Characterization of Alternaria solani and Molecular Mapping of … · 2018-12-29 ·...

Characterization of Alternaria solani and Molecular

Mapping of QTLs for Early Blight Resistance

in Tomato

ABSTRACT

of THESIS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

Doctor of Philosophy

in

Mycology and Plant Pathology

Submitted by

Sanwar Mal Yadav

Supervisor

Dr. Vineeta Singh

DEPARTMENT OF MYCOLOGY AND PLANT PATHOLOGY INSTITUTE OF AGRICULTURAL SCIENCES

BANARAS HINDU UNIVERSITY VARANASI-221005

INDIA

ID. No.PM-1046 2014 Enrolment No.330118

Copyright@Institute of Agricultural Sciences, Banaras Hindu University,

Varanasi, India, 2014. All rights reserved.

UNDERTAKING FROM THE CANDIDATE

I, Sanwar Mal Yadav, a Ph.D. scholar (ID No. PM-1046)

registered and pursued my research work under the supervision of

Dr. Vineeta Singh for full term and hereby submitting the thesis

entitled “Characterization of Alternaria solani and molecular

mapping of QTLs for early blight resistance in tomato” for the

award of Ph.D. degree.

(Sanwar Mal Yadav)

CANDIDATE’S DECLARATION

I, Sanwar Mal Yadav certify that the work embodied in this

Ph.D. thesis is my own bonafied work carried out by me under the

supervision of Dr. Vineeta Singh from the period of September 2010 to

September 2014 at Banaras Hindu University. The matter embodied

in this Ph.D. thesis has not been submitted for the award of any other

degree/diploma.

I, declare that I have faithfully acknowledged, given credit to

and referred to the research workers wherever their works have been

cited in the text and the body of the thesis. I further certify that I have

not willfully lifted up some other’s work, para, text, data, results, etc.

reported in the journals, books, magazines, reports, dissertations,

thesis etc. or available at web-sites and included them in this Ph.D.

thesis and cited as my own work.

Date:

Place: Varanasi (Sanwar Mal Yadav)

CERTIFICATE FROM THE SUPERVISOR

This is to certify that the above statement made by the

candidate is correct to the best of my/our knowledge.

Dr. Vineeta Singh

Assistant Professor (Supervisor)

(Signature of the HOD/Coordinator of School with seal)

COURSE/COMPREHENSIVE EXAMINATION COMPLETION CERTIFICATE

This is to certify that Sri Sanwar Mal Yadav, a bonafide

research scholar of this department, has successfully completed the

course work and comprehensive examination requirement which is a

part of his Ph.D. programme.

Date: Place: Varanasi (Signature of the Head of the Department)

PRE-SUBMISSION SEMINAR COMPLETION CERTIFICATE

This is to certify that Sri Sanwar Mal Yadav, a bonafide

research scholar of this department, has successfully completed the

pre-submission seminar requirement which is a part of his Ph.D.

programme.

Date:

Place: Varanasi (Signature of the Head of the Department)

COPYRIGHT TRANSFER CERTIFICATE

Title of the thesis: “Characterization of Alternaria solani and molecular mapping of QTLs for early blight

resistance in tomato”.

Candidate’s Name: Sanwar Mal Yadav

COPYRIGHT TRANSFER

The undersigned assigns to the Banaras Hindu University all

rights under the copyright that may exist in and for the above thesis submitted for the award of the Ph.D. degree.

Signature of the candidate

Note: However, the author may reproduce or authorize others to

reproduce material extracted verbatim from the thesis of derivative of the thesis for author’s personal use provided that the University’s copyright notice is indicated.

Characterization of Alternaria solani and molecular mapping of

QTLs for early blight resistance in tomato

Thesis submitted in partial fulfilment of the requirements for the award of the degree of

Doctor of Philosophy (Agriculture)

in

Mycology and Plant Pathology

2014

By

Sanwar Mal Yadav

APPROVED BY

Supervisor: Dr. (Mrs.) Vineeta Singh

Assistant Professor

Department of Mycology and Plant Pathology

Internal Subject Expert: Dr. Ramesh Chand

Professor


External Subject Expert: Dr. V. K. Mishra

Professor

Department of Genetics and Plant Breeding

DRC Nominee: Dr. R. K. Singh

Assistant Professor


External Examiner:

ACKNOWLEDGEMENT

Enrolling for a Ph.D. programme in the eponymous holy place Varanasi was indeed a watershed experience in my life for which I am eternally indebted to the Banaras Hindu University and its founder Pandit Madan Mohan Malviya Ji for his life time sacrifice and efforts in establishing such a great temple of learning for the cause of millions of students like me.

The choice of the topic for my research was largely driven by my interest and intellectual commitment to my alma mater, which had turnout to be a eventful accomplishment in my career. At this juncture, it is my prime duty to remember and express my love and indebtedness to those who extended their kind help, moral support and co-operation directly or indirectly in successful completion of this research work. No words adequately express my feeling for them yet these lines are not exaggeration but feelings, which come straight from my heart.

It is really highly fortunate that I go the great opportunity to work under the golden heart personality, Dr. Vineeta Singh, Assistant Professor, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Chairman of my advisory committee. It is beyond my apprehension to express my deep sense of gratitude to her for her skilful and noble guidance, vast experience, unrivalled knowledge, incessant inspiration, stimulating discussion, healthy criticism, unstinting moral support and above all her scientific and humanitarian approach which had left in a everlasting impression on the canvass of my mind and helped me in completing this research work in time. Throughout my study period, she sustained me with her parental care and thoughtful advice which helped me to cope with many problematic situations. It had indeed been my privilege to work under her supervision and I will remain indebted to her.

I greatly acknowledge the technical guidance, encouragement and moral support received from my advisory committee Dr. Ramesh Chand, Professor, Department of Mycology and Plant Pathology, Dr. V. K. Mishra, Professor, Department of Genetics and Plant Breeding and Dr. R. K. Singh, Assistant Professor, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi for every arena of difficulty.

I vest to place on record my heartfelt thanks to Dr. Major Singh, Principal Scientist, Indian Institute of Vegetable Research, Shahanshahpur (Jakhini), Varanasi and Dr. Asha Sinha, Professor & Head, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi for their co-operation during my research work.

Sincerely most recognition are also vocal to Dr. H. B. Singh, Dr. J. S. Srivastava, Dr. S. S. Vaish, Dr. R. K. Singh, Dr. B. K. Sharma, Dr. R. C. Ram, Dr. S. K. Singh and Miss. Ankita Sarkar, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi during the course of study and research work.

I owe my sincere thanks to all the non-teaching staff of the Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi for their keen interest taken in the work providing the necessary and timely research facilities, inspiration and suggestions throughout the work.

I have no words to express my heartfelt gratitude to all my seniors namely, Mr. Sanjay Goswami, Dr. Umesh Singh, Mr. Rai Ajay Kumar, Dr. L. P. Balai, Mr. Prabhat Kumar, Mr. Chatterpal, Dr. O. P. Yadav,. Ms. Smita, Mr. J. M. Sutaliya, Mr. Yogendra Kumar Ghilotiya, Dr. Ved Prakash Rai, Dr. Ashutosh Kumar Rai, Dr. Anil Kumar, Dr. Bineet Sharma, Dr. Pardeep Patel, Dr. Wakar Ahamad Ansari, Mr. Akhilesh kumar Yadav, Dr. Govind Rai, Mr. Rajeev Choudhary, Dr. Moin Khan, Dr. Raju Verma and Dr. Babu Lal Meena.

Juniors namely, Ms. Sunita Yadav, Survesh Singh, Mr. Aanandi Lal Jat, Mr. Manoj Kumar Yadav, Mr. Ratul Moni Ram, Ms. Chinmayee Mohapatra, Mr. Amit Yadav, Mr. Harit, Mr. Saket Kumar, Ms. Arpita, Mr. Jagjit, Mr. Sonu, Mr. Sandeep Choudhary, Mr. Ramawtar Bajiya, Mr. Pradeep Yadav, Mr. Pardeep Joliya, Mr. Ramswaroop Yadav, Mr. Dhara Singh Yadav, Mr. Birbal, Mr. Ram Singh Yadav, Mr. Hawa Singh Yadav, Mr. Abhisekh Singh, Mr. Aanand Rai, Ms. Anjali, Ms. Pallavi, Mr. Krishna, Mr. Animesh Singh, Mr. Vijay Pal, Mr. Santosh Kumar, Mr. Ram Ishwar Yadav, Mr. Akhilesh Vishwakarma, Mr. Mukhram, Mr. Balu Ram, Mr. S.L. Sirvi, Mr. Lakhapati Singh, Mr. Rohit Yadav, Mr. Mukesh Yadav and Ramchandra Yadav for their encouragement and help rendered to me.

Friends namely, Dr. Chandra Prakash, Mr. Imtiyaz Ahmad, Mr. Vivek Pratap Singh, Mr. Chetan Singh Panwar, Mr. Sanjay Kumar Viswakarma, Mr. Himansu Singh, Mr. Saurabh Singh, Dr. Ram Niwas Dhaka, Mr. Umendra Singh, Dr. Sunil Kumar Chongtham, Mr. Hari Ram Choudhary, Dr. D. L. Yadav, Mr. Hukma Ram Choudhary, Mr. Sonu Ram Gujar, Mr. Ram Niwash Yadav, Mr. Bijen, Mr. Vikram Yadav and many well wisher for their everlasting, vibrant, encouragement, co-operation, moral support and valuable assistant at all times with cheerful smiling gestures.

This account will be incomplete without revealing my adorable father Mr. Banshi Dhar Yadav, mother Mrs. Rampyari Devi, elder brother Mr. Sharwan Kumar, Mr. Sita Ram and Mr. Shankar Lal, elder sister Ms. Manbhari and Ms. Sita, younger brother Mr. Lal Chand and younger sister Ms. Santosh for their inspiration, encouragement and sacrifice to fulfil my higher academic achievement.

Love and adorance that had always been showered by family members Nand Kishor Ashok, Mahesh, Suresh, Shankar, Sanju, Komal, Bablu, Guddhu, Harshita, Vishu and my son Krishn and daughter Alka have energized to mount the climax of this exploration.

My personal triumphs and tribulations in achieving me to self-sufficiency to the committed work is not just possible without the active participatiation, pleasant co-operation and affection of my wife Miss. Prem Yadav. But for the blessings by the “God Krishna, Shiva, Khatu Shyam, Bala Ji & Ganesh” without which this piece of work would not have been crafted into the present shape.

Date: ……….. Place: Varanasi (Sanwar Mal Yadav)

Contents Chapter Title Page(s)

No.

1. Introduction 1-7

2. Review of Literature 8-51

3. Materials and Methods 52-77

4. Experimental Findings 78-139

5. Discussion 140-150

6. Summary and Conclusion 151-157

Bibliography i-xxi

Appendices i-xxvii

ABBREVIATIONS AND ACRONYMS

% Per cent

@ At the rate of

µm Micrometer

0C Degree Celsius

A. solani Alternaria solani

ANOVA Analysis of Variance

AUDPC Area Under Disease Progress Curve

BOD Biological Oxygen Demand

CAM Czapek’s Agar Medium

CBM Czapek’s Broth Medium

CD Critical difference

CI Chloroform Isoamyl alcohol

cm Centimetre

CMA Corn Meal Agar

CMB Corn Meal Broth

CODEX Coefficient of Disease Index

CTAB Cetyl Trimethyl Amonium Bromide

CV Coefficient of variation

DAI Days After Inoculation

DAP Days After Planting

DAT Days After Transplanting

DNA Deoxyribo Nucleic Acid

dNTP Deoxyribonucleotide Triphosphate

EB Early Blight

et al. et allii ; and coworkers

Et Br Ethidium Bromide

Fig. Figure

g Gram

ha Hectare

hrs Hours

i.e. That is

IIVR Indian Institute of Vegetable Research

Kg/cm2 Kilogram per centimeter square

m meter

MEA Malt Extract Agar

MEB Malt Extract Broth

mg Milligram

min Minutes

ml Milliliter

mM milli Molar

mm Millimeter

MR Moderately Resistant

MS Moderately Susceptible

MT Metric tone

OMB Oat meal broth

PCI Phenol Chloroform Isoamyl alcohol

PCR Polymerase Chain Reaction

PDA Potato Dextrose Agar

PDI Percentage Disease Index

pH Potential of hydrogen ion

QTL Quantitative Trait Loci

R Resistant

RH Relative humidity

RILs Recombinant Inbred Lines

S Susceptible

S. Em Standard Error of mean

Sr. No. Serial Number

SSR Simple Sequence Repeat

TAE Tris Accetate Extraction buffer

Taq Pol Thermus aquaticus polymerase

Tr. No. Treatment Number

U.P. Uttar Pradesh

viz., Namely

LIST OF TABLES

Table No.

Title Page No.

3.1 Description of disease scale (0-5). 62

3.2 Description of disease scale (0-9). 63

3.3 Details of cultural operations carried out during experiment. 65




3.7 List of the various components of PCR master mixture (25 μl). 73

3.8 The schedule of temperature and duration programmed for PCR

amplification using SNP primers.

74

4.1 List of Alternaria solani collected from different areas of India. 79

4.2 Mycelial growth of A. solani on Potato Dextrose Agar medium

incubated at 25±2ºC.

81

4.3 Cultural and morphological variability of different isolates of A. solani incubated at 25±2ºC.

83

4.4 Pathogenic virulence of A. solani isolates on two

susceptible cultivars of tomato.

85

4.5 Response of 45 and 60 days old tomato cv. Co-3 for early blight

development.

86

4.6 Effect of different solid media on mycelial growth and sporulation of A. solani (Asv-2) incubated at 25 ± 2 ºC.

88

4.7 Effect of different broth on mycelial growth and sporulation of A. solani incubated at 25 ±2ºC.

88

4.8 Effect of different temperature on mycelial growth and sporulation of A. solani isolate (Asv-2) on PDA medium.

90

4.9 Effect of different temperature on mycelial growth and sporulation of A. solani isolate (Asv-2) on PDB medium.

90

4.10 Effect of different pH range with potato dextrose agar on mycelial growth and sporulation of A. solani (Asv-2) incubated at 25±2 ºC.

91

4.11 Effect of different pH range with potato dextrose broth on mycelial growth and sporulation of A. solani (Asv-2) incubated at

25 ± 2 ºC.

92

4.12 Effect of different substrates on A. solani colonization and spore

production at 30 days after incubation at 25 ± 2 ºC.

93

Contd…

Table No.

Title Page No.

4.13 Sporulation efficiency of different isolates on sorghum grains at 10, 20 and 30 DAI (Days After Incubation) at 25 ± 2 ºC.

94

4.14 Effect of sorghum grain and water ratio on A. solani colonization

and spore production at 10, 20 and 30 DAI (Days After Incubation) at 25 ± 2 ºC.

94

4.15 Effect of UV light on spore yield of A. solani incubated at potato

dextrose agar for 12 days at 25 ± 2ºC.

96

4.16 Effect of light and darkness on sporulation of A. solani incubated

at potato dextrose agar for 12 days at 25 ± 2 ºC. 96

4.17 Standardization of inoculation technique for development of A. solani on tomato plants.

97

4.18 Storage effect on spore viability and inoculum quality of A. solani

on sorghum grains.

98

4.19 Effect of spore concentration of A. solani on tomato for early

blight development.

99

4.20 Plant growth type, days to 50 percent flowering after

transplanting, days to fruit setting, days to senescence, AUDPC and Host Reaction of 701 germplasm lines of tomato to A. solani

infection under natural condition in the year 2011-12.

101

4.21 PDI value at 42 days after inoculation (DAI), AUDPC and Host Reaction of 79 determinate core lines of tomato after inoculation with the isolate (Asv-2) of A. solani in the year 2012-13.

103-104

4.22 PDI, AUDPC and Host Reaction of 161 indeterminate core lines of tomato after inoculation with the isolate (Asv-2) of A. solani in the

year 2012-13.

105-107

4.23 Summary of disease reaction of 79 determinate and 161

indeterminate tomato core set lines based on AUDPC, calculated

on the basis of host reaction obtained after inoculation with the isolate (Asv-2) of A. solani in the year 2012-13.

108

4.24 PDI, AUDPC and Host Reaction of determinate 74 core lines of tomato after inoculation with the isolate (Asv-2) of A. solani in the

year 2013-14.

110-111

4.25 PDI Value at 56 DAI, AUDPC and Host Reaction of 152

indeterminate core lines of tomato after inoculation with the isolate (Asv-2) of A. solani in the year 2013-14.

112-114

4.26 Summary of disease reaction of 74 determinate and 152

indeterminate tomato core set lines based on AUDPC, calculated on the basis of host reaction obtained after inoculation with the isolate (Asv-2) of A. solani in the year 2013-14.

115

4.27 PDI, AUDPC and Host Reaction of Recombinant Inbred Lines (Co-

3 × EC-520061) of tomato after inoculation with the isolate (Asv-2) of A. solani in the year 2012-13.

117-119

Contd…

Table No.

Title Page No.

4.28 Summary of disease reaction of RILs (F7 generation) obtained from cross (Co-3 × EC-520061) based on AUDPC value obtained after inoculation with the isolate (Asv-2) of A. solani in the year

2012-13.

120

4.29 PDI, AUDPC and Host Reaction of Recombinant Inbred Lines (Co-

3 × EC-520061) of Tomato after inoculation with the isolate (Asv-2) of A. solani in the year 2013-14.

125-127

4.30 Summary of disease reaction of RILs (F8 generation) obtained

from cross (Co-3 × EC-520061) based on AUDPC value obtained after inoculation with the isolate (Asv-2) of A. solani in the year

2013-14.

128

4.31 Correlation coefficents among phenotypic data under poly house

and field conditions depicted by 151 RILs of Co-3 × EC-520061.

131

4.32 25 informative SSR markers (SGN database) screened in our

polymorphic survey.

131

4.33 Simple interval mapping with final multiple regression analysis

included 3 QTLs for early blight disease resistance.

134

4.34 Composite interval mapping with final multiple regression

analysis included 2 QTLs for early blight disease resistance.

134

LIST OF FIGURES

Figure

No.

TITLE Page No.

4.1 Categorization of germplasm lines of tomato on the basis of AUDPC value (Year 2011- 12) calculated on the basis of host reaction to A. solani, under natural conditions.

101

4.2 Categorization of core set tomato lines based on AUDPC (Year 2012-13) obtained after artificial inoculation under field

conditions.

103

4.3 Categorization of core set tomato lines based on AUDPC (Year 2013-14) obtained after artificial inoculation under field

conditions.

115

4.4 Categorization of RILs (Co-3 × EC-520061) based on AUDPC after artificial inoculation under poly house and field

conditions in the year 2012-13.

120

4.5 Frequency distribution of percent disease index for Early blight in RILs (F7 generation) obtained after inoculation with the

isolate Asv-2 in the year 2012-13.

123

4.6 Categorization of RILs (Co-3 × EC-520061) based on AUDPC after artificial inoculation under poly house and field


128

4.7 Frequency distribution of percent disease index for Early blight in RILs (F8 generation) obtained after inoculation with the

isolate Asv-2 in the year 2013-14.

132

4.8 Linkage map of the S. lycopersicum × S. habrochaites in F7

population showing position of QTLs. The number to the left of

each chromosome indicate map distance (in centi Morgans)

between linked markers. To the right of each chromosome

indicate name of markers, which are linked with particular

chromosome

135

4.9 LOD curve of simple interval mapping of chromosome 2 of

tomato for early blight resistance. It is showing two peaks (at

about 33 and 46.25 cM distance). Ist peak is showing between SSR40 and SSR356 markers (22 cM distance between both

markers) with 13.95 LOD scores. IInd peak is showing between

SSR356 and SSR605 markers (4.5 cM distance between both

markers) with 4.97 LOD scores.

136

Contd…

Figure

No.

TITLE Page No.

4.10 LOD curve of simple interval mapping of chromosome 4 of

tomato for early blight resistance. There is a single peak (at

about 18.5 cM distance). Peak is showing between SSR72 and SSR603 markers (37 cM distance between both markers) with

3.15 LOD scores.

137

4.11 LOD curve of composite interval mapping of chromosome 2 of tomato for early blight resistance. There is a single peak (at

about 33 cM distance). Peak is showing between SSR40 and

SSR356 markers (22 cM distance between both markers) with

6.96 LOD scores

138

4.12 LOD curve of composite interval mapping of chromosome 6 of

tomato for early blight resistance. There is a single peak (at

about 40.5 cM distance). Peak is showing between SSR304 and

SSR45 markers (10 cM distance between both markers) with 3.2 LOD scores.

139

LIST OF PLATES

Plate

No. Title

After

Page No.

3.1 Description of disease scale (0-9) with per cent leaf area infection.

63

4.1 Early blight symptoms on leafs and fruits of tomato. 78

4.2 Cultural growth of virulent isolates of A. solani on PDA, 12 days

after incubation at 25 ± 2 ºC. 84

4.3 Spore production of A. solani on sorghum grains. 91

4.4 Disease reaction of early blight of tomato caused by Alternaria solani (R=Resistant; MR= Moderately Resistant; MS= Moderately

Susceptible; S= Susceptible).

116

4.5 View of experiment for phenotyping of RILs of tomato against early blight under poly house conditions.

116

4.6 Parental screening of RILs (Co-3 × EC-520061) with SSR

primers; The gel shows amplification of DNA of susceptible

parent Co-3 (P1) and resistant parent EC-520061 (P2) obtained

with 7 SSR primers.

130

4.7 Genotyping done in F7 RIL population obtained with SSR 603. L:

1kb DNA Ladder; P1: Parent 1 (Co-3); P2: Parent 2 (EC-520061);

other lanes: F7 RILs.

130

4.8 Genotyping done in F7 RIL population obtained with SSR 72. L: 1kb DNA Ladder; P1: Parent 1 (Co-3); P2: Parent 2 (EC-520061);


130




130




130

PREFACE

Since this thesis is written as the thesis for doctoral degree in Mycology and Plant Pathology and primarily aimed to collection,

isolation and purification of pathogen causing early blight of tomato from different parts of the country; assessment of cultural, morphological and pathogenic variability amongst tomato isolates of

Alternaria solani; standardization of conidial production as well as inoculation technique for Alternaria solani; phenotyping and

genotyping of RILs (F6 generation) using informative/polymorphic markers and mapping of QTLs for early blight resistance in tomato.

The findings of the present study hold a great potential in the field of plant pathology to standardize a ten-point scale (0-9) for early blight scoring and Phenotyping of tomato germplasm lines and RILs against

early blight disease; development of grain based inoculum production technique and its standardization for mass sporulation in A. solani that is required for poly house as well as field inoculation of tomato genotypes (RILs) during phenotyping for disease resistance; standardization of optimum spore load of A. solani for inoculation of

tomato genotypes both under glass house and field conditions; Identification of QTLs for early blight resistance in tomato. The work

described in this thesis was carried out at Department of Mycology and Plant Pathology, Institute of Agricultural Sciencesand Indian Institute of Vegetable Research, Shahanshapur (Jakhini), Varanasi.

The work has been discussed under following chapters.

Chapter I the introduction part which provides much of the

general background and an overview of Information need.

Chapter II the review of literature deals with the findings of

research related work done by scientists from time to time in the past.

Chapter III the research methodology deals with the methodology and systematic procedure adopted for carrying out the

research work.

Chapter IV the results and discussion deals with the results of

this research work. The findings have been discussed with appropriate reasons and support.

Chapter V the summary and conclusion gives brief description

of the results of the investigation and the conclusion drawn from this investigation.

Bibliography deals with citation which has been consulted during the course of investigation.

This is a small contribution in the field of plant pathology and I

hope, tomato growerand researchers will like it.

Chapter I

INTRODUCTION

Tomato (Lycopersicon esculentum Mill, n = 12) belongs to the

family solanaceae and is one of the most remunerable and widely

grown vegetables in the world. Tomato is grown for its edible fruits,

which can be consumed either fresh or in processed form and is a

very good source of vitamins A, B, C and minerals. Tomato cultivation

has become more popular since mid nineteenth century because of its

varied climatic adaptability and high nutritive value. Tomato is being

exported in the form of whole fruits, paste and in canned form to West

Asian countries, U.K., Canada and USA. The crop is grown with an

annual rainfall of 60-150 cm. Tomato ranks third in priority after

potato and onion in India but ranks second after potato in the world.

Being the world's second most cultivated crop, with a

production estimated at 150 million tones and acreage of 5.2 million

hectares, the tomato is an indispensible vegetable crop world over

and, of course, for India. China is the world's largest producer of the

tomato (48.1 mt) followed by India (19.5 mt). Turkey, Italy, Iran,

Egypt, Brazil, Spain, Mexico and Russia are also significant producers

(Sallam et al., 2012). The leading tomato growing areas in India are

Uttar Pradesh, Maharashtra, Karnataka, Haryana, Punjab and Bihar.

The major tomato growing countries are China, USA, Italy, Turkey,

India and Egypt.

There are approximately 12 species within genus Lycopersicon

and many of them are reported to have resistance against various

biotic and abiotic stresses including disease resistance. Tomato is

susceptible to a large number of diseases. Basic and applied research

to minimize the impact of these diseases has resulted in the

Introduction

~2~

characterization of plant responses to numerous disease agents

including bacteria, fungi, viruses, nematodes, chewing insects and

abiotic stresses (Balanchard, 1992).

Early blight caused by Altenaria solani (Ellis & Martin) Sorauer,

is a major disease of tomato, adversely affecting tomato production in

many regions of India. This disease, which in severe cases can lead to

complete defoliation, is most damaging on tomato (Solanum

lycopersicum L. [Peralta et al., 2005]) in regions with heavy rainfall,

high humidity and fairly high temperatures (24-29ºC). Epidemics can

also occur in semiarid climates where frequent and prolonged night

dews occur (Rotem and Reichert, 1964). Apart from the leaf symptoms

that are known as EB, A. solani causes other symptoms on tomato

which are less economically important, including collar rot (basal

stem lesions at the seedling stage); stem lesions in the adult plant

stage and fruit rot (Walker, 1952). Yield losses up to 79% due to EB

damage were reported from Canada, India, USA, and Nigeria (Basu

1974b; Datar and Mayee 1981; Sherf and MacNab 1986; Gwary and

Nahunnaro, 1998). A coefficient of early blight disease index of 71.66

% and 78.51% loss in fruit yield has been reported under severe

epidemic (Datar and Mayee, 1981). Collar rot can cause seedling

losses in the field of about 20 to 40% (Sherf and MacNab, 1986). The

disease appears on leaves, stems, petiole, twig and fruits under

favourable conditions resulting in defoliation, drying off of twigs and

premature fruit drop and thus causing loss from 50 to 86 percent in

fruit yield (Mathur and Shekhawat, 1986).

Spores of the fungi are one of most important means of

dissemination and also used in the identification and classification of

the organism. The ability of the pathogen to survive for a long time in

the diseased plant parts, soil and on alternative/collateral hosts in

the absence of main host, determine the ability of the pathogen to

Introduction

~3~

perpetuate (Moore and Thomas, 1942; Basu, 1971 and Rands,

1917a).

Tomato was the first plant from which a "gene-for-gene" class of

R gene was cloned Pto. In total, more R genes (nine) have been

isolated from tomato than any other plant species. These include

genes conferring resistance to fungi, nematodes, aphids, bacteria and

viruses: Cf-2, Cf-4, Cf-5, Cf-9, Cf-I2, Mi/Meu1, Pto, Prfand Sw-5.

Additional R genes, some of which have been shown to function when

transferred into tomato, have been isolated from related solanaceous

species, including pepper, Bs2; potato, Gpa2 and Rx; and tobacco, N.

Significantly, many tomato R genes encode proteins that have unique

features that have not been observed in R proteins from other plant

species. For example, Pto consists of simply a protein kinase catalytic

domain, Prf contains a large N-terminal region without homology to

other known proteins, the Cf genes encode extracytoplasmic leucine-

rich repeat proteins, and the Mi1/Meu1 gene encodes resistance to

both nematodes and the potato aphid. In addition to yielding many R

genes, tomato has been used to study various plant defense responses

such as expression of "pathogenesis-related" genes, the oxidative

burst, the role of systemin in insect resistance, and signaling events

involving ethylene and jasmonic acid.

The control measures include a 3- to 5-year crop rotation,

routine applications of fungicides, and the use of disease-free

transplants (Sherf and MacNab, 1986). Fungicide treatments are

generally the most effective control measures, but are not

economically feasible in all areas of the world and may not be effective

under weather conditions favorable for epidemics (Herriot et al.,1986).

Resistant cultivars are potentially the most economical control

measure as they can extend the fungicide spray intervals while

maintaining control of the disease (Madden et al., 1978; Shtienberg et

Introduction

~4~

al., 1995; Keinath et al., 1996). The progress in EB resistance

breeding has been limited by the lack of effective resistance genes in

cultivated tomato (Vakalounakis, 1983; Poysa and Tu, 1996; Banerjee

et al., 1998 and Vloutoglou, 1999), quantitative expression and

polygenic inheritance of the resistance (Barksdale and Stoner, 1977;

Maiero et al., 1989; Nash and Gardner, 1988a; Maiero et al., 1990a

and Thirthamalappa and Lohithaswa, 2000). Sources for EB

resistance have been identified in wild relatives of tomato. Some of

these have been utilized through traditional breeding approaches but

an increased level of resistance is negatively correlated to earliness

(Nash and Gardner, 1988a; Maiero, 1989; Foolad and Lin, 2001;

Foolad et al., 2002a) and yield (Barrat and Richards, 1944). The most

resistant breeding lines and hybrid cultivars with acceptable

horticultural characteristics that are currently available have

moderate resistance to EB and are slightly later in maturity (Gardner,

1988; Gardner and Shoemaker, 1999; Gardner, 2000). Therefore,

resistant cultivars with better horticultural traits are still needed.

Classical quantitative genetic analyses have provided estimates of the

number of quantitative trait loci (QTLs) for EB resistance, average

gene action and heritabilities which provided the prospects for

progress in breeding programs based on phenotypic selection (Nash

and Gardner, 1988a; Maiero et al., 1990a and Maiero et al., 1990b).

However, such studies are unable to determine the effects of

individual genes and their locations on the tomato genome. More

recent genetic studies on EB resistance have been directed to the

mapping and characterization of QTLs determining the resistance

with the aid of molecular marker maps (Foolad et al., 2002b; Zhang et

al., 2003).

Most genetic studies on the inheritance of EB resistance using

different sources of resistance (S. lycopersicum, S. habrochaites and S.

Introduction

~5~

pimpinellifolium) arrived at the same conclusion that the resistance is

a quantitative trait which is controlled polygenically. The estimated

minimum number of controlling factors is two (Barksdale and Stoner,

1977) or three (Nash and Gardner 1988a). Analysis using quantitative

genetic methods (generation mean analysis and scaling tests) and

several sources of resistance (C1943, NC EBR-2, IHR 1939 and IHR

1816) revealed additive and dominant genetic control with the

presence of epistatic effects (Maiero, 1990a; Nash and Gardner,

1988a; Thirthamalappa and Lohithaswa, 2000). The EB resistance

genes in C1943 and 71B2 are recessive and not allelic (Barksdale and

Stoner, 1977; Maiero et al., 1989). However, in crosses of these two

resistance sources with another susceptible genotype, the F1 hybrids

were intermediate, indicating additive genetic control or partial

dominance (Maiero et al., 1989). Recessive genes have been reported

in S. lycopersicum 83602029 (Stancheva, 1991), in IHR1939 and

IHR1816 by Thirthamalappa and Lohithaswa (2000). Partially

dominant inheritance has been found in S. pimpinellifolium and S.

habrochaites (Martin and Hepperly, 1987). The line 87B187, derived

from S. habrochaites PI 390662, shared common resistance genes

with NCEBR-2, although this line was developed via C1943 from a S.

lycopersicum source (Maiero et al., 1990a).

Resistance may be difficult to transfer from wild species to

cultivated tomato since it is accompanied by unacceptable

horticultural traits including inferior fruit quality, late maturity, low-

yielding ability and indeterminate growth habit. Moreover, the

quantitative expression and polygenic inheritance of EB resistance

has limited the development of EB resistant cultivars using traditional

breeding approaches. Classical genetic studies revealed at least two

genes with additive and dominance effects and epistatic interactions

that confer resistance to EB symptoms (Barksdale and Stoner, 1977;

Introduction

~6~

Nash and Gardner, 1988; Maiero et al., 1990; Thirthamalappa and

Lohithaswa, 2000).

Some forms of plant disease resistance are genetically simple

and have been analyzed extensively by traditional methods of plant

pathology, breeding and genetics (Flor, 1955; Hulbert and

Michelmore, 1985). Quantitative trait locus (QTL) mapping is a highly

effective approach for studying genetically complex forms of plant

disease resistance. With QTL mapping, the roles of specific resistance

loci can be described, race-specificity of partial resistance genes can

be assessed, and interactions between resistance genes and plant

development can be analyzed. Outstanding examples include:

quantitative resistance to the early blight of tomato, rice blast fungus,

late blight of potato. These studies provide insights into the number of

quantitative resistance loci involved in complex disease resistance,

epistatic and environmental interactions, race-specificity of partial

resistance loci, interactions between pathogen biology, plant

development and the relationship between qualitative and quantitative

loci. QTL mapping also provides a framework for marker-assisted

selection of complex disease resistance characters and the positional

cloning of partial resistance genes (Young, 1996). Given the low to

moderate heritability estimates, a marker-aided selection approach is

potentially useful to accelerate the transfer of EB resistance genes

into new tomato cultivars. Foolad et al. (2002b) were the first to map

QTLs for EB resistance. Zhang et al. (2003) and Chaerani et. al. (2006)

also identified QTLs for early blight resistance in cross between wild

and susceptible tomato lines.

The current study is aimed at identification of QTLs for EB

resistance. Using F7 and F8 populations derived from a cross (Co-3 x

EC-520061) with EC-520061 as the donor parent we have located EB

resistance QTLs. According to our survey no study till date has been

Introduction

~7~

reported on identification of QTLs in tomato materials from India.

Therefore, keeping in view the above fact, the present study was

undertaken with following objectives:

1. Collection, isolation and purification of pathogen causing early

blight of tomato from different parts of the country.

2. Cultural, morphological and pathogenic variability amongst

tomato isolates of Alternaria solani.

3. To standardize conidial production as well as inoculation

technique for Alternaria solani.

4. Phenotyping and Genotyping of RILs (F7 generation) using

informative/polymorphic markers.

5. Mapping of QTLs for early blight resistance.

Chapter ΙΙ

REVIEW OF LITERATURE

2.1 Genus Alternaria

The genus Alternaria belongs to the sub-division

Deuteromycotina, class Hyphomycetes, family Dematiaceae. Species

of the genus are cosmopolitan, surviving both as saprophytes as well

as weak parasites. The genus is characterized by the formation of

conidia either singly or in short or longer chains and provided with

cross, longitudinal as well as oblique septa and having longer or short

beaks. The spores of these fungi occur commonly in the atmosphere

and also in soil. The teleomorphs (sexual stage) are known in a very

few species and placed in the genus Pleospora of class

Loculoascomycetes of sub-division Ascomycotina, in which sleeper-

shaped, muriform ascospores are produced in bitunicate asci.

The genus Alternaria was first recognised by Nees in 1817. In

1836, Berkeley identified the causal fungus on plants belonging to

family Brassicaceae as Macrosporium brassicae Berk, which was later

renamed as Alternaria brassicae (Berk.) Sacc. Thereafter, Elliot

studied the taxonomy of Alternaria in detail. Wiltshire pioneered the

basic studies of this group of hyphomycetes. His descriptive literature

was fundamental to the prevailing concepts of Alternaria,

Macrosporium and Stemphylium. Later, Neergaard made an extensive

study on the taxonomy, parasitism and economic significance of this

genus. The genus Alternaria encompasses a complex group of

saprophytic and pathogenic fungal species (Thomma, 2003).

Frequently, reported as allergenic, food spoilers, mycotoxicogenic,

Alternaria spp. are opportunistic fungi associated with mycosis in

Review of Literature

~9~

animals and humans, and destructive plant pathogens (Rotem, 1994;

Thomma, 2003).

A. solani belongs to the Fungi Imperfecti (Deuteromycotina) in

the class Hyphomycetes and order Pleosporales (Agrios, 2005). Shahi

and Shyam (1993) isolated A. solani and A. alternata f. sp. lycopersici

from tomato plants in Himachal Pradesh, India. Dhal et al. (1997)

observed the association of A. alternata with blossom end rot of

tomatoes for the first time in Orissa, India.

Alternaria spp. from a heterogeneous group of saprophytic and

plant pathogenic fungi widespread in temperate and tropical regions.

However, the relationship between evolutionary processes and genetic

diversity with epidemics is unknown for several plant-pathogenic

Alternaria spp. Species of the genus are ubiquitous and are reported

to occur in different ecosystems and geographic regions, such as

Antarctic soils (Malosso et al., 2006), deserts and the tropics

(Grishkan et al., 2007).

The conidia are dark muriform, pale golden or olivaceous

brown, smooth and usually 150–300 µm in length and 15–19 µm

thick in the broadest part, with 9–11 transverse septa and 1–4

longitudinal or oblique septa; sometimes branched 2.5–5 lm thick

tapering gradually (Ellis, 1971). The mycelium consisted of septate,

branched, light brown hyphae, which turned darker with age. The

conidiophores were short 50 to 90 μm and dark coloured. Conidia

were 120-296 × 12-20 μm in size, beaked, muriform dark coloured

and borne singly. However in culture they formed short chains (Bose

and Som, 1986). According to Singh (1987) the conidia contained 5-10

transverse septa and 1-5 longitudinal septa.


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The conidial morphology of A. solani isolated from blight

infected leaf samples of tomato from 18 localities in and around

Kanpur, Uttar Pradesh, India was studied. The conidia of the isolates

were muriform and dark brown. The conidial length varied from 175.6

to 270.5 µm, whereas the width ranged from 12.5 to 16.5 micro m.

The isolates generally produced beaked conidia, although unbeaked

conidia were also observed. The beak was flexuous, brown and

sometimes branched. The beak was 47.0-65.5 micro m long and 2.5-

5.0 micro m wide, tapering gradually. The number of septa per

conidium was 5-11 (Shahid, 2002).

Conidia were found 12-25 x 120-296 µm, beaked, muriform,

dark in colour, solitary or in chains of 2 (in pure culture), with 9-11

cross-cutting septa and with a few longitudinal or oblique septa

(Schiopu, 2008). The conidia varied in length, breadth, beak size,

septation and in hyphal width. Maximum growth and sporulation was

recorded in Sabouraud's agar followed by PDA supplemented with

CaCO3 by ASB2 isolate (Naik et al., 2010).

2.2 Disease Cycle

Under free moisture or near-saturated humidity at a wide range

of temperatures (8 – 32 °C), conidia germinate to produce one or more

germ tubes. These subsequently penetrate the host epidermal cells

directly by means of appressoria or they enter through stomata or

wounds by hyphal growth (Sherf and MacNab, 1986; Perez and

Martinez, 1995; Agrios, 2005).

Penetration can occur at temperatures between 10 and 25°C

(Sherf and MacNab, 1986). Host colonization is facilitated by enzymes

(cellulases, pectin, methyl galacturonase) that degrade the host cell

wall and by a number of toxins that kill host cells and enable the


~11~

pathogen to derive nutrients from the dead cells (Rotem, 1994).

Lesions become visible 2–3 days after infection, spore production

occurs 3–5 days later and relatively short disease cycle allows for

polycyclic infection (Sherf and MacNab, 1986). The fungus survives

between crops as mycelia or conidia in soil, plant debris and seed

(Sherf and MacNab, 1986).

Chlamydospores can also serve as survival structures (Basu,

1974a; Patterson, 1991). Therefore, the life cycle of A. solani includes

soil and seed as well as air-borne stages, making the pathogen

difficult to control by means of rotation and sanitation. The main

hosts of A. solani are solanaceous crops including tomato, potato,

eggplant and pepper (Neergaard, 1945; Ellis and Gibson, 1975).

2.3 Survey and Losses of Alternaria solani

Among the different fungal diseases infecting tomato crop, early

blight caused by Alternaria solani was most destructive causing heavy

losses in yield of tomato sometimes as high as 78 per cent of fruit loss

(Datar and Mayee, 1981). Karla and Sohi (1985) conducted regular

surveys in Chandigarh markets during 1980-84 which revealed 78

post harvest diseases on 36 hosts including tomato caused by

different fungi and maximum damage was attributed to Fusarium spp.

and Alternaria spp. among the 23 genera isolated.

Chinoko and Nagvi (1989) carried out survey on fungi

associated with post-harvest rot of tomato. Among 243 fungal isolates

from eight marketing sites in Logos and Ohio states, A. solani (Fries)

Keissler and Aspergillus niger van Teighem were most frequently

isolated and were the pathogenic ones.

Fontern (1993) when surveyed 14 nurseries and 67 fields of

tomato in Cameroon, A. solani was the most destructive among eleven


~12~

diseases both on leaves and fruits. Five hundred fungal isolates

obtained from ripened tomato fruits in a market of Faisabad,

Pakistan, seven species were identified of which A. alternata was the

most common (Akhter et al., 1994).

A survey of tomato diseases and disorders in the main tomato

growing regions of South Africa was conducted between 1992-1995.

Early blight, caused by A. solani was the most prevalent leaf disease

followed by the bacterial leaf spots (Uys et al., 1996). The incidence of

fungal rots in tomato fruits from vegetable markets and stores ranged

from 0.5 - 19.7 per cent and the spoilage was due to Alternaria,

Fusarium and Phytophthora spp. (Sharma, 1994). Among 11 fungal

species on tomato fruits collected from different markets of Egypt,

Alternaria alternata was recorded to the extent of 52.7 per cent (Abdul

Mallek et al., 1995).

According to Ramgiry et al. (1997) Alternaria solani and

Pencillium rostatum were the most frequent causal agents of tomato

decay in fields and vegetable markets in Jhabua of Madhya Pradesh.

Bhatt et al. (2000) recorded that the Alternaria alternata is the causal

agent of leaf blight disease of tomato, capsicum and spinach, which

was the first confirmed record of this fungus from Kuma on hills of

Uttar Pradesh.

The survey of field and post harvest diseases of hybrid and desi

cultivars of tomatoes in West Bengal, India, revealed that among

fungal diseases, blight caused by Alternaria sp. was the most

predominant with the crop loss in the field ranging from 70 - 100 %

(Kanjilal et al., 2000). Prasad (2002) conducted a field survey in

northern districts of Karnataka viz., Raichur, Gulbarga and Dharwad

during Kharif 2001 and recorded a percent disease index of 28.60 to

65.36. Abhinandan et al. (2004) conducted a survey in Punjab, India


~13~

during 2001 and reported that maximum disease intensity of 49.5

percent was observed at the Tapa District and minimum disease

intensity of 8.2 per cent was observed at the Babakala District.

The pathogen Alternaria dauci f. sp. solani causes the disease

called leaf blight or tomato alternariosis and may occur in any

growing stage on leaves, stalks and fruits with a damage degree of up

to 48-50 percent (Schiopu, 2008). Early blight incited by Alternaria

solani was found to be major disease of tomato under agro-climatic

conditions of Konkan. Roving survey conducted in Rabi season, 2004-

2005 revealed that, early blight disease intensity in Raigad district

ranged between 20.78 to 42.30 per cent and 35.12 to 55.75 per cent

in Thane district (Kamble, et al., 2009).

2.4 Symptomatology of Early Blight

According to Locke (1949) the early blight on tomato was

characterized by the appearance of brown to dark leathery necrotic

spots first on leaflets producing target board effect. Ramakrishnan

et al., (1971) observed cankerous spots on tomato stems. They were

specially injurious when they occurred at the juncture of the stem

and side branches. Collar rot, another symptom on tomato occurred

as stem lesions on seedlings at soil line extending above and below

that point to form cankers which resulted in girdling of the plants

(Basu, 1971 and McCarter et al., 1976).

Datar and Mayee (1981) showed that A. solani could attack

fruits in the green and ripe stages. According to Datar and Mayee

(1986) the early blight disease of potato was characterized by the

appearance of brown to dark brown coloured necrotic spots.

Appearance of concentric rings inside the spots produced target board

effect. Singh (1987) reported that the spots were oval to angular in


~14~

shape measuring up to 0.3-0.4 cm in diameter and usually with a

chlorotic zone around the spot.

All aboveground parts of plants can be infected by Alternaria

solani and various names have been given for different symptoms,

which often leads to confusion. The first symptoms of EB are small,

dark, necrotic lesions that usually appear on the older leaves and

spread upward as the plants become older (Sherf and MacNab, 1986).

Seedlings are weakened and can die when the stem is completely

girdled by the lesion. On the main stem and side branches of adult

plants, the fungus causes small, dark, slightly sunken areas that

enlarge to form dark brown, elongated spots, which occasionally have

concentric rings like those on the leaves. These spots are scattered

along the stem and branches (Walker, 1952). Some authors make

no distinction between collar rot and stem lesions (Gardner, 1990). In

older literature, collar rot and stem lesions are sometimes referred to

as stem cankers (Barksdale and Stoner, 1977), a term that is

currently reserved for the disease caused by A. alternata (Sherf and

MacNab 1986). On green or ripe fruits, dark, velvety, sunken spots

may occur at the stem end. These spots occasionally develop from

mycelia extending from stem lesions and can reach a considerable

size and also develop distinct concentric markings (Sherf and

MacNab, 1986). Semi-ripe fruits are more susceptible than ripe ones

(Mehta et al., 1975). Heavily infected fruits frequently drop before they

mature. On susceptible genotypes, the calyx and blossom may also be

infected (Pandey et al., 2003).

2.5 Pathogenicity

Andrus et al. (1945) confirmed the pathogenicity on tomato by

using mycelial fragments of A. solani as inoculum. Locke (1949) used

blended mycelial fragments of A. solani for puncture inoculation.


~15~

Brock (1950), Henning and Alexander (1959) used the suspension of

mycelial fragments of A. solani to inoculate the leaves of field or green

house grown plants. Barksdale (1968) and Dhiman et al. (1980) used

suspension containing 20000 spores/ml distilled water for proving

pathogenicity of early blight of tomato caused by A. solani. Further,

they atomized the culture suspension on three leaf stage seedlings at

the rate of 30 ml per seedling for successful inoculation. A research

trial was carried out for determination of pathogens causing damping-

off and their pathogenicity in tomato seedbeds in Ankara Province

(Ayas, Beypazar and Nallhan Districts) in 2003. Two hundred eleven

samples of tomato seedlings, thought to be infected with damping off

were collected from 42 seedbeds. Fusarium spp., Pythium spp.,

Rhizoctonia solani, Alternaria solani and Aspergillus spp. were isolated

from all collected tomato seedlings and prove their pathogenicity

(Askn and Katrcoglu, 2008).

2.6 Cultural, Morphological, Pathogenic, Physiological and Genetic Variability of Alternaria solani.

2.6.1 Cultural variability

Kaul and Saxena (1988) noted the cultural variability of A.

solani isolates on PDA and classified into 4 distinct cultural groups

based on types of growth, colony colour, colour of the substrate and

growth rate. A. solani culture on PDA medium produced hyphae,

which were grey white or grey brown in colour and even yellow

pigment was secreted by some isolates. The pathogenicity varied

significantly among isolates (Tong Yunhui et al., 1994). Babu et al.

(2000b) described the variability in cultural characteristics for isolates

of A. solani, the incitant of early blight of tomato collected from 20

locations in Madurai and Dindigul districts of Tamil Nadu.


~16~

Some researchers have defined races according to cultural

characteristics of various dimensions of spores and virulence (Bonde,

1929; Neergard, 1945). Pandey et al. (2010) observed that fastest

radial growth of A. solani in the So isolate and slowest in the Ka

isolate on PDA, while isolates Dh, Ba-1 and Va-3 were recorded to be

faster in growth on ASM, V-8 juice agar and V-8 juice agar (synthetic)

medium. Significant differences in colony growth were observed

among isolates of A. solani (Kumar and Srivastava, 2013).

The eleven isolates of A. solani designated as So, Dh, Sh, Va-5,

Ka, Ma, Hy, Ba-1, My, Va-3 and Mi were collected from different

agroclimatic conditions and these isolates were characterized for

cultural variations (Pandey et al., 2010). Kaul and Saxena (1988)

reported cultural variability of A. solani isolates on potato dextrose

agar and classified them into four distinct cultural groups based on

type of growth, colony colour, colour of the substrate and growth rate.

Tong-Yunhui et al., 1994 cultured A. solani on PSA medium for

study of biology and pathogenicity on tomato plants. The hyphae were

grey white or grey brown in colour and yellow pigments were secreted

by some isolates. Colonies of 5 day culture had a diameter of 54-70

mm. The optimum temperature for fungal growth was 23-28°C and

pH 6-8 was found to be optimum. Pathogenicity varied significantly

among isolates. There were distinct variations among the colony

characteristics of the isolates. Two isolates (AS-1 and AS-3) showed

light brown coloured colony; one isolate AS-2 had dark brown and two

(AS-4 and AS-5) appeared as greyish white in colour. One isolate (AS6)

was light grey coloured. Pigmentation of the substrate was almost

similar to the colour of the mycelium (Arunakumara, 2006).


~17~

2.6.1.1 Culture media

Several reports in the literature showed PDA as a good medium

for the growth and sporulation of A. solani (Bonde, 1929; Neergaard,

1945 and Rotem, 1966). Barksdale (1968) reported that potato

dextrose agar and lima bean agar were the best media for growth and

sporulation of A. solani.

Cheema et al. (1976) reported that, isolates of A. citri grow

mostly rapidly on PDA followed by yeast extract agar and Czapek’s

Dox agar. According to Mohapatra et al. (1977) maximum growth of A.

solani was recorded on potato dextrose broth followed by Richard’s

medium, Czapek’s dox and oat medium.

Joshi (1981) observed maximum growth of A. gomphrenae on

both potato dextrose agar and Richard’s agar. Mahabaleshwarappa

(1981) recorded maximum growth of A. carthami on potato dextrose

agar followed by Richard’s agar. Fencelli and Kimati (1990) found that

growth and sporulation of A. tenuis was sparse in Czapek’s Dox

synthetic medium, but was high in semi-synthetic and natural carrot

leaf media. Mazzonetto et al. (1996) found that PDA + NaCl were the

best for mycelial growth, followed by PDA, bean leaf and tomato juice.

