Identification and characterisation of diatrypaceae species associeated with declining grapevines and alternative hosts in South Africa

Moyo, Providence (2017-03)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Grapevine trunk diseases have devastating impacts on the sustainability of viticulture, worldwide. Eutypa dieback, in particular, has caused large economic losses and premature mortality of vines. This disease has, for many years, been associated with the Diatrypaceae fungus, Eutypa (E.) lata. Several species of Diatrypaceae were, however, recently discovered to be associated with Eutypa dieback-affected grapevines in different grape growing areas including Australia, Chile, Spain and United States of America. No extensive study has been conducted to identify and characterise the species of Diatrypaceae in South Africa. Surveys were conducted in vineyards located in different grape growing regions of the Western Cape and Diatrypaceae fungi were isolated from grapevines with dying spurs or wood with wedge-shaped necrosis in cross section, as well as from perithecia on dead wood. Isolates were studied using phylogenetic analyses of combined DNA sequences of the internal transcribed spacer regions (ITS1 and ITS2) and 5.8S rRNA gene as well as partial β-tubulin gene. Morphological characteristics of perithecia were also studied. Morphological and phylogenetic analyses revealed the presence of seven Diatrypaceae species to occur on grapevine in South Africa, namely Cryptovalsa (C.) ampelina, C. rabenhorstii, E. consobrina, E. lata, Eutypella (Eu.) citricola, Eu. microtheca and E. cremea, which was described as a new species. The most common species isolated from dying spurs, in order of abundance, were C. ampelina (46.4% of total number of isolates), Eu. citricola (26.8%), E. lata (20.1%), E. cremea (4.3%), Eu. microtheca (1.2%), E. consobrina (0.6%) and C. rabenhorstii (0.6%). On the other hand, from wedge-shaped necrosis, E. lata represented the most frequent species (89.2% of all isolates obtained) followed by Eu. citricola (8.5%), E. cremea (1.4%) and C. ampelina (0.9%). Five species namely, E. lata, C. ampelina, E. cremea, Eu. citricola and Eu. microtheca were found to produce perithecia on dead grapevine wood. These results suggest that Eutypa dieback in South Africa can be associated with several Diatrypaceae species. Different fruit and ornamental trees occurring near vineyards were investigated to determine whether they are colonised by Diatrypaceae species, which are associated with Eutypa dieback of grapevine. Isolates of Diatrypaceae were collected from these trees showing symptoms of dieback, cankers and perithecia. Isolates were analysed by morphological and phylogenetic analyses as described above. Fourteen species namely, C. ampelina, E. consobrina, E. lata, Eu. citricola, Eu. microtheca, E. cremea, Cryptosphaeria (Cr.) multicontinentalis, Cr. ligniota, Diatrypella sp., Eu. leprosa, Eu. australiensis and three undescribed Eutypella species were identified from 29 different fruit and ornamental trees, occurring in close proximity to vineyards. The five most prevalent species were E. lata, C. ampelina, E. cremea, Eu. citricola and Eu. microtheca, which were also the most prevalent on grapevine. These findings suggest that cross infections are possibly occurring between grapevine and other woody hosts growing near vineyards in South Africa. These five species were also the only Diatrypaceae species isolated from stone fruit trees. Pathogenicity of these five Diatrypaceae species on stone fruit trees (apricot and plum) was also determined. In these pathogenicity studies, all five species were pathogenic on both apricot and plum, producing brown-red discolouration, typical of Eutypa dieback of apricot. Finally, pathogenicity of Diatrypaceae species identified from grapevine and other woody hosts in South Africa was evaluated on grapevine, under field conditions. Artificial inoculations of these fungal species were conducted on fresh pruning wounds and lignified shoots of Cabernet Sauvignon as well as green shoots of Cabernet Sauvignon and Sauvignon blanc. After 10 months, all the species caused disease symptoms (brown discolouration) on pruning wounds and lignified shoots of Cabernet Sauvignon. Disease symptoms were also observed on green shoots of both cultivars. Pathogenicity results revealed that several species including C. ampelina, Eu. microtheca, Eu. leprosa, and Eu. citricola were equally virulent as the well-known pathogen, E. lata. Quantitative real-time PCR (qPCR) assays were also developed for the detection and quantification of E. lata and C. ampelina in grapevine wood. The qPCR assays were specific and successfully quantified target taxa in artificially inoculated wood samples. The present study provides knowledge on the identity of Diatrypaceae species associated with declining grapevines and other woody hosts occurring adjacent to vineyards in South Africa. This knowledge, together with qPCR assays can be useful in early diagnosis of infection caused by Diatrypaceae species in vineyards. Furthermore, pathogenicity studies have shown that many Diatrypaceae species, including those obtained from other woody hosts, are pathogenic to grapevine. As such, this study forms the platform for further studies aimed at managing Diatrypaceae species causing disease on grapevine in South Africa.

