseudokoningii + Aspergillus niger + Fusarium
sp. + Alternaria alternata
Figure 2.3.1. A view of the experimental station where pot trials were carried out; the partially controlled conditions can be noted from the available type of wire house applied with glass roof and airy fairy sides being open to natural ambient temperature and moisture regimes.
24
Based on the performance of four pure autochthonous fungal isolates
during their in vitro and in situ interaction studies, two of them namely
Trichoderma pseudokoningii (TS) and Aspergillus niger (AN) were selected
for further experiments.
2.3.3 Verification of TS and AN isolates for HM tolerance by inducting in
phytoextraction with Tagetes patula
Prior to taking TS and AN as ‘ultimate screened fungi’, they were
tested in a phytoextraction pot trial with Tagetes patula for counter verification.
For this purpose, 0, 5, 10 and 20 % (w: w) TSW-Soil mixtures were used as
described in 2.2. Each pot filled with 1.5 kg of respective TSW-Soil mixture.
With three replicates each, one set of pots with said TSW-Soil ratio contained
autoclaved culturing media while the other set of pots was filled with non-
autoclaved mixture.
2.3.3.1 Fungal inoculation
In order to inoculate pots with the selected fungal isolates,
approximately 1.5 ml solution with colony forming units (CFU) 95,000/ml of
Trichoderma pseudokoningii and CFU 75,000/ml of Aspergillus niger was
applied to the rhizosphere of plants by syringe injection through plant
rhizosphere.
The experiment was set up in a wire house with a transparent glass
roof situated at the Department of Botany, University of the Punjab, Lahore
following a randomized complete block design with 96 experimental units (32
× 3). The experimental layout showing combinations of fungi (F) and TSW-
Soil are described in Table 2.1.
The pot capacity was maintained with tap water after every two days
and until the plants were harvested at seeding stage. The activity of the
inoculated fungi was evaluated by estimating their CFU from the soil.
25
Table 2.1.1. Layout for pot experiments showing selected mixtures of tannery solid waste with soil (TSW-Soil) either autoclaved (AS) or non-autoclaved (NAS) and fungi (F1: Aspergillus niger and F2: Trichoderma pseudokoningii) used for inoculation in soil.
Fungal Inoculation
TSW-Soil (% w:w) mixtures
Autoclaved soil (AS) Non-autoclaved soil (NAS)
0 5 10 20 0 5 10 20
C (Control with no
fungus)
0 % AS
with C
5 % AS
with C
10 % AS
with C
20 % AS
with C
0 % NAS
with C
5 % NAS
with C
10 % NAS
with C
20 % NAS
with C
F1 (Aspergillus
niger)
0 % AS with F1
5 % AS with F1
10 % AS with F1
20 % AS with F1
0 % NAS with F1
5 % NAS with F1
10 % NAS with F1
20 % NAS with F1
F2 (Trichoderma
pseudokoningii)
0 % AS with F2
5 % AS with F2
10 % AS with F2
20 % AS with F2
0 % NAS with F2
5 % NAS with F2
10 % NAS with F2
20 % NAS with F2
F1 + F2
0 % AS with
F1+F2
5 % AS with
F1+F2
10 % AS with
F1+F2
20 % AS with
F1+F2
0 % NAS with
F1+F2
5 % NAS with
F1+F2
10 % NAS with
F1+F2
20 % NAS with
F1+F2
2.3.4 Screening of TSW-tolerant plants for HM phytoextraction
For this purpose, a pot trial was set where plants of Helianthus annuus
L., Tagetes patula L., Petunia xhybrida (Hook) Vilm, Zinnia elegans (Jacq.)
and Dahlia coccinea (CAV.) were cultivated on 0, 5, 10, 20, 50 and 100% (w :
w) TSW-Soil mixtures with three replicates each. Each pot had been filled with
1.5 kg of TSW-Soil mixture. For this purpose, certified seeds of the plants
were sterilized with 10 % mercuric chloride solution.
The tolerant plant species Helianthus annuus and Tagetes patula were
selected for actual experiments on the basis of percentage germination. No
germination was observed beyond 20% TSW-soil mixture, due to which 5, 10
and 20% TSW were selected and all other TSW-Soil mixture ratios were
dropped for further experiments.
26
2.4 Pot trial phytoextraction studies based on ultimate
screened fungi and plants
The ultimate screened fungi and plants resulting from the extensive
screening process were procured together for their HM remediation potential.
The pot trials were conducted with Helianthus annuus and Tagetes patula
cultivated on screened TSW-Soil mixtures along with different combinations of
fungi such as, saprobic and arbuscular fungi as well as two saprobic fungi (TS
and AN). Further details are as under:
2.4.1 Pot trial I: Experiments with saprobic fungi
2.4.1.1 Experiments with saprobic TS and AN fungi
The effect of autochthonous fungi TS and AN on phytoextraction
potential of Tagetes patula as well as Helianthus annuus was studied by
setting a pot trial. The details of the combinations of two fungi TS and AN
were similar to the non-autoclaved part of what is described in 2.3.3.1. The
experiment was set in a randomized complete block design with 48
experimental units (16 × 3) for both Tagetes and Helianthus. Approximately
1.5ml solution with CFU 86,900/ml of Trichoderma pseudokoningii and CFU
69,800/ml of Aspergillus niger of the inoculum was applied in the rhizosphere
of the plants. The experiment was set in large sized earthen pots with glazed
inner wall and filled with 10 kg of the soil. The pots were watered after every
two days in order to maintain pot capacity until the plants were harvested. The
experiment was repeated thrice.
2.4.1.2 Live plant analyses-biochemical assays
For live plant analyses based on biochemical assessment of proline
and enzyme assays like superoxide dismutase (SOD) and catalase (CAT), the
plant leaves were plucked, washed with distilled water and immediately
placed in small cylinders filled with liquid nitrogen.
2.4.1.2.1Chlorophyll contents-SPAD values
Chlorophyll content of the plants was determined using a chlorophyll
meter (Model SPAD-502 Singapore). For this purpose, SPAD readings were
taken by selecting 15 plants at random per treatment.
27
2.4.1.2.2 Estimation of protein
The Biuret method of Racusen and Johnstone (1961) was adopted for
the estimation of soluble protein content. The reaction mixture consisted of
2.0 mL of Biuret reagent (3.8 g CuSO4⋅5H2O, 1.0 g KI, 6.7 g Na-EDTA, 200
mL 5 N NaOH in 1,000 mL of solution) and 0.1 mL of supernatant. The control
consisted of 0.1 mL of distilled water instead of supernatant. The optical
density was measured at 545 nm using a Hitachi U-1100 spectrophotometer.
The amount of protein was calculated from a standard curve of known protein
concentrations, which was prepared from bovine serum albumin.
2.4.1.2.3 Estimation of catalase
The catalase (E.C 1.11.1.6) activity was assayed according to Beers
and Sizer (1952) with certain modifications. The reaction was carried out
using two buffer solutions (A and B). Buffer A consisted of 50 mM potassium
phosphate (pH7.0), while buffer B was 0.036% H2O2 solution in 50 mM
potassium phosphate buffer (pH7.0). The reaction mixture consisted of 2.9 mL
buffer B and 0.1 mL of enzyme extract, while control consisted of only 3.0 mL
of buffer A. The enzyme activity was measured by time required for the
absorbance (at 240 nm) to decrease from 0.45 to 0.40 and expressed as units
per milliliter of enzyme.
2.4.1.2.4 Estimation of superoxide dismutase
The superoxide dismutase (SOD; E.C 1.15.1.1) activity was assayed
spectrophotometrically by measuring its ability to inhibit photochemical
reduction of nitroblue tetrazolium (NBT) according to Maral et al. (1977). Two
tubes were taken, each containing 2.0 mL of 1.0 mM sodium cyanide (NaCN),
13 mM methionine, 75 μM NBT, 0.1 mM EDTA, and 2.0 μM riboflavin as a
substrate. One tube was used as sample containing reaction mixture + 5.0 μL
enzyme extract, placed approximately 30 cm below the bank of two 30-W
fluorescent tubes for 15 min. The other tube containing reaction mixture
without enzyme extract was covered with black cloth at the same time. The
absorbance of the illuminated tube was compared to non-illuminated mixture
at 560 nm. SOD activity was expressed as units per milligram of protein.
28
2.4.1.3 Post-harvest analyses
The TSW-Soil mixtures and plants after harvesting were analyzed in
order to compare pre- and post-harvest trends on the basis of selected
parameters.
2.4.1.3.1 Post-harvest soil analyses
The TSW-Soil mixture from the pots after harvesting plants was
digested by the method given in 2.2.6 to assess the level of category-I as well
as category-II metals.
2.4.1.3.2 Post-harvest plant analyses
2.4.1.3.2.1 Morphological parameters and biomass yield
Different morphological parameters like, shoot length (cm), root length
(cm), seedling length (cm), no. of leaves and roots, fresh and dry weight (g)
as well as the plants were harvested, washed with deionized water and
wrapped in blotting paper for oven drying.
2.4.1.3.2.2 Digestion of plant material and HM estimation
The harvested biomass from pot trial was separated into root and
shoot; oven dried and digested after the method given by Huang and Schulte
(1985). The level of (mgkg-1) category-I as well as category-II metals
accumulation in different plant parts was assessed.
2.4.1.3.3 Post-harvest fungal analyses
Parallel to post-harvest physico-chemical analyses of the TSW-Soil
mixtures, the inoculated fungal CFUs (× 105 cfu g-1 soil) as well as no. of
spores (50 gm-1 soil) were determined.
2.4.1.3.4 Meta-analytical assays
The meta-analytical perspective of the proposed phytoextraction
technology assisted with fungi was developed on the basis of the following
parameters.
29
2.4.1.3.4.1 HM translocation index (%)
The HM translocation index (%) as given by Mattina et al., 2003 is used
to determine the efficiency of a plant to translocate HM from root to aerial
parts. The formula used is as under:
2.4.1.3.4.2 HM tolerance index (TI)
The TI value for different HM as given by Wilkins (1978) gives the ratio
of biomass yielded in TSW-Soil mixture to biomass yielded in soil (Control). Its
formula is given below:
2.4.1.3.4.3 Specific HM extraction yield percentage (SEY %)
The SEY (%) as explained by Audet and Charest (2006) represents
percent ratio of plant HM content to soil HM concentration and its formula is
given as under:
2.4.2 Pot trial II: Experiments with both saprobic and arbuscular
mycorrhizal (AM) fungi
The details of this pot experiment were similar to pot trial mentioned in
2.4.1 including live plant analyses, post-harvest and meta-analytical analyses
except for post-harvest fungal analyses in 2.4.1.2.3 where a new parameter of
percentage infection of AM fungi in roots (%) was incorporated. The AM fungi
were inoculated as described below:
2.4.2.1 Fungal inoculum
In order to inoculate TSW-Soil mixtures with AM fungi, a uniform layer
of about 15 gm fresh roots of Cynodon dactylon Pers., naturally infected with
AM fungi, collected from Botanical Garden, Dept. of Botany University of the
Punjab; were applied in pots 3 inches below the soil surface. The applied
grass roots were confirmed microscopically for AM infection prior to
inoculation in pots. The saprobic TS was applied in the form of conidial
30
suspension. For this purpose, TS was grown in 500-ml conical flasks
containing potato dextrose broth for 8 days. The cultures were filtered through
Whatman no. 1 filter paper and the mycelial mat was macerated using a
waring blender for 1 min and mixed with 250 ml of 0.1 M MgSO4.7H2O
solution. Ten ml of this inoculum containing 5 x 104 CFU per ml was used for
inoculating each pot.
2.4.2.2 Mycorrhizal and fungal assessment
The mycorrhizal growth was expressed in terms of extent of infection
while the activity of the saprobic fungus was estimated by the CFU from the
soil. For this purpose, fresh root samples were stained using 0.05% Trypan
Blue as described by Phillips and Hayman (1970) and the percent root
colonization was estimated by adopting the grid-line intersect method of
Giovanetti and Mosse (1980). Extramatrical chlamydospores in root-zone soil
samples were enumerated using the wet sieving and decanting method of
Gerdemann and Nicolson (1963). The population of TS in the root zone soil
was determined by using 2% MEA.
31
2.5 Evaluation of pot-trial findings at field level
For field trials, the selected site was the same from where garden soil
was taken for preparing TSW-Soil mixtures for pot trials. For this purpose, Plot
# 129 of the Botanical Garden, Dept. of Botany University of the Punjab
Lahore, Pakistan was selected (31O49’96.34’’ N, 74O30’08.53’’E).
2.5.1 Field plotting
2.5.1.1 Strip field plotting
Five strip plots (25 × 3 feet each) were prepared by digging and removing
soil up to 2.5 feet depth. There was a soil barrier of 2 feet width between each
of the strip plot (Figure 2.5.1 and 2.5.2). Four of the 5 ditches resulting after
removal of soil were lined with polythene sheet. The soil removed from all of
the ditches was air-dried by spreading it on plastic sheets separately to bring
its moisture level equal to the powdered TSW. One of the ditches with lining
and one without lining were filled back with relevant soil removed from them
without incorporating any TSW taken as Control. One of the ditches was not
lined with geothermal membrane in order to find any effect of lining of the plot
on plant growth. The remaining three ditches were filled back with their
respective soil after mixing appropriate ratios of TSW i.e. 5, 10 and 20% (w :
w) in order to prepare TSW-Soil mixtures similar to the soil mixtures prepared
for pot trials as described earlier in 2.2.
2.5.1.2 Sub-division of the strip plots for fungal inoculation
In order to apply fungal inoculations in strip plots following randomized
complete block design (RCBD), each of the strip plots was sub-divided into
four sub-plots (5 × 3 feet each separated by a soil barrier of 1.25 feet)
categorized as Control with no fungal inoculation, F1, F2 and F1 + F2, as
shown in Figure 2.5.1 and 2.5.2. An individual network of drip irrigation pipes
was applied in each of the sub-plots at about 3 inches depth of the soil
surface for injecting solution of fungal CFU.
The soil mixtures in all field plots were allowed to equate for 30 days.
During that period, there was neither any precipitation nor any pest attack
observed on plants growing in the vicinity of strip plots.
32
Figure 2.5.1. The statistical layout of the experimental units showing distribution of field plots lined with polythene sheet (upper). The vertical grey lines show the separation between experimental plots (25 × 3 feet each) arranged in split plot design with no replication having soil mixed with tannery solid waste (TSW-Soil % w:w) while the horizontal dark black lines show division of each strip plot into sub-plots (5 × 3 feet each) applied with fungal inoculations in a randomized complete block design. The aerial view of experimental station (lower).
Fun
gal i
no
cula
tio
ns
20 10 5 0 0*
TSW-Soil (% w:w) mixtures
0* = experimental plot with only soil as well as without polythene sheet while 0 is similar except being applied with polythene sheet as in the case of other TSW-Soil mixtures.
C = neither of the fungal inoculations applied
F1 = Aspergillus niger
F2 = Trichoderma pseudokoningii
F1 + F2 = one applied with both fungi together
The physical separation between experimental plots
N
33
Figure 2.5.2: Preparation of field plots in order to apply pot trial findings at field level: (A) excavation of soil from strip plots (25 × 3 feet each) up to 2.5 feet depth and lining with polythene sheet; (B) mixing of soil with 2 mm sieved tannery solid waste (TSW); (C) refilling of excavated strip plots with soil mixed with different percentages of TSW; (D) a strip plot refilled with TSW mixed soil; (E) the vertical orientation of 5 strip plots; (F) the sub-plotting of each of the strip plot into four experimental units each for fungal inoculations in a randomized complete block design.
A
E
B
C D
E F
34
2.5.2 Field Trial I: Experiments with Helianthus annuus
After 30 days of equating the field soils, certified seeds of Helianthus
annuus were sown during local peak crop season i.e. last week of Jan 2011.
The field capacity was maintained by irrigating the field with tap water
according to the agricultural recommendations about sunflower crop.
2.5.2.1 Fungal inoculations
When the seedlings were 2 weeks older, approximately 25 liter solution
containing CFU 76,800/ml of TS and CFU 73,800/ml of AN of the inoculum
were applied in each of the sub-plots by injecting through network of drip
irrigation pipes lined at about 3 inches depth of the soil surface.
2.5.2.2 Live plant analysis-Biochemical assays
The detail of live plant analyses based on biochemical assays SPAD
values were similar to the methods given in 2.4.1.2.
2.5.2.3 Harvesting
The sunflower plants were harvested after the sunflower petals started to
wither off and the seeds were mature. The harvested aerial biomass i.e.
leaves, shoots and roots were collected separately in zip-loc bags for lab
analysis after observing morphological parameters and fresh weight. All of the
roots were collected carefully in a 2 mm sieve and washed with distilled water
before blotting and placing in zip-loc bags. The residual water resulting from
washing was returned back to their respective sub-plot altogether in order to
remove experimental error.
2.5.2.4 Post-harvest analyses
The detail of post-harvest analyses was similar to the descriptions
given in 2.4.1.3.
35
2.5.3 Field Trial II: Experiments with Tagetes patula
The certified seeds of Tagetes were sown during its local peak crop
season i.e. last week of Oct 2011. The rest of the details with regard to pre-
and post-harvest analyses was similar to experiment with sunflower as
described in 2.5.2.
2.6 Phytoextraction studies assisted with Trichoderma
harzianum (TH) and TS in a pot trial at Cornell University,
New York USA
The experiment was set in greenhouse at Cornell University in 2012 as
part of PhD research work. The visit was funded by Higher Education
Commission (HEC) Pakistan under International Research Support Initiative
Program (IRSIP). The greenhouse pot trial was aimed at studying effect of
saprobic Trichoderma harzianum (TH) and Trichoderma pseudokoningii (TS)
on phytoextraction potential of Tagetes patula using three TSW-soil mixtures
i-e 0, 5, and 10%. The TH was provided by Dr. Gary E. Harman, Professor at
Dept. of Horticulture, Cornell University Geneva campus; while TS isolates
were similar to those used in the research work done in Pakistan.
2.7 Statistical analyses
The level of significance among treatments was determined using two-
way analysis of variance (ANOVA). In order to compare treatment means,
least significant difference (LSD) test was applied, while to pin-point exact
difference among group of means, Duncan’s multiple range test (DMRT) was
applied. The SPSS software version 11.3 and statistical power package of the
Microsoft Office Excel 2010 was used for all the statistical analyses.
Chapter 3
Results
36
CHAPTER 3
RESULTS
3.1 Soil and TSW analyses
3.1.1 Physico-chemical properties of TSW and garden soil
The physico-chemical properties of TSW, garden soil and their
selected mixtures are given in Table 3.1. The pH of the TSW was significantly
higher (8.9) as compared to garden soil (7.1). On spiking with TSW, the pH of
garden soil start increasing and it reached as high as 8.1 at 20 %. The TSW-
Soil mixtures beyond 20 % were not carried along for further experimentation
and one of the reasons was such a high pH that didn’t let the seeds of
selected plants to germinate. The values of ECe (dScm-1), bicarbonates (mgL-
1) as well as chlorides (mgL-1) were found to be the maximum in TSW and
were significantly higher as compared to garden soil. The ECe increased
significantly in each of the TSW-Soil mixture with increasing amount of TSW
and showing a increasing trend from 0 (garden soil only) to 5, 10 and 20%
mixtures. Similar trends were found for both NaCl (%) and bicarbonates (mgL-
1). The carbonates (mgL-1) were observed to be BDL in both garden soil and
TSW. The bulk density (gcm-3) of TSW was extremely low as compared to soil
and spiking soil with TSW decreased its bulk density. However, organic
matter (OM %) in TSW was significantly higher as compared to garden soil
and blending of soil added OM increments to the soils, as given in Table
3.1.1.
37
Table 3.1.1. The physico-chemical properties of TSW, garden soil and their various (% w:w TSW-Soil) mixtures
Parameters
TSW-Soil mixtures (% w:w)
LSD0.05 0
(Soil only)
5 10 20 50 100
(TSW only)
pH 7.1bc
7.4c 8.0
b 8.1
ab 8.3
ab 8.9
a 0.515
ECe (dScm-1
) 0.02f 0.12
e 0.31
d 0.81
c 1.42
b 2.89
a 0.053
NaCl (%) 2.9e 3.5
e 52.3
d 125.7
c 255.1
b 421.5
a 0.68
Bicarbonates (mgL-
1)
103.7f 125.3
e 152.5
d 189.1
c 262.3
b 359.9
a 1.279
Carbonates (mgL-1
) ND ND ND ND ND ND -
Chlorides (mgL-1
) 62.1ef 76.8
e 255
d 812
c 2,233
b 3,118
a 17.79
Organic matter (%) 2.61c 2.90
bc 3.1
b 3.4
b 3.8
ab 4.5
a 0.657
Bulk Density (gcm-
3)
1.06a 1.04
a 1.02
ab 0.92
b 0.73
c 0.66
d 0.10
The mean values S.D. with common letters (along the row) are not significantly different according to Duncan’s multiple range test (P = 0.05). LSD = Least Significant Difference (P = 0.05)
3.1.2 Category-I metals in TSW and garden soil
The TSW exhibited the maximum concentration of Category-I metals
(detected with flame photometer) i.e. Ca, K and Na; and it was significantly
higher as compared to garden soil, as shown in Table 3.1.2. Blending soil with
TSW elevated the concentration of all the three metals in different TSW-Soil
mixtures. Such a trend of variation was observed for not only the total metal
fraction of Ca, K and Na, but also for the water soluble and DTPA-extractable
part. The Ca found to be the maximum in TSW, being significantly highest
than soil and its mixtures with TSW. Similarly, K and Na were also found to be
the maximum in TSW and significantly highest than any of the soil treatments.
38
Table 3.1.2. The total, water soluble and DTPA-extractable fraction of Category-I metals (mgkg-1
) in TSW, garden soil and their various (% w:w TSW-Soil) mixtures
Category-I metal fraction
TSW-Soil mixtures (% w:w)
LSD0.05 0
(Soil only)
5 10 20 50 100
(TSW only)
Water soluble (mgkg
-1)
Ca 25
e ±
4.08 135
de ±
10.61 190
d ±
16.33 255
c ±
36.74 425
b ±
36.74 940
a ±
40.83 64.84
K 90
ef ±
16.33 125
e ±
12.25 235
d ±
20.41 410
c ±
40.83 690
b ±
40.83 1,105
a ±
85.73 100.83
Na 15
f ±
4.1 35
e ± 4.1 90
d ± 9.8
280b ±
7.4 120
c ± 7.4 890
a ± 13 18.98
DTPA-extractable
(mgkg-1
)
Ca 55
f ±
8.17 450
e ±
32.66 660
d ±
65.32 830
c ±
32.66 990
b ±
65.32 1,240
a ±
81.65 123.94
K 210
e ±
16.33 380
d ±
40.83 545
cd ±
44.9 675
c ±
28.6 1,040
b ±
106.14 1,780
a ±
73.5 137.55
Na 250
f ±
12.2 995
e ±
11.4 1,270
d ±
114.3 1,920
c ±
40.9 2,130
b ±
42 2,685
a ±
61.3 134.3
Total (mgkg
-1)
Ca 95
f ±
8.17 1,625
e ±
20.41 2,345
d ±
110.23 2,910
c ±
89.82 3,420
b ±
179.83 6,320
a ±
97.98 235.02
K 664
e ±
11.4 890
e ±
65.3 1,210
d ±
16.3 1,980
c ±
57.2 2,340
b ±
81.7 4,210
a ±
49 122.5
Na 995
f ±
81.7 1,355
e ±
61.2 2,515
d ±
20.4 4,645
c ±
53.1 6,350
b ±
45 9,440
a ±
57.2 129.29
The mean values S.D. with common letters (along the row) are not significantly different according to Duncan’s multiple range test (P = 0.05). LSD = Least Significant Difference (P =
0.05)
3.1.3 Category-II metals in TSW and garden soil
Likewise, the concentration of Category-II (AAS detected) metals in
TSW was found to be the maximum and significantly highest than soil and its
selected mixtures with TSW. Being a true representative of the cumulative
chrome tanning waste, the TSW showed extremely high concentration of Cr
and it was highly significantly greater than Cr in garden soil, as shown in
Table 3.1.3.
39
Table 3.1.3. The total, water soluble and DTPA-extractable fraction of Category-II metals (mgkg
-1) in TSW, garden soil and their various (% w:w TSW-Soil) mixtures
Category-II metal fraction
TSW-Soil mixtures (% w:w)
LSD0.05 0
(Soil only)
5 10 20 50 100
(TSW only)
Water soluble (mgkg
-1)
Cd 15
f ±
2.5 490
e ±
16.3 610
d ±
12.3 790
c ±
16.3 935
b ±
19.6 2,750
a ±
49 55.4
Cr BDL 1,350
e ±
120 1,970
d ±
52.3 2,250
c ±
50.6 3,450
b ±
65.3 4,570
a ±
106 176.4
Cu 110
e
± 7.3 235
d ±
11.4 580
c ±
21.2 965
c ± 13
1,160b ±
21.2 2,260
a ±
55.5 62
Fe 15
e ±
2.5 70
d ±
10.6 95
cd ±
11.4 115
c ± 13
225b ±
19.6 570
a ±
23.7 34.7
Mg 10
ef ±
3 35
e ±
3.1 65
d ± 7.4 95
c ± 14
270b ±
15.5 630
a ±
19.6 28.1
Ni BDL BDL BDL BDL BDL BDL -
Pb BDL BDL BDL BDL BDL BDL -
Zn 15
f ±
3.8 45
e ±
5.6 85
d ± 7
130c ±
7.3 190
b ± 4
335a ±
11.4 16.2
DTPA-Extractable
(mgkg-1)
Cd 35
f ±
3.3 1,460
e ±
13.9 3,780
d ±
69.4 3,920
c ±
18 4,230
b ±
33.5 5,030
a ±
81.7 107.7
Cr 55
f ±
4.9 3,560
e ±
62.1 4,350
d ±
41.7 5,450
c ±
44.9 6,750
b ±
68.6 9,050
a ±
49.1 114.4
Cu 235
f ±
12.2 780
e ±
22.8 1,350
d ±
49.8 1,790
c ±
59.6 2,750
b ±
53 5,640
a ±
196 205.7
Fe 40
f ±
6.5 120
e ±
6.9 230
d ±
13.9 640
c ±
7.4 870
b ±
14.7 1,030
a ±
25.3 32.5
Mg 30
f ±
7.2 150
e ±
8.7 340
d ±
22.8 540
c ±
23.3 1,240
b ±
24 1,930
a ±
25.9 46.4
Ni BDL BDL 25d ± 1.4 55
c ± 8.3
110b ±
11.2 370
a ±
13.6 18.7
Pb BDL BDL BDL BDL BDL BDL -
Zn 110
e
± 10.6 270
de ±
9 325
d ±
18.9 450
c ±
29.4 960
b ±
33.5 1,035
a ±
37.6 59
Total (mgkg
-1)
Cd 55
f ±
5.7 2,650
e ±
53.1 6,580
d ±
81.7 8,750
c ±
57.2 9,720
b ±
49 10,097
a ±
87.4 142
Cr 110
e
± 7.4 8,250
d ±
48.9 10,250
cd ±
163 15,510
c ±
155 19,520
b ±
139 25,534
a ±
191 310.2
Cu 600
f ±
44 1,350
e ±
57.1 2,100
d ±
8.2 5,250
c ±
57.2 7,510
b ±
81.6 10,554
a ±
125.7 165.5
Fe 50
f ±
6.7 250
e ±
13 510
d ±
28.6 910
c ± 18
1,230b ±
25.3 2,250
a ±
40 56.4
Mg 45
f ±
6.1 310
e ±
21 620
d ± 79
1,020c ±
89 2,870
b ±
101 3,840
a ±
118 196
Ni BDL 35
de ±
1.3 55
d ± 10.7
110c ±
9.2 310
b ±
19.6 590
a ±
20.4 39.8
Pb BDL BDL BDL BDL BDL BDL -
Zn 218
e
± 16.3 1460
d ±
17.1 1890
c ±
19.6 2025
b ±
25.3 5,150
ab ±
57.2 7,590
a ±
15.5 67
The mean values S.D. with common letters (along the row) are not significantly different according to Duncan’s multiple range test (P = 0.05). LSD = Least Significant Difference (P =
0.05)
Similarly, the concentration of Cd and Cu were found to be the maximum in
for the water soluble, DTPA-extractable as well as total metal fraction of the
Category-II metals present in the TSW. The mixing of garden soil with TSW
40
had incremented Cr, Cd and Cu in accordance with increasing percentage of
TSW. The water soluble fraction of Fe and Mg found to be the lowest in TSW,
soil and their various mixtures, followed by DTPA-extractable; however, their
total metal fraction being the highest. The Pb found to be BDL in all the TSW-
Soil mixtures. The water soluble fraction of Ni was also BDL. However,
considerable concentrations of DTPA-extractable and total fraction of Ni were
noticed to be present. As far as Zn is concerned, its total metal fraction was
highest, followed by DTPA-extractable and water soluble fraction, respectively
for TSW, soil and their various mixtures.
41
3.2 Isolation and identification of TSW representative fungi
Using three different fungal nutrient media i.e. 2% MEA, Czapek’s and
MMN medium, 13 autochthonous isolates were purified and confirmed on the
basis of the colony morphology after verification from “The First Fungal Bank
of Pakistan”, University of the Punjab, Lahore Pakistan. There were as many
as nine species of Aspergillus, one species of each of Alternaria, Fusarium,
Rhizopus and Trichoderma. The figure 3.2.1 – 3.2.13 are given to show the
colony morphology of the pure isolates used for further experimentation:
Figure 3.2.1. Alternaria alternata on Czapek’s medium at
250C isolated from TSW.
Figure 3.2.2. Aspergillus flavipus on 2% MEA medium at 25
0C.
Figure 3.2.3. Aspergillus fumigatus on Czapek’s medium
at 250C
Figure 3.2.4. Aspergillus parasiticus on Czapek’s medium at 25
0C
Figure 3.2.5. Aspergillus terreus on MMN medium at 25
0C isolated from TSW
Figure 3.2.6. Fusarium sp. on Czapek’s medium at 25
0C isolated from TSW.
42
Figure 3.2.7. Rhizopus arrhizus on 2% MEA basal
medium at 250C isolated from TSW.
Figure 3.2.8. Trichoderma pseudokoningii on Czapek’s medium at 25
0C isolated from TSW.
Figure 3.2.9. Aspergillus flavus on Czapek’s medium at
250C isolated from TSW.
Figure 3.2.10. Aspergillus japonicus on 2% MEA basal medium at 25
0C isolated from TSW.
Figure 3.2.11. Aspergillus niger on MMN medium at 25
0C
isolated from TSW. Figure 3.2.12. Aspergillus penicilloides on Czapek’s
medium at 250C isolated from TSW.
Figure 3.2.13. Aspergillus versicolor on 2% MEA medium at 25
0C isolated from TSW.
43
3.3 Screening and selection of heavy metal resistant autochthonous
fungi
During screening process of autochthonous fungi on 2 % MEA
prepared in autoclaved extract of TSW, the isolates of Trichoderma
pseudokoningii and Aspergillus niger found to be the best in terms of
tolerance index (TI) as compared to their respective control i.e. isolates of
Trichoderma and Aspergillus cultivated on 2 % MEA prepared in distilled
autoclaved water respectively, as given in Table 3.3.1. The TI value for
Alternaria alternata and Fusarium sp. found to be third and fourth respectively,
among all of the 13 tested fungi. The TI gave an indication of a fungus’
tendency to resist colony radius delimitation caused by the very high level of
heavy metals incorporated in 2 % MEA through autoclaved MSW extract. The
order of heavy metal tolerance of fungi on the basis of TI values observed to
be, Trichoderma pseudokoningii > Aspergillus niger > Alternaria alternata >
Fusarium sp.
Table 3.3.1. The tolerance index (TI) of various fungi cultivated on 2 % MEA prepared in autoclaved extract of TSW along with control of each of the fungus cultivated on 2 % MEA prepared in distilled autoclaved water.
Sr # Fungi Tolerance Index (TI)
1 Alternaria alternata 0.93ab
2 Aspergillus flavipus 0.32
g
3 Aspergillus fumigatus 0.78c
4 Aspergillus Parasiticus 0.21h
5 Aspergillus terreus 0.28gh
6 Fusarium spp 0.89
b
7 Rhizopus arrhizus 0.49ef
8 Trichoderma pseudokoningii 0.97a
9 Aspergillus flavus 0.46ef
10 Aspergillus japonicas 0.69d
11 Aspergillus niger 0.96a
12 Aspergillus penicilloides 0.42f
13 Aspergillus versicolor 0.51e
The values with different letters are significantly different as per Duncan’s multiple range test (n = 3; P = 0.05). The screening experiment repeated thrice.
From the 13 fungi used in screening process, four of them with highest
TI value were selected for further mutual fungal interaction study while
withdrawing rest of them for further experimentation.
44
3.4 In vitro fungal mutual growth interaction studies for Category-II metal
tolerance
Analogous to a tug of war, the two fungi inoculated on opposite sides of
the vertical line crossing the petri plat at its center, tried to overwhelm each
other in terms of radial growth of their respective colony on from point of
origin, across the central vertical line to the opposite fungus’ point of origin.
The relative superseding by one fungus over its counterpart was evaluated on
the basis of radial growth in mutual combination with respect to individual
growth rate (taken as control), maximum colony diameter (grown along with
partner and individually), and mycelial growth towards and away from the
other colony, as given in Table 3.4.1.
3.4.1 Aspergillus niger vs. Trichoderma pseudokoningii
The radial growth area covered by A. niger receded as compared to
radial colony expansion exhibited by T. pseudokoningii across the vertical line
at the center of petriplate to quite near the point of origin of A. niger as can be
observed in Figure 3.4.1. The T. pseudokoningii growth was approximately 65
% while for A. niger it observed to be approximately 35 % of the total area. As
compared to their respective controls, both of the competitor fungi displayed
relatively less growth in mutual interaction plate. Conclusively, T.
pseudokoningii was selected over its counterpart for further study.
Figure 3.4.1. In vitro mutual growth interaction (center) between screened heavy metal tolerant Aspergillus niger (right) vs. Trichoderma pseudokoningii (left)
A. niger (Control) A. niger vs. T. pseudokoningii T. pseudokoningii (Control)
45
3.4.2 Alternaria alternata vs. Trichoderma pseudokoningii
The growth area covered by A. alternata and T. pseudokoningii was
not equal with respect to one another. The T. pseudokoningii overlapped its
opponent A. alternata and suppressed its growth as much as approximately
70 % while limiting A. alternata to as low as approximately 30% of the total
area of the petriplate, as shown in Figure 3.4.2. Conclusively, T.
pseudokoningii found to be better over A. alternata.
Figure 3.4.2. In vitro mutual growth interaction (center) between screened heavy metal tolerant Alternaria alternata (left) vs. Trichoderma pseudokoningii (right)
A. alternata (Control) A. alternata vs. T. pseudokoningii T. pseudokoningii (Control)
3.4.3 Fusarium sp. vs. Alternaria alternata
During this paired mutual fungal interaction study, the Fusarium sp.
displayed growth recession in terms of radial growth as compared to
Alternaria alternata, as given in Figure 3.4.3. The latter autochthonous
saprobic fungal strain overlapped its counterpart up to 80 % and supressed
growth of Fusarium sp. limiting it to only 20 % of the total petriplate area.
Conclusively, A. alternata prevailed over Fusarium sp.
Figure 3.4.3. In vitro mutual growth interaction (center) between screened heavy metal tolerant Fusarium sp. (left) vs. Alternaria alternata (right)
Fusarium sp. (Control) Fusarium sp. vs. A. alternata A. alternata (Control)
46
3.4.4 Aspergillus niger vs. Alternaria alternata
The growth area covered by A. niger was overwhelmingly greater than
A. alternata i.e. the whole petriplate was overlaid by A. niger growth,
suppressing underlying A. alternata, as can be seen in Figure 3.4.4. In the
control of each autochthonous saprobic fungal strain, the growth area was
nearly equal.
Figure 3.4.4. In vitro mutual growth interaction (center) between screened heavy metal tolerant Aspergillus niger (left) vs. Alternaria alternata (right)
A. niger (Control) A. niger vs. A. alternata A. alternata (Control)
3.4.5 Fusarium sp. vs. Trichoderma pseudokoningii
During the mutual growth interaction of Fusarium sp. and T.
pseudokoningii, it was observed T. pseudokoningii overlapped Fusarium sp.
The growth area covered by T. pseudokoningii was approximately 80 % as
compared to Fusarium sp. which covered approximately 20 % of the total
growth area of petriplate as shown in Figure 3.4.5. The rate of colony
expansion resulting from radial growth observed to be extremely quick in
Trichoderma pseudokoningii as compared to Fusarium sp.
Figure 3.4.5. In vitro mutual growth interaction (center) between screened heavy metal tolerant Fusarium sp. (left) vs. Trichoderma pseudokoningii (right)
Fusarium sp. (Control) Fusarium vs. T. pseudokoningii
T. pseudokoningii (Control)
47
In case of Control, both of the mutually interacting fungi displayed
nearly equal growth, as can be seen in the Figure 3.4.5. Conclusively, T.
pseudokoningii observed to be dominant over Fusarium sp. in terms of in vitro
mutual competitive growth.
3.4.6 Fusarium sp. vs. Aspergillus niger
In case of interaction between Fusarium sp. and A. niger, growth area
covered by the latter fungus was about 80 % of the total petri plate area while
the former receding itself to an area of as low as 20 %, as can be observed in
Figure 3.4.6. In case of Control; however, both of the fungi displayed healthy
radial growth. Conclusively, A. niger found to be far better than Fusarium sp.
in terms of in vitro competitive growth.
Figure 3.4.6. In vitro mutual growth interaction (center) between screened heavy metal tolerant Fusarium sp. (left) vs. Aspergillus niger (right)
Fusarium sp. (Control) Fusarium sp. vs. A. niger A. niger (Control)
3.4.7 Comparison of the six paired mutual interactions of screened fungi
The comparison of the six pairs of fungi mutually interacting for
competitive growth under in vitro conditions is given in Table 3.4.1.
Table 3.4.1. Comparison of six pairs of mutually interacting fungi for growth competition on the basis of various morphological parameters observed after 10 days of fungal inoculation.
Pair # Mutually
interacting fungi
Parameters
Colony diameter
(cm)
Relative growth area
(%)
Direction of mutual mycelial growth
(proceed/recede)
Colony morphology (color)
Pair I A. niger vs.
T. pseudokoningii 4 35 Proceed black 4 65 Proceed green
Pair II A. alternata vs. T.
pseudokoningii 3 30 Recede black 5 70 Proceed green
Pair III Fusarium sp. vs.
A. alternata 1 20 Recede white 3 80 Proceed black
Pair IV A. niger vs. A.
alternata 5 100 Proceed black 0 0 Recede black
Pair V Fusarium sp. vs. T.
pseudokoningii 1 20 Recede white 4 80 Proceed green
Pair VI Fusarium sp. vs.
A. niger 1 20 Recede white 4 80 Proceed balck
48
The order of dominance displayed by fungi on the basis of relative
growth area (%) was as: T. pseudokoningii = A. niger > A. alternata >
Fusarium sp.
Consequently, T. pseudokoningii and A. niger were found to be the
best fungi for Category-II metal tolerance after the screening process of metal
tolerance and mutual fungal interaction. Both of them were selected for the
forthcoming pot and field trial aimed at using these fungi as bio-reinforcers for
increasing the biomass production of marigold plant.
49
3.5 In situ mutual growth interaction studies for screened Category-II
metal tolerant fungal isolates with Tagetes patula in soil
On inoculating soil with four fungal isolates either individually or in
combinations, the T. patula responded variably in relation to the type and
number of fungi applied in the pot soil. The plants with F1 + F2 inoculations
yielded maximum root-, shoot- and seedling length; fresh and dry weight; as
well as chlorophyll content (SPAD value), as given in Table 3.5.1. The relative
variation in vegetative growth can also be observed in Figure 3.5.1.
The F1 treatment gave the second highest values for all the vegetative
growth parameters, while F2 treatment being the third among all the fungal
Table 3.5.1. Morphological and biochemical parameters for 50-days old pot cultivated plants of Tagetes
patula cultivated in soil and applied with individual and combined fungal inoculations. The mean values ± SD with different letters are significantly different according to Duncan’s multiple range test (n = 3; P =
0.05).
Treatments Root
length (cm)
Shoot length (cm)
Seedling length (cm)
Chlorophyll content
Fresh weight (g)
Dry weight (g)
Control 14.9
bc ±
1.36 15.7
b ± 2.04 31.5
bc ± 2.04 13.1
b ± 2.53 4.9
b ± 1.14
1.47b ±
0.14
F1 17.2b ± 0.49 16.1
b ± 0.98 33.6
b ± 2.12 15.9
ab ± 2.44 5.6
ab ± 1.31 1.74
ab ± 0.2
F2 16.1b ± 0.9 14.8
b ± 1.47 31.2
bc ± 1.8 14.5
ab ± 2.85 4.7
b ± 1.39
1.41b ±
0.25
F3 6.2cd
± 0.65 4.1e ± 0.65 10.7
ef ± 1.39 9.9
bc ± 2.37 2.1
c ± 0.33
0.63cd
± 0.11
F4 5.5ef
± 0.89 6.4de
± 1.41 12.1e ± 0.89 10.7
b ± 2.20 2.2
c ± 0.49
0.68cd
± 0.15
F1+F2 34.6a ± 1.88 23.3
a ± 1.96 58.1
a ± 1.71 19.5
a ± 2.86 7.2
a ± 1.39
2.16a ±
0.13
F1+F3 10.4d ± 1.31 12.4
c ± 1.14 23
cd ± 2.44 17.3
a ± 1.04 3.4
bc ± 0.32
1.05bc
± 0.12
F1+ F4 14.1c ± 2.45 11.1
c ± 1.72 25.3
c ± 2.69 16.6
ab ± 2.12 4.6
b ± 1.3
1.43b ±
0.26
F2+ F3 9.8d ± 1.63 10.9
c ± 1.55 21.3
cd ± 1.88 9.3
bc ± 1.88 3.2
bc ± 0.57
0.96c ±
0.32
F2+ F4 7.1e ± 0.98
10.6cd
± 1.31
18d ± 1.63 10.2
bc ± 1.79 2.3
c ± 0.73
0.69cd
± 0.16
F3+F4 5.3ef
± 0.73 7.5d ± 1.22 13
e ± 2.44 7.1
c ± 1.71 2.1
c ± 0.41
0.65cd
± 0.12
F1+F2+F3+F4 7.4e ± 1.22 8.2
d ± 0.98 15.9
d ± 2.37 11.9
b ± 2.37 2.4
c ± 0.33 0.74
cd ± 0.2
LSD0.05 3.04 3.13 4.42 4.92 2.02 0.43
The mean values S.D. with common letters (along the row) are not significantly different according to Duncan’s multiple range test (P = 0.05). LSD = Least Significant Difference (P =
0.05)
50
inoculations. Surprisingly, increasing number of isolates in the fungal
inoculant decreased the values of all the parameters, as can be seen in case
of F1 + F2 + F3 + F4 treatment, given in Table 3.5.1.
On the basis of the maximum biomass yield incurred by individual as
well as combined inoculation, both F1 and F2 were selected for further
experimentation.
Figure 3.5.1. Pot cultivated 50-days old plants of Tagetes patula with vegetative growth variation in response to individual and combined fungal inoculations in soil.
51
3.6 Screening of heavy metal tolerant ornamental plant species for
phytoextraction of TSW-Soil mixtures
During screening process, the plants’ suitability for phytoextraction of
TSW-Soil mixtures was judged on the basis of seed germination (%).
3.6.1 Seed germination (%)
The plants of Tagetes patula, Petunia xybrid, Dahlia coccinia, Zinnia
elegans and Helianthus annuus were tested for their germination response on
selected range of TSW-Soil (% w:w) mixtures. It was observed that T. patula
and H. annuus displayed the maximum germination in 20 % TSW:Soil, while
Petunia and Dahlia could come up only in 10 % of the TSW:Soil mixtures.
The Zinnia, however, was able to survive only in simple soil i.e. control. The
relative germination on different TSW-Soil mixtures is given in Table 3.6.1.
Table 3.6.1. Screening of plants for their phytoextraction potential on the basis of percentage germination observed in different TSW-Soil (% w:w) mixtures. The values
± S.D. are mean of three replicates.
TSW-Soil mixtures (% w:w)
Plants considered for screening
Tagetes Patula Helianthus
annuus Petunia xybrid
Dahlia coccinea
Xinia elegans
Soil 100 ± 0 100 ± 0 100 ± 0 100 ± 0.19
100 ± 0
5 88 ± 0.82 88 ± 0.67 55 ± 0.92
55 ± 1.3 NG
10 77 ± 1.73 80 ± 0.65 22 ± 0.38
44 ± 0.98
NG
15 - 80 ± 0.15 NG NG NG 20 77 ± 0.92 75 ± 0.98 NG NG NG 50 NG NG NG NG NG
100 NG NG NG NG NG
The relative germination rate of selected plants in response to the
TSW-Soil mixture is given in Figure 3.6.1.
On the basis of germination (%) results, T. patula and H. annuus were
selected for further experimentation while neglecting rest of the two plants.
52
Figure 3.6.1. Screening of plants for their phytoextraction potential on the basis of percentage germination on different TSW-Soil (% w:w) mixtures. A) Tagetes patula B) Patunia xybrid C)
Dahlia coccinea D) Zinnia elegans
B
C
D
A
E
53
3.7 Pot experiments with Marigold on autoclaved (AS) and non-
autoclaved TSW-Soil mixtures (NAS) to verify bio-reinforcing role of
fungi
3.7.1 Pre-sowing analysis of TSW-Soil mixtures
The physico-chemical properties, concentration of Category-I & II
metals are given in Table 3.1, 3.2 and 3.3 respectively and the details are
described in Chapter 3.1.
3.7.2 Biochemical analyses of Tagetes patula
Overall, inoculating soil and its TSW mixtures with selected fungi
increased the stress alleviation tendency of the plants by increasing its
tendency to produce more chlorophyll, soluble protein and different enzymes
as compared to plants where soil was not applied with fungi. The plants from
NAS treatments performed relatively better as compared to respective AS for
all the TSW-Soil mixtures.
The comparisons for influence of fungal inoculations on phytoextraction
parameters are described along the row of a table while the comparisons for
influence of TSW percentages in soil on phytoextraction parameters are
compared down the columns of the same table. This method of description
has been followed for all the forthcoming data tables.
The specific details of each of the biochemical parameters are as
under:
3.7.2.1 Chlorophyll content
After 50 days of cultivation, the plant chlorophyll content observed to
increase with the application of fungal inoculations to soil, as can be seen
along the row of Table 3.7.1. The maximum quantity within row for soil and
rest of the TSW-Soil mixtures observed where combined fungal inoculations
i.e. the F1 + F2 were applied, as given in Table 3.7.1. The F1 + F2 cause the
greatest (29.1 SPAD value) increase in plants from 5 % NAS while influencing
to the least (11.9 SPAD value) in 20 % AS as compared to any of the
treatments within a row. The AS from C gave the least (8.76 SPAD value)
54
performance in terms of chlorophyll contents. Overall, the autoclaving of soil
and its TSW mixtures significantly decreased plant chlorophyll contents than
their respective non-autoclaved treatments.
Within column, the general trend was decrease in plant chlorophyll
contents with the increase of TSW percentage in soil for all the fungal
treatments. The F1 + F2 from the 5 % NAS while 20 % AS with C gave the
best and the poor (performances respectively like the way it was found in
within-a-row comparison. The application of fungus either individually or in
combination help plants perform better as compared to Control in terms of
chlorophyll contents, as given in Table 3.7.1.
3.7.2.2 Soluble protein contents
Parallel to chlorophyll contents, the values within a row for
soluble protein contents also increased with the application of fungal
inoculations for all the treatments except for the AS from Soil where it was
below the detection limits, as given in Table 3.7.1. The F1 + F2 from 5 % NAS
and the C from 20 % AS exhibited the maximum (21 mgg-1) and the minimum
(4 mgg-1) soluble protein contents as compare to any of the soil or fungal
treatments. The non-autoclaved treatments caused increased in plant soluble
protein contents as compared to their respective autoclaved ones except for
Soil where AS didn’t give any values at all.
Within a column, overall there was decrease in soluble protein contents
with the increasing level of TSW in soil treatments. However, addition of fungi
helped plants to reduce stress by increasing soluble protein contents. The F1
+ F2 overwhelm F1, F2 and C for all of the soil treatments. The 5 % NAS with
F1 + F2 and the 20 % AS with C inoculations observed to have the maximum
and the minimum values respectively. The plants cultivated in pots with F1
inoculations performed better than those with F2 for all the TSW-Soil
mixtures.
3.7.2.3 Superoxide dismutase (SOD) contents
Parallel to the trends found for chlorophyll and soluble protein contents,
the SOD values increased with fungal inoculations within a row for all the
55
treatments, as given in Table 3.7.1. The fungal treatments having both of the
F1 and F2 performed better than control and those applied with either F1 or
F2. The plants in 5 % NAS with F1 + F2 gave maximum SOD values (44 Umg-
1 of protein) while being the least (6 Umg-1 of protein) in case of plants from
Soil AS with no fungal inoculations. The plants from NAS leaded the AS for all
of the treatments by giving significantly large SOD values over their respective
counterparts except for Soil-C treatment.
Within a column, the trend of SOD increase or decrease was similar to
that of chlorophyll and soluble protein contents. There was decrease in SOD
with the increasing fraction of TSW in soil for NAS and AS of all of the
treatments. The F1 + F2 plants performed better than control, F1 as well as
Table 3.7.1. The biochemical parameters observed in 50-days old Tagetes patula cultivated
on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w)
Type
Fungal treatments
LSD0.05 C F1 F2
F1+F2
Chlorophyll content (SPAD value)
Soil NAS 16.8
abB 0.11 19.21
bB 0.80 18.98
bB 0.55
26.49aAB
0.22
4.71
AS 15.8abBC
0.20 16.5bBC
0.20 17.1bC
0.24 20.4aC
0.12 2.7
5 NAS 20.9
bA 0.17 26.1
abA 0.22 25.4
abA 0.14 29.1
aA 0.28 4.2
AS 17.2abB
0.17 18.3abBC
0.19 19.5abB
0.14 21.7aC
0.22 3.98
10
NAS 18.40bAB
0.45 20.1bB
0.14 20.6bB
0.27 25.3aB
0.13 4.54
AS 10.40cC
0.25 12bC
0.22 13.2bD
0.17 16.83aD
0.13 2.28
20 NAS
15.39bBC
0.28
16.98abBC
0.75 16.68
abC
0.28 20.20
aC 0.20 3.59
AS 8.76cCD
0.65 9.8bD
0.87 10.1bE
0.28 11.9aE
0.72 1.21
LSD0.05 3.04 2.99 3.17 3.85
Soluble Protein content
(mgg-1
)
Soil NAS 0.5
aE 0.67 0.4
abE 0.38 0.5
aE 0.03 0.6
aE 0.87 0.22
AS BDL BDL BDL BDL -
5 NAS 16
bA 0.35 18
abA 0.34 17
bA 0.15 21
aA 0.87 3.90
AS 10bcBC
0.98 13abBC
0.91 12bB
0.19 15aB
0.76 3.06
10 NAS 12
bcB 0.71 15
bB 0.61 14
bAB 0.81 19
aAB 0.10 3.33
AS 9abC
0.73 7bCD
0.23 6bcCD
0.82 10aC
0.71 1.18
20 NAS 8
bC 0.53 9
abC 0.45 8
bC 0.19 10
aC 0.72 1.21
AS 4bD
0.91 4.5bD
0.28 5abD
0.45 6aD
0.26 1.13
LSD0.05 2.79 2.18 2.05 2.25
SOD (Umg
-1 of protein)
Soil NAS 7
cD 0.14 11
bcE 0.33 13
bD 0.19 16
aD 1.39 2.47
AS 6cD
0.16 8bF
0.16 7bcE
0.56 11aE
0.19 1.71
5 NAS 32
bcA 0.57 35
bA 0.39 37
bA 0.78 44
aA 1.2 5.19
AS 25bC
0.12 21bcDE
0.67 20cC
0.98 39aB
0.91 4.28
10 NAS 28
cB 0.17 31
bB 0.23 33
bAB 0.45 38
aB 0.19 4.2
AS 24bC
0.45 23bcD
0.87 25bBC
0.34 35aBC
0.75 3.99
20 NAS 30
bAB 0.34 27
bcC 0.45 28
bcB 0.23 34
aBC 0.45 3.83
AS 27bB
0.55 25bcCD
0.89 24bBC
0.78 31aC
0.37 3.11
LSD0.05 2.88 2.43 4.19 4.98
CAT (Uml
-1)
Soil NAS 0.3
bcDE 0.19 0.4
abCD 0.56 0.32
bDE 0.23 0.5
aD 0.56 0.19
AS BDL BDL BDL BDL -
5 NAS 18
bA 0.45 14
cA 0.45 15
cA 0.12 21
aA 0.34 2.35
AS 15abB
0.45 13bAB
0.45 12bB
0.23 17aB
0.45 2.19
10 NAS 11
aC 0.56 7
bcB 0.76 8
bC 0.11 10
abC 0.34 1.17
AS 9.8aCD
0.67 6.5bcB
0.98 7bcCD
0.12 8bCD
0.34 1.04
20 NAS 1.3
abD 0.34 1.4
abC 0.23 1.5
aD 0.12 1.8
aD 0.23 0.71
AS 0.12cDE
0.12 0.14bCD
0.12 0.15abDE
0.11 0.16aDE
0.1 0.02
LSD0.05 1.88 1.81 1.97 2.11
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
56
F2. The plants from NAS performed better than their respective AS treatment
even within a column comparison.
3.7.2.4 Catalase (CAT) contents
The fungal inoculations of TSW-Soil mixtures helped plants improve
their defense mechanism by increasing CAT values. Within a row, there was
an increase in plant CAT value with the individual or combined fungal
inoculations, as compared to control. The plants from 5 % NAS with F1 + F2
and 20 % AS with C had the maximum (21 Uml-1) and the minimum (0.12 Uml-
1) SOD values as compared to any of the treatments except Soil AS where the
values were BDL. The AS prevailed less feasible conditions for plants survival
than NAS for all of the TSW-Soil mixtures by significantly decreasing SOD
values as given in Table 3.7.1.
Down the column, there was decrease in SOD contents with the
increasing percentage of TSW in soil mixtures. The SOD contents of NAS of
every fungal treatment were lower than those from the corresponding AS.
Among the columns for all the fungal treatments where soil were mixed with
TSW, the SOD in plant from NAS of 5 % was highest and being the lowest in
20 % (TSW-Soil). Between the columns, plants from all of the soil treatments
with F1 + F2 inoculations had the maximum SOD and those from the C (with
no fungus applied) had the minimum values.
3.7.3 Post-harvest analysis
3.7.3.1 Growth performance of Tagetes patula
Overall, it was noticed that plants cultivated in soil and its TSW
mixtures inoculated with fungal isolates yielded greater shoot, root and
seedling length, no. of leaves and roots, as well as, fresh and dry weight; as
compared to control. The NAS treatments yielded plants with less vigor and
biomass as compared to those from AS treatments. The exact details of each
of the morphological parameters is given in Table 3.7.2 and described as
under:
57
3.7.3.1.1 Shoot, root and seedling length (cm)
Along the row, the maximum plant shoot (41.76 cm), root (20.43 cm)
and seedling length (62.30 cm) was observed in 5 % NAS with F1 + F2
inoculation while being minimum in 20 % AS with no fungal inoculation i.e. the
C. There was increase in length of all the three vegetative parameters with the
application of fungal inoculations and the order of increase observed to be F1
+ F2 > F2 > F1 > C along the row. In NAS, the plant gave better response
than in AS for all the treatments.
Within column, there was decrease in plant shoot, root and seedling
length with the increasing proportion of TSW in the soil. The different TSW-
Soil mixtures under F1 + F2 column gave best results while those in C column
attained the least height as seen in Figure 3.7.1.
Figure 3.7.1. The vegetative growth variation cab be observed in Marigold (Tagetes patula) in response
to autoclaved soil (AS on right) and non-autoclaved soil (NAS on left) mixed with different percentages of TSW (% w:w) ranging from the maximum in plants from 5 % (TSW:Soil) NAS inoculated with F1 + F2 to the minimum in plants from 20 % (TSW:Soil) AS inoculated with C i.e. no fungi.
3.7.3.1.2 No. of leaves and roots
Along the row, the plants in 5 % NAS with F1 + F2 inoculation
observed to have maximum no. of leaves (18) and roots (29) while being the
minimum (4 and 7 respectively) in 20 % AS without any of the inoculation. The
TSW-Soil mixtures with F2 performed better than those inoculated with F1
and no fungal incorporations.
Within a column except, the increasing ratio of TSW decreased the no.
of leaves and roots. The TSW-Soil mixtures under F1 + F2 gave the best
58
vegetative growth than any of the fungal treatments. The worst growth
response observed to be in C column.
