Cultivable Endophytic Mycobiont Piriformospora indica in...
Transcript of Cultivable Endophytic Mycobiont Piriformospora indica in...
International Journal of Science, Engineering and Technology Research (IJSETR), Volume 3, Issue 7, July 2014
2033 ISSN: 2278 – 7798 All Rights Reserved © 2014 IJSETR
Cultivable Endophytic Mycobiont Piriformospora indica in Millet Growth Promotion
Krishnaveni. N1, Dipika.B
2, Geetha Ramani D
3, Cibi Chakravarthy
4
1. Assistant Professor, Department of Microbiology, PSG College of Arts & Science,
Coimbatore
2. Research Scholar, Department of Microbiology, PSG College of Arts & Science,
Coimbatore
3. Associate Professor, Department of Microbiology, Dr. NGP College of Arts and Science,
Coimbatore
4. Research Scholar, Department of Biotechnology, Bharathiar University, Coimbatore.
ABSTRACT
The novel endophytic fungi Piriformospora
indica has been proven to have vast applications as plant growth promoter. P. indica
significantly increases plant biomass, flowering
and yield, nutrient value etc., during its
colonization and proliferation in the roots of the host plants. Study plants were chosen to be
Sorghum (Sorghum bicolor), Pearl Millet
(Pennisetum glaucum) and Finger Millet (Eleusine coracana). Pot cultures of P. indica
colonized plants were compared for physical
parameters as root, shoot height, dry and fresh weight. The P.indica colonized plants have
shown comparatively higher level of
chlorophyll, protein, phosphate and calcium
content. Enzymic and non-enzymic antioxidants levels were higher in P.indica colonized plants.
Various metabolites produced by
Piriformospora indica was analysed by GC-MS analysis, depicting many medically important
compounds with anti-cancer, insecticidal,
nematicidal, anti parasitic, anti-bacterial and
anti-fungal properties. Thus the present study has added to the proof that Piriformospora
indica is a highly efficient plant growth
promoting endophytic fungi.
Keywords: endophytic fungi, millet endophyte,
Piriformospora indica, plant growth promoter.
INTRODUCTION
Piriformospora indica, the
mycorrhizae (AM)-like fungus, has been
extensively studied to be cultivable in axenic
cultures, where it forms chlamydospores
asexually [1]. The fugus Piriformospora
indica was discovered in the Thar Desert
(1988) from the rhizospheric soil of two
xerophytic plants, namely Prosopis juliflora
and Ziziphus nummularia [1]. It is the first
symbiotic fungus, which was proved to be
inoculated to the roots of a plants [2,3,4] and
found to have stimulated considerable
attention as they form endophytic
associations with most plant species
[22,23]. This is of great interest that they
possess plant growth-promoting
characteristics and contribute several other
benefits to their host plants [1,5, 6, 7, 8, 20,
22, 23]. P. indica colonizes the roots, grows
intracellularly, forming pear-shaped spores
that collect in the roots as well as on the root
surface. The endophyte mediates nutrient
uptake, stimulates growth and seed
production, allows plants to survive in less
water, confers resistance to toxins,
resistance to heavy metal ions, and promotes
salt stress [1, 13, 14, 15, 17, 19, 21]. It
significantly increases plant biomass,
flowering and yield, nutrient value etc.,
during its colonization and proliferation in
the roots of the host plants. [1,7, 14, 18].
International Journal of Science, Engineering and Technology Research (IJSETR), Volume 3, Issue 7, July 2014
2034 ISSN: 2278 – 7798 All Rights Reserved © 2014 IJSETR
Piriformospora indica as a mycofertilizer
has produced a greater keenness among
mycorrhizologists, due to being easily
culturable, plant-growth enhancer and
inducer. In the present work, the recently
discovered root endophytic-fungi
Piriformospora indica has been optimized
and tested for its growth enhancement.
METHODOLOGY
Culture of P.indica
P.indica was procured from the Department
of Microbial technology, Amity University,
Noida. The culture was maintained on Hill
and Kaefer medium [4]. The cultures were
incubated at 30±2°C in incubator for a week.
Characterization of Piriformospora
indica:
Morphological characteristics-Fungal
Wet mount technique
Lacto phenol cotton blue technique was
carried to identify the morphological
structure of the fungi. A small block of
fungal culture from the surface of agar plate
was taken and placed on a clean glass slide.
