The characterisation of basidiomycetes associated with esca disease in South African grapevines

Cloete, Mia (2015-03)

Thesis (PhDAgric)--Stellenbosch University, 2015.


ENGLISH ABSTRACT: Esca is a disease complex of grapevine that includes different foliar and vascular symptoms caused by various fungal pathogens. One of the distinguishing characteristics of the disease on mature vines is the white rot of the wood. Esca-related wood rot is caused by several lignicolous basidiomycetes from the order Hymenochaetales. The Hymenochaetales fungi associated with esca vary depending on geographic location. For example, in Europe and the Mediterranean grape-growing regions, Fomitiporia mediterranea is the prevalent species; in Argentina, Inocutis jamaicensis; in Chile “Fomitiporella vitis”, and in Australia Fomitiporia australiensis. In the United States, Fomitiporia polymorpha has been associated with esca, though not consistently. A previous study identified ten different taxa belonging to the genera Fomitiporella, Fomitiporia, Inocutis, Inonotus, and Phellinus associated with esca in South Africa. The current study was tasked with characterising these taxa and assessing their epidemiology and pathogenicity. The study has characterised three novel species, Fomitiporella viticola, Fomitiporia capensis and Phellinus resupinatus from Vitis vinifera and a first report of Inonotus setuloso-croceus occurring on Vitis vinifera and Salix spp. worldwide and in South Africa. The sporulation of F. viticola was surveyed over two seasons. The pathogenicity of all ten taxa was tested on mature field grown vines and enzymes secreted by all ten taxa were assayed. This study aimed to add in the understanding of the esca complex disease in South Africa and contributed towards the wider knowledge regarding the ecology of the Hymenochaetales. A novel Fomitiporia species, F. capensis, was described based on fruit body morphology and combined internal transcribed spacer rRNA ITS1-5.8S-ITS-2 (ITS) and large sub-unit (LSU) phylogeny, where it formed a clearly delineated and well-supported clade. Morphologically, F. capensis was similar to F. punctata in that both species essentially lack setae. Fomitiporia capensis, F. punctata and F. aethiopica produced similarly sized basidiospores, but differed in terms of host range, pore size and, possibly, fruiting body shape. Phylogenetically, F. capensis appeared to be related to F. tenuis, though morphologically the species differed significantly in that F. tenuis had smaller pores and smaller basidiospores. During all surveys conducted, Fomitiporia capensis was found to occur widely as throughout the Western Cape Province, though fruit bodies were scarce in comparison to mycelium isolated from symptomatic vines. Fruit bodies were also found in a vineyard in the Limpopo region in the north east part of the country. Phellinus resupinatus was described based on fruit body morphology, ITS and LSU phylogenies. It formed a well-supported clade closely related to Phellinus bicuspidatus, a species associated with white rot in oak trees in the United States. Morphologically, P. resupinatus was characterised by its resupinate fruit body shape, straight, ventricose hymenial setae, and broadly ellipsoid hyaline basidiospores. It was only found on diseased grapevines in the summer rainfall regions of South Africa, mainly in the Northern Cape and Limpopo provinces. Fomitiporella viticola was described from Vitis vinifera based on fruit body morphology and ITS phylogeny. It is characterised by a resupinate to effuse-reflexed fruit body with large, loosely spaced pores and fairly small yellowish-brown basidiospores. Inonotus setuloso–croceus was found occurring on Salix and Vitis vinifera and was identified based on fruit body morphology. The ITS region was sequenced from DNA isolated from cultures obtained from rotten wood or fruit bodies, and was matched to the Hymenochaetales species from Vitis previously classified as Taxon 7. The discovery of Inonotus setuloso-croceus on Salix validated the hypothesis that fruit bodies may occur on alternative hosts. Fomitiporella viticola was often isolated from white rot on vines affected by esca and fruit bodies were often found on vines in the Western Cape Province. Twelve fruit bodies of F. viticola were monitored for sporulation weekly over two seasons lasting between winter and early summer. Levels of sporulation had a weak positive correlation with rainfall and a weak negative correlation with average temperature. Sporulation was found to occur throughout the entire monitoring period. Little is known about the pathogenicity and aetiology of the Hymenochaetales taxa associated with esca in South Africa. All ten taxa were subjected to enzyme assays to determine which ligninolytic enzymes were secreted by each taxon. In addition, a field trial was undertaken to determine the pathogenicity of ten South African Hymenochaetales taxa associated with esca in grapevine. Twenty-seven fungal isolates and two negative controls were inoculated into mature grapevines and incubated for 24 months. The results of the enzyme assays indicated a difference in enzyme secretion between taxa and also among isolates of the same taxa. All isolates secreted cellulase and laccase, but there was a difference between isolates‟ ability to secrete manganese peroxidase and lignin peroxidase. The results of the pathogenicity trial showed that all of the isolates used were capable of causing the characteristic white rot symptom in the wood. There were also clear differences in susceptibility to white rot between the two cultivars tested. Cultivars also differed in which taxa proved pathogenic. On Shiraz, Taxon 6 (an Inonotus sp.), Phellinus resupinatus and Inonotus setuloso-croceus were significantly virulent. On Mourvédre, however, Taxon 3, an Inocutis sp. and Taxon 2, a Fomitiporella sp. were significantly virulent. Cultivar differences could be due to various factors, including differences in host response to colonisation and physical differences in wood structure, as well as the differences in enzyme secretion between taxa.