Table 3.7.2. Various morphological parameters observed in 50-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w)
Type
Fungal treatments
LSD0.05 C F1 F2
F1+F2
Shoot Length (cm)
Soil NAS 18.38
cAB 0.11 22.12
bcB 0.80 25.47
bAB 0.55 36.31
aB 0.22 4.02
AS 16.12dB
0.20 19.89cC
0.20 22.56bB
0.24 26.20aDE
0.12 3.13
5
NAS 21.76bcA
0.17 26.54bcA
0.22 28.41bA
0.14 41.76aA
0.28 4.79 AS 18.29
cdAB 0.19 21.18
cB 0.16 29.61
bA 0.23 32.30
aC 0.22 3.28
10
NAS 19.11bcAB
0.45 23.33bAB
0.14 24.39bAB
0.27 34.26aBC
0.93 4.41 AS 14.44
cC 0.25 17.34
bcCD 0.22 19.34
bBC 0.17 28.15
aD 0.13 3.97
20 NAS 15.45
cBC 0.89 18.32
bC 0.34 19.34
bBC 0.62 25.23
aDE 0.48 3.13
AS 11.53bcD
1.0 13.54bcD
0.12 14.74bC
0.19 20.82aE
0.74 3.91
LSD0.05 2.95 3.22 4.62 3.71
Root Length (cm)
Soil NAS 11.50
bA 0.23 12.36
bAB 0.17 14.45
abAB 0.14 16.63
aB 0.17 3.22
AS 7.50bB
0.27 10.36abB
0.42 11.36abB
0.21 14.33aBC
0.24 3.83
5
NAS 11.30cA
0.25 16.60bA
0.26 18.43abA
0.12 20.43aA
0.15 3.49
AS 8.30cAB
0.25 12.60bAB
0.16 13.43bB
0.12 16.43aB
0.15 2.88
10
NAS 8.40bAB
0.27 9.46bB
0.13 7.97abC
0.1 8 14.40aBC
0.26 3.09 AS 6.40
bB 0.22 7.46
bB 0.23 8.40
abC 0.18 10.40
aCD 0.26 2.75
20 NAS 7.45
bB 0.23 8.12
bB 0.87 7.51
bC 0.14 12.47
aC 0.16 3.9
AS 5.67abBC
0.65 7.35bBC
0.17 6.29bCD
0.13 10.63aCD
0.67 2.6
LSD0.05 3.33 3.68 4.11 2.38
Seedling Length (cm)
Soil NAS 30.85
dCD 0.15 34.96
cCD 0.56 40.10
bB + 0.95 52.98
aA 0.84 5.85
AS 23.97bC
0.25 30.26abBC
0.26 34.16aB
0.15 40.93aA
0.14 4.89
5
NAS 33.50cC
0.12 43.32bcBC
0.14 46.82bB
0.10 62.30aA
0.12 6.46
AS 26.94dC
0.22 33.96cB
0.24 43.23bAB
0.10 48.98aA
0.12 5.43
10
NAS 27.80bcC
0.12 33.43bB
0.21 32.63bB
0.27 48.97aA
0.19 5.55 AS 21.17
cC 0.12 24.99
bcBC 0.21 27.93
bB 0.07 38.86
aA 0.09 3.82
20 NAS 23.10
bcBC 0.82 27.06
bB 0.32 26.95
bB 0.15 37.96
aA 0.19 4.88
AS 17.11bcBC
0.12 20.98bB
0.92 21.10bB
0.12 32.12aA
0.34 4.25
LSD0.05 4.45 5.61 3.32 6.44
No. Of Leaves
Soil NAS 9
bB 0.22 12
abB 0.17 11
abB 0.23 14
aB 0.29 4.75
AS 6bC
0.2 7bC
0.1 8abC
0.2 10aC
0.2 2.82
5
NAS 13bA
0.26 14abA
0.18 15abA
0.21 18aA
0.13 4.43
AS 7bcBC
0.26 8bC
0.18 9bBC
0.21 13aB
0.13 3.34
10
NAS 9bB
0.16 15aA
0.33 16aA
0.07 17aA
0.51 4.16 AS 5
aC 0.16 4
abD 0.33 3
bD 0.07 6
aD 0.11 2.75
20 NAS 9
abB 0.16 7
bC 0.16 8
bC 0.16 11
aBC 0.16 2.63
AS 4bCD
0.16 5bD
0.16 6abCD
0.16 8aCD
0.16 2.71
LSD0.05 2.77 2.97 2.89 3.09
No. Of Roots
Soil NAS 16
cAB 0.23 19
bAB 0.30 20
bA 0.09 24
aB 0.16 3.63
AS 12bcB
0.23 14bBC
0.30 16bAB
0.09 21aBC
0.16 4.4
5
NAS 18bcA
0.15 21bA
0.19 19bA
0.30 29aA
0.14 4.27
AS 13bcB
0.16 15bB
0.19 18abA
0.10 21aBC
0.14 3.24
10
NAS 10cBC
0.23 16bB
0.25 17bAB
0.15 26aAB
0.16 4.12 AS 9
bcC 0.23 11
bC 0.25 10
bBC 0.15 17
aC 0.16 3.28
20 NAS 9
cC 0.65 12
bC 0.15 13
bB 0.98 16
aC 0.14 2.57
AS 7cCD
0.12 10bCD
0.16 11bB
0.76 15aCD
0.10 2.81
LSD0.05 3.46 3.62 4.31 3.28
Fresh weight (g)
Soil NAS 4.21
bAB 0.96 5.37
ab 0.13 5.51
a 0.66 6.44
aC 0.19 1.21
AS 3.98bB
0.26 4.95abB
0.13 4.72abBC
0.16 5.89aC
0.19 1.88
5 NAS 6.50
cA 0.27 9.40
bA 0.27 10.70
bA 0.18 14.40
aA 0.55 2.93
AS 4.50bAB
0.17 5.40bB
0.17 5.70bB
0.18 9.40aB
0.15 3.61
10 NAS 3.70
bcB 0.12 5.91
bB 0.15 5.30
bB 0.17 9.22
aB 0.19 2.99
AS 2.70cBC
0.12 4.40bB
0.52 5.30abB
0.71 6.22aC
0.96 1.59
20 NAS 3.52
bcB 0.18 4.23
bBC 0.82 4.76
bBC 0.18 6.78
aC 0.32 1.74
AS 2.76bBC
0.29 2.98bC
0.64 3.12abC
0.76 4.20aD
0.98 1.12
LSD0.05 2.71 2.44 1.86 2.58
Dry weight (g)
Soil NAS 1.96
bAB 0.96 2.30
abB 0.13 2.50
abB 0.66 3.10
aB 0.19 0.88
AS 0.97cD
0.14 1.34bD
0.82 1.41bCD
0.51 2.28aC
0.92 0.24
5 NAS 2.26
bcA 0.18 2.98
bA 0.13 3.20
bA 0.16 5.40
aA 0.19 1.09
AS 1.95bAB
0.17 1.93bBC
0.17 1.70bC
0.18 3.40aB
0.15 0.79
10 NAS 1.70
aB 0.12 1.23
abD 0.15 1.30
abCD 0.17 2.22
aC 0.19 1.17
AS 1.01bCD
0.12 1.12bDE
0.54 1.09bD
0.79 1.52aCD
0.9 0.24
20 NAS 1.34
cC 0.61 1.72
abC 0.27 1.64
bC 0.71 1.96
aC 0.23 0.30
AS 0.67bDE
0.02 0.76abE
0.94 0.78abDE
0.62 0.92aD
0.54 0.21
LSD0.05 0.34 0.38 0.47 1.12
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
59
3.7.3.1.3 Fresh and dry weight (g)
The fresh and dry weight while being inter-dependent observed to be
the maximum and the minimum in accordance with the maximum and the
minimum no. of leaves and roots for both along the row as well as within
column comparisons. In other words, along the row the maximum weight
(14.40 g fresh, 5.40 g dry) observed to be in 5 % NAS with F1 + F2 and the
minimum (2.76 g fresh, 0.67 g dry) in 20 % AS without any fungus.
Within column, the increasing TSW ratio affected the biomass production
negatively except for 5 % TSW-Soil mixture. The TSW-Soil mixtures under F1
+ F2 yielded maximum fresh and dry weight while those in C column yielded
the least.
3.7.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot were variable with respect to fungal inoculations as well as increasing
ratio of TSW in soil, as given in Table 3.7.3.
3.7.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in shoot increased with inoculation
of fungi as compared to C. The NAS of all the treatments had more shoot Ca
contents with respect to that of AS. The maximum (1120 mgkg-1) shoot Ca
observed to be in 10 % NAS with F1 + F2 while being the minimum (5.5 mgkg-
1) in plants from Soil AS with C. The NAS of 10 % observed to have the
maximum Ca in shoot than any of the TSW-Soil mixtures for C, F1, F2 and F1
+ F2 inoculations within row.
Within column, the maximum Ca concentrations in shoot of plants from
TSW-Soil mixtures inoculated with F1 + F2 while being the minimum in those
where no fungi was applied. The plants with F2 inoculation had more Ca in
shoot than those with F1. It was observed that shoot Ca increased with the
increasing TSW ratio in soil mixtures for both NAS and AS.
60
3.7.3.2.2 Potassium (K) in shoot
Along the row, the shoot K uptake increased with fungal applications
for all the TSW-Soil mixtures. The maximum K level (550 mgkg-1) was
observed in 10 % NAS with F1 + F2 while being the minimum (12 mgkg-1) in
soil NAS with no fungi. The values for each AS observed to be lesser than its
respective NAS.
Within column, the K shoot uptake increased with the increasing
concentration of TSW in soil mixtures for all the treatments. For all TSW-Soil
mixtures, the F2 plants showed more K uptake than those with F1 and being
the least where no fungus was applied. The maximum values observed under
F1 + F2 column.
Table 3.7.3. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of 50-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Category-I Metal
TSW-Soil (% w:w)
Type
Fungal treatments
LSD0.05 C F1 F2
F1+F2
Ca
Soil NAS 10.12
cD 0.34 12.22
bcD 0.9 15.9
bE 0.87 22.34
aE 1.1 3.44
AS 5.5cD
0.65 7.6bD
1.1 8.5bE
0.56 12.5aE
1.8 2.19
5
NAS 379cAB
1.2 480bAB
3.5 570bBC
3.1 990aB
2.8 189
AS 230cC
1.4 290bC
2.7 310bDE
2.7 450aDE
2.7 75
10
NAS 475bcAB
1.3 610bA 3.9 660
bA 3.4 1120
aA 4.2 225.6
AS 310bcB
1.6 425bB 2.8 415
bC 2.7 780
aC 2.7 164.2
20 NAS 520
bcA 1.3 590
bA 3.7 610
bB 2.8 960
aB 3.8 178.7
AS 210bC
1.7 390aBC
3.1 345abD
2.9 550aD
2.6 155.1
LSD0.05 59.8 51.2 48.7 116.4
K
Soil NAS 12
bD 0.87 15
abE 0.56 21
bD 1.1 40
aE 1.4 7.52
AS BDL BDL BDL 13E 1.1 -
5
NAS 210cB
1.6 340bB 2.8 310
bB 1.3 490
aAB 2.9 101.8
AS 120cC 1.7 280
bC 2.1 240
bcC 1.6 380
aC 3.7 61.3
10
NAS 325cA 2.1 410
bA 3.6 425
bA 1.8 550
aA 3.3 123.3
AS 225bcB
1.4 290bBC
2.5 310bB 1.7 460
aB 3.2 108.7
20 NAS 310
bcA 2.4 340
bB 2.2 370
abAB 1.8 410
aBC 2.7 54.5
AS 110cC 1.4 190
bcD 1.5 210
bCD 2.1 290
aD 2.6 32.6
LSD0.05 31.7 50.7 56.8 74.4
Na
Soil NAS 240
bcC 1.3 310
bBC 3.3 340
bC 2.1 600
aBC 3.7 154.6
AS 125bcD
2.2 225bC
2.1 210bD
2.7 480aC
2.8 140.5
5
NAS 450bcA
2.6 590bA 2.7 610
bA 2.8 720
aB 3.1 92.4
AS 210bcC
2.2 320bBC
2.5 340abC
2.4 580aBC
2.7 148.7
10
NAS 340cB 1.8 440
bB 1.1 510
bB 3.4 880
aA 3.7 163.5
AS 210cC 1.9 280
bcC 1.4 310
bC 2.7 390
aCD 1.8 41.8
20 NAS 110
cD 1.5 120
bcD 1.6 150
bDE 2.6 210
aCD 1.7 35.5
AS 40cDE
0.99 90bD
1.3 85bE 1.7 125
aD 1.5 31.8
LSD0.05 86.7 141.6 94.8 154.3
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
3.7.3.2.3 Sodium (Na) in shoot
The Na concentration in shoot observed to increase along the row and
it was because of fungal inoculations. The pots with F1 + F2 showed the
greatest Na shoot uptake, the F2 being greater than F1, while those with no
61
fungi being the least. The plants in 10 % NAS with F1 + F2 had the highest
value while those in 20 % AS with no fungi exhibited the lowest Na shoot
contents.
Within column, the increasing ratio of TSW in soil mixtures enhanced
the shoot Na uptake except for 20 % for all the fungal treatments. In case of
20 %, the concentration of shoot Na observed to be the least for all the fungal
treatments.
3.7.3.3 Category-I metals in plant ROOT
The bioavailability of Category-I metals was variable with different fungi
in root also however, it was directly related to the increasing ratio of TSW in
soil mixture, as given in Table 3.7.4.
3.7.3.3.1 Calcium (Ca) in root
The root Ca in both NAS and AS of Soil was BDL for all the fungal
treatments except F1 + F2 of NAS. The application of fungi to the soil helps
increase Ca uptake along the row. The plants in F1 + F2 pots observed to
Table 3.7.4. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of 50-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according
to Duncan’s multiple range test (P = 0.05; n = 3).
Category-I Metals
TSW-Soil (% w:w)
Type
Fungal treatments
LSD0.05 C F1 F2
F1+F2
Ca
Soil NAS BDL BDL BDL 12
CD 0.56 -
AS BDL BDL BDL BDL -
5 NAS 225
dC 1.4 310
cB 2.9 350
bB 1.8 370
aC 2.6 19.6
AS 125cD 1.7 270
bBC 2.8 250
bBC 1.6 310
aC 2.7 57.8
10 NAS 360
bcA 2.7 590
bA 3.6 550
bA 2.6 980
aA 3.4 310.4
AS 230bcC
2.6 350bB 3.1 380
bB 2.5 690
aB 2.8 275.8
20 NAS 310
bB 2.8 380
bB 2.9 360
bB 2.4 775
aB 3.1 388.8
AS 125cD 1.8 250
bBC 2.7 270
bBC 2.3 320
aC 2.6 47.6
LSD0.05 49.1 206.7 165.9 284.2
K
Soil NAS BDL BDL BDL BDL - AS BDL BDL BDL BDL -
5 NAS 125
cC 1.3 230
bcC 2.6 280
bB 1.6 345
aB 2.2 63.5
AS 55bcD
1.4 125bD
1.2 150bC
1.4 290aBC
2.1 137.4
10 NAS 290
bA 2.5 310
bA 2.4 325
bA 1.8 430
aA 2.9 103.2
AS 220bB 2.7 270
bB 1.4 225
bBC 1.6 355
aB 3.1 128.6
20 NAS 130
bcC 1.8 240
bBC 1.3 260
bB 1.9 310
aBC 2.8 45.9
AS 25cD 0.9 90
bDE 1.1 110
bCD 1.7 155
a C 2.6 42.3
LSD0.05 68.8 39.1 43.3 73.3
Na
Soil NAS 120
bC 0.87 225
bC 2.1 280
bBC 2.1 425
aC 2.7 141.2
AS BDL BDL BDL 25G 0.67 -
5 NAS 380
dA 2.6 420
cA 3.2 470
bA 2.5 490
aB 3.1 18.9
AS 225c 2.1 325
aB 3.1 290
bBC 2.6 330
aD 2.1 15.6
10 NAS 290
bB 2.7 380
bA 2.6 350
bB 3.1 550
aA 1.8 198.8
AS 230bBC
2.6 280bBC
1.5 255bBC
3.5 390aCD
1.6 103.4
20 NAS 95
bC 0.98 110
bD 1.1 120
bC 1.1 180
aE 1.2 58.7
AS 30cD 0.79 55
bDE 0.78 60
bCD 0.87 90
aF 0.99 28.1
LSD0.05 85.5 87.8 116.6 56.7
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least
significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
62
have maximum while those with no fungi having the minimum root Ca than
any of the fungal treatments for all of the TSW-Soil mixtures. The highest root
Ca was in 10 % NAS with F1 + F2 while being the minimum in 20 % AS with
no fungal inoculation.
3.7.3.3.2 Potassium (K) in root
The K contents in both NAS and AS of Soil were BDL for all the fungal
inoculations. However, the 10 % NAS with F1 + F2 exhibited the maximum
root uptake than any of the soil treatments while being the minimum in 20 %
AS with no fungal application. The application of fungus as individual
inoculant i.e. F1 and F2 showed better K uptake than TSW-Soil mixtures
where no fungi has been applied.
Within column, the metal uptake in root increased with increasing ratio
of TSW in 5 and 10 %, however, it decreased in 20 %. The NAS treatments
displayed better root metal uptake than those AS for all of the TSW-Soil-fugal
combinations.
3.7.3.3.3 Sodium (Na) in root
Along the row as observed in case of Ca and K, the application of fungi
helps increase Na uptake in roots. The maximum Na in root was observed in
10 % NAS with F1 + F2 while being minimum in 20 % AS despite of the fact
that it was BDL in few of the fungal treatments from Soil AS. Those with F1
and F2 applications also performed better than C i.e. treatment with no fungal
inoculation.
Within column, trend of Na root uptake was also similar to what
observed in case of Ca and K. The increasing ratio of TSW in soil displayed
increased root Na uptake as compared to Soil with C for all the fungal
treatments. However, in case of 20 % the Na root uptake trend reversed and
metal uptake dropped even lower than what observed in case of NAS of Soil
with C. The plants in NAS accumulated more Na than those from AS for all of
the fungal treatments.
63
3.7.3.4 Category-II metals in plant SHOOT
The Category-II metals i.e. the AAS detected metals in shoot were
variable with respect to fungal inoculations as well as increasing ratio of TSW
in soil, as given in Table 3.7.5. The application of fungi enhanced trace metal
uptake tendency of plant for all the TSW-Soil mixtures and both NAS and AS.
However, the increasing level of Category-II metals in shoot was in
accordance with the increasing ratio of TSW in soil mixtures for all the fungal
treatments except for 20 % where it dropped comparatively.
3.7.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in TSW-Soil mixtures with combined and
F2 for both NAS and AS of all treatments. In AS the metal was found to be
BDL except for F1 + F2 treatment where the metal accumulation was recoded
30 mgkg-1. Maximum amount of metal was observed in 10% TSW-Soil mixture
in NAS having combined inoculation of fungi i.e. F1 + F2.
3.7.3.4.2 Cr in shoot
As compared to control with no fungal inoculation, the Cr concentration
in shoot increased in accordance with the application of fungal inoculations
along the row except NAS of soil with F1 inoculation showing low shoot
extraction than the C. The maximum Cr accumulation in shoot was observed
in treatments applied with combined inoculants i.e. F1 + F2, being significantly
higher than any of the treatments along the row.
Within a column for C, the Cr in 5 % NAS plant shoots significantly
increased than any of the treatments. The values found in NAS and AS of Soil
was significantly least. The plants from AS of every TSW-Soil mixture
displayed lower Cr uptake than every corresponding NAS. Similar trend was
observed in plant shoots harvested from F1, F2 and F1 + F2 treatments.
3.7.3.4.3 Cu in shoot
Along the row, the plants harvested from F1 + F2 exhibited maximum
Cu accumulation as compared to any of the treatments with single or no
64
fungal application. Such a trend was observed in all of the TSW-Soil mixtures.
The F2 proved to be better enhancer of Cu uptake in plant shoot as compared
to the F1. The plants from treatments with no fungal inoculations i.e. C
showed the significantly least Cu accumulation as compared to any of the
fungal treatments.
Within a column, the plants from every AS had significantly less Cu
uptake in shoot than those form NAS for all the TSW-Soil mixtures i.e. soil, 5,
10 and 20 %.
3.7.3.4.4 Fe in shoot
The Fe uptake in plant shoots observed to be BDL in AS of Soil with all
the fungal inoculations except F1 + F2. The plants from all the NAS with F1 +
F2 inoculation displayed the maximum Fe uptake in shoot as compare to
plants from the treatments with single or no fungal inoculations. The values of
plant shoot Fe in C i.e. with no fungal inoculation(s) were the minimum as well
as significantly least as compared to plants from treatments with fungal
inoculation(s).
Within a column, in case of C, the value of shoot Fe found to be the
maximum in 5 % TSW-Soil while being the minimum and significantly least in
plant shoots harvested from AS Soil. Similar trends were observed in F1, F2
as well as F1 + F2.
3.7.3.4.5 Mg in shoot
Along the row, the Mg uptake in plant shoot increased with fungal
application in the pots and found to be the maximum in plants harvested from
pots applied with combined fungi i.e. F1 + F2, while being the minimum as
well as significantly least in shoot so of plants cultivated in soil with no fungus.
Such a pattern was observed for all the TSW-Soil mixtures.
Inside column, the value of plant shoot Mg increased with increasing
level of TSW percentage in soil up to 10 % (TSW-Soil). At 20 %, the shoot
uptake decreased drastically and significantly as compared to the treatments
with preceding lower doses of TSW in soil. Such a pattern of shoot Mg
65
variation was observed in C, F1, F2, as well as F1 + F2. The plants from NAS
displayed less Mg uptake in shoot than corresponding AS for all the
treatments.
Table 3.7.5. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 50-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according
to Duncan’s multiple range test (P = 0.05; n = 3).
Category-II Metals
TSW-Soil (% w:w)
Type
Fungal treatments
LSD0.05 C F1 F2
F1+F2
Cd
Soil NAS 10
cD 0.34 25
bcE 0.86 45
bD 1.6 80
aD 1.4 23.6
AS BDL BDL BDL 30DE
1.7 -
5
NAS 320cA 1.4 410
bA 2.5 450
bA 2.6 590
aA 2.7 129.5
AS 210dBC
2.1 350cB 1.9 390
bAB 2.8 425
aBC 4.3 32.8
10
NAS 250bcB
2.2 325bB 1.4 360
bB 2.6 480
aB 4.1 115.5
AS 210cBC
2.5 290bcC
1.6 325bB 2.8 390
aBC 3.3 58.9
20 NAS 110
cC 1.7 190
bcD 1.8 225
bC 2.2 280
aC 2.9 49.9
AS 20bcD
0.67 45bE 1.4 55
bD 1.9 110
aD 2.1 54.4
LSD0.05 68.7 55.4 79.9 118.6
Cr
Soil NAS 45
bcE 0.98 40
bcF 0.99 80
bE 1.8 215
aD 2.1 68.6
AS 15bcE
0.56 30bF 1.5 50
bE 1.1 190
aD 1.7 62.5
5
NAS 990bcA
1.4 1,100bA 3.7 1,550
aA 2.9 1,540
aA 5.1 125.8
AS 420cC
2.3 650bcC
2.2 720bC
2.7 910aBC
3.3 105.5
10
NAS 810cB 2.7 970
bcB 3.1 1,030
bB 2.6 1,120
aB 3.6 89.4
AS 780cB 2.8 855
bcBC 2.4 945
bB 2.3 1,045
aB 3.8 98.2
20 NAS 415
bcC 3.1 480
bD 2.8 510
bCD 2.5 625
aC 2.8 101.7
AS 220cD
3.5 320bE 2.6 345
bD 1.7 510
aCD 4.2 61.9
LSD0.05 96.7 121.1 265.4 215.8
Cu
Soil NAS 60
bcE 1.1 120
bBC 1.6 155
bC 1.3 290
aB 2.4 125.5
AS 45cE 1.3 110
bC 1.5 125
bD 1.4 190
aCD 2.1 60.6
5
NAS 190bB 1.6 210
bB 2.3 225
bA 1.8 350
aA 2.5 120.2
AS 285bA 2.1 410
aA 2.4 160
cC 1.1 190
cCD 2.4 122.8
10
NAS 155cC
1.9 195bcB
2.1 210bA 3.1 230
aC 2.6 18.8
AS 95bcD
0.98 110bC
1,9 115bE 1.1 150
a 1.6 33.3
20 NAS 125
cCD 1.6 175
bB 1,8 180
bB 1.8 210
aC 2.2 27.6
AS 75bDE
1.1 90bC
1.7 85bE 3.9 115
aD 2.7 38.8
LSD0.05 34.8 90.4 24.7 57.4
Fe
Soil NAS 12
cC 0.98 19
bcC 1.1 25
bD 0.85 90
aB 2.5 11.1
AS BDL BDL BDL 25E 1.1 -
5
NAS 45cA 0.68 70
bA 0.98 90
abA 0.91 105
aA 2.1 23.9
AS 30bB 1.2 20
bcC 1.8 35
bCD 0.83 70
aCD 2.6 25.7
10
NAS 30cB 1.8 50
bB 1.6 75
abB 1.2 80
aBC 2.8 5.8
AS 15bcC
0.98 20bC
1.4 25bD
0.57 45aD
1.9 18.4
20 NAS 20
cBC 1.4 35
bcBC 1.2 45
bC 1.8 60
aC 2.1 14.3
AS 15dC
0.49 25cC
0.89 35bCD
1.2 40aD
1.9 4.99 LSD0.05 14.4 18.9 13.3 14.8
Mg
Soil NAS 15
bcD 0.78 25
bD 1.94 35
bDE 1.6 70
aC 1.7 32.4
AS BDL 10bE 0.92 13
bE 1.8 35
aD 1.8 5.3
5
NAS 55cAB
1.1 70bcB
1.2 80bB 1.7 110
aB 1.6 17.8
AS 30bcCD
1.4 50bC
1.5 45bD
1.6 75aC
1.5 18.8
10
NAS 60cA 1.3 90
bA 1.9 125
abA 1.2 135
aA 2.4 18.1
AS 45cB 1.2 70
bB 1.2 65
bC 0.89 110
aB 2.1 11.7
20 NAS 35
cC 1.3 45
bcC 1.7 60
bC 0.93 95
aBC 1.9 14.8
AS 15cD
0.98 40bCD
1.2 35bDE
0.91 65aC
1.2 13.9
LSD0.05 6.7 13.2 14.3 24.4
Ni
Soil NAS BDL BDL BDL BDL - AS BDL BDL BDL BDL -
5
NAS 15bcA
0.93 17bA 1.2 20
abA 0.87 22
aA 1.2 2.98
AS 9cB 0.23 12
bB 1.5 10
bcB 0.86 15
aC 1.5 2.11
10
NAS 8bBC
0.12 15abAB
1.6 10bB 0.91 20
aAB 0.93 5.5
AS 7bcC
0.15 5cD
0.23 9bBC
0.92 16aBC
0.95 2.89
20 NAS 5
cD 0.11 9
bC 0.12 8
bcC 0.84 18
aB 0.98 1.92
AS BDL 8abC
0.15 7bCD
0.96 11aD
0.94 1.3 LSD0.05 1.5 2.8 1.6 2.9
Zn
Soil NAS 10
cCD 0.13 15
bcC 0.19 18
bF 0.93 25
aC 0.90 4.1
AS BDL BDL BDL 15aC
0.76 -
5
NAS 160cA 1.1 190
bcAB 1.1 210
bB 1.6 345
aA 3.1 30.4
AS 135bcAB
1.6 160bB 1.2 170
bC 1.8 280
aAB 2.8 34.8
10
NAS 150cA 2.1 215
bA 1.7 235
bA 1.4 360
aA 2.9 64.6
AS 80bcB
1.5 160bB 1.9 140
bD 1.5 240
aB 2.6 78.8
20 NAS 75
cB 1.6 195
bA 1.8 210
bB 1.1 290
aAB 2.1 76.1
AS 40cC
1.3 120bBC
1.2 95bcE
0.98 190aBC
2.1 44.7 LSD0.05 33.3 45.2 24.3 78.6
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
66
3.7.3.4.6 Ni in shoot
The plant shoot Ni observed in BDL for NAS as well as AS of Soil.
However, its value found to increase with application of fungus and observed
to be the maximum in case of plants cultivated in pots with F1 + F2. The
plants from 5, 10 and 20 % (TSW-Soil) observed to follow the same
sequence.
In a column comparison, the value of plant shoot Ni concentration
decreased with the increasing percentage of TSW in soil and found to be the
maximum in plants harvested from 5 % NAS while being the minimum in
plants from 20 % AS. The plants from NAS showed enhanced shoot Ni uptake
than corresponding AS for all the treatments.
3.7.3.4.7 Zn in shoot
Alongside row, the shoot Zn concentration increased in plants harvest
from pots with fungal inoculations than those harvested from pots applied with
no fungi. Like other metals, the shoot Zn observed to be the maximum in
plants representing soil treatments applied with F1 + F2, those harvested from
the pots filled with autoclaved soil applied with no fungi displayed the
minimum values.
Inside a column, the value of shoot Zn concentration decreased with
increasing fraction of TSW in soil mixture for all the fungal treatments. The
plants from every NAS had significantly greater Zn shoot concentration than
corresponding AS and such a variation observed in C, F1, F2 as well as F1 +
F2.
3.7.3.5 Category-II metals in plant ROOT
The Category-II metals i.e. the AAS detected metals in root were
observed to differ with varying levels of TSW in the soil as well in response to
fungal inoculations, as given in Table 3.7.6.
67
3.7.3.5.1 Cd in root
Along the row, the Cd level in plant roots harvest from Soil observed to
be BDL for NAS and AS of C, F1, F2 and F1 + F2. However, for 5, 10 and 20
% TSW-Soil mixtures, the plant root Cd concentration increased with fungal
inoculations and found to be maximum as well as significantly higher than
those applied with single or no fungal treatments, while being the minimum
and significantly least in C.
Down the columns, the root Cd concentration increased with increasing
percentage of TSW in soil up to 10 % but dropped at 20 % for C, F1, F2 as
well as F1 + F2. The plants from all of the NAS exhibited enhanced Cd uptake
than those harvested from corresponding AS and such a trend was observed
in all of the fungal treatments.
3.7.3.5.2 Cr in root
Alongside the row, the plants from F1 + F1 fungal inoculations showed
the maximum root Cr concentration with C treatment plants having the
minimum uptake for both NAS and AS of Soil, 5, 10 and 20 % TSW-Soil
mixtures. The F2 inoculation enhanced the root Cd concentration better than
F1 as well as C.
Within the columns, the root Cr concentration increased with increasing
percentage of TSW in soil up to 10 % but significantly decreased at 20 % as
compared to 0 %. For C, F1, F2 and F1 + F2, the plants from NAS showed
better root Cr uptake than corresponding AS.
3.7.3.5.3 Cu in root
Alongside the row, the fungal inoculations improvised the root Cu
uptake as compared to C. The plants from F1 + F2 observed to have the
maximum uptake than those cultivated in pots with single or no fungal
inoculations and found to be the minimum in C. The F2 observed to be better
enhancer of root Cu uptake than F1.
Under the columns, the plants from 5 and 10 % TSW-Soil exhibited
better root Cu accumulation than Soil. The 20 % plants had the lower root Cu
68
Table 3.7.6. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 50-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Category-II Metals
TSW-Soil (% w:w)
Type
Fungal treatments
LSD0.05 C F1 F2
F1+F2
Cd
Soil NAS BDL BDL BDL BDL - AS BDL BDL BDL BDL -
5
NAS 290cA 1.1 390
bA 1.8 410
bA 2.1 550
aA 2.5 105.3
AS 190cB 1.6 325
bcB 2.8 350
bB 2.5 390
aB 3.1 38.7
10
NAS 240cAB
2.1 310bBC
2.6 340bB 2.9 410
aB 2.7 68.7
AS 180cB 1.7 270
bcBC 2.5 290
bC 1.9 320
aBC 2.6 29.8
20 NAS 90
cC 0.99 155
bcC 1.9 190
bD 1.6 230
aC 2.1 36.6
AS BDL 15bcD
0.94 25bE 0.69 90
aD 1.1 15.5
LSD0.05 88.5 64.3 56.2 134.7
Cr
Soil NAS 20
bcF 0.23 35
bE 0.67 55
bE 0.56 185
aE 1.6 18.8
AS 10bcF
0.27 25bE 0.93 35
bE 0.94 95
aEF .9 21.2
5
NAS 910cA 1.7 990
bA 2.7 1010
bA 1.8 1320
aA 4.1 77.1
AS 410cD 2.4 620
bC 2.9 695
bC 1.6 890
aBC 3.8 193.5
10
NAS 585cdC
3.1 615cC 3.1 655
bC 2.6 795
aC 2.9 36.4
AS 755bcB
2.7 850bB 2.8 835
bB 3.1 990
aB 3.1 93.7
20 NAS 210
cE 2.9 325
bD 2.1 380
bD 1.6 550
aD 2.8 114.4
AS 185bcEF
1.7 295bD
1.9 270bDE
2.1 480aDE
2.5 107.9
LSD0.05 69.9 92.5 105.2 108.3
Cu
Soil NAS 55
bcC 1.1 95
bC 1.8 100
bD 0.99 255
aA 2.6 75.6
AS 25cD 0.94 75
bD 1.5 80
b 1.0 145
aC 2.7 48.8
5
NAS 130bcA
1.3 155bAB
1.7 160bB 0.98 270
aA 2.9 53.4
AS 95cdB
1.6 110cBC
1.1 130bC
1.7 155aC
1.9 17.5
10
NAS 115dAB
1.2 170cA 0.96 190
bA 1.5 210
aB 1.7 19.8
AS 80bcB
0.94 95bC
0.69 110aCD
1.1 120aD
2.6 23.3
20 NAS 90
cB 0.91 120
bB 0.78 135
abBC 1.7 160
aC 2.8 27.9
AS 55bC
0.56 75abD
0.56 70bE 0.94 110
aD 1.3 38.2
LSD0.05 22.5 17.8 25.2 32.5
Fe
Soil NAS BDL BDL BDL 55
AB 1.3 -
AS BDL BDL BDL 20BC
1.8 -
5
NAS 40cdA
0.93 45cA 0.82 55
bA 0.78 75
aA 1.9 9.5
AS 25bB 0.96 15
cBC 0.29 30
bB 0.58 60
aAB 2.1 13.5
10
NAS 15bcC
0.23 30abAB
0.49 25bB 0.49 40
aB 2.8 11.9
AS 10cD 0.87 15
bcBC 0.67 20
bBC 0.39 35
aB 1.9 8.7
20 NAS 10
cD 0.94 25
bB 0.89 20
bBC 0.56 45
aB 2.6 14.6
AS 5bcE
0.45 10bC
0.93 15bC
0.87 35aB 2.6 12.2
LSD0.05 4.89 17.5 14.9 29.4
Mg
Soil NAS 25
cCD 0.29 50
abBC 1.4 65
bC 0.93 90
aD 1.7 24.3
AS 20cD 0.12 45
bBC 1.4 60
abC 1.1 70
aE 1.2 16.9
5
NAS 70cA 0.96 90
bcA 1.5 115
bA 1.5 165
aA 1.8 34.6
AS 35cC 0.59 80
bA 1.9 110
abA 1.7 140
aB 1.6 30.9
10
NAS 55cB 0.83 75
bcAB 1.1 90
bAB 1.5 120
aC 1.5 25.6
AS 50bB 0.39 65
abB 1.2 45
bD 1.3 95
aD 1.1 32.2
20 NAS 45
cBC 0.31 70
bcAB 0.94 85
bB 0.99 130
aBC 1.4 29.9
AS 15cD 0.12 30
bcC 0.95 50
bCD 1.5 125
aBC 1.3 21.3
LSD0.05 12.4 22.8 18.2 19.8
Ni
Soil NAS BDL BDL BDL BDL - AS BDL BDL BDL BDL -
5
NAS 5cB 0.11 7
bC 0.26 9
abD 0.94 10
aC 0.95 1.7
AS BDL 5bD
0.19 8abDE
0.78 9aC
0.67 1.4
10
NAS 10cA 0.18 12
bcA 0.67 13
bB 1.1 15
aAB 0.95 1.9
AS 8cAB
0.67 10bcB
0.89 11bC
1.6 13aB 0.19 1.8
20 NAS 6
cB 0.18 10
bB 0.28 15
abA 1.8 17
aA 0.95 2.1
AS 5dB 0.31 7
cC 0.83 9
bD 1.6 12
aB 0.78 1.6
LSD0.05 2.1 2 1.5 2.15
Zn
Soil NAS BDL 10
cD 0.94 15
bD 0.78 20
aF 0.29 4.6
AS BDL BDL BDL 12F 1.1 -
5
NAS 125bcA
0.91 150bAB
1.4 195abA
2.6 320aA 2.5 34.6
AS 90cB 1.1 110
bcB 1.9 125
bB 2.1 260
aC 2.6 19.9
10
NAS 130cA
1.8 175bA 1.3 180
bAB 1.7 280
aB 2.9 22.5
AS 70cBC
1.3 120bB 1.7 110
bBC 1.5 210
aDE 2.7 30.1
20 NAS 60
cC 1.2 155
bcAB 1.2 180
bAB 1.8 235
aD 3.1 41.8
AS 25cC 0.92 95
bC 0.91 80
bC 0.93 155
aE 2.7 42.5
LSD0.05 26.5 36.1 28.1 17.2
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
uptake than those harvested from Soil but less than any of the 5 and 10 %.
Such a trend was observed for C, F1, F2 as well as F1 + F2. The plants from
69
NAS showed enhanced root Cu uptake than corresponding AS for all the
treatments.
3.7.3.5.4 Fe in root
Moving alongside the row, the root Fe observed to be BDL in Soil for
NAS and AS of C, F1, F2 but not in F1 + F2. The plants from C had the lowest
root Fe as compared to plants from any of the single or combined application
of fungi. The root Fe accumulation observed to be the maximum in F1 + F2
and significantly higher than those harvested from any of the treatments with
single or no fungal inoculations.
Inside columns, the root Fe concentration decreased with increasing
percentage of TSW in soil for all the fungal treatments. The plants from NAS
showed enhanced root Fe uptake than corresponding AS.
3.7.3.5.5 Mg in root
Beside the rows, the Mg root concentration increased in NAS and AS
with fungal application as compared to C i.e. with no fungal inoculation. The
F1 + F2 plants displayed the maximum root Mg concentration than those from
C as well as F1 and F2. The F2 inoculations gave better results than F1.
Down the columns, except 20 % the root Mg accumulation increased
with increasing percentage of TSW in soil. The plants from every AS
treatment had lower root Mg concentration than corresponding NAS for all the
fungal treatments.
3.7.3.5.6 Ni in root
The root Ni concentration increased along the row with the application
of fungal inoculations and found to be the maximum in plants applied with
combined application of both of the fungi while being the minimum in C with
no fungus added. The F2 application incurred better root Ni uptake effects
than F1. The plants from NAS and AS of Soil had the root Ni accumulation
below the detection limit.
70
Within columns, the plants from 10 % TSW-Soil mixtures had the
maximum root Ni level than those from both of the 5 and 20 % for all of the
fungal treatments. The NAS plants had the better root Ni concentration than
corresponding AS for C, F1, F2 as well as F1 + F2.
3.7.3.5.7 Zn in root
Alongside the rows, the root Zn accumulation observed to increase in
pots applied with fungal inoculations than C and found to be the maximum in
treatments applied with both of the fungi and being the minimum with no
fungal applications.
Down the columns, the root Zn concentration decreased with
increasing percentage of TSW in soil with every AS treatment showing
comparatively less values than corresponding NAS soils. Such a pattern was
observed in all the fungal treatments.
3.7.4 Fungal analyses
Alongside the row, the c.f.u. increased with fungal application than C
and observed to be the maximum in treatments with combined application of
both of the fungi. The order of c.f.u. abundance was F1 + F2 > F2 > F1 > C.
Within a column, the c.f.u. abundance was observed in NAS of all the TSW-
Soil in varying ratio; however, it was altogether absent in all of the
corresponding AS treatments.
Table 3.7.7. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 50-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean
values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
TSW-Soil (% w:w) Type Fungal Treatment
LSD0.05 C F1 F2 F1+F2
0 NAS 0.12
cdD 0.22 1.1
cB 0.17 2.3
bAB 0.23 3.9
aAB 0.29 0.99
AS - 0.9bBC
0.67 1.8abB
0.71 2.7aB 0.43 0.91
5
NAS 0.22cA
0.26 2.4bcA
0.18 2.9bA
0.21 4.6aA
0.13 1.3
AS - 1.4bB 0.12 1.7
bB 0.31 3.2
aB 0.19 0.95
10
NAS 0.19cB
0.16 2.3bA
0.33 2.5bA
0.07 4.2aA
0.51 1.1
AS - 1.2bB 0.76 1.7
abB 0.67 2.1
aBC 0.09 0.84
20 NAS 0.16
cC 0.23 1.6
bAB 0.34 1.8
bB 0.45 2.7
aB 0.76 0.90
AS - 0.7abC
0.82 1.01bBC
0.23 1.8aC
0.16 0.73
LSD0.05 0.2 0.87 0.96 1.2
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
71
3.7.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
3.7.5.1 Category-I metals translocation index (%)
The plant translocation index values were also recorded for category-I
metals those detected by flame photometer i.e. Ca, K and Na.
In case of Ca, maximum value was observed in 5% NAS with F1 + F2
i.e. 267.56 % while least value was recorded (103.38 %) in 10% NAS with F1.
For K, 5% AS showed greater values as compared to NAS, similar trend was
seen in 10% TWS-Soil mixture except with F2 and F 1+ F2. The maximum
translocation index value was calculated in 20% AS with C i.e. 440% being
the minimum in 10% AS (102.06 %).
Table 3.7.8. The Category-I metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Category I Metals
TSW-Soil (% w:w)
Fungal treatments
Plants C F1 F2
F1+F2
Ca
5 NAS 168.44 154.83 162.85 267.56
AS 184 107.40 124 145.16
10 NAS 131.94 103.38 120 114.28 AS 134.78 121.42 109.21 113.04
20 NAS 167.74 155.26 169.44 123.87 AS 168 156 90.74 171.87
K
5 NAS 168 147.82 110.71 142.02
AS 218.18 224 160 131.03
10 NAS 112.06 132.25 130.76 127.90
AS 102.27 107.40 137.77 129.57
20 NAS 238.46 141.66 142.30 132.25
AS 440 211.11 190.90 187.09
Na
5 NAS 118.42 140.47 129.78 146.78
AS 93.33 98.46 117.24 175.75
10 NAS 117.24 115.78 145.71 160
AS 91.30 100 121.56 100
20 NAS 115.78 109.09 125 116.66
AS 133.33 163.63 141.66 138.88
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; NAS: Non-autoclaved soil; AS: Autoclaved soil
Similar trend was seen in case of Na in 20% concentration, while NAS values
showed greater values as compared to AS in 5% except in F1 + F2 and in
10%.
3.7.5.2 Category-II metals translocation index (%)
The plant translocation index observed to be greater in treatments
applied with fungus than that of C and found to increase along the row, being
72
the maximum where F1 and F2 applied together for all metals as given in
Table 3.7.9. As compared to different TWS-Soil concentrations there was
increasing trend observed with increasing TWS-Soil mixture as maximum
values for both AS and NAS soil was observed for 20% then 10% and
minimum values were found for 5% for all metals.
Such a pattern of increase was observed for Cd in almost all the TSW-
Soil mixtures except for F1 treatment in 20% AS soil where the translocation
index value was recorded 300 %.
In case of Cr plants showed better efficiency in NAS and F2 treatment
with 5% TWS-Soil mixture than AS and in all treatments. Similarly in 10% F1
and F2 performed better than C and F1 + F2. However for 20% the plants in C
showed maximum values for translocation index 197.1 % as compared to all
other treatments. For Cr all values were found to be highest for NAS as
compared to AS for all fungal treatment and TWS-soil concentration.
For Cu, the values were found to be greater in AS as compared to NAS
in 5% with C and F1. However the translocation index values were found to
be greater in NAS than AS for all treatments and TWS-Soil mixture The
maximum value was recorded in 5% AS with F1 treatment i.e. 372.72 %.
For Fe, the maximum translocation index values was recorded to be
300 % for 10% NAS with F2 and 20% AS with C treatment, while minimum
value was recorded in 5% NAS with C treatment i.e. 112.5 %.
In case of Mg the plants showed least value in 5% AS with F2 (40.90
%), while the maximum values for this metal was recorded in 10% AS with F2
i.e. 144.44 %.
For Ni, there was 300% translocation index value recorded in 5% NAS
with C treatment then decrease in values was observed in F1 then in F2 and
least value were recorded in combined fungal treatment i.e. 220% for the
same TWS-Soil mixture while for 10% NAS there was maximum value
recorded for F1 + F2 i.e. 123.07 %.
73
As far as the Zn is concerned there was maximum translocation index
recorded in 20% AS with C treatment i.e. 160 %. For 5% plants showed
maximum values with C as compared to F1, F2 or combined treatment i.e. F1
+ F2 for both NAS and AS. Similar kind of trend was seen in 20% TWS-Soil
mixture
Overall NAS performed well as compared to AS with respect to
translocation index values (%) except for some values.
3.7.5.3 Tolerance index (TI)
In shoots TI values were found to be higher in NAS as compared to AS
in 5% with C and F1, and case was reverse with F2 and F1 + F2. Similarly in
Table 3.7.9. The Category-II metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Metals
TSW-Soil (% w:w)
Fungal treatments
Plants C F1 F2
F1+F2
Cd
5 NAS 110.34 105.12 109.75 107.27
AS 110.52 107.69 111.42 108.97
10 NAS 104.16 104.83 105.88 117.07 AS 116.66 107.40 112.06 121.87
20 NAS 122.22 122.58 118.42 121.73 AS - 300 220 122
Cr
5 NAS 108.79 111.11 153.46 116.66
AS 102.43 104.83 103.59 102.24
10 NAS 138.46 157.72 157.25 140.88
AS 103.31 100.58 113.17 105.55
20 NAS 197.61 147.69 134.21 113.63 AS 118.91 108.47 127.77 106.25
Cu
5 NAS 146.15 135.48 140.62 129.62
AS 300 372.72 123.07 122.58
10 NAS 134.78 114.70 110.52 109.52
AS 118.75 115.78 104.54 125
20 NAS 138.88 145.83 133.33 131.25 AS 136.36 120 121.42 104.54
Fe
5 NAS 112.5 155.55 163.63 140
AS 120 133.33 116.66 116.66
10 NAS 200 166.66 300 200
AS 150 133.33 125 128.57
20 NAS 200 140 225 133.33 AS 300 250 233.33 114.28
Mg
5 NAS 78.57 77.77 69.56 66.66
AS 85.71 62.5 40.90 53.57
10 NAS 109.09 120 138.88 112.5
AS 90 107 144.44 115.78
20 NAS 77.77 64.28 70.58 73.07 AS 100 133.33 70 52
Ni
5 NAS 300 242.85 222.22 220
AS 0 240 125 166.66
10 NAS 80 125 76.92 133.33
AS 87.5 50 81.81 123.07
20 NAS 83.33 90 53.33 105.88 AS 0 114.28 77.77 91.66
Zn
5 NAS 128 126.66 107.69 107.81 AS 150 145.45 136 107.69
10 NAS 115.38 122.85 130.55 128.57 AS 114.28 133.33 127.27 114.28
20 NAS 125 125.80 116.66 123.40 AS 160 126.31 118.75 122.58
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; NAS: Non-autoclaved soil; AS: Autoclaved soil
74
10% and 20%, plants showed better TI values in NAS than AS except in F1 +
F2. The highest TI value for shoot (1.31) was recorded in 5% AS with F2
treatment while minimum value (0.65) was observed for 20% AS with F2.
In case of TI in roots 1.34 was recorded for plants grown in 5% NAS
with F1, while 0.51 was recorded as minimum value in 20% NAS with F2.
Table 3.7.10. The tolerance index (TI) analyzed in shoot and root of 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
TSW-Soil % (w:w)
Fungal treatments
Plants C F1 F2
F1+F2
TI Shoot
5 NAS 1.18 1.19 1.11 1.15 AS 1.13 1.06 1.31 1.23
10 NAS 1.03 1.05 0.95 0.94 AS 0.89 0.87 0.85 1.07
20 NAS 0.84 0.82 0.75 0.69 AS 0.71 0.68 0.65 0.79
TI Root
5 NAS 0.98 1.34 1.27 1.22 AS 1.10 1.21 1.18 1.14
10 NAS 0.73 0.76 0.55 0.86 AS 0.85 0.72 0.73 0.72
20 NAS 0.64 0.65 0.51 0.74 AS 0.75 0.70 0.55 0.74
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; NAS: Non-autoclaved soil; AS: Autoclaved soil
3.7.5.4 Category-I metals Specific extraction yield percentage (SEY %)
Overall SEY % for all category-I metals i.e. Ca, K and Na showed a
maximum value in 5% NAS with F1 + F2 in all TWS-Soil mixture while
minimum in 20% AS with C ( having no fungal inoculum), as shown in Table
3.7.11.
Table 3.7.11. The Category-I metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w)
mixtures.
Category I Metals
TSW-Soil (% w:w)
Fungal treatments
Plants C F1 F2
F1+F2
Ca
5 NAS 37.16 31.41 19.80 83.69 AS 21.84 34.46 34.46 46.76
10 NAS 33.20 47.71 48.11 83.49 AS 21.47 30.81 31.61 58.44
20 NAS 1786 20.88 20.88 37.35 AS 7.21 13.77 11.08 18.72
K
5 NAS 37.64 47.10 29.79 93.82 AS 19.66 45.50 43.82 75.28
10 NAS 50.82 59.50 61.98 80.99 AS 36.77 46.28 44.21 67.35
20 NAS 22.22 29.29 31.81 36.36 AS 6.81 14.14 16.16 22.47
Na
5 NAS 61.25 40.15 23.25 89.29 AS 32.10 47.60 46.49 67.15
10 NAS 25.04 32.60 34.19 56.85 AS 17.49 22.26 22.46 31.01
20 NAS 4.41 4.95 5.81 8.39 AS 1.50 3.12 3.12 4.62
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
75
In case of Ca, for all TWS-Soil TWS-Soil mixture i.e. 5, 10 and 20% the
plants showed higher values of SEY % in NAS as compared to AS with all
fungal treatments except in 5% with F1 and F2 where the case was found to
be reversed.
In case of K, plants cultivated in NAS showed higher values of SEY%
as compared to AS in all TWS-Soil mixture and fungal treatments except in
5% NAS with F2 having 29.79 % in NAS and 43.82% in AS.
For Na, the highest value (89.29%) was recorded in 5% NAS with F1 +
F2 while minimum values was found to be 1.50% in case of 20% AS with no
fungal inoculum.
3.7.5.5 Category-II metals Specific extraction yield percentage (SEY %)
The SEY (%) was calculated in category-I metals that were detected by
AAS. Overall a similar kind of trend was seen for all metals in various fungal
treatments along the row, that the SEY % values increased with the
application of fungal inoculums and highest value was observed for F1 + F2
treatment.
In case of Cd the maximum value for SEY % was recorded in 5% NAS
with F1 + F2 treatment i.e. 43.01 %, While minimum (0.22%) was observed in
20% AS with C.
Similarly as in case of Cd, the SEY % value for Cr was found to be
highest (34.66) in 5% NAS with F1 + F2 and minimum (2.61) was recorded in
20% AS with no fungal inoculum i.e. C. Moreover, in 5% and 20% the NAS
have greater values for all fungal treatments as compared to AS but the case
is reverse for 10% where the plants in AS showed better results as compared
to NAS.
For Cu again the maximum value was observed for 5% NAS (45.95%)
with combined fungal inoculum F1 + F2 and least value (2.47%) was recorded
in 20% AS with C.
In case of Fe, the plants grown in NAS showed higher SEY % values
as compared to AS in all fungal treatments and TWS-Soil concentrations i.e.
76
5%, 10% and 20%. As in case of above mentioned metals again the highest
(72%) and lowest (2.19%) values were recorded in 5% NAS with F1 + F2 and
20% AS with C respectively.
As far as the highest and lowest values are concerned there was a
same trend seen in case of Mg, where plants in 5% NAS showed highest
SEY% value (88.70) with F1 + F2 and being minimum (2.94) in 20% AS with
C having no fungal inoculum.
There was maximum value for Ni was calculated (91.42 %) in 5% NAS
with combined fungal inoculum and minimum value (4.54 %) was recorded in
20% AS for C.
Table 3.7.12. The Category-II metals tolerance index (TI) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Metals
TSW-Soil (% w:w)
Fungal treatments
Plants C F1 F2
F1+F2
Cd
5 NAS 23.01 30.01 32.45 43.01
AS 15.09 25.47 27.92 30.75
10 NAS 7.4 9.6 10.63 13.52 AS 5.92 8.51 9.34 10.79
20 NAS 2.28 3.94 4.74 5.82 AS 0.22 0.68 0.91 2.28
Cr
5 NAS 23.03 25.33 31.03 34.66
AS 10.06 15.39 17.15 21.81
10 NAS 13.60 15.46 16.43 18.68
AS 14.97 16.63 17.36 19.85
20 NAS 4.02 5.19 5.73 7.57 AS 2.61 3.96 3.96 6.38
Cu
5 NAS 23.70 27.03 28.51 45.95
AS 15.18 18.51 21.48 25.55
10 NAS 14.76 17.38 19.04 20.95
AS 8.33 9.76 10.71 12.85
20 NAS 4.09 5.61 6.00 7.04 AS 2.47 3.14 2.95 4.28
Fe
5 NAS 34.00 22.54 15.93 72.00
AS 22 14 26 52
10 NAS 8.82 15.68 19.60 23.52
AS 4.90 6.86 8.82 15.68
20 NAS 3.29 6.59 7.14 11.53 AS 2.19 3.84 5.49 8.24
Mg
5 NAS 40.32 25.80 19.11 88.70
AS 20.96 41.93 50 69.35
10 NAS 18.54 26.61 34.67 41.12
AS 15.32 21.77 17.74 33.06
20 NAS 7.84 11.27 14.21 22.05 AS 2.94 6.86 8.33 18.62
Ni
5 NAS 57.14 43.63 26.36 91.42
AS 25.71 48.57 51.42 68.57
10 NAS 32.72 49.09 41.81 63.63
AS 27.27 27.27 36.36 52.72
20 NAS 10 17.27 20.90 31.81 AS 4.54 13.63 14.54 20.90
Zn
5 NAS 19.52 17.98 20 45.54 AS 15.41 18.49 20.20 36.98
10 NAS 14.81 20.63 21.95 33.86 AS 7.93 14.81 13.22 23.80
20 NAS 6.66 17.28 19.25 25.92 AS 3.20 10.61 8.64 17.03
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil
77
For Zn, plants in NAS showed greater SEY% values as compared to
AS for all TWS-Soil mixture and treatments except in 5% with F1 and F2
where AS showed greater values as compared to NAS. However as far as the
maximum and minimum values of SEY% are concerned again there was
similar trend seen i.e. maximum value (45.54 %) in 5% NAS with F1 + F2 and
minimum (3.20 %) in 20% AS with C.
78
3.8 Experiment with saprobic and AM fungi
3.8.1 Pre-sowing analysis
The physico-chemical properties, concentration of Category-I & II
metals are given Table 3.1, 3.2 and 3.3 and the details are described in
Chapter 3.1.
3.8.2 Biochemical analyses of 50-days old Tagetes patula
The biochemical parameters like chlorophyll contents, soluble protein
CAT and SOD were observed. There was increased production of all these
parameters in combined inoculation of fungi as compared to C and single
fungi. The specific details of each of the biochemical parameters are as
under:
3.8.2.1 Chlorophyll content
After 50 days of cultivation, the plant chlorophyll contents within a
fungal treatment were observed to increased by applying combined fungal
inoculation i.e. the F1 + F2, as given in Table 3.8.1.
Table 3.8.1. The biochemical parameters observed in 50-days old Tagetes patula cultivated
on TSW-Soil mixtures applied with different fungi. The mean values S.D. with common
letters (small along the row & capital within a column) are not significantly different according
to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Chlorophyll contents (SPAD value)
0 14.4cB
0.98 16.1bAB
0.89 16.9aAB
0.91 17.2aB
0.34 0.9
10 15.2cA
0.67 16.8bcA
1.09 17.4bA
0.56 19.7aA
0.45 1.6
20 13.8cBC
0.23 15.3bcB
1.1 16. 3bB
0.45 18.5aAB
0.78 1.8
LSD0.05 0.7 0.8 0.6 1.3
Protein content
(mgg-1
)
0 1.2abBC
0.67 0.9bC
0.87 1.4aBC
0.34 1.7aB
0.98 0.6
10 8.9dA
1.1 10.5cA
0.89 12.4bA
0.99 15aA
0.55 1.6
20 4.2bB
1.09 5.6abB
0.14 6.4aB
1.01 3.7bcB
0.66 0.9
LSD0.05 3.6 3.8 5.1 7.8
SOD (Umg
-1 of protein)
0 BDL BDL BDL BDL -
10 18bA
1.78 14cB
1.1 16bcB
1.78 21aA
0.99 2.9
20 17cA
1.79 19.7bA
2.0 20bA
1.89 22aA
1.78 1.8
LSD0.05 0.6 2.5 1.8 0.7
CAT (Uml
-1)
0 0.13bBC
1.09 0.17bC
0.33 0.16bC
0.12 2.3aB
1.5 0.9
10 12.2cA
1.2 13.5bcA
0.78 16bA
1.9 22aA
1.45 2.7
20 4.4bB
1.05 5.9aB
0.93 6.3aB
1.4 3.5bcB
1.09 1.3
LSD0.05 4.8 4.6 5.9 6.7
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
79
The F1 + F2 cause the greatest (19.7 SPAD value) increase in plants
from 10 % while influencing to the least (13.8 SPAD value) in 20 % in C
treatment as compared to any of the treatments within a row. However F2
showed better values with respect to chlorophyll content as compared to F1
values.
Within column, the general trend was decrease in plant chlorophyll
contents with the increase of TSW percentage in soil for all the fungal
treatments. The F1 + F2 from the 10 % NAS while 20 % AS with C gave the
best and the poor (performances respectively like the way it was found in
within-a-row comparison. The application of fungus either individually or in
combination help plants perform better as compared to Control in terms of
chlorophyll contents, as given in Table 3.8.1.
3.8.2.2 Soluble protein contents
Parallel to chlorophyll contents, the values within a row for
soluble protein contents also increased with the application of fungal
inoculations for all the treatment, as given in Table 3.8.1. The F1 + F2 from 10
% and the F1 from 0 % exhibited the maximum (15 mgg-1) and the minimum
(0.9 mgg-1) soluble protein contents as compare to any of the soil or fungal
treatments.
Within a column, overall there was increase in soluble protein contents
with the increasing level of TSW in soil treatments i.e. in 10 % and then
decrease in 20 %. However, addition of fungi helped plants to alleviate stress
by increasing soluble protein contents. The F1 + F2 superseded F1, F2 and C
for all of the soil treatments. The 10 % with F1 + F2 and the 0 % with F1
inoculations observed to have the maximum and the minimum values
respectively. The plants cultivated in pots with F2 inoculations performed
better than those with F1 for all the TSW-Soil mixtures.
3.8.2.3 Superoxide dismutase (SOD) contents
Parallel to the trends found for chlorophyll and soluble protein contents,
the SOD values increased with fungal inoculations within a row for all the
treatments, as given in Table 3.8.1. The SOD values were found to be below
80
detection limits (BDL) in all treatments of 0 (% TSW-Soil). For rest of the
concentrations the fungal treatments having both of the F1 and F2 performed
better than control and those applied with either F1 or F2. The plants in 20 %
with F1 + F2 gave maximum SOD values (22 Umg-1 of protein) while being
the least (14 Umg-1 of protein) in case of plants from 10% with F1 treatment.
Within a column, the plants cultivated in F1 + F2 performed better than
control, F1 as well as F2. The plants from 20% performed better than 10% in
all treatments except control.
3.8.2.4 Catalase (CAT) contents
The fungal inoculations of TSW-Soil mixtures helped plants improve
their defense mechanism by increasing CAT values. Within treatments, there
was an increase in plant CAT value with the individual or combined fungal
inoculations, as compared to control. The plants from 10 % with F1 + F2 and
0 % with C had the maximum (22 Uml-1) and the minimum (0.13 Uml-1) CAT
values as compared to any of the treatments as given in Table 3.8.1.
For different TSW-Soil concentration comparison, there was minimum
CAT contents was observed in 0% concentration. The highest values were
observed for 10% then in 20% TWS-Soil mixture The order from lowest to
highest values for CAT content can be written as 0% >20% >10%.
Cumulatively, the values for plant under F1 + F2 inoculations column had
significantly higher values as compared to values under either of the C, F1 or
F2 columns.
3.8.3 Post-harvest analysis
3.8.3.1 Growth performance of Tagetes patula
The maximum growth of 50-day-old plants of T. patula was observed in
case of soil, being relatively less in 10 % and 20 % as indicated by growth
parameters (Table 3.8.2). It was noticed that plants cultivated in soil and its
TSW mixtures inoculated with fungal isolates yielded greater shoot, root and
seedling length, no. of leaves and roots, as well as, fresh and dry weight; as
compared to control. The statistical analysis of the data showed significant
81
growth in all parameters in lower TSW concentration in soil followed by a
decrease at higher (20 %) concentration. However, the maximum increase in
values was found in F + M treatment over their controls F1 and F2, for each of
the corresponding soil treatments. The details of each of the morphological
parameters is given in Table 3.8.2 and described as under:
3.8.3.1.1 Shoot, root and seedling length (cm)
Along the row, the maximum plant shoot (68.0 cm), root (4.1 cm) and
seedling length (108.2 cm) respectively were observed in 0 % with F1 + F2
inoculation; while being the minimum for plant shoot and seedling length in 20
% with no fungal inoculation i.e. the C. The minimum values for plant root
length was recorded in 20 % with F1 (mycorrhizal inoculation). There was
increase in length of all the three vegetative parameters with the application of
fungal inoculations and the order of increase observed to be F1 + F2 > F2 >
F1 > C along the row, as can be seen in Figure 3.8.1.
