Characterization and Management of Myrothecium Roridum Associated with Myrothecium Leaf Spot Disease of Momordica Charantia L. (Bitter Gourd) in Punjab, Pakistan

CHARACTERIZATION AND MANAGEMENT OF MYROTHECIUM RORIDUM ASSOCIATED WITH MYROTHECIUM LEAF SPOT DISEASE OF MOMORDICA CHARANTIA L. (BITTER GOURD) IN PUNJAB, PAKISTAN

SUMERA NAZ

INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN 2015

CHARACTERIZATION AND MANAGEMENT OF MYROTHECIUM RORIDUM ASSOCIATED WITH MYROTHECIUM LEAF SPOT DISEASE OF MOMORDICA CHARANTIA L. (BITTER GOURD) IN PUNJAB, PAKISTAN

The thesis submitted to the University of the Punjab, Lahore in partial fulfillment of the requirement for the degree of doctor of philosophy in Agricultural Sciences (Plant Pathology)

Sumera Naz

Supervisors

Dr. Salik Nawaz Khan Dr. Ghulam Mohy-ud-Din

INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN 2015

CERTIFICATE This is to certify that the research work entitled entitled “Characterization and management of Myrothecium roridum associated with Myrothecium leaf spot disease of Momordica charantia L. (bitter gourd) in Punjab, Pakistan” described in this thesis by Ms. Sumera Naz, is an original work of Ph.D. scholar and has been carried out under my direct supervision. I have personally gone through all the data, results, materials reported in the manuscript and certify their correctness and authenticity. I further certify that the material included in this thesis has not been used in part or full in a manuscript already submitted or in the process of submission in partial or complete fulfillment of the award of any other degree from any institution. I also certify that the thesis has been prepared under our supervision according to the prescribed format and endorse its evaluation for the award of Ph.D. degree through the official procedure of the University of the Punjab, Lahore, Pakistan. Here, thesis is in pure academic language and it is free from typos and grammatical errors.

Supervisor:

Dr. Salik Nawaz Khan Assistant Professor IAGS, PU, Lahore

Co-supervisor:

Dr. Ghulam Mohy-ud-Din Director/Plant Pathologist AARI, Jhang Road, Faisalabad

Date: ______

DECLARATION CERTIFICATE

This thesis entitled “Characterization and management of Myrothecium roridum associated with Myrothecium leaf spot disease of Momordica charantia L. (bitter gourd) in Punjab, Pakistan” which is being submitted for the award of degree of Ph.D. in the University of the Punjab does not contain any material which has been submitted for the award of Ph.D. degree in any University and, to the best of my knowledge and belief, neither does this thesis contain any material published or written previously by another person, except when due reference is made to the source in the text of the thesis.

(Sumera Naz) Ph.D. Scholar

Dedicated to My Beloved Parents, Mr. & Mrs. Muhammad Ilyas Chattha & Siblings

Qaisar Ilyas (Late), Amara Ilyas & Yasir Ilyas

Who are symbol of my strength & confidence, their prayers & love enabled me to reach this milestone in life

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ACKNOWLEDGMENTS

All the praises and respects to Almighty Allah-the only creator of the Universe, and to His Holy Prophet Muhammad (Peace be upon him) who is forever beacon of knowledge and guidance for humanity as a whole. I have no words to express my deepest sense of gratitude to Almighty Allah, Who, in spite of copious obscurity and acute frustrations, enabled me to complete this thesis. First and foremost, I would like to express my profound gratitude to my respected research supervisors Dr. Salik Nawaz Khan, Assistant Professor, Institute of Agricultural Sciences, University of the Punjab, Lahore and Dr. Ghulam Mohy-ud-Din, Director Plant Pathology Section, Ayub Agriculture Research Institute, Faisalabad for their enthusiastic concern, valuable supervision and invaluable suggestions. Their guidance enabled me to complete my work successfully. I would like to extend my special thanks to Dr. M. Saleem Haider, Professor and Director, Institute of Agricultural Sciences, University of the Punjab, Lahore for his sincere facilitation without which completion of this work was not possible. I am thankful to Dr. Naureen Shahrukh, Assistant Professor and Incharge First Fungal Culture Bank of Pakistan, for critically reviewing the manuscript. The help rendered by all faculty members, especially Dr. Arshad Javaid and Dr. Sajid Ali, Institute of Agricultural Sciences, University of the Punjab, Lahore is gratefully acknowledged. I don’t find words to express my sincerest and limitless thanks to Dr. Muhammad Najeebullah, Ginger Botanist, Vegetable Research Institute, AARI, Faisalabad and Malik Muhammad Fiaz, Ex-Director General, Pest Warning & Quality Control of Pesticides, Punjab, Lahore, without their kind cooperation it was almost impossible to initiate the project. I also thank Phytopathology Group Leader and Dr. Jaffargholi Imani, Institute of Phytopathology and Applied Zoology, Justus-Liebig- University, Giessen, Germany for their contributions to host pathogen interaction studies. I am grateful to the Higher Education Commission, Pakistan, who provided two fellowships for completion of this degree and funding for the research under Indigenous 5000 PhD Fellowship Program (Batch VII November, 2011-October, 2015) and International Research Support Initiative Program (IRSIP January-July, 2014). Thanks are due to Ms. Faiza, Mr. Amjad, and Mr. Iqbal, Lab. Attendants; Mr. Asif Nadeem, Composer, for supporting me in the lab. I also wish to express my appreciation to all those who helped me in one way or the other in completion of my thesis especially Ms. Shumaila Farooq, Mr. Waheed Akram, Ms. Saba Ghazanfar, Ms. Hina Nazli, Ms. Rabia Akram and PhD fellows. Last but not least, I am most gratifying to my Parents, for their prayers, support, and encouragement throughout my educational journey. I have no words to convey my love and affection to my sister, Ms. Amara Ilyas and brother, Mr. Yasir Ilyas for their precious love to me and for their patience.

Sumera Naz

TABLE OF CONTENTS

Title Page # Certificate i Declaration certificate ii Dedications iii Acknowledgments iv Table of contents v List of figures x List of plates xii List of tables xiii List of research papers published from thesis xiv Abbreviations xv Summary xvii

1. Introduction 1.1. Momordica charantia 1 1.1.1 Plant taxonomy 1 1.1.2 Plant description 2 1.1.3 Nutritional value 2 1.1.4 Medicinal value 4 1.1.5 Industrial value 5 1.2. Crop statistics in Pakistan 6 1.3. State Policies 6 1.4. Production constraints 7 1.5. Pathological constraints 7 1.6. Common fungal diseases 7 1.7. Review of literature 8 1.8. Genus Myrothecium 8 1.9. Myrothecium roridum taxonomy 10 1.9.1. Geographical distribution and host range 11 1.9.2. History of Myrothecium leaf spot disease of bitter gourd in 12 Pakistan 1.9.3. Aggressiveness behavior 12 1.9.4. Germplasm screening for resistance 13

1.9.5. Management Strategies 14 1.9.5.1. Intercropping 14 1.9.5.2. Plant aqueous extract 14 1.9.5.3. Chemical control 15 1.9.5.4. IPM strategies 15 Objectives 16

2. Materials and Methods

2.1. Geographical distribution of Myrothecium roridum in Punjab, Pakistan 17

2.1.1. Survey 17 2.1.2. Disease assessment 19 2.1.3. Specimen collection 21 2.1.4. Isolation of associated Fungi 21 2.1.5. Identification of fungus 21 2.1.6. Application of Koch‟s postulates 22 2.1.7. Culture authentication 22 2.2. Characterization of Myrothecium roridum 22 2.2.1. Evaluation of virulence of isolates 22 2.2.2. Morphological studies 23 2.2.3. Physiological studies 25 2.2.3.1. Optimization of nutrients 26 2.2.3.2. Optimization of temperature 26 2.2.3.3. Optimization of pH 26 2.2.3.4. Effect of photoperiod 26 2.2.3.4. Optimization of culture age & Virulence 27 Assessment 2.2.4. Genetic variation studies 27 2.2.4.1. Preparation of solution 27 2.2.4.2. Extraction protocol 28 2.2.4.3. Estimation of Extracted DNA 28 2.2.4.4. DNA Quality Analysis through Agarose Gel 29 electrophoresis 2.2.4.5. Random Amplification of Polymorphic DNA 29 (RAPD) Analysis 2.2.4.5.1. Random Primer Screening 29 vi

2.2.4.5.2. Amplification reaction 31 2.2.4.5.3. RAPD Temperature Cycling Conditions 31 2.2.4.5.4. Analysis of Amplified DNA Fragments 31 2.2.4.5.5. RAPD data analysis 32 2.3. Development pattern of Myrothecium roridum within host leaf 32 and root tissues 2.3.1. WGA-AF 488 staining 32 2.3.1.1. Preparation of ½ MS medium 32 2.3.1.2. Preparation of 1 X PBS buffer 33 2.3.1.3. WGA-AF 488 33 2.3.1.4. Preparation of spore suspension 33 2.3.1.5. Preparation of leaf and root samples 34 2.3.1.6. Staining 34 2.3.2. Transmission electron microscopy 34 2.3.2.1. Sample preparation 34 2.3.2.2. Fixation 35 2.3.2.3. TEM analysis 35 2.4. In vivo Screening of bitter gourd germplasm 35 2.4.1. Soil sterilization 36 2.4.2. Susceptibility reaction in pot under natural environmental 36 conditions 2.4.3. Susceptibility reaction in field under natural 36 environmental conditions 2.5. Disease management strategies 37 2.5.1. Efficacy of plant aqueous extracts against MLS 37 2.5.1.1.Collection of plants 37 2.5.1.2. Preparation of aqueous extract 37 2.5.1.3. In vitroexperiment 37 2.5.1.4. In vivo experiment 38 2.5.2. Intercropping of aromatic plants 38 2.5.3. Efficacy of commercial fungicides against MLS disease 40 2.5.3.1. Commercial Fungicides 40 2.5.3.1.1. In vitro experiment 40 2.5.3.1.2. In vivo experiment 40 2.5.3.2. Technical grade fungicides 41 2.5.3.2.1. In vitro experiment 41

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2.6. Statistical analysis 41 3. Results 42

3.1. Geographical distribution of Myrotheciumroridum in Punjab, Pakistan 42 3.1.1. Survey and disease assessment 42 3.1.2. Isolation and microscopic identification 48 3.1.3. Molecular studies 48 3.2. Characterization of Myrothecium roridum 50 3.2.1. Aggressiveness evaluation 50 3.2.2. Morphological studies 52 3.2.3. Microscopic studies 52 3.2.4. Physiological response 55 3.2.4.1. Growth medium studies 55 3.2.4.2. Optimization of incubation temperature 55 3.2.4.3. Optimization of growth medium pH 55 3.2.4.4. Optimization of photoperiod for fungal growth 56 3.2.4.5. Culture age and virulence relationship 56 3.2.5. Study of genetic variation among selected isolates 60 3.3. Development pattern of Myrothecium roridum within host leaf 65 and root tissues 3.4. In vivo screening of bitter gourd germplasm 72 3.4.1. Pot experiment 72 3.4.2. Effect of MLS susceptibility reaction on agronomic traits 72 of bitter gourd in pot experiment 3.4.3. Field experiment 78 3.4.4. Effect of MLS susceptibility reaction on agronomic traits 78 of bitter gourd under field conditions 3.5. Disease management strategies 84 3.5.1. Efficacy of weed aqueous extracts against Myrothecium. 84 roridum 3.5.1.1. In vitro studies 84 3.5.1.2. Invivo studies 90 3.5.1.2.1 Pot experiment 90 3.5.1.2.2. Field experiment 93 3.5.2. Inter-cropping 96 3.5.2.1 Pot experiment 96 3.5.2.2 Field experiment 99

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3.5.3. Efficacy of different fungicides against Myrothecium 101 roridum 3.5.3.1. Commercial Fungicides 101 3.5.3.2. In vitro studies 101 3.5.3.2. In vivo studies 104 3.5.3.2.1. Pot experiment 104 3.5.3.2.2. Field experiment 107 3.5.3.2. Technical grade fungicide 110 3.5.3.2.1. In vitro studies 110 4. Discussion 113

4.1. Geographical distribution of Myrothecium roridum in Punjab, Pakistan 114 4.2. Characterization studies of Myrothecium roridum 115 4.2.1. Pathogen aggressiveness 115 4.2.2. Morphological studies 117 4.2.2. Physiological studies 117 4.2.3. Genetic characterization 118 4.3. Development pattern of Myrothecium roridum within host leaf 120 and root tissues 4.4. In vivo screening of bitter gourd germplasm 121 4.5. Disease management practices 123 4.5.1. Efficacy of different weed extracts against Myrothecium 123 roridum 4.5.1. Inter-cropping 124 4.5.3. Efficacy of different fungicides against Myrothecium 126 roridum

Conclusions and recommendations 128

5. References 129 Annexures 148 Annexure II 148 Annexure III 155 Annexure IV 157 Annexure V 158 Annexure VI 159 Research Papers Published from Thesis

LIST OF FIGURES

Title Page # Fig. 2.1. Modified PARC agro-ecological zone Map of Punjab, 18 Pakistan observed for MLS of bitter gourd in 2011-13. Fig. 2.2. Illustration of 0-5 visual disease severity rating scale of 20 MLSD of bitter gourd Fig. 2.3. Schematic description of colony growth and sporodochia 25 production in relation to virulence by optimizing fungal cultivation conditions Fig. 3.1. Geographical distribution map of Myrothecium leaf spot of 45 bitter gourd in agro-ecological zones of Punjab, Pakistan during 2011-13. Fig. 3.2. Effect of nutrient medium on colony growth and sporulation 57 of Myrothecium roridum. Fig. 3.3. Effect of different temperatures on colony growth and 57 sporulation ofMyrothecium roridum. Fig. 3.4. Effect of pH on colony growth and sporulation of 58 Myrothecium roridum. Fig. 3.5. Effect of photoperiod on colony growth and sporulation of 58 Myrothecium roridum. Fig. 3.6. Evaluation of culture age on infection development and 59 spore production ofMyrothecium roridum on bitter gourd leaves. Fig. 3.7. Dendrogram of Myrothecium roridum isolates developed 64 from RAPD data using Ward‟s Linkage method. Fig. 3.8. Effect of different aqueous weed extracts on colony growth 87 of Myrothecium roridum. Fig 3.9. Evaluation of growth inhibition of Myrothecium roridum 88 against aqueous weed extract Fig. 3.10. Disease incidence of Myrothecium roridum against weed 91 aqueous extracts in pot experiment. Fig. 3.11. Disease reduction of Myrothecium roridum against weed 92 aqueous extracts in pot experiment. Fig. 3.12. Disease incidence of Myrothecium roridum against weed 94 aqueous extracts in field experiment.

Fig. 3.13. Disease reduction of Myrothecium roridum against weed 95 aqueous extracts in field experiment. Fig. 3.14. Disease incidence of Myrothecium roridum against 98 intercropping of aromatic medicinal and condiment crops in pot experiment. Fig. 3.15. Disease reduction over control of different intercropped 98 plants on Myrothecium roridum infection development. Fig. 3.16. Disease incidence % of Myrothecium roridum against 100 intercropping of aromatic medicinal and condiment crops in field experiment. Fig. 3.17. Disease reduction over control of different intercropped 100 plants on Myrothecium roridum infection development. Fig. 3.18. Effect of different fungicides on colony growth of 103 Myrothecium roridum. Fig. 3.19. Disease reduction over control of fungicides against 103 Myrothecium roridum. Fig. 3.20. Disease incidence of Myrothecium roridum against 105 fungicides in pot experiment. Fig. 3.21. Percent disease reduction % of fungicides against 106 Myrothecium roridumin pot experiment. Fig. 3.22. Disease incidence of Myrothecium roridum against 108 fungicides in field experiment Fig. 3.23. Percent disease reduction % of fungicides against 109 Myrothecium roridum in field experiment. Fig. 3.24. Growth percentage of Myrothecium roridum spores under 112 different concentrations of tebuconazole. Fig. 3.25. Percent inhibition over control of Myrothecium roridum 112 against different concentrations of tebuconazole.

LIST OF PLATES

Title Page #

Plate 1.1. Typical bitter gourd plant 3

Plate 3.1. Symptomatic variations pattern of Myrothecium leaf spot 44 disease in mixed cropping zone of Punjab.

Plate 3.2. Characteristic identification features of Myrothecium 49 roridum.

Plate 3.3. Morphological variations in colony growth of Myrothecium 53 roridum isolated from commercial bitter gourd germplasm.

Plate 3.4. Amplification profiles of Myrothecium roridum isolates 61

Plate 3.5. Infection development of Myrothecium roridum 66 establishment in bitter gourd tissue.

Plate 3.6. Infection development of Myrothecium roridum 67 establishment in bitter gourd tissue.

Plate 3.7. Transmission electron micrographs of the adaxial foliar 69 surface of bitter gourd inoculated with Myrothecium roridum.

Plate 3.8 Transmission electron micrographs of the abaxial foliar 71 surface of bitter gourd inoculated with Myrothecium roridum.

Plate 3.9. Myrothecium roridum colony growth reaction against 86 various weed aqueous extracts

Plate 3.10 Pot and field experimental layout description under natural 97 environmental conditions

Plate 3.11. Effect of commercial fungicides on colony growth of 102 Myrothecium roridum.

Plate 3.12. Fungicidal activity of various concentration of 111 Tebucanazole on Myrothecium roridum.

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LIST OF TABLES

Title Page # Table 2.1. Visual colony characteristics of Myrothecium roridum 24 Table 2.2. Code and Sequence of primers used for PCR analysis. 30 Table 2.3. PCR Reaction mixture 31 Table 2.4. Inventory of seeding material used for intercropping 39 Table 3.1. Geographical distribution of Myrothecium roridum in agro 46 ecological zones of the Punjab, Pakistan Table 3.2. Geographical distribution of Myrothecium roridumduring 47 2011-13 in mixed cropping zone of the Punjab, Pakistan Table 3.3. Aggressiveness of population of Myrothecium roridum 51 against bitter gourd under natural environmental conditions in the pots Table 3.4. Morphological and cultural variation among local 54 population Myrothecium roridum isolated from commercial bitter gourd germplasm Table 3.5. Similarity matrix of local population of Myrothecium 62 roridum isolated from commercial bitter gourd germplasm Table 3.6. RAPD fingerprints data for the molecular characterization 63 of local population of Myrothecium roridum Table 3.7. Susceptibility reaction of tested germplasm against 74 myrothecium leaf spot disease in pot experiment under natural environmental conditions Table 3.8. Effect of MLS susceptibility reaction on agronomic traits of 76 bitter gourd in pot experiment under natural environmental conditions Table 3.9. Correlation of agronomic traits against susceptibility 77 reaction in pot experiment under natural environmental condition Table 3.10. Susceptibility reaction of tested germplasm against 80 myrothecium leaf spot disease under in vivo conditions Table 3.11. Effect of MLS susceptibility reaction on agronomic traits of 82 bitter gourd in field experiment under natural environmental conditions Table 3.12. Correlation of agronomic traits against susceptibility 83 reaction in field experiment under natural environmental condition Table 3.13. Assessment of macroscopic colony characters of 89 Myrothecium roridum under the stress of weed aqueous extracts

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LIST OF RESEARCH PAPERS PUBLISHED FROM THESIS

Sr Vol./P Author/Title Journal Year # age

Salik Nawaz Khan, SumeraNaz, Ghulam Mohy-ud-Din, Shumaila Farooq, Muhammad Najeebullah, Fungicidal activity of aromatic Science 27(3): 1 medicinal plants against Myrothecium International 2015 2169- roridum Tode associated with myrothecium (Lahore) 2172 leaf spot disease of Momordica charantia L. (bitter gourd)

SumeraNaz, Salik Nawaz Khan, Ghulam Pakistan Mohy-Ud-Din and Shumaila Farooq, In vitro Journal of 21(3): 2 fungicidal activity of aqueous extracts of crop Weed 2015 369- and wasteland weeds against Myrothecium Science 379 roridum Tode Research

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LIST OF SIGNS AND ABBREVIATIONS

% Percentage L/mL Micro liter per milliliter ° C Degree Celsius µg/ mL Micro gram per milliliter µL Microliter µm Micrometer AARI Ayub Agriculture Research Institute ANOVA Analysis of variance BGA Bitter gourd agar bp Base pairs CDA Czapekdox agar CL Candidate line cm Centimeter CRD Completely randomized design CTAB Cetyltrimethyl ammonium bromide DAG Days after germination dai Days after inoculation DAS Days after sowing ddH2O Double-distilled water DG Khan Dera Ghazi Khan DI Disease incidence DNA Deoxyribo nucleic acid dNTP Dinitrotriphosphate DR Disease reduction DSR Disease severity rating EDTA Ethylene-diamineteraacetic acid ELISA Enzyme-linked immunosorbent assay Fig Figure g Gram g/L Gram per liter H Hybrid HCl Hydrochloric acid HSD Honest significant difference i.e. That is kPa Kilo Pascal L Liter mcg Microgram MEA Malt extract agar mg Milligram mL Milliliter MLS Myrothecium leaf spot mm Millimeter mM Millimolar 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium MTT bromide

NCBI National Center for Biotechnology Information ng Nanogram nm Nanometer OD Optical density PARC Pakistan agriculture research council PBS Phosphate-buffered saline PCR Polymerase chain reaction PDA Potato dextrose agar pH Hydrogen ion concentration pMol Picomoles PPM Parts per million R&D Research and development RCBD Randomized complete block design rpm Revolutions per minute TAE Tris base:aceticacid:EDTA TEM Transmission electron microscopy VSRS Visual severity rating scale WGA-AF 488 Wheat germ agglutinin-Alexa Fluor 488

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Summary

Momordica charantia (bitter gourd) is among one of the most liked vegetable in world because of its medicinal and nutritive value. Its‟ per hectare yield in Pakistan is much lower than its neighboring countries like China and India. Among various management factors diseases play a major role in low yield. Among the diseases Myrothecium leaf spot (MLS) has been noticed as emerging threat for bitter gourd and other cucurbits. Before initiating for the project investigation, a small scale survey was conducted to investigate disease severity and distribution pattern and for designing of structured questionnaire in collaboration with Pest Warning & Quality Control of Pesticides wing, Punjab, Lahore, and Ayub Agriculture Research Institute Faisalabad. Survey area of the Punjab province was divided into Various zones viz Rice Zone, Cotton Zone, Mixed Cropping Zone, Thal Zone, Barani (Rain fed) Zone and DG Khan zone as described by PARC (Pakistan Agricultural Research Council) zonal allocation system. These zones vary significantly with respect to climate soil, and crop management strategies. Disease index for the survey period ranged 25-30% According to 0-5 visual disease severity Scale (VSRS) disease severity ranged 1-4 on for mixed cropping zone while in DG Khan it ranged from 0-2 during the surveyed years. Cultivation of vegetable in tunnels makes it more susceptible because fungal inoculums and availability of alternate host make situation worst. Myrothecium roridum strain Mr 10 (accession # FCBP 1155) was deposited in first fungal culture bank, Pakistan and M. roridum strain Mr 37 (accession # DSM 28971) in Leibniz- Institute-DSMZ, Germany.

Virulence of Myrothecium roridum population was evaluated against commercial variety “Jaunpuri” @ 1 x 105spore concentration under pot and field conditions. Out of the 54 test isolates, 23 were highly, 17 moderately and 14 were less aggressive and none fall under Avirulent category. The size of spore ranged in length from 5-8 µm and in width to 1.4-1.8µm. Physiological attributes influencing radial growth and subsequently conidia and sporodochia production of M. roridum were analyzed. PDA medium was found best to support the mycelia growth followed by BGA medium. M. roridum produced circular, flat colonies with floccose texture and filiform margins on PDA at 25 °C. The highest radial growth was observed at pH 5.0 followed by pH 5.5 with 16/8 h light/darkness period at 30 °C whereas sporodochia production was highest at 35 °C. xvii

Genetic variation among the test isolates, 13 primers were selectedfor RAPD amplification which produced 93 bands in test isolates. Out of 93 DNA fragments, 28% were monomorphic whereas 72% were polymorphic bands. The isolates Mr31 and Mr54 collected from Faisalabad and DG Khan made a clear subgroup A (genetic distance = 0.68). Similarity of M. roridum isolates originated from distant agro- ecological zones may be due to active movement of infected germplam. The attempts were made to understand interaction of M. roridum with M. charantia. Conidial germination and emergence of germ tube was observed on light microscope while combination of light, transmission electron and fluorescent microscope, germ tube emergence was observed after 6 hours and directly penetrate the host cells. Hyphae and conidia were seen in leaf veins beyond point of infection, leading to assumption that fungus is able to use vascular system for transmission within host. Susceptibility reaction of M. charantia commercial cultivars and candidate lines was investigated in pot and field experiments under natural environmental conditions. During early growth stages there was great similarity in symptomatic development of infection expressions whereas on later stages of plant growth trend of infection development changed under pot and field conditions. The potted plant exhibited 75-90 days life cycle and higher mortality at fruiting stage. Whereas under field conditions no plant mortality was recorded and plant life span was 110-130 days. Under field conditions different growth stages of the plant can be seen on the same time but highest susceptibility was observed at flowering and fruit formation stage. The spectrum of resistance was more clearly defined on potted plants rather than field. Under field conditions, varieties Cross 888 f1 hybrid, Long green, Jhalri, JK Leena, BG-7107, PKBT-1, BSS-616, VRBG 233 and Fsd long exhibited moderately resistant reaction. In case of susceptible varieties sensitivity was higher at flowering and fruiting stages.

Management strategies adopted for Integrated Disease Management (IDM) revealed that Allium sativum inter-cropping lowered disease incidence (63 %), suppressed pathogen at 6-8 leaf stage and remained effective till harvesting while Curcuma longa (11 % under greenhouse and 5 % in field) and Colocasia esculenta (9 % in pots and 8 % in field) were least effective. Variation in mode of action of extracts on physiological response was recorded on macroscopic characters colony

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color, texture, margins, spore production and elevation. Nicotiana plumbaginifolia and Parthenium hysterophorus extracts revealed to inhibit the colony radial growth and possess significant antifungal activity against M. roridum under in vitro conditions. Chemical application intensity for vegetable crops is much higher than on field crops and same is true for M. roridum. Among the test commercial fungicides, Antracol at 0.05% and 0.1% significantly reduced the mycelia growth. In general systemic fungicides Antracol, Score and Cabrio top proved most effective against Mr37 isolate. Tested fungicides proved effective in the present study but could not completely inhibit the M. roridum growth. Tebuconazole cytotoxicity was evaluated and resulted in complete inhibition of spore germination at 5 mg/L whereas significantly reduced the germ tube emergence and mycelium elongation at 0.5 and 1.5 mg/L concentrations under in vitro conditions.

Collected data was subjected to appropriate statistical analysis to measure the significance (P < 0.05) of results, including student‟s t-test, analysis of variance (ANOVA) followed by Tukey‟s HSD and Pearson correlation.

It is concluded that areas apparently looking disease free or exhibiting lesser disease index are because of newly adapted bitter gourd cultivation trend and climatic conditions are suitable for the cultivation of susceptible but high yielding varieties. Candidate lines including MONIKA(7004), LEENA(7005), CBT-36, and cultivars VRBG 233 and Green wonder showed stable resistance during germplasm screening. Local resources like, Nicotiana plumbaginifolia and Parthenium hysterophorus plant extracts along with the inter-cropping of Allium plants could provide effective management of disease in pathogen prevailing areas.

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Chapter 1

INTRODUCTION

Momordica charantia L. (English name: Bitter gourd, bitter melon, balsam pear, bitter squash), locally known as “Karela” in Indo-Pak subcontinent, is among one of the most liked vegetables because of its medicinal and nutritional value. It belongs to family cucurbitaceae and genus Momordica and is a dicotyledonous, monoecious plant. The archeological evidences link its origin with eastern Asia around 2000-200 BC (Robinson & Decker-Walters, 1999). Its small fruit types are cited in ayurvedic texts in Indian Sanskrit and many other parts of the world like Asia, Africa and Caribbean (Robinson & Decker-Walters, 1999). It is being used as vegetable, as ayurvedic medicine or as an ornamental plant. It contains more than 90% oils and twenty five compounds comprising on proteins, carbohydrates, vitamins and minerals (Xiang et al., 2000; Braca et al., 2008). Bitter taste is due to the presence of alkaloid triterpene glycosides and momordicine (known as the most bitter plant originated compounds) but even then this bitterness is considered acceptable for its consumption as food (Maur et al., 2004). Irrespective of its origin in South East Asia, the highest cultivar diversity occurs in Africa (Robinson & Decker-Walters, 1999; Joseph, 2005; Luqman et al., 2013).

