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Final Report Is Submitted Industry allocated project number SATI CFPA SAAPPA/SASPA DFTS Winetech [email protected] [email protected] [email protected] [email protected] [email protected] Tel: 021 872-1438 Tel: 021 872-1501 Tel: 021 882-8470 Tel: 021 870 2900 Tel: 021 807 3387 X Indicate (X) client(s) to whom this final report is submitted. Replace any of these with other relevant clients if required . ______________________________________ FINAL REPORT 2013 Programme & Project Leader Information Research O rganisation Project leader Programme leader Title, initials, surname Roleen Carstens Dr Francois Halleen Present position Acting Research Leader: Plant Specialist Researcher Protection Address ARC Infruitec-Nietvoorbij ARC Infruitec-Nietvoorbij Private Bag X5026 Private Bag X5026 Stellenbosch Stellenbosch 7599 7599 Tel. / Cell no. (021) 809 3023 (021) 809 3040 Fax (021) 809 3002 (021) 809 3002 E-mail [email protected] [email protected] Project Information Research Organisation WW 06-37 Project number Project title The epidemiology and etiology of fungi associated with Esca disease of grapevine Fruit kind(s) Wine grapes Start date (mm/yyyy) 01/04/2009 End date (mm/yyyy) 31/03/2013 Project keywords Wood rot, esca, Basidiomycetes, Hymenochaetales, Vitis vinifera Approved by Research Organisation Programme leader (tick box) x This document is confidential and any unauthorised disclosure is prohibited. Final report 2 THIS REPORT MUST INCLUDE INFORMATION FROM THE ENTIREENTIRE PROJECT Executive Summary Give an executive summary of the total project. Internal and external symptoms associated with esca have been characterised and the fungi associated with each symptom type have been identified. Esca is a grapevine disease believed to be caused by a complex of fungal pathogens, namely Phaeomoniella chlamydospora , Pheaoacremonium spp. and Basidiomycete spp. (wood rot fungi). Before this study, the Basidiomycete spp. associated with the disease in South Africa were unknown. Basidiomycete isolates were obtained from 37 grape growing areas representative of all the main grape growing areas of South Africa. Phylogenetic analysis revealed 10 “unknown” Basidiomycete groups (taxa) all of which needed to be described. Phaeomoniella chlamydospora and six species of Phaeoacremonium including P. aleophilum , P. alvesii , P. parasiticum , P. iranianum, P. mortoniae and P. sicilianum were isolated of which the latter three were reported for the first time in South Africa. Other taxa that were found in association with esca vines include Eutypa lata , Phomopsis viticola , Phomopsis sp. 1, Diaporthe ambigua , Diplodia seriata , Neofusicoccum australe and N. parvum . Attempts to link several Basidiomycete fruiting bodies found in South African vineyards with the 10 taxa in order to conduct the taxonomic studies led to three successfull descriptions, namely Taxon 1, a new Fomitiporella species, Fomitiporia capensis and Phellinus resupinatus , as well as the successful identification of Taxon 7 and 8 as Inonotus setulosocroceus and Inonotus rickii / Ptychogaster cubensis , respectively. Pathogenicity studies with representative isolates of the 10 new Basidiomycete taxa clearly showed that all ten have the ability to survive within grapevine wood after inoculation and cause internal discolouration and rot. Aspects regarding the infection pathway of Basidiomycetes and the toxins and enzymes they produce were also investigated. Leaf scorching was observed on potted glasshouse vines after pruning wounds were inoculated with basidiospores obtained from Taxon 1 and 7. Spore trapping studies conducted in two vineyards in Stellenbosch also showed that basidiospores were released at a high rate from the end of May to the end of September whereafter it declined towards summer. However, this period coincides with the time when farmers prune (July/August) and remove unwanted shoots (September -November). Susceptible wounds could therefore be prone to infection if not treated properly. Representative isolates of all ten Basidiomycete taxa were able to produce 4-hydroxy- benzaldehyde, a toxin thought to be involved in pathogenicity. Basidiomycete species associated with grapevines from around the world were obtained, as well as four Fom. mediterranea isolates with a known history of 4-hydroxy-benzaldehyde for comparison studies. However, after extensive studies were conducted with this compound we came to the conclusion that it is not of importance. Enzyme tests conducted with the new South African taxa and other esca-related pathogens mentioned above showed that these basidiomycetes produce enzymes which are able to degrade cellulose and lignin both of which are structural components of wood. Some of the Basidiomycete isolates were