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Spatial Analysis of Fungicide Resistance Mutations in Botrytis Spp

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Spatial Analysis of Fungicide Resistance Mutations in Botrytis Spp Spatial analysis of fungicide resistance mutations in Botrytis spp. populations Hervé Van der Heyden Master of Science Department of Plant science, McGill University, Ste-Anne de Bellevue, Québec August 2013 A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Master of Science © Hervé Van der Heyden (2013) Table of contents TABLE OF CONTENTS 2 LIST OF TABLES 5 LIST OF FIGURES 6 ABSTRACT 9 RÉSUMÉ 11 ACKNOWLEDGMENT 13 CONTRIBUTION OF AUTHORS 14 1 GENERAL INTRODUCTION 16 1.1 INTRODUCTION 16 1.2 OBJECTIVES 17 1.3 HYPOTHESES 18 2 LITERATURE REVIEW 19 2.1 SPATIAL STATISTICS 19 2.1.1 DISTANCE-BASED APPROACH 20 2.1.2 QUADRAT-BASED APPROACH 22 2.1.3 GEOSTATISTICAL APPROACH 24 2.2 BOTRYTIS SPP. 25 2.2.1 BOTRYTIS 25 2.2.2 BOTRYTIS CINEREA 25 2.2.3 BOTRYTIS SQUAMOSA 28 2.3 FUNGICIDE RESISTANCES 32 2.3.1 RESISTANCE TO ANTI-MICROTUBULE FUNGICIDES 32 2.3.2 RESISTANCE TO DICARBOXIMIDES AND PHENYLPYRROLES 32 2.3.3 RESISTANCE TO ANILINOPYRIMIDINES 33 2 2.3.4 RESISTANCE TO STEROL BIOSYNTHESIS INHIBITORS 33 2.3.5 RESISTANCE TO QUINONE OUTSIDE INHIBITORS 34 2.3.6 RESISTANCE TO SUCCINATE DEHYDROGENASE INHIBITORS 34 2.4 CONNECTING TEXT FOR CHAPTER 3 35 3 COMBINED APPLICATION OF SPATIAL STATISTICS AND MOLECULAR GENETICS FOR CHARACTERIZATION OF SMALL-SCALE SPATIAL RELATIONSHIPS BETWEEN POLYMORPHISMS RELATED TO FUNGICIDE RESISTANCE IN BOTRYTIS CINEREA POPULATIONS 36 3.1 ABSTRACT 36 3.2 INTRODUCTION 37 3.3 MATERIAL AND METHODS 42 3.3.1 INVENTORY OF PHENOTYPIC FUNGICIDE RESISTANCE 42 3.3.2 FUNGICIDES AND CONIDIAL GERMINATION ASSAY 42 3.3.3 INCIDENCE OF SNPS RELATED TO FUNGICIDE RESISTANCE 43 3.3.4 DNA EXTRACTION AND PCR ANALYSIS 43 3.3.5 UNIVARIATE SPATIAL ANALYSIS 44 3.3.6 BIVARIATE SPATIAL ANALYSIS 47 3.4 RESULTS 49 3.5 DISCUSSION 51 3.6 ACKNOWLEDGEMENTS 56 3.7 CONNECTING TEXT FOR CHAPTER 4 67 4 A NOVEL PCR-RFLP ASSAY FOR THE DETECTION OF A SINGLE NUCLEOTIDE POLYMORPHISM RELATED TO DICARBOXIMIDE RESISTANCE IN BOTRYTIS SQUAMOSA FIELD ISOLATES 68 4.1 ABSTRACT 68 4.2 INTRODUCTION 68 4.3 MATERIAL AND METHODS 70 3 4.4 RESULTS 71 4.5 DISCUSSION 72 4.6 ACKNOWLEDGEMENTS 72 4.1 CONNECTING TEXT FOR CHAPTER 5 76 5 SPATIAL DISTRIBUTION OF SINGLE NUCLEOTIDE POLYMORPHISMS RELATED TO FUNGICIDE RESISTANCE AND IMPLICATIONS FOR SAMPLING 77 5.1 ABSTRACT 77 5.2 INTRODUCTION 78 5.3 MATERIALS AND METHODS 82 5.3.1 SAMPLING PROTOCOLS 82 5.3.2 DNA EXTRACTION AND RFLP-PCR 83 5.3.3 GEOSTATISTICAL ANALYSES 85 5.3.4 DISTRIBUTIONAL ANALYSIS 87 5.3.5 SAMPLING CURVES 89 5.4 RESULTS 90 5.4.1 SNP INCIDENCE 90 5.4.2 GEOSTATISTICAL ANALYSES 91 5.4.3 SPATIAL STATISTICS 91 5.4.4 SAMPLING CURVES 92 5.5 DISCUSSION 93 5.6 ACKNOWLEDGEMENTS 98 6 GENERAL CONCLUSION AND CONSIDERATION FOR FUTURE RESEARCH 108 6.1 GENERAL CONCLUSION 108 6.2 CONSIDERATION FOR FUTURE RESEARCH 110 7 LITERATURE CITED 111 4 List of tables TABLE 1: LIST OF FUNGICIDE GROUPS AND ACTIVE INGREDIENTS AND DISCRIMINANT DOSE USED FOR THE PHENOTYPIC ASSAY. ........................................ 