INCIDENCE AND CHARACTERIZATION OF MAJOR FUNGAL PATHOGENS OF STRAWBERRY DISEASES NASIR MEHMOOD 06-arid-109 Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018 INCIDENCE AND CHARACTERIZATION OF MAJOR FUNGAL PATHOGENS OF STRAWBERRY DISEASES by NASIR MEHMOOD (06-arid-109) A thesis submitted in the 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 2018 ivi v vi vii viiiii “IN THE NAME OF ALLAH, THE MOST BENEFICENT AND MERCIFUL” DEDICATION This Humble Effort Is Dedicated To “My Affectionate and Loving Parents” Who always Sacrifice For Me In Every Moment Of Their Life My Loving & Friendly “Brothers, Sister, Nephews and Nieces” Who Are Always A Source Of Happiness, Supports And Backup For Me to achieve my goals. “May Their Hands Ever Praying for Me These Hands may never fall down” iv CONTENTS List of Tables v List of Figures vi List of Abbreviations viii Acknowledgements x ABSTRACT 1 1. INTRODUCTION 3 2. REVIEW OF LITERATURE 10 2.1 STRAWBERRY HISTROY AND IMPORTANCE 10 2.2 MAJOR STRAWBERRY FUNGAL PATHOGENS 11 2.3 Botrytis cinerea (BOTRYTIS FRUIT ROT) 11 2.4 Colletotrichum acutatum AND C. gloeosporioides 15 (ANTHRACNOSE FRUIT ROT) 2.5 Alternaria alternata (ALTERNARIA LEAF SPOT) 19 2.6 Fusarium solani (FUSARIUM FRUIT ROT) 22 2.7 MOLECULAR TOOLS 25 3. MATERIALS AND METHODS 28 3.1 DISEASE SURVEY AND ASSESMENT 28 3.1.1 Survey of Strawberry Fields 28 3.1.2 Field Based Diseases Assessments and Sample Collection 29 3.2 ISOLATION AND PURIFICATION OF PATHOGENS 30 3.3 PRESERVATION OF PURIFIED FUNGAL ISOLATES 31 3.4 PATHOGENICITY TEST 34 3.5 MORPHOLOGICAL CHARACTERIZATION OF FUNGAL 35 v ISOLATES 3.6 MOLECULAR CHARACTERIZATION 36 3.6.1 Genomic DNA Extraction from Fungal Pathogens 36 3.6.2 Polymerase Chain Reaction (PCR) Assay 37 3.6.2.1 Amplification of different gene regions 37 3.6.2.2 PCR reaction mixture 40 3.6.2.3 PCR conditions 40 3.6.3 Agarose Gel Electrophoresis 40 3.6.4 PCR Product Purification 42 3.7 DNA SEQUENCING 42 3.7.1 Sequence Processing and Submission 42 4. RESULTS AND DISCUSSION 44 4.1 DISEASE SURVEY AND ASSESSMENT 44 4.1.1 Prevalence Percentages of Major Diseases 44 4.1.2 Incidence Percentages of Major Diseases 53 4.2 COLLECTION OF DISEASED SAMPLES 61 4.3 ISOLATION, IDENTIFICATION AND PATHOGENICITY 61 4.4 PRESERVATION OF FUNGAL ISOLATES 62 4.5 MORPHO-MOLECULAR CHARACTERIZATION OF 67 FUNGAL PATHOGENS 4.6 MORPHO-MOLECULAR CHARACTERIZATION OF 69 Alternaria alternata 4.6.1 Cultural and Morphological Characterization 69 4.6.1.1 Colony color 69 vi 4.6.1.2 Growth habit 69 4.6.1.3 Colony margins and color 69 4.6.1.4 Concentric rings 69 4.6.1.5 Conidia: color, shape and size 70 4.6.2 Molecular Characterization of A. alternata 79 4.7 MORPHO-MOLECULAR CHARACTERIZATION OF 82 Fusarium solani 4.7.1 Cultural and Morphological Characterization 82 4.7.1.1 Colony color 82 4.7.1.2 Growth habit 85 4.7.1.3 Pigmentation 85 4.7.1.4 Conidiophore and Phialide 85 4.7.1.5 Microconidia shape and size 86 4.7.1.6 Macroconidia shape and size 86 4.7.1.7 Macroconidia apical and basal cells shape 87 4.7.1.8 Macroconidia septation 87 4.7.1.9 Chlamydospores formation and size 88 4.7.2 Molecular Characterization of Fusarium solani 104 4.8 MORPHO-MOLECULAR CHARACTERIZATION OF 104 Colletotrichum spp. 4.8.1 Cultural and Morphological Characterization 104 