ANASTOMOSIS GROUP TYPING OF RHIZOCTONIA SOLANI KÜHN ...
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ANASTOMOSIS GROUP TYPING OF RHIZOCTONIA SOLANI
KÜHN INFECTING SOLANACEOUS VEGETABLE CROPS
AMJAD SHAHZAD GONDLE
13-arid-20
Department of Plant Pathology
Faculty of Crop and Food Science
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
Pakistan
2018
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ANASTOMOSIS GROUP TYPING OF RHIZOCTONIA SOLANI
KÜHN INFECTING SOLANACEOUS VEGETABLE CROPS
by
AMJAD SHAHZAD GONDLE
(13-arid-20)
A thesis submitted in partial fulfilment of
the requirements for the degree of
Doctor of Philosophy
in
Plant Pathology
Department of Plant Pathology
Faculty of Crop and Food Sciences
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
Pakistan
2018
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In the name of Allah,
the beneficent the merciful
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Dedicated to
my Beloved
Father Mother
Brothers & Sisters
Whatever I am and hope to be my life, I love you …..
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CONTENTS
Page
List of Tables xiii
List of Figures xvi
List of Acronyms xx
Acknowledgements xxii
ABSTRACT xxiv
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 6
2.1 SOLANACEOUS VEGETABLES 6
2.1.1 Potato 6
2.1.2 Tomato 8
2.1.3 Chilli 10
2.2 RHIZOCTONIA 13
2.2.1 Rhizoctonia solani 14
2.2.1.1 Origin and taxonomic classification 14
2.2.1.2 General characteristics 15
2.2.1.3 Ecology and epidemiology 16
2.3 ANASTOMOSIS GROUPING IN RHIZOCTONIA SOLANI 17
2.3.1 Categories of Anastomosis Interactions 18
2.3.2 Subgroups within Anastomosis Groups 20
2.3.3 Molecular Methods for AGs Classification 23
2.3.4 Internal Transcribed Spacer (ITS) Sequence Analysis 24
2.3.5 Host Range of Rhizoctonia solani AGs 27
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3 MATERIALS AND METHODS 35
3.1 SURVEILLANCE FOR DISEASE ASSESSMENT 35
3.1.1 Description of the Study Area 35
3.1.2 Disease Assessment and Sample Collection 36
3.2 ISOLATION AND CULTURING OF R. SOLANI 42
3.2.1 Preservation of Rhizoctonia solani Isolates 45
3.3 MORPHOLOGICAL CHARACTERIZATION OF ISOLATES 44
3.3.1 Cultural Characteristics and Microscopic Studies of R. solani 44
3.3.2 Nuclear Number Testing 44
3.4 PATHOGENICITY TESTING 45
3.5 ANASTOMOSIS GROUP TESTING 49
3.5.1 PCR-Restriction Fragment Length Polymorphism (RFLP) 49
3.5.1.1 Culturing, maintenance & lyophilization 49
3.5.1.2 DNA extraction 50
3.5.1.3 Polymerase chain reaction (PCR) amplification 51
3.5.1.4 Confirmation of PCR amplification 52
3.5.1.5 PCR products purification 52
3.5.1.6 PCR–RFLP analysis 54
3.5.1.7 Restriction patterns 54
3.5.1.8 Hyphal anastomosis interactions 54
3.5.2 PCR amplification of ITS-5.8S rDNA 58
3.5.2.1 Sequencing of ITS-5.8S rDNA 59
3.5.2.2 Sequence analysis 59
3.5.2.3 Phylogenetic studies 60
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4 RESULTS 62
4.1 SURVEILLANCE FOR DISEASE ASSESSMENT 62
4.1.1 Surveillance for Disease Assessment on Potato 62
4.1.1.1 Rhizoctonia solani disease prevalence and incidence on potato 62
4.1.2 Surveillance for Disease Assessment on Tomato 65
4.1.2.1 Rhizoctonia solani disease prevalence and incidence on tomato 65
4.1.3 Surveillance for Disease Assessment on Chilli 67
4.1.3.1 Rhizoctonia solani disease prevalence and incidence on chilli 67
4.2 ISOLATION AND CULTURING OF R. SOLANI 71
4.3 MORPHOLOGICAL CHARACTERIZATION OF ISOLATES 71
4.3.1 Morphological Characterization of Isolates from Potato 76
4.3.2 Morphological Characterization of Isolates from Tomato 77
4.3.3 Morphological Characterization of Isolates from Chilli 95
4.4 PATHOGENICITY DETERMINATION 96
4.4.1 Pathogenicity Determination on Potato 96
4.4.2 Pathogenicity Determination on Tomato 107
4.4.3 Pathogenicity Determination on Chilli 107
4.5 ANASTOMOSIS GROUP TESTING 110
4.5.1 PCR-Restriction Fragment Length Polymorphism (RFLP) 113
4.5.2 Hyphal Anastomosis Interaction 116
4.5.3 Sequence Analysis of ITS-5.8S rDNA 122
4.5.4 Frequencies of Different AGs 130
4.5.5 Phylogenetic Analysis of ITS using DNA Sequences 123
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5 DISCUSSION 133
CONCLUSIONS 144
RECOMMENDATIONS 145
SUMMARY 146
LITERATURE CITED 149
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LIST OF TABLES
Table No. Page
2.1 The composition of the primers for amplification of nuclear
internal transcribed spacer (ITS) region.
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2.2 Host ranges and associated disease symptoms, of Rhizoctonia
solani belonging to different anastomosis groups (AGs).
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3.1 Districts and their locations surveyed for Rhizoctonia solani
infection on potato, tomato, and chilli during the crop season
2014-15 and 2015-16.
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3.2 Total number of samples for Rhizoctonia solani infection on
potato, tomato, and chilli from different locations of each
district.
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3.3 Disease rating scale to record stem, stolon, and tuber infection
on potato.
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3.4 Disease rating scale to record stem infection on tomato. 47
3.5 Disease rating scale to record stem infection on chilli. 47
3.6 RFLP types revealed by the restriction analysis of ITS
sequences among Rhizoctonia solani.
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3.7 GenBank accessions of Rhizoctonia solani reference isolates
used in this study.
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4.1 Disease prevalence and incidence percentage of Rhizoctonia
solani on potato in various areas/locations of the districts of
Pothohar region.
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4.2 Disease prevalence and incidence percentage of Rhizoctonia
solani on tomato in various areas/locations of the districts of
Pothohar region.
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4.3 Disease prevalence and incidence percentage of Rhizoctonia
solani on chilli in various areas/locations of the districts of
Pothohar region.
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4.4 Details of Rhizoctonia solani isolates recovered from potato,
tomato, and chilli symptomatic plant samples.
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4.5 Morphological characterization of sixty-three isolates of
Rhizoctonia solani recovered from portions of diseased potato
samples collected from Pothohar region during 2014-15 and
2015-16 crop season.
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4.6 Morphological characterization of sixty-seven isolates of
Rhizoctonia solani recovered from portions of diseased tomato
samples collected from Pothohar region during 2014-15 and
2015-16 crop season.
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4.7 Morphological characterization of fifty-eight isolates of
Rhizoctonia solani recovered from portions of diseased chilli
samples collected from Pothohar region during 2014-15 and
2015-16 crop season.
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4.8 Rhizoctonia solani anastomosis groups (AGs) assigned using
PCR-RFLP and hyphal anastomosis interaction.
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4.9 Type isolates of Rhizoctonia solani representing different
anastomosis groups (AGs) and 07 unknown isolates used for
molecular characterization.
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4.10 Frequency of Rhizoctonia solani anastomosis groups (AGs)
recovered from potato symptomatic samples, collected from
different locations of Pothohar region
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4.11 Frequency of Rhizoctonia solani anastomosis groups (AGs)
recovered from portions of diseased potato samples.
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4.12 Frequency of Rhizoctonia solani anastomosis groups (AGs)
recovered from tomato symptomatic samples, collected from
different locations of Pothohar region.
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4.13 Frequency of Rhizoctonia solani anastomosis groups (AGs)
recovered from chilli symptomatic samples, collected from
different locations of Pothohar region
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LIST OF FIGURES
Figure No. Page
2.1 Structure of nuclear ribosomal DNA repeat unit of fungus. 25
3.1 Map of Pakistan showing Pothohar region the northern part
of Punjab.
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3.2 Map of Pothohar region showing district Attock, Chakwal,
Jhelum, Rawalpindi and Islamabad.
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3.3 (a) Symptomatic potato plant infected with Rhizoctonia solani. 39
3.2 (b) Diseased potato tubers showing visible sclerotia of
Rhizoctonia solani.
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3.4 (a) Symptomatic chilli plant infected with Rhizoctonia solani. 40
3.4 (b) Symptomatic tomato plant infected with Rhizoctonia solani. 40
3.5 Pathogenicity testing of Rhizoctonia solani isolates on potato
plants under pot trials.
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3.6 Extraction of genomic DNA of Rhizoctonia solani isolates. 53
3.7 Restriction patterns revealed by RFLP analysis of internal
transcribed spacers sequences of Rhizoctonia solani with MseI.
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3.8 Restriction patterns revealed by RFLP analysis of internal
transcribed spacers sequences of Rhizoctonia solani with Ava
II+Hinc II and MunI.
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4.1 District wise, mean disease incidence of Rhizoctonia solani
infection on potato, tomato, and chilli.
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4.2 Morphological diversity of Rhizoctonia solani isolates
incubated on malt extract agar (MEA) medium
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4.3 Cultural appearance of Rhizoctonia solani isolate (RWPT5)
under the microscope
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4.4 Inter septal distance of Rhizoctonia solani isolate (JHEP2)
recovered from potato
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4.5 Hyphal diameter of Rhizoctonia solani isolate (ATKP7)
recovered from potato.
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4.6 Nuclear number testing of Rhizoctonia solani isolate
(RWPP9) recovered from potato.
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4.7 Distribution of Rhizoctonia solani isolates recovered from
potato on the basis of hyphal length/ inter septal distance
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4.8 Distribution of Rhizoctonia solani isolates recovered from
potato on the basis of hyphal diameter.
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4.9 Distribution of Rhizoctonia solani isolates recovered from
potato on the basis of number of sclerotia.
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4.10 Distribution of Rhizoctonia solani isolates recovered from
potato on the basis of sclerotia texture.
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4.11 Distribution of Rhizoctonia solani isolates recovered from
potato on the basis of sclerotia topography.
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4.12 Distribution of Rhizoctonia solani isolates recovered from
tomato on the basis of hyphal length/ inter septal distance.
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4.13 Distribution of Rhizoctonia solani isolates recovered from
tomato on the basis of hyphal diameter.
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4.14 Distribution of Rhizoctonia solani isolates recovered from
tomato on the basis of number of sclerotia.
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4.15 Distribution of Rhizoctonia solani isolates recovered from
tomato on the basis of sclerotia texture.
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4.16 Distribution of Rhizoctonia solani isolates recovered from
tomato on the basis of sclerotia topography.
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4.17 Distribution of Rhizoctonia solani isolates recovered from
chilli on the basis of hyphal length/ inter septal distance.
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4.18 Distribution of Rhizoctonia solani isolates recovered from
chilli on the basis of hyphal diameter.
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4.19 Distribution of Rhizoctonia solani isolates recovered from
chilli on the basis of number of sclerotia.
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4.20 Distribution of Rhizoctonia solani isolates recovered from
chilli on the basis of sclerotia texture.
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4.21 Distribution of Rhizoctonia solani isolates recovered from
chilli on the basis of sclerotia topography.
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4.22 Disease index of Rhizoctonia solani infection on potato (cv.
Desiree).
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4.23 Pathogenicity determination of sixty-three Rhizoctonia solani
isolates on Potato (cv. Desiree).
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4.24 Disease index of Rhizoctonia solani infection on tomato (cv.
Rio Grande).
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4.25 Pathogenicity determination of sixty-seven Rhizoctonia
solani isolates on tomato (cv. Rio Grande).
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4.26 Disease index of Rhizoctonia solani infection on tomato (cv.
Sanam).
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4.27 Pathogenicity determination of fifty-eight Rhizoctonia solani
isolates on chilli (cv. Sanam).
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4.28 PCR-RFLP restriction patterns revealed by discriminating
enzymes (MseI, AvaII+HincII, and MunI).
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4.29 Phylogenetic analysis of Rhizoctonia solani isolates infecting
potato in Pothohar region.
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4.30 Phylogenetic analysis of Rhizoctonia solani isolates infecting
tomato in Pothohar region.
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4.31 Phylogenetic analysis of Rhizoctonia solani isolates infecting
tomato in Pothohar region.
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LIST OF ACRONYMS
AARI Ayub Agriculture Research Institute
AG Anastomosis Groups
BARI Barani Agricultural Research Institute
BLAST Basic Local Alignment Search Tool
bp Base pair
cm Centimeter
cv Cultivar
DI Disease Index
Dia Diameter
dNTP deoxyribonucleotide triphosphate
ºC Degrees Celsius (Centigrade)
EDTA Ethylenediaminetetraacetic acid
et al. and others
g Gram
ISGS Interspecific groups
ITS Internal Transcribed Spacer
Kg Kilogram
L Litre
LMW Low Molecular Weight
M Mol
MEA Malt Extract Agar
MEB Malt Extract Broth
MgCl2 Magnesium chloride
Min Minute
mL Milliliter
Mm Millimeter
NARC National Agricultural Research Centre
NCBI National Centre for Biotechnology Information
PCR Polymerase Chain Reaction
PDA Potato dextrose Agar
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pH Proportionate hydrogen ions
% Percent
Psi Pound per square inch
RFLP Restriction fragment length polymorphism
spp. Species
TAE Tris-acetate-EDTA
Tris Hydroxymethyl aminomethane
V Volt
WA Water Agar
xg Relative Centrifugal Force
µg Microgram
µL Microliter
µm Micrometer
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ACKNOWLEDGEMENTS
In the name of almighty Allah, the Merciful and the Beneficent. All praises
(belong) to Allah alone, the Cherisher and Sustainer of the world. He is the First, he
is the Last, He is the Hidden, and He knows about everything. He brings the night
into the day and brings the day into the night, and He knows the thoughts of hearts.
Surely Allah and His angels bless the Prophet; O you who believe! call for
(Divine) blessings on him and salute him with a (becoming) salutation (Al-Quran).
I have the pearls of my eyes to admire the countless blessings of Allah
Almighty because the words are bound, knowledge is limited, and time of life is too
short to express His dignity. It is one of His infinite benedictions that He bestowed
upon me with the potential and ability to complete the present research program and
to make a meek contribution to the deep oceans of knowledge already existing.
I deem it my utmost pleasure to express my heartiest gratitude and deep sense
of obligation to my hardworking, dynamic, and visionary supervisor, Dr. Abdul
Rauf, Professor of Plant Pathology, PMAS Arid Agriculture University Rawalpindi
for his kind behavior, generous knowledge, moral support, constructive criticism,
and enlightened supervision during the whole study period. His available words will
always serve as a beacon of light throughout my life.
I express my deep sense of gratitude to Dr. Iftikhar Ahmad (Late), Former
Chairman, PARC for his helpful discussion, support, availability, and constructive
suggestions, determinant for the accomplishment of the work presented in this thesis.
I wish to extend my sincere gratitude to my supervisory committee, Dr. Farah
Naz, Assistant Professor, Plant Pathology and Dr. Nadeem Akhtar Abbasi, Professor
of Horticultural Sciences & Dean, Faculty of Crop and Food Sciences, PMAS Arid
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Agriculture University Rawalpindi.
A special thanks to Dr. Gulshan Irshad for her kind support and endearment
guidance throughout my doctoral program.
I would also like to mention valuable support of my colleagues Muhammad
Fahim Abbas, Aliya Tariq, Alveena Mumtaz, Muhammad Shahid, Rabia Khurshid,
Asfand Iqbal, and Aamir Bashir, Fungal Plant Pathology Lab. PMAS Arid
Agriculture University Rawalpindi.
I am hugely appreciative to my intimate friend Mahpara Nawaz, especially
for sharing her memorable time during our stay at the university.
I am grateful to my uncle Bashir Ahmed, brothers Amir Shahzad Gondal &
Muhammad Sohail, dearest cousin Ishtiaq Ahmad Gondal, and nephew Gulzar
Ahmad for their support during my studies.
I am also thankful to my sincere friends and class fellows especially, Sajjad
Hyder, Raees Ahmed, Nasir Mehmood, Abdul Sattar, and Muhammad Farooq Aslam
for their consistent help and memorable company during my stay at the university.
I am thankful to Higher Education Commission for giving me financial
support under HEC Indigenous Scholarship Program and International Research
Support Initiative Program to conduct part of this research at Department of Plant,
Soil and Microbial Sciences, Michigan State University (MSU), United States.
Finally, I have no words to express gratitude and deepest thanks to my
parents; Mr. and Mrs. Dost Muhammad Gondal for their moral support, and
supplications during my entire education period. May Allah Bless them, Amin!
Amjad Shahzad Gondle
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ABSTRACT
Rhizoctonia solani Kühn (teleomorph = Thanatephorus cucumeris (Frank) Donk) is an
economically important pathogen of solanaceous vegetables, causing black scurf,
damping-off, stem canker, and root rot in these crops. R. solani is a species complex of
several anastomosis groups (AGs) that exhibit DNA base sequence homology and/or
affinities. To date, thirteen AGs of R. solani have been internationally reported. The
present study determines the occurrence of different AGs of R. solani on Potato, Tomato,
and Chilli together with their morpho-molecular characterization. Survey of different
locations of districts Rawalpindi, Jhelum, Attock, Chakwal, and Federal Capital
Islamabad revealed maximum mean disease incidence on potato was recorded in Attock
(37.4%) followed by Islamabad (35.8%), Jhelum (32.1%), Rawalpindi (30.5%) while
minimum mean disease incidence was recorded in district Chakwal (20.2%). Maximum
mean disease incidence on tomato was observed in Islamabad (38.7%) followed by
Attock (36.3%), Rawalpindi (34.9%), and Jhelum (31.8%) while minimum in district
Chakwal (29.6%). Mean disease incidence on chilli was maximum in Attock (30.9%),
followed by Rawalpindi (30.1%), Islamabad and Jhelum (29.5%), while minimum in
district Chakwal (27.0%). At least 63, 67, and 58 isolates of R. solani were recovered
from potato, tomato, and chilli respectively. Fungal colonies isolated on malt extract
agar (MEA) medium were light grey to brown in colour with plentiful mycelial growth
and branched hyphae. A septum was always present in the branch of hyphae near the
originating point with a slight constriction at the branch. The hyphal distance between
two septa ranged between 66.6 to 150.3 µm and hyphal diameter from 4.8 to 8.3 µm.
Majority of the isolates produced rough sclerotia and were superficially present on the
hyphal mass. No conidia or conidiophores were observed from cultures on malt extract
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agar (MEA) medium. All isolates were multinucleate when subjected to 4’-6 diamidino-
2-phenylindole (DAPI) stain. Based on these morphological characteristics of fungal
hyphae, isolates were identified as R. solani. Recovered isolates subjected to
pathogenicity tests confirmed 47, 42, and 37 isolates from potato, tomato, and chilli
respectively were highly virulent. Restriction analysis of PCR-amplified ribosomal
DNA with four discriminant enzymes (MseI, AvaII, HincII, and MunI) revealed
recovered isolates belong to; AG-2-1, AG-2-2, AG-3 PT, AG-4 HG I, AG-5, and AG-
6. Isolates were further paired with tester strains of R. solani AGs which confirmed the
results of AG composition revealed by RFLP analysis. Amplification of Internal
transcribed spacer (ITS) region of rDNA with primers ITS1/ITS4 and sequence analysis
exhibited 99-100% identity with already reported AGs. Isolates recovered from potato
belong to AG-3 PT (76.5%), AG-5 (8.5%), AG-4 HG I (4.2%), AG-2-1 (6.3%), and
AG-2-2 (4.2%). AG-3 PT was widely distributed to major potato growing areas while
others were confined to distinct locations. Isolates recovered from tomato belong to AG-
3 PT (64.2%), AG-2-1 (14.2%), AG-2-2 (9.5%), AG-5 (7.1%), and AG-4-HGI (4.7%).
AG-3 PT was widely distributed to major tomato growing areas followed by AG-2-1
while other groups were confined to distinct locations. Similarly, AG-4 HGI (59.4%)
was also widely distributed to chilli growing areas. Other AGs recovered from chilli
belong to AG-2-1 (16.2%), AG-6 (10.8%), AG-3 PT (8.1%), and AG-5 (5.4%). This is
the first study on AG composition, genetic variability, virulence, and molecular
characterization of Pakistani isolates of R. solani. These findings will provide the basis
for further understanding the infection of different AGs on differential hosts which will
help in the development of control strategies for management of Rhizoctonia diseases
on solanaceous vegetables and other economic crops being infected by this fungus.
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Chapter 1
1 INTRODUCTION
Solanaceous vegetables; potato (Solanum tuberosum L.), tomato
(Lycopersicon esculentum L.) and chilli (Capsicum annuum L.) are the dominant
vegetable crops, grown worldwide and are generally cultivated in the warm or
tropical climate. They are grown throughout the year in all parts of the country. On
a world scale, these vegetables continue to increase the interest not only for the fresh
market but also as a component in a variety of processed foods and pharmaceutical
products (Barba et al., 2006; Georgé et al., 2011; Rao and Rao, 2007).
Potato is the 4th most important commercial cash crop of the world after
wheat (Triticum aestivum L.), maize (Zea mays L.), and rice (Oryza sativa L.) (Birch
et al., 2012; Moeini et al., 2011; Rauscher et al., 2006). It is a temperate climate
crop with high nutritional value. It contains 79% water, 17% carbohydrates (of
which 88% is starch), and 2% protein. An average sized potato together with its skin
provides 27mg of vitamin C, 600mg of potassium, 0.2mg of vitamin B6, and also
trace amounts of minerals including magnesium, phosphorus, zinc, iron, riboflavin,
thiamine, folate, and niacin (Mu et al., 2017). Potato is a versatile crop that can be
cultivated in diverse environments. With a world production of nearly 475 million
tons (MT) from 25.06 million hectares, the potato is cultivated in 140 countries of
the world (FAO, 2016). Major potato producing countries are China (99.12 MT),
India (43.77 MT), Russia (31.10 MT), USA (19.99 MT), and Germany (10.77 MT).
With an annual production of 4.0 MT, Pakistan Stands 21st in the world’s total
potato production (FAO, 2016).
Tomato is the 2nd most consumed vegetable after potato (Georgé et al., 2011).
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It is a good dietary source of vitamins, minerals, and fibre, which are important for
human nutrition and health. It is not only consumed as a fresh crop but also
incorporated in many prepared foods as canned, frozen, preserved, or dried foods. A
diet including tomato is considered as healthy for many reasons; they are low in fat
and calories, cholesterol free, and a good source of protein. Tomatoes are rich in
several vitamins including vitamin A, C, and K. It also contains carotenoids such as
lycopene and β-carotene, and potassium (Hobson and Grierson, 1993; Shi and
Maguer, 2000; StahlW, 1996). Tomato production in the year 2016 was 233.46
million tons (MT) from 5.78 million hectares. Leading tomato yielding countries are
China (56.42 MT), India (18.39 MT), USA (13.03 MT), Turkey (12.60 MT), and
Egypt (7.94 MT). Pakistan ranked 37th in the tomato producing countries of the world
with an annual production of 576 thousand tons. (FAO, 2016).
Chilli is also an important vegetable and spice crop which is produced and
consumed as fresh or processed. It is cultivated in tropical and sub-tropical domains
of the world. Chilli (red pepper) characterized by tempting colour and arousing
pungency is used to flavour food, make sauces, and is an essential ingredient for
curry especially in subcontinent including Pakistan. Its derivatives are also used in a
variety of medicinal applications. The pool of all chilli cultivars (cvs.) comes from
five species of the genus Capsicum; C. annuum, C. chinense, C. baccatum, C.
frutescens, and C. pubescens (Bosland and Votava, 2000; Smith et al., 1987). Chilli
crop plays a significant socio-economic role and found throughout the world (Joshi
et al., 2015). The growing interest in chilli emanates from increasing awareness of
its nutritional value, medicinal importance, industrial, and also the ornamental
characteristics. It is a source of vitamins (A, B1, B2, B3, C, and E), carotenoids, and
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capsaicinoids (Tateo and Bononi, 2004). It gives relief from more than a dozen
diseases including common cold, headache, and toothache etc. Capsaicin, a type of
capsaicinoid from chillies, is used in pomade to soothe pains from arthritis (Sanogo,
2003). Total chilli production in the year 2016 was 51.95 million tons (MT) from
24.74 million hectares. Leading chilli yielding countries are China (17.45 MT),
Mexico (2.73 MT), Turkey (2.45 MT), Indonesia (1.96 MT), and Spain (1.08 MT).
In Pakistan, the annual chilli production was 133.51 thousand tons and Pakistan
ranked 7th in the chilli producing countries of the world (FAO, 2016).
Over the last couple of years, the demand and supply for agricultural
commodities has increased due to the increasing population. Better use of available
water resources, tunnel farming, market demand-oriented crop selection, hydroponic
cultivation and agricultural extension has played a significant role in increased
vegetable cultivation in Pothohar region.
The yield of these vegetables in Pakistan is far low as compared to other
countries of the world. Several biotic and abiotic factors are attributed to this low
yield of potato, tomato, and chilli. Of all primary food crops, these vegetables are
susceptible to a variety of diseases caused by infectious microorganisms including
fungi, bacteria, viruses, and nematodes enduring the utmost yield losses (Agrios,
2005). Fungal pathogens especially Rhizoctonia solani (Kühn) is the most serious
and worst damaging pathogen that play a vital role in reducing the yield of several
important food crops (Anderson, 1982; Carling et al., 2002a; Carling and Kuninaga,
1990; Morsy et al., 2009). Rhizoctonia solani Kühn (teleomorph = Thanatephorus
cucumeris (Frank) Donk) is one of the most important soil-borne pathogen that has
a wide host range of more than 200 plant species, especially Solanaceae family
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including potato, tomato, and chilli cultivated under the both, greenhouse and field
conditions (Adams, 1988; Prabha et al., 2014; Sneh, 1996). It may also interact with
other pathogens and cause additional damage by forming disease complex (Martin,
2003; Strausbaugh and Gillen, 2009). The fungus is a heterogeneous group of
filamentous fungi identical in their anamorphic, sterile state. It is a non-obligate
parasite and causes damping off, black scurf, stem and stolon canker, and root rot to
vegetables and crop plants (Kareem and Hassan, 2013; Laemmlen, 2004).
Rhizoctonia solani a species complex of several anastomosis groups (AGs)
based on the hyphal fusion of identical isolates which differ in genotypic and
phenotypic characters. Sub-specific groups in R. solani containing both related and
diverse genetics having very diverse life histories make this fungus a complex
species (Adams, 1988; Carling et al., 2002a; Cubeta and Vilgalys, 1997). To date,
thirteen AGs of R. solani designated as AG1-AG13 have been assigned on the basis
of hyphal anastomosis interactions (Carling et al., 2002b; Da-Silva et al., 2010;
Gónzalez et al., 2016).
Different anastomosis groups cause infection on differential hosts. Several
AGs of R. solani such as AG-2 (Misawa and Kuninaga, 2010, 2013), AG-3 (Charlton
and Cubeta, 2007; Misawa and Kuninaga, 2010; Rauf et al., 2007), and AG-4 (Ciampi
et al., 2005; Kuramae et al., 2003; Taheri and Tarighi, 2012) have been reported to be
pathogenic on potato, tomato, and chilli peppers. AG-3, the most widely distributed in
Pakistan designated as homogenous population cause black scurf on potato (Rauf et
al., 2007). It has also been reported to cause disease in other solanaceous vegetables
(Kodama et al., 1982; Misawa and Kuninaga, 2010; Woodhall et al., 2008). Reports
also refer to AG-1 (Carling and Leiner, 1990), AG-2 (subgroup -1 and -2) (Carling and
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Kuninaga, 1990; Chand and Logan, 1983), AG-4 (Anguiz and Martin, 1989; Balali et
al., 1995; Carling and Leiner, 1990; Chand and Logan, 1983), AG-5 (Balali et al.,
1995; Carling and Leiner, 1990), AG-7 (Carling et al., 1998), AG-8 (Carling and
Leiner, 1990), and AG-9 (Carling and Leiner, 1990; Carling et al., 1987) being
pathogenic to solanaceous vegetable crops.