PDA + NaCl was also the best medium for conidial production,

followed by tomato juice, PDA and bean leaf.

Pria et al. (1997) reported that among 3 media (MSTO, V8 agar

and PDA) tested, MSTO media was found to be best for mycelial

growth and sporulation of Colletotrichum lindemuthianum,

Phaeoisariopsis griseola and Alternaria spp. Padmanabhan and

Narayanaswamy (1977) reported that A. macrospora attained

maximum growth after fourteen days of incubation in Czapek’s Dox

medium. Desai (1979) reported progressive increment in the growth of


~18~

A. macrospora on PDA with its peak on the twelfth day and decreased

thereafter. Mahabaleswarappa (1981) recorded that maximum growth

of A. carthami on PDA. Sandhya (1996) reported that A. alternate

attained maximum growth after 16 days of incubation in Czapek’s

Dox medium.

Kulkarni (1998) reported that A. solani attained maximum

growth after 9th and 7th days of incubation in Richard’s and potato

dextrose agar medium respectively. Pawar and Patel (1957) observed,

maltose, xylose and arabinose as good sources of carbon for the

growth of A. ricini. Chaturvedi (1966) reported that A. alternata

utilized fructose, lactose, maltose and arabinose effectively. Gupta et

al. (1970) reported that among the eight monosaccharides tested

fructose supported maximum growth and sporulation of A. brassicae.

Further, they found that, mannitol supported good growth, while the

growth was poor on maltose. Bhandari and Singh (1976) observed

that fructose and mannose supported better growth of A. triticina

compared to ribose, arabinose, xylose, sucrose, glucose, maltose and

lactose. Goyal (1977) found that growth of A. alternata was maximum

on maltose followed by sucrose, starch, glucose and maltose and poor

growth was noticed on galactose and mannitol.

Padmanabhan and Narayanaswamy (1977) reported that A.

macrospora made good growth by using fructose as the carbon source.

Mohapatra et al. (1977) recorded maximum growth of A. sesami on

mannithol followed by lactose and starch. Mathur and Sarboy (1977)

reported that pentose sugars poorly supported the growth of A.

alternata while sucrose supported maximum growth. Rane and Patel

(1956) found ammonium nitrate, potassium nitrate, sodium nitrate

and peptone as the best nitrogen sources for the growth of A.

macrospora. Hasija (1970) reported that A. citri and A. alternata

metabolised three major sources of nitrogen viz., nitrate, ammonical


~19~

and organic form of nitrogen. According to Bhandari and Singh (1976)

among the nitrogen sources, asparagine and glutamine supported

good growth of A. triticina.

Goyal (1977) found that A. alternata utilized nitrate nitrogen

more efficiently than ammonical nitrogen. The maximum growth was

recorded on potassium nitrate followed by sodium nitrate and good

sporulation was found in sodium nitrate followed by potassium

nitrate and calcium nitrate. Padmanabhan and Narayanaswamy

(1977) reported that sodium nitrate and urea supported good growth

of A. macrospora among various inorganic and organic nitrogen

sources tested. Mohapatra et al. (1977) observed that ammonical form

of nitrogen was superior to nitrate form and ammonium chloride was

the best followed by ammonium oxalate in Alternaria sesame in

sesame host. Nine solid media were used to study the growth and

morphological characteristics of A. solani. Maximum growth was

observed on PDA, followed by corn meal agar which may be attributed

to complex nature of natural media supporting good fungal growth

(Arunakumara, 2006).

2.6.1.2 Cultural pH

Rane and Patel (1956) reported that A. macrospora grew well

between pH 4.8 and 5.2. Good growth and sporulation of A. recini

occurred between pH 4.8 and 5.5. Pawar and Patel, 1957; Tandon,

1961 observed that the optimum pH for the growth of A. alternata was

5.0. Taber et al. (1968) reported that A. raphani grew well in the pH

range between 4.8 and 7.2. Hasija (1970) made study on pH

requirement of A. citri and A. alternata and reported that growth of A.

citri was best between pH 4.4 and 6.4, moderate at pH 7.4 and poor at

pH 2.7, 3.4 and 8.0. However A. alternata made good growth from pH

5.4 to 7.4, moderate growth at pH 4.4 and poor growth at pH 2.7, 3.4


~20~

and 8.0 optimum pH for the growth of A. alternata was found to be

6.6 (Verma, 1970). Samuel and Govindaswamy (1972) demonstrated

that good mycelial growth and sporualtion of A. solani was between

pH 4.0 to 8.0 and pH 5.0 was the best for mycelia growth and pH 7.0

for sporulation.

Padmanabhan and Narayanaswamy (1977) reported that pH

range of 5.0 to 7.0 was optimum for the growth of A. macrospora.

Mohapatra et al. (1977) reported that A. sesami grows well in the pH

range of 3-10 and best at 4.5 pH. Mathur and Sarboy (1977) found

maximum growth and sporulation of A. alternata at pH 5.5. Gemawat

and Ghosh (1980) observed that A. solani was capable of growing on

wide range of pH (4.0 to 9.5) and maximum growth and sporulation

were observed at a pH of 6.3. According to Mahabaleswarappa (1981)

A. carthami made fairly good growth between pH range of 5.3 to 8.1

and maximum growth of the fungus was recorded at pH 6.0. Growth

of A. solani was tested at nine different levels of pH and the maximum

dry mycelial weight and mycelial growth were observed at a pH range

of 6.5-7.0 and least at pH 8 in both the isolates (Arunakumara, 2006).

2.6.1.3 Temperature

Effect of temperature on growth of A. solani the temperature

requirement for A. solani was found to be in the range of 5-35°C

(Bonde, 1929; Verma, 1970 and Gemawat and Ghosh, 1980). Kaul

and Saxena (1988) reported that the maximum growth of five isolates

of A. solani was at 25°C followed by 20, 15, 10 and 5°C with least

growth at 35°C. A. solani germinated most rapidly in darkness when

ambient temperature was near 25°C (Stevenson and Packer, 1988).

Growth of A. solani was tested at eight different levels of temperatures

and the maximum dry mycelial weight and mycelial growth were

observed at a temperature of 25°C and least at temperature 5°C

(Arunakumara, 2006).


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2.6.2 Morphological variability

The morphological variations of Alternaria species were

described by Joly and later he divided these in three sections and

proposed a simple key for identification and determination of the most

common species (Arunakumara, 2006). Perez and Martinez (1995)

reported that variability in 4 isolates of A. solani with respect to

morphological characters like colony growth, colony diameter,

mycelial colour, colony texture, pigmentation and conidia size on

medium, differed between isolates and it was concluded that A. solani

exhibited variability.

Morphological variability in colour of colony, substrate colour,

margin of colony, topography of colony, colony growth, sporulation

was observed among six isolates of A. solani. Two isolates namely AS1

(Arabhavi) and AS3 (Amminabhavi) produced good sporulation in

culture media (Arunakumara, 2006). The eleven isolates of A. solani

designated as So, Dh, Sh, Va-5, Ka, Ma, Hy, Ba-1, My, Va-3 and Mi

were collected from different agroclimatic conditions and these

isolates were characterized for morphological variations (Pandey et al.,

2010). Bonde (1929) and Neergaard (1945) classified A. solani into

conidial, mycelial and intermediate types of isolates.

2.6.3 Pathogenic variability

Pathogenic variability among isolates of A. solani has given rise

to claims of the existence of races, although this remains unproven

(Rotem, 1966). Castro et al. (2000) studied the variability of A. solani

under green house conditions based on the inoculation of 7 isolates

on 14 tomato genotypes. The results showed that all the isolates

differed in their virulence on 14 tomato genotypes, demonstrating the

existence of high level of variability in the fungus. The variability of A.

solani studies under greenhouse conditions based on the inoculation


~22~

by several isolates on 14 tomato genotypes, resulted in the finding

that all isolates differed from each other.




pathogenic variations (Pandey et al. 2008). The variability of Alternaria

solani was studied under greenhouse conditions based on the

inoculation of seven isolates on fourteen tomato genotypes.

Inoculation was carried out by spraying the plants 35 days after

germination, with a suspension of 1.25 x 103 conidia/ml. Analysis of

phenotypic stability showed that the PI isolate and the CNPH 417

genotype were the most stable ones, behaving as least virulent and

the most resistant in relation to the other isolates and genotypes

(Castro et al., 2000).

2.6.4 Physiological variability

A. solani belongs to the large-spored group within the genus

Alternaria, which is characterized by separate conidia borne singly on

simple conidiophores (Neergaard, 1945). The conidia of A. solani are

muriform and beaked (Neergaard, 1945; Ellis and Gibson, 1975). Like

other members of the genus Alternaria, A. solani has transverse and

longitudinal septate conidia, multinucleate cells and dark-coloured

(melanized) cells (Rotem, 1994). Melanin gives protection against

adverse environmental conditions including resistance to antagonistic

microbes and their hydrolytic enzymes (Rotem, 1994).

2.6.5 Genetic variability

Weir and Huff (1998) reported high level of genetic diversity

among the 69 isolates of tomato black mould pathogen by RAPD

analysis of genetic variation among isolates of A. solani of tomato and

potato. The RAPD profiling of 55 isolates of Alternaria spp. belonging


~23~

to 13 small-spored species and three large-spored were carried out

using 12 arbitrary primers. The large spored species viz. A. solani, A.

porri and A. lecunthemi were differentiated from small spored species

species by a genetic distance of 0.44 and from each other by the

genetic distance of 0.25, indicating that RAPD analysis can be used to

analyse the phylogenetic relationship of Alternaria spp. (Darakov,

1995).




molecular variations (Pandey et al., 2010). A high genetic diversity

was detected among isolates of A. solani originating from the United

States, South Africa, Cuba, Brazil, Turkey, Greece, Canada, China

and Russia based on vegetative compatibility groups (Vander Waals et

al., 2001) and molecular markers [isozymes, random amplified

polymorphic DNA markers (RAPDs), random amplified microsatellite

markers (RAMs), and amplified fragment length polymorphisms

(AFLPs); Petrunak and Christ 1991; Weir and Huff 1998; Martinez et

al., 2004; Vander Waals et al., 2004]. A. solani isolates cluster

according to country, indicating some degree of genetic isolation. In

contrast, isolates from the same country are not distinctly separated

by geographical origin (Petrunak and Christ, 1992; Weir and Huff,

1998; Martinez et al., 2004; Vander Waals et al., 2004). This can be

ascribed to short or medium distance dispersal of the airborne spores

and movement of plant material within the countries (Weir and Huff,

1998; Vander Waals et al., 2004). In many cases, isolates originating

from tomato and potato clustered according to their hosts based on

RAPD (Weir and Huff, 1998) and AFLP markers (Martinez et al., 2004),

suggesting host specialization. Organ specificity was reported to occur

among Bulgarian isolates by Stancheva (1990), but has not been


~24~

described by other authors. Associations of molecular markers with

variability in physiology, morphology and virulence are not known. So

far, conclusive evidence for the existence of physiological races is

lacking. Physiological races are defined based on differential host

specificity (Mehrotra and Areja, 1990 and Schlegel, 2003). Therefore,

the report of the presence of physiological races of A. solani (Bonde,

1929) is not correct according to the current definition because it

described them in terms of variability in physiological, morphological

and ecological characters in in vitro culture.

Henning and Alexander (1959) characterized isolates on tomato

and related species with quantitative variation in resistance. Some of

these isolates, which showed cultural differences, appeared to be host

specific, but the pattern of infection was not consistent between

experiments. This was attributed to heterogeneity of the host lines

and the unstable nature of the isolate cultures (Henning and

Alexander, 1959). Similarly, Castro et al. (2000) could not

demonstrate consistent host-specific reactions of isolates.

Heterokaryosis could be the driving force for genetic variation in A.

solani (Stall, 1958). Heterokaryosis is the occurrence of genetically

different nuclei in the same cells. This can be the result of hyphal

anastomosis, a process observed in A. solani (Stall and Alexander,

1957 and Stall, 1958). After establishment of heterokaryosis, this

state may be maintained or lost during further cell divisions. Also

nuclear migration is possible through septal pores between cells of

conidia, conidiophores, mycelia, and cells connecting these

structures, allowing dissociation of unlike nuclei leading to

homokaryosis and conversely, also to the reestablishment of

heterokaryosis (Stall, 1958). Therefore, even isolates obtained from

single conidia and hyphal tips are genetically unstable. In their

studies, Stall and Alexander (1957) observed frequent occurrence of


~25~

anastomosis but failed to obtain heterokaryosis as indicated by the

absence of segregation of cultural types. The ability of A. solani to

maintain high genetic variability allows it to react quickly to changing

environments. For example, a recent study demonstrated that isolates

in the mid Western United States have become less sensitive to a

fungicide resulting in significant losses of disease in glasshouse

cultures (Pasche et al., 2004). The high genetic diversity and high

degree of gene flow within countries could break down genetic

resistance in the host; this possibility has been advanced as one of

the reasons for the absence of potato cultivars with complete

resistance to A. solani in South Africa (Vander Waals et al., 2004).

2.7 Conidial Production and Inoculation Technique of Alternaria solani.

2.7.1 Sporulation of Alternaria solani

Rands (1917) produced spores by growing A. solani on potato-

dextrose agar (PDA) for 10 to 12 days, shredding the cultures and

then allowing them to dry in the sun. Charton (1953) and Ludwig et

al. (1962) reported sporulation of A. solani on different media due to

dehydration.

Padhi and Rath (1973) described the effect of sunlight,

nutrition, pH and temperature on sporulation of A. solani. Shahin and

Shepard (1978) reported that A. solani produced spores on water agar

plus calcium carbonate at 18 °C. Vakalounakis (1983a) recorded that

large masses of conidia were formed when mycelial disc of A. solani

was inoculated on to sterile solanaceous leaf discs placed on water

agar plates at 20°C.

Zhu et al. (1985) showed that A. solani sporulated profusely on

corn meal agar when illuminated with fluorescent lamp for 8 hour at


~26~

18°C. Benlioglu and Delen (1996) reported that the media containing

tomato juice was found to be the most suitable with 6 days dark at

23°C; 12 hours light at 26°C and 12 hours dark at 18°C incubation.

Liuchienhui et al. (1997) reported that sporulation was optimal when

mycelia were homogenized and transferred to a second V-8 juice agar

medium after 10 days at 28°C on a similar medium and sporulation

increased in V-8 medium with mycelial wounding at lower

temperature i.e. 18°C.

A. solani does not sporulate readily in culture if left

undisturbed. Factors such as mycelial wounding, temperature and

light affect the formation of spores. Various techniques to induce

sporulation of the fungus have been described (McCallan & Chan,

1944; Barksdale, 1969; Aragaki, 1961; Lukens 1963; Lukens &

Horsfall, 1969; Bashi, 1979). Barksdale (1969); McCallan & Chan

(1944) used a similar technique A. solani was grown on PDA, the

cultures scraped and then placed in moisture chambers in the sun or

under UV light.

Bashi (1979) indicated that spore production is induced by

inhibition of vegetative growth of the fungus. Growing the fungus on

PDA, cutting the culture into 1 mm2 blocks and placing them on a

CaCO3 medium in the dark at room temperature has also been found

to induce spore formation (Shahin and Shepard, 1979). A technique

developed by Barksdale (1969) involved the growing of A. solani on

reconstituted lima bean agar at 23°C. During the day, cultures were

placed under indirect sunlight or for 8 hours under cool-white

fluorescent light. The mycelium of one week old cultures was scraped,

the lids removed and the dishes placed upside down on a rack, 10

mm above the surface, under the same light and temperature

conditions. Although conidiophores are easily damaged by UV light

(Tandon, 1961), their formation is initiated by light. Lukens (1965)


~27~

observed that blue light inhibits sporulation, but red light can reverse

this inhibition. Sporulation in vitro requires a short exposure to near-

UV light. This induces conidiophores to produce conidia, which then

need dark conditions to complete formation (Lukens and Horsfall,

1969). Conidia do not form at temperatures above 20 – 30 °C (Tandon,

1961; Lukens, 1963; and Lilly & Barnett, 1951). An interrupted

wetting period may often be more conducive to producing spores than

a continuous wet period. In vivo, sporulation of the pathogen is

affected by the state of the host and tends to accelerate with an

increase in necrotic tissue formation and a decrease in photosynthesis

(Bashi & Rotem, 1974; Rotem and Bashi 1969).

Prasad and Dutt (1971) found maximum sporulation in six days

old culture with 24 hours of exposure to sunlight, than culture

exposed to incandescent electric light or infrared light. Growth and

sporulation of A. solani was sparse in Czapek’s dox synthetic medium

but the same was high in case of semi-synthetic and natural carrot

leaf media when exposed to light (Fencelli and Kimati, 1990).

Sporulation is inhibited by sugars, which promote vegetative

growth and even the production of conidiophores (Rotem, 1994).

Sporulation in the field requires at least two days. Conidiophores are

produced during wet nights. Light and dryness the next day induce

the production of conidia, which are then formed during the second

wet night (Bashi,1969).

Maximum growth and sporulation was recorded in Sabouraud's

agar followed by PDA supplemented with CaCO3 by ASB-2 isolate.

Temperature of 25 degrees C was optimum with ASB-2 and ASG-3

recording higher growth. ASG3 produced higher growth and

sporulation at 100% RH whereas ASB-2 sporulated even at 65% RH.

Maximum growth and sporulation was obtained at 12 h of alternate


~28~

light and darkness followed by continuous darkness. The pathogenic

reaction indicated ASB-2 as more virulent than others (Naik et al.,

2010).

A procedure to induce sporulation based on mycelial wounding

and dehydration was adapted and validated. Best results were

obtained when fungal colonies were grown in V8 medium at 25

degrees C in the dark with agitation for seven days; the mycelium

mass was ground, poured into potato dextrose agar (pH 6.5) in plates,

and incubated at 25±2 degrees C under near ultraviolet light and 12

h-photoperiod (Rodrigues et al., 2010) .

2.7.2 Inoculum concentrations

The concentration of 104 conidia/ml made it possible to

distinguish resistant and susceptible genotypes of the tomato (Castro

et al., 2000). Vloutoglou (1999) evaluated the effect of inoculum

concentrations, wetness duration and plant age on the development of

tomato early blight in relation to host susceptibility under controlled

environmental condition. The inoculum concentration of 8 × 103

conidia/ml of A. solani gave best result. Castro et al. (2000) studied

the effect of inoculum concentration of Alternaria solani (0.625, 1.25,

5 and 10 × 103 conidia/ml) against resistance to early blight on

tomato cultivars like Santa, Clara (susceptible), CNPH-353 and

NCEBR (resistant) under green house condition. Evaluation was

carried out at 5, 6 and 7 days after inoculation, recording the number

of lesions/cm2 level of coalescence of lesions and percent of necrotic

leaf tissue. Regression analysis showed a linear effect for the

characteristics evaluated for all genotypes studied. Although distinct

lesions were observed at 6.25 ×102 and 1.25 × 103 conidia/ml, this

concentration was not able distinguished resistant and susceptible


~29~

genotypes. The concentration 104 conidia/ml was made possible to

distinguish resistant and susceptible genotypes.

2.8 Screening Methods

In field tests, large populations can be assessed under normal

growth conditions during the whole life cycle of the plants. Artificial

inoculation by (repeated) spraying of inoculums and/or the use of

spreader rows is required to enhance natural infection and to obtain

uniform disease pressure. Prior to inoculation it is often necessary to

prevent or eradicate foliar diseases by scheduled fungicide sprays

(Nash and Gardner, 1988a). Glasshouse tests using spray inoculation

of a spore suspension on seedlings are widely used since the

establishment of efficient screening and spore inoculum production

techniques by Barksdale (1969). Reliable and repeatable techniques

for large-scale screening are necessary to identify host plant

resistance. Techniques have been developed for EB resistance

screening under field and glasshouse conditions.

A droplet inoculation method was used for evaluation of tomato

resistance to early blight. In this test method, leaflets are inoculated

with small droplets of a spore suspension in either water or a 0.1%

agar solution. Early blight resistance was evaluated based on lesion

size. The droplet method better discriminated the level of resistance

(P<0.001) for a range of spore densities in comparison with the more

commonly used spray inoculation method (Chaerani, 2007a).

The EB lesions resulting from spray inoculation are scattered

on the leaves so that the observer must estimate the combined area of

all lesions on all leaflets as a percentage of the total leaf area. Disease

estimates are rapid but rather subjective. Another disadvantage of the

spray inoculation method is that the inoculum may not be uniformly

distributed on the leaves. Furthermore, the method is not sensitive


~30~

enough to discriminate moderately resistant plants from those that

are susceptible (Gardner, 1990).

An alternative method to obtain more precise and reliable

disease readings is offered by placing individual droplets of a fungal

inoculum suspension on the leaflets. This method was first

introduced by Locke (1948) to find sources of resistance to EB.

Detached leaflets were inoculated with a mycelial suspension in a

laboratory assay and the disease reaction was evaluated using a

diagram of a graded series of lesions with known diameters (Locke,

1949). Henning and Alexander (1959) used the droplet method to

investigate the existence of A. solani races by inoculating leaflets still

attached to plants.

Nash and Gardner (1988) applied the method, which they called

point inoculation, on a whole plant assay and measured the EB lesion

diameter. EB resistance of two parents and the F1 were tested in a

glasshouse. Their results correlated well with field tests, but were

based only on a few genotypes. Leaves can be injured prior to spray

by rubbing leaf surfaces between thumb and forefingers (Poysa and

Tu, 1996). Glasshouse or controlled environment chamber

evaluations of young plants were mainly used for preliminary

selection of A. solani resistance sources from large collections

(Barksdale, 1969; Vakalounakis, 1983; Poysa and Tu, 1996;

Vloutoglou, 1999).

The glasshouse screening methods for A. solani resistance are

based on the method established by Barksdale (1969). Generally,

seedlings are spray inoculated with spores at an age of 4 to 6 weeks

(Barksdale, 1969; Marcinkowska, 1982; Nash and Gardner, 1988b;

Banerjee et al., 1998; Vloutoglou, 1999; Foolad et al., 2000).


~31~

Plants are incubated 24 hours under 100 % relative humidity

(RH) followed by 12- 16 hours of 100% RH during the night period for

5-7 days in a mist chamber, mimicking repeated nightly dew in

nature. During the day, plants are exposed to ambient RH to allow the

development of disease symptoms. A leaf wetness period of at least 4

h after inoculation was required for infection (Moore, 1942; Vloutoglou

and Kalogerakis, 2000).

Increasing this period up to 24 h induced progressively higher

EB severity, but more than 24 h humidity periods did not increase

severity further (Vloutoglou, 1999).

2.8.1 Field screening

In field tests, large populations can be assessed under normal

growth conditions during the whole life cycle of the plants. Artificial

inoculation by (repeated) spraying of inoculums and/or the use of

spreader rows is required to enhance natural infection and to obtain

uniform disease pressure. Prior to inoculation it is often necessary to

prevent or eradicate foliar diseases by scheduled fungicide sprays

(Nash and Gardner, 1988a).

EB severity in the field is assessed in terms of percent

defoliation and the average fraction of necrotic leaf area on the plant

(Horsfall and Barrat, 1945). Symptoms on the upper leaves can be

disregarded because the necrotic areas on these leaves are less than

2% of the total damage during the growing season (Basu, 1974b).

Therefore, counting the number of leaves having 75 to 100% necrotic

area on lower half of plants (Basu, 1974b), or estimating the

percentage of necrotic area in the middle third of the plant canopy

(Christ, 1991) are reliable indicators for EB severity.


~32~

Pandey et al. (2003) reported that screening under artificial

conditions was more informative than natural epidemic conditions.

Tomato cultivars CLN-2071-C, CLN-2070-A, BSS-174, and DTH-7

expressed as slow blighting against four pathogenic isolates of A.

solani. Disease intensity increased with the increasing age of plants

under the same inoculum load. The area under the disease progress

curve (AUDPC) was positively correlated with the percentage disease

index and negatively with resistance. Calculation of the apparent

infection rate (r) was more informative for natural epidemics than for

artificial conditions.

Vijaya et al. (2003) evaluated tomato cultivars viz; DT-39, BT-

116-8-1-1, BT-117-5-31-1, RHRT-33-1, RHRT-87-1, RHRT-6-1, HYT-

1, Sel-7 and Panjab Chhuhara (Control) against early blight, tomato

spotted wilt virus and tomato leaf curl virus in field condition at

Andhra Pradesh, India during rabi season of 2000-2001 and 2001-

2002. They found that HYT-1 has the highest yield with mean percent

incidence of early blight disease, tomato spotted wilt virus and tomato

leaf curl virus was 1.3, 10.2 and 16%, respectively. BT-116-8-1-1

recorded 3.4, 12.1 and 15.6 % mean incidence of early blight, tomato

spotted wilt virus and tomato leaf curl virus, respectively with 30.4

t/ha yield.

Foolad et al. (2004) evaluated 29 tomato genotypes (cultivars,

breeding lines and plant introductions) representing three

Lycopersicon species against resistance to early blight caused by

fungus Alternaria solani. Evaluations were conducted in replicate

trials in multiple years under field and green house conditions (with

whole plants) and in growth chamber (with detached leaf let). There

were significant differences among genotypes in their response to

Alternaria solani infection in the field, green house and growth

chamber experiments. Field and green house results were comparable


~33~

across replications and years. There were great correspondence (r/R2)

= 0.71, P<0.01) between field and green house resistance across

genotypes. In contrast results from the detached leaf-let assays were

inconsistence across experiment and non-correlated with either green

house or field result. The overall results indicates the utility of gre en

house evaluation and the inadequacy of detached leaf let assay for

screening tomatoes for early blight resistance.

Prasad and Naik (2004) made intensive roving survey to assess

the incidence and severity of early blight disease of tomato during

Kharif 2000 at Raichur, Gulbarga and Dharwar districts in

Karnataka, India. They surveyed five fields in each five villages of

three districts using 10 plants from each field and severity was

assessed by using 0 to 5 scale based on % of infected leaf area and

PDI (Percent Disease Index). They recorded the highest disease

severity in Raichur (52.55 %) followed by Dharwar (47.87 %) and

Gulbarga (39.39 %). However, the highest incidence 65.36 % was

recorded in Regional Agriculture Research Station in Raichur, while,

the lowest incidence (30.20 %) was recorded in village of Sannurin

Gubay.

Mate et al. (2005) screened 31 tomato genotypes for resistance

to early blight (Alternaria solani) in Maharashtra, India under artificial

inoculation condition. They found none of the cultivars were resistant

to the disease. However, four genotypes viz; ATH-1, ATH-2, Samridhi

and Vaishali were found moderately resistant and nine (EC-321926,

EC-321928, ATV-1, ATV-2, S-28, Devgiri, Roma, HS-101 and

Marglobe) genotypes were expressed moderately susceptible.

Reddy et al. (2006) screened on field and lab environments to

evaluate genotypes for resistance to A. helianthi. Disease severity was

recorded by visually examining each leaf seven days after inoculation


~34~

as percentage of leaf area occupied by the sporulating fungus using

the pictorial key in each replication and the average computed. The

disease intensity for hybrids ranged from 3.73 to 52.33 %. RHA 587

and ARG × RHA 587 were found to be resistant to Alternaria blight

both under field and lab conditions and therefore have the potential to

reduce yield losses.

Chaerani et al. (2007b) assessed a droplet inoculation method

for evaluation of resistance to early blight in tomato. In this test

method, leaflets are inoculated with small droplets of a spore

suspension in either water or a 0.1% agar solution. Early blight

resistance was evaluated based on lesion size. The droplet method

better discriminated the level of resistance (P < 0.001) for a range of

spore densities in comparison with the more commonly used spray

inoculation method. Lesions generated by droplet inoculation at 7

days after inoculation ranged from small flecks to almost complete

blight with an exponential-like distribution of lesion sizes. Significant

correlations (r = 0.52, 0.58 and 0.63, P < 0.001) were observed across

three glass house tests of 54 accessions including wild species using

the droplet method.

Kamble et al. (2007) conducted experiment to screen different

varieties and advance lines against early blight of tomato caused by

Alternaria solani. Twenty one advanced lines and five varieties were

screened under field condition. Early blight was found to be the major

disease of tomato under agro-climatic conditions of Konken region of

Maharashtra state. None of the varieties and advanced lines showed

resistant reaction. Five advanced lines were moderately resistant.

While one variety and four advanced lines were moderately

susceptible and thirteen advanced lines and four varieties were found

susceptible.


~35~

Atik et al. (2007) surveyed for the occurrence of Alternaria spp.

diseases in greenhouses of Lattakia and Tartous provinces, as well as

in the open fields of Lattakia, Tartous, Aleppo and Hama (Al-Ghab)

provinces, were carried out in Syria, during 2005 and 2006. The

survey included 114 greenhouses and 170 fields grown with different

tomato cultivars. Early blight (caused by Alternaria solani) disease

was reported in one greenhouse in each of Lattakia and Tartous only.

However, Alternaria alternata was reported for the first time in Syria

as the casual organism of tomato leaf spot and blight disease in the

open fields. The incidence and severity of infection by the latter

disease were significantly different in all the surveyed fields. Two

virulent isolates of A. solani and 110 isolates of A. alternata (88 non-

pathogenic, 2 highly virulent and 20 moderately virulent) were

isolated. Twenty-four genetic resources of tomato were screened under

growth room conditions. Significant differences were found in the

incidence and severity of infection in all the genotypes, and they

ranged from resistant to susceptible. Five genotypes proved to be

resistant against both the pathogens, and should be considered for

future breeding programmes in Syria.

Upadhyay et al. (2009) evaluated fourteen tomato genotypes,

representing two Solanum species that were screened for resistance

against early blight disease. Evaluations were conducted in growth

chamber for disease severity and host resistance of the plants. EC-

520061 (S. habrochaites) showed resistance, 3 genotypes NCEBR-4,

FEB-4 and VRT-2 were moderately susceptible, while other genotypes

were susceptible to highly susceptible. The resistant material found in

this study was useful in the tomato improvement programme,

especially for early blight disease.

One hundred forty two tomato genotypes including wild and

cultivated lines were screened for resistance against early blight

disease caused by Alternaria solani. Evaluations were conducted in


~36~

vivo and in vitro for disease severity and host resistance of the plants.

Eight lines (EC-520057, EC-520058, EC-520059, EC-520061, EC-

508765, EC-538394, H-88-78-1 and EC-501583) showed highly

resistant reaction against the fungus; three lines were found

resistant, 5 lines moderately resistant whereas 33 lines showed

moderately susceptible besides 57 susceptible and 36 highly

susceptible lines against the disease under natural epiphytotic

condition. Screening under in vitro, revealed that eight genotypes were

highly resistant, 3 resistant and 7 as moderately resistant. It was

found that the accessions of wild relatives of tomato were highly

resistant which may be utilized for the development of prebred lines

or recombinant inbred lines or in other molecular research activities

for the improvement of tomato. Some of the cultivated genotypes were

resistant/moderately resistant under in-vivo and in-vitro conditions.

These include H-86, VRT-2, NCEBR-4 and RCMT-1 that may directly

be promoted for growing in disease prone areas.

Lalit et al. (2013) conducted trial during Rabi season of 2010-

2011 to study phenotyping of Recombinant Inbred Lines that have

been developed for resistance against early blight of tomato caused by

Alternaria solani. 179 Recombinant Inbred Lines were inoculated with

12 days old culture of Alternaria solani and disease severity was

scored on a five-point scale. The data were recorded on four different

dates at 7 days intervals i.e. 7th, 14th, 21th and 28th days after

inoculation. The Percent Disease Index and Area Under Disease

Progress Curve value were calculated on the basis of data recorded.

2.8.2 Glasshouse screening

Glasshouse or controlled environment chamber evaluations of

young plants were mainly used for preliminary selection of A. solani

resistance sources from large collections (Barksdale, 1969;

Vakalounakis, 1983; Poysa and Tu, 1996; Vloutoglou, 1999).


~37~

Foolad et al. (2000) reported for disease symptoms, and area

under the disease progress curve (AUDPC) and final percent

defoliation. In the greenhouse experiments, plants were evaluated for

percent defoliation following spray inoculation with isolates of A.

solani. In the growth chamber experiments, lesion radius, rate of

lesion expansion, and final disease severity were determined for

individual detached leaflets inoculated with isolates of A. solani. In the

field and greenhouse experiments, disease response varied from near-

complete resistance in some accessions of the wild tomato species L.

hirsutum (e.g., PI126445 and LA2099) to complete susceptibility in

tomato cultivar New Yorker and breeding line NC84173. The

previously developed EB-resistant breeding lines 88B231, 89B21,

C1943, NCEBR-1, NCEBR-2, NCEBR-5, NCEBR-6, NC24E, and

NC39E exhibited more resistance than New Yorker and NC84173.

Field and greenhouse results were comparable across replications and

years, and there were great correspondences (r » 0.71, P < 0.01)

between field and greenhouse resistance across genotypes.

The glasshouse screening methods for A. solani resistance are

based on the method established by Barksdale (1969). Generally,

seedlings are spray inoculated with spores at an age of 4 to 6 weeks

(Barksdale, 1969; Marcinkowska, 1982; Nash and Gardner, 1988b;

Banerjee et al., 1998; Vloutoglou, 1999; Foolad et al., 2000). Leaves

can be injured prior to spray by rubbing leaf surfaces between thumb

and forefingers (Poysa and Tu, 1996). Plants are incubated 24 hours

under 100 % relative humidity (RH) followed by 12 - 16 hours of 100

% RH during the night period for 5 - 7 days in a mist chamber,

mimicking repeated nightly dew in nature. During the day, plants are

exposed to ambient RH to allow the development of disease

symptoms. A leaf wetness period of at least 4 h after inoculation was

required for infection (Moore, 1942; Vloutoglou and Kalogerakis,

2000). Increasing this period up to 24 h induced progressively higher


~38~

EB severity, but more than 24 h humidity periods did not increase

severity further (Vloutoglou, 1999).

EB severity is usually determined at seven days after spray

inoculation by estimating the percent necrotic area on leaves which

were present at the time of inoculation (leaves emerging after

inoculation are not affected, Barksdale, 1969; Vloutoglou, 1999). In

the case of a low incidence of necrotic spots, EB severity is expressed

as the number of lesions (Barksdale, 1969). Pandey et al. (2003)

artificially inoculated all the plants with a pure culture of A. solani. A

foolproof inoculation technique was developed and standardized for

inoculation. An inoculum concentration of 125 cfu/ml was used as a

spray. The inoculum was uniformly sprayed on two age groups of

plants (viz., 35 and 50 days old). All inoculated plants were kept in a

moist chamber by maintaining 95% relative humidity (RH) and 20 –

25°C temperature continuously for 5 days. The plants were closely

observed each day after inoculation. Disease was first observed on

each plant 7 days after inoculation. Subsequent observations were

carried out 23 days after inoculation. Most of the inoculated leaves

with primary infection senesced and started to defoliate at this stage.

Screening under artificial conditions was more informative than that

under natural epidemic conditions. Tomato cultivars CLN-2071-C,

LN-2070-A, BSS-174, and DTH-7 with resistance expressed as slow

blighting against four pathogenic isolates of A. solani, were selected

for cultivation in disease-prone areas.

2.9 Genotyping of Alternaria solani

2.9.1 Genetics of disease resistance

Most genetic studies on the inheritance of EB resistance using

different sources of resistance (S. lycopersicum, S. habrochaites and S.

pimpinellifolium) arrived at the same conclusions that the resistance is


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a quantitative trait that is controlled polygenically. Early blight (EB)

resistance is a quantitative trait, which makes selection more difficult

as compared to qualitative traits (Dilip and Feng, 2010). The

estimated minimum number of controlling factors is two (Barksdale,

1971) or three (Nash and Gardner, 1988a). Analysis using

quantitative genetic methods (generation mean analysis and scaling

tests) and several sources of resistance (C1943, NC EBR-2, IHR 1939

and IHR 1816) revealed additive and dominant genetic control with

the presence of epistatic effects (Maiero, 1990a; Nash and Gardner,

1988a; Thirthamalappa and Lohithaswa, 2000).

The first genetic study of EB resistance using S. arcanum as a

donor parent was conducted and the results concur with previous

classical genetic and molecular mapping studies using S. habrochaites

(syn. L. hirsutum) or derived materials and S. pimpinellifolium, which

indicate that EB resistance is under polygenic control. Additive

genetic effects were predominant (Nash and Gardner, 1988a; Maiero

et al., 1990a; Foolad et al., 2002b; Thirthamalappa and Lohithaswa,

2000; Zhang et al., 2003a); in some cases dominant effects (Nash and

Gardner, 1988a; Thirthamalappa and Lohithaswa, 2000) as well as

epistatic interactions (Maiero et al., 1990a; Nash and Gardner, 1988a;

Thirthamalappa and Lohithaswa, 2000) were observed.

The EB resistance genes in C1943 and 71B2 are recessive and

not allelic (Barksdale and Stoner, 1977; Maiero and Barksdale, 1989).

However, in crosses of these two resistance sources with another

susceptible genotype, the F1 hybrids were intermediate, indicating

additive genetic control or partial dominance (Maiero and Barksdale,

1989). Recessive genes have been reported in S. lycopersicum

83602029 (Stancheva, 1991) in IHR1939 and IHR1816 by

Thirthamalappa and Lohithaswa (2000). Partially dominant


~40~

inheritance has been found in S. pimpinellifolium and S. habrochaites

(Martin and Hepperly, 1987).

The line 87B187, derived from S. habrochaites PI 390662,

shared common resistance genes with NCEBR-2, although this line

was developed via C1943 from a S. lycopersicum source (Maiero,

1990a). Also, Thirthamalappa and Lohithaswa (2000) reported

independent genes in IHR 1939 (S. pimpinellifolium L4394) and those

in IHR 1816 (derived from NC EBR-1, which was developed from S.

habrochaites PI 126445).

In contrast to all studies described above, one study reported a

monogenic, dominant inheritance in S. habrochaites PI 134417 (Datar

and Lonkar, 1985). Their conclusion is arguable since a highly

resistant F1 does not necessarily indicate the complete dominance of

EB resistance as was observed by Foolad and Lin (2001). The

resistance phenotypes in F2 population derived from S. habrochaites

PI 134417 were grouped into resistant, intermediate and susceptible

and a 3 : 1 segregation was observed, leading to the conclusion of

monogenic inheritance (Datar and Lonkar, 1985). However, EB

resistance is a quantitatively expressed character and the assignment

of three phenotypic classes is therefore arbitrary and may have led

accidentally to the 3 : 1 segregation (Foolad and Lin, 2001).

Heritability of EB resistance has been estimated in crosses

involving S. habrochaites PI 126445 (Foolad and Lin, 2001) and

derived lines NC EBR-1 and NC39E (Nash and Gardner, 1988a;

Foolad et al., 2002a). Depending on the calculation method,

heritability estimates were low to moderate in two crosses involving

NC EBR-1 (Nash and Gardner, 1988a). Based on parent offspring (PO)

regression narrow sense heritability (h2) for AUDPC was estimated as

0.26 and 0.38 (Nash and Gardner, 1988a). Higher heritability


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estimates were obtained from a cross with S. habrochaites PI126445

(0.70, Foolad and Lin, 2001) and from a cross with S. lycopersicum

NC39E (0.65, Foolad et al., 2002a).

The biological effects of the genes underlying the identified EB

resistance QTLs remain unclear at this moment. A candidate gene

approach, either using genes involved in the pathogen recognition

process (resistance genes [R genes] or R gene analogs [RGAs], Foolad

et al., 2002b) or those involved in the defense response process

(defense response genes [DR genes], Faris et al., 1999) as molecular

markers for QTLs analysis, is potentially useful for the analysis of EB

resistance. Since resistance to A. solani does not seem to be race-

specific and is not mediated by genes with a major effect, R-genes are

unlikely to be involved in this resistance. Therefore, DR genes are

more likely candidate genes for the QTLs involved in EB resistance.

Faris et al., (1999) provided a convincing example. They mapped DR

genes on a wheat linkage map where QTLs for several diseases had

previously been identified. These DR genes were shown to have a

more significant association with disease resistance and explained

more of the phenotypic variation than the original markers used for

QTL detection. Mapping at a higher resolution is also needed,

however, before establishing any functional relationship.

Sinha et al., (2009) worked on sources of resistance against

early blight (Alternaria solani) in tomato (Solanum lycopersicum).

Fourteen tomato genotypes, representing two Solanum species were

screened for resistance against early blight disease. Evaluations were

conducted in growth chamber for disease severity and host resistance

of the plants. EC-520061 (S. habrochaites) showed resistance against

infection, 3 genotypes NCEBR-4, FEB-4 and VRT-2 were moderately

susceptible, while other genotypes were found either susceptible or

highly susceptible. The resistant material found in this study is useful


~42~

in the tomato improvement programme, especially for early blight

disease

2.9.2 Quality traits

Sucheta et al. (1996) analysed 53 varieties of tomato for

chemical constituents such as dry matter content, total soluble solids

(TSS), ascorbic acid, acidity and lycopene. They found that dry matter

ranged from 3.22-6.92%, TSS 3.1 – 5.6 %, ascorbic acid 11.21-53.79

mg/100g. Siviero et al. (2000) studied lycopene content and fresh dry

matter content in fruit of 11 tomato hybrids used for processing

purposes revealed that hybrid DR-10747 had the highest lycopene

content of fruit and the best colour followed by DRD-8133.

Chhabra et al. (2000) studied relationship of ascorbic acid with

Alternaria solani incidence using eight tomato genotypes grouped as

highly resistant (Lycopersicon pimpinilifollium and Lycopersicon

peruvianum), resistant (Lycopersicon esculentum cultivars, Coloubia

and Flora Dade), susceptible (Lycopersicon esculentum cultivars

Anteny and Sel-18) and highly susceptible (Lycopersicon esculentum

cultivars Sel-7 and HS-101) in Haryana condition of India. They found

that at the pre infection stage, there was no significant difference in

ascorbic acid content of leaf of different cultivars. After infection

drastic reduction in ascorbic acid content were observed, being

highest 63.3 % in highly susceptible cultivar HS-101 and the lowest

24.55 % in resistant cultivar Columbia. Ascorbic acid content in the

susceptible cultivars varied significantly 6.13 to 9mg/100g after

infection, while in the resistant cultivars, it varied from 12.33 to 12.56

mg/100g leaf. Overall reduction in ascorbic acid content was nearly

25 % in the resistant cultivars while in the susceptible cultivars, it

ranged from 40 to 63 %.


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Bhatt et al. (2001) made 15 × 15 diallel set of tomato excluding

reciprocal to find out the extent of heterosis, combining ability and

nature of gene action for yield with two important quality traits

vitamin C (ascorbic acid) and total soluble solid (TSS). Significant

difference among genotypes was obtained for all these traits. Positive

high significant heterosis was found for yield (41.97, 157.84 and

28.94 %) for ascorbic acid (16.68, 54.57 and 161.33 %) and for total

soluble solid (TSS) (25.9, 11.93 and 19.02) over the top and better

parent, respectively. The magnitudes of variance due to general as

well as specific combining abilities were highly significant indicating

the importance of both additive and non-additive gene action.

However, the degree of dominance (σ2/G σ2) revealed the prevalence of

a non-additive gene effect. Cross combination Arka Vikas × Sel-12

(13.19), KS-10 × Pant T3 (1.66) and EC-816703 × EC-13402 (0.88)

were best specific combiners for ascorbic acid, TSS and yield per

plant. Preponderance of non-additive gene action plays greater role in

the inheritance of ascorbic acid and TSS in tomato grown in hilly

condition.

Adalid et al. (2005) have characterized the carotenoid and

vitamin C content in 11 accessions of Lycopersicon esculentum var.

cerasiformae and in six L. esculentum traditional Spanish varieties.

Three mutants of L. esculentum with high pigment accumulation, one

commercial hybrid, two experimental breeding lines and one tomato

variety for processing were used as controls in comparisons made

with different tomato types. A very interesting L. esculentum

traditional Spanish variety (UPV17790) was selected with 4 times the

lycopene content of the commercial hybrid control and 1.35 times the

content of the mutant controls. The UPV22353 and UPV22487 L.

esculentum var. cersiforme accessions are of particular interest for

their vitamin C content (similar to UPV20525). Result depicted that


~44~

selected replication and the average computed. The disease intensity

for hybrids ranged from 3.73 to 52.33%. RHA 587 and ARG × RHA

587 were found to be resistant to Alternaria blight both under field

and lab conditions and therefore have the potential to reduce yield

losses because of this disease in the field.

2.9.3 Association of early blight resistance with plant maturity,

yielding ability and determinism

The strong correlation between EB resistance and late maturity,

low yielding ability and indeterminate plant type (Nash and Gardner,

1988; Foolad et al., 2001, Foolad et al., 2002a, b) has limited the

development of lines or cultivars with a high level of resistance. The

QTLs study of Foolad et al. (2002b) described above aimed to identify

QTLs for resistance without an effect on these agronomic traits.

Therefore, they removed plants with poor characteristics from their

population before attempting to map the QTLs (Foolad and Lin, 2001;

Foolad et al., 2002b). However, no one plant with resistance level

equal to that of the donor parent or F1 hybrid was identified in

subsequent generations (Foolad and Lin, 2001; Foolad et al., 2002b).

Substantial work on potato EB also documented the association

of late maturity with EB resistance (e.g. Johanson and Thurston,

1990). As in tomato, it is not yet clear whether this correlation is

caused by closely linked genes or by pleiotropic effects of genes. A

mapping study for EB and maturity in potato identified five EB

resistance QTLs which explained 62 % of the total phenotypic

variation for resistance (Zhang, 2004). Three of these five QTLs

explained 98 % of the total phenotypic variation for maturity. The

other two EB resistance QTLs, which did not have an effect on (foliage)

maturity, explained 33 % of the total phenotypic variation for

resistance (Zhang, 2004).


~45~

In potato therefore about half the genotypic variation for EB

resistance is also linked to maturity; still this may be due to either

close linkage or to pleiotropic effects. A very similar situation occurs

in the potato–late blight (Phytophthora infestans) interaction (Visker et

al., 2003). Even on susceptible plants, the younger, topmost leaves

are usually free of EB symptoms, while the older, lower leaves may be

necrotized by the fungus (Johanson and Thurston, 1990). Several

studies attempted to clarify the physiological mechanisms for this

apparent resistance in young tissues and plants. Low sugar content

has been suggested as the cause of higher EB susceptibility in older

or weakened leaves and plants (Rotem, 1994).

The low sugar content theory might explain the increased

susceptibility of physiologically old plants or those which have a high

fruit to foliage ratio (Barrat and Richards, 1944). Another explanation

of the relative resistance of young tissues is that the concentrations of

three glycoalkaloids (solanine, chaconine and solanidine), which are

capable of inhibiting growth of A. solani in vitro, are higher in young

tomato leaves and steadily decline as leaves and plant matures

(Sinden, 1972). The higher resistance of late maturing cultivars can

similarly be explained in terms of sugar and alkaloid contents. Late

maturing cultivars generally have an indeterminate, vine type growth

habit and continue producing new foliage (Johanson and Thurston,

1990).

In contrast, early maturing types have a determinate growth

habit and do not continue producing new foliage throughout the

season. Therefore, late maturing cultivars might appear resistant as

compared to the early maturing types just because fruit initiation is

delayed and more young leaves are present throughout the season. If

the physiological mechanisms are the only cause of EB resistance

then it might be impossible to find recombinants with a high


~46~

resistance level and highly desirable horticultural characteristics in a

segregating population. In that case, tomato breeders can only expect

to obtain acceptable EB resistance level in varieties with mid- or late

season maturity. However, in potato variation in resistance occurs

between cultivars of the same maturity class, indicating that

differences in resistance are not always and only an artifact of

maturity effect (Holley et al., 1983; Christ, 1991). So far EB resistance

screening in tomato was studied without reference to maturity classes

and yield while the latter traits are taken into consideration in EB

potato research (Douglas and Pavek, 1972).

2.10 Mapping of QTLs for Early Blight Resistance

Foolad et al. (2002b) were the first to map QTLs for EB

resistance. They used backcross progenies of a cross between S.

habrochaites PI 126445 and a susceptible tomato line. Mapping was

done in the BC1 and validated in the BC1S generation. Fourteen QTLs

were identified which together explained 57 % of the total phenotypic

variation. For all QTLs the positive allele originated from the resistant

parent.

In a subsequent study Zhang et al. (2003a) used a selective

genotyping approach on a different part of the same BC1 population.

Seven QTLs were detected, including one previously mapped major

and three minor QTLs. One of the QTLs in this study inherited the

resistance allele from the susceptible parent. Chaerani et al. (2006a)

identified six QTLs for EB resistance in F2 and F3 populations from a

cross between the resistant S. arcanum LA2157 and a susceptible

tomato. Different environments and phenotypic scoring methods were

used in this study, in contrast to the previous mapping studies which

used one type of environment and disease measure. In addition,

resistance to stem lesions was also assessed in the F3 population.


~47~

Interestingly, EB QTLs detected in the F2 population were not always

detected in the F3 population, and vice versa. This indicates the

presence of environment specific or plant age-specific QTLs. Three

QTL regions for stem lesion resistance coincided with EB resistance

QTLs, which allows simultaneous selection for resistance to both

types of disease symptoms.

Foolad et al.(2002b) and Zhang et al.(2003b) recommended

combination of four to six QTLs, which explained more than 40% of

total phenotypic variation for use in marker-assisted breeding, and

Chaerani et al., 2006b suggested two QTLs, which had prominent

effects under different environments and gave both EB and stem

lesion resistance. It still needs to be determined, however, whether the

level of resistance contributed by these QTLs will be of sufficient

practical importance. The EB mapping studies have not yet reached

the stage where QTLs are mapped precisely enough to be included in

a breeding program.

The QTLs on chromosome 7 showed an effect in glasshouse

tests with young plants (56 to 63 days after planting), whereas the

QTLs on chromosomes 1 and 6 were only effective in the field at later

plant stage (90 to 110 days after sowing). The QTLs on chromosome 7,

which inherited the favourable alleles from the susceptible parent,

might not have a true effect on EB resistance. As the susceptible

parent is a cultivated, semi-determinate S. lycopersicum variety and

much better adapted to the glasshouse test environment than the

resistant, indeterminate S. arcanum parent, this suggests that the

QTLs on chromosome 7 may affect the condition of the plants in the

glasshouse rather than the resistance itself. Thus, plants carrying the

S. lycopersicum allele would in general be more vigorous and therefore

better able to withstand infection, which overshadows the effect of

their genotype at the “true” resistance QTLs. The fact that well-


~48~

fertilized plants are more resistant than plants starved for nutrients

and that young plant generally show more apparent resistance to EB

than older plant (Rotem, 1994) support the notion that plant

condition can affect EB resistance. Whether this speculation is true or

not, the QTLs on chromosome 7 is not an interesting target for

breeders, as it doesn’t show an effect on EB severity in the field.