A drop of Lacto phenol cotton blue was
added on the block. The cover slip was
placed on the culture block with a gentle
press. The slide was viewed under
microscope at 45X, to observe the
morphological features of the fungi.
Cultural characteristics
The fungal isolates were inoculated in
different culture media like Nutrient agar,
Malt extract, Malt yeast extract agar,
Sabouraud’s Dextrose agar medium, MYP
Medium, Hill and Kafer medium and Brain
Heart Infusion agar medium to observe its
colony morphology and various cultural
characteristics.
Optimization of Piriformospora indica
To optimize the various growth parameters
of the fungus in laboratory conditions,
several physical parameters such as
Temperature, Incubation time required for
optimum growth, Heavy metals
concentrations, different salt concentrations,
different carbon sources, nitrogen sources,
vitamin sources and growth in various media
have been studied.
Determination of extracellular enzyme
formation by P.indica
To resolve the extracellular enzyme
produced by P.indica, various enzyme
activity such as amlyolytic activity, cellulase
activity, proteolytic activity, laccace activity
and lipolytic activity were performed [10,
21].
Germination studies
In the present study, tissue culture technique
has been used for studying the ability of
Piriformospora indica in pre-determined
conditions to check the plant growth
promoting ability of the fungal
endosymbiont and for this study the Millet
varieties of Sorghum, Pearl Millet
(Pennisetum glaucum) and Finger Millet
(Eleusine coracana) seeds were used for the
growth determination.
The selected seeds of the test plants were
surface-sterilized with sterile distilled water
for three times and rinsed in 0.1% HgCl2 for
about 1 min and finally washed for three
times with sterile distilled water [1,2] to
eliminate the naturally occurring surface
microorganism. The Murashige and Skoog
medium was prepared and dispensed in
sterile petriplates. After solidification the
surface sterilized seeds were aseptically
transferred to Murashige and Skoog medium
[12] and the plates were incubated at room
temperature in presence of light, to observe
the germination of plantlet in tissue culture
media.
International Journal of Science, Engineering and Technology Research (IJSETR), Volume 3, Issue 7, July 2014
2035 ISSN: 2278 – 7798 All Rights Reserved © 2014 IJSETR
Pot culture studies
In pot study, the seeds of chosen plants were
allowed to germinate by inoculating
P.indica on a hard substratum which
contains sand and soil in 3:1 ratio. The soil
was autoclaved thrice on alternate days and
air dried. Riverbed sand was soaked in 10%
HCl overnight and then washed under
running tap water until the pH has reached
near neutrality (pH 6-7 as standardized in
the present study) [8]. This sterile soil has
been used as substratum in pots. Pots were
also surface-sterilized by 70% ethanol and
were then half-filled with this sterile soil
mixture and the fungal inoculum has been
added in the volume as variying
concentrations of 3%, 5% and 7%to the soil
content that is filled in the pot. The fungal
inoculum was mixed well with the sterile
soil so as to enhance even distribution of the
fungal inoculum in the soil in pot. Equal
number of surface sterilized seeds was sown and also in sterile soil for control plants
(without addition of P. indica).The pot study
experiments were carried out in controlled
conditions.
Root Colonization by fungus -
Microscopic Observation
Roots of randomly chosen plants were
washed thoroughly in running tap water, cut
into about 1-cm pieces, transferred to 10%
KOH, boiled for 10 min, then acidified with
1 M HCl for 10 min, and finally stained with
0.02% of Trypan blue in lactophenol
overnight [9, 11]. Samples were destained
with lactic acid:glycerol:water solution in
the ratio of 14:1:1(Volumetric ratio). The
stained root samples were observed under
microscope.
Estimation of plant growth
For assessment of plant growth promotion of
endosymbiotic sorghum, pearl millet and
finger millet plants; shoot and root length,
fresh and dry weight, total protein
estimation and increase in chlorophyll
pigmentation, phosphate content, calcium
content, enzymic and non enzymic
antioxidants were measured. The parameters
were compared with the values in control
plants.
RESULTS AND DISCUSSION
Piriformospora indica cultures were
obtained from Dr. Varma’s laboratory at
Department of Microbial Biotechnology,
Amity University, Noida, India. The culture
was reviewed by using the selective medium
of Hill and Kafer and in this medium the
growth pattern has been shown (Fig 1). P.
indica showed the morphology of pear-
shaped chlamydospores at hyaline hyphae
was examined under light microscope at
45X (Fig 2).
