Characterization and control of Phaeomoniella chlamydospora in grapevines

[摘要] ENGLISH ABSTRACT:Petri grapevme decline, also known as black goo, slow die-back andPhaeoacremonium grapevine decline, causes significant losses of young vines worldwide.Species of Phaeoacremonium, Phaeomoniella chlamydospora and relatedgenera are associated with this grapevine disease. This study investigates thePhaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on thespecies isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and thepossibility of employing molecular techniques for the detection of Pa. chlamydosporain grapevines were also investigated.In an overview of the literature on Petri grapevine decline the disease historyand the relatedness of Petri grapevine decline to esca is discussed. Petri grapvinedecline occurs in propagation material or young vines. Infected material can appearasymptomatic and therefore the possibilities of molecular techniques for identificationwere also investigated in the literature.In South Africa Pa. chlamydospora is the dominant organism causing Petrigrapevine decline and therefore different fungicides were evaluated to control thisfungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, SomersetWest and Malmesbury of Western Cape province, South Africa, were screenedagainst twelve fungicides testing their effect on mycelial inhibition in vitro. Thesefungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione,kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene,tebuconazole and thiram. Results provided the base-line sensitivity of South Africanisolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochlorazmanganese chloride and tebuconazole were the most effective (with EC50 valuesranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa.chlamydospora in vitro. This in vitro test gave a good indication of which fungicidescould be selected for further studies in glasshouses and nurseries.The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolatesfrom grapevines of South Africa, or isolates obtained from the Centraalbureau voorSchimmelcultures (CBS) in the Netherland, were investigated. Sequence data werecreated from the rONA region and partial B-tubulin gene of 33 of these isolates usingthe PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a.An analysis of the sequence data confirmed the genus Phaeomoniella to be distinctfrom Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologicallysimilar, the species status of Pm. aleophi/um and Pm. angustius was confirmed withDNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growthrate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimumgrowth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to besynonymous with Pm. angustius, and a new species, Pm. mortoniae, was newlydescribed from grapevine occurring in California. Futhermore, Pm. aleophilum wasnewly reported from South Africa and grapevine isolates thought to be Pm. inflatipeswere all re-identified as Pm. aleophilum. These findings therefore also shed somedoubt on the possible role of Pm. inflatipes in Petri grapevine decline. It wasconfirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the speciesinvolved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, butits pathogenicity should still be confirmed and the role of Pm. injlatipes in Petrigrapevine decline remains unclear.Pa. chlamydospora has been routinely isolated from symptomless propagationand nursery material. Because the disease can take years to develop, it is crucial thathealthy propagation material is used at planting. Pa. chlamydospora is a slowgrowingfungus, and positive identification from symptomless grapevine tissue cantake up to 4 wks. The possibility of employing molecular techniques for the detectionof Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecificprimers (PCLI and PCL2) based on the regions ITSI and ITS2 were designedfor Pa. chlamydospora. These primers were highly sensitive and amplification wasachieved from genomic DNA of Pa. chlamydospora from as low as 16 pg.Phaeoacremonium spp., related genera and common fungal taxa from grapevineswere tested with these primers, but positive amplification was achieved for Pa.chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevinetissue culture plants was confirmed by PCR within 24 hours. These primers thereforeallow rapid and accurate identification of Pa. c~lamydospora. Testing on a largerscale with nursery material should be conducted to determine the feasibility of usingthese species-specific primers in the grapevine industry.

[发布机构] Stellenbosch University