AFRIKAANSE OPSOMMING: Wingerdstamsiektes het ‘n verwoestende impak op die volhoubaarheid van die wynbou industrie wêreldwyd. Eutypa-terugsterf, in besonder, veroorsaak groot ekonomiese verliese en vroegtydige terugsterf van wingerde. Hierdie siekte was vir baie jare slegs met die Diatrypaceae swam, Eutypa (E.) lata, geassosieer. Verskeie spesies van Diatrypaceae is onlangs ontdek wat verband hou met Eutypa-terugsterf geaffekteerde wingerde in verskillende wingerd produksie areas in Australië, Chili, Spanje en die Verenigde State van Amerika. Tot dusvêr is daar nog geen uitgebreide studie gedoen om die spesies van Diatrypaceae in Suid-Afrika te identifiseer en te karakteriseer nie. Opnames is in wingerde gedoen wat in verskillende wingerdproduksiestreke van die Wes-Kaap geleë is, waartydens Diatrypaceae swamme geïsoleer is vanuit wingerdstokke met sterwende lote of hout met wigvormige nekrose in deursnit, asook van geslagtelike vrugstrukture (perithecia) op dooie hout. Isolate is ondersoek met behulp van filogenetiese analise van gekombineerde DNS volgordes van die interne getranskribeerde spasiëer streke (ITS1 en ITS2), die 5.8S rRNS gene en gedeeltelike β-tubulin gene. Die morfologiese kenmerke van perithecia was ook ondersoek. Die morfologiese en filogenetiese analise het die voorkoms van sewe Diatrypaceae spesies op wingerd in Suid-Afrika bevestig, waaronder Cryptovalsa (C.) ampelina, C. rabenhorstii, E. consobrina, E. lata, Eutypella (Eu). citricola, Eu. microtheca en E. cremea, wat beskryf is as 'n nuwe spesie. Die mees algemene spesie wat geïsoleer is vanuit sterwende lote, in volgorde van voorkoms, was C. ampelina (46,4% van die totale aantal isolate), Eu. citricola (26,8%), E. lata (20,1%), E. cremea (4,3%), Eu. microtheca (1,2%), E. consobrina (0,6%) en C. rabenhorstii (0,6%). Daarinteen was E. lata die mees algemene spesie wat geïsoleer was van wigvormige nekrose (89,2% van alle isolate verkry), gevolg deur Eu. citricola (8.5%), E. cremea (1,4%) en C. ampelina (0,9%). Daar is bevind dat vyf spesies, waaronder E. lata, C. ampelina, E. cremea, Eu. citricola en Eu. microtheca perithecia op dooie wingerdhout kan produseer. Hierdie resultate dui daarop dat Eutypa-terugsterf in Suid-Afrika geassosieer kan word met verskeie Diatrypaceae spesies. Verskillende vrugte en ornamentele bome wat naby aan wingerde voorkom is ondersoek om vas te stel of dit deur Diatrypaceae spesies gekoloniseer word wat verband hou met Eutypa-terugsterf van wingerd. Isolate van Diatrypaceae is ingesamel vanaf hierdie bome wat simptome van terugsterf of kankers of perithecia toon. Die isolate is daarna ontleed deur morfologiese en filogenetiese analise soos wat dit hierbo beskryf is. Veertien spesies, waaronder C. ampelina, E. consobrina, E. lata, Eu. citricola, Eu. microtheca, E. cremea, Cryptosphaeria (Cr.) multicontinentalis, Cr. ligniota, Diatrypella sp., Eu. leprosa, Eu. australiensis en drie onbeskryfde Eutypella spesies, is geïdentifiseer vanaf 29 verskillende vrugte en ornamentele bome wat naby aan wingerde geleë is. Die vyf mees algemene spesie was E. lata, C. ampelina, E. cremea, Eu. citricola en Eu. microtheca, wat ook die mees algemene spesie op wingerd was. Hierdie bevindinge dui daarop dat wedersuidse infeksies in Suid-Afrika moontlik plaasvind tussen wingerd en ander houtagtige gashere in die nabyheid daarvan. Hierdie vyf spesies was ook die enigste Diatrypaceae spesies wat geïsoleer was vanuit steenvrugtebome. Die patogenisiteit van hierdie vyf Diatrypaceae spesies is ook bepaal op steenvrugtebome (appelkoos en pruim). Hierdie studie het bevind dat al vyf spesies patogenies is op beide appelkoos en pruim en veroorsaak bruin-rooi verkleuring, wat ‘n tipiese simptoom van Eutypa-terugsterf op appelkoos is. Ten slotte is die patogenisiteit van Diatrypaceae spesies, wat geïsoleer is vanaf wingerd en ander houtagtige gashere in Suid-Afrika, geëvalueer op wingerd onder veldkondisies. Hierdie swam spesies is kunsmatig geïnokuleer op vars snoeiwonde en gelignifiseerde lote van Cabernet Sauvignon, asook groen lote van Cabernet Sauvignon en Sauvignon Blanc. Al die spesies het siekte simptome (bruin verkleuring) op snoei wonde en gelignifiseerde lote van Cabernet Sauvignon veroorsaak na 10 maande. Siektesimptome is ook waargeneem op groen lote van beide kultivars. Die patogenisiteitsresultate het daarop gedui dat verskeie spesies, insluitend C. ampelina, Eu. microtheca, Eu. leprosa en Eu. citricola ewe virulent is as die welbekende patogeen, E. lata. Kwantitatiewe intyd PKR (kPKR) toetse is ook ontwikkel vir die diagnose en kwantifisering van E. lata en C. ampelina in wingerdhout. Die kPKR analises was spesifiek en kon die teiken taksa suksesvol kwantifiseer in geinokuleerde houtmonsters. Die huidige studie verskaf kennis oor die identiteit van Diatrypaceae spesies in Suid-Afrika, wat verband hou met die terugsterf van wingerde en ander houtagtige gashere in die nabyheid daaraan. Hierdie kennis, tesame met kPKR analises, kan ‘n nuttig hulpmiddel wees in die vroeë diagnose van Diatrypaceae spesies in wingerde. Die patogenisiteitstudies het verder getoon dat baie Diatrypaceae spesies, insluitend dié van ander houtagtige gashere, patogenies is op wingerd. Hierdie studie vorm dus die platform vir verdere navorsing wat gemik is op die bestuur van Diatrypaceae spesies wat siekte op wingerd in Suid-Afrika veroorsaak.

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