Although we used a different resistance source, the 2-LOD

support intervals of five of our QTLs overlapped with the QTLs regions

detected by Foolad et al. (2002b) and Zhang et al. (2003b). The QTLs

on chromosome 7, which we detected only in the glasshouse test

using a single isolate, was not detected in the field studies by Foolad

et al. (2002b) and Zhang et al. (2003b) using a mixture of two isolates

from Pennsylvannia, U.S.. The smaller number of QTLs detected in

our study may be due to a higher LOD threshold employed (3.6 to 3.9

depending on the trait) compared to the previous mapping study

using an S. habrochaites source which used a LOD threshold of 2.4

(Foolad et al., 2002b). Both studies revealed no major QTLs for EB

resistance, but rather showed that resistance is controlled by several

QTLs with small effects: 7 to 16 % explained variance in our study,

and 4 to 22 % in Foolad et al. (2002b).

Previous studies showed that stem lesion resistance was found

in the same sources as EB resistance but the genetic relationship was

not investigated (Barksdale and Stoner, 1973, 1977; Stancheva et al.,

1991a, b). In the present study three EB resistance QTLs coincided

with stem lesion resistance QTLs; one QTLs on chromosome 9 even

had a major effect on the stem lesion resistance (31%).

Given the low to moderate heritability estimates, a marker-

aided selection approach is potentially useful to accelerate the

transfer of EB resistance genes into new tomato cultivars. Foolad et


~49~

al. (2002b) were the first to map QTLs for EB resistance. They used

backcross progenies of a cross between S. habrochaites PI 126445 and

a susceptible tomato line. Mapping was done in the BC1 and validated

in the BC1S generation. Fourteen QTLs were identified which together

explained 57 % of the total phenotypic variation. For all QTLs the

positive allele originated from the resistant parent. In a subsequent

study Zhang et al. (2003a) used a selective genotyping approach on a

different part of the same BC1 population. Seven QTLs were detected,

including one previously mapped major and three minor QTLs. One of

the QTLs in this study inherited the resistance allele from the

susceptible parent.

We assessed EB resistance at the single plant level in the F2

population in glasshouse tests using inoculation with a single isolate

and compared these data to the F3 data from a field test under

artificial inoculations with mixed field isolates. Six QTLs were

detected, two of which (the QTLs on chromosomes 2 and 7) inherited

the resistant allele from the susceptible parent. This is not uncommon

and has been reported in many plant species (e.g. Young et al., 1993;

Lefebvre and Palloix, 1996; Pilet et al., 1998). For EB resistance in

tomato, Zhang et al. (2003a) also detected a QTLs on chromosome 3

for which the resistance allele was inherited from the susceptible

parent. The presence of QTLs with opposite effects to those predicted

by the parents may be responsible for the occurrence of individuals

with transgressive phenotypes (de Vicente and Tanksley, 1993).

Notwithstanding the differences in experimental techniques

(pathogen isolates, inoculation method and resistance assessment

criteria) and environmental conditions between the disease tests, we

detected three EB QTLs in the glasshouse (chromosomes 2, 5, and 9)

which coincided with QTLs for resistance traits in the field. Two QTLs

were detected with a significant effect only on the field-test trait


~50~

RAUDPC on chromosomes 1 and 6, with the second also having an

elevated but non-significant LOD score for PEBI. One QTL on

chromosome 7 was the major QTLs affecting all glasshouse test traits,

while it showed no effect on the field test traits. Especially the QTLs

on chromosome 9 is interesting: it is the major QTLs detected for all

traits in the F3 field test, and it is also an important QTL in the F2

glasshouse tests (Dirlewanger et al., 1994).

The detection of common QTLs at different experimental

locations may be hampered by genotype x environment or genotype x

isolate interactions as was observed in some studies, e.g. by

Lübberstedt et al. (1999). We do not preclude the presence of such

interactions in EB resistance that might further explain the

discrepancy between the F2 glasshouse and F3 field tests; however,

such interactions could not be determined in this study. In the two

environments different isolates were used, so that the effects of the

isolates and experimental conditions were confounded.

2.11 Calculation of Disease Parameters

The percentage disease index (PDI) and area under disease

progress curve (AUDPC) (Campbell and Madden, 1990; Johnson and

Wilcoxson, 1982; Shaner and Finney, 1977) were calculated as follows

PDI = Sum of all ratings 100

Total no. of observation Maximum rating scale

Percent Disease Index (PDI) was worked out by using formula

given by Wheeler (1969).

The mean value of the PDI from the first to the last observations

was calculated.


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(ti+1 – ti )}

Where;

Xi is the disease index expressed as a proportion at the ith

observation.

ti is the time (days after planting) at the ith observations.

And n is the total number of observations.

Chapter ΙΙI

MATERIALS AND METHODS

Present investigation on “Characterization of Alternaria solani

and molecular mapping of QTLs for early blight resistance in tomato”

was carried out in the Department of Mycology and Plant Pathology,

Institute of Agricultural Sciences, Banaras Hindu University, Varanasi

and Indian Institute of Vegetable Research, Adalpura, Varanasi

between the year 2010 to 2014. The materials used and methods

adopted during the studies are described here in this chapter.

3.1 Collection, Isolation and Purification of Pathogen causing Early Blight of Tomato from Different Parts of India.

Leaves stems and fruits of diseased tomato plants showing

typical early blight symptoms were collected from different agro

climatic localities of India (Varanasi, Adalpura, Mirzapur, Allahabad,

Agra, Gorkhpur, Aara, Buxer, Raichur, Banglore, Jaipur, Anand,

Hisar and New Delhi). Samples were washed using tap-water, surface

sterilized with 0.5% sodium hypo-chloride solution for one minute,

then washed three times in sterilized distilled water. Samples were

then dried between two layers of sterilized filter paper to remove the

excess water. The sterilized spotted leaf tissues were cut with adjacent

healthy tissues using a sterile scalpel and placed on plain agar

medium in Petri-dishes. Inoculated dishes were incubated at 25±20C

for five days. Hyphal tips from the outer ends of the growing colonies

were transferred to plates of potato dextrose agar (PDA) medium and

incubated at 25±20C. Pure cultures were obtained for each of the

isolated fungi using the single spore technique according to Hansen

(1926) and or hyphal tip technique according to Brown (1924). The

purified fungi were identified according to their morphological

characters using the description of Ozcelik and Ozcelik (1997), Perez

Materials and Methods

~53~

and Martinez (1997), Patterson (1991) and Kumar et al., (2008). Stock

cultures were maintained on PDA slants and stored in a refrigerator

at 5-100C.

3.2 Cultural, Morphological and Pathogenic Variability amongst Tomato Isolates of Alternaria solani.

3.2.1 Cultural variability of different isolates of A. solani

The cultural character was recorded at 7 DAI (days after

inoculation) of all isolates of A. solani. Characters like pigmentation

on medium, mycelial growth, zonation were recorded (Kumar et. al.,

2008) by direct observation of culture grown in petri plates and

sporulation was recorded on PDA medium by preparing slides 7, 10

and 13 DAI of cultures under the microscope. For this purpose

eighteen selected isolates of A. solani were taken, representing Asv-1,

Asv-2, Asad-1, Asmi-1, Asmi-2, Asal-1, Asag-1, Asgo-1, Asar-1, Asar-

2, Asbx-1, Asrai-1, Asbng-1, Asjai-1, Asan-1, Ashi-1, Asnd-1 and

Asnd-2. All these isolates were tested for their cultural and

morphological variations on PDA. For each isolate five Petri plates

were poured with PDA medium. After solidification of the agar 5 mm

culture bits of each isolates were inoculated onto the above-

mentioned medium. These inoculated petri plates were kept in BOD at

25 ± 2ºC for growth. The data were recorded after 3 days from

inoculation and radial growth was measured 7, 10 and 13 DAI on PDA

medium.

3.2.2 Morphological variability of different isolates of A. solani

Morphology of new strain along with other isolates of A. solani

was studied by scrapping the growth of the pathogen from the

infected leaves and reproductive structure produced in culture

medium. Sporulation was also recorded in cultures grown on PDA


~54~

medium using haemocytometer (Kumar et. al., 2008) at 7, 10 and 13

DAI under the microscope.

3.2.3 Pathogenic variability of different isolates of A. solani

All these isolates were tested for their pathogenic variability on

tomato varieties. Petri plates were poured with PDA medium. After

solidification of the agar, 5 mm culture bits from 12 days old culture

of each isolates were inoculated onto the PDA medium. These

inoculated petri plates were kept in BOD at 25 ± 2ºC for growth. 12

days old culture having spores was used for inoculation. Culture

containing spores were suspended in water and mixed thoroughly and

this suspension was used for inoculation on 30 days old tomato

plants. 5 plants of each variety were inoculated with each isolates. For

the maintenance of temperature around 25-30ºC plants were kept in

green house and high relative humidity was maintained through

spraying of water after every 12 hours for a period of 7 days. The

aggressiveness of different isolates was observed on the basis of leaf

area infection in 0-9 rating scale (Ghosh et. al., 2009) at 7, 14, 21, 28

and 35 DAI.

3.2.3.1 Response of young and old plant against pathogen virulence

Tomato seeds of variety Co-3 were sown (five seeds/pot) on two

different dates. Inoculation was done 45 and 60 days after sowing.

Seedlings were thinned to one per pot 1 week after emergence. The

virulence of eighteen A. solani isolates was tested on susceptible

tomato variety Co-3. Plants were grown in controlled environment

chamber. Plants were spray inoculated with 5 x l03 conidia/ml with

each of the isolates. Five plants per isolate were used as replicates.

Control plants were sprayed with sterile distilled water. All plants

were covered with clear polyethylene for 48 hrs immediately after


~55~

inoculation. A total of five assessments were made at 7 day intervals

(Pandey et. al., 2003).

3.2.4 Effect of media, temperature and pH

3.2.4.1 Media

Total ten media namely Bean meal agar, Corn meal agar,

Glucose peptone agar, Host leaf extract agar, Malt extract agar, Oat

meal agar, Czapek’s dox agar, Potato dextrose agar, Richard’s agar

and V-8 juice agar medium were studied to find out the most suitable

medium for mycelial growth and sporulation of test pathogen.

Twenty milliliter medium was poured into sterilized petri plates

(90 mm diameter). The Petri plates were inoculated with five

millimeter diameter discs of 10 days old mycelial culture from the

periphery with the help of sterilized cork borer and placed at the

centre and incubated at 25 ± 20 C in incubator for 12 days. Three

replicates were maintained for each medium. Observations on

mycelial growth (mm) and sporulation of pathogen on solid media

were recorded after 4, 8 and 12 days of incubation by measuring

colony diameter at right angle to one another. The observations on

sporulation were recorded under microscope (Arunakumara, 2006).

Ten broth media namely Bean meal broth, Corn meal broth,

Glucose peptone broth, Host leaf extract broth, Malt extract broth,

Oat meal broth, Czapek’s dox broth, Potato dextrose broth, Richard’s

broth and V-8 juice broth were studied for the dry mycelial weight and

sporulation of the test pathogen. The compositions of broth were same

as that of solid media except agar-agar.

Twenty milliliter broth medium was poured into 100 ml conical

flasks separately. The flasks were plugged with sterilized cotton and


~56~

autoclaved at 121 0C for 20 min. The flasks were inoculated with five

millimeter disc of 10 days old culture of test pathogen from periphery

with the help of sterilized cork borer and incubated at 25± 20C in

incubator for 12 days. The mycelial mat was filtered through

Whatman’s filter paper No. 42. Filtered mycelial mat on filter paper

was oven dried at 500C for 24 hrs, cooled and then weighed. Three

replicates were maintained for each medium. Observations on dry

mycelial weight (mg) and number of spores produced were recorded

after 12 days of incubation. Dry weight of the mycelium (mg) was

calculated (Arunakumara, 2006) by following formula:

Dry mycelial weight = B-A

Where, A = Weight of filter paper (mg)

B = Weight of filter paper + Weight of dried mycelial mat (mg)

3.2.4.2 Temperature

To study the optimum temperature requirement for the mycelial

growth and sporulation of test pathogen, temperatures ranging from 5

to 400 C were studied on PDA medium. Twenty milliliter sterilized PDA

medium was poured in sterilized Petri plates. The Petri plates were

inoculated with five 5 mm disc of 10 days old culture of test pathogen

and the inoculated plates were separately incubated at 5, 10, 15, 20,

25, 30, 35 and 400 C temperatures. Three replicates were kept for

each treatment. Observations on the mycelial growth (mm) and spore

production of test pathogen were recorded (Arunakumara, 2006) after

12 days of incubation.

Eight temperatures ranging from 5 to 400 C were studied on

PDB medium for the dry mycelial weight and sporulation of the test

pathogen (Arunakumara, 2006). Same methods were followed, that

were used in broth media study.


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3.2.4.3 pH

Different pH levels i.e. 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0

and 8.5 were evaluated to study the influence of pH on the mycelial

growth and sporulation of test pathogen on both solid (PDA) and broth

(PDB) media. The required pH levels were adjusted by adding 0.1 N

HCl/NaOH with the help of electronic digital pH meter. The poured

petri plates were inoculated with 10 days old culture and incubated at

25 ± 20 C in incubator for 12 days. Three replications were

maintained for each pH level. Observations on the mycelial growth

(mm/mg) and sporulation of the pathogen were recorded

(Arunakumara, 2006) after 12 days of incubation.

3.3 To Standardize Conidial Production as well as Inoculation Technique for Alternaria solani.

3.3.1 Standardization of conidial production technique

3.3.1.1 Effect of different grain based medium and moisture content on spore production of A. solani.

Five grain based media were tested for inducing sporulation.

Twenty gram grains of wheat, sorghum, barley, maize and pearl millet

were placed in 100 ml flask (with four replications) and soaked in tap

water for 24 hours and before sterilization, excess water from flask

was drained. Substrates were sterilized at 15 psi for 30 minutes.

Flasks containing grains were inoculated with test pathogen and

incubated at 25±2 °C for sporulation. Colonization and subsequent

sporulation of A. solani in different grain based media was assessed.

For determination of the optimum level of water required for

colonization and sporulation, grains were dried at 60 ºC for 12 h.

After cooling, sterile tap water was added in the ratio of 10:1, 10:2,

10:3, 10:4, 10:5, 10:6, 10:7, 10:8, 10:9 and 10:10 (gm grains : ml

water). Flasks were well shaken and sealed with aluminium foil and

kept for 12 hours at room temperature. Flasks containing grains were


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inoculated with test pathogen and incubated at 25±2 °C for

sporulation (Chaurasia et al., 1998).

3.3.1.2 Effect of UV light on spore production of A. solani

Twenty milliliter potato dextrose agar medium was poured into

sterilized petri plates (90 mm diameter). The Petri plates were

inoculated with five millimeter diameter discs of 10 days old mycelial

culture from the periphery with the help of sterilized cork borer and

placed at the centre and incubated at 25 ± 20C in incubator for 12

days. Four replicates were maintained for each treatment. 5, 7, 9 and

11 days old cultures of A. solani grown on potato dextrose agar were

exposed to UV lights at different concentrations i.e. 0, 10, 20, 40, 60

and 120 seconds each. Observations for sporulation of pathogen on

solid media were recorded 12 days after incubation under microscope

by haemocytometer (Charton, 1953).

Potato dextrose broth was also studied for sporulation of the

test pathogen with UV light exposure for 0, 10, 20, 40, 60 and 120

seconds using 5, 7, 9 and 11 days old culture. Twenty milliliter broth

medium was poured into 100 ml conical flasks, separately. The flasks

were plugged with sterilized cotton and autoclaved at 1210 C for 20

min. The flasks were inoculated with five millimeter disc of 10 days

old culture of test pathogen from periphery with the help of sterilized

cork borer and incubated at 25± 2ºC in incubator for 12 days. Four

replicates were maintained for each treatment. Observations for spore

production was recorded 12 days after incubation.

3.3.1.3 Effect of light and darkness on spore production of A. solani

The Petri plates were inoculated with five millimeter diameter

discs of 10 days old mycelial culture and incubated at 25 + 20 C in

incubator for 12 days. Four replicates were maintained for each


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treatment. A. solani grown on potato dextrose agar media were treated

with twelve different light and darkness treatments i.e. Total

darkness; Total Light (3000 Lux); 12 hrs darkness + 12 hrs light

(3000 Lux); 12 hrs darkness + 24 hrs light (3000 Lux); 12 hrs

darkness + 48 hrs light (3000 Lux); 24 hrs darkness + 12 hrs light


darkness + 48 hrs light (3000 Lux); 48 hrs darkness + 12 hrs light


darkness + 48 hrs light (3000 Lux) and 72 hrs darkness + 72 hrs light

(3000 Lux) for 12 days. The effect of any external source of light was

avoided in the petri dishes that were thoroughly wrapped in black

paper during the incubation period (Luken, 1965). Observations for

sporulation of pathogen on solid media were recorded 12 days after

incubation under microscope by haemocytometer.

3.3.2 Standardization of inoculation technique

The fungus survives on crop, crop debris, seed and soil.

Significant correlations were observed between two susceptible tomato

varieties (Co-3 and Arka Vikash) that have been grown under

glasshouse conditions using four inoculation methods. Six weeks after

germination seedlings were inoculated with different inoculation

methods viz. spray inoculation, root dip inoculation, droplet

inoculation and soil inoculation that were used with conidial

suspension (104/ml). The 30 days old grain based inoculum grown on

sorghum grains was used for inoculation. After inoculation the

temperature 25 ± 20C and humidity 90 – 100 percent were maintained

in poly house with the help of humidifire. The inoculated plants were

regularly examined for appearance of symptoms starting from 24

hours after inoculation (Chaerani, 2007). The data on PDI were

recorded on five different times at 7 days intervals i.e. 7, 14, 21, 28

and 35 days after inoculation (DAI). Disease severity was scored on a

ten-point scale (0-9) as described by Ghosh et. al. 2009.


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3.3.2.1Effect on storage of inoculum and its longevity

The polypropylene bags containing inoculum of A. solani were

stored at 25 ± 20C in the incubator and after every 30 days interval

i.e. 0, 30, 60, 90 and 120 days after inoculation they were checked for

viability and pathogenicity of inoculum that was tested on 42 days old

susceptible varieties of tomato (Co-3 and Arka Vikash), with conidial

suspension having concentration of 104/ml. Control plants were

sprayed with sterile distilled water. The experiment was conducted in

pot condition with five replications. Number of lesions per leaflet was

recorded 15 days after inoculation (Chand et al., 2013).

3.3.2.2 Effect of inoculum concentration on susceptible tomato

varieties

Two susceptible varieties (Co-3 and Arka Vikash) of tomato were

taken for experiment. Six weeks after germination of tomato plants

they were inoculated with different inoculum concentrations viz. 0 x

103, 1 x 103, 2 x 103, 5 x 103 and 10 x 103under pot conditions. There

were five replications for each varieties/conidial concentration

combination. Control plants were sprayed with sterile distilled water.

The 30 days old culture was used for inoculation on sorghum grains.

After inoculation the temperature 25 ± 20C and humidity 90 – 100

percent were maintained in poly house with the help of humidifire.

Number of lesions per leaflet were recorded (Vloutoglou and

Kalogerakis, 2000) after 15 days of inoculation.

3.3.2.3 Effect on growth phase of the pathogen

The growth phase study was conducted on potato dextrose agar

(PDA) and potato dextrose broth (PDB). 20 ml PDA in each petri plates

and 25 ml PDB in each 100 ml flasks were poured. Poured petri plates

were inoculated with 5 mm disc of A. solani culture and flasks were

sterilized at 121 °C for 20 minutes at 15 lb psi pressure and then


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inoculated with 5 mm disc of A. solani culture. The inoculated petri

plates and flasks were incubated at 25 ± 2°C. A set of petri plates and

flasks with three replications were observed starting from first day up

to full growth. The culture was filtered through Whatman No.1 filter

paper. Before filtering, the filter papers were dried to a constant

weight by drying in hot air oven at 50°C. The mycelial mat on the filter

paper was thoroughly washed with distilled water dried in hot air oven

at 50°C. The filter paper with mycelial mat was weighed in a digital

electronic balance. The weight of dry mycelial mat was recorded and

the data were statistically analysed and maximum growth period was

determined (Arunakumara, 2006).

3.4 Phenotyping and Genotyping of Tomato RILs and Germplasm.

3.4.1 Raising of nursery

Seeds of tomato germplasm lines and RILs were taken for

growing in nursery. The mixture of soil, sand and FYM were taken in

nursery and seeds were sown. After sowing, the beds were covered

with a thin film of water thereafter light and frequent irrigation were

given at a regular interval in order to maintain sufficient moisture in

nursery plots for good growth of seedlings. The tomato seedlings were

transplanted. One day before transplanting field or poly house was

irrigated to maintain soft soil to save breaking of seedling roots.

Seedlings were uprooted carefully from the nursery field to transplant

in the field or poly house. A thin film of water was maintained till the

establishment of seedlings. Subsequently, water level in the field or

poly house were maintained during the entire period of crop growth so

that moisture remains in the soil.


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3.4.2 Disease assessment

The 30 days old culture (mycelial mat and spores) were grown

in sorghum grain medium and were harvested. Then the plants were

inoculated with 104 spore concentration with the help of automizer.

After inoculation the moisture was maintained in field or poly house

with the help of irrigation. Plants were examined for appearance of

symptoms. The symptoms appeared 3-7 days after inoculation. The

inoculated plants were regularly examined for appearance of

symptoms starting from 24 hours after inoculation. The data on PDI

were recorded at 7 days intervals. Disease severity was scored by 0-5

scale for natural screening (Vakalounakis, 1983) and 0-9 scale for

artificial screening (Ghosh et. al. 2009).

Table 3.1: Description of disease scale (0-5)

Sr. No. 0-5 Scale Per cent leaf area infected

1 0 No infection

2 1 10

3 2 11-25

4 3 26-50

5 4 51-75

6 5 > 75


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Table 3.2: Description of disease scale (0-9)

Sr. No. 0-9 Scale % leaf area infected

1 0 No infection

2 1 0 – 10

3 2 10 – 20

4 3 20 – 30

5 4 30 – 40

6 5 40 – 50

7 6 50 – 60

8 7 60 – 70

9 8 70 – 80

10 9 80 – 100

The percentage disease index (PDI) and area under disease

progress curve (AUDPC) (Campbell and Madden, 1990; Johnson and

Wilcoxson, 1982; Shaner and Finney, 1977) were calculated as

follows.

PDI =

Sum of all ratings 100

Total no. of observation Maximum rating scale

Percent Disease Index (PDI) was worked out by using formula

given by Wheeler (1969).

𝐴𝑈𝐷𝑃𝐶 = ∑ {[ (𝑋𝑖+1 + 𝑋𝑖)𝑛−1

𝑖=1/2] ∗ (ti+1 – ti)}

Where;

Xi is the disease index expressed as a proportion at the ith

observation.

ti is the time (days after planting) at the ith observations.

And n is the total number of observations.


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3.4.3 Phenotyping

For the early blight screening the tomato growth stages are

described below through chart (FAO, 2000).

3.4.3.1 Phenotyping of 701 germplasm (Year 2011-12)

The seeds of 701 tomato germplasm lines were collected from

IIVR, Varanasi. Some germplasm lines have determinate growth habit

and remaining lines have indeterminate growth habit. 50 percent

flowering, fruit setting and senescence stages were recorded on all

701 germplasm lines (See in appendix table 3 for detailed study of

data).

3.4.3.1.1 Details of field experiment

The details of layout plan are given as follows: Total 701 tomato

germplasm lines were taken with randomized block design (RBD).

Each germplasm lines were taken with ten plants.


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3.4.3.2 Phenotyping of 240 core set lines (Year 2012-13)

The 240 tomato core lines that were selected from 701

germplasm lines were planted in field.

3.4.3.2.1 Details of Layout under field conditions

The details of layout plan are illustrated as follows:

1. Total Area in field = 25 × 70 = 1750 m2

2. Total no. of rows = 240

3. No. of plants per rows = 10

4. Spacing = 40 × 40 cm

5. Design = Randomized Block Design

3.4.3.2.2 Details of cultural operations

Details of various cultural operations done during the

experimentation are presented in table 3.3.

Table 3.3: Details of cultural operations carried out during experiment

S.

No.

Operations Date of operations

1. Nursery preparation

i. Bed preparation

ii. Date of seed sowing

02.09.2012

06.09.2012

2. Field preparation 15.09.2012

3. Transplanting 18.10.2012

4. Hand weeding

i. 1st Hand weeding

ii. 2nd Hand weeding

18.11.2012

18.12.2012


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3.4.3.3 Phenotyping of 226 core set lines (Year 2013-14)

The seeds of tomato 226 core lines were collected from IIVR,

Varanasi.

3.4.3.3.1 Details of Layout in field conditions





4. Spacing = 40 × 40 cm

5. Design = Randomized Block Design




Table 3.4: Details of cultural operations carried out during

experiment

S. No.



i. Bed preparation


10.09.2013

12.09.2013

2. Field preparation 20.10.2013


4. Hand weeding

i. 1st Hand weeding


27.11.2013

17.12.2013


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3.4.3.4 Phenotyping of 151 tomato RILs (Co-3 × EC-520061) in year 2012-13.

The seeds of tomato Recombinant Inbred Lines in F7 generation

were advanced at IIVR, Varanasi.

RILs have been developed from the cross between a susceptible

parent (Co-3) and resistant parent (EC-520061) by single seed descent

method. Co-3 has determinate growth habit, flowering time is 49 DAT,

fruit long and medium in size. EC-520061 has indeterminate growth

habit; flowering time is 64 DAT and fruit round and small berry sized.

3.4.3.4.1 Details of Layout in poly house


1. Total Area of Poly house = 20 × 8.5 = 170 m2



4. Spacing = 45 × 45 cm

5. Design = Alpha Lattice Design

3.4.3.4.2 Details of Layout in field conditions

1. Total area in field = 25 × 50 = 1250 m2



4. Spacing = 45 × 45 cm






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Table 3.5: Details of cultural operations carried out during experiment

S.No. Operations Date of operations


i. Bed preparation


20.09.2012

22.09.2012

2. 1. Field preparation

2. Poly house preparation

i. Harrowing

ii. Layout

20.10.2012

25.10.2012

07.11.2012


4. Hand weeding

i. 1st Hand weeding


08.12.2012

09.01.2013

3.4.3.5 Phenotyping of 151 tomato RILs (Co-3 × EC-520061) in year 2013-14.

The seeds of tomato Recombinant Inbred Lines in F8 generation

were advanced at IIVR, Varanasi.

3.4.3.5.1 Details of layout in poly house


1. Total Area of Poly house = 20 × 8.5 = 170 m2



4. Spacing = 45 × 45 cm



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3.4.3.5.2 Details of layout in field conditions




4. Spacing = 45 × 45 cm





Table 3.6: Details of cultural operations carried out during

experiment

Sr. No.



i. Bed preparation


02.09.2013

03.09.2013

2. 1. Field preparation

2. Poly house preparation

i. Harrowing

ii. Layout

20.10.2013

15.10.2013

20.10.2013


4. Hand weeding

i. 1st Hand weeding


08.12.2013

28.12.2013


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3.4.3.6 Statistical analysis

The experiment was laid out in Alpha Lattice Design (ALD) with

two replication. The values of data obtained from the poly house were

subjected to following statistical analysis.

1. Analysis of variance (ANOVA)

2. DNMRT analysis by SAS

3.4.3.6.1 Analysis of variance

Analysis of variance was done on the basis of available data.

The skeleton of ANOVA is given in table form.

3.4.3.6.2 Test of significance

If the variance ratio (or) F-calculated value was greater than the

F-table value at 5% level of significance, the differences between

treatments was considered to be significant. If the F-calculated value

is less than F-tabulated value, the differences between treatments

were considered to be non-significant.

3.4.4 Genotyping of RILs

3.4.4.1DNA extraction

DNA was extracted from fresh seedling leaves of each RILs

following the method of Doyle and Doyle (1990).

3.4.4.1.1 Requirements for DNA extraction

Leaf sample

Cetyl Trimethyl Ammonium Bromide (CTAB) extraction buffer

(100 ml)


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Chilled isopropanol

Phenol : chloroform : isoamylalcohol =25:24:1(v/v)

Ethanol

RNase A: RNase A was dissolved in TE buffer and boiled for 15

minutes at 100 °C to destroy RNase and stored at -20 °C.

3.4.4.1.2 Protocol

Up to 100 mg of frozen leaf tissues were taken in autoclaved

mortar pestle.

The leaf tissues were ground in liquid nitrogen and extracted

with 500 μl of CTAB buffer and transferred to 2 ml centrifuge

tube.

Incubate at 65°C for at least one hour, mixing once after 30

minutes. They can be left in water bath for a few hours if

necessary.

The tubes were removed from the water bath and equal volume

of Phenol: chloroform: Isoamylalcohol mixture (25:24:1 v/v) was

added and mixed by using shaker for 15 minutes.

It was centrifuged at 14,000 rpm for 10 minutes at room

temperature.

Transfer 800 μl aqueous phase (top layer) into the new labeled

tube.

800 μl of Chloroform: Isoamylalcohol mixture was added,

shaken for 10-15 minutes on shaker and centrifuged for 10

minutes at 14,000 rpm.

Supernatant was taken in 1.5 ml autoclaved centrifuge tube.


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2/3 volume of chilled isopropanol was added and mixed gently

by inversion and then kept in the deep freezer until DNA was

precipitated out.

It was centrifuged at 14,000 rpm for 10 minutes at 4 °C

temperature.

Discard the supernatant and pellet was washed by using 200 μl

70% of ethanol and kept it overnight at room temperature for

drying.

Then the DNA pellet was dissolved in 50 μl of TE and stored at -

20 °C.

3.4.4.2 DNA quality check

50 mg of agarose was added to 100 ml of 1X TAE buffer in a

conical flask melted and then cooled. The combs were placed in gel

plate. When the gel temperature was around 50ºC 2 µl of ethidium

bromide was added. The gel was then poured into the gel plate slowly

to avoid air bubbles. After polymerisation of gel, the plate was placed

in electrophoresis tank and 1X TBE buffer was added. Gently combs

were removed without damaging the wells. The samples were then

loaded by mixing 3μl-dissolved DNA with 5μl of loading dye and run

till the bromophenol blue has reached the 3/4th

of the gel. The gel was

visualised under transilluminator where DNA is visualised as orange

band under UV. The gel was photographed using gel doc system.

Depending on the molecular standard, the quality and quantity of

extracted DNA was estimated.

3.4.4.3 SSR primer selection

A total of 447 SSR primers were selected for identification of

early blight resistance genes/QTLs in tomato.


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3.4.4.4 Optimization of polymerase chain reaction (PCR)

PCR amplification using SSR primers was carried out using

sterilized thin-walled PCR tubes (0.2 ml) according to the protocol of

Loridon et al., 2005 with minor modifications. After optimizing the

reaction conditions, Polymerase Chain Reaction was carried out in 25

µl reaction mixture (Table 3.7) in a thermal cycler.

Table 3.7: List of the various components of PCR master mixture (25 μl).

S.No. Reagents Stock

Concentration

Required

concentration

Quantity

(μl)

1. Assay buffer 10x 1x 2.50

2. Mgcl2 25Mm .3mM 0.75

3 DNTPs 10Mm 133µM 2.00

4 Taq polymerase 5 U 1 U 0.50

5 Primer(Forward) 10µM 1.25µM 1.0

6 Primer(Reverse) 10µM 1.25µM 1.0

7 Genomic DNA 15 ng/µl 15 ng/µl 2.0

8 HPLC water - - 15.25

Total - - 25.00

This master reaction mixture was distributed into individual

PCR tubes at the rate of 25µl/tube. The tubes containing reaction

mixture were placed in the wells of the thermal cycler block, and

amplification reaction was carried out with the temperature

programme summarized in table 3.8, and schedule of temperature

and duration for PCR amplification using SSR primers are presented

in table 3.8. List of 447 SSR primers is given in appendix table 5.


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Table 3.8: The schedule of temperature and duration programmed for PCR amplification using SNP primers.

S.No. Objective Temperature (0C)

Duration Number of cycles

1. Initial denaturation 94 3 min 1

2. Denaturation 94 30 sec 39

3. Annealing Ta 30 sec 39

4. Extension 72 1 min 39

5. Final extension 72 5 min 1

Where,

Tm = Optimum annealing temperature; varies according to GC

content of the primers.

After the completion of the PCR, the products were stored at

40C until the gel electrophoresis was done.

3.4.4.5 Separation of amplified products on agarose gel electrophoresis

3.4.4.5.1 Requirements

10X TAE (Tris Acetate EDTA buffer)

Tris Base 48.4 g

Acetic acid 11.42 ml

0.5M EDTA 20 ml

(Dissolved in 800 ml of sterile water and made up to 1000 ml)

Loading Dye

Glycerol 30% (v/v)

Bromophenol blue 0.5% (w/v)


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3.4.4.5.2 Protocol

Agarose gel of 2.5% concentration was prepared in 1x TAE

buffer (electrophoresis buffer) and then heated in microwave to

melt the agarose completely till the solution becomes clear.

The agarose solution was cooled to 600C and then ethidium

bromide was added at a concentration of 0.5 μg/ml of gel and

mixed well.

The agarose solution was poured in gel casting tray slowly to

avoid any air bubble formation.

The gel was then allowed to set completely before removing the

comb. The comb was removed carefully and the gel was placed

in gel electrophoresis tank which is half filled with 1x TAE

buffer.

2μl of 6x gel loading dye (Bromophenol blue 0.25%; Xylene

cyanol 0.25% and Glycerol 30.0%) was added to 15μl of PCR

products and mixed well before loading into the wells. Then the

amplified product loaded in each well using micropipette. Care

was taken to prevent mixing of samples between the wells.

After loading of all samples 4μl of 100bp DNA size ladder

(Fermentas, India) was loaded in first well as a reference for

molecular weight of amplified products.

A constant voltage of 65V was given for a time period of four

hours for separation of PCR fragments using a power pack (BIO

RAD, USA).

After the run, the amplified products were visualized and

photographed under UV light source in a gel documentation

system (Gel Doc TM XR+, BIO RAD, USA) and image of band

patterns were stored in a computer for future use.


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3.4.4.6 Band scoring for QTLs

The banding patterns obtained from PCR amplification of

various SSR primers in the F7 Individuals were scored as follows:

A = Homozygote for allele a from parental strain P1 at this locus

B = Homozygote for allele b from parental strain P2 at this locus

- = Missing data for the individual at this locus

After scoring, the individual progeny genotypes were typed in a

Microsoft Excel spread sheet in a format suitable for linkage analysis

by Map Maker I Exp. (i.e., rows =genotype score at a given locus;

columns = F7 individual of the mapping population)

3.4.4.7 Chi-square test

Chi- square was done to test the goodness of fit of data to 1:1

ratio among the genotypic data of the 25 SSR markers in F7

segregating population. The segregation ratios of the marker loci were

calculated using Chi-square formula t2 = (Observed -

Expected)/Expected. The calculated χ² (Chi-square) values were then

compared with tabulated values for 1 degree of freedom.

3.5 QTL Analysis

The genetic linkage map was constructed using primers

identified polymorphic in different cross combination during the

parental polymorphism survey. For each segregating marker, a Chi-

square analysis was performed to test for deviation from the expected

segregation ratio (1:1). Linkage analysis of SSR markers was

conducted using the Kosambi (1944) mapping function with a

minimum log10 odds ratio (LOD) of 2.0 and maximum recombination


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frequency of 0.4 performed by Map-Maker/EXP 3.0 (Lander et al.

1987).

Quantitative trait loci analyses for combined one across all two

environments were performed by composite interval mapping using

Windows QTL Cartographer 2.5 (Basten et al, 2003). A QTL was

considered significant when the LOD (log10 of the likelihood of odds

ratio) value that derived from permutation analysis was large than 2.

Additive and dominance effects for detected QTLs were estimated

using the Zmapqtl procedure of QTL Cartographer. The R2 value, the

percentage of the phenotypic variance explained by marker genotype

at the QTL, (coefficient of de-termination) was taken from the peak

QTL position as estimated by QTL Cartographer. Gene action was

determined by the ratio of the absolute value of the estimated

dominance effect divided by the absolute value of the estimated

additive effect (d)/(a) following Stuber et al., (1987); (additive = 0 to

0.20; partial dominance = 0.21 to 0.80; dominance = 0.81 to 1.20;

and over dominance > 1.20).

Plate 3.1: Description of disease scale (0-9) with per cent leaf area infection

Scale-0 Scale-1 Scale-2

Scale-6 Scale-7 Scale-8 Scale-9

Scale-3 Scale-4

Scale-5

Chapter ΙV

EXPERIMENTAL FINDINGS

The present investigations on “Characterization of Alternaria

solani and molecular mapping of QTLs for early blight resistance in

tomato” was carried out in the Department of Mycology and Plant

Pathology, Institute of Agricultural Sciences, Banaras Hindu

University, Varanasi and Indian Institute of Vegetable Research,

Adalpura, Varanasi. The results obtained on various aspects are

presented here in this chapter.

4.1 Collection, Isolation and Purification of Pathogen causing Early Blight of Tomato from Different Parts of the Country.

4.1.1 Collection of samples

Freshly infected diseased leafs and fruits showing typical

symptoms of A. solani were collected from different localities of India

(Varanasi, Adalpura, Mirzapur, Allahabad, Agra, Gorkhpur, Aara,

Buxer, Raichur, Banglore, Jaipur, Anand, Hisar and New Delhi) (Table

4.1).

4.1.2 Symptomatology

The disease symptoms appeared on all the plant parts viz., leaf,

stem, petioles, calyx and fruit (Plate-4.1). First symptoms appeared on

older leaves as minute dark brown usually round necrotic spots of one

to two mm in diameter. Later, the spots enlarged with characteristic

concentric rings in the center to produce a target board effect and the

colour of the spots changed from brown to dark brown. The adjacent

spots eventually coalesced to form large irregular spots leading to

drying and defoliation.

Experimental Findings

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Table 4.1: List of Alternaria solani collected from different areas of India.

S.No. Location Host Date of Collection Name of collector Date of Isolation Code of culture Species identified

1. BHU Farm, Varanasi, U. P Tomato 03-02-11 S. M. Yadav 03-02-11 Asv-1 A. Solani

2 Ganga Bank, Varanasi, U. P Tomato 05-02-11 S. M. Yadav 05-02-11 Asv-2 A. Solani

3 IIVR, Adalpura, U. P Tomato 15-02-11 S. M. Yadav 16-02-11 Asad-1 A. Solani

4 Farmer Field, Mirzapur, U. P Tomato 07-03-11 S. M. Yadav 09-03-11 Asmi-1 A. Solani

5 Farmer Field, Mirzapur, U. P Tomato 07-03-11 S. M. Yadav 09-03-11 Asmi-2 A. Solani

6 Farmer Field, Allahabad, U. P Tomato 29-11-11 S. M. Yadav 30-11-11 Asal-1 A. Solani

7 Farmer Field, Agra, U. P. Tomato 04-12-11 S. M. Yadav 06-12-11 Asag-1 A. Solani

8 Farmer Field, Gorkhpur, U. P. Tomato 03-02-12 S. M. Yadav 05-02-12 Asgo-1 A. Solani

9 Farmer Field, Aara, Bihar Tomato 10-03-11 S. M. Yadav 11-03-11 Asar-1 A. Solani

10 Farmer Field, Aara, Bihar Tomato 10-03-11 S. M. Yadav 11-03-11 Asar-2 A. Solani

11 Farmer Field, Buxer, Bihar Tomato 03-01-13 S. M. Yadav 05-01-13 Asbx-1 A. Solani

12 Raichur,Karnataka Tomato 03-02-13 Vineeta Singh 05-02-13 Asrai-1 A. Solani

13 Banglore, Karnataka Tomato 03-02-13 Vineeta Singh 05-02-13 Asbng-1 A. Solani

14 Farmer Field, Jaipur, Raj. Tomato 04-04-12 S. M. Yadav 08-04-12 Asjai-1 A. Solani

15 Farmer Field, Anand, Gujarat Tomato 06-03-12 S. M. Yadav 10-03-12 Asan-1 A. Solani

16 CCSHAU, Hisar, Hariyana Tomato 25-02-13 S. M. Yadav 28-02-13 Ashi-1 A. Solani

17 IARI, New Delhi Tomato 13-02-11 S. M. Yadav 15-02-11 Asnd-1 A. Solani

18 IARI,New Delhi Tomato 13-02-11 S. M. Yadav 15-02-11 Asnd-2 A. Solani


~80~

When plants were 60-90 days old, symptoms also appeared on

stem and petioles as brown to dark brown elongated cankerous target

board type spots. These spots enlarged and covered the entire stem

and petioles leading to withering of the plants. Symptoms also

developed on calyx and flower buds in the form of minute brown to

dark brown spots which enlarged later and spread to sepals and fruits

resulting in pre-mature dropping of fruits. The symptoms on fruits

appeared first at stem end as black or brown sunken spots both on

green and ripe fruits which enlarged within eight days involving most

of the fruits, finally the fruits were rotted.

4.1.3 Isolation and identification of the pathogen

Standard tissue isolation technique was followed to obtain A.

solani from the tomato leaves showing typical early blight symptoms.

The culture thus obtained was further purified by single spore

isolation technique. The pure culture of the pathogen was maintained

throughout the investigations by periodical transfer on PDA. The

description of the fungus isolated is as follows. The conidiophores

were formed singly or in groups, straight or flexuous brown to

olivaceous brown. The conidia we have observed muriform with 6-8

transverse and 2-3 longitudinal septa (Alexopoulos et al., 1996).

4.2 Cultural, Morphological and Pathogenic Variability amongst Tomato Isolates of Alternaria solani.

4.2.1 Cultural variability

4.2.1.1 Radial Growth

Radial growth observed for eighteen isolates (Table 4.2) were

significantly different for most of the isolates. Highest radial growth

was observed in isolate Asnd-2 (41.00 mm) which was at par with

isolates Asnd-1 (40.33 mm) and Ashi-1 (38.67 mm) at 7 DAI; Asnd-1

(72.67 mm) was at par with isolates Asnd-2 (70.67 mm) and Ashi-1


~81~

(69.33 mm) at 10 DAI and Asnd-1 (88.67 mm) was at par with isolates

Asnd-2 (88.00 mm) and Ashi-1 (88.00 mm) at 13 DAI.

Table 4.2: Mycelial growth of A. solani on Potato Dextrose Agar medium

incubated at 25±2ºC.

Isolates

Mycelial growth of different isolates (mm)

Sporulation

7 DAI 10 DAI 13 DAI

Asv-1 33.33 60.67 83.33 +

Asv-2 29.67 54.33 80.00 +

Asad-1 26.00 50.63 78.33 -

Asmi-1 25.67 56.33 79.33 -

Asmi-2 29.67 59.67 82.33 +

Asal-1 34.67 63.67 84.00 -

Asag-1 31.67 60.67 79.67 -

Asgo-1 33.33 64.67 82.33 -

Asar-1 24.33 50.33 78.00 -

Asar-2 26.33 53.33 79.00 -

Asbx-1 32.33 54.67 78.33 -

Asrai-1 34.33 60.67 80.67 -

Asbng-1 37.33 67.00 86.67 -

Asjai-1 32.00 62.67 84.00 -

Asan-1 34.33 57.67 81.67 +

Ashi-1 38.67 69.33 88.00 -

Asnd-1 40.33 72.67 88.67 +

Asnd-2 41.00 70.67 88.00 -

C.D. (1 %) 2.99 3.43 4.05

CV % 5.52 3.41 2.95

+ = Present; - = Absent


~82~

Minimum mycelial growth was observed in isolate Asar-1

(24.33, 50.33 & 78.00 mm) at 7, 10 and 13 DAI, respectively. The

mean mycelial growth observed at these different time intervals i. e. 7,

10, & 13 DAI was not significantly different to each other. But the

maximum mean mycelial growth was observed in isolate Asnd-1

(67.22 mm) followed by Asnd-2 (66.55 mm) and Ashi-1 (65.33 mm)

and minimum mean mycelial growth was observed in isolate Asar-1

(51.22 mm) followed by Asad-1 (51.55 mm) and Asar-2 (52.88 mm).

4.2.1.2 Sporulation

Conidia production was observed only in five isolates (Asv-1,

Asv-2, Asmi-2, Asan-1 and Asnd-2) with low quantity and found that,

out of 18 isolates remaining 13 isolates produced mycelial growth in

petri plates. Highest number of conidia (1.00 × 104) were produced in

isolate Asv-2 and minimum (0.75 × 104) in isolate Asan-1.

4.2.2 Morphological variability

4.2.2.1Pigmentation

Isolates of A. solani depicted great variability in pigment

production on PDA medium. Three isolates (Asv-1, Asnd-1 & Asnd-2)

produced reddish black; two isolates (Asv-2 & Asag-1)produced light

black; three isolates (Asad-1, Asjai-1 &Ashi-1) produced greenish

black; five isolates (Asmi-1, Asmi-2, Asbng-1, Asgo-1&Asrai-1)

produced brownish black; one isolate (Asal-1) produced reddish; one

isolate (Asar-1) produced blackish; one isolate (Asar-2) produced dirty

white, while two isolates (Asbx-1 & Asan-1) produced light yellow

pigment on PDA after 12 days of inoculation at 25±2 ºC (Table 4.3).


~83~

Table 4.3: Cultural and morphological variability of different isolates of A. solani incubated at 25±2ºC.

Isolates Pigmentation Sporulation

on PDA media

Mycelial growth/Colony character

Margin of growth

Colony growth

Zonation

Asv-1 Redish black Yes Irregular Smooth Without Zonation

Asv-2 Light black Yes Circular Rough Without Zonation

Asad-1 Greenish black No Circular Smooth Without Zonation

Asmi-1 Brownish black No Irregular Smooth Concentric Zonation

Asmi-2 Brownish black Yes Irregular Rough Without Zonation

Asal-1 Redish No Circular Rough Without Zonation

Asag-1 Light black No Circular Smooth Concentric Zonation

Asgo-1 Brownish black No Circular Smooth Concentric Zonation

Asar-1 Blackish No Irregular Smooth Without Zonation

Asar-2 Dirty white No Irregular Smooth Concentric Zonation

Asbx-1 Light yellow No Irregular Rough Concentric Zonation

Asrai-1 Brownish black No Irregular Smooth Concentric Zonation

Asbng-1 Brownish black No Circular Smooth Concentric Zonation

Asjai-1 Greenish black No Circular Rough Without Zonation

Asan-1 Light yellow Yes Circular Rough Without Zonation

Ashi-1 Greenish black No Irregular Rough Without Zonation

Asnd-1 Redish black No Circular Smooth Concentric Zonation

Asnd-2 Redish black Yes Circular Smooth Concentric Zonation

4.2.2.2 Mycelial growth pattern

Mycelial growth patterns were observed on PDA where eight

isolates grew with irregular growth patterns and ten isolates grew with

circular growth patterns. Colony characters on PDA were also

observed where eleven isolates depicted smooth surfaced colony and

seven isolates depicted rough surfaced colony growing. Eight isolates


~84~

depicted growth without zonation and ten isolates were with

concentric zonation (Plate 4.2).

These isolates exhibited significant variation for their cultural

characters, pigmentation and growth rate per day. Few isolates grew

very fast in the initial 4–5 days of observation and few were fast in the

middle-age and rest grew fast at a later age. As the isolates of A.

solani were collected from different agro climatic zones of the country.

4.2.3 Pathogenic variability

The pathogenicity test of A. solani isolates was conducted in

poly house conditions using two highly susceptible varieties of

tomato. Based on the recorded data (PDI), five isolates were found to

be virulent, causing severe disease in both tested varieties. Other

thirteen isolates were rated as less virulent (Table 4.4). At 35 DAI

percent disease index of virulent isolates ranged between 85.06 % and

93.83 % (Mean AUDPC ranged between 1392.26 & 1477.78) while in

other isolates that were categorized as less virulent, the percent

disease index ranged between 69.14 % to 87.65 % (Mean AUDPC

ranged between 1011.11 & 1322.22).

4.2.4 Effect of plant age for early blight development

The effect of age of tomato plants on disease incidence was

studied after artificial inoculation in pot condition. As the age of

plants i.e. variety Co-3 increased, the percentage of leaf area showing

symptoms and the percentage of defoliation also increased. Linear

regressions provided a good description of the relationship between

percentage leaf area infected and plant age, and the percentage of

defoliation and plant age. Sixty days after sowing, plants of Co-3

developed greater area under disease progress curve i.e. 1322.22 than

in 45-days-old plants showing AUDPC value of 1477.78 when

inoculated with the isolates Asv-2.


~85~

Table 4.4: Pathogenic virulence of A. solani isolates on two

susceptible cultivars of tomato.

S.No. Isolates

Co-3 Arka Vikash

IP

(Days)

35 DAI (PDI Value)

AUDPC IP

(Days)

35 DAI (PDI Value)

AUDPC

1 Asv-1 5 87.65 1416.67 4 85.06 1361.11

2 Asv-2 4 93.83 1477.78 4 89.00 1438.89

3 Asad-1 6 83.95 1244.44 6 81.89 1205.56

4 Asmi-1 6 87.65 1392.26 7 83.83 1342.02

5 Asmi-2 4 87.65 1322.22 4 78.22 1244.44

6 Asal-1 6 77.78 1166.67 7 70.37 1088.89

7 Asag-1 7 76.54 1011.11 6 66.67 1127.78

8 Asgo-1 7 74.07 1166.67 7 71.60 894.44

9 Asar-1 6 74.07 1244.44 6 66.67 972.22

1`0 Asar-2 5 74.07 1153.16 6 66.67 1127.78

11 Asbx-1 6 69.14 1244.44 6 66.67 1127.78

12 Asrai-1 7 74.07 1127.78 7 66.67 1050.00

13 Asbng-1 7 74.07 1127.78 6 67.90 1244.44

14 Asjai-1 7 77.78 1088.89 6 66.67 1050.00

15 Asan-1 5 85.06 1438.89 4 83.95 1344.44

16 Ashi-1 6 74.42 1322.22 6 69.14 1166.67

17 Asnd-1 7 85.06 1438.89 6 83.95 1400.00

18 Asnd-2 6 78.44 1322.22 7 75.32 1205.56

C.D. (1 %) 8.230 9.128

CV % 95.042 103.06

The results clearly indicated that 45-day-old plants were less

susceptible to A. solani infection than were the 60-day-old plants with

all the isolates. The final PDI and AUDPC values for most of isolates

on Co-3 variety were much higher in 60-day-old plants than in 45-


~86~

day-old plants. No symptoms developed and no defoliation was

observed on uninoculated plants (Table 4.5).

