CHARACTERIZATION OF FUNGAL PATHOGEN(S) CAUSING WILT OF LENTIL AND THEIR MANAGEMENT KHOLA RAFIQUE 03-arid-47 Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2015 CHARACTERIZATION OF FUNGAL PATHOGEN(S) CAUSING WILT OF LENTIL AND THEIR MANAGEMENT by KHOLA RAFIQUE (03-arid-47) A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Pathology Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2015 ii CERTIFICATION I hereby undertake that this research is an original one and no part of this thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences. Student’s Name: Khola Rafique Signature: ____________ Registration No: 03-arid-47 Date: ____________ Certified that the contents and form of thesis entitled “Characterization of Fungal Pathogen(s) Causing Wilt of Lentil and their Management” submitted by Ms . Khola Rafique have been found satisfactory for the requirement of the degree. Supervisor: ______________________________ (Prof. Dr. Abdul Rauf) Member: ______________________________ (Dr. Farah Naz) Member: ______________________________ (Dr. Ghulam Shabbir) Chairman: _________________________ Dean: __________________________ Director Advanced Studies: __________________________ iii iv v CONTENTS Page List of Tables x List of Figures xi List of Abbreviations xvi Acknowledgement xviii Abstract xx 1. INTRODUCTON 1 2. REVIEW OF LITERATURE 8 2.1 THE IMPORTANCE OF LENTIL 8 2.2 LENTIL DISEASES 9 2.3 LENTIL WILT AND THE CAUSAL ORGANISMS 11 2.4 BIOLOGY AND MORPHOLOGICAL CHARACTERIZATION 12 OF FUSARIUM SPECIES ASSOCIATED WITH LENTIL WILT 2.4.1 Fusarium oxysporum 13 2.4.2 Fusarium redolens 15 2.4.3 Fusarium nygamai 15 2.4.4 Fusarium commune 16 2.4.5 Fusarium equiseti 17 2.5 SURVIVAL AND DISEASE CYCLE OF FUSARIUM WILT OF 17 LENTIL 2.6 SYMPTOMS OF FUSARIUM WILT OF LENTIL 18 2.7 SILICA GEL PRESERVATION OF FUSARIUM ISOLATES 19 vi 2.8 MOLECULAR CHARACTERIZATION AND 20 PHYLOGENETIC ANALYSIS OF FUSARIUM ISOLATES THROUGH DNA SEQUENCING 2.9 INCIDENCE, DISTRIBUTION AND YIELD LOSSES OF 24 LENTIL FUSARIUM WILT 2.10 PATHOGENICITY TEST 25 2.11 SCREENING FOR HOST RESISTANCE AGAINST LENTIL 26 WILT 2.12 BIOLOGICAL MANAGEMENT OF LENTIL WILT 28 2.13 CHEMICAL MANAGEMENT OF LENTIL WILT 30 3. MATERIALS AND METHODS 33 3.1 DISEASE SURVEY AND ASSESSMENT 33 3.1.1 Disease Survey 33 3.1.2 Disease Assessment and Sampling 35 3.2 ISOLATION AND IDENTIFICATION OF THE PATHOGENS 36 3.3 PRESERVATION OF FUSARIUM ISOLATES 37 3.4 MORPHOLOGICAL CHARACTERIZATION 38 3.5 PATHOGENICITY TEST 38 3.5.1 Disease Parameters 42 3.6 MOLECULAR CHARACTERIZATION 43 3.6.1 Mycelium Production and DNA Extraction 43 3.6.2 Polymerase Chain Reaction (PCR) Amplification of TEF-1α 45 Gene Region 3.6.3 PCR Product Analysis 47 vii 3.6.4 DNA Sequencing 47 3.6.5 Phylogenetic Analysis 48 3.7 MANAGEMENT OF FUSARIUM WILT 48 3.7.1 Management Through Host Plant Resistance 49 3.7.1.1 Disease parameters 49 3.7.2 Biological Management 50 3.7.2.1 Disease parameters 51 3.7.3 Chemical Management 51 3.7.3.1 In vitro evaluation of fungicides 51 3.7.3.2 In vivo evaluation of fungicides 53 3.7.3.2.1 Disease parameters 54 4. RESULTS AND DISCUSSION 55 4.1 DISEASE SURVEY AND ASSESSMENT 55 4.1.2 Disease Sampling 65 4.2 