1.1. Bitter gourd (Momordica charantia)

1.1.1. Taxonomic description

Kingdom: Plantae Phylum: Tracheophyta Class: Magnoliopsida Order: Cucurbitales Family: Cucurbitaceae Genus: Momordica Species: Momordica charantia L.

1.1.2. Plant description

M. charantia is a perennial climbing vine that usually grows up to 5-6 m and has simple tendrils (Figure 1.1). It bears simple, alternate leaves 4-12 cm across, with 3-7 deeply separated lobes. The lobes are mostly blunt, but have small marginal points. Stipules are absent. Bitter gourd is a plant with actinomorphic (radial symmetry), yellow colored flowers. Both staminate and pistillate flowers are produced on the same plant. Staminate flowers are usually larger in size than pistillate flowers. Fruit are ovoid, ellipsoid or spindle shaped usually distinct warty looking exterior and surface covered with jagged, triangular “teeth” and ridges and green to white coloration. Between these two color extremes is any number of intermediate forms. It is hollow in cross-section with a relatively thin layer of flesh surrounding a central seed cavity filled with large flat seed and pith. Ripened fruit is orange red in color and usually dehiscent into three valves. Seeds are few to numerous in numbers, 8-13mm in size, long compressed, corrugate on the margin, sculptured on both faces.

1.1.3. Nutritional value

It is an excellent source of phenolic compounds, antioxidants, and antimutagen. The high nutritive value ranks it first among the cucurbits in iron and vitamin C contents (Islam et al., 2011; http://momordica.allbio.org/). This can find application in food products and dietary supplements (Grover and Yadav, 2004). One cup (93 g) of ½″ sliced bitter gourd contains 930 mg of proteins, 87.4 g water, 1 g ash, 158 mg fat, 3.4 g of carbohydrates, 2.6 g of dietary fiber, 438 IU vitamin A, 78 mg of vitamin C, 37 mcg thiamin, 37 mcg riboflavin, 372 mcg niacin, 40 mcg vitamin B6, 67 mcg folate and 197 mcg pantothenic acid (http://www.foodofy.com/bitter- gourd.html). It also has considerable amount of minerals including iron (400 mcg), potassium (275 mcg), calcium (18 mg), phosphorus (29 mg), magnesium (16 mg), sodium (4.7 mg), zinc (744 mcg), copper (32 mcg), manganese (83 mcg) and selenium (0.19 mcg) (http://www.foodofy.com/bitter-gourd.html; Wills et al., 1984).

Plate 1.1. Typical bitter gourd plant.

1.1.4. Medicinal value

Bitter gourd contains a wide range of biologically active compounds including tripenes and steroids that contributes in its anti-inflammatory, antioxidant, antimicrobial, anti-diabetic, anti-hepatotoxic, anti-cancer, anti-viral and anti- ulcerogenic properties (Grover and Yadav, 2004; Paul and Raychaudhuri, 2010). Traditionally its extracts are being used for the treatment of fever, measles, rheumatism, gout, diabetes, ulcers, wounds and hepatitis (Grover and Yadav, 2004). The fruit juice and leaf decoctions have been used for a wide variety of conditions, from laxative to contraceptive, diabetes and hypertension to malaria, and also for intestinal parasites and worms (Beloin et al., 2005; Behera et al., 2008). Studies based on scientific lines being conducted all over the world for the verification of traditional remedies and results revealed the potential of bitter gourd against various diseases. Crude preparations of Momordica charantia have been evaluated during most of the studies without mentioning the active chemical constituents. Capsules and tablets containing concentrated fruit or seed extracts are available in market.

Extensive research work has been carried out all over the world to investigate the anti-diabetic potential of Momordica charantia. All parts of the plant including fruit pulp, seed, leaves and whole plant have shown antihyperglycemic activity in alloxan and hypoglycemic activity in normal animals or streptozotocin-induced as well as genetic models of diabetes (Akhtar, 1982; Kedar and Chakrabarti, 1982; Bailey et al., 1985; Day et al., 1990; Higashino et al., 1992; Ali et al., 1993; Shibib et al., 1993; Sarkar et al., 1996; Jayasooriya et al., 2000; Ahmed et al., 2001a; Ahmed et al., 2001b; Miura et al., 2001; Pari et al., 2001; Grover et al., 2002; Rathi et al., 2002a; Kar et al., 2003; Grover and Yadav, 2004). A significant reduction in blood glucose, glycosylated haemoglobin, and an increase in plasma insulin and total haemoglobin in animals was observed by poly-herbal preparation of M. charantia (Pari et al., 2001). Antihyperglycemic activity of M. charantia fruit was as good as to glibenclamide in diabetic rats (Virdi et al., 2003). Hypoglycemic activity of phytochemicals such as charantin, momordin Ic, oleanolic acid 3-O-glucuronide, oleanolic acid 3-O-monodesmoside and a Polypeptide-p, isolated from M. charantia have been reported (Matsuda et al., 1998). M. charantia has been shown to increase number of beta cells that help in insulin release or akin to insulin (Ahmed et al., 1998;

Higashino et al., 1992). Diabetes increases the risks of serious health problems and may result in irreversible functional and structural changes in various organs particularly eyes, kidneys, nerves, and blood vessels (American Diabetes Association, 1998; Grover and Yadav, 2004). Not a single medicine is yet available to prevent these complications but onset of these could be slowed down. In a variety of experiments on animals, M. charantia has shown promising effects in prevention as well as delay in progression of diabetic complications like nephropathy, neuropathy, gastroapresis, cataract and insulin resistance (Grover et al., 2001, 2002; Rathi et al., 2002b; Vikrant et al., 2001). Clinical trial supplementing a water-soluble extract of the fruits of M. charantia significantly reduced blood glucose concentrations. Daily consumption of fried M. charantia fruits as diet produced a small but significant improvement in glucose tolerance without any increase in serum insulin levels in diabetic subjects (Ahmad et al., 1999; Grover and Yadav, 2004).

M. charantia extracts and some of its isolated phytochemicals, e.g. MAP 30, lectins and alpha and beta-momorcharin have been recognized to exhibit in vitro antiviral activity against HIV, polio, coxsackieviruse B3, Epstein–Barr and herpes (Grover and Yadav, 2004). M. charantia protein; MAP30, (MW, 30 kDa) has shown promising anti-HIV activity by hampering HIV-1 integrase in several in vivo and in vitro studies (Lee-Huang et al., 1990; Lee-Huang et al., 1995; Huang et al., 1999). Alpha- and beta-momorcharin proteins isolated from seeds, fruit, and leaves have been reported to show anti-HIV activity under in vitro conditions (Zheng et al., 1999; Au et al., 2000). Bitter gourd seed yields clear brown oil that has high concentration (60 %) of 9 cis, 11 trans, 13 trans-conjugated linolenic acid (9c, 11t, 13t-CLN) and its potential application against colon cancer adds up to its significance among various scientific communities (Yasui et al., 2005; Khan, 2007).

1.1.5. Industrial value

Conventional chemical and physical methods of dye degradation / decolorization are actually outdated due to some unresolved problems. Recent studies indicate that an enzymatic immobilization approach has attracted much interest in the removal of phenolic pollutants from aqueous solutions as an alternative strategy to exploit the enzymes at the industrial level (Duran and Esposito, 2000; Husain and Jan, 2000). Peroxidases from bitter gourd (Momordica charantia) have been reported with

very high potential in the degradation of textile and other industrially important dyes. Potential of Con A-Sephadex bound immobilized bitter gourd peroxidases was investigated in the removal of twenty one reactive textile dyes (Akhtar et al., 2005). The immobilized enzyme exhibited more than 90% of the original activity while the soluble enzyme lost 33% of the initial activity when stored for 40 d at room temperature (Akhtar et al., 2005). Calcium alginate-starch gel entrapped Con A- bitter gourd peroxidases and bitter gourd peroxidases immobilized on the surface of Con A layered calcium alginate– starch beads were found to remove more than 70 and 90% effluent color in a stirred batch process after 3 h of incubation, respectively. Entrapped bitter gourd peroxidases retained 59% effluent decolorization reusability even after its tenth repeated use (Matto et al., 2009; Matto and Husain, 2009). Disperse Red 17 and Disperse Brown 1 (Water insoluble-disperse dyes) were decolorized by partially purified bitter gourd peroxidases in the presence of bromophenol, 2,4-dichlorophenol, guaiacol, HOBT, m-cresol, quinol, syringaldehyde, VLA, and vanillin (Satar and Husain, 2009 a,b).

1.2. Crop statistics

It is normally grown as an annual crop in the subcontinent and sown from January to June. In Pakistan the total area under bitter gourd cultivation during 2009- 2010 was 6565 hectares with a total production of 56994 tones while in Punjab, total area under cultivation was 3897 hectares with a production of 43489 tones (MINFAL, 2009-10). Bitter gourd yield can vary depending on variety and crop management. Average marketable yields for hybrid bitter gourd crop in Pakistan are 8-10 t/ha whereas in India it is 20-30 t/ha (Pal et al., 2005; Singh et al., 2006; AVRDC, 2011).

1.3. State policies

Leakages in seed quarantine trade and dominance of private sector in seed import and distribution can be claimed as major reason in introduction of new pathogens in the country. The other contributing factors are high cost of input materials (land preparation, hybrid seeds, plant protection measures to reduce the incidence of diseases and labor scarcity), unpredictable market and high transporting charges play major role in limiting the adoption of vegetables as cash crops (Riaz, 2008). 6

1.4. Production constraints

A number of constraints have been reported which appear at different growth stages of the plant and affect its physiology and plant health and responsible for deterioration in quality and yield. These include germplasm availability, improper cultural practices and pests and diseases. The hard seed coat is major problem responsible for its lower germination rate. The crop is also sensitive to lack of micronutrients (e.g., boron), and micronutrients are often needed to incorporate to improve plant growth (Njoroge and Luijk, 2004).

1.5. Pathological constraints in bitter gourd growing areas of Punjab, Pakistan

Bitter gourd is attacked by a number of insect pests, viral, fungal and bacterial plant pathogen. So far more than 15 fungal, bacterial and viral plant pathogens have been reported to attack bitter gourd plant in Pakistan. Among insect pests, Bactrocera cucurbitae (melon fruit fly) infest the bitter gourd fruits and produce larva inside the fruits resulting in serious losses (Gogi et al., 2010). Bacterial wilt, gummy stem blight and several viral diseases causing leaf distortion, chlorosis, mosaic, stunted growth of plants, are also reported infecting bitter gourd crop (Agrios, 2005; AVRDC, 2011). Among these some have regular pattern and can attack any growth stage and some appear at particular growth stage of the plant and some are specific to some plant part (Webster, 2006; Noble, 2009).

1.6. Common fungal diseases

Fungal infections often occur during prolonged wet periods. Serious fungal diseases of the bitter gourd plant are Cercospora leaf spot (Cercospora sp.), downy mildew (Pseudoperonospora cubensis), Powdery Mildew (Sphaerotheca fuliginea), Fusarium Wilt (Fusarium oxysporum f. sp. niveum) and Myrothecium leaf spot (Myrothecium roridum) (Khan & Kamal, 1962; 1963; Manthachitra, 1971; Nair, 1982; Maholay, 1986; Ali et al., 1988; Mathur, 1990). M. roridum has been recognized as a seriously damaging leaf spot and crown rot pathogen and reported as fruit rot pathogen of bitter gourd from India (Sharma and Bhargara, 1978; Chase,

1983). It was reported to occur in small pockets in adjacent districts of Faisalabad and became a serious pathogen affecting the quality and yield of bitter gourd crop throughout the Punjab Pakistan (unpublished reports of Punjab Agriculture Department).

Little work has been reported in Pakistan on distribution and management of MLS therefore present project was aimed to conduct detailed systematic investigations on M. roridum, host-pathogen characterization and designing of integrated disease management strategies.

1.7. REVIEW OF LITERATURE

Bitter gourd is widely cultivated in plane and sub mountainous regions of Pakistan and is preferred for its edible and medicinal value. Being a short duration and consumer demand pressure it has got the status of cash crop. The local and exotic germplasm is easily available with local distribution companies. Improved hybrid seeds usually preferred by the growers due to higher yield potential. Its average per unit holding yield in Pakistan is much lower than many developing countries of the world (BiG, 2014). Low yield in Pakistan is attributed to many factors ranging from individual farmer crop management strategies to state policies. Among the plant protection issues diseases are the most serious concern for field and tunnel crop. Wide range of issues interconnected with crop production and protection are covered with the objectives of highlighting the background of host and pathogen.

1.8. Genus Myrothecium

Genus Myrothecium is a small genus comprising on twelve species namely Myrothecium atrum, M. carmichaelü, M. cinctum, M. gramineum, M. inundatum, M. leucotrichum, M. penicilloides, M. prestonii, M. roridum, M. setiramosum, M. tongaense, and M. verrucaria (Tulloch, 1972; Nguyen et al., 1973; Ahrazem et al., 2000; Quezado, 2010). These are mostly saprophytic and consist of the imperfect stages with reports that the perfect state of Myrothecium species belongs to Nectria (Tulloch, 1972; Quezado, 2010). Myrothecium species produce sporodochia with differentiated marginal hyphae and phialidic spores and belongs to the order Hypocreales (Kirk, 2008).

Myrothecium species are common soil inhabitants in temperate and tropical regions and infect aerial parts of many plant species. Among these Myrothecium roridum has appeared a serious plant pathogen (Ahrazem et al., 2000; Domsch et al., 2007). Besides pathogenic behavior, some isolates of myothecium species are reported as endophytes such as Myrothecium roridum isolated from a gymnosperm, Pinus albicaulis needles (Worapong et al., 2009). Few isolates of Myrothecium species have been used as bioherbicides such as Myrothecium verrucaria could control kudzu, sicklepod and several other herbaceous weeds (Walker and Tilley, 1997; Boyette et al., 2002) whereas M. roridum is found effective for water hyacinth management (Piyaboon et al., 2014). Some isolates of M. carmichaelü, M. cinctum, M. roridum, M. tongaense and M. verrucaria were found to have an antagonistic activity either through antibiosis or mycoparasitism against fungi such as Pythium debaryanum and Sclerotinia sclerotiorum (Gülay and Grossmann, 1994). Mycelium extracts and culture filtrates of the selected Myrothecium isolates using the agar-well method were found to have both antifungal and antibacterial properties (Gülay and Grossmann, 1994). Gees and Coffey (1989), reported the antifungal potential of M. roridum strain against Phytophthora cinnamomi.

Endophytic isolates exhibited trichothecenes (biologically active mycotoxins) production such as verrucarins and roridins (Tamm and Breitenstein, 1980; Grove, 1993.). Most common trichothecenes molecule is deoxynivalenol (T toxin) in culture medium and up to 11 genes have been involved during its production (Agrios, 2005). T toxin is a mixture of linear, long (35 to 45 carbon) polyketols that apparently acts specifically on mitochondria of susceptible cells, which are rendered nonfunctional, and inhibits ATP synthesis. These toxins have shown to have a potential antibiotic effect against a wide range of clinical and environmental bacteria and fungi such as: Bacillus subtilis, Clavibacter michiganensispv. Michiganesis, Botrytis cinerea, Colleotricum acutatum, Pyricularia oryzae Cavara (rice blast fungus) and Sclerotinia sclerotiorum (Lib.) de Bary (Turhan and Grossmann, 1994; Murakami et al., 1999; Wang et al., 2007; Xie et al., 2008). M. roridum appeared on Oryza sativa during the latter part of the storage and is proved mycotoxigenic (Surekha et al., 2011). Mortimer et al., (1971) described roridin A and verrucarin A myrotheciotoxicosis and poisoning in ruminants. Addition of muskmelon cell wall extracts into liquid media affected mycotoxin production by M. roridum strain indicates that cell wall

polysaccharides from susceptible host tissue provide a better substrate for toxin production (Kuti et al., 1989). Myrothecium species are known to produce a wide range of amylase, cellulolytic and xylanolytic enzymes (Reddy, 1989; Moreira et al., 2005; Okunowo et al., 2010). These enzymes have biotechnological use in food and pharmaceutical industries, paper industries, biomass conversion of agricultural and industrial wastes to chemical feedstock, biofuels, animal feeds and pollution control (Ikram-ul-Haq et al., 2006; Okunowo et al., 2010).

1.9. Myrothecium roridum Kingdom: Fungi Phylum: Ascomycota Class: Sordariomycetes Order: Hypocreales Family: Incertaesedis Genus: Myrothecium Species: Myrothecium roridum Tode

Myrothecium roridum was first described by Tode in 1790 and lately found two homotypic synonyms as Dacrydium roridum (Tode) Link (1809) and Myrothecium advena Sacc. whereas heterotypic synonym is Gliocladium nigrum Moreau (Tode, 1790; Link, 1809; Moreau and Moreau, 1941; Mycobank # 142164). M. roridum cultures produce white to hyaline mycelium on PDA medium. Hyphae are branched, tapered towards ends, septate and cell size is between 20-30 x 1-2.5 µm. Conidiophores arise directly from the mycelium, bear 2-5 phialides in whorls and slightly olivaceous in color at the ends. Sporulation is in concentric rings and throughout the colony. Sporodochia are olive green, wet and slimy at the beginning that turns into black hard structures on later stages. Conidia are olive green in color, rod shaped with rounded ends and 4.5 – 7.0 × 1.0 - 2.5 µm in size. Conidia germinate well at 25-30 ºC and grow better in alternate 16/8 hours of light and dark periods (Talukdar and Dantre, 2013).

1.9.1. Geographical distribution and host range

M. roridum is a facultative parasite with a large number (around 263 plant species) of plant hosts, including vegetables, fruits and ornamental plants (Mendes et al., 1998; Murakami and Shirata, 2005; Silva and Meyer, 2006).It is widely distributed in hot and humid tropical climates of the world and has been recognized as a seriously damaging leaf, crown and fruit of the plant (Chase, 1983). Distribution frequency of Myrothecium conidia is affected by soil surface temperature and viewed that mono cropping culture, field sanitation and rain contribute in disease epidemics (Murakami et al., 1998; Burton and Fish, 2012). M. roridum has been detected for the first time on Peperomia quadrangularis causing leaf and stem rot (Han et al., 2014) and in Anthurium plant culture medium (Kwon et al., 2014) causing leaf spots in Korea. Zhao et al., (2010) presented the first report of Myrothecioum leaf spot of common bean in China caused by M. roridum. Dong, (2003) first observed Myrothecium leaf spot on watermelon (Citrullus vulgaris Schrad) under polyethylene film-covered greenhouse. Leath and Kendall, (1983) described tha pathogenic association of M. roridum to roots of alfalfa and red clover. M. roridum reported causing Myrothecium leaf spot on Salvia spp. (Mangandi et al., 2007); on garden hydrangea (Mmbaga et al., 2010) and on water melon (Seebold and Langston, 2005) in the United States. Worapong et al., (2009) found endophytic association of M. roridum with Pinus albicaulis. It has been isolated from seeds of cucurbits such as bitter gourd (Sultana and Ghaffar, 2007), bottle-gourd, Indian gourd, red gourd, sponge gourd (Wahid et al., 1991; Shakir et al., 1995), pumpkin (Manthachitra, 1971) and melon (Lima et al., 1997). Munjal (1960) reported spreading intensity and increasing host range of Myrothecium roridum in India. Talukdar and Jagdish, (2013) isolated M. roridum from soyyabean while Nguyen et al., (1973) reported seed borne association of M. roridum on Dahlia and Nasturtium, M. verrucaria in soybean and rice and also reported the role of fungal toxins in virulence. Whereas Kuti et al., (1989) demonstrated role on non host specific toxin trichothecene metabolite in Myrothecium leaf spot disease development against muskmelon.

Increasing number of reports of Myrotheium spp. attacking on several plant species around the world indicate that pathogen is under continuous evolutionary

changes and expanding its pathogenicity to other plant families and if favorable conditions persist it has a potential threat to result in epidemic.

1.9.2. History of Myrothecium leaf spot disease of bitter gourd in Pakistan

In Pakistan, Myrothecium leaf spot (MLS) disease was first reported during 1988 at Faisalabad district of Punjab province (Ali et al., 1988). Later on it was found associated with seeds of bitter gourd and isolated frequently from rotted and un- germinated seeds during seed health testing (Shakir and Mirza, 1992; Sultana and Ghaffar, 2007). It reduced seed germination and caused seed rot, damping off, root rot and spots on aerial parts of bitter gourd. According to the official reports of year 2007 of Pest Warning and Quality control of Pesticides, Punjab Agriculture Department, it is distributed throughout the bitter gourd growing areas in Punjab, Pakistan.

The disease is characterized by appearance of water soaked minute spots that are dark brown to black in color. These spots vary in shapes from round to irregular and may present anywhere on leaves. These spots coalesce on later stages to form blighted areas on the leaves (Belisario et al., 1999). Irregularly shaped black sporodochia can form with a white fringe of mycelium. These spore structures appear in concentric rings within the necrotic areas and seen on the leaf undersides. Myrothecium leaf spot most frequently appears on wounded areas of leaves such as tips and breaks in the main vein which occurs during handling.

1.9.3. Aggressiveness behavior

Aggressiveness plays a role in adaptation of plant pathogens and divided it into elementary quantitative traits of the pathogen life cycle, such as infection efficiency, latent period, sporulation rate, infectious period or lesion size. Infection efficiency is usually measured as a percentage of successful infections resulting from a controlled number of deposited spores (Pariaud et al., 2009). Practically, infection efficiency may be indirectly measured by the observed numbers of lesions or chlorotic flecks per unit of leaf area (Pariaud et al., 2009). Nguyen et al., (1973) described the role of fungal toxins in virulence of Myrothecium. Carter (1980) noticed significant differences in virulence among individual isolates of M. roridum on cantaloup, but not

among leaf, stem, and fruit groups of isolates. Duval et al., (2010) observed variability in aggressiveness among isolates of M. roridum on tomato and cucumber. This variability in virulence agreed to those observed by Taneja et al., (1990) and suggested the presence of different pathotypes among M. roridum. Ploetz and Englehard (1980) demonstrated the virulence of five isolates of M. roridum on Sinningia speciosa.

1.9.4. Germplasm screening for resistance

There is scarcity of available information to find new sources of resistance in plants against Myrothecium leaf spot disease and none of the studies on bitter gourd germplasm against M. roridum have been reported. Yum and Park (1990) found that only two among 20 cultivars appeared to be resistant to M. roridum in seedling inoculation tests in the greenhouse. Srivastava and Khan, (1994) conducted field trials to screen the soybean genotypes against M. roridum and they reported only two of the 26 cultivars as resistant for three consecutive years (1984-86), three were moderately resistant and the rest were moderately to highly susceptible. Norman et al., (2003) screened commercial germplasm of 16 Syngonium species and 5 cultivars and reported that all cultivars were susceptible to Myrothecium leaf spot. However variation in resistance was observed among different species and this genetic diversity could be used to produce hybrids resistant to M. roridum. Mahesha (2006) screened soya bean germplasm for multiple resistances against foliar diseases and illustrated that none of the tested cultivars was found resistant against Myrothecium leaf spot. Differential resistance was exhibited among the watermelon cultivars and cucurbit types during greenhouse inoculations of M. roridum whereas severe defoliation of watermelon in Southern plains was observed (Fish et al., 2012). Peris et al., (2012) evaluated five mulberry accessions for the incidence and severity of foliar diseases and found lower incidence of brown leaf spot caused by M. roridum in Kenya. Nunues et al., (2015) screened available parent germplasm and inbred lines of yellow melon for new sources of resistance against M. roridum and Podosphaeria xanthii and reported selection of resistant inbred lines as a multifaceted phenomena.

1.9.5. Management strategies

1.9.5.1. Intercropping

Study of allelopathic potential of plants in managing several pathogens may leads to cost effective and environmental friendly approach. Intercropping different non-host plants with known antimicrobial activity may help in reducing the pathogen build up by providing either physical barriers or releasing volatile chemical constituents that retard the fungal growth. Gomez-Rodriguez et al., (2003) studied the marigold and pigweed allelopathy by intercropping for the management of tomato early blight disease. Suman et al., (2000) reported reduced Septoria leaf spot disease by Septoria lycopersici in tomato-maize inter-cropping. Chickpea blight caused by Ascochyta sp was significantly lowered by inter-cropping chickpea with wheat and barley (Gaur and Singh, 1994; 1996; Gan et al., 2006). Due to the phenolic substances, inter-cropping of Lactuca sativa in tomato and cucumber crops whereas Helianthus annuus and Tagetes indica in gladiolus exhibited significant reduction in foot and root rot caused by F. oxysporum (Jarvis, 1989; Pavlou and Vakalounakis, 2005; Riaz, 2008).

1.9.5.2. Plant aqueous extracts

Little information is available for management of Myrothecium leaf spot disease of bitter gourd. Maji et al., (2005) screened several plant species for their antifungal potential against Myrothecium roridum causing brown leaf spot in Morus spp. and found that extracts of Datura metel, Allium sativum, Chromoleana odorata and Eucalyptus citriodora inhibited colony growth up to 33% under in vitro conditions. Chattopadhyay et al., (2002) evaluated the antifungal potential of 5 plant species against Myrothecium roridum and found that Allium spp. and Eucalyptus spp. aqueous extracts of which plant part are effective in controlling the observed fungus. Little is known about the factors developing disease and effecting its cultivation. Therefore, research is needed to explore ways and means to put this remunerative vegetable industry on scientific lines and to ensure that it can bring prosperity to the growers and the country.

1.9.5.3. Chemical control

There is scarcity of registered fungicides against MLS disease, besides, only few published papers worldwide on chemical control of this pathogen are available (Dighule et al., 2011; Duval et al., 2010). But growing reports of disease highly suggested the need of in vitro and in vivo evaluation of some fungicides on the mycelial growth of Myrothecium. It would provide useful information for future in vivo screenings and the registration of some fungicides. Dighule et al., (2011) investigations revealed that chemical fungicides Mancozeb, Propiconazole and bioagent Trichoderma viride proved efficacy against Myrothecium leaf spot disease of cotton. McMillan (2010) reported Myrothecium leaf spot as serious disease of Dieffenbachia picta Schott cv. Compacta in a shade house and elucidate that iprodione, trifloxystrobin, chlorothalonil, azoxystrobin, fludioxonil and myclobutanil were significantly more effective in controlling the Myrothecium leaf spot than copper. Preliminary assays showed that fungicides with quaternary ammonium and copper were highly effective in inhibiting the mycelial growth of M. roridum (Duval et al., 2010).

1.9.5.4. IPM Strategies

Application of salicylic acid to control M. roridum determined that it is effective at higher doses (30 mg/10 mL) but boric acid was found less efficient (Shakoor et al., 2011). Tetraconazole resulted in significant Cercospora leaf spot control, root yield, and recoverable sucrose compared to fenbuconazole with an adjuvant (Khan and Smith, 2005). Maji et al., (2004) isolated six mulberry phylloplane bacterial strains and in vitro screened against M. roridum. These six phylloplane bacterial strains exhibited antibiosis against M. roridum under in vitro conditions whereas all test bacterial strains reduced MLS disease severity more than 44% even 30 days after inoculation under field conditions.

Carter, (1980) investigated incidence of M. roridum on cantaloup and its control in relation to time of fungicide application. Mixture of benomyl and zinc ion- maneb complex significantly controlled M. roridum infecting cantaloup leaves and stems but author reported that it was difficult to find any relationship among rainfall, time of fungicide application, and incidence of M. roridum (Carter, 1980). Soil

drenching of iprodione 50W gave effective, non phytotoxic control of the leaf spot and crown rot phases of the disease (Ploetz and Englehard, 1980). In vitro resistance of a gloxinia isolate of M. roridum to benomyl and ethazole + thiophanate-methyl fungicides was also determined (Ploetz and Englehard, 1980).

Objectives

Objectives of the study are:

 Development of disease distribution map and its associated socio economic factors of crop production and protection

 Characterization of host and pathogen for its potential application in disease management strategies

 Histpathological study of tissue infection process

 Identification of reliable sources of disease resistance in available commercial germplasm and breeding material of bitter gourd.

Chapter 2

MATERIALS AND METHODS

Present study was conducted at Seed and Postharvest Pathology Laboratory, Institute of Agricultural Sciences (IAGS), University of the Punjab, Lahore and Plant Pathology Section, Ayub Agriculture Research Institute (AARI), Faisalabad. Host pathogen interaction study and antifungal efficacy of technical grade tebuconazole was carried out at Institute of Phytopathology and Applied Zoology, Justus-Liebig- University, Giessen, Germany.