Table 4.5: Response of 45 and 60 days old tomato cv. Co-3 for early blight development.

S.No. Isolates

45 DAS 60 DAS

IP*

(Days)

35 DAI

(PDI Value)

AUDPC IP*

(Days)

35 DAI

(PDI Value)

AUDPC

1 Asv-1 7 85.19 1192.59 5 96.30 1464.81

2 Asv-2 6 92.59 1283.33 5 100.00 1464.81

3 Asad-1 8 85.19 1153.70 6 88.89 1205.56

4 Asmi-1 7 85.19 1244.44 5 92.59 1296.30

5 Asmi-2 7 85.19 1231.48 5 92.59 1322.22

6 Asal-1 8 66.67 933.33 6 77.78 1062.96

7 Asag-1 8 66.67 881.48 7 74.07 1101.85

8 Asgo-1 7 70.37 907.41 7 77.78 1075.93

9 Asar-1 9 66.67 816.67 7 77.78 1050.00

10 Asar-2 7 66.67 894.44 7 77.78 1075.93

11 Asbx-1 8 66.67 894.44 6 74.07 1140.74

12 Asrai-1 7 66.67 907.41 7 77.78 1088.89

13 Asbng-1 7 70.37 881.48 7 77.78 1088.89

14 Asjai-1 7 66.67 933.33 7 81.48 1127.78

15 Asan-1 6 85.19 1218.52 5 96.30 1335.19

16 Ashi-1 8 74.07 1011.11 6 81.48 1231.48

17 Asnd-1 7 85.19 1205.56 6 96.30 1387.04

18 Asnd-2 6 88.89 1322.22 5 100.00 1477.78

CV % 6.050 7.596

LSD (1 %) 105.48 154.04

*IP=Incubation period


~87~

4.2.5 Effect of media, temperature and pH

4.2.5.1Media

The results revealed that the highest mycelial growth of A.

solani was recorded on Potato dextrose agar medium (31.00, 50.67 &

88.33 mm) over all other media but it was at par with V-8 agar (29.67,

48.67 & 86.00 mm) at 4, 8 and 12 days after incubation, respectively.

Significantly lowest mycelial growth was observed in host leaf extract

agar (15.33, 30.00 & 45.33 mm) at 4, 8 and 12 days after incubation,

respectively as compared to other treatments. The sporulation of A.

solani was observed on Potato dextrose agar and V-8 agar media. But

all other media failed to produce sporulation of A. solani (Table 4.6).

The highest dry mycelial weight of A. solani was also recorded in

Potato dextrose broth medium (78.67, 169.67 & 514.00 mg) which

was at par with V-8 juice broth medium (77.33, 158.33 & 509.67 mg)

at 4, 8 and 12 days after incubation, respectively. Significantly lowest

mycelial growth in liquid media was observed in host leaf extract agar

(32.33, 104.00 & 210.67 mg) at 4, 8 and 12 days after incubation,

respectively as compared to other treatments. The sporulation of A.

solani was also observed on Potato dextrose broth and V-8 broth

media. But all other media failed to produce sporulation of A. solani

(Table 4.7).

4.2.5.2 Temperature

Influence of temperature on mycelial growth and sporulation of

A. solani were determined on potato dextrose agar. The petri plates

inoculated with A. solani were exposed to different temperatures i. e.

5, 10, 15, 20, 25, 30, 35 and 400C. Observations on the mycelial

growth (mm) and sporulation were recorded after seven days of

incubation.


~88~

Table 4.6: Effect of different solid media on mycelial growth and

sporulation of A. solani (Asv-2) incubated at 25 ± 2 ºC.

Name of Medium Mycelial Growth (mm) Sporulation*

(12 DAI) 4 DAI 8 DAI 12 DAI

Bean meal agar 23.67 44.00 79.00 -

Corn meal agar 25.00 44.67 80.67 -

Glucose peptone agar 18.67 40.33 77.67 -

Host leaf extract agar 15.33 30.00 45.33 -

Malt extract agar 27.00 41.67 76.33 -

Oat meal agar 23.67 41.33 75.67 -

Czapek’s Dox agar 26.67 42.67 77.67 -

Potato dextrose agar 31.00 50.67 88.33 +

Richard’s agar 26.00 41.33 80.67 -

V-8 juice agar 29.67 48.67 86.00 +

C.D. (1 %) 2.71 4.01 3.15

CV % 6.41 5.50 2.39

*Absent (-) and Present (+); DAI=Days After Incubation

Table 4.7: Effect of different broth on mycelial growth and sporulation of A. solani incubated at 25 ±2ºC.

Name of Medium Dry mycelial weight (mg) Sporulation*

(12 DAI) 4 DAI 8DAI 12 DAI

Bean meal broth 64.67 138.67 473.67 -

Corn meal broth 67.33 138.00 487.33 -

Glucose peptone broth 62.67 133.33 419.67 -

Host leaf extract broth 32.33 104.00 210.67 -

Malt extract broth 54.00 131.67 431.00 -

Oat meal broth 63.00 135.33 440.67 -

Czapek’s Dox broth 69.00 145.33 449.33 -

Potato dextrose broth 78.67 169.67 514.00 +

Richard’s broth 61.00 139.67 413.33 -

V-8 juice broth 77.33 158.33 509.67 +

C. D. (1 %) 5.29 5.14 8.20

CV % 4.89 2.15 1.10



~89~

It is clear from the results in table 4.8 that A. solani could grow

well at 25 and 30ºC temperatures. Highest mycelial growth (30.00,

51.67 & 85.00 mm) was recorded at 25ºC which was not significantly

different to temperature 30ºC (27.33, 50.67 & 81.00 mm) at 4, 8 and

12 days after incubation, respectively. Lowest mycelial growth of A.

solani was obtained at 5ºC (7.67, 13.33 &19.33 mm) without

sporulation at 4, 8 and 12 days after incubation, respectively as

compared to other treatments. It is revealed from the result that A.

solani sporulated at 25 and 30ºC on PDA medium among the

temperatures tested. All other temperatures except 25, 30 and 35ºC

failed to produce sporulation of A. solani.

To study the effect of temperatures on growth of A. solani,

potato dextrose broth was used as the basal medium (Table 4.9).

There was more dry mycelial weight along with sporulation of A. solani

recorded at 25°C i.e. 127.67, 316.67 & 603.33 mg which, was not

significantly different to temperature at 30°C i.e. 123.67, 31.00 &

595.67 mg at 4, 8 and 12 days after incubation, respectively. Lowest

dry mycelial weight of A. solani was obtained at 5ºC (21.67, 35.33 &

60.33 mg) at 4, 8 and 12 days after incubation, respectively as

compared to other treatments. The results of experiment indicated

that A. solani preferred a temperature range of 25°C and 30°C for its

growth and sporulation.

4.2.5.3 pH

It is clear from the results presented in table 4.10 that the

highest mycelial growth with sporulation was obtained at pH 7.0

(34.67, 53.67 & 86.67 mm) which was at par with pH 6.5 (33.67,

53.00 & 83.67 mm). While, pH 4.0 (15.33, 21.67 & 34.00 mm) that

gave least mycelial growth and no sporulation of A. solani at 4, 8 and

12 days after incubation, respectively. The moderate pH was found

more favourable for the mycelial growth and sporulation of A. solani

as compared to acidic and alkaline pH.


~90~

Table 4.8: Effect of different temperature on mycelial growth and

sporulation of A. solani isolate (Asv-2) on PDA medium.

Temperatures Mycelial Growth (mm) Sporulation*


5 7.67 13.33 19.33 -

10 12.67 21.67 28.33 -

15 15.67 25.33 37.33 -

20 23.67 35.67 61.67 -

25 30.00 51.67 85.00 +

30 27.33 50.67 81.00 +

35 15.33 21.00 37.67 +

40 9.67 14.00 19.67 -

C.D. (1 %) 3.56 3.27 5.20

CV % 11.50 6.41 6.44


Table 4.9: Effect of different temperature on mycelial growth and sporulation of A. solani isolate (Asv-2) on PDB medium.

Temperatures Dry mycelial weight (mg) Sporulation*


5 21.67 35.33 60.33 -

10 33.67 57.00 108.00 -

15 67.00 110.33 238.67 -

20 109.33 243.67 475.00 -

25 127.67 316.67 603.33 +

30 123.67 311.00 595.67 +

35 73.67 108.33 303.33 -

40 28.67 48.00 106.67 -

C.D. (1 %) 8.10 7.70 11.70

CV % 6.34 2.87 2.15



~91~

Table 4.10: Effect of different pH range with potato dextrose agar on mycelial growth and sporulation of A. solani (Asv-2) incubated at

25±2 ºC.

pH range Mycelial Growth (mm) Sporulation*


4.0 15.33 21.67 34.00 -

4.5 15.67 24.00 39.67 -

5.0 17.00 28.33 59.00 -

5.5 22.67 35.33 73.00 -

6.0 28.33 46.67 79.00 -

6.5 33.67 53.00 83.67 -

7.0 34.67 53.67 86.67 +

7.5 32.00 49.33 80.67 +

8.0 20.67 34.33 57.00 -

8.5 15.67 23.00 40.33 -

C.D. (1 %) 2.64 3.53 5.08

CV % 6.53 5.57 4.68


The results presented in table 4.11 indicate that the highest dry

mycelial weight with sporulation was also obtained at pH 7.0 (117.00,

340.00 & 620.33 mg) which was not significantly different to pH 6.5

(114.33, 339.33 & 612.67 mg)at 4, 8 and 12 days after incubation,

respectively. pH 4.0 (57.33, 131.67 & 362.00 mg) gave least mycelial

growth of A. solani at 4, 8 and 12 days after incubation, respectively.

Only the pH 7.0 and 7.5 depicted sporulation of A. solani as compared

to other pH treatments.

4.3 To Standardize Conidial Production as well as Inoculation Technique for Alternaria solani.

4.3.1 Effect of different grain based medium on spore production of A. solani

Substrates differed significantly for days to colonization and

number of spores g-1 by the A. solani isolate (Plate 4.3).


~92~

Table 4.11: Effect of different pH range with potato dextrose broth on

mycelial growth and sporulation of A. solani (Asv-2) incubated at

25 ± 2 ºC.

pH range

Dry mycelial weight (mg) Sporulation*


4.0 57.33 131.67 362.00 -

4.5 68.00 162.33 407.33 -

5.0 75.67 228.67 510.00 -

5.5 85.33 303.00 563.00 -

6.0 100.33 318.00 602.33 -

6.5 114.33 339.33 612.67 -

7.0 117.00 340.00 620.33 +

7.5 112.33 336.67 512.00 +

8.0 88.00 206.67 416.00 -

8.5 74.00 158.00 302.67 -

C.D. (1 %) 5.40 8.39 17.52

CV % 3.53 1.94 2.06


Sorghum grains took significantly minimum time (30 days) for

complete colonization and also produced significantly higher number

of spores (4.50 x 103) g-1 of grains as compared to other grains

including wheat, barley, pearl millet and maize. Sorghum and wheat

grains produced sporulation but other three grains (barley, pearl

millet and maize) did not depict sporulation. Only sorghum and wheat

grains supported colonization whereas barley, pearl millet and maize

showed no mycelial growth at 10 DAI (days after inoculation). The

colonization of A. solani at 20 DAI was good in wheat, very good in

sorghum and poor in all the rest grains. Colonization at 30 DAI was

excellent, very good, good and poor on sorghum; wheat, barley and

pearl millet; maize, respectively (Table 4.12).


~93~

Table 4.12: Effect of different substrates on A. solani colonization and spore

production at 30 days after incubation at 25 ± 2 ºC.

Substrates Colonization

Sporulation/gm SRTD 10 DAI 20 DAI 30 DAI

Wheat Poor Good Very Good 5.20 × 102 22.823

Barley Nil Poor Good Nil 1.049

Maize Nil Poor Poor Nil 1.049

Sorghum Good Very Good Excellent 4.50 × 103 67.046

Pearl Millet Nil Poor Good Nil 1.049

C.D. (1%) 1.612

CV % 6.524

*Mean of five replications; SRTD=Square root transformed data

Five most virulent isolates (Asv-1, Asv-2, Asnd-1, Asmi-2 and

Asan-1) of A. solani were also studied for conidial production (Table

4.13). The isolate Asv-2 gave maximum number (1.0 x 103) of conidia

at 20 and also at 30 DAI (1.00 X 104). Minimum number (0.5 x 103) of

conidia was recorded at 20 DAI in the isolates Asv-1 and Asmi-2 while

the isolates Asv-1 and Asan-1 depicted minimum (0.50 X 104) at 30

DAI.

4.3.2 Effect of sorghum grain and water ratio for spore

production

The moisture content has an important role in conidial

production of A. solani. Colonization of test pathogen did not occur

with sorghum grain and water ratio of 10:1 & 10:2 while, poor

colonization was observed in treatment containing sorghum grains

and water ratio of 10:3. All the other grains and water ratio depicted

colonization of A. solani. Significantly higher number (1.0 X 104/gm)

of conidia were obtained from sorghum grains and water ratio of 10:8

followed by 10:7 (0.85 X 104/gm) and 10:9 (0.8 X 104/gm). The

conidiophores are formed under high humidity and light, whereas

conidial formation is favoured by alternating high and low humidity

along with darkness (Table 4.14).


~94~

Table 4.13: Sporulation efficiency of different isolates on sorghum grains at

10, 20and 30 DAI (Days After Incubation) at 25 ± 2 ºC.

Isolates

Conidia/gram of sorghum grain

10 DAI 20 DAI SRTD 30 DAI SRTD

Asv-1 Nil 0.5 × 103 22.4 0.50 × 104 70.7

Asv-2 Nil 1.0 × 103 31.5 1.00 × 104 100

Asnd-1 Nil 0.7 × 103 26.4 0.75 × 104 86.6

Asmi-2 Nil 0.5 × 103 22.4 0.60 × 104 77.4

Asan-2 Nil 0.6 × 103 24.5 0.50 × 104 70.7

C.D. (1%) 3.42 4.23

CV % 7.3 2.83


Table 4.14: Effect of sorghum grain and water ratio on A. solani colonization

and spore production at 10, 20and 30 DAI (Days After

Incubation) at 25 ± 2 ºC.

Sorghum grain : Water

Colonization Sporulation/gm

SRTD

10 DAI 20 DAI 30 DAI

10:1 Nil Nil Nil Nil 1.049

10:2 Nil Nil Nil Nil 1.049

10:3 Nil Very Poor Poor Nil 1.049

10:4 Poor Good Good 2.20 × 103 46.907

10:5 Poor Good Very Good 4.40 × 103 66.32

10:6 Good Very Good Excellent 6.50 × 103 80.622

10:7 Good Excellent Excellent 0.85 × 104 92.197

10:8 Good Excellent Excellent 1.00 × 104 99.667

10:9 Good Good Very Good 0.80 × 104 89.358

10:10 Poor Poor Good 0.80 × 103 28.152

C.D. (1%) 6.552

CV % 7.546



~95~

4.3.3 Effect of UV light on spore production of A. solani

The 5, 7, 9 and 11 days old cultures of A. solani grown on

potato dextrose agar and dextrose broth media were exposed to UV

light for 0, 10, 20, 40, 60 and 120 seconds. The optimum time of UV

light exposure for maximum sporulation 5800/ml in PDA and

5600/ml in PDB medium was noted 20 seconds. As the time of

exposure was increased there was fall in the amount of A. solani

sporulation. Zero second indicates control, which did not produce the

sporulation of A. solani in both the medium. Prolonged exposure of

cultures to ultraviolet light retards sporulation but the inhibitory

effect can be modified by decreasing the intensity of irradiation (Table

4.15).

4.3.4 Effect of light and darkness on spore production ofA. solani

The effect of dark and light was evaluated singly and in

combination on spore production of A. solani. When cultures of

Alternaria solani were kept in treatment 24 hrs darkness + 24 hrs

light (3000 Lux) it gave maximum mycelial growth i.e. 88.00 mm at 12

DAI with 4900 sporulation. However, the treatment; 24 hrs darkness

+ 48 hrs light (3000 Lux) gave higher spore production i.e. 5700/ml

(Table 4.16).

4.3.5 Standardization of inoculation technique

Four inoculation methods were used (spray inoculation, root dip

inoculation, droplet inoculation and soil inoculation) with spore

concentration of 104 spore/ml for better disease development on two

susceptible varieties (Co-3 & Arka Vikash).


~96~

Table 4.15: Effect of UV light on spore yield of A. solani incubated at potato dextrose agar for 12 days at 25 ± 2ºC.

Sr. No. Time of Exposure

(Seconds)

Spore Yield (×103)

PDA PDB

1 0 0.00 0.00

2 10 0.70 0.40

3 20 5.80 5.60

4 40 3.40 3.10

5 60 1.27 1.12

6 120 0.00 0.00

C.D. (1%) 0.427 0.161

CV % 15.331 6.300

Table 4.16: Effect of light and darkness on sporulation of A. solani incubated at potato dextrose agar for 12 days at25 ± 2 ºC.

Sr. No. Light and Darkness Mycelial growth

in mm (12 DAI)

Spore Con.

(103 ×) on PDA

1 Total darkness 76.66 0.0

2 Total Light (3000 Lux) 85.66 1.0

3 12 hrs darkness + 12 hrs light (3000 Lux) 79.00 2.4










C.D. (1%) 3.095

CV % 2.587


~97~

The droplet method gave maximum PDI on two varieties i.e. Co-

3 (85.19%) and Arka Vikash (81.48 %) followed by spray inoculation

method that gave 74.07 % PDI on both susceptible varieties. The

incubation period was also minimum in droplet method (4 days in

both varieties) followed by spray inoculation method (5& 4 days) on

both susceptible varieties (Co-3 & Arka Vikash), respectively (Table

4.17).

Table 4.17: Standardization of inoculation technique for development of A.

solani on tomato plants.

Techniques

Co-3 Arka Vikash

IP (Days) 35 DAI

(PDI value)

AUDPC IP (Days) 35 DAI

(PDI value)

AUDPC

Spray inoculation 5 74.07 1309.26 4 74.07 1374.07

Root Dip Inoculation 8 37.04 596.30 8 37.04 570.37

Droplet Inoculation 4 85.19 1620.37 4 81.48 1542.59

Soil Inoculation 8 25.93 427.78 7 29.63 479.63

C.D. (1%) 8.53 6.13

CV % 12.544 10.434

4.3.6 Storage of inoculum and its efficiency

There was a significant loss in the number of spores/gm of

sorghum grains and inoculum efficiency when inoculum was stored

for more than 30 days (Table 4.18). But these grain based media were

found best for long term storage of A. solani as compared to other

artificial media. Significantly higher spore concentration was recorded

at 8400/gm of sorghum grains after 30 days incubation period. When

30 days old sorghum grain culture was inoculated on two susceptible

tomato varieties viz. Co-3 and Arka Vikash, 14.50 and 13.25

lesions/leaflet were recorded after 15 days of inoculation on both of


~98~

them, respectively. Linear relationships were found between storage

period and incubation period, and between storage period and

number of lesions/leaflets. No symptom developed and no defoliation

was noted on uninoculated plants.

Table 4.18: Storage effect on spore viability and inoculum quality of A.

solani on sorghum grains.

Storage period

Spore g-1 (×103)

IP* Number of Lesion 15 DAI

Co-3 Arka Vikash Co-3 Arka Vikash

0 0 0 0 0.00 0.00

30 8.4 5 6 14.50 13.25

60 4.7 7 8 9.50 10.00

90 2.8 10 11 7.00 7.00

120 0.8 14 13 4.50 4.25

C.D. (1%) 1.416 1.075

CV % 13.112 10.248


4.3.7 Standardization of inoculum concentration for early blight development

Under controlled conditions, all inoculated plants depicted

disease symptoms on leaves after inoculation, even with the lowest

conidial concentration i.e. 1 x 103 conidia/ml (4.00 & 3.50

lesions/leaflets 15 DAI) on both susceptible varieties (Co-3 & Arka

Vikash), respectively. Maximum number of lesions/leaflets were

recorded with spore concentration of 10 x 103 i. e. 15.25 and 13.75 on

two susceptible tomato varieties (Co-3 & Arka Vikash) which was not

significantly different to spore concentration of 5 x 103 giving 13.25

and 12.25 lesions/leaflets. Minimum incubation period of 5 and 6

days on Co-3 and Arka Vikash, respectively were noted with spore

concentration of 10 x 103. Linear relationships were found between

inoculum concentration and number of lesions/leaflets, and between


~99~

inoculum concentration and incubation period. No symptoms

developed and no defoliation was noted on water sprayed plants

(Table 4.19).

Table 4.19: Effect of spore concentration of A. solani on tomato for early

blight development.

Spore Density (103/ml)

IP Number of Lesion 15 DAI

Co-3 Arka Vikash Co-3 Arka Vikash

0 0 0 0.00 0.00

1 12 13 4.00 3.50

2 10 11 5.75 4.75

5 7 8 13.25 12.25

10 5 6 15.25 13.75

C.D. (1%) 2.230 2.050

CV % 19.167 19.676


4.4 Phenotyping and Genotyping of RILs and germplasm

4.4.1 Phenotyping of 701 germplasm

Natural screening of 701 germplasm lines of tomato has been

done for resistance to early blight caused by A. solani under field

conditions. The 50 percent flowering, fruit setting and senescence

stages were also recorded (Table 3 in appendices).

4.4.1.1 Area Under Disease Progress Curve (AUDPC)

Under field condition with natural inoculum, the AUDPC value

were calculated on the basis of PDI value. All the germplasm lines

were grouped in four categories (Resistant, Moderately Resistant,

Moderately Susceptible and Susceptible) on the basis of AUDPC value.

Out of 701 germplasm lines, two hundred six resistant lines were

identified (AUDPC ranged from 102.00 to 447.25); two hundred


~100~

twenty three moderately resistant lines (AUDPC ranging from 447.26

to 792.50); one hundred twenty nine moderately susceptible lines

(AUDPC ranging from 792.51 to 1137.75) and one hundred forty three

susceptible lines (AUDPC ranging from 1137.76 to 1483.00) were

recorded in the year 2011-2012 (Table 4.20 and figure 4.1).

Maximum AUDPC value (1483.00) was recorded in germplasm

line PS-1, which was categorized in susceptible (S) group with

flowering time 21 days after transplanting; fruit setting time 9 days

after flowering and senescence stage 20 days after fruit setting. The

minimum AUDPC value (102.00) was recorded in germplasm line BT-

10, which was categorized in resistant (R) group with flowering time

21 days after transplanting; fruit setting time 13 days after flowering

and senescence stage 30 days after fruit setting. Over all in 701

germplasm lines the AUDPC range has been recorded between 102.00

to 1483.00 in natural screening under field condition.

4.4.1.2 Flowering, fruit setting and senescence stage:

Flowering time were recorded between 35 to 66 days after

seeding in all germplasm lines; fruit setting time were observed

between 8 to 15 days after flowering in all germplasm lines and

senescence stages were also recorded between 17 to 35 days after

fruit setting in all germplasm lines. Minimum time for flowering i. e.

35 days after seeding was observed in indeterminate line EC-620501

and maximum time for flowering i.e. 48 days after seeding was

observed in determinate line DARL-66. Minimum time for fruit setting

i. e. 8 days after flowering were observed in indeterminate line G-4-2

and in determinate line PB- Kesari and maximum time for fruit setting

i. e. 16 days after flowering was observed in determinate line DVRT-

14. Minimum time for senescence i. e. 16 days after fruit setting was

observed in indeterminate line TLS-30 and maximum time for

senescence i. e. 35 days after fruit setting was observed in

determinate line Arka Alok.


~101~

Table 4.20: Plant growth type, days to 50 percent flowering after transplanting, days to fruit setting, days to senescence, AUDPC

and Host Reaction of 701 germplasm lines of tomato to A. solani infection under natural condition in the year 2011-

12.

Disease Reaction Field conditions

AUDPC range No. of core lines

Resistant 102.00 - 447.25 206

Moderately Resistant 447.26 - 792.50 223

Moderately Susceptible 792.51 - 1137.75 129

Susceptible 1137.76 -1483.00 143

*Range is based on minimum value of the group plus LSD value

Figure 4.1: Categorization of germplasm lines of tomato on the basis of AUDPC value (Year 2011- 12) calculated on the basis of host reaction to A. solani, under natural conditions.

29.38 %31.81 %

18.40 %20.39 %

102.00 - 447.25 447.26 - 792.50 792.51 - 1137.75 1137.76 -1483.00

Resistant Moderately Resistant Moderately Susceptible Susceptible

10

60

110

160

210

Nu

mb

er o

f gen

oty

pes

Host Reaction against A. solani


~102~

4.4.2 Phenotyping of 240 core set lines

Screening of 240 core lines (79 Determinate and 161

Indeterminate) have been done for resistance to early blight of tomato

caused by A. solani. After inoculation with highly virulent isolate of A.

solani, the phenotypic reactions in field condition were obtained with

wide variation (Table 4.21, 4.22 & 4.23; Figure 4.2).

4.4.2.1 Percent disease index (PDI)

The 79 determinate lines were screened for resistance to early

blight under field conditions with artificial inoculum in the year 2012-

13. The maximum PDI i. e. 100 per cent was recorded in core tomato

lines no. 4, 5, 6, 8, 11, 13, 25, 30, 34, 35, 43, 58, 59 and 78 and

minimum PDI 8.89 per cent were recorded in core lines no. 54.

The 161 indeterminate core lines were also screened for

resistance to early blight under field conditions with artificial

inoculum in the year 2012-13. The maximum PDI i.e. 100 per cent

was recorded in core set tomato lines no. 2, 12, 13, 22, 32, 33, 35, 36,

37, 39, 40, 41, 50, 54, 58, 66, 73, 74, 76, 79, 80, 81, 83, 91, 92, 93,

94, 97, 99, 100, 103, 109, 110, 111, 112, 113, 116, 118, 119, 120,

124, 127, 129, 131, 135, 136, 141, 142, 143, 144, 145, 146 and 160

and minimum PDI 8.89 per cent were recorded in core lines no. 62.


Out of 79 core lines under field conditions with artificial

inoculum the AUDPC value of thirty four resistant core lines ranged

from 256.67 to 966.39; six moderately resistant core lines ranged

from 966.40 to 1676.12; seventeen moderately susceptible core lines

ranged from 1676.13 to 2385.84 and twenty two susceptible core set

tomato lines ranged from 2385.85 to 3095.56 that were recorded in

the year 2012-2013. The range of AUDPC value of 79 determinate core

set tomato lines ranged between 256.67 to 3095.56.


~103~

Table 4.21: PDI value at 42 days after inoculation (DAI), AUDPC and Host

Reaction of 79 determinate core lines of tomato after

inoculation with the isolate (Asv-2) of A. solani in the year 2012-13.

No. of

core lines Name of tomato core lines

Determinate (Field trial)

42 DAI

(PDI value)

Host Reaction

AUDPC

1 Arka Vikas (Sel-22) 66.67 S 2807.78

2 EC- 521039 75.56 S 3080.00

3 EC- 528374 84.44 S 2831.11

4 EC- 605694 100.00 S 2978.89

5 EC- 620455 100.00 S 2983.33

6 EC- 620533 100.00 S 2512.22

7 EC- 620241 42.22 MR 1057.78

8 Kashi Hemant (IIVR Sel-1) 100.00 S 3080.00

9 MUKTHI (LE-79-5) 15.56 R 381.11

10 Money Maker 22.22 R 404.44

11 Pant T-3 100.00 S 2983.33

12 Pusa-120 20.00 R 303.33

13 C-26-1 100.00 S 3095.56

14 D-3-2 71.11 S 2807.78

15 D-5-1 73.33 S 2978.89

16 DVRT-2 20.00 R 474.44

17 EC- 538405 62.22 S 2800.00

18 EC- 620386 71.11 S 2473.33

19 EC- 620514 64.44 MS 2364.44

20 EC- 538441 13.33 R 326.67

21 EC- 552141 33.33 R 583.33

22 Arka Alok (BWR-5) 44.44 MR 1648.89

23 Azad T-2 (KS-2) 20.00 R 350.00

24 Bhillai 66.67 S 2403.33

25 CO-3 (Marutham) 100.00 S 2877.78

26 D-1-1 11.11 R 560.00

27 DVRT-1 13.33 R 311.11

28 EC- 381554 20.00 R 396.67

29 EC- 520046 26.67 R 560.00

30 EC- 570028 100.00 S 2761.11

31 EC- 620398 46.67 R 661.11

32 EC- 620401 71.11 MR 1485.56

33 EC- 620438 22.22 R 326.67

34 EC- 620444 100.00 S 2496.67

35 EC- 620469 100.00 MS 1820.00

36 EC- 620480 17.78 R 264.44

37 H-88-78-1 17.78 R 311.11

38 H-88-78-4 24.44 R 521.11

39 Kashi Vishesh (H-86) 48.89 MR 1415.56


~104~

40 N-2-2 11.11 R 381.11

41 N-2-3 68.89 MS 1897.78

42 NDT-1 75.56 MS 2037.78

43 Pb-Chhuhara 100.00 S 2963.33

44 PDT-3-1 57.78 MS 1874.44

45 Pusa Gaurav 37.78 MR 1571.11

46 Roma 66.67 S 2387.78

47 Sioux 60.00 MS 1913.33

48 Solan Gola 62.22 MS 2325.56

49 Swarna Naveen 11.11 R 280.00

50 TLBR-6 20.00 R 381.11

51 TLH-17 40.00 MR 1602.22

52 TLH-30 62.22 MS 2193.33

53 Utkal Pragyan 13.33 R 381.11

54 97/384 8.89 R 280.00

55 EC- 620556 88.89 S 2823.33

56 EC- 620598 88.89 MS 1781.11

57 Kashi Amrit (DVRT-1) 24.44 R 536.67

58 97/753 100.00 MS 1960.00

59 Utkal Urvashi 100.00 S 3048.89

60 EC- 519730 26.67 R 497.78

61 EC- 538404 (NCEBR-4) 57.78 MS 2084.44

62 Persia Bed 57.78 MS 1952.22

63 PKM-1 66.67 MS 2278.89

64 PS-1 42.22 R 661.11

65 C-4-1 17.78 R 280.00

66 C-11-3 15.56 R 381.11

67 C-20-1 20.00 R 334.44

68 C-20-2 33.33 R 583.33

69 CLN - 2116 15.56 R 381.11

70 CLN-2366 15.56 R 256.67

71 DARL-66 11.11 R 661.11

72 EC-13904 13.33 R 280.00

73 EC- 620413 62.22 MS 1905.56

74 EC- 620446 64.44 MS 1905.56

75 G-5-4 53.33 MS 1967.78

76 NDT-8 15.56 R 256.67

77 NDTVR-73 13.33 R 280.00

78 EC- 620474 100.00 S 3095.56

79 FEB.-02 46.67 MS 1695.56

Max. 100.00 S 3095.56

Min. 8.89 R 256.67

CV % 21.82

LSD (1 %) 709.722


~105~

Table 4.22: PDI, AUDPC and Host Reaction of 161 indeterminate core lines

of tomato after inoculation with the isolate (Asv-2) of A. solani

in the year 2012-13.

No. of core lines

Name of tomato core lines

Indeterminate (Field trial)

56 DAI

(PDI value)

Host

Reaction AUDPC

1 Ageta-32 22.22 R 420.00

2 Azad T-5 (KS-17) 100.00 MS 2488.89

3 C-11-2 17.78 R 473.33

4 CLN 1621 57.78 MS 2131.11

5 Dhrubya 26.67 R 941.11

6 EC- 2791 22.22 R 557.78

7 EC- 273966 33.33 MR 1221.11

8 EC- 501580 71.11 MS 2146.67

9 EC- 520059 28.89 R 536.67

10 EC- 526139 42.22 MR 1578.89

11 EC- 529083 48.89 MR 1687.78

12 EC- 538419 100.00 S 3064.44

13 EC- 538423 100.00 MR 1812.22

14 EC- 538439 66.67 MS 1952.22

15 EC- 538440 13.33 R 502.22

16 EC- 538455 17.78 R 626.67

17 EC- 605695 24.44 R 450.00

18 EC- 620370 24.44 R 521.11

19 EC- 620375 33.33 R 1112.22

20 EC- 620403 13.33 R 495.56

21 EC- 620409 28.89 R 474.44

22 EC- 620470 100.00 S 2644.44

23 EC- 620486 26.67 R 513.33

24 EC- 620500 51.11 MR 1835.56

25 F-5020 37.78 MR 1151.11

26 IC-469626 11.11 R 694.44

27 Kashmiriya 13.33 R 513.33

28 Pant T-5 13.33 R 482.22

29 Pusa Ruby 40.00 MR 1637.78

30 TLH-27 32.22 MR 1466.67

31 Utkal Raja (BT-20-2-1) 20.00 R 665.56

32 15 SB 100.00 S 2877.78

33 S.Lalima 100.00 S 2698.89

34 LA-3772 51.11 MS 2325.56

35 PDVT-14 100.00 S 3305.56

36 IC-427766 100.00 MS 2566.67

37 INDAM-2102 100.00 S 2730.00

38 EC- 520074 26.67 R 466.67

39 EC-620362 100.00 S 2877.78

40 Indam-2103 100.00 S 3021.11

41 Jawahar-99 100.00 S 2854.44

42 Monte Favet 73.33 MS 2162.22

43 Sanjeevani 28.89 MR 1521.11

44 Sun-cherry 24.44 R 412.22

45 Tripura local 48.89 MR 1726.67

46 WIR-3957 22.22 R 488.89

47 WIR-5032 33.33 MR 1223.33

48 WIR-13706 37.78 MR 1676.67

49 Swetzerland 17.78 R 542.22

50 WIR-13717 100.00 S 2792.22

51 Angoorlata 26.67 R 528.89

52 Arka Abha 28.89 R 505.56


~106~

53 Avinash-2-2-1 (VRT-102) 40.00 MR 1563.33

54 B-4-1 100.00 S 3251.11

55 BL-1208 48.89 MR 1734.44

56 C-3-2 28.89 R 536.67

57 C-11-1 11.11 R 482.22

58 CH-155 100.00 S 3142.22

59 CLN- 2026 17.78 R 457.78

60 DT-10 28.89 R 583.33

61 EC- 501576 20.00 R 466.67

62 EC- 520075 8.89 R 457.78

63 EC- 521056 11.11 R 482.22

64 EC- 538380 22.22 R 466.67

65 EC- 538408 62.22 MS 1967.78

66 EC- 620373 100.00 S 2947.78

67 EC- 620374 55.56 MS 2115.56

68 EC- 620383 20.00 R 518.89

69 EC- 620406 24.44 R 490.00

70 EC- 620410 22.22 R 482.22

71 EC- 620411 15.56 R 458.89

72 EC- 620456 20.00 R 434.44

73 EC- 620464 100.00 MS 2411.11

74 EC- 620540 100.00 S 2885.56

75 EC- 625644 53.33 MS 2387.78

76 EC- 625651 100.00 S 3014.44

77 EC- 625652 53.33 MS 2037.78

78 EC- 625660 48.89 MS 2068.89

79 F-6022 100.00 S 2955.56

80 F-6050-1 100.00 S 3145.56

81 F-6059 100.00 MS 2123.33

82 F-7012 46.67 MR 1757.78

83 F-7025 100.00 S 3060.00

84 F-7028 15.56 R 458.89

85 FEB.-04 44.44 MR 1812.22

86 FLA-7171 26.67 R 482.22

87 FLA-7421 26.67 R 575.56

88 GT-2 33.33 R 521.11

89 GT-3 33.33 MR 1278.89

90 H-88-78-3 20.00 R 404.44

91 H-88-78-5 100.00 S 3184.44

92 Hisar Anmol (H-24) 100.00 S 3213.33

93 Hisar Lalit (NT-8) 100.00 S 2624.44

94 I-4-4 100.00 S 2725.56

95 IC-373378 20.00 R 534.44

96 IC-447708 26.67 R 560.00

97 IIHR-01 100.00 S 2655.56

98 INDAM-2103-6-1 15.56 R 458.89

99 INDAM-2103-6-4 100.00 S 3142.22

100 Kashi Sharad (IIVR Sel-2) 100.00 S 3048.89

101 Kajla 55.56 MS 2123.33

102 Kalyanpur type -1 24.44 R 458.89

103 LA-3957 100.00 S 2652.22

104 LA-3997 46.67 MS 2348.89

105 M-1-4 75.56 S 2885.56

106 NF37SB-8 75.56 S 3212.22

107 Parul 71.11 S 2745.56

108 Pb. Upma 71.11 MS 2302.22

109 Prestige 100.00 S 3110.00

110 Punjab Barkha Bahar-2 100.00 S 3068.89

111 Pusa hybrid-2 100.00 S 2836.67


~107~

112 Sankranti 100.00 S 3150.00

113 Sel-18 100.00 S 2854.44

114 VRT-32-1 66.67 S 2706.67

115 97/754 (Kewalo) 97.78 S 3037.78

116 Punjab Keshri 100.00 S 3140.00

117 B-7-2 22.22 R 668.89

118 EC- 620502 100.00 MS 2286.67

119 LA-3957 100.00 S 2901.11

120 Nandhi 100.00 S 3076.67

121 EC- 520078 22.22 R 420.00

122 EC- 521078 11.11 R 532.22

123 Hawai 57.78 MS 2333.33

124 INDAM-2103-1 100.00 MS 2092.22

125 VRT-101A (Mutent) 28.89 R 583.33

126 WIR-13708 28.89 R 723.33

127 Rio Grande 100.00 MS 2566.67

128 EC-528372 24.44 R 692.22

129 Palam Pink 100.00 MR 1788.89

130 BTH-9 Male 68.89 MS 2177.78

131 C-1-4 100.00 MS 2380.00

132 C-10-2 44.44 R 606.67

133 EC- 381263 42.22 R 723.33

134 EC- 501574 33.33 R 552.22

135 EC- 501575 100.00 S 3142.22

136 EC- 501577 100.00 MS 2232.22

137 EC- 501582 33.33 R 692.22

138 EC- 501583 71.11 S 2800.00

139 EC-529080 62.22 MS 2450.00

140 EC- 538138 33.33 R 614.44

141 EC- 620419 100.00 S 3266.67

142 EC- 620421 100.00 S 3013.33

143 EC- 620476 100.00 S 3078.89

144 EC- 620519 100.00 S 2780.00

145 EC- 620568 100.00 S 2710.00

146 EC- 620575 100.00 MS 1944.44

147 EC- 625645 66.67 MS 1975.56

148 Flora-Dade 51.11 MS 2100.00

149 G-4-5 22.22 R 435.56

150 G-6-3 28.89 R 536.67

151 GT-1 37.78 R 941.11

152 H-88-78-2 33.33 R 676.67

153 Hisar Arun 51.11 MS 1944.44

154 IIHR-2202 55.56 MS 2193.33

155 INDAM-2103 55.56 MR 1726.67

156 Indam-2103 15.56 R 456.67

157 NDT-4 57.78 MS 2543.33

158 NDTVR-60 55.56 MS 1928.89

159 Swarna Vaibhav 26.67 R 435.56

160 EC- 620530 100.00 MS 2512.22

161 EC-520061 22.22 R 404.44

Maximum 100.00 S 3305.56

Minimum 8.89 R 404.44

CV % 14.17

LSD (1 %) 725.28


~108~

Table 4.23: Summary of disease reaction of 79 determinate and 161 indeterminate tomato core set lines based on AUDPC,

calculated on the basis of host reaction obtained after inoculation with the isolate (Asv-2) of A. solani in the year

2012-13.

Disease Reaction Determinate Indeterminate

AUDPC range No. of RILs AUDPC range No. of RILs

Resistant 256.67 - 966.39 34 404.44 - 1129.72 63

Moderately Resistant 966.40 - 1676.12 06 1129.73 - 1855.00 19

Moderately Susceptible 1676.13 - 2385.84 17 1855.01 - 2580.28 33

Susceptible 2385.85 - 3095.56 22 2580.29 - 3305.56 46

*Range is based on minimum value of the group plus LSD

Figure 4.2: Categorization of core set tomato lines based on AUDPC (Year 2012-13) obtained after artificial inoculation under field

conditions.

43.03 %

7.59 %

21.51 %

27.84 %

39.13 %

11.80 %

20.49 %

28.57 %

0

10

20

30

40

50

60

70

Num

ber

of

core

set

lin

es

R MR MS S



~109~

Out of 161 indeterminate core set tomato lines under field

conditions with artificial inoculum the AUDPC value of sixty three

resistant core lines ranged from 404.44 to 1129.72; nineteen

moderately resistant core lines ranged from 1129.73 to 1855.00;

thirty three moderately susceptible core lines ranged from 1855.01 to

2580.28 and forty six susceptible core lines ranged from 2580.29 to

3305.56 that were recorded in the year 2012-2013. The range of

AUDPC value of total 161 indeterminate core set tomato lines ranged

between 404.44 to 3305.56.

4.4.3 Phenotyping of 226 core set lines

Screening of 226 core lines (74 Determinate and 152

Indeterminate) have been done for resistance to early blight of tomato

caused by A. solani in the year 2013-14. After inoculation with highly

virulent isolate of A. solani the phenotypic reaction in field condition

were obtained with wide variation (Table 4.24, 4.25 & 4.26; Figure

4.3).

4.4.3.1Percent disease index (PDI)

The 74 determinate lines were screened for resistance to early

blight under field conditions with artificial inoculum in the year 2013-

14. The maximum PDI i.e. 100 per cent was recorded in core set

tomato lines no. 3, 4, 5, 14, 15, 17, 18, 25, 43, 44, 48, 70 and 73 and

minimum PDI i.e. 15.56 per cent were recorded in core set tomato

lines Utkal Pragyan, D-1-1 and DVRT-1.

The 152 indeterminate core set tomato lines were also screened

for resistance to early blight under field conditions with artificial

inoculum in the year 2013-14. The maximum PDI i.e. 100 per cent

was recorded in core set tomato lines no. 2, 12, 13, 32, 33, 35, 36, 37,

44, 48, 49, 52, 60, 61,68, 70, 73, 74, 77, 85, 86, 87, 88, 91, 93, 94,

97, 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 114, 115,

118, 120, 121, 122, 126, 127, 129, 132, 133, 134, 135, 136, 137,

138, 139, 145, 146 and 148 and minimum PDI i.e. 11.11 per cent was

recorded in core set tomato lineEC-520061.


~110~

Table 4.24: PDI, AUDPC and Host Reaction of determinate 74 core lines of

tomato after inoculation with the isolate (Asv-2) of A. solani in

the year 2013-14.

No. of

core lines Name of tomato core lines

Determinate (Field trial)

42 DAI

(PDI value)

Host

Reaction AUDPC

1 Arka Vikas 66.67 MS 1555.56

2 EC- 521039 66.67 MS 1804.44

3 EC- 528374 100.00 S 2006.67

4 EC- 605694 100.00 S 1827.78

5 EC- 620455 100.00 S 1952.22

6 EC- 620533 66.67 MS 1812.22

7 EC- 620241 56.43 MS 1765.56

8 Kashi Hemant (IIVR Sel-1) 66.67 MS 1812.22

9 MUKTHI (LE-79-5) 26.67 R 326.67

10 Money Maker 24.44 R 334.44

11 Pant T-3 66.67 MS 1524.44

12 Pusa-120 24.44 R 287.78

13 C-26-1 74.32 MS 1610.00

14 D-3-2 100.00 S 2286.67

15 D-5-1 100.00 S 2364.44

16 DVRT-2 20.00 R 210.00

17 EC- 538405 100.00 S 2076.67

18 EC- 620386 100.00 S 2045.56

19 EC- 620514 66.67 MS 1820.00

20 EC- 538441 37.78 R 614.44

21 EC- 552141 24.44 R 412.22

22 Arka Alok (BWR-5) 48.89 MR 1120.00

23 Azad T-2 (KS-2) 37.78 R 676.67

24 Bhillai 66.67 MS 1594.44

25 CO-3 (Marutham) 100.00 S 1921.11

26 D-1-1 15.56 R 241.11

27 DVRT-1 15.56 R 256.67

28 EC- 381554 24.44 R 318.89

29 EC- 520046 35.56 R 435.56

30 EC- 570028 43.32 MR 1236.67

31 EC- 620398 26.67 R 482.22

32 EC- 620401 33.33 R 427.78

33 EC- 620438 17.78 R 342.22

34 EC- 620444 54.32 MS 1610.00

35 EC- 620469 66.67 MS 1703.33

36 EC- 620480 33.33 R 630.00

37 H-88-78-1 26.67 R 295.56

38 H-88-78-4 17.78 R 233.33

39 Kashi Vishesh (H-86) 26.67 R 357.78

40 N-2-2 40.00 MR 982.22


~111~

41 N-2-3 66.67 MR 937.78

42 NDT-1 58.33 MS 1765.56

43 Pb- Chhuhara 100.00 S 1905.56

44 PDT-3-1 100.00 S 1827.78

45 Pusa Gaurav 66.67 MS 1400.00

46 Roma 74.67 MS 1687.78

47 Sioux 66.67 MS 1742.22

48 Solan Gola 100.00 S 1967.78

49 Swarna Naveen 17.78 R 202.22

50 TLBR-6 20.00 R 256.67

51 TLH-17 40.00 MR 863.33

52 TLH-30 66.67 MS 1625.56

53 Utkal Pragyan 15.56 R 194.44

54 97/384 20.00 R 256.67

55 EC- 620556 58.88 MS 1664.44

56 EC- 620598 66.67 MS 1454.44

57 Kashi Amrit (DVRT-1) 34.17 R 676.67

58 97/753 56.63 MR 987.78

59 Utkal Urvashi 86.22 S 2100.00

60 C-4-1 20.00 R 303.33

61 C-11-3 17.78 R 217.78

62 C-20-1 20.00 R 256.67

63 C-20-2 22.22 R 295.56

64 CLN - 2116 20.00 R 256.67

65 CLN-2366 17.78 R 202.22

66 DARL-66 34.44 MR 887.78

67 EC-13904 20.00 R 458.89

68 EC- 620413 66.67 MS 1400.00

69 EC- 620446 66.67 MS 1470.00

70 G-5-4 100.00 S 1882.22

71 NDT-8 24.44 R 287.78

72 NDTVR-73 17.78 R 233.33

73 EC- 620474 100.00 S 2037.78

74 FEB.-02 86.22 S 1827.78

Max. 100.00 S 2364.44

Min. 15.56 R 194.44

CV % 21.82

LSD (1 %) 408.7


~112~

Table 4.25: PDI Value at 56 DAI, AUDPC and Host Reaction of 152 indeterminate core lines of tomato after inoculation with the

isolate (Asv-2) of A. solani in the year 2013-14.