57 TABLE 2: CHEMICAL GROUPS, ACTIVE INGREDIENTS AND SEQUENCES OF THE PRIMERS AND PROBES USED FOR THE DETECTION OF THE MUTANT ALLELES ASSOCIATED WITH FUNGICIDE RESISTANCE. ....................................................................... 58 TABLE 3: INDEX OF DISPERSION D AND PARAMETERS ESTIMATE OF THE BETA- BINOMIAL AND BINOMIAL DISTRIBUTION. ............................................................................. 59 TABLE 4: AMINO ACID SUBSTITUTIONS OF BCOS-1 HOMOLOGOUS IN 18 SINGLE- SPORE ISOLATES OF BOTRYTIS SQUAMOSA USED FOR DEVELOPMENT. ............... 73 TABLE 5: RESULTS OF CRAD ANALYSIS (COREGIONALIZATION ANALYSIS WITH A DRIFT) AND VARIOGRAM MODEL PARAMETER ESTIMATES (RANGE, NUGGET EFFECT, SILL OF SPHERICAL STRUCTURE) FOR SNP DISTRIBUTIONS WITHIN B. CINEREA AND B. SQUAMOSA POPULATIONS IN TWO VINEYARDS AND TWO ONION FIELDS SAMPLED IN 2009-2011. ........................................................................................ 99 TABLE 6: OBSERVED VALUES OF THE INDEX OF DISPERSION AND PARAMETER ESTIMATES FOR THE BETA-BINOMIAL AND BINOMIAL DISTRIBUTIONS ........ 102 TABLE 7: MINIMUM SAMPLE SIZE (N) REQUIRED TO ESTIMATE THE MEAN SNP INCIDENCE (P) FOR LOW, INTERMEDIATE AND HIGH VALUES OF THE LOCAL AGGREGATION INDEX (Θ) AND INCREASING VALUES OF THE CV (EXCERPTS FROM FIGURE 3; SEE TEXT FOR DETAILS) ............................................................................. 104 5 List of figures FIGURE 1: SCHEMATIC REPRESENTATION OF THE THREE POSSIBLE OUTCOMES IN THE ANALYSIS OF INTER-SNP DISTANCES A) ABSENCE OF A SPATIAL RELATIONSHIP, B) EXCLUSIVE RELATIONSHIP AND C) INCLUSIVE RELATIONSHIP. THE CONTINUOUS LINE REPRESENTS THE OBSERVED CUMULATIVE RELATIVE FREQUENCY DISTRIBUTION, WHILE THE DASHED LINES REPRESENT THE 2.5TH- AND 97.5TH-PERCENTILE ENVELOPES. ...................... 60 FIGURE 2: PROPORTION OF RESISTANT ISOLATES TO SIX FUNGICIDES OBTAINED IN A CONIDIAL GERMINATION ASSAY FOR 232 BOTRYTIS CINEREA SAMPLES, COLLECTED FROM 23 PLOTS IN 18 VINEYARDS FROM DIFFERENT PRODUCTION AREAS IN THE PROVINCE OF QUÉBEC. ...................................................... 61 FIGURE 3: A) FREQUENCY DISTRIBUTION OF THE SINGLE NUCLEOTIDE POLYMORPHISM (SNPS) FOUND IN TWO VINEYARDS IN 2011 AND B) NUMBER OF SNPS PER ISOLATES. GREY BARS: FIRST FIELD; BLACK BARS: SECOND FIELD. ................................................................................................................................................................ 62 FIGURE 4: UNIVARIATE 2-D SPATIAL POINT PATTERNS OBSERVED ON THE FIRST FIELD. A POINT INDICATES THE PRESENCE OF A SNP FOR MUTATION G143A (A), H272R (B), DAF1 (C), H272Y (D), N230I (E), F412I (F), H272L (G), G143A- INTRON (H) AND F412S (I), RESPECTIVELY. .............................................................................. 63 FIGURE 5: UNIVARIATE 2-D SPATIAL POINT PATTERNS OBSERVED ON THE SECOND FIELD. A POINT INDICATES THE PRESENCE OF A SNP FOR MUTATION G143A (A), H272R (B), DAF1 (C), H272Y (D), H272L (E) AND F412S (F), RESPECTIVELY. 64 FIGURE 6: BIVARIATE ANALYSIS OF THE 2-D SPATIAL POINT PATTERNS OF FIGURE 4. THE CONTINUOUS LINES REPRESENT THE OBSERVED CUMULATIVE RELATIVE FREQUENCY DISTRIBUTIONS OF INTER-SNP DISTANCES, WHILE THE DASHED LINES REPRESENT THE 2.5TH- AND 97.5TH-PERCENTILE ENVELOPES. .................................................................................................................................................. 