Although, some contributions to R. solani anastomosis groups and their
infection on potato is reported from Pakistan (Rauf et al., 2007) however, detailed
work with regard to solanaceous vegetables is lacking. Therefore, there is a need for
detailed information about R. solani AGs that could help in devising potato, tomato,
and chilli crop improvement programs. Keeping in view the above scenario, the
present research is aimed to investigate;
a. The incidence of Rhizoctonia solani incited infection on selected solanaceous
vegetable crops (potato, tomato, and chilli)
b. The occurrence of anastomosis groups of R. solani on potato, tomato, and
chilli
c. Characterization of recovered isolates of R. solani representing different
anastomosis groups.
6
Chapter 2
2 REVIEW OF LITERATURE
2.1 SOLANACEOUS VEGETABLES
Solanaceae, the nightshades or potato family include an economically
important group of flowering plants that has 102 genera and more than 2700 plant
species comprising annual and perennial herbs, shrubs, trees, agricultural food crops,
and medicinal plants (Olmstead and Bohs, 2007). Members of the family originated
in the tropical regions of Latin America and are found throughout the world. At least,
40 genera of this family are endemic, while a few members are also found in the
temperate regions (Lotha, 2014). Economically important genera of the family are;
Solanum, Lycopersicon and Capsicum (Rubatzky and Yamaguchi, 1997).
2.1.1 Potato
Potato is a starch enriched tuberous crop. The English word potato was
derived from the Spanish word “patata”. The Spanish Royal Academy used the word
that was composed of “Taino batata” means sweet potato and “Quechua papa” means
potato (Hawkes and Francisco-Ortega, 1993). The potato was originated as food crop
around Lake Titicaca in Peruvian Andes mountains in South America about 8,000
years ago (Burton, 1989; Dodds, 1965; Singh and Kaur, 2016) and was introduced
to Indo-Pak subcontinent by Portuguese traders when they came north of Bombay
(Kokab and Smith, 1989). Being moderately frost tolerant and cool season crop, it
has a broad range of seasonal adaptability. During growing seasons, the temperature
has been referred to as one of the most important factors affecting its yield. Young
plants grow best at a temperature of 24oC while the later growth is favoured at 18oC.
Tuber production reaches a maximum at 20oC, decrease with increase in temperature
7
and reaches a maximum and stops at about 30oC (Pereira and Shock, 2007). Three
crops of potato are grown in Pakistan in a year. Autumn and spring crops are grown
in plains of Punjab and KPK, while summer crop is grown in hilly areas. Autumn
crop is cultivated in September and harvested in the last week of December, while
spring crop is cultivated in December-January and harvested in the month of May.
In hilly areas, summer crop is cultivated in May-June and harvested in late October.
The shares of autumn, spring, and summer crops in the annual production of the
country are estimated at 75, 10, and 15 percent, respectively.
In Pakistan, it is grown over an area of 178 thousand hectares with an annual
production of 4000 thousand tons (FAO, 2016). The area under the cultivation in
Punjab is 86% of the total potato growing area in the country with 88.3% of the total
potato production. The area under potato cultivation in Sindh is 0.5% of the total
area under potato cultivation with 0.3% of the country’s production. KPK contributes
7.2% of the total potato production of the country from an area of 9% of the total
area under potato cultivation. The area under potato cultivation in Baluchistan is
4.5% contributing 4.2% of the total potato production of the country.
Of all primary food crops, potato endures significant losses due to numerous
fungal, bacterial, viral, and nematode diseases owing to high starch and sugar
contents (Agrios, 2005; Chakraborty, 2016; Kelman, 1984). Heavy losses occur due
to poor management practices, disease and pest attack, non-availability of improved
higher yielding, and better resistant germplasm (Ghebreslassie et al., 2014). More
than eighteen potato diseases have been reported in Pakistan, of which thirteen occur
commonly in almost all potato growing areas (Khalid et al., 2000). Most prevalent
diseases of potato in our country are early blight, common scab, black scurf or
8
rhizoctoniasis, wilts, stem rot, soft rot, brown rot, potato cyst nematode, and root rot
nematode. Other diseases caused by mycoplasmas and soil borne pathogens are also
serious problems in major potato growing areas. Among soil borne fungal pathogens,
Rhizoctonia solani (Kühn) is amongst most serious and the worst damaging cause
black scurf and sprout killing of potato (Brierley et al., 2016; Das et al., 2014; Rauf
et al., 2007). R. solani is a natural soil habitant to the potato growing areas. The
infection to the potato plants is mainly due to soilborne or tuberborne inoculum and
disease severity varies year to year (Hide et al., 1973). Soil borne inoculum of R.
solani cause stolon canker while tuberborne inoculum effect significantly to the
sprout emergence (Frank and Leach, 1980).
2.1.2 Tomato
Tomato is an edible fruit and one of the most consumed vegetables in the
world. It is an adaptable crop used for both fresh market and processing in prepared
foods as canned, ketchup, sauce, juice, paste, powder, puree, salad dressings, soups,
vegetable and juice cocktails, frozen tomatoes, and preserved or dried foods. It is
now considered to be a part of the daily diet (Onyambus et al., 2011). The word
“tomato” originated from Spanish word “tomate” derived from a Nahuatlic word
tomatl. Wild tomatoes originated during 700 A.D; in South and Central America as
the relatives of the cultivated tomatoes are native of western South America along
the coastal high Andes from central Ecuador, through Peru, to northern Chile in the
Galapagos Islands (Peralta et al., 2008; Razdan, 2006). It is originated in Mexico as
food and spread throughout the world following the Spanish colonization of the
Americas. It was introduced to Indo-Pak during the sixteenth century by Spanish
explorers. Tomato is a short-lived perennial crop by nature however, it is universally
9
grown as an annual crop of 05-06 months (Kaul, 1991). It is grown in temperate
climates across the world. The optimum temperature for growth and development is
between 21-24oC (Naika et al., 2005). Temperature tolerance for extreme heat and
cold is important for blossoms development and subsequent fruit set. (Barten et al.,
1992; Gould, 2013). Fruit setting is poor when the temperature falls below 10oC or
exceeds 32oC.
Pakistan has varying climatic conditions. Three crops of tomato; early, mid-
season, and mains season are being grown in Pakistan. For an early crop, the nursery
is sown in July-August, transplanted in August-September, and harvesting starts in
November. The nursery is sown in September for the mid-season crop, transplanted
in October, and harvested in December/January. Main season crop is sown in mid-
November, transplanted in February and harvested in May-June (Khan, 2012b).
Tomato varieties; Moneymaker, Rio Grande, Roma VF, and Tropic are being grown
in Pakistan (Sajjad et al., 2011).
Locally, it is grown over an area of 62 thousand hectares with a production
of 575 thousand tons. For the last few years, there has been a progressive increase in
the area and production of tomato in Pakistan. (FAO, 2016). The area under the
cultivation in Punjab is about 12% of the total tomato growing area in the country
with 15% of the whole tomato production of the country. The area under tomato
cultivation in Sindh is 15% with 8% of the country’s total production. KPK
contributes 34% of the total tomato production with of the country over an area of
38% of the total area under tomato production. The area under tomato cultivation in
Baluchistan is about 35% of the total potato growing area of the country contributing
43% of the total potato production of the country.
10
Diseases are a major limiting factor in tomato production throughout the
world. Tomato plant is prone to numerous fungal, bacterial, viral, and nematode
diseases (Agrios, 2005; Jones et al., 2014; Scofield et al., 1996). At present, tomato
is susceptible to more than 200 diseases (Rashid et al., 2016). Most commonly
occurring disease of tomato are; Early and Septoria Leaf Blight, Gray mold,
Fusarium and Verticillium Wilt, Powdery Mildew, Damping off, root rot, stem
canker, Bacterial Canker, Bacterial wilt, Blossom Drop, Anthracnose, different viral
diseases, and Root knot nematode. Fungi are considered an important group of
microorganisms responsible for various diseases of tomato and cause significant
yield losses (Shrestha, 2015). Among soilborne fungal pathogens, Fusarium spp.,
Phytophthora spp., R. solani, and Sclerotium rolfsii cause significant yield losses
(Abdel-Monaim, 2012; Moataza, 2006). R. solani cause seedling damping off and
foot rot of mature tomato (Montealegre et al., 2003; Traquair et al., 2013). The
pathogen is also responsible for stem canker, collar rot and root rot, and eventual
death of diseased plants leading to the significant yield losses (Anderson, 1982;
Arora et al., 2008). Different anastomosis groups of R. solani has been reported to
infect tomato (Kuramae et al., 2003; Misawa and Kuninaga, 2010).
2.1.3 Chilli
Chilli, also red pepper or chilli pepper is one of the most popular vegetables
after potato and tomato used to add flavour and spiciness to food. It is used as fresh
and/or processed in by-products; sauces, vinegar, pickles, soups, chilli powder, and
condiments as curry powder across the world. Chilli derivatives possess medicinal
values and are being used as carminatives, antiseptics anti-rheumatic, antispasmodic,
and appetite stimulant. The principal flavouring component Capsaicin, is used in
11
ointments, nasal sprays, and dermal patches to relieve pain (AVRDC, 2002; Fattori et
al., 2016).
The word “chilli” derived from a Nahuatlic word chīlli (Kunnumakkara et
al., 2009). It was originated 7500 BC ago in the tropical regions of Latin America
(New Mexico and Guatemala) (Eshbaugh, 1980; González, 1991). Mexico is
considered as the native home of chilli peppers as they were domesticated for more
than 6000 years ago (Bosland, 1996). The primary source of origin of domesticated
chillies is semi-tropical domains of Mexico (Hernández-Verdugo et al., 1999;
Hernández-Verdugo et al., 2001; Whitmore and Turner, 2001). Portuguese traders,
prior to 1585 introduced chilli peppers to Asia (Collingham, 2006). By the end of
15th century, they were introduced to Indo-Pak subcontinent. Chillies are now an
integral part of Indian cuisines (Mini Raj et al., 2007).
It is cultivated in tropical and sub-tropical areas across the world (Somos,
1984). The domesticated species of chillies are; C. annuum, C. frutescens, C.
chinense, C. pubescens, and C. baccatum (Heiser and Smith, 1953; Heiser Jr, 1985;
Moscone et al., 2006). Most commonly grown species in Asia are C. annum and C.
frutescens (Berke, 2002). Chilli is a warm season crop and highly vulnerable to frost
(Rey et al., 2000). The total duration of chilli crop is 05-06 months that depends on
cultivated variety, growing season, climate conditions, soil type with its fertility, and
water management (Hosmani, 1993). The optimum temperature for vegetative
growth is 21-23oC and fruit growth 21oC. (Lorenz and Maynard, 1980). In Punjab
plains of Pakistan, two crops are being grown. The nursery is sown in October/
February and March, transplanted in February/ April/ May and harvesting starts in
May to August/ July to October. In Sindh province, Nursery is sown in December/
12
June-July/ September-October, transplanted in Last week of January/ July-August/
November, and harvested in April-August/ November-December/ February-April.
In KPK province, the crop is sown in mid-November, transplanted in February-
March, and harvested in June-November while in the plains of Baluchistan, the
nursery is sown in October-February, transplanted in May-June and harvested in
September to December. Two chilli crops are being grown in hilly areas. The
nursery is sown in April/ December, transplanted in May-June/Last week of January
and harvested in September-December/ April to August. (Khan, 2012a).
In Pakistan, chilli is grown over an area of 188 thousand hectares with an
annual production of 73 thousand tons (FAO, 2016). The area under the cultivation
in Punjab is 4.57% of the total chilli growing area of the country with 7.1% of the
total chilli production. The area under chilli cultivation in Sindh is 91.7% of the total
area under chilli cultivation with 83% of the country’s production. KPK contributes
0.85% of the total chilli production from 0.39% of the total area under chilli
cultivation. The area under chilli cultivation in Baluchistan is 3.29% of the total chilli
growing area contributing 5.72% of the country’s total chilli production. There has
been a progressive decline in the chilli production due to several abiotic factors;
heavy rains and floods in the country and biotic factors including plant pathogens.
Chilli crop endures substantial yield losses due to numerous diseases and pests
attack (Agrios, 2005). More than fifty pathogens have been reported to cause infection
on chilli (Saha and Singh, 1988). Most commonly occurring disease of chilli are; leaf
curl virus, cucumber mosaic virus, collar rot, Pythium and Rhizoctonia damping off,
Fusarium wilt, chilli anthracnose, bacterial leaf spot, and grey mould. Among
soilborne fungal pathogens, R. solani is an important yield-limiting factor in chilli
13
(Velásquez Valle et al., 2001) that cause damping off disease at the seedling stage,
root rot and stem canker of young transplants (Kucharek and Pernezny, 2003). It also
infects mature chilli plants and causes root rot and stem canker at soil line level of the
stem that may lead to wilting and plant death (Sanogo, 2003). R. solani may also
interact with other pathogens and cause disease complex. Almost, all commercially
available chilli cultivars are susceptible to R. solani (Rather et al., 2012).
2.2 RHIZOCTONIA
The Rhizoctonia, a genus of anamorphic fungi was first described by French
mycologist Augustin Pyramus De Candolle in 1815 to accommodate root pathogens;
Rhizoctonia crocorum (teleomorph: Helicobasidium purpureum) (Pers.) DC.
(Parmeter, 1970; Tu and Kimbrough, 1975). The “Rhizoctonia” is an Ancient Greek
word, ῥίζα (rhiza, "root") + κτόνος (ktonos, "murder") means “root killer”. The genus
became a heterogeneous assemblage of mycelia of ascomycetes, basidiomycetes,
and imperfect fungi which allowed inclusion of several unrelated species. (Andersen
and Stalpers, 1994; Parmeter Jr et al., 1967). However, with the revised concept of
the genus, Rhizoctonia was restricted to the type species and their relatives, with
unrelated species moved to other genera (Moore, 1987).
In general, members of this genus are characterized by the possession of
hyphae, branched near the distal septum at an acute angle when young but right
angles at later, the presence of septum, and constriction of hyphae at the junction.
The mycelia may be capable of producing inter woven mass of hyphae called
sclerotia (Parmeter, 1970; Tu and Kimbrough, 1975).
Members of the genus Rhizoctonia were classified into three basidiomycete
groups based on teleomorphic states (i) the Rhizoctonia complex, contains
14
multinucleate species with Thanatephorus teleomorph, (ii) binucleate Rhizoctonia
species with Ceratobasidium teleomorph, and (iii) multinucleate Rhizoctonia zeae
and R. oryzae with Waitea teleomorph (De la Cerda et al., 2007; Mihail et al., 1992;
Sneh et al., 1991; Toda et al., 2007).
The most widely studied species is R. solani which is most virulent and
polyphagous species across the world capable of infecting several plant species.
2.2.1 Rhizoctonia solani
Rhizoctonia solani Kühn, the mycelial state of Thanatephorus cucumeris
(Frank) Donk is the most widely recognized species of the genus Rhizoctonia
discovered more than 100 years ago (Ogoshi, 1987). It is a cosmopolitan, soil-borne
plant pathogenic fungus which develops in both cultured and non-cultured soils. It
has worldwide distribution and significant diversity of host plants (Anderson, 1982;
Sneh et al., 2013; Thornton et al., 2004).
The fungus is a facultative parasite found in the agricultural soils and survives
on crop residues as microsclerotia (Laemmlen, 2004). It has high survival due to its
establishment in the soil and difficult to eliminate (Almasia et al., 2008; Papavizas,
1970). R. solani can attack to a wide range of host plants, causing seed death, stem
and stolon canker, aerial leaf blight, foliage yellowing, root rot, and damping off
(Harikrishnan and Yang, 2004; Parmeter, 1970; Thornton et al., 2004).
2.2.1.1 Origin and taxonomic classification
Rhizoctonia solani Kühn was first described by Julius Kühn during 1858 on
diseased potato (Ceresini, 1999). It belongs to the group “Mycelia sterilia” which
does not produce asexual spores (conidia) and reproduce by vegetative hyphae
(Carroll, 2004). The fungus occasionally produces sexual spores, basidiospores that
15
are not enclosed in a fleshy fruiting body. The sexual reproductive stage; teleomorph
in R. solani (Thanatephorus cucumeris) was first described by Prillieux and
Delacroiz in 1891 (Ceresini, 1999).
The mycelium of the fungus consists of hyphae divided into individual cells
by a dolipore septum (Menzies, 1970). The nuclear condition in the individual cells
forms the basis of the classic taxonomical scheme. Multinucleate conditions in the
vegetative hyphal cells distinguish R. solani from binucleate, Rhizoctonia like fungi
with similar cultural and morphological appearance (Ruppel, 1972; Sharon et al.,
2006). Multinucleate species may contain 03-28 nuclei in their young cells (Sneh et
al., 1991; Sneh et al., 2013).
Kingdom: Fungi
Division: Basidiomycota
Class: Agaricomycetes
Order: Cantharellales
Family: Ceratobasidiaceae
Genus: Rhizoctonia
Species: Rhizoctonia solani
(Tsukiboshi, 2002).
2.2.1.2 General characteristics
The mycelium of R. solani is colourless when young and transformed to
brown as grow and mature. The mycelium consists of basal hyphae partitioned into
individual cells by a dolipore septum that allows the movement of cytoplasmic
contents and nuclei from cell to cell. Each cell contains more than three nuclei. The
young vegetative hyphae are branched at right angles near the distal septum of the
16
cells and constricted at their junction or at a short distance from the septum. (Ogoshi,
1975; Sneh et al., 1991; Tu and Kimbrough, 1975).
Rhizoctonia solani can survive saprophytically in the soil for years as it forms
dormant resting structures called sclerotia (Papavizas, 1970). These sclerotia are
irregular in shape, light to dark brown in colour, not differentiated into the rind, and
medula and have Thanatephorus cucumeris as their teleomorph (Sneh et al., 1991).
Rhizoctonia solani is a multinucleate basidiomycete fungus that generally
reproduces asexually in nature and exists mostly as a thread like mycelium and/or
sclerotia on plants or in culture. This is the imperfect state of the basidiomycete fungus
that does not produce conidia (asexual spores) however, occasionally produces
basidiospores (sexual spores) (Ceresini, 1999; Naito, 1996; Parmeter, 1970).
Rhizoctonia solani is a soilborne pathogen that generally attacks roots and
lower stems of plants, best known to infect a range of crop plants, and causes
damping off, collar rot, root rot, stem and stolon canker (Parmeter, 1970; Thornton
et al., 2004).
2.2.1.3 Ecology and epidemiology
Rhizoctonia solani is a soil-inhabiting fungus that exhibits physiological
characters associated with highly competitive saprophytic ability however it can also
survive parasitically as distinct clones (Garrett, 1956; Papavizas and Davey, 1962).
The fungus can remain active as vegetative mycelium in plant debris, by colonizing
the soil organic matter (Olsen and Young, 2011). It can survive unfavourable
conditions and remain dormant in the form of visible sclerotia for a varying period
of time (Boosalis and Scharen, 1969; Ceresini, 1999; Liddell et al., 2001). The main
sources of inoculum are contaminated soils, host plants; weeds or rotation crops,
17
plant debris, and infected seeds (Parmeter, 1970). Factors including soil temperature,
soil pH, and competitive activity with associated organism influence the pathogen
survival and its inoculum potential (Jones et al., 1997).
Fungal mycelium or sclerotia are the main sources of infection however,
several important diseases also result from basidiospores that serve as a source of
long distance dispersal of fungus (Naito, 1996).
The fungus can be found in cool and warm soils and can remain active at a
range of temperatures (Olsen and Young, 2011). Soil temperature and moisture
greatly influence the R. solani disease development. The optimum temperature range
for disease development is 24-32oC. (Harikrishnan and Yang, 2004; Parmeter, 1970).
Rhizoctonia solani is well adapted to life outside the host plants. The host
plants are merely a source of food for R. solani however, its infection allows
exploiting plants as a food source (Keijer et al., 1997). Sclerotia and fungal
mycelium germinate by producing hyphae that attack host plants. (Ceresini, 1999).
The optimum temperature range for sclerotia production is between 18-25ºC
(Harikrishnan and Yang, 2004).
2.3 ANASTOMOSIS GROUPING IN RHIZOCTONIA SOLANI
Rhizoctonia solani a species complex of several anastomosis groups (AGs)
based on the hyphal fusion of the identical isolates differ in genotypic, phenotypic,
and pathogenic characters (Ogoshi, 1987; Vilgalys and Cubeta, 1994). Sub-specific
groups in R. solani containing both; related and diverse genetics having very diverse
life histories that make this fungus a complex species (Adams, 1988; Carling et al.,
2002a; Cubeta and Vilgalys, 1997).
In hyphal anastomosis interactions, isolate of an unknown strain is paired with
18
known strain/ anastomosis group (AG) of R. solani on cellophane membranes placed
on nutrient medium, water agar in Petri dish (Ogoshi, 1975) or water agar coated clean
glass slides (Kronland and Stanghellini, 1988), and incubated until growing hyphae
overlapped to form zones of confrontation (Carling, 1996). Fusion between the
confronting hyphae confirms the unknown isolate belongs to the same AG.
Anastomosis reactions are classified from C0 to C3; C0 (no reaction=
incompatibility), C1(contact fusion), C2 (killing reaction= somatic fusion), and C3
(perfect fusion) (Carling et al., 2002a; Matsumoto, 1932). The perfect fusion
between the distantly related R. solani isolates involves the death of the
anastomosing cells, a phenomenon known as “killing reaction” which do not occur
in self-anastomosis or anastomosis between clonal isolates (Carling, 1996).
To date, thirteen AGs; AG1 to AG13 and AG-BI have been assigned on the
basis of hyphal anastomosis interactions (Carling et al., 2002b; Gónzalez et al.,
2016). Seven AGs (AG-1, AG-2, AG-3, AG-4, AG-6, AG-8, and AG-9) have been
further classified into subgroups to reflect differences observed in frequency of
anastomosis interactions, pathogenicity, thiamine requirement, cultural appearance,
fatty acids, and isozyme patterns (Baird et al., 2000; Godoy-Lutz et al., 2008;
González et al., 2002; Johnk and Jones, 1993; Kuninaga et al., 1997; Kuninaga et
al., 2000; Nicoletti et al., 1999; Ogoshi, 1987; Sharon et al., 2008a; Sharon et al.,
2006; Tewoldemedhin et al., 2006).
2.3.1 Categories of Anastomosis Interactions
The concept of hyphal anastomosis was reported by Matsumoto (1921) and
the first natural subdivision of R. solani isolates into five distinct groups on the basis
of hyphal anastomosis reactions was made by Schultz (1936).
19
Flentje and Stretton (1964) revised the concept of hyphal anastomosis and
categorized the hyphal interactions based on killing reactions followed by hyphal
fusion. Four terminologies (S = self, K = killing, WF= wall fusion, and NR = no
reaction) were used. The “S” reaction involving the fusion of the cell membrane with
no cell death. This type of reaction was observed between the hyphae of the same
isolates or those having a close relationship. Fusion of the cell wall followed by
cytoplasmic contents with the death of the anastomosing hyphae was defined as “K”.
“WF” defined as the cell wall attachment with no hyphal fusion and the term “NR”
was assigned to the condition where colliding hyphae didn’t interact.
Parmeter et al. (1969) reported that cell death may be observed in both cases;
perfect and imperfect fusions. Parmeter et al. (1969) assigned numerical categories
(0-2) to the anastomosis interactions as; “0 = no reaction”, “1 = hyphal contact” and
“2 = hyphal fusion accompanied by with cell death”.
Carling et al. (1988) categorized anastomosis reactions from C0 to C3. The
C3 reaction occurs either between the hyphae of the same isolate or (self-
anastomosis) or two closely related isolates of the same AG. The C2 reaction occurs
between the distantly related isolates of the same AG. The C1 reaction is defined as
the bridging reaction that may either occur between distantly related isolates of the
same AG or between the closely related isolates of the different AGs. C1 type
reaction may or may not confirm the AG identity. C0 reaction indicates no hyphal
interactions between isolates confirming them belong to different AGs. Matsumoto
(1932) and Parmeter et al. (1969) proposed the occurrence of at least four AGs (AG-
1 to AG-4) of R. solani based on these categories of hyphal anastomosis interactions.
Ogoshi (1975) grouped isolates of R. solani and their perfect stages, and also
20
identified AG-5. Subsequently, (Kuninaga et al., 1979) identified AG-6 and AG-B1.
This is followed by the characterization of AG-7 by Homma et al. (1983), AG-8 by
Neate and Warcup (1985), AG-9 by Carling et al. (1987), AG-10 by MacNish et al.
(1995), AG-11 by Carling et al. (1994), AG-12 Carling et al. (1999), and AG-13
Carling et al. (2002a).
2.3.2 Subgroups within Anastomosis Groups
Considerable variations in anastomosis frequency, culture morphology,
nutritional requirement, pathogenicity, biochemical properties, and genetical traits
exist within isolates of the same AGs. Ogoshi (1987) introduced the term
interspecific group (ISG) to determine molecular variability within AG-2 and AG-
2-1, AG-2-2 IIIB, AG-2-2 LP, AG-2-2 IV, AG-2 E, AG-2 F were recognized based
on host specificity (Hyakumachi et al., 1998; Kuninaga et al., 2000; Sneh et al.,
1991). ISGs have also been determined on the basis of DNA sequence homology
(Kuninaga and Yokosawa, 1984a) and Isoenzyme analysis (Liu et al., 1990; Liu et
al., 1992). Zymogram groups determination on the basis of pectic enzymes
expression by Cruickshank and Wade (1980) also supports interspecific grouping
in R. solani.
AG-1 has been divided into three subgroups based on pathogenic
variability; AG-1-A (sheath blight of rice), AG-1-B (web blight of rice) and AG-
1-C (damping-off of rice) (Ogoshi, 1987). Kuninaga et al. (1997) further
distinguished all three groups of AG-1 based on the sequence analysis of internal
transcribed spacer (ITS) region of their rDNA. Priyatmojo et al. (2001) recovered
distant isolate within AG-1 from necrotic spots of coffee leaves and designate as
AG-1-ID on the basis of its comparative virulence, fatty acids, restriction fragment
21
length polymorphism (RFLP) of ITS regions, and random amplified polymorphism
DNA (RAPD) analysis.
AG-2 is the most diverse among all recovered AGs having considerable
variability within the isolates recovered to date and have a greater number of
subgroups (Carling et al., 2002b). Ogoshi (1987) differentiated AG-2 into two distant
subgroups AG-2-1 and AG-2-2 based on anastomosis frequency, cultural
morphology, and thiamine requirement for growth in the culture. Isolates of AG-2-2
showed diverse response towards cultural appearance and pathogenic behaviour to
sugar beet and mat rush and were further classified as AG-2-2 IIIB and AG-2-2 IV.
Later, Johnk and Jones (1993) also reported that both these subgroups; AG-2-2 IIIB
and AG-2-2 IV could be differentiated by their cellular fatty acid profiles. Liu et al.
(1990) supported the differentiation of AG-2-1, AG-2-2 IIIB, and AG-2-2 IV by
isozyme profiles. Based on cultural characteristics, virulence, and rDNA RFLP
(restriction fragment length polymorphism) profiles, another subset of isolates within
AG-2-2 was identified as AG-2-2 LP (Hyakumachi et al., 1998). Subsequently, AG-
2-3 by Naito and Kanematsu (1994), AG-2t by Schneider et al. (1997), and AG-2-4
Carling et al. (2002b) were identified.
AG-3 is the most widely distributed among all recovered AGs designated as
homogenous population causing black scurf on potato. However, it has been reported
to cause disease in other solanaceous crops (Kodama et al., 1982; Meyer et al., 1990;
Misawa and Kuninaga, 2010; Rauf et al., 2007; Woodhall et al., 2008). AG-3 has
been assigned three subgroups; AG-3 PT (potato type), AG-3 TM (tomato type), and
AG-3 TB (tobacco type) (Kuninaga et al., 2007; Misawa and Kuninaga, 2010).