No. of core lines

Name of tomato core lines

Indeterminate (Field trial)

56 DAI

(PDI value)

Host

Reaction AUDPC

1 Ageta-32 20.00 R 303.33

2 Azad T-5 (KS-17) 100.00 MS 1407.78

3 C-11-2 22.22 R 280.00

4 CLN 1621 44.32 MR 956.67

5 Dhrubya 37.78 R 490.00

6 EC- 2791 40.00 R 544.44

7 EC- 273966 44.44 MR 777.78

8 EC- 501580 58.52 MS 1407.78

9 EC- 520059 42.22 R 458.89

10 EC- 526139 76.22 MS 1400.00

11 EC- 529083 76.22 MS 1322.22

12 EC- 538419 100.00 S 1874.44

13 EC- 538423 100.00 S 1773.33

14 EC- 538439 66.47 MS 1532.22

15 EC- 538440 24.44 R 256.67

16 EC- 538455 22.22 R 248.89

17 EC- 605695 22.22 R 497.78

18 EC- 620370 28.89 R 412.22

19 EC- 620375 37.78 MR 816.67

20 EC- 620403 17.78 R 528.89

21 EC- 620409 37.78 MR 692.22

22 EC- 620470 66.47 MS 1438.89

23 EC- 620486 24.44 R 350.00

24 EC- 620500 66.47 MS 1392.22

25 F-5020 48.89 R 606.67

26 IC-469626 22.22 R 528.89

27 Kashmiriya 22.22 R 560.00

28 Pant T-5 15.56 R 194.44

29 Pusa Ruby 35.56 R 513.33

30 TLH-27 48.89 MR 785.56

31 Utkal Raja (BT-20-2-1) 35.56 R 637.78

32 15 SB 100.00 S 2208.89

33 S.Lalima 100.00 S 2177.78

34 EC-620362 82.14 MS 1680.00

35 Indam-2103 100.00 S 1890.00

36 Jawahar-99 100.00 S 2107.78

37 Monte Favet 100.00 S 2107.78

38 Sanjeevani 42.22 R 474.44

39 Tripura local 48.89 MS 1314.44

40 WIR-3957 33.33 MR 1034.44

41 WIR-5032 42.22 R 536.67

42 WIR-13706 48.89 R 560.00

43 Swetzerland 22.22 R 311.11

44 WIR-13717 100.00 S 2084.44

45 Angoorlata 35.56 R 435.56

46 Arka Abha 33.33 R 443.33

47 Avinash-2-2-1 (VRT-102) 40.00 MR 1166.67

48 B-4-1 100.00 S 2045.56

49 BL-1208 100.00 S 2068.89

50 C-3-2 32.22 R 474.44


~113~

51 C-11-1 17.78 R 248.89

52 CH-155 100.00 S 1998.89

53 CLN- 2026 35.56 R 435.56

54 DT-10 22.22 R 373.33

55 EC- 501576 17.78 R 248.89

56 EC- 520075 11.11 R 147.78

57 EC- 521056 17.78 R 186.67

58 EC- 538380 22.22 R 668.89

59 EC- 538408 74.31 MS 1516.67

60 EC- 620373 100.00 S 1975.56

61 EC- 620374 100.00 S 1843.33

62 EC- 620383 37.78 R 661.11

63 EC- 620406 20.00 R 350.00

64 EC- 620410 20.00 R 256.67

65 EC- 620411 15.56 R 194.44

66 EC- 620456 20.00 R 350.00

67 EC- 620464 74.31 MS 1742.22

68 EC- 620540 100.00 S 1858.89

69 EC- 625644 66.22 MS 1392.22

70 EC- 625651 100.00 S 2146.67

71 EC- 625652 74.31 MS 1368.89

72 EC- 625660 66.22 MS 1485.56

73 F-6022 100.00 S 1750.00

74 F-6050-1 100.00 S 2030.00

75 F-6059 74.31 MS 1726.67

76 F-7012 74.31 MS 1516.67

77 F-7025 100.00 S 2263.33

78 F-7028 15.56 R 567.78

79 FEB.-04 62.13 MS 1462.22

80 FLA-7171 24.44 R 318.89

81 FLA-7421 24.44 R 318.89

82 GT-2 26.67 R 668.89

83 GT-3 28.89 R 427.78

84 H-88-78-3 17.78 R 217.78

85 H-88-78-5 100.00 S 1998.89

86 Hisar Anmol (H-24) 100.00 S 1944.44

87 Hisar Lalit (NT-8) 100.00 S 1890.00

88 I-4-4 100.00 S 1796.67

89 IC-373378 28.89 R 303.33

90 IC-447708 22.22 R 295.56

91 IIHR-01 100.00 S 1827.78

92 INDAM-2103-6-1 13.33 R 186.67

93 INDAM-2103-6-4 100.00 S 2045.56

94 Kashi Sharad (IIVR Sel-2) 100.00 S 1936.67

95 Kajla 74.31 MS 1664.44

96 Kalyanpur type -1 26.67 R 357.78

97 LA-3957 100.00 S 1991.11

98 LA-3997 40.00 MR 972.22

99 M-1-4 100.00 S 2006.67

100 NF37SB-8 100.00 S 1804.44

101 Parul 100.00 S 1975.56

102 Pb. Upma 74.31 MS 1617.78

103 Prestige 100.00 S 2037.78


~114~

104 Punjab Barkha Bahar-2 100.00 S 2138.89

105 Pusa hybrid-2 100.00 S 2201.11

106 Sankranti 100.00 S 2170.00

107 Sel-18 100.00 S 1804.44

108 VRT-32-1 100.00 S 1788.89

109 97/754 (Kewalo) 100.00 S 2224.44

110 Punjab Keshri 100.00 S 2278.89

111 B-7-2 26.67 R 357.78

112 EC- 620502 76.12 MS 1703.33

113 LA-3957 74.31 MS 1687.78

114 Nandhi 100.00 S 1820.00

115 INDAM-2103-1 100.00 S 1998.89

116 VRT-101A (Mutant) 31.11 R 404.44

117 WIR-13708 28.89 R 396.67

118 Rio Grande 100.00 S 2061.11

119 EC-528372 28.89 MR 863.33

120 Palam Pink 100.00 S 2030.00

121 BTH-9 Male 100.00 S 1835.56

122 C-1-4 100.00 S 1913.33

123 C-10-2 20.00 R 365.56

124 EC- 381263 35.56 R 528.89

125 EC- 501574 57.78 MR 1088.89

126 EC- 501575 100.00 S 1882.22

127 EC- 501577 100.00 S 1890.00

128 EC- 501582 48.89 R 560.00

129 EC- 501583 100.00 S 1998.89

130 EC-529080 74.31 MS 1493.33

131 EC- 538138 53.33 R 528.89

132 EC- 620419 100.00 S 1858.89

133 EC- 620421 100.00 S 2006.67

134 EC- 620476 100.00 S 1882.22

135 EC- 620519 100.00 S 2053.33

136 EC- 620568 100.00 S 2131.11

137 EC- 620575 100.00 S 2177.78

138 EC- 625645 100.00 S 1757.78

139 Flora-Dade 100.00 S 1812.22

140 G-4-5 57.78 MR 824.44

141 G-6-3 28.89 R 396.67

142 GT-1 33.33 R 575.56

143 H-88-78-2 42.13 MR 832.22

144 Hisar Arun (Sel-7) 42.13 MR 1174.44

145 IIHR-2202 100.00 MS 1337.78

146 INDAM-2103-1-1 100.00 MS 1353.33

147 Indam-2103-4 45.56 MR 676.67

148 NDT-4 100.00 S 1827.78

149 NDTVR-60 74.31 MS 1718.89

150 Swarna vaibhav 28.89 R 381.11

151 Ec-620530 74.31 MS 1594.44

152 EC-520061 11.11 R 132.22

Maximum 100.00 S 2278.89

Minimum 11.11 R 132.22

CV % 17.244

LSD (1 %) 536.67


~115~

Table 4.26: Summary of disease reaction of 74 determinate and 152 indeterminate tomato core set lines based on AUDPC,

calculated on the basis of host reaction obtained after inoculation with the isolate (Asv-2) of A. solani in the year

2013-14.

Disease Reaction Determinate Indeterminate


Resistant 194.44 - 736.94 32 132.22 - 668.89 57

Moderately Resistant 736.95 - 1279.44 07 668.90 - 1205.56 16

Moderately Susceptible 1279.45 - 1821.94 20 1205.57 - 1742.22 26

Susceptible 1821.95 - 2364.44 15 1742.23 - 2278.89 53

*Range is based on minimum value of the group plus LSD

Figure 4.3: Categorization of core set tomato lines based on AUDPC (Year 2013-14) obtained after artificial inoculation under field

conditions.

43.24

9.46

27.03

20.27

37.50

10.53

17.11

34.87

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

1 2 3 4

Determinate Indeterminate

R MR MS SHost Reaction against A. solani

Perc

en

t of

core

set

lin

es


~116~


Out of 74 core set tomato lines under field conditions with

artificial inoculum the AUDPC value of thirty two resistant core set

tomato lines ranged from 194.44 to 736.94; seven moderately

resistant core set tomato lines ranged from 736.95 to 1279.44; twenty

moderately susceptible core set tomato lines ranged from 1279.45 to

1821.94 and fifteen susceptible core lines ranged from 1821.95 to

2364.44 in the year 2013-2014. The range of AUDPC value of total 74

determinate core set tomato lines ranged between 194.44 to 2364.44.

Out of 152 indeterminate core set tomato lines under field

conditions with artificial inoculum the AUDPC value of fifty seven

resistant core set tomato lines ranged from 132.22 to 668.89; sixteen

moderately resistant core set tomato lines ranged from 668.90 to

1205.56; twenty six moderately susceptible core set tomato lines

ranged from 1205.57 to 1742.22 and fifty three susceptible core set

tomato lines ranged from 1742.23 to 2278.89in the year 2012-2013.

The range of AUDPC value of total 152 indeterminate core set tomato

lines ranged between 132.22 to 2278.89.

4.4.4 Phenotyping of 151 tomato RILs (Co-3 X EC-520061) in the

year 2012-13.

Phenotyping of 151 RILs in F7 generation with parents has been

done for resistance to early blight of tomato caused by A. solani in the

year 2012-13 (Plate 4.4 & 4.5). After inoculation of 151 RILs along

with parents using one highly virulent isolate of A. solani (Asv-2), the

phenotypic reactions were obtained with wide variation (Table 4.27 &

4.28; Figure 4.4).


~117~

Table 4.27: PDI, AUDPC and Host Reaction of Recombinant Inbred Lines (Co-3 × EC-520061) of tomato after inoculation with the isolate (Asv-

2) of A. solani in the year 2012-13.

PDI & AUDPC (Year 2012-2013)

RILs

No.

Name of RILs

Poly house Field

42 DAI (PDI

value)

Host Reaction

AUDPC 56 DAI (PDI

value)

Host Reaction

AUDPC

1 W- EB-1 67.18 MS 1525.01 77.78 MS 1859.26

2 W- E-3-1 72.22 MS 1605.56 72.59 MS 2074.07

3 W- E-3-2 72.22 MS 1483.33 65.19 MS 1825.93

4 W- 10-B-3 77.78 S 1944.44 85.19 S 2670.37

5 W-10-EB 61.11 MS 1605.56 59.26 MS 2051.85

6 W- 16-A 67.18 MS 1483.33 64.07 MS 1951.85

7 W- 17 77.78 S 2210.32 88.89 S 2677.78

8 W- 17-1 45.13 MR 1188.89 48.15 MR 1207.41

9 W- 19-C-1 61.11 MS 1544.44 59.26 MS 2103.70

10 W- 24-3 55.56 MS 1750.00 62.96 MS 1937.04

11 W- 26 66.67 MS 1605.56 58.07 MS 1818.52

12 W- 27-1 72.22 MS 1483.33 42.96 MR 1496.30

13 W- 27-C-1 37.78 MR 833.33 33.33 MR 1222.22

14 W- 27-C-2 37.78 MR 1027.78 48.15 MR 1666.67

15 W- 27-D-1 22.22 R 583.33 22.22 R 481.48

16 W- 27-EB 50.00 MR 1342.13 59.26 MR 1225.93

17 W- 28-2 16.67 R 722.22 25.93 MR 1074.07

18 W- 28-4 42.22 MR 1150.00 46.67 MR 1207.41

19 W- 30-1 83.33 S 2027.78 100.00 S 2462.96

20 W- 35-1 55.56 MS 1342.13 76.34 MS 1985.56

21 W- 35-2 55.56 MS 1342.13 66.67 MS 2092.59

22 W- 35-3 55.56 MS 1777.78 48.89 MR 1277.78

23 W- 38-2 57.78 MS 1750.00 44.44 MR 1123.12

24 W- 40-A 88.89 S 2210.32 100.00 S 2688.89

25 W- 40-A-1 66.67 MS 1544.44 62.96 MS 1923.32

26 W- 40-B-1 22.22 R 444.44 17.41 R 333.33

27 W- 40-B-4 27.78 R 750.00 29.63 MR 1185.19

28 W- 41-1 41.11 MR 1027.78 40.37 MR 1237.04

29 W- 44-B 38.89 MR 1277.78 22.22 MR 1111.11

30 W- 46-B-1 44.44 MR 1123.12 55.56 R 744.44

31 W- 47-1 41.11 MR 1012.22 46.67 MR 1237.04

32 W- 47-2 83.33 S 2210.32 100.00 S 2500.00

33 W- 48-1 88.89 S 2027.78 100.00 S 2611.11

34 W- 48-2 11.11 R 472.22 18.52 R 462.96

35 W- 49-B-1 11.11 R 314.32 16.39 R 481.48

36 W- 49-C-3 16.67 R 611.11 18.52 R 722.22

37 W- 50-3 55.56 MS 1750.00 40.74 MR 1311.11

38 W- 50A-2 16.67 R 527.78 25.93 R 537.04

39 W- 53-A-3 22.22 R 638.89 29.63 R 752.12

40 W- 55-A-1 27.78 MR 888.89 51.85 R 911.11

41 W- 55-A-2 44.44 MR 1166.67 40.74 MR 1285.19

42 W- 55-B-1 16.67 R 416.67 11.11 R 351.85

43 W- 59-B-1 57.78 MS 1629.63 70.37 MS 2044.44

44 W- 61-4 61.11 MS 1740.74 74.07 MS 1948.15

45 W- 59-B-3 52.22 MS 1629.63 64.00 MS 2029.63

46 W- 75-1 66.67 MS 1722.22 62.96 MS 2102.42

47 W- 60-EB 44.44 MR 1222.22 48.15 MR 1385.19

48 W- 71-1 50.00 MS 1583.33 51.85 MS 1851.85

49 W- 84-7 88.89 S 2083.33 100.00 S 2562.96

50 W- 84-EB 83.33 S 2166.67 100.00 S 2696.30


~118~

51 W- 82-B-2 44.44 MR 1138.89 48.15 MR 1648.15

52 W- 85-1 66.67 S 2166.67 76.43 MS 1948.15

53 W- 82-1-1 38.89 MR 1027.78 40.74 MR 1129.63

54 W- 61-3 66.67 MS 1694.44 42.34 MR 1303.70

55 W- 69-B-2 83.33 S 2166.67 100.00 S 2651.85

56 W- 69-B-1 88.89 S 2083.33 74.07 S 2321.65

57 W- 82-B-3 37.78 MR 1000.00 37.04 MR 1222.22

58 W- 137-2 45.18 MR 1294.44 37.04 MR 1425.93

59 W- 125-3 55.56 MS 1666.67 61.85 MS 1929.63

60 W- 110-1 83.33 S 2083.33 66.67 S 2681.48

61 W- 125-2 72.22 S 2083.33 100.00 S 2500.00

62 W- 114-B-1 63.33 MS 1518.52 62.96 MS 1944.44

63 W- 108-H-3 63.33 MS 1518.52 76.30 MS 2111.11

64 W- 88-2 57.78 MS 1666.67 64.32 MS 1833.33

65 W-95-B-2 33.33 MR 861.11 37.04 MR 1370.37

66 W- 95-B-3 38.89 MR 1261.11 62.96 MR 1777.78

67 W- 105-A-4 52.20 MS 1694.44 59.26 MS 1825.93

68 W- 101-1 72.22 S 2111.11 85.96 S 2229.63

69 W- 90-2 16.67 R 527.78 18.52 R 611.11

70 W- 152-B-1 72.22 MS 1518.52 59.26 MS 1925.93

71 W- 142-A-3 33.33 MR 1027.78 40.74 MR 1240.74

72 W- 143-B-2 72.22 S 2055.56 70.37 S 2533.33

73 W- 144-A-1 66.67 MS 1518.52 59.26 MS 2051.85

74 W- 144-B-1 57.78 MS 1666.67 62.96 S 2614.81

75 W- 143-1-1 16.67 R 416.67 18.52 R 462.96

76 W- 142-B-1 45.56 MR 1038.89 62.96 MR 1442.16

77 W- 146-B-2 50.00 MR 911.11 48.15 MS 1611.11

78 W- 146-1-1 61.11 MS 1683.33 66.67 MS 1948.15

79 W- 151-C-2 50.00 MS 1555.56 59.26 MS 2022.22

80 W- 151 61.11 MS 1351.85 66.67 MS 2092.59

81 W- 152-A-2 63.33 MS 1655.56 78.22 MS 1814.81

82 W- 148-1-1 88.89 S 2138.89 100.00 S 2648.15

83 W- 136-1 83.33 S 2138.89 100.00 S 2517.43

84 W- 137-6-1 88.89 S 2055.56 100.00 S 2670.37

85 W- 138-2 16.67 R 611.11 18.12 R 370.37

86 W- 138-3 66.67 MS 1655.56 100.00 MS 1817.43

87 W- 137-B-3 52.22 MS 1502.18 67.82 MS 1848.15

88 W- 140-1 83.33 S 2012.22 100.00 S 2648.15

89 W- 141-A-2 16.67 R 611.11 18.52 R 314.81

90 W- 141-6 77.78 S 2012.22 100.00 S 2517.43

91 W- 109-B-1 83.33 S 2055.56 100.00 S 2648.15

92 W- 203-1 88.89 S 2194.44 100.00 S 2488.89

93 W- 201-B-1 16.67 R 583.33 25.93 R 833.33

94 W- 197-A-2 76.50 S 2055.56 59.26 MS 1800.23

95 W- 201-A-4 11.11 R 314.32 18.52 R 314.81

96 W- 188-B 50.00 MS 1527.78 51.85 MS 2022.22

97 W- 183-B-2 27.78 R 722.22 25.93 R 796.30

98 W- 175-B-1 33.33 R 861.11 22.22 R 777.78

99 W- 184-4 55.56 MS 1722.22 48.15 MS 1870.37

100 W- 202-1 83.33 S 2194.44 74.07 S 2648.15

101 W- 154-2 16.67 R 416.67 29.63 R 703.70

102 W- 174-B-2 72.22 S 2194.44 100.00 S 2555.56

103 W- 156-1 52.00 MS 1603.44 100.00 MS 2044.44

104 W- 155-B-1 66.67 MS 1433.33 59.26 MS 2018.52

105 W- 177-B-2 61.11 MS 1603.44 48.15 MS 1985.19

106 W- 197-A-1 16.67 R 611.11 18.52 R 425.93

107 W- 242-1 22.22 R 750.00 22.22 R 685.19

108 W- 216-1 50.00 MS 1722.22 81.48 MS 1855.56


~119~

109 W- 215-B-2 83.33 S 2110.11 100.00 S 2722.22

110 W- 214-1 67.78 MS 1583.33 100.00 MS 2107.41

111 W- 213-3 55.56 MS 1722.22 62.96 MS 1851.85

112 W- 207-A-3 50.00 MR 1194.44 59.26 MS 1603.70

113 W- 247-A-1 88.89 S 2277.78 100.00 S 2651.85

114 W- 230-2 16.67 R 361.11 29.63 R 848.15

115 W- 247-A-3 50.00 MR 1083.33 49.26 MR 1470.37

116 W- 247-B-1 50.00 MR 1194.44 52.96 MR 1407.41

117 W- 250-1 33.33 MR 1194.44 37.04 MR 1185.19

118 W- 259-B-1 11.11 R 361.11 25.93 R 648.15

119 W- 250-2 22.22 R 750.00 22.22 R 870.37

120 W- 258-1 61.11 MS 1722.22 70.37 MS 2096.30

121 W- 274-A 42.50 MR 1188.89 46.67 MR 1370.37

122 W- 258-2 22.22 R 694.44 33.33 MR 1166.67

123 W- 284-4 22.22 R 722.22 29.63 R 774.07

124 W- 259-E-1 45.56 MR 1166.13 62.96 MR 1359.26

125 W- 242-2 44.44 MR 1161.11 33.33 MR 1314.81

126 W- 226-1 45.56 MR 1044.44 44.44 MR 1266.67

127 W- 216-2 22.22 R 555.56 22.22 R 740.74

128 W- 228 16.67 R 555.56 22.22 R 814.81

129 W- 216-3 55.56 MR 1161.11 48.15 MR 1333.33

130 W- 225 56.50 MS 1777.78 48.15 MR 1574.07

131 W- 312 72.22 MS 1666.13 74.07 S 2681.48

132 W- 307-B-1 83.33 S 2166.67 59.26 MS 2090.74

133 W- 307-A-1 83.33 S 2110.11 70.37 S 2725.93

134 W- 288-B-3 27.78 MR 833.33 48.15 MR 1870.37

135 W- 305-2 11.11 R 361.11 33.33 R 351.85

136 W- 301-1 22.22 R 694.44 25.93 MR 1129.63

137 W- 302-A-3 47.02 MR 922.22 22.22 R 777.78

138 W- 296-2 55.56 MS 1750.00 51.85 MS 2166.67

139 W- 293 44.44 MR 1250.00 49.26 MR 1403.70

140 W- 296-1 16.67 R 361.11 18.52 R 462.96

141 W- 311-B-1 16.67 R 472.22 14.81 R 518.52

142 W- 288-A 41.11 MR 1133.33 42.96 MR 1259.26

143 W- 384-3 37.72 MR 1050.00 22.22 R 888.89

144 W- 354-1 27.78 MR 833.33 22.22 R 740.74

145 W- 354-2 27.78 MR 1138.89 29.63 MR 1259.26

146 W- 390-1-2 42.22 MR 905.56 29.63 R 851.85

147 W- 328-1 73.33 MS 1750.00 74.07 MS 1790.26

148 W- 351-C-1 11.11 R 388.89 18.52 R 425.93

149 W- 311-B-2 44.44 MR 1205.56 33.33 MR 1351.85

150 W- 312-1 38.89 MR 1194.44 33.33 MR 1166.67

151 W- 307-B-2 47.78 MR 1205.56 66.67 MS 2722.22

152 Co-3 88.89 S 2250.00 100.00 S 2790.26

153 EC-520061 11.11 R 314.32 14.81 R 370.37

Max. 88.89 S 2277.78 100.00 S 2790.26

Min. 11.11 R 314.32 11.11 R 314.81

CV % 12.40 18.81

LSD (1 %) 490.87 618.86


~120~

Table 4.28: Summary of disease reaction of RILs (F7 generation) obtained from cross (Co-3 × EC-520061) based on AUDPC value

obtained after inoculation with the isolate (Asv-2) of A. solani in the year 2012-13.

Disease Reaction Poly house conditions Field conditions


Resistant 314.32-805.19 34 314.81-933.67 35

Moderately Resistant 805.20-1296.05 42 933.68- 1552.53 43

Moderately Susceptible 1296.06-1786.92 48 1552.54- 2171.39 46

Susceptible 1786.93-2277.78 30 2171.40 -2790.26 29


Figure 4.4: Categorization of RILs (Co-3 × EC-520061) based on AUDPC after artificial inoculation under poly house and field


22.22

27.45

30.72

19.61

22.88

28.1030.07

18.95

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

1 2 3 4

Poly house conditions Field conditions

Perc

en

t of

RIL

s

R MR MS S



~121~

4.4.4.1Percent disease index (PDI)

A total of 151 RILs along with parents were screened for

resistance to early blight under artificial conditions (Poly house) in the

year 2012-2013. The maximum PDI i.e. 88.89 per cent was recorded

in RILs No. 24, 33, 49, 56, 82, 84, 92 and 113 and minimum PDI i.e.

11.11 per cent was recorded in RIL No. 34, 35, 95, 118, 135 and 148.

The susceptible parent Co-3 depicted 88.89 per cent PDI and resistant

parent EC-520061 depicted 11.11 per cent, which is equal to

maximum and minimum PDI value in RILs, respectively.

Same 151 RILs with parents were also screened for resistance

to early blight under field conditions with artificial inoculation in the

year 2012-2013. The maximum PDI i.e. 100 per cent was recorded in

RILs No. 19, 24, 32, 33, 49, 50, 55, 61, 82, 83, 84, 86, 88, 90, 91, 92,

102, 103, 109, 110 and 113 and minimum PDI i.e. 11.11 per cent

was recorded in RILs No. 42. The susceptible parent Co-3 depicted

100 per cent PDI and resistant parent EC-520061 depicted 14.81 per

cent PDI.


Poly house conditions: The AUDPC value of the thirty four resistant

RILs ranged from 314.32 to 805.19; forty two moderately resistant

RILs ranged from 805.20 to 1296.05; forty eight moderately

susceptible RILs ranged from 1296.06 to 1786.92 and fifty one

susceptible RILs ranged from 1786.93 to 2277.78 which was recorded

in the year 2012-2013. AUDPC of parents ranged from 314.32 to

2250.00.

Field conditions: The AUDPC value of the thirty five resistant RILs

ranged from 314.81 to 933.67; forty three moderately resistant RILs

ranged from 933.68 to 1552.53; forty six moderately susceptible RILs

ranged from 1552.54 to 2171.39 and twenty nine susceptible RILs


~122~

ranged from 2171.40 to 2790.26 which was recorded in the year

2012-2013. AUDPC of parents ranged from 370.37 to 2790.26.

4.4.4.3 Gene prediction on the phenotypic basis

Number of genes predicted on the bases of phenotypic data

analysis (F7 generation) of 151 RILs (Co-3 X EC-520061) under

artificial conditions in field trial. We have predicted three major genes

in 151 RILs (Co-3 X EC-520061) that are responsible to early blight

resistance in tomato under field conditions. Under poly house

conditions in the same 151 RILs (Co-3 X EC-520061) we have

predicted two major genes on the basis of phenotypic data analysis (F7

generation). For gene prediction the statistical analysis was done by

the help of Chi Square Test.

4.4.4.4 Disease frequency on the basis of PDI

Normal disease frequency was observed in poly house as well as

field condition on the basis of percent disease index in the year 2012-

13. In poly house condition, the PDI value 42 DAI of 22 RILs ranged

from 11.11 to 27.00; 32 RILs ranged from 27.78 to 42.22; 39 RILs

ranged from 42.50 to 56.60; 35 RILs ranged from 57.78 to 73.33 and

25 RILs ranged from 76.50 to 88.89.Under field conditions the PDI

value 56 DAI of 26 RILs ranged from 11.11 to 25.93; 32 RILs ranged

from 29.63 to 44.44; 47 RILs ranged from 46.67 to 64.32; 27 RILs

ranged from 65.19 to 81.48 and 21 RILs ranged from 82.21 to 100.00

percent (Figure 4.5).

4.4.4.5 Correlation coefficient between field and poly house data in the year 2012-13.

Relationship was observed between field and poly house

evaluation of 151 tomato RILs in the year 2012-13 for early blight

disease. There was significant (P=0.05) correlation between field and

poly house phenotypic evaluation data for early blight resistance

depicting correlation coefficient of 0.888.


~123~

A. Poly House Conditions B. Field Conditions

Figure 4.5: Frequency distribution of percent disease index for Early blight in RILs (F7 generation) obtained after inoculation with

the isolate Asv-2 in the year 2012-13.

0 10 20 30 40 50 60 70 80 90

Percent Disease Index

0

5

10

15

20

25

30

35

40

45

Fre

qu

ency

12 24 36 48 60 72 84 96 108


5

10

15

20

25

30

35

40

45

Fre

qu

ency


~124~

4.4.5 Phenotyping of 151 tomato RILs (Co-3 X EC-520061) in year the 2013-14.

Phenotyping of 151 RILs in F8 generation along with parents

has been done for resistance to early blight of tomato caused by A.

solani. After inoculation of 151 RILs with parents using one highly

virulent isolate of A. solani, the phenotypic reactions were obtained

with wide variation (Table 4.29& 4.30; Figure 4.6).

4.4.5.1 Percent disease index (PDI)

Total 151 RILs with parents were screened for resistance to

early blight under artificial conditions (Poly house) in the year 2013-

2014. The maximum PDI 96.30 per cent was recorded in RILs no. 50

and 61, and minimum PDI 14.81 per cent was recorded in RIL no. 35

and 118. But the susceptible parent Co-3 depicted 96.30 per cent PDI

that was equal to maximum PDI value in RILs. The resistant parent

EC-520061 depicted 11.11 per cent PDI as compared to minimum PDI

value (14.81 per cent) in RILs.

Same 151 RILs along with parents were also screened for

resistance to early blight under field conditions with artificial

inoculation in the year 2013-2014. The maximum PDI (94.44 per

cent) was recorded in RIL no. 19 and minimum PDI (18.52 per cent)

was recorded in RILs no. 117 and 148. But the susceptible parent Co-

3 depicted 100 per cent PDI that was more than maximum PDI (88.89

per cent) recorded in RILs. The resistant parent EC-520061 depicted

18.52 per cent PDI equal to minimum PDI value 18.52 per cent in

RILs.


~125~

Table 4.29: PDI, AUDPC and Host Reaction of Recombinant Inbred Lines (Co-3 × EC-520061) of Tomato after inoculation with the isolate (Asv-

2) of A. solani in the year 2013-14.

PDI & AUDPC (Year 2013-2014)

RILs No.

Name of RILs

POLY HOUSE TRIAL FIELD TRIAL

42 DAI (PDI

value)

Host Reactio

n

AUDPC 56 DAI (PDI

value)

Host Reaction

AUDPC

1 W- EB-1 74.07 MS 1833.33 75.93 MS 1842.59

2 W- E-3-1 74.07 MS 1759.26 85.19 MS 1853.70

3 W- E-3-2 77.78 MS 1796.30 81.48 MS 1907.41

4 W- 10-B-3 88.89 S 2148.15 83.33 S 2185.19

5 W-10-EB 37.04 MR 962.96 57.41 MS 1412.96

6 W- 16-A 62.96 MS 1796.30 72.22 S 1925.93

7 W- 17 70.37 S 2074.07 81.48 S 2305.56

8 W- 17-1 29.63 MR 925.93 46.30 MR 1148.15

9 W- 19-C-1 44.44 MR 1314.81 57.41 MS 1572.22

10 W- 24-3 70.37 MS 1759.26 61.11 MS 1759.26

11 W- 26 44.44 MR 1333.33 32.22 MR 918.81

12 W- 27-1 45.56 MR 1314.81 41.11 MR 970.37

13 W- 27-C-1 35.93 MR 822.22 33.33 MR 840.74

14 W- 27-C-2 29.63 MR 796.30 44.44 MR 996.30

15 W- 27-D-1 22.22 R 555.56 38.52 MR 811.11

16 W- 27-EB 37.04 MR 1037.04 55.56 MR 1342.59

17 W- 28-2 45.93 MR 848.15 25.93 MR 805.56

18 W- 28-4 51.85 MR 1314.81 64.81 MS 1842.59

19 W- 30-1 85.19 S 2055.56 94.44 S 2037.04

20 W- 35-1 51.85 MS 1462.96 57.04 MS 1362.96

21 W- 35-2 77.78 MS 1962.96 61.11 MS 1861.11

22 W- 35-3 48.15 MR 1148.15 49.26 MR 1003.70

23 W- 38-2 48.15 MR 1148.15 62.96 MR 1342.59

24 W- 40-A 59.26 MS 1833.33 88.89 S 2185.19

25 W- 40-A-1 62.96 MS 1611.11 59.26 MS 1740.74

26 W- 40-B-1 25.93 R 277.78 44.81 MR 970.37

27 W- 40-B-4 39.63 MR 844.44 41.48 MR 818.52

28 W- 41-1 55.56 MR 907.41 57.41 MR 820.37

29 W- 44-B 33.33 MR 822.22 27.78 MR 870.37

30 W- 46-B-1 22.22 R 296.30 32.00 R 379.63

31 W- 47-1 51.85 MR 962.96 61.11 MR 1075.93

32 W- 47-2 85.19 S 2000.00 78.22 S 1957.41

33 W- 48-1 88.89 S 2129.63 83.33 S 2018.52

34 W- 48-2 18.52 R 240.74 25.93 R 666.67

35 W- 49-B-1 14.81 R 185.19 27.78 R 661.11

36 W- 49-C-3 25.93 R 314.81 37.78 MR 950.00

37 W- 50-3 62.96 MR 1129.63 54.44 MR 1009.26

38 W- 50A-2 18.52 R 240.74 33.33 R 305.56

39 W- 53-A-3 22.22 R 296.30 29.63 R 425.93

40 W- 55-A-1 39.63 MR 844.44 48.15 MR 885.19

41 W- 55-A-2 51.85 MR 1240.74 38.89 R 361.11

42 W- 55-B-1 18.52 R 240.74 20.37 R 194.44

43 W- 59-B-1 74.07 MS 1870.37 66.67 MS 1750.00

44 W- 61-4 74.07 MS 1629.63 57.41 MS 1694.44

45 W- 59-B-3 74.07 MS 1740.74 62.59 MS 1435.19

46 W- 75-1 70.37 MS 1592.59 81.48 MS 1685.19

47 W- 60-EB 37.04 MR 881.48 38.52 MR 944.44

48 W- 71-1 51.85 MS 1422.22 60.37 MS 1624.07

49 W- 84-7 88.89 S 2111.11 79.63 S 2018.52

50 W- 84-EB 96.30 S 2185.19 82.15 S 2064.81


~126~

51 W- 82-B-2 51.85 MR 1000.00 57.41 MR 1240.74

52 W- 85-1 66.67 MS 1518.52 73.33 MS 1842.59

53 W- 82-1-1 37.04 MR 781.48 46.30 MR 981.48

54 W- 61-3 51.85 MR 1092.59 48.33 MR 1014.81

55 W- 69-B-2 77.78 S 2092.59 71.11 S 2005.56

56 W- 69-B-1 92.59 S 2222.22 79.63 S 1996.30

57 W- 82-B-3 39.63 MR 940.74 39.63 MR 962.96

58 W- 137-2 62.96 MR 1351.85 56.04 MR 1281.48

59 W- 125-3 70.37 MS 1425.93 83.33 S 2166.67

60 W- 110-1 92.59 S 2222.22 77.78 S 2018.52

61 W- 125-2 96.30 S 2000.00 83.33 S 1972.22

62 W- 114-B-1 88.89 MS 1777.78 61.11 MS 1679.63

63 W- 108-H-3 85.19 MS 1814.81 81.48 MS 1675.93

64 W- 88-2 85.19 MS 1962.96 83.33 MS 1837.04

65 W-95-B-2 29.63 R 407.41 27.78 R 688.89

66 W- 95-B-3 44.44 MR 1092.59 43.33 MR 851.85

67 W- 105-A-4 74.07 MS 1703.70 75.93 MS 1787.04

68 W- 101-1 77.78 MS 1740.74 62.96 MS 1570.37

69 W- 90-2 18.52 R 296.30 44.44 R 490.74

70 W- 152-B-1 74.07 MS 1740.74 75.19 MS 1725.93

71 W- 142-A-3 40.74 MR 1111.11 37.78 MR 1127.78

72 W- 143-B-2 81.48 MS 1944.44 77.78 S 1952.59

73 W- 144-A-1 74.07 MS 1648.15 75.93 S 1938.89

74 W- 144-B-1 81.48 MS 1870.37 77.78 S 2083.33

75 W- 143-1-1 18.52 R 240.74 19.26 R 416.67

76 W- 142-B-1 50.37 MR 1351.85 48.52 MR 968.52

77 W- 146-B-2 77.78 MS 1611.11 76.67 MS 1894.44

78 W- 146-1-1 85.19 MS 1925.93 77.78 S 1975.93

79 W- 151-C-2 62.96 MS 1425.93 53.70 MS 1500.00

80 W- 151 81.48 MS 1944.44 83.33 S 2029.63

81 W- 152-A-2 92.59 MS 1888.89 88.89 S 1996.30

82 W- 148-1-1 74.07 S 2185.19 72.96 MS 1737.04

83 W- 136-1 81.48 S 2037.04 90.74 S 2025.93

84 W- 137-6-1 85.19 S 2296.30 89.63 S 1925.93

85 W- 138-2 38.52 MR 755.56 37.41 MR 850.00

86 W- 138-3 44.44 MS 1407.41 59.26 MS 1842.59

87 W- 137-B-3 81.48 MS 1814.81 66.67 MS 1870.37

88 W- 140-1 88.89 S 2185.19 86.30 S 2055.56

89 W- 141-A-2 22.22 R 518.52 31.11 R 555.56

90 W- 141-6 81.48 S 2074.07 75.56 S 2081.48

91 W- 109-B-1 88.89 S 2388.89 85.19 S 2018.52

92 W- 203-1 88.89 S 2000.00 87.04 S 2083.33

93 W- 201-B-1 22.22 R 444.44 24.44 R 583.33

94 W- 197-A-2 62.96 MS 1592.59 50.00 MS 1750.00

95 W- 201-A-4 25.93 R 277.78 37.41 R 396.30

96 W- 188-B 62.96 MS 1574.07 58.52 MS 1431.48

97 W- 183-B-2 33.33 R 351.85 25.93 R 324.07

98 W- 175-B-1 22.22 R 296.30 24.07 R 259.26

99 W- 184-4 62.96 MS 1425.93 54.07 MS 1379.63

100 W- 202-1 77.78 S 2111.11 74.07 MS 1750.00

101 W- 154-2 18.52 R 240.74 20.52 R 401.85

102 W- 174-B-2 77.78 S 2000.00 69.26 S 1942.59

103 W- 156-1 88.89 MS 1796.30 87.04 S 1990.74

104 W- 155-B-1 66.67 MS 1444.44 75.93 MS 1683.33

105 W- 177-B-2 62.96 MS 1759.26 48.15 MS 1509.26

106 W- 197-A-1 18.52 R 240.74 22.22 R 342.59

107 W- 242-1 25.93 R 314.81 33.70 R 487.04


~127~

108 W- 216-1 59.26 MS 1500.00 55.93 MS 1740.74

109 W- 215-B-2 81.48 S 2296.30 72.22 S 1996.30

110 W- 214-1 74.07 MS 1981.48 87.04 S 2212.96

111 W- 213-3 66.67 MS 1611.11 59.63 MS 1853.70

112 W- 207-A-3 62.96 MS 1629.63 50.00 MS 1777.78

113 W- 247-A-1 85.19 S 2611.11 78.15 S 2412.48

114 W- 230-2 22.22 R 259.26 28.15 R 555.56

115 W- 247-A-3 51.85 MR 1277.78 51.48 MR 1333.33

116 W- 247-B-1 48.15 MR 1129.63 43.33 MR 820.37

117 W- 250-1 45.93 MR 914.81 18.52 MR 953.70

118 W- 259-B-1 14.81 R 148.15 20.37 R 314.81

119 W- 250-2 39.63 MR 966.67 31.48 R 416.67

120 W- 258-1 55.56 MS 1444.44 60.74 MS 1518.52

121 W- 274-A 44.44 MR 940.74 49.26 MR 837.04

122 W- 258-2 18.52 R 240.74 42.96 MR 948.15

123 W- 284-4 22.22 R 296.30 24.07 R 314.81

124 W- 259-E-1 51.85 MR 1240.74 45.07 MR 1259.26

125 W- 242-2 48.15 MR 1092.59 51.48 MR 1259.26

126 W- 226-1 70.37 MS 1388.89 51.85 MR 1240.74

127 W- 216-2 22.22 R 296.30 31.48 MR 759.26

128 W- 228 22.22 R 259.26 31.11 R 705.56

129 W- 216-3 37.04 MR 1037.04 47.41 MR 1133.33

130 W- 225 51.85 MS 1500.00 54.07 MS 1388.89

131 W- 312 66.67 MS 1870.37 74.81 MS 1840.74

132 W- 307-B-1 77.78 S 2018.52 73.70 MS 1848.15

133 W- 307-A-1 81.48 S 2129.63 77.41 S 2077.78

134 W- 288-B-3 39.63 MR 848.15 46.30 MR 842.59

135 W- 305-2 18.52 R 240.74 22.96 R 624.07

136 W- 301-1 18.52 R 277.78 25.56 R 420.37

137 W- 302-A-3 43.33 MR 825.93 37.78 MR 933.33

138 W- 296-2 59.26 MS 1537.04 49.63 MS 1444.44

139 W- 293 48.15 MR 833.33 48.15 R 657.41

140 W- 296-1 18.52 R 240.74 32.96 R 687.04

141 W- 311-B-1 22.22 R 296.30 21.11 R 574.07

142 W- 288-A 51.85 MR 1222.22 48.89 MR 1111.11

143 W- 384-3 37.04 MR 829.63 42.22 MR 833.33

144 W- 354-1 33.33 MR 833.33 29.63 MR 842.59

145 W- 354-2 40.74 MR 1000.00 42.59 MR 898.15

146 W- 390-1-2 45.93 MR 974.07 42.22 MR 871.11

147 W- 328-1 66.67 MS 1481.48 74.07 MS 1916.67

148 W- 351-C-1 18.52 R 240.74 18.52 R 407.41

149 W- 311-B-2 48.15 MR 907.41 45.93 MR 824.07

150 W- 312-1 40.74 MR 1018.52 27.78 MR 796.30

151 W- 307-B-2 40.74 MR 1259.26 49.63 MS 1592.59

152 CO-3 96.30 S 2407.41 100.00 S 2490.74

153 EC-520061 11.11 R 129.63 18.52 R 194.44

CV % 12.70 17.92

LSD (1 %) 620.14 574.07


~128~

Table 4.30: Summary of disease reaction of RILs (F8 generation) obtained from cross (Co-3 × EC-520061) based on AUDPC value

obtained after inoculation with the isolate (Asv-2) of A. solani in the year 2013-14.

Disease Reaction Poly house conditions Field conditions


Resistant 129.63 - 750.-00 32 194.44-768.52 30

Moderately Resistant 750.01 - 1370.37 47 768.53– 1342.59 48

Moderately Susceptible 1370.38 - 1990.74 49 1342.60- 1916.67 43

Susceptible 1990.75 – 2611.11 25 1916.68 -2490.74 32


Figure 4.6: Categorization of RILs (Co-3 × EC-520061) based on AUDPC after artificial inoculation under poly house and field conditions in the year 2013-14.

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

1 2 3 4

Poly house conditions Field conditions

Perc

en

t of

RIL

s

R MR MS S



~129~


Under poly house conditions the AUDPC value of the thirty two

resistant RILs ranged from 129.63 to 750.00; forty seven moderately

resistant RILs ranged from 750.01 to 1370.37; forty nine moderately

susceptible RILs ranged from 1370.38 to 1990.74 and twenty five

susceptible RILs ranged from 1990.75 to 2611.11. AUDPC of patents

ranged from 129.63 to 2407.41 in the year 2013-2014.

Under field conditions, the AUDPC value of thirty resistant RILs

ranged from 194.44 to 768.52; forty eight moderately resistant RILs

ranged from 768.53 to 1342.59; forty three moderately susceptible

RILs ranged from 1342.60 to 1916.67 and thirty two susceptible RILs

ranged from 1916.68 to 2490.74. AUDPC of parents ranged from

194.44 to 2490.74 in the year 2013-2014.

4.4.5.3 Gene prediction on the phenotypic basis

Number of genes were predicted on the basis of phenotypic data

analysis (F8 generation) of 151 RILs (Co-3 × EC-520061) after artificial

inoculation in field trial. We have predicted two major genes in RILs

that are responsible for early blight resistance in tomato under field

condition. Under poly house condition in the same 151 RILs (Co-3 ×

EC-520061) we have predicted two major genes on the basis of

phenotypic data analysis (F8 generation). For gene prediction the

statistical analysis was done by the help of Chi Square Test.

4.4.5.4 Disease frequency on the basis of PDI

Normal disease frequency was observed in poly house as well as

field data on the basis of 42 DAI percent disease index in the year

2013-14. Under poly house conditions the PDI value of 15 RILs

ranged from 18.52 to 25.56; 18 RILs ranged from 25.93 to 33.33; 20


~130~

RILs ranged from 33.70 to 45.07; 28 RILs ranged from 45.93 to 57.07;

28 RILs ranged from 57.41 to 72.96; 18 RILs ranged from 73.33 to

78.15; 15 RILs ranged from 78.22 to 83.33 and 11 RILs ranged from

88.89 to 100.00 percent. Under field conditions the PDI value of 16

RILs ranged from 18.52 to 25.56; 24 RILs ranged from 25.93 to 38.52;

29 RILs ranged from 38.89 to 49.26; 33 RILs ranged from 49.63 to

64.81; 22 RILs ranged from 66.67 to 77.41; 18 RILs ranged from

77.78 to 83.33 and 11 RILs ranged from 85.19 to 100.00 percent

(Figure 4.7).

4.4.5.5 Correlation coefficient between field and poly house data in the year 2013-14.

Relationship was observed between field and poly house

evaluation of 151 tomato RILs in the year 2013-14 for early blight

disease. There was significant (P=0.05) correlation between field and

poly house phenotypic evaluation data for early blight resistance

depicting correlation coefficient of 0.908.

4.5 Mapping of QTLs for early blight resistance.

Most commercial cultivars of tomato, Solanum lycopersicum, are

susceptible to early blight (EB), a devastating fungal (Alternaria solani

Sorauer) disease of tomato in the northern and eastern parts of India,

and elsewhere in the world. The highly resistant source of EB within

the indeterminate species i.e. S. habrochaites was used in traditional

breeding program at IIVR to develop high-yielding, early-maturing

tomatoes with improved EB resistance. Four hundred forty seven

simple sequence repeat markers were screened within F7 population

of a cross between a susceptible tomato breeding line (Co-3; maternal

and recurrent parent) of S. lycopersicum with a resistant S.

habrochaites accession (EC-520061) for EB resistance. Results were

obtained with 25 polymorphic SSR markers (Table 4.32; Plate 4.6 to

4.10).


~131~

Table 4. 31: Correlation coefficents among phenotypic data under poly house

and field conditions depicted by 151 RILs of Co-3 × EC-520061.

Environments Poly house 2012-13

Field2012-13 Poly house 2013-14

Field2013-14

Poly house 2012-13 1

Field 2012-13 0.888* 1

Poly house 2013-14 0.877 0.843 1

Field 2013-14 0.866 0.860 0.908* 1

*Significant at 5 % (P=0.05) level of significance

Table 4.32: 25 informative SSR markers (SGN database) screened in our polymorphic survey

Marker Consensus Tomato Maps Chr. Position (cM) Confidence

SSR316 Tomato-EXPIMP 2008 1 66.00 uncalculated

SSR308 Tomato-EXPEN 2000 1 121.00 CF(LOD3)

SSR341 Tomato-EXPEN 2000 1 137.50 I



SSR40 Tomato-EXPEN 2000 2 22.00 F(LOD3)




SSR11 Tomato-EXPEN 2000 3 164.00 I(LOD2)











SSR73 Tomato-EXPEN 2000 9 32.00 CF(LOD3)


SSR526 Tomato-LXCHM 2007 10 23.00 uncalculated




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A. Poly House Conditions B. Field Conditions

Figure 4.7: Frequency distribution of percent disease index for Early blight in RILs (F8 generation) obtained after inoculation with

the isolate Asv-2 in the year 2013-14.

12 24 36 48 60 72 84 96 108


3

6

9

12

15

18

21

24

27F

requ

ency

12 24 36 48 60 72 84 96 108


4

8

12

16

20

24

28

32

36

Fre

qu

ency


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The F7 individuals were screened for disease symptoms for EB

lesion on the basis of (0-9) disease scale in glass house experiment,

the area under disease progress curve (AUDPC) and the final disease

percentage index (disease severity) were determined for each plant.

Linkage analysis was performed with simple interval mapping (SIM)

and composite interval mapping (CIM) to identify QTLs for EB

resistance (Figure 4.9 to 4.12). Five markers (SSR448, SSR40,

SSR356, SSR605 and SSR5) on chromosome 2; three markers

(SSR72, SSR603 and SSR310) on chromosome 4 and four markers

(SSR103, SSR285, SSR304 and SSR45) on chromosome 6 showed

linkage (Table 4.33& 4.34; Figure 4.8). The closest marker for the

trait was SSR605 that showed linkage at 46.1 cM distance at

chromosome 2 in close proximity to probable EBR locus responsible

for early blight disease resistance with LOD value 3.2 and maximum

distance 53.00cM. Both the mapping approaches revealed a possible

epistatic interaction between the QTLs identified, a significant additive

gene action was seen for the early blight resistance whereas positive

dominance gene action resulted in only one QTL on chromosome 2,

the mean phenotypic variation was estimated up to 3 %. The

preliminary results obtained through QTL mapping for EB resistance

showed only few loci related to the early blight disease. The number of

polymorphic SSR markers were also very small, may be due to narrow

genetic base of tomato.


~134~

Table 4.33: Simple interval mapping with final multiple regression analysis included 3 QTLs for early blight disease resistance.

Trait/QTLs Marker interval Interval

(cM) Chr. LOD Add. Domi. R²

Qdis.bhu-2.1 SSR40-SSR356 22 2 13.95 +21.48 -4.92 0.55

Qdis.bhu-2.2 SSR-356- SSR-605

4.5 2 4.97 +4.9 -5.82 0.35

Qdis.bhu-4 SSR72-SSR603 37 4 3.15 +32.23 -2.82 0.76

Chr.=chromosome number; SIM=simple interval mapping; CIM=composite interval mapping; cM=centimorghan; LOD=Logarithm of odds; Add.= Additive; Domi.= Dominance; R2= Phenotypic variance in percentage (%).

Table 4.34: Composite interval mapping with final multiple regression

analysis included 2 QTLs for early blight disease resistance.

Trait/QTLs Marker interval Interval (cM)

Chr. LOD Add. Domi. R²

Qdis.bhu-2.1 SSR40-SSR356 22 2 6.96 +30.49 8.74 0.59

Qdis.bhu-6 SSR304-SSR45 10 6 3.2 +21.39 -2.78 0.26

Chr.=chromosome number; SIM=simple interval mapping; CIM=composite interval mapping; cM=centimorghan; LOD=Logarithm of odds; Add.= Additive; Domi.= Dominance; R2= Phenotypic variance in percentage (%).


~135~

Figure 4.8: Linkage map of the S. lycopersicum × S. habrochaites in F7 population showing position of QTLs. The number to the

left of each chromosome indicate map distance (in centi Morgans) between linked markers. To the right of each chromosome indicate name of markers, which are linked with particular chromosome.


~136~

Figure 4.9: LOD curve of simple interval mapping of chromosome 2 of tomato for early blight resistance. It is showing two peaks (at about 33 and 46.25 cM distance). Ist peak is showing between SSR40 and SSR356 markers (22 cM distance between

both markers) with 13.95 LOD scores. IInd peak is showing between SSR356 and SSR605 markers (4.5 cM distance

between both markers) with 4.97 LOD scores.


~137~

Figure 4.10: LOD curve of simple interval mapping of chromosome 4 of tomato for early blight resistance. There is a single peak

(at about 18.5 cM distance). Peak is showing between SSR72 and SSR603 markers (37 cM distance between both

markers) with 3.15 LOD scores.


~138~

Figure 4.11: LOD curve of composite interval mapping of chromosome 2 of tomato for early blight resistance. There is a single

peak (at about 33 cM distance). Peak is showing between SSR40 and SSR356 markers (22 cM distance between both markers) with 6.96 LOD scores.


~139~

Figure 4.12: LOD curve of composite interval mapping of chromosome 6 of tomato for early blight resistance. There is a single

peak (at about 40.5 cM distance). Peak is showing between SSR304 and SSR45 markers (10 cM distance between

both markers) with 3.2 LOD scores.

Plate 4.6: Parental screening of RILs (Co-3 × EC-520061) with SSR primers;

The gel shows amplification of DNA of susceptible parent Co-3 (P1)

and resistant parent EC-520061 (P2) obtained with 7 SSR primers.

Plate 4.7: Genotyping done in F7 RIL population obtained with SSR 603. L: 1kb

DNA Ladder; P1: Parent 1 (Co-3); P2: Parent 2 (EC-520061); other lanes: F7 RILs.

Plate 4.8: Genotyping done in F7 RIL population obtained with SSR72. L: 1kb

DNA Ladder; P1: Parent 1 (Co-3); P2: Parent 2 (EC-520061); other

lanes: F7 RILs.



lanes: F7 RILs.



lanes: F7 RILs.

Plate 4.1: Early blight symptoms on leafs and fruits of tomato

Plate 4.2: Cultural growth of virulent isolates of A. solani on PDA, 12 days after incubation at 25 ± 2 ºC.

(A) A. solani sporulated sorghum grains (B) Close view of A. solani sporulated

grains

(C) Spore production on sorghum grains (D) Microscopic photograph of A.

solani on sorghum grains

Plate 4.3: Spore production of A. solani on sorghum grains

Plate 4.4: Disease reaction of early blight of tomato caused by Alternaria

solani (R=Resistant; MR= Moderately Resistant; MS=Moderately Susceptible; S= Susceptible)

Plate 4.5: View of experiment for phenotyping of RILs of tomato against early

blight under poly house conditions.

