Secondary metabolite production by the fungal pathogen Eutypa lata: Analysis of extracts from grapevine cultures and detection of those metabolites in planta

Eutypa dieback of grapevines is caused by the fungal pathogen Eutypa lata and reduces vineyard longevity worldwide. Early detection could reduce losses due to this disease, so our aim was to identify acetylenic phenol metabolites of E. lata that could prove suitable as chemical markers in an early diagnostic test for the pathogen. Accordingly, secondary metabolite production by 30 isolates of E. lata grown on media derived from canes of three grapevine cultivars was analysed using HPLC. Six metabolites, namely eutypinol, methyl eutypinol, eulatachromene, eutypine, 2-iso-propenyl-5-formylbenzofuran and eulatinol, were detected in culture filtrates. Most abundant were eutypinol and methyl eutypinol, produced by 97 and 83% of isolates, respectively. There was no apparent correlation between secondary metabolite production on media containing milled canes from the three cultivars of grapevine, and the field tolerance of these same cultivars to Eutypa dieback. When various other fungi commonly isolated from grapevine trunks in Australia were grown on milled cane, no secondary metabolites characteristic of E. lata were detected, suggesting such compounds are specific to E. lata. To examine the detection of secondary metabolites in planta, micropropagated grapevine plantlets were treated with purified or crude culture filtrates from nine isolates of E. lata grown on malt yeast broth. Various secondary metabolites were identified in treated plantlets, however, no single compound was detected consistently. Eutypinol was detected in micropropagated grapevine plantlets inoculated with mycelium of E. lata, however, no metabolites were detected in the sap of vines which had been artificially inoculated with the pathogen. 108 Secondary metabolite production by E. lata Australian Journal of Grape and Wine Research 12, 107–114, 2006 foliar symptoms are most evident. Hence, diagnosis based on identifying specific metabolites of E. lata in planta may enable early and more reliable detection of the pathogen before it spreads extensively throughout the vine. The phytotoxin eutypine was originally implicated as the compound responsible for the foliar symptoms of Eutypa dieback (Tey Rulh et al. 1991). However, we previously found that eutypine is not produced by all isolates of E. lata, and that it readily undergoes facile cyclisation to a benzofuran compound (Molyneux et al. 2002). Furthermore, E. lata produced a range of other metabolites in artificial culture. Previous studies have shown that these metabolites have varying degrees of phytotoxicity, with some compounds, for example eutypinol and siccayne, showing no phytotoxicity when applied to grapevine leaf discs and others, including eutypine and eulatachromene, causing widespread necrosis of leaf discs (Mahoney et al. 2003, Smith et al. 2003). We also found that the amount and type of metabolite produced varied according to the fungal isolate and growth medium (Mahoney et al. 2003, Mahoney et al. 2005). Originally, we hoped that a chemical marker could be detected remote from the site of infection, in the sap or foliage of infected vines. We anticipated that low levels of metabolites of E. lata would be present before foliar symptoms became visible, and that this could be used as an early diagnostic tool for the pathogen. However, working in vivo, metabolites of E. lata have so far only been found within the woody tissues of infected vines. Those metabolites could nevertheless prove diagnostic, and here we report on a series of experiments conducted to clarify what metabolites were consistently produced by E. lata in artificial culture and to obtain information on detecting these metabolites in planta. Because E. lata is an out-crossing species that displays considerable genetic and pathogenic variation (Péros and Berger 2003, Sosnowski et al. 2006), we analysed 30 isolates to obtain a representative picture of secondary metabolite production by the pathogen. Firstly, we analysed secondary metabolite production by 30 isolates of E. lata which were grown on milled cane from three cultivars of grapevine. Our goals were (1) to identify a chemical marker that was produced by the majority of isolates of E. lata and (2) to determine whether cultivar tolerance to Eutypa dieback influenced the amount or type of secondary metabolite produced. We also analysed secondary metabolite production by 18 isolates of other grapevine-inhabiting fungi after growth on milled grapevine cane to confirm that the metabolites detected here were specific to E. lata. Secondly, we grew nine isolates of E. lata on malt yeast broth, previously shown to be conducive to the production of a range of metabolites by E. lata. Micropropagated grapevine plantlets were then treated with purified or crude filtrates taken from these cultures. Our aim was to see whether metabolites of E. lata could be detected in grapevine tissue, and to gather information about the fate of such metabolites in planta. Thirdly, to determine whether chemical markers indicative of E. lata could be detected in infected vines, we used HPLC to identify such metabolites in both micropropagated and potted vines which had been previously inoculated with mycelium from E. lata.