Figure 3.8.1. The vegetative growth variation in Marigold (Tagetes patula) in response to soil mixed with
different percentages of TSW (% w:w) and inoculated with different fungi.
Within column, there was decrease in plant shoot, root and seedling
length with the increasing proportion of TSW in the soil. The different TSW-
Soil mixtures under F1 + F2 column gave best results while those in C column
attained the least height.
3.8.3.1.2 No. of leaves and roots
Along the row, the plants in 0% with F1 + F2 inoculation observed to
have maximum no. of roots (27) and leaves (21) while being the minimum (6
82
and 6 respectively) in 20 % without any of the inoculation i.e. C. The TSW-Soil
mixtures with F2 performed better than those inoculated with F1 and no fungal
incorporations.
Table 3.8.2. Various morphological parameters observed in 50-days old Tagetes patula
cultivated on TSW-Soil mixtures applied with different fungi. The mean values S.D. with
common letters (small along the row & capital within a column) are not significantly different
according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Shoot Length (cm)
0 45.1cdA
0.11 50.5cA
0.21 60.2bA
0.20 68.0aA
0.21 6.9
10 44.2bA
0.17 42.5bB
0.45 43.9bB
0.52 65.10aA
0.35 5.7
20 25.9cA
0.45 31.2bcC
0.12 34.5bBC
0.41 40.3aB
0.30 4.4
LSD0.05 6.2 6.7 9.8 9.5
Root Length (cm)
0 30.1cA
0.23 35.2bA
0.55 39.4aA
0.44 40.1aA
0.21 3.1
10 29.0abA
0.24 21.5cB
0.25 25.4bB
0.27 31.6aB
0.28 2.9
20 18.9bB
0.36 15.2cC
0.25 17.5bcC
0.16 21.4aC
0.27 1.9
LSD0.05 4.1 6.8 7.5 6.5
Seedling Length (cm)
0 75.4cA
0.28 86.0cA
0.16 99.7bA
0.34 108.2aA
0.66 8.5
10
73.4bA
0.34 64.2cB
0.52 69.4bcB
0.36 97.2aAB
0.17 6.3
20 44.8cB
0.21 46.5cC
0.47 52.1bC
0.23 61.9aB
0.23 4.7
LSD0.05 10.7 13.4 16.8 15.9
No. of roots
0 21cA
O.12 20cA
0.22 24bA
0.43 27aA
0.32 2.9
10 13bcB
0.12 11cB
0.56 14bB
0.31 16aB
0.45 1.7
20 6dC
0.16 8cBC
0.13 10bBC
0.48 12aBC
0.27 1.9
LSD0.05 5.6 4.8 4.9 5.8
No. of leaves
0 14cA
0.11 16bcA
0.21 18bA
0.42 21aA
0.33 2.1
10 7cB
0.34 9bB
0.23 10bB
0.63 13aB
0.41 1.8
20 6cB
0.09 8bB
0.14 9abB
0.12 10aBC
0.54 1.2
LSD0.05 2.8 2.9 3.2 3.6
Fresh wt. (g)
0 26.5cA
0.17 29.1bA
0.16 30.7bA
0.08 35.2aA
0.36 3.5
10 19.2bB
0.36 18.4bcB
0.16 19.1bB
0.46 24.4aB
0.24 2.1
20 12.4bC
0.34 11.3bcC
0.29 12.1bC
0.34 14.2aC
0.22 1.4
LSD0.05 4.9 6.1 6.5 7.1
Dry wt. (g)
0 10.4bA
0.12 9.5bcA
0.13 10.1bA
0.65 13.5aA
0.34 0.98
10 8.1bcB
0.22 8.5bcAB
0.52 8.9bAB
0.38 9.7aB
0.27 0.80
20 5.2cC
0.41 6.1bB
0.12 6.7abB
0.37 7.1aC
0.26 0.56
LSD0.05 1.9 1.6 1.3 2.5
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
Within different TWS-Soil mixture of TWS-Soil, the increasing ratio of
TSW decreased the no. of leaves and roots. The TSW-Soil mixtures under F1
+ F2 gave the best vegetative growth than any of the fungal treatments. The
worst growth response observed to be in C column.
3.8.3.1.3 Fresh and dry weight (g)
The fresh and dry weight was observed to be the maximum and the
minimum in accordance with the maximum and the minimum no. of leaves
83
and roots for both along the row as well as within column comparisons. In
other words, along the row the maximum weight (35.2 g fresh, 13.5 g dry)
observed to be in 0 % with F1 + F2 and the minimum (11.3 g fresh, 5.2 g dry)
in 20 % with F1 and C, without any fungus respectively.
Within column, the increasing TSW ratio affected the biomass
production negatively. The TSW-Soil mixtures under F1 + F2 yielded
maximum fresh and dry weight while those in C column yielded the least.
3.8.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot were variable with respect to fungal inoculations as well as increasing
ratio of TSW in soil, as given in Table 3.8.3.
Table 3.8.3. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied with different fungi. The mean
values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Ca
0 110cB
0.34 190bB
1.23 210bB
0.98 325aB
0.23 56
10 220cA
0.67 280bcA
2.2 305bA
2.6 390aA
0.25 45
20 95cB
0.56 145bC
1.09 170bBC
0.98 385aA
0.67 75
LSD0.05 46 49 48 25
K
0 55cC
0.12 120bcBC
0.23 150bC
0.55 225aB
0.17 55
10 125cB
1.4 165bcB
0. 210bB
0.33 355aAB
0.83 62
20 250cA
0.12 295bcA
0.94 310bA
0.97 390aA
0.45 38
LSD0.05 45 60 56 57
Na
0 440cBC
1.4 510bBC
1.8 525bC
1.9 690aC
1.1 65
10 550cB
2.3 610bcB
0.9 650bB
1.5 760aB
0.23 55
20 810dA
1.6 850cA
1.09 910bA
1.3 950aA
1.6 38
LSD0.05 128 118 130 90
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
3.8.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in shoot increased with inoculation
of fungi as compared to C. The maximum (390 mgkg-1) shoot Ca observed to
be in 10 % with F1 + F2 while being the minimum (95 mgkg-1) in 20% with C.
The plants with F2 inoculation had more Ca in shoot than those with F1.
Within column, the maximum shoot concentrations were in TSW-Soil
mixtures with F1 + F2 while being the minimum in those where no fungi was
applied. It was observed that shoot Ca increased with the increasing TSW
ratio in soil mixtures for 10% and then decreased for 20%.
84
3.8.3.2.2 Potassium (K) in shoot
Along the row, the shoot K uptake increased with fungal applications
for all the TSW-Soil mixtures. The maximum K level (390 mgkg-1) was
observed in 0 % with F1 + F2 while being the minimum (55 mgkg-1) in soil with
no fungi i.e. C.
Within column, the K shoot uptake increased with the increasing
concentration of TSW in soil mixtures for all the concentrations. For all TSW-
Soil mixtures, the F2 plants showed more K uptake than those with F1 and
being the least where no fungus was applied. The maximum values observed
under F1 + F2 column.
3.8.3.2.3 Sodium (Na) in shoot
The Na concentration in shoot observed to increase along the row and
it was because of fungal inoculations. The pots with F1 + F2 showed the
greatest Na shoot uptake, the F2 being greater than F1, while those with no
fungi being the least. The plants in 20 % with F1 + F2 had the highest value
(950 mgkg-1) while those in 0 % with no fungi exhibited the lowest Na shoot
contents (440 mgkg-1).
Within column, the increasing ratio of TSW in soil mixtures enhanced
the shoot Na uptake for all the fungal treatments. In case of 0 %, the
concentration of shoot Na observed to be the least for all the fungal
treatments.
3.8.3.3 Category-I metals in plant ROOT
The bioavailability of Category-I metals was variable with different fungi
in root also however, it was directly related to the increasing ratio of TSW in
soil mixture, as given in Table 3.8.4.
3.8.3.3.1 Calcium (Ca) in root
The application of fungal inoculum to the soil helps to increase Ca uptake
along the row i.e. various fungal treatments. The plants in F1 + F2 pots
observed to have maximum while those with no fungi having the minimum
root Ca than any of the fungal treatments for all of the TSW-Soil mixtures. The
85
highest root Ca (365 mgkg-1) was in 10 % with F1 + F2 while being the
minimum (55 mgkg-1) in 0 % with no fungal inoculation.
Table 3.8.4. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of 50-days old Tagetes patula cultivated on TSW-Soil mixtures and applied with different fungi. The
mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Ca
0 55cC
0.2 150bD
0.23 170aC
1.09 295aB
0.87 62
10 200cA
0.97 240bcA
0.23 275bA
1.1 365aA
1.8 45
20 70cC
0.46 130bcD
0.35 150bC
1.5 275aBC
1.5 54
LSD0.05 50 40 43 35
K
0 30cDE
1.12 95bcD
1.3 110bD
1.7 210aD
0.12 48
10 90cD
1.05 160bcC
1.45 190bBC
2.01 310aAB
0.98 60
20 230cdA
0.23 255cA
0.09 290bA
1.6 345aA
0.12 35
LSD0.05 71 56 65 52
Na
0 710cD
0.65 850bC
1.67 890bD
1.51 995aD
1.7 85
10 750cCD
0.12 950bBC
1.12 1000bBC
1.71 1240aB
1.9 130
20 940dA
0.67 1150bcA
2.12 1210bA
1.01 1310aA
2.01 98
LSD0.05 82 115 120 120
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
3.8.3.3.2 Potassium (K) in root
The K contents in the 20 % with F1 + F2 exhibited the maximum root
uptake (345 mgkg-1) than any of the soil treatments while being the minimum
(30 mgkg-1) in 0 % with no fungal application. The application of fungus as
individual inoculant i.e. F1 and F2 showed better K uptake than TSW-Soil
mixtures where no fungi has been applied. However, F2 showed better uptake
as compared to F1 treatment.
Within column, the metal uptake in root increased with increasing ratio
of TSW in 10 and 20 %, however, it was found to be minimum in 0 %.
3.8.3.3.3 Sodium (Na) in root
Along the row as observed in case of Ca and K, the application of fungi
helps increase Na uptake in roots. The maximum Na in root (1310 mgkg-1)
was observed in 20 % with F1 + F2 while being the minimum in 0 %
amounting 710 mgkg-1. Those with F1 and F2 applications also performed
better than C i.e. treatment with no fungal inoculation.
86
Within column, trend of Na root uptake was also similar to what
observed in case of Ca and K. The increasing ratio of TSW in soil displayed
increased root Na uptake as compared to Soil with C for all the fungal
treatments.
3.8.3.4 Category-II metals in plant shoot
The Category-II metals i.e. the AAS detected metals in shoot were
variable with respect to fungal inoculations as well as increasing ratio of TSW
in soil, as given in Table 3.8.5. The application of fungi enhanced trace metal
uptake tendency of plant for all the TSW-Soil mixtures. However, the
increasing level of Category-II metals in shoot was in accordance with the
increasing ratio of TSW in soil mixtures for all the fungal treatments.
3.8.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in TSW-Soil mixtures with combined
fungal treatments. Maximum amount of metal (850 mgkg-1) was observed in
20% TSW-Soil mixture in combined inoculation of fungi i.e. F1 + F2, while
being minimum in 0% amounting 110 mgkg-1.
Within different concentrations there is increasing trend of metal
accumulation with TSW-Soil TWS-Soil mixture
3.8.3.4.2 Cr in shoot
With different fungal treatments the maximum Cr accumulation in shoot
was observed in treatments applied with combined inoculants i.e. F1 + F2,
being significantly higher than any of the treatments along the rows. Cr was
found to be as low as 1 mgkg-1 in 0% with C and maximum 1,450 mgkg-1 were
recorded in 20% with F1 + F2.
Within different TWS-Soil mixture of TWS-soil there was increasing
trend of accumulation of metal as the TWS-Soil mixture increased i.e.
maximum accumulation was observed in 20%, while minimum accumulation
of metal was observed in 0%.
87
Statistical analysis showed the significant difference of accumulation of
Cr in 0 % and 20 %.
Table 3.8.5. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 50-days
old Tagetes patula cultivated on TSW-Soil mixtures applied with different fungi. The mean
values S.D. with common letters (small along the row & capital within a column) are not
significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 110bD
1.5 100bcC
1.6 120abCD
1.09 140aD
1.1 25
10 410cAB
0.98 590bA
0.91 620bA 1.04 710
aAB 0.76 80
20 520efA
0.34 590eA
0.47 610dA
1.1 850aA
0.23 85
LSD0.05 145 165 170 205
Cr
0 1cCD
0.01 3bC
0.02 4aBC
0.01 5aC
0.01 1.5
10 1,050cA
2.03 990cdAB
1.8 1,120bA
2.1 1,210aA
2.1 65
20 1,110dA
1.2 1,190cdA
1.3 1,345bA
3.2 1,450aA
2.6 95
LSD0.05 375 398 455 485
Cu
0 85cD
0.43 90cCD
0.59 110abCD
0.23 125aC
0.98 25
10 310dB
0.23 425bcAB
0.12 490bcA
0.18 610aAB
1.1 85
20 450dA
0.68 510cA
0.89 550bcA
1.1 690aA
3.1 75
LSD0.05 105 115 120 140
Fe
0 BDL 12bcCD
0.23 18bC
0.45 30aCD
0.86 12
10 75cdB
0.89 90cA
0.12 110bcAB
0.24 275aA
0.98 55
20 85deA
0.65 115dA
0.75 145cdA
0.89 325aA
0.23 75
LSD0.05 8.8 45 50 110
Mg
0 20cCD
0.11 30cD
0.19 45bcCD
0.89 85aD
0.99 25
10 125cdA
0.89 190cA
0.23 220bcA
1.1 345aA
0.19 70
20 130dA
0.56 155cAB
0.49 170cB
0.89 275aB
0.45 45
LSD0.05 40 45 48 72
Ni
0 BDL BDL BDL BDL -
10 10cB
0.13 15bcB
0.19 20bB
0.99 30aBC
0.33 8
20 15cA
0.15 25bA
0.45 30bA
0.28 50aA
0.73 12
LSD0.05 4 6 9 12
Zn
0 15dDE
0.13 65bcBC
0.72 90bCD
0.89 135aC
1.2 38
10 120cA
0.39 230bA
1.3 250aA
1.5 280aAB
0.99 49
20 70dC
0.38 170cB
0.89 245bA
0.38 320aA
1.5 70
LSD0.05 25 43 69 75
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
3.8.3.4.3 Cu in shoot
Along the row, the plants harvested from F1 + F2 exhibited maximum
Cu accumulation as compared to any of the treatments with single or no
fungal application. Such a trend was observed in all of the TSW-Soil mixtures.
The F2 proved to be better enhancer of Cu uptake in plant shoot as compared
to the F1. The plants from treatments with no fungal inoculations i.e. C
showed the significantly least Cu accumulation as compared to any of the
fungal treatments as shown in Table 3.8.5.
88
Within different TWS-Soil TWS-Soil mixture 0 % showed significantly less Cu
uptake in shoots than those form 10 and 20 % as minimum accumulation (85
mgkg-1) was seen in 0 % with C and maximum accumulation (690 mgkg-1) in
20 % with F1 + F2.
3.8.3.4.4 Fe in shoot
The Fe uptake in plant shoots observed to be BDL in Soil (0 %) with C,
but F1 + F2 inoculation displayed the maximum Fe uptake in shoot as
compare to plants from the treatments with single or no fungal inoculations.
The values of plant shoot Fe was minimum 12 mgkg-1 in 0 % with F1 and
maximum 325 mgkg-1 in 20 % with F1 + F2.
Within a column, a similar kind of trend for metal accumulation was
observed as with Cu that there was increasing trend of metal accumulation by
the plants as the TWS-Soil TWS-Soil mixture increased as shown in Table
3.8.5.
3.8.3.4.5 Mg in shoot
Along various fungal treatments, the Mg uptake in plant shoot
increased with fungal application in the pots and found to be the maximum
345 mgkg-1 in plants harvested from pots applied with combined fungi i.e. F1
+ F2 in 10 % TWS-Soil mixture, while being the minimum as well as
significantly least in shoot of plants cultivated in soil with no fungus i.e. 20
mgkg-1 as shown in Table 3.8.5. Such a pattern was observed for all the TSW-
Soil mixtures.
For different TWS-Soil mixture, the value of plant shoot Mg increased
with increasing level of TSW percentage in soil up to 10 % (TSW-Soil). At 20
%, the shoot uptake decreased drastically and significantly as compared to
the treatments with preceding lower dose of TSW in soil
3.8.3.4.6 Ni in shoot
The plant shoot Ni observed to be BDL in 0 % i.e. Soil. However, its
value found to increase with application of fungus and observed to be the
maximum in case of plants cultivated in pots with F1 + F2. The plants from 10
89
and 20 % (TSW-Soil) observed to follow the same sequence. The maximum
accumulation was observed in 20 % for F1 + F2 while minimum value 10
mgkg-1 in 10 % with C having no fungal application.
In a TWS-Soil mixture comparison, the value of plant shoot Ni
concentration increased with the increasing percentage of TSW in soil and
found to be the maximum in plants harvested from 20 % being the minimum in
plants from 10 %.
3.8.3.4.7 Zn in shoot
The Zn concentration in shoots increased in plants harvest from pots
with fungal inoculations than those harvested from pots applied with no fungi.
Like other metals, the shoot Zn observed to be the maximum in plants
inoculated with F1 + F2 while those harvested from the pots with no fungi
displayed the minimum values. There was increasing trend of accumulation of
metal as the application of fungi. However F2 showed better results as
compared to F1 in terms of accumulation of metal as shown in Table 3.8.5.
For different TWS-Soil mixtures the value of shoot Zn concentration
was found to be maximum 320 mgkg-1 in 20 % with F1 + F2 being minimum
15 mgkg-1 in 0 % with C as shown in Table 3.8.5. The order of accumulation
of metal accumulation within different TWS-Soil mixture from maximum to
minimum was as 10 % >20 % > 0 %.
3.8.3.5 Category-II metals in plant root
The Category-II metals i.e. the AAS detected metals in root were
observed to differ with varying levels of TSW in the soil as well in response to
fungal inoculations, as given in Table 3.8.6.
3.8.3.5.1 Cd in root
For different fungal treatments within the rows, the Cd level in plant
roots harvest from Soil observed to be minimum 150 mgkg-1 in 0% with C and
found to be maximum 1,190 mgkg-1 in 20 % with F1 + F2 as shown in Table
3.8.6. The plant root Cd concentration increased with fungal inoculations and
found to be maximum as well as significantly higher than those applied with
90
single or no fungal treatments, while being the minimum and significantly least
in C.
Inside the columns, for different TWS-Soil TWS-Soil mixture the root
Cd concentration increased with increasing percentage of TSW in soil being
maximum in 20 % for C, F1, F2 as well as F1 + F2.
Table 3.8.6. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied with different fungi. The mean
values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 150cdCD
0.89 190cCD
0.12 210bcD
0.67 290aCD
0.98 42
10 710dAB
0.99 850cA
1.2 890bcA
1.1 1,050aA
1.4 90
20 840dA
0.36 950cA
1.5 990cA
0.89 1,190aA
1.3 95
LSD0.05 238 265 280 320
Cr
0 3abCD
0.01 2bC
0.03 4aC
0.05 3abBC
0.23 1.2
10 1,500cdB
1.5 2,550bAB
0.88 2,910aA
0.67 3,100aAB
0.28 425
20 2,500eA
2.5 3,150cdA
0.91 3,410bcA
0.19 3,950aA
0.20 375
LSD0.05 835 1025 1145 1330
Cu
0 110dDE
0.89 90cdCD
0.91 125bCD
0.92 140aC
0.82 12
10 510dC
1.1 640cB
0.29 590cdBC
2.4 890aAB
0.81 115
20 910deA
2.9 1,100cA
2.3 1,250bA
0.89 1,390aA
1.62 135
LSD0.05 280 355 390 440
Fe
0 BDL BDL BDL BDL -
10 45dB
0.92 70cAB
0.29 85cAB
0.89 155aC
0.91 35
20 65dA
0.29 90dA
0.39 120cdA
0.40 270aA
0.23 59
LSD0.05 10 25 45 55
Mg
0 BDL BDL 30bCD
0.29 55aCD
0.39 8
10 75dA
0.39 135bA
0.12 170aA
0.19 110cB
0.1 25
20 65eB
0.49 110dB
0.90 165cA
0.38 260aA
1.1 45
LSD0.05 5 12 55 75
Ni
0 BDL. BDL BDL BDL -
10 6bB
0.22 8abAB
0.23 10abB
0.49 15aC
0.1.3 8
20 10cA
0.34 15bcA
0.95 20bcA
0.48 45aA
2.6 14
LSD0.05 2.5 11 4 15
Zn
0 10cdDE
0.45 30cCD
0.39 45bC
0.59 80aC
0.92 25
10 90dA
0.29 185abA
0.32 210aA
0.38 220aA
2.6 48
20 65dB
0.92 125cBC
0.99 215aA
0.45 245aA
1.1 55
LSD0.05 22 35 70 80
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
3.8.3.5.2 Cr in root
For different fungal treatments i.e. along the row, the plants from F1 +
F1 fungal inoculations showed the maximum 3,950 mgkg-1 root Cd
concentration in 20 %, while with F1 treatment plants having the minimum
uptake 2 mgkg-1 in 0%. The F2 inoculation enhanced the root Cd
concentration better than F1 as well as C as shown in Table 3.8.6.
91
Within the columns, the root Cd concentration increased with
increasing percentage of TSW in soil having the maximum accumulation of
metal in 20 % for all fungal treatments like C, F1, F2 and F1 + F2.
3.8.3.5.3 Cu in root
The fungal inoculations enhanced the root Cu uptake as compared to
C. The plants from F1 + F2 observed to have the maximum (1,390 mgkg-1)
uptake in 20 % than those cultivated in pots with single or no fungal
inoculations and found to be the minimum 90 mgkg-1 in 0% with F1. The F2
observed to be better enhancer of root Cu uptake than F1.
Under the columns, the plants from 10 and 20 % TSW-Soil exhibited
better root Cu accumulation than Soil. The 20 % plants had the maximum root
Cu uptake than those harvested from Soil and 10%.
3.8.3.5.4 Fe in root
Moving alongside the row while analyzing effect of different fungal
treatments, the root Fe observed to be BDL in Soil with all fungal treatments.
The plants from C had the lowest root Fe as compared to plants from any of
the single or combined application of fungi. The root Fe accumulation
observed to be the maximum in F1 + F2 and significantly higher than those
harvested from any of the treatments with single or no fungal inoculations.
Inside columns, the root Fe concentration decreased with increasing
percentage of TSW in soil for all the fungal treatments. The maximum Fe
uptake (270 mgkg-1) by roots was observed in 20% with F1 + F2 treatment
while the least value of metal uptake (45 mgkg-1) was observed in 10 % with C
as shown in Table 3.8.6.
3.8.3.5.5 Mg in root
While analyzing the fungal application effect on plants, it was observed
that the Mg root concentration increased in plant roots with fungal application
as compared to C i.e. with no fungal inoculation. The F1 + F2 plants displayed
the maximum root Mg concentration than those from C as well as F1 and F2.
92
For 0% the values for metal uptake were found to be BDL with C and F1. The
F2 inoculations gave better results than F1.
For the columns, the root Mg accumulation decreased with increasing
percentage of TSW in soil for all fungal treatments except for F1 + F2 where
the metal accumulation was increased i.e. 260 mgkg-1 in 20%, as compared to
110 mgkg-1 in 10% as shown in Table 3.8.6.
3.8.3.5.6 Ni in root
The root Ni concentration was found to be BDL for all fungal treatments
in 0% i.e. soil. However there was increased metal accumulation along the
row with the application of fungal inoculations and found to be the maximum
in plants applied with combined application of both of the fungi while being the
minimum in C with no fungus added. The F2 application incurred better root
Ni uptake effects than F1.
Within columns, the plants from 20 % TSW-Soil mixtures had the
maximum root Ni level than 10 % for all of the fungal treatments. There was
maximum (45 mgkg-1) accumulation of metal was noted in 20% with F1 + F2
while minimum uptake (6 mgkg-1) was observed in 10% with C.
3.8.3.5.7 Zn in root
The root Zn accumulation observed to increase in pots applied with
fungal inoculations than C and found to be the maximum in treatments applied
with both of the fungi and being the minimum with no fungal applications.
Again as with most of the above discussed metals F2 performed better than
F1 as far as metal accumulation efficiency of the plant is concerned.
For different TWS-Soil TWS-Soil mixture, the root Zn concentration
increased with increasing percentage of TSW in soil with every fungal
treatment except F1 where the uptake was reduced in 20% (125 mgkg-1) as
compared to TWS-Soil mixture in 10 % (185 mgkg-1) with the same fungal
treatment i.e. F1 as shown in Table 3.8.6.
93
3.8.4 Fungal analyses
The results of assessment of fungal inoculum both in soil and in plants
are shown in Table 3.8.7. For analysis of various fungal treatments it is
observed that for the C treatment (without any inoculum) plants showed 0 %
AM infection and no c.f.u for T. pseudokoningii and no spores in soil. In 10 %
concentration of TWS-Soil, F1 + F2 showed maximum percentage of infection
(95 %) while the minimum infection (35 %) was observed in 20 %
concentration in F2 treatment. Similarly, maximum c.f.u. per gram of soil was
observed in 20 % TWS-Soil mixture in F1 + F2 treatment 3.5 X 105 c.f.u. of T.
pseudokoningii per gram of soil and the minimum value, 0.1 X 105 was
observed for F2 treatment. The maximum spore number of 256 spores per 50
g of soil was observed in F1 + F treatment, while the minimum number of 50
spores per 50 g of soil was observed for 20 % TWS-Soil mixture in F2
treatment.
Table 3.8.7. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 50-days old Tagetes
patula cultivated on TSW-Soil mixtures applied with different fungi. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Percentage infection of AM fungi (in roots)
0 - 86.0aA
23 60cA
0.91 90aA
0.33 10.6
10 - 88abA
1.4 64cA
0.67 95aA
1.6 8.3
20 - 55aCD
1.6 35dCD
2.3 59aCD
1.2 7.4
LSD0.05 - 11.9 10.2 12.8
T. pseudokoningii (× 10
5 c.f.u. g
-1 soil)
0 - 0.30bcA
0.34 0.96aCD
0.78 1.20aC
0.23 0.87
10 - 0.16bcC
0.50 1.1aB
0.23 1.40aC
0.56 0.67
20 - 0.20deAB
0.33 1.6bA
0.98 3.5aA
0.93 1.3
LSD0.05 - 0.12 0.43 0.98
Spore No. 50 g-1
soil
0 - 190bcA
0.29 64dA
0.92 256aA
2.1 55
10 - 185bAB
0.23 60dAB
1.2 250aA
1.6 50
20 - 170aD
0.93 50dCD
1.6 188aCD
2.3 42
LSD0.05 8.9 5.8 25
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference
3.8.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
3.8.5.1 Category-I metals translocation index (%)
94
The plant translocation index (%) values were recorded for Category-I
metals those detected by flame photometer i.e. Ca, K and Na shown in Table
3.8.8.
Table 3.8.8. The Category-I metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 (M) F2 F1+F2
Ca 10 110 116.66 110.90 106.84
20 135.71 111.53 113.33 140
K 10 138.88 103.12 110.52 114.51
20 108.69 115.68 106.89 113.04
Na 10 73.33 64.21 65 61.29
20 86.17 73.91 75.20 72.51
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii;
In case of Ca, maximum value was observed in 20 % with F1 + F2 i.e.
140 % while the minimum value was recorded (110 %) in 10% with C.
For K, the maximum translocation index value was calculated in 10 %
with C i.e. 138.88 % being the minimum in 10 % with F1 (103.12%).
In case of Na in for both 10 and 20 % (TSW-Soil) with C treatment
showed maximum values as compared to any other treatment. Within row, in
10 % (TSW-Soil) the maximum values were noted with C i.e. 73.33 % and for
also for 20 % (TSW-Soil) with C the maximum values (86.17 %) along the row
were noted while the minimum values were recorded for corresponding F1 +
F2.
3.8.5.2 Category-II metals translocation index (%)
The plant translocation index found to be greater in treatments applied
with fungus than that of C, being the maximum where F1 and F2 applied
together for all metals as given in Table 3.8.9. As compared to different TWS-
Soil concentrations there was decreasing trend observed with increasing
TWS-Soil mixture of TWS-Soil with few exceptions.
For Cd, the maximum Translocation index (71.42 %) was noted for F1
+ F2 in 20 % (TSW-Soil) while minimum value was recorded 57.74 % in 10%
with C having no fungal application.
95
In case of Cr plants showed better metal translocation efficiency (70 %)
in 10 % with C. similarly for 20 % the plants in C showed maximum values for
translocation index 44.4 % as compared to all other treatments.
For Cu, the maximum translocation index values was recorded to be
83.05 % for 10 % (TSW-Soil) with F2, while minimum value was recorded in
20 % (TSW-Soil) with F2 treatment i.e. 44 %.
Table 3.8.9. The Category-II metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 (M) F2 F1+F2
Cd 10 57.74 69.41 69.66 67.61
20 61.90 62.10 61.61 71.42
Cr 10 70 38.82 38.48 39.39
20 44.4 37.77 39.44 36.70
Cu 10 60.78 66.40 83.05 68.53
20 49.45 46.36 44 49.64
Fe 10 166.66 128.57 129.41 177.41
20 130.76 127.77 120.83 313.63
Mg 10 166.66 140.74 129.41 118.96
20 200 140.90 103.03 105.76
Ni 10 166.66 187.5 200 200
20 150 166.66 150 111.11
Zn 10 133.33 124.32 119.04 127.27
20 107.69 136 113.95 130.61
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii;
For Fe, the maximum translocation index values were recorded to be
313.63 % for 20 % (TSW-Soil) with F1 + F2, while the minimum value was
recorded in 20 % (TSW-Soil) with F2 treatment i.e. 120.83 %. For both TWS-
Soil mixtures, the F1 + F2 inoculations showed greater values than any other
fungal treatment.
In case of Mg the plants showed least value in 20 % (TSW-Soil) with
F2 (103.03 %), while the maximum values for this metal was recorded in 20 %
(TSW-Soil) with C i.e. 200 %. For Mg plants showed the maximum values in C
(without fungal treatment) in both 10 and 20 % (TSW-Soil) mixtures.
For Ni, there was as much as 200 % translocation index value
recorded in 10 % (TSW-Soil) with F2 and F1 + F2 treatment and it decreased
in F1 then in C, being the least in combined fungal treatment of 20 % (TSW-
Soil) i.e. 111.11 %.
96
As far as the Zn is concerned, the maximum translocation index
recorded in 20 % (TSW-Soil) with F1 treatment i.e. 136 %. For plants from 10
% (TSW-Soil), the maximum value was with C (133.3 %) as compared to F1
(124.32 %), F2 (119.04 %) or F1 + F2 (127.27 %).
3.8.5.3 Tolerance index (TI)
In shoot, the TI values were found to be the highest in 10 % (TSW-Soil)
with C (0.98). In case of 20 % (TWS-Soil), the maximum value was 0.61
noted in F1 treatment as shown in Table 3.8.10.
Table 3.8.10. The root and shoot tolerance index (TI) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 (M) F2 F1+F2
TI Shoot 10 0.98 0.84 0.72 0.95
20 0.57 0.61 0.57 0.59
TI Root 10 0.96 0.61 0.64 0.78
20 0.62 0.43 0.44 0.53
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii;
In case of TI in roots, 0.96 was recorded as the maximum TI for plants
grown in 10 % (TSW-Soil) with C, while 0.43 was recorded as the minimum
value in 20 % (TSW-Soil) with F2.
3.8.5.4 Category-I metals specific extraction yield (SEY %)
The SEY (%) for Category-I metals i.e. Ca, K and Na is given in Table
3.8.11.
Table 3.8.11. The Category-I metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 (M) F2 F1+F2
Ca 10 17.91 22.17 24.73 32.19
20 5.6 9.4 10.99 22.68
K 10 17.76 26.85 33.05 54.95
20 24.24 27.77 30.30 37.12
Na 10 51.68 62.02 65.60 79.52
20 37.67 43.05 45.64 48.65
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii;
97
In case of Ca, the maximum value (32.19 %) was recorded for plants
grown in 10 % (TSW-Soil) with F1 + F2, and the minimum (5.6 %) in 20 %
(TSW-Soil) with C.
In case of K, plants cultivated in 10 % (TWS-Soil) with F1 + F2 showed
the highest value of SEY (54.95 %) and the minimum (17.76 %)
corresponding C treatment.
For Na, the highest value (679.52 %) was recorded in 10 % (TSW-Soil)
with F1 + F2 while the minimum values (37.67 %) were found to be in case of
20% (TSW-Soil) with C i.e. no fungal inoculums.
3.8.5.5 Category-II metals Specific extraction yield (SEY %)
The SEY (%) was calculated for the Category-II metals is given in
Table 3.8.12. A trend of SEY (%) variation for Category-II similar to Category-
I was observed for all the fungal treatments along the row i.e. the SEY (%)
values increased with the application of fungal inoculums and the highest
values were observed for F1 + F2 treatments.
In case of Cd, the maximum value for SEY (%) was recorded in 10 %
(TSW-Soil) with F1 + F2 treatment being 26.74 %, while the minimum (15.54
%) was observed in 20% (TSW-Soil) with C.
Similarly the SEY % value for Cr was found to be the highest (42.04 %)
in 10 % (TSW-Soil) with F1 + F2 and the minimum (23.27 %) was recorded in
20 % with no fungal inoculums i.e. C. However, plants with F2 inoculums
showed greater SEY values as compared to those from F1.
98
Table 3.8.12. The Category-II metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 (M) F2 F1+F2
Cd 10 17.02 21.88 22.94 26.74
20 15.54 17.6 18.28 23.31
Cr 10 24.87 34.53 39.31 42.04
20 23.27 27.98 30.65 34.81
Cu 10 39.04 50.71 51.42 71.42
20 25.90 30.66 34.28 39.61
Fe 10 23.52 31.37 38.23 84.31
20 16.48 22.52 29.12 65.38
Mg 10 32.25 52.41 62.90 73.38
20 19.11 25.98 32.84 52.45
Ni 10 29.18 41.81 54.54 81.81
20 22.72 36.36 45.45 86.36
Zn 10 11.11 21.95 24.33 26.45
20 6.66 14.56 22.71 27.90
C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii;
For Cu, again the maximum value (71.42 %) was observed for 10 %
(TSW-Soil) with combined fungal inoculums i.e. F1 + F2 and the minimum
value (25.90 %) was recorded in 20 % (TSW-Soil) with C.
For Fe, the highest (84.31 %) and the lowest (16.48 %) SEY values
were recorded in 10 % (TSW-Soil) with F1 + F2 and 20 % (TSW-Soil) with C
respectively.
The SEY for Mg observed to be highest (73.38 %) in plants from 10 %
(TSW-Soil) with F1 + F2 and being the minimum (10.11 %) in 20 % (TSW-
Soil) with C i.e. having no fungal inoculums.
The maximum value (86.36 %) of SEY (%) for Ni was calculated in 20
% (TSW-Soil) with combined fungal inoculums and the minimum value (22.72
%) was recorded in 20 % (TSW-Soil) for C. The 10 % (TSW-Soil) treatments
showed higher SEY (%) values as compared to 20 % (TSW-Soil) mixtures.
A similar kind of SEY (%) variation was seen for Zn and Ni. The
maximum value (27.90 %) was noted for 20 % (TSW-Soil) with F1 + F2 and
the minimum value (6.66 %) was found in 20 % (TSW-Soil) with C.
99
3.9 Experiments with saprobic fungi
3.9A. Experiment with Tagetes patula inoculated with saprobic fungi
3.9A.1 Pre-sowing analysis
The physico-chemical properties, concentration of Category-I & II
metals are given Table 3.1, 3.2 and 3.3 respectively and their details are
described in Chapter 3.1.
3.9A.2 Biochemical analyses of 55-days old Tagetes patula
The biochemical parameters like chlorophyll contents, soluble protein
CAT and SOD were observed in 55-days old marigold. There was increased
production of all these parameters in plants from soil mixtures applied with
combined inoculation of fungi as compared to those from applied with no or
single fungus. The specific details of each of the biochemical parameters are
as under:
3.9A.2.1 Chlorophyll content
The plant chlorophyll content within a fungal treatment increased by
applying combined fungal inoculation i.e. the F1 + F2, as given in Table
3.9A.1. The F1 + F2 incurred the greatest (19.3 SPAD value) increase in
plants from 10 % (TSW-Soil) while influencing to the least (13.5 SPAD value)
in 20 % in C treatment as compared to any of the treatments within a row.
However F2 showed better values with respect to chlorophyll content as
compared to F1 values for all TWS-soil TWS-Soil mixture except for 10%
where F1 showed greater value (17.3) as compared to F2 (16.8) as shown in
Table 3.9A.1.
Within column, there was increase in plant chlorophyll contents with the
increase of TSW percentage in soil for all the fungal treatments but the values
were dropped being the minimum in 20% TWS-soil mixture. The application of
fungus either individually or in combination help plants perform better as
compared to C in terms of chlorophyll contents, as given in Table 3.9A.1.
100
3.9A.2.2 Soluble protein contents
Parallel to chlorophyll contents, the values within a row for soluble
protein contents also increased with the application of fungal inoculations for
all the treatment, as given in Table 3.9A.1.
Table 3.9A.1. The biochemical parameters observed in 55-days old Tagetes patula cultivated on TSW-Soil mixtures.
The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Chlorophyll content (SPAD value)
0 14.3abB
0.38 14.9abAB
0.37 15.4aAB
0.12 15.8aB
0.39 2.1
5 15.5bA
0.71 16.6abA
0.22 17.2abA
0.17 19.3aA
0.28 3.2
10 16.4abA
0.88 17.3aA
0.11 16.8abA
0.28 18.7aA
0.19 3.6
20 13.5abCd
0.38 13.9abAB
0.18 14.3aB
0.91 15.4aB
0.11 3.8
LSD0.05 1.9 2.8 2.4 2.8
Soluble Protein content
(mgg-1
)
0 0.3cdD
0.03 0.6bE
0.04 0.8aF
0.12 0.9aE
1.02 0.23
5 12cA
0.38 9deB
0.18 14bB
0.91 17aB
1.04 2.1
10 13dA
0.48 14cA
0.14 18abA
0.49 21aA
0.10 3.5
20 8bB
0.38 6bcC
0.04 7bD
0.13 11aC
0.13 2.8
LSD0.05 2.9 3.2 2.9 3.8
SOD (Umg
-1 of protein)
0 02bF
0.01 01cdD
0.02 03aC
0.21 01cdD
0.02 0.78
5 33deA
0.38 36cdA
0.38 38cA
0.32 45aA
0.88 3.4
10 31cdA
0.59 29dAB
o.45 30cAB
0.76 36aAB
0.16 2.9
20 22cdC
0.18 26bcB
0.12 28bAB
0.98 32aB
0.55 3.7
LSD0.05 4.7 10.2 12.4 9.6
CAT (Uml
-1)
0 0.1cD
0.02 0.3abE
0.12 0.3abF
0.12 0.4aE
0.17 0.13
5 16deA
0.07 18cdA
0.33 21bcA
0.38 26aA
0.38 2.8
10 12cdAB
0.23 15bcAB
0.08 14cBC
0.78 20aB
0.37 2.6
20 09cB
0.38 11bcB
0.45 10cD
0.38 16aBC
0.67 1.9
LSD0.05 4.1 5.1 3.9 4.5
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
The F1 + F2 from 10 % and the C from 0 % exhibited the maximum (21
mgg-1) and the minimum (0.3 mgg-1) soluble protein content as compare to
any of the soil or fungal treatments. Within Fungal treatments C showed
minimum values as compared to other fungal applications like F1, F2 and F1
+ F2 except in 5% (TSW-Soil) where the value is higher (12) than F1 (9).
However in F2 the value found to be 14 and 21 in F1 + F2.
Within a column, overall there was increase in soluble protein contents
with the increasing level of TSW in soil i.e. in 10 % and then decrease in 20%
(TSW-Soil). However, addition of fungi helped plants to assuage stress by
increasing soluble protein contents. The F1 + F2 superseded F1, F2 and C for
all of the soil treatments. The 10 % with F1 + F2 and the 0 % with F1
inoculations observed to have the maximum and the minimum values
101
respectively. The plants cultivated in pots with F2 inoculations performed
better than those with F1 for all the TSW-Soil mixtures.
3.9A.2.3 Superoxide dismutase (SOD) contents
Following the trends found for chlorophyll and soluble protein contents,
the SOD values increased with fungal inoculations within a row for all the
treatments, as given in Table 3.9A.1. The SOD values were found to be 01
mgg-1 in F1 and F1 + F2 treatments of 0% (TSW-Soil). For the rest of the
concentrations the fungal treatments having both of the F1 and F2 performed
better than control and those applied with either F1 or F2. The plants in 20 %
with F1 + F2 gave maximum SOD values.
While analyzing the response of plant cultivated in different TWS-Soil
mixtures it was observed that the 0% showed least values, while 5% showed
maximum SOD values in all fungal treatments, then decrease in 10% and
20%. The order from highest to lowest SOD values can be written as 5%
>10% > 20% > 0%.
3.9A.2.4 Catalase (CAT) contents
Within different treatments, there was an increase in plant CAT value
with the individual or combined fungal inoculations, as compared to control.
The plants from 5 % with F1 + F2 and 0 % with C had the maximum (26 Uml-
1) and the minimum (0.1 Uml-1) CAT values as compared to any of the
treatments as given in Table 3.9A.1.
For different TSW-Soil mixtures comparison, there was minimum CAT
contents was observed in 0% (TSW-Soil). The highest values were observed
for 5% then in 10 and 20 %. Cumulatively, the values for plant under F1 + F2
inoculations column had significantly higher values as compared to values
under either of the C, F1 or F2 columns.
3.9A.3 Post-harvest analysis
3.9A.3.1 Growth performance of Tagetes patula
Better growth of 55-day-old plants of T. patula was observed in case of
lower TWS-soil mixture i.e. 5% and 10%, being relatively less in soil (0%) and
102
20% as indicated by growth parameters (Table 3.9A.2). It was noticed that
plants cultivated in soil and its TSW mixtures inoculated with fungal isolates
yielded greater shoot, root and seedling length, no. of leaves and roots, as
well as, fresh and dry weight; as compared to control. The statistical analysis
of the data showed significant growth in all parameters in lower TSW mixtures
in soil followed by a decrease at higher (20%) level. However, the maximum
increase in values was found in F + M treatment over their controls F1 and F2,
for each of the corresponding soil treatments. The details of each of the
morphological parameters is given in Table 3.8.2 and described as under:
3.9A.3.1.1 Shoot, root and seedling length (cm)
The shoot, root and seedling length (cm) of the marigold are given in
Table 3.9A.2. Along the row in comparison with different fungal treatments,
the maximum plant shoot (32.1 cm), root (40.1 cm) and seedling length (35.2
cm) was observed in 5 % with F1 + F2 inoculation while being minimum
values for plant shoot (16.3 cm) and seedling length (28.5 cm) in 20 % with
no fungal inoculation i.e. the C. The minimum value for plant root length (11.7
cm) was recorded in 20% with control having no fungal inoculation. There was
increase in length of all the three vegetative parameters with the application of
fungal inoculations and the order of increase observed to be F1 + F2 > F2 >
F1 > C along the row.
While in comparison with different TWS-Soil mixture there was
increase in plant shoot, root and seedling length with the increasing proportion
of TSW in the soil. The different TSW-Soil mixtures under F1 + F2 column
gave best results while those in C column attained the least height. However
the highest TWS-Soil TWS-Soil mixture i.e. 20% showed least growth as
shown in Table 3.9A.2.
3.9A.3.1.2 No. of leaves and roots
Along the row, the plants in 5% with F1 + F2 inoculation observed to
have maximum no. of roots (27) and leaves (19) while being the minimum (12
and 5 respectively) in 20 % without any of the inoculation i.e. C.
Within different TWS-Soil mixture of TWS-Soil, the increasing ratio of TSW
increased the no. of leaves and roots upto 10% but there was decreased for
103
these parameters in 20% TWS-Soil mixture The TSW-Soil mixtures under F1
+ F2 gave the best vegetative growth than any of the fungal treatments. The
least growth response observed to be in C column as shown in Figure 3.9A.1.
Table 3.9A.2. Various morphological parameters observed in 55-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Shoot Length (cm)
0 18.9cAB
0.67 20.4bBC
0.12 21.1bAB
1.1 24.7aC
1.03 2.8
5 19.7dA
0.99 22.5cB
0.98 23.3cA
1.05 32.1aA
1.4 4.7
10 17.6cB
0.78 23.8bA
0.23 21.9bAB
0.33 29.3aAB
0.34 5.2
20 16.3cC
0.56 18.3bCD
0.45 19.0bBC
0.36 23.7aC
0.65 2.9
LSD0.05 1.3 2.2 1.5 3.8
Root Length (cm)
0 12.4cB
0.39 18.1bC
1.02 20.3bBC
1.2 26.7aBC
0.87 4.2
5 15.2dA
0.59 24.7bA
0.29 26.1bA
1.04 35.2aA
0.91 2.9
10 14.9cA
1.4 19.2bBC
0.34 20.7bcBC
1.4 27.1aBC
0.39 4.3
20 11.7dB
1.04 14.5bcC
0.99 15.1bCD
1.2 19.4aCD
1.22 2.8
LSD0.05 2.2 3.2 3.9 4.5
Seedling Length (cm)
0 31.6dC
1.3 38.8cBC
0.78 41.9bcAB
0.36 51.8aC
0.47 6.2
5 35.3eA
1.5 47.5cdA
1.54 49.7cdA
0.28 67.6aA
0.94 9.3
10 32.9deBC
0.98 43.4bcAB
1.9 42.9bcAB
0.19 56.8aBC
1.5 10.2
20 28.5dD
0.38 33.1cCD
1.02 34.5cC
1.03 43.6aDE
1.39 5.8
LSD0.05 3.1 4.2 4.8 6.9
No. of roots
0 13bcCD
0.27 15bBC
1.07 18abB
0.27 21aCD
0.23 2.9
5 16cdA
0.56 19cA
1.6 20bcA
0.45 27aA
0.99 3.1
10 15cAB
0.98 14cdC
1.89 18bcB
1.2 24aB
1.8 3.9
20 12cdD
0.27 14bcC
2.6 17abB
1.7 19aD
1.46 2.7
LSD0.05 1.4 1.9 1.2 2.9
No. of leaves
0 6cBC
0.27 8bcB
0.23 10bAB
0.56 13aBC
0.78 2.1
5 8cdA
0,21 10cA
1.06 11cA
0.22 19aA
0.27 2.8
10 7cdA
0.21 9cA
0.11 12bcA
0.20 17aAB
0.33 3.8
20 5cdC
0.23 7cB
0.56 9bcBC
0.08 14aB
0.31 3.5
LSD0.05 1.2 1.7 1.5 2.1
Fresh wt. (g)
0 2.1bBC
0.27 2.7abC
0.91 3.0aB
0.12 3.5aB
0.27 1.6
5 3.4bA
0.38 3.9abA
0.34 4.1aA
0.87 4.3aA
1.09 0.7
10 2.3bBC
0.20 2.9abBC
0.56 3.2aB
0.67 3.8aA
1.45 1.4
20 1.9abC
0.99 2.4aCD
0.77 2.6aBC
0.29 2.9aCD
0.27 1.1
LSD0.05 0.53 0.39 0.7 0.28
Dry wt. (g)
0 0.9bBC
0.27 1.1bCD
0.28 1.4aC
0.27 1.9aBC
0.12 0.7
5 1.6abA
1.78 2.0aA
0.34 2.3aA
0.20 2.4aA
0.18 0.8
10 1.0bBC
1.09 1.2bCD
0.27 1.5abC
0.44 2.1aA
0.13 0.7
20 0.7bC
0.99 0.92abD
1.11 1.02aCD
0.02 1.2aC
0.73 0.4
LSD0.05 0.17 0.21 0.24 0.41
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.3.1.3 Fresh and dry weight (g)
The fresh and dry weight were observed to be the maximum and the
minimum in accordance with the maximum and the minimum no. of leaves
and roots for both along the row as well as within column comparisons. In
104
other words, along the row the maximum weight (4.3 g fresh, 2.4 g dry)
observed to be in 5 % with F1 + F2 and the minimum (1.9 g fresh, 0.7 g dry) in
20 % with C, without any fungus respectively.
Within column, the increasing TSW ratio affected the biomass production
positively for lower TWS-Soil mixture i.e. 5% and 10 % and negatively for 20
% (TSW-Soil). The TSW-Soil mixtures under F1 + F2 yielded maximum fresh
and dry weight while those in C column yielded the least.
Figure 3.9A.1. The vegetative growth variation in Marigold (Tagetes patula) in response to soil mixed
with different percentages of TSW (% w:w) and inoculated with different fungi.
3.9A.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot were variable with respect to fungal inoculations as well as increasing
ratio of TSW in soil, as given in Table 3.9A.3.
3.9A.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in shoot increased with inoculation
of fungi as compared to C. The maximum (455 mgkg-1) shoot Ca observed to
be in 10 % with F1 + F2 while being the minimum (13 mgkg-1) in 0% with C.
Within different concentrations of TSW in soil, the maximum shoot
concentrations were observed with F1 + F2 while being the minimum in those
where no fungi was applied. It was observed that shoot Ca increased with the
105
increasing TSW ratio in soil mixtures for 5 and 10 % and then decreased for
20 %.
Table 3.9A.3. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of
55-days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Ca
0 13bcD
0.38 16
bDE
0.11 11
bcE 0.09 25
aDE 0.16 4.8
5 110dB
0.12 390
abA
0.22 360
bA 0.41 425
aA 0.33 85
10 145cdA
0.17 410
abA
0.29 380
bA 0.38 455
aA 0.66 75
20 95cdB
0.88 335
bBC
0.56 340
bAB
0.61 395
aAB 0.91 80
LSD0.05 35 110 98 125
K
0 14cD
0.38 20
bDE
0.87 22
bD 0.18 35
aD 1.09 6.2
5 220cdA
0.78 310
bA
0.38 325
bA 0.68 380
aAB 0.56 49
10 245dA
0.18 295
cdA
0.18 345
bA 0.12 410
aA 0.97 45
20 195dB
0.99 305
bcA
0.67 330
bA 1.09 400
aA 0.38 55
LSD0.05 62 75 85 110
Na
0 5cdDE
0.09 12
bcDE
0.05 18
aE 0.1 22
aDE 0.56 4.8
5 140dA
0.18 260
bA
1.09 275
bA 0.49 355
aB 0.67 55
10 90eBC
0.44 280
cdA
0.67 295
cdA 0.38 425
aA 0.98 45
20 55deC
0.12 160
cCD
0.39 190
bcBC
0.56 270
aC 1.6 60
LSD0.05 38 75 78 109
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.3.2.2 Potassium (K) in shoot
Along the row, the shoot K uptake increased with fungal applications
for all the TSW-Soil mixtures. The maximum K level (410 mgkg-1) was
observed in 10 % with F1 + F2 while being the minimum (14 mgkg-1) in soil
with no fungi i.e. C.
Within column, the K shoot uptake increased with the increasing
concentration of TSW in soil mixtures for all the concentrations except for
20% where the values were observed to be decreased. For all TSW-Soil
mixtures, the F2 plants showed more K uptake than those with F1 and being
the least where no fungus was applied. The maximum values observed in
plants cultivated in 10% TWS-Soil mixture.
3.9A.3.2.3 Sodium (Na) in shoot
The Na concentration in shoot observed to be increased along the row
and it was because of fungal applications. The pots with F1 + F2 showed the
106
greatest Na shoot uptake, the F2 being greater than F1, while those with no
fungi being the least. The plants in 10 % with F1 + F2 had the highest value
(425 mgkg-1) while those in 0 % with no fungi exhibited the lowest Na shoot
contents (5 mgkg-1).
Within column, the increasing ratio of TSW in soil mixtures enhanced
the shoot Na uptake for all the fungal treatments except 20 %. In case of 0 %,
the concentration of shoot Na observed to be the least for all the fungal
treatments and maximum for 10% as shown in Table 3.9A.3.
3.9A.3.3 Category-I metals in plant ROOT
The bioavailability of Category-I metals was variable with different fungi
in root also however, it was directly related to the increasing ratio of TSW in
soil mixture, as given in Table 3.9A.4.
3.9A.3.3.1 Calcium (Ca) in root
The application of fungal inoculum to the soil helps to increase Ca
uptake along the row i.e. various fungal treatments. The plants in F1 + F2 pots
observed to have maximum while those with no fungi having the minimum
root Ca than any of the fungal treatments for all of the TSW-Soil mixtures. The
highest root Ca (410 mgkg-1) was in 5 and 10 % with F1 + F2 while being the
minimum (6 mgkg-1) in 0 % with no fungal inoculation.
3.9A.3.3.2 Potassium (K) in root
The K contents in the 10 % with F1 + F2 exhibited the maximum root
uptake (315 mgkg-1) than any of the soil treatments while being the minimum
(8 mgkg-1) in 0 % with no fungal application. The application of fungus as
individual i.e. F1 and F2 showed better K uptake than TSW-Soil mixtures
where no fungi has been applied. However, F2 showed better uptake as
compared to F1 treatment except for 0 % where F1 showed slightly better
value (12 mgkg-1) as compared to F2 (11 mgkg-1).
Within column, the metal uptake in root was found to be maximum in
10% while being minimum in 0 %.
107
Table 3.9A.4. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of 55-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Ca
0 6cdDE
0.28 12bE
0.77 9bcE
0.33 18aDE
0.16 3.9
5 55dBC
0.38 355bcA
0.49 335bcA
0.28 410aA
0.89 92
10 125deA
0.45 390bA
1.6 340bcA
0.36 410aA
0.34 110
20 40deC
0.81 180cC
1.4 210bcCD
0.19 305aBC
0.29 95
LSD0.05 35 99 105 85
K
0 8cDE
0.12 12bD
0.58 11bDE
0.91 20aD
0.17 4.2
5 110deAB
0.38 210bA
0.87 225abA
0.45 290aA
0.92 55
10 160dA
1.6 230bA
0.13 260bA
0.38 315aA
0.45 60
20 75cdC
0.38 195bAB
0.099 220aAB
0.11 270aAB
0.19 58
LSD0.05 40 65 80 75
Na
0 3cDE
0.67 6bcDE
0.098 9aE
0.34 12aE
0.96 8.3
5 90deA
0.95 220cA
0.25 245bcA
0.38 320aAB
1.3 90
10 70dAB
0.38 190bcA
0.76 210bcAB
0.96 380aA
1.9 80
20 45cdC
0.45 140bcBC
1.08 155bcCD
2.5 245aCD
2.7 65
LSD0.05 28 65 75 90
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.3.3.3 Sodium (Na) in root
Along the row as observed in case of Ca and K, the application of fungi
helps increase Na uptake in roots. The maximum Na in root (380 mgkg-1) was
observed in 10 % with F1 + F2 while being minimum in 0 % amounting 3
mgkg-1. Those with F1 and F2 applications also performed better than C i.e.
treatment with no fungal inoculation.
Within column, trend of Na root uptake was also similar to what
observed in case of Ca and K. The increasing ratio of TSW in soil displayed
increased root Na uptake as compared to Soil with C for all the fungal
treatments.
3.9A.3.4 Category-II metals in plant shoot
The Category-II metals i.e. the AAS detected metals in shoot were
variable with respect to fungal inoculations as well as increasing ratio of TSW
in soil, as given in Table 3.9A.5. The application of fungi enhanced trace
metal uptake tendency of plant for all the TSW-Soil mixtures. However, the
increasing level of Category-II metals in shoot was in accordance with the
increasing ratio of TSW in soil mixtures for all the fungal treatments.
108
Table 3.9A.5. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 55-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 2deD
0.23 8cDe
0.12 12cE
0.34 25aDE
0.78 8.9
5 210efAB
0.22 390dBC
0.99 425cdCD
1.1 970aAB
1.3 205
10 290eA
1.03 510cA
1.03 590cA
1.5 1,020aA
2.3 190
20 280deA
1.6 490cA
0.12 530cA
1.9 1,050aA
2.6 220
LSD0.05 78 127 155 265
Cr
0 2cdDE
0.04 5cDE
0.3 8bcD
0.11 15aE
0.91 3.8
5 245efB
0.12 560dAB
0.82 625cA
1.4 1,415aCD
1.3 280
10 315eA
1.12 610dA
0.72 670cdA
1.32 1,380aA
2.1 275
20 295efA
0.12 590dA
0.38 600dB
1.91 1,265aB
1.9 255
LSD0.05 83 168 178 365
Cu
0 20cdE
0.29 60bD
0.022 55bDE
0.38 90aE
1.23 19.2
5 450cB
0.19 630aBC
0.11 670aA
1.1 690aC
1.4 55
10 510deA
0.28 760bcA
0.38 710bcA
1.3 990aA
2.9 125
20 325dCD
0.28 410cCD
1.1 485cC
1.9 990aA
1.2 175
LSD0.05 128 185 168 235
Fe
0 BDL BDL BDL BDL -
5 40cB
0.31 70bcC
0.23 110aB
0.12 220aB
1.5 48
10 60cA
0.22 105aAB
0.34 110aB
1.4 120aCD
0.91 25
20 55eA
0.11 130cA
0.31 145cA
1.2 325aA
0.94 72
LSD0.05 6.3 18 9 53 13
Mg
0 BDL BDL BDL BDL -
5 45cC
0.23 90bcBC
0.29 115bB
0.23 170aC
0.321 35
10 80cdA
0.78 130bA
0.56 155bA
0.12 290aA
0.38 55
20 70dA
0.99 145cA
0.38 165cA
0.19 310aA
0.29 65
LSD0.05 4 15 15 38
Ni
0 BDL BDL BDL BDL -
5 8bC
1.2 10bCD
0.11 12aD
0.38 15aC
0.31 3
10 11bcC
0.38 12bcCD
0.20 15bD
0.91 20aC
1.51 4
20 35bA
0.32 40bA
0.98 55aA
0.93 60aA
1.44 8
LSD0.05 8 9 12 13
Zn
0 70bD
0.11 90bD
0.37 110abDE
0.22 145aD
0.74 20
5 220deBC
0.38 425cdB
1.5 470cdB
0.11 875aA
0.19 165
10 340dA
0.18 510cA
1.3 550cA
0.45 920aA
0.29 148
20 290eB
0.36 465cdA
1.2 490cdAB
0.78 895aA
0.75 154
LSD0.05 69 107 114 195
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in TSW-Soil mixtures with combined
fungal treatments. Maximum amount of metal (1,020 mgkg-1) was observed in
10% TSW-Soil mixture in combined inoculation of fungi i.e. F1 + F2, while
being minimum in 0% amounting 2 mgkg-1.