Chapter V

DISCUSSION

An investigation was carried out on various aspects of the

pathogen viz. isolation, purification and pathogencity; cultural,

morphological and pathogenic variability of different isolates;

standardization of conidial production as well as inoculation

technique; phenotyping and genotyping of RILs and tomato

germplasm; and Mapping of QTLs for early blight resistance in RILs

(Co-3 X EC-520061). The results of the investigation have been

discussed in this chapter.

The symptoms on tomato plants in the field were first noticed

on the older leaves as minute brown to black necrotic spots

measuring one to two mm in diameter. These spots often enlarged

with concentric rings to produce characteristic target board effect.

Later upward progress of the disease was observed and leaves dried

up and drooped down. Similar description of symptoms on tomato

were made by Walker (1952) and Singh (1987). The disease on fruits

and at stem end spots started as black or brown sunken lesions

which later enlarged to considerable extent and they covered the

whole fruits which ultimately led to their decay. Datar and Mayee

(1986) and Ramakrishnan et al. (1971) also recorded such symptoms

on stem, petioles and fruits. The fungus was isolated from leaves

showing typical early blight symptoms by following standard tissue

isolation technique. The pathogenicity tests were carried out by

inoculating spore and mycelial bit suspensions on 30 days old tomato

varieties Co-3 and Arka Vikash. First symptoms appeared on older

leaves 15 days after inoculation as brown necrotic spots with

concentric rings at the center. Similar technique was followed by

Kumar et al., (2008) and Hassanein et al., (2008) to prove the

Discussion

~141~

pathogenicity of A. solani on tomato. The reisolation yielded A. solani

fungus, which exhibited similar characters as in the originally isolated

culture.

The cultural characteristics (mycelial growth, sporulation) differ

in various isolates. These isolates exhibited significant variation for

their cultural characters, pigmentation and growth rate per day. Few

isolates grew very fast in the initial 5–7 days of observation and few

were fast in the middle-age and rest grew fast at a later stage.

Therefore, the variation was recorded in the average radial growth per

day after inoculation. It may be due to the reason that the isolates of

A. solani were collected from different regions of the country. Similar

results were reported by Sodlauskiene (2003) and Kumar et al. (2008).

Kaul and Saxena (1988) noted the cultural variability of A. solani

isolates on PDA and classified into 4 distinct cultural groups based on

type of growth, colony colour and colour of the substrate and growth

rate. Sporulation was observed only in five isolates; remaining

thirteen isolates did not produce sporulation on PDA media.

Morphological variability was studied among eighteen isolates

collected from different regions. Morphological variability like colour of

colony, margin of colony, colony growth and sporulation, were studied

among eighteen isolates on potato dextrose agar. The isolates had

distinct morphological variability. Kumar et al., (2008) also studied

morphological characteristics of A. solani of tomato.

The pathogenic variability of Alternaria solani was studied

under greenhouse conditions based on the inoculation of eighteen

isolates on two susceptible tomato genotypes. The present study is

confined to test differential infection potential by mycelial culture and

conidial suspensions. Sporulating isolates showed early and more

severe infection than non-sporulating isolates. Approximately 125

Discussion

~142~

c.f.u./ml inoculum concentration from a 12 day old culture was used

to spray on healthy plants. Co-3 showed early and more severe

infection than Arka Vikash. This type of experiment for the pathogenic

variability was conducted by Castro et al., (2000). Pathogenic

variability of eleven isolates of Alternaria solani was studied under

greenhouse conditions on three highly susceptible tomato varieties, in

which six isolates were found to be virulent and five less virulent

(Pandey et al. 2008).

The isolates of Alternaria solani obtained on PDA media showed

typical characteristics of the species. The conidiophores were straight

and brown to olivaceous brown. The description of the fungus agreed

with the description given for A. solani sorauer by Common Wealth

Mycological Institute, Kew, Surrey, England (Ellis, 1971). Growth of

the fungus on different solid media and the cultural characteristics

were recorded. A. solani isolated from tomato was grown on ten solid

media to study the variation in growth and cultural characteristics.

Among the media tested, the pathogen preferred non synthetic media

for its growth as compared to synthetic media. Maximum growth was

observed on potato dextrose agar (PDA) followed by V-8 juice agar

media after 12 days of inoculation which may be attributed to

complex nature of natural media supporting good fungal growth. The

results are at par with the results obtained in an experiment

conducted by Arunakumara, (2006); Gemawat and Ghosh (1920).

Rotem (1966) and Mazzonetto et al. (1996) recommended potato

dextrose agar as the best medium for Alternaria spp. Morphological

variations such as colony colour, margin of colony and substrate

colour were noticed on A. solani. Majority of isolates produced brown

and grey pigmentation in the culture media. Several workers notably

Bonde (1929), Henning and Alexander (1959), Rotem (1966) and Kaul

and Saxena (1988) also observed differences in cultural characters

Discussion

~143~

like growth rate, type of growth, colony colour, colour of the substrate

and sporulation on different media. Determination of optimum growth

period is essential to study the physiology of fungi.

Maximum dry mycelial weight in the present study was

obtained 12 days after inoculation on potato dextrose broth, which is

indicative of the optimum growth of the fungus. Afterwards the growth

declined gradually with the increment in number of days of

incubation. This may be possibly due to autolysis of the fungus and

exhaustion of nutrients in the medium as suggested by Lilly and

Barnett (1951) who also pointed out that the growth of the fungus as

in other organisms follow a definite pattern which depend on species,

environmental and nutritional conditions. Potato dextrose broth was

used to study the growth phase of A. solani. Maximum dry mycelial

weight was recorded on 12th day of inoculation (Arunakumara, 2006).

Waghunde (2008) observed the maximum dry mycelial weight of A.

alternata on 10 DAI.

Temperature is the most important physical environmental

factor for regulating vegetative and reproductive activity of the fungi.

In the present study on the influence of temperature, A. solani showed

maximum growth at 25 °C. Kaul and Saxena, (1988) also reported the

temperature of 25 °C being good for the growth of A. solani. Bonde,

(1929) reported that growth rate of A. solani ranged from 15 - 40 °C

and he observed maximum growth of the fungus at 25 °C.

Ten pH levels were studied to determine the effect of hydrogen

ion concentrations on the mycelial growth and sporulation of A. solani

of tomato. The results of the present study indicated that optimum pH

for the growth and sporulation of A. solani was in the range of 6.5 to

7.0. However, good growth of the fungus was recorded at pH 6.0, 6.5

and 7.0. This shows that A. solani prefers acidic pH to alkaline pH

Discussion

~144~

indicating its acid tolerance. The results obtained in the present study

are in accordance with the results obtained by Verma (1970).

Gemawat and Ghosh (1980) who reported that pH 6.3 was best for A.

solani and Arunakumara, (2006) also found that pH 6.0 to 7.0 gave

best results in comparison to other pH range.

This study showed that only sporulating isolates produced

spores consistently on different media. A. solani is a slow growing

pathogen; spore production in culture media takes 12-13 days with

less number of spores due to slow growth of fungus. The sorghum

grain is most suitable substrate for inoculum production, because it

supports relatively faster colonization (within 30 days) and the highest

spore production (4.5 X 103 g-1 of grain). The present inoculum

production technique is cheap and environmentally safe. Similar

results of present study was also recorded by Chand et al. (2013) on

the production of conidia in C. canescens of mungbean with sorghum

grains. The grain based inoculum production technique is one step

process and easy to adopt. The inoculum produced on the grain can

be stored in the polypropylene bag. The technique would facilitate

both field and greenhouse screening of different tomato varieties for

the selection of Alternaria resistant genotypes.

In present study the significantly higher number (1.0 X 104/gm)

of conidia were obtained from sorghum grains and water ratio of 10:8

as compared to others. Similar results was observed by Chaurasia et

al., (1998) on Alternaria triticina of wheat with sorghum grains and

water ratio of 10 : 9. The conidiophores are formed under high

humidity, whereas conidia formation is favoured by alternating high

and low humidity along with darkness (Waggoner & Horsfall, 1969).

The in vivo conditions that favor sporulation under controlled

conditions has not been successful in previous attempts to develop a

protocol to induce sporulation in A. solani on artificial media. Other

Discussion

~145~

factors, besides light conditions and levels of humidity may be

required to stimulate conidia formation in colonies developed in vitro

(Prabhu and Prasada, 1966).

The 5, 7, 9 and 11 days old cultures of A. solani grown on

potato dextrose agar and dextrose broth were exposed to UV light for

20 seconds which gave maximum sporulation i.e. 5800/ml in PDA

and 5600/ml in PDB medium. As the time of exposure increased

there was fall in the amount of A. solani sporulation. Prolonged

exposure of cultures to ultraviolet light retards sporulation but the

inhibitory effect can be modified by decreasing the intensity of

irradiation. Prolonged exposure of cultures to ultraviolet light retards

sporulation but the inhibitory effect can be modified by decreasing the

intensity of irradiation. This explains the different optima for

maximum sporulation obtained by McCallan and Chan (1944) and

Charton (1953).

When cultures of Alternaria solani were kept at 24 hrs darkness

+ 24 hrs light (3000 Lux) they gave maximum mycelial growth at 12

DAI with 4900 spore/ml. However, the treatment in which cultures

were kept for 12 days at 25 ± 2 ºC for 24 hrs darkness + 48 hrs light

(3000 Lux) gave higher spore production i.e. 5700/ml. In relation to

the above results, Luken (1965) demonstrated a reversal of blue light

induced suppression by a subsequent short exposure to red light in

the photo sporulation of A. solani, suggesting a possibility that the

phytochrome system participated in this reversion.

Four inoculation methods were used (spray inoculation, root dip

inoculation, droplet inoculation and soil inoculation) with spore

concentration of 104 spore/ml for better disease development on two

susceptible varieties (Co-3 & Arka Vikash). The droplet method gave

maximum PDI and minimum incubation period on both susceptible

Discussion

~146~

varieties (Co-3 & Arka Vikash). A droplet inoculation method was

used for evaluation of tomato resistance to early blight and better

discriminated the level of resistance (P<0.001) for a range of spore

densities in comparison with the more commonly used spray

inoculation method (Chaerani, 2007). The method has been also used

to evaluate EB resistance components (O’Leary and Shoemaker,

1983). Glasshouse tests using spray inoculation of conidial

suspension on seedlings are widely used following the establishment

of efficient screening and conidial inoculum production techniques.

An alternative method to obtain more precise and reliable disease

readings is offered by a method in which individual droplets of fungal

inoculum suspension are inoculated on leaflets. This method was first

introduced by Locke (1948) to find sources of resistance to EB (Locke

1949). The considerable amount of time required to measure lesions

make this method less attractive for large scale screening (Chaerani,

2007). So, the adoptability of spray inoculation method is more as

compared to droplet inoculation method.

There was a significant loss in the number of spores/gm of

sorghum grains and inoculum efficiency when inoculum was stored

for more than 30 days. But these grain based media were found best

for long term storage of A. solani as compared to other artificial media.

Higher spore concentration was recorded in sorghum grains after 30

days incubation period. Similar study was also done by Chand et al.,

2013 on Cercospora canescens of mungbean and observed that there

was significant loss in the number of spores when inoculum was

stored for 60 days and inoculum efficiency was reduced from 16

lesions leaflet-1 to 9 lesions leaflet-1 on susceptible cultivar Kopergoan.

Under glass house conditions, all inoculated plants showed

disease symptoms on leaves after inoculation, even with the lowest

conidial concentration of 1 x 103 conidia/ml. Maximum number of

Discussion

~147~

lesions/leaflets (15.25) and minimum incubation period (5 days) was

recorded with spore concentration of 10 x 103 on susceptible tomato

varieties Co-3 followed by the variety Arka Vikash in which 13.75

lesions/leaflets and 6 days incubation period were recorded. The

effects of inoculum concentration on symptom development and

defoliation of tomato plants in these controlled-environment

experiments support the observations of Coffey et al. (1975), who

showed that early blight severity on young tomato plants increased as

conidial concentration increased from 5 x 103 conidia/ml. A positive

relationship between inoculum concentration and symptom

development has also been demonstrated for other Alternaria species

(Vloutoglou and Kalogerakis, 2000).

In the present study, both PDI and AUDPC were used to

evaluate and compare tomato RILs. Screening of large-sized

population, may be sufficient to conduct only a single evaluation

(Foolad, 2002). Field evaluation has been the principal procedure and

one that has resulted in reliable information. Field screening, has

limitations, as it depends on the presence of proper environmental

conditions such as humidity, temperature and the pathogen

inoculums. Furthermore, field screening can often be carried out only

once a year. Such limitations would restrict breeding progress

(Foolad, 2000). Various methods have been used to evaluate tomato

RILs for disease resistance under field conditions (Barksdale, 1971).

Glasshouse or controlled-environment chamber assays with

seedlings or small plants provide uniform, favourable, repeatable

environmental conditions and permit several cycles of screening per

year, thus offering more reliable results. Glasshouse and field test

results correspond well (Banerjee et al., 1998; Foolad et al., 2000).

Glasshouse tests have the advantage that conditions are more

reproducible than in the field and that the duration of the test is

Discussion

~148~

shorter and that, especially after droplet inoculation, more objective

and precise data can be obtained. Still, conditions in the glasshouse

cannot be fully controlled and some genotypes are not well adapted to

glasshouse conditions. Results from poly house experiment were

correlated or highly similar to those from the field, as judged by the

significant correlation between poly house and field data (percent

disease index), with correlation coefficient 0.888 and 0.908 for early

blight disease in 151 tomato RILs in the years 2012-13 and 2013-14,

respectively.

Various screening test methods have been developed, which

differ with respect to test environment (field, glasshouse, or

laboratory), biological materials (detached leaflets, intact young or old

plants) and inoculum (conidia, mycelium or toxin). In few cases

different methods have been compared. Reasonable correlations have

been found between various types of glasshouse and field tests.

The complex genetic control of EB resistance in several sources

of resistance has been studied using quantitative genetic methods.

The loci underlying the resistance have been further dissected using a

QTL mapping approach in S. habrochaites (Foolad et al., 2002; Zhang

et al., 2003). The detection of common QTLs at different experimental

locations may be hampered by genotype × environment or genotype ×

isolate interactions as was observed in some studies (Lubberstedt et

al., 1999).

The inoculation technique is critical for this epidemiological and

screening work; hence, only sporulated cultures of the pathogen were

used. Periodic observation of PDI is essential to assess the pathogenic

reaction on RILs. Based on a single observation, only the PDI at that

instance and tolerance level of RILs can be known. It is difficult to

evaluate the disease severity and pathogen reaction of the RILs at

Discussion

~149~

later stages. So, we took 5 to 7 observations on seven days interval

each to know the progress of disease on RILs. Therefore, it is

important to know the complete cycle of disease to evaluate a

particular host reaction; moreover, screening must be based on

several, periodical observations to know the ultimate fate of any

variety. A phenomenon that was observed in most of the susceptible

to highly susceptible plants is that the lower leaves rapidly defoliated

after rapid, severe infection under artificial and natural epidemic

conditions. The most of the inoculated varieties had a high PDI after

natural infection; screening for early blight resistance should use

artificial inoculation rather than natural inoculation in the field to

find resistance sources. With the AUDPC the host, pathogen, and

environmental effects occurring during the epidemic are integrated

(Pandey et al., 2003).

Large numbers of tomato germplasm lines (701) were

preliminary screened under natural conditions in the year 2011-12.

On the basis of disease severity phenotypic data obtained in

preliminary screening, a core set of tomato lines (240) was selected

which depicted total phenotypic variance based on disease severity

obtained under natural field screening. The 240 core set lines were

again screened for disease reaction after artificial inoculation under

field conditions continuously for two years i.e. 2012-13 and 2013-14.

The mean phenotypic variation was estimated up to 3 percent

in our study, which was very less for good result. The smaller number

of QTLs detected in this study may be due to a higher LOD threshold

employed (3.5–3.7) as compared to the previous mapping study using

S. habrochaites source which used a LOD threshold of 2.4 (Foolad et

al. 2002). The QTL on chromosome 7, which was detected only in the

glasshouse test using a single isolate, was not detected in the field

studies by Foolad et al. (2002b) and Zhang et al. (2003b). During

Discussion

~150~

present investigation, the closest marker for the trait was SSR605

that showed linkage at 46.1 cM distance at chromosome 2 in close

proximity to probable EBR locus responsible for early blight disease

resistance with LOD value 3.2. Acceptable marker interval (4.5 cM)

was observed only between SSR356 and SSR605 markers with simple

interval mapping at chromosome 2; and at chromosome 4 and 6 no

acceptable linked distance was observed between two markers.

We are continuously searching polymorphic SNP (Single

nucleotide polymorphism) markers to saturate the linkage map, so

that fine mapping of QTLs for early blight resistance can be done. The

phenotypic disease screening data generated with tomato core set

lines will be used for association mapping and validation of markers

for early blight resistance.

Chapter VI

SUMMARY AND CONCLUSION

Tomato is the world’s largest vegetable crop and known as

protective food both because of its special nutritive value and also

because of its wide spread production. Tomato is one of the most

important vegetable crops cultivated for its fleshy fruits. Tomato is

considered as important commercial and dietary vegetable crop. As it

is short duration crop and gives high yield, it is important from

economic point of view and hence area under its cultivation is

increasing day by day.

Early blight (EB) is widely distributed in the world and can

cause substantial yield loss of tomato in endemic areas. The yield loss

of tomato fruit was 78 % recorded at 72 % disease intensity of A.

solani and each 1 % increase reduced tomato yield by 1.36 % (Dater

and Mayee, 1985). The disease appears first on the lower leaves and

intensifies as the plant matures. The frequent application of

fungicides needed to control the disease might be reduced if cultivars

with a sufficient level of resistance and satisfactory horticultural

characteristics become available.

In the present investigation cultural, morphological and

pathogenic variability of different isolates was assessed; mass

sporulation technique as well as inoculation technique of the

pathogen was standardized. Phenotyping and genotyping of RILs and

germplasm was done along with mapping of QTLs for early blight

resistance caused by Alternaria solani.

Radial growth observed for eighteen isolates were significantly

different for most of the isolates. Highest radial growth was observed

in isolate Asnd-2 (41.00 mm) that was at par with isolates Asnd-

Summary and Conclusion

~152~

1(40.33 mm) and Ashi-1 (38.67 mm) at 7 days after inoculation (DAI).

The mean mycelial growth was not significantly different in different

isolates. The maximum mean mycelial growth was observed in isolate

Asnd-1 (67.22 mm) and minimum mean mycelial growth was

observed in isolate Asar-1 (51.22 mm).

Based on the disease severity data, five isolates (Asv-1, Asv-2,

Asnd-1, Asmi-2 and Asan-1) were found to be virulent, causing severe

disease in both susceptible varieties (Co-3 and Arka Vikash). Other

thirteen isolates were rated as less virulent. The mean AUDPC in

virulent isolates ranged between 1380.56 to 1503.70 while in less

virulent ones mean AUDPC lied between 939.81 to 1270.37.

Evaluation of solid media and broth, pH and temperature on

the growth and sporulation of A. solani was studied in vitro. It was

observed that the Potato dextrose agar and broth medium proved best

for the mycelial growth and sporulation of A. solani and it was at par

with V-8 juice agar and broth, respectively. The temperature 25 ºC to

30 ºC were found most congenial for growth and sporulation of A.

solani on Potato dextrose agar and broth medium. The fungus could

grow on wide range of pH i.e. from 4.0 to 9.0, among which pH 7.0

and 6.5 gave significantly highest mycelial growth and dry mycelial

weight on Potato dextrose agar and Potato dextrose broth media,

respectively. The pH 6.5 to 7.5 was found suitable for the mycelial

growth and sporulation of A. solani.

The five substrates used in the study were colonized by A.

solani. Earliest colonization within 10 days was found with wheat and

sorghum grains at 25±2°C. A maximum duration of 30 days was

required for pearl millet, maize and barley when incubated at 25±2°C.

Mycelial growth and sporulation of A. solani was excellent on sorghum


~153~

grains (4.50 x 103 spores/gm) and poor on pearl millet at 30 DAI

(Days after inoculation).

The varying moisture content of substrates gave diversified

colonization and sporulation of test pathogen on all the grain based

media. 8 ml water : 10 gm grains gave best result as compared to

other treatments. This technique is cost effective since it does not

require use of agar and chemicals that are costly and the technique is

simple, rapid and reliable. This inoculum production technique is

easily adoptable to many laboratories where facilities and resources

are limited.

The optimum time of UV light exposure for maximum

sporulation i. e. 5800/ml in PDA and 5600/ml in PDB was 20

seconds as compared to other treatments. But when, the time of

exposure was increased there was fall in the amount of A. solani

sporulation. The When cultures of Alternaria solani were kept under

treatment i.e. 24 hrs darkness + 24 hrs light (3000 Lux) they gave

maximum mycelial growth i.e. 88.00 mm at 12 DAI with 4900

spores/ml. However, the treatment 24 hrs darkness + 48 hrs light

(3000 Lux) gave higher spore production (5700/ml) as compared to

other treatments.

In the droplet inoculation method leaflets of intact plants were

inoculated with droplets of A. solani conidial suspension (104) and

gave better results in comparison to other inoculation methods.

Screening of large numbers of accessions in the glasshouse has never

been conducted using the droplet inoculation method, because this

method is time taking and cumbersome to inoculate individual plants.

Therefore, the spray inoculation technique which is next in efficiency

for disease development to droplet inoculation technique was used


~154~

both under poly house and field conditions. Phenotyping during

present investigation was done using spray inoculation technique.

Grain based inoculum on sorghum grains was found best for

long period storage of A. solani compared to other artificial media. It’s

easy, cheap and non-chemical method for growth of A. solani and its

long time storage.

The early blight disease severity on 45 days old tomato plants

increased as conidial concentration increased from 5 x 103 conidia/ml

in susceptible variety, Co-3. A positive relationship between inoculum

concentration and lesion/leaflets was observed.

240 tomato lines of a core set were selected from 701

germplasm, which was natural screened for early blight resistance

under field condition in the year i.e. 2011-12. Selected 240 core set

lines again screened for disease reaction after artificial inoculation

under field conditions continuously for two years i.e. 2012-13 and

2013-14.

Similarity was observed between poly house and field

screenings of tomato recombinant inbreed lines for early blight

resistance in both the year 2012-13 and 2013-14. Field and poly

house evaluation was found to be useful for screening tomatoes for

EB resistance, so it may be employed to facilitate EB resistance

breeding. The AUDPC value of RILs in poly house condition was

observed less than the field study. Limited early blight resistance was

found in the recombinant inbred lines of tomato in F7 and F8

generation. Results from poly house experiment were correlated or

highly similar to those from the field, as judged by the significant

correlation between poly house and field data in both the years 2012-

13 and 2013-14.


~155~

Out of 447 SSR primers we have found out 25 informative SSR

markers screened in our polymorphic survey. Linkage analysis was

performed with simple interval mapping (SIM) to identify QTLs for EB

resistance. Twelve markers showed linkage to chromosomes 2, 4 and

6. The closest marker for the trait was SSR 608 that showed linkage

at 46.1 cM distance at chromosome 2 in close proximity to EBR gene

responsible for early blight disease resistance.

The mapping approaches revealed a possible epistatic

interaction between the QTLs identified, a significant additive gene

action was seen for the early blight resistance whereas, positive

dominance gene action resulted in only one QTL on chromosome 2,

the mean phenotypic variation was estimated up to 3 %. It would be

useful for breeders to make use of the QTL on chromosomes 2 and 6

(as they are effective in environment) by development of a population

of each containing parts of the S. harbochaites QTL regions in a

cultivated tomato background.


~156~

Conclusions

Based on above findings, following conclusions may be drawn:

1. Cultural media, pH and temperature have played an important

role for growth and sporulation of A. solani.

2. Grain based media is cost effective since it does not require use

of agar and chemicals that are costly and the technique is

simple, rapid and reliable. We are continuously exploring the

possibilities for inducing high sporulation in A. solani which is

an important pathogen in most of the vegetable crops. This

inoculum production technique can be easily adoptable to many

laboratories where facilities and resources are limited.

3. The droplet inoculation method in which leaflets of intact plants

are inoculated with droplets of A. solani conidial suspension

gave better results in comparison to other inoculation methods.

The considerable amount of time required to measure lesions

make this method less attractive for large scale screening. So,

the adoptability of spray inoculation method is more as

compared to droplet inoculation method.

4. Among grain based media sorghum was found best for long

period storage of A. solani as compared to other artificial media.

It’s easy, cheap and non chemical method for A. solani growth

and long time storage.

5. Similarity was observed between poly house and field

screenings of tomato recombinant inbreed lines for early blight

resistance. Field and poly house evaluation was found to be

useful for screening tomato genotypes and RILs for EB

resistance, so it may be employed to facilitate EB resistance

breeding.


~157~

6. Linkage analysis was performed with simple interval mapping

(SIM) and composite interval mapping to identify QTLs for EB

resistance. Twelve markers showed linkage to chromosomes 2,

4 and 6. The closest marker for the trait was SSR 605 that

showed linkage at 46.1 cM distance at chromosome 2 in close

proximity to EBR gene responsible for early blight disease

resistance.

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APPENDICES

Table 1: Mean weekly meteorological data during year 2012-13.

Week No.

Month & Date Rainfall

(mm)

Temperature (0C) RH (%) Sun-shine hours

Evaporation (mm) Max. Min. Max. Min.

1. June 11-17 0.0 42.6 30.6 42 22 8.5 9.7

2. June 18-24 69.3 34.3 27.1 79 62 1.8 5.0

3. June 25-1 14.0 40.8 29.5 54 33 5.0 7.3

4. July 02-08 83.3 33.4 27.9 81 66 4.5 5.9

5. July 09-15 70.0 33.7 27.4 85 68 3.3 3.7

6. July 16-22 105.2 32.3 26.2 87 73 2.6 4.4

7. July 23-29 43.4 32.1 26.9 89 81 4.4 3.1

8. July 30-05 57.8 32.2 26.5 89 74 3.5 3.6

9. August 06-12 38.4 32.0 26.2 87 76 5.6 4.1

10. August 13-19 16.4 32.2 29.1 86 74 3.5 3.8

11. August 20-26 30.9 32.3 26.1 87 73 4.0 3.7

12. August 27-02 16.7 34.3 27.8 89 68 7.0 4.4

13. September 03-09 18.0 31.4 25.9 89 75 4.9 3.7

14. September 10-16 112.2 29.6 24.9 94 82 2.5 3.2

15. September 17-23 9.5 32.1 25.4 90 68 4.4 3.5

16. September 24-30 0.0 33.6 24.0 82 54 9.0 3.6

17. October 01-07 6.4 33.7 24.0 85 37 7.2 3.6

18. October 08-14 0.0 31.6 21.6 86 56 8.2 3.6

19. October 15-21 0.0 31.8 19.9 85 52 7.9 2.5

20. October 22-28 0.0 30.4 17.5 84 54 8.0 2.4

21. October 29-04 0.0 29.4 16.3 87 49 6.7 2.2

22. Nov 12-18 0.0 27.9 13.1 90 57 7.0 1.7

23. Nov 19-25 0.0 27.0 11.4 90 61 7.6 1.7

24. Nov 26-02 0.0 25.9 10.7 87 35 7.9 1.8

25. Dec 03-09 0.0 25.9 9.8 79 27 8.5 2.1

26. Dec 10-16 0.0 22.1 14.1 89 67 3.7 1.6

27. Dec 17-23 0.0 21.5 10.7 81 45 6.5 1.8

28. Dec 24-31 0.0 18.8 6.8 91 53 3.8 1.1

29. Jan 1-07 0.0 17.1 7.5 89 53 4.3 1.3

30. Jan 08-14 0.0 24.3 12.1 83 47 6.4 1.7

31. Jan 15-21 0.0 24.3 12.1 83 47 4.9 1.6

32. Jan 22-28 0.0 19.3 5.8 89 50 6.8 1.4

33. Jan 29-04 0.0 24.0 9.1 87 45 1.4 6.2

34. Feb 05-11 0.0 23.7 11.1 88 53 6.7 2.2

35. Feb 12-18 65.2 23.8 12.9 89 64 4.7 2.2

36. Feb 19-25 1.6 23.4 15.2 92 60 7.6 2.2

37. Feb 26-04 0.0 26.9 13.4 82 44 9.9 3.4

38. March 05-11 0.0 30.0 14.8 74 37 9.8 3.2

39. March 12-18 3.6 31.3 17.7 73 38 8.8 4.4

40. March 19-25 0.0 33.6 18.5 64 30 9.3 4.4

41. March 26-01 6.0 33.3 18.6 65 35 7.8 4.7

Source: Meteorological observatory, Indian Institute of Vegetable Sciences, Varanasi.

Appendices

~ii~

Table 2: Mean weekly meteorological data during year 2013-14.

Week No.

Month & Date Rainfall (mm)

Temperature (0C) RH (%) Sun-

shine hours

Evaporation (mm)

Max. Min. Max. Min.

1. June 11-17 6.7 38.0 28.0 73 48 5.8 5.9

2. June 18-24 31.8 35.3 27.3 82 65 3.9 4.6

3. June 25-1 134.4 30.7 25.7 82 74 2.8 3.7

4. July 02-08 39.4 33.3 26.7 82 71 4.0 3.2

5. July 09-15 81.5 32.2 26.6 86 72 3.4 3.5

6. July 16-22 0.0 34.8 28.3 83 67 6.9 5.0

7. July 23-29 48.2 32.2 26.3 86 77 5.8 4.3

8. July 30-05 69.5 32.5 25.5 84 75 6.7 4.3

9. August 06-12 28.6 32.5 26.0 91 76 4.6 4.1

10. August 13-19 37.4 31.8 25.7 88 80 6.9 3.8

11. August 20-26 32.3 32.1 26.2 83 74 6.9 4.2

12. August 27-02 150.3 28.7 25.7 92 87 1.5 2.5

13. September 03-09 3.2 32.5 25.3 80 66 7.6 3.7

14. September 10-16 0.0 33.7 26.9 85 66 7.2 3.7

15. September 17-23 4.6 31.6 25.3 81 76 8.0 3.9

16. September 24-30 12.2 31.6 25.5 86 77 6.8 2.9

17. October 01-07 83.9 28.8 24.3 93 85 3.9 3.3

18. October 08-14 44.0 29.1 23.8 84 78 6.5 2.6

19. October 15-21 17.0 26.7 21.1 92 80 4.2 1.7

20. October 22-28 0.0 29.6 21.1 88 72 8.9 2.9

21. October 29-04 0.0 28.6 17.3 80 76 7.8 2.3

22. Nov 12-18 0.0 26.2 11.5 90 48 7.9 1.5

23. Nov 19-25 0.0 26.7 25.0 89 41 8.5 1.8

24. Nov 26-02 0.0 26.4 13.7 87 46 8.1 1.6

25. Dec 03-09 0.0 25.4 13.7 89 43 8.2 1.5

26. Dec 10-16 0.0 24.0 10.6 88 48 8.3 1.7

27. Dec 17-23 0.0 23.5 11.5 83 57 7.5 1.6

28. Dec 24-31 0.0 21.1 10.3 85 49 7.8 1.4

29. Jan 1-07 0.0 20.7 11.3 91 59 6.9 1.3

30. Jan 08-14 22.4 17.9 10.7 92 68 2.0 1.1

31. Jan 15-21 37.1 18.1 10.6 92 76 0.6 0.8

32. Jan 22-28 0.0 20.4 11.7 94 63 2.4 1.4

33. Jan 29-04 0.0 20.1 9.8 89 63 4.6 1.2

34. Feb 05-11 0.0 24.9 12.3 81 45 9.1 2.7

35. Feb 12-18 24.4 20.4 11.0 83 54 3.1 1.3

36. Feb 19-25 15.5 23.5 12.3 82 64 7.6 1.8

37. Feb 26-04 34.5 23.9 15.6 87 68 3.6 1.8

38. March 05-11 0.0 27.0 13.0 83 45 8.1 3.0

39. March 12-18 0.5 28.5 15.9 82 55 8.3 3.5

40. March 19-25 0.0 31.9 17.5 66 38 9.5 4.7

Source: Meteorological observatory, Indian Institute of Vegetable Sciences, Varanasi.

Appendices

~iii~

Table 3: Plant growth type, days to 50 percent flowering after

transplanting, days to fruit setting, days to senescence, AUDPC

and Host Reaction of 701 germplasm lines of tomato to A. solani infection under natural condition.