Fig 1
Fig 2
Fig 1- Colonies of Piriformospora indica on
Hill & Kafer medium
Fig 2-Fungal culture showing hyphae with
spores on Fungal mount technique
The cultural characteristics of the P. indica
was determined in different types of
medium, showed puffed white chalky
powdery appearance with the pointed tip at
International Journal of Science, Engineering and Technology Research (IJSETR), Volume 3, Issue 7, July 2014
2036 ISSN: 2278 – 7798 All Rights Reserved © 2014 IJSETR
the centre of the each colony in the different
medium (Fig 3) .
Nutrient agar SDA
Brain heart infusion agar Malt extract agar
Malt yeast extract agar MYP agar
Hill & Kafer agar
Fig 3- Growth of Piriformospora indica on
different medium
Optimization of Piriformospora indica
The cultures were revived and optimized to
grow in laboratory conditions. P. indica
showed optimum growth at a temperature of
270C and dry weight of 1.2 g/l, the optimum
incubation time for the initiation of growth
was found to be 3 days and it was also noted
that the maximum level of growth was
obtained on the 33rd
day and dry weight of
0.48g/5ml, Hill and Kafer medium have
shown to demonstrate better growth among
all the other nutrient media which showed a
maximum growth of 0.66g/5ml dry weight.
P.indica culture showed more resistance to
Nickel and Lead when compared to that of
Zinc and Cadmium, this proves that only
minimal amount of Heavy metal tolerance
has been observed in P.indica culture. The
culture showed a reduced growth when the
concentration of heavy metal and salt was
increased but there was still a viability of the
culture observed at lowest concentration of
heavy metals and salt used for the present
study, which shows that they are tolerant to
heavy metals and salt stress may be at more
lower concentrations which has to be
explored.
The fungal culture has the ability to grow in
the high concentration of glucose in which,
the dry weight was higher in the
concentration of 5% and the poor carbon
source was straw, in this it shows less
amount of dry weight when compared to
other carbon sources, has ability to grow in
the high concentration of peptone in which,
the dry weight was higher in the
concentration of 1% and lesser growth was
observed when nitrogen source was casein
and also has the ability to grow in the high
concentration of nicotinamide in which, the
dry weight was higher in the concentration
of 1% and the poor growth was observed
when vitamin source was riboflavin, it
shows less amount of dry weight when
compared to other vitamin sources.
Determination of extracellular enzyme
production by P.indica
The extracellular enzyme activity produced
by Piriformospora indica culture were
studied and found to be amylolytic,
cellulase, laccase and lipolytic activity
which were positive. Proteolytic activity was
not positive for the fungi and the results
were shown (Fig 4).
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Amylolytic activity Cellulase activity
Proteolytic activity Laccase activity
Lipolytic activity
Fig 4- Enzyme production by
Piriformospora indica
Germination and Pot culture studies
Germination studies of P. indica in MS
medium have shown considerable
advancement in growth compared to control
plant (Fig 5a. & 5b and Table 1). Pot
culture studies have also revealed better
plant growth promotion which is evidenced
by comparing the physical parameters of
endosymbiotic plant with the control plant
(Fig 6). In all the three types of plants the
growth rate was excellent in P.indica
colonized plants in pot culture.
Pearl millet plant
Sorghum plant
Finger millet plant
Fig 5a- Germination studies on the study
plant in plates
Pi
Pi C
Pi c
c
C
C
Pi
C
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PI
Pearl millet plant Sorghum plant Finger millet plant
Fig 5b- Germination studies on the study plant in bottles
Table 1-Shows the difference in germination between the inoculated and uninoculated plants
Sorghum plant Pearl millet plant Finger millet plant
Fig 6- Pot culture studies in the study plants
S.No
Study
Plants
Day of germination
Measurement of germination
in cm
P.indica
Inoculated
Control P.indica
Inoculated
Control
1 Sorghum 2nd
day 3rd
day 0.6 0.4
2 Pearl
Millet
4th day 5
thday 0.3 0.05
3 Finger
millet
2nd
day 4thday 0.4 0.15
Pi Pi Pi
C C C
Control 3%
5% 7% Control 33%
5% 7% Control 3%
33% 5%
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Microscopic Observation of Root
Colonization by Fungus
The stained root samples were observed under
light microscope (100X). The colonization of root by P. indica in the study plants has been
shown in Plate 6, Figure 4.15.This shows the
difference between the P.indica colonized study
plants with that of control plants.