2.1. Geographical distribution of Myrothecium roridum in Punjab, Pakistan

2.1.1. Survey

The surveys were conducted on a scheduled plan from April 2011 to September 2013 at seedling, vegetative, flowering and fruiting growth stages of the crop. It was conducted on early spring (February-March) and late summer (mid June – mid July) seasoned crops. Information on cropping history, input source, and crop production and protection technology was gathered on a structured questionnaire (Annexure-I). Structured questionnaire was distributed among the stakeholders. Information collected from the surveys was analyzed and expressed in percentage to evaluate the consumption, quality census of the consumers and market status. Preliminary survey was conducted during July-September 2010. The key objective of this survey was to construct questionnaire, survey scheduling, route, basic information on crop and disease with respect to its stake holders as farmer, representatives‟ public and private sector organization engaged in marketing and R&D sector for vegetable.

In Pakistan vegetables are grown on small pieces of land and sometimes small fields are not in proper sequence or rectangular shape so 0.25 hectare was considered as basic sample unit. A total of 319 locations from 11 sub agro-ecological zones covering 29 districts and 117 fields comprising on 0.25 hectares from farmers‟ fields, demonstration plots of Agriculture Extension Wing of Punjab Agriculture Department

1: DG Khan 11: Okara 21: Sialkot 2: Rajanpur 12: Kasur 22: Gujrat 3: Rahim Yar Khan 13: Lahore 23: Jehlum 4: Bahawalpur 14: Faisalabad 24: Rawalpindi 5: Lodhran 15: Jhang 25: Attock 6: Muzaffargarh 16: Sargodha 26: Chakwal 7: Multan 17: Hafizabad 27: Mianwali 8: Vehari 18: Sheikhupura 28: Khushab 9: Bahawalnagar 19: Gujranwala 29: Bhakkar 10: Pakpatan 20: Narowal

Fig. 2.1. Modified PARC agro-ecological zone map of Punjab, Pakistan observed for MLS of bitter gourd in 2011-13. (http://old.parc.gov.pk/Maps/AgroEcoPunjab.html )

were visited (Fig. 2.1). To achieve real field representation, fields were examined in cross, zigzag or diagonal fashion depending upon geometry of the field.

2.1.2 Disease assessment

Disease assessment was made on disease prevalence, incidence, severity and percent disease index. Formulae used for the calculations are given below. Prevalence percentage of the disease was calculated on the basis of the number of locations showing disease in an area; whereas, Percent disease incidence was noted on leaves/plant infected from five spots in a field. Data for severity of disease was recorded on a 0-5 visual severity rating scale (VSRS, Fig. 2.2). Disease index of an area was calculated on sum of all numerical categories of disease severity divided by number of samples (Annexure-II).

Equation 1:

Equation 2:

Equation 3:

Fig. 2.2. Illustration of 0-5 visual severity rating scale (VSRS) of MLS disease of bitter gourd

2.1.3. Specimen collection

To find out the association of Myrothecium roridum with Myrothecium leaf spot disease of bitter gourd, leaves showing the characteristic Myrothecium leaf spot disease symptoms were collected, sorted individually, packed in cellulose bags and labeled with necessary information regarding location, time, date and grower identification. Collected specimens were transported to Seed and Postharvest Pathology Laboratory, Institute of Agricultural Sciences (IAGS), University of the Punjab, Lahore where isolation, identification and lab scale multiplication of the single spore cultures of the associated fungi were carried out.

2.1.4. Isolation of associated fungi

Collected samples were examined under stereoscope and infected leaf, stem and fruits with typical MLS symptoms were processed further. Potato dextrose agar medium (PDA) was used for isolation in 5 replicates for each test sample. Samples were washed under running tap water and placed overnight at 4°C, cut into 1 cm2 pieces, surface sterilized with 1% sodium hypochlorite solution for five minutes followed by thorough washing with autoclaved double distilled water and aseptically transferred on 90mm Petri plates containing PDA medium. These Petri plates were incubated at 25 °C for 3-5 days. The isolated fungus was purified by transferring actively growing mycelium from the colony margins.

2.1.5. Identification of fungus

Single spore cultures were produced on fresh PDA and examined for its morphology and taxonomy. Detailed taxonomic studies were carried out under compound microscope at 100X and 400X. Key identification features of Myrothecium roridum Tode i.e., colony color and shape, mycelia morphology, sporodochia production, conidia formation, conidia size and shape were studied as described by Mycobank, Korea (Mycobank # 142164). The confirmed colonies of M. roridum were transferred on 90mm PDA Petri plates, incubated on PDA at 25 °C for culture submission to First Fungal Culture Bank of Pakistan, Institute of Agricultural Sciences, University of the Punjab, Lahore.

2.1.6. Application of Koch’s postulates

Pathogenicity was confirmed by application of Koch‟s postulates before initiating towards the series of experiments. Twelve centimeter disposable glasses were filled with sandy loam soil and sterilized by 40 % commercial formaldehyde. Isolated fungus was evaluated for its pathogenicity on four weeks old plants of Momordica charantia. Leaves were sprayed with spore suspension till run off and examined for symptoms development after every 24 hrs for 7 days. The experiments were carried out in green house at temperatures of 25-30 °C.

2.1.7. Culture authentication

Genomic DNA was extracted from fungal mycelia using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). DNA amplification was performed using Polymerase Chain Reaction (PCR) in three biological replicates. Universal primer pairs, ITS4 (TTCCTCCGCTTATTGATATGC) and NS1 (AACTTAAAGGAATTGACGGAAG) following standard PCR procedures with minor modifications. Sequence analysis was done by the LGC AGOWA Ltd. at LGC Genomics GmbH, Berlin (http://www.lgcgroup.com). Sequences obtained were compared with all sequences of ITS region in the GenBank closest sequence similarity by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).

2.2. Characterization of Myrothecium roridum

A total of 54 Myrothecium roridum isolates were purified to single spore culture from the disease specimens collected during field scouting (Annexure-III). Population was selected for investigations on virulence, morphological, physiological and genetic characterization of M. roridum.

2.2.1. Evaluation of virulence of isolates

Myrothecium roridum isolates from different geographical origin and plant source were evaluated for its virulence against four weeks old plants of bitter gourd in green house at a temperature of 25-30 °C. The mass culture was prepared by mixing of one week old cultures in 300 mL sterilized ddH2O with house hold blender. The suspension was then passed through muslin cloth and spore count was adjusted to 1 ×

105 spore/mL. Afterwards the spore suspension sprayed on the plants with hand atomizer at 5pm in the evening for better establishment of the infection. Control plants were sprayed with sterilized water. Infection development process was examination initiated 48 hours after the inoculums application. Data was recorded at 72 hrs interval on the basis of disease incidence and severity. The leaf samples were taken at day 8 for re-isolation of fungus to confirm virulence.

2.2.2. Morphological studies

Nine isolates (Mr10, Mr21, Mr28, Mr30, Mr34, Mr37, Mr49, Mr51 and Mr54) out of 54 field isolates having stable resistance were sub-cultured on PDA and incubated at 25°C for fourteen days. Macroscopic and morphological characters that were selected for each isolate are summarized in Table (2.1). Conidia geometry (length and width) of 20 conidia of each isolate were measured in water mounts under compound microscope. The data matrix was produced enlisting cultural, morphological and microscopic characters for each isolate.

Table 2.1.Visual colony characters of Myrothecium roridum

Sr # Character Category

1 Circular Colony form Filamentous 2 Raised Flat Colony elevation Convex Umbonate 3 White Colony color Off white observe Cream

4 White Off white Colony color Yellow reverse Yellowish brown Pale

2.2.3. Physiological studies

Physiological requirements of the pathogen were optimized thorough experimental layout and practiced using completely randomized design (CRD) (Fig. 2.3).

Selection of best growth medium (A) BGA, CDA, GA, MEA, NA, PDA

A + selection of suitable temperature (B)

15, 20, 25, 30, 35, 40 °C

A + B + optimization of pH (C)

5.0, 5.5, 6.0, 6.5, 7.0, 7.5

A + B + C + Photoperiod (D)

Light, Dark, Alternate light and dark

A + B + C + D + relation of culture age with virulence

Inoculation of 2, 4, 6, 8, 10 & 12 day old culture on leaves of bitter gourd

Fig. 2.3. Schematic description of colony growth and sporodochia production in relation to virulence by optimizing fungal cultivation conditions. BGA: Bitter gourd dextrose, CDA: Czapekdox agar, GA: Glucose agar, MEA: Malt extract agar, NA: Nutrient agar medium, PDA: Potato dextrose agar

2.2.3.1. Optimization of nutrients

To evaluate the nutrients provisions of the pathogen, different culture media i.e. Nutrient agar (NA; Beef Extract,3.0 g; Peptone, 5.0 g; Agar, 15.0 g; pH, 6.8), Potato dextrose agar (PDA; Potato infusions, 4.0 g; Dextrose, 20.0 g; Agar, 15.0 g, pH, 5.6), Czapek dox agar (CDA; Sucrose, 30.0 g; Sodium nitrate, 2.0 g; Dipotassium phosphate, 1.0 g; Magnesium sulphate, 0.5 g; Potassium chloride, 0.5 g; Ferrous sulphate, 0.01 g; Agar 15.0 g, pH, 7.3), Glucose agar (GA; Glucose, 20.0 g; agar, 15.0 g), Malt extract agar (MEA; Malt extract, 20.0 g; Agar, 15.0 g; pH,) and Bitter gourd agar (BGA; Bitter gourd fresh leaves, 20.0 g; Agar, 15.0 g) medium were used. Under aseptic conditions Pyrex glass Petri plates (90 mm) containing test media were inoculated in the center with a 5 mm disc of 5 days old culture of the M. roridum and incubated at 25 °C. Mycelia growth of each Petri plate was measured at 72 hours till the maximum growth achieved on a treatment. The most suitable medium yielding maximum colony growth of the pathogen and No. of spore/mL was used for onward studies.

2.2.3.2. Optimization of incubation temperature

Temperature was optimized for M. roridum by plating the fungus (5 mm plug) on the most suitable selected medium, incubating on 15, 20, 25, 30, 35 and 40 °C for ten days. Response of the fungus was measured by measuring the colony growth of the pathogen and No. of spore/mL.

2.2.3.3. Optimization of growth medium pH

Optimum pH for the maximum growth of the M. roridum was studied on most suitable medium incubated at best selected temperature for ten days. Optimum pH level was made by adjusting pH of the medium adjusted to 5, 5.5, 6, 6.5, 7 and 7.5 by HCl and NaOH before autoclaving. Data was recorded for the maximum colony growth and No. of spore/mL.

2.2.3.4. Effect of photoperiod

The best selected medium, pH and temperature was employed for impact of photoperiod on the growth of M. roridum, The test Petri plates were incubated under

continuous darkness, continuous light and 16/8 hrs dark and light conditions for ten days. Optimization was measured on colony growth and No. of spore/mL.

2.2.3.4. Optimization of culture age & Virulence Assessment

Fresh leaves of commercial variety of bitter gourd (Jaunpuri) were placed on plane agar medium. A 3 mm plug from actively growing colony of 2 to 14 day old culture multiplied under best selected conditions was placed on center of the leaf and incubated at 25 ± 1°C in 12hrs alternate light and darkness. The observations were recorded at 12 hrs interval for infection development stages (latent period to disease severity by observing various stages of infection initiation, development and colonization in marked 1 cm2area along with spore density.

2.2.4. Genetic variation studies 2.2.4.1. Preparation of solutions  CTAB (Cetyltrimethyl ammonium bromide) buffer 100 mL of 1 M Tris HCl (pH 8.0), 280 mL of 5 M NaCl, 40 ml of 0.5 M EDTA and 20 g of CTAB was added in 500 mL water and final volume was raised to 1 L. β-mercaptoethanol (0.2%) was added just before use.  1 M Tris HCl pH 8.0 121.1 g of Tris base was dissolved in 800 mL of double distilled water

(ddH2O). pH level was adjusted to 8 by adding concentrated HCl. Final volume was made up to 1 L with ddH2O.  TE Buffer

10 mL of 1 M Tris HCl (pH 8.0) and 2 mL of 0.5 M EDTA was added to 800 mL ddH2O and volume was raised up to 1 L.

 0.5 M EDTA

186.12 g EDTA was added in 700 mL H2O and pH was adjusted to 8.0 with

16-18 g of NaOH pellets. Total volume was raised to 1 L with ddH2O.

 5 M NaCl

292.2 g of NaCl was dissolved in 700 mL H2O and volume was raised to 1 L.

 TAE buffer

Stock (50X) was prepared by adding 242 g of Tris-base, 57.1 mL of Acetate (100 % acetic acid) and 100 mL of 0.5M sodium EDTA in 700 mL ddH2O and volume was raised to 1L. To make 1X TAE from 50X TAE stock by diluting 20 mL of stock into 980 mL of deionised water.

2.2.4.2. Extraction protocol

Fungal strains were grown on PD broth incubated at 27 °C for 10-12 days and mat was collected for genomic DNA extraction. The total genomic DNA was extracted by using CTAB method (Doyle and Doyle, 1991). Fungal mycelium (1-2 g) was grinded in a sterile pestle and mortar using liquid nitrogen (-196°C). Powdered fungal tissue was transferred to 2 mL Eppendorf tubes. The extraction solution (1.5 mL) was added to the samples and incubated at 65°C in pre-warmed water bath for 45 minutes. The Eppendorf tubes were gently inverted two to three times during incubation. After incubation samples were centrifuged at 13000 rpm for 10 minutes. Supernatant was transferred to another Eppendorf and add equal volume of solution Choloform isoamyl alcohol (24:1) was added. The mixture was again centrifuged at 13000 rpm for 10 minutes. After centrifugation separate the aqueous supernatant layer in another sterilized Eppendorf and incubated in ice for 10 minutes after adding 1.5 volume of chilled isopropanol Afterwards samples were centrifuged at 13000 rpm for 10 minutes and DNA pellet was obtained. DNA pellet was washed with 70 % ethanol; air dried in the laminar air flow cabinet until the whole ethanol evaporated from the Eppendorf. Pellet was resuspended in 50 µL of syringe water for further qualitative and quantitative analysis. The RNA contamination in extracted DNA was nullified by incubating the samples with 5 µL of RNase (20 mg/mL) for 30 minutes at 37°C.

2.2.4.3. Estimation of Extracted DNA

DNA concentration was determined by spectrophotometer measurement using Techne Spec gene Spectrophotometer (140801-2, UK). Absorbance was taken at 260nm and 280 nm and amount of DNA was quantified by using the following formula (Sambrook et al., 1989).

DNA concentration (µg/mL) = OD260 × Dilution factor × 50

2.2.4.4. DNA Quality Analysis through Agarose Gel electrophoresis

Agarose (1 g) was dissolved 100 mL 1X TAE electrophoresis buffer, heated in microwave oven for 2 minutes and was swirled regularly to ensure proper mixing. Gel casting tray was prepared and a suitable comb was adjusted on it to create wells for loading samples. The melted agarose was cooled to 45°C before adding 5µL ethidium bromide (10 mg/mL). The agarose containing ethidium bromide was poured in the gel casting tray and left for solidification at room temperature. The gel casting tray containing the gel was placed horizontally in an electrophoresis tank, containing 500ml 1X TAE buffer. The comb was removed carefully to avoid tearing of wells. DNA samples were prepared by mixing 3 µL of 6X gel loading dye, Bromophenol blue (Fermentas) before loading in to wells of the gel. The electrophoresis apparatus was connected to power supply and the immersed electrophoretic gel was run at 100V for 45 minutes or till the dye migrated one third of the gel length. DNA bands were visualized using UV trans-illuminator (WiseDoc MUV-M20). The DNA bands were compared with the 1Kb DNA marker (Fermentas) for quality estimation.

2.2.4.5. Random Amplification of Polymorphic DNA (RAPD) Analysis

Random amplified polymorphic DNA (RAPD) analysis was employed to study the DNA polymorphism in different fungal isolates for genetic diversity and phylogenetic relation among them (Ranganath et al., 2002).

2.2.4.5.1. Random Primer Screening

Thirteen primers were used for RAPD analysis. These decamers were supplied by school of Biological Sciences (SBS) Genetech Co. Ltd-Beijing, China in lyophilized form stored at -20°C. These primers were diluted up to 100 picomole concentration before use in RAPD analysis.

Table 2.2. Details of primers used for RAPD analysis.

Sr. Primer Sequence (5΄-3΄) No. 1 SBSA01 CAG GCC CTT C

2 SBSA02 TGC CGA GCT G

3 SBSA03 AGT CAG CCA G

4 SBSA04 AAT CGG GCT G

5 SBSA05 AGG GGT CTT G

6 SBSA06 GGT CCC TGA C

7 SBSA07 GAA ACG GGT G

8 SBSA09 GGG TAA CGC C

9 SBSA10 GTG ATC GCA G

10 SBSA11 CAA TCG CCG T

11 SBSA13 CAG CAC CCA C

12 SBSA14 TCT GTG CTG G

13 SBSA16 AGC CAG CGA A

2.2.4.5.2. PCR Amplification reaction

RAPD reaction was carried out by using standard reagents (Fermentas). All the process for the preparation of reaction mixture was carried out on ice under sterile conditions. The following reagents with optimized concentrations/amount were used.

Table 2.3. PCR Reaction mixture

Chemicals Stock conc. Used conc. Volume used

PCR Buffer 10 X 1.0 X 5.0 µL

MgCl2 25 mM 1.5-3.0 mM 5.0 µL

dNTPs 0.2 mM 0.2 mM 5.0 µL

Primer 100 pMole/µL 5.0 µL

Template DNA 0.5 - 1µg 5.0 µL

Taq Polymerase 2.5 Ư/ µL 1Ư/ μL 0.5 µL

Deionized H2O 28 µL

Total volume 50 µL

2.2.4.5.3. RAPD Temperature Cycling Conditions

RAPD amplification were carried out in Master cycle gradient PCR (TECHNE 412) with initial denaturation at 98 °C for 5 minutes followed by 40 cycles of denaturation at 94 °C for 1 minute , annealing at 56 °C for 1 minute. Final extension was set for 10 minutes at 72 °C. The reaction was terminated at 4 °C in 2-3 hours.

2.2.4.5.4. Analysis of Amplified DNA Fragments

The analysis of RAPD amplified bands were performed on 1 % agarose gel. Before loading the RAPD amplified DNA in the wells of gel, 5 µL of 6X gel loading dye was added to the 50 µL RAPD sample. DNA ladder of 1Kb was also loaded on both sides of gel to compare the size of different amplified fragment in RAPD

analysis. The gel was run at 100 volts for 45 minutes at room temperature. DNA bands were examined under UV trans illuminator (Wise Doc MUV-M20) and photographed using the Gel Documentation system.

2.2.4.5.5. RAPD Data analysis Data matrix was constructed with 1 or 0 based on the presence or absence of the band respectively (Halmschlager et al., 1994). Only reproducible bands were considered for analysis. Similarity coefficients (S) between isolates were calculated using the formula.

Similarity coefficients (S) = 2Nxy / (Nx+ Ny)

Where Nx and Ny are the number of fragments amplified in samples X and Y, respectively, and Nxy is the number of bands shared by the two isolates (Nei and Li, 1979). Similarity coefficients were converted to genetic distances (D) using the equation: D = 1 - S. A genetic distance matrix was used to construct a dendrogram by Ward‟s linkage using MINITAB software (MINITAB, 2004).

2.3. Development pattern of M. roridum within host leaf and root tissues

2.3.1. WGA-AF 488 staining

2.3.1.1. Preparation of ½ X Murashige & Skoog (MS) medium

MS medium including vitamins 2.2 g/L

Sucrose 5 g/L

pH 5.8

Gelrite 4 g/L

Distilled H2O 1 L

MS medium and sucrose was added to 800 mL distilled water and pH was optimized to 5.8 using 0.1 M HCl/NaOH. Gelrite (4gm) was added. Volume was raised up to 1L and autoclaved at 121°C temperature and 100 kPa pressure for 15 minutes.

2.3.1.2. Preparation of 1X PBS buffer

KCl 0.2 g/L

KH2PO4 0.2 g/L

Na2HPO4 1.15 g/L pH 7.4 Tween 20 0.05 %

All chemicals were added in 800 mL water, mixed well and pH 7.4 was adjusted. Volume was raised to 1000 mL and autoclaved at 121 °C temperature and 100 kPa pressure for 15 min.

2.3.1.3. Preparation of Wheat germ agglutinin-Alexa Fluor 488

One milligram (1 mg) of Wheat germ agglutinin-Alexa Fluor (WGA-AF) 488 was dissolved in 1mL deionized water and stored at 4 °C.

2.3.1.4. Preparation of spore suspension

Myrothecium roridum plates were prepared 15 days before spore isolation. 10 mL of autoclaved Tween 20 water was added to the fully grown culture plate to cover the spores and gently scrubbed with round ends glass rod. Solution was filtered through mira cloth. Spore suspension was transferred to the 15 mL falcon tube and centrifuged at 4000 rpm. Supernatant was discarded and palette was re-suspended with 50 mL of tween 20 water. Spore number was counted using spore counting chamber and adjusted to 1 × 105. Sterilized conditions were maintained throughout the isolations protocol.

2.3.1.5. Preparation of leaf and root samples

Nutrient media, ½ MS was prepared, autoclaved and poured into 12 × 18 cm sterilized glass jars (approx. 100 mL) and solidified at room temperature (20 °C). Seeds were surface sterilized with 1 % NaOCl for 15 minutes followed by 2-3 times washing with sterilized water. 3 seeds were placed in each jar and incubated at 25 °C for 7-10 day for germination. Roots of same sized seedlings were selected, dip inoculated in spore suspension for 2 hrs and transferred to fresh ½ MS jars. For leaf infection development, seedlings were sprayed with spore suspension. Jars were incubated at 25 °C temperature, 16 h light/8h dark period for 10 days. Data was recorded and samples were collected to study the infection development pattern.

2.3.1.6. Staining

Hyphae in the root and leaf tissue sections were stained with chitin specific dye WGA-AF 488 as described by Deshmukh et al., (2006). Tissue sections were transferred to 0.15 % trichloroacetic acid fixation solution. From fixation solution, sections were transferred to 1X PBS buffer containing 5 mg/mL WGA-AF 488 dye and incubated at room temperature for 10 min. During incubation vacuum infiltration was done for 2 min at 25 mmHg. After incubation segments were rinsed with PBS buffer and mounted on glass slide. All samples were analyzed with axioplan 2 microscope excited at 470/20 and detected at 505-530 nm for WGA-AF 488.

2.3.2. Transmission Electron Microscopy

For TEM studies of host-pathogen interaction, samples were prepared at Institute of Phytopathology and Applied Zoology, Justus-Liebig-University, Giessen, Germany and harvested after 0 (control), 6, 24, 48, 72, 96 and 120 hrs incubation period for fixation. TEM work was performed at the Zentrale Biotechnische Betriebeinheit, Justus-Liebig-University, Giessen, Germany.

2.3.2.1 Sample preparation

For sample preparation, fresh leaves (medium sized) were detached from bitter gourd plants and placed on solidified agar plates (80 mm). 100 µL of spore suspension (1000 spore/mL) was inoculated on the center of leaves and plates were

incubated in growth chamber at 16/8 h light/dark with 25/18 °C temperature respectively.

2.3.2.2. Fixation

After 0 (control), 6, 24, 48, 72, 96 and 120 hrs incubation period, fixation of leaf samples for standard TEM observation was performed by following Fuchs et al., (2010). Leaf tissue inoculated with M. roridum (1 cm2) was fixed for standard TEM observation in 0.05 M sodium cacodylate buffer, pH 7.2 [1.5 % paraformaldehyde (w/v) and 3 % glutardialdehyde (v/v)] at room temperature for 2 h. Tissue was cut into approx. 4 mm × 4 mm sections and incubated in fresh fixative for 3 h at room temperature, washed with 0.05 M sodium cacodylate buffer, pH 7.2, on ice, post-fixed with 1 % osmium tetroxide (w/v) overnight on ice, washed with demineralized water and stained with 0.5% aqueous uranyl acetate (w/v) on ice. Dehydration through a graded ethanol series was followed by treatment with dried propylene oxide. After embedding through several steps of Spurr‟s resin (Spurr, 1969), polymerization was performed at 68 °C for 24 h. Sections, 90 nm, were cut from samples, collected on Formvar-coated, single-slot copper grids (2 mm × 1 mm) and post-stained with Reynolds‟ lead citrate (Reynolds, 1963) and 2 % aqueous uranyl acetate (w/v).

2.3.2.3. TEM analysis

After fixation, sections were examined in a LEO EM 912 (Omega AB, Zeiss, Oberkochen, Germany) transmission electron microscope at 120 kV. An integrated CCD camera (slow-scan CCD camera; Proscan, Lagerlechfeld, Germany) was used for digital micrographs.

2.4. In vivo screening of bitter gourd germplasm

Momordica charantia germplasm consisting 36 commercial varieties and breeding material was investigated for its resistance reaction against MLS both under in pots and under field conditions. The trials were repeated at the same time at the experimental fields of Institute of Agricultural Sciences, University of the Punjab and at the fields of Plant Pathology Section, Ayub Agriculture Research Institute (AARI) Faisalabad. Crop production technology for input application developed by Vegetable

Section AARI Faisalabad was observed. Randomized complete block design (RCBD) was used in triplicate to conduct experiment.

2.4.1. Soil sterilization

Commercial formalin (10 %) was mixed in moist heap of soil and covered with polythene sheets. After 48 hours polythene sheets removed and pulverized the soil so that fumes should not retain in soil (Khan, 2002).

2.4.2. Susceptibility reaction in pot under natural environmental conditions

For pot experiment, four week old plants of bitter gourd of thirty six varieties were planted in earthen pots (12 × 18 cm). Plants were inoculated with freshly prepared spore suspension of M. roridum (1 × 105) at 8-10 leaf stage. Ten plants randomly tagged for data recording from a set of fifteen plants of each replication. Data was recorded on disease incidence and disease reduction over control was recorded at 15 days interval up two months. Agronomic performance of bitter gourd germplasm in pot experiment under natural environmental conditions against MLS was measured for; vine length, number of branches, days to first flowering, number of fruits and fruit yield (g) per plant.

2.4.3. Susceptibility reaction in field under natural environmental conditions

Thirty six bitter gourd varieties were included in the in vivo trials. Plants were inoculated with freshly prepared spore suspension of M. roridum (1 × 105) at 8-10 leaf stage. Fifteen plants of each variety represent a replication in the in vivo screening trials. Disease reaction was recorded on disease incidence and disease reduction over control at fifteen days interval up two months. Agronomic performance of bitter gourd germplasm under field conditions against MLS was measured on following traits; vine length, no. of branches, days to first flowering, number of fruits and yield per plant.

2.5. Disease management practices

Investigations on management of Myrothecium leaf spot disease were conducted by working on plant aqueous extracts, intercropping of aromatic plants and commercial fungicides under in vitro and in vivo conditions. For in vivo (pot and field) experiments, soil was sterilized with 10 % commercial formalin as described in section 2.4.1. Crop production technology for input application developed by vegetable section AARI was observed and experiments were conducted by using RCBD in triplicate.

2.5.1. Efficacy of plant aqueous extracts against MLS

2.5.1.1. Collection of plants

Weed plants [Chenopodium album (local name: Bathu), Parthenium hysterophorus (local name: Congress booti), Trianthema portulacastrum L. (local name: Itsit), Malvestrum coromendelianum (local name: Malvestrum), Coronopus didymus (local name: Jangli halo),Sphaeranthus indicus (local name: Mundi booti), Digera arvensis (local name: Tandala), Solanum nigrum (local name: Mako) and Nicotiana plumbaginifolia (local name: Giddar tumbako)] were collected from the crop and wasteland fields of the University of the Punjab, Lahore, Pakistan. Plants were thoroughly washed with tap water and surface dried on the blotter paper.

2.5.1.2. Preparation of aqueous extract

Aqueous extract of weeds was prepared by taking 20 g of fresh leaves and macerated in 20 mL of distilled water and kept for 48 hrs at room temperature. The extract was double filtered through muslin cloth layers and filter paper.

2.5.1.3. In vitro experiments

Extracts were added (10 %) in sterilized PDA medium before pouring. Control plates were poured with only PDA medium. A disc of 3mm from the actively growing colony margins of 10 days old Myrothecium roridum cultures were transferred to the PDA plates amended with the weed extracts. Inoculated Petri plates were incubated at 27±2 °C and colony growth was measured at an interval of 2 days up to 14 days. All

the treatments were replicated thrice. Growth inhibition percentage was measured by the following formula.

