Characterisation of Cylindrocarpon spp. associated with black foot disease of grapevine

[摘要] ENGLISH ABSTRACT: During the past few years a drastic reduction has been noted in the survival rate ofgrafted grapevines in nurseries, as well as in young vineyards in the Western Cape Province ofSouth Africa. Circumstantial evidence suggested that Cylindrocarpon spp., which cause blackfoot disease of grapevine, were associated with this decline. Black foot disease of grapevine is arelatively new, and as yet poorly known disease affecting vines in various countries wheregrapevines are cultivated. Primary aims of this research have been (1) to conduct nurserysurveys in order to determine which fungi are involved in the decline phenomenon, with specialreference to the involvement of Cylindrocarpon spp., (2) to identify and characterise theorganisms believed to be the causal organisms of black foot disease, and (3) the development ofcontrol and/or management strategies to prevent or eradicate Cylindrocarpon infections.Nursery grapevines were sampled at different stages from three commercial nurseries inthe Wellington area of the Western Cape Province and were investigated during the 19992000season by means of destructive sampling. The first samples were taken in September fromcallused cuttings prior to planting in nurseries. After planting, asymptomatic rooted cuttingswere selected from nurseries after 3, 6 and 9 months. Isolation studies clearly demonstrated thatdifferent 'Cylindrocarpon spp. infected cuttings from nursery soils. These species rarelyoccurred in rootstock propagation material prior to planting. At the time of planting, thesusceptible basal ends (especially the pith area) of most of the nursery cuttings are partly or evenfully exposed. Callus roots also break during the planting process, resulting in small woundssusceptible to infection by soilborne pathogens. The isolation studies revealed that the firstinfections occurred in the roots, followed by infections of the rootstocks. These infectionsincreased progressively during the course of the growing season.Substantial variation in cultural and morphological characters was observed among theCylindrocarpon isolates obtained from the nursery survey, as well as from isolations that weremade from diseased grapevines. Morphological and phylogenetic studies were conducted toidentify these 'Cylindrocarpon spp. and to establish their association with black foot disease.Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial β-tubulin geneintrons and exons were used for phylogenetic inference. Phylogenetic analyses confirmed thediversity observed among the isolates and four Cylindrocarpon-like species were identified.One of these species was initially identified as Cylindrocarpon destructans. However, furtherresearch revealed C. destructans to represent a species complex. Grapevine isolates of 'C.destructans proved to be identical to the ex-type strain of Cylindrocarpon liriodendri, whichalso produced a teleomorph, Neonectria liriodendri in culture. A second species was newlydescribed in this study as Cylindrocarpon macrodidymum (Neonectria macrodidyma). The tworemaining Cylindrocarpon-like species were placed in a new genus, Campylocarpon. The twospecies were named Campylocarpon fasciculare and Campylocarpon pseudofasciculare.Pathogenicity studies confirmed that all four species were able to reduce root and shoot masssignificantly.Knowledge obtained pertaining to the disease cycle of black foot disease suggest that suitablemanagement strategies should focus on prevention of primary infection in nurseries. However,at present, no fungicides are registered for control of this disease in South African vineyards ornurseries. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens.Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effectivefungicides tested, and were subsequently included in semi-commercial field trials. Basal ends ofgrafted cuttings were dipped (1 min) in various chemical and biological treatments prior toplanting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttingsprior to planting in nurseries. Additional treatments involved soil amendments withTrichoderma formulations and hot water treatment (50°C for 30 min) of dormant nurserygrapevines. Field trials were evaluated after a growing season of eight months. The incidence ofblack foot pathogens was not significantly and/or consistently reduced by the majority ofchemical or biological treatments. However, these pathogens were not isolated from uprootedplants that were subjected to hot water treatment. It is therefore recommended that hot watertreatment of dormant nursery plants be included in an integrated strategy for the proactivemanagement of black foot disease in grapevine nurseries.

[发布机构] Stellenbosch University