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

Wessels, Andries Bernardus (2012-03)

Thesis (MScAgric)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Botrytis cinerea Pers. Fr. [teleomorph Botryotinia fuckeliana (de Bary) Whetzel] causes serious losses of over 200 crops worldwide, including rooibos seedlings and pears. This pathogen is characterized by morphological, physiological and genetic diversity. The genetic diversity and population structure have not been investigated for B. cinerea populations in South Africa. Botrytis cinerea collected from rooibos seedlings and in pear orchards in the Western Cape of South Africa were investigated in the present study. The study was done with the aid of microsatellite markers, the amplification of mating type alleles MAT1-1 and MAT1-2 and determination of resistance towards various fungicides. Population dynamics was inferred and a similar picture emerged in both production systems. Botrytis cinerea annually causes severe losses of rooibos seedlings (Aspalathus linearis) in nurseries situated in the Clanwilliam region. Sampling was done in five nurseries and the cryptic species status of the isolates obtained was determined through restriction enzyme digestion of the Bc-hch gene. All but one (206 out of 207) of the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the B. cinerea Group II population, using seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.67. Two of the nurseries populations’ sample sizes were severely limited after clone correction, yet 100 genotypes were discerned among the 206 isolates genotyped. The percentage of maximal genotypic diversity (G) ranged between 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 but significant 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 significantly from the ratio of 1:1 for the total population plus two of the nurseries’ populations. Three nursery populations had an equal mating type distribution. The index of association (IA) analyses suggests that the populations are asexually reproducing. Analysis of molecular variance (AMOVA) indicated that 97% of the total genetic variation is distributed within subpopulations. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates displayed highly varying levels of resistance amongst the five nurseries. The mean total incidence of resistance towards iprodione was 43%, ranging from 0% to 81% for the five nurseries. Baseline sensitivity towards pyrimethanil yielded an average EC50 value of 0.096 mg/L. Botrytis cinerea isolates were collected from pear blossoms (Pyrus communis) in four orchards. 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 infection in the orchards ranged from 3% to 17%. Overall, there was a high incidence of isolates that had only the Boty transposable element (74%) compared to those harbouring both (Boty and Flipper), simultaneously (transposa, 24%). One isolate examined had the Flipper element only. Cryptic species status according to restriction enzyme digestion of the Bc-hch gene indicated that all the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the Group II population, through the use of seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Although two of the subpopulations displayed a high clonal proportion, overall 91 genotypes were discerned among the 181 isolates. The percentage of maximal genotypic diversity (G) ranged between 18 and 33 for the four populations, with a total value of 14 for the 91 genotypes. One genotype, represented by 27 clones, was isolated from all orchards. Moderate, but significant population differentiation was present in total among orchards (mean FST = 0.118, P = 0.001). The distribution of the mating types, MAT1-1 and MAT1-2, did not differ significantly from a 1:1 ratio for the total population as well as the subpopulations. Index of association (IA) analyses, on the other hand, suggests that the populations reproduce asexually. Analysis of molecular variance (AMOVA) indicated that 88% of the total genetic variation is distributed within subpopulations, 9% between subpopulations and only 3% between production areas. Fungicide resistance frequency against fenhexamid, iprodione and benomyl varied, with the highest levels of resistance present against benomyl and low levels of resistance seen towards iprodione and fenhexamid. In conclusion, this study has shown that there exist within the studied populations of B. cinerea, obtained from rooibos nurseries and pear orchards, an adaptive capacity to overcome current means of control. The use of population genetics to further our understanding of how plant pathogens interact and spread throughout a given environment is of cardinal importance in aiding the development of sustainable and integrated management strategies. Knowledge of the dispersal of B. cinerea in the two studied cropping systems has shed light on the inherent risk that it poses, and this together with knowledge of the levels of resistance that occurs should serve as an early warning to help divert possible loss of control in future.

