Inoculum ecology of petri disease fungi in grapevines of South Africa

Baloyi, Mahlatse Annabella (2016-12)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Petri disease is among the important grapevine trunk diseases affecting lifespan and productivity of young vines worldwide. Infection result in poor vine stand in newly established vineyards and a general vine decline. Pathogens causing this disease are known invaders of susceptible pruning wounds. The knowledge of when aerial spore inoculum of these pathogens are released in vineyards has not been reported in South Africa, and this result in growers pruning without the knowledge of whether that would coincide with periods of high aerial spore concentration. This study aimed at investigating when aerial spores of Petri disease pathogens are released, and to determine their source of inoculum. Knowledge regarding spore release in South African vineyards was determined for two seasons in 2012 and 2013. Spore traps were affixed to arms of infected vines in six vineyards and two rootstock mother blocks. Results showed the occurrence of Petri disease pathogens throughout the year and Phaeomoniella chlamydospora and Pm. minimum were trapped in all vineyards. A total of 14 Phaeoacremonium species were identified from the different blocks. Spore release was shown to coincide with pruning and suckering activities, however, there was no positive correlation between rainfall and spore release events. The occurrence of Petri disease pathogens fruiting bodies was determined by surveying six vineyards and two rootstock mother blocks between 2012 and 2014. Dead wood from diseased vines were collected for microscopic examination. Phaeomoniella chlamydospora pycnidia were found in all vineyards and rootstock mother blocks surveyed. Perithecia of Pm. minimum were only found in vineyards of Stellenbosch P2 and B3, Rawsonville and a rootstock mother block in Slanghoek. Additionally, mating studies with isolates of Pm. australiense and Pm. scolyti were conducted in vitro. After seven and eleven months fertile perithecia of Pm. australiense and Pm. scolyti were observed, respectively. Crosses of both species corresponded to a heterothallic mating system. This study gives the first report of the occurrence of pycnidia of Pa. chlamydospora and perithecia of Pm. minimum in South African vineyards and rootstock mother blocks and also the first description of sexual morphs of Pm. australiense and Pm. scolyti. The pathogenic status of 10 Phaeoacremonium species found in South African vineyards was studied. Fresh pruning wounds of a nine-year-old Cabernet Sauvignon vineyard were inoculated with 104 conidia/ml of each fungus per wound and assessed after 18 months. All inoculated isolates successfully colonized pruning wounds causing lesions significantly different from the negative control and were re-isolated at varying percentages ranging from 28.57% to 85.71%. The study confirmed the capability of all tested Phaeoacremonium species to infect grapevine pruning wounds and cause lesions. The genetic diversity and mode of reproduction were assessed using microsatellite markers and also by determining the mating type distribution of aerial trapped spores of Pm. minimum. In total 320 Pm. minimum isolates were assessed with Mat1-2 specific-primers. Both mating types of Pm. minimum were found in all eight vineyards. An equal distribution of MAT1-1 and MAT1-2 were found in six of the vineyards, but not in the Paarl A and Wellington populations. Primers for dinucleotide microsatellite loci were designed and 15 microsatellite loci were identified to be polymorphic and could thus be used to assess the genetic diversity of the Pm. minimum isolates. A total of 134 multilocus genotypes (MLGs) were observed of which 115 were observed once and 19 genotypes were observed either two or more times. The presence of the same MLG in a vineyard at different collection times, supports the presence of asexual reproduction, and the widespread distribution of MLGs is most probably due to infected nursery planting material. The total gene diversity (H) was high with a mean of 0.58 across all populations. Analysis of molecular variance indicated that 94% of the genetic variation was distributed within populations and only 6% between populations. High and significant population differentiation values were only obtained when Paarl Z was compared to Stellenbosch P2. This study confirms the importance of infected planting material that can distribute similar MLGs over long distances. Therefore, the management of Petri disease needs to focus on ensuring clean mother vines and nursery plants.

