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Surveillance and Pathogen Characterization Of SURVEILLANCE AND PATHOGEN CHARACTERIZATION OF BACTERIAL CANKER OF STONE FRUITS RAEES AHMED 10-arid-1770 Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018 2 SURVEILLANCE AND PATHOGEN CHARACTERIZATION OF BACTERIAL CANKER OF STONE FRUITS by RAEES AHMED (10-arid-1770) A thesis submitted in partial fulfillment of the requirement 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 3 4 5 6 7 CONTENTS Page List of Tables ix List of Figures x List of Appendices xiv List of Abbreviations Xv Acknowledgements Xvii ABSTRACT xix 1. INTRODUCTION 1 1.1 STONE FRUITS 1 1.2 NUTRITIONAL VALUE OF STONE FRUITS 3 1.3 STONE FRUITS IN WORLD 3 1.4 STONE FRUITS IN PAKISTAN 4 1.5 DISEASES OF STONE FRUITS 6 1.6 BACTERIAL CANKER OF STONE FRUITS 7 2. REVIEW OF LITERATURE 13 2.1 BACTERIAL CANKER OF STONE FRUITS 13 2.2 BIOCHEMICAL CHARACTERIZATION 14 2.3 MOLECULAR CHARACTERIZATION 15 3. MATERIALS AND METHODS 17 3.1 COLLECTION OF SAMPLES 17 3.2 DETERMINATION OF DISEASE INCIDENCE AND 17 DISEASE PREVALENCE 8 3.3 ISOLATION AND PURIFICATION OF PATHOGENIC 18 BACTERIA FROM DISEASES SAMPLES 3.4 INITIAL SCREENING OF BACTERIAL CULTURES 18 3.4.1 Gram Staining 18 3.4.2 KOH (Loop) Test 19 3.4.3 Florescent Under UV Light 19 3.5 PRESERVATION OF CULTURE 19 3.5.1 Preservation in Glycerol 19 3.5.2 Preservation in Mineral Oil 20 3.6 PATHOGENICITY TESTS 20 3.7 SYRINGOMYCIN BIOASSAY 20 3.8 BIOCHEMICAL CHARACTERIZATION OF BACTERIAL 21 CANKER PATHOGENS 3.8.1 Levan Production Test 21 3.8.2 Oxidase Test 21 3.8.3 Potato Soft Rot Test 22 3.8.4 Arginine Dihydrolase Test 22 3.8.5 Hypersensitivity Test 22 3.8.6 Gelatine Hydrolysis Test 23 3.8.7 Aesculin Hydrolysis Test 23 3.8.8 Tyrosinase Activity 23 9 3.8.9 Tartrate Utilization Test 23 3.8.10 L-lactate Utilization Test 24 3.9 MOLECULAR CHARACTERIZATION AND GENETIC 24 VARIABILITY 3.9.1 Primers Used in Study 24 3.9.2 Extraction of Genomic DNA 24 3.9.3 Plasmid Extraction 25 3.9.4 PCR Amplification 26 3.9.4.1 PCR for 16S rRNA 26 3.9.4.2 PCR for virB1 and virD4 Genes 27 3.9.4.3 PCR for gyrB Gene 27 3.9.5 PCR Purification 28 3.9.6 DNA Clean and Concentration 29 3.9.7 Quantification of Purified PCR Product 29 3.9.8 Sequencing and Phylogenetic Analysis 29 4 RESULTS AND DISCUSSION 32 4.1 SURVEYS FOR DISEASE INCIDENCE 32 4.2 DISEASE INCIDENCE AND DISEASE PREVALENCE OF 32 BACTERIAL CANKER OF STONE FRUITS 4.2.1 Punjab Province 32 4.2.2 KPK Province 37 10 4.3 ISOLATION AND PURIFICATION 48 4.4 INITIAL SCREENING OF BACTERIAL ISOLATES 48 4.5 PATHOGENICITY TEST 54 4.6 SYRINGOMYCIN BIOASSAY 58 4.7 BIOCHEMICAL CHARACTERIZATION OF 59 PATHOGENIC ISOLATES 4.8 MOLECULAR CHARACTERIZATION 71 4..8.1 16S rRNA Gene 71 4.8.2 gyrB Gene 72 4.8.3 virB1 and virD4 Gene 73 SUMMARY 82 CONCLUSIONS 85 FUTURE DIRECTIONS 86 LITERATURE CITED 87 APPENDICES 96 11 List of Tables Table No. Page 1 Nutritional values of some stone fruits 8 2 According to FAOSTAT, 2014 stone fruits production tons 8 3 According to FAOSTAT, 2014 stone fruits production area in hectares 8 4 Primer sequence used for PCR analysis 31 5 Screened isolates with their labeled name, their host and isolation 64 source 6 Pathogenicity of bacterial isolates on fresh peach fruits using pin 69 prick method 7 Syringomycin bioassay of pathogenic bacterial isolates 75 8 LOPAT scheme for the identification of P. syringae according to 79 Lelliott and Stead (1987) 9 GATTa and Lactate test for the confirmation of pathovars in P. 82 syringae according to Lelliott and Stead (1987) 12 List of Figures Figure No. Page 1 Map of Pakistan showing Khyber-Pakhtunkhwa, Punjab 9 and Baluchistan Provinces 2 Map showing districts of Province Baluchistan, Pakistan 9 3 Map showing districts of KPK Province and Northern 10 areas of Pakistan 4 Doing molecular work on thermocycler, gel appratus, 31 gel documentation system and nano drop 5 Disease incidence of bacterial canker on peach, apricot 34 and plum in district Attock in year 2015. 6 Disease incidence of bacterial canker on peach, apricot 34 and plum in district Attock in year 2016. 7 Disease incidence of bacterial canker on peach, apricot and 35 plum in district Rawalpindi in year 2015. 8 Disease incidence of bacterial canker on peach, apricot and 35 plum in district Rawalpindi in year 2016. 9 Disease incidence of bacterial canker on peach, apricot 36 and plum in district Khushab in year 2015. 10 Disease incidence of bacterial canker on peach, apricot 36 and plum in district Khushab in year 2016. 11 Disease incidence of bacterial canker on peach, apricot 38 and plum in district Abbottabad in year 2015. 