Isolation and characterisation of phytotoxins produced by the Botryosphaeriaceae and their role in grapevine trunk diseases

Botryosphaeria dieback (BD) is considered a serious problem worldwide causing cankers, dieback and eventually death of vines. Foliar symptoms due to BD have been reported in some vineyards overseas. The foliar symptoms caused by grapevine trunk diseases are usual-ly associated with the production of phytotoxic metabo-lites (PMs) produced by the pathogen that are translocat-ed into the leaves. However, foliar symptoms due to BD have not been reported in Australian vineyards to date. This study investigated the production of PMs by the most virulent and widespread Botryosphaeriaceae spe-cies associated with BD in Australia. Isolates of Diplodia mutila, D. seriata, Dothiorella vidmadera, Neofusicoccum australe, N. luteum and Spencermartinsia viticola were grown in liquid media and PMs were extracted from the culture filtrates. The PMs were purified using a chroma-tographic method while their chemical characterization was performed by spectroscopic method, essentially 1D and 2D NMR. The purified PMs from all species showed different degrees of toxicity when tested on grapevine leaves at different concentrations. R-(-)-mellein and other well-known PMs were isolated from D. mutila, D. seriata, N. australe and N. luteum culture filtrates. Tw o novel toxic quinones (diploquinone A and B) were isolat-ed from the culture filtrates of D. mutila while two novel compounds, spencertoxin and spencer acid, were isolat-ed from the culture filtrates of S. viticola. Furthermore, six polyphenols were isolated from the culture filtrate of D. vidmadera, one being characterized as 5-hydroxym-ethyl-2-isopropoxyphenol, a novel compound. The isola-tion and characterization of the PMs secreted by these fungi are fundamental to understanding the potential role of these PMs in the virulence and symptomatology of Botryosphaeria dieback pathogens in grapevines.