Population structure, sex and spatial distribution of phyllosticta citricarpa, the citrus black spot pathogen

Carstens, Elma (2018-03)

Thesis (PhDAgric)—Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Citrus Black Spot (CBS), caused by Phyllosticta citricarpa, is a fungal disease that influences citrus industries worldwide. All commercial Citrus spp. are susceptible to the disease. The pathogen was first described 117 years ago from Australia; subsequently, from summer rainfall citrus production regions in China, Africa, and South America; and, recently, the United States. Limited information is available on the pathogen’s population structure, mode of reproduction, and introduction pathways at a global scale and at a regional (provincial) scale in South Africa. This is also true for the effect of distance (spatial), season (temporal) and Citrus spp. on population structure at the orchard scale. The aforementioned aspects were investigated in the current study. Since limited co-dominant markers are available for P. citricarpa population genetic analyses, one of the first aims of the study was to develop new simple-sequence repeat (SSR) markers. The population structure of P. citricarpa was investigated at a global scale in 12 populations from South Africa, the United States, Australia, China, and Brazil. Seven published and eight newly developed polymorphic SSR markers were used for genotyping populations. The Chinese and Australian populations had the highest genetic diversities, whereas populations from Brazil, the United States, and South Africa exhibited characteristics of founder populations. Based on population differentiation and clustering analyses, the Chinese populations were distinct from the other populations. High connectivity was found, and possibly linked introduction pathways, between South Africa, Australia and Brazil. With the exception of the clonal United States populations that only contained one mating type, the other populations contained both mating types in a ratio that did not deviate significantly from 1:1. Although most populations exhibited sexual reproduction, linkage disequilibrium analyses indicated that asexual reproduction is also important. The effects of distance (spatial) and season (temporal) on the population structure of P. citricarpa were investigated over two seasons, in two lemon orchards in South Africa; one in the Mpumalanga province and the other in the North West province. Spatial analyses indicated that subpopulations separated by a short distance (within 200 m) were typically not significantly genetically differentiated, but that those separated by longer distances were sometimes significantly differentiated. Temporal analyses in the North West orchard showed that seasonal populations were not significantly genetically differentiated. In contrast, seasonal populations from the Mpumalanga orchard were significantly differentiated, most likely due to higher rainfall and disease pressure, and the spatial scale of sampling. Based on linkage disequilibrium analyses, sexual and asexual reproduction occurred in both orchards. In each orchard, two dominant multilocus genotypes (MLGs) were identified in most of the subpopulations, as well as in the seasonal populations. Pycnidiospores are therefore important in the development of CBS at the temporal and spatial scales in South African lemon orchards. Population genetic studies on a regional (provincial) scale in South Africa showed that ten P. citricarpa populations, representing five provinces (North West, Mpumalanga, Limpopo, KwaZulu-Natal and Eastern Cape), were not significantly genetically differentiated. Based on gene and genotypic diversities and private allele richness, the KwaZulu-Natal or the Limpopo provinces are likely the provinces where the pathogen was first introduced. There might have been at least two separate introductions of the pathogen into the country. The Eastern Cape province was confirmed as being the province where the latest introduction occurred in South Africa. Despite lemon trees having overlapping fruit crops, potentially providing increased opportunities for clonal reproduction, Citrus spp. (lemons vs. oranges) did not have an effect on population structure; not all lemon populations were significantly genetically differentiated from all orange populations. The current study has revealed novel information on the population structure of P. citricarpa at global and regional (South Africa) scales, which have implications for the epidemiology and management of the disease. The finding that pycnidiospores, in addition to ascospores, are also important in the epidemiology of the disease in South Africa, contradicts previous reports that pycnidiospores are of minor significance. Future studies should reinvestigate the role of these spore types in the epidemiology of CBS in South Africa using conventional orchard inoculation and leaf removal studies, combined with a population genetic data analyses. The role that distance and season have on the population structure should also be considered in orchard trial designs. Ascospore spore trap data should be generated that involve the differentiation of P. citricarpa from P. capitalensis.