Equation 4:

2.5.1.4. In vivo experiments

Seeds of moderately resistant cultivars Jaunpuri were seeded in 12 × 18 cm sized pots keeping 1/3 free board, on first week of March 2012 & 2013. For in vivo studies, bitter gourd crop was seeded on ridges with 75 cm P × P and 45 cm R × R distance. Moderately resistant variety Jaunpuri was seeded on beds in 7 × 7 meter plots using RCBD design with three replicates. Spore suspension of M. roridum (1 × 105) was sprayed at 8-10 leaf stage of plant and incubated under natural environmental conditions for a week. Three sprays of selected plant (Nicotiana plumbaginifolia, Parthenium hysterophorus, Solanum nigrum, Coronopus didymus and Sphaeranthus indicus) extracts were applied at 10 % concentration at fifteen days intervals between two sprays. Data was recorded on percent disease incidence and percent disease reduction over control.

2.5.2. Inter-cropping of aromatic plants

Pot experiments were performed at Experimental Station of Institute of Agricultural Sciences, University of the Punjab (IAGS, PU) Lahore, Pakistan. Field trials were conducted at fields of plant pathology section, Ayub Agriculture Research Institute (AARI) Faisalabad, Pakistan. Seeds of bitter gourd, chili, capsicum, onion bulbs and rhizomes of turmeric, ginger were obtained from AARI Faisalabad, Pakistan (Table 2.4). For pot experiments, 30 × 45 cm sized pots were used, filled ¾ with the sandy loam soil while for field experiments, 76-91 cm ridges with 61 cm distance between the rows was prepared. Plant to plant distance was maintained at 45 cm. Production technology prescribed by the Punjab Agriculture Department was followed. The four week old seedling growth was evaluated after the germination of 75% germplasm of all the tested plants. Spore suspension of M. roridum (1 × 105) was

sprayed at 8-10 leaf stage of plant with hand atomizer for artificial inoculation of four weeks old seedlings. All the treatments were replicated thrice and each replication contains ten plants. Disease incidence was recorded at weekly intervals up to 7 weeks (bitter gourd plant maturity).

Table 2.4.Inventory of seeding material used for intercropping

Plant Planting Sr # Botanical name Plant family intercropped material 1 Garlic Allium sativum Amaryllidaceae Cloves

2 Onion Allium cepa Amaryllidaceae Bulb

3 Ginger Zingiberofficinale Zingiberaceae Rhizome

4 Green chili Capsicum frutescens Solanaceae Seeds

5 Capsicum Capsicum annuum Solanaceae Seeds

6 Turmeric Curcuma longa Zingiberaceae Rhizome

7 Arvi Colocasia esculenta Araceae Cornels

2.5.3. Efficacy of commercial fungicides against MLS disease

2.5.3.1. Commercial fungicides

2.5.3.1.1. In vitro experiment

Food poison technique was used to evaluate the in vitro effect of different commercial fungicides against the M. roridum. The experiments were arranged in completely randomized design (CRD) with fifteen treatments and four replications. A total of five fungicides [Aliette (Fosetyl-Aluminium), Antracol (Propineb), Dithane M 45 WP 80 (Mancozeb 80 %), Score 250 EC (Difenoconazole) and Cabrio top (Metiram 92 % + Pyraclostrobin 8 %)] were tested each with three dose i.e. 0.01, 0.05 and 0.1 % (100, 500 and 1000 PPM respectively) concentrations. Stock solutions of all tested fungicides were prepared freshly when required. Tested concentrations were added to the pre-autoclaved molten medium at 45 °C. 20 ml of amended medium each having different fungicide concentrations in 250 mL of flask was poured into the 9 cm sterilized Petri plates. The medium without fungicide was served as control. 5 mm plug from the actively growing culture was used for inoculation. All the inoculated plates were incubated at 25 °C till the control achieve the maximum growth i.e. 90 mm.

2.5.3.1.2. In vivo experiment

For pot experiments, 12 × 18 cm sized pots keeping 1/3 free board, moderately resistant variety Jaunpuri was seeded. For field experiment, a distance of 76 cm P × P and 45 cm R × R was maintained on ridges. Three most effective chemicals (antracol, score and cabrio top) were selected from the in vitro trials. Bitter gourd seeds were sown at a depth of 2-3 cm on first week of March 2012 and 2013. Spore suspension of M. roridum (1 × 105) was sprayed at 8-10 leaf stage of plant and incubated under natural environmental conditions for a week. Three sprays of selected fungicides at recommended dose were applied at fifteen days intervals between two sprays. Data was recorded on percent disease incidence and percent reduction over control.

2.5.3.2. Technical grade fungicides

2.5.3.2.1. In vitro experiment

Efficacy of technical grade tebuconazole was evaluated by MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay under In vitro conditions. Experiment was carried out in 96 well, flat bottomed, transparent, sterile micro titer plate with lid cover (TPP, Sigma Aldrich). M. roridum spore concentration was adjusted to 1 × 103 in Potato Dextrose broth medium and 100 µL was poured in wells. Tebuconazole stock (5 mg/mL ethanol) was added into the wells at 0.5, 1.25, 2.5 and 5 mg/L concentration with four replications whereas control treatment was without tebuconazole. Micro titer plate was incubated at room temperature (25 °C) at 160 rpm for 48 hours for spore germination and hyphae growth. Stock solution of MTT (5mg/mL deionized water) was prepared and filter sterilized. After 48 hours incubation, 10 µL of MTT stock was added to the wells and incubated at 160 rpm for 16 hours at room temperature. Micro titer plate was centrifuged at 4000 rpm for 10 min. Medium was pipetted out of the wells and 100 µL of isopropanol was added to each well. Blue coloration appeared due to the reduction of MTT to formazon. Plate was kept on shaker at 160 rpm for 30 min to dissolve all blue dye, formazon. Optical density (OD) was measured at 595 nm with ELISA plate reader (Infinite 200, Tecan) and isopropanol control was taken as blank. Percentage growth inhibition was measured by the equation 4 (pp. 38).

2.6. Statistical analysis

All the data was used to calculate mean value for each replicate in all experiments. Data means were used to calculate standard deviation and standard error. Pearson correlation was calculated among agronomic traits recorded for germplasm screening whereas management data was subjected to analysis of variance (ANOVA) followed by Tukey‟s HSD (honest significant difference) using SPSS v 15.0 for windows. Significance of tebuconazole concentrations was measured by Student‟s T Test (Steel and Torrie, 1980).

Chapter 3

RESULTS

The increasing trend for the Myrothecium leaf spot disease distribution was observed on commercial germplasm of bitter gourd in various agro-ecological zones of the Punjab Province. None of the growth stage was observed disease free however seedling mortality of the nursery stock has become an emerging threat for the commercial vegetable growers. It was also noticed that if the proper and practicable solution of this menace is not worked out, the disease could be spread to other provinces and favorable climatic conditions could lead the epidemics.

3.1. Geographical distribution of M. roridum in Punjab, Pakistan

3.1.1. Survey and disease assessment

Disease was widely distributed and none of the area or cultivar was found disease free in canal irrigated plains and Barani areas (Fig 3.1). The disease was observed at every growth stage of the plant (Plate 3.1) and cumulative description of the surveys conducted during 2011, 2012 & 2013 reveals the highest prevalence, disease incidence and severity and disease index for Myrothecium leaf spot (MLS) disease in mixed cropping zone of Punjab (Table 3.1).Mean prevalence of MLS disease was consecutively recorded 100 % for the surveyed years in mixed cropping zone whereas in rice zone, 36, 32 and 42% for the year 2011, 2012 and 2013 respectively were recorded. Barani and zone showed prevalence range of 14 to 17% and the highest 17% was recorded during 2013 whereas cotton zone exhibited range of 14 to 22% and the highest 22% was recorded in during 2012. The least prevalence recorded in DG Khan Zone was 11, 19 and 14 % during the years 2011, 2012 and 2013 respectively. Disease index ranged 3-31%during 2011-2013 with the highest index of 31 % in mixed cropping zone and the least of 3 % in DG Khan zone during 2013. Disease incidence ranged between 8-13% with severity range of 0-2 on 0-5 visual severity rating scale (2.2). MLS disease was not observed in Thal irrigated

region comprising of Mianwali, Bhakkar and Khushab districts and Marginal land comprising on Bahawalpur district throughout the survey period (Annex-II).

Detailed monitoring of specific sites was conducted in five major bitter gourd producing districts of central Punjab viz i.e., Lahore, Kasur, Faisalabad, Sargodha and Jhang, considered as major bitter gourd production areas of mixed cropping zone. Disease incidence ranged between 42-50% with severity range of 1-4and 28 % disease index for mixed cropping zone (Table 3.2). Among the districts within mixed cropping zone, Lahore showed the highest disease incidence i.e., 59% and disease severity range of 1-4 on scale. Sargodha, Kasur and Faisalabad showed a moderate response with 48, 45 and 33 % disease incidence and 1-4, 1-3 and 1-2 severity scale respectively. The least disease incidence of 29% with 0-2 disease severity and 10% disease index was recorded at District Jhang in mixed cropping zone (Table 3.2).

Plate 3.1. Symptomatic variation pattern of Myrothecium leaf spot disease in mixed cropping zone of Punjab. A: Lahore; B: Kasur; C: Sargodha; D: Faisalabad

Fig. 3.1. Geographical distribution map of Myrothecium leaf spot of bitter gourd in agro- ecological zones of Punjab, Pakistan during 2011-13. (Modified PARC, http://old.parc.gov .pk /Maps/AgroEcoPunjab.html )

Table 3.1. Geographical distribution of Myrothecium roridum in agro ecological zones of the Punjab, Pakistan

Zones Year PP PDI DSR (0-5) PDIn

2011 11.35 8.05 0-2 3.27

DG Khan 2012 19.42 13.30 0-2 4.13

2013 14.26 11.23 0-1 3.23

2011 16.15 12.28 1-3 4.28

Barani (Rain 2012 14.11 9.21 1-2 6.1 fed) 2013 17.41 13.24 0-2 5.83

2011 36.43 33.16 1-3 14.78

Rice zone 2012 32.14 26.08 1-2 15.12

2013 42.07 35.22 1-3 16.47

2011 100 45.20 2-4 28.97

Mixed zone 2012 100 42.31 1-4 25.43

2013 100 49.22 1-4 30.72

2011 14.31 14.09 1-2 5.32

Cotton zone 2012 21.19 18.19 0-2 5.66

2013 19.23 15.19 1-2 4.07

2011 0 0 0 0

Marginal 2012 0 0 0 0 land 2013 0 0 0 0

2011 0 0 0 0

Thal region 2012 0 0 0 0

2013 0 0 0 0

PP= Percent prevalence; PDI= Percent disease incidence; DSR= Disease severity range; PDIn= Percent disease index.

Table 3.2. Geographical distribution of Myrothecium roridum during 2011-13 in districts of mixed cropping zone of the Punjab, Pakistan

District PDI ± SE DSR (0-5) PDIn ± SE

Lahore 59.07 ± 0.23 1-4 31.43 ± 0.19

Kasur 44.56 ± 0.11 1-3 27.04 ± 0.07

Faisalabad 33.17 ± 0.28 1-3 23.14 ± 0.20

Sargodha 48.22 ± 0.29 1-4 31.05 ± 0.25

Jhang 28.73 ± 0.13 0-3 10.27 ± 0.17

PDI= Percent disease incidence; DSR= Disease severity range; PDIn= Percent disease index; SE= standard error

3.1.2. Isolation and identification studies of M. roridum

A total of 317 diseased bitter gourd specimens showing characteristics symptoms of infection on stems, leaves and fruit as well as rhizospheric soil of infected plants were collected from different location of the Punjab province. Diseased samples were inoculated on PDA medium for isolation of associated fungi Single spore cultures of the isolated fungi were obtained and studied for their characteristics (Plate 3.2). Pathogenicity of the pathogen M. roridum was confirmed by adopting Koch‟s postulates following the leaf inoculation technique.

3.1.3. Molecular studies

DNA was extracted and PCR product was prepared according to the manufacturer direction (Qiagen, Hilden, Germany). The sequence of 1200 bp fragments of ITS rDNA PCR product was determined by LGC AGOWA (Annexure III). The sequence was used to search Genbank database of NCBI and we found that it has 99% similarity with M. roridum gb: strains BBA 71015 (AJ3020010) and BBA 67679 (AJ301995). 3-5 days old cultures were submitted to First Fungal Culture Bank of Pakistan (FCBP accession no. 1155) and Leibniz-institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Accession # DSM 28971).

Plate 3.2. Characteristics identification features of Myrothecium roridum. A: colony on PDA medium; B: mycelia at 100X; C: conidia formation; D & E: sporodochia at 40X and spores; F and G: single spore of M. roridum at 1000X (Axioplan 2, Germany).

3.2. Characterization of M. roridum

3.2.1. Aggressiveness evaluation

A total of 54 isolates of M. roridum isolated from bitter gourd plant parts and rhizospheric soil were investigated for their aggressiveness behavior against bitter gourd cultivar Jaunpuri (MCV01) under green house conditions (Table 3.3). Among the test population of isolates twenty three isolates were found highly aggressive; seventeen were moderately aggressive whereas fourteen were less aggressive and none of the isolate was foundnon aggressive or avirulent. A group of nine isolates (Mr10, Mr21, Mr28, Mr30, Mr34, Mr37, Mr49, Mr51 and Mr54) showing stable aggressiveness behavior under green house conditions was selected for microscopic, molecular and physiological characterization.

Table 3.3. Aggressiveness of population of Myrothecium roridum against bitter gourd under natural environmental conditions in the pots

Sr# Isolate code Aggressiveness behavior

1 None -

2 Mr03, Mr08, Mr09, Mr14,Mr15, Mr16, + Mr32, Mr40, Mr41, Mr45, Mr50, Mr51, Mr52, Mr53, Mr54

3 Mr01, Mr02, Mr04, Mr05, ++ Mr06,Mr07,Mr10, Mr11, Mr12, Mr13, Mr25, Mr26, Mr30, Mr31, Mr42, Mr43, Mr44

4 Mr17, Mr18, Mr19, Mr20, Mr21, Mr22, +++ Mr23, Mr24, Mr27, Mr28, Mr29, Mr33, Mr34, Mr35, Mr36, Mr37, Mr38, Mr39, Mr46, Mr47, Mr48, Mr49 “-”= avirulent, “+” = Less aggressive, “++” =moderately aggressive, “+++” = highly aggressive

3.2.2. Morphological studies Due to slow growing nature on growth medium, fourteen day old M. roridum isolates (Mr10, Mr21, Mr28, Mr30, Mr34, Mr37, Mr49, Mr51 and Mr54) were observed for their macroscopic and cultural variations on potato dextrose agar medium (Plate 3.3). Macroscopic features like color, size, growth pattern, elevation and colony color were examined under stereoscope (Table 3.4). Colony color varied from white to dirty-white from observes side and from off-white to pale on reverse side of the Petri plate (Table 3.4). On observe side Mr10, Mr30, Mr34, Mr37 isolates produce white color,Mr28 produce dirty white to off-white whereas Mr21, Mr49 and Mr54 exhibited off white to cream.Mr10 and Mr51isolate exhibited white color on reverse side, Mr30, Mr34 and Mr37 produce off white while Mr21, Mr28, Mr49 and Mr54 produced yellowish brown to pale color.

3.2.3. Microscopic studies

Shape of conidia of all isolates was cylindrical/rod with round and tapered ends for all isolates. All isolates produced hyaline to olive green spores while size of conidia ranged in length from 5-8 µm and in width to 1.4-1.8µm (Table 3.4). Maximum average length of conidia (7.65 × 1.8 µm) was produced by isolate Mr28 whereas Mr21 produced the smallest spores (5.16 × 1.4 µm) among the tested isolates. There was no significant difference in the length and width of spores among different tested isolates.

Mr30

Plate 3.3. Morphological variation in colony growth of Myrothecium roridum isolated from commercial bitter gourd germplasm.

Table 3.4. Morphological and cultural variation among local population of M. roridum isolated from commercial bitter gourd germplasm

Isolate Colony Colony color Colony color Spore Spore width Sr # Colony form code elevation observe reverse length (µm) (µm)

01 Mr10 Circular Convex White White 6.07 1.6

02 Mr21 Circular Raised Yellowish white Pale 5.16 1.4

03 Mr28 Circular Flat Off white Yellowish brown 7.63 1.8

04 Mr30 Circular Flat White Off white 6.65 1.7

05 Mr34 Circular Flat White Off white 6.42 1.6

06 Mr37 Circular Flat White Off white 5.29 1.5

07 Mr49 Circular Raised Cream Yellow 6.97 1.6

08 Mr51 Circular Umbonate White White 6.87 1.7

09 Mr54 Circular Flat Cream Yellowish brown 7.24 1.8

3.2.4. Physiological response

A wide range of variations among the test treatments on colony growth (mm) and sporulation (no. of spores/mL) for medium, temperature, pH and photoperiod was observed.

3.2.4.1. Growth medium studies

Among the test growth medium, highest radial growth (77 mm) was observed on PDA whereas on BGA, MEA, GA and CDA colony diameter was 68, 64, 56, 43 respectively (Fig. 3.3). The least radial growth 37 mm was recorded on NA medium. But spore production trend was not in line with radial growth development. Among the all tested growth media, when grown on PDA, highest number of spores (239 × 106 spores/mL) were counted while on GA, least number of spores (49 × 106 spores/mL) were observed.

3.2.4.2. Optimization of incubation temperature

Temperature optimization studies were conducted on PDA which proved best growth medium among all tested media. Role of temperature seemed to be more dominant towards the radial growth and sporodochia production. The highest colony growth (87 mm) was observed at 30 °C whereas highest spore production (315 × 106 spores/mL) was at 35 °C (Fig. 3.4). Colony growth was 46, 84 and 73 mm at 20, 25 and 35 °C respectively. Least colony growth (28 mm) and minimum spore production (39 × 106 spores/mL) was recorded at 15 °C.

3.2.4.3. Optimization of growth medium pH

Studies for optimization of growth medium pH were conducted at pH 5, 5.5, 6, 6.5, 7 and 7.5 levels on PDA medium and incubated at 30 °C which was best growing temperature for M. roridum. A decreasing trend in colony growth and spore production was observed with increase in pH level. The highest colony growth (87 mm) was observed at pH 5 and least (45 mm) at pH 7.5 whereas 5.5, 6.0, 6.5 and 7.0 exhibited intermediate level of growth with 74, 71, 62 and 54 mm colony diameter respectively

(Fig. 3.5). Highest spore production (504 × 106 spores/mL) was recorded at pH 5.0 whereas minimum spore production (119 × 106 spores/mL) was at pH 7.5.

3.2.4.4. Optimization of photoperiod for fungal growth

Photoperiod optimization studies were conducted at 24h light, 24h dark and 12h alternate light and dark period and incubated at 30 °C. The highest mycelial growth (88 mm) was observed at 16/8 h alternate light and dark period whereas least was observed at 24h light (Fig. 3.6). The maximum spore production (524 × 106 spores/mL) at 16/8 h alternate light and dark period and minimum (268 × 106 spores/mL) at 24h dark.

3.2.4.5. Culture age and virulence relationship

The relationship between virulence and age of the culture was evaluated by inoculating 2-10 day old culture on detached leaves. The investigations were conducted by placing young leaves of bitter gourd on plain agar medium and incubated at 25 ± 2 °C to cover 1 cm area. A 3 mm plug of 2-10 day old culture (in previous studies 15 day old culture was used developed on optimum nutrient medium, temperature, pH and light/darkness conditions. The virulence was measured on the basis of infection development, conidia and sporodochia production on 1cm2 area of the leaf (Fig. 3.7). According to the data recorded at 12 h interval, 5-6 days old culture proved the most virulent as it covered 1cm2 prescribed area of the leaf in 82 h whereas least growth rate was observed for 2 day old culture which covered prescribed 1cm2 leaf areas in 126 h.

Colony growth Spores

100 300

a a 250 10

80 b

b × b b 200 60 c c 150 d 40 100 d d 20 d

Colony growth (mm) Colonygrowth 50 No. of spores/mL spores/mL of No. 0 0 BGA CDA GA MEA NA PDA Nutrient medium

Fig. 3.2. Effect of nutrient medium on colony growth and sporulation of Myrothecium roridum. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method. Primary Y axis= colony growth (mm); Secondary Y axis= number of spores/ mL; NA; nutrient agar, PDA; potato dextrose agar, CDA; czapekdox agar, MEA; malt extract agar, BGA; bitter gourd agar

Colony growth Spores 100 350

a a 6 a a

300 10 80 b b × 250 60 200 c c cd 40 d 150 d 100 20 e

Colony growth (mm) growth Colony No. of spores/mL spores/mL of No. 0 0 15 20 25 30 35 40 Temperatre °C

Fig. 3.3. Effect of different temperatures on colony growth and sporulation of Myrothecium roridum was measured using PDA medium. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method. Primary Y axis= colony growth (mm); Secondary Y axis= number of spores/ mL

Colony growth Spores 100 600

a 6 a

500 10 80 b b b × b c 400 60 d e c 300 40 d 200 e

20 100

Colony growth (mm) Colonygrowth No. of spores/mL spores/mL of No. 0 0 5 5.5 6 6.5 7 7.5 pH level

Fig. 3.4. Effect of pH on colony growth and sporulation of Myrothecium roridum was measured using PDA medium. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method. Primary Y axis= colony growth (mm); Secondary Y axis= number of spores/ mL

Colony growth Spores 100 600 a

6 80 b 500 b 10 b 400 × 60 c 300 40 200

20 Colony growth (mm) Colonygrowth

100 spores/ml of No.

0 0 Dark Light Light/Dark

Fig. 3.5. Effect of photoperiod on colony growth and sporulation of Myrothecium roridum. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method. Primary Y axis= colony growth (mm); Secondary Y axis= number of spores/ mL

Time Spores 140 180

a a ab a a

120 ab 150

b 6 bc bc bc c 10 100

c 120 × c c 80 90 60 60

40 No. of spores/ml spores/ml of No.

20 30 Infection development time (h) development time Infection

0 0 2 4 6 8 10 12 14 Cultue age (day old)

Fig. 3.6. Evaluation of culture age on infection development and spore production of Myrothecium roridum on bitter gourd leaves. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method. Primary Y axis= colony growth (mm); Secondary Y axis= number of spores/ mL

3.2.5. Study of genetic variation among selected isolates

A total of 14 decamer random primers were used and all the primers produced amplicons in all the tested isolates. PCR was repeated three times and reproducible amplicons were considered for the genetic variation studies and dendrogram construction. A total of 93 DNA fragments were amplified, with an average of 7.15 RAPD markers per primer. Out of 93 DNA-amplified fragments, 28% were found to be monomorphic. The rest of 72% were polymorphic. The number of DNA bands ranged from 4 with primers SBSA07 to 9 with SBSA03, SBSA11 and SBSA14. The extent of polymorphism per primer ranged from 20-88.9% with average of 72.04%. Approximate size of the largest fragment produced was 1.3 kb and the smallest easily recognizable fragment produced was approximately 0.1 kb. Among the 9 test isolates studied, genomic DNA of Isolate Mr37 produced the maximum number of amplified fragments i.e., 67 while Isolate Mr54 produced 43 bands, which is the minimum number. Other isolates produced between 49 and 61 bands in general. The similarity matrix is based upon Nei and Li‟s similarity coefficient. The genetic distances (Nei‟s similarity) ranged from 0.17 to 0.68. Maximum similarity (83%) was observed between Isolate Mr34 and Mr49. Isolates Mr54 and Mr30 formed cluster A whereas isolate Mr37, Mr49, Mr34 and Mr21 were placed into cluster B irrespective of their different geographical origin. Isolates Mr51, Mr28 and Mr10 formed Cluster C (genetic distance = 0.54).

Plate 3.4. Amplification profiles of Myrothecium roridum isolates produced by random primers (SBSA02, SBSA05, SBSA06, SBSA07, SBSA09, SBSA11, SBSA13 and SBSA14); Lane 1 – 9 = Isolates Mr10, Mr21, Mr28, Mr30, Mr34, Mr37, Mr49, Mr51 and Mr54

Table 3.5. Similarity matrix of of local population of Myrothecium roridum isolated from commercial bitter gourd germplasm

Mr10 Mr21 Mr28 Mr30 Mr34 Mr37 Mr49 Mr51 Mr54 1 Mr10 1.00 2 Mr21 0.65 1.00 3 Mr28 0.73 0.66 1.00 4 Mr30 0.65 0.62 0.72 1.00 5 Mr34 0.57 0.52 0.62 0.60 1.00 6 Mr37 0.60 0.61 0.75 0.79 0.63 1.00 7 Mr49 0.62 0.63 0.71 0.81 0.57 0.80 1.00 8 Mr51 0.65 0.54 0.60 0.66 0.66 0.63 0.63 1.00 9 Mr54 0.66 0.61 0.67 0.77 0.57 0.80 0.74 0.63 1.00

Table 3.6. RAPD fingerprints data for the molecular characterization of local population of Myrothecium roridum.

Sr # Primer Total bands Polymorphic Percentage bands polymorphism

1 SBSA01 7 6 85.7

2 SBSA02 7 5 71.4 SBSA03 3 9 8 88.9 SBSA04 4 6 4 66.7 SBSA05 5 5 1 20 SBSA06 6 6 4 66.7

7 SBSA07 4 1 25 SBSA09 8 7 3 42.8 SBSA10 9 8 7 87.5 SBSA11 10 9 8 88.9 SBSA13 11 8 6 75 SBSA14 12 9 7 77.8 SBSA16 13 7 6 83.3

Total 93 67 72.04

Fig. 3.7. Cluster analysis based dendrogram depicting genetic relationship among Myrothecium roridum isolates developed from RAPD data using Ward’s Linkage method.

3.3. Studies on infection development by M. roridum within host tissues

Interaction mechanism of M. roridum with Momordica charantia examined by Combination of light, fluorescent (Axioplan 2; 505-530 nm) and transmission electron microscopy. Conidia of M. roridum initially adhere to the epidermal cell wall and invasion is directly through the epidermal cell wall and into the leaf interior most often at the junction of two epidermal cells. Figures show selected views of a regularly monitoring of infection development site. Sometimes fungal hyphae appeared to penetrate at epidermal cell wall junctions. The fungal hyphae appeared to spread throughout the leaf and root surface (Plate 3.4). An extensive mycelium network was being observed on the leaf and root surface. Penetration of M. roridum in the leaf tissues is through cuticle with formation of appressoria (Plate 3.5). Germ tubes tip swell and develop to form an appressorium which penetrate the host cuticle directly. Close examination of the vascular tissues (xylem and phloem) using light and electron microscopy was performed to elucidate whether the vascular tissue was involved in transportation. M. roridum hyphae and spores penetration in xylem vessels was observed through fluorescent microscope (Plate 3.4). Presence of hyphae within the leaf xylem appeared within eight days after colonization in leaf veins. The M. roridum hyphae move within the vascular elements of the xylem. The surface of colonized leaves exhibit macroscopic symptoms 4 day after inoculation indicating ill health compared to normal, uninfected leaves. A large no of mycelia were protrude out of leaf veins and stained with WGA AF 488 dye. These erected mycelia produce sporodochia on later stages. Light and electron microscopy confirmed the production of extracellular matrix by M. roridum (Plate 3.6).

Plate 3.5. Infection development of M. roridum in bitter gourd plant leaf tissue. (A) By 5 dai, hyphae form extensive mycelia network on leave surface. (B) By 8 dai, hyphae seems to be spread across leaf veins beyond the point of infestation. (C) Erected hyphae protrude out of leaf veins cell. [Scale bar C = 10 μm]

Plate 3.6. Infection development of M. roridum in bitter gourd plant root tissue. (D) By 5 dai, hyphae excessively occupy root. The root cap is heavily infested with hyphae. (E) To better visualize the position of hyphae with the fluorescent signal of the wheat germ agglutinin-stained (WGA-AF 488) fungal hyphae. (F) The penetration site is indicated by an arrow. [Scale bar F = 10 μm]

Plate 3.7 to be continued

Plate 3.7. Transmission electron micrographs of the adaxial surface of bitter gourd inoculated with M. roridum. (A) germ tube initiation at 6 hour. (B) elongation of germ tube. (C & D) mycelia clump formation. (E) network of mycelia. (F) mat formation over the leaf surface. Magnification bar = A&B= 50, C= 200, D= 20, E= 50, F= 200μm.

Plate 3.8 to be continued

Plate 3.8. Transmission electron micrographs of the abaxial surface of bitter gourd leaves inoculated with M. roridum. (G) metabolites excretion could be observed by fungal mycelia mat and production of spores. (H & I) deplasmolysis of cell protoplast on adaxial and abaxial surface of leaf. (J) conidia production. (K) germination of conidia. (L) appresoria penetrating the cell. Magnification bar G=20, H=50, I-K = 10, L= 20μm.