AFRIKAANSE OPSOMMING: Esca is „n siekte-kompleks op wingerd wat gekarakteriseer word deur verskeie blaar- en houtsimptome. Die siekte word veroorsaak deur verskeie patogene. Een van die onderskeidende kenmerke van die siekte op ouer wingerd is die voorkoms van wit hout-verrotting. Hout-verrotting wat met esca geässosieer word, word veroorsaak deur verskeie houtverrottende basidiomycete wat behoort aan die orde Hymenochaetales. Die Hymenochaetales spesie wat met esca geässosieer word, verskil na gelang van geografiese area. In Europa en die Mediterreense area, is Fomitiporia mediterranea die hoofspesie geässosieer met esca, terwyl Inocutis jamaicensis en “Fomitiporella vitis” hoofsaaklik in Argentinië en Chile, onderskeidelik, voorkom. In Australië is Fomitiporia australiensis die hoofspesie, en in die Verenigde State is Fomitiporia polymorpha al vantevore met esca geässosieer. „n Voorafgaande studie het ten verskillende taxa in die genera Fomitiporia, Phellinus, Inocutis, Inonotus and Fomitiporella gevind vanuit esca-geïnfekteerde wingerd. Die doel van hierdie studie was om hierdie taxa te karakteriseer, sowel as om hulle epidemiologie en patogenisiteit te ondersoek. Die studie het drie nuwe spesie gekarakteriseer, nl. Fomitiporella viticola, Fomitiporia capensis en Phellinus resupinatus. Die studie het ook „n verdere spesie, Inonotus setuloso-croceus, vir die eerste keer op Vitis vinifera en Salix spp. in Suid Afrika en wereldwyd aangemeld. Die sporulasie van F. viticola is ondersoek gedurende twee seisoene. Die patogenisiteit van al tien taxa is ondersoek op wingerd in die veld, en ensiem-vrystelling deur al tien taxa is ondersoek. Hierdie studie het ten doel gehad om „n bydrae te lewer tot verdere kennis van die aard van die esca siekte-kompleks in Suid Afrika, asook die breër kennis rakende die ekologie van die Hymenochaetales. „n Nuwe Fomitiporia spesie, Fomitiporia capensis, is beskryf op grond van vrugliggaam morfologie en „n gekombineerde rRNA ITS1-5.8S-ITS2 (ITS) en groot subeenheid (LSU) filogenie, waar dit „n duidelike en goed ondersteunde groep gevorm het. Morfologies was F. capensis eenders aan F. punctata in dat albei spesies basies geen setae gehad het nie. Fomitiporia capensis, F. punctata en F. aethiopica het eenderse basidiospore gevorm, maar het verskil in terme van gasheer, porie-grootte en vrugliggaam vorm. F. capensis is waarskynlik filogeneties verwant aan F. tenuis, maar die spesies verskil in dat F. tenuis kleiner porieë en basidiospore het. Gedurende die studie is F. capensis in die hele Wes Kaap provinsie gevind. Vrugliggame was skaars in vergelyking met miselium wat vanuit simptomatiese wingerdstokke geïsoleer is. Vrugliggame is ook in „n wingerd in Limpopo gevind. Phellinus resupinatus is beskryf op grond van vrugliggaam morfologie en ITS en LSU filogenieë. Dit het „n goed-ondersteunde groep gevorm wat naby verwant aan P. bicuspidatus, „n spesie wat geässosieer word met wit vrot in eikebome in die