anastomosis group typing of rhizoctonia solani kÜhn

i

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

ii

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

vii

In the name of Allah,

the beneficent the merciful

viii

Dedicated to

my Beloved

Father Mother

Brothers & Sisters

Whatever I am and hope to be my life, I love you …..

ix

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

x

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

xi

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

xii

5 DISCUSSION 133

CONCLUSIONS 144

RECOMMENDATIONS 145

SUMMARY 146

LITERATURE CITED 149

xiii

LIST OF TABLES

Table No. Page

2.1 The composition of the primers for amplification of nuclear

internal transcribed spacer (ITS) region.

26

2.2 Host ranges and associated disease symptoms, of Rhizoctonia

solani belonging to different anastomosis groups (AGs).

29

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.

41

3.2 Total number of samples for Rhizoctonia solani infection on

potato, tomato, and chilli from different locations of each

district.

43

3.3 Disease rating scale to record stem, stolon, and tuber infection

on potato.

47

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.

57

3.7 GenBank accessions of Rhizoctonia solani reference isolates

used in this study.

61

4.1 Disease prevalence and incidence percentage of Rhizoctonia

solani on potato in various areas/locations of the districts of

Pothohar region.

64

xiv


solani on tomato in various areas/locations of the districts of

Pothohar region.

66


solani on chilli in various areas/locations of the districts of

Pothohar region.

69

4.4 Details of Rhizoctonia solani isolates recovered from potato,

tomato, and chilli symptomatic plant samples.

72

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.

78

4.6 Morphological characterization of sixty-seven isolates of

Rhizoctonia solani recovered from portions of diseased tomato



86

4.7 Morphological characterization of fifty-eight isolates of

Rhizoctonia solani recovered from portions of diseased chilli



97

4.8 Rhizoctonia solani anastomosis groups (AGs) assigned using

PCR-RFLP and hyphal anastomosis interaction.

117

xv

4.9 Type isolates of Rhizoctonia solani representing different

anastomosis groups (AGs) and 07 unknown isolates used for

molecular characterization.

124

4.10 Frequency of Rhizoctonia solani anastomosis groups (AGs)

recovered from potato symptomatic samples, collected from

different locations of Pothohar region

131


recovered from portions of diseased potato samples.

131


recovered from tomato symptomatic samples, collected from

different locations of Pothohar region.

132


recovered from chilli symptomatic samples, collected from

different locations of Pothohar region

132

xvi

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.

37

3.2 Map of Pothohar region showing district Attock, Chakwal,

Jhelum, Rawalpindi and Islamabad.

38

3.3 (a) Symptomatic potato plant infected with Rhizoctonia solani. 39

3.2 (b) Diseased potato tubers showing visible sclerotia of

Rhizoctonia solani.

39

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.

48

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.

55

3.8 Restriction patterns revealed by RFLP analysis of internal

transcribed spacers sequences of Rhizoctonia solani with Ava

II+Hinc II and MunI.

56

4.1 District wise, mean disease incidence of Rhizoctonia solani

infection on potato, tomato, and chilli.

70

4.2 Morphological diversity of Rhizoctonia solani isolates

incubated on malt extract agar (MEA) medium

73

xvii

4.3 Cultural appearance of Rhizoctonia solani isolate (RWPT5)

under the microscope

73

4.4 Inter septal distance of Rhizoctonia solani isolate (JHEP2)

recovered from potato

74

4.5 Hyphal diameter of Rhizoctonia solani isolate (ATKP7)

recovered from potato.

74

4.6 Nuclear number testing of Rhizoctonia solani isolate

(RWPP9) recovered from potato.

75

4.7 Distribution of Rhizoctonia solani isolates recovered from

potato on the basis of hyphal length/ inter septal distance

81


potato on the basis of hyphal diameter.

82


potato on the basis of number of sclerotia.

83


potato on the basis of sclerotia texture.

84


potato on the basis of sclerotia topography.

85


tomato on the basis of hyphal length/ inter septal distance.

90


tomato on the basis of hyphal diameter.

91


tomato on the basis of number of sclerotia.

92

xviii


tomato on the basis of sclerotia texture.

93


tomato on the basis of sclerotia topography.

94


chilli on the basis of hyphal length/ inter septal distance.

100


chilli on the basis of hyphal diameter.

101


chilli on the basis of number of sclerotia.

102


chilli on the basis of sclerotia texture.

103


chilli on the basis of sclerotia topography.

104

4.22 Disease index of Rhizoctonia solani infection on potato (cv.

Desiree).

105

4.23 Pathogenicity determination of sixty-three Rhizoctonia solani

isolates on Potato (cv. Desiree).

106

4.24 Disease index of Rhizoctonia solani infection on tomato (cv.

Rio Grande).

108

4.25 Pathogenicity determination of sixty-seven Rhizoctonia

solani isolates on tomato (cv. Rio Grande).

109

4.26 Disease index of Rhizoctonia solani infection on tomato (cv.

Sanam).

111

xix

4.27 Pathogenicity determination of fifty-eight Rhizoctonia solani

isolates on chilli (cv. Sanam).

112

4.28 PCR-RFLP restriction patterns revealed by discriminating

enzymes (MseI, AvaII+HincII, and MunI).

118

4.29 Phylogenetic analysis of Rhizoctonia solani isolates infecting

potato in Pothohar region.

127


tomato in Pothohar region.

128


tomato in Pothohar region.

129

xx

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

xxi

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

xxii

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

xxiii

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

[email protected]

xxiv

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

xxv

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.

1

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).