3.4. In vivo screening of bitter gourd germplasm

After an extensive survey of different bitter gourd growing areas of the Punjab Province it was noticed that the disease is prevailing in about all the fields, its severity varies on different varieties. A total of thirty seven cultivars were screened under in vivo conditions. Data regarding disease severity on different varieties indicated the variance among the reaction of different bitter gourd varieties. Screening germplasm was mentioned as local and hybrid commercial varieties and candidate lines based on the seed procured source. 3.4.1. Pot experiment The test germplasm exhibited significant variation at P<.05 in resistance reaction against MLS disease. Variation of resistance reaction measured on to 0-5 rating scale. None of the variety was found disease free or immune and in general reaction ranged 1-4. On the basis of cumulative resistance reaction for the years 2012 and 2013 germplasm is classified in the following groups; 0=Immune, 1= Resistant, 2=Moderately resistant, 3=Moderately Susceptible, 4= Susceptible, and 5=Highly Susceptible. The varieties MONIKA(7004), LEENA(7005), CBT-36, VRBG 233, Green wonder, Fsd long, Cross 888 f1 hybrid, Long green, JK Lena exhibited moderately resistant reaction. Desi karela, BG-7107, PKBT-1, BSS-616, VRBG 233, SHBG-48, Lamba karela, Karela, No 361 f1 hybrid, Advanta 103, Jaunpuri showed moderately susceptible reaction and Indian karela, Sachal black4722, Jaunpuri long, TIPU, KIRAN, Jhalri, VRBG 227 , VRBG 230, VRBG 231, Jhalri, , BG-34, RAJA, Early green, Runfeng, Preeti were found susceptible (Table 3.6).

3.4.2. Effect of MLS susceptibility reaction on agronomic traits of bitter gourd in pot experiment

Wide range of variation in agronomic parameters viz. vine length, number of branches, days to flowering, number of fruits and yield per plant were recorded (Table 3.9). The tested germplasm exhibited a great variation among the vine length, number of branches and yield per plant. While days to flower and number of fruits showed comparatively least variations.

Vine length ranged between 42-188 cm and longest vine length was recorded in MONIKA (7004) followed by LEENA (7005) and CBT-36 at 90 days after germination (DAG) i.e., 187, 184 and 176 cm respectively. Whereas BG-7107 exhibited the smallest (i.e., 42.2 cm) vine length (Table 3.9). Number of branches ranged 1-8 with the highest (7.3) number of branches observed in Jaunpuri cultivar. The lowest number of branches i.e., 1.5 was found in BG-7107 and KIRAN while rest of the cultivars ranged between 2.3 to 5.5. Day to first flowering ranged between 39-47 DAG among the tested germplasm; desi karela showed early flower emergence (i.e., 39.3) followed by BSS-616 and BG- 7107 with 39.6 DAG. Lamba karela exhibited late flowering and first flower emergence was recorded on 45.6 DAG. MONIKA (7004) cultivar bore the highest (8.3) number of fruits per plant followed by LEENA(7005) and CBT-36 ( i.e., 7 and 6.6 respectively). Indian karela and BG-7107 cultivars exhibited the least number (i.e., 2.3) of fruits. Number of fruits per plant showed inconsistency ranging from 3 to 6 for the rest of the cultivars. A vast range of variation was observed among the cultivars in terms of yield per plant. The highest yield was recorded in Fsd long followed by MONIKA (7004) and Preeti with yield of 1511, 1288, and 1271g per plant respectively. Minimum recorded yield (140 g per plant) was for BG-7107. Pearson correlation revealed a significant positive correlation (P ≤ 0.01) between vine length and number of branching (0.368), vine length and first flowering (0.404), number of branches and number of fruits per plant (0.303), number of fruits per plant and yield per plant (0.631) under in vitro conditions (Table 3.10). Correlation between vine length and number of fruits (0.241), vine length and yield per plant (0.224) was significant at P≤0.05. A non significant correlation between number of branches and first flowering (0.444), first flowering and number of fruits (- 0.065) and first flowering and yield per plant (- 0.167) was observed.

Table 3.7. Susceptibility reaction of tested germplasm against myrothecium leaf spot disease in pot experiment under natural environmental conditions

Disease severity Cultivar response Varieties scale (0-5) category

0 Nil Immune

1 Nil Resistant

2 MONIKA(7004), LEENA(7005), CBT-36, VRBG 233, Green wonder, Moderately resistant Fsd long, Cross 888 f1 hybrid, Long green, JK Lena

3 Desi karela, BG-7107, PKBT-1, BSS-616, VRBG 233, SHBG-48, Moderately susceptible Lamba karela, Karela, No 361 f1 hybrid, Advanta 103, Jaunpuri

4 Indian karela, Sachal black4722, Jaunpuri long, TIPU, KIRAN, Jhalri, Susceptible VRBG 227 , VRBG 230, VRBG 231, Jhalri, , BG-34, RAJA, Early green, Runfeng, Preeti

5 Nil Highly susceptible

Table 3.8. Effect of MLS susceptibility reaction on agronomic traits of bitter gourd in pot experiments under natural environmental conditions

Sr Vine length Number of Number of Variety Days to flower Yield (g/plant) # (cm) branches fruits/plant 1 Jaunpuri 109.3 h-l 7.3 a 41.3 a-c 6 a-d 966.6 b-h 2 Jhalri 120.2 hi 4.2 d-g 43.3 a-c 3.3 c-e 359.3 lm 3 Indian karela 131.8 f-h 3.3 g-i 42.3 a-c 2.3 e 311lm 4 Green wonder 171 a-e 2.7i-m 43.6 a-c 4 b-e 686.6 g-m 5 Chaman 159.1 b-e 4.2 d-f 43.3 a-c 4 b-e 700.3 g-m 6 Fsd long 173.3 a-d 5.4bc 42.6 a-c 6 a-d 1511.6 a 7 VRBG 227 46.5pq 3.4 f-i 40.6 a-c 4 b-e 630.3 h-m 8 VRBG 230 111.2 h-k 3.2 h-k 42.3 a-c 4.6 b-e 496 j-m 9 VRBG 231 92.7 j-o 3.6 e-h 43 a-c 3.6 b-e 415.3 k-m 10 VRBG 233 150.1 d-g 5.5 b 40.6 a-c 5.6 a-e 865.3 d-j 11 SHBG-48 171.8 a-e 3.3 h-j 45.3 ab 3.6 b-e 648.6 h-m 12 Early green 110.8 h-k 2.4 k-m 41.3 a-c 4.6 b-e 727.6 f-l 13 JK Lena 82.6 m-o 2.5i-m 40 a-c 5.3 a-e 776.6 e-k 14 BG-7107 42.2 q 1.5 n 39.6 bc 2.3 e 140.3 m 15 CBT-36 88 k-o 2.3 l-n 41.3 a-c 6.6 a-c 1079.3 b-g 16 PKBT-1 128.2gh 3.6 e-h 43 a-c 5 a-e 773.3 e-k 17 Preeti 117.133 2.6i-m 41 a-c 5.6 a-e 1271.3 a-c 18 KIRAN 95.2i-n 1.5 n 42.3 a-c 4.3 b-e 882.6 c-j 19 Palli 151.2 c-g 5.4bc 41.6 a-c 4 b-e 1168 a-e Contd…

20 Cross888F1hybrid 86.3 k-o 4.2 de 41.3 a-c 5.3 a-e 1237.6 a-d 21 Runfeng 97.3i-n 3.4 f-i 42 a-c 4.6 b-e 1077 b-g 22 No361F1 hybrid 146.4 e-g 3.2 h-l 41.6 a-c 4.6 b-e 771.3 e-k 23 Advanta 103 94.8i-n 4.6 cd 42.6 a-c 5 a-e 618 h-m 24 Sachal black 4722 157.5 c-f 3.3 h-k 44.6 a-c 3 de 456.3 k-m 25 Long green 93.1 j-o 3.3 h-k 44 a-c 4 b-e 631 h-m 26 Jhalri 84.7 l-o 2.5 j-m 42.6 a-c 3.3 c-e 548.6i-m 27 Jaunpuri long 115.1 h-j 2.3mn 42 a-c 4.3 b-e 649.6 h-m 28 Desikarela 87.2 k-o 3.2 h-k 39.3 c 5 a-e 720.6 f-l 29 Lambakarela 115.1 h-j 4.3 de 45.6 a 4.3 b-e 598 h-m 30 Karela 106.8 h-m 3.4 f-i 43 a-c 4.6 b-e 669.6 h-m 31 BG-34 116 h-j 3.2 h-l 42 a-c 3.6 b-e 502 i-m 32 BSS-616 68.5 op 4.4 de 39.6bc 5.3 a-e 898.6 b-i 33 TIPU 97.7i-n 4.3 de 40 a-c 4.6 b-e 704 g-m 34 RAJA 74.7 no 3.1 h-m 42.6 a-c 4.3 b-e 878.3 c-j 35 CBT-36 176.1 a-c 4.3 de 44 a-c 6.6 a-c 1111.6 a-f 36 LEENA(7005) 183.7 ab 5.3bc 43.6 a-c 7 ab 980 b-h 37 MONIKA(7004) 187.1 a 5.4bc 43 a-c 8.3 a 1288.6 ab Values in columns with different letters show significant difference (P≤0.05) as determined by Tukey‟s HSD method.

Table 3.10. Correlation of agronomic traits against susceptibility reaction in pot experiment under natural environmental condition

Vine length No of First Yield No of fruits (cm) Branches flowering (g/plant) Vine length (cm) Pearson Correlation 1 Sig. (2-tailed) N 111 No of Branches Pearson Correlation 0.386(**) 1 Sig. (2-tailed) 0.000 N 111 111 First flowering Pearson Correlation 0.404(**) 0.073 1 Sig. (2-tailed) 0.000 0.444 N 111 111 111 No of fruits Pearson Correlation 0.241(*) 0.303(**) -0.065 1 Sig. (2-tailed) 0.011 0.001 0.499 N 111 111 111 111 Yield (g/plant) Pearson Correlation 0.224(*) 0.283(**) -0.167 0.631(**) 1 Sig. (2-tailed) 0.018 0.003 0.081 0.000 N 111 111 111 111 111 * Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed). N= total no of treatment replicates

3.4.3. Field experiment

The test germplasm exhibited significant variation in resistance reaction against myrothecium leaf spot (MLS). The data describes that none of the germplasm was found immune or disease free. None of the variety was found disease free or immune and general reaction ranged 1-4. On the basis of cumulative resistance reaction for the years 2012 and 2013 germplasm is classified in the following groups; Immune, Highly resistant, moderately resistant, moderately susceptible and susceptible. The varieties Cross 888 f1 hybrid, Long green, Jhalri, JK Lena, BG-7107, PKBT-1, BSS-616, VRBG 233 exhibited moderately resistant reaction. During early growing season six cultivars (MONIKA(7004), LEENA(7005), CBT-36, CBT-36, VRBG 233, Green wonder) exhibited the resistant reactions, eight cultivars (Cross 888 f1 hybrid, Long green, Jhalri, JK Lena, BG-7107, PKBT-1, BSS-616, VRBG 233 ) were moderately resistant, 16 cultivars (Desikarela, Lamba karela, Karela, No 361 f1 hybrid, Advanta 103, Jhalri, VRBG 227 , VRBG 230, VRBG 231, Jhalri, Jaunpuri, BG-34, RAJA, Early green, Runfeng, Preeti, ) were moderately susceptible and 7 cultivars (Jaunpuri, Indian karela, Sachal black4722, Fsd long, Jaunpuri long, TIPU, KIRAN) were susceptible for the pathogen (Table 3.7).

3.5.4. Effect of MLS susceptibility reaction on agronomic traits of bitter gourd under field conditions

Variation in agronomic parameters among the cultivars was observed under field conditions. Agronomic parameters comprised of vine length, number of branches, days to first flowering, number of fruits and yield per plant were recorded (Table 3.11). The tested germplasm exhibited a great variation among the vine length, number of fruits per plant and yield per plant parameters. While number of branches and day to flower parameters showed little variation. Vine length ranged 132-191 cm among the tested germplasm. The longest vine length (190 cm) was recorded in KIRAN at 90 days after germination (DAG). It was followed by MONIKA(7004) and LEENA(7005) (188 and 185cm respectively) whereas Lamba karela exhibited the smallest (132.7 cm) vine length (Table 3.11). Number of branches ranged 5-9 under field conditions and the highest number of branches (8-8.4)

with significant difference was observed in chaman, green wonder, Fsd long, VRBG 233, SHBGG-48, KIRAN, Runfeng, LEENA(7005) AND MONIKA(7004) cultivars. The lowest number of branches (5.2) was found in desi karela whereas rest of cultivars showed number of branches in a range of 7.6-5.3. Days to first flowering ranged 46 to 57 among the tested germplasm. Desi karela (46 DAG) showed early flower emergence followed by lamba karela and jaunpuri long (47 DAG). Indian karela exhibited late flowering and first flower emergence was recorded on 56 DAG. CBT-36 cultivar bore the highest (15) number of fruits per plant followed by MONIKA (7004), LEENA (7005) and Fsd long with 14, 12.6 and 12 respectively. Indian karela exhibited the least number (i.e., 5) of fruits while a variance of 6-11 was observed for the rest of the cultivars. A vast range of variation was observed among the cultivars in terms of yield per plant. The highest yield (2394g) was recorded in MONIKA (7004) followed by CBT-36 and Fsd long with 2235 and 2220 g per plant respectively. The minimum yield (700 g) per plant was recorded for Indian karela. Pearson correlation revealed a significant positive correlation (P≤0.01) between vine length and number of branching (0.617), vine length and number of fruits per plant (0.522), number of fruits per plant and yield per plant (0.893) under in vivo conditions (Table 3.12). Correlation between first flowering and number of fruits per plant (0.242) was significant at P≤0.05.

Table 3.10.Susceptibility reaction of test germplasm against myrothecium leaf spot disease under in vivo conditions

Disease severity Cultivar response Varieties scale (0-5) Category

0 Nil Immune

1 MONIKA(7004), LEENA(7005), CBT-36, VRBG 233, Green wonder Resistant

2 Cross 888 f1 hybrid, Long green, Jhalri, JK Lena, BG-7107, PKBT-1, Moderately resistant BSS-616, VRBG 233, SHBG-48, Fsd long,

3 Desikarela, Lambakarela, Karela, No 361 f1 hybrid, Advanta 103, Jhalri, Moderately VRBG 227 , VRBG 230, VRBG 231, Jhalri, Jaunpuri, BG-34, RAJA, susceptible Early green, Runfeng, Preeti,

4 Jaunpuri, Indian karela, Sachal black4722, Jaunpuri long, TIPU, KIRAN Susceptible

5 Nil Highly susceptible

Table 3.11. Effect of MLS susceptibility reaction on agronomic traits of bitter gourd in field experiment under natural environmental conditions

Sr Vine length Number of Number of Variety Days to flower Yield/plant g # Cm branches fruits/plant 1 Jaunpuri 165.3 f-j 6.0 gh 49.6 d-g 10 d-f 1580 h-l 2 Jhalri 160. 2 g-l 7.2 b-d 55 a-c 6 hi 864 rs 3 Indian karela 157.5 h-m 6.0 gh 56.3 a 5 i 700 s 4 Green wonder 147.2 mn 8.0 a 50.6 b-g 8 f-h 1320 k-o 5 Chaman 162.2 g-l 8.2 a 50 c-g 9 e-g 1431 j-m 6 Fsd long 167.3 e-h 8.1 a 49.3 e-g 12 b-d 2220 a-c 7 VRBG 227 162.4 g-j 7.0 cd 50.3 c-g 7 g-i 1057 q-r 8 VRBG 230 149.5 l-n 6.2 e-h 52.3 a-f 8 f-h 1296 l-o 9 VRBG 231 147.3 mn 6.9 d 49 e-g 7 g-i 973 q-s 10 VRBG 233 154.5 i-m 8.2 a 47.6 fg 9 e-g 1332 k-o 11 SHBG-48 167.3 e-i 8.3 a 50.6 b-g 8 f-h 1232 m-p 12 Early green 160.8 g-l 6.4 e-g 50 c-g 11 c-e 1661 f-j 13 JK Lena 172.7 d-g 6.5 e 51.6 a-g 10 d-f 1630 g-j 14 BG-7107 170.1 d-g 7.2 b-d 53 a-e 9 e-g 1602 h-k 15 CBT-36 180.6 a-d 7.3 b-d 55.6 ab 15 a 2235 ab 16 PKBT-1 162.5 g-k 6.3 e-h 51.3 a-g 9 f-h 1494 i-m 17 Preeti 172.5 d-g 7.6 b 50.3 c-g 12 c-e 2028 b-d 18 KIRAN 190.1 a 8.2 a 54.6 a-d 9 f-h 1719 e-i 19 Palli 160.5 g-l 7.4 b-d 50 c-g 11 c-e 1837 d-h Contd….

20 Cross888F1hybrid 182.6 a-d 7.2 b-d 53.3 a-e 11 c-e 1892 d-g 21 Runfeng 177.4 b-e 8.4 a 52.6 a-f 11 c-e 1804 d-h 22 No361F1 hybrid 170.4 d-g 7.2 b-d 49.3 e-g 9 e-g 1422 j-m 23 Advanta 103 149.5 k-n 5.9 h 48.3 e-g 8 f-h 1208 n-p 24 Sachal black 4722 154.4 i-m 6.3 e-h 48.3 e-g 6 hi 738 s 25 Long green 172.5 d-g 7.3 b-d 52.6 a-f 8 f-h 1128 o-q 26 Jhalri 162.4 g-k 6.5 ef 49 e-g 7 g-i 952 q-s 27 Jaunpuri long 154.5 i-m 6.3 e-h 47.6 fg 9 e-g 1413 j-n 28 Desikarela 134.5 o 5.2 i 46.6 g 10 d-f 1340 k-o 29 Lambakarela 132.7 o 5.3 i 47 g 8 f-h 1112 p-r 30 Karela 139.3 no 6.0 f-h 48.3 e-g 8 f-h 968 q-s 31 BG-34 152.4 j-m 6.2 e-h 51.6 a-g 9 e-g 1332 k-o 32 BSS-616 160.4 g-l 7.4 bc 49.6 d-g 11 c-e 1859 d-h 33 TIPU 154.6 h-m 7.3 b-d 48.3 e-g 11 c-e 1694 e-j 34 RAJA 154.2 i-m 6.0 f-h 48.6 e-g 10 d-f 1590 h-k 35 CBT-36 175.4 c-f 7.3 b-d 53 a-e 13 a-c 1963 b-e 36 LEENA(7005) 185.6 a-c 8.3 a 55 a-c 12 b-d 1944 c-f 37 MONIKA(7004) 188.5 ab 8.4 a 55 a-c 14 ab 2394 a Values in columns with different letters show significant difference (P≤0.05) as determined by Tukey‟s HSD method.

Table 3.12. Correlation of agronomic traits against susceptibility reaction in field experiment under natural environmental condition

Vine length No of First Yield No of fruits (cm) Branches flowering (g/plant) Vine length Pearson Correlation 1 (cm) Sig. (2-tailed) N 111 No of Pearson Correlation 0.617(**) 1 Branches Sig. (2-tailed) 0.000 N 111 111 First Pearson Correlation 0.640(**) 0.353(**) 1 flowering Sig. (2-tailed) 0.000 0.000 N 111 111 111 No of fruits Pearson Correlation 0.522(**) 0.365(**) 0.242(*) 1 Sig. (2-tailed) 0.000 0.000 0.010 N 111 111 111 111 Yield Pearson Correlation 0.616(**) 0.488(**) 0.278(**) 0.893(**) 1 (g/plant) Sig. (2-tailed) 0.000 0.000 0.003 0.000 N 111 111 111 111 111 * Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed). N= total number of replicates in all treatments

3.5. Disease management practices

3.5.1. Efficacy of weed aqueous extracts against M. roridum 3.5.1.1. In vitro studies

Colony growth of M. roridum was observed against tested weed extracts after 4, 7, 10 and 14 incubation day (Plate 3.9). There was a significant increase in colony growth with increase in incubation period. The highest colony growth (i.e. 87 mm) was observed in control treatment during all incubation periods (Fig. 3.12). Among the treatment at day 4, N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus and S. indicus extracts did not exhibit any colony growth. This suppression was maintained in N. plumbaginifolia up to day 7. Weed extracts of C. album, T. portulacastrum, M. coromendelianum, D. muricata were proved least effective as they enhanced the radial growth after 4, 7, 10 and 14 days of incubation. At 14 day incubation period, N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus, S. indicus, C. album, T. portulacastrum, M. coromendelianum and D. muricata extract treatments exhibited M. roridum colony growth 11, 26, 30, 31, 32, 36, 42, 72 and 81 mm respectively (Fig. 3.12). Among tested aqueous extracts, the highest antifungal potential was found in N. plumbaginifolia extract that inhibited the colony growth up to 88% followed by P. hysterophorus that reduced the growth up to 71% over control (Fig. 3.13). S. nigrum, C. didymus, S. indicus and T. portulacastrum restrained the colony growth up to 66%, 65%, 64% and 60% respectively. C. album extracts slowed down the M. roridum colony growth up to 54% whereas D. muricata extract was least effective (11%) in inhibiting M. roridum colony growth.

Variations were observed in macroscopic characters of the colony growth and morphology among various treatments but not in microscopic characters. Macroscopic characters like colony color, texture, margins, sporodochia production and elevation were recorded (Table 3.13). M. roridum produce circular, flat colonies with floccose texture and filiform margins on PDA at 25°C. A large number of conidia produce after 3-4 days on colony surface while mycelium keeps growing from margins. N. plumbaginifolia and P. hysterophorus extracts revealed to inhibit the colony radial growth in the present studies. N. plumbaginifolia did not exhibit any spore production till last reading. This

might because of presence of potent antifungal compounds in aqueous extracts that inhibited in the fungal growth. S. indicus and M. coromendelianum produce submerged colonies as compared to the control treatment. C. album and M. coromendelianum extracts produce irregular shapes colonies with lobate margins.

Plate 3.9. Response of Myrothecium roridum colony growth against various weeds aqueous extracts

Fig. 3.8. Effect of different aqueous weed extracts on colony growth of Myrothecium roridum. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method.

Fig 3.9. Evaluation of aqueous weed extracts for colony growth inhibition of Myrothecium roridum

Table 3.13. Assessment of macroscopic colony characters of M. roridum under the stress of weed aqueous extracts

Weed extract Colony form Colony Colony texture Colony Spore elevation margins production Control Circular Flat Floccose Filiform +

N. plumbaginifolia Irregular Umbonate Filamentous Filiform -

P. hysterophorus Filamentous Raised Floccose Filiform -

S. nigrum Filamentous Raised Floccose Filiform +

C. didymus Filamentous Flat Floccose Undulate +

S. indicus Irregular Submerged Sparsely filamentous Entire +

T. portulacastrum Filamentous Crateriform Sparsely floccose Filiform +

C. album Filamentous Crateriform Floccose Undulate +

M. coromendelianum Irregular Submerged Sparsely filamentous Lobate +

D. muricata Circular Flat Floccose Filiform +

“+”: Produce spores, “-” : No spore production

3.5.1.2. In vivo studies 3.5.1.2.1. Pot experiment Percent disease incidence (PDI) was recorded after each of 3 sprays of tested plant extracts viz; Nicotiana plumbaginifolia, Parthenium hysterophorus, Solanum nigrum, Coronopus didymus and Sphaeranthus indicus against M. roridum (Fig. 3.14). All treatments exhibited significantly lower disease incidence (%) when compared over control. A continuous increase of (%) PDI was observed in control over time whereas test treatments showed either static or lower PDI. After 1st, 2nd and 3rd spray, PDI recorded for control was 4, 57 and 71% whereas N. plumbaginifolia exhibited 15, 17 and 21% PDI respectively. All of the tested extracts viz; N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus and S. indicus caused a significant (P < 0.05) reduction in infection development over control (Fig 3.15). N. plumbaginifolia was the most effective aqueous extract and reduced the spots enlargement rate by 70% followed by P. hysterophorus with 61%. S. nigrum (52%) and C. didymus (43%) showed intermediate reduction while S. indicus proved least effective and reduced (36%) spots enlargement.

80 After 1st spray 70 60 50 a 40 b b 30 c de 20 e 10

Disease (%) Disease incidence 0

80 70 After 2nd spray a 60 50 b b 40 c 30 cd e 20 10

0 Disease (%) Disease incidence

80 a After 3rd spray 70 60 b 50 c 40 d e 30 f 20 10

Disease (%) Disease incidence 0

Fig. 3.10. Disease incidence of Myrothecium roridum against weed aqueous extracts in pot experiment. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method.

80 70

70 61 60 52 50 43 40 36 30 20

Disease reduction (%) Disease reduction 10 0 0

Fig. 3.11. Disease reduction of Myrothecium roridum against weed aqueous extracts in pot experiment. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method.

3.5.1.2.2. Field experiment Effect of different weed aqueous extracts spray on the infection development of M roridum in field condition was evaluated. Three sprays of N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus and S. indicus extracts were completed with 15 days intervals and percent disease incidence (PDI) was recorded after each spray (Fig 3.13). Percent disease incidence exhibited in all test treatments was significantly lower when compared over control. A continuous increase of PDI was observed in control over time whereas test treatments showed either static or lower PDI. After 1st, 2nd and 3rd spray, PDI recorded for control was 41, 48 and 59% whereas N. plumbaginifolia exhibited 14, 17 and 20 % PDI respectively. The results presented in Fig 3.13 indicate the percent reduction of all test extracts as compared to control. N. plumbaginifolia was the most effective aqueous extract under field conditions and reduced the spots enlargement rate by 66% compared to control followed by P. hysterophorus with 57%. S. nigrum (35%) and C. didymus (40%) showed intermediate reduction among the tested plant extracts. S. indicus was the least effective and reduced (28%) spots enlargement.

70 After 1st spray 60 50 a 40 b bc 30 cd 20 e de 10

0 Disease incidence (%) incidence Disease

70 After 2nd spray 60 a 50 b 40 bc 30 cd de d 20 10

0 Disease incidence (%) incidence Disease

70 a After 3rd spray 60 50 b bc 40 cd 30 e f 20 10

Disease incidence (%) incidence Disease 0

Fig. 3.12. Disease incidence (%) of Myrothecium roridum against weed aqueous extracts in field experiment. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method.

70 66

60 57

50 40 40 35 28 30

Disease reduction (%) Disease reduction 10 0 0

Fig. 3.13. Disease reduction over control of Myrothecium roridum against weed aqueous extracts in field experiment. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method.

3.5.2. Inter-cropping

3.5.2.1. Pot experiment

Antifungal potential of seven vegetable and condiment crops were assessed by adopting intercropping strategy and data was recorded at weekly interval on percent disease incidence and percent disease reduction over control. Plants were monitored up to 7 week after inoculation (Fig 3.14). First reading of disease incidence of myrothecium leaf spot was recorded after 48 hours of artificial inoculation as appearance of pinhead sized infection spots. Initial disease incidence ranged 18-26% did not significantly differ among the intercropped treatments compared to the bitter gourd monoculture (control). Final reading taken at week 7 showed a remarkable reduction in disease incidence over control (79%). Disease incidence in Bitter gourd-Garlic inter-cropping was 29% followed by Bitter gourd-Chilli (38.20%) and Bitter gourd-Onion (47%) inter-cropping. Bitter gourd-Ginger inter-cropping exhibited 58%, B-Capsicum 60%, Bitter gourd-turmeric 70% and Bitter gourd-Colocasia inter-cropping disease incidence was 72%. Percent disease reduction over control was calculated and revealed that bitter gourd-garlic intercropping treatment significantly lowers (P < 0.05) the incidence of myrothecium leaf spot disease by 63% under greenhouse conditions (Fig 3.15). Chilies- bitter gourd intercropping also reduced the myrothecium leaf spot disease incidence, 52%, significantly followed by onion-bitter gourd intercropping that showed an inhibition percentage of 41 than the control treatment. Bitter gourd-ginger and bitter gourd- capsicum intercropping treatments were less significantly inhibited the myrothecium leaf spot disease i.e., 27% and 24% respectively. Bitter gourd-turmeric and bitter gourd- Colocasia intercropping treatments reduce the disease incidence non-significantly by 11% and 9% respectively than the bitter gourd alone.

Plate 3.10. Pot and field experimental layout description under natural environmental conditions. A= disease management practices in pot experiment; B= germplasm screening under field conditions; C= disease management strategies under field conditions.