AFRIKAANSE OPSOMMING: Botrytis cinerea Pers. Fr. [teleomorf Botryotinia fuckeliana (de Bary) Whetzel] veroorsaak ernstige verliese van meer as 200 gewasse wêreldwyd, insluitende rooibossaailinge en pere. Hierdie patogeen word deur morfologiese, fisiologiese, asook genetiese diversiteit gekenmerk. Die genetiese diversiteit en populasie-struktuur van B. cinerea populasies wat in Suid-Afrika voorkom, is nog nie ondersoek nie. Botrytis cinerea verkryg vanaf rooibossaailinge en in peerboorde in die Wes-Kaap van Suid-Afrika is ondersoek. Hierdie studie is met behulp van mikrosatellietmerkers, amplifikasie van die twee paringstipe gene (MAT1-1 en MAT1-2), asook die bepaling van weerstandsvlakke teenoor verskeie swamdoders, uitgevoer. Populasie-dinamika is afgelei en ‘n soortgelyke tendens is in beide produksie-sisteme waargeneem. Botrytis cinerea veroorsaak jaarliks ernstige verliese van rooibossaailinge (Aspalathus linearis) in kwekerye in die Clanwilliam-area. Monsters is in vyf kwekerye versamel en die kriptiese spesiestatus van die verkrygde isolate is deur restriksie-ensiemvertering van die Bc-hch geen bepaal. Almal behalwe een (206 uit 207) isolaat het aan Groep II of B. cinerea ‘sensu stricto’ behoort. Analise van die B. cinerea Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om die genetiese populasiestruktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.67. Alhoewel twee van die kwekerye se monstergrootte erg ingeperk is ná kloonverwydering, is daar nogtans 100 genotipes onder die 206 isolate wat geïsoleer is, waargeneem. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 16 en 68, vir die vyf populasies, gewissel, met ‘n totaal van 17 vir die 100 genotipes. Een genotipe, verteenwoordig deur 27 klone, is uit vier kwekerye geïsoleer. Relatief lae dog noemenswaardige populasie-differensiasie is in totaal tussen kwekerye waargeneem (gem. FST = 0.030, P = 0.001). Die verspreiding van die twee paringstipes (MAT1-1 en MAT1-2) het beduidend verskil van ‘n 1:1 verhouding vir die totale populasie, asook twee van die kwekerye se populasies. Die drie oorblywende kwekerye se populasies het egter ‘n gelyke verdeling van die twee paringstipes getoon. Die indeks van assosiasie (IA) analises toon dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 97% van die totale genetiese variasie binne die subpopulasies versprei is. Hoogs variërende vlakke van weerstand tussen die vyf kwekerye teenoor die swamdoder iprodioon, is vir die 198 isolate wat getoets is, gevind. Die totale gemiddelde frekwensie van weerstand teenoor iprodioon was 43%, wat tussen 0% en 81% vir die vyf kwekerye gevarieer het. Fondasie-vlak-sensitiwiteit vir pyrimethanil het ‘n gemiddelde EC50 waarde van 0.096 mg/L opgelewer. Botrytis cinerea isolate is ook vanuit peerbloeisels (Pyrus communis L.) vanuit vier boorde versamel, twee uit elk van die Ceres- en Grabouw-areas. In totaal is 181 isolate vanuit die vier boorde versamel. Die frekwensie van bloeiselinfeksie het tussen 3% en 17% gewissel. Oor die algemeen was daar ‘n hoë frekwensie van isolate wat slegs die Boty transponeerbare element teenwoordig gehad het (74%) in vergelyking met dié wat tegelykertyd beide (Boty en Flipper) teenwoordig gehad het. Een isolaat het slegs die Flipper element gehad. Bepaling van die kriptiese spesiestatus met behulp van restriksie-ensiemvertering van die Bc-hch geen het aangedui dat alle versamelde isolate tot Groep II of B. cinerea ‘sensu stricto’ behoort het. Analise van die Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om genetiese populasie-struktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.69 oor alle populasies. Alhoewel twee subpopulasies ‘n hoë klonale fraksie getoon het, is 91 genotipes tussen die 181 isolate wat verkry is, onderskei. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 18 en 33 vir die vier populasies gewissel, met ‘n totale waarde van 14 vir die 91 genotipes. Een genotipe, verteenwoordig deur 27 klone, was in al vier boorde teenwoordig. Gematigde dog beduidende populasie differensiasie was in totaal tussen boorde teenwoordig (gem. FST = 0.118, P = 0.001). Die verspreiding van die paringstipes (MAT1-1 en MAT1-2) het nie betekenisvol van ‘n 1:1 verhouding vir die totale populasie, insluitende die subpopulasies, verskil nie. Indeks van assosiasie (IA) analises het egter aangedui dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 88% van die totale genetiese variasie in subpopulasies te vinde was, 9% tussen subpopulasies en slegs 3% tussen produksie-areas. Frekwensie van swamdoder weerstandbiedendheid vir fenhexamid, iprodioon en benomyl het gewissel, met die hoogste vlakke teenoor benomyl waargeneem, maar baie lae vlakke teenoor fenhexamid en iprodioon. Samevattend het hierdie studie getoon dat die populasies van B. cinerea wat in hierdie twee produksie-sisteme, op rooibossaailinge en in peer boorde, ondersoek is, ‘n aanpasbaarheid toon om huidige metodes van beheer te oorkom. Die gebruik van populasiegenetika as ‘n hulpmiddel om ons kennis van patogeen-interaksies en -verspreiding te verbreed, is van kardinale belang in die ontwikkeling van geïntegreerde en volhoubare beheermaatreëls. Kennis van die verspreiding van B. cinerea in die bestudeerde gewasproduksiestelsels, werp lig op die inherente risiko wat dié patogeen inhou. Dít, tesame met kennis van die weerstandsvlakke wat voorkom, kan as ‘n vroegtydige waarskuwing dien ten einde moontlike verlies van beheer in die toekoms te help teenwerk.

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