Control root Pi colonized root
Fig 7- Root staining of the study plants
showing SEM picture of Pi colonization
Estimation of physical parameters
The colonization of the study plants
by P.indica has led to its increased growth.
Due to the growth promotional activities in
the study plants when compared to un-
inoculated plants, plants inoculated with
P.indica showed greater increase in root and
shoot length and increase in fresh and dry
weight. In all the three plants the
endosymbiont colonized plants have shown
excellent growth, and gain in weight and
length when compared to that of control
plants in pot studies and their differences
have been shown in Table 2a &2b and Fig
8a & 8b.
Table 2a- Fresh and Dry weight of plants
studied with Piriformospora indica
Table 2b- Shoot and Root length of plants
studied with Piriformospora indica
1
2
Plants
Sorghum
Pearl millet
Finger millet
Control
plants
Fresh
weight
in gms
Dry
weight
in gms
Fresh
weight
in gms
Dry
weight
in gms
Fresh
weight
in gms
Dry
weight
in gms
5.06
1.92
3.45
1.05
4.23
0.93
P.indica
colonized
plants
7.94
2.53
5.5
1.85
6.09
1.34
1
2
Plants
Sorghum
Pearl millet
Finger millet
Control
plants
Shoot
length
in cms
Root
length
in cms
Shoot
length
in cms
Root
length
in cms
Shoot
length
in cms
Root
length
in cms
34
3.5
26
2.8
30
3.1
P.indica
colonized
plants
49
4.6
32
3.7
43
4.2
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Fig 8a- Shoot length of the Pi colonized
study plants with control plants
Sorghum plant Finger millet plant
Pearl millet plant
Sorghum plant Finger millet plant
Pearl millet plant
Fig 8b- Root length of the Pi colonized
study plants with control plants
The P.indica colonized plants have
shown comparatively higher chlorophyll
content when compared to that of control
plants which indicates that P.indica helps to
enhance the chlorophyll production in the
plants, there is net gain in total protein
content in P. indica colonized plants which
might help for the various plant growth
promoting and protecting characteristics of
the fungus that has been demonstrated
earlier. The proteins were further
characterized by Native-PAGE to determine
the difference in molecular weight of the
proteins present in P. indica colonized and
control plants, when compared to control
plants there is net gain in total phosphate
content in P. indica colonized plants which
might help for diverse regulatory, structural,
and energy transfer roles the various plant
growth promoting and protecting
characteristics of the fungus, increasing
amount of calcium content present in
P.indica colonized plant when compared to
that of control plants which helps for the
plant growth promotional and building
activities.
P.indica colonized plants showed
increasing level of ascorbic acid which helps
in protecting the plants and also helps in
plant growth promoting properties, increased proline content which helps the
plants to grow under stress conditions and
also helps in plant growth promoter, increased reduced glutathione content which
favours the plant–fungal association and
increases resistance to the pathogen and
also promotes plant growth, increased level
of catalase content which responsible for
preventing and neutralizing the free radical
induced damages on tissues. This result has
been shown (Table 3).
Pi C Pi
C
Pi C
Pi C
Pi
C
Pi C
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Table 3 - Biochemical, Enzymic and non-enzymic antioxidant studies on Pi colonized plants
Gas Liquid Chromatography-Mass Spectrometry
Piriformospora indica was subjected to gas liquid chromatography mass spectrometry analysis.
The identification of compounds is based on the peak area, molecular weight and molecular
formula. Peak area is directly proportional to quantity of the compound present in the culture.
S.No
Estimations
Sorghum plant
Pearl millet plant
Finger millet plant
1
Chlorophyll
content
Control plant
Pi colonized
plant
Control plant
Pi colonized
plant
Control plant
Pi colonized
plant
1.42 g/tissue
2.11 g/tissue
0.86 g/tissue
1.33 g/tissue
1.27 g/tissue
2.06 g/tissue
2
Protein content
0.7mg/g
0.9 mg/g
0.2 mg/g
0.5 mg/g
0.4 mg/g
0.7 mg/g
3
Phosphate content
7.5µg/tissue
10 µg/tissue
3.5 µg/tissue
5 µg/tissue
5 µg/tissue
8.5 µg/tissue
4
Calcium content
180mg/g
220 mg/g
120 mg/g
170 mg/g
150 mg/g
190 mg/g
5
Non enzymic
aantioxidants
Ascorbic acid
0.46mg/g
0.6 mg/g
0.32 mg/g
0.42 mg/g
0.36 mg/g
0.48 mg/g
6
Proline
11.08µmoles /g
of tissue
15.93 µmoles
/g of tissue
5.19µmoles /g
of tissue
919µmoles /g
of tissue
9.35 µmoles /g
of tissue
13.16 µmoles
/g of tissue
7
Enzymic
antioxidants
Reduced
glutathione
0.48mg/g
0.72 mg/g
0.36 mg/g
0.6 mg/g
0.24 mg/g
0.48 mg/g
8
Catalase
860units/ml of
extract
1220 units/ml
of extract
503 units/ml of
extract
719 units/ml of
extract
647 units/ml
of extract
1000 units/ml
of extract
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The GC-MS results shows the various metabolites produced by the fungi, there are many
medically important compounds that have been referred to show ant-bacterial, anti-fungal, anti-
parasitic, anti-cancer, insecticidal, nematicidal properties etc., shown in the Table 4. This study
gives a better understanding of the protective effect that it confers on its host plant against
invading pathogenic organisms. The components may further be analyzed to establish a better
understanding about these secondary metabolites produced by the fungi.