70 Ctrl (B) 60 B-Gar 50 B-Chi B-Oni 40 B-Gin 30 B-Cap

Disease incidence(%) 20 B-Cur B-Col 10

0 1 2 3 4 5 6 7 Weeks after inoculation

Fig. 3.14. Disease incidence of Myrothecium roridum against intercropping of aromatic medicinal and condiment crops in pot experiment. Ctrl(B)= bitter gourd; B-Gar=Bitter gourd-Garlic; B- Chi= Bitter gourd-Chilli; B-Oni= Bitter gourd-Onion; B-Gin= Bitter gourd-Ginger; B- Cap= Bitter gourd-Capsicum;B-Tur= Bitter gourd-turmeric; B-Col= Bitter gourd- Colocasia

59 60 54 50 40 36 37

30 24 20 14 9 10 Disease reduction (%) Disease reduction 0 0

Fig. 3.15. Disease reduction over control of different intercropped plants on Myrothecium roridum infection development.

3.5.2.2. Field experiment

Under field conditions, similar data recording strategy was adopted as in pot experiment to evaluate the effect of intercropping of different vegetables and condiments against Myrothecium leaf spot disease. First reading of disease incidence of Myrothecium leaf spot was recorded after 48 hours of artificial inoculation at appearance of pinhead sized spots (Fig. 3.16). Initial disease incidence ranged 19-26% within treatments and no significant difference was observed from the control treatment. Final reading recorded 7 weeks after inoculation, showed a remarkable reduction in disease incidence over control (79%). Disease incidence in Bitter gourd-Garlic treatment was 33% followed by Bitter gourd-Chilies (40%) and Bitter gourd-Onion (48%) treatments. Bitter gourd-Ginger treatment exhibited 62%, Bitter gourd-Capsicum 66%, Bitter gourd-Turmeric 75% and Bitter gourd-Colocasia treatment disease incidence was 72%.

Bitter gourd-garlic intercropping treatment significantly (P < 0.05) lowers the incidence of myrothecium leaf spot disease, by 58% than in bitter gourd solo cultivation (Fig. 3.17). Chilies-bitter gourd intercropping also reduced the myrothecium leaf spot disease incidence, 49%, significantly followed by onion-bitter gourd intercropping that showed an inhibition percentage of 39 than the control treatment. Bitter gourd-ginger and bitter gourd-capsicum intercropping treatments less significantly inhibited the myrothecium leaf spot disease i.e., 22% and 17% respectively. Bitter gourd-turmeric and bitter gourd-arvi intercropping treatments reduce the disease incidence non-significantly by 5% and 8% respectively.

70 Ctrl (B) 60 B-Gar 50 B-Chi

40 B-Oni B-Gin 30 B-Cap

Disease (%) Disease incidence 20 B-Cur

10 B-Col

0 1 2 3 4 5 6 7 Weeks after inoculation

Fig. 3.16 Disease incidence of Myrothecium roridum against intercropping of aromatic medicinal and condiment crops in field experiment. Ctrl(B)= bitter gourd; B-Gar=Bitter gourd-Garlic; B-Chi= Bitter gourd-Chilli; B-Oni= Bitter gourd-Onion; B-Gin= Bitter gourd-Ginger; B-Cap= Bitter gourd-Capsicum;B- Tur= Bitter gourd-turmeric; B-Col= Bitter gourd-Colocasia

58 60 49 50 39 40

30 22 20 17 8 Disease reduction (%) Disease reduction 10 5 0 0

Fig. 3.17. Disease reduction over control of different intercropped plants on M. roridum infection development.

100

3.5.3. Efficacy of different fungicides against M. roridum

3.5.3.1. Commercial fungicides

3.5.3.1.1. In vitro studies

The effect of 5 different foliar fungicides viz Alliette, Antracol, Score, Cabrio Top and Dithane M and @ 0.01, 0.05 and 0.1 % (100, 500 and 1000 PPM concentrations respectively) on the growth of M. roridum (isolate Mr37) on PDA was tested. Generally, pathogen was more tolerant to some fungicides and none on the tested fungicide completely inhibited the mycelial growth (Plate 3.10, Fig 3.18). The majority of the fungicides significantly (P < 0.05) reduced the growth of causal fungus. Antracol @ 0.05% and 0.1% significantly reduced the mycelial growth (Fig. 3.19). Cabrio top at 0.1% level significantly reduced the growth of pathogen. Alliette concentrations were least effective against mycelia growth. Overall, the fungicides that were most effective in controlling mycelia growth of isolate Mr37 of M. roridum were Antracol, Score and Cabrio top.

101

Plate 3.11. Effect of commercial fungicides on colony growth of Myrothecium roridum.

102

Control 0.01% 0.05% 0.10% 3500 a a a a a

3000

) 2 2500

2000 b b

1500 c b c

Colony area (mm Colonyarea 1000 c d d d b bc c b 500 c c

0 Alliette Dithane M Cabrio top Score Antracol

Fig. 3.18. Effect of different fungicides on colony growth of Myrothecium roridum. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method.

100 92

80 81 81

78 80

Disease reduction (%) Disease reduction 20

0 0 Control Aliette Dithane M Cabrio top Score Antracol

Fig. 3.19. Disease reduction (%) of fungicides against M. roridum. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method.

103

3.5.3.2. In vivo studies 3.5.3.2.1. Pot experiment Three fungicides viz; Antracol, Score, and Cabrio top selected on the basis of their performance in mycelial growth experiments for further pot and field trials. Inoculum suspension of isolate Mr37 was sprayed on bitter gourd plants at 8-10 leaf stage, initially pin head spots were developed after 48 hours and continued to enlarge until 7 days after inoculation (DAI). Percent disease incidence was recorded after each spray (Fig. 3.20). Percent disease incidence (PDI) exhibited in all treatments was significantly lower when compared over control. A continuous increase of PDI was observed in control over time whereas test treatments showed either static or lower PDI. After 1st, 2nd and 3rd spray, cabrio top exhibited 38, 32 and 29% PDI respectively. All of the test fungicides (Score @ 1 ml/ litre of water,) caused a significant reduction over control in infection development (Fig 3.21). Cabrio top proved most effective fungicide with above said concentration reduced the spots enlargement rate by 60% compared to control followed by score (57%). Whereas Antracol was the least effective and reduced (54%) spots enlargement.

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80 After 1st spray 70 60 50 a b b b 40 30 20

Disease (%) Disease incidence 10 0 Control Score Antracol Cabrio top

70 After 2nd spray a 60 50 b b 40 b 30 20

Disease (%) Disease incidence 10 0 Control Score Antracol Cabrio top

80 a

70 After 3rd spray 60 50

40 b b b 30 20

Disease (%) Disease incidence 10 0 Control Score Antracol Cabrio top

Fig. 3.20. Disease incidence of M. roridum against fungicides in pot experiment. Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method.

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70 60 60 57 54 50

20 Disease reduction (%) Disease reduction 10 0 0 Control Score Antracol Cabrio top

Fig. 3.21. Percent disease reduction (PDR) of M. roridum against fungicides in pot experiment. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method.

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3.5.3.2.2. Field experiment

Effect of different fungicidal spray was investigated on the infection development of M roridum in field condition after cultural practices. Three spray of fungicides i.e. Antracol, score and cabrio top were completed with 15 days intervals. Percent disease incidence (PDI) exhibited in all treatments was significantly lower when compared over control (Fig 3.22). A continuous increase of PDI was observed in control over time whereas test treatments showed either static or lower PDI. After 1st, 2nd and 3rd spray, score exhibited 43, 42 and 39% PDI respectively. Score showed best performance and decreased (45%) the percent incidence after 3rd spray (Fig 3.23). Antracol decreased percent incidence by 36 after 3rd spray. Cabrio top was comparatively less effective than other fungicides as it reduced the population by 33% over control.

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70 After 1st spray

60 a b b 50 c 40 30 20

10 Disease (%) Disease incidence 0 Control Score Antracol Cabrio top

70 After 2nd spray

a 60 b 50 b c 40

Disease (%) Disease incidence 10

0 Control Score Antracol Cabrio top

80 a After 3rd spray

70 60 b 50 b c 40 30

20 Disease (%) Disease incidence 10 0 Control Score Antracol Cabrio top

Fig. 3.22. Disease incidence of Myrothecium roridum against fungicides in field experiment. Vertical Vertical bar illustrate standard error of means. Values with different letters on top of bar show significant difference (P≤0.05) as determined by Tukey’s HSD method.

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40 34 33 30

10 Disease reduction (%) Disease reduction 0 0 Control Score Antracol Cabrio top

Fig. 3.23. Disease reduction (%) of M. roridum against fungicides in field experiment. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method.

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3.5.3.2. Technical grade fungicide

3.5.3.2.1. In vitro studies

The effect of tebuconazole fungicides at different concentrations 0.5, 1.25, 2.5 and 5 mg/L was investigated at 595 nm optical density (OD) using MTT ELISA plate assay. Fungal physiology response recorded on the basis of germination and density of mycelia network formation and spore viability (Plate 3.12). There was increasing trend in inhibition of the radial growth with the increase of dose. Spore germination and mycelia network was observed under microscope (Fig 3.24). In the control treatment where media was not amended with Tebuconazole clumped mycelium network was formed at 0.5 and 1.25 mg/ L concentration levels. No spore germination and mycelia growth was observed at 2.5 and 5 mg/ L concentrations of tebuconazole. These results were verified by quantification of living cell using MTT assay and measuring optical density at 595nm using ELISA plate reader. Inhibition % was measured by using formula and found that it reduced growth up to 58, 84, 91 and 97% against 0.5, 1.25, 2.5 and 5 mg/ L dose levels respectively (Fig 3.25).

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Plate 3.12. Fungicidal activity of various concentrations of Tebuconazole on spore germination of M. roridum.

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120

100

60 **

Growth (%) Growth 40 *** 20

0 Control 0.5 1.25 2.5 5 Concentration (mg/L)

Fig. 3.24. Growth percentage of Myrothecium roridum spores under different concentrations of tebuconazole. Asterisk indicates statistically significant difference by Students’ T-test. ** P < 0.01, ***P < 0.001

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97 100 91 85 80 58 60

20 Growth reduction (%) reduction Growth 0 0 Control 0.5 1.25 2.5 5 Concentration (mg/L)

Fig. 3.25. Percent inhibition of different concentrations of tebuconazole against M. roridum. Vertical bars show standard errors of means of five replicates.

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Chapter 4

DISCUSSION

Bitter gourd is one of the most popular edible pod vegetable in many Asian countries including Pakistan. It is a rich source of phytonutrients like polypeptide-P (a plant insulin known to lower blood sugar levels), hypoglycemic agent (charantin), dietary fiber, minerals, vitamins (folates, vitamin-A, B-complex and C) and anti-oxidants. The average yield of bitter gourd in Punjab Pakistan is much lower than its neighboring countries, China and India. In Pakistani agro-ecosystem, description of yield attributes is a complex phenomenon and no single factor can be claimed for lower yield. Among various factors diseases play a dominating role. In Pakistan so far, more than 15 fungal, bacterial and viral diseases has been reported. Very little documented information is available on bitter gourd diseases in Pakistan. In the light of personal communications with farmers and agriculture department field staff, it is concluded that Myrothecium leaf spot (Myrothecium roridum) Angular leaf spot (Pseudomonas syringae), Bacterial leaf spot (Xanthomonas campestris), Alternaria leaf blight (Alternaria cucumerina) Downy mildew (Pseudoperonospora cubensis), Powdery mildew (Sphaerotheca fuligniea) are of usual occurrence. Mildews are important for tunnel forming however it is difficult to comment on distribution and index of a particular disease.

Myrothecium leaf spot (MLS) disease has been emerged as a serious threat for bitter gourd crop during last decade. Earlier consideration for Myrothecium roridum in Punjab was a weak parasite for bitter gourd. This hypothesis was attributed to monoculture cropping and poor management strategies especially the unjustified use of agronomic inputs has made sporadic occurrence of MLS on bitter gourd. Very little information is available on disease epidemiology and distribution pattern. Keeping in view economic importance of the disease a project based on the series of interlinked investigations on disease distribution and severity index, host pathogen interaction, germplasm susceptibility, pathogen population virulence analysis and development of strategies for integrated disease management (IDM) was designed.

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4.1. Geographical distribution of M. roridum in Punjab, Pakistan

Survey is considered as an important step for initiation of a project, it provides reliable information on statistical updates and involvement of socio-economic factors associated with the issue. While designing initial project draft, reliability of survey findings depends upon survey methodology, sample size, fluctuation intensity and measuring scale. Key objectives for present field investigations were to update disease statistics, analyzing role of production technology and social factors in disease spread. During field scouting disease specimens were also collected to get single spore culture population of the isolates for onward investigations mentioned for disease diagnostics and management. In the present investigation, guidelines of Hoagland et al., (2007) and Michereff et al., (2011; 2012) were observed. In addition to above guide lines reliability of socioeconomic factors was cross examined by distribution of a structural questionnaire (Annexure-I), formal and informal interviews with farmers, public and private sector stake holders engaged with crop production and marketing. Major attention was focused on spring crop which is sown during mid February to mid April and harvested May-July.

Vegetable are cultivated as regular feature on 10 days interval on small holdings. This interval is in continuous cultivation on smaller units to keep in touch with the market for sustainable farm income. Therefore at the same time different growth stages of the crop can be seen in the adjacent fields. Before initiation of the survey Punjab province was divided in four agro-ecological zones viz irrigated planes, Barani (Rain fed) region, Thal region and Marginal Land (Fig. 3.1). A total of 319 fields from 117 locations were surveyed in collaboration with Agriculture Extension Wing of Punjab Agriculture Department and Plant Pathology Section, AARI Faisalabad. While conducting the survey, attempts were made to ensure maximum representation of a soil type and production technology observed in a region. Keeping in view holding unit 0.25 ha was considered as basic sample unit. Mix cropping zone which is the major pocket for crop production and marketing, 15 sites were marked for regular monitoring of the crop for

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disease development and its associated factors so that in depth understanding of the MLS could be made.

The cumulative assessment of surveys conducted during 2011, 2012 and 2013 exhibited highest disease index of 29, 25 and 30% respectively in the mixed cropping zone. Whereas the least disease index of 3.27, 4.13 and 3.23 % during 2011, 2012 and 2013 respectively was recorded in DG Khan zone. Disease severity was ranged 1-4 on visual severity rating scale (VSRS) for mixed cropping zone while in DG Khan irrigated, it ranged from 0-2 during the surveyed years. The mixed cropping zone consists of Lahore, Faisalabad, Kasur, Gujranwala and Jhang districts. Significant variation prevails for meteorological conditions, production technology, market approach investment trends and crop protection strategies. In mixed cropping zone soil is fertile and intensive cultivation culture prevails in general. Cultivation of vegetable in tunnels makes it more susceptible because fungal inoculums and availability of favorable environmental conditions make situation worst (Powell et al., 2013). Due higher return because market hub farmers prefer to cultivate and invest on crop protection. On the other hands DG khan and Bhakar, areas at certain sites we could not observe the disease or it was is low intensity. In these areas no strong background for cultivation of vegetables on commercial scale exists and dry hot climate with sandy loam soils exists. On the west bank of DG khan link canal, low rainfall and poor quality sandy loam soil is found generally. Keeping in view disease statistics and analysis of field and market sociology it is suggested that these disease free or lower index areas should be promoted for vegetable especially bitter gourd cultivation.

4.2. Characterization of M. roridum

4.2.1. Aggressiveness evaluation

Green house experiments were conducted to evaluate the virulence pattern of local population of Myrothecium roridum isolates collected during 2011-2012 survey. Virulence was evaluated against Jaunpuri cultivar by inoculation of 1 × 105 spore suspension. The control plants were sprayed with water without inoculums.

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Aggressiveness spectrum of the isolate population was classified in to Non aggressive, Less aggressive, Moderately Aggressive, Highly Aggressive designated with “0”, “+”, “+ +” and “+ + +” signs. Out of the 54 test isolates, 23 were highly aggressive, 17 were moderately aggressive and 14 were less aggressive. None of the isolate was found for each group in “0 class”. The isolate collected from mixed cropping zone and rice zone showed highest level of virulence whereas low rainfall zone and marginal land isolates were less virulent. The relation of aggressiveness of soil borne pathogens with its geographical origin has been reported (Jamil et al., 2000). Mix cropping zone has specialized features of cultural practices, comparatively low temperature than other zones and high humidity. Due to extensive cultivation, inoculums remains active than DG Khan irrigated zone where less inoculums survive due to unfavorable conditions, slower infection development and isolate exhibited lesser aggressiveness pattern. However aggressiveness of an isolate against its host is also related with its compatibility with the carbohydrates, sugars, amino acids and phenols available in the host. Cabral et al., (2009) examined five M. roridum isolates collected from Amazonas State, Brazil against different cucurbits and found some variability in their pathogenicity and aggressiveness. Watermelon was slightly less susceptible to M. roridum isolates whereas cucumber was highly susceptible maybe because of some degree specialization in the host-pathogen interaction (Cabral et al., 2009). Taneja et al., (1990) suggested the existence of different pathotypes of M. roridum, based on the pathogenicity on distinct plant species and on the variability in aggressiveness of isolates to different hosts.

Bitter gourd plant showed weak or poorly detectable expression of infestations against less aggressive isolates. Disease expression was more pronounced in case of highly aggressive isolates and if plants survive through seedling mortality, they exhibited yellowing and distorted leaves, over ripening and early shedding of fruit with clearly visible fungal mycelium patches. Due to some irregular variation in virulence levels, it was difficult to correlate virulence with geographical origin of the isolate. As all of the isolates were collected from bitter gourd plant or rhizosphere soil therefore the source got least importance. General trend describes that isolates collected from DG Khan zone were less aggressive while isolates collected from mixed cropping zone were highly aggressive. 116

4.2.2. Morphological studies

Characteristic white color colonies with concentric rings of sporodochia that turned olive green to black with maturity were formed. The recovered isolates on PDA medium exhibited varying pigmentation and abundant conidial production with least phenotypic variation, respectively. Identification to the species was verified by typical sporodochia production, spore size and mycelium morphology. Nine representative isolates (i.e., Mr10, Mr21, Mr28, Mr30, Mr34, Mr37, Mr49, Mr51 and Mr54) were studied for morphological variations from different areas of the Punjab. All the test isolates possessed morphological features of M. roridum confirming to the attributes of species. Minor differences in morphological characteristics among isolates were detected. Ahrazem et al., (2000) has been reported minimal differences in cell wall polysaccharide contents among the various Myrothecium species. The colonies originating from isolates exhibited an initial white color on PDA, subsequently turning off-white and pale on reverse. As for as color and shape of spores is concerned, color was olive green and shape was rod with rounded edges for all isolates. Worapong et al., (2009) reported color variation among the conidia. The size of spore ranged in length from 5-8 μm and in width to 1.4-1.8 μm. A notable feature of M. roridum colony was concentric rings pattern among different isolates varied from compact rings to irregularly spread throughout the colony. Morphological characteristics get influenced by environment and race differences. These findings are supported by Worapong et al., (2009) and Hong et al., (2013) who reported that size of conidia on maturity ranged usually 6-8 × 1.6-2 μm.

4.2.3. Physiological response

Physiological attributes influencing colony growth and subsequently conidia and sporodochia production of M. roridum were analyzed. The optimization process was based on a series of experiments; best growth medium, temperature, pH of nutrient medium and photoperiod for the evaluation of radial growth and sporulation per mm2 (Fig. 2.3). Temperature and nutrient availability play crucial role in inducing the diseases caused by different microorganisms. Among the test growth media PDA medium was found best to support the mycelia growth followed by BGA medium. The difference in

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fungal colony diameter was found significant at P < 0.05. In present studies, Nutrient agar medium exhibited the least number of spore production whereas Okunowo et al., (2010) showed lower number of spore production in Czapek dox agar medium. Presence of chloride ions in Czapek dox medium was the suggested reason for lesser spore development and same might be true for nutrient agar medium (Okunowo et al., 2010). High temperatures (28-30 °C) and humidity triggers the rapid mycelial growth and sporulation of M. roridum. These finding are in line with previous studies by Talukdar and Dantre, (2013). Different pH levels in fungal growth medium also influence the fungal mycelia growth significantly. The highest radial growth was observed at pH 5.0 followed by pH 5.5 with 8h darkness period whereas sporodochia production per 5 mm2 was highest at 35°C. Different studies confirmed that pH levels between 5.0 and 6.5 are suitable for maximum radial growth of Myrothecium (Okunowo et al., 2010; Talukdar and Dantre, 2013). It is evident that PDA with pH levels 5.0-6.0 proved best growth medium with 16/8 h light and dark periods at 30°C.

Mycelia growth is not a reliable tool for measuring virulence of the pathogen. From the present investigations, it is evident that virulence directly relates with spore production. Aggressive behavior was measured by taking 3 mm plug from the periphery of the culture, raised on the optimum growth conditions, placed on young leaves of bitter gourd to cover 1 cm2 area. Fungus remained dormant for 24 hrs but covered 1 cm area in 3-5 days. The key difference observed in 1-4 day old culture and 5-10 day old culture was production of conidia and sporodochia. Infection development pattern for 7-8 and 9- 10 day old culture was not significantly different than 5-6 day old culture. The present investigations revealed that virulence factor is more related with spore production rather than radial growth of the colony. These findings might help in understanding the physiology of the pathogen that could lead to develop proper management strategies for the disease.

4.2.4. Study of genetic variation among selected isolates

Genetic relatedness of fungal population with its ecology and source is essential for its identification and virulence. RAPD markers serves as a powerful diagnostic tool

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that can be used to distinguish genetic variation among different species, varieties, forma speciales or biotypes within population of plant pathogens (Muthusamy et al., 2008; Abadio et al., 2012; Pedroso et al., 2012; Parveen et al., 2013). Extensive genetic diversity of the test population was confirmed by RAPD markers analysis and High levels of genotypic diversity within a plot, a field, and a location suggested evolving of new genotypes is a regular occurrence phenomenon Therefore diversity remains high even in a limited spatial area (Walker et al., 2001). Existence of different pathotypes of M. roridum has been suggested on the basis of pathogenicity on distinct plant species and on the variability in aggressiveness of isolates to different hosts (Taneja et al., 1990; Cabral et al., 2009). Present studies conducted with objectives of characterization of M. roridum isolates collected from different cultivars of bitter gourd and geographical origins and to find out the interaction amongst them were effectively achieved.

The rapid availability of diagnostic PCR for fungal genomic DNA has gained importance in epidemiologic sub-typing and isolates characterization. Quality of DNA was determined on compactness of the band on 1% agarose gel by Gel electrophoresis. An aggregate of 13 decamer random primers were used which produced amplicons in test isolates. The reproducible amplicons were considered for the genetic variation studies and dendrogram construction. Out of 93 DNA fragments an average of 7 bands per RAPD primer were amplified and produced 28% monomorphic whereas 72% polymorphic bands. The isolates Mr31 and Mr54 collected from Faisalabad and DG Khan made a clear subgroup A (genetic distance = 0.68). Similarity of M. roridum isolates between these areas may be due to transportation of infected germplam. However, Mr28 and Mr34 belonging to neighboring districts Sheikhupura and Lahore exhibited dissimilar profile and fell in different clusters. It is evident that some isolates from distant areas were also closely related e.g. in subcluster C, Mr10, Mr51 from Pakpattan and Attock (genetic distance = 0.54), formed a distinct related group. Therefore it is difficult to conclude that different isolates are area specific. Distribution across the country through infected germplasm or air borne conidia from their place of origin to other places may be the major misleading factor (Rivas et al., 2004; Pande et al., 2005; Ghewande, 2009). Some strains could be grouped according to host or geographic origin however other strains may not be. The long distance dispersal is efficient due to 119

supply of seeds and seedling nursery to different parts of province. So adoption of bitter gourd isolates carried along with infected germplasm to remote places is possible.

4.3. Studies on infection development by M. roridum within host tissues

Understanding of infection process in plant diseases is a complex phenomenon as it involves host physiology, cell biology, and metabolism of the host physiological activities. The Successful infection interferes with photosynthesis, respiration, cell wall composition and metabolism, nucleic acid and protein metabolism, secondary metabolites, growth regulator metabolism, transcellular and vascular transport, toxins, and resistance to infection (van der Plank, 2013). Little information is available on infection process of M. roridum though various scientists reported its soil, seed borne nature but produce symptoms on above ground plant parts (Domsch et al., 1980; Bharath et al., 2006; Sultana and Ghaffar, 2009). Therefore attempts were made to understand interaction of M. roridum with Momordica charantia. Conidial germination and emergence of germtube was observed on light microscope while combination of light, transmission electron and fluorescent microscopy to examine infection of bitter gourd by M. roridum. M. roridum hyphae grow across the surface of bitter gourd leaves. Penetration of M. roridum in the leaf tissues is through cuticle with formation of appressoria. Germ tubes tip swell and develop to form an appressorium which penetrate the host cuticle directly. M. roridum does not require precise orientation as do rust fungi to invade the plant through stomata. M. roridum hyphae and spores penetration in xylem vessels observed through fluorescent microscope. Movement of this fungus within bitter gourd is poorly understood probably because of presence of hyphae within the leaf xylem appear within four days after colonization in leaf veins. M. roridum could spreads actively within the plant through mycelial growth, or move passively by conidial transfer through the veins. The M. roridum hyphae move within the vascular elements of the xylem. Theoretically this can move throughout the plant via the interconnecting xylem tissues to colonize the entire plant; however all of the leaf veins do not contain hyphae and spores. In view of the low numbers of penetrating hyphae from the leaf surface, the

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presence of hyphae within the leaf xylem cells shows that colonization may occur from just a few successful penetration sites and the fungus can access this transport system rather rapidly and successfully. The surface of colonized leaves exhibit macroscopic symptoms 4 day after inoculation indicating ill health compared to normal, uninfected leaves. Light and Transmission Electron Microscopy confirmed the production of extracellular matrix by M. roridum. Plant pathogenic fungi can produce an extracellular matrix either prior to or after germination. This extracellular matrix involves secretion from spores, germ tubes, and appressoria are believed to facilitate the fungus adhering to the plant surface. Further histopathological investigations on mode of penetration and tissue colonization at different parts of bitter gourd are needed.

4.4. In vivo screening of bitter gourd germplasm

Evaluation of breeding material against a known virulence status isolate is crucial for plant breeders to develop reliable sources of resistance against plant diseases. So far, very little and scattered information is available for varietal resistance of bitter gourd against MLS in Pakistan. Bitter gourd germplasm evaluation studies for MLS resistance was conducted in the pot and field environments on spring crop during 2012 and 2013. Resistance of available commercial germplasm was evaluated against moderately resistant isolate Mr37. In some previous studies it is pointed out that planting environment plays a significant role in infection development expressions and complexities of seeding environment cannot duplicated in controlled environment but controlled conditions bitterly help in understanding infection development mechanism (Olaya et al., 1996; Michel, 2000)

There was a remarkable difference in infection development pattern and intensity under pot and field conditions. The spectrum of resistance and disease symptoms were more clearly defined on pots experiment. During early growing season The varieties MONIKA(7004), LEENA(7005), CBT-36, VRBG 233, Green wonder, Fsd long, Cross 888 f1 hybrid, Long green, JK Leena exhibited moderately resistant reaction. Desikarela, BG-7107, PKBT-1, BSS-616, VRBG 233, SHBG-48, Lambakarela, Karela, No 361 f1 hybrid, Advanta 103, Jaunpuri showed moderately susceptible

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reaction. Under field conditions, varieties Cross 888 f1 hybrid, Long green, Jhalri, JK Leena, BG-7107, PKBT-1, BSS-616, VRBG 233 and Fsd long exhibited moderately resistant reaction whereas cultivars Desikarela, Lambakarela, Karela, No. 361 f1 hybrid, Advanta 103, Jhalri, VRBG 227 , VRBG 230, VRBG 231, Jhalri, Jaunpuri, BG-34, RAJA, Early green, Runfeng, Preeti were moderately susceptible reaction. The varieties MONIKA(7004), LEENA(7005), CBT-36, VRBG 233, Green wonder observed stable resistance reaction of category 2 exhibited for flowering and fruit formation. Whereas Jaunpuri, Indian karela, Sachal black4722, Jaunpuri long, TIPU, KIRAN varieties exhibited highly susceptible reaction (category 4) and in these varieties flowering and fruiting stages which proved highly sensitive stages for disease development.

Strong link between age of the plant and diseases expression development and crop phenology has been reported. Grulke, (2011) observed association of crop phenology and disease epidemiology by identifying the link between stress physiology and host resistance while Capik and Molner, (2014) described that little information is available on link between flowering stage and resistance however different events like pollen shedding, bloom period are linked with the temperature which is common point between aggressiveness of the pathogen and host susceptibility reaction. During early growth stages there was great similarity in symptomatic development of infection expressions whereas on later stages of plant growth trend of infection development changed. The potted plant exhibited 75-90 days life cycle and higher mortality at fruiting stage whereas under field conditions no plant mortality was recorded and plant life span was 110-130 days. Under field conditions different growth stages of the plant can be seen on the same time but highest susceptibility was observed at flowering and fruit formation stage. The relationship between growth period, phenology and resistance was also observed.