109
Within different concentrations there is increasing trend of metal
accumulation with TSW-Soil TWS-Soil mixture
3.9A.3.4.2 Cr in shoot
With different fungal treatments the maximum Cr accumulation in shoot
was observed in treatments applied with combined inoculants i.e. F1 + F2,
being significantly higher than any of the treatments along the rows. Cr was
found to be as low as 2 mgkg-1 in 0% with C and maximum 1,415 mgkg-1 in
5% with F1 + F2.
Within different TWS-soil mixtures there was maximum accumulation of
metal was observed in 10% for all fungal treatments except F1 + F2, as for
this treatment the maximum accumulation was observed in 5% as shown in
Table 3.9A.5.
3.9A.3.4.3 Cu in shoot
Along the row, the plants harvested from F1 + F2 exhibited maximum
Cu accumulation as compared to any of the treatments with single or no
fungal application. Such a trend was observed in all of the TSW-Soil mixtures.
The plants from treatments with no fungal inoculations i.e. C showed the
significantly least Cu accumulation as compared to any of the fungal
treatments as shown in Table 3.9A.5.
Within different TWS-Soil mixtures, 0% showed significantly less Cu
uptake in shoots than those form 5, 10 and 20 % as minimum accumulation
20 mgkg-1 was seen in 0% with C and maximum accumulation 990 mgkg-1 in
10 and 20% with F1 + F2.
3.9A.3.4.4 Fe in shoot
The Fe uptake in plant shoots observed to be BDL in Soil (0%) with C,
but F1 + F2 inoculation displayed the maximum Fe uptake in shoot as
compare to plants from the treatments with single or no fungal inoculations.
The values of plant shoot Fe was minimum 40 mgkg-1 in 5% with C and
maximum 325 mgkg-1 in 20 % with F1 + F2.
110
Within a column, maximum accumulation was observed in 10 % as
shown in Table 3.9A.5.
3.9A.3.4.5 Mg in shoot
Along various fungal treatments, the Mg uptake in plant shoot
increased with fungal application in the pots and found to be the maximum
310 mgkg-1 in plants harvested from pots applied with combined fungi i.e. F1
+ F2 in 20 % (TSW-Soil), while being the minimum as well as significantly
least in shoot of plants cultivated in soil with no fungus i.e. 45 mgkg-1 as
shown in Table 3.9A.5. Such a pattern was observed for all the TSW-Soil
mixtures.
For different TSW-Soil mixtures the value of plant shoot Mg was found
to be BDL in 0%. For 10 %, the shoot uptake found to be highest as
compared to 5 and 20 % TSW-Soil mixture.
3.9A.3.4.6 Ni in shoot
The Ni concentration observed to be BDL in 0% for all the fungal
treatments along the row. However, for all the TSW-Soil mixtures the shoot Ni
concentration increased with the application of fungus and observed to be the
maximum in plants inoculated with F1 + F2 and being the least in those from
C i.e. where no fungus was applied.
Within the columns comparison, the value of plant shoot Ni
concentration increased with the increasing percentage of TSW in soil and
found to be the maximum in plants harvested from 20 % being the minimum in
plants from 5 %.
3.9A.3.4.7 Zn in shoot
The Zn concentration in shoots increased in plants harvest from pots
with fungal inoculations than those harvested from pots applied with no fungi.
Like other metals, the shoot Zn observed to be the maximum in plants
inoculated with F1 + F2 while those harvested from the pots with no fungi
displayed the minimum values. There was increasing trend of accumulation of
111
metal as the application of fungi. However F2 showed better results as
compared to F1 in terms of accumulation of metal as shown in Table 3.9A.5.
For different TWS-Soil mixtures, the value of Zn concentration in shoot
was found to be maximum 920 mgkg-1 in 10 % with F1 + F2 being minimum
70 mgkg-1 in 0% with C as shown in Table 3.9A.5. The order of accumulation
of metal accumulation within different TWS-Soil mixtures from maximum to
minimum was as 10 % >20 % > 5% > 0%.
3.9A.3.5 Category-II metals in plant root
The Category-II metals i.e. the AAS detected metals in root were
observed to differ with varying levels of TSW in the soil as well in response to
fungal inoculations, as given in Table 3.9A.6.
3.9A.3.5.1 Cd in root
For different fungal treatments within the rows, the Cd level in plant
roots harvest from Soil observed to be minimum 5 mgkg-1 in 0% with F1 and
found to be maximum 990 mgkg-1 in 20 % with F1 + F2 as shown in Table
3.9A.6. The plant root Cd concentration increased with fungal inoculations
and found to be maximum as well as significantly higher than those applied
with single or no fungal treatments, while being the BDL in 0 % with C.
Inside the columns, for different TWS-Soil mixtures the root Cd
concentration was found to be highest in 10% with C, F1 and F2 except F1 +
F2. The highest concentration was noted in 20% with F1 + F2 i.e. 990 mgkg-1.
3.9A.3.5.2 Cr in root
For different fungal treatments i.e. along the row, the plants from F1 +
F1 fungal inoculations showed the maximum 1,320 mgkg-1 root Cr
concentration in 5 % (TSW-Soil), while with F2 treatment plants having the
minimum uptake 2 mgkg-1 in 0%. The F2 inoculation enhanced the root Cr
concentration better than F1. However for C and F1 fungal treatments the
values found to be BDL as shown in Table 3.9A.6.
112
Within the columns, the root Cr concentration increased with increasing
percentage of TSW in soil having maximum accumulation of metal in 10 % for
all fungal treatments like C, F1, F2 except F1 + F2.
Table 3.9A.6. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 55-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 BDL 5cdD
0.02 7cdE
0.09 21aDE
0.34 5.6
5 190efB
0.89 355dAB
0.38 390dC
0.56 910aAB
0.38 140.8
10 250eA
0.69 485dA
0.86 510cdA
0.88 980aA
0.78 185
20 210dAB
0.45 455cA
0.12 490cA
0.99 990aA
0.98 179
LSD0.05 18 124 128 245
Cr
0 BDL. BDL 2cD
0.01 8aE
0.2 1.3
5 225deB
0.38 390dA
0.99 420cdA
1.23 1,320aA
0.38 205
10 275dA
1.28 425cA
1.2 455cA
0.38 1,290aA
1.23 195
20 235cdA
1.37 370cA
0.38 445bcA
1.11 1,050aB
1.4 215
LSD0.05 14 15 115 330
Cu
0 BDL 15deDE
0.78 20dD
0.36 45aE
0.26 4.9
5 380bcA
1.03 410abA
0.96 425aA
1.2 430aC
1.33 13.4
10 295efBC
1.05 525cA
0.78 505dA
1.07 685aA
1.36 79
20 210eCD
1.3 390dB
1.2 455cdA
0.97 860aA
1.22 165
LSD0.05 45 128 124 205
Fe
0 BDL BDL BDL BDL -
5 35dA
0.45 55cdCD
1.8 85bcBC
0.38 120aC
0.13 24
10 45eA
0.23 80dBC
0.07 110cdA
0.34 235aA
0.32 49
20 30dB
0.35 115bA
0.38 125bA
1.2 210aA
0.29 68
LSD0.05 5 18 12 30
Mg
0 BDL BDL BDL BDL -
5 30cBC
0.12 55abC
0.73 60aC
0.44 65aBC
0.31 7.4
10 45dAB
1.2 95bAB
0.94 120aA
0.51 130aAB
0.22 23
20 55deA
0.99 115cA
0.12 135cA
0.88 290aA
0.44 60
LSD0.05 8 18 20 58
Ni
0 BDL BDL BDL BDL -
5 5cdDE
0.12 5cdCD
0.12 8bC
0.19 10aB
0.16 1.53
10 12cdC
0.38 10dC
0.76 13cdBC
0.31 18aAB
0.35 1.6
20 30bA
0.43 32abA
0.87 35aA
0.44 30bA
1.5 1.3
LSD0.05 6.8 7 9 8
Zn
0 55cdC
0.23 75bcD
0.25 90bCD
0.87 115aC
1.3 18
5 190cA
0.56 380aA
0.31 410aA
0.34 425aAB
1.7 58.8
10 230eA
0.98 415cA
0.23 435cA
0.11 670aA
1.2 112
20 210dA
0.48 395bcA
0.46 455bcA
0.38 690aA
0.99 155
LSD0.05 46 90 95 145
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.3.5.3 Cu in root
Along the row for various fungal inoculations the root Cu uptake was
enhanced as compared to C. The value was found to be BDL in 0% for C
treatment. The plants from F1 + F2 observed to have the maximum (860
113
mgkg-1) uptake in 20 % than those cultivated in pots with single or no fungal
inoculations and found to be the minimum 15 mgkg-1 in 0% with F1.
For different columns, the plants from 5 and 10 % TSW-Soil exhibited
better root Cu accumulation than soil (0 %) and 20 %.
3.9A.3.5.4 Fe in root
Moving alongside the row while analyzing effect of different fungal
treatments, the root Fe observed to be BDL in soil (0 %) with all fungal
treatments. The plants from C had the lowest root Fe as compared to plants
from any of the single or combined application of fungi. The root Fe
accumulation observed to be the maximum (235 mgkg-1) in 10 % with F1 + F2
and significantly higher than those harvested from any of the treatments with
single or no fungal inoculations.
Inside columns, the maximum Fe uptake by roots was observed in 10
% while the least value of metal uptake was observed in 5 % except the C
treatment as shown in Table 3.9A.6.
3.9A.3.5.5 Mg in root
While analyzing the fungal application effect on plants along the rows,
it was observed that the Mg root concentration increased in plant roots with
fungal application as compared to C i.e. with no fungal inoculation. The F1 +
F2 plants displayed the maximum root Mg concentration than those from C as
well as F1 and F2. For 0% the values for metal uptake were found to be BDL
for all fungal treatments. However the F2 inoculations gave better results than
F1.
For the columns, the root Mg accumulation increased with increasing
percentage of TSW in soil for all fungal treatments being maximum in 20%
with F1 + F2 where the metal accumulation was observed to be 290 mgkg-1
while minimum concentration 30 mgkg-1 in 5% with C treatment was noted as
shown in Table 3.9A.6.
3.9A.3.5.6 Ni in root
114
The root Ni concentration was found to be BDL for all fungal treatments
in 0% i.e. soil. However there was increased metal accumulation along the
row with the application of fungal inoculations and found to be the maximum
in plants applied with combined application of both of the fungi while being the
minimum in C with no fungus added.
Within columns, the plants from 20 % TSW-Soil mixtures had the
maximum root Ni level than 10 % for all of the fungal treatments. There was
maximum (35 mgkg-1) accumulation of metal was noted in 20% with F2 while
minimum uptake (5 mgkg-1) was observed in 5% with C and F1.
3.9A.3.5.7 Zn in root
The root Zn accumulation observed to increase in pots applied with
fungal inoculations than C and found to be the maximum in treatments applied
with both of the fungi and being the minimum with no fungal applications.
Again as with most of the above discussed metals F2 performed better than
F1 as far as metal accumulation efficiency of the plant is concerned.
For different TWS-Soil mixtures, the root Zn concentration increased
with increasing percentage of TSW in soil with every fungal treatment except
in 20% where the metal uptake was reduced. However the maximum
accumulation was noted in 20 % with F1 + F2 i.e. 690 mgkg-1 and minimum
(55 mgkg-1) in 0 % with C treatment as shown in Table 3.9A.6.
3.9A.4 Fungal analyses
The results of estimation of the post-harvest fungal analyses (× 105
c.f.u. g-1 soil) of 55-days old Tagetes patula cultivated on TSW-Soil mixtures
are shown in Table 3.9A.7. Alongside the row, the c.f.u. increased with fungal
application than C and observed to be the maximum in treatments with
combined application of both of the fungi. The order of c.f.u. abundance was
F1 + F2 > F2 > F1 > C.
Within a column, the c.f.u. abundance was observed in 5% with 11.1 ×
105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2. Statistical
115
analysis indicated that there is significant difference of 5% with other TWS-
Soil mixtures.
Table 3.9A.7. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 55-days old Tagetes
patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
TSW-Soil (% w:w) mixture and its type
Treatment LSD0.05
C F1 F2 F1+F2
Soil 1.5deBC
0.22 3.6dAB
0.17 4.2cdA
0.23 9.8aB
0.29 2.3
5 1.8cdA
0.26 4.1bcA
0.18 4.5bcA
0.21 11.1aA
0.13 4.6
10 1.6dA
0.16 3.2cB
0.33 3.9bcAB
0.07 7.5aCD
0.51 1.98
20 1.4cdBC
0.24 2.8bCD
1.2 2.9bBC
0.38 3.8aD
0.98 0.57
LSD0.05 0.19 0.36 1.8 2.9
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
3.9A.5.1 Category-I metals translocation index (%)
The plant translocation index values were also recorded for Category-I
metals those detected by flame photometer i.e. Ca, K and Na shown in Table
3.9A.8.
Table 3.9A.8. The Category-I metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca
5 200 109.85 101.40 103.65
10 116 105.12 111.76 110.97
20 237.5 186.11 161.90 129.50
K
5 200 147.61 144.44 131.03
10 153.12 128.28 132.69 130.15
20 260 156.41 150 148.14
Na
5 155.55 118.18 112.24 110.93
10 128.57 147.36 140.47 111.84
20 122.22 114.28 122.58 110.20
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
In case of Ca, maximum value was observed in 20% (TSW-Soil) with C
i.e. 237.5 % while the minimum value was recorded (101.40 %) in 5 % (TSW-
Soil) with F2.
116
For K, the maximum translocation index value was calculated in 20%
with C i.e. 260 % being the minimum 138.28 % in 10% with F1.
In case of Na, the maximum value 155.55 % was observed in 5% with
C treatment and least value was recorded in 20 % (TSW-Soil) for F1 + F2
(110.20 %).
3.9A.5.2 Category-II metals translocation index (%)
The plant translocation index found to be greater in treatments applied
with fungal inoculum than that of C, being the maximum where F1 and F2
applied together for all metals as given in Table 3.9A.9. As compared to
different TWS-Soil concentrations there was decreasing trend observed with
increasing percentage of TWS-Soil with few exceptions.
Table 3.9A.9. The Category-II metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd
5 110.52 109.86 108.97 106.59
10 116 105.15 115.69 104.08
20 133.33 107.69 108.16 106.06
Cr
5 108.88 141.02 148.8 107.2
10 114.55 143.53 147.25 106.98
20 125.53 159.45 134.83 120.47
Cu
5 118.42 153.65 157.64 160.47
10 172.88 144.76 140.59 144.52
20 154.54 141.37 106.59 115.11
Fe
5 114.28 127.27 129.41 183.33
10 133.33 131.25 100 51.06
20 183.33 113.04 116 154.76
Mg
5 150 163.63 191.66 261.53
10 177.77 136.84 129.16 223.07
20 127.27 126.08 122.22 106.89
Ni
5 160 200 150 150
10 91.66 120 115.38 111.11
20 116.66 125 157.14 200
Zn
5 115.78 111.84 114.63 205.88
10 147.82 122.89 126.43 137.31
20 138.09 117.72 107.69 129.71
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
For Cd, the maximum Translocation index (133.33 %) was noted in
20% (TSW-Soil) with C, while minimum value was recorded 104.08 % in 10%
with F1 + F2.
117
In case of Cr plants showed maximum metal translocation efficiency
(159.45 %) in 20% with F1 and minimum 106.98 % in 10 % with F1 + F2.
For Cu, the maximum translocation index values was recorded to be
172.88 % for 10% with C, while minimum value was recorded in 5 % with C
treatment i.e. 118.42 %.
For Fe, the maximum translocation index values was recorded to be
183.33 % for 5 % with F1 + F2 and for 20 % with C, while minimum value was
recorded in 10% with F1 + F2 treatment i.e. 51.06 %.
In case of Mg the plants showed least value in 20% with F1 + F2
(106.89 %), while the maximum values for this metal was recorded in 10%
with F1 + F2 i.e. 223.07 %.
For Ni, there was 200% translocation index value recorded in 5% with
F1 and in 20% with F1 + F2 treatment then least value was observed in 10%
with C i.e. 91.66 %.
As far as the Zn is concerned there was maximum translocation index
recorded in 5 % with F1 + F2 treatment i.e. 205.88 % while minimum value
was recorded in 20% with F2 i.e. 107.69 % as shown in Table 3.9A.9.
3.9A.5.3 Tolerance index (TI)
In shoots TI values were found to be highest in 5 % with F1 + F2
(1.29) while minimum value was recorded to be 0.86 in 20% with C as shown
in Table 3.9A.10.
Table 3.9A.10. The tolerance index (TI) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
TI Shoot
5 1.04 1.10 1.10 1.29
10 0.93 1.16 1.03 1.18
20 0.86 0.89 0.90 0.95
TI Root
5 1.22 1.36 1.28 1.31
10 1.60 1.06 1.01 1.01
20 0.94 0.80 0.74 0.72
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
118
In case of TI in roots 1.60 was recorded as maximum for plants grown
in 10% with C, while 0.72 were recorded as minimum value in 20% with F1 +
F2.
3.9A.5.4 Category-I metals Specific extraction yield percentage (SEY %)
SEY % for Category-I metals i.e. Ca, K and Na showed in Table
3.9A.11.
In case of Ca, there was maximum value 51.38 % was recorded for
plants grown in 5 % (TSW-Soil) with F1 + F2, and minimum 4.63 % in 20 %
(TSW-Soil) with C.
In case of K, plants cultivated in 5 % TWS-soil showed highest value of
SEY% (75.28 %) with F1 + F2 and minimum in 20 % with C i.e. 13.63 %.
As in case of Ca and K, the highest value for Na was recorded in 5 %
with F1 + F2 (49.81 %) while minimum values was found to be 2.15 % in case
of 20% with no fungal inoculum i.e. C.
Table 3.9A.11. The Category-I specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca
5 10.15 45.84 44.0 51.38
10 11.51 34.11 30.70 36.88
20 4.63 17.69 18.90 24.05
K
5 37.07 58.42 61.79 75.28
10 33.47 43.33 50 59.91
20 13.63 25.25 27.77 33.83
Na
5 16.97 35.42 38.37 49.81
10 6.36 18.68 20.07 32.00
20 2.15 6.45 7.42 11.08
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9A.5.5 Category-II metals Specific extraction yield percentage (SEY %)
The SEY (%) was calculated in Category-II metals that were detected
by AAS as shown in Table 3.9A.12. Overall a similar kind of trend was seen
for all metals in various fungal treatments along the row, that the SEY %
values increased with the application of fungal inoculum and highest value
was observed for F1 + F2 treatment.
119
In case of Cd the maximum value for SEY % was recorded in 5 %
(TSW-Soil) with F1 + F2 treatment i.e. 70.94 %, while minimum (5.6 %) was
observed in 20% (TSW-Soil) with C.
Similarly the SEY % value for Cr was found to be highest (33.15 %) in
5 % (TSW-Soil) with F1 + F2 and minimum (3.41 %) was recorded in 20%
with no fungal inoculum i.e. C. However F2 showed greater values as
compared to F1.
For Cu again the maximum value was observed for 5 % (79.76 %) with
combined fungal inoculum F1 + F2 and least value (10.19 %) was recorded in
20% (TSW-Soil) with C.
Table 3.9A.12. The Category-II metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd
5 15.09 28.11 30.75 70.94
10 8.20 15.12 16.71 30.39
20 5.6 10.8 11.65 23.31
Cr
5 5.69 11.39 12.66 33.15
10 5.75 10.09 10.97 26.04
20 3.41 6.18 6.73 14.92
Cu
5 61.48 77.03 81.11 82.96
10 38.33 61.19 57.85 79.76
20 10.19 13.33 17.90 35.23
Fe
5 30 50 78 136
10 20.58 36.27 43.13 69.60
20 9.34 26.92 29.67 58.79
Mg
5 24.19 46.77 56.45 75.80
10 20.16 36.29 44.35 67.74
20 12.25 25.49 29.41 58.82
Ni
5 37.14 42.85 57.14 71.42
10 41.81 40 50.90 69.09
20 59.09 65.45 81.81 81.81
Zn
5 28.08 55.13 60.27 89.04
10 30.15 48.94 52.11 84.12
20 24.69 42.46 46.66 78.27
C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
As in case of above mentioned metals again the highest (136 %) and
lowest (9.34 %) values were recorded in 5% with F1 + F2 and 20 % with C
respectively for Fe. As far as the highest and lowest values are concerned
there was a same trend seen in case of Mg, where plants in 5 % (TSW-Soil)
120
showed highest SEY% value (75.80 %) with F1 + F2 and being minimum
(12.25 %) in 20% (TSW-Soil) with C having no fungal inoculum.
There was maximum value for Ni was calculated (81.81 %) in 20%
(TSW-Soil) with combined fungal inoculum and F2, while minimum value
(37.14 %) was recorded in 5 % (TSW-Soil) for C.
In case of Zn the maximum value was noted (89.04 %) for 5 % with F1
+ F2 and minimum value (24.69 %) in 20 % (TSW-Soil) with C.
121
3.9B. Experiments with Helianthus annuus inoculated with saprobic
fungi
3.9B.1 Pre-sowing analysis
The physico-chemical properties, concentration of Category-I & II
metals are given Table 3.1, 3.2 and 3.3 and the details are described in
Chapter 3.1.
3.9B.2 Biochemical analyses of 52-days old Helianthus annuus
The biochemical parameters like chlorophyll contents, soluble protein
CAT and SOD were observed. There was increased production of all these
parameters in combined inoculation of fungi as compared to C and single
fungi. The specific details of each of the biochemical parameters are as
under:
3.9B.2.1 Chlorophyll content
After 52 days of cultivation, the plant chlorophyll content within fungal
treatments increased by applying combined fungal inoculation i.e. the F1 +
F2, as given in Table 3.9B.1. The greatest (28.7 SPAD value) was observed
in 10 % with F1 + F2 while influencing to the least (14.1 SPAD value) in 20 %
in C treatment as compared to any of the treatments within a row. However
F2 showed better values with respect to chlorophyll content as compared to
F1 values for all TWS-soil mixtures.
Within column, there was increase in plant chlorophyll contents with the
increase of TSW percentage in soil i.e. 10 % for all the fungal treatments but
the values were dropped being the minimum in 20%. The application of
fungus either individually or in combination help plants perform better as
compared to C in terms of chlorophyll contents, as given in Table 3.9B.1.
3.9B.2.2 Soluble protein contents
Parallel to chlorophyll contents, the values within a row for
soluble protein contents also increased with the application of fungal
inoculations for all the treatment, as given in Table 3.9B.1. The F1 + F2 from
10 % and the F1 from 0 % exhibited the maximum (21.8 mgg-1) and the
122
minimum (0.8 mgg-1) soluble protein contents as compare to any of the soil or
fungal treatments. Within Fungal treatments C showed minimum values as
compared to other fungal applications like F1, F2 and F1 + F2.
Within a column, there was increase in soluble protein contents with
the increasing level of TSW in soil treatments i.e. in 10 % and then decrease
in 20%. However, addition of fungi helped plants to lessen stress by
increasing soluble protein contents. The plants cultivated in pots with F2
inoculations performed better than those with F1 for all the TSW-Soil
mixtures.
3.9B.2.3 Superoxide dismutase (SOD) contents
Following the trends found for chlorophyll and soluble protein contents,
the SOD values increased with fungal inoculations within a row for all the
treatments, as given in Table 3.9B.1. The SOD values were found to be 0.5
mgg-1 in 0% with C bring minimum. For the rest of the concentrations the
Table 3.9B.1. The biochemical parameters observed in 52-days old Helianthus annuus
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Chlorophyll content
(SPAD value)
0 17.7bcBC
1.1 18.5abC
2.1 19.1abCD
0.97 21.9aD
1.1 1.2
10 19.9dA
2.1 22.5cA
1.5 23.3cA
0.22 28.7aA
1.3 2.2
20 14.1bcDE
1.4 15.4aE
0.94 15.9aE
0.34 16.1aEF
1.2 1.5
LSD0.05 0.85 1.9 0.83 1.8
Soluble Protein content
(mgg-1
)
0 0.9bcF
1.5 0.8bcF
0.43 1.1abEF
0.22 1.6aF
0.22 0.19
10 16.1cA
0.66 17.2bcA
1.5 17.8bcA
2.1 21.8aA
0.32 1.4
20 12.7dC
0.34 14.3bcBC
1.7 14.5bcC
0.37 15.9aCD
0.33 0.27
LSD0.05 3.2 4.9 4.3 5.2
SOD (Umg
-1 of protein)
0 0.5cDE
0.97 0.8abE
0.34 1.9abDE
1.3 1.1aCD
0.12 0.15
10 22deA
0.54 37bcA
0.97 39bcA
0.24 44aA
0.34 5.1
20 24cdA
1.2 35bA
0.33 37bA
0.29 41aA
0.97 4.2
LSD0.05 6.8 11.1 14 15
CAT (Uml
-1)
0 BDL BDL BDL BDL -
10 18eA
0.99 24deA
1.4 28cdA
0.92 39aA
0.66 3.4
20 12deBC
1.3 21bcCD
0.12 26aB
0.38 28aDE
0.13 4.3
LSD0.05 1.1 0.98 0.89 3.9
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
123
fungal treatments having both of the F1 and F2 performed better than control
and those applied with either F1 or F2. The plants in 10 % with F1 + F2 gave
maximum SOD values (44 Umg-1 of protein).
While analyzing the response of plant cultivated in different TWS-Soil
mixtures, it was observed that the 0% showed least values, while 10 %
showed maximum SOD values in all fungal treatments except C treatment
where highest value was recorded for 20 %.
3.9B.2.4 Catalase (CAT) contents
Within different treatments along the row, there was an increase in
plant CAT value with the individual or combined fungal inoculations, as
compared to control. The plants from 0 % showed CAT value to be BDL for all
fungal treatments. The maximum value was noted (39 Uml-1) in 10 % with F1
+ F2 while the minimum (12 Uml-1) CAT value was noted in 20 % with C
treatment as given in Table 3.9B.1.
For different TSW-Soil mixtures comparison, greater CAT content was
observed in 10% as compared to 20 % for all fungal treatments.
3.9B.3 Post-harvest analysis
3.9B.3.1 Growth performance of Helianthus annuus
Better growth of 52-day-old plants of H. annuus was observed in case
of lower TWS-soil TWS-Soil mixture i.e. 10%, being relatively less in soil (0%)
and 20% as indicated by growth parameters (Table 3.9B.2). It was noticed
that plants cultivated in soil and its TSW mixtures inoculated with fungal
isolates yielded greater shoot, root and seedling length, no. of leaves and
roots, as well as, fresh and dry weight; as compared to control. The statistical
analysis of the data showed significant growth in all parameters in lower TSW
concentration in soil followed by a decrease at higher (20%) concentration.
However, the maximum increase in values was found in F + M treatment over
their controls F1 and F2, for each of the corresponding soil treatments. The
details of each of the morphological parameters is given in Table 3.9B.2 and
described as under:
124
3.9B.3.1.1 Shoot, root and seedling length (cm)
Along the row in comparison with different fungal treatments, the
maximum plant shoot (70.2 cm), root (61.1 cm) and seedling length (131.7
cm) was observed in 10 % with F1 + F2 inoculation while being minimum
values for plant shoot (33.1 cm) and seedling length (71.3 cm) in 20 % with no
fungal inoculation i.e. the C. The minimum value for plant root length (32.5
cm) was recorded in 0% with control having no fungal inoculation. There was
increase in length of all the three vegetative parameters with the application of
fungal inoculations and the order of increase observed to be F1 + F2 > F2 >
F1 > C along the row as shown in Figure 3.9B.1.
Figure 3.9B.1. The vegetative growth variation in sunflower (Helianthus annuus) in response to soil
mixed with different percentages of TSW (% w:w) and inoculated with different fungi.
While in comparison with different TWS-Soil mixture there was increase in
plant shoot, root and seedling length with the increasing proportion of TSW in
the soil. The different TSW-Soil mixtures under F1 + F2 column gave best
results while those in C column attained the least height.
3.9B.3.1.2 No. of leaves and roots
Along the row, the plants in 10 % with F1 + F2 inoculation observed to
have maximum no. of roots (39) and leaves (18) while being the minimum (13
and 8 respectively) in 20 % without any of the inoculation i.e. C and 0% with
C.
125
Within different percentage of TWS-Soil, the increasing ratio of TSW
increased the no. of leaves and roots upto 10% but there was decreased for
these parameters in 20%. The TSW-Soil mixtures under F1 + F2 gave the
best vegetative growth than any of the fungal treatments. The least growth
response observed to be in C column.
Table 3.9B.2. Various morphological parameters observed in 52-days old Helianthus annuus
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Shoot Length (cm)
0 44.1efB
2.9 51.2dB
1.2 56.3bcA
1.1 67.8aA
1.3 5.68
10 48.1eA
1.4 59.5cA
1.3 61.2cA
1.05 70.2aA
1.22 5.1
20 33.1efCD
1.1 41.2cdDE
0.99 43.8cdC
1.03 54.1aD
0.99 4.97
LSD0.05 3.8 4.7 5.7 4.2
Root Length (cm)
0 32.5dD
0.98 38.7cC
1.1 41.1bcBC
1.5 49.7aCD
1.4 4.49
10 43.3deA
2.8 50.3bcA
1.4 52.3bA
1.1 61.1aA
1.07 5.3
20 36.8dC
2.5 38.3cdC
0.88 39.5cdC
1.09 56.1aB
1.02 5
LSD0.05 3.1 3.9 4.7 3.4
Seedling Length (cm)
0 76.9dBC
0.99 90.2cC
1.4 97.7bcB
1.3 117.9aB
1.4 11.5
10 91.4cdA
1.9 110.1bA
1.2 113.8bA
1.2 131.7aA
1.31 11.1
20 71.3cdC
2.3 79.8cDE
0.98 83.6cCD
1.6 110.6aBC
0.93 10.7
LSD0.05 5.72 7.82 11.3 12.6
No. of roots
0 18dAB
2.1 23bcB
3.3 26bcA
2.1 33aB
1.6 4.2
10 21cdA
3.1 28bcA
2.2 31bA
2.2 39aA
1.9 3.7
20 13cCD
0.45 16bD
0.89 18bC
2.4 22aCD
2.1 2.5
LSD0.05 2.63 3.7 5.1 5.6
No. of leaves
0 8dC
0.99 12bBC
1.3 13bAB
2.1 16aB
2.1 2.1
10 11dA
0.45 14bcA
1.1 15bcA
1.2 18aA
2.5 1.93
20 9cdC
0.33 11cdC
1.9 14bA
0.22 17aA
1.9 1.88
LSD0.05 0.44 0.59 0.62 0.9
Fresh wt. (g)
0 9.5cAB
1.01 11.4bcA
2.09 13.2aA
1.1 15.1aBC
2.09 1.8
10 11.5cA
1.1 12.1cA
2.2 14.3bcA
2.3 19.9aA
0.99 2.9
20 9.1cdAB
1.09 10.4cB
0.11 11.6cAB
2.2 16.7aBC
0.22 2.1
LSD0.05 1.2 1.1 2.1 2.6
Dry wt. (g)
0 2.3cdAB
1.1 2.9cA
0.23 3.2abA
1.2 3.6aC
1.9 0.38
10 2.9bcA
1.2 3.1bcA
0.99 3.6bcA
2.1 4.8aA
0.76 0.72
20 2.1cAB
0.98 2.4bcAB
0.34 2.6bcBC
2.09 3.8aC
0.33 0.49
LSD0.05 0.8 0.9 1.2 2.1
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9B.3.1.3 Fresh and dry weight (g)
The fresh and dry weight were observed to be the maximum and the
minimum in accordance with the maximum and the minimum no. of leaves
and roots for both along the row as well as within column comparisons. In
other words, along the row the maximum weight (19.9 g fresh, 4.8 g dry)
observed to be in 10 % with F1 + F2 and the minimum (9.1 g fresh, 2.1 g dry)
in 20 % with C, without any fungus respectively.
126
Within column, the increasing TSW ratio affected the biomass
production positively for lower percentage i.e. 10 % and negatively for 20 %
TWS-Soil mixture The TSW-Soil mixtures under F1 + F2 yielded maximum
fresh and dry weight while those in C column yielded the least.
3.9B.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot were variable with respect to fungal inoculations as well as increasing
ratio of TSW in soil, as given in Table 3.9B.3.
3.9B.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in shoot increased with inoculation
of fungi as compared to C. The maximum (430 mgkg-1) shoot Ca observed to
be in 20 % with F1 + F2 while being the minimum (16 mgkg-1) in 0% with C.
Within different concentrations of TSW in soil, the maximum shoot
concentrations were observed with F1 + F2 while being the minimum in those
where no fungi was applied. It was observed that shoot Ca increased with the
increasing TSW ratio in soil mixture for 10 % and then decreased for 20 %.
3.9B.3.2.2 Potassium (K) in shoot
Along the row, the shoot K uptake increased with fungal applications
for all the TSW-Soil mixtures as in case with Ca. The maximum K level (765
mgkg-1) was observed in 20 % with F1 + F2 while being the minimum (30
mgkg-1) in 0% with no fungi i.e. C.
Within column, the K shoot uptake increased with the increasing
concentration of TSW in soil mixtures for all TSW-Soil mixtures, the F2 plants
showed more K uptake than those with F1 and being the least where no
fungus was applied i.e. the C treatment. The maximum values observed in
plants cultivated in 20% TWS-Soil mixture.
3.9B.3.2.3 Sodium (Na) in shoot
The Na concentration in shoot observed to be increased along the row
and it was because of fungal applications. The pots with F1 + F2 showed the
127
highest Na shoot uptake, the F2 being greater than F1, while those with no
fungi being the least. The value for Na in 0 % with C treatment found to be
BDL. The plants in 20 % with F1 + F2 had the highest value (520 mgkg-1)
while those in 0 % with F1 exhibited the lowest Na shoot contents (35 mgkg-1).
Table 3.9B.3. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of 52-
days old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Ca
0 16dE
0.12 22cDE
0.76 28bcD
0.17 44aDE
0.23 8.4
10 125cdBC
0.34 230cA
1.3 245cA
0.19 380aB
0.44 65
20 190dA
0.67 250cdA
1.1 270cdA
0.99 430aA
1.6 63
LSD0.05 64 78 86 135
K
0 30deD
0.65 60cE
0.76 75cDE
1.01 145aD
1.1 32
10 210eA
1.2 390cA
2.1 410cAB
1.6 670aA
1.7 125
20 270eA
1.3 455cdA
2.4 480cdA
1.7 765aA
2.1 140
LSD0.05 82 132 143 225
Na
0 BDL 35eDE
0.99 55bcD
0.78 75aDE
1.1 11
10 155dA
2.3 180cA
1.5 225cA
1.1 430aAB
2.7 72
20 145deA
3.1 170dA
1.4 210dA
1.02 520aA
2.4 98
LSD0.05 5 54 68 155
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
Within column, the increasing ratio of TSW in soil mixtures enhanced
the shoot Na uptake for all the fungal treatments except 20 % where the
values for Na were decreased as compared to 10 %. However in 20% with F1
+ F2 the Na accumulation was found to be greater than in 10 %. In case of 0
%, the concentration of shoot Na observed to be the least for all the fungal
treatments as shown in Table 3.9B.3.
3.9B.3.3 Category-I metals in plant ROOT
The bioavailability of Category-I metals was variable with different fungi
in root also however, it was directly related to the increasing ratio of TSW in
soil mixture, as given in Table 3.9B.4.
3.9B.3.3.1 Calcium (Ca) in root
The application of fungal inoculum to the soil helps to increase Ca
uptake along the row i.e. various fungal treatments. The plants in F1 + F2 pots
observed to have maximum while those with no fungi having the minimum
root Ca than any of the fungal treatments for all of the TSW-Soil mixtures. The
128
highest root Ca (390 mgkg-1) was in 20 % with F1 + F2 while being the
minimum (10 mgkg-1) in 0 % with no fungal inoculation.
3.9B.3.3.2 Potassium (K) in root
The K contents in the 20 % with F1 + F2 exhibited the maximum root
uptake (455 mgkg-1) than any of the soil treatments while being the minimum
(15 mgkg-1) in 0 % with no fungal application. The application of fungus as
individual i.e. F1 and F2 showed better K uptake than TSW-Soil mixtures
where no fungi has been applied. However, F2 showed better uptake as
compared to F1 treatment.
Within column, the metal uptake in root was found to be increased with
increasing TWS-Soil mixtures i.e. being the maximum values in 20% than in
10 % and least values were observed in 0 %.
Table 3.9B.4. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of 52-days
old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Ca
0 10cdD
0.1 15cDE
0.11 20abD
1.1 35aE
1.6 8.4
10 55eBC
0.23 120cdC
1.1 135cdBC
1.12 310aB
2.6 67
20 95deA
0.37 210cA
1.12 225cA
1.5 390aA
1.12 78
LSD0.05 35 74 81 128
K
0 15dDE
0.23 40cDE
0.99 55bcE
0.18 90aE
0.95 22
10 135deBC
0.26 255bcB
1.31 290bA
2.6 380aAB
1.12 68
20 180A 1.1 310
bcA 1.4 325
bcA 1.5 455
aA 2.2 76
LSD0.05 64 78 105 134
Na
0 BDL 15cdDE
0.41 20bcCD
0.12 45aD
1.2 9.4
10 90eB
1.4 125dA
1.12 145dAB
1.5 325aA
1.12 65
20 110dA
1.12 110dB
0.23 190cdA
0.98 355aA
1.05 72
LSD0.05 7.6 6.9 64 115
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9B.3.3.3 Sodium (Na) in root
Along the row as observed in case of Ca and K, the application of fungi
helps increase Na uptake in roots. The maximum Na in root (355 mgkg-1) was
observed in 20% with F1 + F2 while being least in 0 % amounting 15 mgkg-1
with F1. Those with F1 and F2 applications also performed better than C i.e.
treatment with no fungal inoculation. For 0% with C treatment the values was
found to be BDL.
129
Within column, trend of Na root uptake was also similar to what
observed in case of Ca and K. The increasing ratio of TSW in soil displayed
increased root Na uptake as compared to Soil with C for all the fungal
treatments, however the value was found to be decreased in F1 for 20 % (110
mgkg-1) as compared to 10% (125 mgkg-1).
3.9B.3.4 Category-II metals in plant shoot
The Category-II metals i.e. the AAS detected metals in shoot were
variable with respect to fungal inoculations as well as increasing ratio of TSW
in soil, as given in Table 3.9B.5. The application of fungi enhanced trace
metal uptake tendency of plant for all the TSW-Soil mixtures. However, the
increasing level of Category-II metals in shoot was in accordance with the
increasing ratio of TSW in soil mixtures for all the fungal treatments.
3.9B.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in TSW-Soil mixtures with combined
fungal treatments. Maximum amount of metal (1,030 mgkg-1) was observed in
10% TSW-Soil mixture in combined inoculation of fungi i.e. F1 + F2, while
being minimum in 0% amounting 35 mgkg-1 for C treatment.
Within different TWS-Soil mixtures there is increasing trend of metal
accumulation with 10 % and then the values was decreased in 20 % for all
fungal treatments.
3.9B.3.4.2 Cr in shoot
With different fungal treatments the maximum Cr accumulation in shoot
was observed in treatments applied with combined inoculants i.e. F1 + F2,
being significantly higher than any of the treatments along the rows. Cr was
found to be as low as 3 mgkg-1 in 0% with C and maximum 1,135 mgkg-1 in
10% with F1 + F2.
Within different columns, there was maximum accumulation of metal
was observed in 10% for all fungal treatments as shown in Table 3.9B.5.
130
Table 3.9B.5. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 55-
days old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 35dDE
0.94 70cE
0.23 95bcDE
2.1 145aE
1.4 32
10 380eA
1.4 665cA
3.4 790cA
4.1 1,030aA
2.8 176
20 220eC
1.7 540cB
3.7 610cA
2.4 980aA
2.1 215
LSD0.05 128 205 237 310
Cr
0 3dD
0.1 6cE
0.34 9abDE
0.34 12aE
0.12 2.6
10 415deA
1.8 645cA
2.3 685cAB
2.7 1,135aA
2.5 210
20 395eA
2.1 525cdA
2.6 595cdA
2.3 985aBC
2.1 155
LSD0.05 143 228 232 390
Cu
0 BDL 15dE
0.21 10dDE
0.13 45aE
0.86 10.7
10 210eA
1.6 355cdA
1.6 335cdA
1.71 875aA
1.1 168
20 115efBC
1.41 230dCD
2.1 265dA
1.35 660aBC
0.23 145
LSD0.05 35 122 130 155
Fe
0 6dD
0.94 15cE
0.93 20bcDE
0.43 45aDE
0.87 12.7
10 55deBC
0.91 110cAB
1.01 145bA
1.1 170aBC
1.1 32
20 80efA
0.23 155cdA
1.1 170cdA
1.01 270aA
1.6 48
LSD0.05 28 45 65 72
Mg
0 20eDE
0.78 45dD
0.14 65cDE
0.45 110aD
1.12 27
10 160bcA
1.2 190abA
0.99 220aA
1.5 240aB
1.65 38
20 180dA
1.54 225bcA
1.4 265bA
1.2 310aA
2.1 42
LSD0.05 22 65 72 78
Ni
0 BDL BDL BDL BDL -
10 10cBC
0.12 15bcB
1.6 25aA
0.12 30aA
0.34 7.8
20 15cA
0.41 20abA
0.33 15cBC
0.21 25aA
0.76 3.2
LSD0.05 3.4 2.9 3.1 3.5
Zn
0 25dD
0.12 45bcDE
0.88 55bcD
0.67 80aDE
0.98 16.3
10 435deA
0.13 660bcA
2.6 710bcA
2.6 990aA
1.65 142
20 390eA
0.45 480cBC
2.9 510cB
2.1 860aBC
2.1 128
LSD0.05 142 218 227 327
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.9B.3.4.3 Cu in shoot
Along the row, the plants harvested from F1 + F2 exhibited maximum
Cu accumulation as compared to any of the treatments with single or no
fungal application. Such a trend was observed in all of the TSW-Soil mixtures.
The plants from treatments with no fungal inoculations i.e. C showed the
significantly least Cu accumulation as compared to any of the fungal
treatments as shown in Table 3.9B.5.
Within different TWS-Soil mixtures, 0% showed significantly less Cu
uptake in shoots than those form 10 and 20 % as minimum accumulation 15
mgkg-1 was seen in 0% with F1 and maximum accumulation 875 mgkg-1 in 10
% with F1 + F2. The value was found to be BDL in 0% with C treatment.
131
3.9B.3.4.4 Fe in shoot
The Fe uptake in plant shoots observed to be least in Soil (0%) with C
i.e. 6 mgkg-1, but F1 + F2 inoculation displayed the maximum Fe uptake in
shoot as compared to plants from the treatments with single or no fungal
inoculations. The values of plant shoot Fe was maximum 270 mgkg-1 in 20 %
with F1 + F2.
Within a column, maximum accumulation was observed in 20 % as
compared to 10 % and found to be least in 0% for all fungal applications as
shown in Table 3.9B.5.
3.9B.3.4.5 Mg in shoot
Along various fungal treatments, the Mg uptake in plant shoot
increased with fungal application in the pots and found to be the maximum
310 mgkg-1 in plants harvested from pots applied with combined fungi i.e. F1
+ F2 in 20 % TWS-Soil mixture, while being the minimum as well as
significantly least in shoot of plants cultivated in soil with no fungus i.e. 20
mgkg-1 as shown in Table 3.9B.5.
Along the columns, the value of plant shoot Mg was found to be least in
0%. For 20 %, the shoot uptake found to be highest as compared to 10 %
TSW-Soil mixture.
3.9B.3.4.6 Ni in shoot
The Ni concentration observed to be BDL in 0% for all the fungal
treatments along the row. However, for all the TSW-Soil mixtures the shoot Ni
concentration increased with the application of fungus and observed to be the
maximum in plants inoculated with F1 + F2 and being the least in those from
C i.e. where no fungus was applied.
Within the columns comparison, the value of plant shoot Ni
concentration increased in 20% with C and F1 treatment while decreased with
F2 and F1 + F2.
3.9B.3.4.7 Zn in shoot
The Zn concentration in shoots increased in plants harvest from pots
with fungal inoculations than those harvested from pots applied with no fungi.
132
Like other metals, the shoot Zn observed to be the maximum in plants
inoculated with F1 + F2 while those harvested from the pots with no fungi
displayed the minimum values. There was increasing trend of accumulation of
metal as the application of fungi. However F2 showed better results as
compared to F1 in terms of accumulation of metal as shown in Table 3.9B.5.
For different TWS-Soil mixtures, the value of Zn concentration in shoot
was found to be maximum 990 mgkg-1 in 10 % with F1 + F2 being minimum
25 mgkg-1 in 0% with C as shown in Table 3.9B.5. The order of accumulation
of metal accumulation within different TWS-Soil mixtures from maximum to
minimum was as 10 % >20 % > 0% for all fungal treatments.
3.9B.3.5 Category-II metals in plant root
The Category-II metals i.e. the AAS detected metals in root were
observed to differ with varying levels of TSW in the soil as well in response to
fungal inoculations, as given in Table 3.9B.6.
3.9B.3.5.1 Cd in root
For different fungal treatments within the rows, the Cd level in plant
roots harvest from Soil observed to be minimum 20 mgkg-1 in 0% with C and
found to be maximum 930 mgkg-1 in 10 % with F1 + F2 as shown in Table
3.9B.6. The plant root Cd concentration increased with fungal inoculations
and found to be maximum as well as significantly higher than those applied
with single or no fungal treatments.
Inside the columns, for different TWS-Soil mixtures the root Cd
concentration was found to be highest in 10% and decreased values were
noted in 20%.
3.9B.3.5.2 Cr in root
For different fungal treatments i.e. along the row, the plants from F1 +
F1 fungal inoculations showed the maximum 875 mgkg-1 root Cr concentration
in 10 %, while with C treatment plants having the minimum uptake 290 mgkg-1
in 20%. The F2 inoculation enhanced the root Cd concentration better than
F1. However in 0 % the values were found to be BDL for all fungal treatments
as shown in Table 3.9B.6.
133
Table 3.9B.6. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 52-days
old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 20deEF
0.23 60bcDE
1.12 90aE
1.33 110aE
1.24 28
10 310eA
1.1 520cdA
2.45 550cdA
1.23 930aA
2.53 163
20 210deC
1.34 490cA
1.23 510cA
2.1 745aBC
2.81 143
LSD0.05 65 158 162 284
Cr
0 BDL BDL BDL BDL -
10 325eA
1.65 430cdA
2.11 480cdA
2.4 875aA
2.3 145
20 290efAB
1.87 365dAB
2.3 390dBC
2.6 770aC
2.6 129
LSD0.05 13.2 28 33 39.3
Cu
0 BDL 8cEF
0.86 5cE
0.1 25aEF
0.23 5.5
10 90efA
1.5 245cdA
1.32 260cdA
2.1 670aA
1.3 164
20 65eCD
1.4 195cdC
1.3 210cdAB
2.7 455aC
1.4 111
LSD0.05 9.3 82 88 238
Fe
0 BDL 10cdF
0.45 13cdDE
0.95 30aD
0.94 6.5
10 20deD
0.44 90adCD
1.01 135bcA
1.1 210aA
1.66 98
20 60dA
0.98 120bcA
1.1 155aA
1.6 180aA
1.6 45
LSD0.05 17 39.2 55 62
Mg
0 15dDE
0.45 30cDE
0.54 45bcD
0.94 75aE
0.99 18
10 110cA
0.56 145abAB
1.54 150aBC
1.02 170aC
1.3 23
20 75dBC
0.87 190bcA
1.1 215bcA
1.01 290aA
1.1 78
LSD0.05 34 58.9 64 76
Ni
0 BDL BDL BDL BDL -
10 5bcBC
0.1 10aA
0.66 9aA
0.11 10aBC
0.2 2.9
20 8cA
0.13 12bcA
0.65 8cA
0.23 15aA
0.03 2.7
LSD0.05 1.8 2.5 1.4 3.1
Zn
0 15deDE
0.33 30cD
1.1 35cDE
0.34 45aD
1.1 3.1
10 225cdAB
1.1 265cA
1.6 280cA
0.99 480aAB
2.7 67.8
20 295dA
1.4 310cdA
1.4 325cdA
1.66 535aA
2.5 62
LSD0.05 98 102 112 118
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
Within the columns, the root Cr concentration was found to be greater
in 10 % as compared to 20 % for all fungal treatments.
3.9B.3.5.3 Cu in root
Along the row for various fungal inoculations the root Cu uptake was
enhanced as compared to C. The value was found to be BDL in 0% for C
treatment. The plants from F1 + F2 observed to have the maximum (670
mgkg-1) uptake in 10 % than those cultivated in pots with single or no fungal
inoculations and found to be the minimum 5 mgkg-1 in 0% with F2.
For different columns, the plants from 10 % TSW-Soil exhibited better
root Cu accumulation than 20 %.
134
3.9B.3.5.4 Fe in root
Moving alongside the row while analyzing effect of different fungal
treatments, the root Fe observed to be BDL in soil (0 %) with C treatment. The
plants from C had the lowest root Fe as compared to plants from any of the
single or combined application of fungi for the rest of TWS-Soil mixtures i.e.
10 % and 20 %. The root Fe accumulation observed to be the maximum (210
mgkg-1) in 10 % with F1 + F2 and significantly higher than those harvested
from any of the treatments with single or no fungal inoculations.
Inside columns, the maximum Fe uptake by roots was observed in 10
% with F1 + F2 while the least value of metal uptake was observed in 0 % as
shown in Table 3.9B.6.
3.9B.3.5.5 Mg in root
While analyzing the fungal application effect on plants along the rows,
it was observed that the Mg root concentration increased in plant roots with
fungal application as compared to C i.e. with no fungal inoculation. The F1 +
F2 plants displayed the maximum root Mg concentration than those from C as
well as F1 and F2. However the F2 inoculations gave better results than F1.
For the columns, the root Mg accumulation was found to be highest in
20% with F1 + F2 where the metal accumulation was observed to be 290
mgkg-1 while minimum concentration 15 mgkg-1 in 0 % with C treatment was
noted as shown in Table 3.9B.6.
3.9B.3.5.6 Ni in root
The root Ni concentration was found to be BDL for all fungal treatments
in 0% i.e. soil. However there was increased metal accumulation along the
row with the application of fungal inoculations and found to be the maximum
in plants applied with combined application of both of the fungi while being the
minimum in C with no fungus added.
Within columns, the plants from 20 % TSW-Soil mixtures had the
maximum root Ni level than 10 % for all of the fungal treatments. There was
135
maximum (15 mgkg-1) accumulation of metal was noted in 20% with F1 + F2
while minimum uptake (5 mgkg-1) was observed in 10 % with C.
3.9B.3.5.7 Zn in root
The root Zn accumulation observed to increase in pots applied with
fungal inoculations than C and found to be the maximum in treatments applied
with both of the fungi and being the minimum with no fungal applications.
Again as with most of the above discussed metals F2 performed better than
F1 as far as metal accumulation efficiency of the plant is concerned.
For different TWS-Soil mixtures along the columns, the root Zn
concentration increased with increasing percentage of TSW in soil with every
fungal treatment. The maximum accumulation was noted in 20 % with F1 + F2
i.e. 535 mgkg-1 and minimum (15 mgkg-1) in 0 % with C treatment as shown in
Table 3.9B.6.
3.9B.4 Fungal analyses
The results of estimation of the post-harvest fungal analyses (× 105
c.f.u. g-1 soil) of 52-days old Helianthus annuus cultivated on TSW-Soil
mixtures are shown in Table 3.9B.7. Alongside the row, the c.f.u. increased
with fungal application than C and observed to be the maximum in treatments
with combined application of both of the fungi. The order of c.f.u. abundance
was F1 + F2 > F2 > F1 > C.
Within a column, the c.f.u. abundance was observed to be highest in %
with 10.9 × 105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2.
Table 3.9B.7. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 52-days old
Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).
TSW-Soil (% w:w) Treatment
LSD0.05 C F1 F2 F1+F2
0 2.7deBC
0.22 4.8cA
0.17 5.5cB
0.23 10.9aBC
0.29 3.1
10 3.1cdAB
0.16 4.2cAB
0.33 4.9cB
0.07 9.5aC
0.51 1.8
20 2.4bcC
0.92 2.9bcDE
0.52 3.1bcCD
0.83 4.9aE
0.20 1.54
LSD0.05 0.38 0.64 0.97 2.5
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
136
3.9B.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
3.9B.5.1 Category-I metals translocation index (%)
The plant translocation index values were also recorded for category-I
metals those detected by flame photometer i.e. Ca, K and Na shown in Table
3.9B.8.
In case of Ca, maximum value was observed in 10% with C i.e. 227.27
% while the minimum value was recorded (110.25 %) in 20 % with F1 + F2.
For K, the maximum translocation index value was calculated in 10%
with F1 + F2 i.e. 176.31 % being the minimum 141.37 % in 10% with F2.
Table 3.9B.8. The Category-I metals translocation index (%) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca 10 227.27 191.66 181.48 122.58
20 200 119.04 120 110.25
K 10 155.55 152.94 141.37 176.31
20 150 146.77 147.69 168.13
Na 10 172.22 144 155.17 132.30
20 131.81 154.54 110.52 146.47
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Na, the maximum value 172.22 % was observed in 10% with
C treatment and least value was recorded in 20 % for F2 (110.52 %).
3.9B.5.2 Category-II metals translocation index (%)
The plant translocation index found to be greater in treatments applied
with fungal inoculum than that of C, being the maximum where F1 and F2
applied together for all metals as given in Table 3.9B.9. As compared to
different TWS-Soil mixtures there was decreasing trend observed with
increasing percentage of TWS-Soil with few exceptions.
For Cd, the maximum Translocation index (143.63 %) was noted in
10% with F2, while minimum value was recorded 104.76 % in 20% with C.
137
In case of Cr plants showed maximum metal translocation efficiency
(152.56 %) in 20% with F2 and minimum 127.69 % in 10 % with C.
For Cu, the maximum translocation index values was recorded to be
233.33 % for 10% with C, while minimum value was recorded in 20 % with F1
treatment i.e. 117.94 %.
For Fe, the maximum translocation index values was recorded to be
275 % for 10 % with C, while minimum value was recorded in 10 % with F1 +
F2 treatment i.e. 80.95 %.
In case of Mg the plants showed least value in 20% with F1 + F2
(106.89 %), while the maximum values for this metal was recorded in 20%
with C i.e. 240 %.
For Ni, there was 300% translocation index value recorded in 10% with
F1 + F2 and least value was observed in 10% with F1 i.e. 150 %.
As far as the Zn is concerned there was maximum translocation index
recorded in 10 % with F2 treatment i.e. 253.57 % while minimum value was
recorded in 20% with C i.e. 132.20 % as shown in Table 3.9B.9.
Table 3.9B.9. The Category-II metals translocation index (%) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd 10 122.58 127.88 143.63 110.75
20 104.76 110.20 119.60 131.54
Cr 10 127.69 150 142.70 129.71
20 136.20 143.83 152.56 127.92
Cu 10 233.33 144.89 136.53 130.59
20 176.92 117.94 126.19 145.05
Fe 10 275 122.22 107.40 80.95
20 133.33 129.16 109.67 150
Mg 10 145.45 131.03 146.66 141.17
20 240 118.42 123.25 106.89
Ni 10 200 150 277.77 300
20 187.5 166.66 187.5 166.66
Zn 10 193.33 249.05 253.57 206.25
20 132.20 154.83 156.92 160.74
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
138
3.9B.5.3 Tolerance index (TI)
In shoots TI values were found to be highest in 10 % with F1 (1.16)
while minimum value was recorded to be 0.75 in 20% with C as shown in
Table 3.9B.10.
Table 3.9B.10. The tolerance index (TI) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
TI Shoot 10 1.09 1.16 1.08 1.03
20 0.75 0.80 0.77 0.79
TI Root 10 1.33 1.29 1.27 1.22
20 1.13 0.98 0.96 1.12
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of TI in roots 1.33 was recorded as maximum for plants grown
in 10% with C, while 0.96 was recorded as minimum value in 20% with F2.
3.9B.5.4 Category-I metals specific extraction yield percentage (SEY %)
The SEY % for Category-I metals i.e. Ca, K and Na showed in Table
3.9B.11.
Table 3.9B.11. The Category-I specific extraction yield (SEY %) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca 10 17.91 22.17 24.73 32.19
20 5.6 9.4 10.99 22.68
K 10 17.76 26.85 33.05 54.95
20 24.24 27.77 30.30 37.12
Na 10 51.68 62.02 65.60 79.52
20 37.67 43.05 45.64 48.65
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Ca, there was maximum value 32.19 % was recorded for
plants grown in 10 % with F1 + F2, and minimum 5.6 % in 20 % with C.
In case of K, plants cultivated in 10 % TWS-soil showed highest value
of SEY% (54.95 %) with F1 + F2 and minimum in 10 % with C i.e. 17.76 %.
The highest value for Na was recorded in 10 % with F1 + F2 (79.52 %)
while minimum values was found to be 37.67 % in case of 20% with no
fungal inoculum i.e. C.
139
3.9B.5.5 Category-II metals specific extraction yield percentage (SEY %)
The SEY (%) was calculated in Category-II metals that were detected
by AAS as shown in Table 3.9B.12. Overall a similar kind of trend was seen
for all metals in various fungal treatments along the row, that the SEY %
values increased with the application of fungal inoculum and highest value
was observed for F1 + F2 treatment.
Table 3.9B.12. The Category-II specific extraction yield (SEY %) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd 10 17.02 21.88 22.94 26.74
20 15.54 17.6 18.28 23.31
Cr 10 24.87 34.53 39.31 42.04
20 23.27 27.98 30.65 34.81
Cu 10 39.04 50.71 51.42 71.42
20 25.90 30.66 34.28 39.61
Fe 10 23.52 31.37 38.23 84.31
20 16.48 22.52 29.12 65.38
Mg 10 32.25 52.41 62.90 73.38
20 19.11 25.98 32.84 52.45
Ni 10 29.18 41.81 54.54 81.81
20 22.72 36.36 45.45 86.36
Zn 10 11.11 21.95 24.33 26.45
20 6.66 14.56 22.71 27.90
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Cd the maximum value for SEY % was recorded in 10 %
with F1 + F2 treatment i.e. 26.74 %, while minimum (15.54 %) was observed
in 20% with C.
Similarly the SEY % value for Cr was found to be highest (42.04 %) in
10 % with F1 + F2 and minimum (23.27 %) was recorded in 20% with no
fungal inoculum i.e. C. However F2 showed greater values as compared to
F1.
For Cu again the maximum value was observed for 10 % (71.42 %)
with combined fungal inoculum F1 + F2 and least value (25.90 %) was
recorded in 20% with C.
140
As in case of above mentioned metals again the highest (84.31 %) and
lowest (16.48 %) values were recorded in 10% with F1 + F2 and 20 % with C
respectively for Fe.
As far as the highest and lowest values are concerned there was a
same trend seen in case of Mg, where plants in 10 % showed highest SEY%
value (73.38 %) with F1 + F2 and being minimum (19.11 %) in 20% with C
having no fungal inoculum.
There was maximum value for Ni was calculated (86.36 %) in 20% with
combined fungal inoculum, while minimum value (22.72 %) was recorded in
20 % for C.
In case of Zn the maximum value was noted (27.90 %) for 20 % with
F1 + F2 and minimum value (6.66 %) in 20 % with C.