AG-4 has been subdivided to at least two heterogeneous groups (HGs), AG-
22
4-HGI and AG-4 HG II, based on cultural appearance, protein electrophoretic
patterns, and DNA-DNA reassociation kinetics (Kuninaga et al., 1997; Kuninaga
and Yokosawa, 1984a; Ogoshi, 1987). Cultural appearances and cellular fatty acid
methyl ester profiling of the isolates recovered from sugar beet, peanut, and soybean
confirmed another subgroup AG-4 HG III within AG-4 (Johnk and Jones, 2001).
AG-6 has also been subdivided to two heterogeneous subgroups (HGs), AG-
6-HGI and AG-6-GV based on DNA-DNA reassociation kinetics (Kuninaga and
Yokosawa, 1984b). Carling and Kuninaga (1990) reported AG-6-GV to be more
heterogeneous than AG-6-HGI. Sharon et al. (2006) further subdivided AG-6-GV
into four subsets; AG-6-GV1, AG-6-GV2, AG-6-GV3, and AG-6-GV4 based on
sequence analysis of their ITS region.
AG-7 isolates exhibited variations in the frequency of anastomosis reactions
and fatty acid profiling and were designated as AG-7-1 and AG-7-2 (Baird et al.,
2000). Sequence analysis of the ribosomal DNA ITS region of AG-7 isolates
suggests the existence of further subgroups within this AG (Sharon et al., 2006).
AG-8 has been subdivided into five zymogram groups; ZG-1, ZG-2, ZG-3,
ZG-4, and ZG-5 based on zymography, an electrophoretic method for measuring
proteolytic activity (MacNish and Sweetingham, 1993). Anastomosis interactions
confirmed the existence of vegetative compatibility populations (VCP) within
zymogram groups of AG-8 (MacNish et al., 1997). MacNish and O’Brien (2005)
confirmed the existence of five zymogram groups using RAPD-PCR analysis.
AG-9 isolates showed diverse response towards thiamine requirement and
DNA-DNA hybridization studies and were divided into two subgroups AG-9 TP and
AG-9 TX. Subgroup AG-9 TP was prototrophic while AG-9 TX was auxotrophic to
23
thiamine. To date, no subgroups have been reported within other AGs (Carling and
Kuninaga, 1990).
2.3.3 Molecular Methods for AGs Classification
Generally, isolates of the same anastomosis groups share similar morphology
and pathogenic profiles as well as physiological and ecological features. Their
identification is based on the hyphal anastomosis reactions, however, recent studies
confirmed considerable variability within and between the same AGs. Hyphal
anastomosis reactions are considered insufficient for determination of specific AG.
Furthermore, reproducibility of anastomosis interactions needs experiences, is a
time-consuming process, and can be affected by factors including laboratory
environment, nutritional conditions, and genetic stability (Carling et al., 2002b;
Stodart et al., 2007).
Characteristics including pathogenicity, biochemical and genetic makeup,
molecular approaches including DNA based sequence homology, and restriction
analysis of ribosomal DNA have been confirmed as reliable tools to differentiate
isolates of R. solani into distinct clades corresponding to different AGs and
subgroups (Fang et al., 2013; Sharon et al., 2008b). Different molecular
techniques have been employed to support genetic groups within R. solani and its
teleomorphs Thanatephorous better than morphological characters, number of
nuclei, and host range (González et al., 2006; Lees et al., 2002; Ophel-Keller and
Kirkwood, 2006).
Restriction fragment length polymorphism (RFLP) analysis of ribosomal
DNA (rDNA) sequences has successfully been used to characterize isolates of R.
solani into respective AGs or subsets within AGs (Hyakumachi et al., 1998; Liu et
24
al., 1993; Vilgalys and Gonzales, 1990). Isolates belonging to different AGs exhibit
polymorphism in their 18S, 28s rDNA sequences, and internal transcribed spacers
(ITS) sequences (Kuninaga et al., 1997; Liu et al., 1995; Matsumoto et al., 1996;
Meyer et al., 1998). ITS sequence analysis of the rDNA has added genetic support
to the AGs and ISGs determination together with the investigation of their
evolutionary relationships (Gonzalez et al., 2001; Pope and Carter, 2001).
2.3.4 Internal Transcribed Spacer (ITS) Sequence Analysis
Most of the fungi have three rRNA genes (28s, 5.8s and 18s rDNA) as
repeated units separated by intergenic spacer (IGS) regions (Sharon et al., 2006) as
shown in Figure 2.1. Vilgalys and Gonzalez (1990) confirmed R. solani AG-4 rDNA
repeats to a maximum length of 8.8kb with an estimated number of rDNA copies to
be 59 per haploid genome. The 5.8s rDNA gene is flanked by the internal transcribed
spacer regions (ITS1 and ITS2). These two regions have become important
molecular targets for fungal taxonomy, identification, and phylogenetic relationships
(Bruns et al., 1991; Cubeta et al., 1996; Iwen et al., 2002). ITS domains are more
suited for fungal identification at species level because of their higher rate of
molecular evolution and proximity to highly conserved rDNA regions (Gonzalez et
al., 2001; Iwen et al., 2002).
The rDNA sequences are more reliable for phylogenetic studies of R. solani
as most of the sequences present in publicly available nucleotide databases consists
of ITS (ITS1-5.8S-ITS2), 18s and 28s subunit regions (Lübeck, 2004). The tested
primers for ITS regions of the fungus are easily available. The nucleotide
composition of the primers designed by White et al. (1990) for sequence analysis of
R. solani ITS region is given in table 2.1.
25
Figure 2.1: Structure of nuclear ribosomal DNA repeat unit of fungus.
26
Table 2.1: Composition of the internal transcribed spacer (ITS) region primers used
for amplification. (White et al., 1990).
27
The rDNA ITS sequence analysis can be employed to establish taxonomic
and phylogenetic relationships between different AGs and AG subgroups (Carling et
al., 2002a; Pope and Carter, 2001; Sharon et al., 2006).
2.3.5 Host Range of Rhizoctonia solani AGs
The fungus R. solani has distinct host range however, the working definition
of the host range is different from other fungus as the adaptability of the fungus is
more apparent than real because of variations of different AGs that constitutes R.
solani species complex (Parmeter, 1970). Isolates belonging to some AGs are widely
distributed and capable of infecting a large number of host plants while others have
a limited host range (Sneh et al., 2013). However, specific AGs preferentially infect
particular host plant species (Keijer et al., 1997). Information on the host range of
each AG of R. solani with typical symptoms is given in Table 2.2.
Rhizoctonia solani AG-3 has been reported to be mainly associated with
Rhizoctonia diseases; black scurf, stem and stolon canker of potato. The most
commonly isolated AG from sclerotia on infected potato tubers is AG-3 (Bolkan and
Ribeiro, 1985; Campion et al., 2003; El Bakali et al., 2000; Rauf et al., 2007; Truter
and Wehner, 2004; Virgen-Calleros et al., 2000; Woodhall et al., 2007). However, a
number of other AG populations including AG-1, AG-2-1, AG-2-2, AG-4, AG-5, and
AG-9 have also been reported to be pathogenic to potato (Balali et al., 1995; Campion
et al., 2003; Carling et al., 1998; Rauf et al., 2007; Woodhall et al., 2007; Woodhall
et al., 2008; Yanar et al., 2005). Carling and Leiner (1990) reported that AG-8 cause
infection to the root portions of potato plants while AG-7 only infects stem portions,
stolon, and tubers (Carling et al., 1998). AG-8 has been reported to cause severe
infections to root portions of potato plants (Balali et al., 1995; Woodhall et al., 2008).
28
AG-3 has been assigned three subgroups; AG-3 PT (potato type), AG-3 TM
(tomato type) and AG-3 TB (tobacco type) (Kuninaga et al., 2007). AG-3 PT has
been reported to cause foot rot disease in tomato (Misawa and Kuninaga, 2010).
Mikhail et al. (2010) reported the infection of AG-2 causing foot root disease. This
has also been supported by the findings of Misawa and Kuninaga (2010). Other AGs
(AG-2, AG-4, and AG-5) (Kuramae et al., 2003; Mikhail et al., 2010; Yildiz and
Döken, 2002) have also been reported to be pathogenic on tomato.
AG-4 is the major cause of root rot disease in chilli (Bolkan and Ribeiro,
1985; Demirci and Doken, 1995; Elias-Medina et al., 1997; Meza-Moller et al.,
2007; Mikhail et al., 2010; Tuncer and Eken, 2013). Katan and Eshel (1974) reported
AG-3 from damping-off in directly seeded pepper (Capsicum frutescens L.) fields.
R. solani isolates belonging to AG-1, AG-2, AG-6, and AG-8 have also been reported
to infect chilli pepper (Bolkan and Ribeiro, 1985; Meza-Moller et al., 2007; Tuncer
and Erdiller, 1990; Tuncer and Eken, 2013).
29
Table 2.2: Host ranges and associated disease symptoms, of Rhizoctonia solani belonging to different anastomosis groups (AGs).
AG Described by Hosts Symptoms Information source
AG-1 Parmeter et al. (1969) Carrot Damping-off Grisham and Anderson (1983)
Cabbage Foliar blight (Abawi and Martin, 1985)
Soybean Aerial and web blight (Rico, 1990)
Pepper Hypocotyl (Bolkan and Ribeiro, 1985)
Common bean Leaf and web blight Muyolo et al. (1993)
Coffee Necrotic foliar disease Priyatmojo et al. (2001)
Lettuce Bottom rot Kuramae et al. (2003)
Rice Web blight Groth and Bond (2006)
Rice Sheath blight Sayler and Yang (2007)
Pepper Root rot (Meza-Moller et al., 2007)
Corn Leaf blight Tomaso-Peterson and Trevathan (2007)
Bristle basket grass Blight Aghajani et al. (2008)
AG-2 Parmeter et al. (1969) Radish Root canker Grisham and Anderson (1983)
carrot Root canker, damping-off (Grisham and Anderson, 1983)
Pepper Root rot (Tuncer and Erdiller, 1990)
Soybean Foliar blight Naito and Kanematsu (1994)
30
Corn Root rot Nelson et al. (1996)
Soybean Damping-off Nelson et al. (1996)
Tulip Bare patch Schneider et al. (1997)
Barley Crown lesion, damping-off Demirci (1998)
Wheat Crown lesion, damping-off Carroll (2004)
Turfgrass Large-patch Hyakumachi et al. (1998)
Common bean Web blight Godoy-Lutz et al. (2003)
Potato Tuber deformation Campion et al. (2003)
Pea Damping-off, root rot Hwang et al. (2007)
Potato Damping-off Rauf et al. (2007)
Potato Stem, stolon and root canker Woodhall et al. (2008)
Tomato Foot rot Misawa and Kuninaga (2010)
Tomato Foot rot (Mikhail et al., 2010)
Sugar beet Root and crown rot Bolton et al. (2010)
Tobacco Target Spot Cardenas et al. (2012)
Pepper Root rot (Tuncer and Eken, 2013)
AG-3 Parmeter et al. (1969) Pepper Seedling damping-off (Katan and Eshel, 1974)
Eggplant Brown spot Kodama (1982)
Tobacco Leaf spot (Meyer et al., 1990)
31
Barley Crown lesion, damping-off Demirci (1998)
Wheat Crown lesion, damping-off Demirci (1998)
Potato Damping-off Rauf et al. (2007)
Potato Stem, stolon and root canker, black scurf Woodhall et al. (2008)
Tomato Foot rot Misawa and Kuninaga (2010)
Pepper Root rot (Tuncer and Eken, 2013)
AG-4 Parmeter et al. (1969) Carrot Damping off Grisham and Anderson (1983)
Pepper Hypocotyl (Bolkan and Ribeiro, 1985)
Pepper Root rot (Demirci and Doken, 1995)
Potato Stem and stolon canker Balali et al. (1995)
Soybean Tissue necrosis, damping-off Nelson et al. (1996)
Pepper Root rot (Elias-Medina et al., 1997)
Barley Crown lesion, damping-off Demirci (1998)
Wheat Crown lesion, damping-off Demirci (1998)
Tomato Stem and foot rot Kuramae et al. (2003)
Mellon Hypocotyl and root rot Kuramae et al. (2003)
Broccoli Hypocotyl and root rot Kuramae et al. (2003)
Spinach Hypocotyl and root rot Kuramae et al. (2003)
Onion Root rot Erper et al. (2006)
32
Potato Damping-off Rauf et al. (2007)
Pepper Root rot (Meza-Moller et al., 2007)
Pepper Stem canker (Mikhail et al., 2010)
Cotton Damping-off Abd‐Elsalam et al. (2010)
Pea Root rot Mathew et al. (2012)
Pepper Root rot (Tuncer and Eken, 2013)
AG-5 Ogoshi (1975) Soybean Root rot, damping-off Nelson et al. (1996)
Apple Root rot Mazzola (1997)
Barley Crown lesion, damping-off Demirci (1998)
Potato Damping-off Rauf et al. (2007)
Potato Stem, stolon and root canker, black scurf Woodhall et al. (2008)
Cotton Damping-off Abd‐Elsalam et al. (2010)
Wheat Stem and root lesion Woodhall et al. (2012)
Pea Root rot Mathew et al. (2012)
AG-6 Kuninaga et al. (1979) Apple Root rot Mazzola (1997)
Wheat Crater disease Meyer et al. (1998)
Cauliflower Seedling damping-off Carling et al. (1999)
Lucerne Root canker Anderson et al. (2004)
Pepper Root rot (Tuncer and Eken, 2013)
33
AG-7 Homma et al. (1983) Potato Stem canker Carling et al. (1988)
Cotton Damping-off, root rot Abd‐Elsalam et al. (2010)
Wheat Root rot Ogoshi et al. (1990)
Barley Root rot and stunning Ogoshi et al. (1990)
Potato Root canker Woodhall et al. (2008)
AG-8 Neta and Warcup (1985) Wheat Root rot (Ogoshi et al., 1990)
Barley Root rot and stunning (Ogoshi et al., 1990)
Pepper Root rot (Tuncer and Erdiller, 1990)
Potato Root canker (Woodhall et al., 2008)
AG-9 Carling et al. (1987) Cauliflower Damping-off Yang et al. (1996)
Flax Damping-off Yang et al. (1996)
Canola Root discolouration Yang et al. (1996)
Potato Stem and stolon canker Yanar et al. (2005)
Potato Damping-off Rauf et al. (2007)
AG-10 MacNish et al. (1995) Canola Root rot Schroeder and Paulitz (2012)
AG-11 Carling et al. (1994) Cotton Seedling discolouration Carling et al. (1994)
Radish Seedling discolouration Carling et al. (1994)
Wheat Seedling discolouration Carling et al. (1994)
Potato Minor stem and root lesions Carling et al. (1994)
34
Barley Crown lesion, damping-off Demirci (1998)
Wheat Crown lesion, damping-off Demirci (1998)
Lupin Damping-off, hypocotyl rot Kumar et al. (1999)
AG-12 Carling et al. (1999) Cauliflower Seedling damage Carling et al. (1999)
Radish Seedling damage Carling et al. (1999)
AG-13 Cotton Minor lesions on shoot and root Carling et al. (2002a)
Corn Weakly pathogenic on seedling Tomaso-Peterson and Trevathan (2004)
35
Chapter 3
3 MATERIALS AND METHODS
The study in question involves an extensive survey of selected solanaceous
vegetable growing areas of Pothohar region for Rhizoctonia solani infection,
isolation and identification, pathogenicity testing, cultures preservation, morpho-
molecular characterization, and anastomosis group typing of the recovered isolates.
Studies were made at Fungal Plant Pathology Lab., Department of Plant Pathology,
PMAS Arid Agriculture University, Rawalpindi. Part of this research project was
accomplished at the Department of Plant, Soil and Microbial Sciences, Michigan
State University, East Lansing, Michigan.
3.1 SURVEILLANCE FOR DISEASE ASSESSMENT AND SAMPLE
COLLECTION
3.1.1 Description of the Study Area
The studies on disease documentation of R. solani on three solanaceous
vegetables; potato, tomato, and chilli were made in Pothohar region including districts;
Jhelum, Chakwal, Attock, Rawalpindi, and Islamabad Capital Territory (Figure 3.1).
Pothohar region is a high plain forming the northern part of Punjab province of Pakistan
(Figure 3.1). It is situated between latitude 32.5o 00’N to 34o 00’N and altitude 72o 00’E
to 74o 00’E in the Asian sub-continent with an elevation of 517m from sea level and
experiences semi-arid to humid climate (Chaudhry and Rasul, 2004).
Pothohar region features a humid and subtropical climate with hot summers,
monsoon and short, mild, and wet winters. The climate of the region has considerable
temperature variations. Daytime temperature reaches above 40oC during the summer
from April to September while June is the hottest month. During winter from
36
November to March, the average temperature ranges between 0-10oC. December, and
January are considered as the coldest months where night time temperatures fall
below 0oC (Rashid and Ayaz, 2016). The average annual rainfall is abundant at 1,249
millimetres (49.2 in) of which 65% is received in the monsoon season. Summer
monsoon produces more rainfall as compared to winter (Rashid and Ayaz, 2016).
3.1.2 Disease Assessment and Sample Collection
Three solanaceous vegetables; potato, tomato, and chilli growing areas in
four districts; Jhelum, Chakwal, Attock, Rawalpindi, and Federal Capital Islamabad
were surveyed during the cropping season 2014-15 and 2015-16 to record the disease
incidence and prevalence percentage. The detail on different locations surveyed from
each district for all three crops is given in figure 3.2 and table 3.1.
Rhizoctonia solani cause black scurf, stem and stolon canker disease in
potato. For potato, the survey was conducted in X-plus manner. Fields were
diagonally visited for foliage yellowing and infected tubers with visible symptoms
of sclerotia and plants with characteristic symptoms of stem canker were collected
(Figure 3.3). At least five samples from each field were collected. 1kg of infected
potato tubers were collected from each field with the consent of the grower.
Depending on the field size, quadrate of 1m2 was thrown 5-8 times in each field.
Healthy and infected plants within the specific area were counted to determine
disease incidence percentage.
For tomato, and chilli, positive sampling was done. R. solani cause foot rot
of tomato and root rot in chilli plants. Tomato, and chilli fields were diagonally
visited for R. solani infection on soil line level of the stem and root infections
respectively, and symptomatic plants samples were collected (Figure 3.4).
37
Figure 3.1: Map of Pakistan showing Pothohar region the northern part of Punjab.
38
Figure 3.2: Map of Pothohar region showing district Attock, Chakwal, Jhelum,
Rawalpindi and Islamabad.
39
Figure 3.3(a): Symptomatic potato plant infected with Rhizoctonia solani.
Figure 3.3(b): Diseased potato tubers showing visible sclerotia of Rhizoctonia solani.
a
b
40
Figure 3.4(a): Symptomatic chilli plant infected with Rhizoctonia solani.
Figure 3.4(b): Symptomatic tomato plant infected with Rhizoctonia solani.
a b
41
Table 3.1: Districts and their locations surveyed for Rhizoctonia solani infection on
potato, tomato, and chilli during the crop season 2014-15 and 2015-16.
42
Depending on the field size, quadrate of 1m2 was thrown 5-8 times in each
field. Healthy and infected plants within the specific area were counted to determine
disease incidence percentage. Disease prevalence and incidence percentage was
calculated using formula;
Disease Prevalence (%)=Locations showing R. solani infection
Total locations examined×100
Disease Incidence (%)=No. of infected plants
Total no. of plants ×100
A total of 438 samples from potato, 457 from tomato, and 426 from chilli
were collected in rubber band tightened bags, properly labelled with sample
number, collection date, and location and were stored at 4oC in the refrigerator at
Fungal Plant Pathology Lab., Department of Plant Pathology, PMAS-Arid
Agriculture University Rawalpindi for further processing. Detail on number of
samples from each location is given in table 3.2.
3.2 ISOLATION AND CULTURING OF RHIZOCTONIA SOLANI
Rhizoctonia solani isolates were recovered from infected plant portions on
potato dextrose agar (PDA) medium. Sclerotia were excised from the surface using a
sterilized scalpel and infected stem and root portions were cut into 5mm segments,
surface disinfected for two minutes in 2.5% sodium hypochlorite, rinsed with sterile
tap water, and were allowed to dry on sterilized filter paper in the laminar-flow bench.
The surface sterilized pieces were plated on Petri plates containing PDA medium
amended with 0.3 g/L streptomycin sulfate (Sigma Chemical Co., St. Louis, MO)
incubated at 25oC. After 48 hours of incubation, hyphae resembling Rhizoctonia
(Ogoshi, 1987) were identified under microscope and hyphal tips from each isolate
were transferred to a fresh Petri plate containing PDA medium.
43
Table 3.2: Total number of samples for Rhizoctonia solani infection on potato,
tomato, and chilli from different locations of each district.
Sr. No. District Location(s) surveyed Potato Tomato Chilli
1 Rawalpindi Taxila, Adayla, Kallar
Syedan, Kotli Sattian, Gujar
Khan, Murree, Rawalpidi
163 109 93
2 Chakwal Barani Agriculture Research
Institute, Talagang, Lava,
Tamman, Bhon
18 93 97
3 Jhelum Dina, Sohawa, Pind Dadan
Khan, Amra Kalan, Bhelowal
92 123 112
4 Attock Fateh Jang, Bhatar, Hassan
Abdal, Pindi Gheb
147 117 107
6 Islamabad NARC, Islamabad 18 15 17
Total 1321 438 457 426
44
3.3 MORPHOLOGICAL CHARACTERIZATION OF RHIZOCTONIA
SOLANI ISOLATES
3.3.1 Cultural Characteristics and Microscopic Studies of Rhizoctonia solani
Recovered isolates were morphologically characterized by cultural and
microscopic observations as described by Ogoshi (1975). The mycelium of R.
solani is colourless when young and transformed to brown as grow and mature.
The mycelium consists of basal hyphae partitioned into individual cells by a
dolipore septum and each cell contains more than three nuclei. The young
vegetative hyphae are branched at right angles near the distal septum of the cells
and constricted at their junction or at a short distance from the septum (Ogoshi,
1987; Sneh et al., 1991).
Isolates were grown on 30mm × 15mm Petri plates containing malt extract
agar (MEA) medium incubated at 25oC. Cultural characteristics including growth
patterns, colony colour, colony diameter, number of sclerotia, texture, and
topography of sclerotia were noted. Isolates were grown on a separate set of 30mm
× 15mm Petri plates containing 2% water agar (WA) incubated at 25oC for 4 days
stained with 0.05% lactophenol blue and examined under microscope to observe
hyphal morphology, inter-septal distance, and hyphal diameter. Inter-septal distance
and hyphal diameter were determined by measuring 20 cells per plate for each of the
isolates in microscopic fields.
3.3.2 Nuclear Number Testing
Number of nuclei per cell of R. solani were counted by staining hyphae with
1ug/ml of DAPI (4’-6 diamidino-2-phenylindole) stain. Petri plates were examined
under fluorescent microscope at 400X magnification to count number of nuclei per cell.
45
3.3.3 Preservation of Rhizoctonia solani Isolates
Rhizoctonia spp. isolates can be preserved by colonizing on cereal grains
including barley, wheat, oat, and rice (Kuznia and Windels, 1994; Naito, 1993; Sneh et
al., 1986). Isolates were purified using hyphal tipping method on PDA medium. Purified
isolates were grown on water agar (WA) medium for use in the short-term. Isolates were
colonized on sterile barley grains and maintained at 4 °C for long-term preservation.
Hulled barley grains were hydrated by soaking in double distilled water
(ddH2O) overnight. The hydrated barley grains were autoclaved at 121oC and 15 PSI
pressure for 60 minutes, allowed to cool for 24 hours, and autoclaved again.
Sterilized Petri plates were almost more half filled with barley grains. Mycelial disks
from margins of the actively growing cultures of R. solani were placed in the Petri
plates incubated at 25oC for 7 days for colonization. Few drops of ddH20 were added
to the plates in order to keep barley grains moist during the period of colonization.
The Petri plates were then placed in the desiccator for complete drying. The
colonized barley grains were transferred to sterilized screw cap vials stored at -20oC.
3.4 PATHOGENICITY TESTING
Recovered isolates were subjected to pathogenicity testing to confirm Koch’s
Postulates in greenhouse conditions at 25 ± 2oC. For potato, pot trial was conducted
using method previously described by Balali et al. (1995). Six sprouted potato tubers
cv. Desire were planted in 5L plastic pots filled with sterilized potting mixture i.e.
sand: clay: farmyard manure at the rate of 1:1:1 (Naz et al., 2008). 10g of barley grains
colonized with each isolate of R. solani for 14 days were placed 10mm above the tuber
covered with a layer of potting mixture. Uninoculated pots were used as a control.
The trial was conducted with three replicates for each isolate arranged in a
46
randomized complete block design (RCBD). Three plants from each replicate were
uprooted after four weeks of inoculation to record stem and stolon infection. Four
months after inoculation plants were removed to record tuber infection. 1-4 disease
rating scale by Balali et al. (1995) was used for disease assessment (Table 3.3).
For tomato, pathogenicity tests were performed following the method described
by Misawa and Kuninaga (2010). Plastic cell trays (53.49cm L x 26.82cm W) having
32 cells/ tray were filled with sterilized potting mixture i.e. sand: clay: farmyard manure
at the rate of 1:1:1 (Naz et al., 2008). Three weeks old tomato plants cv. Rio Grande
were transplanted in the cells and soil inoculum containing 10g of barley grains
colonized with each isolate of R. solani for 14 days was mixed in the upper 2cm layer
of soil. Uninoculated cells were used as a control. Plants were grown at 25 ± 2oC for 28
days. Infection on soil line level of the stem was categorized as -, no symptom; ±, brown
lesion on part of the stem; +, brown lesion girdled the stem; ++, brown lesion girdled
the stem and plants wilted (Table 3.4). The trial was conducted with three replicates for
each isolate arranged in a completely randomized design (CRD).
For chilli, pathogenicity test was performed in a greenhouse experiment
following the modified method described by Tuncer and Eken (2013). Seeds of chilli
cv. Sanam were surface sterilized by dipping in 1% Sodium hypochlorite (NaOCl)
for 5 minutes followed by washing twice with sterilized distilled water and air dried.
Plastic cell trays (53.49cm L x 26.82cm W) having 32 cells/ tray were filled with
sterilized potting mixture i.e. sand: clay: farmyard manure at the rate of 1:1:1 (Naz
et al., 2008). Five seeds were planted in each cell. Two weeks after sowing, 10g of
blended barley grains colonized with each isolate of R. solani for 14 days was mixed
in the upper 2cm layer of soil.
47
Table 3.3: Disease rating scale to record stem, stolon, and tuber infection on potato.
Rating Disease reaction Symptoms of stem and
stolon infection
Symptoms of tuber
infection
0 Avirulent No canker present No sclerotia present
1 Slightly virulent Superficial canker less
than 10%
Less than 10% tuber
covered with sclerotia
2 Moderately
virulent
Superficial canker 10-
25%
10-25% tuber covered
with sclerotia
3 Virulent Deep canker 26-50% 26-50 % tuber covered
with sclerotia
4 Highly virulent More than 50%
canker, sprout or
stolon girdled or killed
More than 50% tuber
covered with sclerotia
(Balali et al., 1995).
Table 3.4: Disease rating scale to record stem infection on tomato.
Rating Disease reaction Symptoms of stem infection
- No disease no symptom
± Slightly diseased brown lesion on part of the stem
+ Moderately diseased brown lesion girdled the stem
++ Severely diseased brown lesion girdled the stem and plants wilted
(Misawa and Kuninaga, 2010).
Table 3.5: Disease rating scale to record stem infection on chilli.
Rating Disease reaction Symptoms of root infection
- No disease no symptom
± Slightly diseased lesions on the part the hypocotyl
+ Moderately diseased lesions girdling the hypocotyl and root portions
++ Severely diseased lesions girdling the hypocotyl and dead seedlings
(Tuncer and Eken, 2013).