Library Search Results
International Journal of Science, Engineering and Technology Research (IJSETR), Volume 3, Issue 7, July 2014
2043 ISSN: 2278 – 7798 All Rights Reserved © 2014 IJSETR
Compound Name Probabilit
y
Molecular
Formula
Molecular
Weight Structure Commen Comments
Cholestan-3-ol,
2-methylene-,
(3á,5à)- (CAS)
4.73 C28H48O 400
Antimicrobial Antiinflammatory
Anticancer Diuretic
Antiasthma Antiarthritic
Ergosta-5,7,22-
trien-3-ol,
(3á,22E)- (CAS)
12.89 C28H44O 396
Synaptogenin R 0.68 C30H48O3 456 Anti fungal Property
Lanosta-
7,9(11),20(22)-
triene-3á,18-diol,
diacetate
(CAS)
9.08 C34H52O4 524
A plant derived secondary
metabolite with anti-Tuberculosis
product, anti-cancer properties
7-Oxogedunin 3.89 C26H30O6 438 Anti fungal Property & Insecticidal
activity
Neurosporaxanth
in methyl ester
6.19 C36H48O2 512 Major carotenoid product in F.
fujikuroi is neurosporaxanthin (b-
apo-4¢-carotenoic acid), a
carboxylic
xanthophyll formerly identified in
Neurosporacrassa
8-Norurs-9(11)-
en-12-one-28-oic
acid, 3-acetoxy-,
methyl ester (14á,20á)
7.00 C32H48O5 512 Cytotoxic activity
6,7-
Isopropylidenedi
oxy-1,15-
dihydroxy-8,15-
seco-mao
erystals A
3.62 C23H36O5 392
Herbicidal and Nematicidal
Property
Cholic acid 9.08 C24H40O5 408
An elicitor of defence response
expressed in plants
Cyclotrisiloxane, hexaphenyl-
6.62
C36H30O3 Si3
594
Plant active compound
Hexatriacontane 11.34 C36H74 506
Antioxidant Property
International Journal of Science, Engineering and Technology Research (IJSETR), Volume 3, Issue 7, July 2014
2044 ISSN: 2278 – 7798 All Rights Reserved © 2014 IJSETR
Table 4- Compounds present in the fungal culture was analyzed by GC-MS.
CONCLUSION
Endophytic fungi are an excellent model
organism for plant growth promotion studies
and the most widespread symbionts on earth
are receiving attention because of the
increasing range of their application in
sustainable agriculture and ecosystem
management. Development of P.indica may
not only complement crop-growth
promotion strategies but will also serve as
model representation to study its
physiological functions, molecular behaviors
that result in disease resistance against biotic
and abiotic stress including root and leaf
infecting pathogens, insect invaders, grain
yielders and production of many additional
growth mediating component as
biofertilizers in nutrient deficient soils. All
these impressive traits make P. indica very
valuable for applied research and also can be
easily proliferated on a large scale in axenic
culture in the absence of host plant. It could
thus be suggested to consider this endophyte
as a tool for endless sustainable agriculture.
ACKNOWLEDGMENT
The authors acknowledge Dr.Varma’s
laboratory, AMITY University, for the
culture and the Management PSG College of
Arts And Science, Coimbatore, for the
laboratory facilities.
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Corresponding Author:
Krishnaveni. N - Assistant Professor,
Department of Microbiology, PSG College
of Arts & Science, Coimbatore, Tamil nadu,
India.