Sr. No. Name of lines PGT* DF* DFS* DSS* AUDPC* HR*

1 971384 D 30 12 29 520.20 MR

2 Ajeet T-2 D 31 15 29 1479.00 S

3 971753 D 32 14 25 642.60 MR

4 Angoorlata ID 30 11 23 122.40 R

5 Arka Alok D 30 12 35 183.60 R

6 971754 ID 29 12 34 224.40 R

7 Azad T-5 ID 22 12 21 224.40 R

8 Ageta-32 ID 23 14 19 122.40 R

9 BT-10 ID 21 13 30 102.00 R

10 B-9-2 ID 40 10 30 112.20 R

11 B-10-2 ID 23 9 26 183.60 R

12 Arka Saurabh D 24 12 21 1479.00 S

13 Arka Meghali D 33 13 32 438.60 R

14 B-7-1 ID 39 10 27 1234.20 S

15 B-7-2 ID 36 9 30 326.40 R

16 Arka Vikas D 19 12 31 1479.00 S

17 Azad T-6 D 28 11 32 1193.40 S

18 Azad T-5 ID 24 11 27 1269.90 S

19 B-9-3 D 31 11 34 346.80 R

20 BT-121 D 32 11 29 377.40 R

21 B-4-1 ID 36 14 29 550.80 MR

22 BT-120 D 31 12 25 132.60 R

23 BT-11 ID 36 10 23 173.40 R

24 Bhilai-2 D 34 9 35 122.40 R

25 B-5-1 ID 28 9 34 122.40 R

26 Avinash-2-2-1 ID 30 15 21 1142.40 S

27 Arka Abka ID 31 14 19 571.20 MR

28 B-9-1 D 40 15 30 693.60 MR

29 C-2-2 D 37 15 30 887.40 MS

30 C-14-1 D 43 13 26 1254.60 S

31 C-2-3 D 40 13 21 887.40 MS

32 C-3-2 ID 37 12 32 1116.90 MS

33 C-6-3 ID 41 14 27 887.40 MS

34 C-20-2 D 40 12 30 326.40 R

35 C-22-1 ID 37 15 31 887.40 MS

36 C-20-1 D 43 14 32 1142.40 S

37 C-15-1 ID 35 11 27 918.00 MS

38 C-2-1 D 28 12 34 459.00 MR

39 C-14-2 ID 35 12 24 260.10 R

40 C-10-3 ID 38 12 17 550.80 MR

41 BTH-9 ID 42 11 19 1264.80 S

42 C-1-1 ID 43 10 24 1254.60 S

43 C-11-4 ID 28 10 24 255.00 R

44 C-21-1 ID 40 9 28 663.00 MR

45 C-12-2 D 34 12 21 1071.00 MS

46 C-3-3 D 40 13 20 663.00 MR

47 C-12-1 D 37 10 26 1468.80 S

48 C-5-2 D 40 9 23 1254.60 S

49 C-10-1 ID 37 12 32 234.60 R

50 C-10-4 ID 40 11 29 214.20 R

51 C-10-2 ID 41 11 31 1254.60 S

52 C-11-1 ID 32 11 30 1254.60 S

53 C-11-3 D 39 11 27 550.80 MR

54 BT-1-36 D 35 14 33 448.80 MR

55 C-11-2 ID 27 12 27 459.00 MR

56 C-13-1 ID 35 10 27 1479.00 S

57 C-19-1 ID 31 9 18 1366.80 S

58 C-1-4 ID 42 9 29 132.60 R

59 C-4-1 D 38 15 23 132.60 R

Appendices

~iv~

60 C-6-2 D 28 14 28 275.40 R

61 C-7-1 D 38 15 23 224.40 R

62 CLN - 2116 D 43 11 26 1234.20 S

63 CLN-2011 ID 38 13 30 255.00 R

64 CLN-2366 D 40 13 20 438.60 R

65 C-7-2 ID 36 12 22 1234.20 S

66 C-9-1 ID 36 14 26 214.20 R

67 C-8-1 D 36 12 29 1234.20 S

68 C-9-2 ID 41 9 28 214.20 R

69 CHART-4 ID 39 9 30 438.60 R

70 CH-155 ID 28 13 26 132.60 R

71 CLN-2026-C D 28 10 26 1132.20 MS

72 C-8-2 D 40 9 32 326.40 R

73 CGT ID 36 12 21 346.80 R

74 DCT-2 ID 36 13 28 530.40 MR

75 D-7-1 D 35 10 17 244.80 R

76 Shrubby ID 23 9 24 244.80 R

77 D-5-2 ID 37 12 19 459.00 MR

78 D-7-2 D 35 11 18 1346.40 S

79 DMT-1 D 46 8 18 663.00 MR

80 D-1-2 D 43 9 18 1234.20 S

81 D-2-2 ID 30 11 30 663.00 MR

82 D-1-1 D 35 14 22 326.40 R

83 D-2-1 ID 35 12 19 438.60 R

84 D-2-3 D 35 10 23 1234.20 S

85 D-2-1-1 D 36 9 22 326.40 R

86 C-26-1 D 23 9 18 1224.00 S

87 C-14-4-2 ID 37 15 20 469.20 MR

88 D-3-1 ID 38 14 19 550.80 MR

89 D-3-2 D 20 15 18 550.80 MR

90 DARL-66 D 48 15 17 1132.20 MS

91 D-5-1 D 27 13 25 346.80 R

92 D-2-2-1 D 39 13 24 326.40 R

93 EC-13904 D 35 12 25 306.00 R

94 EC-538156 ID 26 14 19 1147.50 S

95 EC-12692 D 39 12 21 1234.20 S

96 EC-126902 ID 35 15 23 214.20 R

97 EC-15127 ID 35 14 26 234.60 R

98 EC-3526 D 42 11 24 326.40 R

99 EC-539450 D 25 12 23 1244.40 S

100 EC-141887 ID 38 12 25 234.60 R

101 EC-13736 ID 35 12 29 1132.20 MS

102 EC-10304 ID 39 14 25 214.20 R

103 EC-135580 ID 25 13 23 566.10 MR

104 E-4-3 D 40 10 23 265.20 R

105 E-2-1 ID 40 9 24 438.60 R

106 DT-10 ID 35 12 19 428.40 R

107 E-8-1 ID 39 13 20 1234.20 S

108 DVRT-2 ID 27 10 20 234.60 R

109 EC-3527828 ID 24 9 24 1479.00 S

110 EC-14078 ID 39 12 22 1264.80 S

111 E-6-2 D 38 11 27 561.00 MR

112 E-4-1 ID 27 11 21 1275.00 S

113 E-2-2 ID 36 11 25 540.60 MR

114 E-3-1 D 35 11 21 1244.40 S

115 E-5-1 D 40 14 22 459.00 MR

116 E-6-1 D 39 12 20 142.80 R

117 E-1-1 ID 36 10 28 591.60 MR

118 EC-35293 ID 39 9 23 1162.80 S

119 E-4-2 D 40 9 23 530.40 MR

120 EC-31761 ID 35 15 26 459.00 MR

121 EC-317-6 ID 35 14 27 408.00 R

122 EC- 2977 ID 39 15 25 459.00 MR

123 EC- 1914 D 26 15 27 591.60 MR

124 EC- 299335 ID 31 13 21 550.80 MR

125 EC- 27995 ID 42 12 22 530.40 MR

Appendices

~v~

126 EC- 168290 D 30 15 24 1448.40 S

127 EC- 322634 ID 36 14 19 510.00 MR

128 EC- 326106 D 25 11 23 520.20 MR

129 EC- 35322 D 27 12 20 428.40 R

130 EC- 326146 ID 37 12 26 214.20 R

131 EC- 381554 D 35 12 21 244.80 R

132 EC- 339066 D 38 14 22 459.00 MR

133 EC- 32933 ID 37 13 25 510.00 MR

134 EC- 339057-A D 39 10 22 693.60 MR

135 EC- 16788 ID 35 9 17 816.00 MS

136 EC- 2517 D 35 12 18 1101.60 MS

137 EC- 164660 ID 35 13 30 867.00 MS

138 EC- 251649 ID 35 10 27 550.80 MR

139 EC- 2798 ID 38 9 26 591.60 MR

140 EC- 501574 ID 46 12 27 693.60 MR

141 EC- 27251 ID 39 11 23 999.60 MS

142 EC- 170047 ID 40 11 24 1009.80 MS

143 EC- 29914 ID 35 11 30 428.40 R

144 EC- 273966 ID 23 11 30 122.40 R

145 EC- 25265 ID 39 14 18 142.80 R

146 EC- 501580 ID 20 12 28 428.40 R

147 EC- 50075 D 35 10 20 122.40 R

148 EC- 501577 ID 42 9 28 428.40 R

149 EC- 381263 ID 39 9 31 1448.40 S

150 EC- 501583 ID 41 15 33 285.60 R

151 EC- 501575 ID 40 14 28 418.20 R

152 EC- 362933 D 35 15 26 255.00 R

153 EC- 362942 ID 38 15 26 550.80 MR

154 EC- 177371 ID 26 13 24 357.00 R

155 EC- 366899 ID 38 13 32 357.00 R

156 EC- 362941 ID 27 12 25 550.80 MR

157 EC- 368883 ID 40 14 17 408.00 R

158 EC- 362948 ID 26 12 31 510.00 MR

159 EC- 251881 D 25 15 20 591.60 MR

160 EC- 2791 ID 21 14 30 612.00 MR

161 EC- 241446 D 40 11 20 663.00 MR

162 EC- 52077 ID 40 12 20 234.60 R

163 EC- 520046 D 35 12 22 1142.40 S

164 EC- 520053 ID 35 12 22 1254.60 S

165 EC- 501576 ID 31 11 27 1479.00 S

166 EC- 501061 ID 36 10 24 1208.70 S

167 EC- 521038 ID 35 10 24 1025.10 MS

168 EC- 521068 ID 35 9 20 122.40 R

169 EC- 501582 ID 42 12 29 1122.00 MS

170 EC- 520075 ID 35 13 32 1315.80 S

171 EC- 521071 ID 23 10 32 775.20 MR

172 EC- 521039 D 26 9 26 907.80 MS

173 EC- 521047 ID 38 12 19 1132.20 S

174 EC- 521055 ID 39 11 18 877.20 MS

175 EC- 521056 ID 28 11 21 805.80 MS

176 EC- 521069 ID 39 11 21 346.80 R

177 EC- 526139 ID 25 11 20 214.20 R

178 EC- 520059 ID 23 14 20 326.40 R

179 EC- 546727 ID 22 12 20 810.90 MS

180 EC- 538444 D 22 10 33 765.00 MR

181 EC- 538455 ID 26 9 27 816.00 MS

182 EC- 538439 ID 22 9 27 1132.20 MS

183 EC- 538156 ID 40 15 27 1254.60 S

184 EC- 568940 ID 26 14 20 346.80 R

185 EC- 538440 ID 27 15 18 535.50 MR

186 EC- 531805 D 22 11 23 428.40 R

187 EC- 538146 ID 26 13 32 224.40 R

188 EC- 538419 ID 24 13 33 1300.50 S

189 EC- 538405 D 20 12 21 1377.00 S

190 EC- 538380 ID 35 14 21 122.40 R

191 EC- 538411 D 21 12 28 596.70 MR

Appendices

~vi~

192 EC- 538423 D 21 9 30 566.10 MR

193 EC- 538148 ID 28 9 20 214.20 R

194 EC- 538373 ID 38 13 29 224.40 R

195 EC- 529080 ID 38 10 19 183.60 R

196 EC- 531803 ID 39 9 22 443.70 R

197 EC- 560340 ID 19 12 25 627.30 MR

198 EC- 531801 D 35 13 22 1392.30 S

199 EC- 529085 ID 35 10 30 1025.10 MS

200 EC- 529083 ID 27 9 27 550.80 MR

201 EC- 528374 D 25 12 27 198.90 R

202 EC- 538422 ID 26 11 24 571.20 MR

203 EC- 528362 ID 27 8 24 693.60 MR

204 EC- 528365 ID 27 9 29 1458.60 S

205 EC- 520061 ID 37 11 28 122.40 R

206 EC- 538155 D 35 14 32 428.40 R

207 EC- 552141 ID 28 12 17 244.80 R

208 EC- 620380 D 35 12 22 550.80 MR

209 EC- 620377 D 35 15 20 558.45 MR

210 EC- 620378 ID 38 14 24 326.40 R

211 EC- 620381 ID 35 11 27 540.60 MR

212 EC- 620383 ID 35 12 26 887.40 MS

213 EC- 620379 ID 35 12 28 1239.30 S

214 EC- 620-03 ID 26 12 27 234.60 R

215 EC- 620-3 ID 24 14 23 581.40 MR

216 EC- 568943 ID 35 13 22 214.20 R

217 EC- 578422 ID 37 10 28 438.60 R

218 EC- 620361 ID 39 9 26 418.20 R

219 EC- 610092 ID 26 12 29 367.20 R

220 EC- 538408 ID 37 13 20 244.80 R

221 EC- 57442 ID 35 10 27 520.20 MR

222 EC- 570019 ID 37 9 18 1438.20 S

223 EC- 620385 D 26 12 21 1387.20 S

224 EC- 605696 D 34 11 33 234.60 R

225 EC- 5863 ID 34 11 34 438.60 R

226 EC- 5888 ID 37 11 21 234.60 R

227 EC- 620374 ID 37 11 30 775.20 MS

228 EC- 620365 ID 22 14 22 1458.60 S

229 EC- 560262 D 25 12 31 775.20 MS

230 EC- 620363 D 23 10 21 652.80 MR

231 EC- 605694 D 24 9 26 438.60 R

232 EC- 620-1 ID 24 9 30 122.40 R

233 EC- 620376 ID 24 15 20 142.80 R

234 EC- 620371 ID 23 14 34 122.40 R

235 EC- 620369 ID 21 15 30 382.50 R

236 EC- 620370 ID 24 15 32 688.50 MR

237 EC- 620375 ID 24 13 21 887.40 MS

238 EC- 620368 ID 18 13 24 367.20 R

239 EC- 620367 D 19 12 27 459.00 MR

240 EC- 620373 ID 34 14 26 382.50 R

241 EC- 620-2 ID 38 12 28 316.20 R

242 EC- 605695 ID 24 15 30 550.80 MR

243 EC- 620386 D 23 14 30 489.60 MR

244 EC- 620395 ID 38 11 29 122.40 R

245 EC- 538138 ID 39 12 27 122.40 R

246 EC- 620366 ID 29 12 19 321.30 R

247 EC- 620404 ID 23 12 23 1086.30 MS

248 EC- 620415 ID 26 11 24 428.40 R

249 EC- 620403 ID 25 10 32 652.80 MR

250 EC- 620410 ID 28 10 32 535.50 MR

251 EC- 6202041 D 24 9 30 122.40 R

252 EC- 620398 D 30 12 31 413.10 R

253 EC- 620393 ID 27 13 28 765.00 MR

254 EC- 620409 ID 26 10 26 1315.80 S

255 EC- 620402 ID 24 9 25 887.40 MS

256 EC- 620412 D 34 12 20 397.80 R

257 EC- 620397 ID 28 11 32 290.70 R

Appendices

~vii~

258 EC- 570018 ID 24 11 26 999.60 MS

259 EC- 620416 ID 35 11 22 1346.40 S

260 EC- 620401 D 37 11 23 1009.80 MS

261 EC- 620400 ID 38 14 35 214.20 R

262 EC- 620391 ID 23 12 34 1178.10 S

263 EC- 620396 ID 35 10 21 775.20 MR

264 EC- 620408 ID 26 9 19 693.60 MR

265 EC- 620407 ID 35 9 30 351.90 R

266 EC- 620390 ID 25 15 30 535.50 MR

267 EC- 620411 ID 35 14 26 663.00 MR

268 EC- 620406 ID 37 15 21 229.50 R

269 EC- 620405 ID 36 11 32 550.80 MR

270 EC- 620413 D 38 13 27 122.40 R

271 EC- 620387 ID 39 13 30 122.40 R

272 EC- 570028 D 35 12 31 867.00 MS

273 EC- 620455 D 27 14 32 1479.00 S

274 EC- 620446 D 40 12 27 887.40 MS

275 EC- 620431 ID 40 9 34 877.20 MS

276 EC- 620453 D 35 9 29 663.00 MR

277 EC- 620456 ID 36 13 29 550.80 MR

278 EC- 620427 ID 26 10 25 571.20 MR

279 EC- 620443 ID 39 9 23 775.20 MR

280 EC- 620428 ID 37 12 35 504.90 MR

281 EC- 620442 ID 35 13 34 688.50 MR

282 EC- 620439 D 25 10 21 1458.60 S

283 EC- 620458 ID 35 9 19 765.00 MR

284 EC- 620459 D 36 12 30 1479.00 S

285 EC- 620414 ID 43 11 30 214.20 R

286 EC- 620-4 ID 42 8 26 224.40 R

287 EC- 620422 ID 39 9 21 474.30 MR

288 EC- 620440 ID 35 11 32 581.40 MR

289 EC- 620430 ID 38 14 27 1269.90 S

290 EC- 620434 ID 36 12 30 459.00 MR

291 EC- 620438 D 37 10 31 459.00 MR

292 EC- 620452 D 38 9 32 428.40 R

293 EC- 620421 ID 39 9 27 550.80 MR

294 EC- 620441 ID 35 15 34 214.20 R

295 EC- 620419 ID 38 14 24 673.20 MR

296 EC- 620429 ID 37 15 17 224.40 R

297 EC- 570028 ID 35 15 19 198.90 R

298 EC- 620385 ID 31 13 24 214.20 R

299 EC- 620417 ID 35 13 24 1193.40 S

300 EC- 620444 D 37 12 28 122.40 R

301 EC- 620448 ID 36 14 21 153.00 R

302 EC- 620045 ID 37 12 20 1208.70 S

303 EC- 620447 ID 35 15 26 346.80 R

304 EC- 620449 ID 37 14 23 367.20 R

305 EC- 620445 ID 32 11 32 1479.00 S

306 EC- 620424 ID 24 12 29 1448.40 S

307 EC- 620437 ID 39 12 31 367.20 R

308 EC- 620432 ID 29 12 30 224.40 R

309 EC- 620437 ID 40 14 27 1479.00 S

310 EC- 620426 ID 35 13 33 428.40 R

311 EC- 620573 ID 34 10 27 1147.50 S

312 EC- 620447 ID 40 9 27 428.40 R

313 EC- 620476 ID 39 12 18 1285.20 S

314 EC- 620467 D 39 13 29 1448.40 S

315 EC- 620468 D 40 10 23 877.20 MS

316 EC- 620463 ID 39 9 28 785.40 MR

317 EC- 620464 ID 37 12 23 642.60 MR

318 EC- 620470 ID 25 11 26 397.80 R

319 EC- 620469 D 36 11 30 663.00 MR

320 EC- 620465 ID 39 11 20 1387.20 S

321 EC- 620494 ID 23 11 22 994.50 MS

322 EC- 620471 ID 39 14 26 994.50 MS

323 EC- 620484 ID 34 12 29 627.30 MR

Appendices

~viii~

324 EC- 620498 ID 38 10 28 550.80 MR

325 EC- 620529 ID 38 9 30 1086.30 MS

326 EC- 620532 ID 39 9 26 550.80 MR

327 EC- 620518 ID 38 15 26 1422.90 S

328 EC- 620493 ID 40 14 32 1466.60 S

329 EC- 620480 D 34 15 21 1479.00 S

330 EC- 620478 ID 38 15 28 550.80 MR

331 EC- 620485 ID 38 13 17 688.50 MR

332 EC- 620460 ID 38 12 24 1239.30 S

333 EC- 620-5 ID 34 15 19 336.60 R

334 EC- 620463 ID 39 14 18 688.50 MR

335 EC- 620474 D 39 11 18 1147.50 S

336 EC- 620466 ID 37 12 18 326.40 R

337 EC- 620531 ID 39 12 30 688.50 MR

338 EC- 620539 ID 34 12 22 1454.20 S

339 EC- 620527 ID 39 14 19 214.20 R

340 EC- 620521 ID 38 13 23 1208.70 S

341 EC- 620522 ID 39 10 22 224.40 R

342 EC- 620519 ID 38 9 18 520.20 MR

343 EC- 620525 ID 37 12 20 775.20 MR

344 EC- 620515 ID 38 13 19 459.00 MR

345 EC- 620530 ID 39 10 18 1448.40 S

346 EC- 620502 ID 35 9 17 428.40 R

347 EC- 620520 ID 38 12 25 907.80 MS

348 EC- 620513 ID 34 11 24 1009.80 MS

349 EC- 620564 ID 34 11 25 397.80 R

350 EC- 620571 D 39 11 19 550.80 MR

351 EC- 620538 ID 34 11 21 1448.40 S

352 EC- 620573 ID 37 14 23 1285.20 S

353 EC- 620533 D 24 12 26 1300.50 S

354 EC- 620534 ID 34 10 24 688.50 MR

355 EC- 620536 ID 34 9 23 1040.40 MS

356 EC- 620517 ID 33 9 25 1479.00 S

357 EC- 620516 ID 32 15 29 688.50 MR

358 EC- 620523 ID 25 14 25 1300.50 S

359 EC- 620514 D 24 15 23 438.60 R

360 EC- 620535 ID 39 15 23 550.80 MR

361 EC- 620570 ID 34 13 24 244.80 R

362 EC- 621667-B ID 28 13 19 918.00 MS

363 EC- 620569 ID 38 12 20 1208.70 S

364 EC- 620568 ID 40 14 20 550.80 MR

365 EC- 620559 ID 39 12 24 688.50 MR

366 EC- 620558 ID 38 15 22 504.90 MR

367 EC- 620560 ID 28 14 27 275.40 R

368 EC- 620552 ID 32 11 21 1448.40 S

369 EC- 620561 ID 37 12 25 1479.00 S

370 EC- 620574 ID 34 12 21 688.50 MR

371 EC- 620565 ID 38 12 22 994.50 MS

372 EC- 620554 ID 39 11 20 346.80 R

373 F-2-4 ID 28 10 28 1009.80 MS

374 EC- 620505 ID 41 10 23 1132.20 MS

375 EC- 620507 ID 36 9 23 382.50 R

376 EC- 620462 ID 32 12 26 1479.00 S

377 EC- 620478 ID 33 13 27 688.50 MR

378 EC- 620481 ID 33 10 25 612.00 MR

379 EC- 620492 ID 30 9 27 688.50 MR

380 EC- 620491 ID 24 12 21 887.40 MS

381 EC- 620597 D 32 11 22 1479.00 S

382 EC- 620598 D 34 11 24 688.50 MR

383 EC- 620555 ID 25 11 19 1361.70 S

384 EC- 620540 ID 35 11 23 902.70 MS

385 EC- 620548 ID 23 14 20 1361.70 S

386 EC- 620510 ID 32 12 26 1448.40 S

387 EC- 620509 ID 35 10 21 1422.90 S

388 EC- 620511 ID 43 9 22 550.80 MR

389 EC- 620508 ID 33 9 25 561.00 MR

Appendices

~ix~

390 EC- 620575 ID 33 15 22 443.70 R

391 EC- 620512 ID 41 14 17 326.40 R

392 EC- 620551 ID 42 11 18 1208.70 S

393 EC- 620544 ID 35 11 30 688.50 MR

394 EC- 620648 ID 31 13 27 1361.70 S

395 EC- 620563 ID 39 13 26 571.20 MR

396 EC- 620567 ID 37 12 27 1392.30 S

397 EC- 620442 ID 35 14 23 1331.10 S

398 EC- 625651 ID 30 12 24 224.40 R

399 EC- 620495 ID 36 9 30 1208.70 S

400 EC- 620488 ID 31 9 30 1392.30 S

401 EC- 620500 ID 25 13 18 688.50 MR

402 EC- 620486 ID 26 10 28 1448.40 S

403 EC- 620504 ID 23 9 20 688.50 MR

404 EC- 620553 ID 34 12 28 1479.00 S

405 EC- 620501 ID 17 13 31 688.50 MR

406 EC- 620549 ID 35 10 33 1269.90 S

407 EC- 620556 D 34 9 28 1392.30 S

408 EC- 620541 ID 33 12 26 1468.80 S

409 EC- 620545 D 33 11 26 132.60 R

410 EC- 625665 ID 37 8 24 1142.40 S

411 EC- 625657 ID 37 9 32 734.40 MR

412 F-2-2 ID 32 11 25 214.20 R

413 EC- 625642 ID 41 14 17 1208.70 S

414 EC- 624077 ID 36 12 31 224.40 R

415 EC- 625644 ID 31 12 20 1132.20 MS

416 EC- 625652 ID 32 15 30 234.60 R

417 EC- 63531 ID 34 14 20 688.50 MR

418 EC- 6004 ID 24 11 20 612.00 MR

419 EC- 6486 ID 37 12 22 504.90 MR

420 EC- 625645 ID 40 12 22 306.00 R

421 EC- 625643 ID 39 12 27 459.00 MR

422 EC- 9046 ID 34 14 24 688.50 MR

423 F-1-1 ID 24 13 24 663.00 MR

424 F-5020 ID 25 10 20 877.20 MS

425 EC-2822 D 35 9 29 663.00 MR

426 EC-8591 ID 18 12 32 561.00 MR

427 EC-625660 ID 36 13 32 1040.40 MS

428 F-3-1 D 23 10 26 688.50 MR

429 EC-625647 ID 37 9 19 1422.90 S

430 EC-625-486 ID 19 12 18 367.20 R

431 EC-7912 ID 31 11 21 1101.60 MS

432 EC-625653 D 32 11 21 561.00 MR

433 F-1-2 ID 23 11 20 1300.50 S

434 EC-625658 ID 35 11 20 321.30 R

435 EC-6792 ID 34 14 20 1116.90 MS

436 F-9-2 ID 37 12 33 561.00 MR

437 F-8-2 ID 42 10 27 428.40 R

438 Flora date ID 40 9 27 459.00 MR

439 FLA-7421 ID 31 9 27 673.20 MR

440 G-1-1 ID 38 15 20 183.60 R

441 G-2-2 D 40 14 18 1366.80 S

442 F-8-4 ID 31 15 23 326.40 R

443 G-1-2 ID 33 15 32 561.00 MR

444 F-7-3 ID 31 13 33 510.00 MR

445 F-9-1 ID 37 13 21 1366.80 S

446 G-2-1 ID 39 12 21 550.80 MR

447 F-4-1 ID 31 14 28 1458.60 S

448 EC-62-5659 ID 32 12 30 1458.60 S

449 Feb.-02 D 38 15 20 336.60 R

450 F-8-5 ID 33 14 29 1458.60 S

451 FLA-7171 ID 28 11 19 112.20 R

452 F-6022 ID 30 12 22 688.50 MR

453 F-7012 ID 31 12 25 1479.00 S

454 F-7028 ID 32 12 22 112.20 R

455 F-6-4 ID 33 11 30 571.20 MR

Appendices

~x~

456 F-6-3 ID 33 10 27 795.60 MS

457 F-7-1 ID 39 10 27 688.50 MR

458 F-6050-1 ID 29 9 24 1479.00 S

459 F-7-4 ID 26 12 24 1458.60 S

460 F-8-2 ID 36 13 29 214.20 R

461 G-2-3 ID 37 10 28 612.00 MR

462 F-6-1 ID 38 9 32 224.40 R

463 G-3-1 ID 37 12 17 719.10 MR

464 G-4-1 ID 33 11 22 428.40 R

465 F-8-1 ID 31 11 20 459.00 MR

466 F-6059 ID 37 11 24 571.20 MR

467 JT-3 ID 31 11 27 703.80 MR

468 Indam-2105-4 ID 33 14 26 214.20 R

469 IC-427006 ID 31 12 28 224.40 R

470 IC-373378 ID 33 10 27 459.00 MR

471 F-6-2 ID 32 9 23 994.50 MS

472 Feb.- 04 ID 33 9 22 290.70 R

473 F-7-2 ID 23 15 28 535.50 MR

474 F-7025 ID 33 14 26 703.80 MR

475 G-4-4 ID 36 15 29 428.40 R

476 G-5-3 ID 37 11 20 408.00 R

477 G-5-2 ID 36 13 27 367.20 R

478 HATH-8 ID 33 13 18 489.60 MR

479 HATH-T-4 ID 34 12 21 1142.40 S

480 GT-1 ID 38 14 33 183.60 R

481 GT-12 ID 37 12 34 397.80 R

482 G-4-3 ID 36 9 21 122.40 R

483 H-2-2 ID 33 9 30 459.00 MR

484 H-1-4 ID 39 13 22 214.20 R

485 G-5-1 ID 28 10 31 265.20 R

486 G-4-5 ID 38 9 21 326.40 R

487 G-5-4 D 39 12 26 775.20 MR

488 G-7-1 ID 35 13 30 1009.80 MS

489 GT-2 ID 33 10 20 459.00 MR

490 G-6-3 ID 38 9 34 438.60 R

491 H-1-1 ID 35 12 30 775.20 MR

492 G-7-2 ID 36 11 32 775.20 MR

493 G-4-2 ID 36 8 21 673.20 MR

494 G-6-1 ID 35 9 24 734.40 MR

495 H-88-78-2 ID 43 11 27 142.80 R

496 H-1-3 D 37 14 26 122.40 R

497 H-1-2 ID 33 12 28 224.40 R

498 H-88-78-1 D 33 10 30 224.40 R

499 H-2-1 ID 33 9 30 1009.80 MS

500 H-3-1 ID 33 9 29 795.60 MS

501 H-88-78-5 ID 32 15 27 897.60 MS

502 H-24 ID 35 14 19 489.60 MR

503 H-23 ID 33 15 23 887.40 MS

504 IC-447708 ID 32 15 24 382.50 R

505 LH-301-SPS ID 19 13 32 1009.80 MS

506 IC-439542 ID 35 13 32 224.40 R

507 Kajla bulk ID 33 12 30 244.80 R

508 I-4-4 ID 32 14 31 448.80 MR

509 Indam-2102-1-7 ID 33 12 28 306.00 R

510 I-1-4 ID 38 15 26 581.40 MR

511 I-1-2 ID 36 14 25 663.00 MR

512 I-4-3 ID 37 11 20 642.60 MR

513 I-4-2 ID 40 12 32 1009.80 MS

514 HATH-8 ID 41 12 26 1009.80 MS

515 I-4-8 ID 35 12 22 999.60 MS

516 Hisar Arun ID 38 14 23 571.20 MR

517 I-181 ID 33 13 35 775.20 MR

518 I-2-1 ID 39 10 34 887.40 MS

519 I-2-2 D 37 9 21 244.80 R

520 I-1-1 ID 36 12 19 504.90 MR

521 IC-469517 ID 35 13 30 1479.00 S

Appendices

~xi~

522 Hisar Lalit ID 33 10 30 963.90 MS

523 I-1-3 ID 32 9 26 663.00 MR

524 Kashi Hemant ID 26 12 21 1178.10 S

525 Kashi Vishesh ID 35 11 32 1269.90 S

526 KS-16 ID 36 11 27 897.60 MS

527 Kasmiriya ID 19 11 30 999.60 MS

528 LA-3997 ID 35 11 31 1009.80 MS

529 LA-3957 ID 33 14 32 688.50 MR

530 Kal. type-1 ID 32 12 27 907.80 MS

531 LA-3772 ID 33 10 34 907.80 MS

532 NDTVR-60 ID 39 9 29 413.10 R

533 NDT-4 ID 39 9 29 897.60 MS

534 Indam-2103-6 ID 33 15 25 183.60 R

535 IC-469626 ID 21 14 23 663.00 MR

536 Indam-2103-1-1 ID 38 15 35 887.40 MS

537 IIHR-2202 ID 39 15 34 979.20 MS

538 IIHR-1 ID 33 13 21 1025.10 MS

539 Indam-2103-6-4 ID 32 12 19 795.60 MS

540 IC-469603 ID 33 15 30 887.40 MS

541 Indam-2103-1-1 ID 38 14 30 867.00 MS

542 Indam-2102 ID 26 11 26 867.00 MS

543 NDTVR-73 ID 39 12 21 663.00 MR

544 NDT-8 D 39 12 32 663.00 MR

545 I-4-5 D 33 12 27 260.10 R

546 NF-21-5B-8 ID 35 14 30 683.40 MR

547 Indam-2103 ID 33 13 31 785.40 MS

548 NDT-3 ID 32 10 32 1009.80 MS

549 N-2-1 D 33 9 27 933.30 MS

550 M-4-2 D 33 12 34 887.40 MS

551 M-4-3 ID 32 13 24 535.50 MR

552 Nandhi D 32 10 17 566.10 MR

553 N-2-3 ID 36 9 19 673.20 MR

554 N-2-2 D 33 12 24 550.80 MR

555 M-4-1 D 34 11 24 663.00 MR

556 NDT-1 ID 33 11 28 673.20 MR

557 M-3-2 D 33 11 21 1448.40 S

558 M-3-1 D 40 11 20 887.40 MS

559 M-2-3 D 27 14 26 688.50 MR

560 MUKTI ID 26 12 23 688.50 MR

561 M-1-4 D 33 10 32 775.20 MR

562 M-2-4 ID 31 9 29 887.40 MS

563 M-1-1 D 33 9 31 989.40 MS

564 M-1-3 D 33 15 30 1009.80 MS

565 M-1-2 ID 36 14 27 887.40 MS

566 Bulk-Sel-7-6 ID 38 15 33 775.20 MR

567 DVRT-20 ID 32 15 27 775.20 MR

568 WIR-13706 ID 31 13 27 571.20 MR

569 WIR-13717 D 31 13 18 469.20 MR

570 EC-538380 D 32 12 29 652.80 MR

571 EC-538381 D 36 14 23 540.60 MR

572 VRT-32-1 D 35 12 28 540.60 MR

573 WIR-4360 D 35 15 23 1458.60 S

574 WIR-3928 ID 26 14 26 663.00 MR

575 EC-1161-4-2-1 ID 25 11 30 785.40 MR

576 WIR-3996 ID 26 12 20 897.60 MS

577 WIR-4361 D 21 12 22 999.60 MS

578 DT-10 D 33 12 26 1458.60 S

579 PDVR-14 D 29 11 29 214.20 R

580 LB-292 D 25 10 28 1152.60 S

581 F-1150-P-60 ID 27 10 30 1009.80 MS

582 PDT-3-1 ID 26 9 26 887.40 MS

583 EC-521082 ID 24 12 26 897.60 MS

584 EC-520044 ID 26 13 32 1162.80 S

585 H-86 ID 32 10 21 1173.00 S

586 EC-520074 ID 33 9 28 887.40 MS

587 49-C-3 ID 34 12 17 856.80 MS

Appendices

~xii~

588 LB-283 ID 33 11 24 663.00 MR

589 50-A-2 ID 34 11 19 785.40 MR

590 152-B-1 ID 25 11 18 1009.80 MS

591 LB-209 ID 23 11 18 1009.80 MS

592 105-A-4 ID 34 14 18 887.40 MS

593 LB-286 ID 34 12 30 775.20 MR

594 155-B-1 ID 37 10 22 663.00 MR

595 110-1 ID 36 9 19 663.00 MR

596 156-1 ID 32 9 23 1346.40 S

597 40-A-1 D 32 15 22 1346.40 S

598 71-1 D 28 14 18 703.80 MR

599 49-B-1 D 21 15 20 897.60 MS

600 175-B-1 D 23 11 19 897.60 MS

601 27-D D 23 13 18 887.40 MS

602 28-Feb D 32 13 17 1152.60 S

603 142-A-3 ID 33 12 25 1132.20 MS

604 55-A-2 ID 35 14 24 1244.40 S

605 69-B-1 ID 37 12 25 112.20 R

606 61-4 ID 42 9 19 234.60 R

607 125-2 D 41 9 21 887.40 MS

608 59-B-2 D 26 13 23 775.20 MR

609 59-B-3 D 33 10 26 887.40 MS

610 174-A-1 D 35 9 24 775.20 MR

611 140-1 ID 35 12 23 683.40 MR

612 138-3 ID 36 13 25 683.40 MR

613 136-1 ID 35 10 29 1009.80 MS

614 28-4. ID 41 9 25 948.60 MS

615 PDT-3-1 D 31 12 23 306.00 R

616 PB-Chuhara D 32 11 23 1458.60 S

617 PB- Kesari D 32 8 24 688.50 MR

618 Parul D 31 9 19 1239.30 S

619 PBC-2010 D 32 11 20 550.80 MR

620 Pant T-3 D 25 14 20 367.20 R

621 Pant T-5 ID 26 12 24 688.50 MR

622 Pusa Hybrid-2 D 29 12 22 688.50 MR

623 Punjab Upma D 31 15 27 688.50 MR

624 Pusa Hybrid-2 D 30 14 21 688.50 MR

625 PT-11 ID 32 11 25 897.60 MS

626 Prestige ID 31 12 21 688.50 MR

627 Punjab BB-2 ID 30 12 22 775.20 MR

628 Pusa Hybrid -4 D 32 12 20 1208.70 S

629 Pusa Gaurav D 31 14 28 688.50 MR

630 PDT-3-1-1 D 32 13 23 627.30 MR

631 Punjab B-1 D 38 10 23 1183.20 S

632 S-Laima D 31 9 26 1208.70 S

633 Utkal Pallavi D 31 12 27 1055.70 MS

634 S. Gola D 31 13 25 948.60 MS

635 Sel.-18 D 34 10 27 673.20 MR

636 S. Sampada D 30 11 21 775.20 MR

637 SB-15 ID 27 12 22 673.20 MR

638 VRT-2 ID 31 11 24 877.20 MS

639 X-33 D 32 11 19 867.00 MS

640 EC-620550 D 31 11 23 382.50 R

641 ST-4 D 32 11 20 719.10 MR

642 Swarna Naveen D 32 14 26 448.80 R

643 Swarna Vaibhav D 19 12 21 459.00 MR

644 Swarna Lalima D 25 10 22 887.40 MS

645 Sioux D 32 9 25 877.20 MS

646 Roma ID 31 9 22 897.60 MS

647 Sel.-12 ID 31 15 17 897.60 MS

648 C-22-2 ID 30 14 18 1147.50 S

649 Swarna Samridi ID 32 15 30 183.60 R

650 EC-620354 D 32 15 27 1451.30 S

651 Sankranti D 33 13 26 346.80 R

652 WIR-13706 ID 33 13 27 1468.80 S

653 PT-20-1 ID 32 12 23 550.80 MR

Appendices

~xiii~

654 Utkal Raja ID 33 14 24 260.10 R

655 BT-20-2-1 ID 31 12 30 581.40 MR

656 DVRT-12 D 32 15 30 438.60 R

657 Type Y-1 D 31 14 18 438.60 R

658 Utkal Pragyan D 33 11 28 224.40 R

659 TLS-27 D 26 12 20 933.30 MS

660 EC-168283 D 33 12 28 448.80 MR

661 VRT-32-1 D 33 12 31 571.20 MR

662 WIR-3957 ID 33 11 33 504.90 MR

663 WIR-13708 ID 26 10 28 897.60 MS

664 U-301 ID 23 10 26 688.50 MR

665 T-1-1 ID 35 9 26 887.40 MS

666 TLBR-6 ID 34 12 24 918.00 MS

667 Utkal Urvashi ID 34 13 32 999.60 MS

668 TLC-1 ID 33 10 25 963.90 MS

669 TLS-30 ID 32 9 16 877.20 MS

670 TLBR-3 D 33 12 31 877.20 MS

671 TLH-17 D 33 11 20 933.30 MS

672 BL-120 ID 27 11 30 571.20 MR

673 F-2-1 ID 36 11 20 663.00 MR

674 EC-501576 D 32 11 20 663.00 MR

675 EC-528372 D 32 14 22 571.20 MR

676 H-88-78-3 D 32 12 22 438.60 R

677 EC-6552-1 D 29 10 27 550.80 MR

678 CO-3 D 32 9 24 1458.60 S

679 H-88-74-4 D 29 9 24 448.80 MR

680 DVRT-14 D 33 16 20 443.70 R

681 EC-570018 D 33 14 29 805.80 MS

682 F-7025 D 23 15 32 1009.80 MS

683 Pusa Ruby D 25 11 32 1142.40 S

684 Pusa - 120 D 23 13 26 1458.60 S

685 EC- 570028 D 24 13 19 1331.10 S

686 M-1-2 D 18 12 18 1142.40 S

687 Swarna Gola D 19 14 21 214.20 R

688 EC-620545 ID 22 12 21 275.40 R

689 PS-1 ID 21 9 20 1483.00 S

690 CHART-4 ID 21 9 20 571.20 MR

691 BL-1207 ID 23 13 20 550.80 MR

692 PDT-3-1 ID 25 10 33 1142.40 S

693 Utkal Pallavi ID 22 9 27 1254.60 S

694 Prestige ID 22 12 27 1234.20 S

695 EC-538138 D 23 13 27 489.60 MR

696 Money Maker D 23 10 20 326.40 R

697 Utkal Raja D 27 9 18 326.40 R

698 EC-620366 D 26 12 23 1234.20 S

699 EC-120573 ID 18 11 32 663.00 MR

700 EC-620385 D 18 12 33 397.80 R

701 I-4-5 D 25 9 21 489.60 MR

Maximum (PS-1) ID 21 8 20 1483.00 S

Minimum (BT-10) ID 21 13 30 102.00 R

CV % 19.293

LSD (1 %) 345.25

PGT=Plant Growth Type; DF=Days of Flowering; DFS= Days of Fruit Setting (After flowering); DSS= Days of senescence Stage (After fruit setting); AUDPC= Area Under Disease Progressive Curve; HR=Host

Reaction.

Appendices

~xiv~

Table 4: Concentration of DNA samples isolated from tomato RILs (Co-3 × EC-520061) as estimated using nanophotometer at factor 50.

Sample ID Name of DNA

samples Conc. (ng/µl) A260 A280 260/280 260/230

1 W- EB-1 2167.50 43.349 20.680 2.10 2.05

2 W- E-3-1 1222.90 24.459 11.603 2.11 2.14

3 W- E-3-2 2102.80 42.055 19.722 2.13 2.06

4 W- 10-B-3 938.70 18.774 9.116 2.06 2.01

5 W-10-EB 1293.70 25.874 12.484 2.07 2.05

6 W- 16-A 2770.90 55.418 27.323 2.03 1.45

7 W- 17 2031.00 40.620 20.189 2.01 1.75

8 W- 17-1 2504.60 50.091 25.209 1.99 1.55

9 W- 19-C-1 2335.70 46.714 21.632 2.16 1.88

10 W- 24-3 1779.20 35.584 18.612 1.91 1.51

11 W- 26 2435.90 48.719 23.734 2.05 1.83

12 W- 27-1 2013.70 40.275 21.476 1.88 1.48

13 W- 27-C-1 3514.50 70.290 32.215 2.18 1.97

14 W- 27-C-2 2107.00 42.139 20.091 2.10 1.98

15 W- 27-D-1 1603.00 32.060 17.712 1.81 1.65

16 W- 27-EB 1044.10 20.883 10.870 1.92 1.75

17 W- 28-2 1357.10 27.142 12.894 2.11 1.61

18 W- 28-4 1959.60 39.192 18.094 2.17 1.95

19 W- 30-1 1092.90 21.859 10.201 2.14 1.67

20 W- 35-1 1198.50 23.969 11.218 2.14 2.01

21 W- 35-2 332.90 6.659 3.321 2.00 1.32

22 W- 35-3 1256.50 25.130 11.701 2.15 1.91

23 W- 38-2 837.70 16.754 7.930 2.11 1.92

24 W- 40-A 1377.70 27.554 13.264 2.08 1.96

25 W- 40-A-1 652.20 13.043 6.162 2.12 1.95

26 W- 40-B-1 2028.70 40.573 18.592 2.18 2.02

27 W- 40-B-4 853.10 17.062 7.982 2.14 1.84

28 W- 41-1 2334.60 46.693 21.842 2.14 1.89

29 W- 44-B 1325.60 26.512 12.414 2.14 1.90

30 W- 46-B-1 1158.10 23.162 10.685 2.17 1.73

31 W- 47-1 1912.40 38.248 17.753 2.15 1.80

32 W- 47-2 658.50 13.169 6.825 1.93 1.79

33 W- 48-1 472.70 9.453 4.486 2.11 2.05

34 W- 48-2 301.30 6.026 2.881 2.09 2.02

35 W- 49-B-1 340.60 6.812 3.239 2.10 1.99

36 W- 49-C-3 319.10 6.383 2.844 2.24 1.96

37 W- 50-3 399.80 7.996 3.833 2.09 2.14

38 W- 50A-2 329.80 6.595 3.106 2.12 1.85

39 W- 53-A-3 316.20 6.325 2.986 2.12 1.84

40 W- 55-A-1 798.30 15.966 7.684 2.08 1.99

41 W- 55-A-2 686.90 13.737 6.603 2.08 1.79

42 W- 55-B-1 958.70 19.175 9.488 2.02 2.02

43 W- 59-B-1 595.50 11.910 5.750 2.07 2.01

44 W- 61-4 700.80 14.015 7.109 1.97 1.86

45 W- 59-B-3 776.70 15.534 7.734 2.01 2.09

46 W- 75-1 529.90 10.598 5.120 2.07 2.05

47 W- 60-EB 440.80 8.817 4.175 2.11 1.96

48 W- 71-1 225.40 4.509 2.120 2.13 1.89

49 W- 84-7 283.30 5.665 2.666 2.13 1.82

50 W- 84-EB 381.10 7.623 3.615 2.11 1.81

51 W- 82-B-2 402.30 8.046 3.816 2.11 2.03

52 W- 85-1 546.30 10.926 5.173 2.11 2.06

53 W- 82-1-1 406.50 8.130 3.927 2.07 2.01

54 W- 61-3 1064.70 21.293 9.994 2.13 1.86

55 W- 69-B-2 800.90 16.017 7.709 2.08 1.95

56 W- 69-B-1 814.80 16.295 7.858 2.07 1.79

57 W- 82-B-3 221.60 4.432 2.095 2.12 1.96

58 W- 137-2 414.70 8.294 3.900 2.13 1.87

59 W- 125-3 339.70 6.794 3.216 2.11 1.88

Appendices

~xv~

60 W- 110-1 596.60 11.931 5.708 2.09 2.03

61 W- 125-2 432.30 8.646 4.080 2.12 1.87

62 W- 114-B-1 684.90 13.699 6.431 2.13 1.70

63 W- 108-H-3 489.30 9.786 4.589 2.13 1.65

64 W- 88-2 528.20 10.564 5.090 2.08 1.98

65 W-95-B-2 424.10 8.481 3.966 2.14 1.60

66 W- 95-B-3 477.20 9.543 4.504 2.12 1.96

67 W- 105-A-4 474.80 9.495 4.494 2.11 1.98

68 W- 101-1 663.30 13.265 6.445 2.06 1.91

69 W- 90-2 388.30 7.765 3.650 2.13 1.99

70 W- 152-B-1 1718.50 34.370 16.209 2.12 1.96

71 W- 142-A-3 1132.20 22.644 10.796 2.10 2.08

72 W- 143-B-2 1165.60 23.311 11.015 2.12 2.08

73 W- 144-A-1 346.40 6.927 3.281 2.11 1.86

74 W- 144-B-1 910.20 18.203 8.558 2.13 2.06

75 W- 143-1-1 696.30 13.927 6.899 2.02 2.03

76 W- 142-B-1 1159.60 23.192 10.975 2.11 2.02

77 W- 146-B-2 368.30 7.367 3.487 2.11 1.78

78 W- 146-1-1 1415.80 28.315 13.379 2.12 2.00

79 W- 151-C-2 1213.70 24.274 11.488 2.11 1.98

80 W- 151 1780.50 35.611 16.843 2.11 1.93

81 W- 152-A-2 853.20 17.065 8.334 2.05 2.02

82 W- 148-1-1 653.90 13.078 6.291 2.08 1.80

83 W- 136-1 431.70 8.633 4.050 2.13 1.76

84 W- 137-6-1 494.50 9.890 4.710 2.10 1.96

85 W- 138-2 452.70 9.054 4.327 2.09 2.06

86 W- 138-3 976.50 19.530 8.978 2.18 1.75

87 W- 137-B-3 693.40 13.868 6.542 2.12 2.11

88 W- 140-1 756.30 15.125 7.081 2.14 1.97

89 W- 141-A-2 828.20 16.564 7.825 2.12 1.95

90 W- 141-6 531.80 10.635 5.091 2.09 2.12

91 W- 109-B-1 822.20 16.444 7.772 2.12 1.86

92 W- 203-1 1464.30 29.286 13.420 2.18 1.66

93 W- 201-B-1 818.40 16.368 7.810 2.10 1.67

94 W- 197-A-2 468.60 9.372 4.521 2.07 1.90

95 W- 201-A-4 1070.70 21.414 9.982 2.15 1.88

96 W- 188-B 579.70 11.595 5.840 1.99 1.70

97 W- 183-B-2 576.10 11.522 5.459 2.11 1.84

98 W- 175-B-1 1582.20 31.644 14.756 2.14 1.98

99 W- 184-4 1676.00 33.519 15.739 2.13 2.05

100 W- 202-1 583.00 11.661 5.526 2.11 1.53

101 W- 154-2 642.70 12.853 6.071 2.12 1.86

102 W- 174-B-2 1190.70 23.813 11.330 2.10 1.57

103 W- 156-1 759.10 15.181 7.444 2.04 1.57

104 W- 155-B-1 972.70 19.454 9.230 2.11 1.85

105 W- 177-B-2 329.00 6.580 3.198 2.06 1.86

106 W- 197-A-1 377.20 7.543 3.554 2.12 1.88

107 W- 242-1 1088.30 21.767 10.297 2.11 1.98

108 W- 216-1 936.70 18.733 8.687 2.16 1.59

109 W- 215-B-2 1334.40 26.688 12.550 2.13 1.41

110 W- 214-1 548.20 10.963 5.344 2.05 2.04

111 W- 213-3 1165.60 23.311 11.015 2.12 2.08

112 W- 207-A-3 1313.90 26.279 12.461 2.11 1.71

113 W- 247-A-1 2077.50 41.549 19.318 2.15 1.72

114 W- 230-2 1392.70 27.853 13.221 2.11 2.16

115 W- 247-A-3 865.10 17.303 8.175 2.12 1.96

116 W- 247-B-1 1232.40 24.647 11.265 2.19 1.53

117 W- 250-1 1346.70 26.934 12.402 2.17 1.62

118 W- 259-B-1 1175.20 23.503 11.040 2.13 1.97

119 W- 250-2 968.60 19.372 8.963 2.16 1.82

120 W- 258-1 1890.30 37.807 17.786 2.13 2.03

121 W- 274-A 1539.40 30.788 14.756 2.09 1.90

122 W- 258-2 433.40 8.668 4.066 2.13 1.81

123 W- 284-4 71.70 1.434 0.607 2.36 2.11

124 W- 259-E-1 532.00 10.640 5.032 2.11 2.02

125 W- 242-2 852.30 17.045 8.237 2.07 2.00

Appendices

~xvi~

126 W- 226-1 521.50 10.431 4.754 2.19 1.52

127 W- 216-2 693.20 13.864 6.694 2.07 1.66

128 W- 228 751.50 15.030 7.341 2.05 1.79

129 W- 216-3 1592.90 31.858 15.032 2.12 1.78

130 W- 225 1110.60 22.212 10.565 2.10 1.89

131 W- 312 764.00 15.280 7.444 2.05 2.00

132 W- 307-B-1 2154.20 43.083 20.034 2.15 1.90

133 W- 307-A-1 1190.70 23.813 11.330 2.10 1.57

134 W- 288-B-3 2842.00 56.839 27.340 2.08 1.85

135 W- 305-2 2400.60 48.013 23.249 2.07 1.83

136 W- 301-1 1244.30 24.887 11.899 2.09 1.89

137 W- 302-A-3 1004.50 20.090 9.516 2.11 1.94

138 W- 296-2 661.60 13.232 6.267 2.11 1.99

139 W- 293 353.10 7.062 3.377 2.09 2.11

140 W- 296-1 1054.00 21.081 10.036 2.10 1.93

141 W- 311-B-1 948.60 18.972 9.209 2.06 1.70

142 W- 288-A 425.90 8.518 3.970 2.15 1.88

143 W- 384-3 912.10 18.243 8.710 2.09 1.90

144 W- 354-1 1253.60 25.073 11.893 2.11 1.93

145 W- 354-2 859.50 17.189 8.250 2.08 1.66

146 W- 390-1-2 827.30 16.546 7.942 2.08 1.96

147 W- 328-1 547.90 10.958 5.335 2.05 1.86

148 W- 351-C-1 1082.50 21.650 9.886 2.19 1.52

149 W- 311-B-2 265.20 5.304 2.517 2.11 1.73

150 W- 312-1 712.20 0.343 0.288 1.19 0.81

151 W- 307-B-2 386.70 7.734 3.619 2.14 1.97

Parent (S) CO-3 1325.60 26.512 12.414 2.14 1.90

Parent (R) EC-520061 1158.10 23.162 10.685 2.17 1.73

Appendices

~xvii~

Table 5: Selected SSR primers are listed below.

S.No. SSR

Name Start Primer End Primer

Length (Start)

Length (End)