48
Figure 3.5: Pathogenicity testing of Rhizoctonia solani isolates on potato plants
under pot trials.
b
49
In control plants, 10g of sterilized blended barley grains was mixed. The
trial was conducted with three replicates for each isolate arranged in a completely
randomized design (CRD). Four weeks after inoculation, plants were removed,
washed and disease severity was evaluated using disease rating scale (Table 3.5),
where - = healthy, no lesions on the hypocotyls; ± = lesions on part the hypocotyl;
+ = lesions girdling the hypocotyl and root portions; ++ = lesions girdling the
hypocotyl, roots and dead seedlings. Pathogenicity tests for each crop were
repeated twice.
3.5 ANASTOMOSIS GROUP TESTING
Generally, isolates of R. solani are identified on the basis of hyphal
interactions reactions, however, reproducibility of anastomosis interactions for a
large number of populations is a time-consuming process. The resolution of this
method at subgroup level is insufficient (Muzhinji et al., 2015; Muzhinji et al., 2014;
Sharon et al., 2006). Restriction fragment length polymorphism (RFLP) analysis of
ribosomal DNA (rDNA) sequences has successfully been used to characterize a large
number of R. solani isolates into respective AGs or subsets within AGs (Hyakumachi
et al., 1998; Liu et al., 1993; Vilgalys and Gonzales, 1990).
Virulent isolates were subjected to PCR-Restriction Fragment Length
Polymorphism (RFLP) analysis for their categorization at AGs level. The results of
AG composition were further confirmed by hyphal anastomosis interactions.
3.5.1 PCR-Restriction Fragment Length Polymorphism (RFLP)
3.5.1.1 Culturing, maintenance & lyophilization
Rhizoctonia solani isolates were inoculated in malt extract broth (MEB)
medium in 9cm Petri plates incubated at 25oC for 5 days. Mycelium mat for each
50
isolate was harvested and washed with double distilled water (ddH2O) to remove
excessive broth medium. Blot tried hyphal mats were transferred to 20ml falcon
tubes stored at -80oC for few days before lyophilization.
Falcon tubes were freeze dried in the lyophilization chamber for 05 days.
Sterilized iron beads were placed in the falcon tubes containing lyophilized hyphal
mats ground to fine powder in the mechanical shaker. This tissue powder was
transferred to 2ml microcentrifuge tubes stored at room temperature and was used
for DNA extractions.
3.5.1.2 DNA extraction
DNA from each isolate was extracted using the standard protocol of
Omniprep for fungi extraction kit (G-Biosciences) (Cat. # 786-399) modified by
Linda Hansen, Michigan State University.
20-25mg ground, lyophilized tissues were added to a 2ml microcentrifuge
tube having 500µL genomic lysis buffer and vortexed. 5µL (1µL/100µL lysis buffer)
proteinase K was added and incubated at 60-65oC for about 2 hours. Tubes were
periodically shaken during incubation to mix the contents and were then allowed to
cool by placing several minutes on ice. 500µL chloroform isoamyl alcohol (24:1)
was added and mixed by inverting tubes several times followed by centrifugation at
14,000xg for 10 minutes. The upper aqueous phase (450µL) was transferred to a
clean 1.5ml microcentrifuge tube. 5µL RNase A was added to it and incubated at
room temperature for 30 minutes. 300µL chloroform isoamyl alcohol (24:1) was
added and mixed by inverting tubes several times. Tubes were centrifuged for 10
minutes at 14,000xg. 50µL DNA stripping solution was added and mixed incubated
at 60oC for 10 minutes. 150µL precipitation solution was added and mixed by
51
inverting the tubes several times. A white precipitation was formed if not, additional
50µL was added to and placed in ice for several minutes form precipitation. Tubes
were centrifuged for 5 minutes at 14,000xg and supernatant was transferred to a clean
1.5µL microcentrifuge tube. 500µL isopropanol was added and well mixed by
inverting tubes 10-15 times to precipitate DNA. Tubes were centrifuged for 5
minutes at 14,000xg to collect DNA pellet at the bottom and supernatant was
discarded. DNA pellet was dissolved in 450µL ddH2O and 100µL 100% ethanol was
added. Tubes were placed on ice for 10-15 minutes followed by centrifugation at 4oC
for 2 minutes at 5,000xg. The tubes were kept at chilling temperature during this step
to avoid re-suspension of contaminants. The supernatant was transferred to clean
15µL microcentrifuge tube and 45µL 3M sodium acetate (pH 5.5) was added and
mixed. 900µL 100% ethanol was added and mixed by inverting tubes for several
times. The tubes were kept on chilling temperature for 24 hours. Tubes were
centrifuged for 10 minutes at 10,000xg to pelletize DNA. DNA pellet was washed
with 500µL 70% ethanol and centrifuged for 1 minute at 10,000xg. The supernatant
was discarded, and DNA pellet was washed again by following the same procedure.
Tubes were inverted for few minutes on a clean absorbent surface to drain excess
ethanol followed by placing tubes by their sides for 1 hour until all ethanol
evaporated. DNA pellet was dissolved in 50µL TE buffer.
3.5.1.3 Polymerase chain reaction (PCR) amplification
The extracted DNA was subjected to PCR amplification of the ITS region
with primers RS1 (5′-CCTGTGCACCTGTGAGACAG-3′) and RS4 (5′-
TGTCCAAGTCAATGGACTAT-3′) (Camporota et al., 2000). PCR reaction
mixture was prepared by adding 10µL 5X Buffer, 1µL dNTPs, 2.5µL each forward
52
and reverse primers, 1µL MgCl2, 0.5µL Taq Polymerase 2.5µL DNA template in the
final volume of 50µL. A negative control (without DNA template) was always
included in PCR reactions. Amplifications were performed in MJ Research Tetrad
PTC-225 Thermal Cycler system (Bunker Lake Blvd. Ramsey, Minnesota, USA).
The initial denaturation was done at 94°C for 2min followed by 35 cycles of
denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 60s,
and a final extension at 72°C for 5 min.
3.5.1.4 Confirmation of PCR amplification
Aliquots (5μL) of each PCR products were analyzed in 2% agarose gel (high
resolution agarose, Q-BIOgen) in TAE buffer containing 40 mmol/L Tris–HCl (pH
7.9), 4 mmol/L sodium acetate, and 1 mmol/L EDTA (pH 7.9).
5µL of PCR amplified products were mixed with 2µL gel loading dye and
the mixture was loaded to the wells. 1kb DNA ladder (0.5 µg/µL) (Thermo
Scientific, Waltham, Massachusetts, USA) was used for detecting the size of the
amplified products. The loaded gel was electrophoresed at 100V for 45 minutes and
fragments were visualized over using a gel documentation system.
3.5.1.5 PCR products purification
Each PCR product was cleaned up using Sephadex G-50. 250mg Sephadex G-
50 was added in 500µL double distilled water in 15ml falcon tube and was vortexed.
Spin columns were placed in 2ml water collections tubes and 300µL Sephadex
solution was added to each column followed by spinning at 1500rpm/m to remove
excess water. Spin columns were then transferred to 1.5m centrifuge tubes and PCR
product was added to each spin column followed by spinning at 200rpm/m. Cleaned
PCR product collected in each centrifuge tube was used for restriction analysis.
53
Figure 3.6: Extraction of genomic DNA of Rhizoctonia solani isolates.
54
3.5.1.6 PCR–RFLP analysis
Cleaned PCR products of RS1 & RS4 amplifications were characterized by
PCR-RFLP with selected enzymes (MseI, AvaII+HincII, and MunI). Restriction
digest mixtures were prepared by adding 1µL of each enzyme (MseI, AvaII+HincII,
and MunI), 13µL Template (PCR Product) and 5µL Buffer in final volume of 50µL.
The PCR conditions were as described previously. 10µL 6X gel loading dye (Thermo
Scientific, Waltham, Massachusetts, USA) (1µL 6X gel loading dye/5µL sample
unit) was added to each well.
3.5.1.7 Restriction patterns
The restriction fragments were separated by electrophoresis in 3% agarose
gel (high resolution agarose, Q-BIOgen) in TAE buffer containing 40 mmol/L Tris–
HCl (pH 7.9), 4 mmol/L sodium acetate, and 1 mmol/L EDTA (pH 7.9). 15µL of
each PCR amplified product mixed with 6X gel loading dye was loaded to the wells.
100bp low molecular weight (LMW) DNA ladder (0.5 µg/µL) (Thermo Scientific,
Waltham, Massachusetts, USA) was used to detect the size of the amplified products.
The loaded gel was electrophoresed at 100V for 180 minutes and fragments were
visualized over using a gel documentation system. Restrictions patterns of each
isolate were compared RFLP type determined by Guillemaut et al. (2003) (Figure
3.7, 3.8, and Table 3.6) and anastomosis groups were defined.
3.5.2 Hyphal anastomosis interactions
Anastomosis group identities by restriction analysis of their ribosomal DNA
with discriminating enzymes were further confirmed by the hyphal anastomosis
interactions. Cultures of the individual isolates and tester strains of the respective
AGs were maintained on PDA medium at 25oC for 96 hours.
55
Figure 3.7: Restriction patterns revealed by RFLP analysis of internal transcribed spacers sequences of Rhizoctonia solani with MseI.
(Guillemaut et al., 2003).
56
Figure 3.8: Restriction patterns revealed by RFLP analysis of internal transcribed spacers sequences of Rhizoctonia solani with Ava II+Hinc
II and MunI.
(Guillemaut et al., 2003).
57
Table 3.6: RFLP types revealed by the restriction analysis of ITS sequences among
Rhizoctonia solani.
(Guillemaut et al., 2003).
58
An agar disc (5mm) was excised from the edge of the actively growing
hyphae and placed on 1.5% WA coated clean glass slides having similar agar disc of
tester strain of the known AG. After 48-72 hours when hyphae from each isolate
overlapped, slides were stained with lactofuchsin and were examined under 400x
magnification for hyphal anastomosis. Anastomosis reactions were classified from
C0 to C3 where, C0 = no reaction, C1 = contact fusion, C2 = somatic fusion or perfect
anastomosis, and C3 = auto-anastomosis as described by Carling (1996). C3 type
interactions; auto-anastomosis or self pairing were used as positive control.
Twenty random locations were selected to observe hyphal interactions
between unknown strain and the tester strain of respective AG and percentage fusion
frequency (% FF) was determined as;
% 𝐹𝐹 =𝐴 × 100
𝐵
Where,
A = Sum of fusion locations (in C1, C2, C3) in 20 microscopic fields
B = Sum of contact points in 20 microscopic fields
Isolates pairing at more than 80% locations were confirmed as belonging to
respective anastomosis group.
3.5.3 PCR amplification of ITS-5.8S rDNA
Molecular identification of the type isolates belonging to various AGs was
accomplished by extracting DNA following procedures described by Sambrook
and Russell (2001). ITS region of each isolate was amplified using universal sense
ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-
TCCTCCGCTTATTGATATGC-3´) (Qiagen) encoding ITS-1-5.8S-ITS-2 of the
59
DNA samples (White et al., 1990). PCR mixtures were prepared in a total volume
of 50µL containing 2.5µL of the total DNA, 2.5µL each forward and reverse
primers, (Sigma-Aldrich), 10µL 5X Buffer, 1µL MgCl2, 1µL dNTPs, and 0.5µL of
Taq DNA polymerase. A negative control (without DNA template) was always
included in PCR reactions. Amplifications were performed in MJ Research Tetrad
PTC-225 Thermal Cycler system (Bunker Lake Blvd. Ramsey, Minnesota, USA).
The initial denaturation was done at 94°C for 2min followed by 35 cycles of
denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1
min, and a final extension at 72°C for 5 min. The PCR amplified products were
analyzed in 2% agarose gel (high resolution agarose, Q-BIOgen) in TAE buffer
containing 40 mmol/L Tris–HCl (pH 7.9), 4 mmol/L sodium acetate, and 1 mmol/L
EDTA (pH 7.9).
3.5.3.1 Sequencing of ITS-5.8S rDNA
8µL of PCR amplified products were mixed with 2µL gel loading dye and the
mixture was loaded to the wells. 1kb DNA ladder (0.5 µg/µL) (Thermo Scientific,
Waltham, Massachusetts, USA) was used for detecting the size of the amplified
products. The loaded gel was electrophoresed at 100V for 45 minutes and fragments
were visualized over using a gel documentation system. PCR products were purified
using Sephadex G-50 as described earlier and were sequenced in both directions
using sequence facility at Michigan State University.
3.5.3.2 Sequence analysis
The ITS sequence of the type isolates were manipulated in sense and
antisense directions using BioEdit software (Hall, 1999) and were aligned with
Clustal W program (Thompson et al., 1994). The Basic Local Alignment Search Tool
60
(BLAST) was used to determine the percentage genetic nucleotide homology of the
ITS regions with those of related genera available in the database of the National
Center for Biotechnology Information (NCBI) GenBank.
3.5.3.3 Phylogenetic studies
Phylogenetic and molecular evolutionary analyses were accomplished by
constructing Maximum Likelihood tree with the ITS sequences for R. solani obtained
from GenBank (Table 3.7) using Mega (Tamura and Nei, 1993) and MrBayes
software (Ronquist and Huelsenbeck, 2003). The positions containing gaps and
missing data were eliminated. Bootstrapping was performed at 1000 replications of
the data being analyzed.
61
Table 3.7: GenBank accessions of Rhizoctonia solani reference isolates used in this
study.
Sr. Isolate Origin Host AG Accession No
1 HeN-16H China Potato AG-3 PT KR006060
2 LN-3 China Potato AG-2-1 KJ170317
3 HLJ-22 China Potato AG-4 KX468085
4 HL-16 China Potato AG-5 JQ946294
5 Rh 86 South Africa Potato AG-2-2 KJ777632
6 GX-2H China Potato AG-3 PT KP013070
7 LN-3 China Potato AG-2-1 KJ170317
8 HLJ-20 China Potato AG-5 KX468083
9 F521 USA Beta vulgaris AG-2-2 FJ492157
10 RR8 China Sugarbeet AG-4HGI KR259925
11 HLJ-154 China Potato AG-2-1 KX631360
12 HLJ-127 China Potato AG-3 PT KX631333
13 HL-10-1 China Potato AG-5 JQ946293
14 CR 6 Egypt ELS Cotton AG-6 KT362072
15 KXC21004 China Spinach AG4-HGI KY189917
16 FJ766520 Outgroup Rhizoctonia oryzae FJ766520
62
Chapter 4
4 RESULTS
4.1 SURVEILLANCE FOR DISEASE ASSESSMENT AND SAMPLE
COLLECTION
4.1.1 Surveillance for Disease Assessment on Potato
Survey for Rhizoctonia solani disease prevalence and incidence of selected
solanaceous vegetables; potato, tomato, and chilli was conducted in two consecutive
two years i.e. 2014-15 and 2015-16 crop season. Pothohar region falls in potato
production zone 3. In districts Rawalpindi, Jhelum, Attock, Chakwal, and Federal
Capital Islamabad potato crop is planted in mid-September/ October and harvested in
mid - December/ January. The survey was conducted during early December - mid-
January. In tehsil Murree of district Rawalpindi potato crop is cultivated in May-June
and harvested in late October. The survey was conducted in mid-October. A total of 438
symptomatic plant samples were collected from private potato farms and research
stations.
4.1.1.1 Rhizoctonia solani disease prevalence and incidence on potato
During the survey of potato growing areas for R. solani infection, it was found
that the disease was 100% prevalent in all locations visited. Potato plants exhibited
typical symptoms of black scurf however, considerable variations were found in
disease incidence (DI) percentage of the surveyed locations.
In district Rawalpindi, the main potato production areas are in tehsil Taxila
and Rawalpindi. Some scattered locations of tehsil Kahuta and Kotli Sattian were
also surveyed. In tehsil Murree, summer crop was investigated for R. solani disease
incidence. The maximum mean disease incidence was found in Taxila (35.4%)
63
followed by Rawalpindi (34.4%) while minimum mean disease incidence (24.2%)
was recorded in tehsil Murree. In district Attock, potato production is concentrated
in tehsil Attock, Hazro, Fateh Jang, and Hasan Abdal. The maximum mean disease
incidence was found in tehsil Attock (39.0%) followed by Hazro (37.9%) and
(37.6%) while minimum mean disease incidence was recorded at Fateh Jang (35.1%).
In district Jhelum, potato production was concentrated in tehsil Jhelum, Sohawa, and
Pind Dadan Khan. The maximum mean disease incidence was calculated in Jhelum
areas (34.4%) followed by Pind Dadan Khan (31.1%) while minimum disease
incidence was recorded in tehsil Sohawa (30.85%). In district Chakwal, potato
cultivation was observed only at scattered locations and only two locations were
visited. Disease incidence was recorded 21.2% in 2014 while 19.3% in 2015. Mean
disease incidence recorded in Chakwal was 20.2%. The mean disease incidence in
Islamabad (Federal Capital Territory) was recorded as 35.8%.
Overall in the Pothohar region; maximum mean disease incidence was
recorded in tehsil Attock (39.0%) followed by Hazro (37.9%) Hassan Abdal
(37.65%), National Agricultural Research Center (NARC) (35.8%), (Taxila 35.4%),
Fateh Jang (35.1%), Rawalpindi (34.4%), Jhelum (34.4%), Pind Dadan Khan
(31.1%), Sohawa (30.8%), Kotli Sattian (29.7%) Kahuta (28.8%) and Murree
(24.2%) while minimum mean disease incidence was recorded in Chakwal (20.2%)
as shown in Table 4.1.
District wise, maximum mean disease incidence was observed in district
Attock (37.4%) followed by Islamabad (35.8%), district Jhelum (32.1%) and district
Rawalpindi (30.5%) while minimum mean disease incidence was recorded in district
Chakwal (20.2%) as shown in Figure 4.1.
64
Table 4.1: Disease prevalence and incidence percentage of Rhizoctonia solani on potato in various areas/locations of the districts of Pothohar
region (crop season 2014-15 and 2015-16).
Districts Location (s)
Surveyed
Locations Surveyed Disease Prevalence Disease Incidence %
2014-15 2015-16 2014-15 2015-16 Mean 2014-15 2015-16 Mean
Rawalpindi Taxila 9 9 100 100 100 36.5 34.3 35.4 Rawalpindi 5 5 100 100 100 32.3 36.5 34.4 Gujar Khan - - - - - - - -
Kallar Syedan - - - - - - - - Kahuta 4 4 100 100 100 26.3 31.3 28.8 Kotli Sattian 5 5 100 100 100 27 32.5 29.7 Murree 9 9 100 100 100 21.7 26.7 24.2
Jhelum Jhelum 8 8 100 100 100 37.5 31.3 34.4 Pind Dadan Khan 5 5 100 100 100 29.7 32.5 31.1 Sohawa 5 5 100 100 100 27.2 34.5 30.8 Dina - - - - - - - -
Attock Attock 6 6 100 100 100 37.6 40.5 39.0 Hazro 7 7 100 100 100 36.5 39.3 37.9 Fateh Jang 9 9 100 100 100 32.8 37.5 35.1 Hassan Abdal 7 7 100 100 100 35.9 39.4 37.6 Jand - - - - - - - - Pindi Gheb - - - - - - - -
Chakwal Chakwal 2 2 100 100 100 21.2 19.3 20.2 Kallar Kahar - - - - - - - - Choa Saidan Shah - - - - - - - - Talagang - - - - - - - - Lawa - - - - - - - - BARI - - - - - - - -
Islamabad NARC 3 3 100 100 100 34.3 37.3 35.8
65
4.1.2 Surveillance for Disease Assessment on Tomato
Tomato is a warm season crop and varying climatic conditions in Pothohar
region provide optimum temperature conditions for tomato growth and development.
Survey for disease assessment of R. solani infection on tomato was conducted in crop
season 2014-15 and 2015-16. A total of 457 symptomatic plant samples of tomato
were collected from scattered locations in district Rawalpindi, Jhelum, Attock,
Chakwal, Rawalpindi, and Islamabad.
4.1.2.1 Rhizoctonia solani disease prevalence and incidence on tomato
Tomato production in Pothohar region is mostly at scattered locations in. R.
solani infection was 100% prevalent in all the locations visited. In district
Rawalpindi, the maximum mean disease incidence was recorded in Taxila (41.0%)
followed by tehsil Rawalpindi (39.4%), Kallar Syedan (35.8%), and Gujar Khan
(31.2%) while minimum mean disease incidence was recorded in Kahuta (30.6%). In
district Jhelum, the maximum mean disease incidence was recorded in tehsil Jhelum
(38.0%) followed by Sohawa (32.9%) while minimum mean disease incidence was
observed in Pind Dadan Khan and Dina (28.3%). In district Attock, the maximum
mean disease incidence was recorded in Fateh Jang (40.3%) followed by Hazro
(39.9%), Hassan Abdal (38.5%), Attock (34.5%) and Jand (33.9%) while minimum
mean disease incidence was recorded in Pindi Gheb (31.0%). In district Chakwal, the
maximum mean disease incidence was recorded at Barani Agricultural Research
Institute (35.5%) followed by Lawa (31.2%), Choa Saidan Shah (30.0%), Chakwal
(28.3%) while the minimum mean disease incidence was recorded at Talagang and
Kallar Kahar (26.3%). The mean disease incidence in Islamabad (Federal Capital
Territory) was recorded as 38.7% (Table 4.2).
66
Table 4.2: Disease prevalence and incidence percentage of Rhizoctonia solani on tomato in various areas/locations of the districts of Pothohar
region (crop season 2014-15 and 2015-16).
Districts Location (s)
Surveyed
Locations Surveyed Disease Prevalence Disease Incidence %
2014-15 2015-16 2014-15 2015-16 Mean 2014-15 2015-16 Mean
Rawalpindi Taxila 5 5 100 100 100 42.3 39.7 41.0 Rawalpindi 6 6 100 100 100 39.5 39.3 39.4 Gujar Khan 4 4 100 100 100 33.2 29.3 31.2
Kallar Syedan 3 3 100 100 100 35.4 36.3 35.8 Kahuta 3 3 100 100 100 32.1 29.1 30.6 Kotli Sattian 2 2 100 100 100 33.3 30 31.6 Murree - - - - - - - -
Jhelum Jhelum 8 8 100 100 100 36.7 39.4 38.0 Pind Dadan Khan 4 4 100 100 100 29.3 27.3 28.3 Sohawa 5 5 100 100 100 34.5 31.4 32.9 Dina 4 4 100 100 100 27.3 29.3 28.3
Attock Attock 8 8 100 100 100 36.7 32.4 34.5 Hazro 7 7 100 100 100 42.3 37.5 39.9 Fateh Jang 7 7 100 100 100 39.5 41.2 40.3 Hassan Abdal 6 6 100 100 100 37.4 39.6 38.5 Jand 4 4 100 100 100 32.3 35.6 33.9 Pindi Gheb 4 4 100 100 100 29.7 32.3 31.0
Chakwal Chakwal 6 6 100 100 100 27.3 29.3 28.3 Kallar Kahar 5 5 100 100 100 26.3 26.3 26.3 Choa Saidan Shah 3 3 100 100 100 29.4 30.6 30.0 Talagang 6 6 100 100 100 26.3 26.4 26.3 Lawa 7 7 100 100 100 29.7 32.7 31.2 BARI 2 2 100 100 100 36.5 34.6 35.5
Islamabad NARC 3 3 100 100 100 38.5 39 38.7
67
Overall in the Pothohar region; maximum mean disease incidence was
recorded in Taxila (41.0) followed by Fateh Jang (40.3%), Hazro (39.9%),
Rawalpindi (39.4%), National Agricultural Research Center (38.7%), Hassan Abdal
(38.5%), Jhelum (38.0%), Kallar Syedan (35.8%), Barani Agricultural Research
Institute (35.5%), Attock (34.5%), Jand (33.9%), Sohawa (32.9%), Kotli Sattian
(31.6%), Gujar Khan and Lawa (31.2%), Pindi Gheb (31.0%), Kahuta (30.6%), Choa
Saidan Shah (30.0%), Pind Dadan Khan and Dina (28.3%), Chakwal (28.3%) and
Talagang (26.3%) while minimum mean disease incidence was recorded in Kallar
Kahar (26.3%) as shown in Table 4.2.
District wise, maximum mean disease incidence was observed in Islamabad
(38.7%) followed by district Attock (36.3%), district Rawalpindi (34.9%) and district
Jhelum (31.8%) while minimum mean disease incidence was recorded in district
Chakwal (29.6%) as shown in Figure 4.1.
4.1.3 Surveillance for Disease Assessment on Chilli
Chilli is also a warm season crop and is mostly cultivated with tomato. Varying
climatic conditions in Pothohar region provide optimum temperature conditions for
chilli growth and development. Survey for disease assessment of R. solani infection
chilli was conducted in crop season 2014-15 and 2015-16. A total of 426 symptomatic
plant samples of chilli were collected from scattered locations in district Rawalpindi,
Jhelum, Attock, Chakwal, and Islamabad.
4.1.3.1 Rhizoctonia solani disease prevalence and incidence on chilli
Like tomato, chilli production is mostly at scattered locations in Pothohar
region. Survey of different locations of Pothohar region revealed that R. solani
infection was 100% prevalent in all the locations visited. In district Rawalpindi, the
68
maximum mean disease incidence was recorded in Taxila (30.9%) followed by
Kahuta (35.9%), Gujar Khan (29.9%), tehsil Rawalpindi (29.4%) and Kotli Sattian
(28.0%) while minimum mean disease incidence was recorded in Kallar Syedan
(27.5%). In district Jhelum, the maximum mean disease incidence was recorded in
tehsil Jhelum (32.6%) followed by Sohawa (29.4%) and Dina (28.3%) while
minimum mean disease incidence was observed in Pind Dadan Khan (27.8%). In
district Attock, the maximum mean disease incidence was recorded in Hassan
Abdal (33.7%) followed by Jand (33.5%), Fateh Jang (32.9%), tehsil Attock
(32.4%), and Pindi Gheb (27.8%) while minimum mean disease incidence was
recorded in Hazro (25.4%). In district Chakwal, the maximum mean disease
incidence was recorded at Chakwal and Kallar Kahar (30.8%) followed by tehsil
Talagang (26.5%), Choa Saidan Shah (25.4%), and Barani Agricultural Research
Institute (25.3%) while the minimum mean disease incidence was recorded at Lawa
(23.7%). The mean disease incidence in Islamabad (Federal Capital Territory) was
recorded as 29.5%.
Overall in the Pothohar region; maximum mean disease incidence was
recorded in Kahuta (35.0%) followed by Hassan Abdal (33.7%), Jand (33.5%), Fateh
Jang (32.9%), Jhelum (32.6%), Attock (32.4%), Taxila (30.9%), Chakwal and Kallar
Kahar (30.8%), Gujar Khan (29.9%), National Agricultural Research Center
(29.5%), Sohawa, and Rawalpindi (29.4%), Dina (28.3%), Kotli Sattian (28.0%),
Pind Dadan Khan and Pindi Gheb (27.8%), Kallar Syedan (27.5%), Talagang
(26.5%), Hazro and Choa Saidan Shah (25.4%), Barani Agricultural Research
Institute (25.3%) while minimum mean disease incidence was recorded in Lawa
(23.7%) as shown in Table 4.3.
69
Table 4.3: Disease prevalence and incidence percentage of Rhizoctonia solani on chilli in various areas/locations of the districts of Pothohar
region (crop season 2014-15 and 2015-16).