141
3.10 Experiment with French marigold
3.10.1 Pre-sowing analysis
The physico-chemical properties of TSW, Caldwell field soil and their
various (% w:w TSW-Soil) mixtures is shown in Table 3.10.1. The pH of TSW
was extremely high as compared to the Caldwell soil and mixing soil with
TSW increased its pH. Likewise, the OM of soil increased with increasing the
percentage of TSW. The concentration of Category-I as well as Category-II
metals were also extremely in TSW as compare to the soil. However, mixing
of TSW in soil added doses of both of the categories of the metals to the soil
with maximum fractions observed in 10 %.
Table 3.10.1. The physico-chemical properties, Category-I and Category-II metals determined
in TSW, Caldwell field soil and their various (% w:w TSW-Soil) mixtures; The mean values
with common letters (small along the row & capital within a column) are not significantly
different according to Duncan’s multiple range test (P = 0.05; n = 3).
Parameters 0
(Soil only) 5 10
100
(TSW only) LSD0.05
pH 5.7bc
6.1b 6.5
b 8.9
a 2.4
Organic matter (OM %) 3.65ab
3.91ab
4.2a 4.5
a 1.2
Ca 699.5f 1,800
e 2,450
cd 6,320
a 1,525
Na 23.92gh
880f 1,550
e 9,440
a 2,398
K 118.5e 290
de 1,330
c 4,210
a 1,088
Cd BDL 2,320e 4,230
cd 10,097
a 2,545
Cr 13.3fg 7,560
de 9,910
d 25,534
a 6,378
Cu 9.32ef 1,210
d 1,930
cd 10,554
a 2,672
Fe 18.62f 280
de 550
cd 2,250
a 678
Mg 75.44f 530
de 945
d 3,840
a 789
Ni 16.21f 80
ef 125
de 590
a 165
Pb 16.5a 13.3
b 11.8
bc BDL 2.1
Zn 7.29f 280
ef 430
ef 7,590
a 1,810
LSD: Least significance difference
3.10.2 Biochemical analyses of 45-days old Tagetes patula
The biochemical parameters like chlorophyll contents, soluble protein
CAT and SOD were observed. There was increased production of all these
parameters in combined inoculation of fungi as compared to C and single
fungi. The specific details of each of the biochemical parameters are as
under:
142
3.10.2.1 Chlorophyll content
The plant chlorophyll content within a fungal treatment increased by
applying combined fungal inoculation i.e the F1 + F2, as given in Table 3.10.2.
The F1 + F2 cause the greatest (22.3 SPAD value) increase in plants from 5
% while having the least 12.2 SPAD value in 0 % with C treatment as
compared to any of the treatments within a row. However F2 showed better
values with respect to chlorophyll content as compared to F1 values.
Within column, there was increase in plant chlorophyll contents in 5 %
for all the fungal treatments but the values were dropped in 10%. The
application of fungus either individually or in combination help plants perform
better as compared to C in terms of chlorophyll contents, as given in Table
3.10.2.
Table 3.10.2. The biochemical parameters observed in 45-days old Tagetes patula cultivated
on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range
test (P = 0.05; n = 4).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Chlorophyll content (SPAD value)
0 12.2cC
0.67 13.5abC
0.99 13.9aCD
0.67 14.8aBC
1.2 0.9
5 16.5dA
0.11 18.1cA
1.4 19.2bcA
0.69 22.3aA
1.87 2.2
10 14.3abAB
1.3 14.9abBC
0.67 15.4aC
0.23 15.9aBC
0.62 2.8
LSD0.05 1.5 3.2 3.7 4.2
Soluble Protein content
(mgg-1
)
0 0.6bDE
1.5 0.9abCD
0.78 1.1abD
0.60 1.9aDE
0.45 1.7
5 11cdA
1.8 13bcA
0.66 14bcA
0.77 18aA
0.38 1.6
10 12cdA
0.67 15bA
0.34 16bA
0.67 19aA
0.92 2.8
LSD0.05 4.2 4.7 4.5 4.9
SOD (Umg
-1 of protein)
0 BDL BDL BDL BDL -
5 16cA
0.68 18cA
0.56 21bcA
0.45 28aA
0.33 4.1
10 17cA
0.44 19cA
0.61 23abA
0.98 27aA
0.67 2.9
LSD0.05 2.1 2.5 2.6 2.8
CAT (Uml
-1)
0 BDL BDL BDL BDL -
5 13deA
0.6 16cA
0.55 18cA
0.24 27aA
0.23 3.7
10 9cBC
0.63 11bcC
0.89 12bcBC
0.45 16aC
0.45 2.7
LSD0.05 2.3 1.9 2.8 3.8
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
3.10.2.2 Soluble protein contents
Same as the case with chlorophyll contents, the values within a
row for soluble protein contents also increased with the application of fungal
inoculations for all the treatment, as given in Table 3.10.2. The F1 + F2 from
10 % and the C from 0 % exhibited the maximum (19 mgg-1) and the minimum
(0.6 mgg-1) soluble protein contents respectively, as compare to any of the soil
143
or fungal treatments. Within Fungal treatments C showed minimum values as
compared to other fungal applications like F1, F2 and F1 + F2.
Within a column, overall there was increase in soluble protein contents
with the increasing level of TSW in soil. However, addition of fungi helped
plants to lessen stress by increasing soluble protein contents. The 10 % with
F1 + F2 and the 0 % with C observed to have the maximum and the minimum
values respectively. The plants cultivated in pots with F2 inoculations
performed better than those with F1 for all the TSW-Soil mixtures.
3.10.2.3 Superoxide dismutase (SOD) contents
Following the trends found for chlorophyll and soluble protein contents,
the SOD values increased with fungal inoculations within a row for all the
treatments, as given in Table 3.10.2. The SOD values were found to be BDL
for all fungal treatments of 0%. For the rest of the two TWS-Soil mixtures i.e. 5
and 10 % the fungal treatments having both of the F1 and F2 performed
better than control and those applied with either F1 or F2. The plants in 10 %
with F1 + F2 gave greater SOD values except with F1 + F2 where 5% showed
slightly better value i.e. 28 Umg-1 of protein.
3.10.2.4 Catalase (CAT) contents
Within different treatments along the row, there was an increase in
plant CAT value with the individual or combined fungal inoculations, as
compared to control. CAT values for 0 % were found to be BDL for all fungal
treatments. The plants from 5 % with F1 + F2 had the maximum (27 Uml-1)
CAT values. The minimum (9 Uml-1) CAT value was observed in 10 % in C
treatment as given in Table 3.10.1.
For different TSW-Soil mixture comparison, there was minimum CAT
contents was observed in 10% as compared to 5% for all fungal treatments.
3.10.3 Post-harvest analysis
3.10.3.1 Growth performance of Tagetes patula
Better growth of 45-day-old plants of T. patula was observed in case of
lower TWS-soil percentage i.e. 5% being relatively less in 10% as indicated
by growth parameters (Table 3.10.3). It was noticed that plants cultivated in
144
soil and its TSW mixtures inoculated with fungal isolates yielded greater
shoot, root and seedling length, no. of leaves and roots, as well as, fresh and
dry weight; as compared to control. The statistical analysis of the data showed
significant growth in all parameters in lower TWS-Soil mixtures in soil followed
by a decrease at higher (10%) level. However, the maximum increase in
values was found in F + M treatment over their controls F1 and F2, for each of
the corresponding soil treatments. The details of each of the morphological
parameters is given in Table 3.10.3 and described as under:
3.10.3.1.1 Shoot, root and seedling length (cm)
Along the row in comparison with different fungal treatments, the
maximum plant shoot (22.3 cm), root (18.1 cm) and seedling length (40.8 cm)
was observed in 0 % with F1 + F2 inoculation while being minimum values for
plant shoot (9.7 cm) and seedling length (18.6 cm) in 10 % with no fungal
inoculation i.e. the C. The minimum value for plant root length (9.7 cm) was
recorded in 10% with control having no fungal inoculation. There was increase
in length of all the three vegetative parameters with the application of fungal
inoculations and the order of increase observed to be F1 + F2 > F2 > F1 > C
along the row as shown in Figure 3.10.1.
While in comparison with columns, there was decrease in plant shoot,
root and seedling length with the increasing proportion of TSW in the soil. The
different TSW-Soil mixtures under F1 + F2 column gave best results while
those in C column attained the least height. However the highest TWS-Soil
mixture i.e. 10% showed least growth as shown in Table 3.10.3.
3.10.3.1.2 No. of leaves and roots
Along the row, the plants in 5% with F1 + F2 inoculation observed to
have maximum no. of roots (21) and leaves (15) while being the minimum (07
and 04 respectively) in 10 % without any of the fungal inoculation i.e. C.
Within columns, the increasing ratio of TSW increased the no. of roots
upto 5 % but there was decreased for these parameters in 10%. The TSW-
Soil mixtures under F1 + F2 gave the best vegetative growth than any of the
fungal treatments. The least growth response observed to be in C column.
145
Table 3.10.3. Various morphological parameters observed in 45-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).
Parameters TSW-Soil
(% w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Shoot Length (cm)
0 12.1dA
0.34 16.4bcA
1.2 17.8bcA
0.78 22.3aA
0.67 2.7
5 11.7dA
1.33 14.9bcAB
1.4 15.5bcB
0.45 19.3aBC
1.5 3.2
10 9.7cdBC
1.45 12.3bC
1.1 13.7bBC
0.11 17.1aC
0.79 3.8
LSD0.05 0.7 1.5 1.4 1.8
Root Length (cm)
0 11.3bcA
2.4 13.8bA
0.14 14.5bcA
0.14 18.1aA
0.77 1.8
5 9.6bAB
1.5 10.3aBC
0.44 10.9aBC
0.87 12.1aC
.1 2.7
10 8.5abB
0.99 8.1abC
0.57 8.7abC
0.33 10.4aC
1.07 3.2
LSD0.05 0.9 1.7 1.9 2.6
Seedling Length
(cm)
0 23.7dA
1.93 30.5bcA
0.66 32.6bcA
1.2 40.8aA
1.88 1.5
5 21.6dA
2.38 25.5bcC
0.32 26.7bBC
1.5 31.7aC
1.34 1.6
10 18.6dBC
1.66 20.8bcD
0.24 22.7bcC
1.7 27.8aD
1.23 2.5
LSD0.05 1.7 1.8 3.5 4.8
No. of roots
0 09cdB
1.22 13bA
0.98 15aA
0.84 17aC
0.18 2.2
5 12dA
1.54 15bcA
0.54 16bcA
0.38 21aA
0.34 2.7
10 07cdBC
0.95 09cBC
1.3 11cBC
0.32 19aAB
0.56 3.3
LSD0.05 1.8 1.7 1.9 2.2
No. of leaves
0 07dA
0.32 10bcA
0.34 09bA
0.78 13aA
0.45 3.1
5 08bcA
0.11 07cB
0.89 06cA
0.67 15aA
0.86 2.8
10 04cdBC
0.20 06cB
1.3 05cdAB
0.45 12aB
N0.46 4.2
LSD0.05 0.9 1.5 1.7 2.6
Fresh wt. (g)
0 5.6abA
0.12 6.4aB
1.1 6.9aA
1.3 7.2aBC
0.43 2.4
5 6.4cA
0.32 8.1abA
0.26 7.8bA
1.2 9.7aA
0.64 2.9
10 4.2abB
0.67 4.9aCD
0.78 5.1aC
0.92 5.9aC
0.89 1.5
LSD0.05 0.8 1.1 2.6 2.9
Dry wt. (g)
0 2.1abA
0.23 2.4aAB
1.1 2.5aA
1.3 2.9aA
0.44 1.7
5 2.5abA
0.75 3.1aA
0.24 2.8abA
1.1 3.5aA
0.65 1.9
10 1.2bBC
0.61 1.5abBC
0.87 1.8abB
0.95 2.3aAB
0.91 2.6
LSD0.05 1.4 2.2 2.7 3.1
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
3.10.3.1.3 Fresh and dry weight (g)
The fresh and dry weight were observed to be the maximum and the
minimum in accordance with the maximum and the minimum no. of leaves
and roots for both along the row as well as within column comparisons. In
other words, along the row the maximum weight (9.7 g fresh, 3.5 g dry)
observed to be in 5 % with F1 + F2 and the minimum (4.2 g fresh, 1.2 g dry) in
10 % with C, respectively.
Within column, the increasing TSW ratio affected the biomass
production positively for lower TWS-Soil mixture i.e. 5%. The TSW-Soil
mixtures under F1 + F2 yielded maximum fresh and dry weight while those in
C column yielded the least.
146
Figure 3.10.1. The vegetative growth variation in marigold (Tagetes patula) in response to Caldwell field soil mixed with different percentages of TSW (% w:w) and inoculated with different fungi; (upper) the representative pots of each of the treatments with best growth of marigold; (lower) all of the experimental units with replicates of all the treatments.
3.10.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot were variable with respect to fungal inoculations as well as increasing
ratio of TSW in soil, as given in Table 3.10.4.
3.10.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in shoot increased with inoculation
of fungi as compared to C. The maximum (225 mgkg-1) shoot Ca observed to
be in 5 % with F1 + F2 while being the minimum (07 mgkg-1) in 0% with C.
Within different mixtures of TSW in soil, the maximum shoot
concentrations were observed with F1 + F2 while being the minimum in those
147
where no fungi was applied. It was observed that shoot Ca increased with the
increasing TSW ratio in soil mixtures for 5 % and then decreased for 10 %.
Table 3.10.4. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of 45-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Ca
0 07deC
0.23 10dCD
0.66 15cD
0.22 25aDE
0.99 4
5 45efA
0.45 70dA
0.45 110cdA
0.84 225aA
2.3 48
10 30cAB
0.45 55bcAB
0.98 80bB
1.1 145aC
1.2 32
LSD0.05 29 23 34 78
K
0 20dD
0.96 35cCD
0.94 45bcC
1.0 65aCD
1.1 17
5 70cBC
0.09 90abAB
1.5 95aA
1.04 110aAB
2.5 16
10 105cA
0.45 120bcA
1.3 125bcA
2.4 155aA
2.03 18
LSD0.05 36 47 52 65
Na
0 BDL BDL BDL BDL -
5 80deA
0.99 170cA
1.3 210bcA
1.4 290aA
2.5 54
10 60deBC
2.5 135cC
0.98 155cCD
1.2 260aAB
2.1 49
LSD0.05 8 16 27 18
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
3.10.3.2.2 Potassium (K) in shoot
Along the row, the shoot K uptake increased with fungal applications
for all the TSW-Soil mixtures. The maximum K level (155 mgkg-1) was
observed in 10 % with F1 + F2 while being the minimum (20 mgkg-1) in soil
with no fungi i.e. C.
Within column, the K shoot uptake increased with the increasing
percentage of TSW in soil mixtures for all the TWS-Soil mixtures. For all TSW-
Soil mixtures, the F2 plants showed more K uptake than those with F1 and
being the least where no fungus was applied. The maximum values observed
in plants cultivated in 10% TWS-Soil mixture.
3.10.3.2.3 Sodium (Na) in shoot
The Na concentration in shoot observed to be increased along the row
and it was because of fungal applications. The pots with F1 + F2 showed the
greatest Na shoot uptake, the F2 being greater than F1, while those with no
fungi being the least. The plants in 5 % with F1 + F2 had the highest value
(290 mgkg-1) while those in 10 % with no fungi exhibited the lowest Na shoot
contents (60 mgkg-1).
148
Within column, 0% showed Na values BDL for all fungal treatments.
For the rest of two TWS-Soil mixtures i.e. 5 and 10 %, the concentration of
shoot Na observed to be higher in 5% as compared to 10% as shown in Table
3.10.4.
3.10.3.3 Category-I metals in plant ROOT
The bioavailability of Category-I metals was variable with different fungi
in root also however, it was directly related to the increasing ratio of TSW in
soil mixture, as given in Table 3.10.5.
3.10.3.3.1 Calcium (Ca) in root
The application of fungal inoculum to the soil helps to increase Ca
uptake along the row i.e. various fungal treatments. The plants in F1 + F2 pots
observed to have maximum while those with no fungi having the minimum
root Ca than any of the fungal treatments for all of the TSW-Soil mixtures. The
highest root Ca (160 mgkg-1) was in 5 % with F1 + F2 while being the
minimum (4 mgkg-1) in 0 % with F1. The value was found to be BDL in 0%
with C.
Table 3.10.5. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of
45-days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Ca
0 BDL 4eD
0.23 10cdE
0.34 20aE
0.99 7
5 35cA
1.1 55cA
0.55 90bcA
0.43 160aA
1.5 64
10 20dBC
1.2 35cdB
0.99 60cCD
0.54 110aBC
1.4 32
LSD0.05 8 18 33 48
K
0 10cC
0.2 15cCD
1.5 25bcD
0.65 45aC
1.4 9
5 55bcA
0.34 80aA
1.2 85aA
0.77 90aA
2.5 15
10 50cA
0.99 70bcA
1.1 65bcB
0.35 80aA
1.5 17
LSD0.05 19 26 18 22
Na
0 BDL BDL BDL BDL -
5 35deA
0.44 125bcA
0.76 145bcA
1.1 220aA
2.5 35
10 20efBC
0.56 90dB
0.65 125bcAB
1.5 185aB
2.3 42
LSD0.05 6 17 16 27
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
149
3.10.3.3.2 Potassium (K) in root
The K contents in the 5 % with F1 + F2 exhibited the maximum root
uptake (90 mgkg-1) than any of the soil treatments while being the minimum
(10 mgkg-1) in 0 % with no fungal application i.e. C.
Within column, the metal uptake in root was found to be highest in 5 %
while being least in 0 % for all fungal treatments.
3.10.3.3.3 Sodium (Na) in root
Along the row as observed in case of Ca and K, the application of fungi
helps increase Na uptake in roots. The maximum Na in root (220 mgkg-1) was
observed in 5 % with F1 + F2 while being least in 10 % amounting 20 mgkg-1
with C. Those with F1 and F2 applications also performed better than C i.e.
treatment with no fungal inoculation.
Within column, in 0% the values for Na were found to be BDL in all
fungal treatments. The increasing ratio of TSW in soil displayed increased
root Na uptake for 5 % as compared to 10%.
3.10.3.4 Category-II metals in plant shoot
The Category-II metals i.e. the AAS detected metals in shoot were
variable with respect to fungal inoculations as well as increasing ratio of TSW
in soil, as given in Table 3.10.6. The application of fungi enhanced trace metal
uptake tendency of plant for all the TSW-Soil mixtures. However, the
increasing level of Category-II metals in shoot was in accordance with the
increasing ratio of TSW in soil mixtures for all the fungal treatments.
3.10.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in TSW-Soil mixtures with combined
fungal treatments. Maximum amount of metal (785 mgkg-1) was observed in
5% TSW-Soil mixture in combined inoculation of fungi i.e. F1 + F2, while
being minimum in 0% amounting 12 mgkg-1 with C.
150
Within columns comparison, there is increasing trend of metal
accumulation with TSW-Soil mixtures for 5 % then decrease in values were
observed for 10%.
Table 3.10.6. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 45-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Cd
0 12cdD
0.12 18cDe
0.91 20cDE
0.83 45aE
0.97 12.2
5 110efA
0.23 230dA
1.4 260cdA
1.4 785aA
2.7 170
10 90deA
0.56 185dAB
1.1 165dBC
1.7 625aC
2.2 124
LSD0.05 38 76 28 235
Cr
0 BDL 3cEF
0.94 5cE
0.78 15aEF
0.11 4.4
5 340eA
0.56 625bcA
3.4 655bcA
2.4 930aA
2.6 132
10 280efBC
0.88 515cdC
2.3 545cdB
2.5 765aCD
1.4 127
LSD0.05 25 210 225 275
Cu
0 10cDE
0.78 12cE
0.93 20bcDE
0.99 45aE
1.02 9.7
5 120cdA
1.3 290bA
1.3 315bA
1.5 450aAB
1.5 78
10 145deA
1.4 310cA
1.03 345cA
2.1 520aA
1.3 98
LSD0.05 55 95 105 170
Fe
0 3dE
0.55 5bcDE
0.39 9bE
0.94 14aE
0.92 7.6
5 30cdCD
0.87 90abAB
0.92 110aA
1.04 115aD
1.6 22
10 65deA
0.99 125cA
1.02 145cA
1.5 255aA
1.4 51
LSD0.05 32 45 65 122
Mg
0 10eDE
0.76 20cdD
0.98 25cdDE
0.92 45aE
0.99 9
5 90cA
0.85 130abA
1.32 145aA
1.3 160aA
2.1 67
10 70bcAB
0.83 110bA
1.2 155abA
1.4 190aA
1.4 22
LSD0.05 30 38 55 63
Ni
0 BDL BDL BDL BDL -
5 10abC
0.45 12aBC
1.3 13aB
0.93 15aB
1.2 6
10 17abA
0.64 19aA
1.1 18aA
0.82 22aA
1.3 9.8
LSD0.05 4.7 3.1 5.5 3.8
Zn
0 25dB
0.87 55bcCD
0.98 80aBC
0.91 95aA
1.1 23
5 35cA
2.2 90abA
2.54 105aA
2.2 120aA
2.1 34
10 20bcBC
2.6 55aCD
2.45 60aC
3.1 70aBC
3.2 17
LSD0.05 8 33 40 80
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
3.10.3.4.2 Cr in shoot
With different fungal treatments along the rows, the maximum Cr
accumulation in shoot was observed in treatments applied with combined
inoculants i.e. F1 + F2, being significantly higher than any of the fungal
treatments. Cr was found to be as low as 3 mgkg-1 in 0% with F1 and
maximum 930 mgkg-1 in 5% with F1 + F2.
151
Within different mixtures of TWS-soil there was maximum accumulation
of metal was observed in 5 % for all fungal treatments. the value for Cr was
found to be BDL in 0% with C.
3.10.3.4.3 Cu in shoot
Along the row, the plants harvested from F1 + F2 exhibited maximum
Cu accumulation as compared to any of the treatments with single or no
fungal application. Such a trend was observed in all of the TSW-Soil mixtures.
The plants from treatments with no fungal inoculations i.e. C showed the
significantly least Cu accumulation as compared to any of the fungal
treatments as shown in Table 3.10.6.
Within different TWS-Soil mixtures along the column, 0% showed
significantly less Cu uptake in shoots than those form 5 and 10 %. The
minimum accumulation 10 mgkg-1 was seen in 0% with C and maximum
accumulation 520 mgkg-1 in 10 % with F1 + F2.
3.10.3.4.4 Fe in shoot
The Fe uptake in plant shoots observed to be least in Soil (0%) with C,
but F1 + F2 inoculation displayed the maximum Fe uptake in shoot as
compare to plants from the treatments with single or no fungal inoculations.
The values of plant shoot Fe was minimum 3 mgkg-1 in 0 % with C and
maximum 255 mgkg-1 in 10 % with F1 + F2.
Within a column, maximum accumulation was observed in 10 % as
shown in Table 3.10.5.
3.10.3.4.5 Mg in shoot
Along various fungal treatments, the Mg uptake in plant shoot
increased with fungal application in the pots and found to be the maximum
190 mgkg-1 in plants harvested from pots applied with combined fungi i.e. F1
+ F2 in 10 %, while being the minimum as well as significantly least in shoot of
plants cultivated in soil with no fungus i.e. 10 mgkg-1 as shown in Table
3.10.6.
152
For different TWS-Soil mixtures, the value of plant shoot Mg was
found to be least in 0%. For 10 %, the shoot uptake found to be highest as
compared to 5 and 0 % TSW-Soil mixture.
3.10.3.4.6 Ni in shoot
The Ni concentration observed to be BDL in 0% for all the fungal
treatments along the row. However, for all the TSW-Soil mixtures the shoot Ni
concentration increased with the application of fungus and observed to be the
maximum in plants inoculated with F1 + F2 and being the least in those from
C i.e. where no fungus was applied.
Within the columns comparison, the value of plant shoot Ni
concentration increased with the increasing percentage of TSW in soil and
found to be the maximum in plants harvested from 10 % being the minimum in
plants from 5 %.
3.10.3.4.7 Zn in shoot
The Zn concentration in shoots increased in plants harvest from pots
with fungal inoculations than those harvested from pots applied with no fungi.
Like other metals, the shoot Zn observed to be the maximum in plants
inoculated with F1 + F2 while those harvested from the pots with no fungi
displayed the minimum values. There was increasing trend of accumulation of
metal as the application of fungi. However F2 showed better results as
compared to F1 in terms of accumulation of metal as shown in Table 3.10.6.
For different TWS-Soil mixtures, the value of Zn concentration in shoot
was found to be maximum 120 mgkg-1 in 5 % with F1 + F2 being minimum 20
mgkg-1 in 10% with C as shown in Table 3.10.6.
3.10.3.5 Category-II metals in plant root
The Category-II metals i.e. the AAS detected metals in root were
observed to differ with varying levels of TSW in the soil as well in response to
fungal inoculations, as given in Table 3.10.7.
153
3.10.3.5.1 Cd in root
For different fungal treatments within the rows, the Cd level in plant
roots harvest from Soil observed to be minimum 10 mgkg-1 in 0% with C and
found to be maximum 670 mgkg-1 in 5 % with F1 + F2 as shown in Table
3.10.7. The plant root Cd concentration increased with fungal inoculations and
found to be maximum as well as significantly higher than those applied with
single or no fungal treatments.
Inside the columns, for different TWS-Soil mixtures the root Cd
concentration was found to be highest in 10% with C and F1 as compared
with 5 and 0 %. However the 5 % showed greater values with F2 and F1 + F2
as compared to 10 and 0%.
3.10.3.5.2 Cr in root
For different fungal treatments i.e. along the row, the plants from F1 +
F1 fungal inoculations showed the maximum 875 mgkg-1 root Cd
concentration in 5 %, while with C treatment plants having the minimum
uptake 190 mgkg-1 in 10%. The F2 inoculation enhanced the root Cd
concentration better than F1. However in 0% for all the fungal treatments the
values found to be BDL as shown in Table 3.10.7.
Within the columns, the root Cr concentration decreased with
increasing percentage of TSW in soil having maximum accumulation of metal
in 5 % for all fungal treatments like C, F1, F2 except F1 + F2 than 10%.
3.10.3.5.3 Cu in root
Along the row for various fungal inoculations the root Cu uptake was
enhanced as compared to C. The plants from F1 + F2 observed to have the
maximum (435 mgkg-1) uptake in 10 % than those cultivated in pots with
single or no fungal inoculations and found to be the minimum 5 mgkg-1 in 0%
with C.
For different columns, the plants from 5 and 10 % TSW-Soil exhibited better
root Cu accumulation than soil (0 %).
154
Table 3.10.7. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 45-days
old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0 10dCD
0.44 15bcC
0.56 18bcDE
0.98 35aE
0.67 6.6
5 65deA
0.91 155dA
1.34 190dA
1.34 670aA
1.6 155
10 70deA
0.98 160cA
1.45 110cdBC
1.67 520aC
1.8 128
LSD0.05 22.5 54 67 232
Cr
0 BDL BDL BDL BDL -
5 255deA
1.2 425cA
1.7 550cA
1.34 875aA
1.9 162
10 190eBC
1.1 410cA
1.1 505abAB
2.1 630aC
2.3 115
LSD0.05 23 9 18 88
Cu
0 5aD
0.87 8cD
0.89 15bcDE
1.1 30aD
1.02 6.9
5 90deA
0.81 180dA
0.99 245cA
1.6 315aBC
2.05 59
10 55efBC
0.67 215dA
1.01 265cdA
1.2 435aA
2.1 98
LSD0.05 32 209 85 138
Fe
0 BDL BDL BDL BDL -
5 20cBC
0.98 65abA
1.07 70aC
1.15 80aBC
1.04 19
10 45cdA
1.1 70bA
0.99 95aA
1.12 110aA
1.02 21
LSD0.05 9.4 2.4 9.1 11
Mg
0 5cdDE
0.91 9cE
0.99 13cDE
0.43 30aCD
0.82 7
5 85bA
0.98 95abA
1.2 110aA
0.92 125aA
0.29 13
10 40cC
0.91 65bcBC
0.99 70bcBC
0.94 125aA
0.39 22
LSD0.05 28 32 35 43
Ni
0 BDL. BDL BDL BDL -
5 8aA
0.78 4bCD
0.98 5aC
0.9 7aBC
0.8 2
10 10cdA
1.1 15bcA
0.56 14bcA
1.2 18aA
1.4 3.4
LSD0.05 1.1 2.5 3.4 4.6
Zn
0 20dA
0.99 35cAB
0.98 55bcA
1.3 75aA
2.1 17
5 15cdBC
1.23 40cA
2.4 55bA
3.4 70aA
1.5 15
10 10cdCD
2.4 25bC
2.7 30aBC
1.5 35aC
1.1 6.8
LSD0.05 3.2 5.7 7.6 9.2
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
3.10.3.5.4 Fe in root
Moving alongside the row while analyzing effect of different fungal
treatments, the root Fe observed to be BDL in soil (0 %) with all fungal
treatments. The plants from C had the lowest root Fe as compared to plants
from any of the single or combined application of fungi. The root Fe
accumulation observed to be the maximum (110 mgkg-1) in 10 % with F1 + F2
and significantly higher than those harvested from any of the treatments with
single or no fungal inoculations.
Inside columns, the maximum Fe uptake by roots was observed in 10
% while the least value of metal uptake was observed in 5 % as shown in
Table 3.10.7.
155
3.10.3.5.5 Mg in root
While analyzing the fungal application effect on plants along the rows,
it was observed that the Mg root concentration increased in plant roots with
fungal application as compared to C i.e. with no fungal inoculation. The F1 +
F2 plants displayed the maximum root Mg concentration than those from C as
well as F1 and F2. However the F2 inoculations gave better results than F1.
For the columns, the root Mg accumulation increased with increasing
percentage of TSW in soil for all fungal treatments being maximum in 5 and
10% with F1 + F2 where the metal accumulation was observed to be 125
mgkg-1 while minimum concentration 5 mgkg-1 in 0 % with C treatment was
noted as shown in Table 3.10.6.
3.10.3.5.6 Ni in root
The root Ni concentration was found to be BDL for all fungal treatments
in 0% i.e. soil. However there was increased metal accumulation along the
row with the application of fungal inoculations and found to be the maximum
in plants applied with combined application of both of the fungi while being the
minimum in C with no fungus added.
Within columns, the plants from 10 % TSW-Soil mixture had the
maximum root Ni level than 5 % for all of the fungal treatments. There was
maximum (18 mgkg-1) accumulation of metal was noted in 10% with F1 + F2
while minimum uptake (4 mgkg-1) was observed in 5% with F1.
3.10.3.5.7 Zn in root
The root Zn accumulation observed to increase in pots applied with
fungal inoculations than C and found to be the maximum in treatments applied
with both of the fungi and being the minimum with no fungal applications.
Again as with most of the above discussed metals F2 performed better than
F1 as far as metal accumulation efficiency of the plant is concerned. The
maximum accumulation of metal was observed in 0 % with F1 + F2 i.e. 75
mgkg-1 while minimum value for the metal accumulation was noted in 10 %
with C i.e. 10 mgkg-1.
156
3.10.4 Fungal analyses
The results of estimation of the post-harvest fungal analyses (× 105
c.f.u. g-1 soil) of 45-days old Tagetes patula cultivated on TSW-Soil mixtures
are shown in Table 3.10.8. Alongside the row, the c.f.u. increased with fungal
application than C and observed to be the maximum in treatments with
combined application of both of the fungi. The order of c.f.u. abundance was
F1 + F2 > F2 > F1 > C.
Table 3.10.8. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 45-days old Tagetes
patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).
TSW-Soil (% w:w) mixture and its type
Treatment LSD0.05
C F1 F2 F1+F2
0 0.8cdC
0.22 1.7cD
0.17 2.8bcC
0.23 5.9aCD
0.29 1.5
5 2.7deA
0.26 3.8cdA
0.18 4.9cdA
0.21 9.1aA
0.13 1.9
10 2.4cdA
0.16 3.2cdA
0.33 4.2cA
0.07 7.5aB
0.51 2.6
LSD0.05 0.9 0.8 0.7 1.3
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
Within a column, the c.f.u. abundance was observed in 5% with 9.1 ×
105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2. Statistical
analysis indicated that there is significant difference of 5% with other TWS-
Soil mixtures.
3.10.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
3.10.5.1 Category-I metals translocation index (%)
The plant translocation index values were also recorded for category-I
metals those detected by flame photometer i.e. Ca, K and Na shown in Table
3.10.9.
In case of Ca, maximum value was observed in 10% with F1 i.e. 157.1
% while the minimum value was recorded (122.2 %) in 5 % with F2.
For K, the maximum translocation index value was calculated in 10%
with C i.e. 210 % being the minimum 111.7 % in 5 % with F2.
157
In case of Na, the maximum value 300 % was observed in 10 % TWS-
Soil mixture with C treatment and least value was recorded in 10 % for F2
(124 %).
3.10.5.2 Category-II metals translocation index (%)
The plant translocation index found to be greater in treatments applied
with fungal inoculum than that of C, being the maximum where F1 and F2
applied together for all metals as given in Table 3.10.10.
Table 3.10.10. The Category-II metals translocation index (%) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd 5 169.2 148.3 136.8 117.1
10 128.5 115.6 150 120.1
Cr 5 133.3 147.0 119.0 106.2
10 147.3 125.6 107.9 121.4
Cu 5 133.3 161.1 128.5 142.8
10 263.6 144.1 130.1 119.5
Fe 5 150 138.4 157.1 143.7
10 144.4 178.5 152.6 231.8
Mg 5 105.8 136.8 131.8 128
10 175 169.2 221.4 152
Ni 5 125 300 260 214.2
10 170 126.6 128.5 122.2
Zn 5 233.3 225.0 190.9 171.4
10 200.0 220.0 200.0 200.0
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
For Cd, the maximum Translocation index (169.2 %) was noted in 5 %
with C, while minimum value was recorded 115.6 % in 10% with F1.
In case of Cr plants showed maximum metal translocation efficiency
(147.3 %) in 10% with C and minimum 106.2 % in 5 % with F1 + F2.
Table 3.10.9. The Category-I metals translocation index (%) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca 5 128.5 127.2 122.2 140.6
10 150 157.1 133.3 131.8
K 5 127.2 112.5 111.7 122.2
10 210 171.4 192.3 193.7
Na 5 228.5 136 144.8 131.8
10 300 150 124 140.5
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
158
For Cu, the maximum translocation index values was recorded to be
263.6 % for 10% with C, while minimum value was recorded in 10 % with F1 +
F2 treatment i.e. 119.5 %.
For Fe, the maximum translocation index values was recorded to be
231.8 % for 10 % with F1 + F2, while minimum value was recorded in 5 %
with F1 treatment i.e. 138.4 %.
In case of Mg the plants showed least value in 5 % with C (105.8 %),
while the maximum value for this metal was recorded in 10% with F2 i.e.
221.4 %.
For Ni, there was 300% translocation index value recorded in 5% with
F1 to be the highest and least value was observed in 10% with F1 + F2 i.e.
122.2 %.
As far as the Zn is concerned there was maximum translocation index
recorded in 5 % with C treatment i.e. 233.3 % while minimum value was
recorded in 5 % with F1 + F2 i.e. 171.4 % as shown in Table 3.10.9.
3.10.5.3 Tolerance index (TI)
In shoots TI values were found to be highest in 5 % with C
(0.966) while minimum value was recorded to be 0.75 in 10% with F1 as
shown in Table 3.10.11.
Table 3.10.11. The tolerance index (TI) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
TI Shoot 5 0.966 0.908 0.870 0.865
10 0.801 0.75 0.76 0.76
TI Root 5 0.84 0.74 0.75 0.66
10 0.75 0.58 0.60 0.57
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
In case of TI in roots 0.84 was recorded as highest for plants grown in
5 % with C, while 0.57 was recorded as minimum value in 10% with F1 + F2.
3.10.5.4 Category-I metals specific extraction yield (SEY %)
The SEY % for Category-I metals i.e. Ca, K and Na showed in Table
3.10.12. In case of Ca, there was maximum value 21.3 % was recorded for
plants grown in 5 % with F1 + F2, and minimum 2.0 % in 10 % with C.
159
Table 3.10.12. The Category-I metals specific extraction yield (SEY %) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca 5 4.4 6.9 11.1 21.3
10 2.0 3.6 5.7 10.4
K 5 43.1 58.6 62.0 68.9
10 11.6 14.2 14.2 17.6
Na 5 13.0 33.5 40.3 57.9
10 5.1 14.5 18.0 28.7
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
In case of K, plants cultivated in 5 % TWS-soil showed highest value of
SEY% (68.9 %) with F1 + F2 and minimum in 10 % with C i.e. 11.6 %.
As in case of Ca and K, the highest value for Na was recorded in 5 %
with F1 + F2 (57.9 %) while minimum values was found to be 5.1 % in case of
10% with no fungal inoculum i.e. C.
3.10.5.5 Category-II metals specific extraction yield (SEY %)
The SEY (%) was calculated in Category-II metals that were detected
by AAS as shown in Table 3.10.13. Overall a similar kind of trend was seen
for all metals in various fungal treatments along the row, that the SEY %
values increased with the application of fungal inoculum and highest value
was observed for F1 + F2 treatment.
Table 3.10.13. The Category-II metals specific extraction yield (SEY %) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd 5 7.5 16.5 19.3 62.7
10 3.7 8.1 6.5 27.0
Cr 5 7.8 13.8 15.9 23.8
10 4.74 9.3 10.5 14.0
Cu 5 17.3 38.8 46.2 63.2
10 10.3 27.2 31.6 49.4
Fe 5 17.8 55.3 64.2 69.6
10 20.0 35.4 43.6 66.3
Mg 5 33.0 42.45 44.3 46.2
10 11.6 18.5 23.8 33.3
Ni 5 22.5 20 22.5 27.5
10 21.6 27.2 25.6 32.0
Zn 5 17.8 46.4 57.1 67.8
10 6.9 18.6 20.9 24.4
C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference
160
In case of Cd the maximum value for SEY % was recorded in 5 % with
F1 + F2 treatment i.e. 62.7 %, while minimum (3.7 %) was observed in 10%
with C.
Similarly the SEY % value for Cr was found to be highest (23.8 %) in 5
% with F1 + F2 and minimum (4.74 %) was recorded in 10% with no fungal
inoculum i.e. C. However F2 showed greater values as compared to F1.
For Cu again the maximum value was observed for 5 % (63.2 %) with
combined fungal inoculum F1 + F2 and least value (10.3 %) was recorded in
10% with C.
In case of Fe the highest (69.6 %) and lowest (17.8 %) values were
recorded in 5% with F1 + F2 and 5 % with C respectively.
As far as the highest and lowest values are concerned there was a
same trend seen in case of Mg, where plants in 5 % showed highest SEY%
value (46.2 %) with F1 + F2 and being minimum (11.6 %) in 10% with C
having no fungal inoculum.
There was maximum value for Ni was calculated (32.0 %) in 10% with
combined fungal inoculum, while minimum value (20 %) was recorded in 5 %
for F1.
In case of Zn the maximum value was noted (67.8 %) for 5 % with F1 +
F2 and minimum value (6.9 %) in 10 % with C.
161
3.11 Field experiments
3.11A: Experiment with Helianthus annuus
3.11A.1 Pre-sowing analysis
The physico-chemical properties, concentration of Category-I & II
metals are given Table 3.1, 3.2 and 3.3 and the details are described in
Chapter 3.1.
3.11A.2 Biochemical analyses of Helianthus annuus
The chlorophyll contents, soluble protein, CAT and SOD were
observed in H. annuus while being cultivated in field soil amended with
different levels of TSW. The specific details of each of the biochemical
parameters are as under:
3.11A.2.1 Chlorophyll content
Along the row, the plant chlorophyll contents based on SPAD value
observed in 50-days old sunflower increased in soil treatments applied with
fungus as compared to the C (with no fungus). The plants from F2 showed
enhanced chlorophyll contents than F1, being the maximum in F1 + F2
(applied with combined fungal inoculation), as given in Table 3.11A.1. Such a
trend was observed in all of the TSW-Soil mixtures.
Within column, the general trend was decrease in plant chlorophyll
contents with the increase of TSW percentage in soil for all the fungal
treatments. In all fungal treatments, the plants from all the TSW-Soil 0 (%
w:w) had more chlorophyll contents than every corresponding 0* (% w:w). The
plants from 5 % (% w:w) of all the fungal treatments showed the maximum
SPAD value within a column while those from 20 (% w:w) had the least
chlorophyll contents.
3.11A.2.2 Soluble protein contents
Likewise chlorophyll contents, the soluble protein contents along the
row increased with the application of fungal inoculations for all the fungal
treatments, as given in Table 3.11A.1. The plants from C (with no fungal
162
treatment) had the least protein contents than any of the treatment with single
or combine fungal inoculations, being the maximum in F1 + F2.
Table 3.11A.1. The biochemical parameters observed in 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery solid waste (TSW-Soil
mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Chlorophyll contents (SPAD value)
0* 23.5aBC
0.98 23.7aC
1.2 24.1aBC
2.5 24.8aC
2.1 1.1
0 23.6abBC
1.5 22.7abC
2.1 23.3abBC
0.92 25.2aC
3.3 2.1
5 29.7abA
1.4 31.6abA
0.98 32.1aA
2.6 34.5aA
3.9 2.9
10 24.3aBC
2.7 20.4bcCD
3.1 24.5aBC
2.9 23.9aC
1.7 1.3
20 18.8abD
1.8 19.4aD
3.2 19.7aD
3.5 20.2aCD
1.98 0.9
LSD0.05 2.2 3.2 3.9 3.6
Protein content
(mgg-1
)
0* 2.4deCD
0.98 13.6bcC
2.1 14.2bcCD
2.2 25.1aD
3.2 7.4
0 3.2deB
2.9 14.1cC
0.99 13.7cCD
1.9 34.9aBC
0.98 9.1
5 6.7dA
2.7 27.9bA
1.8 24.7bcA
1,5 38.9aA
1.99 10.1
10 4.1cB
3.2 9.7abD
1.7 8.5abE
0.34 12.6aE
2.5 4.4
20 2.1bcCD
3.6 7.6aDE
2.5 6.1aEF
1.2 8.1aE
3.1 3.3
LSD0.05 1.9 6.2 4.3 7.6
SOD
(umg-1
of protein)
0* 4.3aDE
3.6 3.7aD
2.8 4.6aE
2.7 2.8cDE
2.1 1.6
0 1.9aE
2.9 1.8aD
2.1 1.6aE
0.98 1.2bDE
1.5 0.8
5 25.6dAB
0.98 34.7cA
3.2 44. 3abA
2.5 48.6aA
1.9 7,9
10 29.6bcA
2.7 31.1bcA
3.3 33.2bcBC
3.0 45.7aA
3.7 6.3
20 12.3cC
2.2 15.9bBC
2.1 14.9bD
2.6 18.4aC
0.97 3.4
LSD0.05 7.6 8.6 10.6 11.5
CAT (Uml
-1)
0* 1.2bE
0.95 1.5abDE
1.6 1.9aE
0.87 1.1bDE
4.2 0.6
0 1.1aE
2.6. 0.9abDE
0.34 1.0aE
2.1 1.2aDE
3.8 0.2
5 22.1cA
2.4 28.2abA
1.4 32.2aA
0.98 29.7aA
3.2 4.7
10 18.1bcB
2.7 21.3aAB
2.1 22.1aBC
1.7 19.3bcBC
4.1 2.2
20 11.1abCD
3.1 10.3bC
2.7 12.4abD
1.5 14.5aC
0.95 1.2
LSD0.05 7.4 7.5 9.1 7.5
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
The soluble protein contents increased down the column up to 5 (%
w:w); however, it decreased in 10 and 20 %. Such a variation was observed
for all the fungal treatments’ down the column analyses. The plants from 5 %
had the maximum protein contents in all the columns.
3.11A.2.3 Superoxide dismutase (SOD) contents
Parallel to the trends found for soluble protein contents, the SOD
contents in plants increased with fungal inoculations within a row for all the
treatments; however, it decreased in case of combined fungal application in
both 0* and 0 (% TSW-Soil) mixtures, as given in Table 3.11A.1. For 5, 10
and 20 % (TSW-Soil) mixture, the SOD values increased with the application
163
of single or combined fungal inoculation, being the maximum in plants applied
with F1 + F2.
Within a column, the plants from 0* (control with no geothermal
membrane lining) had shown enhanced SOD contents than 0 (with lining) for
all the fungal treatments. However, it decreased with increasing level of TSW
percentage in soil, being the maximum in 5 % and the minimum in 20 %
except 5 % C.
3.11A.2.4 Catalase (CAT) contents
The CAT contents of the plant exhibited variable pattern along the row.
Other than the 0 (% TSW-Soil) where plants with F1 had lesser values than
the C, the CAT values increased in plants applied with individual fungal
inoculations for F1 and F2 but decreased in treatments inoculated with F1 +
F2. For 5, 10 and 20 % (TSW-Soil), the plants inoculated with F2 had the
maximum CAT contents while those applied with no fungus had the least
values.
Down the column, the plants from 0* showed enhanced CAT contents
than corresponding 0 (% TSW: Soil) for all the fungal treatments except F1 +
F2. However it decreased down the column with increasing percentage of
TSW in soil for all of the fungal treatments, being the maximum in 5 %
(TSW:Soil) and the minimum in 0 for all the treatments.
3.11A.3 Post-harvest analysis
3.11A.3.1 Growth performance of Tagetes patula
The growth of 78-days old sunflower varied in response to different
TSW percentages as well as fungal inoculations, as given in Table 3.11A.2.
The details are as under:
3.11A.3.1.1 Shoot, root and seedling length (cm)
Along the row, the sunflower shoot, root and seedling length increased
in treatments applied with fungus than those applied with no fungus i.e. C. It
164
was the maximum in F1 + F2 being the minimum in C for all the TSW-Soil
mixtures. The plants with F2 inoculation performed better than those from F1.
Table 3.11A.2. Various morphological parameters observed in 78-days old Helianthus annuus cultivated
on field soil mixed with different percentages of tannery solid waste (TSW-Soil mixtures). The mean
values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Shoot Length (cm)
0* 54.86cC
2.3 57.91cCD
3.1 64.0bcC
2.9 85.34aBC
2.4 7.9
0 57.9dC
1.6 51.82dCD
2.8 67.05cdC
2.5 88.39aBC
1.5 8.7
5 97.53bcA
1.8 112.77abA
2.5 118.87abA
2.7 146.30aA
2.3 13.1
10 81.44cAB
2.2 100.58bcA
3.2 106.68bcA
4.1 140.20aA
2.7 16.8
20 45.72cdCD
2.7 51.81bcCD
3.1 57.91bCD
3.3 67.05aD
3.4 5.6
LSD0.05 12.2 17.5 21 19
Root Length (cm)
0* 40.34abAB
2.5 43.92aA
4.3 42.21aA
1.7 45.24aA
3.2 3.2
0 47.45aA
3.1 40.92bAB
4.6 35.78bcAB
2.4 46.12aA
3.1 5.4
5 50.12aA
2.8 50.78aA
3.8 47.20abA
3.2 52.67aA
4.1 6.7
10 49.29aA
2.4 45.98abA
2.2 39.22cAB
2.6 53.23aA
3.3 6.5
20 25.98cdD
3.5 35.37bC
1.7 32.38aB
2.1 37.27aBC
2.6 8.5
LSD0.05 6.9 7.8 9.2 7.3
Seedling Length (cm)
0* 95.5cdCD
3.3 101.86cC
4.1 106.4cCD
2.8 130.8aC
2.5 6.8
0 105.5cdC
2.4 92.8dCD
3.8 102.9cdCD
4.1 134.7aC
2.2 9.2
5 147.8dA
1.7 163.7bcA
4.5 166.12bcA
3.3 199.1aA
2.7 11.3
10 130.9dBC
1.9 146.7cdAB
3.9 146.1cdB
3.6 193.5aA
3.4 12.2
20 71.9bcDE
2.2 87.21abCD
4.1 90.31abD
3.8 104.5aDE
3.1 10.9
LSD0.05 5.5 8.3 5.8 3.4
No. of roots/plant
0* 44bcC
3.4 54aB
3.1 58aAB
2.3 61aBC
1.2 5.5
0 42bcC
3.1 48bcBC
1.6 57aAB
3.1 62aBC
2.2 6.2
5 65abA
2.1 68abA
3.7 71aA
2.6 78aA
1.5 4.5
10 72aA
2.3 64abA
1.9 68aA
3.7 71aA
2.7 3.2
20 40aD
2.7 38aCD
2.9 36abC
2.8 41aD
1.8 1.9
LSD0.05 3.5 6.5 8 8.6
No. of leaves/plant
0* 8abAB
1.4 9abBC
1.6 8abC
1.1 11aC
2.1 3.5
0 9aAB
2.1 8aBC
1.5 9aC
2.2 10aC
2.8 4.9
5 14bA
2.2 18abA
2.5 21aA
2.9 24aA
3.1 7.7
10 12aA
3.2 11abB
1.2 10abC
3..7 16aBC
1.7 6.5
20 6abC
1.1 7aBC
1.4 8aC
1.6 9aC
1.2 2.1
LSD0.05 2.7 3.2 2.9 3.8
Fresh wt./plant (g)
0* 305.1aE
2.2 295.6abC
3.1 303.6aCD
1.6 312.3aC
2.4 4.3
0 290.5bcCD
3.3 310.4abC
2.7 314.5abCD
4.6 333.5aC
3.8 5.4
5 470.7bcA
2.4 495.1abA
1.9 505.09aA
3.7 525.8aA
4.5 13.6
10 255.3bD
1.7 263.6abCD
1.8 271.1abD
3.3 291.1aC
3.9 11.6
20 122.9bcF
1.9 131.8aE
2.5 135.2aEF
2.9 149.7aDE
4.1 16.5
LSD0.05 12.6 13.5 17.8 21.3
Dry wt. (g)
0* 65.5aA
2.3 72.1aA
2.6 74.2aA
2.8 76.7aA
3.1 8.9
0 61.9abAB
1.3 68.9aAB
4.1 71.2aA
3.7 73.6aAB
2.6 14.3
5 78.9aA
4.3 81.5aA
3.3 82.7aA
3.1 85.6aA
1.1 16.5
10 57.2abAB
2.2 62.2aAB
2.1 61.9aBC
2.5 67.3aAB
3.3 18.7
20 48.2aC
3.2 52.2aCD
1.1 53.7aC
2.2 54.9aCD
4.2 12.2
LSD0.05 7.6 8.7 7.5 9.2
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
165
Down the column, the plants from 0* had less efficient vegetative
growth in terms of shoot, root and seedling length than the 0 (TSW-Soil)
except those applied with F1. For 5, 10 and 20 % (TSW-Soil), the plant height
decreased down the column with increasing percentage of TSW, being the
maximum in 5 % and the minimum in 20 %. Such a variability trend was
observed for all the fungal treatments as shown in Figure 3.11A.1.
Figure 3.11A.1. Phytoextraction field trials with sunflower (Helianthus annuus) cultivated on soil
amended with different levels of tannery solid waste (TSW:Soil w:w); 0 % the only treatment without geothermal membrane allowing leaching (A), 0 % with geothermal membrane to avoid leaching (B), 5 % (C), 10 % (D) and 20 % (E). The white lines across the strip plots (25 × 3 ft) indicate soil barriers (1.25 ft) subdividing each strip plot into four subplots (5 × 3 ft each) for fungal inoculations viz. C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger + T. pseudokoningii, applied in a randomized complete block design.
3.11A.3.1.2 No. of leaves and roots
Parallel to the vegetative growth components of plant height, the
number of leaves and roots observed to increase along the row with fungal
inoculation than C (with no fungus applied), being the maximum in F1 + F2
and the minimum in C. The plants with F2 showed more number of leaves
and roots than plants applied with F1.
Within a column, the root and leaf number were lesser for plants
harvested from 0* than 0 (% TSW-Soil) for all the fungal treatments except F1
and F1 + F2 treatments. For 5, 10 and 20 % TSW-Soil mixtures, the trend of
root and leaf number increase was from 5 % towards 20 % being the
A
B
C
D
E
N
166
maximum in 5 % and the minimum in 20 %. Such a variation pattern was
observed for all the fungal treatments.
3.11A.3.1.3 Fresh and dry weight (g)
Similar to the plant vegetative growth parameters, the fresh and dry
plant biomass found the increase along the row in plants applied with fungi
than those applied with no fungi. The maximum sunflower biomass production
was observed in F1 + F2 treatments and the minimum in C. The plants
inoculated with F2 showed enhanced biomass production than F1. Such a
variability pattern was observed for all the TSW-Soil mixtures.
Down the column, the biomass production decreased with increasing
TSW percentage in soil, being the maximum in 5 % and the minimum in 20 %.
The plants from 0* had yielded less biomass than those from the 0 (% TSW-
Soil) for all the fungal treatments.
3.11A.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot observed to vary in response to the variability of TSW percentage in soil
as well as fungal inoculations, as given in Table 3.11A.3.
3.11A.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in sunflower shoot increased with
inoculation of fungi as compared to C, being the maximum in plants from F1 +
Figure 3.11A.2. The sunflower stem girth variation in response to soil amended with different levels of tannery solid waste (% w:w) inoculated with fungi; (clockwise from upper left) 10 % with F2, 20 % with F1 + F2; 5 % with F1 + F2, 10 % with F1 + F2, 20 % with C, 5 % with F1 + F2.
167
F2 treatments while being the minimum in C. The F2 plants showed enhanced
Ca shoot uptake than F1. Such a variability trend was observed for all the
TSW-Soil mixtures.
Down the column, the shoot Ca observed to increase with increasing
percentage of TSW in soil and found to be the maximum in plants harvested
from 10 % except F2. The minimum shoot Ca concentration within a column
observed to be in plants harvested from those mixed with no TSW. The 0*
plants showed lesser shoot Ca uptake than corresponding 0 (% TSW-Soil) for
all the fungal treatments except F2.
Table 3.11A.3. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery
solid waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Ca
0* 25bE
3.9 30aDE
1.3 28aE
2.6 32aEF
3.2 2.5
0 29aE
1.7 31aDE
3.9 27bE
2.3 35aEF
3.3 4.7
5 355cB
3.2 370cAB
1.7 410bcA
1.7 550aBC
2.1 119
10 405dA
3.3 445cdA
3.1 390dA
2.1 625aA
3.9 155
20 290cdC
2.1 310cdB
3.2 295cdC
2.7 440aD
1.7 95
LSD0.05 83 95 78 128
K
0* 20dE
3.8 45bE
3.2 35bcE
2.4 65aE
3.9 18
0 22cE
1.4 33bE
3.3 23cE
3.9 55aE
1.7 15
5 210dA
2.1 330aA
2.6 290bcA
3.7 355aA
3.2 94
10 190bcA
2.7 205bcBC
3.9 210bcB
4.1 280aB
3.3 79
20 125abCD
1.9 145aC
1.7 130aCD
1.7 155aCD
2.1 48
LSD0.05 48 54 47 58
Na
0* 25bcE
3.2 35bE
3.2 40aE
3.1 55aDE
3.9 18
0 20bE
3.3 30abE
3.9 35aE
3.2 45aDE
1.7 13
5 210dA
2.1 325bcA
1.7 300bcA
3.3 445aA
1.4 117
10 110bcC
2.7 145aCD
2.5 165aC
2.1 170aC
2.1 48
20 70cD
1.8 95bcD
1.6 110bcCD
2.7 190aC
2.7 52
LSD0.05 43 46 51 56
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
3.11A.3.2.2 Potassium (K) in shoot
Alongside row, the shoot K uptake increased with fungal inoculations
as compared to C (with no fungus applied), being the maximum in F1 + F2
and the minimum in C. The plants harvest from F1 showed enhanced K
uptake than F2. Such a variability pattern was observed for all the TSW-Soil
mixtures.
168
Within column, the K shoot uptake decreased with the increasing
percentage of TSW in soil mixtures for all TSW-Soil mixtures (5, 10 and 20
%). However, the 5 % plants showed significantly higher shoot K uptake than
soil mixed with no TSW. The 0* plants showed enhanced shoot k uptake than
corresponding 0 (% TSW-Soil) except for C.
3.11A.3.2.3 Sodium (Na) in shoot
Alongside row, the shoot Na concentration increased with fungal
inoculation as compared to C (with no fungus applied) and found to be the
maximum in F1 + F2 being the minimum in C. The plants from F2 exhibited
enhanced shoot Na uptake than F1. Such a variability trend was observed for
all of the TSW-Soil mixtures except 5 %, where F1 plants had greater shoot
Na than F2.
Within columns, the shoot Na concentration decreased with increasing
TSW percentage in soil, being the maximum in 5 % and the minimum in soil
treatments mixed with no TSW. The 0* plants showed greater shoot Na
concentration than corresponding 0 (% TSW-Soil).
3.11A.3.3 Category-I metals in plant ROOT
The sunflower root uptake of Category-I metals was variable with
respect to different fungal inoculations as well as TSW percentage in soil. The
details are as under:
3.11A.3.3.1 Calcium (Ca) in root
Like the Ca variability trend observed plant shoot, the root Ca
concentration increased in plants inoculated with fungi than those applied with
no fungi, being the maximum in F1 + F2 treatments and the minimum in C
(with no fungus applied). Such a variability trend was observed for all the
TSW-Soil mixtures. The F2 inoculations incurred enhanced root Ca uptake
than those inoculated with F1.
Down the column, the root Ca concentration observed to be lesser in
0* than corresponding 0 (TSW-Soil) of all the fungal treatments. Among 5, 10
and 20 % TSW-Soil, the plant from 10 % showed the maximum root Ca while
169
those from 20 % exhibited the least root Ca uptake for all of the fungal
treatments.
3.11A.3.3.2 Potassium (K) in root
Alongside row, the K contents in plant roots increased with fungal
inoculation as compared to those applied with no fungi i.e. C. The plants from
F1 + F2 exhibited the maximum while those from the C showed the minimum
K root concentration. The F2 inoculation caused greater root K accumulation
than corresponding F1 for all the TSW-Soil treatments except 5 % where F1
plants had more root K than those from F2.
Down the column, the 0* plants showed lesser root K concentration
than those from the corresponding 0 (% TSW-Soil) and such a trend was
observed for all the fungal treatments. Among 5, 10 and 20 (% TSW-Soil), the
root K concentration decreased down the column with increasing percentage
of TSW in soil, being the maximum in 5 % and the minimum in 20 %. Such a
variability tendency was observed for all the fungal treatments.
Table 3.11A.4. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery
solid waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Ca
0* 5cE
1.1 8bcE
1.2 10bcEF
1.0 15aE
1.8 1.2
0 5deE
1.9 10cdE
2.9 15bEF
1.9 20aE
1.8 3.7
5 110cCD
1.2 155abBC
1.9 160aC
2.1 180aC
2.9 47
10 245abA
2.2 260aA
1.3 265aA
2.4 280aA
1.2 73
20 95bcCD
1.6 110bcCD
1.5 115bcD
2.3 175aC
2.4 48
LSD0.05 48 52 51 46
K
0* 5bcD
0.9 8bE
2.1 10aDE
2.7 12aD
2.2 6.5
0 10bD
2.2 12bE
2.9 14abDE
2.2 18aD
1.6 5.9
5 90bcA
2.3 110bA
2.1 105bA
3.5 125aA
2.9 32
10 80cA
1.2 95bcA
1.9 110bA
2.4 130aA
1.8 37
20 45cdC
2.1 55cCD
1.2. 65bcC
3.2 85aB
2.1 29
LSD0.05 18 22 21 25
Na
0* 7cdD
0.6 6cdDE
3.2 10bcE
1.2 15a 2.2 7.6
0 8cD
1.5 10bcDE
0.9 12bcE
2.9 20aE
2.8 5.9
5 65cdA
2.8 90bA
2.2 110aA
2.5 125aA
1.9 32
10
35cBC
2.6 40cC
1.1 50bcCD
0.7 75aBC
1.5 28
20 20bcC
3.1 25bcCD
1.5 30bD
1.3 45aD
2.1 18
LSD0.05 11 15 16 25
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
170
3.11A.3.3.3 Sodium (Na) in root
Along the row as observed in case of Ca and K, the application of fungi
enhanced the Na uptake in roots. The maximum Na in root observed to be in
plants harvested from F1 + F2 while the minimum in plants cultivated on soil
inoculated with no fungi. The F2 incurred better root Na uptake effects than
F1. The plants from all of the TSW-Soil treatments followed the above
mentioned variability pattern.