4.8.1.1 Colony color 104 4.8.1.2 Growth habit 104 4.8.1.3 Pigmentation 105 vii 4.8.1.4 Conidia: shape, size and color 105 4.8.1.5 Appressoria: shape, size and color 106 4.8.1.6 Setae: shape, size and color 106 4.8.2 Molecular Characterization of Colletotrichum spp. 119 4.9 MORPHO-MOLECULAR CHARACTERIZATION OF 124 Botrytis cinerea 4.9.1 Cultural and Morphological Characterization 124 4.9.1.1 Colony color 124 4.9.1.2 Growth habit 124 4.9.1.3 Sclerotia color, diameter and formation 125 4.9.1.4 Conidiophore color and length 126 4.9.1.5 Shape, size and colour of conidia 127 4.9.2 Molecular Characterization of Botrytis cinerea 138 SUMMARY 144 LITERATURE CITED 150 viii LIST OF TABLES Table No. Page 3.1 Important strawberry growing area surveyed during crop seasons 33 2014-15 & 2015-16 3.2 Gene/Gene Region, Primers used with detail and PCR conditions 41 and references 4.1 Disease Prevalence Percentage of 4 major fungal diseases in 48 surveyed locations from 12 Districts during crop seasons 2014- 15 and 2015-16 4.2 Disease Incidence Percentage of 4 major fungal diseases in 56 surveyed locations from 12 Districts during crop seasons 2014- 15 and 2015-16 4.3 Alternaria alternata isolates ID with culture identification and 73 morphological characterization 4.4 Details of Alternaria alternata isolates and Accession number 83 with ITS and EndoPG primers used in molecular study 4.5 Fusarium solani isolates ID with culture identification and 91 morphological characterization 4.6 Details of Fusarium solani isolates and Accession number with 102 ITS and TEF-1α primers used in molecular study 4.7 Colletotrichum spp. isolates ID with culture identification and 110 morphological characterization 4.8 Details of Colletotrichum acutatum isolates and Accession 122 number with ITS and Bt primers used in molecular study 4.9 Details of Colletotrichum gloeosporioides isolates and 122 Accession number with ITS and Bt primers used in molecular study 4.10 Botrytis cinerea isolates ID with culture identification and 131 morphological characterization 4.11 Details of Botrytis cinerea isolates and Accession number with 142 ITS and G3PDH primers used in molecular study ix LIST OF FIGURES Fig. No. Page 3.1 Surveyed districts with number of fields visited for major 32 fungal diseases of strawberry (2014-15 & 2015-16) 3.2 Diagrammatic representation of Steps of DNA Extraction 38 using PrepMan Ultra Sample Preparation Reagent 3.3 Schematic diagrams indicating genes regions and the primer 39 positions (a) Inter transcribed space region (ITS), (b) Translation elongation factor (TEF-1α), (c) Beta-tubulin (BT) (d) endopolygalacturonase (endoPG) and (e) Glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) 4.1 During survey across different districts: (a) Sialkot, (b) 45 Mardan, (c) Multan (d) Islamabad 4.2 Field samples collected during survey ofr fungal diseases: 63 (a) Alternaria leaf spot (ALS), (b) Fusarium fruit rot (FFR) (c) Anthracnose fruit rot (AFR) and (d) Botrytis fruit rot (BFR) 4.3 Pathogenicity test performed on the respective strawberry 64 plant part from where the fungal pathogen was isolate: (a) Alternaria alternata on leaf (b) Fusarium solani on fruit (c) Colletotrichum spp. on fruits (d) Botrytis cinerea on fruit 4.4 Preserved fungal cultures