Districts Location (s)
Surveyed
Locations Surveyed Disease Prevalence Disease Incidence %
2014-15 2015-16 2014-15 2015-16 Mean 2014-15 2015-16 Mean
Rawalpindi Taxila 4 4 100 100 100 29.3 32.5 30.9 Rawalpindi 5 5 100 100 100 32.5 26.3 29.4 Gujar Khan 5 5 100 100 100 33.5 26.3 29.9
Kallar Syedan 4 4 100 100 100 26.4 29.3 27.5 Kahuta 4 4 100 100 100 34.4 35.6 35.0 Kotli Sattian 2 2 100 100 100 26.5 29.6 28.0 Murree - - - - - - - -
Jhelum Jhelum 8 8 100 100 100 32.1 33.2 32.6 Pind Dadan Khan 5 5 100 100 100 29.3 26.3 27.8 Sohawa 4 4 100 100 100 26.6 32.2 29.4 Dina 4 4 100 100 100 27.3 29.3 28.3
Attock Attock 6 6 100 100 100 32.5 32.3 32.4 Hazro 5 5 100 100 100 26.5 24.3 25.4 Fateh Jang 4 4 100 100 100 32.6 33.2 32.9 Hassan Abdal 4 4 100 100 100 33.6 33.9 33.7 Jand 4 4 100 100 100 34.5 32.5 33.5 Pindi Gheb 4 4 100 100 100 26.3 29.3 27.8
Chakwal Chakwal 6 6 100 100 100 32.3 29.3 30.8 Kallar Kahar 6 6 100 100 100 29.3 32.3 30.8 Choa Saidan Shah 4 4 100 100 100 26.4 24.5 25.4 Talagang 4 4 100 100 100 26.3 26.8 26.5 Lawa 4 4 100 100 100 21.2 26.3 23.7 BARI 2 2 100 100 100 26.4 24.3 25.3
Islamabad NARC 3 3 100 100 100 29.6 29.5 29.5
70
Figure 4.1: District wise, mean disease incidence of Rhizoctonia solani infection on potato, tomato, and chilli.
0
5
10
15
20
25
30
35
40
45
Rawalpindi Jhelum Attock Chakwal Islamabad
Potato Tomato Chilli
Per
centa
ge
dis
ease
in
ciden
ce
71
District wise, maximum mean disease incidence was observed in district
Attock (30.9%) followed by district Rawalpindi (30.1%), Islamabad and district
Jhelum (29.5%) while minimum mean disease incidence was recorded in district
Chakwal (27.0%) as shown in Figure 4.1
4.2 ISOLATION AND CULTURING OF RHIZOCTONIA SOLANI
Isolates were recovered on WA medium following by culturing on PDA
medium using hyphal tipping method and morphological features of all recovered
isolates were recorded using MEA medium. A total 63 isolates from potato, 67 from
tomato, and 58 isolates were recovered from chilli symptomatic plant samples
(Table 4.4).
4.3 MORPHOLOGICAL CHARACTERIZATION OF RHIZOCTONIA
SOLANI ISOLATES
Isolates of R. solani recovered from potato, tomato, and chilli symptomatic
plant samples were morphologically characterized according to the descriptions of
R. solani by Ogoshi (1975) and Sneh et al. (1991). Isolates recovered on water agar
(WA) medium started the hyphal growth from the second day of incubation. The
hyphal tips of the actively growing mycelium were cultured on Malt Extract Agar
(MEA) medium. The hyphal growth on MEA medium started on the second day
however, the growth was more vigorous than WA medium. All isolates exhibited
typical R. solani colony and cultural characteristics. Colony colour, mycelial growth
rate, inter septal distance, hyphal diameter, nuclear number, colour, texture, and
topography of sclerotia were considered (Figure 4.2 to 4.6). Cultural and
morphological characteristics revealed considerable variations among different R.
solani isolates.
72
Table 4.4: Details of Rhizoctonia solani isolates recovered from potato, tomato, and chilli symptomatic plant samples.
Location Potato Tomato Chilli R
awal
pin
di RWPP1, RWPP2, RWPP3, RWPP4, RWPP5,
RWPP6, RWPP7, RWPP8, RWPP9, RWPP10,
RWPP11, RWPP12, RWPP13, RWPP14,
RWPP15, RWPP16, RWPP17, RWPP18,
RWPP19
RWPT1, RWPT2, RWPT3, RWPT4, RWPT5,
RWPT6, RWPT7, RWPT8, RWPT9, RWPT10,
RWPT11, RWPT12, RWPT13, RWPT14,
RWPT15
RWPC1, RWPC2, RWPC3, RWPC4, RWPC5,
RWPC6, RWPC7, RWPC8, RWPC9, RWPC10,
RWPC11, RWPC12, RWPC13, RWPC14
Isla
mab
ad
ISBP1, ISBP2, ISBP3, ISBP4, ISBP5, ISBP6,
ISBP7 ISBT1, ISBT2, ISBT3, ISBT4, ISBT5 ISBC1, ISBC2, ISBC3, ISBC4
Jhel
um
JHEP1, JHEP2, JHEP3, JHEP4, JHEP5, JHEP6,
JHEP7, JHEP8, JHEP9, JHEP10, JHEP11,
JHEP12, JHEP13
JHET1, JHET2, JHET3, JHET4, JHET5,
JHET6, JHET7, JHET8, JHET9, JHET10,
JHET11, JHET12, JHET13, JHET14, JHET15,
JHET16, JHET17, JHET18, JHET19
JHEC1, JHEC2, JHEC3, JHEC4, JHEC5,
JHEC6, JHEC7, JHEC8, JHEC9, JHEC10,
JHEC11, JHEC12, JHEC13
Att
ock
ATKP1, ATKP2, ATKP3, ATKP4, ATKP5,
ATKP6, ATKP7, ATKP8, ATKP9, ATKP10,
ATKP11, ATKP12, ATKP13, ATKP14,
ATKP15, ATKP16, ATKP17, ATKP18,
ATKP19, ATKP20, ATKP21
ATKT1, ATKT2, ATKT3, ATKT4, ATKT5,
ATKT6, ATKT7, ATKT8, ATKT9, ATKT10,
ATKT11, ATKT12, ATKT13, ATKT14,
ATKT15, ATKT16, ATKT17
ATKC1, ATKC2, ATKC3, ATKC4, ATKC5,
ATKC6, ATKC7, ATKC8, ATKC9, ATKC10,
ATKC11, ATKC12, ATKC13, ATKC14,
ATKC15, ATKC16
Ch
akw
al
CHKP1, CHKP2, CHKP3
CHKT1, CHKT2, CHKT3, CHKT4, CHKT5,
CHKT6, CHKT7, CHKT8, CHKT9, CHKT10,
CHKT11
CHKC1, CHKC2, CHKC3, CHKC4, CHKC5,
CHKC6, CHKC7, CHKC8, CHKC9, CHKC10,
CHKC11
Total 63 67 58
73
Figure 4.2: Morphological diversity of Rhizoctonia solani isolates incubated on malt
extract agar (MEA) medium.
Figure 4.3: Cultural appearance of Rhizoctonia solani isolate (RWPT5) under the
microscope.
74
Figure 4.4: Inter septal distance of Rhizoctonia solani isolate (JHEP2) recovered
from potato.
Figure 4.5: Hyphal diameter of Rhizoctonia solani isolate (ATKP7) recovered from
potato.
75
Figure 4.6: Nuclear number testing of Rhizoctonia solani isolate (RWPP9) recovered
from potato.
76
4.3.1 Morphological Characterization of Rhizoctonia solani Isolates from
Potato
Sixty-three isolates of R. solani were recovered from diseased potato samples
collected from different locations of Pothohar region. All isolates had typical of R.
solani colony and hyphal characteristics as shown in Table 4.5. Fungal colonies
incubated on MEA medium were light grey or medium to dark brown with plentiful
mycelial growth and branched hyphae. A septum was always present in the branch of
hyphae near the originating point with a slight constriction at the branch that is of
immense taxonomic importance. No conidia or conidiophores were observed. The
hyphal distance between two septa varied from 67.2 to 149.2μm (average 109.4 μm).
On the basis of inter septal distance, the isolates were categorized into three groups
viz; Small (<90µm), Medium (91-120µm) and Long (120>µm). Out of sixty-three
isolates, 19% isolates had short length (ranged from 67.2-73.5µm), 25% medium
(ranged from 92.4-119.6µm) while the majority of the isolates (56%) exhibited long
(ranged from 121.5-149.2µm) length between two septations (Figure 4.7). The hyphal
diameter ranged between 5.3 to 7.9μm (average 6.3μm). On the basis of hyphal
diameter, the isolates were categorized into three groups viz; Narrow (<5.5µm),
Moderate (5.6 - 7.0µm) and Wide (>7.0µm). Only 11% of the isolates exhibited
narrow (ranged from 5.3-5.5µm), 21% moderate (ranged from 5.5-6.9µm) while 68%
of the isolates had wide (ranged from 7.1-7.9µm) hyphal diameter (Figure 4.8).
Number of nuclei per cell of R. solani were counted by staining hyphae of each isolate
with 1ug/ml of DAPI (4’-6 diamidino-2-phenylindole) stain. All the isolates were
multinucleate. After seven days of incubation, most of the isolates produced sclerotia.
The sclerotia developed from the middle to the edges of the colonies and were light
77
to dark brown in the start and later turned dark brown to black in colour.
Out of sixty-three isolates, 16% of the isolates didn’t produce any sclerotia,
19% produced less than 30 sclerotia per cm2 while 65% of the isolates produced high
(>30) number of sclerotia per cm2 as shown in Figure 4.9. The sclerotia were either
rough or smooth.
Most of the isolates produced rough sclerotia. 63% of the isolates produced
rough while 21% of the isolates produced smooth sclerotia as shown in Figure
4.10. The sclerotia were either immersed or superficially available on the hyphae.
In 27% of the isolates, the sclerotia were immersed in the hyphal mass while in
most of the isolates (57%) sclerotia were superficially available on the hyphae
(Figure 4.11). Formation of the dark brown to black exudates was also observed
in some of the isolates.
4.3.2 Morphological Characterization of Rhizoctonia solani Isolates from
Tomato
Sixty-seven isolates of R. solani were recovered from diseased stem portions
of tomato showing characteristic symptoms of foot root. With considerable variations,
all isolates had typical of R. solani colony and hyphal characteristics as shown in
Table 4.6. R. solani colonies incubated on MEA medium were light grey or medium
to dark brown with plenteous mycelial growths. Fungal hyphae were branched at right
angles and a septum was always present in the branch of hyphae near the originating
point with a slight constriction at the branch. No conidia or conidiophores were
observed. The hyphal distance between two septa varied from 67.6 to 149.8μm
(average 109.5μm). Based on the septal distance, the isolates were categorized into
three groups viz; Small (<90µm), Medium (91-120µm) and Long (120>µm).
78
Table 4.5: Morphological characterization of sixty-three isolates of Rhizoctonia solani recovered from portions of diseased potato samples
collected from Pothohar region during 2014-15 and 2015-16 crop season.
Isolate Plant Portion Colony Colour Constriction
Colony
Diameter
cm
Hyphal
Length
µm
Hyphal
Width
µm
Nuclear
Condition
Sclerotia
Number Sclerotia Colour
Sclerotia
Texture
Sclerotia
Topography
ATKP1 Tuber Hyaline to light brown Present 7.3 93.4 6.9 Multinucleate 43 Light brown to dark brown Rough Immersed
ATKP2 Tuber Hyaline to light brown Present 7.8 149.2 7.3 Multinucleate 29 Medium to dark brown Smooth Superficial
ATKP3 Stolon Hyaline to light brown Present 7.6 139.3 6.9 Multinucleate 40 Light brown to dark brown Rough Superficial
ATKP4 Stem Medium brown Present 7.5 110.6 5.4 Multinucleate 27 Light brown to dark brown Rough Superficial
ATKP5 Tuber Medium brown Present 8.3 121.5 5.7 Multinucleate 40 Light brown to dark brown Rough Superficial
ATKP6 Stolon Dark brown Present 7.7 148.2 7.9 Multinucleate 51 Light brown to dark brown Rough Immersed
ATKP7 Tuber Medium brown Present 7.0 72.6 5.3 Multinucleate 39 Medium to dark brown Rough Superficial
ATKP8 Root Dark brown Present 8.3 117.6 5.8 Multinucleate 27 Dark brown Rough Superficial
ATKP9 Stem Dark brown Present 7.7 136.5 6.8 Multinucleate 29 White to light brown Rough Superficial
ATKP10 Tuber Dark brown Present 7.8 118.2 6.9 Multinucleate 38 Medium to dark brown Rough Superficial
ATKP11 Tuber Dark brown Present 8.2 146.3 7.2 Multinucleate 33 Medium to dark brown Rough Superficial
ATKP12 Tuber Hyaline to light brown Present 7.2 105.3 7.1 Multinucleate 46 Light brown to dark brown Rough Superficial
ATKP13 Root Medium brown Present 7.6 102.5 6.3 Multinucleate 0 - Not present Not present
ATKP14 Tuber Hyaline to light brown Present 7.3 96.7 6.9 Multinucleate 43 Light brown to dark brown Rough Immersed
ATKP15 Tuber Medium brown Present 7.5 110.6 5.5 Multinucleate 27 Light brown to dark brown Rough Superficial
ATKP16 Tuber Dark brown Present 7.7 148.2 7.9 Multinucleate 51 Light brown to dark brown Rough Immersed
ATKP17 Tuber Dark brown Present 7.3 141.3 6.9 Multinucleate 27 White to light brown Rough Immersed
ATKP18 Stem Hyaline to light brown Present 7.8 149.2 7.3 Multinucleate 32 Medium to dark brown Smooth Superficial
ATKP19 Tuber Dark brown Present 7.7 136.5 6.8 Multinucleate 32 White to light brown Rough Superficial
ATKP20 Tuber Dark brown Present 8.2 146.3 7.2 Multinucleate 33 Medium to dark brown Rough Superficial
79
ATKP21 Tuber Hyaline to light brown Present 7.2 105.3 7.1 Multinucleate 46 Light brown to dark brown Rough Superficial
CHKP1 Tuber Dark brown Present 7.7 72.6 6.1 Multinucleate 46 Light brown to dark brown Rough Superficial
CHKP2 Root Dark brown Present 7.3 141.3 6.9 Multinucleate 27 White to light brown Rough Immersed
CHKP3 Tuber Dark brown Present 8.0 73.5 5.9 Multinucleate 43 Light brown to dark brown Smooth Superficial
ISBP1 Tuber Hyaline to light brown Present 8.3 100.3 6.3 Multinucleate 52 Medium to dark brown Rough Immersed
ISBP2 Stolon canker Hyaline to light brown Present 7.7 96.9 6.2 Multinucleate 0 - Not present Not present
ISBP3 Tuber Medium brown Present 7.8 92.4 6.2 Multinucleate 33 Medium to dark brown Rough Superficial
ISBP4 Root lesion Hyaline to light brown Present 8.2 107.5 6.2 Multinucleate 52 Light brown to dark brown Rough Superficial
ISBP5 Tuber Hyaline to light brown Present 7.7 112.0 6.3 Multinucleate 46 Medium to dark brown Smooth Immersed
ISBP6 Stem Hyaline to light brown Present 7.3 117.5 6.9 Multinucleate 33 Medium to dark brown Smooth Superficial
ISBP7 Tuber Dark brown Present 7.8 101.5 6.8 Multinucleate 0 - Not present Not present
JHEP1 Root lesion Medium brown Present 8.3 136.6 7.1 Multinucleate 40 Brown Rough Immersed
JHEP2 Tuber Hyaline to light brown Present 7.7 136.3 7.5 Multinucleate 51 Brown Rough Immersed
JHEP3 Stem canker Hyaline to light brown Present 7.8 134.5 7.2 Multinucleate 42 Brown Rough Immersed
JHEP4 Root lesion Dark brown Present 8.2 117.4 5.9 Multinucleate 0 - Not present Not present
JHEP5 Tuber Medium brown Present 7.7 114.5 5.6 Multinucleate 51 Dark brown Smooth Immersed
JHEP6 Stolon canker Dark brown Present 7.3 113.2 5.9 Multinucleate 32 Dark brown Smooth Superficial
JHEP7 Tuber Dark brown Present 8.0 97.8 6.6 Multinucleate 43 Medium to dark brown Rough Superficial
JHEP8 Tuber Hyaline to light brown Present 7.1 136.2 7.2 Multinucleate 27 Medium to dark brown Smooth Superficial
JHEP9 Stolon canker Dark brown Present 7.5 119.6 6.5 Multinucleate 0 - Not present Not present
JHEP10 Tuber Dark brown Present 7.1 106.5 6.1 Multinucleate 22 Medium to dark brown Smooth Superficial
JHEP11 Root lesion Dark brown Present 8.2 117.3 5.6 Multinucleate 0 - Not present Not present
JHEP12 Tuber Dark brown Present 7.1 113.5 5.6 Multinucleate 33 Medium to dark brown Rough Superficial
JHEP13 Tuber Dark brown Present 7.7 72.6 6.1 Multinucleate 46 Light brown to dark brown Rough Superficial
RWPP1 Tuber Hyaline to light brown Present 7.6 69.3 5.9 Multinucleate 39 Medium to dark brown Rough Superficial
80
RWPP2 Tuber Medium brown Present 7.9 102.3 6.3 Multinucleate 39 Medium to dark brown Smooth Immersed
RWPP3 Stem canker Hyaline to light brown Present 7.3 67.2 5.3 Multinucleate 42 Dark brown Rough Superficial
RWPP4 Root lesion Medium brown Present 8.0 107.5 6.1 Multinucleate 28 Medium to dark brown Rough Immersed
RWPP5 Tuber Hyaline to light brown Present 7.6 69.6 5.3 Multinucleate 0 - Not present Not present
RWPP6 Tuber Dark brown Present 7.1 106.5 6.1 Multinucleate 22 Medium to dark brown Smooth Superficial
RWPP7 Stolon canker Dark brown Present 8.3 99.5 5.9 Multinucleate 39 Medium to dark brown Rough Immersed
RWPP8 Root lesion Hyaline to light brown Present 7.7 72.6 5.7 Multinucleate 46 Dark brown Rough Superficial
RWPP9 Tuber Hyaline to light brown Present 7.8 67.9 5.3 Multinucleate 48 Dark brown Smooth Immersed
RWPP10 Tuber Dark brown Present 8.2 117.3 5.6 Multinucleate 46 Medium to dark brown Rough Superficial
RWPP11 Stem canker Hyaline to light brown Present 7.2 114.3 6.2 Multinucleate 52 Medium to dark brown Rough Superficial
RWPP12 Root lesion Medium brown Present 7.3 71.0 5.9 Multinucleate 0 - Not present Not present
RWPP13 Tuber Medium brown Present 7.8 71.6 5.8 Multinucleate 0 - Not present Not present
RWPP14 Stem canker Dark brown Present 7.6 69.3 5.9 Multinucleate 0 - Not present Not present
RWPP15 Tuber Dark brown Present 7.1 113.5 5.6 Multinucleate 33 Medium to dark brown Rough Superficial
RWPP16 Stolon canker Dark brown Present 8.3 114.5 5.3 Multinucleate 32 Medium to dark brown Rough Superficial
RWPP17 Tuber Medium brown Present 7.7 117.5 6.9 Multinucleate 38 White to light brown Rough Immersed
RWPP18 Stem canker Hyaline to light brown Present 8.3 99.3 6.1 Multinucleate 43 Dark brown Smooth Superficial
RWPP19 Tuber Medium brown Present 7.7 115.3 7.2 Multinucleate 26 Brown Rough Superficial
81
Small (60-90µm) RWPP3, RWPP9, RWPP1, RWPP14,
RWPP5, RWPP12, RWPP13, ATKP7,
CHKP1, JHEP13, RWPP8, CHKP3
Medium (91-120µm) ISBP3, ATKP1, ATKP14, ISBP2, JHEP7,
RWPP18, RWPP7, ISBP1, ISBP7, RWPP2,
ATKP13, ATKP12, ATKP21, JHEP10,
RWPP6, ISBP4, RWPP4, ATKP4, ATKP15,
ISBP5, JHEP6, JHEP12, RWPP15,
RWPP11, JHEP5, RWPP16, RWPP19,
JHEP11, RWPP10, JHEP4, ISBP6,
RWPP17, ATKP8, ATKP10, JHEP9
Long (120>µm) ATKP5, JHEP3, JHEP8, JHEP2, ATKP9,
ATKP19, JHEP1, ATKP3, ATKP17,
CHKP2, ATKP11, ATKP20, ATKP6,
ATKP16, ATKP2, ATKP18
Maximum = 149.2µm
Minimum = 67.2µm
Figure 4.7: Distribution of Rhizoctonia solani isolates recovered from potato on the basis of hyphal length/ inter septal distance.
19%
56%
25%
Isolates distribution on the basis of hyphal length
Small (60-90µm) Medium (91-120µm)
Long (120>µm)
82
Narrow (<5.5µm)
ATKP7, RWPP3, RWPP5, RWPP9,
RWPP16, ATKP4, ATKP15
Moderate (5.6 -
7.0µm)
JHEP5, JHEP11, JHEP12, RWPP10,
RWPP15, ATKP5, RWPP8, ATKP8,
RWPP13, CHKP3, JHEP4, JHEP6, RWPP1,
RWPP7, RWPP12, RWPP14, CHKP1,
JHEP10, JHEP13, RWPP4, RWPP6,
RWPP18, ISBP2, ISBP3, ISBP4, RWPP11,
ATKP13, ISBP1, ISBP5, RWPP2, JHEP9,
JHEP7, ATKP9, ATKP19, ISBP7, ATKP1,
ATKP3, ATKP10, ATKP14, ATKP17,
CHKP2, ISBP6, RWPP17
Wide (>7.0µm) ATKP12, ATKP21, JHEP1, ATKP11,
ATKP20, JHEP3, JHEP8, RWPP19, ATKP2,
ATKP18, JHEP2, ATKP6, ATKP16
Maximum = 7.9µm
Minimum = 5.3µm
Figure 4.8: Distribution of Rhizoctonia solani isolates recovered from potato on the basis of hyphal diameter.
11%
68%
21%
Isolates distribution on the basis of hyphal
diameter
Narrow (<5.5µm) Moderate (5.6 - 7.0µm)
Wide (>7.0µm)
83
Sclerotia not present ATKP7, ISBP7, JHEP9, JHEP11, RWPP1,
RWPP3, RWPP10, RWPP12, RWPP13,
RWPP14
Moderate (1-30)
JHEP10, RWPP6, RWPP19, ATKP4,
ATKP8, ATKP15, ATKP17, CHKP2,
JHEP8, RWPP4, ATKP2, ATKP9
High (>30) ATKP18, ATKP19, JHEP6, RWPP16,
ATKP11, ATKP20, ISBP3, ISBP6, JHEP12,
RWPP15, ATKP10, JHEP4, RWPP17,
ATKP13, RWPP2, RWPP7, ATKP3,
ATKP5, JHEP1, JHEP3, RWPP5, ATKP1,
ATKP14, CHKP3, JHEP7, RWPP18,
ATKP12, ATKP21, CHKP1, ISBP2, ISBP5,
JHEP13, RWPP8, RWPP9, ATKP6,
ATKP16, JHEP2, JHEP5, ISBP1, ISBP4,
RWPP11
Figure 4.9: Distribution of Rhizoctonia solani isolates recovered from potato on the basis of number of sclerotia.
16%
19%
65%
Isolates distribution on the basis of number of
sclerotia
No Sclerotia Moderate (1-30) High (>30)
84
Sclerotia not present
ATKP7, ISBP7, JHEP9, JHEP11, RWPP1,
RWPP3, RWPP10, RWPP12, RWPP13,
RWPP14
Rough
ATKP1, ATKP3, ATKP4, ATKP5, ATKP6,
ATKP8, ATKP9, ATKP10, ATKP11,
ATKP12, ATKP13, ATKP14, ATKP15,
ATKP16, ATKP17, ATKP19, ATKP20,
ATKP21, CHKP1, CHKP2, ISBP1, ISBP2,
ISBP3, ISBP4, JHEP1, JHEP2, JHEP3,
JHEP4, JHEP7, JHEP12, JHEP13, RWPP4,
RWPP5, RWPP7, RWPP8, RWPP11,
RWPP15, RWPP16, RWPP17, RWPP19
Smooth ATKP2, ATKP18, CHKP3, ISBP5, ISBP6,
JHEP5, JHEP6, JHEP8, JHEP10, RWPP2,
RWPP6, RWPP9, RWPP18
Figure 4.10: Distribution of Rhizoctonia solani isolates recovered from potato on the basis of sclerotia texture.
16%
63%
21%
Isolates distribution on the basis of sclerotia
texture
Sclerotia not present Rough Smooth
85
Sclerotia not present RWPP13, ATKP7, ISBP7, JHEP9, JHEP11,
RWPP1, RWPP3, RWPP10, RWPP12,
RWPP14
Immersed
ATKP1, ATKP6, ATKP14, ATKP16,
ATKP17, CHKP2, ISBP1, ISBP5, JHEP1,
JHEP2, JHEP3, JHEP5, RWPP2, RWPP4,
RWPP7, RWPP9, RWPP17
Superficial
ATKP2, ATKP3, ATKP4, ATKP5, ATKP8,
ATKP9, ATKP10, ATKP11, ATKP12,
ATKP13, ATKP15, ATKP18, ATKP19,
ATKP20, ATKP21, CHKP1, CHKP3,
ISBP2, ISBP3, ISBP4, ISBP6, JHEP4,
JHEP6, JHEP7, JHEP8, JHEP10, JHEP12,
JHEP13, RWPP5, RWPP6, RWPP8,
RWPP11, RWPP15, RWPP16, RWPP18,
RWPP19
Figure 4.11: Distribution of Rhizoctonia solani isolates recovered from potato on the basis of sclerotia topography.
27%
57%
16%
Isolates distribution on the basis of sclerotia
topography
Immersed Superficial Sclerotia not present
86
Table 4.6: Morphological characterization of sixty-seven isolates of Rhizoctonia solani recovered from portions of diseased tomato samples
collected from Pothohar region during 2014-15 and 2015-16 crop season.
