DETECTION AND QUANTIFICATION OF SOILBORNE PATHOGENS IN GRAPEVINE NURSERIES by SHAUN DENVER LANGENHOVEN Thesis presented in partial fulfilment of the requirements for the degree of Master of Science in the Faculty of AgriSciences at the University of Stellenbosch Supervisor: Dr L. Mostert Co-supervisor: Prof F. Halleen December 2017 The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. Stellenbosch University https://scholar.sun.ac.za DECLARATION By submitting this thesis/dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: December 2017 Copyright © 2017 Stellenbosch University All rights reserved II Stellenbosch University https://scholar.sun.ac.za SUMMARY Black-foot disease (BFD) and crown and root rot (CRR) are important decline diseases of grapevines, worldwide. These diseases occur in both nurseries and vineyards. Black-foot disease is caused by species in the genera: Dactylonectria, Ilyonectria, Campylocarpon, Cylindrocladiella and Thelonectria, whereas CRR is caused by Phytophthora, Pythium and Phytopythium species. Plant stress, caused by improper planting holes, waterlogging, drought, nutrient deficiencies and high temperatures, is a major predisposing factor for BFD and CRR. The symptoms of these two diseases are similar and include necrotic root lesions, reduced root biomass, leaf chlorosis and ultimately, plant death. The first aim of this study was to conduct a survey of BFD and CRR pathogens in nursery grafted plants over a 3 year period (2013-2015) as well as from weeds and rotation crops in the alternate season (2014). Furthermore, pathogen detection using quantitative real- time polymerase chain reaction (qPCR) was conducted on soil samples which were collected in close proximity to the sampled grapevine plants, weeds and rotation crops. Soil was collected up to a depth of 30 cm and 60 cm. The decline pathogens that were quantified in the soil with qPCR were Dactylonectria and Ilyonectria genera, Phytophthora species and Pythium irregulare. The predominant BFD pathogens isolated from grapevines were Dactylonectria macrodidyma, Campylocarpon pseudofaciculare and Ca. fasciculare. The predominant CRR pathogens were Pythium irregulare followed by Phytopythium vexans. Furthermore, Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, Py. irregulare, Py. ultimum and Py. heterothallicum were isolated from Triticale roots. Dactylonectria was also isolated from the weed corn spurry, while Py. irregulare and Py. ultimum were isolated from numerous weeds and rotation crops. Dactylonectria, Ilyonectria, Phytophthora and Py. irregulare DNA was detected in all nurseries across all years at varying concentrations with Nursery E having the lowest concentrations. Ilyonectria and Dactylonectria mean soil DNA concentrations ranged from 0.04 pg.µL-1 to 37.14 pg.µL-1, while the Py. irregulare mean soil DNA concentrations ranged from 0.01 pg.µL-1 to 3.77 pg.µL-1. The Phytophthora mean soil DNA concentrations ranged from 0.01 pg.µL-1 to 33.48 pg.µL-1. This study successfully used existing SYBR green I qPCR assays to quantify BFD and CRR pathogens in grapevine nursery soil. During this study nursery soils were confirmed as a sources of infection for BFD and CRR pathogens, and that weeds may serve as alternative hosts to these pathogens. This is also the first report of D. alcacerensis and D. pauciseptata in South African nurseries. The second aim was to describe a putative new Phytopythium species which was isolated from nursery grapevine roots and to determine if it is a pathogenic species. Six isolates of an unknown Phytopythium species were isolated from the roots of an asymptomatic III Stellenbosch University https://scholar.sun.ac.za plant. Morphological observations and phylogenetic analyses, based on the ITS rDNA and cytochrome oxidase subunit 1 gene regions, proved that it was indeed a novel species. Together with an undescribed Japanese isolate, the six South African isolates were named Phytopythium paucipapillatum and displayed occasionally papillate, direct sporangial germination as well as sporangia which proliferates internally. The sporangia were globose, subglobose, ovoid, obovoid, limoniform to ellipsoid or distorted. The oogonia were small, globose, with some oogonia also displaying short papillae protruding from the surface. This putative new species was also insensitive to the isoxazole fungicide, hymexazol. A pathogenicity trial, on the rootstock 110-Richter, revealed that Pp. paucipapillatum was not pathogenic towards