in skimmed milk and silica gel (a) 66 Glass vials (b) Eppendorf tubes 4.5 Revival of pure fungal colonies from preserved cultures in 66 silica gel 4.6 Alternaria alternata morphological characters: Colony 72 color and margins (a) Light brown, irregular (b) Dark brown, regular (c) Olivaceous black, regular (d) Reverse color (e) Spores showing longitudinal and transverse septation (d) Conidia in short chains 4.7 Molecular phylogenetic tree inferred by using Maximum 84 Likelihood analysis from (a) ITS and (b) EndoPG gene sequences of 12 representative A. alternata isolates x 4.8 Fusarium solani Colony color and Growth habit (a) Cottony 89 & fluffy white (b) White & flat (c) Creamy white & compact; Pigmentation (d) Colorless, (e) Pale yellow 4.9 Fusarium solani morphological characters: (a) Long 90 Monophialides (b) Long & branched Monophialides (c) Microconidia (oval and reniform) and Macroconidia (Fusiform curved and sepatation) (d) Fusiform straight & septation; Formation of chlamydospores (e) Singly (f) Singly & paired 4.10 Molecular phylogenetic tree inferred by using Maximum 103 Likelihood analysis from (a) ITS and (b) TEF-1α gene sequences of 11 representative F. solani isolates 4.11 Colletotrichum acutatum cultural and morphological 108 characters: Colony color and Growth habit (a) Creamy white (b) Pale yellowish; Pigmentation (c) Reddish yellow (d) Salmon (e) Elliptic-fusiform conidia (f) Ovate appressoria 4.12 Colletotrichum gloeosporioides cultural & morphological 109 characters: Colony color and Growth habit (a) Gray & dense aerial mycelium (b) Grayish white; Pigmentation (c) Grayish (d) Pinkish (e) Oblong, obtuse ends conidia (f) Setae (g) Irregular appressoria 4.13 Molecular phylogenetic tree inferred by using Maximum 123 Likelihood analysis from (a) ITS and (b) BT gene sequences of 19 representative Colletotrichum spp. 4.14 Botrytis cinerea morphological characters: Colony color 130 and Growth habit (a) Dark grey (b) Cloudy white (c & d) Sclerotia formation (e) Conidiophore with conidia in grape bunch shape, (d) Conidia of lemon or pear shaped 4.15 Molecular phylogenetic tree inferred by using Maximum 143 Likelihood analysis from (a) ITS and (b) G3PDH gene sequences of 12 representative B. cinerea isolates xi List of Abbreviations % Percentage @ At the rate ± Plus Minus ° Degree °C Centigrade µg Microgram µL Micro Liter AFR Anthracnose Fruit Rot ALS Alternaria Leaf Spot BFR Botrytis Fruit Rot BLAST Basic Local Alignment Search Tool Bp Base pairs BS Boot Strape BT/TUB2 Beta Tubulin DNA Deoxyribose nucleic acid PmoL Peco Mole DEPC Diehtylpyrocarbonate EDTA Ethylenediaminetetraacetic acid EndoPG Endo-polygalacturonase et al., And others FAO Food and Agriculture Organization FFR Fusarium Fruit Rot G3PDH Glyceraldehyde 3-phosphate dehydrogenase xii GM Gray Mold Ha Hectare ICT Islamabad Capital Territory ITS Internal Transcribed Spacer Region ISU Iowa State University KPK Khyber Pakhtunkhwa L Liter M Molar MgCl2 Magnesium Chloride Mins Minutes Mm Millimeter NCBI National Center for Biotechnological Information PCR Polymerase Chain Reaction PDA Potato Dextrose Agar PMAS-AAUR Pir Mehr Ali Shah Arid Agriculture University Rawalpindi PSI Pound-force per square inch rDNA Ribosomal DNA rpm Revolutions per minute spp.