Down the column, root Na concentration in 0* observed to be lesser
than the corresponding 0 (% TSW-Soil). Among 5, 10 and 20 % (TSW-Soil),
the root Na concentration decreased with increasing percentage of TSW in
soil and found to be the maximum in 5 % and the minimum in 20 %. The
plants from all of the fungal treatments followed the similar variability pattern.
3.11A.3.4 Category-II metals in plant shoot
The Category-II metals i.e. the AAS detected metals in shoot varied
with respect to fungal inoculations as well as increasing percentage of TSW in
soil, as given in Table 3.11A.5. The details are as under:
3.11A.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in F1 + F2 treatments and being the
minimum in C i.e. where no fungi was applied. The F2 inoculations incurred
enhanced shoot Cd concentration effects than F1. The plants from all of the
TSW-Soil treatment observed to follow the same variability pattern.
Down the column, the shoot Cd concentration observed to be lesser in
0* than those from the 0 (% TSW-Soil) for all of the fungal treatments. Among
5, 10 and 20 % (TSW-Soil), the plant shoot Cd concentration decreased with
increasing percentage of TSW in soil and found to be the maximum in 5 %
and the minimum in 20 % of all the fungal treatments.
3.11A.3.4.2 Cr in shoot
Alongside row similar to the trend observed for shoot Cd concentration,
the shoot Cr concentration increased with fungal inoculation being the
171
maximum in F1 + F2 while being the minimum in C i.e. with no fungal
inoculation.
Table 3.11A.5. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery
solid waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Cd
0* 5cE
0.8 10bcEF
2.1 15abE
2.2 20aEF
3.2 4.3
0 8bcE
2.9 12abEF
0.9 14aE
1.9 16aEF
0.98 4.1
5 110dA
2.7 290abA
1.8 310aA
1.5 345aA
1.99 55
10 90bAB
3.2 110abCD
1.7 125aCD
0.34 140aD
2.5 34
20 40bcCD
3.6 55bDE
2.5 60aDE
1.2 75aE
3.1 28
LSD0.05 29 62 68 71
Cr
0* 10cEF
0.93 15bcF
3.4 20aE
1.9 22a 2.4 5.6
0 8bcEF
2.2 10bcF
3.79 12bcE
3.5 23a 2.8 7.9
5 280eA
2.9 455dA
3.8 390dA
2.3 880aA
3.9 159
10 190eBC
2.1 395cdAB
3.5 410cdA
2.3 990aA
3.5 174
20 110efD
4.1 290dD
2.1 325dB
3.2 855aA
2.8 143
LSD0.05 58 98 78 85
Cu
0* 15cF
3.9 20abF
2.8 22aE
1.3 25aF
2.7 7
0 10cdF
1.9 15bcF
3.9 18bcE
3.2 30aF
3.8 12
5 285dA
2.4 455bcA
2.8 430bcA
2.9 550aA
2.9 47
10 245cdB
2.4 390abAB
1.9 355bAB
3.4 425aB
3.9 51
20 110deDE
3.1 215bcD
1.3 245bCD
2.4 310aCD
2.4 39
LSD0.05 58 88 76 115
Fe
0* 3cCD
0.12 6aDE
0.9 5abD
1.2 7aD
1.4 5.4
0 5bcCD
2.1 6aDE
1.8 6aD
1.5 8aD
0.32 8.2
5 45dA
3.7 70bcA
1.5 65bcA
1.2 80aA
1.9 23
10 40cA
3.5 50bcB
2.7 40cBC
0.3 70aA
2.5 37
20 10cC
3.1 20bCD
3.5 25abC
2.5 35aBC
2.1 24
LSD0.05 8.7 9.7 10.2 13.8
Mg
0* 8bcC
1.2 10abBC
2.4 12aB
0.8 15aBC
1.5 9
0 5cCD
1.1 7bBC
1.9 8bBC
2.1 12aC
0.9 6.8
5 15bAB
0.9 17bAB
2.8 20aA
3.2 25aA
2.9 5.6
10 20bcA
2.3 22bcA
1.4 18bcA
2.1 30aA
1.2 7.2
20 10bC
2.1 12abB
1.1 11bB
1.2 15aBC
1.5 6.4
LSD0.05 3.2 9.6 11.1 6.5
Ni
0* BDL BDL BDL BDL -
0 BDL BDL BDL BDL -
5 5bcA
1.9 7bAB
2.8 8aB
2.5 10aB
2.7 4.3
10 8cA
1.5 10bcA
2.7 13aA
2.4 15aA
2.2 7.6
20 4cAB
1.1 7bAB
1.5 5bcBC
1.2 10aB
1.6 5.6
LSD0.05 0.8 1.1 1.7 3,1
Zn
0* 15dD
0.8 35bcE
2.3 30bcF
2.7 55aG
3.2 12
0 18cD
2.6 22bcE
1.2 28bcF
1.1 45aG
1.9 22
5 210dA
2.1 490bcA
1.8 510bcA
1.5 620aA
1.9 156
10 235cdA
1.2 310cBC
1.7 290cC
1.4 425aCD
1.3 134
20 190bB
2.6 225aC
1.5 210aCD
1.2 230aE
1.3 127
LSD0.05 46 98 115 165
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
172
The plants from F2 showed enhanced shoot Cr uptake than
corresponding F1 for all of the TSW-Soil treatments except 5 % where F1
plants performed better than those from F2. The plants from all of the TSW-
Soil mixtures followed the same shoot Cr variability pattern.
Within columns, the shoot Cr concentration observed to be greater in
plants harvested from 0* than corresponding 0 (% TSW-Soil) for all the fungal
treatments apart from F1 + F2. For 5, 10 and 20 %, the shoot Cd
concentration decreased with increasing percentage of TSW in soil for all the
fungal treatments; being the maximum in sunflower shoots harvested from 5
% and the minimum in 20 % except F2 and F1 + F2.
3.11A.3.4.3 Cu in shoot
Along the row, the variability pattern of shoot Cu uptake followed the
same pattern as observed for Cd. The shoot Cu concentration increased with
fungal inoculations as compared to those where no fungus was applied; being
the maximum in plants harvested from F1 + F2 and the minimum C. Such a
shoot Cu variability trend was observed for all of the TSW-Soil treatments.
Down the column, the plants from 0* showed enhanced shoot Cu
uptake than those from the corresponding 0 (% TSW-Soil). Such a variability
pattern was observed for all the fungal treatments except F1 + F2 where 0*
plants showed lesser shoot Cu uptake than the corresponding 0. Among 5, 10
and 15 % (TSW-Soil), the shoot Cu concentration decreased with increasing
percentage of TSW in soil; being the maximum in 5 % and the minimum in 20
%. All of the fungal treatments observed to show the similar shoot Cu uptake
variability.
3.11A.3.4.4 Fe in shoot
Along the row, the sunflower shoot Fe concentration observed to
increase with fungal inoculations as compared to treatments where no fungus
was applied. The maximum value of shoot Fe was observed to be in plants
harvested from F1 + F2 while being the minimum in C i.e. with not fungus
applied. Such a variability of shoot Fe uptake was observed for all of the
173
TSW-Soil mixtures. The F2 incurred lesser shoot Fe uptake than
corresponding F1 for all the TSW-Soil mixtures except 0 and 20 %.
Within columns, the plants from 0* had lesser shoot Fe uptake than
corresponding 0 (% TSW-Soil) for all of the fungal treatments except for F1. In
case of 5, 10 and 20 (% TSW-Soil), the shoot Fe uptake decreased with
increasing percentage of TSW in soil; being the maximum in plants harvested
from 5 % and the minimum in 20 % for all the fungal treatments.
3.11A.3.4.5 Mg in shoot
Along the row, the shoot Mg concentration increased with fungal
inoculations as compared to those where no fungus was applied and such a
trend was observed for all of the TSW-Soil mixtures. The maximum shoot Mg
concentration was observed for plants harvest from F1 + F2 while being the
minimum in C i.e. where no fungus was applied. The plants with F2
inoculations showed enhanced shoot Mg uptake than corresponding F1 plants
except 10 and 20 (% TSW-Soil) mixtures.
Down the column, the value of plant shoot Mg was greater in 0* than
corresponding 0 (% TSW-Soil). Among 5, 10 and 15 %, the maximum shoot
Mg concentration was observed in plants harvested from 10 % while being the
minimum in those harvest from 20 % for all of the fungal treatments.
3.11A.3.4.6 Ni in shoot
Moving along the row, the sunflower shoot Ni observed to be BDL in 0*
as well as 0 %. i.e. Soil. In case of 5, 10 and 20 %, it was observed to
increase where fungus was inoculated than where no fungus was applied;
being the maximum in F1 + F2 and the minimum in C.
Down the column, the plants from 10 % observed to have the
maximum value of shoot Ni concentration while being the minimum in those
harvested from 20 %.
3.11A.3.4.7 Zn in shoot
Alongside row, the shoot Zn concentration increased with fungal
inoculations than those applied with no fungi. The maximum shoot Zn
174
concentration was observed in plants inoculated with F1 + F2 while being the
minimum in C where no fungus was applied. The F2 inoculations incurred
enhanced shoot Zn concentration than F1 for all of the TSW-Soil mixtures
except 0*, 10 and 20 %.
Down the column, the shoot Zn concentration was greater in 0* % than
corresponding 0 % for all the fungal treatments except C. The 10 % plants
showed the maximum shoot Zn concentration than plants from any of the
TSW-Soil mixtures for all the fungal treatments.
3.11A.3.5 Category-II metals in plant root
The Category-II metals i.e. the AAS detected metals in root were
observed to differ with varying percentage of TSW in the soil as well in
response to different fungal inoculations, as given in Table 3.11A.6.
3.11A.3.5.1 Cd in root
Along the row, the Cd root concentration increased with application of
fungi and found to be the maximum in F1 + F2 treatments and being the
minimum in C i.e. where no fungi was applied. The F2 inoculations incurred
enhanced root Cd concentration than F1. The plants from all of the TSW-Soil
treatment observed to follow the same variability pattern.
Down the column, the root Cd concentration observed to be lesser in
plants from 0* than those from the 0 (% TSW-Soil) for all of the fungal
treatments except F1. Among 5, 10 and 20 % (TSW-Soil), the plant root Cd
concentration decreased with increasing percentage of TSW in soil and found
to be the maximum in 5 % and the minimum in 20 % of all the fungal
treatments.
3.11A.3.5.2 Cr in root
Alongside row similar, the root Cr concentration increased with fungal
inoculation being the maximum in F1 + F2 while being the minimum in C i.e.
with no fungal inoculation. The plants from F2 showed enhanced root Cr
uptake than corresponding F1 for all of the TSW-Soil treatments except 10 %
where F1 plants performed better than those from F2.
175
Table 3.11A.6. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery
solid waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Cd
0* BDL 5bD
3.1 3bcDE
1.2 8aCD
2.9 0.8
0 BDL 4bcD
1.9 5bcDE
1.1 10aCD
1.8 0.7
5 35cdA
1.8 45cA
2.8 60bA
1.9 80aA
4.4 12.4
10 20dBC
4.2 35cB
3.7 40cBC
2.7 70aA
3.1 11.6
20 10cC
2.8 12cDE
3.2 15bcD
2.7 25aC
3.6 3.3
LSD0.05 4.5 6.5 5.7 9.8
Cr
0* 3cE
4.1 5bD
2.7 6bE
3.2 10aE
2.9 1.1
0 BDL 7bD
1.6 8abE
2.1 11aE
3.8 0.9
5 180deA
2.2 225cA
2.8 255cA
2.4 340aC
2.9 34.2
10 95fC
3.7 210deA
3.2 190deB
3.3 640aA
3.5 94.5
20 55deD
2.9 90dC
3.5 110dCD
1.8 310aC
3.5 35.2
LSD0.05 35 47 52 68
Cu
0* BDL 5bcE
2.1 8aDE
3.2 10aDE
2.1 3.1
0 BDL 8cE
2.9 10bcDE
2.5 15aDE
3.8 1.8
5 155bcA
1.3 210aA
1.8 190aA
1.6 210aA
2.9 30.5
10 110bcB
3.5 125abC
2.7 130abB
3.3 155aBC
2.3 5.9
20 40cdD
2.1 80bCD
3.5 90bC
1.2 115aC
1.1 39.7
LSD0.05 22 27 32 44
Fe
0* BDL 3aE
1.4 2abD
1.2 4aD
1.1 0.5
0 2bD
2.9 3abE
1.12 2bD
1.1 5aD
2.8 0.7
5 20bcA
2.4 25bcA
3.8 30aA
1.3 35aA
2.9 7.6
10 15cAB
3.1 20bcB
2.7 18cBC
1.4 35aA
2.2 7.3
20 8cBC
1.9 12abCD
1.5 13aC
1.1 15aC
1.3 3.5
LSD0.05 4.3 5.7 6.3 8.7
Mg
0* 3cC
0.8 5bcD
1.2 6aD
1.3 8aCD
2.3 1.1
0 4bcBC
1.9 5bD
1.9 6bD
1.7 10aCD
2.9 1.3
5 13cA
2.1 20bcA
1.5 18bcA
1.5 28aA
2.9 2.3
10 10cA
2.5 15bB
1.8 20aA
1.4 22aAB
2.8 2.7
20 5cdBC
1.4 10bcC
1.7 12bcBC
2.2 17aC
3.1 3.8
LSD0.05 1.5 1.7 2.1 3.9
Ni
0* BDL BDL BDL BDL -
0 BDL BDL BDL BDL -
5 2bcAB
2.7 4bA
2.4 5bA
1.5 8aA
1.9 0.8
10 4bcA
3.4 5bcA
2.7 3cAB
2.4 10aA
2.5 1.2
20 2cAB
2.9 3bcAB
2.5 2cB
1.2 6aB
1.1 1.1
LSD0.05 0.6 0.4 0.2 0.7
Zn
0* 10dD
1.3 25bDE
1.8 28bD
1.2 35aE
1.2 1.5
0 12dD
1.9 15cdDE
1.3 17cdDE
2.9 35aE
1.8 4.5
5 125dA
1.7 210abA
1.2 190bA
1.3 230aA
1.99 23.2
10 130cdA
1.2 145cBC
1.9 155cB
1.3 225aA
2.5 17.7
20 110bcB
1.6 125bC
2.1 135abBC
2.2 160aC
2.1 19.4
LSD0.05 22.2 24.1 27.3 28.2
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant
difference
Within columns, the root Cr concentration observed to be lesser in
plants harvested from 0* than those from corresponding 0 (% TSW-Soil) for all
the fungal treatments. Among all the fungal treatments apart from soil with no
176
TSW, the root Cr concentration decreased with increasing percentage of TSW
in soil for all of the TSW-Soil mixtures; being the maximum in sunflower roots
harvested from 5 % and the minimum in 20 % except F1 + F2 where it was
the maximum in 10 %.
3.11A.3.5.3 Cu in root
Along the row, the variability pattern of root Cu uptake followed the
same pattern as observed for root Cd. The root Cu concentration increased
with fungal inoculations as compared to those where no fungus was applied;
being the maximum in plants harvested from F1 + F2 and the minimum in
plants belonging to C. Such a root Cu variability trend was observed for all of
the TSW-Soil mixtures.
Down the column for all of the fungal treatmetns, the plants from 0*
showed reduced root Cu uptake than those from the corresponding 0 (%
TSW-Soil) C where it was BDL. For all of the fungal treatments for soils mixed
with TSW, the root Cu concentration decreased with increasing percentage of
TSW in soil; being the maximum in 5 % and the minimum in 20 %.
3.11A.3.5.4 Fe in root
Along the row, the sunflower root Fe concentration was increased with
fungal inoculations as compared to treatments where no fungus was applied.
The maximum value of root Fe was observed to be in plants harvested from
F1 + F2 while being the minimum in C i.e. with not fungus applied for all of the
TSW-Soil mixtures.
Within columns for all of the fungal treatments, the plants from 0* had
lesser root Fe uptake than corresponding 0 (% TSW-Soil) for all of the fungal
treatments except for F1 and F2. For all the fungal treatments where soils
were mixed with TSW, the root Fe uptake decreased with increasing
percentage of TSW in soil; being the maximum in plants harvested from 5 %
and the minimum in 20 %.
3.11A.3.5.5 Mg in root
Along the row, the root Mg concentration increased with fungal
inoculations as compared to those where no fungus was applied and such a
177
trend was observed for all of the TSW-Soil mixtures. The maximum root Mg
concentration was observed for plants harvest from F1 + F2 while being the
minimum in C i.e. where no fungus was applied. The plants with F2
inoculations showed enhanced root Mg uptake than corresponding F1 plants
except 5 (% TSW-Soil) mixtures.
Down the column, the value of plant root Mg in each of 0* was either
equal to or greater than the corresponding 0 (% TSW-Soil). For all of the
fungal treatments where soil was mixed with TSW, the shoot Mg
concentration decreased with increasing percentage of TSW down the
column; being the maximum in plants harvested from 5 % while being the
minimum in those harvest from 20 % for all of the fungal treatments.
3.11A.3.5.6 Ni in root
While comparing along the row, the sunflower root Ni was observed to
increase where fungus was inoculated than where no fungus was applied;
being the maximum in F1 + F2 and the minimum in C. The plant from both F1
and F2 had almost similar Ni concentration in root.
Down the column for all of the fungal treatments, the root Ni was BDL
in plants harvested from 0* as well as 0 (% TSW-Soil). However, the plants
from 10 % observed to have the maximum value of root Ni concentration
while being the minimum in those harvested from 20 %.
3.11A.3.5.7 Zn in root
Alongside row, the root Zn concentration increased with fungal
inoculations than those applied with no fungus. The maximum root Zn
concentration was observed in plants inoculated with F1 + F2 while being the
minimum in C where no fungus was applied. The F2 inoculations incurred
enhanced root Zn concentration than F1 for all of the TSW-Soil mixtures
except 5 % where F1 plants had more root Zn concentration than
corresponding F2.
Down the column for all of the fungal treatments, the root Zn
concentration was greater in 0* % than corresponding 0 % except C. For all
178
fungal treatments where soil was mixed with TSW, the root Zn concentration
decreased down the column with increasing percentage of TSW in soil; being
the maximum in 5 % and the minimum in 20 % except C treatment where the
maximum root Zn was observed in 10 % while being the minimum in 20 %.
3.11A.4 Fungal analyses
The c.f.u. (× 105 c.f.u. g-1 soil) counts of the soils after the harvest of
78-days old plants of sunflower are given in Table 3.11A.7.
Along the row, the c.f.u. number increased in soil treatments inoculated
with fungus than those applied with no fungus; being the maximum in F1 + F2
treatments and the minimum in C i.e. where no fungus was applied. This trend
of variability was observed for all of the TSW-Soil mixtures. The soil
treatments applied with F2 fungus had greater number of c.f.u. than
corresponding F1 for all the TSW-Soil mixtures except 5 %.
Down the column for all the fungal treatments, the soils from 0* had
lesser number of c.f.u. than corresponding 0 (% TSW-Soil). For all the fungal
treatments where soils were mixed with TSW, the c.f.u. in soil decreased
down the column with increasing percentage of TSW in soil; being the
maximum in 5 % and the minimum in 20 %.
3.11A.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
Table 3.11A.7. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of soil used for the cultivation of
78-days old sunflower (Helianthus annuus) on field soil mixed with different percentages of tannery solid
waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n =
6). TSW-Soil (% w:w)
mixture and its type
Treatment LSD0.05
C F1 F2 F1+F2
0* 0.2dBC
0.6 0.5cdC
1.5 0.7cdCD
1.1 2.5aCD
1.6 0.9
0 0.3bcBC
1.2 0.8bC
1.8 1.1aCD
1.2 1.5aD
2.8 0.5
5
2.9dA
2.3 4.1cA
1.6 3.1cdAB
1.3 6.3aA
2.7 1.5
10
2.8cA
2.6 3.2bcAB
1.4 3.9bcA
2.0 5.7aA
2.4 1.2
20 2.1bcA
2.8 2.6bBC
2.4 2.7bBC
2.5 3.8aC
2.3 1.1
LSD0.05 1.2 0.8 0.6 0.7
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference
179
3.11A.5.1 Category-I metals translocation index (%)
The plant translocation index values analyzed for Category-I metals
varied in response to the fungal inoculations as well as with different
percentages of TSW, as given in Table 3.11A.8.
Table 3.11A.8. Meta-analytical phytoextraction indices of sunflower: the Category-I translocation index
(%) analyzed for sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca
5 322.7 238.7 256.2 305.5
10 165.3 171.1 147.1 223.2
20 305.2 281.8 256.5 251.4
K
5 233.3 300.0 276.1 284.0
10 237.5 215.7 190.9 215.3
20 277.7 263.3 200.0 182.3
Na
5 323.0 361.1 272.7 356.0
10 314.2 362.5 330.0 226.6
20 350.0 380.0 366.6 422.2
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Ca, in terms of influence incurred by mixing of TSW in soil
and fungal inoculations, the 5 % C treatment found to be having the highest
translocation index value (322.7 %) and that of 10 % F2 being the least (147.1
%).
For K, the maximum translocation index value (300 %) was found to be
in 5 % F1 while being the least (182.3 %) in 20 % F1 + F2.
In case of Na, the 20 % F1 + F2 had the maximum translocation index
value (422.2 %) while being the least (226.6 %) in 10 % F1 + F2.
3.11A.5.2 Category-II metals translocation index (%)
The Category-II metals translocation index values showed greater
variability in response to the influence of TSW mixing in soil and inoculations
with different fungi, as given in Table 3.11A.9. For Cd, the maximum
translocation index value (644.4 %) was noted for 5 % F1 while the minimum
value (200 %) was recorded in 10% F1 + F2.
In case of Cr, the highest translocation efficiency (322.2 %) was
observed in 20 % F1 and the minimum (152.9 %) was observed in 5 % F2.
180
For Cu, the maximum translocation index value (312 %) was calculated
for 10 % F1 and the minimum (183.8 %) was recorded in 5 % C.
Table 3.11A.9. Meta-analytical phytoextraction indices of sunflower: the Category-II translocation index
(%) analyzed for sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd
5 314.2 644.4 516.6 431.2
10 450.0 314.2 312.5 200.0
20 400.0 458.3 400.0 300.0
Cr
5 155.5 202.2 152.9 258.8
10 200.0 188.0 215.7 154.6
20 200.0 322.2 295.4 275.8
Cu
5 183.8 216.6 226.3 261.9
10 222.7 312.0 273.0 274.1
20 275.0 268.7 272.2 269.5
Fe
5 225.0 280.0 216.6 228.5
10 266.6 250.0 222.2 200.0
20 125.0 166.6 192.3 233.3
Mg
5 115.3 85.0 111.1 89.2
10 200.0 146.6 90.0 136.3
20 200.0 120.0 91.6 88.2
Ni
5 250.0 175.0 160.0 125.0
10 200.0 200.0 433.3 150.0
20 200.0 233.3 250.0 166.6
Zn
5 168.0 233.3 268.4 269.5
10 180.7 213.7 187.0 188.8
20 172.7 180.0 155.5 143.7
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
For Fe, the maximum translocation index value (280 %) was found in 5
% F1, while the minimum value (125 %) was observed in 20 % C.
In case of Mg, the highest (200 % each) translocation efficiency was
observed in C of both 10 and 20 % while the lowest value (85 %) was
recorded for 5 % F1.
For Ni, the translocation index recorded to be the highest (433 %) in 10
% F2 and the least (125 %) of it was observed to be in 5 % F1 + F2.
As far as the Zn is concerned, there was the maximum (269.5 %)
translocation index in 5 % F1 + F2 while the lowest value (143.7 %) was
recorded in 20 % F1 + F2.
181
3.11A.5.3 Tolerance index (TI) for shoot and root
The tolerance index values for both root and shoot and their variation in
response to TSW mixing in soil and inoculation with different fungi are given in
Table 3.11A.10.
Table 3.11A.10: Meta-analytical phytoextraction indices of sunflower: the tolerance index (TI) analyzed
for shoot and root of sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
TI Shoot
5 1.68 2.17 1.77 1.65
10 1.40 1.94 1.59 1.58
20 0.78 0.99 0.86 0.75
TI Root
5 1.05 1.24 1.31 1.14
10 1.03 1.12 1.09 1.15
20 0.54 0.86 0.90 0.80
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
For shoot, the TI found to be the highest (2.17) in 5 % F1 while being
the lowest (0.75) in 20 % F1 + F2.
In case of root, the highest value (1.31) of TI observed to be in 5 % F2
while being the lowest (0.54) in 20 % C.
3.11A.5.4 Category-I metals specific extraction yield (SEY %)
The SEY (%) for Category-I metals and its variability with respect to
TSW mixing in soil and inoculation with different fungi is given in Table
3.11A.11.
Table 3.11A.11: Meta-analytical phytoextraction indices of sunflower: the specific extraction yield (SEY
%) for Category-I metals in sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca
5 28.6 32.3 35.0 44.9
10 27.7 30.6 27.9 38.5
20 13.2 14.4 14.0 21.1
K
5 33.7 49.4 44.3 53.9
10 22.3 24.7 26.4 33.8
20 8.5 10.1 9.8 12.1
Na
5 20.2 30.6 30.2 42.0
10 5.7 7.3 8.5 9.7
20 1.9 2.5 3.0 5.0
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
182
In case of Ca, the maximum SEY (44.9 %) was found to be in 5 % F1 +
F2 while being the minimum (13.2 %) in 20 % C.
The SEY value for K found to be the highest (53.9 %) in 5 % F1 + F2
and the lowest (8.5 %) of it was recorded in 20 % C.
For Na, the highest value of SEY (42 %) was calculated for 5 % F1 +
F2 and the lowest of it (1.9 %) was found to be in 20 % C.
3.11A.5.6 Category-II metals specific extraction yield (SEY %)
The SEY percentages calculated for Category-II metals and their
variability in response to spiking of soils with variable levels of TSW and
inoculations with different fungi, are given in Table 3.11A.12.
Table 3.11A.12. Meta-analytical phytoextraction indices of sunflower: the specific extraction yield (SEY
%) for Category-II metals in sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd
5 5.4 12.6 13.9 16.0
10 1.6 2.2 2.5 3.1
20 0.5 0.7 0.8 1.1
Cr
5 5.5 8.2 7.8 14.7
10 2.7 5.9 5.8 15.9
20 1.0 2.4 2.8 7.5
Cu
5 32.5 49.2 45.9 56.2
10 16.9 24.5 23.0 27.0
20 2.8 5.6 6.3 8.0
Fe
5 26.0 38.0 38.0 46.0
10 10.7 13.7 11.3 20.5
20 1.9 3.5 4.1 5.4
Mg
5 9.0 11.9 12.2 17.0
10 4.8 5.9 6.1 8.3
20 1.4 2.1 2.2 3.1
Ni
5 20.0 31.4 37.1 51.4
10 21.8 27.2 29.0 45.4
20 5.4 9.0 6.3 14.5
Zn
5 22.9 47.9 47.9 58.2
10 19.3 24.0 23.5 34.3
20 14.8 17.2 17.0 19.2
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
For Cd, the highest SEY percentage was found to be 16 in case of 5 %
F1 + F2 while the lowest of it was observed to be 0.5 % for 20 % C.
183
In case of Cr, the maximum SEY value 15.9 % was recorded 10 % F1
+ F2 and the minimum of it being 1 % in 20 % C.
Likewise for Cu, 5 % F1 + F2 exhibited the maximum SEY value (56 %)
with corresponding minimum value (2.8 %) in 20 % C.
Similarly for Fe, the highest SEY value (46 %) was recorded in 5 % F1
+ F2 and that of lowest was found to be 1.9 % in 20 % C i.e. with no fungal
inoculum.
Following the similar trends, the SEY calculated for Mg found to be the
highest (17 %) in 5 % F1 + F2 while the lowest of it (1.4 %) found to be in 20
% C.
As well as, the SEY for Ni calculated to be the highest (51.4 %) in case
of 5 % F1 + F2 while the lowest of it (5.4 %) being in 20 % C.
Also for Zn, the maximum SEY percentage was calculated to be 58.2 in
5 % F1 + F2 while the minimum of it was observed to be 14.8 % in 20 % C.
184
3.11B. Experiment with Tagetes patula
3.11B.1 Pre-sowing analysis
The analyses of the soil in field plots mixed with different percentages
of TSW and inoculated with different fungi after harvesting sunflower
(Helianthus annuus) and before cultivating French marigold (Tagetes patula)
are given in Table 3.11B.1.
3.11B.1.1 Pre-sowing Category-I metals in field soil
For the Category-I metals among the fungal treatments, the
concentration of Ca was significantly greater in all of the TSW-Soil mixtures
than corresponding soil treatments mixed with no TSW i.e. 0* and 0 (% TSW-
Soil). It increased with increasing percentage of TSW in soil for all the fungal
treatments; being the maximum in 20 % and the least in 5 %. Similar trends of
soil Ca variability observed for K as well as Na.
For the Category-I metals between the fungal treatments, the Ca
concentration in 0* of all the fungal treatments found to the maximum in case
of C and the minimum in corresponding F1 + F2. Similar trend of variation was
observed for K as well as Na in 0* of all the fungal treatments. Likewise, the 0,
5, 10 and 20 % (TSW-Soil) of all the fungal treatments had the minimum
values of Category-I metals in C treatment and the corresponding minimum
values in F1 + F2, as given in Table 3.11B.1.
3.11B.1.2 Pre-sowing Category-II metals in field soil
For the Category-II metals among the fungal treatments, the
concentration of Cd was significantly greater in all of the TSW-Soil mixtures
than corresponding soil treatments mixed with no TSW i.e. 0* and 0 (% TSW-
Soil). It increased with increasing percentage of TSW in soil for all the fungal
treatments; being the maximum in 20 % and the least in 5 %. Similar trends of
soil Cd variability observed for rest of the Category-II metals.
For the Category-II metals between the fungal treatments, the Cd
concentration in 0* of all the fungal treatments was found to be the maximum
in case of C and the minimum in corresponding F1 + F2. Similar trends of
variation were observed for Cd in 0* of all the fungal treatments.
185
Table 3.11B.1. The concentration of category-I category-II metals (mgkg-1
) observed in soil amended with different concentration of tannery solid waste
(TSW-Soil % w:w) determined after the harvesting sunflower (Helianthus annuus) and prior the sowing French marigold (Tagetes patula). The mean values
S.D. with common letters are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Type of
metals
TSW-Soil (% w:w) mixtures with different fungal inoculations
0* 0 5 10 20 0* 0 5 10 20 0* 0 5 10 20 0* 0 5 10 20
LSD0.05
C F1 F2 F1+F2
Cate
go
ry-I
(mg
kg
-1)
Ca 50
+ 2.4 80
+ 2.3 1,570 + 4.1
1,811 + 2.1
2,700 + 2.6
45 + 2.4
75 + 3.2
1,450 + 3.7
1,640 + 3.7
2,610 + 2.4
38 + 3.4
70 + 2.6
1,410 + 2.3
1,750 + 1.3
2,600 + 3.2
35 + 3.1
65 + 1.8
1,190 + 2.1
14,40 + 3.4
2,290 + 3.2
K 550 + 3.1
640 + 3.1
810 + 3.5
940 + 1.8
1,850 + 2.7
525 + 3.7
610 + 3.3
720 + 2.5
820 + 3.8
1,750 + 2.1
510 + 3.1
590 + 2.4
755 + 3.1
910 + 2.5
1,790 + 2.8
490 + 3.1
550 + 2.6
670 + 3.1
720 + 2.1
1,680 + 3.0
Na 670 + 3.2
950 + 2.4
1,290 + 2.4
2,410 + 2.6
4,600 + 3.3
640 + 3.1
890 + 3.1
1,255 + 2.6
2,320 + 4.4
4,450 + 4.1
630 + 2.5
895 + 2.8
1,235 + 2.9
2,290 + 3.3
4,440 + 3.1
590 + 2.3
740 + 2.1
1,125 + 2.5
2,195 + 3.5
4,350 + 4.0
Cat
ego
ry-I
I
(mg
kg
-1)
Cd 40
+ 2.1 45
+ 1.9 2,530 + 3.1
6,490 + 3.9
8,740 + 3.8
35 + 2.0
40 + 2.5
2,410 + 2.8
6,450 + 4.1
8,735 + 4.4
30 + 1.7
40 + 1.9
2,390 + 3.9
6,430 + 4.4
8,720 + 3.6
25 + 1.8
35 + 2.1
2,200 + 3.5
6,370 + 3.3
8,670 + 4.1
Cr 55
+ 2.3 90
+ 1.8 7,790 + 4.1
10,050 + 4.8
15,410 + 4.6
45 + 2.1
80 + 2.3
7,550 + 2.8
9,610 + 3.4
15,270 + 4.7
50 + 1.4
85 + 2.1
7,610 + 2.2
9,620 + 4.1
15,110 + 4.1
35 + 3.2
70 + 1.8
7,030 + 3.7
8,650 + 3.1
13,400 + 4.4
Cu 510 + 3.1
560 + 2.7
1,025 + 3.7
1,810 + 2.1
5,110 + 3.8
450 + 2.2
490 + 2.5
970 + 2.9
1,730 + 3.5
4,950 + 4.6
430 + 1.4
480 + 2.4
990 + 2.1
1780 + 4.8
4.890 + 3.6
410 + 2.1
450 + 1.9
780 + 3.1
1,690 + 2.1
4,810 + 3.4
Fe 30
+ 2.0 50
+ 1.1 210
+ 2.3 480
+ 2.7 890
+ 2.7 35
+ 1.0 45
+ 2.1 190
+ 1.6 485
+ 2.5 840
+ 3.2 38
+ 1.1 45
+ 2.1 185
+ 2.4 490
+ 3.1 830
+ 3.3 25
+ 1.1 30
+ 1.5 170
+ 1.9 470
+ 1.8 810
+ 2.7
Mg 25
+ 1.9 40
+ 1.5 290
+ 2.6 590
+ 2.1 920
+ 3.6 20
+ 0.89 35
+ 1.4 270
+ 1.7 580
+ 2.1 870
+ 2.2 22
+ 2.2 38
+ 2.1 260
+ 2.6 565
+ 2.8 865
+ 2.9 15
+ 2.2 30
+ 2.1 250
+ 2.3 530
+ 3.1 830
+ 2.1
Ni BDL BDL 28
+ 1.4 45
+ 1.8 95
+ 2.1 BDL BDL
25 + 1.1
40 + 1.1
70 + 2.7
BDL BDL 22
+ 1.1 38
+ 1.3 85
+ 1.4 BDL BDL
19 + 1.6
30 + 1.5
65 + 1.5
Zn 140 + 2.1
190 + 1.8
1230 + 2.4
1620 + 2.5
1,890 + 1.1
135 + 1.1
170 + 2.1
960 + 2.7
1,560 + 3.3
1,720 + 2.5
130 + 1.2
185 + 2.2
980 + 2.1
1,590 + 3.1
1,725 + 2.7
120 + 1.1
160 + 2.4
890 + 3.1
1,210 + 3.3
950 + 2.2
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least
significant difference
186
Likewise, the 0, 5, 10 and 20 % (TSW-Soil) of all the fungal treatments
had the minimum values of Category-II metals in C treatment and the
corresponding minimum values in F1 + F2, as given in Table 3.11B.1.
3.11B.2 Biochemical analyses of Tagetes patula in field
The biochemical parameters observed in live T. patula and its variation
in response to mixing of soil with different TSW percentages and inoculations
with different fungi are given in 3.11B.2. The specific details of each of the
biochemical parameters are as under:
3.11B.2.1 Chlorophyll content
Along the row, the plant chlorophyll contents based on SPAD value
observed in 50-days old French marigold increased in soil treatments applied
with fungus as compared to the C (with no fungus). The plants from F2
showed enhanced chlorophyll contents than F1, being the maximum in F1 +
F2 (applied with combined fungal inoculation) and the least in C, as given in
Table 3.11A.1. Such a trend of variability was observed in all of the TSW-Soil
mixtures.
Within column, the general trend was decrease in plant chlorophyll
contents with the increase of TSW percentage in soil for all the fungal
treatments. In all fungal treatments, the plants from all the TSW-Soil 0 (%
w:w) had more chlorophyll contents than every corresponding 0* (% w:w)
except for F1 and F2 where plants from 0* exhibited the higher value than
those from corresponding 0 (% TSW-Soil). The plants from 5 % (% w:w) of all
the fungal treatments showed the maximum SPAD value within a column
while those from 20 (% w:w) had the least chlorophyll contents.
3.11B.2.2 Soluble protein contents
Likewise chlorophyll contents, the soluble protein contents along the
row increased with the application of fungal inoculations for all the fungal
treatments, as given in Table 3.11B.2. The plants from C (with no fungal
treatment) had the least protein contents than any of the treatment with single
or combine fungal inoculations, being the maximum in F1 + F2. Such a trend
of variability was observed for all of the TSW-Soil mixtures.
187
Table 3.11B.2. The biochemical parameters observed in 82-days old Tagetes patula
cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Chlorophyll contents
0* 19.5bB
1.8 21.2aC
2.1 22.4aAB
2.3 22.3aC
2.6 0.8
0 20.6bB
1.2 20.4bC
2.4 19.3bB
1.3 23.1aC
2.3 1.1
5 24.1bcA
1.3 26.6bcA
2.8 25.3bcA
2.7 32.5aA
2.9 2.3
10 22.3abA
1.7 23.4aAB
1.1 23.5aA
1.9 24.9aC
1.6 2.8
20 13.8cD
1.8 14.4cDE
2.2 13.7cCD
2.5 19.2aD
1.8 3.1
LSD0.05 1.2 1.5 2.5 2.8
Protein content
0* 2.6dC
1.9 9.6bBC
2.4 9.2bC
3.2 12.1aC
2.2 2.4
0 3.1dC
2.3 10.1abBC
1.9 9.7bC
2.9 12.9aC
1.8 2.1
5 7.4cdA
1.7 17.1aA
2.8 16.7aA
2,5 18.9aA
1.9 3.4
10 3.8dC
2.2 6.5bcC
2.3 7.2bcCD
1.4 11.6aC
2.9 4.1
20 1.9cdCD
2.1 2.9cDE
1.5 3.1cE
1.5 6.1aDE
3.1 4.7
LSD0.05 3.1 2.8 3.2 4.1
SOD
0* 2.1bcD
1.9 3.6aDE
1.8 2.9aEF
1.4 3.1aE
2.1 1.2
0 1.3cD
1.2 2.3aDE
1.4 2.5aEF
1.8 1.1cE
0.89 0.9
5 12.6abA
1.8 13. 2aA
1.2 14.63aA
1.5 14.8aA
2.2 2.6
10 11.1cA
2.7 13.7abA
2.3 13.2abA
2.3 15.1aA
2.7 3.5
20 9.3bcB
1.2 8.9aBC
1.1 7.8aCD
1.6 8.4aCD
1.7 1.6
LSD0.05 1.4 2.3 2.5 3.6
CAT
0* BDL BDL BDL BDL -
0 0.1bcDE
0.6 0.4aE
0.1 0.3aEF
0.2 0.2bEF
0.8 0.2
5 12.2deA
1.4 16.2cA
1.3 19.2abA
1.8 21.4aA
2.2 2.1
10 10.2cA
1.3 11.3cC
1.4 12.3cC
1.7 17.4aB
2.1 4.2
20 9.4bcB
1.3 10.6bC
1.7 11.2bC
1.2 13.2aC
1.5 4.7
LSD0.05 2.1 3.2 3.4 5.4
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference
The soluble protein contents was increased down the column observed
to be greater in 0 than corresponding 0* (% w:w) for all the fungal treatments.
For all the fungal treatments and soils mixed with TSW, the soluble protein
contents decreased down the column being the least in 5 % and the highest in
20 % (TSW-Soil). The plants from 5 % had the maximum protein contents in
all the columns.
3.11B.2.3 Superoxide dismutase (SOD) contents
Parallel to the trends found for soluble protein contents, the SOD
contents in plants increased with fungal inoculations within a row for all the
treatments; however, it decreased in case of combined fungal application in
both 0 (% TSW-Soil) mixture, as given in Table 3.11A.2. For 5, 10 and 20 %
(TSW-Soil) mixture, the SOD values increased with the application of single or
188
combined fungal inoculation, being the maximum in plants applied with F1 +
F2.
Within a column, the plants from 0* (control with no geothermal
membrane lining) had shown enhanced SOD contents than 0 (with lining) for
all the fungal treatments. However, it decreased with increasing level of TSW
percentage in soil, being the maximum in 5 % and the minimum in 20 %
except 5 % F1.
3.11B.2.4 Catalase (CAT) contents
The CAT contents of the French marigold exhibited variable pattern
along the row. Other than the 0* (% TSW-Soil) where observed values were
BDL, the CAT values increased along the row with fungal inoculations and
being the maximum in plants applied with F1 + F2 and being the minimum in
TSW-Soil mixtures applied with no fungus.
Down the column, the plants from 0 showed enhanced CAT contents
than corresponding 0* (% TSW: Soil) for all the fungal treatments. However it
decreased down the column with increasing percentage of TSW in soil for all
of the fungal treatments, being the maximum in 5 % (TSW:Soil) and the
minimum in 0 for all the treatments.
3.11B.3 Post-harvest analysis
The growth of 50-days old French marigold cultivated in field varied in
response to different TSW percentages as well as fungal inoculations, as
given in Table 3.11B.3. The details are as under:
3.11B.3.1 Growth performance of Tagetes patula
The details of each of the morphological parameters observed in
French marigold are given in Table 3.11B.3 and described as under:
3.11B.3.1.1 Shoot, root and seedling length (cm)
Along the row, the sunflower shoot, root and seedling length increased
in treatments applied with fungus than those applied with no fungus i.e. C. It
was observed to be the maximum in F1 + F2 and being the minimum in C for
all the TSW-Soil mixtures.
189
Table 3.11B.3. Various morphological parameters observed in 82-days old Tagetes patula cultivated on
TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (%
w:w)
Fungal treatments LSD0.05
C F1 F2 F1+F2
Shoot Length (cm)
0* 24.2bA
2.4 27.3aBC
2.1 24.0bBC
1.9 28.6aBC
2.7 2.1
0 23.5bcAB
1.7 21.8bcBC
2.3 22.0bcC
2.9 28.9aBC
2.5 3.4
5 27.5dA
2.8 38.7bcA
2.6 33.7cA
2.8 46.3aA
3.3 7.2
10 25.4cA
2.9 23.8cbC
2.5 26.8cBC
3.1 40.2aA
2.3 5.7
20 21.2bcAB
2.2 22.8bcC
2.8 21.9bcC
2.1 27.5aBC
2.4 3.4
LSD0.05 2.6 4.7 5.2 5.8
Root Length (cm)
0* 20.4bB
2.8 23.2abB
2.3 22.1abB
2.7 25.4aB
2.2 2.6
0 20.5bcB
2.1 21.9bcBC
2.6 21.8bcB
2.4 26.1aB
2.8 3.8
5 27.2bA
1.7 28.8bA
2.8 27.9bA
2.1 32.6aA
3.1 2.9
10 22.2bcB
2.1 25.9bB
2.6 23.2bAB
2.1 27.3aB
2.3 3.4
20 19.1bB
2.5 15.7cdCD
1.7 18.5aB
2.2 19.7aC
2.7 3.1
LSD0.05 3.7 4.8 2.9 4.8
Seedling Length (cm)
0* 44.9bcB
2.3 50.7abBC
2.2 46.4bcbC
2.4 54.2aC
2.8 4.6
0 44.3bcB
2.8 44.0bcC
2.8 44.1bcBC
3.1 55.2aC
2.7 3.4
5 55.0cdA
2.5 67.8bcA
3.5 65.9bcA
3.1 79.1aA
2.4 9.1
10 47.9bcB
1.6 50.0bcBC
2.8 50.3bcB
2.6 67.9aB
. 3.1 7
20 40.6bBC
2.8 38.8bC
2.1 40.7bC
2.3 47.5aC
2.8 6.1
LSD0.05 4.1 6.1 8.1 7.8
No. of roots/plant
0* 24aA
2.4 22aB
2.1 20abBC
2.6 21aBC
2.2 4
0 22abA
2.1 23aB
2.7 24aA
2.4 25aB
2.4 8.3
5 25abA
2.4 28aA
2.7 26abA
2.9 30aA
2.8 3.1
10 21abAB
2.4 23aB
2.5 22aB
2.7 24aB
2.3 1.9
20 20bAB
2.1 18bC
2.6 16bcCD
2.3 21aBC
2.8 3.5
LSD0.05 4 3 6 3.1
No. of leaves/plant
0* 7bcC
1.5 8aC
1.8 9abC
1.3 11aBC
2.1 1.4
0 8abC
2.1 9aC
1.5 8abC
2.2 10aBC
2.8 2.1
5 15bcA
2.9 17bA
2.7 16bA
2.6 21aA
2.5 3.4
10 9bC
1.2 10bbC
1.7 9bC
2.6 13aBC
1.9 2.9
20 5cCD
1.5 7bC
1.6 8aC
1.9 9aC
1.8 2.6
LSD0.05 4 6 3.3 4.2
Fresh wt./plant (g)
0* 97.1bB
2.5 115.1aB
3.4 123.8aAB
2.2 112.7aBC
2.5 3.7
0 120.1aA
3.5 110.3aB
2.9 114.1aB
4.1 113.5aBC
3.8 3.6
5 125.3abA
2.9 138.3aA
2.8 135.9aA
3.3 145.2aA
2.5 4.5
10 115.1aA
2.2 113.6aB
2.8 111.7aB
3.7 121.1aB
3.5 2.9
20 102.3aB
2.4 109.8aB
2.5 105.2aB
3.1 114.7aBC
4.2 3.2
LSD0.05 5.4 7.9 9.2 8.8
Dry wt. (g)
0* 30.5bA
2.9 32.3aA
2.7 34.1aA
3.6 36.7aA
3.6 2.2
0 31.4aA
2.3 33.1aA
3.1 31.2aA
3.9 33.9aA
2.8 2.8
5 34.9abA
4.1 36.5aA
3.8 33.7abA
3.3 37.8aA
2.1 5.9
10 27.6aB
2.4 22.8bBC
3.2 21.7bBC
3.4 27.5aBC
3.1 7.9
20 18.8bC
3.7 21.2abBC
2.1 23.7aBC
2.7 24.9aBC
3.2 9.2
LSD0.05 3.4 4.1 4.8 4.2
No. of flowers /plant
0* 10bcB
3.1 12bB
2.7 14aA
2.2 16aAB
2.4 4.8
0 11bB
2.8 13aB
3.3 11bAB
2.8 14aB
2.1 5.3
5 14bcA
4.6 16bA
3.7 13bcA
2.5 20aA
2.9 8.6
10 13bA
2.6 11bBC
3.5 12bA
2.7 17aAB
3.7 5.2
20 9bcB
3.2 12abB
2.8 13aA
2.6 14aB
3.9 4.1
LSD0.05 4.2 3.6 3.1 3.7
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference
190
The plants from 0*, 5 and 20 % with F1 inoculation performed better than
those from corresponding F2 treatments. However; from 0 and 10 % with F2
performed better than those from corresponding F1 treatments as shown in
figure 3.11B.1.
Figure 3.11B.1: Phytoextraction field trials with French Marigold (Tagetes patula) cultivated on soil amended with different levels of tannery solid waste (TSW-Soil % w:w); upper: (A) 0* % the only treatment without geothermal membrane allowing leaching, (B) 0 % with geothermal membrane to avoid leaching, (C) 5 %, (D) 10 % and (E) 20 %. The white lines across the strip plots separated by bricked walk ways (horizontal in above and vertical in lower; 25 × 3 ft) in upper and the white arrows in the lower picture indicate soil separations (1.25 ft) subdividing each strip plot into four subplots (5 × 3 ft each) for fungal inoculations viz. C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger + T. pseudokoningii, applied in a randomized complete block design.
A
B
C
D E
N
N
191
Down the column, the plants from 0* had more efficient vegetative
growth in terms of shoot, root and seedling length than corresponding 0
(TSW-Soil) except those applied with F1 + F2. For all the fungal treatments
where soil was mixed with TSW, the plant height decreased down the column
with increasing percentage of TSW, being the maximum in 5 % and the
minimum in 20 %.
The stem girth variations in different TSW-Soil mixtures and fungal
treatment can be observed in Figure 3.11B.2
Figure 3.11.2B: The Tagetes patula stem girth variation in response to soil mixed with different levels of tannery solid waste (TSW : soil w:w) inoculated with fungi (A- 0*% with F1+F2; B- 0% with F1+F2; C- 5% with F2; D- 5% with F1+F2 ; E- 5% with C ;F-10% with F1+F2; G- 10% with F2; H- 20% with F2; I- 20% with F1+F2.
3.11B.3.1.2 No. of leaves, roots and flowers per plant
Along the row, the plants from pots inoculated with fungi showed better
vegetative growth than those applied with no fungi. Except 0*, the maximum
no. of root, leaves as well as flowers observed in plants inoculated with F1 +
F2 and the minimum of which being in C. The F2 inoculation incurred better
vegetative growth than F1.
A B
D
G H
E
I
F
C
192
Down the columns, plants from 0* of all the fungal treatments exhibited
better growth than those from corresponding 0 (5 TSW-Soil). For fungal
treatments where soil was mixed with TSW, the increasing percentage of
TSW decreased the no. of leaves, roots as well as flowers per plant; being the
maximum in 5 % and the minimum in 20 % as shown in Figure 3.11B.1.
3.11B.3.1.3 Fresh and dry weight (g)
The fresh and dry weight was observed to be the maximum and the
minimum in accordance with the maximum and the minimum no. of leaves
and roots for both along the row as well as within column comparisons.
3.11B.3.2 Category-I metals in plant SHOOT
The Category-I metals i.e. the flame photometer detected metals in
shoot were variable with respect to fungal inoculations as well as increasing
ratio of TSW in soil, as given in Table 3.11B.4.
193
Table 3.11B.4. The concentration of Category-I Metals (mgkg-1
) observed in SHOOT of 82-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Ca
0* 15bcDE
1.3 20aD
1.1 18abD
2.2 22aDE
2.1 2.2
0 18bDE
1.7 22abD
2.5 19bD
2.3 25aDE
1.3 2.6
5 145cBC
3.4 240aA
2.4 210bA
1.7 250aA
2.9 32
10 210bcA
1.9 245abA
2.2 210bcA
2.6 275aA
2.3 49
20 130cBC
2.6 220aA
2.2 235aA
2.3 240aA
2.5 55
LSD0.05 36 42 51 58
K
0* 10bcD
1.8 15aDE
1.2 12bE
1.4 15aDE
2.9 3.4
0 12bcD
1.2 13bDE
1.1 14bE
1.4 17aDE
1.3 2.6
5 110cA
2.3 130cA
2.5 170bA
2.7 215aA
2.2 43
10 90bcA
2.3 115bA
2.9 130aB
2.1 150aBC
3.3 34
20 65bcBC
1.6 75bcC
1.7 60bcCD
1.3 115aC
2.4 28
LSD0.05 45 56 38 47
Na
0* 5cD
1.2 10bD
1.3 8bcDE
1.1 15aDE
1.9 4.1
0 7cD
1.2 10bcD
1.9 11bcDE
2.4 18aDE
1.5 3.8
5 105bA
1.1 125abA
1.7 110bA
2.3 145aA
1.7 26
10 105bcA
1.8 115bA
2.5 105bcA
2.6 155aA
3.1 31
20 40cdC
1.8 55cCD
1.2 50cC
2.1 90aBC
2.2 46
LSD0.05 22 28 26 31
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference
3.11B.3.2.1 Calcium (Ca) in shoot
Along the row, the Ca concentration in shoot increased with inoculation
of fungi as compared to C. For 0*, the maximum (22 mgkg-1) shoot Ca
observed to be in F1 + F2 while being the minimum (15 mgkg-1) in C. Similar
trends were observed for 0, 5, 10 and 20 %.
Within columns, the plants from 0* had the lower shoot Ca than those
from corresponding 0 (% TSW-Soil) for all the fungal treatments. For those
fungal treatments where soil was mixed with TSW, the shoot Ca increased
(10 %) and then decreased (20 %) down the column as compared to the
shoot Ca observed in plants from 5 %.
3.11B.3.2.2 Potassium (K) in shoot
The trend of variation of shoot K was similar to that of shoot Ca i.e. it
increased along the row with application of fungi and was greater in plants
with fungal inoculations than those without fungi. Plant shoot K observed to
follow the same trend along row for all the TSW-Soil mixtures.
194
Likewise, the K shoot concentration increased down the column with
the increasing concentration of TSW in soil mixtures for all the fungal
treatments.
3.11B.3.2.3 Sodium (Na) in shoot
The Na concentration in shoot observed to increase along the row and
it was because of fungal inoculations. The pots with F1 + F2 showed the
greatest Na shoot uptake, the F1 being greater than F2, while those with no
fungi being the least. The plants in 10 % with F1 + F2 had the highest value
(155 mgkg-1) while those in 0* % with no fungi exhibited the lowest Na shoot
contents (5 mgkg-1).
Within column, the increasing ratio of TSW in soil mixtures enhanced
the shoot Na uptake for 5 and 10 % but decreased for 20 %. In case of 0 %,
the concentration of shoot Na observed to be the least for all the fungal
treatments.
3.11B.3.3 Category-I metals in plant ROOT
The bioavailability of Category-I metals was variable with different fungi
in root also however, it was directly related to the increasing ratio of TSW in
soil mixture, as given in Table 3.11B.5.
3.11B.3.3.1 Calcium (Ca) in root
The application of fungal inoculums to the soil helped increase Ca root
uptake along the row. The plants from F1 + F2 pots observed to have
maximum while those from where no fungi was applied having the minimum
root Ca for all the TSW-Soil mixtures. The highest root Ca (110 mgkg-1) was in
5 % with F1 + F2 while being the minimum (4 mgkg-1) in 0* % with C i.e. no
fungal inoculation.
Down the columns, the root Ca decreased with increasing percentage
of TSW in soil being the maximum in plants from 5 % and minimum in those
from 20 %. Such variation was observed for all the fungal treatments.
195
3.11B.3.3.2 Potassium (K) in root
The K root contents in the 5 % with F1 + F2 exhibited the maximum
value (65 mgkg-1) than any of the soil treatments while being the minimum (2
mgkg-1) in 0 % with C i.e. no fungal application. The application of fungus as
individual inoculant i.e. F1 and F2 showed better K uptake than TSW-Soil
mixtures where no fungi has been applied. However, F2 showed better uptake
as compared to F1 for all the soil treatments except 5 %.
Within column, the metal uptake in root increased with increasing ratio
of TSW in 5 and 10 %, however, it was decreased in 20 %.
Table 3.11B.5. The concentration of Category-I Metals (mgkg-1
) observed in ROOT of 82-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Ca
0* 4cE
0.1 5cE
1.1 7bcDE
1.4 11aE
1.5 1.2
0 5bcE
1.4 8bE
2.9 6bcDE
1.3 10aE
1.8 1.7
5 70cA
1.3 95abA
1.4 90bA
1.7 110aA
2.7 32
10
45cC
2.3 60bcBC
1.3 55bcC
2.1 80aB
2.7 25
20 35cC
1.8 40bcD
1.5 45bC
2.1 55aCD
2.2 38
LSD0.05 12 17 21 26
K
0* 2dD
0.9 5bcD
2.6 7bD
2.3 10aDE
1.2 2.4
0 4cD
1.2 6bcD
1.4 8bD
1.2 12aDE
1.3 3.2
5 40cA
1.4 50bcA
1.6 45bcA
1.5 65aA
2.1 16
10 20dBC
1.5 35bcBC
1.7 40bcA
1.2 50aB
1.4 27
20 15cdC
1.1 20cC 1.5 35
abB 2.7 45
aB 1.6 32
LSD0.05 7 10 9 14
Na
0* 5bcCD
0.4 7bcD
1.2 5cD
0.2 10aD
1.2 1.6
0 6cCD
0.5 8bD
0.6 9bD
1.5 13aD
1.4 1.9
5 45bA
1.2 50abA
1.2 45bA
1.5 55aA
1.3 23
10
30bcB
1.2 35bcB
1.3 32bcB
0.9 45aAB
1.5 34
20 16cC
1.1 19bcCD
1.8 22bC
1.6 30aC
1.7 29
LSD0.05 13 14 8 11
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference
3.11B.3.3.3 Sodium (Na) in root
Along the row, as observed in case of Ca and K, the application of
fungi helped increase Na uptake in roots. The maximum Na in root (55 mgkg-
1) was observed in 5 % with F1 + F2 while being the minimum in 0 % with no
fungi (5 mgkg-1). Those with F1 and F2 applications also performed better
than C i.e. treatment with no fungal inoculation.
196
Within column, the increasing ratio of TSW in soil decreased root Na
uptake as compared to 5 %. And this trend was observed for all the fungal
treatments.
3.11B.3.4 Category-II metals in plant shoot
The Category-II metals i.e. the AAS detected metals in shoot were
variable with respect to fungal inoculations as well as increasing ratio of TSW
in soil, as given in Table 3.11B.6.
3.11B.3.4.1 Cd in shoot
Along the row, the Cd shoot concentration increased with application of
fungi and found to be the maximum in TSW-Soil mixtures with combined
fungal treatments while being minimum where no fungi was applied.
Maximum amount of metal (85 mgkg-1) was observed in 5 % TSW-Soil
mixture with combined inoculation of fungi i.e. F1 + F2 and the minimum of
which was observed in 0 % with C (BDL).
Down the columns, for fungal treatments where soil was mixed with
TSW, the shoot Cd significantly decreased as compared to 5 % with
increasing percentage of TSW in soil.
3.11B.3.4.2 Cr in shoot
Like Cd in shoot, the plant Cr also exhibited the same variation pattern
i.e. along the row it increased with fungal application and found to be the
maximum in plants where both of the fungi were inoculated. The minimum of it
was observed in plants where no fungi were applied. This trend was observed
for all of the TSW-Soil mixtures.
Alongside columns, the increasing percentage of TSW in soil
decreased the shoot Cr and such variation was observed in plants from all of
the fungal treatments.
3.11B.3.4.3 Cu in shoot
The shoot Cu concentration increased along the row in plants with the
application of fungal inoculations and the observed increase was with respect
to the C treatments where no fungi were applied. Down the column, the shoot
197
Cu decreased with increasing fraction of TSW in soil and was the most in
plants cultivated on 5 % while being the least in those cultivated on 20 %.