Total length

1. SSR53 CCCTTCTTCGCTCCTCTTCT ATGGCAAGCCACTGGTTATC 20 20 40

2. SSR54 CCACCGCAACAAACCTTATT GGGTGGTGAGAAGGATCTGA 20 20 40

3. SSR55 CAAAGGATGCAAGAAGGAGG CAAGAATTGGTAACGGCTTTG 20 21 41

4. SSR56 GGCAACATGTCACACTTGGT GCTCCGGTCCAACATAAACA 20 20 40

5. SSR57 TCCATCTAAGGTCTTTGCCG ACAAAGGAAGTGGGAGAGCA 20 20 40

6. SSR58 TTGGTGCATTCATAACTCGC ATTGCTACCGTCTGTTTGGA 20 20 40

7. SSR59 CTTCACTTGATGACCATTCTGA TCATTGGCACACAACAGTGA 22 20 42

8. SSR60 ATGAGGCAATCTTCACCTGG TTCAGCTGATAGTTCCTGCG 20 20 40

9. SSR62 TGCAAATGAATGTCCAGGAT TCAGCAGAGTTATGCCATGC 20 20 40

10. SSR64 GGCAACAGGTGATGGAGATA CAGCCTGAGTAAGGTAGCCC 20 20 40

11. SSR68 AGCAGAGGCAGTAAGGTGTG CCGATTCACTCGGAAACATT 20 20 40

12. SSR71 AAATGGCATGGAGAATGGAA CATCCACTGAGAGCCCAAAG 20 20 40

13. SSR77 GAGGATTCCTTCTCTTGTGGG TTTAGTCCGAGCACGTTGTG 21 20 41

14. SSR78 AATGTCAACCTCAACGAGGC AGTGGCAGATTGATCAGCAT 20 20 40

15. SSR79 TCTGCCGCCATAAGAGATTT TTTGGAATAATTGCGGAGGA 20 20 40

16. SSR81 TGCCTATCGCGTTATGTTGA GCCTCCCACAACAATCATCT 20 20 40

17. SSR82 ACCATCGAGGCTGCATAAAG TGCCTCAATCCTTCTTACCC 20 20 40

18. SSR83 GCAAATGGGTCTTTAGCCCT CCATCACACAAACAGCAACC 20 20 40

19. SSR84 CAACAATTCAATCCATGACCC CCTGACCATCGAGGTCTTTC 21 20 41

20. SSR87 TAGCTCCATTGCCAATTTCC CCAATTCATGCCAGTAACTTGA 20 22 42

21. SSR88 TCCTAAGGTAGGGTTAAAGTCTGC ACTGCTCTACTGCACAAATCA 24 21 45

22. SSR89 ACTGCGTAATTTAATGGCGG TGACAAGGTAAAGCCAACCC 20 20 40

23. SSR90 AGGCAGCTTACGACTGGATAA CGCCAGCCACAAATTCTTT 21 19 40

24. SSR91 TACCGAAACTCAGAGACCGC GGGACGCTGTCGATGTTACT 20 20 40

25. SSR93 AAACGCCATATAACACGTTGC TCGGCATAATAGCGTTGAAA 21 20 41

26. SSR94 AATCAGATCCTTGCCCTTGA AGCTGAGAAAGAGCAGCCAT 20 20 40

27. SSR95 CAATCCAACAAGCAATCCCT CCACATAACTAAGCCCACAACTT 20 23 43

28. SSR97 ACAGGGTGCTATACGTTCGG CAAATCCTTCTCTTCGGTGC 20 20 40

29. SSR98 CAACCCATCAATAATAACACCC CCACTGCAGTAGGTAGCTGG 22 20 42

30. SSR100 GCAAGCCTGTGACAATTGAG GTCGTCGTGTTCCGTCATAA 20 20 40

31. SSR101 GGTCCAGTATCTGCCATCGT TGACATTCATGCTACTCAGTTCAG 20 24 44

32. SSR102 AAGGGAGGTTCTGTTGGTGA GGTCGGTTCATAAGCCAGAG 20 20 40

33. SSR105 GAGCGGCTTCGAATTCATC CATTTGAGCAGAAGCGAACA 19 20 39

34. SSR106 CATGCTTAAGGAATGGTTTGC GTCTCCTCAATTTAATGGCA 21 20 41

35. SSR107 TCTGCATTTCTGAGCACCAG TGTGGAGAATTTGGAAGTTGAC 20 22 42

36. SSR109 TATGGAACTCTGCACTTGCG AATCCAGCACCTCCATCAAG 20 20 40

37. SSR113 TGGCCGGAGAATTCAAGTAG TGCTCAATCGGAAGAGCAG 20 19 39

38. SSR114 TGGACACATTCCTAGTGCCA CACGCAATCTCATGATTGAA 20 20 40

39. SSR116 GGAATAACCTCTAACTGCGGG CTCCCAATACAACACCCACC 21 20 41

40. SSR118 AGTGGTTCCACTTGTTTAGACC GAATCCGATCTGGGTCAGTG 22 20 42

41. SSR119 GCATTCGCTGTAGCTCGTTT GGGAGCTTCATCATAGTAACG 20 21 41

42. SSR120 GCGTTTCACAGAGCAACAAC GGATTAGTGCAGTGGCAGGT 20 20 40

43. SSR121 CTCGGCAAATGGCTGATTAT ATTTGACCAACTCCCAGCAA 20 20 40

44. SSR123 GATGTGGCAAGAGAGATACG CTGCTTGAAGTTCTGCTGGA 20 20 40

45. SSR126 TTCTCTCTGTCGCCATTGTG TTCCAACTCCAGAATCTCCG 20 20 40

46. SSR127 TGGAGAAACCAATTCAAGATCC AACATCCACCTCCAACTGGT 22 20 42

47. SSR129 TTCGAACTGCACTTGAAACG TCCTGAGGTTGATTTCCCTG 20 20 40

48. SSR130 AAACCCTCAACACCATCACC CATCATTTCTTTCATGGCTCC 20 21 41

49. SSR131 CTCTCTTCACTCTTTACAATTGCC CGTTTGCTGTTGTTGTTGCT 24 20 44

50. SSR132 AAGGCGTGAGAGGCAACTAA ACTTGTCCTGGCAATGAACC 20 20 40

51. SSR133 CCGTTCTTGGTGGATTAGGA AAAGAGTGAAATGTGCACAGAC 20 22 42

52. SSR137 TGGATTCTTCTTCACTAGAAGGG TTCCGAATTCGATGAGGTTT 23 20 43

53. SSR138 GTGGTAACGGCAAAGGGACT CTTATGGCCTTAGCAGCCAG 20 20 40

54. SSR139 TGGGTATGGGATTTACACCAA AAACGAAGGCAACAACGAAG 21 20 41

55. SSR140 GTTTCAGCAGCCTGTCCAAT TGACAGCATTTGGGTTTGAG 20 20 40

56. SSR141 GTTCCCGCTTGAGAAACAAC CCAATGCTGGGACAGAAGAT 20 20 40

57. SSR142 CGAACAGATGCATAGATGAAGA ACGGTGTGGACTCATGTGAA 22 20 42

58. SSR143 CACCATTCTCGAGCATCATTC CATTGTTAGTCACGCCCTCG 21 20 41

59. SSR144 TTCATATCGGGATTGTAGACCC AGGGATATGCCCATATTGTGA 22 21 43

60. SSR145 CCCGATGATCGATTGTACCT GATCGACGAAATGCATGAAA 20 20 40

61. SSR147 TCTGCTTGCATTAGGGTGC GATTTGGCCAACATACCCAC 19 20 39

62. SSR148 CCCAATTCGTCAGTATCGTATTC CATGGCCATAACATGAGCAC 23 20 43

63. SSR149 GCAAAGCCACGAATTTCATT TGTTGTGCTTGATGAGGAGC 20 20 40

64. SSR151 CCATTCTGAGAAGTTTCCCG CGTCTCTTCCATCCCTCCTA 20 20 40

65. SSR152 GCACACGCACTCTCTCACTC CCACAATGGTGATGAACACAG 20 21 41

66. SSR153 GCTCCGCCATACTCCTCTTA GACGGCAACTTTGTAACGGT 20 20 40

Appendices

~xviii~

67. SSR154 GAGAACTGGCCTATCTGAATTA ACGAACTTGGAGCTGACCTG 22 20 42

68. SSR157 CGGGCCCGTATAACCTATTC CATCATGTTGGCAGGAGAAA 20 20 40

69. SSR158 GGGCGACAACAGTAGCATAA TCATTGAGAATGGAGAGCCC 20 20 40

70. SSR160 GGTTTGAAACAATTCTTCTCCG TCAGAGGACCAGAAACTGCC 22 20 42

71. SSR161 TCAGATATGATGCCAAATGC TTTGACAACCAAGGTTATGGG 20 21 41

72. SSR163 CCAGCAACAGCTCCACTTTA TTCTTGGATCCACAAGTCTTC 20 21 41

73. SSR164 CCACGGATGGAACCAATTTA ATTAGGGCGGGATACTGGAT 20 20 40

74. SSR165 GCTGCTAAACACTCAAGCAGAA GCTCAGCTTTCAGAAGAAACCA 22 22 44

75. SSR166 TTGCCATTTCATTCTCCTCC AATCAACGCTCGTTTCTGGT 20 20 40

76. SSR167 TGCTTCGGAATTACCCTCTG TACGCGCACGTGCATAAATA 20 20 40

77. SSR168 TGGAGGAGGTTAGGAGGAAGA AAGCTGAAACCAAAGCCAAA 21 20 41

78. SSR169 ACCCGAATCCAAACCCTAAC AACCCGTGTCAACTTCTGCT 20 20 40

79. SSR170 TGCTCTCTCCTCCATCCTTC GAGGCATCCTCAGCTTCATC 20 20 40

80. SSR171 CACTCAACGACCCTTCCTGT TGACTTGCATCACCATACCC 20 20 40

81. SSR172 TCCGACTTTCTCTGTCAAGG CAGATCAGAAGAAGACAAGCTGA 20 23 43

82. SSR173 CGTAGCTTCCAATTCTCCTCA AGCAGCAATCAGCTCTGTGA 21 20 41

83. SSR174 GGTGAGAACCCAAGATACTCCA TGAGCCATGAACCTCATTCA 22 20 42

84. SSR175 GGAATTTCACAGCCCATGTC GAAGGTAGTCGGGTACGGGT 20 20 40

85. SSR176 ACAGCCACTAGGTTTCCGTC CACCCTGACTTCAGAAGAAGC 20 21 41

86. SSR177 CTTATCGATGGCCCATTTCA ATCAAATCAAGCTGAAGCCC 20 20 40

87. SSR178 GGGAAACCTTAGATCTCACTGAA CACAGGGCTAGGCATCTTTC 23 20 43

88. SSR179 TCGCTCTATTCCTCTCGCAT CTCCTCATCATGGGCTTCAT 20 20 40

89. SSR180 CACAACGCTCTGATCTTCTCA ACCCATCCTGCTTGTGGATA 21 20 41

90. SSR181 CGGAAGACTCCTTCCTTCCA GAAGCCTGTTCTTCGATTGC 20 20 40

91. SSR182 CAAACAAGGTTGGGTGATCG AAATGGGCAGTGGTAGATGG 20 20 40

92. SSR183 GAGTCTGCAAGCAAGCAGTG AAGCTGATTGCTCTGGCTTC 20 20 40

93. SSR184 CAACATTCCGATGCTATTCCT GTTCATTCAAATGCTCCGGT 21 20 41

94. SSR185 CCCTTTCTTCTTCTCCTCTGTG TGTTCAAACTCCGAAGACGA 22 20 42

95. SSR186 AGCCACCCATATTAATGATCC AAATTCCTTTCTGCTGCCC 21 19 40

96. SSR187 AATGAAGGCAACAATTTCCC TGGGACATCTAATGGTGGTG 20 20 40

97. SSR189 CAAACAAGGTTGGGTGATCG AAATGGGCAGTGGTAGATGG 20 20 40

98. SSR190 CAATTGCTCAATCAGTTCGC TTCCAGCCATACTCTCACCA 20 20 40

99. SSR191 GCTGAGTCAAATCACAACAAGA TCCAACATAGCCAGGATTCA 22 20 42

100. SSR193 TCTGGTTTGACCCACAAAGC AACCTTAGGCGTAGATGCCC 20 20 40

101. SSR194 TCAGGAGAACCTTCATCGGT TGATGAAGAAGGTGGTTCGC 20 20 40

102. SSR195 TCCATCTCGTTGCTGTGTTC ATACCTTTGTCGTTGGCGTC 20 20 40

103. SSR196 TGGGTTGGTTTCCATGTTCT ATTTGACCAGACTCGCCATC 20 20 40

104. SSR197 CAATGGTAGCTGATCTGGCA GCCTTTGAGGATTCCATTCA 20 20 40

105. SSR199 CAGCACCCTGCGCGT CTAATGCCTCCATCGTTCGT 15 20 35

106. SSR200 TCCATGGCTTCAACAAGACC CTGAAGGTGGCATAGGTGGT 20 20 40

107. SSR202 GCCCAAAGGAGCAAATACAT CATTGCTACTCTAAATTTGACACCC 20 25 45

108. SSR203 CAGATCATTACGTAGTTCATGTGGA CCGAAAGCTTTGTTCAAGGT 25 20 45

109. SSR204 CAACTTTGAATTTGGGAGCA TTCCTCCTCAACGCTCCTTA 20 20 40

110. SSR205 GCAGCTCACCATCTTCATCA GGTGCACTTCCATTGCTCTT 20 20 40

111. SSR206 ATCCAAAGACTCCCATTGAA CGAACCCATAATTCCACCAC 20 20 40

112. SSR207 TGCTCTCTCCTCCATCCTTC GAGGCATCCTCAGCTTCATC 20 20 40

113. SSR208 CAAAGGATGCAAGAAGGAGG CAAGAATTGGTAACGGCTTTG 20 21 41

114. SSR209 AGCTGCTACTGCCACCACTT ACCATAAGACTGCACTGCCC 20 20 40

115. SSR210 GTCTCCCACCAAGAACCAAA AAGTAGCAGATTGTGCAAGGC 20 21 41

116. SSR211 TGGCCGGAGAATTCAAGTAG TGCTCAATCGGAAGAGCAG 20 19 39

117. SSR212 CCAAATGAGGCTAAGGGTGA ATCGAGTCCCTTAAGGCCAA 20 20 40

118. SSR213 CGTCAAGAAATTAAGTGGGA CAGACCTTCGCACTTTCCTC 20 20 40

119. SSR215 AAGCTGCTTCTGCTGGATTG GAGGAGAAGAAGGTGCACGA 20 20 40

120. SSR216 CCATGCTCTCATGCATGTCT GCCTTCAACCAAGCAACAAT 20 20 40

121. SSR217 CTTCACTTGATGACCATTCTGA AAGAATCATTGGCCCACAAC 22 20 42

122. SSR218 GTGGTTATCCCAAGACCCAA CGCCAGTCTTCCTCTGACTT 20 20 40

123. SSR220 GCACCTCAACAGAGCAACAA CGCCGGAGATGAAGTAGAAA 20 20 40

124. SSR221 TCTTCTTTGAAGGGACTGTGC ACATGAAGTGGCTCCTCCAC 21 20 41

125. SSR224 GCGATGGTCTGAGACACTGA CAGCTGGTGATCCTCCTCTT 20 20 40

126. SSR225 GGATCCTAGATTCCGCGAAC GCGTCTTCTGTTTCTCACCC 20 20 40

127. SSR226 TTTACTACCCTCATGCAACGC CGCCGGTAAAGCTTCAATAA 21 20 41

128. SSR227 GAAACCTACTCGTCCCGTTG CTGCTTTCTCGCTGAGGAGT 20 20 40

129. SSR228 TCAACAGAATAGAGGGTTCCA CCATGCCCAACTTCATATAGTGT 21 23 44

130. SSR229 CAAATTGTTACCTAACCGCCC TGGAGATGTAAGATTTGGGTG 21 21 42

131. SSR230 AAAGGAGCTCTAGGATTTGGG TAAATGCATTCAAAGCGCAC 21 20 41

132. SSR232 CCCACCTTGTTGGAAGAAAT ATGTTTCGGTACCCATTGGA 20 20 40

133. SSR233 CACTCGAGTACACGGCAATTT CGGTAAACCTAGGAAAGGGC 21 20 41

134. SSR234 TGACGTGCGTCTATTGACATC CGTTCCCTATTGGGTTTCCT 21 20 41

135. SSR235 GTTTACGTCGAGTAGCGGC TGGTCCTTGCTCTCTTGGTT 19 20 39

136. SSR236 CTGTTTATTGACCCTCCAACT CCAGCAAGAACACGAGATGA 21 20 41

137. SSR238 AGCCAATTGGGCTACTGAGA TGCCAGACACAAGGGTACAA 20 20 40

Appendices

~xix~

138. SSR239 GCCAATGAAGTGAGTGTTGC AGGCACGAGAATTTAGCCAC 20 20 40

139. SSR240 TGCACCGATCTCTCATCTTG TGTGCAATATGGTTGGCTGT 20 20 40

140. SSR242 GCTCGAGACCAGCCTCTGT ATTTCCTTCCCTCCTGCAAT 19 20 39

141. SSR243 CTCTTCGATCCCGCAATG TAGCCAGGACAGGCCTTATG 18 20 38

142. SSR245 CCTGCAGGTCGTTCTAGTGG GGTGCATCTTGGAATTTGCT 20 20 40

143. SSR246 CATGGATACATACATACGGACA CGATGTTTATCGTGATTGCG 22 20 42

144. SSR247 TGTTGAGTCTAGGAGGGCAAA AACATGGCATCCTTCACCTC 21 20 41

145. SSR249 GGAAGATGAGCGGTACAGACC CTCACACTCACACACCCACA 21 20 41

146. SSR250 AGGTCGGTAGAAGTGGAACCT TGTGCTTTGGTAAGAAAGGGA 21 21 42

147. SSR251 CCATTGACAGCTCTCATCCA TCCAAAGTCCAAGCTCAGGT 20 20 40

148. SSR252 AGAGGAGCTCGTAAGCAAGC GCTTGTCCGTTGGTTGTTCT 20 20 40

149. SSR253 CCACAAACAATTCCATCTCA GCTTCCGCCATACTGATACG 20 20 40

150. SSR254 CCATTGGATAGCGTCTCCC ATTGTGCATGTTGATACCCA 19 20 20

151. SSR255 TGTGAATACAATTTGCACCC GGGTTACTAATGCACAAGCGA 20 21 41

152. SSR256 GGTTAGATAATCTCCCACAGAAAC GGAGTATGCAAGTTACAATGC 24 21 45

153. SSR257 GAATGAGCACATCGATACGG TACTTCCCTATGGGCCTCCT 20 20 40

154. SSR258 TCTTGACAGCCATTTCTCCC TATTCACAACCACCGATCCA 20 20 40

155. SSR259 CGGCCGTTCTAGAAGAACTC AGGACATCAAGTCTTGGGCA 20 20 40

156. SSR260 TCGACACCATTGTCAACGAC ATGTCACAGACAGACAGCCG 20 20 40

157. SSR261 AGCTTCCACCATTTACCGTG TGGGAGAAGGATCATTGAGG 20 20 40

158. SSR262 AACGCTGACATTTCAGTTCG CTTGGAGACTTCCCTCCCTC 20 20 40

159. SSR263 GCACGAGCTCTGAGCCTATT CTGACTACCTTTCACTGGCAA 20 21 41

160. SSR264 GCTACCTCATCCAATTATCCC AACAAGCCTTAGGCGATGAA 21 20 41

161. SSR265 TCTCATATCCACAAATCACCCT GGGTAATAGCACGAGATTTGG 22 21 43

162. SSR267 GAGCAAGAGCAAGAGCAAGG CACCTCCAGTAGCAGCAACA 20 20 40

163. SSR268 CTGAAGCTGAGAAAGGCGAC CTGGCATTTAAGGCAAAGAA 20 20 40

164. SSR269 GTGAACAAGAGAGTGGACGC CTCGCTTGTCTCTTCACGCT 20 20 40

165. SSR271 GAAGGTGAATCAGAGCAGGC GGCATGTAAACTGGGTGCTT 20 20 40

166. SSR272 TGGGTTGTTGGGAAATTCAT TAATCCGAGCAGCAGGAAAT 20 20 40

167. SSR273 CACAGGGAATGAAACATGTGA TTGACCCAATTCATCAACTCC 21 21 42

168. SSR274 TACCGAAACTCAGAGACCGC GGGACGCTGTCGATGTTACT 20 20 40

169. SSR275 ACCAAAGACAACGCACGG GTTGGGTTGGTTGTTTCTCG 18 20 38

170. SSR277 CGGACACCTGTACACACCAC CATGTCATTGGAACATTGGG 20 20 40

171. SSR278 TTTACCCTGCCGTCGTTATC ATCGATCGAACCCACATCAT 20 20 40

172. SSR279 CTTGAGGAGGATGAAGGTGG AGTTACCCTCCCTTGCCATT 20 20 40

173. SSR280 CTTGTATGCTTCCCTCAGTGC GGGTTTAATGTGGAAATGGG 21 20 41

174. SSR281 CTGTTTCTGATCAGGTATGTCTTTC CACTGATCAAAGCAACTCAAG 25 21 46

175. SSR282 TAATCCGTCAAGCCAGATCC AAATCAGGATGGCAAACTCG 20 20 40

176. SSR283 CATGGAGGTGCAAAGATTGA AAACGAAGGCAACAACGAAG 20 20 40

177. SSR284 ACCACCACTACCCGTTCCTC GTCTCCATAGTGAGCTCCGC 20 20 40

178. SSR286 AGCTATGGAGTTTCAGGACCA ATTCAGGTAGCATGGAACGC 21 20 41

179. SSR289 AACAATGGCAGGAATCATCC TGTCCGTCATGTTTCTTCTCC 20 21 41

180. SSR291 GGCACGAGCACATATAGAAGAG TGGCTTCAACCATTTCATCA 22 20 42

181. SSR292 TTCAGCACTCTCCTCCACCT TCCAGCAGAAACATTAGCACA 20 21 41

182. SSR294 GGCTGACCTGAGATCCAACT CGGCATTACATCCTCCACTT 20 20 40

183. SSR295 CTCCAGAAGGAACTCGATGC CAATTCCTTTCACCTGCCAC 20 20 40

184. SSR296 CCGGAACAAGTCCCTTCATA TCAGCCAAGTTCATGGTACATC 20 22 42

185. SSR297 AGCCACCAACTAAGGCTCAA TCATTCCTTGGGACCCATAA 20 20 40

186. SSR298 CCCAAAGATGATCGTGACAG CGACTGAGGGAAACACCATT 20 20 40

187. SSR299 CCAGCTCATTCCTCTCTCGT GGACACTCTATACGGCAGGC 20 20 40

188. SSR302 GTAGTAGAATCCTCAAACGCTAGT CCTCATTGATAATGCGGCTT 24 20 44

189. SSR303 AATGCGATGCCACATCATAA CATTGGCACCATCTGCATAC 20 20 40

190. SSR305 CGACTAGGCGGCCTCTTT CGCAATACTTGCTGGAACTG 18 20 38

191. SSR307 CTCCTCCTCCAAGGTGTTCA TGCTCTTTCTCCGCATTTCT 20 20 40

192. SSR309 GGCAATCGGCTGAGAAATTA ACTCTCATCGGGAAGTGGTG 20 20 40

193. SSR311 CCTGCCAATTAACAGCAACA CATTGTTAGTCACGCCCTCG 20 20 40

194. SSR312 CCCTCCTCATTCTCCAATCA CGTCGCTGTATTTCCTCCAT 20 20 40

195. SSR313 GTTTGACCCACCAATCCAAC TGGATGCCATGTGATGAAGT 20 20 40

196. SSR314 AATCTTACACGGCCAGCATC TGCTAACCAAATGACAGTCCC 20 21 41

197. SSR315 GGAGGTCCCAATTCAACAAA CACCATCAGCAAGAGTCCAA 20 20 40

198. SSR317 TTTGGCCACCACGGC TGAGGAGGAGGTGGTTCATC 15 20 35

199. SSR319 TTCGAACTGCACTTGAAACG TCCTGAGGTTGATTTCCCTG 20 20 40

200. SSR321 AAGGCGTGAGAGGCAACTAA ACTTGTCCTGGCAATGAACC 20 20 40

201. SSR322 TGCTCAAACTTCAATGCAGC ATCTCGTCCTCCTCCTCGTC 20 20 40

202. SSR323 CAGCCCAAATAACACGTCTCT GGCAACGAAACTGTCATCAA 21 20 41

203. SSR324 TTGAACTTCACTCCAAATGGG CTTGAGTCCACCCGTATCGT 21 20 41

204. SSR328 CCCTCTTTCCCTTTAGCCA TGTGAACCTTTGGGTGTTGA 19 20 39

205. SSR329 GGCGCAGAAGCACAATAATA ATCAGGGTAACCACGGAGTG 20 20 40

206. SSR332 GTTTGATGTGGCTCCTGGTT AACTCCATTCAGGATGTGGC 20 20 40

207. SSR333 GTTCCCGCTTGAGAAACAAC CCAATGCTGGGACAGAAGAT 20 20 40

208. SSR334 GGTTCTACAGAGGACGGCAC AAGAACAGCTTGGGCAAAGA 20 20 40

Appendices

~xx~

209. SSR337 CTCGGCAAATGGCTGATTAT ATTTGACCAACTCCCAGCAA 20 20 40

210. SSR338 TGGAACGGTGACAAACAAGA CGTTCGAATATGGTATCCAA 20 20 40

211. SSR339 TTTCTGGTTTAATCGCCCTG GCTGTATGGTGCCAGCTTCT 20 20 40

212. SSR342 TCGTCGACCTAATCACACGA TCAACTGCGACACTCTCCAC 20 20 40

213. SSR343 TTTGCTTTGATTTCCGTGTG TACTGCCAAAGTGAGCACCC 20 20 40

214. SSR347 GAACCAAACACGGCCTTAAA TCTTGGCTAGGGTTCTTTCG 20 20 40

215. SSR348 AGTGGTTCCACTTGTTTAGACC GAATCCGATCTGGGTCAGTG 22 20 42

216. SSR351 CCTTCAATTGACCTCCCTCA GCATCTGGAAATTAGAGGCG 20 20 40

217. SSR352 AGTCAGAAGAGATGGCGGAA AATCGATGGATGGAATTGGA 20 20 40

218. SSR353 TCCACATGTCAAGTGAGGGA GATCGACGAAATGCATGAAA 20 20 40

219. SSR354 TGGGAGCTTTAACATTAGGAGG TGCATTTCATCACTTGGTGC 22 20 42

220. SSR355 CAACAATTCAATCCATGACCC CCTGACCATCGAGGTCTTTC 21 20 41

221. SSR357 AACTGCTGAACTGGATGACG CCAGCGATGATAAAGATGAAGA 20 22 42

222. SSR358 TGGAGAGTGCTGCGTGTACT TGGGAAATGATCCGATGAG 20 19 39

223. SSR359 TCGGGAGATCACTTACACACA TCCACTAGAACCACCCTCCA 21 20 41

224. SSR361 CACGGTTGGAATCATCACTG AATGAACCGGTATGCACCTC 20 20 40

225. SSR362 GCACGGTGAGCACACTAGAC CCATAAGATGGGTTCATGGG 20 20 40

226. SSR363 CGCGAAGAGAGTCACACATC GCTGCCCTCACTTGTCATTT 20 20 40

227. SSR364 TATCTTCTTGCTGCACCCAC TTTGGCTGACTGACATTTCG 20 20 40

228. SSR365 AAAGAACACGCTTGGCTCAT AACATATGACGTGGCACGAG 20 20 40

229. SSR366 CCCGACGTATGAGCGACT CGGTCCACAGGGTTTATAGC 18 20 38

230. SSR367 CCCTGCTAGCCATAGAGACG GTGAGGTCTTGCTCTGGGTC 20 20 40

231. SSR368 GGGATGATGATCGAAGCG GCATAAGCCCGTCATTACCT 18 20 38

232. SSR369 GCATCTATAAGCACGCCTCC TCTGTTGCTGTTGTCTTGGC 20 20 40

233. SSR370 AGATCTCTAAGCAACCGCGA TTTGCGTCCATTCGGTTAAT 20 20 40

234. SSR371 CCCAAGATCGATAGTATGCAA CGTAAAGGGACAAATGTGGG 21 20 41

235. SSR372 GCCAGTGCTACTATAGACTCTCCA GTGTAATGTGTGGATTGGCG 24 20 44

236. SSR373 AAGTCTGAGCTCATTATGACATGC TGCATTATGCCAGGAATTGA 24 20 44

237. SSR374 CGTACTGAATTCGTGACTTTCG TTATAAGAACGCGCCTCTCG 22 20 42

238. SSR375 GAGTATCTCTGAGACGCGCA CGGTGGCTTAGCACAGTACA 20 20 40

239. SSR376 CGGAGACGCCCTCGTA CAAAGCCTGGGTTTAGGTGA 16 20 36

240. SSR377 ATGAGCTCGTAAGGCACAGC TTTCCATCCTCACTCTCGCT 20 20 40

241. SSR378 TTACGCAACCACCCTTTCTC TCATGTAGGCCACACTCGAT 20 20 40

242. SSR379 CGTTGCAGCTGAAGAAGATG GGGAGACAGATTTCTCGGG 20 19 39

243. SSR380 GATGGGCTCGTAAGCATGAC TGTATTAGACCAAAGGGCGG 20 20 40

244. SSR381 CATGCACCCATCCTTTCTCT GCACACTAATAGGACCGGGA 20 20 40

245. SSR382 CCACTGATCTCTGAGTCCTGC TACCCAGGGATTCATTTCCA 21 20 41

246. SSR384 GGAAAGTGTGACGCTCGAAT GCTCGTGGTTGCACACACTT 20 20 40

247. SSR385 GAAGAGCCCTGAGACAGCAT CGAGTGGTGGTTCCTTGG 20 18 38

248. SSR386 GCTGGACTTCATTGGGTCAT TTTCCAGAAGCCGGTATCAC 20 20 40

249. SSR387 AGACTCTCAGGGTCATGTGC GTCCCTAATGCGTCCTGTGT 20 20 40

250. SSR388 GAGCTGCTCCTCGTAAGCAC AGAGTTGAAGCGTTGCCAGT 20 20 40

251. SSR389 ATCACATTTGAGGCACACCA GGTTCCATCAGCTTAGTCGC 20 20 40

252. SSR390 AAGTGCACCTTGTAAACAGCC CCCTTTAAGGCAATGAGGGT 21 20 41

253. SSR391 TGTGCGTCTAGCCATATCTCTC AGAATAAGGAAACGCCCTGG 22 20 42

254. SSR392 GGGAGAAGATCTCCGTAGCC GGTCTCGCGACAAATGTTCT 20 20 40

255. SSR393 GGATCATTTGACCGCATCTC GCTTCTTTGTGCGTTTGGAT 20 20 40

256. SSR394 GGAGCGCTGATGAGACTCTA TATTTGCTTTCCGCGCTAGT 20 20 40

257. SSR395 TGGAAAGATCGACTGACCAC AATCAAGGGTGAGCATTTGG 20 20 40

258. SSR396 TGATGACTGCTACCCATTCC CACGCATGGGTGAGTATTTG 20 20 40

259. SSR397 CCGCGCGCTCTGAAGTA GCCTTGGACTTTCCCAATTT 17 20 37

260. SSR398 GTGAGCAGCTCCGACCC TCCGGCCTTTACGCTTAGTA 17 20 37

261. SSR399 CGTCGTCTAGTCAACCAACC GATCCACGGAATTCGACAAA 20 20 40

262. SSR400 GCAAAGGTTTACACGACATGC CCTTGTCCAGTTCTGCCATT 21 20 41

263. SSR401 CCACCCTTTGAGCTAGTTCTC TGTGTGTCTGTGTCCGTGTG 21 20 41

264. SSR402 CCCACCTTTGCTAGTTCTCTTT TGCTGGTGTGTCCCACTTTA 22 20 42

265. SSR403 GGCTGACCCTGCAGACAC TGGGTCGGTTTAAGGTGTTC 18 20 38

266. SSR404 ATGACCTCTGAACGCACCAC GACGATTGCTCTGACGACAA 20 20 40

267. SSR405 TGTAGCCTCAGACTTCTTCCTTG AACCCTGGTAAAGGGTTTGG 23 20 43

268. SSR406 ACCTGTGGGATCGACCTAGT GCTTGTGGGTGCATAACCTT 20 20 40

269. SSR407 ATTTCTGACTTCGCCACTGC GCCGCATATCCATTCTGTCT 20 20 40

270. SSR408 AGGCGTTGCGCTCGTA GTCGGTCAACTCGTAAAGGC 16 20 36

271. SSR409 GGCACCTAGAGAACTGCGTC GCACACACGCCTTTAGCATA 20 20 40

272. SSR410 GGGCGAGAACTACATCAAA GGTATTGCGTGGTTCAGGTT 19 20 39

273. SSR411 TTATGCCGTAGAAGCCCGT CGGGTCAAACTGGTAAGACG 19 20 39

274. SSR412 GGCGATGACGCTCTTACTCT CTTTGCCAGTCTCGAGTTCC 20 20 40

275. SSR413 ACTGACCCGCTCGAGACC TCCGCCTACTGTCTTTGCTT 18 20 38

276. SSR414 CCAACAACCAAGACATCACG GTCGTTGGTTTGGCTTTGTC 20 20 40

277. SSR415 GAGCGCACACAAACAGAGAT TCTCTCGCTCTTTCTCTCGG 20 20 40

278. SSR416 ACCACCCGTTGGAAATCAC TGTGGGTGGATGTCACTTTG 19 20 39

279. SSR417 TGAATGATGACGACCAACC TTGCTCTTGTGATGGTGCTC 19 20 39

Appendices

~xxi~

280. SSR418 TATTGTGGCCAAATCTGACG GGACGGGACAATTGGTTAGA 20 20 40

281. SSR419 TCTCAGCTTGAGGAACTTGGA TGCCACTTAAGTCCGTGATG 21 20 41

282. SSR420 TCGCAAATTGTTACCTAACCG GGGTGTAGATTGATGGTGAGTG 21 22 43

283. SSR421 CTGAGTCGCGATTGGTCAC TCACACCGAAAGCTGCTATG 19 20 39

284. SSR422 AAGGTGAGCACATCGCACTA GTGCAGGTTTACCCTCGTGT 20 20 40

285. SSR423 TCGATACGGCGATTTCTCTC TTTCATTAACCGTTGCTCCC 20 20 40

286. SSR424 AAGGACTTGTGCTGGATTGG TAGTACGCCATGGACCTTCC 20 20 40

287. SSR425 TGAGAACGTCCTAGGAGAGAGTG ACCAAATAGGTTCGGTGCAA 23 20 43

288. SSR426 CTTCTGCATCCCTCCTCAAG ATCGGTGTATGTGGATGTGG 20 20 40

289. SSR427 AAGCTCAAACCACTAGGCCA TGTTGCTCGAACTCTCCAAA 20 20 40

290. SSR428 AGAACGTTATGAGGCACAGC CGTGCATGCATATATTTCGG 20 20 40

291. SSR429 AGGTGTAATGCAGCCTCA GCAGCGCATATCCAGACATA 18 20 38

292. SSR430 TGACCATCTTAACGCGAATG CCGCTAGTTAGATCATCGGC 20 20 40

293. SSR431 GTGTGTCTTGGGAGGTTGCT GTTTAGGTGCCACTCCGTTT 20 20 40

294. SSR432 TGGGCGCTAGCCCTATTT TTCCTGTAGAGTGCTGCGTG 18 20 38

295. SSR433 AGACCGTTCTACCTGCATTT AAGTTGTTGAGTTGAAGGAGCC 20 22 42

296. SSR434 CAGATAGACGCACTCGCAGA CGGACTCAGGGTCTATAGCG 20 20 40

297. SSR435 ATCGGTGCTTGATAAACGGT TAGCTGCAATTGCCAAGAAA 20 20 40

298. SSR436 AGTAGCACTCCACTACCGGC CAATTTAGGCATAGCGGGAA 20 20 40

299. SSR437 GCTGATCTGACCTCGCTCTC TCCGGTCAAACAAGGTAAGG 20 20 40

300. SSR438 GCGCGGATAGATAGATGGAG TTCCTGCTTGTCAACGAATG 20 20 40

301. SSR439 GTCTGCAAGGTGATCAGCAA GTGGCTTCACTTGCTAAGGC 20 20 40

302. SSR440 GGAAAGACGATCAGTACGGC AGGAATTGTGAGTGGTTGGC 20 20 40

303. SSR441 GAGCGAGCACTTATAACGCC GGGATGTTTACGGGTGCTAA 20 20 40

304. SSR442 GGGCGGCAAATTACATTACA CATTGCGTCATGGAGTTGTT 20 20 40

305. SSR443 GGAGGAGCCCATGAGGAC CTTCCTTTGACCTTTCGCTG 18 20 38

306. SSR444 ACTGACTGTGTGTGCGGGT TGCATCACTGTCGTGGCTAT 19 20 39

307. SSR445 AGCCTCAGCAAGAGCGTAGA TCGCTAACGCAATACTGTGG 20 20 40

308. SSR446 AGCCACTAGATGGCCGC TCTCTGACCTCCTGTGCCTT 17 20 37

309. SSR447 CAGCTAGATCCACAGCCACA GAGCGTCGTGAGCAATACAA 20 20 40

310. SSR449 TGGATGAGCCTCGGCA CGAATAGTGAGCGACTATGTTCA 16 23 39

311. SSR451 TGACAGTAGACCGCATAGGG TTTCCGACTTTGGGTTTGTC 20 20 40

312. SSR452 GGGAGATCATCCGAGCAT TGCATAATTTCTCGCTCGTG 18 20 38

313. SSR453 TGAAATGATCTGATGCTCCTCT CTGGTTCCTTCTGTGTGGGT 22 20 42

314. SSR454 ACTCCACCTGCAGAAATGCT GGGTTTCCGGGATATCAAAT 20 20 40

315. SSR455 TCAAGGTCCTTATGAACGGC AAGAACAAGGTGGGCGAATA 20 20 40

316. SSR456 TGAGACGGCAATAGAGGTCA TTAGGTGGGAACTCTCACGG 20 20 40

317. SSR457 GGAGATGCTTGCCCGC CCCACTTGTCACACGCATAG 16 20 36

318. SSR458 TGAATGACGCCTTTCTGCAT CCATCACTCAATTCCTCGGT 20 20 40

319. SSR459 TGATCTCATAGACAACGGCG CGTTCCCAGTGTTCATTCCT 20 20 40

320. SSR460 CGAGTGAACTCTAGAACCTGCC GGCACAAGTGATCTATGCCA 22 20 42

321. SSR461 AAAGTTGACCTCGAGCCG GAGGGTTTCCTTTAGCAGGG 18 20 38

322. SSR462 CAAAGACAACGCACGGC TGCGTGTTTGGTTCGTTATC 17 20 37

323. SSR463 CCCAACGGAGCACCA TCCACTTTCTCCCTCTTTCTG 15 21 36

324. SSR464 GACCACCGGATGTTGATCTC AGCTTCTCTCTTGTCAGCGG 20 20 40

325. SSR465 AGCTGTCCTGACCTGCATCT CCTTATGGCTGTGCTCTCCT 20 20 40

326. SSR466 CCCGATGAGCTACTCGAGAC CAGGCTATGGGCTTTCTCTG 20 20 40

327. SSR467 GACCATTGTAACGGCGATTC CATCGGCCTAGTCAAGCAAT 20 20 40

328. SSR468 AGATGACTTATTGACTCGGCG TCCATAAGACGCAATAGGGC 21 20 41

329. SSR469 CTAGCACATTTCTATCGGCG GTAACTCCCTTTGCGGGACT 20 20 40

330. SSR470 ATGCGAGTCTGATCTGCGT GTACCCGCTATTGCATCCAT 19 20 39

331. SSR471 TTATGAGCCTATCGAGACTGC AAAGGTGAGGCTTACGGAAC 21 20 41

332. SSR472 AGCCCTCATAACCATACGGC AAATATGGGAGACAAACACCC 20 21 41

333. SSR473 ATCGAGCCTGCGATTGTTAC ATCCAGTCATGCTTATGGGC 20 20 40

334. SSR474 AAAGCTAAGACTCGTTTCACGC CTTGTTAGCCCAAAGGGTCC 22 20 42

335. SSR475 GCTAGCCCGATCGACCC TTGCTTGCTCGTGCTCTCTA 17 20 37

336. SSR476 AAATGTTCCTGAGGACGCC ATGAGTCGCGTAAACTCGGT 19 20 39

337. SSR477 AAGGTTGATCCTGTTCCGTTT AATATAGCGCGTTTGGGTTG 21 20 41

338. SSR478 GCAGCATATATCACCTTGGCT CGTGCTCTCCAATAGTTCACC 21 21 42

339. SSR480 CCTCATCAGCAACTGACCTG GCTCTCGTGATGTGCTGTGT 20 20 40

340. SSR481 GATAGCTCCTGCAAAGACGC GGTACCGGTAGTCCATGGTG 20 20 40

341. SSR482 TTCTCATATCCACAAATCACCC GCGAGGTGTAGGGTAATAGCAC 22 22 44

342. SSR483 ACGGACACCTGTACACACCA GCCTTGCATCATGACATCAG 20 20 40

343. SSR484 GCGGCAGTACAACACGTAAC ATCAGCGCCAATAAACTGGA 20 20 40

344. SSR485 CATATCCAGCCAACACACCA AAGGAGAGCGTACAGGAGAGG 20 21 41

345. SSR486 CCCAGATATCTTTATAGCACACG GCCAATTTCTGTAAGCCAGC 23 20 43

346. SSR487 GGTTGACTCTATATGCAAGCCA CAAGGGCTCCTTACTCATGG 22 20 42

347. SSR488 CCGATGGTCCACTTGAAAC CGGAAGAAGAATCCTGAGCA 19 20 39

348. SSR489 TGACTCTACCCATTCTTTCTCTCTC CAAGGGTTTCAATGCAAGGT 25 20 45

349. SSR490 ACCCGAAGCACAGCTCTAAT TTAGAATTGATCGCGTGCAG 20 20 40

350. SSR491 CTGGACTGATACTAGACCACCC GTGAACGTTCCCACCTGTTT 22 20 42

Appendices

~xxii~

351. SSR492 TGGATACTTTGATCGTTTCCAC AAGGCAGATCTGATGGCAAG 22 20 42

352. SSR493 TGGACGTAAGCAGCACCAC GCCTGACCAGGTTCTCTCAG 19 20 39

353. SSR494 GCTCTCATAGTGGGCCATTG ACCCACAGATATAATGCCCG 20 20 40

354. SSR495 GGGCGCGCTCACTACATAC AATGCCTTATGGGCCTTTCT 19 20 39

355. SSR496 GCATAGAGACATCGACACATACTG TTGTGCTCGTGCGCTTATAG 24 20 44

356. SSR497 GGCTGAGTCGTGTAAGCACA TCGTGCATGCTTAGACTTGG 20 20 40

357. SSR498 CGAACTCGATACGACTCTATCTCTC CCAGCTGTTTGTTCTCCCTC 25 20 45

358. SSR499 GCGGCGATGGTAAGCTATC TAGATAGCCGGACAGAGGGA 19 20 39

359. SSR500 GCCGCCGTTGTAGAACTATT TTCGTCATGAAGACTGCTGG 20 20 40

360. SSR501 TTGTAGATGCGAAGTTGGGA GCAGCATTGGAGGAATTTGT 20 20 40

361. SSR502 GACAGCTGACACCGCCAT TGGCAGCAGATCTTTGAGTG 18 20 38

362. SSR503 ATCGATCGCACCACCCT AAGCAGGACGCCCTATGAC 17 19 36

363. SSR504 GGGTCCATGACACCGACAT GTCAAAGGGCGATTAACCAA 19 20 39

364. SSR505 GCTGACACTGATACGCCATT CCTGACCGATCCCACTAGTTA 20 21 41

365. SSR506 AGATGTTTCAGTTCGCCAGG CTCTTTGCCTGTGTGGCTTT 20 20 40

366. SSR507 GCGAATCGACCCGCAT TCTATCTTAGAGCTCGCCGC 16 20 36

367. SSR508 TAAGCAACCACCAATGTTCA GCTCCCTGGAGTTTGTGAAG 20 20 40

368. SSR509 TCGACCATTGAGTACACACAGA TAAGCAAGGGTCCGTCAACT 22 20 42

369. SSR510 CCATGGGATGCTGGGAT ATAGTATGTCCCAAGTACGGGC 17 22 39

370. SSR511 CAGGGAGATAGATCGGAACG ACTATTTCTCCCGCTCTCGC 20 20 40

371. SSR512 CGAGCTCATAGATCGGCGT TTGCGGTCACCAGTGTTCTA 19 20 39

372. SSR513 GGCTTTGAACCGCTCTATACC CCGGCCATAAGACCAGTAAA 21 20 41

373. SSR514 AAATACATGATGCGGCGATT ATGGATAGCCAGCAGGTCAC 20 20 40

374. SSR515 AAGAACAGCTTGGGCAAAGA GGTTCTACAGAGGACGGCAC 20 20 40

375. SSR516 TACAGACAAGGGCAAGAGCA CGCCAAGCCTGAAATTAAAC 20 20 40

376. SSR517 AAACCACAACCAGGAGACAAA TTTAACTATGTGGTGGGAAGGG 21 22 43

377. SSR518 CCGATCCTTAGTAACGGCCT GGAACGTCCCTTGCAATCT 20 19 39

378. SSR519 CTTCAAAGATGCCTGGTGCT GGGAAAGAGAGAAAGAGGGAA 20 21 41

379. SSR520 CGCTTGAGGACGCTAGAACT ACCTTGGATGGTGGTGTGAT 20 20 40

380. SSR521 CTCTCTCGTCTGCAACCACC ATGGTGAGAATCGGTAACGC 20 20 40

381. SSR522 TGTACGAAAGGGTGCAGAAA TGCGTCCTTGTCTGTGTCTC 20 20 40

382. SSR523 GGCCGGACGACACACTA TCCATCGGCTTGTTAGCTTT 17 20 37

383. SSR524 CGATCGACTGTACCCACCTT TTTCCGTCACTCTAGGCGTT 20 20 40

384. SSR525 GGAGCGAAGCCGATGATA TATTGGCCTTTGTTTACGCC 18 20 38

385. SSR527 GAGCAGAAATGGCGACTCTT CGGACAGAGAAATGCACAGA 20 20 40

386. SSR528 GGAAATGTTGGTGGAATTGG ACCAAACGTGCATGAAACAA 20 20 40

387. SSR529 TTTGATAGCGAGTCTGGCG AATGAGCTCGGCATCACTCT 19 20 39

388. SSR530 GCCGTCGTTATCTTGTGGAT ACATCGTTCCGCCTCTATTG 20 20 40

389. SSR531 GGAATGCGTGTGAGGCA TTCGAGGGCTTACTGCATTT 17 20 37

390. SSR532 CGCCCAACAGTAGAAGAAGC GGAGCGAGACCTGTGTAAGC 20 20 40

391. SSR533 TTCTCCATGGTGCAGTTCTTT GGTTCATTAGTGGGCGAGAA 21 20 41

392. SSR534 GCAGGTTGATACTGGTGGCT ATAGCTCGCTCGCTCTTGAA 20 20 40

393. SSR535 TCTCCCTACAATGGGAGCAC CTGGCGATAGTGGGTTGTCT 20 20 40

394. SSR536 GATATGCGGCAACCACTTA GGGTCTGGGTGTAGAAATGTG 19 21 40

395. SSR537 TGCAACGCTAGCTGTCTATCA GTTGTGAGAGCGCACGTCTT 21 20 41

396. SSR538 CAATTGGTCGGTTCACTAATCA ATGCCTAAGCCTGTTCCTCA 22 20 42

397. SSR539 CCACACATACCAACACACCC TGTGTGATTGGTGGTTAAGGG 20 21 41

398. SSR540 ACGACGACAACAACATCGAC TGGACTCACAACTCAGCCAG 20 20 40

399. SSR541 GAGGCACACAAAGCTGATGA AGATCAGATTGATGGGCTGG 20 20 40

400. SSR542 ATGATGGCTGTCAGGTCGTA AGCTTCCGGAGTCTGTACCA 20 20 40

401. SSR543 TTCCCATGGACAAGGACAGT CCTAACTGCGGGTATCCAGA 20 20 40

402. SSR544 ACCGGTGGATCCAAGAGTTC GCTGAGGTAGCGTACTTGGC 20 20 40

403. SSR545 AATGAAGGCAACAATTTCCC TGGGACATCTAATGGTGGTG 20 20 40

404. SSR546 AGCCACCCATATTAATGATCC AAATTCCTTTCTGCTGCCC 21 19 40

405. SSR547 CCCTTTCTTCTTCTCCTCTGTG TGTTCAAACTCCGAAGACGA 22 20 42

406. SSR548 GAGTCTGCAAGCAAGCAGTG AAGCTGATTGCTCTGGCTTC 20 20 40

407. SSR549 CGGAAGACTCCTTCCTTCCA GAAGCCTGTTCTTCGATTGC 20 20 40

408. SSR550 CACAACGCTCTGATCTTCTCA AAGAGCCCTTGCCATACAGA 21 20 41

409. SSR551 CTTATCGATGGCCCATTTCA ATCAAATCAAGCTGAAGCCC 20 20 40

410. SSR552 ACAGCCACTAGGTTTCCGTC CACCCTGACTTCAGAAGAAGC 20 21 41

411. SSR553 CGTAGCTTCCAATTCTCCTCA AGCAGCAATCAGCTCTGTGA 21 20 41

412. SSR554 ATGCAAACAACACCAGATCA GATCAAAGATGTTTCACCGGA 20 21 41

413. SSR556 TGGAGGAGGTTAGGAGGAAGA AAGCTGAAACCAAAGCCAAA 21 20 41

414. SSR558 TTGCCATTTCATTCTCCTCC TAAGTTCCAGCATCATGCCA 20 20 40

415. SSR559 CCAGCAACAGCTCCACTTTA TTCTTGGATCCACAAGTCTTC 20 21 41

416. SSR560 GCTCTCTACAAGTGGAACTTTCTC CAACAGCCAGGAACAAGGAT 24 20 44

417. SSR561 GGGCGACAACAGTAGCATAA GGTCCATTCTTCAGGTCCAA 20 20 40

418. SSR562 GAGAACTGGCCTATCTGAATTA ACGAACTTGGAGCTGACCTG 22 20 42

419. SSR563 GCTCCGCCATACTCCTCTTA GACGGCAACTTTGTAACGGT 20 20 40

420. SSR564 GCACACGCACTCTCTCACTC CCACAATGGTGATGAACACAG 20 21 41

421. SSR566 TCTGCTTGCATTAGGGTGC GATTTGGCCAACATACCCAC 19 20 39

Appendices

~xxiii~

422. SSR567 TGGATTCTTCTTCACTAGAAGGG TTCCGAATTCGATGAGGTTT 23 20 43

423. SSR568 CTCTCTTCACTCTTTACAATTGCC CGTTTGCTGTTGTTGTTGCT 24 20 44

424. SSR569 TGGAGAAACCAATTCAAGATCC AACATCCACCTCCAACTGGT 22 20 42

425. SSR570 CCTAAAGAAGATAGGAAGAAATGCC CTTCCTCTACCCATCCCTCC 25 20 45

426. SSR571 TCAATCCATCACACCTTGGA GAGGAAGAAGACCACGCAAA 20 20 40

427. SSR573 TATGGAACTCTGCACTTGCG AATCCAGCACCTCCATCAAG 20 20 40

428. SSR574 ATGCAAATCGGTCATAAGGC TTTGACAATTAAGCTCGGCA 20 20 40

429. SSR575 GCAAGCCTGTGACAATTGAG GTCGTCGTGTTCCGTCATAA 20 20 40

430. SSR576 ACAGGGTGCTATACGTTCGG CAAATCCTTCTCTTCGGTGC 20 20 40

431. SSR577 TAGCTCCATTGCCAATTTCC CCAATTCATGCCAGTAACTTGA 20 22 42

432. SSR579 CTACCCTGGCATGGTCTCTG CCACAAGCGTTGTGAAGAAA 20 20 40

433. SSR581 GCTGCAGCTTCTGCG GTGGAACTCCAACAGGGTGT 15 20 35

434. SSR582 GGCAGGAGATTGGTTGCTTA TTCCTCCTGTTTCATGCATTC 20 21 41

435. SSR583 CGCGTCATAGAGAGAAAGGG TCATCTTGAGGAGGAGGATCA 20 21 41

436. SSR584 CAGCATCAACGGCTGAGAT TCAGCAGAGTTATGCCATGC 19 20 39

437. SSR585 CCCTTCTGTGAAACTACTCTTATC CCTCGTAGAGGTAAAGATGCAGA 24 23 47

438. SSR587 GGCAACATGTCACACTTGGT GAAGCTCCGGTCCAACATAA 20 20 40

439. SSR588 AAGAACAACAATCGCGAACC GCACAACGATTTGACTGCAC 20 20 40

440. SSR591 ATCTCCTTGGCCTCCTGTTT GTCATGGCCACATGAATACG 20 20 40

441. SSR592 CCGAGGCGAATCTTGAATAC CCACCACCTTACTTTCCTCG 20 20 40

442. SSR597 GTCTTTACACAATCTCTCACTTGG CTGGTCACTGAACTGCCAGA 24 20 44

443. SSR600 CGGAAAGTTCTCTCAAAGGAG CACAAAGAGAGCATATGTGAGCA 21 23 44

444. SSR604 CAGCAGCATCAGCATCAAAT TTGCAATTGACTCATCCTCG 20 20 40

445. SSR607 TTGTGGAAGTGTATGGAAGCTG GCCGAATGGTGACTTCAAA 22 19 41

446. SSR608 TTGCCCTCCTTGAAATATCG GGAATGCTCTTGAAAGTGGG 20 20 40

447. SSR609 GGCACGAGCACATATAGAAGAG CCCTTCTTGCTCCGATTACA 22 20 42

Appendices

~xxiv~

Table 6: Genotypic ratio of parent (P1 and P2) types with RILs.

S.No. Primers

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

P-1 a a a a a a a a a a a a a a a a a a a a a a a a a

P-2 b b b b b b b b b b b b b b b b b b b b b b b b b

1 a b a b a a b a a b b b a b b a a b a a a b b b a

2 b b a a b a a b a b a b a b b a b a b a a b a b a

3 b a b a b a b a a b b b a b a b b a b a a b b b a

4 b b a a b b a a b b b b a b b b a a b b b b b b a

5 b a b a b a b b a a b b b b a b a b b b a a b b b

6 a a b a a b a b a b b a b b a a a b a b a b b a b

7 b a b b a b b b b a b b a b b b a b b a b a b b a

8 b b b a a b a a b a b b b b a b a b a b b a b b b

9 a a a a b a b a a b b b a b a b b b a b a b b b a

10 a a b a a b a b a a b b b b a b b a b a a a b b b

11 b a a a b a a a a a a b b a a b b a a b a a a b b

12 a a a b a b a b a a a a b a a b b b a a a a a a b

13 b b b a b a a b b b a b a a b b b b b b b b a b a

14 b b b b a b a a b a b b a b b a b a a a b a b b a

15 a a a a b a b a a b b a b b a a a b a b a b b a b

16 b a b a a b a b a b b a b b a b a b b b a b b a b

17 a b a a b a b a b a b a b b b b a a b a b a b a b

18 a b b a b a b a b a b b a b b b a a a b b a b b a

19 a b a b a a b b b b a b b b b b a b b b b b a b b

20 a a b a a b a b a b a a b b b a a b a b a b a a b

21 b a b a a b a b b a b b a b a b a b a b b a b b a

22 a a b a a b b a a a a a a b a a b a a b a a a a a

23 - b a a a b a - a a b a a b a b a b a - a a b a a

24 b b a b b a b b a b b b a b b b a b b b a b b b a

25 a a b b b a - b a b b b a b b a b b b a a b b b a

26 b b b b a b - a b a b b a b b a b a b a b a b b a

27 a a a b b a - b a b b a - b b a b a b b a b b a -

28 b b b b b b b a b b b a - b b b a b a b b b b a -

29 b b a b a a b a b b b a b a a b a b a b b b b a b

30 b a b a b b - b b b b a b a a b a b b a b b b a b

31 a a b a b a b b b b - b - a b a a b b a b b - b -

32 a b a b a a a b a b a b b a b a b b a b a b a b b

33 b b b b a b b a b b b b a a b b a a b a b b b b a

Appendices

~xxv~

34 - a b a b a b a b a b b b a b a b a b - b a b b b

35 a b b b b a a b a a a b a b a b b a b a a a a b a

36 a b b a b a b b a b b a b b b a b b a a a b b a b

37 a b a b b b a a b a b b b a b a b b a b b a b b b

38 b b b b a b b a a b - b a b b a b a b a a b - b a

39 - b b - b b a b b b b b a b b b b b b b b b b b a

40 b a b b b a b a b b a b b a a b b a b a b b a b b

41 a b a a b a a a b a b b a a b a b b a b b a b b a

42 b a a a b a a a b b a b b a b a b b b a b b a b b

43 b b a a b b a b a b a b a a b a b b a b a b a b a

44 a a b a b b b b a b - a b a b b b b a b a b - a b

45 b b a a b a b b a b b a a b b a a b a b a b b a a

46 a a a a b b b b a b b b - b a b a a b b a b b b -

47 - b b - b b - b b b b - - a b b b b b b b b b - -

48 b b a a b b a b a a b a b b a b b a b a a a b a b

49 a a b b b a a b b b a b b a b a a b a a b b a b b

50 b b a b b a a b a b a b a b a b b a a b a b a b a

51 b a b b a b a b b b b b a b a b b b b b b b b b a

52 b b b b b b b b b a b b b b a b b b a b b a b b b

53 - a b a b b a b b a a b b - b a - a b b b a a b b

54 - b b b b - a b b b - - b b b b b a b b b b - - b

55 - b a a b a b b a - b b a b a b a b b b a - b b a

56 a a b b b b a b a b a b a a a b a b a b a b a b a

57 a b a a b b a b a b b b - b b b a a b b a b b b -

58 b b b a b b b a b a b a b b b b a b b b b a b a b

59 b b b a b b a b a a b b a b b b b a b b a a b b a

60 a a b a b b a a b a b b b a b a b a a b b a b b b

61 b b a a b b a b a a b a b a b a b a b b a a b a b

62 a a b a b b a a b a b a b b a b a b b b b a b a b

63 b b b b b b a b a a b a b b b a b b a b a a b a b

64 a a b b b a b b b a b a b b a b a a b b b a b a b

65 b b b a b b b b a b b a b a b b b b b b a b b a b

66 b b a b b b a b b a a b a a b a b a a b b a a b a

67 - b a a a b a - a a b a a a a b a b a a a a b a a

68 a b a a b a b a b b a a b a b a b a b a b b a a b

69 a a b a b b b a b b a a b b a b a b b a b b a a b

70 a a a b b b a a b b a a b b a a b a b a b b a a b

71 a a b b b b b b a b b a b b a b a b b a a b b a b

72 b b a b b b a b a a b b b b b a a b a a a a b b b

73 b b b b b a a a b b a a b a b b b b a a b b a a b

74 a b a b b a b b b a a a b a b a a b a b b a a a b

Appendices

~xxvi~

75 b b a b a b a b a a a b a b b a b a a b a a a b a

76 a a a b a b a b a a b a b b a b a b a b a a b a b

77 b a b b b a b b a b a b a b a b a b b b a b a b a

78 b b b a b a b a b a b b a b b a a b b b b a b b a

79 b b b a b a b b b a b a b a b a a b b a b a b a b

80 b a a b a a a a b b a b a a b a a b a b b b a b a

81 a b b b a a b a b a a a a a b a b a b a b a a a a

82 b a a b a b b a b a b a a b b b a b b b b a b a a

83 b b a b a b b b a b a b - b a a a b a b a b a b -

84 b b a a b b b b a b b b b a b b a b a b a b b b b

85 b b a a b a b b a b b a b a b b b b b b a b b a b

86 a b a b b a a b a a a b b a b a b a b a a a a b b

87 - a a a a a b a a a a b a b a a b b a a a a a b a

88 b b b b b b b b a b b b b b b a b b b a a b b b b

89 a b a b b a a a a b a b a b b a a b a a a b a b a

90 b a a a b b a a b a b a b a b a a b a b b a b a b

91 a b b a a b a b b a b a b b a b b b a b b a b a b

92 b b a b b b a b a b b a a b b b b a a b a b b a a

93 - b b a b - a b b a - - - b a b - a b b b a - - -

94 - a a a a b a a b a b a - a a b a a a a b a b a -

95 a b a a b a a a a b b a a a b b a a a a a b b a a

96 b b b a a a b a a b a a a a b a b b a a a b a a a

97 b a b b a b b b b a a b a b a b a b b a b a a b a

98 b b b b b b b b a b a b a b b b a b b a a b a b a

99 b a a b a a b a a a b a b b a a b a b a a a b a b

100 b b b b a a b a b a a a b a b a b a a a b a a a b

101 a a a b a a b a b a b a b a b a b b a b b a b a b

102 b b b a b b b b a b b b a a b b b a b b a b b b a

103 - a b b - - a b b a b b a b b a - a b b b a b b a

104 a b a a b b b a b a b b a a b b b a b a b a b b a

105 a a b a b a b a b a b a b a a b b a b a b a b a b

106 a b b b a a b a b b a a b a b a b a b a b b a a b

107 a a a b a b b a b a b a b a a a b b a b b a b a b

108 a b b a a b - b a b b a b a b b a a b a a b b a b

109 b b a a b a a b b a b a b a b b b b b a b a b a b

110 a a b b b b b b a b a b a b a b a b a b a b a b a

111 a b a a b a b b a b a b a b a b a b b b a b a b a

112 a a a b a a b a b a b a a b a b a b a a b a b a a

113 - b b b a b a a b - a b a b a b a b a - b - a b a

114 a a a b b a a b b a b a b a b a a b a b b a b a b

115 b b a b b a b b a b a b a a b b a a b a a b a b a

Appendices

~xxvii~

116 a b b b b a a a b a - a a b b a a b a a b a - a a

117 a a b a b a a b a b a a b a a a b a a a a b a a b

118 a a b a a a a b b a b a - a a a b b a a b a b a -

119 b a a a b a b b a b a b a b a a a a b b a b a b a

120 a a b a a b a b a b a b a b a a a a b a a b a b a

121 b b a a b b a b a b a b a b a b a a a b a b a b a

122 a b b a b a b b b b a b b b b a a b b b b b a b b

123 a a a a a b a b b a b b b a a b b a b b b a b b b

124 b a b b a b a b a b b b b a a b b b a b a b b b b

125 b a a b a b a b a a b a b b a a b b a b a a b a b

126 a a b a b a - b a b a b a b a a b a b a a b a b a

127 b b b b b a - b b a b a b a b a b a b a b a b a b

128 a a a b a b - b a a b a b a a b b a b a a a b a b

129 b a b b b a - b a b a b a b a a b b a b a b a b a

130 a a a b b a b a b a - b b a a b a a b a b a - b b

131 b b b b b b b a b a b b b a b b b b a b b a b b b

132 b a b b a b - b a a b a b a b a b a b a a a b a b

133 b a b b b b - b a b b a b a a a b a b b a b b a b

134 - a a a a - b a a a a a b a - b a a a a a a a a b

135 b b a b a a b b a b a b a a b a b b a a a b a b a

136 b b a b b a b b a b a b a a b a a b a a a b a b a

137 a a b b b a a b a b a a a b a a b b a b a b a a a

138 a a a b b a a b a b a b b a a b a b a b a b a b b

139 b b a b a b a b a b a b a b a b a b a b a b a b a

140 - a a a b a a a a a a b a a a b a a a a a a a b a

141 - a a a a - b a a a b a b a a b a a a a a a b a b

142 a a a b a b - b b a a b a b a a b b a b b a a b a

143 b a b b a a - b a a b a b a a b b a b a a a b a b

144 a b a b b a b a b a - b b a a b a a b a b a - b b

145 b A b b b a b b a b b b b a a b b b b b a b b b b

146 b A b b a b - b a a b a b a b a b a a b a a b a b

147 b A b b b a - b b a b b a a a b b a a b b a b b a

148 - A a a a - a a a a b a b a - b a a a a a a b a b

149 b B a b a b a b b a b a a b b a b a a b b a b a a

150 b B a b a b a b b a b a a b a a b a a b b a b a a

151 a A b b b a a b a b a a a b a a b b a b a b a a a

Primers (1=SSR316; 2= SSR308; 3= SSR341; 4= SSR65; 5= SSR448; 6= SSR40; 7= SSR356; 8= SSR605; 9= SSR5; 10= SSR11; 11= SSR27; 12= SSR72; 13= SSR603; 14=

SSR310; 15= SSR13; 16= SSR108; 17= SSR286; 18= SSR304; 19= SSR45; 20= SSR335; 21= SSR73; 22= SSR34; 23= SSR526; 24= SSR46; 25= SSR20)

Characterization of Alternaria solani and Molecular Mapping of … · 2018-12-29 ·...

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