Isolate Colony Colour Constriction
Colony
Diameter
cm
Hyphal
Length
µm
Hyphal
Width
µm
Nuclear
Condition
Sclerotia
Number Sclerotia Colour
Sclerotia
Texture
Sclerotia
Topography
RWPT1 Medium brown Present 7.6 69.1 6.1 Multinucleate 51 Medium to dark brown Rough Immersed
RWPT2 Dark brown Present 7.9 145.7 6.7 Multinucleate 38 Dark brown Rough Superficial
RWPT3 Dark brown Present 7.3 96.4 5.9 Multinucleate 31 Dark brown Rough Immersed
RWPT4 Dark brown Present 8.0 99.4 6.7 Multinucleate 42 Medium to dark brown Rough Immersed
RWPT5 Medium brown Present 7.6 101.8 6.4 Multinucleate 37 Medium to dark brown Smooth Superficial
RWPT6 Medium brown Present 7.1 108.0 6.1 Multinucleate 27 Medium to dark brown Rough Superficial
RWPT7 Hyaline to light brown Present 8.3 67.6 5.3 Multinucleate 47 Dark brown Smooth Immersed
RWPT8 Dark brown Present 7.7 122.6 7.2 Multinucleate 38 Light brown to dark brown Rough Superficial
RWPT9 Dark brown Present 8.3 146.9 7.2 Multinucleate 32 Medium to dark brown Rough Superficial
RWPT10 Hyaline to light brown Present 8.2 149.2 7.2 Multinucleate 31 Medium to dark brown Smooth Superficial
RWPT11 Medium brown Present 7.2 96.2 6.4 Multinucleate 45 Medium to dark brown Rough Superficial
RWPT12 Hyaline to light brown Present 7.1 148.7 7.5 Multinucleate 31 Medium to dark brown Smooth Superficial
RWPT13 Hyaline to light brown Present 8.1 70.9 5.9 Multinucleate 26 Medium to dark brown Smooth Superficial
RWPT14 Dark brown Present 7.7 99.9 6.8 Multinucleate 42 Medium to dark brown Rough Superficial
RWPT15 Hyaline to light brown Present 7.8 107.2 5.9 Multinucleate 51 Light brown to dark brown Rough Superficial
CHKT1 Hyaline to light brown Present 8.2 71.4 5.3 Multinucleate 42 Dark brown Rough Immersed
CHKT2 Dark brown Present 7.3 149.8 7.8 Multinucleate 39 Dark brown Smooth Superficial
CHKT3 Dark brown Present 7.6 119.1 6.8 Multinucleate 39 White to light brown Rough Superficial
CHKT4 Hyaline to light brown Present 7.6 114.9 6.4 Multinucleate 0 - Not present Not present
CHKT5 Medium brown Present 6.9 97.0 6.7 Multinucleate 45 Medium to dark brown Rough Superficial
87
CHKT6 Dark brown Present 8.3 117.7 6.9 Multinucleate 37 Medium to dark brown Rough Superficial
CHKT7 Hyaline to light brown Present 7.7 99.8 6.2 Multinucleate 40 Dark brown Smooth Superficial
CHKT8 Hyaline to light brown Present 7.8 105.9 7.7 Multinucleate 45 Light brown to dark brown Rough Immersed
CHKT9 Hyaline to light brown Present 8.2 117.2 6.6 Multinucleate 32 Medium to dark brown Smooth Superficial
CHKT10 Dark brown Present 7.2 111.1 7.1 Multinucleate 29 Light brown to dark brown Rough Superficial
CHKT11 Hyaline to light brown Present 7.1 74.5 5.4 Multinucleate 0 - Not present Not present
ATKT1 Dark brown Present 7.8 71.9 5.8 Multinucleate 0 - Not present Not present
ATKT2 Dark brown Present 7.8 146.3 7.2 Multinucleate 32 Medium to dark brown Rough Superficial
ATKT3 Medium brown Present 8.3 103.0 5.2 Multinucleate 39 Medium to dark brown Rough Superficial
ATKT4 Dark brown Present 7.7 118.2 6.9 Multinucleate 37 Medium to dark brown Rough Immersed
ATKT5 Hyaline to light brown Present 7.8 136.8 7.3 Multinucleate 50 Brown Rough Superficial
ATKT6 Dark brown Present 7.2 148.5 8.1 Multinucleate 0 - Not present Not present
ATKT7 Medium brown Present 7.3 89.6 6.2 Multinucleate 44 Light brown to dark brown Rough Superficial
ATKT8 Medium brown Present 7.8 122.1 5.4 Multinucleate 39 Light brown to dark brown Rough Superficial
ATKT9 Medium brown Present 6.9 121.2 5.9 Multinucleate 39 Light brown to dark brown Rough Superficial
ATKT10 Medium brown Present 8.3 99.7 5.4 Multinucleate 48 Dark brown Rough Immersed
ATKT11 Dark brown Present 7.8 106.4 6 Multinucleate 24 Medium to dark brown Smooth Immersed
ATKT12 Hyaline to light brown Present 6.8 78.9 5.5 Multinucleate 45 Dark brown Rough Superficial
ATKT13 Dark brown Present 7.7 113.1 6.5 Multinucleate 0 - Not present Not present
ATKT14 Dark brown Present 7.7 99.9 6.8 Multinucleate 42 Medium to dark brown Rough Superficial
ATKT15 Medium brown Present 7.8 114.3 6.8 Multinucleate 25 Brown Rough Immersed
ATKT16 Dark brown Present 7.7 101.2 6.5 Multinucleate 50 Light brown to dark brown Rough Superficial
ATKT17 Hyaline to light brown Present 8.2 117.2 6.6 Multinucleate 32 Medium to dark brown Smooth Superficial
JHET1 Dark brown Present 7.7 136.2 6.5 Multinucleate 31 White to light brown Rough Superficial
JHET2 Dark brown Present 7.8 99.3 6.2 Multinucleate 26 Medium to dark brown Rough Superficial
88
JHET3 Hyaline to light brown Present 7.7 129.4 6.2 Multinucleate 42 Medium to dark brown Smooth Immersed
JHET4 Dark brown Present 7.8 100.9 5.8 Multinucleate 27 Dark brown Smooth Immersed
JHET5 Dark brown Present 8.2 94.5 6.7 Multinucleate 32 Dark brown Rough Superficial
JHET6 Dark brown Present 7.2 89.5 5.5 Multinucleate 45 Light brown to dark brown Smooth Superficial
JHET7 Medium brown Present 7.3 137.2 7.2 Multinucleate 39 Brown Rough Superficial
JHET8 Dark brown Present 7.8 114.0 5.1 Multinucleate 31 Medium to dark brown Rough Superficial
JHET9 Dark brown Present 6.8 76.7 5.7 Multinucleate 0 - Not present Not present
JHET10 Medium brown Present 7.7 115.3 7.3 Multinucleate 26 Brown Rough Immersed
JHET11 Hyaline to light brown Present 7.8 135.9 6.9 Multinucleate 26 Medium to dark brown Smooth Superficial
JHET12 Dark brown Present 8.2 96.0 6.1 Multinucleate 22 - Rough Immersed
JHET13 Medium brown Present 8.3 102 6.3 Multinucleate 38 Medium to dark brown Rough Immersed
JHET14 Hyaline to light brown Present 6.9 135.9 7.3 Multinucleate 0 - Not present Not present
JHET15 Dark brown Present 8.3 117.8 5.3 Multinucleate 23 - Rough Superficial
JHET16 Medium brown Present 7.7 117 7.1 Multinucleate 37 White to light brown Rough Immersed
JHET17 Hyaline to light brown Present 7.8 110.3 5.4 Multinucleate 26 Light brown to dark brown Rough Superficial
JHET18 Hyaline to light brown Present 7.1 111.9 6.1 Multinucleate 45 Medium to dark brown Smooth Immersed
JHET19 Dark brown Present 7.8 73.2 5.8 Multinucleate 23 - Rough Superficial
ISBT1 Hyaline to light brown Present 6.8 86.7 5.8 Multinucleate 45 Dark brown Rough Superficial
ISBT2 Medium brown Present 7.7 103.3 6.3 Multinucleate 39 Medium to dark brown Rough Superficial
ISBT3 Medium brown Present 8.2 99.3 6.8 Multinucleate 50 Medium to dark brown Rough Superficial
ISBT4 Hyaline to light brown Present 7.2 113.2 5.9 Multinucleate 31 Dark brown Smooth Superficial
ISBT5 Medium brown Present 7.3 145.2 7.5 Multinucleate 42 Light brown to dark brown Rough Superficial
89
Out of sixty-seven isolates, 18% isolates were found short length (ranged from
67.6-89.5μm), 55% medium (ranged from 94.5-118.2μm) while 27% isolates had long
(ranged from 121.2-149.8µm) length between two septations (Figure 4.12). Hyphal
diameter of the isolates ranged between 5.1 to 8.1μm (average 6.40μm). On the basis of
hyphal diameter, the isolates were categorized into three groups viz; Narrow (<5.5µm),
Moderate (5.6 - 7.0µm) and Wide (>7.0µm). Out of sixty-seven isolates, 14% of the
isolates exhibited narrow (ranged from 5.1 to 5.4μm), 64% moderate (ranged from 5.7-
6.9μm) while 22% of the isolates had a wide hyphal diameter (ranged from 7.1-8.1μm)
(Figure 4.13). DAPI (4’-6 diamidino-2-phenylindole) stain was used to count number
of nuclei per cell of R. solani. Microscopic studies under fluorescent light microscope
revealed all isolates were multinucleate. Seven days after incubation on MEA medium
most of the isolates produced sclerotia however, some isolates failed to produce
sclerotia. The sclerotia developed from the middle to the edges of the colonies and were
light to dark brown in the start and later turned dark brown to black in colour. Out of
sixty-seven isolates, 11% of the isolates didn’t produce any sclerotia, 19% produced
less than 30 sclerotia per cm2 (ranged from 22-29 sclerotia/cm2) while 70% of the
isolates produced high (>30) number of sclerotia per cm2 (ranged from 31-51
sclerotia/cm2) as shown in Figure 4.14. The sclerotia were either rough or smooth. Most
of the isolates produced rough sclerotia. 66% of the isolates produced rough while 24%
of the isolates produced smooth sclerotia as shown in Figure 4.15. Most of the sclerotia
were superficially available on the hyphal mass. Among all isolates, 64% of the isolates
produced superficial sclerotia while in most 25% isolates, sclerotia were immersed in
the mass of hyphae (Figure 4.16). Formation of the dark brown to black exudates was
also observed in some of the isolates recovered from diseased tomato plant portions.
90
Small (60-90µm) RWPT7, RWPT1, RWPT13, CHKT1,
ATKT1, JHET19, CHKT11, JHET9,
ATKT12, ISBT1, ATKT7, JHET6
Medium (91-120µm) JHET5, JHET12, RWPT11, RWPT3,
CHKT5, JHET2, ISBT3, RWPT4, ATKT10,
CHKT7, RWPT14, ATKT14, JHET4,
ATKT16, RWPT5, JHET13, ATKT3,
ISBT2, CHKT8, ATKT11, RWPT15,
RWPT6, JHET17, CHKT10, JHET18,
ATKT13, ISBT4, JHET8, ATKT15, CHKT4,
JHET10, JHET16, CHKT9, ATKT17,
CHKT6, JHET15, ATKT4
Long (120>µm) CHKT3, ATKT9, ATKT8, RWPT8, JHET3,
JHET11, JHET14, JHET1, ATKT5, JHET7,
ISBT5, RWPT2, ATKT2, RWPT9, ATKT6,
RWPT12, RWPT10, CHKT2
Maximum = 149.2µm
Minimum = 67.2µm
Figure 4.12: Distribution of Rhizoctonia solani isolates recovered from tomato on the basis of hyphal length/ inter septal distance.
18%
55%
27%
Isolates distribution on the basis of hyphal length
Small (60-90µm) Medium (91-120µm)
Long (120>µm)
91
Narrow (<5.5µm)
JHET8, ATKT3, RWPT7, CHKT1, JHET15,
CHKT11, ATKT8, ATKT10, JHET17
Moderate (5.6 -
7.0µm)
JHET6, ATKT12, JHET9, ISBT1, ATKT1,
JHET4, JHET19, RWPT3, RWPT13,
RWPT15, ATKT9, ISBT4, ATKT11,
RWPT6, JHET12, JHET18, RWPT1,
CHKT7, ATKT7, JHET2, JHET3, JHET13,
ISBT2, RWPT5, RWPT11, CHKT4,
ATKT13, ATKT16, JHET1, CHKT9,
ATKT17, RWPT4, RWPT2, CHKT5,
JHET5, RWPT14, CHKT3, ATKT14,
ISBT3, ATKT15, CHKT6, ATKT4, JHET11
Wide (>7.0µm) CHKT10, JHET16, RWPT8, JHET7,
RWPT9, RWPT10, ATKT2, ATKT5,
JHET10, JHET14, RWPT12, ISBT5,
CHKT8, CHKT2, ATKT6
Maximum = 8.1µm
Minimum = 5.1µm
Figure 4.13: Distribution of Rhizoctonia solani isolates recovered from tomato on the basis of hyphal diameter.
14%
64%
22%
Isolates distribution on the basis of hyphal
diameter
Narrow (<5.5µm) Moderate (5.6 - 7.0µm)
Wide (>7.0µm)
92
Sclerotia not present
RWPT1, RWPT13, CHKT11, ATKT1,
ATKT13, ATKT16, JHET9
Moderate (1-30)
JHET12, JHET15, JHET19, ATKT11,
ATKT15, JHET2, JHET10, JHET4, JHET11,
JHET14, JHET17, RWPT6, CHKT10
High (>30) RWPT3, RWPT10, RWPT12, JHET1,
JHET8, ISBT4, RWPT9, CHKT9, ATKT2,
ATKT17, JHET5, RWPT5, CHKT6,
ATKT4, JHET16, RWPT2, RWPT8,
JHET13, CHKT2, CHKT3, ATKT3,
ATKT8, ATKT9, JHET7, ISBT2, CHKT7,
RWPT4, RWPT14, CHKT1, ATKT14,
JHET3, ISBT5, ATKT7, RWPT11, CHKT5,
CHKT8, ATKT12, JHET6, JHET18, ISBT1,
RWPT7, ATKT10, ATKT5, ATKT6, ISBT3,
RWPT15, CHKT4
Figure 4.14: Distribution of Rhizoctonia solani isolates recovered from tomato on the basis of number of sclerotia.
11%
19%
70%
Isolates distribution on the basis of number of
sclerotia
No Sclerotia Moderate (1-30) High (>30)
93
Sclerotia not present
RWPT1, RWPT13, CHKT11, ATKT1,
ATKT13, ATKT16, JHET9
Rough
RWPT2, RWPT3, RWPT4, RWPT6,
RWPT8, RWPT9, RWPT11, RWPT14,
RWPT15, CHKT1, CHKT3, CHKT4,
CHKT5, CHKT6, CHKT8, CHKT10,
ATKT2, ATKT3, ATKT4, ATKT5, ATKT6,
ATKT7, ATKT8, ATKT9, ATKT10,
ATKT12, ATKT14, ATKT15, JHET1,
JHET2, JHET5, JHET7, JHET8, JHET10,
JHET12, JHET13, JHET15, JHET16,
JHET17, JHET19, ISBT1, ISBT2, ISBT3,
ISBT5
Smooth RWPT5, RWPT7, RWPT10, RWPT12,
CHKT2, CHKT7, CHKT9, ATKT11,
ATKT17, JHET3, JHET4, JHET6, JHET11,
JHET14, JHET18, ISBT4
Figure 4.15: Distribution of Rhizoctonia solani isolates recovered from tomato on the basis of sclerotia texture.
10%
66%
24%
Isolates distribution on the basis of sclerotia
texture
Sclerotia not present Rough Smooth
94
Sclerotia not present RWPT1, RWPT13, CHKT11, ATKT1,
ATKT13, JHET9, ATKT16
Immersed
RWPT3, RWPT4, RWPT7, CHKT1,
CHKT4, CHKT8, ATKT10, ATKT11,
ATKT15, JHET4, JHET10, JHET12,
JHET18, ATKT4, JHET3, JHET13, JHET16
Superficial
RWPT2, RWPT5, RWPT6, RWPT8,
RWPT9, RWPT10, RWPT11, RWPT12,
RWPT14, RWPT15, CHKT2, CHKT3,
CHKT5, CHKT6, CHKT7, CHKT9,
CHKT10, ATKT2, ATKT3, ATKT5,
ATKT6, ATKT7, ATKT8, ATKT9,
ATKT12, ATKT14, ATKT17, JHET1,
JHET2, JHET5, JHET6, JHET7, JHET8,
JHET11, JHET14, JHET15, JHET17,
JHET19, ISBT1, ISBT2, ISBT3, ISBT4,
ISBT5
Figure 4.16. Distribution of Rhizoctonia solani isolates recovered from tomato on the basis of sclerotia topography.
25%
64%
11%
Isolates distribution on the basis of sclerotia
topography
Immersed Superficial Sclerotia not present
95
4.3.3 Morphological Characterization of Rhizoctonia solani Isolates from
Chilli
Fifty-eight isolates of R. solani were recovered from diseased chilli root
portions collected from different locations of Pothohar region. Morphological
characteristics of the recovered isolates showed considerable dissimilarities however,
exhibited typical R. solani colony and hyphal characteristics as shown in Table 4.7.
Fungal colonies incubated on MEA medium were light grey or medium to
dark brown in appearance and had plentiful mycelial growth. The young vegetative
hyphae were branched at right angles near the distal septum of the cells and a
constriction was observed at their junction or at a short distance from the septum. All
the isolates failed to produce conidia or conidiophores. The septal distance for each
isolate varied from 66.7 to 150.3μm (average 111.8μm). Based on the hyphal distance
between two septa, the isolates were categorized into three groups viz; Small
(<90µm), Medium (91-120µm) and Long (120>µm). Out of fifty-eight isolates, 16%
isolates were found short length (ranged from 66.7-73.8μm), 50% medium (ranged
from 94.7-119.8μm) while rest of the 34% isolates had long (ranged from 121.8-
150.3μm) length between two septations (Figure 4.17). The hyphal diameter ranged
between 5.3 to 8.2μm (average 6.4μm). The isolates were categorized into three
groups viz; Narrow (<5.5µm), Moderate (5.6 - 7.0µm) and Wide (>7.0µm) based on
their hyphal diameter. Among all, 14% of the isolates exhibited narrow (ranged from
5.3-5.4μm), 59% moderate (ranged from 5.8-7μm) while 27% of the isolates had
wide (ranged from 7.0-8.2μm) hyphal diameter (Figure 4.18). Number of nuclei per
cell of R. solani were counted by staining hyphae of each isolate with 1ug/ml of DAPI
(4’-6 diamidino-2-phenylindole) stain. Each cell contained more than three nuclei.
96
Seven days after incubation, most of the isolates produced sclerotia however,
few isolates failed to produce sclerotia. The sclerotia developed from the middle to
the edges of the colonies and were light to dark brown in the start and later turned
dark brown to black in colour. Out of fifty-eight isolates, 13% of the isolates didn’t
produce any sclerotia, 22% produced less than 30 sclerotia per cm2 while 65% of the
isolates produced high (>30) number of sclerotia per cm2 as shown in Figure 4.19.
The sclerotia were either rough or smooth. Most of the isolates produced
rough sclerotia. Among all, 59% of the isolates produced rough sclerotia while 27%
of the isolates produced smooth sclerotia (Figure 4.20). The sclerotia were either
superficially available or immersed on the hyphae. Mostly superficial sclerotia were
observed. In 31% of the isolates, the sclerotia were immersed on the hyphae while
55% of the isolates had superficial sclerotia (Figure 4.21). Formation of the dark
brown to black exudates was also observed in some of the isolates.
4.4 PATHOGENICITY DETERMINATION
4.4.1 Pathogenicity Determination on Potato
Pathogenicity of each isolate was confirmed under greenhouse conditions at 25
± 2oC. Isolates of R. solani were artificially inoculated on healthy potato tubers under
pot trials. A set of uninoculated plants were used as control. Observations were made
for stem and stolon canker four weeks after plantation and tuber infection
(development of sclerotia), four months after plantation. A significant variation in
aggressiveness toward stem, stolon, and tuber infection on potato was observed among
R. solani isolates, as reflected in disease index (DI) ranging from 4.0 to 67.0% (Figure
4.22). Isolates were classified into five categories based on the virulence i.e. avirulent,
slightly virulent, moderately virulent, virulent, highly virulent (Figure 4.23).
97
Table 4.7: Morphological characterization of fifty-eight isolates of Rhizoctonia solani recovered from portions of diseased chilli samples
collected from Pothohar region during 2014-15 and 2015-16 crop season.
Isolate Colony Colour Constriction
Colony
Diameter
cm
Hyphal
Length
µm
Hyphal
Width
µm
Nuclear
Condition
Sclerotia
Number Sclerotia Colour
Sclerotia
Texture
Sclerotia
Topography
RWPC1 Medium brown Present 7.3 68.8 6.3 Multinucleate 21 Dark brown Rough Superficial
RWPC2 Dark brown Present 8.0 144.7 7.5 Multinucleate 42 Light brown to dark brown Rough Immersed
RWPC3 Hyaline to light brown Present 7.6 115.8 7.3 Multinucleate 0 - Not present Not present
RWPC4 Dark brown Present 7.1 96.6 6.1 Multinucleate 0 - Not present Not present
RWPC5 Dark brown Present 8.3 135.6 7.2 Multinucleate 26 Medium to dark brown Rough Superficial
RWPC6 Hyaline to light brown Present 7.7 94.7 6.7 Multinucleate 32 Medium to dark brown Rough Superficial
RWPC7 Medium brown Present 8.3 119.8 6.8 Multinucleate 39 Dark brown Rough Immersed
RWPC8 Hyaline to light brown Present 8.2 146.4 7.3 Multinucleate 32 Light brown to dark brown Rough Superficial
RWPC9 Hyaline to light brown Present 7.2 112.9 5.9 Multinucleate 31 Medium to dark brown Rough Immersed
RWPC10 Dark brown Present 7.1 139.9 6.9 Multinucleate 47 Medium to dark brown Rough Superficial
RWPC11 Hyaline to light brown Present 8.1 73.8 5.9 Multinucleate 44 Medium to dark brown Rough Superficial
RWPC12 Medium brown Present 7.7 150.3 7.8 Multinucleate 39 Medium to dark brown Rough Superficial
RWPC13 Dark brown Present 7.8 102.5 5.4 Multinucleate 29 Light brown to dark brown Rough Immersed
RWPC14 Medium brown Present 8.2 73.5 5.4 Multinucleate 45 White to light brown Rough Superficial
CHKC1 Hyaline to light brown Present 7.3 70.3 5.3 Multinucleate 25 Light brown to dark brown Rough Superficial
CHKC2 Hyaline to light brown Present 7.8 98.9 5.9 Multinucleate 0 - Not present Not present
CHKC3 Dark brown Present 7.6 66.7 5.4 Multinucleate 0 - Not present Not present
CHKC4 Hyaline to light brown Present 7.1 96.2 5.9 Multinucleate 31 Brown Rough Superficial
98
CHKC5 Medium brown Present 8.3 98.9 6.4 Multinucleate 45 White to light brown Rough Immersed
CHKC6 Medium brown Present 7.7 111.7 7.1 Multinucleate 27 Medium to dark brown Rough Superficial
CHKC7 Dark brown Present 8.3 96.5 6.3 Multinucleate 32 Medium to dark brown Rough Superficial
CHKC8 Medium brown Present 7.7 111.4 6.1 Multinucleate 45 Dark brown Smooth Immersed
CHKC9 Hyaline to light brown Present 7.8 149.7 7.2 Multinucleate 31 Medium to dark brown Rough Superficial
CHKC10 Hyaline to light brown Present 8.2 71.0 6.2 Multinucleate 0 - Not present Not present
CHKC11 Dark brown Present 7.7 129.5 6.2 Multinucleate 42 Dark brown Smooth Immersed
ATKC1 Hyaline to light brown Present 7.6 136.3 7.3 Multinucleate 50 Brown Rough Immersed
ATKC2 Hyaline to light brown Present 7.3 118.6 6.1 Multinucleate 37 Light brown to dark brown Smooth Superficial
ATKC3 Dark brown Present 7.8 116.6 5.3 Multinucleate 26 Medium to dark brown Smooth Superficial
ATKC4 Dark brown Present 7.6 114.7 6.4 Multinucleate 51 Dark brown Rough Superficial
ATKC5 Hyaline to light brown Present 7.1 101.5 6.3 Multinucleate 38 Medium to dark brown Smooth Superficial
ATKC6 Medium brown Present 8.3 114.4 5.3 Multinucleate 31 Medium to dark brown Rough Immersed
ATKC7 Dark brown Present 7.6 148.0 8.2 Multinucleate 50 Dark brown Smooth Superficial
ATKC8 Hyaline to light brown Present 7.3 96.9 6.4 Multinucleate 45 Brown Smooth Superficial
ATKC9 Hyaline to light brown Present 7.8 139.3 6.9 Multinucleate 39 Medium to dark brown Rough Immersed
ATKC10 Dark brown Present 7.6 121.9 5.8 Multinucleate 39 Medium to dark brown Smooth Superficial
ATKC11 Medium brown Present 7.1 122.1 7.2 Multinucleate 38 Dark brown Smooth Immersed
ATKC12 Dark brown Present 8.2 99.1 5.8 Multinucleate 22 Dark brown Smooth Superficial
ATKC13 Dark brown Present 8.2 99.1 5.8 Multinucleate 22 Dark brown Smooth Superficial
ATKC14 Medium brown Present 8.3 119.8 6.8 Multinucleate 39 Dark brown Smooth Immersed
ATKC15 Medium brown Present 8.2 69.9 6.2 Multinucleate 0 - Not present Not present
ATKC16 Hyaline to light brown Present 7.6 136.3 7.3 Multinucleate 50 Brown Rough Immersed
99
JHEC1 Dark brown Present 8.3 121.8 5.9 Multinucleate 39 Brown Smooth Superficial
JHEC2 Hyaline to light brown Present 7.7 137.0 7.0 Multinucleate 39 Light brown to dark brown Rough Superficial
JHEC3 Dark brown Present 8.3 141.2 7.6 Multinucleate 26 Medium to dark brown Rough Superficial
JHEC4 Dark brown Present 7.7 136.6 7.3 Multinucleate 26 Medium to dark brown Rough Immersed
JHEC5 Hyaline to light brown Present 7.8 114.7 6.8 Multinucleate 22 Medium to dark brown Smooth Superficial
JHEC6 Medium brown Present 8.2 101.0 6.4 Multinucleate 51 Brown Rough Superficial
JHEC7 Dark brown Present 7.7 144.7 7.5 Multinucleate 42 Light brown to dark brown Rough Immersed
JHEC8 Hyaline to light brown Present 7.7 115.8 7.3 Multinucleate 25 Light brown to dark brown Rough Superficial
JHEC9 Hyaline to light brown Present 8.3 100.1 5.4 Multinucleate 48 Light brown to dark brown Rough Immersed
JHEC10 Medium brown Present 8.2 69.9 6.2 Multinucleate 0 - Not present Not present
JHEC11 Dark brown Present 7.2 101.3 6.1 Multinucleate 37 Medium to dark brown Rough Superficial
JHEC12 Hyaline to light brown Present 7.1 112.8 6.5 Multinucleate 38 Medium to dark brown Smooth Immersed
JHEC13 Medium brown Present 7.6 137.2 6.9 Multinucleate 50 Medium to dark brown Rough Superficial
ISBC1 Medium brown Present 7.1 116.7 6.6 Multinucleate 32 Light brown to dark brown Rough Superficial
ISBC2 Hyaline to light brown Present 7.7 134.3 6.9 Multinucleate 41 Medium to dark brown Rough Immersed
ISBC3 Dark brown Present 7.8 71.0 5.4 Multinucleate 0 - Not present Not present
ISBC4 Dark brown Present 8.2 99.1 5.8 Multinucleate 22 Dark brown Smooth Superficial
100
Small (60-90µm) CHKC3, RWPC1, ATKC15, JHEC10,
CHKC1, CHKC10, ISBC3, RWPC14,
RWPC11
Medium (91-120µm) RWPC6, CHKC4, CHKC7, RWPC4,
ATKC8, CHKC5, CHKC2, ATKC12,
ATKC13, ISBC4, JHEC9, JHEC6, JHEC11,
ATKC5, RWPC13, CHKC8, CHKC6,
JHEC12, RWPC9, ATKC6, ATKC4, JHEC5,
RWPC3, JHEC8, ATKC3, ISBC1, ATKC2,
RWPC7, ATKC14
Long (120>µm) JHEC1, ATKC10, ATKC11, CHKC11,
ISBC2, RWPC5, ATKC1, ATKC16, JHEC4,
JHEC2, JHEC13, ATKC9, RWPC10,
JHEC3, RWPC2, JHEC7, RWPC8, ATKC7,
CHKC9, RWPC12
Maximum = 150.3µm
Minimum = 66.7µm
Figure 4.17: Distribution of Rhizoctonia solani isolates recovered from chilli on the basis of hyphal length/ inter septal distance.
16%
50%
34%
Isolates distribution on the basis of hyphal length
Small (<90 µm) Medium (91-120 µm)
Long (120> µm)
101
Narrow (<5.5µm)
CHKC1, ATKC3, ATKC6, ISBC3, CHKC3,
JHEC9, RWPC13, RWPC14
Moderate (5.6 -
7.0µm)
ATKC10, ATKC12, ATKC13, ISBC4,
RWPC9, RWPC11, CHKC2, CHKC4,
JHEC1, RWPC4, CHKC8, ATKC2,
JHEC11, CHKC10, CHKC11, ATKC15,
JHEC10, RWPC1, CHKC7, ATKC5,
JHEC6, CHKC5, ATKC4, ATKC8, JHEC12,
ISBC1, RWPC6, RWPC7, ATKC14, JHEC5,
RWPC10, ATKC9, JHEC13, ISBC2
Wide (>7.0µm) JHEC2, CHKC6, ATKC11, RWPC5,
CHKC9, RWPC3, RWPC8, ATKC1,
ATKC16, JHEC4, JHEC8, RWPC2, JHEC7,
JHEC3, RWPC12, ATKC7
Maximum = 8.2µm
Minimum = 5.3µm
Figure 4.18: Distribution of Rhizoctonia solani isolates recovered from chilli on the basis of hyphal diameter.