Table 3.11B.6. The concentration of Category-II Metals (mgkg-1
) observed in SHOOT of 82-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Cd
0* BDL 5bDE
1.1 7aDE
1.2 8aDE
1.2 4.2
0 BDL 6aDE
0.7 6aDE
1.4 7aDE
0.8 3.7
5 60bA
1.7 70abA
1.9 65bA
2.5 85aA
1.9 26
10 15cdCD
1.2 30bC
1.9 25bcCD
1.4 40aC
2.1 11
20 10dCD
1.2 25bC
2.8 30aCD
1.8 35aC
2.1 9
LSD0.05 11 18 21 28
Cr
0* 5cDE
1.3 7bDE
1.4 10aE
1.1 12aE
1.1 2.6
0 4cdDE
1.1 8bDE
1.7 12aE
1.5 13aE
1.2 3.1
5 180dA
2.9 230cB
2.3 280cA
2.9 580aA
2.4 115
10 115dBC
2.2 295cA
2.5 270cA
2.9 490aAB
2.5 98
20 70dCD
1.7 190bcBC
2.9 165bcC
2.2 375aBC
2.3 85
LSD0.05 45 57 59 95
Cu
0* 5cDE
0.9 8bcE
1.1 10bE
0.8 15aDE
1.7 2.2
0 3cdDE
0.7 5cE
0.8 8bE
0.5 10aDE
1.2 3.1
5 180dB
2.8 225cBC
2.3 210cBC
2.7 350aAB
2.5 87
10 245cdA
2.4 390aA
1.9 355bA
3.4 425aA
3.9 105
20 70cCD
1.7 115bD
1.3 95bD
2.8 140aCD
2.6 78
LSD0.05 55 62 58 67
Fe
0* BDL 2bDE
0.7 3bDE
0.4 5aD
1.1 2.9
0 BDL 3bcDE
0.6 4bcDE
0.5 8aD
0.9 1.9
5 20dA
1.7 40cB
1.8 35cAB
1.2 70aA
1.8 21
10 15cdB
1.5 55aA
2.2 40bcA
2.3 60aA
2.2 18
20 10dC
1.1 30bcC
2.5 25bcC
1.9 55aAB
2.7 11
LSD0.05 2.6 11 18 21
Mg
0* BDL 8bcCD
1.9 10aC
1.5 12aCD
1.8 3.2
0 BDL 7cCD
1.6 8cC
1.2 15aCD
1.3 4.1
5 20dA
0.9 30cdA
1.8 25cdA
1.2 70aA
1.9 27
10 15dB
2.3 20cdB
1.1 15cdB
1.5 65aA
1.9 19
20 10dC
1.4 25cAB
1.2 20cAB
1.6 45aBC
1.4 16
LSD0.05 2.5 4.6 5.9 8.2
Ni
0* BDL BDL BDL BDL -
0 BDL BDL BDL BDL -
5 BDL 5bcAB
0.8 7aA
1.5 8aB
1.7 1.8
10 5bcAB
0.5 7bA
0.7 8abA
0.4 10aA
1.2 1.9
20 7cA
0.8 5cAB
0.6 6cAB
0.5 12a 1.3 3.5
LSD0.05 0.9 0.6 0.8 1.7
Zn
0* 5dCD
0.8 15cD
2.3 10cCD
2.7 25aD
3.2 3.5
0 10cdCD
1.4 20bcD
1.7 15cCD
1.4 30aD
1.3 4.1
5 90dB
2.3 170cA
1.2 160cAB
1.3 310aA
1.7 78
10 125cdA
1.4 150cdA
1.5 190cA
1.5 320aA
1.1 85
20 80cdB
2.1 125cB
1.9 170cA
1.6 280aAB
1.7 89
LSD0.05 28 32 38 42
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference
198
3.11B.3.4.4 Fe in shoot
The application of fungal inoculations to the soil cause increased
uptake of Fe in plant shoot and such an increase was observed along the row
in Table 3.11b.6. The maximum shoot Fe uptake effects was incurred by the
F1 + F2 than any of the individual fungal inoculations.
Down the columns for fungal treatments where soil was mixed with
TSW, the shoot Fe decreased with increasing percentage of TSW in soil.
3.11B.3.4.5 Mg in shoot
The shoot Mg concentration observed in plants after they were
harvested and it appeared that application of fungi in soil cause an increased
uptake as compared to those plant harvested form soil with no fungi. The
combined application of fungi incurring maximum uptake effect and those with
no fungi had the least tendency for phytoextraction.
Moving along the column, the shoot Mg observed to decrease with
increasing percentage of TSW in soil and thus it was accumulated to the most
in plants from 5 % and the least being extracted by plants from 20 %. Such
variation was observed for all the fungal treatments.
3.11B.3.4.6 Ni in shoot
The shoot Ni was BDL in plants from 0* and 0 (% TSW-Soil) with any
of the fungi inoculated in soil. However, for 5, 10 and 15 %, its concentration
increased in plant shoots along the row of table with the application of fungal
inoculums.
Down the column of Table 3.11B.6, the shoot Ni uptake decreased with
increasing percentage of TSW in soil, although negligible amounts of uptake
were observed.
3.11B.3.4.7 Zn in shoot
The Zn shoot concentration increased in plants applied with fungal
inoculums and this trend could be observed along the row for all the TSW-Soil
mixtures.
199
The increasing percentage of TSW in soil caused increase in plant
shoot Zn uptake in 10 % but a decrease was observed in 20 % as compared
to plants from 5 %. Such variations were observed for all the fungal
treatments down the column of Table 3.11B.6.
3.11B.3.5 Category-II metals in plant ROOT
The Category-II metals i.e. the AAS detected metals in plant root
exhibited variation in response to the fungal inoculations as well as to the
increasing percentages of TSW in soil, as given in Table 3.11B.7.
200
Table 3.11B.7. The concentration of Category-II Metals (mgkg-1
) observed in ROOT of 82-
days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
Parameters TSW-Soil (% w:w) Fungal treatments
LSD0.05 C F1 F2 F1+F2
Cd
0* BDL BDL 2bD
0.6 4aD
1.2 0.5
0 BDL BDL 2bD
0.8 5aD
0.8 0.3
5 30bcA
1.3 40abA
2.4 25cA
1.5 50aA
2.2 11
10 10bcC
1.2 15bCD
1.7 20abB
1.3 25aC
1.4 18
20 8bC
1.8 10bD
1.2 12aBC
1.3 15aCD
1.4 9
LSD0.05 3.8 3.9 5,7 9.6
Cr
0* BDL 2cD
0.7 4bcD
1.2 8aE
1.3 1.6
0 BDL 3cdD
0.6 5cD
1.1 10aE
1.1 2.2
5 110cA
1.8 125cA
1.4 115cA
1.9 240aA
2.2 78
10 50dC
1.3 110bcA
2.4 90cAB
2.3 210aAB
2.3 56
20 25dD
1.9 60cC
1.5 55cC
1.3 110aCD
2.5 49
LSD0.05 21 27 31 54
Cu
0* BDL 2cdD
0.4 4cDE
0.2 8aDE
0.6 3.2
0 BDL 4abD
0.5 5aDE
0.5 6aDE
0.8 2.8
5 85bAB
1.7 110abA
1.3 90bB
1.9 120aBC
2.1 34
10 110bA
3.5 125abA
2.7 130abA
3.3 155aA
2.3 49
20 30cdCD
2.8 70bBC
2.5 65bBC
1.8 85aC
1.9 28
LSD0.05 22 29 38 43
Fe
0* BDL BDL BDL 2DE
0.6 -
0 BDL BDL BDL 4DE
0.8 -
5 15cdA
2.4 25cA
2.8 20cAB
1.6 45aA
2.9 8
10 10cB
3.1 30aA
2.7 25bA
1.4 35aB
2.2 19
20 5dC
1.9 20bB
1.5 15bcBC
1.1 25aC
1.3 11
LSD0.05 2.9 3.8 5.2 9.3
Mg
0* BDL 2bcCD
0.6 4aC
0.7 5aCD
0.8 2.1
0 BDL 3bcCD
0.9 5bC
0.6 8aC
0.9 1.8
5 15cA
1.1 15cA
1.1 13cA
1.3 30aA
2.5 8
10 10cdB
1.5 12cdAB
1.6 10cdA
1.2 35aA
1.8 6
20 5dC
0.8 10cB
1.7 12bcAB
1.2 20aB
1.1 5.7
LSD0.05 2.1 3.1 5.3 7.6
Ni
0* BDL BDL BDL BDL -
0 BDL BDL BDL BDL -
5 BDL 2cB
0.4 2cA
0.9 5aA
0.8 2.2
10 3cA
0.4 4bcA
0.7 3cA
0.8 6aA
0.5 3.8
20 2cdA
1.2 3cA
1.5 2cdA
1.8 6aA
1.3 1.8
LSD0.05 0.4 0.8 0.5 0.6
Zn
0* BDL 10bcD
0.8 8cD
1.1 15aE
1.2 1.7
0 2dDE
0.8 10aD
1.2 12aD
1.2 10aE
1.1 1.5
5 55cdBC
1.5 110abA
1.1 90bcA
1.4 130aC
1.9 37
10 70dA
1.3 115cdA
1.8 95cdA
1.6 275aA
2.7 75
20 50cdBC
1.7 80bcBC
2.5 75bcB
2.3 140aC
2.2 33
LSD0.05 5.4 4.2 17.2 19.2
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant
difference
201
3.11B.3.5.1 Cd in root
In 0* and 0 (% TSW-Soil) with C and F1 inoculations, the plant root Cd
observed to BDL; however, detected in soil treatments of F2 and F1 + F2. For
5, 10 and 20 %, the root Cd was higher in plants applied with fungi than those
applied with no fungi. The F 1 + F2 showed the best assistance in root Cd
uptake than any individual or no fungal application.
The increasing percentage of TSW in soil lowered down the root Cd
concentration resulting maximum accumulation in plants from 5 % and
minimum uptake in plants from 20 %.
3.11B.3.5.2 Cr in root
Like root Cd, the concentration of Cr in root increased along the row of
Table 3.11B.7.and it was due to application of fungi in rhizoshpere of plants.
Those applied with no fungi, showed the least Cr accumulation in root.
The increasing level of TSW in soil added to the soil Cr; however,
plants were able to uptake the maximum concentration from 5 % as compared
to 10 and 20 % treatments of soil. Such variation in plant root was observed
for all of the fungal treatments.
3.11B.3.5.3 Cu in root
An increase in the root Cu was observed in plants harvested from soil
inoculated with fungi as compared to those harvested from soil with no fungi.
The F1 + F2 incurred the maximum root Cu accumulation than any of the
individual fungi as well as C.
The root Cu observed to decrease in 20 % treatments but increased in
10 % as compared to 5 %; although its level in plant roots from all the TSW-
Soil mixtures was significantly higher than those harvested from C.
3.11B.3.5.4 Fe in root
Moving alongside the row while analyzing effect of different fungal
treatments, the root Fe observed to be BDL in soil (0 %) with C, F1 and F2
treatments. The plants from 0* % with F1 + F2 had the lowest root Fe as
compared to plants from any of the single or combined application of fungi for
202
the rest of TWS-Soil mixtures i.e. 10 % and 20 %. The root Fe accumulation
observed to be the maximum (45 mgkg-1) in 10 % with F1 + F2 and
significantly higher than those harvested from any of the treatments with
single or no fungal inoculations.
Inside columns, the maximum Fe uptake by roots was observed in 10
% with F1 + F2 while the least value of metal uptake was observed in 0 % as
shown in Table 3.11B.7.
3.11B.3.5.5 Mg in root
While analyzing the fungal application effect on plants along the rows,
it was observed that the Mg root concentration increased in plant roots with
fungal application as compared to C. for both of 0 % i.e. in soil, the value was
found to be BDL with C. The F1 + F2 plants displayed the maximum root Mg
concentration than those from C as well as F1 and F2. However the F2
inoculations gave better results than F1.
For the columns, the root Mg accumulation was found to be highest in
10% with F1 + F2 where the metal accumulation was observed to be 35
mgkg-1 while minimum concentration 2 mgkg-1 in 0* % with F1 treatment was
noted as shown in Table 3.11B.7.
3.11B.3.5.6 Ni in root
The root Ni concentration was found to be BDL for all fungal treatments
in 0* and 0 % i.e. soil. However there was increased metal accumulation
along the row with the application of fungal inoculations and found to be the
maximum in plants applied with combined application of both of the fungi
while being the minimum in C with no fungus added.
Within columns, the plants from 10 and 20 % TSW-Soil mixtures had
the maximum root Ni level than 5 % for all of the fungal treatments. There was
maximum (6 mgkg-1) accumulation of metal was noted in 10 and 20% with F1
+ F2 while minimum uptake (2 mgkg-1) was observed in 5 % with F1.
203
3.11B.3.5.7 Zn in root
The root Zn accumulation observed to increase in pots applied with
fungal inoculations than C and found to be the maximum in treatments applied
with both of the fungi and being the minimum with no fungal applications.
Again as with most of the above discussed metals F2 performed better than
F1 as far as metal accumulation efficiency of the plant is concerned.
For different TWS-Soil mixtures along the columns, the root Zn
concentration increased with increasing percentage of TSW in soil with every
fungal treatment. The maximum accumulation was noted in 10 % with F1 + F2
i.e. 275 mgkg-1 and minimum (8 mgkg-1) in 0* % with F2 treatment as shown
in Table 3.11B.7.
3.11B.4 Fungal analyses
The results of estimation of the post-harvest fungal analyses (× 105
c.f.u. g-1 soil) of 82-days old Helianthus annuus cultivated on TSW-Soil
mixtures are shown in Table 3.11B.8. Alongside the row, the c.f.u. increased
with fungal application than C and observed to be the maximum in treatments
with combined application of both of the fungi. The order of c.f.u. abundance
was F1 + F2 > F2 > F1 > C.
Within a column, the c.f.u. abundance was observed to be highest in 5
% with 8.4 × 105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2.
Table 3.11B.8. The post-harvest fungal analyses (× 105 c.f.u. g
-1 soil) of 82-days old Tagetes
patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).
TSW-Soil (% w:w) mixture and its type
Treatment LSD0.05
C F1 F2 F1+F2
0* 0.3cdD
0.27 0.7cdDE
1.1 0.9cdD
1.3 2.9aDE
1.9 0.9
0 0.4cdD
1.5 1.0bDE
1.9 1.2bD
1.6 1.8aDE
2.2 0.8
5 3.2cA
2.2 4.8bcA
1.8 5.1bcA
1.21 8.4aA
2.13 1.9
10 3.4cA
2.4 3.8cB
2.3 4.9cA
2.07 7.8aA
2.51 1.5
20 2.4cBC
2.3 3.1bBC
3.2 3.7aBC
2.83 4.7aCD
3.20 0.7
LSD0.05 0.8 1.1 1.4 1.8
0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference
204
3.11B.5 Meta-analytical perspective
The meta-analytical indices of plant-metal-TSW interactions for
Category-I and Category-II metals are as under:
3.11B.5.1 Category-I metals translocation index (%)
The plant translocation index values were also recorded for Category-I
metals those detected by flame photometer i.e. Ca, K and Na shown in Table
3.11B.9.
In case of Ca, maximum value was observed in 10% with C i.e. 446.6
% while the minimum value was recorded (207.1 %) in 5 % with C.
For K, the maximum translocation index value was calculated in 10%
with C i.e. 450 % being the minimum 171.4 % in 20% with F2.
Table 3.11B.9. The Category-I metals translocation index (%) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca
5 207.1 252.6 233.3 227.2
10 446.6 408.3 381.8 343.7
20 371.4 550 522.2 436.3
K
5 275 260 377.7 330.7
10 450 328.5 325 300
20 433.3 375 171.4 255.5
Na
5 233.3 250 244.4 263.6
10 350 328.5 328.1 344.4
20 250 289.4 227.2 300
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Na, the maximum value 350 % was observed in 10% with C
treatment and least value was recorded in 20 % for F2 (227.2 %).
3.11B.5.2 Category-II metals translocation index (%)
The plant translocation index is given in Table 3.11B.10. For Cd, the
maximum Translocation index (260 %) was noted in 5% with F2, while
minimum value was recorded 125 % in 20% with C and in 10% with F2.
In case of Cr plants showed maximum metal translocation efficiency
(340.9 %) in 20% with F1 + F2 and minimum 163.9 % in 5 % with C.
205
For Cu, the maximum translocation index values was recorded to be
312 % for 10% with F1, while minimum value was recorded in 20 % with F2
treatment i.e. 146.1 %.
For Fe, the maximum translocation index values was recorded to be
220 % for 20 % with F1 + F2, while minimum value was recorded in 5 % with
C treatment i.e. 133.3 %.
Table 3.11B.10. The Category-II metals translocation index (%) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd
5 200 175 260 170
10 150 200 125 160
20 125 250 250 233.3
Cr
5 163.6 184 243.4 241.6
10 230 268.1 300 233.3
20 280 316.6 300 340.9
Cu
5 211.7 204.5 233.3 291.6
10 222.7 312 273 274.1
20 233.3 164.2 146.1 164.7
Fe
5 133.3 160 175 155.5
10 150 183.3 160 171.4
20 200 150 166.6 220
Mg
5 133.3 200 192.3 233.3
10 150 100 150 185.7
20 200 250 166.6 225
Ni
5 0 250 350 160
10 166.6 175 266.6 166.6
20 350 166.6 300 200
Zn
5 163.6 154.5 177.7 238.4
10 178.5 130.4 200 116.3
20 160 156.2 226.6 200
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Mg the plants showed highest value in 20% with F1 (250 %),
while the least value for this metal was recorded in 10% with F1 i.e. 100 %.
For Ni, there was 350% translocation index value recorded in 20% with
C and 0 value was observed in 5 % with C.
As far as the Zn is concerned there was maximum translocation index
recorded in 5 % with F1 + F2 treatment i.e. 238.4 % while minimum value was
recorded in 10% with F1 + F2 i.e. 116.3 % as shown in Table 3.11B.10.
206
3.11B.5.3 Tolerance index (TI)
In shoots TI values were found to be highest in 5 % with F1 + F2 (1.61)
while minimum value was recorded to be 0.83 in 20% with F1 as shown in
Table 3.11B.11.
Table 3.11B.11. The translocation index (%) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
TI Shoot*
5 1.13 1.41 1.40 1.61
10 1.04 0.87 1.11 1.40
20 0.87 0.83 0.91 0.96
TI Root*
5 1.33 1.24 1.26 1.28
10 1.08 1.18 1.06 1.04
20 0.93 0.67 0.83 0.77
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of TI in roots 1.33 was recorded as maximum for plants grown
in 5 % with C, while 0.67 was recorded as minimum value in 20% with F1.
3.11B.5.4 Category-I metals specific extraction yield (SEY %)
The SEY % for Category-I metals i.e. Ca, K and Na showed in Table
3.11B.12.
Table 3.11B.12. The Category-I metals specific extraction yield (SEY %) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Ca
5 13.69 23.10 21.27 30.25
10 14.08 18.59 15.14 24.65
20 6.11 9.96 10.76 12.88
K
5 18.51 25 28.47 41.79
10 11.70 18.29 18.68 27.77
20 4.32 5.42 5.30 9.52
Na
5 11.62 13.94 12.55 17.77
10 5.60 6.46 5.98 9.11
20 1.21 1.66 1.62 2.75
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Ca, there was maximum value 24.65 % was recorded for
plants grown in 10 % with F1 + F2, and minimum 6.11 % in 20 % with C.
In case of K, plants cultivated in 5 % TWS-soil showed highest value of
SEY% (41.79 %) with F1 + F2 and minimum in 20 % with C i.e. 4.32 %.
207
The highest value for Na was recorded in 5 % with F1 + F2 (17.77 %)
while minimum values was found to be 1.21 % in case of 20% with no fungal
inoculum i.e. C.
3.11B.5.5 Category-II metals specific extraction yield (SEY %)
The SEY (%) was calculated in Category-II metals that were detected
by AAS as shown in Table 3.11B.13. Overall a similar kind of trend was seen
for all metals in various fungal treatments along the row, that the SEY %
values increased with the application of fungal inoculum and highest value
was observed for F1 + F2 treatment.
Table 3.11B.13. The Category-II metals specific extraction yield (SEY %) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.
Parameters TSW-Soil (% w:w) Fungal treatments
C F1 F2 F1+F2
Cd
5 3.55 4.56 3.76 6.13
10 0.38 0.69 0.69 1.02
20 0.20 0.40 0.48 0.57
Cr
5 3.72 4.70 5.19 11.66
10 1.64 4.21 3.74 8.09
20 0.61 1.63 1.45 3.61
Cu
5 25.85 34.53 30.30 60.25
10 19.61 29.76 27.24 34.31
20 1.95 3.73 3.27 4.67
Fe
5 16.66 34.21 29.72 67.64
10 5.20 17.52 13.26 20.21
20 1.68 5.95 4.81 9.87
Mg
5 12.06 16.66 14.61 40
10 4.23 6.89 4.42 18.86
20 1.63 4.02 3.69 7.83
Ni
5 0 28 40.90 68.42
10 17.77 27.5 28.94 53.33
20 9.47 11.42 9.41 27.69
Zn
5 11.78 29.16 25.51 49.43
10 12.03 16.98 17.92 49.17
20 6.87 11.91 14.20 44.21
C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii
In case of Cd the maximum value for SEY % was recorded in 5 % with
F1 + F2 treatment i.e. 6.13 %, while minimum (0.20 %) was observed in 20%
with C.
208
Similarly the SEY % value for Cr was found to be highest (11.66 %) in
5 % with F1 + F2 and minimum (0.61 %) was recorded in 20% with no fungal
inoculum i.e. C.
For Cu again the maximum value was observed for 5 % (60.25 %) with
combined fungal inoculum F1 + F2 and least value (1.95 %) was recorded in
20% with C.
As in case of above mentioned metals again the highest (67.64 %) and
lowest (1.68 %) values were recorded in 20% with F1 + F2 and 20 % with C
respectively for Fe.
As far as the highest and lowest values are concerned there was a
same trend seen in case of Mg, where plants in 5 % showed highest SEY%
value (40 %) with F1 + F2 and being minimum (1.63 %) in 20% with C having
no fungal inoculum.
There was maximum value for Ni was calculated (68.42 %) in 5 % with
combined fungal inoculum, while minimum value (9.47 %) was recorded in 20
% for C. The value was found to be 0 in 5 % with C.
In case of Zn the maximum value was noted (49.43 %) for 5 % with F1
+ F2 and minimum value (6.87 %) in 20 % with C.
Chapter 4
Discussion
209
CHAPTER 4
DISCUSSION
4.1 Physico-chemical properties of TSW and garden soil
Leather processing with several salts contained with Category-I and II
metals have been used to convert raw leather into tanned leather. Pre-tanning
has been involved with numerous operations, such as curing by dehydration
of skins, soaking for rehydration of skins, liming of skins for swelling, deliming
for deswelling, pickling involving acidification of skins and depickling carried
out by basification (Aravindhan et al., 2007). Furthermore, the skins or hides
are subjected to wide variations of pH. A huge amount of salts with and
without metals, used during different steps of tanning process, become part of
TSW. The TSW originating from Cr tanning plants has mainly been composed
of tanned residue, hair, lime, and salts of Cr (Amir et al., 2008). All of the solid
wastes resulting from tanneries of Kasur have predominantly been contained
with such salts and metals. The lack of sophistication and environmental
consideration in handling of TSW from tanning factories has been
accelerating discharge of untreated heavily polluted TSW on to the
mismanaged dumping sites. Even tannery wastewater when accidently get
spilled into TSW heaped within a tanning factory, high concentrations of
chlorides, sulfates, Cr or tannins and other minerals can be carried to the
TSW (Thanikaivelan et al., 2000).
The high valued physico-chemical properties of TSW such as, pH,
ECe, bicabonates, and chlorides has been resulting due to the incorporation
of salts in the TSW and its untreated disposal at the dumping site. The mixing
of TSW in soil aggravates the values of physic-chemical properties of soil and
putting it to stressed situation. The increasing quantity of TSW in soil
intensified the soil stress and further additions of TSW beyond 20 % (TSW-
Soil) made the soil completely unfit for germination of selected plant seeds.
The high metal (Category-I and II) contents of the TSW has also been
alarming in terms of polluting soil. The higher values of Cr and other heavy
metals in representative samples of TSW, collected from the dumping site,
indicate that majority of tanneries working in Kasur have been the chrome
tanning methods due to its high speed and cost effectiveness. But the
210
environmental hazards associated with heavy metals released from tanning
have been left unnoticed. The high content of heavy metals in TSW has been
reported to make their treatment by biological processes difficult (Amir et al.,
2008).
Plants cultivated on heavy metal polluted soils cannot usually access
the total pool of a metal present in the growth substrate. The fraction of a
metal which plants can absorb is known as the available or bioavailable
fraction (Fageria et al., 1991; Marschner, 1995; Whitehead, 2000). The metals
soluble in the soil solution are the metals directly available for plant uptake
and other soil metal pools are less available (del Castilho et al., 1993). The
metals present in soil are in dynamic equilibrium and have been divided into a
number of fractions including; the soluble metal in the soil solution, metal-
precipitates, metal sorbed to clays, hydrous oxides and organic matter, and
metals within the matrix of soil minerals (Norvell, 1991).
Metal speciation, on the basis of kind of solvent used to check the
metal availability, has been helpful in determining the fraction of plant
available metals (Iwegbue et al., 2007). In the current study, the different
pools of metal in TSW have been carried out to find out the plant available
fraction of different metals. There are several factors that can affect the
concentration and speciation of metals in the soil solution including the plant
available fraction of metals such as, the total metal present in the soil, pH,
clay and hydrous oxide content, bulk density, organic matter and redox
conditions. In the current study, the water soluble and DTPA-extractable
fractions of metals have been found to increase in accordance with increase
in pH, ECe, NaCl (%), bicarbonates as well as chlorides. Parallel to that, a
shift in total metal contents of the TSW as well as its mixtures with soil has
been reported (Reichman, 2002). However, the increasing percentage of
TSW in soil significantly reduces the bulk density of the resulting TSW-Soil
mixtures. The water soluble, DTPA-extractable and total fraction of both
Category-I and II metals have been observed to increase with decreasing bulk
density. Perhaps, the increase in metal fractions could have been due to
increasing volume of air spaces in the soil substratum as well as presence of
inert fibrous leather hide ingredients of crushed TSW.
211
The simple linear regression between bulk densities (gcm-3) and
Category-I metals’ water-soluble as well as DTPA-extractable fractions
showed that both of the fractions of Category-I metals were strongly
dependent on variations in bulk density, as given in Figure 4.1.1. The high
Figure 4.1.1: Simple non-linear regression between bulk density (gcm-3
) of TSW-soil mixture and
different fractions of Category-I metals (mgkg-1
): (A) between bulk density and water-soluble fraction, (B) between bulk density and DTPA-extractable fraction.
Co
nce
ntr
atio
n (
mgk
g-1)
of
wat
er-s
olu
ble
fra
ctio
n
Co
nce
ntr
atio
n (
mgk
g-1)
of
DTP
A-e
xtra
ctab
le f
ract
ion
Variation in bulk density (gcm-3
) of soil mixed with TSW
B
A
212
values of regression co-efficient R2 for both fractions of Category-I metals
indicates that they were highly dependent on the bulk density variations.
Similarly for Category-II metals, both of the fractions were strongly
dependent on bulk density variations, as shown in Figure 4.1.2. The values of
regression co-efficient R2 for both fractions (i.e. water-soluble and DTPA-
extractable) of Category-I were highly dependent on variations on bulk density
variations.
Figure 4.1.2: Simple linear regression between bulk density (gcm-3
) of TSW-soil mixture and different fractions of Category-II metals (mgkg
-1): (A) between bulk density and water-soluble fraction, (B)
between bulk density and DTPA-extractable fraction.
Co
nce
ntr
atio
n (
mgk
g-1)
of
wat
er-s
olu
ble
fra
ctio
n
Co
nce
ntr
atio
n (
mgk
g-1)
of
DTP
A-e
xtra
ctab
le f
ract
ion
Variation in bulk density (gcm-3
) of soil mixed with TSW
A
B
213
The total fraction of Category-I and II metals was directly dependent on
percentage of TSW in soil mixture, as given in Figure 4.1.3. The correlation
between TSW percentage (% w:w) in soil and total fraction of Category-I and
II metals indicated that the total fraction of both metals increased in
accordance with the increase in TSW percentage, as is obvious from the high
values of coefficient of correlation (r2).
Figure 4.1.3: Correlation between TSW (% w/w) of TSW-soil mixture and different fractions of Category-II metals (mgkg
-1): (A) between bulk density and water-soluble fraction, (B) between bulk
density and DTPA-extractable fraction.
Co
nce
ntr
atio
n (
mgk
g-1)
of
tata
l fra
ctio
n o
f m
etal
s
TSW (% w:w) mixed in soil
The total metal has been reported to indicate the maximum pool of
metal in the soil with other factors being important in determining how much of
214
this soil pool will be available to plants (Wolt, 1994). In addition, researchers
have found that the total metal may correlate with the bioavailable soil pools
of metal but is still inadequate by itself to reflect bioavailability (Lexmond,
1980; Sauve et al., 1996; McBride et al., 1997; Sauve et al., 1997;
Peijnenburg et al., 2000).
Such studies of tannery effluents in relation to soil and water have
been carried out in Pakistan (Tariq et al., 2005; Tariq et al., 2006) but the
research work has been carried out for the first time for TSW. Overall, the
average concentrations of Category-II metals were within the above range of
the permissible limits of EPA (USEPA, 1999; Bosnic et al., 2000). Any shift in
the heavy metals carried as TSW by natural hazard or anthropogenic
activities can expand the exposure spectrum heavy metals to the biotic life.
215
4.2 Isolation and identification of TSW representative fungi
The isolation of a fungus on PDA from an industrial waste
contaminated with heavy metals (Ezzouhri et al., 2009) and application of
such isolated fungi back into the same system (autochthonous fungi) for
fungal bioaccumulation purposes has been carried out (Zapotoczny et al.,
2007). The isolation of fungi such as Trichoderma from TSW of KTWMA,
Kasur, has been done (Bareen and Nazir, 2010). Earlier, isolation from other
KTWMA tannery wastes such as, tannery effluent has been carried out
(Bareen et al., 2011). There have been different criteria to define a newly
isolated fungal species. The most common of which has been phenotypic,
being the most classic approach describing a fungus species on the basis of
the morphological characteristics only (Inui et al., 1965). In the current study,
the identification and characterization of fungi isolated from TSW was carried
out according the selected (only of the morphological) formal requirements
and best practices for the publication of descriptions of new fungal species
(Seifert and Rossman, 2010); while bringing legitimacy and validity at the First
Fungal Bank of Pakistan, University of the Punjab, Lahore, Pakistan. The
isolated fungi were obtained after rigorous procurement of powdered TSW on
selected fungal nutrient media. The identifications were mainly based on
color, physical growth attributes of fungal colonies as well as morphological
structures following Schipper and Stalpers (1984).
The TSW representative fungal species, among genera of Aspergillus
and Fusarium, isolated during the current study have also been isolated from
heavy metal polluted soils by Iram et al., (2009) while using similar fungal
nutrient media. As the microbiological and molecular marker requirements for
the identification of isolated fungi were not fulfilled, the rigorous isolation with
repeated appearance of the same 13 species of fungi, during the isolation
process, was taken as the criteria for claiming isolated fungi as the
representative fungal flora of TSW from KTWMA, Kasur. The described
number of fungal species during current work is not been the true proportion
of actually existing fungal flora, as the fungal flora of a contaminated site is
never limited to a few species. (Hawksworth, 2001). However, future research
will be focused on expanding the process of isolation on the bases of testing
216
further fungal nutrient media, identifications based on microbiological features
as well as validations based on DNA markers.
4.3 Screening and selection of heavy metal resistant autochthonous
saprobic fungi
The fungi have known to be heavy metal tolerant (Baldrian, 2003;
Gavrilesca, 2004). The screening process of fungi during mutual interaction
studies have been more appropriate on selected nutrient media taken in petri
plates (Zapotoczny et al., 2006). During the current study, the screening
process of fungi on the basis of mutual interaction has been carried on 2 %
MEA taken in petri plates.
The variability of fungal micoflora in the contaminated growth media
and their adaptation to the heavy metal stress has mainly been driven by
availability of nutrients, pH, concentration of metal ions as well as physical
conditions of the contaminated media (Okoronkwo et al., 2005). During the
current study, the TSW representative fungi tested for their heavy metal stress
tolerance i.e. Trichoderma pseudokoningii, Aspergillus niger, Alternaria
alternata and Fusarium sp. were found to be the best adapted to the multi-
metal contaminated growth media on the basis of their highest tolerance index
(TI), while Aspergillus parasiticus were found the least tolerant to the provided
nutrient conditions. The most reported fungi from the heavy metal pollutes
sites that seems to have adapted themselves to such situation include
Aspegillus, Fusarium and Penicillium, and thus may be called heavy-metal-
stress-adapted fungal flora of Pakistan (Zafar et al., 2007).
The origin, type, source and duration of exposure of fungi are the main
factors that regulate the adaptation of fungi to the heavy metal stressed media
(Anand et al., 2006). The fungi isolated during the current study had taken
their origin from TSW. The highest acclimatization of the four fungal species
to the heavy metals on the basis of their origin enabled them to show high TI
values throughout their recurrent cultivation on growth media impregnated
with TSW extracts taken with water. The species of Aspergillus (Valix et al.,
2001; Anahid et al., 2011); Aspergillus and Fusarium (Iram et al., 2009);
Aspergillus and Trichoderma (Parameswari, et al., 2010); Aspergillus,
Fusarium and Alternaria (Ezzouhri et al., 2009); Aspergillus, Fusarium,
217
Alternaria and Trichoderma (Zafar et al., 2007) have been reported to show
high TI when cultivated on PDA impregnated with multi-metal stress. The
least reduction in colony size of tolerant fungi on TSW impregnated growth
media as compared to their corresponding replicates on growth media with no
TSW extract (i.e. control) indicated that A. niger was highly tolerant to multi-
metal stress including Cu and Cd. Similar findings have been reported by
Saleh and Al-Sohaibani (2011) where A. niger was identified as tolerant to Cu
and Cd stress; and by Shugaba et al. (2010) where A. niger and A. flavus, A.
parasiticus has been shown as tolerant to Cr (VI).
The screened autochthonous saprobic fungi during current study were
able to show maximum fungal biomass under heavy metal stress. Therefore,
the procurement of final selected fungi during the screening process would
show a high potential for the bio-reinforcing tendency on their inoculation in
the phytoextraction trials to be conducted with multi-metal (from TSW mixing)
contaminated soils.
218
4.4 In vitro fungal mutual interaction studies for Category-II metal
tolerance
Although based on hyphal length variation and not on radial colony
distribution, the in vitro interaction studies have been conducted by Arriagada
et al. (2009). During in vitro interaction studies, the prevalence of Aspergillus
niger, Trichoderma pseudokoningii, Alternaria alternata and Fusarium sp. over
rest of the 9 fungal sp. could be attributed to several features. Aspergillus
niger and T. pseudokoningii had acquired a better tendency to utilize the
available nutrients in growth media, more readily and efficiently, than their
respective competitors fungi under the prevalent culturing conditions. Thus,
their mycelial growth occupied greater area of the petri plate during the
observation period. Similarly, Alternaria alternata and Fusarium sp., although
not as efficient as A. niger and T. pseudokoningii, were able to supersede
their competitor fungi and consumed the nutrients from the growth media at
faster pace.
The antagonistic potential of a fungus could also be helpful in
suppressing the growth of competitor fungi. It has been reported that
Trichoderma can be a strong antagonist and completely suppresses the
growth of Fusarium sp. (Dubey et al., 2007; Gupta and Mishra, 2009),
Aspergillus (Lone et al., 2012; Usha et al., 2012) and Alternaria alternata
(Gveroska and Ziberoski, 2012). The fungi that showed radial colony
expansion than their competitor fungi could also be more sensitive to the
growth media and incubation conditions than those which thrived extensively
under the same conditions. Thus it could be a matter of conduciveness of
conditions under the same culturing conditions making these fungi become
overlap or become overlapped by the other.
Conclusively, Trichoderma and Aspergillus proved to be the best
antagonists in suppressing the growth of other fungi (Usha et al., 2012). Thus,
both of them were selected for their assistance potential in phytoextraction
studies with Tagetes patula.
219
4.5 In situ mutual growth interaction studies for screened Category-II
metals tolerant fungal isolates with Tagetes patula in soil
The autochthonous saprobic fungi have been found to show significant
in situ mutual interaction studies with Eucalyptus globulus compared to their
competitor fungi (Arriagada et al., 2009). During the current study, the
interaction of Trichoderma pseudokoningii and Aspergillus niger (F1 + F2)
showed highly significant results in terms of mutual interaction by enhancing
plant biomass and chlorophyll content in Tagetes patula encircled red (Figure
4.5.1). Synergistic effects have been shown by saprobic fungi through plant
dry biomass increase when applied in situ to the soil (Fracchia et al., 1998;
Arriagada et al., 2004). In the present study, the production of higher biomass
in marigold due to inoculation with F1 + F2 on repetitive basis in soil provided
strong grounds for selecting both the fungi for phytoextraction trials in
greenhouse and in the field.
Figure 4.5.1: The growth variation observed in Tagetes patula in response to different fungal
inoculations in soil
220
4.6 Screening of heavy metal tolerant ornamental plant species for
phytoextraction of TSW-Soil mixtures
Over 500 Angiosperms have found to be hyperaccumulators
constituting 0.2 % of the total heavy metal (Sarma, 2011), after passing
through the screening process for heavy metal tolerance. Such screening of
plants for heavy metal tolerance has been performed by several scientists
(Hakmaoui et al., 2006; Kachenko et al., 2007). The adaptation of plants to
the heavy metal polluted soil starts from a healthy germination response,
contained in the inherited, physiological, molecular, genetic and ecological
traits. In the present study, marigold and sunflower showed the maximum
germination as compared to the rest of the test plants. According to Sarma
(2011), there have been several important criteria in selecting any plant as a
suitable candidate for phytoremediation such as high tolerance for targeted
heavy metals, tendency for adequate uptake, accumulation and translocation,
high biomass yield, tolerance for abiotic stress, preferred habitat
acclimatization and tolerance for high pH and salinity. During this study,
results have indicated that both marigold and sunflower had the requirements
for being multi-metal hyperaccumulators, as has been given by Cutright et al.
(2010) for sunflower and Sun et al. (2011) for marigold. Both sunflower and
marigold were selected as hyperaccumulator and taken along for further
experimentations.
221
4.7 Experiments with marigold cultivated on autoclaved and non
autoclaved TSW-Soil mixtures and inoculated with selective
autochthonous saprobic fungi
The experiments with autoclaved soil (AS) were conducted to find
whether inoculation of TSW-Soil mixtures with screened autochthonous
saprobic fungi i.e. F1 (Aspergillus niger) and F2 (Trichoderma pseudokoningii)
have been responsible for bio-reinforcing of marigold phytoextraction or not.
The autoclaving of TSW-Soil mixtures killed all of the soil microbes in the
experimental pots and had only two of the inoculated fungi in plant
rhizosphere.
A trend of increase in all selected biochemical parameters was
observed in plants harvested from AS applied with fungal inoculations as
compared to those harvested from AS with no inoculations and such
variability was observed in all the soil treatments. The increase in production
of antioxidant enzymes SOD and CAT in plants with inoculation was due to
the application of autochthonous saprobic fungi. These findings have been
parallel to the activation of the antioxidant machinery in plants under stress
when inoculated with Trichoderma sp. (Brotman et al., 2013). The plants
applied with Trichoderma in their rhizosphere undergo stimulation of growth
and resistance to a wide range of adverse environmental conditions (Mastouri
et al., 2010; Soresh et al., 2010). The application of combined inoculation of
Trichoderma pseudokoningii and Aspergillus niger enabled the marigold
plants to become tolerant to heavy metals by activating its antioxidant enzyme
system. This is why, the increase in SOD and CAT was observed in plants
from pots with elevated levels of Category-I and II metals. However, those
provided with F1 + F2 inoculations exhibited the highest levels of both of the
antioxidant activities than any of the AS with single or no fungal inoculations.
The experiments with non-autoclaved soil (NAS) were conducted to
find i) validity of both of the screened fungi for inoculation under field
conditions with possible mutual synergism, ii) involvement of other
(unreported) soil microbes other than the inoculated fungi in enhancing the
phytoextraction tendency of marigold, iii) hyperaccumulator tendencies of the
screened fungi for the screening process while being in TSW-Soil mixtures in
222
natural conditions as in in-vitro conditions, and iv) the implications of
application of screened fungi under field conditions. The plants from NAS pots
inoculated with fungi exhibited elevated levels of all the biochemical
parameters than those harvested from NAS with no fungi. Such results have
been reported by Song et al. (2011) where inoculation of saprobic fungus
(Aspergillus niger) enhanced the plant SOD and CAT production as compared
to the un-inoculated control plants. In the present study, chlorophyll contents
observed to decrease with increasing level of metals in TSW-Soil mixture. The
plants from NAS with or without fungal inoculations performed better than
those from AS with or without fungi, respectively in terms of chlorophyll,
soluble protein, SOD and CAT. In this study, the combined application of F1 +
F2 in both NAS and AS pots showed elevated levels of all the biochemical
parameters than those from both NAS and AS with either of the single or no
fungal treatments for of all the TSW-Soil mixtures. The SOD and CAT
production in marigold has been reported to increase by increasing the level
of abiotic stress (Tian et al., 2012). Increase in SOD activity under metal
stress (Cho and Sohn 2004; Zhang et al., 2006) as well as of both SOD and
CAT (Wang et al., 2009) has been reported.
In the present study, the plant chlorophyll content decrease with the
increase in heavy metals concentration in soil. These results are parallel with
decrease in chlorophyll production with increasing concentration of metal in
soil (Zengin and Munzuroglu, 2006; Elloumi et al., 2007). The soluble protein
contents were also noticed to decrease parallel to the decrease in chlorophyll
contents due to increase in metal concentration in soil. It has been reported
that the soluble protein contents decrease with increasing concentration of
metals in soil (Ahmad and John, 2005; Ahmad et al., 2006).
It has also been reported that the free radical species (forms of active
oxygen) may be increased in stress condition, which will enhance the
activities of these detoxifying enzymes (Bhattacharjee, 1997–98; Gallego et
al., 1999). Also, the activities of SOD, CAT, and POD are induced in plants
due to heavy metal exposure (Pereira et al., 2002; Fornazier et al., 2002; Lee
and Shin, 2003; Sk´orzy´nska-Polit et al., 2003–2004; Li et al., 2006).
223
Correlation is the statistical measurement of the relationship among
two variables (Aslam et al., 2012). The substantial mutual variability
relationships between biochemical parameters, dry weight, Na and Cr based
on Pearson correlation coefficient values for T. patula cultivated on non-
autoclaved (NAS) TSW:Soil mixtures was found (Table 4.7.1).
Table 4.7.1. Pearson correlation among selected biochemical properties, dry weight, Cr and Na uptake observed in Tagetes patula cultivated on non-autoclaved soil (NAS) mixed with tannery solid waste (TSW).
Chlorophyll Soluble Protein
SOD CAT Dry
weight Cr Na
Chlorophyll
Pearson correlation
Sig. (2-tailed)
N
1 - 8
0.643*
0.043
8
0.523*
0.046 8
0.524*
0.041 8
0.745**
0
8
0.781**
0 8
0.682*
0.038 8
Soluble Protein
Pearson correlation
Sig. (2-tailed)
N
0.643*
0.043
8
1 -
8
0.213
0.543 8
0.734** 0 8
0.435
0.61
8
0.802** 0 8
0.784**
0.01 8
SOD
Pearson correlation
Sig. (2-tailed)
N
0.523*
0.046 8
0.213
0.543
8
1 - 8
-0.023
0.861 8
0.451
0.54
8
0.572*
0.042 8
0.631*
0.027 8
CAT
Pearson correlation
Sig. (2-tailed)
N
0.524*
0.041
8
0.734**
0
8
-0.023
0.861 8
1 - 8
0.341
0.124
8
0.623*
0.015 8
0.567*
0.031 8
Dry weight
Pearson correlation
Sig. (2-tailed)
N
0.745** 0 8
0.435
0.61
8
0.451
0.54 8
0.341
0.124 8
1 -
8
0.871** 0 8
0.785** 0 8
Cr
Pearson correlation
Sig. (2-tailed)
N
0.781** 0 8
0.802**
0
8
0.572*
0.489 8
0.623*
0.015 8
0.871**
0
8
1 - 8
0.789** 0 8
Na
Pearson correlation
Sig. (2-tailed)
N
0.682*
0.038 8
0.784**
0.01
8
0.631*
0.027 8
0.567*
0.031 8
0.785**
0
8
0.789** 0 8
1 - 8
**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)
The variation in chlorophyll contents with respect to soluble protein
contents, dry weight, Cr and Na uptake showed considerably stronger
224
correlation; however, the chlorophyll contents found have weak correlation
with SOD and CAT values. A relatively strong correlation was found between
soluble protein contents and uptake of Cr and Na. The SOD contents found to
have little bit strong mutual variability relationship with Na while showing weak
correlation with rest of the selected variables. The CAT contents showed
stronger correlation with soluble proteins only, while dry weight of the plant
showed strong correlation with chlorophyll contents, Cr and Na uptake. The
uptake of Cr showed stronger relationship with chlorophyll production of plant,
CAT, dry weight and Na uptake while giving small correlation value (0.572)
with SOD. Likewise, the Na uptake exhibited relatively strong correlation with
all of the selected variables except CAT.
The mutual variability between selected variables on the basis of
Pearson correlation values for T. patula cultivated on autoclaved (AS)
TSW:Soil mixtures is given in Table 4.7.2.
225
Table 4.7.2. Pearson correlation among selected biochemical properties, dry weight, Cr and Na uptake in Tagetes patula cultivated in autoclaved soil (AS) mixed with tannery solid waste (TSW)
Chlorophyll Soluble Protein
SOD CAT Dry
weight Cr Na
Chlorophyll
Pearson correlation
Sig. (2-tailed)
N
1 - 8
0.546*
0.043 8
0.456*
0.039 8
0.786** 0 8
0.451*
0.045
8
0.534
0.12 8
0.623*
0.035 8
Soluble Protein
Pearson correlation
Sig. (2-tailed)
N
0.546*
0.043 8
1 - 8
0.523*
0.042 8
0.342
0.32 8
0.651**
0.012
8
0.451*
0.038 8
0.410*
0.048 8
SOD
Pearson correlation
Sig. (2-tailed)
N
0.456*
0.039 8
0.523*
0.042 8
1 - 8
-0.012
0.67 8
0.234
0.56
8
0.452
0.23
8
0.523*
0.032 8
CAT
Pearson correlation
Sig. (2-tailed)
N
0.786** 0 8
0.342
0.32 8
-0.012
0.67 8
1 - 8
0.456
0.14
8
0.523*
0.031 8
0.512*
0.041 8
Dry weight
Pearson correlation
Sig. (2-tailed)
N
0.451*
0.045
8
0.651**
0.012 8
0.234
0.56 8
0.456
0.14 8
1 -
8
0.561*
0.021 8
0.612** 0 8
Cr
Pearson correlation
Sig. (2-tailed)
N
0.534
0.12
8
0.451*
0.038 8
0.452
0.23 8
0.523*
0.031 8
0.561*
0.021
8
1 - 8
0.578*
0.023 8
Na
Pearson correlation
Sig. (2-tailed)
N
0.623*
0.036 8
0.410*
0.048 8
0.523*
0.032 8
0.512
0.041 8
0.612*
0.041
8
0.578*
0.023 8
1 - 8
**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)
The chlorophyll contents were observed to vary with strong correlation
with CAT and Na uptake, while soluble protein contents displayed weak
correlation with all the variables except dry weight production. The SOD found
226
weakly correlated with all of the selected variables, likewise CAT except its
relation with chlorophyll production. The dry weight production showed
relatively strong correlation with soluble protein and Na uptake, while Cr
uptake had weak correlation with all the variables. The Na uptake found to
vary with mutual correlation with chlorophyll content and dry weigh production.
Correlation between different plants was also studied by Aslam et al. (2012).
227
4.8 Experiments with marigold cultivated on TSW-Soil mixtures and
inoculated with selective autochthonous saprobic fungi and AM fungi
under greenhouse conditions
The AM fungal associations of the plants have been reported to
decrease their sensitivity to the heavy metal stress by increasing production of
antioxidant defense enzymes (Blaudez et al., 2000; Frey et al., 2000;
Jentschke and Godbold, 2000; Auge, 2001; Schützendübel et al., 2001;
Schützendübel and Polle, 2002; Ehsanpour and Amini, 2003; Hernandez et
al., 2003; Cho et al., 2006; Kohler et al., 2009). In the present study, the
application of AM inoculum in trial 3.8 has been observed to show significant
increase in the production of SOD and CAT, enabling marigold plants to
withstand the stress exerted in soil due to mixing of TSW. It has been
reported that the antioxidant defense system of plants is strengthened by the
elevation of SOD and CAT production in soils where AM fungi are applied
either individually (Azcon et al., 2009) or in combination with saprobic fungi
(Medina et al, 2006). Thus in this study (Experiment 3.8), the application of
both AM and saprobic fungi in the same soil were responsible for maximum
production of SOD and CAT in marigold. Other than the antioxidant defense
system of the plant, several other defense strategies against heavy metals
may be there in AM inoculated plants (Vivas et al., 2006). However the
autochthonous saprobic fungi (Trichoderma pseudokoningii) performed better
as compared to AM fungi when inoculated to the marigold individually. This
finding prompted the use of autochthonous saprobic fungi for experimentation
with marigold and sunflower in greenhouse and field trials (3.9-3.11).
On finding Pearson correlation between chlorophyll contents, soluble
protein, SOD, CAT, dry weight production as well as Cr and Na uptake in
plants (Table 4.8.1); it was found that chlorophyll contents had strong
correlation with soluble protein contents while having weak variability
relationship with rest of the variables. The soluble protein showed relatively
strong correlation with chlorophyll; however its variation with respect to other
variables was weakly correlated.
228
Table 4.8.1. Pearson correlation among selected biochemical properties and metals for Tagetes patula cultivated in soil mixed with tannery solid waste (TSW) and inoculated with AM (arbuscular mycorrhizal) and saprobic fungi.
Chlorophyll Soluble Protein
SOD CAT Dry
weight Cr Na
Chlorophyll
Pearson correlation
Sig. (2-tailed)
N
1 - 8
0.674*
0.011
8
0.563
0.431
8
0.546
0.23 8
0.129
0.78
8
0.023
0.91 8
0.034
0.67 8
Soluble Protein
Pearson correlation
Sig. (2-tailed)
N
0.674*
0.011 8
1 -
8
0.453
0.21
8
0.512*
0.023 8
0.220
0.42
8
0.124
0.34 8
0.151
0.62 8
SOD
Pearson correlation
Sig. (2-tailed)
N
0.563
0.431
8
0.453
0.21
8
1 -
8
0.324
0.41 8
0.435
0.71
8
0.241
0.56 8
0.034
0.70 8
CAT
Pearson correlation
Sig. (2-tailed)
N
0.546
0.23 8
0.512*
0.023
8
0.324
0.41
8
1 - 8
0.134
0.391
8
0.142
0.56 8
0.120
0.67 8
Dry weight
Pearson correlation
Sig. (2-tailed)
N
0.129
0.78 8
0.220
0.42
8
0.435
0.71
8
0.134
0.391 8
1 -
8
0.651** 0 8
0.578*
0.034 8
Cr
Pearson correlation
Sig. (2-tailed)
N
0.023
0.91 8
0.124
0.34
8
0.241
0.56
8
0.142
0.56 8
0.651**
0
8
1 - 8
0.789** 0 8
Na
Pearson correlation
Sig. (2-tailed)
N
0.034
0.67 8
0.151
0.62
8
0.034
0.70
8
0.120
0.67 8
0.578*
0..034
8
0.789** 0 8
1 - 8
**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)
The SOD, CAT and dry weight found to have weak correlation between
them and with all other variables. The Cr uptake in plant exhibited strong
mutual correlation with Na uptake.
229
4.9 Experiments with marigold and sunflower inoculated with selective
autochthonous saprobic fungi under greenhouse and field conditions
During the current study, the SOD and CAT in sunflower and marigold
was found to increase with increasing level of heavy metal concentration in
soil, due to increasing percentage of TSW. Our studies are parallel to the
elevation in production of antioxidant defense enzymes on increasing the
concentration of tannery waste in soil (Halliwell and Gutteridge, 2004; Singh
et al., 2004; Ahmad et al., 2009; Gupta and Sinha, 2009). On the contrary,
reduced CAT production in sunflower with increasing stress has also been
reported (Quartacci and Navari-Izzo, 1992). However, due to increasing level
of tannery sludge, a decrease in soluble protein content and chlorophyll
content (Singh et al., 2004) and total chlorophyll content (Gupta and Sinha,
2009) has been reported. In the current study, the soluble protein and
chlorophyll content was found to decrease down the column in all the cases
where fungal inoculations were not applied and it was because of increasing
level of multi-metal stress in soil.
The Pearson correlation between selected biochemical parameters, dry
weight, Cr and Na uptake in plant showed that the chlorophyll showed strong
correlation with Cr and Na uptake. The soluble protein contents showed
strong correlation with CAT and dry weight production, while SOD had strong
mutual variation with soluble protein, CAT and dry weight production. The
SOD was strongly variable with soluble protein, CAT and dry weight. The CAT
production in plant strongly correlated with chlorophyll contents, soluble
protein, CAT and dry weight. The CAT in plant was strongly variable with
respect to the soluble protein, SOD, Cr and Na uptake. The dry weight was
found to have strong correlation with soluble protein, SOD, Cr and Na uptake.
The Cr uptake varied strongly with respect to chlorophyll contents, CAT, dry
weight production; however very strongly correlated with Na uptake in plant.
The Na uptake in plant showed strong correlation with chlorophyll contents,
CAT dry weight and Cr uptake in plant, as given in Table 4.9.1.
230
Table 4.9.1. Pearson correlation among selected biochemical properties, CAT, SOD, dry weight production, Cr and Na uptake in Tagetes patula cultivated on TSW:Soil taken in pots and inoculated with saprobic fungi.
Chlorophyll Soluble Protein
SOD CAT Dry
weight Cr Na
Chlorophyll
Pearson correlation
Sig. (2-tailed)
N
1
8
0.453
0.67
8
0.567
0.34
8
0.613*
0.042 8
0.571
0.56
8
0.614
0.067 8
0.678*
0.023 8
Soluble Protein
Pearson correlation
Sig. (2-tailed)
N
0.453
0.67
8
1 -
8
0.651*
0.042
8
0.645*
0.032 8
0.713**
0
8
0.561*
0.043 8
0.589*
0.039 8
SOD
Pearson correlation
Sig. (2-tailed)
N
0.567
0.34
8
0.651*
0.042
8
1 -
8
0.671*
0.023 8
0.651*
0.035
8
0.456
0.142 8
0.478
0.23 8
CAT
Pearson correlation
Sig. (2-tailed)
N
0.613*
0.042
8
0.645*
0.032
8
0.671*
0.023
8
1 - 8
0.569*
0.034
8
0.761** 0 8
0.691*
0.042 8
Dry weight
Pearson correlation
Sig. (2-tailed)
N
0.571
0.56
8
0.713**
0
8
0.651*
0.035
8
0.569*
0.034 8
1 -
8
0.681*
0.041 8
0.718** 0 8
Cr
Pearson correlation
Sig. (2-tailed)
N
0.614
0.067
8
0.561*
0.043
8
0.456
0.142
8
0.761** 0 8
0.681*
0.041
8
1 - 8
0.812** 0 8
Na
Pearson correlation
Sig. (2-tailed)
N
0.678*
0.023
8
0.589*
0.039
8
0.478
0.23
8
0.691*
0.042 8
0.718**
0
8
0.812** 0 8
1 - 8
**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)
Thus, in the present study, the inoculation of marigold and sunflower
with saprobic fungi in pot and field trials has led both of the plants to withstand
the stress exerted by the Category-I and II metals. The sparobic Trichoderma
sp. has also been reported to involve in improving the phytoextraction
231
efficiency of Brassica juncea (Cao et al., 2008) and in clover (Medina et al.,
2006). The saprobic fungus Trichoderma harzianum has been reported to
involve in Cd removal from contaminated soils (Lima et al., 2011). In this
work, the application of T. harzianum in marigold phytoextraction trials with
Caldwell field soil has also manifested the similar results.
The Pearson correlation values for chlorophyll with respect to soluble
protein contents, dry weight and Cr uptake in plant showed strong correlation,
as given in Table 4.9.2. The soluble protein contents varied strongly with
respect to chlorophyll contents; however, SOD and CAT didn’t show strong
correlation with other variables. The dry weight production showed strong
mutual variation with chlorophyll production, SOD and Cr uptake in plant. The
Cr uptake in plant showed strong correlation with chlorophyll contents, dry
weight production and Na uptake in plant. The Na uptake in plant observed to
vary strongly with respect to CAT production and Na uptake in plant.
232
Table 4.9.2. Pearson correlation among selected biochemical properties and metals for Tagetes patula cultivated in Caldwell field soil mixed with tannery solid waste (TSW) and inoculated with saprobic fungi (incl. Trichoderma harzianum).
Chlorophyll Soluble Protein
SOD CAT Dry
weight Cr Na
Chlorophyll
Pearson correlation
Sig. (2-tailed)
N
1 - 8
0.647*
0.038 8
0.645*
0.046
8
0.478
0.34
8
0.610*
0.032
8
0.637*
0.046 8
0.563
0.31 8
Soluble Protein
Pearson correlation
Sig. (2-tailed)
N
0.647*
0.038 8
1 - 8
0.564*
0.039
8
0.453
0.56
8
0.356
0.76
8
0.467
0.56 8
0.574
0.24 8
SOD
Pearson correlation
Sig. (2-tailed)
N
0.645*
0.046 8
0.564*
0.039 8
1 -
8
0.235
0.78
8
0.645*
0.034
8
0.545
0.34 8
0.564
0.54 8
CAT
Pearson correlation
Sig. (2-tailed)
N
0.478
0.34 8
0.453
0.56 8
0.235
0.78
8
1 -
8
0.572
0.23
8
0.578
0.32 8
0.610*
0.041 8
Dry weight
Pearson correlation
Sig. (2-tailed)
N
0.610*
0.032 8
0.356
0.76 8
0.645*
0.034
8
0.572
0.23
8
1 -
8
0.671*
0.031 8
0.571
0.43 8
Cr
Pearson correlation
Sig. (2-tailed)
N
0.637*
0.046 8
0.467
0.56 8
0.545
0.34
8
0.578
0.32
8
0.671*
0.031
8
1 - 8
0.678*
0.029 8
Na
Pearson correlation
Sig. (2-tailed)
N
0.563
0.31 8
0.574
0.24 8
0.564
0.54
8
0.610*
0.041
8
0.571
0.43
8
0.678*
0.029 8
1 - 8
**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)
Both sunflower and marigold were able to accumulate significant
concentrations of metals in their root and shoot. Such reports for sunflower
have shown effective accumulation of multi-metals in both shoot and root
233
while grown on tannery waste amended soils (Singh et al., 2004). It was
observed that the metal accumulation ability of both of the plants increased
with increasing level of heavy metal concentration in soil i.e. due to increasing
percentage of TSW in soil. Similar results have been reported where metal
accumulation increased with increasing level of tannery waste in soil (Barman
et al., 2000; Armienta et al., 2001). It was also noted during this study that the
metal uptake in both the selected plants increased with increasing
concentration of multi-metals present in soil. Such studies have been reported
by AlHamdani and Blair, (2004); Hoffman et al., (2004) and Sune et al., (2007)
where plant exposure to the increasing concentration of metals such as, Cr,
Cd, and Pb has been reported to elevate metal uptake in plants.