able to produce lignin peroxidase, and the majority of the isolates were able to produce manganese peroxidase and laccase. All the basidiomycete isolates were able to produce cellulase and none were able to produce xylanase. Therefore, South African isolates compare well with other well-known esca and esca-related pathogens . A PCR detection system was also developed for the discrimination between the 10 Basidiomycete taxa found in South African vineyards as well as other Basidiomycetes associated with esca and esca-related diseases from around the world. This document is confidential and any unauthorised disclosure is prohibited. Final report 3 This was the first in-depth study of the grapevine disease esca in South Africa. Problem identification and objectives State the problem being addressed and the ultimate aim of the project. Esca is a grapevine disease caused by a complex of fungal pathogens, namely Phaeomoniella chlamydospora , Pheaoacremonium spp. and Basidiomycete spp. (wood rot fungi) (Mugnai et al ., 1999). Previously it was believed that Stereum hirsitum and Phellinus igniarius were responsible for this disease in local vineyards (Marais, 1981), although no studies have ever been conducted to confirm this. Leaf symptoms are not common in South African vineyards. Consequently, the wrong assumption was made that esca does not occur very frequently and that it is not of great economic importance. Different wood rotting fungi produce different toxins and therefore it is possible that it could influence symptom expression (Sparapano et al ., 2000; Tabacchi et al ., 2000). It is therefore of cardinal importance to determine to what extent esca occurs in the various grape growing areas of South Africa and to identify the different fungi associated with the disease. The toxins produced by the Basidiomycete spp. must be identified and their role in symptom expression needs to be clarified. Wood rot fungi can have a huge impact on production and can reduce the productive lifespan of a grapevine significantly. The disease also has a direct effect on the quality of wine made from affected vines. Knowledge of the causal organisms and their infection pathways will also help to implement effective management strategies to control the disease. The aim of this project is therefore to identify the Basidiomycete fungi associated with Esca disease in local vineyards and to study their biology. Other fungal species directly associated with wood rot symptoms will also be identified (ie. Phomopsis , Phaeoacremonium and Botryosphaeriaceae). The pathogen status of the Basidiomycetes must be determined as well as the factors that contribute to pathogenesis, ie the production of secondary metabolites and hydrolytic enzymes involved in wood degradation. A molecular identification technique must also be developed for quick and accurate detection of the Basidiomycetes involved with esca. Workplan (materials and methods) List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage and examination stages and parameters. Milestone 1: Symptoms and fungi associated with esca in South African vineyards Sampling of esca diseased vines Vineyards showing foliar symptoms of esca, esca proper or general decline were identified in all the major wine, raisin and table grape production areas of South Africa between 2001 and 2008. The presence of external symptoms was not always recorded, since several vines were collected during winter when farmers traditionally remove old or unproductive vines. Vines showing typical tiger stripe foliar symptoms and also declining vines were cut open and those showing the whole range of internal symptoms (white rot and different types of discolourations) were removed and immediately taken to the laboratory where fungal isolations were made. Fungal isolations from diseased vines Cross and longitudinal sections were made at various places in the cordons and trunk of each plant to investigate internal necrosis. For fungal isolations, wood sections with internal necrosis were selected and cut into two smaller sections adjacent to each other, in order to obtain two mirror images of the same symptom type. This was also done to facilitate the use of two sterilisation techniques to ensure fungal isolation from soft, spongy material. A photograph of each wood section showing the various symptom types was taken. One section was flame sterilized by holding the wood with sterile forceps, lightly spraying it with 70% ethanol and passing it through a flame. The other piece was triple sterilized as follows: 30 s in 70% ethanol, 2 min in 3.5% NaOCl and 30 s in 70% ethanol. Twelve small sections of wood (1×1×2 mm) from This document is confidential and any unauthorised disclosure is prohibited. Final report
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