Genetic characterization and fungicide resistance profiles of Botrytis cinerea in rooibos nurseries and pear orchards in the Western Cape of South Africa

[摘要] ENGLISH ABSTRACT:Botrytis cinerea Pers. Fr. [teleomorph Botryotinia fuckeliana (de Bary) Whetzel] causesserious losses of over 200 crops worldwide, including rooibos seedlings and pears. Thispathogen is characterized by morphological, physiological and genetic diversity. The geneticdiversity and population structure have not been investigated for B. cinerea populations inSouth Africa. Botrytis cinerea collected from rooibos seedlings and in pear orchards in theWestern Cape of South Africa were investigated in the present study. The study was donewith the aid of microsatellite markers, the amplification of mating type alleles MAT1-1 andMAT1-2 and determination of resistance towards various fungicides. Population dynamicswas inferred and a similar picture emerged in both production systems.Botrytis cinerea annually causes severe losses of rooibos seedlings (Aspalathuslinearis) in nurseries situated in the Clanwilliam region. Sampling was done in five nurseriesand the cryptic species status of the isolates obtained was determined through restrictionenzyme digestion of the Bc-hch gene. All but one (206 out of 207) of the isolates belonged toGroup II or B. cinerea 'sensu stricto'. Analysis of the B. cinerea Group II population, usingseven microsatellite loci, was performed to assess the genetic population structure. Totalgene diversity (H) was high, with a mean of 0.67. Two of the nurseries populations' samplesizes were severely limited after clone correction, yet 100 genotypes were discerned amongthe 206 isolates genotyped. The percentage of maximal genotypic diversity (G) rangedbetween 16 and 68 for the five populations, with a total value of 17 for the 100 genotypes.One genotype, represented by 27 clones, was isolated from four nurseries. Relatively low butsignificant population differentiation was observed in total between nurseries (mean FST =0.030, P = 0.001). The distribution of mating types MAT1-1 and MAT1-2 differed significantlyfrom the ratio of 1:1 for the total population plus two of the nurseries' populations. Threenursery populations had an equal mating type distribution. The index of association (IA)analyses suggests that the populations are asexually reproducing. Analysis of molecularvariance (AMOVA) indicated that 97% of the total genetic variation is distributed withinsubpopulations. Fungicide resistance frequency against iprodione for 198 of the genotypedisolates displayed highly varying levels of resistance amongst the five nurseries. The meantotal incidence of resistance towards iprodione was 43%, ranging from 0% to 81% for the fivenurseries. Baseline sensitivity towards pyrimethanil yielded an average EC50 value of 0.096mg/L.Botrytis cinerea isolates were collected from pear blossoms (Pyrus communis) in fourorchards. Two orchards in the Ceres area and two in the Grabouw area were sampled from.A total of 181 isolates were collected from the four orchards. Incidence of blossom infectionin the orchards ranged from 3% to 17%. Overall, there was a high incidence of isolates thathad only the Boty transposable element (74%) compared to those harbouring both (Boty andFlipper), simultaneously (transposa, 24%). One isolate examined had the Flipper element only. Cryptic species status according to restriction enzyme digestion of the Bc-hch geneindicated that all the isolates belonged to Group II or B. cinerea 'sensu stricto'. Analysis ofthe Group II population, through the use of seven microsatellite loci, was performed toassess the genetic population structure. Total gene diversity (H) was high, with a mean of0.69 across all populations. Although two of the subpopulations displayed a high clonalproportion, overall 91 genotypes were discerned among the 181 isolates. The percentage ofmaximal genotypic diversity (G) ranged between 18 and 33 for the four populations, with atotal value of 14 for the 91 genotypes. One genotype, represented by 27 clones, was isolatedfrom all orchards. Moderate, but significant population differentiation was present in totalamong orchards (mean FST = 0.118, P = 0.001). The distribution of the mating types, MAT1-1and MAT1-2, did not differ significantly from a 1:1 ratio for the total population as well as thesubpopulations. Index of association (IA) analyses, on the other hand, suggests that thepopulations reproduce asexually. Analysis of molecular variance (AMOVA) indicated that88% of the total genetic variation is distributed within subpopulations, 9% betweensubpopulations and only 3% between production areas. Fungicide resistance frequencyagainst fenhexamid, iprodione and benomyl varied, with the highest levels of resistancepresent against benomyl and low levels of resistance seen towards iprodione andfenhexamid.In conclusion, this study has shown that there exist within the studied populations ofB. cinerea, obtained from rooibos nurseries and pear orchards, an adaptive capacity toovercome current means of control. The use of population genetics to further ourunderstanding of how plant pathogens interact and spread throughout a given environment isof cardinal importance in aiding the development of sustainable and integrated managementstrategies. Knowledge of the dispersal of B. cinerea in the two studied cropping systems hasshed light on the inherent risk that it poses, and this together with knowledge of the levels ofresistance that occurs should serve as an early warning to help divert possible loss of controlin future.

[发布机构] Stellenbosch University