14%
59%
27%
Isolates distribution on the basis of hyphal
diameter
Narrow (< 5.5 µm) Moderate (< 5.6 - 7.0 µm)
Wide (> 7.0 µm)
102
No Sclerotia
RWPC1, RWPC4, CHKC2, CHKC3,
CHKC10, ATKC15, JHEC10, ISBC3
Moderate (1-30)
CHKC1, ATKC12, ATKC13, JHEC5,
ISBC4, RWPC3, JHEC8, RWPC5, ATKC3,
JHEC3, JHEC4, CHKC6, RWPC13
High (>30) RWPC9, CHKC4, CHKC9, ATKC6,
RWPC6, RWPC8, CHKC7, ISBC1, ATKC2,
JHEC11, ATKC5, ATKC11, JHEC12,
RWPC7, RWPC12, ATKC9, ATKC10,
ATKC14, JHEC1, JHEC2, ISBC2, RWPC2,
CHKC11, JHEC7, RWPC11, RWPC14,
CHKC5, CHKC8, ATKC8, RWPC10,
JHEC9, ATKC1, ATKC7, ATKC16,
JHEC13, ATKC4, JHEC6
Figure 4.19: Distribution of Rhizoctonia solani isolates recovered from chilli on the basis of number of sclerotia.
13%
22%
65%
Isolates distribution on the basis of number of
sclerotia
No Sclerotia Moderate (1-30) High (>30)
103
Sclerotia not present
RWPC1, RWPC4, CHKC2, CHKC3,
CHKC10, ATKC15, JHEC10, ISBC3
Rough
RWPC2, RWPC3, RWPC5, RWPC6,
RWPC9, RWPC13, RWPC14, CHKC1,
CHKC4, CHKC5, CHKC6, CHKC9,
ATKC1, ATKC4, ATKC6, ATKC9,
ATKC16, JHEC2, JHEC3, JHEC4, JHEC6,
JHEC7, JHEC8, JHEC9, JHEC11, JHEC13,
ISBC1, ISBC2, RWPC7, RWPC8, RWPC10,
RWPC11, RWPC12, CHKC7
Smooth CHKC8, CHKC11, ATKC2, ATKC3,
ATKC5, ATKC7, ATKC8, ATKC10,
ATKC11, ATKC12, ATKC13, ATKC14,
JHEC1, JHEC5, JHEC12, ISBC4
Figure 4.20: Distribution of Rhizoctonia solani isolates recovered from chilli on the basis of sclerotia texture.
14%
59%
27%
Isolates distribution on the basis of sclerotia
texture
Not present Rough Smooth
104
Sclerotia not present RWPC1, RWPC4, CHKC2, CHKC3,
CHKC10, ATKC15, JHEC10, ISBC3
Immersed
CHKC8, CHKC11, ATKC2, ATKC3,
ATKC5, ATKC7, ATKC8, ATKC10,
ATKC11, ATKC12, ATKC13, ATKC14,
JHEC1, JHEC5, JHEC12, ISBC4
Superficial
RWPC2, RWPC3, RWPC5, RWPC6,
RWPC9, RWPC13, RWPC14, CHKC1,
CHKC4, CHKC5, CHKC6, CHKC9,
ATKC1, ATKC4, ATKC6, ATKC9,
ATKC16, JHEC2, JHEC3, JHEC4, JHEC6,
JHEC7, JHEC8, JHEC9, JHEC11, JHEC13,
ISBC1, ISBC2, RWPC7, RWPC8, RWPC10,
RWPC11, RWPC12, CHKC7
Figure 4.21: Distribution of Rhizoctonia solani isolates recovered from chilli on the basis of sclerotia topography.
31%
14%
55%
Isolates distribution on the basis of sclerotia
topography
Immersed Not present superficial
105
Figure 4.22. Disease index of Rhizoctonia solani infection on potato (cv. Desiree).
0
10
20
30
40
50
60
70
80A
TK
P10
RW
PP
6
RW
PP
13
AT
KP
6
AT
KP
11
ISB
P2
ISB
P6
JHE
P5
RW
PP
5
RW
PP
16
AT
KP
18
JHE
P4
JHE
P9
JHE
P12
RW
PP
10
RW
PP
19
AT
KP
1
AT
KP
12
AT
KP
13
AT
KP
14
AT
KP
15
AT
KP
16
AT
KP
17
AT
KP
19
AT
KP
2
AT
KP
20
AT
KP
21
AT
KP
3
AT
KP
4
AT
KP
5
AT
KP
7
AT
KP
8
AT
KP
9
ISB
P1
ISB
P3
ISB
P4
ISB
P5
ISB
P7
JHE
P1
JHE
P10
JHE
P11
JHE
P13
JHE
P2
JHE
P3
JHE
P6
JHE
P7
JHE
P8
RW
PP
1
RW
PP
11
RW
PP
12
RW
PP
14
RW
PP
15
RW
PP
17
RW
PP
18
RW
PP
2
RW
PP
3
RW
PP
4
RW
PP
7
RW
PP
8
RW
PP
9
CH
KP
2
CH
KP
3
CH
KP
1
Mea
n D
isea
se I
ndex
Rhizoctonia solani isolates
Disease index of Rhizoctonia solani infection on potato (cv. Desiree)
Mean Disease Index
106
Avirulent -
Slightly virulent ATKP10, RWPP6, RWPP13
Moderately virulent ATKP6, ATKP11, ISBP2, ISBP6, JHEP5,
RWPP5, RWPP16
Virulent ATKP18, JHEP4, JHEP9, JHEP12,
RWPP10, RWPP19
Highly virulent ATKP1, ATKP2, ATKP3, ATKP4, ATKP5,
ATKP7, ATKP8, ATKP9, ATKP12,
ATKP13, ATKP14, ATKP15, ATKP16,
ATKP17, ATKP19, ATKP20, ATKP21,
CHKP1, CHKP2, CHKP3, ISBP1, ISBP3,
ISBP4, ISBP5, ISBP7, JHEP1, JHEP2,
JHEP3, JHEP6, JHEP7, JHEP8, JHEP10,
JHEP11, JHEP13, RWPP1, RWPP2,
RWPP3, RWPP4, RWPP7, RWPP8,
RWPP9, RWPP11, RWPP12, RWPP14,
RWPP15, RWPP17, RWPP18
Figure 4.23: Pathogenicity determination of sixty-three Rhizoctonia solani isolates on Potato (cv. Desiree).
5%
11%
9%
75%
Pathogenicity determination R. solani isolates on
Potato (cv. Desiree)
Slightly virulent Moderately virulent
Virulent Highly virulent
107
Out of sixty-three R. solani, 75% of the isolates were highly virulent. Among
rest of the isolates, 5% isolates were slightly virulent, 11% moderately virulent while
9% isolates were found virulent. None of the isolates showed an avirulent response.
4.4.2 Pathogenicity Determination on Tomato
Pathogenicity of the isolates recovered from tomato symptomatic samples
was tested in plastic cell trays (53.49cm L x 26.82cm W) having 32 cells/ tray under
greenhouse conditions at 25 ± 2oC. Isolates of R. solani were artificially inoculated
on 3 weeks old tomato seedlings with a set of uninoculated plants as control.
Observations were made for stem infections, four weeks after inoculation.
A significant variation in aggressiveness toward tomato seedlings was
observed among R. solani isolates, as reflected in DI ranging from 0 to 51% for
stem damage (Figure 4.24). Infection on soil line level of the stem was categorized
as -, no symptom (avirulent); ±, brown lesion on part of the stem (moderately
virulent); +, brown lesion girdled the stem (virulent); ++, brown lesion girdled
the stem and plants wilted (highly virulent) (Figure 4.25). Out of sixty-seven
isolates, 8% of the isolates showed an avirulent response, 10% showed
moderately virulent, 19% showed virulent while 63% of the isolates showed a
highly virulent response.
4.4.3 Pathogenicity Determination on Chilli
For pathogenicity determinization of the isolates recovered from chilli
symptomatic plant samples, a greenhouse experiment was conducted in plastic cell
trays (53.49cm L x 26.82cm W) having 32 cells/ tray. Seeds of chilli (cv. Sanam)
were planted in each cell and each isolate of R. solani was artificially inoculated 2
weeks after sowing with a set of control plants.
108
Figure 4.24. Disease index of Rhizoctonia solani infection on tomato (cv. Rio Grande).
0
10
20
30
40
50
60R
WP
T6
RW
PT
15
AT
KT
4
JHE
T9
JHE
T16
RW
PT
10
JHE
T13
RW
PT
3
AT
KT
12
JHE
T10
CH
KT
7
ISB
T1
AT
KT
1
CH
KT
10
JHE
T3
RW
PT
9
CH
KT
3
CH
KT
6
JHE
T6
JHE
T18
AT
KT
5
AT
KT
8
AT
KT
15
ISB
T3
RW
PT
13
CH
KT
1
CH
KT
8
CH
KT
2
CH
KT
4
CH
KT
9
CH
KT
5
CH
KT
11
JHE
T11
ISB
T2
RW
PT
11
AT
KT
16
JHE
T5
JHE
T15
JHE
T2
RW
PT
1
RW
PT
5
AT
KT
2
AT
KT
7
AT
KT
11
JHE
T14
ISB
T5
JHE
T4
RW
PT
2
AT
KT
3
RW
PT
14
JHE
T1
JHE
T8
JHE
T12
JHE
T19
ISB
T4
RW
PT
4
AT
KT
6
AT
KT
10
JHE
T7
JHE
T17
RW
PT
8
RW
PT
12
AT
KT
14
AT
KT
17
RW
PT
7
AT
KT
9
AT
KT
13
Mea
n D
isea
se I
ndex
Rhizoctonia solani isolates
Disease index of Rhizoctonia solani infection on tomato (cv. Rio Grande)
Mean Disease Index
109
Avirulent RWPT6, RWPT15, ATKT4, JHET9,
JHET16
Moderately virulent RWPT3, RWPT10, CHKT7, ATKT12,
JHET10, JHET13, ISBT1
Virulent RWPT9, RWPT13, CHKT3, CHKT6,
CHKT10, ATKT1, ATKT5, ATKT8,
ATKT15, JHET3, JHET6, JHET18, ISBT3
Highly virulent RWPT1, RWPT2, RWPT4, RWPT5,
RWPT7, RWPT8, RWPT11, RWPT12,
RWPT14, CHKT1, CHKT2, CHKT4,
CHKT5, CHKT8, CHKT9, CHKT11,
ATKT2, ATKT3, ATKT6, ATKT7, ATKT9,
ATKT10, ATKT11, ATKT13, ATKT14,
ATKT16, ATKT17, JHET1, JHET2, JHET4,
JHET5, JHET7, JHET8, JHET11, JHET12,
JHET14, JHET15, JHET17, JHET19, ISBT2,
ISBT4, ISBT5
Figure 4.25: Pathogenicity determination of sixty-seven Rhizoctonia solani isolates on tomato (cv. Rio Grande).
8%
10%
19%
63%
Pathogenicity determination R. solani isolates on
Tomato (cv. Rio Grande)
Avirulent Moderately virulent
Virulent Highly virulent
110
Four weeks after inoculation, observations on plant roots were recorded and
categorized as; -, no lesions on the hypocotyls, healthy (avirulent); ±, lesions on part
the hypocotyl (moderately virulent); +, lesions girdling the hypocotyl and root
portions (virulent); ++, lesions girdling the hypocotyl, roots and dead seedlings
(highly virulent).
A Significant variation in aggressiveness of R. solani isolates was noted, as
reflected in DI ranging from 0 to 57% for root infection (Figure 4.26). All isolates
varied in virulence to root infections on chilli. Out of fifty-eight isolates, 7% of the
isolates were avirulent. 14% of the isolates showed moderately virulent, 15% virulent
while 64% isolates showed a highly virulent response (Figure 4.27).
One hundred and twenty-six highly virulent isolates (forty-seven from potato,
forty-two from tomato, and thirty-seven from chilli) were further tested for AG
composition.
4.5 ANASTOMOSIS GROUP TESTING
Classification of R. solani isolates to anastomosis groups (AGs) is widely
accepted as the first way of categorizing this heterogeneous spp. to a more
homogeneous subspecific group. In general, AG typing is based on the hyphal
fusion of two vegetatively compatible isolates however, reproducibility of
anastomosis interactions for a large number of populations is a time-consuming
process. Restriction fragment length polymorphism (RFLP) and analysis of
ribosomal DNA (rDNA) sequences of the recovered isolates was performed to
characterize these isolates into respective AGs or subsets within AGs. The results
of AG composition by RFLP analysis were further confirmed by hyphal
anastomosis interactions.
111
Figure 4.26. Disease index of Rhizoctonia solani infection on tomato (cv. Sanam).
0
10
20
30
40
50
60C
HK
C7
CH
KC
9
JHE
C1
0
RW
PC
2
AT
KC
1
CH
KC
3
ISB
C2
JHE
C1
RW
PC
8
AT
KC
12
AT
KC
7
JHE
C5
AT
KC
11
AT
KC
14
AT
KC
5
CH
KC
11
CH
KC
5
JHE
C1
1
JHE
C1
2
JHE
C7
RW
PC
5
RW
PC
1
RW
PC
10
RW
PC
11
RW
PC
12
RW
PC
13
RW
PC
14
RW
PC
3
RW
PC
4
RW
PC
6
RW
PC
7
RW
PC
9
CH
KC
1
CH
KC
2
CH
KC
4
CH
KC
6
CH
KC
8
CH
KC
10
AT
KC
2
AT
KC
3
AT
KC
4
AT
KC
6
AT
KC
8
AT
KC
9
AT
KC
10
AT
KC
13
AT
KC
15
AT
KC
16
JHE
C2
JHE
C3
JHE
C4
JHE
C6
JHE
C8
JHE
C9
JHE
C1
3
ISB
C1
ISB
C3
ISB
C4
Mea
n D
isea
se I
ndex
Rhizoctonia solani isolates
Disease index of Rhizoctonia solani infection on chilli (cv. Sanam)
Mean Disease Index
112
Avirulent CHKC7, CHKC9, JHEC10, RWPC2
Moderately virulent ATKC1, CHKC3, ISBC2, JHEC1, RWPC8,
ATKC12, ATKC7, JHEC5
Virulent ATKC11, ATKC14, ATKC5, CHKC11,
CHKC5, JHEC11, JHEC12, JHEC7, RWPC5
Highly virulent RWPC1, RWPC10, RWPC11, RWPC12,
RWPC13, RWPC14, RWPC3, RWPC4,
RWPC6, RWPC7, RWPC9, CHKC1,
CHKC2, CHKC4, CHKC6, CHKC8,
CHKC10, ATKC2, ATKC3, ATKC4,
ATKC6, ATKC8, ATKC9, ATKC10,
ATKC13, ATKC15, ATKC16, JHEC2,
JHEC3, JHEC4, JHEC6, JHEC8, JHEC9,
JHEC13, ISBC1, ISBC3, ISBC4
Figure 4.27: Pathogenicity determination of fifty-eight Rhizoctonia solani isolates on chilli (cv. Sanam).
7%
14%
15%
64%
Pathogenicity determination R. solani isolates on
Tomato (cv. Sanam)
Avirulent Moderately virulent
Virulent Highly virulent
113
4.5.1 PCR-Restriction Fragment Length Polymorphism (RFLP)
Genomic DNA of a total of forty-seven isolates from potato (highly virulent),
forty-two isolates from tomato (highly virulent) and thirty-seven isolates from chilli
(highly virulent) amplified with a set of RS1/RS4 primers;
RS1 (5′-CCTGTGCACCTGTGAGACAG-3′) and
RS4 (5′-TGTCCAAGTCAATGGACTAT-3′)
(Camporota et al., 2000)
generated a fragment of approximately 540 bp on agarose gel. The resulting PCR
products were cleaned up using Sephadex G-50 and further subjected to RFLP
analysis with four discriminating enzymes (MseI, AvaII+HincII, and MunI)
(Guillemaut et al., 2003). The amplified products of the rDNA-ITS regions obtained
using RS1 and RS4 primers varied for their accessible restriction sites to
discriminating restriction enzymes. The RFLP patterns with these discriminating
enzymes were observed in agarose gel (Figure 4.28). Specific markers were assigned
in accordance with the restriction patterns observed and the combination of these
markers as described by Guillemaut et al. (2003) was used to designate specific
anastomosis group to each isolate as shown in Table 4.8.
A total of sixty-six isolates were assigned AG-3 PT as they shared BNAN
and FNAN RFLP type. Sixty-one isolates shared BNAN and five isolates shared
FNAN RFLP type. MseI restricted the fragments at two locations; 106-119, 188-
223bp for maker B, and 194, 327bp for marker F. The restriction patterns for
combination of Ava II and HInc II were 189-203 and 306-337bp for marker NA while
the restriction patterns corresponding to MunI were 22 and 475-550bp for marker N.
Most of the isolates belong to AG-3 PT as expected strains for crop types.
114
Twenty-five isolates were designated as AG-4 HGI based on the RFLP type;
IEAA they shared. The restriction patterns corresponding to MseI were 21-26, 34, 53-
72, 71-90, 126-149, and 213-219bp for marker I while patterns conforming
combination of Ava II and HInc II were 19, 40-50, 199, and 258-273bp for marker EA.
MunI restricted the fragments at two locations; 184-215, 306-340bp for maker A.
Sixteen isolates were assigned AG-2-1 and six isolates as AG-2-2 based on
the restriction patterns. The restriction patterns corresponding to MseI were 106-119
and 188-223bp for marker B, 44-57, 58-71, and 193-219bp for maker C while 15-23,
84-94, and 192-233bp for marker D. The restriction patterns for combination of Ava
II and HInc II were 20, 62-71, 175-224, and 255-272bp for marker AA, 71, 448-
475bp for marker AN while 20, 70, 149, and 287-295bp for marker BN. MunI
restricted the fragments at two locations; 184-215, 306-340bp for maker A, and
22,475-550bp for marker N. Out of sixteen isolates belonging to AG-2-1, ten isolates
shared BBNA while six isolates shared DANA RFLP type. Isolates assigned AG-2-
2 shared BAAN and CANN RFLP type. Out of six isolates belonging to AG-2-2, five
isolates shared BAAN while one isolate shared CANN RFLP type.
Nine isolates were assigned AG-5 while four isolates were designated as AG-
6 as they shared HAAC and ANAA, AENA RFLP types respectively. The restriction
patterns corresponding to MseI were 44, 192, and 272bp for marker H while 10-25,
20-48, and 190-217bp for maker A. The restriction patterns for a combination of Ava
II and HInc II were 20, 62-71, 175-224, and 255-272bp for marker AA, 189-203 and
306-337bp for marker NA while 44-50, 448-489bp for marker EN. MunI restricted
the fragments at two locations; 51, 186-188, 271-275bp for marker C, and 184-215,
306-340bp for marker A.
115
Figure 4.28. PCR-RFLP restriction patterns revealed by discriminating enzymes
(MseI, AvaII+HincII, and MunI).
116
4.5.2 Hyphal Anastomosis Interaction
Anastomosis group identities of PCR-RFLP analysis were further confirmed
by the hyphal anastomosis interactions on 1.5% WA coated glass slides. Each isolate
was paired with the corresponding tester strain of known AGs. Four types of
anastomosis reactions; C0 to C3 were observed where, C0 = no reaction, C1 = contact
fusion, C2 = somatic fusion or perfect anastomosis, and C3 = auto-anastomosis as
described by Carling (1996). C3 type interactions; auto-anastomosis or self-pairing
were used as positive control. Percentage of fusion frequency at twenty random
locations were used to determine AG typing. Isolates pairing at more than 80%
locations were confirmed as belonging to respective anastomosis group.
One hundred and twenty-six isolates of R. solani assigned different AGs
through PCR-RFLP analysis were paired with the tester strain of AG-3. Somatic
interactions were randomly taken at twenty different locations.
A considerable variation in the hyphal interactions as no reaction (C0), only
contact fusion (C1), somatic fusion or perfect anastomosis (C2) and auto anastomosis
(C3) were observed. Hyphal fusion frequency varied from 27 to 93% suggesting a
high level of heterogeneity among the isolates. A total of sixty-five isolates formed
a C2 = somatic fusion of perfect anastomosis interactions with the tester strain AG-
3. C3 type or self-anastomosis interactions were not taken into consideration. The
hyphal fusion frequency among these isolates was more than 80%. Selected isolates
were identified as members of AG-3.
A total of sixty-one isolates had either C0 = no reaction or C1 = only contact
fusion with the tester strain of AG-3. These isolates were further paired with the tester
strains of AG-2-1, AG-2-2, AG-4, AG-5, and AG-6.
117
Table 4.8: Rhizoctonia solani anastomosis groups (AGs) assigned using PCR-RFLP
and hyphal anastomosis interaction.
118
119
120
121
122
Twenty-four isolates paired with tester strains of AG-4, fourteen isolates
paired with AG-2-1, eight isolates paired with AG-5, six isolates paired with AG-2-
2 while two isolates paired with AG-6 at more than 80% fusion frequency. All these
isolates (except seven isolates) had either C0 = no reaction or C1 = only contact
fusion with the tester strains other than their respective AGs (Table 4.8).
Four isolates showed strong C2 = somatic fusion of perfect anastomosis with
the tester strains of AG-3, AG-4, and AG-5 while three isolates showed C2 = somatic
fusion of perfect anastomosis with the tester strains of AG-3, AG-2-2, AG-4, and
AG-6 at 45-69% fusion frequency. The identity of these seven isolates was subjected
to molecular characterization.
4.5.3 Sequence Analysis of ITS-5.8S rDNA
Type isolates representing anastomosis groups identified by PCR-RFLP and
hyphal anastomosis interactions and seven isolates showing hyphal interactions with
more than one tester strain R. solani AGs (Table 4.9) were subjected to PCR
amplification with a set of universal sense primers; ITS1 (5´-
TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-
3´) (Qiagen) encoding ITS-1-5.8S-ITS-2 (White et al., 1990) generated a fragment
of approximately 700 bp on agarose gel. The ITS region (ITS1, 5.8S rDNA, and
ITS2) of each isolate was sequenced in both sense and antisense directions. BLAST
analysis of these sequences with the known sequences of R. solani AGs from NCBI
GenBank confirmed the identity of respective AGs (99-100% sequence identities)
previously revealed by PCR-RFLP and hyphal interactions. All the isolates showed
heterogeneity in their ITS sequences. Sequence analysis of seven isolates paired
with more than tester strains of R. solani AGs revealed two isolates (RWPP12 and
123
RWPT14) were closely related to AG-2-1, two isolates (CHKC6 and ATKC8) AG-
6, one isolate (JHEP11) AG-5, one isolate (ATKC16) AG-3, and one isolate
(ATKC13) AG-4 HGI with 99-100% identities to the sequences of respective AGs.
4.5.4 Phylogenetic Analysis of ITS using DNA Sequences
DNA sequences of the type isolates representing different AGs from potato,
tomato, and chilli and the known sequences of R. solani AGs from NCBI GenBank
were compared for phylogenetic analysis.
The phylogenetic tree to infer the evolutionary history of the isolates from
each crop type separately was made by using the Maximum Likelihood method based
on the Tamura-Nei model (Tamura and Nei, 1993). The percentage of trees in which
the associated taxa clustered together is shown next to the branches. Initial tree(s) for
the heuristic search were obtained automatically by applying Neighbor-Join and
BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum
Composite Likelihood (MCL) approach, and then selecting the topology with
superior log likelihood value. Each tree is drawn to scale, with branch lengths
measured in the number of substitutions per site (next to the branches). The analysis
involved 21 nucleotide sequences. Codon positions included were
1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were
eliminated. There was a total of 492 positions in the final dataset. Evolutionary
analyses were conducted in MEGA7 (Kumar et al., 2016).
DNA sequences of the fifteen type isolates from potato representing AG-
3 PT, AG-2-1, AG-4 HGI, AG-5, and AG-2-2 formed distinct clades (Figure 4.26)
supported by bootstrapping indices 99, 98, 99, 99, and 95% respectively and an
outgroup clade for Rhizoctonia oryzae.
124
Table 4.9: Type isolates of Rhizoctonia solani representing different anastomosis
groups (AGs) and 07 unknown isolates used for molecular characterization.
125
Isolates ISBP4, JHEP8, CHKP3, ATKP9, and RWP11 shared the same clade
with the reference isolate of R. solani AG-3 PT with 99% bootstrap support. Isolates
RWPP12, JHEP6, and ISBP3 shared the clade with AG-2-1 with 98% bootstrap
support. Within the clad of AG-2-1, isolates; RWPP12 and ISBP3 formed a distinct
cluster from JHEP6 isolate with bootstrap support of 72%. Isolate RWPP4 shared
clade with the reference isolate of AG-4 HGI with bootstrap support of 99%. Isolates;
JHEP11, ATKP13, RWPP14, and ISBP7 shared a clade with reference isolate of AG-
5 with 99% bootstrap support. Within this clade, isolate JHEP11 made a distinct clad
with the bootstrap support of 66% from all other isolates. Isolates RWPP18 and
ATKP4 shared a clade with the reference isolate of AG-2 with the bootstrap support
of 95%. DNA sequence of Rhizoctonia oryzea as an outgroup formed a distinct clade
from all clusters of R. solani.
DNA sequences of eighteen type isolates from tomato representing AG-3
PT, AG-2-1, AG-5, AG-2-2, and AG-4 HGI formed different clades with 99, 90,
93, 99, and 99% bootstrap support respectively (Figure 4.27). Isolates; CHKT5,
RWPT5, CHKT1, ATKT9, JHET8, and ISBT4 shared the clade of reference
isolate AG-3 PT. Within this clade, two isolates; CHKT5 and RWPT5 made a
distinct clad from all other isolates with a bootstrap support of 49%. ATKT10,
RWPT14, ISBT5, and JHET4 shared the cluster of AG-2-1 with a bootstrap
support of 90%. Within this clad ATKT10 formed a distinct clad from all other
isolates with 97% bootstrap support. RWPT14 formed a different clad at 93%
bootstrap support from ISBT5 and JHET4 forming another sub clad with 93%
bootstrap support. Isolates; JHET14, CHKT4, and ATKT6 formed a clade with
reference isolate of AG-5 with a bootstrap support of 93%. Within this clad
126
CHKT4 and ATKT6 formed a distinct clad from JHET14 with a bootstrap support
of 87%. Isolates; RWPT8, CHKT8, and ATKT17 share the same clade with
reference isolate AG-2-2 with 99% bootstrap support from the main three. Within
this clade, CHKT8 and ATKT17 formed a distinct clad from RWPT8 with 75%
bootstrap support. Reference isolate AG-4 HGI shared a clade with JHET11 and
RWPT4. Within this clad RWPT4 formed a distinct clad from JHET11 with 69%
bootstrap support. DNA sequence of Rhizoctonia oryzea as an outgroup formed a
distinct clade from all clusters of R. solani.
DNA sequences of eighteen type isolates from chilli representing AG-2-1, AG-
3 PT, AG-5, AG-6, and AG-4 HGI, formed distinct clades with 98, 100, 99, 99 and
100% bootstrap support respectively (Figure 4.28). Isolates; RWPC13, JHEC3,
CHKC8, and ATKC4 shared a clade with reference isolate AG-2-1 with 99% bootstrap
value representing all four isolates belonging to AG-2-1. Isolates; RWPC6, ATKC16,
and JHEC8 shared a clade with reference isolate of AG-3 PT with a bootstrap support
of 99%. Within this clade, RWPC6 formed a distinct cluster from JHEC8 and ATKC16
isolates with 57% bootstrap support. ATKC2, ATKC8, CHKC1, and CHKC6 formed
a distinct clad with 99% bootstrap value and shared with the clad of AG-3. The
reference isolate AG-6 shared a clade with isolates; RWPC10 and ISBC4 with 99%
bootstrap support. Isolates; ISBC3, JHEC9, RWPC12, CHKC10, and ATKC13 shared
a clade with reference isolate AG-4 HG I with 100% bootstrap support. Within the clad
of isolates belonging to AG-4, JHEC9 and RWPC12 formed a distinct clad from
CHKC10 and ATKC 13 with 64 and 55% bootstrap support respectively while ISBC3
clustered separately. The reference isolate for an outgroup isolate Rhizoctonia oryzea
formed a distinct clade from all isolates of R. solani.