2

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

3

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

4

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

5

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


Wheat Crown lesion, damping-off Demirci (1998)


Potato Stem, stolon and root canker, black scurf Woodhall et al. (2008)

Tomato Foot rot Misawa and Kuninaga (2010)


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)



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


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)


AG-5 Ogoshi (1975) Soybean Root rot, damping-off Nelson et al. (1996)

Apple Root rot Mazzola (1997)



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)


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)


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



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.


57

Table 3.6: RFLP types revealed by the restriction analysis of ITS sequences among

Rhizoctonia solani.


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



Surveyed


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


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%.


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



Surveyed


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,


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,



JHET16, JHET17, JHET18, JHET19

JHEC1, JHEC2, JHEC3, JHEC4, JHEC5,


JHEC11, JHEC12, JHEC13

Att

ock

ATKP1, ATKP2, ATKP3, ATKP4, ATKP5,


ATKP11, ATKP12, ATKP13, ATKP14,


ATKP19, ATKP20, ATKP21

ATKT1, ATKT2, ATKT3, ATKT4, ATKT5,


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.


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


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


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,


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,


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,


RWPP14

Rough





ATKP21, CHKP1, CHKP2, ISBP1, ISBP2,

ISBP3, ISBP4, JHEP1, JHEP2, JHEP3,

JHEP4, JHEP7, JHEP12, JHEP13, RWPP4,


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,


RWPP14

Immersed


ATKP17, CHKP2, ISBP1, ISBP5, JHEP1,

JHEP2, JHEP3, JHEP5, RWPP2, RWPP4,


Superficial




ATKP20, ATKP21, CHKP1, CHKP3,

ISBP2, ISBP3, ISBP4, ISBP6, JHEP4,


JHEP13, RWPP5, RWPP6, RWPP8,


RWPP19

Figure 4.11: Distribution of Rhizoctonia solani isolates recovered from potato on the basis of sclerotia topography.

27%

57%

16%


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


Isolate Colony Colour Constriction

Colony

Diameter

cm

Hyphal

Length

µm

Hyphal

Width

µm

Nuclear

Condition

Sclerotia


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



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


ATKT7 Medium brown Present 7.3 89.6 6.2 Multinucleate 44 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


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%


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%


diameter

Narrow (<5.5µm) Moderate (5.6 - 7.0µm)

Wide (>7.0µm)

92


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,


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%


sclerotia


93



ATKT13, ATKT16, JHET9

Rough



RWPT15, CHKT1, CHKT3, CHKT4,

CHKT5, CHKT6, CHKT8, CHKT10,



ATKT12, ATKT14, ATKT15, JHET1,


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%


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



RWPT14, RWPT15, CHKT2, CHKT3,


CHKT10, ATKT2, ATKT3, ATKT5,


ATKT12, ATKT14, ATKT17, JHET1,


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%


topography

Immersed Superficial Sclerotia not present

95


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


Isolate Colony Colour Constriction

Colony

Diameter

cm

Hyphal

Length

µm

Hyphal

Width

µm

Nuclear

Condition

Sclerotia


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



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%


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%


diameter

Narrow (< 5.5 µm) Moderate (< 5.6 - 7.0 µm)

Wide (> 7.0 µm)

102

No Sclerotia


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%


sclerotia


103




Rough

RWPC2, RWPC3, RWPC5, RWPC6,

RWPC9, RWPC13, RWPC14, CHKC1,

CHKC4, CHKC5, CHKC6, CHKC9,


ATKC16, JHEC2, JHEC3, JHEC4, JHEC6,


ISBC1, ISBC2, RWPC7, RWPC8, RWPC10,

RWPC11, RWPC12, CHKC7

Smooth CHKC8, CHKC11, ATKC2, ATKC3,



JHEC1, JHEC5, JHEC12, ISBC4

Figure 4.20: Distribution of Rhizoctonia solani isolates recovered from chilli on the basis of sclerotia texture.

14%

59%

27%


texture

Not present Rough Smooth

104

Sclerotia not present RWPC1, RWPC4, CHKC2, CHKC3,


Immersed

CHKC8, CHKC11, ATKC2, ATKC3,



JHEC1, JHEC5, JHEC12, ISBC4

Superficial





ATKC16, JHEC2, JHEC3, JHEC4, JHEC6,


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%


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,




CHKP1, CHKP2, CHKP3, ISBP1, ISBP3,

ISBP4, ISBP5, ISBP7, JHEP1, JHEP2,


JHEP11, JHEP13, RWPP1, RWPP2,




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


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,


RWPT14, CHKT1, CHKT2, CHKT4,




ATKT16, ATKT17, JHET1, JHET2, JHET4,


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%


Tomato (cv. Rio Grande)

Avirulent Moderately 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


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,




CHKC10, ATKC2, ATKC3, ATKC4,


ATKC13, ATKC15, ATKC16, JHEC2,


JHEC13, ISBC1, ISBC3, ISBC4

Figure 4.27: Pathogenicity determination of fifty-eight Rhizoctonia solani isolates on chilli (cv. Sanam).

7%

14%

15%

64%


Tomato (cv. Sanam)

Avirulent Moderately 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.

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.

129

Figure 4.31: Phylogenetic analysis using ITS region of Rhizoctonia solani isolates infecting chilli in Pothohar region drawn using Mega 7.

130

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).

131

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


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

132


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


Jhelum 2 0 8 1 1 12

Chakwal 1 1 4 0 1 7


Total 6 4 27 2 3 42


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


Jhelum 1 1 5 0 0 10

Chakwal 1 0 3 1 1 6


Total 6 3 22 2 4 37

133

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

134

(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

136

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

137

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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