AFRIKAANSE OPSOMMING: Sitrus Swartvlek (SSV) is 'n swamsiekte wat deur Phyllosticta citricarpa veroorsaak word, en wat sitrusbedrywe wêreldwyd beïnvloed. Alle kommersiële Sitrus spp. is vatbaar vir die siekte. Die patogeen is 117 jaar gelede vir die eerste maal in Australië beskryf en daarna van sitrus produserende streke in somerreënval gebiede in Sjina, Afrika en Suid-Amerika en mees onlangs van die Verenigde State. Beperkte inligting oor die patogeen se populasie-struktuur, wyse van voortplanting en introduksie roetes is op ‘n globale vlak beskikbaar, sowel as op ‘n provinsiale vlak in Suid-Afrika. Op ʼn boordvlak, is inlgiting ook beperk oor die effek wat afstand (“spatial”), seisoen (temporaal) en Sitrus spp. op die populasie-struktuur het. Voorafgenoemde aspekte is in die studie ondersoek. Aangesien beperkte dominante merkers vir P. citricarpa populasie genetiese analises beskikbaar is, was een van die eerste doelstellings van die studie om nuwe mikrosatelliet merkers te ontwikkel. Die populasie-struktuur van P. citricarpa is op ‘n globale vlak in 12 populasies van SuidAfrika, die Verenigde State, Australië, Sjina en Brasilië ondersoek. Sewe gepubliseerde en agt nuut ontwikkelde polimorfiese mikrosatelliet merkers is gebruik om die populasies te genotipeer. Die Sjinese en Australiese populasies het die hoogste genetiese diversiteit getoon, terwyl populasies van Brasilië, die Verenigde State en Suid-Afrika eienskappe van stigterspopulasies toon. Gebaseer op populasie-differensiasie en groeperings-analises verskil die Sjinese populasies van die ander populasies. Hoë konnektiwiteit en moontlik gedeelde introduksie roetes is tussen Suid-Afrika, Australië en Brasilië gevind. Met die uitsondering van die klonale populasies van die Verenigde State, met net een paringstipe, het die ander populasies beide paringstipes gehad in 'n verhouding wat nie beduidend van 1:1 afwyk nie. Alhoewel die meeste populasies geslagtelike voortplanting getoon het, het “linkage disequilibrium” analises getoon dat ongeslagtelike voortplanting ook belangrik is. Die effek van afstand (ruimtelik) en seisoen (temporaal) op die populasie-struktuur van P. citricarpa is oor twee seisoene in twee suurlemoenboorde in Suid-Afrika ondersoek; een boord in die Mpumalanga-provinsie en die ander in die Noordwes-provinsie. Ruimtelike analises het getoon dat subpopulasies wat deur 'n kort afstand (binne 200 m) geskei word, tipies nie betekenisvol geneties gedifferensieerd was nie, maar dat die wat deur langer afstande geskei is, soms betekenisvol gedifferensieerd was. Temporale analises in die Noordwes boord het getoon dat seisoenale populasies nie betekenisvol geneties gedifferensieerd was nie. In teenstelling hiermee, was seisoenale populasies van die Mpumalanga-boord betekenisvol gedifferensieerd, waarskynlik weens hoër reënval en siektedruk en die ruimtelike skaal van monsterneming. Gebaseer op “linkage disequilibrium” analises, het geslagtelike en ongeslagtelike voortplanting in beide boorde plaasgevind. In elke boord het twee dominante multi-lokus genotipes (MLG's) in die meeste van die subpopulasies, sowel as in die seisoenale populasies, voorgekom. Piknidiospore is dus belangrik in die ontwikkeling van SSV op temporale en ruimtelike vlakke in Suid-Afrikaanse suurlemoenboorde. Populasie genetika studies op 'n streeks- (provinsiale) vlak in Suid-Afrika het getoon dat tien P. citricarpa-populasies wat vyf provinsies verteenwoordig (Noordwes, Mpumalanga, Limpopo, KwaZulu-Natal en Oos-Kaap), nie betekenisvol geneties gedifferensieerd was nie. Gebaseer op geen- en genotipiese diversiteit en die aantal privaat allele, is die KwaZulu-Natal provinsie of die Limpopo provinsie waarskynlik die provinsies waar die patogeen eerste gevestig het. Daar is moontlik ten minste twee afsonderlike introduksies van die patogeen. Daar is bewys dat die Oos-Kaap die provinsie is waar die laaste introduksie in Suid-Afrika plaasgevind het. Ten spyte daarvan dat suurlemoenbome wat oorvleuende oeste het, moontlik verhoogde geleenthede vir klonale voortplanting bied, het Citrus spp. (suurlemoene vs. lemoene) nie 'n effek op die populasie-struktuur gehad nie, omdat nie al die suurlemoenpopulasies betekenisvol geneties gedifferensieerd van al die lemoenpopulasies was nie. Die studie het nuwe inligting oor die populasie-struktuur van P. citricarpa op ‘n globale en streeks- (Suid-Afrika) vlak gebring, wat implikasies vir die epidemiologie en bestuur van die siekte inhou. Die bevinding dat piknidiospore, bykomend tot askospore, ook belangrik in die epidemiologie van die siekte in Suid-Afrika is, weerspreek vorige verslae dat piknidiospore van geringe belang is. Verdere studies moet die rol van hierdie spoortipes in die epidemiologie van SSV in Suid-Afrika deur middel van konvensionele boord-inokulasies en blaarverwyderingstudies ondersoek. Dit moet met 'n populasie genetika studie gekombineer word. Die rol wat afstand en seisoen op die populasie-struktuur het, moet ook vir die ontwerp van boordproewe oorweeg word. Askospoor lokval-data moet gegenereer word wat tussen P. citricarpa en P. capitalensis kan onderskei.

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