On finding Pearson correlation between chlorophyll and rest of the
selected variables, it was found that chlorophyll strongly correlated with Na
uptake in plant. The soluble protein contents of the plant strongly varied with
respect to dry weight production in plant and Na uptake. The SOD contents
observed to have strong correlation with Cr uptake in plant, while CAT
production observed to correlate weakly with all of the selected variables. The
dry weight production in plant exhibited strong correlation with Cr and Na
uptake in plant. The Cr uptake in plant strongly varied with respect to Na
uptake, while Na uptake found to have strong correlation with chlorophyll
contents, soluble protein, dry weight and Cr uptake in plant, as given in Table
4.9.3. Increased level of SOD activity as a result of oxidative stress caused by
surfeit of heavy metals is well documented (Dey et al., 2007; Zhang et al.,
2007).
234
Table 4.9.3. Pearson correlation among selected biochemical properties and metals for Helianthus annuus cultivated in soil mixed with tannery solid waste (TSW) and inoculated with saprobic fungi (FIELD TRIAL).
Chlorophyll Soluble Protein
SOD CAT Dry
weight Cr Na
Chlorophyll
Pearson correlation
Sig. (2-tailed)
N
1 - 8
0..536
0.43
8
0.456
0.56 8
0.563
0.61
8
0.568
0.32
8
0.563
0.41 8
0.673*
0.038 8
Soluble Protein
Pearson correlation
Sig. (2-tailed)
N
0.536
0.43 8
1 -
8
0.562
0.45 8
0.462
0.67
8
0.615*
0.023
8
0.525
0.56 8
0.681*
0.034 8
SOD
Pearson correlation
Sig. (2-tailed)
N
0.456
0.56 8
0.562
0.45
8
1 - 8
0.013
0.89
8
0.456
0.56
8
0.615*
0.021 8
0.578
0.46 8
CAT
Pearson correlation
Sig. (2-tailed)
N
0.563
0.61 8
0.462
0.67
8
0.013
0.89 8
1 -
8
0.561
0.23
8
0.518
0.34 8
0.591
0.43 8
Dry weight
Pearson correlation
Sig. (2-tailed)
N
0.568
0.32 8
0.615*
0.023
8
0.456
0.56 8
0.561
0.23
8
1 -
8
0.781** 0 8
0.699*
0.031 8
Cr
Pearson correlation
Sig. (2-tailed)
N
0..563
0.41 8
0.525
0.56
8
0.615*
0.021 8
0.518
0.34
8
0.781**
0
8
1 - 8
0.767** 0 8
Na
Pearson correlation
Sig. (2-tailed)
N
0.673*
0.038 8
0.681*
0.034
8
0.578
0.046 8
0.591
0.43
8
0.699*
0.031
8
0.767** 0 8
1 - 8
**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)
Some studies have also reported that increasing the level of tannery
waste to a certain limit adds the necessary nutrients to soil (Singh and Sinha,
2004). As a result, the production of biomolecules such as chlorophyll is
235
increased in plants. In the current study, the 5 % TSW:Soil mixture in all the
cases not only improved the chlorophyll content of both marigold and
sunflower but also the biomass production. Such results have also been found
by Singh et al. (2004) where increase in the chlorophyll content of H. annuus
have been reported due to mixing of soil with tannery waste.
The distribution and variation of heavy metals in soil due to amendment
with tannery wastes have been reported (Vankar and Bajpai, 2008; Aceves et
al., 2009). In this study, the plants from TSW-Soil mixtures with fungi where
soil had high levels of Category-I and II metals, showed elevated levels of
biochemical parameters as compared to those harvested from the
corresponding soil treatments without fungal inoculation.
Literature suggests increased plant antioxidant enzymes in response
to increasing level of heavy metals in soils (Pietrini et al., 2003; Al-Hamdani
and Blair, 2004; Ederli et al., 2004; Hu et al., 2007; Dhir et al., 2009).The level
of toxicity caused by heavy metals has been found to affect several processes
in plants (Siedlecka et al., 2001). In the current study, an increase in CAT
production was observed relative to the increase in heavy metal concentration
in TSW-Soil mixture. An increase in CAT with increase in stress level has
been reported (Zhang et al., 2006). In contrast, however, CAT activity has
been reported to decrease on exposing plants to high levels of Cr (Aravind
and Prasad, 2005; Shankar et al., 2005). As the biochemical parameters in
this study were noticed for leaves of both marigold and sunflower, the
assessment of the antioxidant defense system was validated. In leaves of
heavy metal-stressed plants (SOD) activity fluctuated in different stress levels
compared to the control, while CAT activity increased with stress levels
(Zhang et al., 2007).
The decrease in plant growth with increasing level of heavy metals has
been reported by John et al., (2009). During this study, the growth of
marigold was observed to decrease with increasing concentration of
Category-I and II metals, being the minimum in 20 % TSW-Soil mixtures in
most of the cases. The increasing toxicity due to heavy metals has been
found to lower plant growth and tolerance (Mahmood et al., 2007). Thus in
236
this study, the elevated level of Category-I and II concentration levels in soils
for both pot and field trials has been found to lower down the plant growth and
biomass yield. The elevated level of metals in the growing medium has been
reported to cause a decrease in chlorophyll production (Prasad et al., 2001;
Pietrini et al., 2003; Panda and Chaudhary, 2005; Hu et al., 2007; John et al.,
2009). The soluble protein content has also been reported to decrease with
increasing concentration of metal in soil (John et al., 2009). The excess of
heavy metals in soils minimize the tendency of plants to produce the key
biochemical molecules such as, chlorophyll and soluble proteins, which
ultimately result in increasing sensitivity to stress environments. Thus in this
study, the growth variation of marigold was subjected to the variation in
concentration of Category-I and II metals in the soil.
The meta-analytical perspective of Category-I and II in TSW-Soil
mixtures helped analyze the trend of metals in soil, plant and plant parts. In
the literature, the metal analytical parameters have been used to analyze the
metal distribution pattern in soil to plant during different phytoextraction
studies such as tolerance index (TI %) (Bauddh and Singh, 2011); relative
growth rate (RGR) and TI (Umebese and Motajo 2008) and only TI (Mahmood
et al., 2007), translocation index (Zehra et al., 2009); translocation index in
field experiments (Liu et al., 2009) and translocation index and mobility index
of heavy metals (Tukura et al., 2012). In this study, the metal-analytical
comparisons for circulation of Category-I and II metals from TSW to soil and
from soil to sunflower and marigold were carried out.
Conclusion
The present study demonstrates the potential of Helianthus annuus
and Tagetes patula in phytoremediation of multimetal contaminated TSW.
Exposure to metals with fungal inoculation individually or in combinations
resulted in enhanced metal accumulation in the roots and shoots of these
plants, with comparatively high biomass production indicating no toxicity
symptoms, shows that these plants can tolerate high metal concentrations.
On the basis of high values of SEY (%), Tolerance index and translocation
index, it can be concluded that H. annuus and T. patula have a great ability to
237
phytoextract the metals from TSW. Moreover, results suggests that both the
plants have a high capability to detoxify the ROS produced in response to
metal stress throughout their growth and developmental stages, indicated by
high activities of antioxidant enzymes like SOD and CAT. Hence, H. annuus
and T. patula along with autochthonous fungi, proved their phytoremediational
efficiency for novel phytoremediation strategies for metals.
In general, the benefits and disadvantages of phytoremediation must
be assessed by finding the most appropriate organism for the task.
Combining technologies like phytoremediation and mycoremediation offers
the greatest potential to decontaminate the multimetal contaminated TSW.
The application of latest innovations in biotechnology has made it easy to find
a workable and cost effective method of solving a problem. Disposal of
tannery solid waste is not a problem if it is converted into a useful by-product
or fertilizer free of heavy metals. Phytoremediation is a miracle and can be
used to transform byproduct of leather industry into a non toxic or non toxic
material.
Future perspective
A considerable biotechnological approach for increasing the potential
for metal phytoextraction may be to improve the hyperaccumulator growth
rate through selective plants, or by the transfer of metal hyperaccumulation
genes to the high biomass species. The transgenic plant approach has shown
to be promising, but only very few studies have proved authentic till now
under field conditions. Genetic manipulations will involve to change the
expression levels in a number of genes, and also to determine the number of
genes involved and their characteristics. Functions and regulations of genes
involved in metal hyperaccumulation, uptake, root-to-shoot translocation,
detoxification, sequestration mechanisms need to be fully understood to
provide transgenic approach less competent to solve the problem.
In spite of progress made in recent years by numerous studies, the
complexity of hyperaccumulation is far from being understood and several
aspects of this astonishing feature still await explanation. The recent idea that
heavy metals would provide an elemental defense to the plant through joint
effects with organic defense compounds requires much experimental
238
evidence. More elements and a larger number of hyperaccumulator species
need to be examined to validate the hypothesis of defensive effects of heavy
metals.
Furthermore, it is of pivotal importance to increase the understanding
of hyperaccumulator-based remedial mechanisms because they will be able
to provide clues for optimizing the effectiveness of phytoextraction with
appropriate agronomic practices. In addition, knowledge acquired on genes
involved in hyperaccumulation mechanisms will open the opportunity to use
biotechnology to transfer specific genes to high-biomass promising species.
Much research is still needed on rhizosphere and soil microbial composition
under field conditions, in order to identify micro-organisms associated with
metal solubility or precipitation.
There is also an urgent need to find and characterize other
hyperaccumulators, to cultivate them and investigate agronomic practices and
management to enhance plant growth and metal uptake. Even then, metal
uptake might create environmental risks, unless the biomass produced during
the phytoremediation process could be rendered economical by burning it to
produce bio-ore or converting it into bioenergy. Before the commercialization
of phytoextraction using high-biomass hyperaccumulator plants it needs to be
determined that they will not only remediate contaminated sites but also
generate income from agricultural lands not utilized otherwise.
References
239
REFERENCES
Aceves, M.B., Santos, H.S., Berber, J.D.R., Mota, J.L.O. and Vázquez, R.R.
2009. Distribution and mobility of Cr in tannery waste amended semi-
arid soils under simulated rainfall. J. Hazard. Mater. 171: 851–858.
Afsar, N., Özgür, E., Gürgan, M., Akköse, S., Yücel, M., Gündüz, U. and
Eroðlu, I. 2011. Hydrogen productivity of photosynthetic bacteria on
dark fermenter effluent of potato steam peels hydrolysate. Int. J.
Hydrog. Ener. 36(1): 432-438.
Ahluwalia, S.S. and Goyal, D. 2007. Microbial and plant derived biomass for
removal of heavy metals from wastewater. Biores. Technol. 98: 2243–
2257.
Ahmad, I. and Hellebust, A. 1988. The relationship between inorganic
nitrogen metabolism and proline accumulation in osmoregulatory
responses of two euryhaline micro algae. Plant Physiol. 88: 348–354.
Ahmad, P. and Jhon, R. 2005. Effect of Salt stress on growth and biochemical
parameters of Pisum sativum L. Arch. Agro. Soil Sci. 51: 665-672.
Ahmad, P., Sharma, S. and Srivastava, P.S. 2006. Differential physio-
biochemical responses of high yielding varieties of Mulberry (Morus
alba) under alkalinity (Na2CO3) stress in vitro. Physiol. Mol. Biol. Plant
12: 59-66.
Ahmed, C.B., Rouina, B.B., Sensoy, S., Boukhriss, M. and Abdullah, F.B.
2009. Saline Water Irrigation Effects on Antioxidant Defense System
and Proline Accumulation in Leaves and Roots of Field-Grown Olive. J.
Agric. Food Chem. 57: 11484–11490.
Alexander, M. 1977. Introduction to Soil Microbiology, 2nd Edn. New York:
Wiley, 467 pp.
240
Al-Hamdani, S.H. and Blair, S.L. 2004. Influence of copper on selected
physiological responses in Salvinia minima and its potential use in
copper remediation. Am. Fern J. 94: 47–56.
Alia, Mohanty, P. and Matysik, J. 2001. Effect of proline on the production of
singlet oxygen. Amino Acid 21: 195–200.
Alscher, R.G. 1989. Biosynthesis and antioxidant function of glutathione in
plants. Physiol. Plant 77: 457–464.
Amir, S., Benlboukhtb, F., Cancianc, N., Wintertond, P. and Hafidi, M. 2008.
Physico-chemical analysis of tannery solid waste and structural
characterization of its isolated humic acids after composting. J. Hazard.
Mater. 160: 448–455.
Ammarati, J.F. and Michelle, T.S. 2005. Uses of fungi, Fungus encyclopedia,
Department of Botany, University of Washington. USA
Anahida, S., Yaghmaeia, S. and Ghobadinejad, Z. 2011. Heavy metal
tolerance of fungi. Scientia Iranica C. 18(3): 502–508.
Anand, P., Isar, J., Saran, S. and Saxena, R.K. 2006. Bioaccumulation of
copper by Trichoderma viride. Biores. Technol. 97: 1018-1025.
Aravind, P. and Prasad, M.N.V., 2005. Cadmium–zinc interactions in a
hydroponic system using Ceratophyllum demersum L.: adaptive
ecophysiology, biochem- istry and molecular toxicology. Braz. J. Plant
Physiol. 17(1): 3-20
Aravindhan, R., Saravanabhavan, S., Thanikaivelan, P., Raghava Rao, J. and
Nair, B.U. 2007. A chemo-enzymatic pathway leads towards zero
discharge tanning. J. Clean. Prod. 15: 1217–1227.
Armienta, M.A., Morton, O., Rodriguez, R., Cruz, O., Aguayo, A.,Ceniceros,
N., 2001. Chromium in a tannery wastewater irrigated area Lion Valley,
Mexico. Bull. Environ. Contam. Toxicol. 66: 189–195.
Arriagada, C., Aranda, E., Sampedro, I., Garcia-Romera, I. and Ocampo, J.A.
2009. Interactions of Trametes versicolor, Coriolopsis rigida and the
241
Arbuscular mycorrhizal fungus Glomus deserticola on the copper
tolerance of Eucalyptus globules. Chemosphere 77: 273–278.
Arriagada, C.A., Herrera, M. A. and Ocampo, J.A. 2005. Contribution of
arbuscular mycorrhizal and saprobe fungi to the tolerance of
Eucalyptus globulus to Pb. Water Air Soil Pollut. 166: 31–47.
Arriagada, C.A., Herrera, M.A., Borie, F. and Ocampo, J.A. 2007. Contribution
of arbuscular mycorrhizal and saprobe fungi to the aluminium
resistance of Eucalyptus globulus. Water Air Soil Pollut. 182: 383-394.
Arriagada, C.A., Herrera, M.A., Garcia-Romera, I. and Ocampo, J.A. 2004.
Tolerance of cadmium of soybean (Glycine max) and eucalyptus
(Eucalyptusglobules) inoculated with Arbuscuar mycorrhiza and
saprobe fungi. Symbiosis 36: 285–299.
Asada, K. 1984. Chloroplasts: formation of active oxygen and its scavenging.
Methods Enzymol. 10: 422–429.
Aslam, M., Verma, D.K., Dhakerya, R., Rais, S., Alam, M. and Ansari, F.A.
2012. Bioindicator: A Comparative Study on Uptake and Accumulation
of Heavy Metals in Some Plant`s Leaves of M.G. Road, Agra City,
India. J. Environ. Earth Sci. 4(12): 1060-1070.
Aslan, A. 2009. Determination of Heavy Metal Toxicity of Finished Leather
Solid Waste. Bull. Environ. Contam. Toxicol. 82:633–638.
Aslan, A., Gülümser, G. and Ocak, B., 2006. Increasing the Efficiency of
Chromium Tanning by Using Collagen Hydrolysates from Shavings. J.
Soc. Leather Technol. Chem. 90: 201-204.
Aslan, A., Karavana, H.A., Gulumser, G., Yasa, I. and Cadirci, B.H. 2007.
Utilization of collagen hydrolyzate in keratinase production from
Bacillus subtilis ATCC 6633. J American Leather Chem. Assoc. 102:
129–134.
Audet, P. and Charest, C. 2006. Effects of AM colonization on “wild tobacco”
plants grown in zinc- contaminated soil. Mycorrhiza 16: 277-283.
242
Audet, P. and Charest, C. 2007. Heavy metal phytoremediation from a meta-
analytical perspective. Environ. Pollut. 147: 231e237.
Augé, R.M., 2001. Water relations, drought and vesicular-arbuscular
mycorrhizal symbiosis. Mycorrhiza 11, 3–42.
Augustus, T. 1996. The Pakistan Leather Industry (LEATHER).
http://www.american.edu/projects/mandala/TED/leather.htm.
Azco´n, R., del Carmen Pera´ lvarez, M., Biro´, B., Rolda´n, A., Ruı´z-Lozano,
J.M. 2009. Antioxidant activities and metal acquisition in mycorrhizal
plants growing in a heavy-metal multicontaminated soil amended with
treated lignocellulosic agrowaste. Appl. Soil Ecol. 41: 16 8 – 177.
Azcón, P., Perálvarez, M.C., Roldán, A. and Barea, J.M. 2010. Arbuscular
Mycorrhizal Fungi, Bacillus cereus, and Candida parapsilosis from a
Multicontaminated Soil Alleviate Metal Toxicity in Plants. Microb. Ecol.
59: 668–677.
Azom, M.R., Mahmud, K., Yahya, S.M. Sontu, A. and Himon, S. B. 2012.
Environmental Impact Assessment of Tanneries: A Case Study of
Hazaribag in Bangladesh. Int. J. Environ. Sci. Develop. 3(2): 152-156.
Babula, P., Adam, V., Opatrilova, R., Zehnalek, J., Havel, L. and Kizek, R.
2008. Uncommon heavy metals, metalloids and their plant toxicity: a
review. Environ. Chem. Lett. 6: 189-213.
Baldrian, P. 2003. Interactions of heavy metals with white-rot fungi. Enzym.
Microb. Technol. 32: 78-91.
Baldrian, P. 2010. Effect of Heavy Metals on Saprotrophic Soil Fungi. In: Soil
Heavy Metals. Soil Biology (Sherameti and Varma, Eds.), Vol. 19.
Springer, Germany: 263-279.
Bareen, F. and Nazir, A. 2010. Metal decontamination of tannery solid waste
using Tagetes patula in association with saprobic and mycorrhizal
fungi. Environmentalist 30: 45-53.
243
Bareen, F., Nazir, A. and Ahmad, S. 2011. Role of live autochthonous fungi in
removing toxic metals from tannery and textile effluents. Afr. J.
Biotechnol. 10 (32): 6072-6081
Bareen, F., Shafiq, M. and Jamil, S. 2012. Role of plant growth regulators and
a saprobic fungus in enhancement of metal phytoextraction potential
and stress alleviation in pearl millet. J. Hazard. Mater. 237-238:186-
193.
Barman, S.C., Sahu, R.K., Bhargava, S.K. and Chaterjee, C., 2000.
Distribution of heavy metals in wheat, mustard and weed grown in field
irrigated with industrial effluents. Bull. Environ. Contam. Toxicol. 64:
489–496.
Bauddh, K. and Singh, R.P. 2011. Differential toxicity of cadmium to mustard
(Brassica juncia L.) genotypes under higher metal levels. J. Environ.
Biol. 32: 355-362.
Beers, R.F.and Sizer, I.W. 1952. A spectrophotometric method for measuring
the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:
133–140.
Beyer, W., Imlay, J. and Fridovich, I. 1991. Superoxide dismutase progress in
nucleic acids. Proc. Nucleic Acid Res. 40: 221–253.
Bhattacharjee, S. 1997–98. Membrane lipid peroxidation, free radical
scavengers and ethylene evolution in Amaranthus as affected by lead
and cadmium. Biol. Plant 40 (1): 131–135.
Blacksmith Institute’s World´s Worst Pollution Problems Report 2010, Top Six
Toxic Threats: Six pollutants that jeopardize the health of tens of
millions of people, pp. 39.
Blaudez, D., Botton, B. and Chalot M, 2000. E_ects of heavy metals on
nitrogen uptake by Paxillus involutus and mycorrhizal birch seedlings.
FEMS Microbiol. Ecol. 33(1): 61– 67.
244
Bosnic, M., Buljan, J. and Daniels, R. P. 2000. Regional program for pollution
control in the Tanning industry US/RAS/92/120 in South-East Asia pp
1-14.
Brotman, Y., Landau, U., Cuadros-Inostroza, A., Takayuki, T. and Fernie, A.R.
2013. Trichoderma-Plant Root Colonization: Escaping Early Plant
Defense Responses and Activation of the Antioxidant Machinery for
Saline Stress Tolerance. PLoS Pathog. 9(3): 1003221.
Cao, L., Jiang, M., Zeng, Z., Du, A., Tan, H. and Liu, Y. 2008. Trichoderma
atroviride F6 improves phytoextraction efficiency of mustard (Brassica
juncea L. Coss. Var. foliosa Bailey) in Cd, Ni contaminated soils.
Chemosphere 71: 1769-1773.
Huang, L. Y. C. and Schulte, E. E. 1985. Digestion of plant tissue for analysis
by ICP emission spectroscopy. Commun. Soil Sci. Plant Analysis 16
(9): 943-958.
Chatterjee, S., Chattopadhyay, B. and Mukhopadhyay, S. K. 2010. Monitoring
waste metal pollution at ganga Estuary via the East Calcutta Wetland
areas. Environ. Monit. Assess. 170: 1-4, 23-31.
Cho, K., Toler, H., Lee, J., Owenley, B., Stutz, J.C., Moore, J.L. and Augé,
R.M., 2006. Mycorrhizal symbiosis and response of sorghum plants to
combined drought and salinity stresses. J. Plant Physiol. 163: 517–
528.
Cho, U.H. and Sohn, J.Y. 2004. Cadmium-Induced Changes in Antioxidative
Systems, Hydrogen Peroxide Content, and Lipid Peroxidation in
Arabidopsis thaliana. J. Plant Biol. 47(3): 262-269.
Citterio, S., Prato, N., Fumagalli, P., Aina, R., Massa, N., Santagostino, A.,
Sgorbati, S. and Berta, G. 2005. The Arbuscular mycorrhizal fungus
Glomus mosseae induces growth and metal accumulation changes in
Cannabis sativa L. Chemosphere 59: 21-29.
245
Cutright, T., Gunda, N. and Kurt, F. 2010. Simultaneous hyperaccumulation of
multiple heavy metals by Helianthus annuus grown in a contaminated
sandy-loam soil. Int. J. Phytorem. 12(6): 562-73.
del Castilho, P., Chardon, W.J. and Salomons, W. 1993. Influence of cattle-
manure slurry application on the solubility of cadmium, copper, and
zinc in a manured acidic, loamy-sand soil. J. Environ. Qual. 22: 689-
697.
Denton, B. 2007. Advances in phytoremediation of heavy metals using plant
growth promoting bacteria and fungi. MMG 445 Basic Biotechnol. 3: 1-
5.
Devi, S.R. and Prasad, M.N.V. 1998. Copper toxicity in Ceratophyllum
demersum L. (coontail), a free-floating macrophyte: response of
antioxidant enzymes and antioxidants. Plant Sci. 138: 157–165.
Dey, S.K., Dey, J., Patra, S. and Pothal, D. 2007. Changes in the antioxidative
enzyme activities and lipid peroxidation in wheat seedlings exposed to
cadmium and lead stress. Braz. J. Plant Physiol. 19: 14-26.
Dhir, B. 2010. Use of aquatic plants in removing heavy metals from
wastewater. Int. J. Environ. Eng. 2: 185-201.
Dhir, B., Nasim, A. S., Sharmila, P. and Saradhi, P.P. 2010. Heavy metal
removal potential of dried Salvinia biomass. Int. J. Phytorem. 12: 133–
141.
Dhir, B., Sharmila, P., Saradhi, P.P. and Nasim, S.A. 2009. Physiological and
antioxidant responses of Salvinia natans exposed to chromium-rich
wastewater. Ecotoxicol. Environ. Saf. 72: 1790–1797.
Dix, N. J. and Webster, J. 1995. Fungal Ecology. Chapman & Hall, London,
UK. 594 pp.
Dox, A.W. 1910. The intracellular enzymes of Penicillium and Aspergillus with
special reference to those of P. camemberti. U. S. Dept. Age. Bur.
Animal Ind. Bull. pp. 120-170.
246
Dubey, S.C., Suresh, M. and Singh, B. 2007. Evaluation of Trichoderma spp
against Fusarium oxysporum f sp ciceri for integrated management of
chickpea wilt. Biol.Control. 40: 118-127.
Ederli, L., Reale, L., Ferranti, F. and Pasqualini, S. 2004. Responses induced
by high concentration of cadmium in Phragmites australis roots.
Physiol. Plant 121: 66–74.
Ehsanpour, A.A., Amini, F., 2003. Effect of salt and drought stress on acid
phosphatase activities in alfalfa (Medicago sativa L.) explants under in
vitro culture. Afr. J. Biotechnol. 2, 133–135.
El-Kassas, H.Y. and El-Taher, E.M. 2009. Optimization of Batch Process
Parameters by Response Surface Methodology for Mycoremediation of
Chrome-VI by a Chromium Resistant Strain of Marine Trichoderma
viride. American-Eurasian J. Agric. Environ. Sci. 5 (5): 676-681.
Elloumi, N., Ben, F., Rhouma, A., Ben, B., Mezghani, I. and Boukhris, M.
2007. Cadmium induced growth inhibition and alteration of biochemical
parameters in almond seedlings grown in solution culture. Acta Physiol.
Plant 29: 57-62.
EM Research Organization for Central and Eastern Asia, Pakistan. 2003.
Removal of existing pollution problems odor and concentration of
pollution at Kasur Tannery Pollution Control Project using EM
technology http//www.embiotech.org.
Ezzouhri, L., Castro, E., Moya, M., Espinola, F. and Lairini, K. 2009. Heavy
metal tolerance of filamentous fungi isolated from polluted sites in
Tangier, Morocco. Afr. J. Microbiol. Res. 3(2): 35-48.
Fageria, N.K., Baligar, V.C. and Jones, C.A. 1991. Growth and Mineral
Nutrition of Field Crops. Marcel Dekker, New York.
Federici, E., Leonardi, V., Giubilei, M.A., Quaratino, D., Spaccapelo, R., D'Annibale,
A. and Petruccioli, M. 2007. Addition of allochthonous fungi to a
historically contaminated soil affects both remediation efficiency and
bacterial diversity. Appl. Microbiol. Biotechnol. 77(1): 203-11.
247
Feed International. April 1993 and Sept. 2003. Chromium in Broiler Diets.
Supplementation more critical under stressful conditions.
Fornazier, R.F., Ferreira, R.R., Pereira, G.J.G., Molina, S.M.G., Smith, R.J.,
Lea, P.J., Azevedo, R.A., 2002. Cadmium stress in sugar cane callus
cultures: effect on antioxidant enzymes. Plant Cell Tissue Organ Cult.
71: 25–131.
Foyer, C.H., Lelandais, M. and Kunert, K.J. 1994. Photoxidative stress in
plants. Physiol. Plant 92: 696–717.
Fracchia, S., Mujica, M.T., Garcia-Romera, I., Garcia-Garrido, J.M., Martin, J.,
Ocampo, J.A. and Godeas, A., 1998. Interactions between Glomus
mosseae and Arbuscular mycorrhizal sporocarp-associated
saprophytic fungi. Plant Soil 200: 131–137.
Freitas, H., Prasad, M.N.V. and Pratas, J. 2004. Plant community tolerant to
trace elements growing on the degraded soils of Sao Domingos mine
in the south east of Portugal: environmental implications. Environ. Int.
30: 65-72.
Frey, B., Zierold, K. and Brunner, I. 2000. Extracellular complexation of Cd in
the Hartig net and cytosolic Zn sequestration in the fungal mantle of
Picea abies–Hebeloma crustuliniforme ectomycorrhizas. Plant Cell
Environ. 23: 1257–1265.
Gadd, G.M. 1993. Interactions of fungi with toxic metals. New Phytol. 124: 25-
60.
Gallego, S.M., Benavides, M.P. and Tomaro, M.L. 1999. Effect of cadmium
ions antioxidant defense system in sunflower cotyledons. Biol. Plant
42(1): 49–55.
Garbisu, C. and Alkorta, I. 2001. Phytoextraction: A cost effective plant-based
technology for the removal of metals from the environment. Biores
Technol. 77(3): 229–236.
248
García-Romera, I., García-Garrido, J. M., Martín, J., Fracchia, S., Mujica, M.T.
and Godeas, A. 1998. Interactions between saprotrophic Fusarium
strains and Arbuscular mycorrhizas of soybean plants. Symbiosis 24:
235–246.
Gaur, A. and Adholeya, A. 2004. Prospects of Arbuscular mycorrhizal fungi in
phytoremediation of heavy metal contaminated soils. Curr. Sci. 86:
528–534.
Gavrilesca, M. 2004. Removal of heavy metals from the environment by
biosorption. Eng. Life Sci. 4(3): 219-232.
Gerdemann, J.W. and Nicolson, T.H. 1963. Spores of mycorrhizal Endogone
species extracted from the soil by wet sieving and decanting. Trans.
Brit. Mycol. Soc. 46: 235–244.
Giovannetti, M. and Mosse, B. 1980. Evaluation of techniques for measuring
vesicular Arbuscular mycorrhizal infection in roots. New Phytol. 84:
489–500.
Gisbert, C., Ros, R., de Haro, A., Walker, D.J., Pilar Bernal, M., Serrano, R.
and Avino, J.N. 2003. A plant genetically modified that accumulates Pb
is especially promising for phytoremediation. Biochem. Biophys. Res.
Commun. 303(2): 440–445.
Gohre, V. and Paszkowski, U. 2006. Contribution of arbuscular mycorrhizal
symbiosis to heavy metal phytoremediation. Planta 223:1115-1122.
Gratao, P.L., Prasad, M.N.V., Cardoso, P.F., Lea, P.J. and Azevedo, R.A.
2005. Phytoremediation: green technology for the clean up of toxic
metals in the environment. Braz. J. Plant Physiol. 17: 53-64.
Gupta, A.K. and Sinha, S. 2009. Antioxidant response in sesame plants
grown on industrially contaminated soil: Effect on oil yield and
tolerance to lipid peroxidation Biores. Technol. 100: 179–185.
Gupta, V.K. and Mishra, A.K. 2009. Efficacy of biogents against Fusarium wilt
of guava. J. Mycol Plant Pathol. 39(1): 101-106.
249
Gveroska, B. and Ziberoski, J. 2012. Trichoderma harzianum as a biocontrol
agent against Alternaria alternata on tobacco. ATI – Appl. Technol.
Innov. 7(2): 67-76.
Hakmaoui, A., Barón, M. and Ater, M. 2006. Environmental Biotechnology
Screening Cu and Cd tolerance in Salix species from North Morocco.
Afr. J. Biotechnol. 5(13): 1299-1302.
Hall, J.L. 2002. Cellular mechanisms for heavy metal detoxification and
tolerance. J. Exp. Bot. 53: 1–11.
Halliwell, B. and Gutteridge, J.M. 1984. Oxygen toxicity, oxygen radicals,
transition metals and disease. Biochem. J. 219: 1–14.
Halliwell, B. and Gutteridge, J.M.C. 2004. Free Radicals in Biology and
Medicine. Clarendon Press, Oxford.
Hatvani, N., and Mecs, L. 2003. Effects of certain heavy metals on the growth,
dye, decolorization and enzyme activity of Lentinula edodes.
Ecotoxicol. Environ. Saf. 55(2): 199-203.
Hawksworth, D.L. 2001. The magnitude of fungal diversity: the 1.5 million
species estimate revisited. Mycol. Res. 105: 1422–1432.
Hernández, J.A., Aguilar, A., Portillo, B., López-Gómez, E., Mataix Beneyto, J.
and García- Legaz, M.F., 2003. The effect of calcium on the antioxidant
enzymes from salt treated loquat and anger plants. Funct. Plant Biol.
30: 1127–1137.
Hildebrandt, U., Regvar, M and Bothe, H. 2007. Arbscular mycorrhiza and
heavy metal tolerance. Phytochemistry. 68: 139–146.
Hoffman, T., Kutter, C. and Santamaria, J.M. 2004. Capacity of Salvinia
minima Baker to tolerate and accumulate As and Pb. Eng. Life Sci. 4:
61–65.
Hu, C., Zheng, L., Hamilton, D., Zhou, W., Yang, T. and Zhu, D. 2007.
Physiological responses induced by copper bioaccumulation in
Eichhornia crassipes. Hydro- biologia 579: 211–218.
250
Inam ul Huq, S.M. 1998. Critical Environmental Issues Relating to Tanning
Industries in Bangladesh. In: Towards Better Management of Soils
Contaminated with Tannery waste (Naidu et al., Eds), in Proceedings
of a workshop held at the Tamil Nadu Agricultural University,
Coimbatore, India.
International Union of Leather Technologists and Chemists Societies
(IULTCS), IU Commission Environment (IUE) 2008. Technical
guidelines for environmental protection aspects for the world leather
industry. Pembroke, UK: IULTCS. Available at
http://www.iultcs.org/environment.asp.
Inui, T., Takeda, Y. and Iizuka, H. 1965. Taxonomical studies on genus
Rhizopus. J. Gen. and Appl. Microbiol. 11: 1-121.
Iram, S., Ahmad, I., Javed, B., Yaqoob, S., Akhtar, K., Kazmi, M.R. and
Zaman, B. 2009. Fungal tolerance to heavy metals. Pak. J. Bot. 41(5):
2583-2594.
ISO 11466. 1995. Soil quality: extraction of trace elements soluble in aqua
regia. ISO, Geneva, Switzerland.
Iwegbue, C. M. A., Emuh, F. N., Isirimah, N. O. and Egun, A. C. 2007.
Fractionation, characterization and speciation of heavy metals in
composts and compost-amended soils. Afr. J. Biotechnol. 6(2): 067-
078.
Jenkins, R., Barton, J. and Hesselberg, J. 2004. The Global Tanning Industry:
a Commodity Chain Approach. Environmental Regulation in the New
Global Economy: the impact on industry and competitiveness: Edward
Elgar Publishing, pp. 157–172.
Jentschke, G. and Godbold, D.L. 2000. Metal toxicity and ectomycorrhizas.
Physiol. Plant. 109: 107–116.
Ji, G.L., Wang, J.H. and Zhang, X.N., 2000. Environmental problems in soil
and groundwater induced by acid rain and management strategies in
251
China. In: Soils and Groundwater Pollution and Remediation (Huang,
P.M. and Iskandar, I.K., Eds.), CRC Press, London, pp. 201-224.
John, R., Ahmad, P., Gadgil, K. and Sharma, S. 2009. Heavy metal toxicity:
Effect on plant growth, biochemical parameters and metal
accumulation by Brassica juncea L. Int. J. Plant Prod. 3(3): 65-76.
Kachenko, A.G., Singh, B. and Bhatia, N.P. 2007. Heavy metal tolerance in
common fern species. Aust. J. Bot. 300: 207–219.
Kadpal, R.P. and Rao, N.A. 1985. Alteration in the biosynthesis of proteins
and nucleic acid in finger millet (Eleucine coracana) seedling during
water stress and the effect of proline on protein biosynthesis. Plant Sci.
40: 73–79.
Kanagaraj, J., Velappan, K.C., Chandra Babu, N.K and Sadulla, S. 2006.
Solid wastes generation in the leather industry and its utilization for
cleaner environment- A review. J. Sci. Ind. Res. 65: 541-548.
Khade, H.W. and Adholeya, A. 2009. Arbuscular mycorrhizal association in
plants growing on metal-contaminated and non contaminated soils
adjoining Kanpur tanneries, Uttar Pradesh, India. Water Air Soil Poll.
202: 45-56.
Khan, A.G. 2001. Relationship between chromium biomagnification ratio,
accumulation factor and mycorrhizae in plants growing a tannery
effluents- polluted soil. Enviorn. Intern. 26: 417-423.
Khan, A.G. 2005. Role of soil microbes in the rhizospheres of plants growing
on trace metal contaminated soils in phytoremediation. J. Trace. Elem.
Med. Biol.18: 355–64.
Khwaja, M.A., Jan, R.M. and Irshad, A. 1995. Survey of Tanneries and
Leather Products Manufacturing Units in NWFP. J. Analytical Environ.
Chem. 3: 87.
Kohler, J., Hernandez, J.A., Caravaca, F. and Roldan, A. 2009. Induction of
antioxidant enzymes is involved in the greater effectiveness of a PGPR
252
versus AM fungi with respect to increasing the tolerance of lettuce to
severe salt stress. Environ. Exp. Bot. 65: 245–252.
Korda, P.A., Santas, A. and Tenente, R.S. 2004. Petroleum hydrocarbon
bioremediation: Sampling and analytical techniques, in situ treatments
and commercial and micoroorganisms currently used. Appl. Microb.
Biotech. 48: 677-686.
Laliberte, G. and Hellebust, J.A. 1989. Regulation of proline content of
Chlorella autopica in response to change in salinity. Can. J. Bot. 67:
1959–1965.
Lebeau, T., Braud, A. and Jezequel, K. 2008. Performance of ioaugmentation
-assisted phytoextraction applied to metal contaminated soil: A review.
Environ. Pollut., 153, 497-522.
Lee, M.Y. and Shin, H.W. 2003. Cadmium-induced changes in antioxidant
enzymes from the marine algae Nannochloropsis oculata. J. Appl.
Phycol. 15, 13–19.
Lexmond, T.M. 1980. The effect of soil pH on copper toxicity to forage maize
grown under field conditions. Netherlands J. Agric. Sci. 28: 164-183.
Li, M., Hu, C.W., Zhu, Q., Chen, L., Kong, Z.M., Liu, Z.L., 2006. Copper and
zinc induction of lipid peroxidation and effects on antioxidant enzyme
activities in the microalga Pavlova viridis (Prymnesiophyceae).
Chemosphere 62: 565–572.
Lima, A.F., de Moura, G.F., de Lima, M.A.B., de Souza, P.M., da Silva,
C.A.C., de Campos Takaki, G.M. and do Nascimento, A.E. 2011. Role
of the Morphology and Polyphosphate in Trichoderma harzianum
Related to Cadmium Removal. Molecules 16: 2486-2500.
Lindsay, W.L. and Norvell, W.A., 1978. Development of a DTPA soil test for
zinc, iron, manganese and copper. Soil Sci. Soc. Amer. J. 42: 421–
428.
253
Liu, W.X., Liu, J.W., Wu, M.Z., Li, Y., Zhao, Y. and Li, S.R. 2009.
Accumulation and Translocation of Toxic Heavy Metals in Winter
Wheat (Triticum aestivum L.) Growing in Agricultural Soil of
Zhengzhou, China. Bull. Environ. Contam. Toxicol. 82:343–347.
Lone, M.A., Wani, M.R., Sheikh, S.A., Sahay, S. and Dar, M.S. 2012.
Antagonistic Potentiality of Trichoderma harzianum Against
Cladosporium spherospermum, Aspergillus niger and Fusarium
Oxysporum. J. Biol. Agric. Healthcare 2(8):.72-76.
Macek, T., Francova, K., Kochankova, L., Lovecka, P., Ryslava, E., Rezek, J.,
Triska, J., Demnerova, K. and Mackova, M. 2004. Phytoremediation:
biological cleaning of a polluted environment. Rev. Environ. Hlth. 19: 63-
82.
Madrid, F., De La Rubia, T. and Martinez, J. 1996. Effect of Phanerochaete
flavido-alba on aromatic acids in olive oil mill waste waters. Technol.
Environ. Chem. 51: 161–168.
Mahmood, T., Islam, K.R. and Muhammad, S. 2007. Toxic effects of heavy
metals on early growth and tolerance of cereal crops. Pak. J. Bot.
39(2): 451-462.
Malik, A. 2004. Metal bioremediation through growing cells. Environ. Int. 30:
261– 278.
Maral, J., Puget, K.and Michelson, A.M. 1977. Comparative study of
superoxide dismutase, catalase and glutathione peroxidase levels in
erythrocytes of different animals. Biochem. Biophys. Res. Commun.
77: 1525–1535; 1977.
Marschner, H. 1995. Mineral Nutrition of Higher Plants. 2nd Edn. Academic
Press, London.
Mastouri, F., Bjorkman, T. and Harman, G.E. 2010. Seed treatment with
Trichoderma harzianum alleviates biotic, abiotic, and physiological
stresses in germinating seeds and seedlings. Phytopathology. 100:
1213–1221.
254
Mattina, M.J.I., Lannucci-Berger, W., Musante, C. and White, J.C. 2003.
Concurrent plant uptake of heavy metals and persistent organic
pollutants from soil. Environ. Pollut. 124 : 375–378.
McBride, M., Sauve, S. and Hendershot, W. 1997. Solubility control of Cu, Zn,
Cd and Pb in contaminated soils. Europ. J. Soil Sci. 48: 337-346.
Medina, A., Vassileva, M., Barea, J.M. and Azco´n, R. 2006. The growth-
enhancement of clover by Aspergillus-treated sugar beet waste and
Glomus mosseae inoculation in Zn contaminated soil. Appl. Soil Ecol.
33: 87–98.
Miao, Q. and Yan, J. 2013. Comparison of three ornamental plants for
phytoextraction potential of chromium removal from tannery sludge. J.
Mater. Cycles Waste Manage. 15:98–105.
Morx, D.H. 1969. The influence of ectotrophic mycorrhizal fungi on resistance
of Pine root to pathogenic infections. I. Antagonism of mycorrhizal
fungi to roots. Pathogenic fungi and soil bacteria. Phytopathology 59:
153-163.
Mungasavalli, D.P., Viraraghavan, T. and Jin, Y.C. 2007. Biosorption of
chromium from aqueous solutions by pretreated Aspergillus niger:
batch and column studies. Colloid Surf. A-Physicochem. Eng. Asp.
301: 214–223.
Nazir, A. and Bareen, F. 2011. Synergistic effect of Glomus fasciculatum and
Trichoderma pseudokoningii on Heliathus annuus to decontaminate
tannery sludge from toxic metals. Afr. J. Biotechnol. 10: 4612-4618.
Neagoe, A., Iordache, V., Bergmannt, H. and Kothe, E. 2013. Patterns of
effects of Arbuscular mycorrhizal fungi on plants grown in
contaminated soil. J. Plant Nut. Soil Sci. 176: 273-286.
Nehnevajova, E., Herzig, R., Bourigault, C., Bangerter, S. and Schwitzguébel,
J.P. 2009. Stability of enhanced yield and metal uptake by sunflower
mutants for improved phytoremediation. Int. J. Phytoremed. 11(4):329–
346.
255
Nikolopoulos, D. and Manetas, Y. 1991. Compatible solutes and in vitro
stability of Salsola soda enzyme: proline incompatibility.
Phytochemistry 30: 411–413.
Norvell, W.A. 1991. Reactions of metal chelates in soils and nutrient solutions.
In: Micronutrients in Agriculture (Mortvedt, J.J., Cox, F.R., Shuman,
L.M. and Welch, R.M., Eds.), 2nd Edn. pp. 187-227. Soil Science
Society of America, Madison.
Ohkawa, H., Ohishi, N. and Yagi, K. 1979. Assay for lipid peroxidation in
animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351.
Okoronkwo, N.E. Igwe, J.C. and Onwuchekwa, E.C. 2005. Riskand health
implication of polluted soils for cropproduction. Afr. J. Biotechnol. 4:
1521-1524.
Paleg, L.G., Steward, G.R. and Bradbeer, J.W. 1984. Proline and glycine
betaine influence proline salvation. Plant Physiol. 75: 974–978.
Panda, S.K. and Chaudhary, S. 2005. Chromium stress in plants. Braz. J.
Plant Physiol. 17: 95–102.
Parameswari, E. Lakshmanan, A. and Thilagavathi, T. 2010. Biosorption and
metal tolerance potential of filamentous fungi isolated from metal
polluted ecosystem. Elec. J. Environ. Agric. Food Chem. 9(4):664-671.
Peijnenburg, W., Baerselman, R., de Groot, A., Jager, T., Leenders, D.,
Posthuma, L. and Van Veen, R. 2000. Quantification of metal
bioavailability for lettuce (Lactuca sativa L.) in field soils. Arch. Environ.
Contam. Toxicol. 39: 420-430.
Pereira, G.J.G., Molian, S.M.G., Lea, P.J. and Azevedo, R.A., 2002. Activity of
antioxidant enzymes in response to cadmium in Crotalaria juncea.
Plant Soil. 239: 123–132.
Phillips, J.M. and Hayman, D.S. 1970. Improved procedure of clearing roots
and staining parasitic and vesicular-arbuscular mycorrhizal fungi for
rapid assessment of infection. Trans. Brit. Mycol. Soc. 55: 159–161.
256
Pietrini, F., Iannelli, M.A., Pasqualini, S. and Massaci, A. 2003. Interaction of
cadmium with glutathione and photosynthesis in developing leaves and
chloroplasts of Phragmites australis Trin. exSteudel. Plant Physiol.
133: 829–837.
Prasad, M.N.V., Malec, P., Waloszek, A., Bojko, M. and Strzalka, K. 2001.
Physiological responses of Lemna trisulca to cadmium and copper
bioaccumulation. Plant Sci. 161: 881–889.
Prigione, V., Zerlottin, M., Pefosco, D., Tigini, V., Anastasi, A. and Varese,
G.C. 2009. Chromium removal from a real tanning effluent by
autochthons and allochthonous fungi. Biores. Technol. 100: 2770-
2776.
Quartacci, M.G. and Navari-Izzo, F. 1992. Water stress and free radical
mediated changes in sunflower seedlings. J. Plant Physiol. 139: 621–
626.
Racusen, D. and Johnstone, D.B. 1961. Estimation of protein in cellular
material. Nature. 191: 292–493.
Rajkumar, M., Prasad, M.N.V., Freitas, H. and Ae, N. 2009. Biotechnological
applications of serpentine bacteria for phytoremediation of heavy
metals. Crit. Rev. Biotech. 29: 120-130.
Rajkumar, M., Sandhya, S., Prasad, M.N.V. and Freitas, H. 2012.
Perspectives of plant associated microbes in heavy metal
phytoremediation. Biotechnol. Adv. 30: 1562–74.
Raman, N. and Sambandan, S. 1998. Distribution of VAM fungi in tannery
effluent polluted soils Tamil Nadu, India. Environ. Contam. Toxicol. 60:
142-150.
Reboreda, R. and Cacador, I. 2008. Enzymatic activity in the rhizosphere of
Spartina maritima, Potential contribution for phytoremediation of
metals. Marine Environ. Res. 65: 77–84.
257
Reichman, S. M. 2002. The Responses of Plants to Metal Toxicity: A review
focusing on Copper, Manganese and Zinc, Published by the Australian
Minerals and Energy Environment Foundation, Melbourne, Australia.
Review on Pakistan Exports by Trade Development Authority of Pakistan
(TDAP): July-June 2007-08: http://www.epb.gov.pk/v1/statistics/
Rhoades, J.D. 1982. Soluble salts. In: Methods of soil analysis: Part 2, pp.
167-179. Chemical and microbiological properties. Monograph
Number 9 (Second Edition). (A. L. Page et al. Eds.) ASA, Madison,
WI, pp. 149-157.
Saeed, G. 1980. Technical Guide for chemical analysis of soil water samples.
Soil survey of Pakistan, Lahore.
Saleh, A. and Al-Sohaibani 2011. Heavy metal tolerant filamentous fungi from
municipal sewage for bioleaching. Asian J. Biotechnol. 3(3): 226-236.
Sarma and Hemen 2011. Metal hyperaccumuulation in plants: A Review
focusing on phytoremediation technology. J. Environ. Sci. Technol.
4(2): 118–138.
Sauve, S., Cook, N., Hendershot, W.H. and McBride, M.B. 1996. Linking plant
tissue concentrations and soil copper pools in urban contaminated
soils. Environ. Pollut. 94: 153-157.
Sauve, S., McBride, M.B., Norvell, W.A. and Hendershot, W.H. 1997. Copper
solubility and speciation of in situ contaminated soils: effects of copper
level, pH and organic matter. Water, Air Soil Pollut. 100: 133-149.
Schipper, M.A.A. and Stalpers, J.A. 1984. A revision of genus Rhizopus. II
The Rhizopus microsporus-group. Stud. Mycol. 25: 20-34.
Schützendübel, A. and Polle, A. 2002. Plant responses to abiotic stresses:
heavy metal-induced oxidative stress and protection by mycorrhization.
J. Exp. Bot. 53(372): 1351-1365.
Schützendübel, A., Schwanz, P., Teichmann, T., Gross, K., Langenfeld-
Heyser, R., Godbold, D.L. and Polle, A. 2001. Cadmium-induced
258
changes in antioxidative systems, H2O2 content and differentiation in
pine (Pinus sylvestris) roots. Plant Physiol. 127: 887–892.
Seifert, K.A. and Rossman, A.Y. 2010. How to describe a new fungal species.
IMA Fungus 1(2): 109–116.
Shanker, A.K., Cervantes, C., Lon Tavera, H. and Arudainayagam, S. 2005.
Chromium toxicity in plants. Environ. Int. 31: 739–753.
Shoresh, M., Harman, G.E. and Mastouri, F. 2010. Induced systemic
resistance and plant responses to fungal biocontrol agents. Ann. Rev.
Phytopathol. 48: 21–43.
Shugaba, A., Nok, A.J., Ameh, D.A. and Lori, J.A. 2010. Studies on the
growth of some filamentous fungi in culture solutions containing
hexavalent chromium. Int. J. Biotechnol. Biochem. 6(5): 715-722.
Siedlecka, A., Tukendorf, A., Sk´orzy´nska-Polit, E., Maksymiec, W., W’ojcik,
M., Baszy’nski, T. and Krupa, Z., 2001. Angiosperms (Asteraceae,
Convolvulaceae, Fabaceae and Poaceae; other than Brassicaceae).
In: Metals in the Environment. Marcel (Prasad, M.N.V., Ed.), Dekker
Inc., New York, pp. 171–215.
Singh, J.S., Pandey, V.C. and Singh, D.P. 2011. Efficient soil microorganisms:
a new dimension for sustainable agriculture and environmental
development. Agric., Ecosys. Environ. 140: 339–353.
Singh, K.P., Mohan, D., Sinha, S. and Dalwani, R. 2004. Impact assessment
of treated/ untreated wastewater toxicants discharged by sewage
treatment plants on health agricultural and environmental quality in the
wastewater disposal area. Chemosphere 55: 227–255.
Singh, S. and Sinha, S. 2004. Scanning electron microscopic studies and
growth response of the plants of Helianthus annuus L. grown on
tannery sludge amended soil. Environ. Int. 30: 389–395.
Singh, S., Saxena, R., Pandey, K., Bhatt, K. and Sinha, S., 2004. Response
of antioxidants in sunflower (Helianthus annuus L.) grown on different
259
amendments of tannery sludge: its metal accumulation potential.
Chemosphere 57: 1663–1673.
Sivasankar, R., Kalaikandhan, R. and Vijayarengan, P. 2012.
Phytoremediating capability of four plant species under zinc stress,
Intern. J. Res. Environ. Sci. Technol. 2 (1): 1-9.
Sk´orzy´nska-polit, E., Dra´zkiewicz, M. and Krupa, Z. 2003–04. The activity
of the antioxidantive system in cadmium-treated Arabidopsis thaliana.
Biol. Plant 47(1): 71–78.
Smirnoff, N. and Cumbes, Q.J. 1989. Hydroxyl radical scavenging activity of
compatible solute. Phytochem. 28: 1057–1060.
Song, L., Yu-Xi, D. and Xiao-Feng, Z. 2011. The Effects of Adding secondary
metabolites of Aspergillus niger on Disease Resistance to Root-knot
Nematode of Tomato. China Vegetables 1(4): 44-49.
Sun, Y., Zhou, Q., Xu, Y., Wang, L. and Liang, X. 2011. Phytoremediation for co-
contaminated soils of benzo[a]pyrene (B[a]P) and heavy metals using
ornamental plant Tagetes patula. J. Hazard. Mater. 186(2-3): 2075-82.
Sune, N., Sanchez, G., Caffaratti, S. and Maine, M.A. 2007. Cadmium and
chromium removal kinetics from solution by two aquatic macrophytes.
Environ. Pollut. 145: 467–473.
Suresh, B. and Ravishankar, G.A. 2004. Phytoremediation- a novel and
promising approach for environmental clean- up. Crit. Rev. Biotechnol.
24: 97-124.
Tariq, S.R., Shah, M.H., Shaheen, N., Khalique, A., Manzoor, S. and Jaffar,
M. 2005. Multivariate analysis of selected metals in tannery effluents
and related soil. J. Hazard. Mater. A122: 17–22.
Tariq, S.R., Shah, M.H., Shaheen, N., Khalique, A., Manzoor, S. and Jaffar,
M. 2006. Multivariate analysis of trace metal levels in tannery effluents
in relation to soil and water: A case study from Peshawar, Pakistan. J.
Environ. Manage. 79: 20–29.
260
Terry, N. and LeDuc, D. 2005. Phytoremediation of Selenium and Other Trace
Elements. Souvenir, (ICPEP-3), Lucknow.
Thanikaivelan, P., Raghava Rao, J. and Nair, B.U. 2000. Development of a
leather processing method in narrow pH profile. Part 1. Standardization
of unhairing process. J. Soc. Leather Technol. Chem. 84: 276–284.
Tian, Z., Wang, F., Zhang, W., Liu, C. and Zhao, X. 2012. Antioxidant
Mechanism and Lipid Peroxidation Patterns in Leaves and Petals of
Marigold in Response to Drought Stress. Hort. Environ. Biotechnol.
53(3):183-192.
Transfer of Technology for Development (TOOL). 2003. Environmentally
sound leather tanning. 1018 AV Amsterdam, the Nether Lands.
Tuite, J.F. 1969. Plant pathological method; fungi and bacteria. Burgress
Publications Co. Minneapolis, Minn. USA.
Tukura, B.W., Anhwange, B.A., Mohammed, Y. and Usman, N.L., 2012.
Translocation of Trace Metals in Vegetable Crops Grown on Irrigated
Soil along Mada River, Nasarawa State, Nigeria. Int. J. Modern Analy.
Sep. Sci. 1(1): 13-22.
Umebese, C.E. and Motajo, A.F. 2008. Accumulation, tolerance and impact of
aluminium, copper and zinc on growth and nitrate reductase activity of
Ceratophyllum demersum (Hornwort). J. Environ. Biol. 29(2) 197-200.
USEPA. 1999. Volunteer lake monitoring: A methods manual. EPA 440/4-91-
002. Office of water. U.S Environmental Protection Agency,
Washington, DC. file://A\Hydrology and Water Quality of Lake
Merced.htm
Usha, E., Reddy, S.A., Manuel, S.G.A. and Kale, R.D. 2012. In-vitro control of
Fusarium oxysporum by Aspergillus sp and Trichoderma sp isolated
from vermin compost. J. Bio. Innov. (5): 142-147.
261
Vadkertiova, R. and Slavikova, E. 2006. Metal tolerance of yeasts isolated
from water, soil and plant environments. J. Basic Microbiol. 46: 145-
152.
Valix, M., Tang, J.Y. and Malik, R. 2001. Heavy metal tolerance of fungi.
Miner. Eng. 14(5): 499–505.
Vankar, P.S. and Bajpai, D. 2008. Phyto-remediation of chrome-VI of tannery
effluent by Trichoderma species. Desalination 222: 255–262.
Venekemp, J.H. 1989. Regulation of cytosolic acidity in plants under condition
of drought. Plant Physiol. 76: 112–117.
Verma, T., Srinath, T., Gadpayle, R.U., Ramteke, P.W., Hans, R.K. and Garg,
S.K. 2001. Chromate tolerant bacteria isolated from tannery effluent.
Biores. Technol. 78: 31-35.
Vivas, A., Biro, B., Ruı´z-Lozano, J.M., Barea, J.M. and Azco´n, R. 2006. Two
bacterial strains isolated from a Zn-polluted soil enhance plant growth
and mycorrhizal efficiency under Zntoxicity. Chemosphere 62: 1523–
1533.
Vivas, A., Vörös, A., Biró, B., Barea, J.M., Ruíz-Lozano, J.M. and Azcón, R.
2003. Beneficial effects of indigenous Cd-tolerant and Cd sensitive
Glomus mosseae associated with a Cd-adapted strain of Brevi bacillus
sp. in improving plant tolerance to Cd contamination. Appl. Soil Ecol.
24:177–186.
Wang, D.Q., Zhang, A.L., Zhang, X.H., Liu, J., You, S.H., Zhao, W.Y. and
Jiao, Y.X. 2009. Effect of Cr (III) Stress on Activities of Antioxidant
Enzymes in L. hexandra Swartz. The Guangxi Key Laboratory of
Environmental Engineering, Protection and Assessment, Guilin
University of Technology Guilin, China.
Weckx, J.E.J. and Clijsters, H.M.M. 1996. Oxidative damage and defense
mechanisms in primary leaves of Phaseolus vulgaris as a result of root
assimilation of toxic amounts of copper. Physiol. Plant 96: 506–512.
262
Whitehead, D.C. 2000. Nutrient Elements in Grasslands: Soil-Plant-Animal
Relationships. CABI Publishing, Wallingford.
Wilkins, D.A. 1978. The measurement of tolerance to edaphic factors by
means of root growth. New Phytol. 80: 623–633.
Wolt, J.D. 1994. Soil Solution Chemistry: Applications to Environmental
Science. John Wiley and Sons, New York.
Wong, M.H. 2003. Ecological restoration of mine degraded soils, with
emphasis on metal contaminated soils. Chemosphere 50: 775-780.
Wright, A. L., Wang, Y. and Reddy, K. R. 2008. Loss-on-ignition method to
assess soil organic carbon in calcareous Everglades wetlands.
Commun. Soil Sci. Plant Analysis 39: 3074–3083.
Zafar, S., Aqil, F. and Ahmad, I. 2007. Metal tolerance and biosorption
potential of filamentous fungi isolated from metal contaminated
agricultural soil. Biores. Technol. 98: 2557–2561.
Zapotoczny, S., Jurkiewicz, A., Tylko, G., Anielska, T. and Turnau, K. 2007.
Accumulation of copper by Acremonium pinkertoniae, a fungus isolated
from industrial waste. Microbiol. Res. 162(3): 219-228.
Zehra, S.S., Arshad, M., Mahmood, T. and Waheed, A. 2009. Assessment of
heavy metal accumulation and their translocation in plant species. Afr.
J. Biotechnol. 8(12): 2802-2810.
Zengin, F.K. and Munzuroglu, O. 2006. E_ects of heavy metals (Pb++, Cu++,
Cd++, Hg++) on total protein and abscisic acid content of bean
(Phaseolus vulgaris L. cv. Strike) seedlings. Fresenius Environ. Bull.
15(4): 277–282.
Zhang, F.Q., Wang, Y.S., Lou, Z.P., Dong, J.D. 2007. Effect of heavy metal
stress on antioxidative enzymes and lipid peroxidation in leaves and
roots of two mangrove plant seedlings (Kandelia candel and Bruguiera
gymnorrhiza). Chemosphere 67: 44–50.
263
Zhang, X. H., Lin, A.J., Chen, B.D., Wang, Y.S., Smith, S.E. and Smith, F.A.
2006. Effects of Glomus mosseae on the toxicity of heavy metals to
Vicia faba. J. Environ. Sci. 18(4): 721–726.
264
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