AFRIKAANSE OPSOMMING: Petri-siekte is ‘n belangerike wingerdstamsiekte wat die leeftyd en produktiwiteit van jong wingerde wêreldwyd affekteer. Infeksie deur hierdie siekte veroorsaak dat nuwe wingerdaanplantings swak vestig tesame met ‘n algemene afname in die wingerd. Die patogene wat hierdie siekte veroorsaak is bekend vir hul vermoë om vatbare snoeiwonde te infekteer. Kennis rakende die periode waarin luggedraagde spoorinokulum van hierdie patogene vrygestel word, is nog nie in Suid-Afrika gerapporteer nie. Produsente snoei gevolglik sonder om te weet of dit saam met periodes sou val waarin die konsentrasies van luggedraagde spore hoog is. Die doel van hierdie studie was om vas te stel wanneer luggedraagde spore van Petri-siekte patogene vrygestel word, asook om die bron van die inokulum te bepaal. Inligting aangaande die spoorvrystelling in Suid-Afrikaanse wingerde was vir twee seisoene in 2012 en 2013 versamel. Spoorlokvalle was aaangebring op die stamme van geïnfekteerde wingerdstokke in ses wingerde en twee onderstokmoederblokke. Die resultate het aangedui dat Petri-siekte patogene reg deur die jaar voorkom en het ook Phaeomoniella chlamydospora en Pm. minimum in al die wingerde gevang. In totaal is 14 Phaeoacremonium spesies geïdentifiseer vanuit verskeie blokke. Daar is bevind dat spoorvrystelling in dieselfde periode voorkom as snoei- en suieraktiwiteite. Daar was egter geen positiewe korrelasie tussen die reënval en spoorvrystelling gevind. Opnames was in ses wingerde en twee onderstok moederblokke gedoen tussen 2012 en 2014 om die voorkoms van die vrugliggame van Petri-siekte patogene vas te stel. Dooie hout van geïnfekteerde wingerde was ingesamel om te ondersoek op mikroskopiese vlak. Vrugstrukture van Phaeomoniella chlamydospora was gevind in al die wingerde en onderstokmoederblokke waarvoor opnames gedoen was. Vrugstrukture van Pm. minimum was slegs gevind in wingerde van Stellenbosch P2 en B3, Rawsonville en ‘n onderstok moederblok in Slanghoek. Daarbenewens is in vitro paringstudies ook uitgevoer met isolate van Pm. australiense en Pm. scolyti. Vrugbare geslagtelike vrugstrukture van Pm. australiense en Pm. scolyti is na sewe en elf maande onderskeidelik, waargeneem. Kruisings van beide spesies het met ‘n heterotalliese paringstelsel ooreengestem. Hierdie studie lewer die eerste verslag van die voorkoms van vrugstrukture van Pa. chlamydospora en Pm. minimum in Suid-Afrikaanse wingerde en onderstokmoederblokke, asook die eerste beskrywing van die geslagtelike vrugstrukture van Pm. australiense en Pm. scolyti. Die patogeniese status van 10 Phaeoacremonium spesies wat in Suid-Afrikaanse wingerde voorkom was bestudeer. Vars snoeiwonde van ‘n nege-jaar-oue Cabernet Sauvignon wingerd was geïnokuleer met 104 kondida/ml van elke swam per wond en 18 maande later geëvalueer. Al die isolate wat geïnokuleer was, was suksesvol daarin om die snoeiwonde te koloniseer en letsels te vorm wat noemenswaardig verskil het van die negatiewe kontrole, en kon geherisoleer word teen persentasies wat gewissel het tussen 28.57% en 85.71%. Die studie het die vermoë van die Phaeoacremonium spesies wat ondersoek is, om wingerd snoeiwonde te infekteer en letsels te veroorsaak, bevestig. Die genetiese diversiteit en tipe voortplanting is geëvalueer met behulp van mikrosatelliet merkers, asook om die paringstipe-verspreiding van die luggedraagde spore van Pm. minimum wat gevang is, vas te stel. In totaal is 320 Pm. minimum isolate geëvalueer met Mat1-2 spesifieke-inleiers. Beide paringstipes van Pm. minimum is gevind in al agt wingerde. ‘n Gelyke verspreiding van MAT1-1 en MAT1-2 is gevind in ses van die wingerde, maar nie in die Paarl A of Wellington populasies nie. Inleiers vir dinukleotied mikrosatelliet-lokusse is ontwerp en 15 mikrosatelliet-lokusse was gevind om polimorfies te wees en kon daarom gebruik word om die genetiese diversiteit van die Pm. minimum isolate te bepaal. ‘n Totaal van 134 multilokus genotipes (MLG’s) is waargeneem, waarvan 115 een keer voorgekom het en 19 daarvan twee of meer kere voorgekom het. Die voorkoms van dieselfde MLG in ‘n wingerd op verskillende versamelingstye ondersteun die voorkoms van ongeslagtelike voortplanting, terwyl die wye verspreiding van dieselfde MLG’s waarskynlik toegeskryf kan word aan besmette kwekery plantmateriaal. Die totale geendiversiteit (H) was hoog in alle bevolkings met ‘n gemiddeld van 0.58. Die ontleding van molekulêre variansie het daarop gedui dat 94% van die genetiese variasie binne-in bevolkings verspreid is en slegs 6% tussen bevolkings verspreid is. Hoë en noemenswaardige bevolkingsdifferensiasie-waardes was net gevind toe die Paarl Z en Stellenbosch P2 met mekaar vergelyk is. Hierdie studie bevestig die bydrae van besmette plantmateriaal wat soortgelyke MLGs oor lang afstande kan versprei. Die bestuur van Petri-siekte moet gevolglik daarop fokus om skoon moederwingerde en kwekeryplante te verseker.

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