grapevines. In accordance with other studies regarding Phytopythium species, Pp. paucipapillatum may be a soil inhabitant. IV Stellenbosch University https://scholar.sun.ac.za OPSOMMING Swartvoet en kroon- en wortelvrot is ernstige siektes wat ‘n afname in wingerd wêreldwyd te weeg bring. Hierdie siektes kom in beide kwekerye en wingerde voor. Swartvoet word veroorsaak deur spesies in die Dactylonectria, Ilyonectria, Campylocarpon, Cylindrocladiella en Thelonectria genera, terwyl kroon- en wortelvrot veroorsaak word deur Phytophthora, Pythium en Phytopythium spesies. Verskeie faktore kan stres op ‘n wingerd plaas, soos swak voorbereide plantgate, waterdeurdrenkte gronde, droogte, ‘n tekort aan voedingstowwe asook hoë temperature, wat die wingerd meer vatbaar maak vir swartvoet en kroon- en wortelvrot. Die simptome van hierdie twee siektes is soortgelyk en sluit nekrotiese wortelletsels, verminderde wortelmassa en blaarchlorose in, wat gesamentlik na die uiteindelike dood van die wingerd lei. Die eerste doel van hierdie studie was om ‘n opname te doen van swartvoet en kroon- en wortelvrotpatogene in kwekery entplante oor 'n tydperk van 3 jaar (2013-2015), asook van onkruide en rotasiegewasse in die afwisselende seisoen (2014). Grondmonsters, wat in die nabyheid van wingerdplante, onkruide en rotasiegewasse geneem is tydens die steekproef, is ontleed met behulp van kwantitatiewe intyd polimerase kettingreaksies (kPKR) om die patogene wat daarin voorkom op te spoor. Die grondmonsters was tot op ‘n diepte van 30 cm en 60 cm geneem. Die siekteveroorsakende patogene wat gekwantifiseer is in die grond deur middel van hierdie tegniek sluit spesies in die Dactylonectria en Ilyonectria genera in, asook Phytophthora spesies en Pythium irregulare. Dactylonectria macrodidyma, Campylocarpon pseudofaciculare en Ca. fasciculare was die oorheerstende swartvoetpatogene wat vanuit wingerd geïsoleer was. Die mees algemene kroon- en wortelvrotpatogeen was Pythium irregulare, gevolg deur Phytopythium vexans. Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, Py. irregulare, Py. ultimum en Py. heterotallicum is vanuit Triticale wortels geïsoleer. Dactylonectria is ook vanuit die onkruid ‘corn spurry’ geïsoleer, terwyl Py. irregulare en Py. ultimum vanuit verskeie ander onkruide en rotasiegewasse geïsoleer is. Wisselende konsentrasies van DNS van Dactylonectria, Ilyonectria, Phytophthora en Py. irregulare was gevind in alle kwekerye oor alle jare, met Kwekery E wat die laagste konsentrasies gehad het. Die gemiddelde grond DNS konsentrasies van Ilyonectria en Dactylonectria het gewissel tussen 0.04 pg.μL-1 en 37.14 pg.μL-1, terwyl dit tussen 0.01 pg.μL-1 en 3.77 pg.μL-1 gewissel het vir Py. irregulare. Die gemiddelde grond DNS konsentrasies van Phytophthora was tussen 0.01 pg.μL-1 en 33.48 pg.μL-1. Hierdie studie kon reeds betsaande SYBR groen I kPKR-toetse met sukses gebruik om swartvoet en kroon- en wortelvrotpatogene in wingerdkwekerygronde te kwantifiseer. Dit het bevestig dat kwekerye as bron van infeksie vir hierdie patogene dien, V Stellenbosch University https://scholar.sun.ac.za terwyl dit ook daarop gedui het dat onkruide as alternatiewe gashere kan dien. Boonop is dit die eerste verslag van D. alcacerensis en D. pauciseptata in Suid-Afrikaanse kwekerye. Die tweede doel van hierdie studie was om 'n moontlike nuwe Phytopythium-spesie te beskryf wat vanuit wingerdwortels geïsoleer is, en om die patogenisiteit daarvan te bepaal. Ses isolate van 'n onbekende Phytopythium spesie is geïsoleer vanuit die wortels van ‘n plant wat geen simptome getoon het nie. ‘n Morfologiese studie tesame met filogenetiese ontledings, wat gebaseer was op die ITS-rDNS en COI geenareas, het bewys dat dit inderdaad 'n nuwe spesie was. Hierdie ses isolate, tesame met ‘n onbeskryfde isolaat van Japan, is Phytopythium paucipapillatum genoem. Dit het soms papillate en direkte ontkieming van die sporangia vertoon, asook sporangia wat intern vermeerder. Die sporangia was bolvormige, subglobose, ovoid, obovoid, limoniform tot ellipsoid of verwring. Die oögonia was klein en bolvormige, terwyl sommige oögonia ook kort papilla vertoon het wat by die oppervlak uitsteek. Hierdie nuwe spesie is weerstandbiedend teen hymexazool, ‘n isoxazool swamdoder. ‘n Patogenesiteitstoets wat op die 110-Richter onderstok uitgevoer is, het egter daarop gedui dat Pp. paucipapillatum nie patogenies is tot wingerd nie. Pp. paucipapillatum is dus moontlik net 'n grondbewoner, wat ooreenstem met resultate van