127
Figure 4.29: Phylogenetic analysis using ITS region of Rhizoctonia solani isolates infecting potato in Pothohar region drawn using Mega 7.
128
Figure 4.30: Phylogenetic analysis using ITS region of Rhizoctonia solani isolates infecting tomato in Pothohar region drawn using Mega 7.
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Figure 4.31: Phylogenetic analysis using ITS region of Rhizoctonia solani isolates infecting chilli in Pothohar region drawn using Mega 7.
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4.5.5 Frequencies of Different AGs
Forty-seven highly virulent isolates recovered from diseased portions of
the potato samples were subjected to anastomosis group typing using; PCR-
RFLP, hyphal interactions, and sequence analysis. Among the 47 R. solani
isolates, thirty-six isolates belong to AG-3 PT, four isolates AG-5, three isolates
AG-2-1, two isolates AG-2-2, and remaining two isolates belonged AG-4 HG I
respectively (Table 4.10).
Most of the AG-3 PT (25), AG-5 (4) and AG-2-1 (2) isolates were recovered
from the sclerotia formed on potato tubers while frequencies of the AG-3 PT (4),
AG-2-2 (2), AG -HGI (2), and AG-2-1 (1) recovered from stem canker were less as
compared to tuber. Isolates of AG-3 PT recovered from stolon canker and root
lesions were 4 and 3 respectively (Table 4.11).
Among forty-two virulent isolates recovered from stem portions of the
diseased tomato plants; twenty-seven isolates belonged to AG-3 PT, six isolates AG-
2-1, four isolates AG-2-2, three isolates AG-5, and two isolates belonged AG-4 GI
(Table 4.12).
Among thirty-seven virulent isolates recovered from root portions of
diseased chilli plants; twenty-two isolates belonged to AG-4 HG I, six isolates AG-
2-1, four isolates AG-6, three isolates AG-3 PT, and two isolates belonged AG-5
(Table 4.13).
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Table 4.10: Frequency of Rhizoctonia solani anastomosis groups (AGs) recovered
from potato symptomatic samples, collected from different locations of
Pothohar region.
Districts AG-3 PT AG-2-1 AG-2-2 AG-4 HG I AG-5 Total
Rawalpindi 9 1 1 1 1 13
Jhelum 6 1 0 0 1 8
Attock 15 0 1 1 1 18
Chakwal 3 0 0 0 0 3
Islamabad 3 1 0 0 1 5
Total 36 3 2 2 4 47
Table 4.11: Frequency of Rhizoctonia solani anastomosis groups (AGs) recovered
from portions of diseased potato samples.
Plant Portions AG-2-1 AG-2-2 AG-3 PT AG-4 HG I AG-5 Total
Black Scurf 2 0 25 0 4 31
Stem Canker 1 2 4 2 0 9
Stolon Canker 0 0 4 0 0 4
Root Lesion 0 0 3 0 0 3
Total 3 2 36 2 4 47
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Table 4.12: Frequency of Rhizoctonia solani anastomosis groups (AGs) recovered
from tomato symptomatic samples, collected from different locations of
Pothohar region.
Districts AG-2-1 AG-2-2 AG-3 PT AG-4 HG I AG-5 Total
Attock 1 2 7 0 1 11
Rawalpindi 1 1 6 1 0 9
Jhelum 2 0 8 1 1 12
Chakwal 1 1 4 0 1 7
Islamabad 1 0 2 0 0 3
Total 6 4 27 2 3 42
Table 4.13: Frequency of Rhizoctonia solani anastomosis groups (AGs) recovered
from chilli symptomatic samples, collected from different locations of
Pothohar region.
Districts AG-2-1 AG-3 PT AG-4 HG I AG-5 AG-6 Total
Attock 2 1 5 1 1 11
Rawalpindi 2 1 7 0 1 7
Jhelum 1 1 5 0 0 10
Chakwal 1 0 3 1 1 6
Islamabad 0 0 2 0 1 3
Total 6 3 22 2 4 37
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Chapter 5
5 DISCUSSION
Rhizoctonia solani Kühn is a widespread soilborne fungal pathogen of both
cultivated and noncultivated soils capable of infecting a range of plant species
including solanaceous vegetables. It is a species complex of several anastomosis
groups (AGs) based on the hyphal fusion of the identical isolates, differ in genotypic,
and phenotypic characters. Different AGs cause infection on their differential hosts.
R. solani is a commonly occurring pathogen of Pakistani soils (Ahmad, 1998; Bhutta,
2008).
Although, some contributions to R. solani infection and AG typing on
potato is reported from Pakistan (Rauf et al., 2007), however damping-off of young
tomato and wilt of adult plants are mostly mixed with other soil borne pathogens
including Fusarium, Pythium, and Phytophthora spp. No literature is available on
R. solani infection on tomato, and chilli from Pakistan. This is the first study to
document the disease incidence, AG composition and somatic compatibility of R.
solani isolates infecting selected solanaceous vegetables; potato, tomato, and chilli
in the Pothohar region.
In the present studies, an overall 31.2, 34.2, and 29.4% mean disease
incidence of R. solani in Pothohar region was recorded on potato, tomato, and chilli
crops respectively. R. solani infection was prevalent in all the locations visited.
District Attock and Rawalpindi are the major potato while Attock and Jhelum are the
major tomato, and chilli growing areas under study in Pothohar region. These crops
are also grown on scattered locations of other districts. Mean disease incidence in
different districts varied for each crop. For potato, maximum mean disease incidence
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(37.4%) was recorded in district Attock followed by Islamabad while in district
Chakwal (20.2%). For tomato, the mean disease incidence was maximum in
Islamabad (38.7%) followed by district Attock (36.3%) and minimum in district
Jhelum (27.5%). The same variations were observed for chilli as disease incidence
was maximum (30.9%) in district Attock while minimum in district Chakwal and
Rawalpindi (27.3%). Since the climate of the region has considerable temperature
variations including semi-arid and sub-humid (Shamshad, 1988), the fungus can
survive under both cool and warm soils. It can remain active at a range of
temperatures (Olsen and Young, 2011) and is well adapted to survive unfavourable
conditions as it remains dormant as sclerotia. (Ceresini et al., 2002). The optimum
temperature ranges 24-32oC for potato tuber development, vegetative growth in
chilli, and the emergence of tomato seedlings also provide optimum temperatures for
R. solani disease development; 24-32oC (Harikrishnan and Yang, 2004). Pothohar
region receives an average of 1,249 millimetres (49.2 in) rainfall of which more than
65% is received in monsoon. In addition to temperature, soil moisture greatly
influences the amount of R. solani inoculum in the soil (Frank, 1978) that ultimately
favour the disease development (Kyritsis and Wale, 2002; Naz et al., 2008; Shehata
et al., 1984).
Multicropping and the intercropping are the common practices adopted by
the farmers of the region. Mostly the farmers are not progressive. They have small
land holdings and do not follow the same cropping pattern and crop rotation. It is
well accepted that the occurrence of soil borne pathogens including R. solani is
greatly influenced by intensive cropping (Gilligan et al., 1996; Hooker, 1981; Kluth
et al., 2010). Selected solanaceous vegetables cultivation on the same fields also help
135
in the inoculum multiplication however, this pathogen is also well adapted for life
outside the host plants (Keijer et al., 1997; Olsen and Young, 2011).
The use of noncertified and poor quality seeds is commonly practised as the
same germplasm of the few local varieties is used for cultivation year after years.
The most commonly used potato varieties are Desire, Cardinal, and Diamant. The
reports of Rauf et al. (2007) and Ahmad et al. (1995) confirmed these varieties to be
highly susceptible to R. solani infection. The commonly used tomato varieties are;
Rio Grande, Money Maker, and Roma while chilli are Dandi Cut, Gola Peshawari,
and Tata Puri.
In the present study, a total of 63, 67, and 58 isolates of R. solani were
recovered from potato, tomato, and chilli symptomatic plant samples on malt extract
agar (MEA) medium. With considerable variations, all recovered isolates exhibited
typical R. solani colony and cultural characteristics. All isolates were multinucleate
with 3-8 nuclei per cell and had hyphal branching at a right angle to the constriction
found at the point of branching mycelium, a known feature for R. solani described
by Sneh et al. (1991). A septum, that is of immense taxonomical importance was
always present near the branching junction. All 188 isolates were morphologically
differentiated and classified on the basis of septal distance, hyphal diameter, no. of
sclerotia/ cm2, texture, and topography of sclerotia.
Morphological variations between isolates from different geographical
regions have previously been studied by Parmeter et al. (1969), Sharma et al. (2005)
and Goswami et al. (2010). Neeraja et al. (2002) and Vineeta et al. (2002) reported
the significant importance of the mycelial and sclerotial characteristics in
categorizing R. solani isolates into distinct groups. The septal distance ranged
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between 66.7 to 150.3µm. The hyphal diameter ranged between 5.1 to 8.2μm.
Hansen (1963) also found that hyphal diameter ranged from 4.3-8.0µm. These
findings were also in line with the findings of Vijayan and Nair (1985). All recovered
isolates were incubated on Malt Extract Agar (MEA) medium for seven days for
sclerotial production. Most of the isolates produced sclerotia that were either rough
or smooth. Most of the isolates produced rough sclerotia. Some isolates failed to
produce any sclerotia. Meyer et al. (1998) also found some R. solani isolates may
not produce sclerotia under different cultural conditions. Therefore, the absence of
sclerotia should not be criteria for the mycelium to be excluded from R. solani. The
sclerotia were either immersed or superficially available on the hyphae. Location of
the sclerotial production as superficial or immersed was also supported by the
findings of Vineeta et al. (2002). Anderson (1982) and Hoa (1994) also differentiated
sclerotia from different isolates on the basis of colour. The findings of Sinha and
Ghufran (1988) also supported the variations in colony colour, number, size, and the
colour of sclerotia formed. The Results of the variations in cultural characteristics
were in line with Kuiry et al. (2014). Categorizing isolates based on the cultural and
morphological features confirmed the diversity among the isolates was not correlated
with their origin of the collection as supported by Baird et al. (1996). The
morphological classes based on the present studies were however, conservative since
only MEA medium was used for this study. Boosalis and Scharen (1969)
differentiated distinct clones of morphologically similar isolates on potato dextrose
agar (PDA) medium using other media. The initial screening of the isolates based on
the morphological characteristics was used to select type isolates for further
molecular identifications. Sunder et al. (2003) reported colony colour varies from
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brown, light brown, dark brown, and yellowish brown.
The isolates were subjected to pathogenicity determination under shade-
house experiments. Significant variations were noted in disease index of each isolate
against respective crop type, they were recovered from. The pathogenicity results
showed isolates within the same AG had variability in virulence, which may be
isolate dependent rather than AG dependent.
Out of 63 isolates tested against potato cv. Desire, 75% of the isolates were
highly virulent, 5% isolates were slightly virulent, 11% moderately virulent while
9% isolates were virulent towards stem infections. These findings confirmed the
findings of Balkan and Wenham (1973). Similar results were also reported by
Goswami et al. (2010). Among sixty-seven isolates tested for pathogenicity on
tomato cv. Rio Grande under greenhouse conditions, 8% of the isolates showed an
avirulent response, 10% showed moderately virulent, 19% showed virulent while
63% of the isolates showed a highly virulent response. These findings are in
accordance with the findings of Taheri and Tarighi (2012) who compared the same
susceptible cv. Rio Grande with cv. Sunny against R. solani infection. Similar results
of R. solani infection on tomato were reported by Misawa and Kuninaga (2010). Out
of fifty-eight isolates recovered from chilli were tested for their virulence against
chilli cv. Sanam. 7% of the isolates were avirulent, 9% slightly virulent, 5%
moderately virulent, 15% virulent while 64% isolates showed a highly virulent
response. There was no correlation between mycelial growth and virulence. This was
also supported by the results of Basu et al. (2004). These results were in line with
the findings of Mannai et al. (2018), and Güney and Güldür (2018).
Isolates purified using hyphal tipping on PDA medium were preserved by
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colonizing on hulled barley grains maintained at 4 °C. Sneh et al. (1986) and Webb
et al. (2011) also used the cryogenic storage method for long-term preservation of R.
solani isolates.
Hyphal anastomosis interactions are considered to be a more accurate method
for accommodating isolates of R. solani into AGs however, reproducibility of this
method for a large number of populations is difficult and its reliability at subgroup
identification is unsatisfactory (Fang et al., 2013; Muzhinji et al., 2015). Molecular
approaches including DNA based sequence homology, restriction analysis of
ribosomal DNA have confirmed their reliability to differentiate isolates of R. solani
into different AGs and subgroups. Bounou et al. (1999) and Salazar et al. (2000)
used PCR based assays for categorizing isolates of R. solani to different AGs and
subgroups. A total of 126 virulent isolates (47, 42, and 37 isolates from potato,
tomato, and chilli respectively) were subjected to PCR-RFLP analysis with four
discriminating enzymes (MseI, AvaII+HincII, and MunI) to categorize them into
different at AGs. Results of the PCR-RFLP analysis revealed 66 isolates belonged
to AG-3 PT while 25, 16, 9, 6, and 4 isolates belonged to AG-4 HGI, AG-2-1, AG-
5, AG-2-2, and AG-6 respectively. Hyakumachi et al. (1998) and Vilgalys and
Gonzales (1990) also used PCR-RFLP technique to categorize isolates of R. solani
into different AGs. Guillemaut et al. (2003) used the same technique to differentiate
isolates of R. solani into different anastomosis groups.
Each isolate was paired with the tester strain of respective AG identified by
PCR-RFLP. Environmental factors including temperature variations and nutritional
stress may greatly influence the vegetative compatibility of the isolates (Julián et al.,
1996). In present studies, compatibility of the isolates with tester strains was tested
139
on MEA medium with growth conditions. Among four types of anastomosis
reactions; C0 to C3 only C2 reactions were considered as somatic fusion or perfect
anastomosis as described by Carling (1996). Out of 126 isolates tested for hyphal
interactions, 119 isolates confirmed the AG identity revealed by PCR-RFLP.
Farrokhi-Nejad et al. (2007) used hyphal anastomosis interactions with the known
tester strains of R. solani to differentiate AGs identities of unknown R. solani
isolates.
Seven isolates showed hyphal anastomosis interactions with more than one
tester strain of known AGs, so their identity was not confirmed. The bridging
interactions between distantly related isolates of the same AG and closely related
isolates belonging to different AGs give ambiguous results leading to
misidentifications. Carling et al. (2002b) also found bridging interactions between
isolates belonging to different AGs. The compatibility of hyphal anastomosis
interactions of the unknown isolates with more than one tester strain supports the
unsatisfying results of anastomosis identifications. The AGs determination for these
isolates was confirmed using sequence analysis of their ITS-5.8S rDNA. ITS
sequence analysis technique has previously been used to determine R. solani AGs
and subgroups (Carling et al., 2002b; Fiers et al., 2011; Lehtonen et al., 2008).
Type isolates representing anastomosis groups identified by PCR-RFLP and
confirmed by hyphal anastomosis interactions together with seven unknown isolates
were subjected to PCR amplification with a set of universal sense primers; ITS1 and
ITS4 encoding ITS-1-5.8S-ITS-2 which generated a fragment of approximately 700
bp on agarose gel. BLAST analysis of the obtained sequences with the known
sequences of R. solani AGs from NCBI GenBank confirmed the identity of
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respective AGs (99-100% sequence identities). All the isolates showed heterogeneity
in their ITS sequences. This was not unexpected as R. solani is multi-nucleate and
heterokaryotic. This also indicates the existence of a complex population structure
of the fungus in this region. Hyphal anastomosis interactions have not been
considered a reliable tool for AG composition because of the existence of the
bridging interactions between members of different AGs. Sequence analysis
confirmed the identity of unknown isolates interacting more than one test stains.
Sequencing of the ITS region and phylogenetic analysis have been confirmed to be
a reliable tool classifying isolates into distant clades corresponding to different AGs,
subgroups and further subsets of subgroups (Fang et al., 2013; Justesen et al., 2003;
Lehtonen et al., 2008; Sharon et al., 2008b). Thus, AG determination study was
confirmed using ITS sequence analysis. Sequencing of the ITS region and
phylogenetic analysis to differentiate AGs of R. solani have also been used by
Kuninaga et al. (2000); Justesen et al. (2003); Lehtonen et al. (2008); Pannecoucque
and Höfte (2009); Tsror (2010) and Fiers et al. (2011).
AG composition of the highly virulent isolates recovered potato revealed
76.5% isolated belonged to AG-3 PT and they were predominant to all potato
production areas of Pothohar region concordant with the previous findings by Rauf
et al. (2007). In our findings The predominance of AG-3 PT with R. solani infection
on potato was also in line with the findings of the other researchers from Cyprus
(Kanetis et al., 2016), New Zealand (Das et al., 2014), Finland (Lehtonen et al.,
2008) Great Britain (Woodhall et al., 2007), France (Campion et al., 2003; Fiers et
al., 2011) and Ireland (Chand and Logan, 1983). The association of AG-3 with black
scurf of potato has also been reported by other researchers as well (Anderson, 1982;
141
Bolkan and Ribeiro, 1985; Ogoshi, 1985). The predominance and high virulence of
AG-3 PT could be due to the ability to produce sclerotia on potato tubers. Lehtonen
et al. (2009) also reported the specialized nature of AG-3 PT infection and its ability
to produce sclerotia on potato tubers is higher than other AGs.
The frequency of the other AGs was far less than AG-3 as AG-5 8.5%, AG-
2-1 6.3%, AG-2-2 and AG-4 HGI 4.2% whereas none belonged to AG-1, AG-7, and
AG-9. Previously, Rauf et al. (2007) reported the association of AG-1-1 and AG-9
with R. solani infection on potato. Most of the AG-3 PT isolates (25) and few isolates
of AG-5 (4) and AG-2-1 (2) were isolated from sclerotia on infected tubers. Four
isolates of AG-3 PT, two isolates of AG-2-2 and AG-4 HG I, and one isolate of AG-
2-1 was recovered from stem canker. Four isolates of AG-3 PT were associated with
stolon canker while three isolates of the same AG were recovered from root lesions.
The presence of AG-3 PT and AG-5 with black scurf of potato was also supported
by the findings of Muzhinji et al. (2015) however the association was AG-2-1 was
in contradiction.
R. solani has also been reported to cause tomato foot rot, damping off, and
root infections by different researchers (Conover, 1949; McCarter, 1991; Small,
1927). Mitidieri (1994) reported infection of R. solani infection on tomato, and chilli
pepper. In our studies, isolates recovered from tomato belong to AG-3 PT (64.2%),
AG-2-1 (14.2%), AG-2-2 (9.5%), AG-5 (7.1%) and AG-4-HGI (4.7%). AG-3 PT
was also predominant to tomato growing areas followed by AG-2-1 while other
groups were confined to distinct locations. The association of AG-3 PT with tomato
foot rot have also been supported by (Misawa and Kuninaga, 2010).
Mikhail et al. (2010) reported AG-2 from foot rot while Kuramae et al.
142
(2003) confirmed the association of AG-4 with stem and foot rot of tomato. AG-2-1
and AG-2-2 have also been reported to cause foot rot of tomato by Misawa and
Kuninaga (2010). AG-1 and AG-6 were not detected in our studies despite the fact
that it has been reported to be associated with tomato infection by Charlton et al.
(2008). The association of AG-5 with tomato foot root was in lined with the findings
of Kuramae et al. (2003). The results of the present studies were also in line with the
findings of Karaca et al. (2002); Bartz et al. (2010); Misawa and Kuninaga (2010)
and Solanki et al. (2012).
The relative proportion of AG-3 PT, AG-5, and AG-2-1 in our findings are
also supported by the findings of and Chand and Logan (1983); Campion et al. (2003);
Truter and Wehner (2004) and Woodhall et al. (2007).
AG-4 HG I was most prevalent on chilli with the frequency of 59.4%
followed by AG-2-1 (16.2%), AG-6 (10.8%), AG-3 PT (8.1%) and AG-5 (5.4%).
The predominance of AG-4 on chilli was also in line with the findings of Mikhail et
al. (2010). Bolkan and Ribeiro (1985) from Brazil, Elias-Medina et al. (1997) from
Mexico and Wu et al. (2008) from China also confirmed the predominance of R.
solani AG-4 from chilli. Reports also confirmed the association of AG-4 with root
rot of chilli by Demirci and Doken (1995); Meza-Moller et al. (2007) and (Tuncer
and Eken, 2013).
The association of AG-2-1 in our studies is in accordance with the findings
of other researchers (Tuncer and Erdiller, 1990; Tuncer and Eken, 2013). Katan and
Eshel (1974) reported AG-3 cause seedling damping-off in chilli while the
association of other AG-4, AG-5, and AG-6 was also reported by Tuncer and Eken
(2013).
143
To our knowledge, this study is the first comprehensive report of AG
composition and genetic diversity of R. solani associated potato, tomato, and chilli
diseases in the Pothohar region. R. solani AG-3 PT was most widely distributed to
all the locations surveyed for the selected crop types especially in potato and may be
a potential threat to the tomato as reported in many countries of the world. In
Pakistan, seeds of these crops are being imported since many years for commercial
cultivation. Majority of the AG-2-1, AG-2-2, and AG-4 HGI isolates were found in
the three districts; Attock, Rawalpindi, and Jhelum. The localized occurrence of
these two AGs could be attributed to the susceptible preceding crops.
144
CONCLUSIONS
i. Study reports status of Rhizoctoniasis with reference to our country along with
the occurrence of at least 05 anastomosis groups of R. solani on selected
solanaceous vegetable crops with various levels of intensities.
ii. Variations have been observed in the disease incidence of R. solani infection on
potato, tomato, and chilli in Pothohar region. The disease was prevalent in all
locations surveyed.
iii. AG-3 PT was found to be most prevalent and aggressive as compared to other
AGs in all potato as well as tomato growing areas while AG-4 HGI was
prevalent in chilli growing areas.
iv. The present study is the first report of Rhizoctonia solani infection on tomato
(AG-3 PT, AG-2-1) and chilli (AG-4 HGI) from Pakistan.
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RECOMMENDATIONS
i. Rhizoctonia solani is widely distributed in the potato, tomato, and chilli growing
areas of Pothohar region, stringent surveillance is needed through the country.
ii. While evolving new varieties of potato, tomato, and chilli, R. solani isolates
belonging to the reported anastomosis groups (AGs) may be used in breeding
program.
iii. The long duration of crop rotation if possible, may be adopted as to reduce the
inoculum level of R. solani in the fields especially in seed production system.
iv. Seed should be inspected at the time of sowing and only healthy seed from
approved varieties should be used.
v. Preparation and distribution of education material pertaining to R. solani
infection may be used for the awareness of this disease among the farmers.
vi. Workshops and training may be arranged to enhance the professional capability
of extension staff and empowerment of seed growers about R. solani disease
identification and its management.
146
SUMMARY
Solanaceous crops viz; potato, tomato, and chilli endure significant yield
losses owing to numerous fungal, bacterial, nematode, and viral diseases. Among
soil-borne fungal pathogens, Rhizoctonia solani is a devastating pathogen causing
black scurf, damping-off, stem canker, and root rot on these crops. R. solani is a
ubiquitous soil-borne fungus with broad host range and diverse genetic makeup. It is
a species complex of several anastomosis groups that exhibit DNA base sequence
homology and/or affinities. So for now, thirteen anastomosis groups have been
internationally reported however, infection of R. solani AGs on solanaceous
vegetables with reference to our country has not been reported. Therefore, the present
study was designed to investigate the occurrence of different AGs of R. solani on
potato, tomato, and chilli together with molecular characterization of R. solani
isolates representing different AGs.
For the above purpose, a survey of different locations of districts
Rawalpindi, Jhelum, Attock, Chakwal, and Federal Capital Islamabad was
conducted. For potato, field survey in an X-plus manner was done and infected tubers
were collected, while for tomato, and chilli positive sampling was done to collect
symptomatic plants. Maximum mean disease incidence on potato was recorded in
Attock (37.4%) followed by Islamabad (35.8%), Jhelum (32.1%), Jhelum (31.76%)
and Rawalpindi (30.5%) while minimum mean disease incidence was recorded in
district Chakwal (20.2%). Maximum mean disease incidence on potato was observed
in Islamabad (38.7%) followed by Attock (36.3%), Rawalpindi (34.9%) and Chakwal
(29.6%) while minimum in district Jhelum (27.5%). Mean disease incidence on Chilli
was maximum in Attock (30.9%), followed by Islamabad and Jhelum (29.5%), while
147
minimum in district Chakwal and Rawalpindi (27.3%).
A total of 1321 samples were collected during 02 years survey (2014-15 and
2015-16 cropping season). The pathogen was isolated on water agar and cultures
were maintained on Malt Extract Agar (MEA) medium. At least 63 isolates from
potato, 67 from tomato, and 58 isolates were recovered from chilli. Fungal colonies
isolated on MEA were light grey to brown in colour with plentiful mycelial growth
and branched hyphae. A septum was always present in the branch of hyphae near the
originating point with a slight constriction at the branch. The hyphal distance
between two septa ranged between 66.7 to 150.3µm and hyphal diameter from 5.1
to 8.2µm. Majority of the isolates produced rough sclerotia and were superficially
present on the sclerotia. No conidia or conidiophores were observed from cultures
on MEA. All isolates were multinucleate when subjected to DAPI stain. Based on
these morphological characteristics of fungal hyphae, isolates were identified as R.
solani. Recovered isolates subjected to pathogenicity test confirmed 47 isolates from
potato, 42 from tomato, and 37 from chilli were highly virulent.
Restriction analysis of PCR-amplified ribosomal DNA was used for rapid
characterization of R. solani isolates at AG level. DNA using the standard protocol
of Omniprep for fungi extraction kit (G-Biosciences) was extracted and was
subjected to PCR amplification with two sets of primers; RS1R/RS4 and
ITS1/ITS4.Cleaned PCR product amplified with RS1 and RS4 was subjected to
restriction digest with four discriminant enzymes (MseI, AvaII, HincII, and MunI).
Restriction patterns were used to assign AGs to each isolate. RFLP analysis revealed
recovered isolates belong to; AG-2-1, AG-2-2, AG-3 PT, AG-4 HG I, AG-5, and
AG-6. Isolates were further paired with tester strains of R. solani anastomosis groups
148
(AGs). All isolates confirmed the results of AG composition as of RFLP analysis.
ITS region of ribosomal DNA was amplified using ITS1/ITS4 primers and
subsequent sequencing which had a 99-100% identity with already reported AGs.
PCR-RFLP, hyphal anastomosis reactions, and phylogenetic analysis
revealed isolates recovered from potato belong to AG-3 PT (76.5%), AG-5 (8.5%),
AG-4 HG I (4.2%), AG-2-1 (6.3%) and AG-2-2 (4.2%). AG-3 PT was widely
distributed to major potato growing areas while others were confined to distinct
locations. Isolates recovered from tomato belong to AG-3 PT (64.2%), AG-2-1
(14.2%), AG-2-2 (9.5%), AG-5 (7.1%) and AG-4-HGI (4.7%). AG-3 PT was widely
distributed to major tomato growing areas followed by AG-2-1 while other groups
were confined to distinct locations. Similarly, AG-4 HGI (59.4%) was also widely
distributed to Chilli growing areas. Other AGs recovered from Chilli belong to AG-2-
1 (16.2%), AG-6 (10.8%), AG-3 PT (8.1%) and AG-5 (5.4%).
The study reports AG composition, genetic variability, virulence, and
molecular characterization of Pakistani isolates of R. solani. This is the first report
on the occurrence of at least 05 different AGs of R. solani on solanaceous vegetables
from Pakistan. Findings will provide the basis for further understanding infection of
different AGs on differential hosts which will help in the development of novel
control strategies for management of Rhizoctonia diseases on solanaceous
vegetables and other economic crops being infected by this fungus.
149
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