12 Disease incidence of bacterial canker on peach, apricot and 38 13 plum in district Abbottabad in year 2016. 13 Disease incidence of bacterial canker on peach, apricot 39 and plum in district Mansehra (Tehsil Mansehra) in year 2015. 14 Disease incidence of bacterial canker on peach, apricot 39 and plum in district Mansehra (Tehsil Mansehra) in year 2016. 15 Disease incidence of bacterial canker on peach, apricot 40 and plum in district Mansehra (Tehsil Oghi) in year 2015. 16 Disease incidence of bacterial canker on peach, apricot 40 and plum in district Mansehra (Tehsil Oghi) in year 2016. 17 Disease incidence of bacterial canker on peach, apricot and 41 plum in district Mansehra (Tehsil Balakot) in year 2015. 18 Disease incidence of bacterial canker on peach, apricot and 41 plum in district Mansehra (Tehsil Balakot) in year 2016. 19 Disease incidence of bacterial canker on peach, apricot and 44 plum in district Swat in year 2015. 20 Disease incidence of bacteria canker on peach, apricot and 44 plum in district Swat in year 2016. 21 Disease incidence of bacterial canker on peach, apricot 45 and plum in district Peshawar in year 2015. 22 Disease incidence of bacterial canker on peach, apricot 45 and plum in district Peshawar in year 2016. 23 Disease incidence of bacterial canker on peach, apricot and 46 plum in district Nowshera in year 2015. 24 Disease incidence of bacterial canker on peach, apricot and 46 14 plum in district Nowshera in year 2016. 25 Peach Orchards with clear symptom of gummosis on 47 stem 26 Apricot fruits with bacterial canker symptoms collected 47 from field 27 Isolation of bacteria on nutrient agar media 53 28 Florescence of bacterial isolates under UV light 53 29 Formation of loop in KOH test 53 30 Disinfection of fruits with 50% ethanol 57 31 Inoculation of bacterium using pin prick method 57 32 Formation of canker symptoms after 4-5 days of incubation 57 33 Re-isolation of pathogen from developed symptoms 57 34 Syringomycin bioassay showing zone of inhibition 60 35 Inoculation of bacterial isolate into the leaf of tobacco plant 64 36 Hypersensitive response on tobacco plant 64 37 Phylogenetic tree constructed using maximum likelihood 74 method of 32 Pseudomonas syringae pv. syringae isolates from stone fruits using 16S rRNA primers 38 Phylogenetic tree constructed using maximum likelihood 75 method of 11 Pseudomonas syringae pv. morsprunorum isolates from stone fruits using 16S rRNA primers 39 Phylogenetic tree constructed using maximum likelihood 76 method of Pseudomonas syringae isolates from stone fruits using 16S rRNA primers with other isolates submitted in NCBI from the world 40 Phylogenetic tree of 8 Pss and 2 Psm isolates constructed 78 using gyrB primer 15 41 Dendrogram of P. syringae pv. syringae isolates with 79 other isolates collected from NCBI data base using gyrB primers 42 Amplified PCR product with approximately 1453 bp 80 fragments using virD4 primers and Plasmid DNA was used as template 43 Amplified PCR product with approximately 513 bp 81 fragments using virB1 primers and Plasmid DNA was used as template 16 List of Appendices Appendix No. Page 1 Preparation of Nutrient Agar (NA) Medium 96 2 Preparation of Potato Dextrose Agar (PDA) 96 Medium 3 Preparation of King’s B (KB) Medium 96 4 Preparation of Levan Media 97 5 Preparation of Oxidase Solution 97 6 Preparation of Arginine dihydrolase Solution 97 7 Preparation of Gelatine Media 98 8 Preparation of Aesculin Media 98 9 Preparation of Tyrosinase Media 98 10 Preparation of L-Tartrate Media 99 11 Aligned Sequences obtained from NCBI 100 17 List of Acronyms µl Micro liter DNA Deoxyribose Nucleic Acid CFU Colony Forming Units cm Centimeter d Days Fe Iron Pss Pseudomonas syringae pv. syringae Psm Pseudomonas syringae pv. morsprunorum g/gm Gram h Hour L Liter L. Linnaeus NA Nutrient Agar lbs Pounds M Molar min Minute ml/mL Milliliter mM Millimolar 18 KB King’s B Media NB Nutrient Broth oC Degree Celsius P Phosphorus PCR Polymerase Chain Reaction psi Per Square Inch RNA Ribose Nucleic Acid rpm Revolution Per Minute RT-qPCR Real Time Quantitative Polymerase Chain Reaction vol. Volume wt. Weight DI Disease Incidence MEGA Molecular Evolutionary Genetic Analysis 19 ACKNOWLEDGEMENTS Allah Almighty, the eternal of this universe. The most Beneficent, The Merciful, The Gracious and The Compassionate whose bounteous blessings gave me potential, thoughts, talented teachers, helping friends and opportunity to make this humble effort and enabled me to pursue and perceive higher ideas of life.
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