Studies on the resistance against M. roridum were conducted in various parts of the world. Varietal resistance against the invasion of M. roridum has been reported by Mahesha, (2006) against soybean. The physiological bases of resistance are reported as higher amount of reducing sugars, amino acids and phenols (Jeun and Hwang, 1991).

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Besides these factors thickness of epidermis in corn and mung bean was also reported as a structural basis of resistance against M. roridum. Moderately susceptibility variety observed in screening process was forwarded to host-pathogen interaction studies and application of disease management strategies.

4.5. Disease management practices

4.5.1. Efficacy of different weed extracts against M. roridum

Antifungal potential of the weed plants is due to the presence of several chemical constituents like phenols, alkaloids, terpenoids, coumarins and tannins (Levin, 1976; Gandhiraja et al., 2009). Some of them already have been discovered and known for their antifungal potential but many needed to be explored (Macías et al., 2007). The plant parts and their extracts suppressed Penicillium spp., inhibited germination of spores of Drechslera rostrata, Fusarium oxysporum, Alternaria alternata, Corynespora cassiicola, Aspergillus fumigatus, A. niger, A. sulphureus and Microsporum gypseum (Luke, 1976; Kumar et al., 1979; Shrivastava et al., 1984; Sharma and Gupta, 2012). Colony growth of M. roridum was observed against 9 test weed extracts at 4, 7, 10 and 14 incubation day. Among test aqueous extracts, Nicotiana plumbaginifolia exhibited highest antifungal activity by inhibiting 88% mycelial growth, P. hysterophorus and D. muricata showed least effective by reducing 11% of colony growth.

Five highly effective weed extracts viz; N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus and S. indicus were selected for in vivo studies against M. roridum under natural environmental conditions. N. plumbaginifolia (70 % in pot experiment and 66 % in field) and P. hysterophorus (61 % in pot experiment, 57 % in field) were proved effective for the management of Myrothecium leaf spot disease of bitter gourd.

Ibaraki and Murakami (2006) co-related virulence and toxin production characteristic of the fungus with conidia production. Variation in mode of action of extracts on physiological response was recorded on macroscopic characters colony color, texture, margins, spore production and elevation. M. roridum produced circular, flat colonies with floccose texture and filiform margins on PDA at 25°C. A large number of

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conidia produced after 3-4 days on colony surface while mycelium continues growing from margins. Nicotiana plumbaginifolia and P. hysterophorus extracts revealed to inhibit the colony radial growth whereas N. plumbaginifolia did not exhibit any spore production till last reading. This might be due to presence of potent antifungal compounds in aqueous extracts that provide inhibition in the fungal growth. S. indicus and M. coromendelianum produce submerged colonies as compared to the control treatment. C. album and M. coromendelianum extracts produce irregular shapes colonies with lobate margins.

Phytochemical evaluation of leaves of N. plumbaginifolia revealed the presence of alkaloids, saponin, tannin, flavonoides, cardiac glycosides, phenolic compounds, steroids, terpenoides and carbohydrates (Singh et al., 2010). Stukkens et al., (2005) reported terpenoids compounds in N. plumbaginifolia are responsible for its antifungal properties. P. hysterophorus is known to have chemical constituents like parthenin, p- coumaric acid, ferrulic acid, vanillic acid and caffeic acid that act as antifungal compounds (Kanchan and Jayachandra, 1980; Das and Das, 1995). Plant extract inhibited mycelial growth and sporulation in pathogen Aspergillus flavus (Lokesha et al., 1986). D. muricata is reported to contain antifungal compounds and showed significant reduction in growth of Fusarium oxysporum and Aspergillus niger (Kohli et al., 1998; Sharma and Vijayvergia, 2013). While in the present study it exhibited least inhibition against M. roridum. This may be attributed to resistance reaction of the test fungus to its compounds but detailed investigations are needed. It is concluded that aqueous extracts of N. plumbaginifolia and P. hysterophorus possess significant antifungal activity against M. roridum under in vitro and in vivo conditions.

4.5.2. Inter-cropping

Aromatic plants are well known for their characteristics release of several volatile compounds in their surroundings and most of these compounds have been reported as antimicrobials. Inter-cropping with aromatic plants is cost effective and environment friendly approach against fungal and bacterial plant pathogens management that can easily be incorporated in production technology (Gomez-Rodriguez et al., 2003; Gurjar et

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al., 2012). These may involve the nutrient uptake processes, creating microclimate or escaping the disease either as non-host crop or librating chemical constituents to fight with the pests (Gan et al., 2006). In the present investigations vegetable and candimum crops were focused so that it should improve farm income besides disease management. These crops secrete allelopathic compounds which interfere with the physiology of the fungus by suppressing its growth and sporulation process. Present studies show that Allium sativum intercropping significantly lowers (58%) the disease incidence followed by chili (49%) as compared to the +ve control treatment. It was observed that A. sativum, C. frutescens and A. cepa treatments, appearance and development of disease was much lower than other treatments during initial stages. A. sativum and A. cepa suppressed pathogen at 6-8 leaf stage when M. roridum was inoculated and suppression remained effective till harvesting. A. sativum lowers the disease incidence up to 63% under greenhouse and 58% in field experiments followed by the Capsicum frutescens (52% in pot and 49% in field) and Allium cepa (41% in pot and 39% in field). Zingiber officinale (27% in pot and 22% in field) and Capsicum annuum (24% in pot and 17% in field) shows a moderate antifungal potential while Curcuma longa (11% in pot and 5 % in field) and Colocasia esculenta (9% in pots and 8% in field) were least effective. Many sulphide compounds have been identified from A. sativum as antifungal and antimicrobial agents. A. sativum oils are enriched with diallylmonosulphides, diallyldisulphides and diallyltrisulphides and reported to be effective against Candida albicans and Aspergillus spp. (Tsao and Yin, 2001; Naganawa et al., 1996). Many scientists confirmed antimicrobial activity of A. sativum and A. cepa against pathogenic bacteria and fungi (Tajkarimi et al., 2010; Ceylan and Fung, 2004; Lanzotti, 2006). In A. sativum intercropping, disease severity was ranged 1-2 on scale throughout the vegetative growth, flowering, fruit formation and harvesting phases of the bitter gourd crop. While in +ve control treatment, MLS disease was scored 1-3 on scale during vegetative growth and flowering initiation whereas scored 2-4 during harvesting. Disease severity was higher on older leaves. Reduced disease severity levels might be contributed towards the accumulation of volatile constituents on the plant surface that retard the further infections, sporulation and development of fungus (Trenbath, 1993). Plants in +ve control treatment where 100% incidence with 1-4 severity rating was observed exhibited lesser

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number of fruits setting with smaller size and low weight. Early maturity was compared to the –ve control and A. sativum intercropping. The results suggest that intercropping of A. sativum, C. frutescens and A. cepa can significantly reduce the infection of Myrothecium leaf spot under field conditions and can reduce significantly cost of fungicides. Further studies for evaluation and extraction of the active chemical constituents responsible for their antifungal potential may help in the development of effective fungicides.

4.5.3. Efficacy of different fungicides against M. roridum

Due to lack of awareness for hazards of chemical application among the farmer community in Pakistan, application of chemicals is considered as reliable and effective management strategy against insects and diseases. Chemical application intensity for vegetable crops is much higher than on field crops and same is true for M. roridum. The commercial fungicides; [Aliette (Fosetyl-Aluminium), Antracol (Propineb), Dithane M 45 WP 80 (Mancozeb 80 %), Score 250 EC (Difenoconazole) and Cabrio top (Metiram 92 % + Pyraclostrobin 8 %)] were examined under in vitro and in vivo conditions against the M. roridum. Fungitoxicity was measured on colony growth and conidial germination of M. roridum. In general, test fungicides suppressed colony growth and conidial germination the effect was directly linked with the increase in concentration of the fungicide. Among the test fungicides, Antracol at 0.05% and 0.1% concentrations significantly reduced (92 %) the mycelia growth. Cabrio top at 0.1% significantly reduced the growth (81 %) of pathogen. Dithane M concentrations were least effective to reduce the mycelia growth. In general systemic fungicides Antracol (92 %), Score (81 %) and Cabrio top (81 %) proved most effective against Mr37 isolate. Findings of present investigations are supported by the findings of Sultana and Ghaffar (2009). Systemic fungicides have strong protective and curative activity. In vitro effectiveness of Alliette, Topsin-M, Benlate, Mancozeb and Carbendazim in checking the growth of mycelium of M. roridum on PDA has been reported earlier. Sultana and Ghaffar (2009) found that Topsin-M (3 %) is most effective and suppressed the mycelial growth of M. roridum. Ploetz and Englehard (1980) reported that Iprodione 50W at 1.0 g/liter gave effective, nonphytotoxic control of the leaf spot and crown rot phases of the disease.

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Shakoor et al., (2011) applied systemic fungicides, ridomil gold MZ, bavistin, and score; and reported that Ridomil gold MZ gave good results at various concentrations against M. roridum and confirmed effectiveness of higher concentration dose 40 mg/10 ml. The use of fungicides in the laboratory in comparison to field depends on its in vitro efficacy at minimal, economically acceptable dosages and their proficient and rapid transport to the infection site. Score, Antracol and Cabrio top proved effective fungicides in the present study but could not completely inhibit the M. roridum growth. Therefore active ingredient of tebuconazole was tested for its antifungal potential. It completely inhibited the growth at 0.5 mg/L whereas significantly reduced the germ tube emergence and mycelium elongation at 1 and 1.5mg/L concentrations under in vitro conditions. Further investigations on mode of action and phytotoxicity under in vivo conditions are needed.

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Conclusions and Recommendations

Following are the conclusions and recommendations on the basis of these studies;

 Monoculture cultivation, varietal junk in the market, self-determined crop production and protection technology, development of inoculum by burying infected crop residue in the field.  Areas apparently looking disease free or exhibiting lesser disease index are because of newly adapted bitter gourd cultivation trend and climatic conditions.  Crop cultivation in lesser infected zones should be encouraged by providing better marketing facilities and increasing awareness for crop agronomy and protection.  Myrothecium roridum directly penetrate the host cell through germ tube during infection development.

 Candidate lines including MONIKA(7004), LEENA(7005), CBT-36, and cultivars VRBG 233 and Green wonder showed stable resistance during germplasm screening.

 Utilization of local resources by adopting cultural methods of disease control in Punjab.

 Exploitation of aqueous extracts of local weeds can provide ecofriendly disease management alternates.  Intercropping of Allium plants significantly reduced the MLS disease incidence.  Dire need to investigate the antifungal activity of newly derived active compounds against MLS  Coordination between academia, research and extension to exploit indigenous resources.

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ANNEXURE-1 Date------QUESTIONARE FOR ASSESSMENT OF MAJOR DISEASES OF BITTER GOURD IN AGRO-ECOLOGICAL ZONES OF PUNJAB, PAKISTAN

Identity of Subject ------Name (Farmer or institution) ------Address Village ------Union Council ------Tehsil ------District ------

Farming as source of income Major Compensatory Extra Academic qualification Primary Secondary Graduation Other Cropping History Total holding (acr) Area under bitter gourd cultivation Previous crop Since when cultivating bitter gourd Agronomic inputs Source of seed a. Prev. crop b. local dealer c. Seed Company d. Res. Institute e. Agriculture department

Chemical Fertilizer (Bag acr-1 or kg acr-1) N P K Organic fertilizer FYM Green manure Plant protection Insecticide Fungicide Herbicide Marketing system Direct Middleman Disease Total plant inspected Plant infected (%)

Key for measuring incidence and severity of infection 0 = no infection 1 = 1-10% 2 = 11-20% 3 = 21-30% 4 = 31-50% 5 = above 50%

Sample collected a. Leaf b. Fruit c. Soil d. All Sample code a b c d Sample unit Kanal Sample size 10 plants No. of replicates 4 Remarks ------Sumera Naz

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Annexure: II

Sr District Village Cropping Previous Bitter gourd Disease # pattern crop variety Index (%) 1 Narowal Khara Wheat- Fellow Black Diamond 6 – 20 Vegetable- Fellow Bassra Jala Wheat-Vegetable Wheat Parachi, Palli 6 – 20 Bassra Jala Wheat- Fodder- Barseem Fsd Long 1 – 5 Vegetable Bheari Wheat-rice- Fellow Black Dimend 6 – 20 Khurd Vegetable Baddo Vegetable- Fellow Palli 6 – 20 Malhi Fellow 2 Faisalabad 39 JB Wheat- Wheat Fsd-Long 1 – 5 Vegetables- Sugarcane 36 JB Vegetables- Cauliflower Fsd-Long 1 – 5 Sugar cane 92 GB Vegetables- Radish Hybrid-888 1 – 5 Sugar cane 263 RB Vegetables- Radish Fsd-Long 1 – 5 Sugar cane 254 RB Vegetables- Cauliflower Fsd-Long 6 – 20 Sugar cane 470 GB Sugarcane- Wheat Palli 6 – 20 Wheat-Vegetable 437 GB Vegetables- Cucumber Jhon Puri 6 – 20 Sugar cane 547 GB Cotton-Wheat- Wheat Fsd-Long 1 – 5 Vegetable 550 GB Cotton-Wheat- Cucumber, Hybrid-200 1 – 5 Vegetable Capsicum 165 RB Wheat-Vegetable Pumpkin Fsd-Long 6 – 20 145 RB S. Cane-Wheat- Peas Chowinda 6 – 20 Vegetable 165 RB Wheat-Vegetable Pumpkin Fsd-Long 1 – 5 145 RB Wheat-Vegetable Peas Chowinda 1 – 5 186 RB Wheat-Vegetable Carrot Parachi 6 – 20 72 GB Wheat-Vegetable Wheat Jhon Puri 6 – 20

148

643 GB Rice-Wheat Wheat Fsd-Long 1 – 5 3 Gujranwal Aroop Potato, Bitter Potato Hybrid 6 – 20 a gourd, Sorghum Nawan Pind Bitter gourd, Onion Hybrid 1 – 5 Onion Mughal Rice, Vegetable Rice Hybrid 485 6 - 20 Chak Kalan Mughal Rice, Vegetable Rice Hybrid 485 6 – 20 Chak Kalan Mughal Turnip, Rice, Turnip Hybrid 1 – 5 Chak Kalan Bitter Gourd Gajju Chak Rice, Peas, Bitter Peas Hybrid 1 - 5 Gourd Gondlanwal Rice, Sorghum, Potato Hybrid 1 - 5 a Village Bitter Gourd Kot Qazi Bitter Gourd, Peas Hybrid 1 - 5 Rice 4 Sialkot Golo Phalla Vegetables Cucumber Hybrid (241) 1 - 5 Mandair Vegetables Cucumber Rama Krishna 6 - 20 Sharif KishanGarh Vegetables Peas Hybrid (Kiran) 6 - 20 Randheer Vegetables Potato Hybrid 6 - 20 Nangal Vegetables Peas Hybrid (208) 1-5 Mirza Bambawala Vegetables Potato Hybrid 1 - 5 Radiyal Vegetables Cauliflower Desi 1 – 5 Sadhanwali Vegetables Peas Hybrid (Kiran) 21 - 50 Dingey Vegetables Turnip Desi (Jahania) 1-5 Sarhali Vegetables Potato Desi 1-5 Kakuwal Vegetables Cocumber Hybrid 6-20 5 Bahawalpu Chak Wheat-Cotton- Peas Rama Krishna 1 - 5 r Loharan Vegetable Chak 72 DB Wheat-Cotton- Spinach Hybrid Nil Vegetable Mubarakpur Wheat-Cotton- Peas Rama Krishna 1 - 5 Vegetable Ahmadpur Wheat-Cotton- Carrot Hybrid (Kiran) 6 - 20 East Vegetable Rajkan Vegetables Peas Hybrid 1 - 5 6 Chakwal Karyaala Wheat- Jawar Rama Krishna 1 - 5 vegetable-fodder

149

Bhoon Wheat- Spinach Hybrid Nil vegetable Dhok Agri Wheat-Oil seed- Mung Rama Krishna 1 - 5 Vegetable Pipli Wheat- Wheat Hybrid 6 - 20 vegetable Chak Wheat-Oil seed- Wheat Hybrid 1 - 5 Malook Vegetable Chak Umra Wheat- Wheat Palli (Hybrid) 1 - 5 vegetable Khokhar Zer Wheat-Oil seed- Cauliflower Palli (Hybrid) 1 - 5 Vegetable 7 Gujrat Kolian Shah Wheat- rice- Cauliflower Hybrid (Kiran) 1 - 5 Hussain vegetable Khotaha Wheat-Rice- Spinach Hybrid Nil Arian vegetable Dharowal Rice-vegetable Peas Rama Krishna 1 - 5 Jallal Pur Wheat-Rice- Rice Hybrid 6 - 20 Jattan vegetable Mehmood Rice-vegetable Peas Hybrid 1 - 5 Abad Syeda Rice-vegetable Peas Palli (Hybrid) 1 - 5 Bhehran Chanu Rice-vegetable Cauliflower Palli (Hybrid) 1 - 5 Bhoja 8 Hafizabad Hafizabad Rice-vegetable Fenugreek Rama Krishna 1 - 5 Hafizabad Vegetable- Spinach Rama Krishna 6 –20 vegetable Hafizabad Vegetable- Cauliflower Rama Krishna 6 – 20 vegetable Dhingranwa Rice-vegetable Potato Jaunpuri 6 – 20 li Dhingranwa Vegetable- Peas Hybrid (241) 1 – 5 li vegetable Dhingranwa Vegetable- Peas Hybrid (208) 1 – 5 li vegetable Dhingranwa Vegetable- Cauliflower Hybrid (Kiran) 1 - 5 li vegetable Dhingranwa Vegetable- Peas Desi 21 - 50 li vegetable Bangla Vegetable- Peas Hybrid 6 - 20 Maghiani vegetable Pindi Vegetable- Cauliflower Desi 1 - 5

150

Bhattian vegetable

Kot Nakka Vegetable- Spinach Hybrid 6 - 20 vegetable Kot Nakka Vegetable- Cauliflower Hybrid 1 - 5 vegetable 9 Jehlum Dina Wheat- Cauliflower Hybrid 1 - 5 vegetable Chak Jamal Wheat-Oil seed- Spinach Hybrid Nil Vegetable Pind Matay Wheat-Oil seed- Peas Fsd-Long Nil Khan Vegetable Sultanpur Wheat-Oil seed- Wheat Hybrid 6 - 20 Vegetable Kotla Faqir Wheat- Wheat Hybrid 1 - 5 vegetable-fodder 10 Jhang Chak 269 Wheat- Wheat Rama Krishna 1 - 5 vegetable Khokhran Wheat- cotton- Spinach Hybrid 6 - 20 Chak vegetable Kot Khaira Wheat- Peas, gram Krishna 1 - 5 vegetable Bagh Wheat- Gram Hybrid 6 - 20 vegetable 11 Kasur Khudian Wheat- Cauliflower Rama Krishna 1 - 5 Khas vegetable Roday Vegetable- Spinach Hybrid Nil vegetable Tatra Kamil Vegetable- Peas Hybrid (Kiran) 1 - 5 vegetable Mahlam Rice-vegetable Rice Hybrid (Kiran) 6 -20 Klan Gohar Jagir Vegetable- Peas Hybrid 1 - 5 vegetable 12 Lahore Shahdra Wheat- Cauliflower Hybrid 1 - 5 vegetable Harbanse Vegetable- Spinach Hybrid Nil Pura vegetable Mughalpura Vegetable- Peas Rama Krishna 1 - 5 vegetable Begum Kot Wheat-Rice- Wheat Hybrid (Kiran) 6 - 20 vegetable Ravi Town Vegetable- Peas Hybrid (Kiran) 1 - 5 vegetable

151

Shadbagh Vegetable- Peas Palli (Hybrid) 1 - 5 vegetable Baghban Rice-vegetable Cauliflower Palli (Hybrid) 1 - 5 Pura 13 Multan 19 Kassi Wheat- cotton- Fellow Hybrid 1 - 5 vegetable Kot Wheat- cotton- Wheat Hybrid Nil Rabnawaz vegetable Lutfabad Wheat- cotton- Peas Hybrid (Kiran) 1 - 5 vegetable Chak Wheat- cotton- Wheat Hybrid 6 - 20 140/10.R vegetable Chak 7/MR Wheat- cotton- Fellow Hybrid 1 - 5 vegetable 14 Muzaffarg Basti Wheat- cotton- Fellow Rama Krishna 1 - 5 arh Jalalabad vegetable Basti Wheat- Spinach Hybrid 6 - 20 Jagatpur vegetable-cotton Basti Peer Wheat- Peas Rama Krishna 1 - 5 Jahanian vegetable 15 Rahim Yar Machi Goth Wheat- cotton- Cauliflower Hybrid 1 - 5 Khan vegetable Bhutta Wheat- cotton- Cotton Hybrid Nil Wahan vegetable Bhutta Wheat- cotton- Peas Rama Krishna Nil Wahan vegetable Islam Garh Wheat- cotton- Cotton Hybrid 6 - 20 vegetable Islam Garh Vegetable- Peas Hybrid 1 - 5 vegetable Khair Garh Wheat- cotton- Cotton Palli (Hybrid) 1 - 5 vegetable Khair Garh Wheat- Cauliflower Palli (Hybrid) 1 - 5 vegetable 16 Rajanpur Lal Garh Wheat- Cauliflower Hybrid 1 - 5 vegetable Lal Garh Wheat- Spinach Hybrid Nil vegetable Lal Garh Wheat-cotton- Peas Krishna 1 - 5 vegetable Adachiragh Wheat- cotton- Cotton Hybrid 6 - 20 Shah vegetable AdaChiragh Vegetable- Peas Hybrid 1 - 5

152

Shah vegetable

AdaChiragh Wheat- cotton- Peas Hybrid (Kiran) 1 - 5 Shah vegetable Adachiragh Wheat- cotton- Cauliflower Palli (Hybrid) 1 - 5 Shah vegetable 17 Rawalpindi Rawalpindi Wheat- Cauliflower Rama Krishna 1 - 5 vegetable-oil seed Rawalpindi Wheat- Spinach Hybrid Nil vegetable-oil seed Rawalpindi Vegetable-oil Peas Rama Krishna 1 - 5 seed 18 Sargodha Bhera Wheat- Cauliflower Hybrid (Kiran) 1 - 5 vegetable Bhera Wheat- Spinach Fsd-Long Nil vegetable Sargodha Vegetable- Peas Desi 1 - 5 vegetable Sargodha Rice-vegetable Peas Hybrid (Kiran) 6 - 20 Sial Sharif Vegetable- Peas Hybrid 1 - 5 vegetable Kot Momin Vegetable- Peas Palli (Hybrid) 1 - 5 vegetable Kot Momin Rice-vegetable Cauliflower Hybrid (Kiran) 1 - 5 19 Sheikhupu Sheikhupura Wheat- rice- Wheat Rama Krishna 1 - 5 ra vegetable Sheikhupura Wheat- rice- Spinach Hybrid Nil vegetable Ferozwala Vegetable- Peas Rama Krishna 1 - 5 vegetable Ferozwala Rice-vegetable Rice Hybrid 6 - 20 Sharaq Pur Rice-vegetable Peas Hybrid 1 - 5 Sharaq Pur Rice-vegetable Peas Desi 1 - 5 Sharaq Pur Rice-vegetable Capsicum Palli (Hybrid) 1 - 5 20 Toba Tek Chak 393 Jb Wheat-vegetable Cucumber Desi karela 1 - 5 Singh Khanpur Chak 324 JB Wheat- cotton- Spinach Hybrid Nil vegetable Chak 296 Wheat-vegetable Cucumber Rama Krishna 1 - 5 GB Chak 150 Wheat- Cucumber Hybrid 6 - 20 GB vegetable 153

Jagguwala

21 Vehari Chak 24 Wheat- Cucumber Fsd-Long 1 - 5 WB vegetable Chak 63 Wheat- cotton- Spinach Hybrid Nil WB vegetable Chak 67 Vegetable- Peas Desi 1 - 5 WB vegetable Qadir Wah Wheat- cotton- Cucumber Hybrid 6 - 20 vegetable Adda Wheat- cotton- Gram Desi 1 - 5 Chakrala vegetable Adda Pull Wheat- cotton- Gram Palli (Hybrid) 1 - 5 MohsinShah vegetable 22 Pakpattan Tiba Sher Vegetables Carrot FSD Long Nil Pur 37 SP Vegetables Bitter gourd FSD Long Nil in Tunnel 37 SP Vegetables Spinach FSD Long Nil Arif Abad Wheat- Radish FSD Long Nil Vegetables 23 Khushab Khushab Wheat- Mung Desi Nil mungbean- Vegetables Piplan Wheat- Mung FSD Long Nil mungbean- Vegetables Kamer Wheat- Cucumber FSD Long Nil Mashani mungbean- Vegetables Mianwali Wheat- Cauliflower Palli (Hybrid) Nil mungbean- Vegetables 24 Attock Pindigheb Wheat- Wheat Palli (Hybrid) Nil vegetable-fellow Jand Wheat- Barseem FSD Long Nil vegetable-fodder Hassan Wheat-vegetable Cucumber FSD Long Nil Abdal

Geological position of surveyed area = 28.42°N to 33.46°N and 70.30°E to 72.22°E

154

Annexure-III: Isolates inventory

Sr. Code District Village Isolate source no. assigned 1 Bahawalnagar Ahmadpur East Infected leaves Mr01 2 Rajkan Infected leaves Mr02 3 Khanewal Kabirwala Infected leaves Mr03 4 Multan Kot Rabnawaz Infected leaves, Mr04 5 19 Kassi Soil Mr05 6 Muzaffargarh Basti Jalalabad Infected leaves Mr06 7 Basti Jagatpur Infected leaves Mr07 8 Okara Basir pur Infected leaves Mr08 9 Haveli lakha Fruit Mr09 10 Pakpatan 37 SP Infected leaves Mr10 11 Rahim yar khan Machi goth Infected leaves, Mr11 12 Bhutta wahan Fruit Mr12 13 Islam garh Fruit Mr13 14 Rajan pur Lal garh Infected leaves Mr14 15 Vehari Chak 24 WB Infected leaves Mr15 16 Qadir wah Infected leaves Mr16 17 Gujranwala Mughal Chak Kalan Infected leaves Mr17 18 Gondlan wala Fruit Mr18 19 Nawan Pind Infected leaves Mr19 20 Kot Qazi Fruit Mr20 21 Hafizabad Hafizabad Infected leaves Mr21 22 Hafizabad Fruit Mr22 23 Dhingranwali Fruit Mr23 24 Bangla Maghiani Infected leaves Mr24 Mandi 25 Phalia Infected leaves Mr25 bahauddin 26 Malikwal Infected leaves Mr26

155

27 Sheikhupura Sheikhupura Infected leaves Mr27 28 Sharaq pur Infected leaves Mr28 29 Ferozwala Infected leaves Mr29 30 Faisalabad 145RB Infected leaves Mr30 31 139RB Guhmmi Fruit Mr31 32 Jhang Chak 269 Infected leaves Mr32 Mr33 33 Lahore Shahdra Infected leaves

34 Lahore Infected leaves Mr34 35 Kot abdulmalik Fruit Mr35 36 Kasur Gohar jagir Fruit Mr36 37 Sargodha Sargodha Infected leaves Mr37 38 Bhera Soil Mr38 39 Kot momin Fruit Mr39 40 Toba tek singh Chak 297GB Infected leaves Mr40 41 Rajana Infected leaves Mr41 42 Gujrat Chanu Bhoja Infected leaves Mr42 43 Mehmood Abad Infected leaves Mr43 44 Jallal Pur Jattan Infected leaves Mr44 45 Rawalpindi Rawalpindi Infected leaves Mr45 46 Sialkot Mandair Sharif Infected leaves, Mr46 47 Randheer Fruit Mr47 48 Sadhanwali Infected leaves Mr48 49 Sadhanwali Soil Mr49 50 Kakuwal Fruit Mr50 51 Attock Dhok Fateh Infected leaves Mr51 52 Dhok Waraich Infected leaves Mr52 53 Chakwal Khokhar Zer Infected leaves Mr53 54 DG khan DG khan Infected leaves Mr54