65 6 FIGURE 7: BIVARIATE ANALYSIS OF THE 2-D SPATIAL POINT PATTERNS OF FIGURE 5. THE CONTINUOUS LINES REPRESENT THE OBSERVED CUMULATIVE RELATIVE FREQUENCY DISTRIBUTIONS OF INTER-SNP DISTANCES, WHILE THE DASHED LINES REPRESENT THE 2.5TH- AND 97.5TH-PERCENTILE ENVELOPES. .................................................................................................................................................. 66 FIGURE 8: PREDICTED AMINO-ACID PARTIAL SEQUENCE OF B. SQUAMOSA BCOS-1 HOMOLOGOUS, ALIGNED WITH BCOS-1 OF B. CINEREA. THE ASTERISK (*) AT POSITION 210 INDICATES THE I86S SUBSTITUTION SITE AND THE DOTS (.) REPRESENT GAPS INTRODUCED TO MAXIMIZE ALIGNMENT. .................................... 74 FIGURE 9: VALIDATION RESULTS OF THE PCR-RFLP ASSAY USING 24 SINGLE- COLONY ISOLATES. THE PRESENCE OF TWO FRAGMENTS OF 400 AND 160 BP FOLLOWING DIGESTION WITH TAQΑ1 INDICATES THE ABSENCE OF THE I86S MUTATION. THE PRESENCE OF AN ENTIRE 560-BP FRAGMENT IMPLIES THE PRESENCE OF THE I86S MUTATION. WILD-TYPE STRAINS SENSITIVE TO IPRODIONE ARE IN LANES 1 TO 12, 13, 15 TO 17, AND 24, WHILE MUTANT STRAINS RESISTANT TO IPRODIONE ARE IN LANES 14 AND 18 TO 23. THE PCR NEGATIVE CONTROL IS IN LANE N. .............................................................................................. 75 FIGURE 10: RAW COUNT DATA MAPS FOR B. CINEREA IN VINEYARD OR FOR A) H272R (OBSERVED MEAN INCIDENCE, 0.68, ± 0.023, STANDARD ERROR), B) H272Y (0.21 ± 0.021), AND C) I86S (0.55 ± 0.024), AND IN VINEYARD TE FOR E) H272R (0.61 ± 0.021) F) H272Y (0.20 ± 0.018), AND G) I86S (0.59 ± 0.020), AND FOR B. SQUAMOSA FOR I86S HOMOLOGOUS IN D) 2009 (0.16 ± 0.020) AND H) 2010 (0.42 ± 0.025). .............................................................................................................................................................. 105 FIGURE 11: FREQUENCY DISTRIBUTION OF THE OBSERVED NUMBER OF BOTRYTIS ISOLATES CARRYING A GIVEN SNP PER QUADRAT (EMPTY BARS), TOGETHER WITH THE EXPECTED NUMBERS OF BOTRYTIS ISOLATES CARRYING THE SNP PER QUADRAT UNDER THE BETA-BINOMIAL DISTRIBUTION MODEL (FILLED BARS) AND THE BINOMIAL DISTRIBUTION MODEL (DASHED BARS), FOR B. 7 CINEREA IN VINEYARD OR FOR A) H272R, B) H272Y AND C) I86S, IN VINEYARD TE FOR E) H272R, F) H272Y AND G) I86S, AND FOR B. SQUAMOSA FOR I86S IN D) 2009 AND H) 2010. .................................................................................................................................... 106 FIGURE 12: SAMPLING CURVES CALCULATED UNDER THE ASSUMPTION OF A BETA-BINOMIAL DISTRIBUTION, USING LOW, INTERMEDIATE AND HIGHER VALUES OF THE PARAMETER Θ THAT CORRESPOND TO THE MINIMUM, MEDIAN AND MAXIMUM VALUES OF Θ -ESTIMATES OBTAINED IN OUR STUDY, FOR THREE LEVELS OF PRECISION GIVEN BY CV VALUES OF 10%, 20% AND 30%. THE CONTINUOUS LINE REPRESENTS THE MINIMUM SAMPLE SIZE REQUIRED TO ESTIMATE MEAN SNP INCIDENCE WITH A CV OF 10%; THE DOTTED LINE, WITH A CV OF 20%; AND THE DASHED LINE, WITH A CV OF 30%. ............................................................................................................................................................................ 107 8 Abstract
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