ISOLATION AND IDENTIFICATION OF THE PATHOGENS 67 4.3 PRESERVATION OF FUSARIUM ISOLATES 69 4.4 MORPHOLOGICAL CHARACTERIZATION 71 4.4.1 Colony Color 85 4.4.2 Growth Habit 85 4.4.3 Pigmentation 88 4.4.4 Days to Fill 9 cm Dish 88 4.4.5 Concentric Rings 90 4.4.6 Conidiophore and Phialide 90 4.4.7 Shape and Size of Micro-conidia 92 viii 4.4.8 Shape and Size of Macro-conidia 95 4.4.9 Shape of Apical and Basal Cells of Macro-conidia 104 4.4.10 Septation in Macro-conidia 106 4.4.11 Chlamydospores: Formation and Diameter 106 4.4.12 Interseptal Distance 108 4.5 PATHOGENICITY TEST 116 4.6 MOLECULAR CHARACTERIZATION 126 4.7 MANAGEMENT OF FUSARIUM WILT 150 4.7.1 Management Through Host Plant Resistance 150 4.7.2 Biological Management 155 4.7.3 Chemical Management 158 4.7.3.1 In vitro evaluation of fungicides 158 4.7.3.2 In vivo evaluation of fungicides 164 SUMMARY 171 CONCLUSION AND RECOMMENDATIONS 178 LITERATURE CITED 179 APPENDICES 205 ix List of Tables Table No. Page 3.1 Fungicides used for the management of lentil Fusarium wilt 52 4.1 Location-wise percent disease prevalence and incidence 60 4.2 Fusarium culture identification and morphological 72 characterization checklist 4.3 Mean/ standard deviation (S.D.) in four morphological 96 characteristics of Fusarium species 4.4 Virulence of morphologically identified and characterized 117 Fusarium species tested on lentil germplasm NARC-08-1 and Masoor-93 4.5 Identification of Fusarium isolates based on DNA sequencing 139 of the translation elongation factor 1-α gene region 4.6 Involvement of major Fusarium species in lentil wilt and plant 144 mortality 4.7 Screening of lentil germplasm against Fusarium wilt 151 4.8 Effect of fungicides at different concentrations on mycelia 160 radial growth of Fusarium isolate x List of Figures Figure No. Page 1.1 Lentil production zones 2 3.1 Map of Punjab Province showing major lentil districts 34 surveyed for the assessment of wilt prevalence, incidence and collection of Fusarium isolates 3.2 Lentil seedlings in germinators (a) 8-days old, and (b) 15-days 40 old 3.3 Lentil roots dipping in fungal spore suspension (1 x 41 10 7conidia/ mL) 3.4 Inoculated lentil seedlings after transplantation to pots in 41 screen house 3.5 Schematic of part of translation elongation factor (TEF-1α) 46 gene region showing primer positions 4.1 Map of Pakistan and major lentil growing districts and sites of 56 Punjab province surveyed for the assessment of Fusarium wilt prevalence, incidence and distribution 4.2 Wilted lentil fields: (a) Layyah-Fateh Pur, and (b) Bhakkar- 57 Garh Morr 4.3 District-wise disease incidence of lentil wilt at two plant 58 growth stages during 2011-12 and 2012-13 crop seasons. 4.4 Patches of wilted lentil plants in fields: (a) Chakwal-Piplee 66 xi field, and (b) Bhakkar-Garh Morr field 4.5 Lentil plant samples: (Right) Wilted lentil plant, and (Left) 68 Healthy lentil plant (left) 4.6 Preserved Fusarium isolates on silica gel in glass vials stored 70 at 4±2 oC (a), Gel crystals coated with fungal growth (b), and Revived Fusarium culture using silica gel crystals after 5 days of incubation at 25 oC (c) 4.7 Isolates showing distinct White (a), Creamy