156

Annexure-IV

157

Annexure-V : ITS4/NS5 Gene sequence

>Myrothecium_roridum_Mr 37

GCTTAATTGCGATAACGAACGAGACCTTWACCTGCTAAATAGCCCGTATTGCTTTGG CAGTACGCTGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAA TAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACGGA GCCAGCGAGTACTCCCTTGGCCGGAAGGTCCGGGTAATCTTGTTAAACTCCGTCGTG CTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAA GTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTA CCGATTGAATGGCTCAGTGAGGCGTTCGGACTGGCCCAGAGAGGTGGGAAACTACC ACTCAGGGCCGGAAAGTTCTCCAAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAAC AAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACCGAGTTTACAAACTCCCAAA CCCTTTGTGAACCTTACCTATCGTTGCTTCGGCGGGACCGCCCCGGCGCCTTCGGGCA ACGGAACCAGGCGCCCGCCGGAGAACCCAAACTCTTATGTCTTTAGTGGTTTTCTCCT CTGAGTGACACATAAACAAATAAATAAAAACTTTCAACAACGGATCTCTTGGTTCTG GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGT GAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCT GTTCGAGCGTCATTTCAACCCTCAGGCCCCCAGTGCCTGGCGTTGGGGATCGGCGTG GGCCGGGGCTGTCCTCCGGGACGGTCCCCGCGCCTGCCGGCCCCGAAATTCA

158

Annexure-VI: Bitter gourd germplasm inventory

Code Code Sr Sr Variety Source assigne Variety Source assigne # # d d Cross 888 f1 1 Jaunpuri FSCRD MCV01 20 China hybrid MCV20 hybrid 2 Jhalri FSCRD MCV02 21 Runfeng China hybrid MCV21 Indian 3 FSCRD MCV03 22 No 361 f1 hybrid China hybrid MCV22 karela Green 4 Auriga MCV04 23 Advanta 103 ICI MCV23 wonder Sachal 5 Chaman AARI Fsd MCV05 24 ICI MCV24 black4722 Subeej 6 Fsd long AARI Fsd MCV06 25 Long green MCV25 nuziveedu VRBG 7 AARI Fsd MCV07 26 Jhalri Nutech MCV26 227 VRBG 8 AARI Fsd MCV08 27 Jaunpuri long Nutech MCV27 230 VRBG 9 AARI Fsd MCV09 28 Desi karela Nayab MCV28 231 VRBG 10 AARI Fsd MCV10 29 Lamba karela Nayab MCV29 233 11 SHBG-48 AARI Fsd MCV11 30 Karela Nayab MCV30 Early 12 AARI Fsd MCV12 31 BG-34 AARI Fsd MCV31 green 13 JK Lena AARI Fsd MCV13 32 BSS-616 AARI Fsd MCV32 14 BG-7107 AARI Fsd MCV14 33 TIPU AARI Fsd MCV33 15 CBT-36 AARI Fsd MCV15 34 RAJA AARI Fsd MCV34 16 PKBT-1 AARI Fsd MCV16 35 CBT-36 AARI Fsd MCV35 17 Preeti AARI Fsd MCV17 36 LEENA(7005) AARI Fsd MCV36 18 KIRAN FSCRD MCV18 37 MONIKA(7004) AARI Fsd MCV37 19 Jaunpuri Proline MCV19

159

Pak. J. Weed Sci. Res., 21(3): 369-379, 2015

IN VITRO FUNGICIDAL ACTIVITY OF AQUEOUS EXTRACTS OF CROP AND WASTELAND WEEDS AGAINST Myrothecium roridum TODE

Sumera Naz1 * , Salik Nawaz Khan1 , Ghulam Mohy-Ud-Din2 and Shumaila Farooq1

ABSTRACT Application of aqueous weed extracts is an environment friendly approach to manage destructive plant pathogens and is an emerging tool in biological control of pathogens. In the present study, aqueous extracts of nine weeds Chenopodium album L., Parthenium hysterophorus L., Trianthema portulacastrum L., Malvestrum coromandelianum (L.) Garcke, Coronopus didymus (L.) Sm., Sphaeranthus indicus L., Digera muricata (L.) Mart., Solanum nigrum L. and Nicotiana plumbaginifolia Viv. were applied against Myrothecium roridum Tode strain Mr 10 (accession no. 1155) by food poison technique. Aqueous extracts of weeds were prepared by macerating 20g of fresh leaves in 20 mL of sterilized distilled water (100% w/v stock). The extracts were double filtered through muslin cloth and Whatman filter paper no. 1 and added in PDA medium under asceptic conditions before pouring. The extract of N. plumbaginifolia exhibited growth inhibition of 88%, P. hysterophorus (71%) and S. nigrum, C. didymus, S. indicus and T. portulacastrum L. restrained the colony growth up to 66, 65, 64 and 60%, respectively. Digera muricata was least effective with 11% of colony growth.

Key words: Antifungal potential, aqueous extract, crop and waste land weeds, Myrothecium roridum.

Citation: Naz, S., S.N. Khan, G. Mohy-Ud-Din and S. Farooq. 2015. In vitro fungicidal activity of aqueous extracts of crop and wasteland weeds against Myrothecium roridum Tode. Pak. J. Weed Sci. Res. 21(3): 369-379.

INTRODUCTION Myrothecium roridum is a seed- and soil-borne fungus with a wide host range of vascular plants. It has been isolated frequently

1 Institute of Agricultural Sciences, Quaid-e-Azam Campus, University of the Punjab, Lahore 2 Plant Pathology Section, Ayub Agriculture Research Institute, Jhang Road, Faisalabad *Corresponding author’s email: [email protected] 370 Sumera Naz et al., In vitro fungicidal activity of ...

from seeds of bitter gourd (Momordica charantia L.) and found associated with rotted and un-germinated seeds. It has become a problematic pathogen affecting the yield and quality of bitter gourd crop in Punjab, Pakistan (Sultana and Ghaffar, 2007). Appearances of dark brown leaf spots with concentric rings of olive green to black colored sporodochia are signs of the presence of myrothecium leaf spot disease. At later stage, these spots coalesce to form blighted areas on the leaves (Belisario et al., 1999). Very little information is available on myrothecium leaf spot disease and associated promoting factors of climate and need experimental elaboration. Score (Difenoconazole DMI group), and zinc and copper based fungicides like Captan and Maneb are generally recommended to control M. roridum but their efficacy can be affected by climatic conditions and growth stage of the plants (Sultana and Ghaffar, 2009; McMillan, 2010). Application of commercial synthetic fungicides may also have negative effect on produce quality and grower environment. There is a need to investigate efficient, effective and economical ways for environmental friendly management strategies against plant pathogens. Study of allelopathic potential of plants in managing several pathogens may lead to cost effective and environmental friendly approach and provides an excellent alternative to synthetic chemical applications (Vyvyan, 2002). Scientists are working on the aqueous and organic solvent extracts of flowering plants like Azadirachta indica, Eucalyptus spp., Syzygium cumuni, Curcuma longa, C. didymus, C. album, and Aloe vera to control wide range of fungal plant pathogens (Davicino et al., 2007; Dellavalle et al., 2011; Javaid and Iqbal, 2014). The archeological references reveal that the concept of application of phyto pesticides is centuries old in the Indian subcontinent and Africa. This phenomenon is supported due to presence of essential compounds which can further be exploited for managing plant pathogens (Srivastava and Lawton, 1998; Root, 1973). The inhibitory effect of S. indicus has been reported against Alternaria solani, Fusarium oxysporum and Penicillium pinophilum (Dubey et al., 2000; Galani et al., 2010). Parthenium hysterophorus was reported to have antifungal potential against soil borne pathogens (Bajwa et al., 2001). Antifungal potential of D. muricata and C. didymus under in vitro and in vivo conditions was found to reduce the incidence of Alternaria alternata and Sclerotium rolfsii against vegetable and cereals (Shafique et al., 2006; Sharma and Vijayvergia, 2013). Myrothecium roridum is an emerging threat for bitter gourd crop in Pakistan. Few synthetic fungicides have been evaluated against M. roridum but there is scarcity of information on exploring antifungal activity of weeds. Host susceptibility and broader Pak. J. Weed Sci. Res., 21(3): 369-379, 2015 371

host range of M. roridum demands for exploring new strategies for integrated management of the disease. Therefore attempt is made to explore the antifungal potential of aqueous extracts of endemic crop and wasteland weeds.

MATERIALS AND METHODS Fungal culture Myrothecium roridum strain Mr 10 (FCBP accession no. 1155) isolated from bitter gourd leaves in Seed and Post Harvest Pathology Lab, Institute of Agricultural Sciences, University of the Punjab, Lahore was maintained on potato dextrose agar medium (potatoes, 200g; dextrose, 20g; agar, 16g and distilled water; 1L) at 25°C. Collection of weed plants Tender plants of N. plumbaginifolia, C. album, P. hysterophorus, T. portulacastrum, M. coromendelianum, C. didymus, S. indicus, D. muricata and S. nigrum were collected from the crop and wasteland fields of Lahore and its suburbs. Preparation of aqueous extracts Plants were thoroughly washed under running tap water to remove dust and other contaminants and surface dried on the blotter paper. Aqueous weed extracts were prepared by macerating 20g of fresh leaves in 20 mL of distilled water (100% w/v stock) and double filtered through muslin cloth and filter paper (Javaid et al., 2010). Stock extracts were stored at 4 °C and used within 2-3 days. Food poison assay The experiment was laid out in CRD with five replicates and five 90 mm Petri plates in each replicate. Each of the tested weed extract was added @ 10% in 2% PDA medium before pouring under sterilized conditions. Control PDA plates were not amended with weed extracts. A disc of 3mm diameter from the actively growing colony margins of 10 days old M. roridum culture was transferred to the PDA plates amended with the weed extracts. The plates were incubated at 28±2 °C and colony growth was measured after 4, 7, 10 and 14 day interval. Inhibition percentage was measured at day 14 by the following formula given below.

Growth in control - growth in weed amended medium Radial growth inhibition % = x 100 Growth in control Morphological response was assessed on colony macroscopic characters i.e., colony texture, colony margins, colony form, colony elevation and physiological response on microscopic character i.e., spore production were recorded. Data were subjected to analysis of variance (ANOVA) followed by Tukey’s HSD test using computer software SPSS version 15.0. 372 Sumera Naz et al., In vitro fungicidal activity of ...

RESULTS AND DISCUSSION Radial mycelia growth of M. roridum was observed against tested weed extracts at 4, 7, 10 and 14 incubation day (Fig. 1). There was a significant increase in radial growth with increase in incubation period. The highest radial growth (i.e. 87 mm) was observed in control treatment during all incubation periods (Fig. 2). Among the tested weed extracts, N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus and S. Indicus did not exhibit any radial growth at day 4. This suppression was maintained in N. plumbaginifolia up to day 7. Weed extracts of C. album,T. portulacastrum, M. coromendelianum, D. muricata were proved least effective as they showed increasing pattern in radial growth at 4, 7, 10 and 14 day of incubation. At 14 day incubation period, N. plumbaginifolia, P. hysterophorus, S. nigrum, C. didymus, S. indicus, C. album, T. portulacastrum, M. coromendelianum and D. muricata exhibited 11, 26, 30, 31, 32, 36, 42, 72 and 81 mm radial growth respectively (Fig. 2). Among tested aqueous extracts, the highest antifungal potential was found in N. plumbaginifolia extract that inhibited the colony radial growth up to 88% followed by P. hysterophorus that reduced the growth up to 71% over control (Fig. 3). S. nigrum, C. didymus, S. indicus and T. portulacastrum L restrained the colony growth up to 66%, 65%, 64% and 60% respectively. C. album slows down the colony growth up to 54%. D. muricata was least effective with 11% of colony growth. Variation in physiological response was recorded on the basis of macroscopic characters like colony color, texture, margins, spore production and elevation (Table-1). M. roridum produce circular, flat colonies with floccose texture and filiform margins on PDA at 25°C. A large number of conidia produced after 3-4 days on colony surface while mycelium continues growing from margins. Nicotiana plumbaginifolia and P. hysterophorus extracts revealed to inhibit the colony radial growth whereas N. plumbaginifolia did not exhibit any spore production till last reading. This might due toof presence of potent antifungal compounds in aqueous extracts that provide inhibition in the fungal growth. S. indicus and M. coromendelianum produce submerged colonies as compared to the control treatment. C. album and M. coromendelianum extracts produce irregular shapes colonies with lobate margins. Inhibition potential of the weed plants is due to the presence of several chemical constituents like phenols, alkaloids, terpenoids, coumarins and tannins. Some of them already have been discovered and known for their antifungal potential but many needed to be explored. Singh et al. (2010) observed antibacterial activity of aqueous and methanol extracts of N. plumbaginifolia on five human pathogenic bacteria Viz Bacillus cereus, Bacillus fusiformis, Salmonella Pak. J. Weed Sci. Res., 21(3): 369-379, 2015 373

typhimurium Staphylococcus aureus and Pseudomonas aeruginosa. Phytochemical evaluation of leaves of N. plumbaginifolia revealed the presence of alkaloids, saponin, tannin, flavonoides, cardiac glycosides, phenolic compounds, steroids, terpenoides and carbohydrates (Singh et al., 2010). Stukkens et al. (2005) reported terpenoids compounds in N. plumbaginifolia are responsible for its antifungal properties. P. hysterophorus is known to have chemical constituents like parthenin, p-coumaric acid, ferrulic acid, vanillic acid and caffeic acid that act as antifungal compounds (Kanchan and Jayachandra, 1980; Das and Das 1995). The plant parts and their extracts suppressed Penicillium spp., inhibited germination of spores of Drechslera rostrata, Fusarium oxysporum, Alternaria alternata, Corynespora cassiicola, Aspergillus fumigatus, A. niger, A. sulphureus and Microsporum gypseum (Luke, 1976; Kumar et al., 1979; Shrivastava et al., 1984; Sharma and Gupta, 2012). Plant extract inhibited mycelial growth and sporulation in pathogen Aspergillus flavus (Lokesha et al., 1986). Digera muricata is reported to contain antifungal compounds and showed significant reduction in growth of Fusarium oxysporum and Aspergillus niger but in the present study it exhibited least inhibition against M. roridum (Kohli et al., 1998; Sharma and Vijayvergia, 2013). This may be attributed resistance reaction of the test fungus to its compounds but detailed investigations are needed. Further, in vivo investigations of highly effective concentration of aqueous extracts and exploitation of their organic fractions are in progress.

CONCLUSION It is concluded that aqueous extracts of N. plumbaginifolia and P. hysterophorus possess significant antifungal activity against M. roridum under in vitro conditions.

Sumera Pak.Naz J.et Weed al., In Sci vitro. Res., fungicidal 21(3): activity369-379 of, ...201 5 374

Figure 1. Effect of different aqueous weed extracts on colony growth of M. roridum.

12 Pak.Sumera J. Weed Naz Sci. et Res., al., 2In1 (vitro3): 369fungicidal-379, 201 activity5 of ... 375

Figure 2. Effect of different aqueous weed extracts on colony growth of M. roridum. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by Tukey’s HSD method. Sumera NazPak. et J. al.,Weed In vitroSci. Res., fungicidal 21(3 ):activity 369-379 of ,... 201 135 376

Figure 3. Evaluation of colony growth inhibition of M. roridum against aqueous weed extract

Pak. J. Weed Sci. Res., 21(3): 369-379, 2015 377 14 Sumera Naz et al., In vitro fungicidal activity of ...

Table-1. Assessment of macroscopic colony characters of M. roridum under the stress of weed aqueous extracts Weed extract Colony form Colony Colony texture Colony Spore elevation margins production Control Circular Flat Floccose Filiform + N. plumbaginifolia Irregular Umbonate Filamentous Filiform - P. hysterophorus Filamentous Raised Floccose Filiform - S. nigrum Filamentous Raised Floccose Filiform + C. didymus Filamentous Flat Floccose Undulate + S. indicus Irregular Submerged Sparsely filamentous Entire +

T. portulacastrum Filamentous Crateriform Sparsely floccose Filiform +

C. album Filamentous Crateriform Floccose Undulate + M. coromendelianum Irregular Submerged Sparsely filamentous Lobate +

D. muricata Circular Flat Floccose Filiform + +: Produce spores, - : No spore production

Sumera Naz et al., In vitro fungicidal activity of ... 378 Pak. J. Weed Sci. Res., 21(3): 369-379, 2015

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Keywords: MyrotheciumroridumTode, aromatic medicinal plants, fungicidal activity, in vivo. INTRODUCTION alternative strategies for incorporation in Integrated Disease Vegetable plays an important role in meeting with nutrition Management (IDM). Study of allelopathic potential of plants needs and fighting against ailments. Among vegetables, bitter against fungal and bacterial pathogens is getting acceptance gourd (Momordica charantia Linn) has a unique medicinal among the production and consumption chain stakeholders and nutritional value and belongs to family Cucurbitaceae. It because its cost effectiveness and user safety. Intercropping is among the popular vegetables in Asia and other part of the of different non-host plants with known antimicrobial activity world. It is normally grown as an annual crop in Pakistan may help in reducing the pathogen build up by providing with the total area under bitter gourd cultivation during 2009- either physical barriers or releasing volatile chemical 2010 was 6565 hectares and total production of 56994 tones constituents that retard the fungal growth. [3] studied the [6]. Bitter gourd fruit is medicinal and nutritious vegetable. marigold and pigweed allelopathy by intercropping for the The high nutritive value ranks it first among the cucurbits in management of tomato early blight disease. [8] reported iron and vitamin C contents. It is an excellent source of reduced Septoria leaf spot disease by Septoria lycopersici in phenolic compounds, antioxidants, and antimutagen [5]. The tomato-maize intercropping. Chickpea blight caused by fruit has considerable amount of potassium, calcium, Ascochyta sp was significantly lowered by intercropping magnesium, protein, and dietary fiber as compared with other chickpea with wheat and barley [2,4]. commercial vegetables [10]. Little work has been reported in Pakistan on diseases of bitter Myrothecium. sp. is soil borne as well as seed borne pathogen gourd. The present studies are, therefore, aimed for the and attacks on a wide range of plant species. On cucurbits it management of Myrothecium leaf spot disease by causes round dark-brown leaf spot which on later stage intercropping medicinal aromatic plants with bitter gourd coalesces to form blighted areas on the leaves [1]. It requires crop which will enable us to protect bitter gourd crop from a prolonged wet period for perpetuation and epidemics. Myrothecium leaf spot disease. Though Myrothecium roridum is frequently observed on above ground parts especially the leaves but it is primarily METHODOLOGY seed and soil borne in nature. [7] Reported that fungus is The investigations protocol comprises on greenhouse and associated with rotted and un-germinated seeds. Yield and field trails. Greenhouse trials were performed at experimental quality loss of bitter gourd crop is usual phenomenon in station of institute of agricultural sciences, university of the Punjab, Pakistan. Periodic occurrence of myrothecium leaf Punjab (IAGS, PU) Lahore, Pakistan. Field trials were spot disease on the bitter gourd and several other crops conducted at research forms of plant pathology section, Ayub belonging to different families and even isolations from Agriculture Research Institute (AARI) Faisalabad, Pakistan. gymnosperms needs the detailed study of the pathogen Seeds of bitter gourd, chili, capsicum, onion bulbs and biology, physiology and management. rhizomes of turmeric, ginger were procured from vegetable Application of fungicides is conventional tool for disease section AARI Faisalabad, Pakistan (Table 1). Single spore management and highly practiced due to effective disease culture of Myrothecium roridum was isolated from the field management. Due to health hazards and other economic grown bitter gourd and maintained on potato dextrose agar concerns attention has been diverted for evaluation of medium. May-June 2170 ISSN 1013-5316; CODEN: SINTE 8 Sci.Int.(Lahore),27(3),2169-2172,2015 For green house experiments, 18x24 cm earthen pots were artificial inoculation as pinhead sized infections (Fig. 2). The used. The pots were filled ¾ with the sandy loam soil while initial disease incidence readings (ranged 18.63-36.08) less for field experiments; 75-90 cm ridges with 60cm distance significantly differ within the intercropped treatments between the rows was prepared. Plant to plant distance was compared to the bitter gourd monoculture (control). Further maintained at 45cm. Crop Production technology prescribed readings were taken at weekly intervals. The final reading by the Punjab agriculture department for farmers were taken at week 7 shows a remarkable reduction in disease followed. The four week old seedling growth was evaluated incidence against control (78.96). Disease incidence in B-Gar after the germination of 75% germplasm of all the tested treatment was 33.1% followed by B-Chi (40.29%) and B-Oni plants. Ten days old Myrothecium roridum cultures were (48.19%) treatments. B-Gin treatment exhibited 61.62%, B- sprayed (@2x103spores/ml) with hand atomizer for artificial Cap 65.75%, B-Cur 75.08% and B-Col treatment disease inoculation of four weeks old seedlings. The spray was incidence was 72.43%. repeated at 30 minutes interval. All the treatments were 100 replicated thrice and each replication contains ten plants. Ctrl (B) 80 Data regarding disease incidence and disease severity was B-Gar taken after two days of spray and then at weekly intervals up to 7 weeks (bitter gourd plant maturity) by using the disease 60 B-Chi rating scale. B-Oni Data recording for infection development in pots was 40 initiated 48 hours after inoculation and for field trails 20 B-Gin inoculation was made at germination, vegetative growth,

disease incidence%age B-Cap maturity, harvest and for some selective plants at seed 0 development stage of the plant and data recording was 1 2 3 4 5 6 7 B-Cur initiated at germination stage. Data recorded was subjected No of weeks after inoculation to analysis of variance (ANOVA) followed by students T test Fig. 1: Disease incidence percentage of Myrothecium leaf spot using Microsoft excel 2010. disease in intercropping treatments Table 1: Inventory of plants used in intercropping against MyrotheciumroridumTode under greenhouse and field 100 conditions

Ctrl (B) Plant Botanical name Sowing material 80 intercropped B-Gar Garlic Allium sativum Cloves 60 Onion Allium cepa Bulb B-Chi Ginger Zingiber Rhizome 40 officinale B-Oni

incidence%age 20 Green chili Capsicum Seeds B-Gin frutescens 0 Capsicum Capsicum Seeds B-Cap 1 2 3 4 5 6 7 annuum weeks Turmeric Curcuma longa Rhizome Fig. 2: Disease incidence percentage of Myrothecium leaf spot Arvi Colocasia Cornels disease in intercropping treatments under field conditions esculenta during 2013. All the readings are mean values of three replicates and each replication contains ten plants. The readings are RESULTS subjected to ±5% SE. First reading of disease incidence of myrothecium leaf spot under greenhouse was recorded after 48 hours of artificial inoculation as pinhead sized infections and then at weekly Inhibition %age intervals up to 7 weeks (Fig.1). The initial disease incidence 100 readings (ranged 17.33-26.07) did not significantly differ among the intercropped treatments compared to the bitter 50 gourd monoculture (control). The final reading taken at week 0 7 shows a remarkable reduction in disease incidence against control (79.18). Disease incidence in B-Gar treatment was

29.2% followed by B-Chi (38.20%) and B-Oni (46.66%) inhibition%age plants intercropped in bitter gourd treatments. B-Gin treatment exhibited 57.62%, B-Cap 60%, B-Cur 70.03% and B-Col treatment disease incidence was 71.84%. under greenhouse conditions during 2013. All the Fig. 3: Disease inhibition percentage of Myrothecium leaf spot readings are mean values of three replicates and each disease in intercropping treatments under greenhouse replication contains ten plants. The readings are subjected to conditions during 2013. All the readings are mean values of three replicates and each replication contains ten plants. The ±5% SE. readings are subjected to ±5% SE. Under field conditions, first reading of disease incidence of myrothecium leaf spot was recorded after 48 hours of May-June Sci.Int.(Lahore),27(3),2169-2172,2015 ISSN 1013-5316; CODEN: SINTE 8 2171 have been reported activity as antimicrobials. They may be used for the management of several insects, bacteria, fungi 2013 and nematodes. Garlic and onion are well-known to release allicin compound belonging to solfoxide class that is active

80 against fungi and bacteria [4]. Curcumin, a terpenoid; is 60 reported in turmeric and is biologically active against fungi, 40 bacteria and protozoa. Present studies show that garlic and 20 onion intercropping significantly lowers the disease incidence 0 of myrothecium leaf spot disease of bitter gourd as compared to the monoculture bitter gourd. This reduction might be inhibition%age contributed towards the accumulation of volatile constituents on the plant surface that retard the further infections, sporulation and development of fungus [9]. The results plants intercropped in bittergourd field suggest that intercropping of garlic, green chili and onion can significantly reduce the Myrothecium leaf spot disease Fig. 4: Disease inhibition percentage of Myrothecium leaf spot incidence in farmer fields without increasing their input cost disease in intercropping treatments under greenhouse in the form of fungicides. Further studies for evaluation and conditions during 2013. All the readings are mean values of extraction of the active chemical constituents responsible for three replicates and each replication contains ten plants. The readings are subjected to ±5% SE. their antifungal potential may help in the development of Under greenhouse conditions @(P < 0.05), bitter gourd-garlic effective fungicides. intercropping treatment significantly lowers the incidence of myrothecium leaf spot disease by 63% over control (Fig. 3). REFERENCES Chilies-bitter gourd intercropping also reduce the Belisario, A., Forti, E., Corazza, L. and Kestsren, HAV. First myrothecium leaf spot disease incidence, 52%, significantly report of Myrothecium verrucaria from muskmelon followed by onion-bitter gourd intercropping that shows an seeds. Plant Pathology, 83: 589 (1999) inhibition percentage of 41 than the control treatment. Bitter Gan Y.T. , Siddique K.H.M., MacLeod W.J., Jayakumar P. gourd-ginger and bitter gourd-capsicum intercropping Management options for minimizing the damage by treatments were less significantly inhibit the myrothecium ascochyta blight (Ascochyta rabiei) in chickpea (Cicer leaf spot disease incidence i.e., 27% and 24% respectively. arietinum L.) Field Crops Research 97: 121–134(2006) Bitter gourd-turmeric and bitter gourd-arvi intercropping Go´mez-Rodrı´guez O., Zavaleta-Mejı´ E, Gonza´lez- treatments reduce the disease incidence non-significantly by Herna´ndez V.A., Livera-Mun˜oz M, Ca´rdenas-Soriano 11% and 9% respectively than the bitter gourd alone. E. Allelopathy and microclimatic modification of Under field conditions, the results were much similar to those intercropping with marigold on tomato early blight with greenhouse conditions except for their degree of disease development. Field Crops Research 83: 27– inhibition slightly decrease. Bitter gourd-garlic intercropping 34(2003) treatment significantly (P < 0.05) lowers the incidence of Gurjar M.S., Ali S, Akhtar M, Singh K.S. Efficacy of plant myrothecium leaf spot disease, by 58% than in bitter gourd extracts in plant disease management. Agricultural solo cultivation (Fig. 4). Chilies-bitter gourd intercropping Sciences, 3(3); 425-433 (2012) also reduce the myrothecium leaf spot disease incidence, Islam, S., Jalaluddin, M. and Hettiarachchy, NS. Bio-active 49%, significantly followed by onion-bitter gourd compounds of bitter melon genotypes (Momordica intercropping that shows an inhibition percentage of 39 than charantia L.) in relation to their physiological functions, the control treatment. Bitter gourd-ginger and bitter gourd- Functional Foods in Heals and Disease, 2:61-74. capsicum intercropping treatments were less significantly (2011) inhibit the myrothecium leaf spot disease incidence i.e., 22% MINFAL,. Agricultural statistics of Pakistan, Ministry of and 17% respectively. Bitter gourd-turmeric and bitter gourd- Food, Agricultural and Co-operative, Islamabad, arvi intercropping treatments reduce the disease incidence Pakistan. (2010) non-significantly by 5% and 8% respectively than the bitter Sultana, N. and Ghaffar, A. Seed Borne Fungi Associated gourd alone. with Bitter Gourd (Momordica charantia Linn..) Pak. J. Bot., 39(6): 2121-2125. (2007) DISCUSSION Suman, K., Sugha, S.K., Kumar, S.. Role of cultural practices Intercropping with different aromatic and medicinal plants in the management of Septoria leaf spot of tomato. can lead to mutual economically cost effective as well as IndianP hytopath. 53, 105–106. , (2000) environment friendly approach in adopting the production Trenbath B.R., Intercropping-Bases of Productivity technology of the crop of interest [3]. These may involve the Intercropping for the management of pests and diseases, nutrient uptake processes, creating microclimate or escaping Field Crops Research, 34(3–4): 381–405 (1993) the disease either as non-host crop or liberating chemical Wills, RBH., Wong, AWK. Scriven, FM. Greenfield, H. constituents to fight with the pests [2]. Aromatic plants are Nutrient composition of Chinese vegetables. Journal of well known for their characteristics release of several volatile Agricultural and Food Chemistry. 32:413-416. (1984). compounds in their surroundings. Most of these compounds

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