Verenigde State. P. resupinatus word morfologies gekarakteriseer deur sy resupinate vrugliggaam-vorm, reguit-vormige ventricose setae en ellipsoid, hyaline basidiospore. Dit is skegs gevind op sieklike wingerdstokke in die somer-reënval areas in Suid Afrika, meestal in die Noord Kaap, maar ook in Limpopo. Fomitiporella viticola is beskryf vanaf Vitis vinfera gebaseer op vrugliggaam morfologie en ITS filogenie. Dit word gekarakteriseer deur „n resupinate tot effuse-reflexed vrugliggaam met groot, wyd-gespasieërde porieë en redelike klein gelerige basidiospore. Inonotus setuloso-croceus is op Salix en Vitis vingera gevind en is geïdentifiseer deur middel van vrugliggaam-morfologie. Die ITS area is gekarakteriseer vanuit DNA wat geïsoleer is vanaf miselium kulture of vrugliggame, en is dieselfde as die Hymenochaetales spesie vroeër bekend as Taxon 7. Die ontdekking van I. setulosocroceus op wingerd en Salix ondersteun die hipotese dat vrugliggame dalk op alternatiewe gashere gevind kan word. Fomitiporella viticola is gereeld geïsoleer uit wit vrot op wingerd geäffekteer deur esca en vrugliggame is algemeen gevind in die Wes Kaap. Twaalf vrugliggame van F. viticola is weekliks gemonitor vir sporulasie gedurende twee seisoene tussen winter en vroeë somer. Vlakke van sporulasie het „n vlou positiewe korrelasie met reënval, en „n swak negatiewe korrelasie met temperatuur gehad. Sporulasie het plaasgevind gedurende die hele moniterings-periode. Min is bekend rakende die patogenisiteit en etiologie van die Hymenochaetales taxa geässosieerd met esca. Al tien taxa is onderwerp aan ensiem toetse om te bepaal watter ensieme deur elke taxon afgeskei word. „n Veld-proef is ook onderneem om patogenisiteit te bepaal vir al tien taxa. Sewe en twintig swam-isolate en twee negatiewe kontroles is geïnokuleer in wingerdstokke en geïnkubeer vir 24 maande. Die resultate van die ensiem-toetse het aangedui dat daar „n verskil in ensiem-sekresie tussen die verskeie taxa, asook tussen verskillende isolate binne dieselfde taxa, is. Alle isolate het cellulase en laccase afgeskei, maar daar was „n verskil in isolate se vermoë om manganese peroxidase en lignin peroxidase af te skei. Die resultate van die patogenisiteitstoets het gedui daarop dat alle isolate wat in die toets gebruik word wel die vermoë het om die wit vrot te vorm. Daar was duidelike verskille in vatbaarheid teen wit vrot tussen die twee cultivars in die toets. Die patogeniese taxa het ook verskil tussen die twee cultivars. In Shiraz was Taxon 6 („n Inonotus spesie), Phellinus resupinatus en Inonotus setuloso-croceus beduidend virulent. In Mourvédre was Taxon 3 („n Inocutis spesie) en Taxon 2, „n Fomitiporella spesie, beduidend virulent. Cultivar-verskille kan as gevolg van verskeie faktore wees, insluitende verskille in gasheer-respons op kolonisering en fisiese verskille in hout-struktuur, sowel as die verskille in ensiem sekresie tussen taxa.

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