white (b), and 86 Pinkish white (c) colony color 4.8 Petriplates showing variation in growth patterns of Fusarium 87 isolates: (a) Fluffy, (b) Compact, and (c) Flat 4.9 Petriplates showing presence and absence of pigmentation in 89 Fusarium isolates: (a) Isolate FWC1 without any colored pigmentation, (b) Isolate FWC5 with dark violet pigmentation, (c) Isolate FWB3 with violet pigmentation, (d) Isolate FWC3 with Pale brown pigmentation, (e) Isolate FWM1 with Light brown pigmentation, and (f) Isolate FWB11 with Pink pigmentation 4.10 Petriplates (9 cm) with mycelia at varied radial growth after 7 91 days of incubation at 25 °C 4.11 Isolates showing production of distinguished concentric rings 91 in 12 hours light/ darkness cycle: (a) With 1 concentric ring, and (b) With 2 concentric rings 4.12 Phialide characteristics observed in Fusarium isolates under 93 xii light microscope at100X magnification: (a, b) Short and plump monophialides, (c) Long monophialdes, and (d) Polyphialides, and (a-d) Scale bar = 50 µm 4.13 Micro-conidia of Fusarium isolates under light microscope at 94 100X magnification: (a) Oval single-celled and two-celled micro-conidia, (b) Obovoid micro-conidia, (c) Measurement of length of a micro-conidium with a scale bar, (d) Measurement of width of a micro-conidium with a scale bar, and (a-d) Scale bar = 25 µm 4.14 Macro-conidia (a) and Macro-conidial shapes observed under 103 light microscope at 100X magnification (b-f): (b) Slightly curved macro- conidia of isolate FW, (c) Straight-shape macro-conidia of isolate FWC10, (d, e, f) Slender macro- conidia of isolate, and (a-f) Scale bar = 25 µm 4.15 Macro-conidia with curved-shaped apical and foot-shaped 105 basal cells under light microscope at 100X magnification (a), and Septation of macro-conidia (b-d): (b) Three-septate, (c) Four-septate, (d) Five-septate, and (a-d) Scale bar = 25µm 4.16 Formation of Chlamydospores: (a) Singly, (b) Pairs, (c) Short 107 chains, (d) Clusters, (e) Rough-walled, (f) Smooth-walled, and (a-d) Scale bar = 25 µm 4.17 Production of Chlamydospores: (a, b, c) Terminally, (d, e) 109 Intercalary, (f) Measurement of diameter of chlamydospore with a scale bar at 100X magnification under light microscope, xiii and (a-f) Scale bar = 25 µm 4.18 Septate, hyaline and branched hyphae and mycelia (a and b), 110 measurement of Interseptal distance using a scale bar under light microscope at 100X magnification (c), and Scale bar = 50 µm (a-c) 4.19 Pathogenicity testing of Fusarium isolates: (a) Screen house 120 pot experiment showing characteristic wilt symptoms on lentil germplasm NARC-08-1 and Masoor-93, (b-e) Variation in wilt incidence and severity on NARC-08-1: (b) Highly virulent reaction with wilted and dead plants, (c) Moderately virulent reaction, (d) Low virulent reaction, and (e) Avirulent reaction with healthy plants 4.20 PCR amplification products (700bp) of genomic DNA of 67 128 (a-e) Fusarium isolates using TEF-1α primers 4.21 Phylogenetic tree based on Maximum likelihood analysis 130 generated from the translation elongation factor-1α gene sequences of 67 Fusarium isolates from lentil along with the sequences of GenBank accessions.