Investigating the production and translocation of phytotoxic metabolites by Australian Botryosphaeriaceae spp. in artificially-inoculated and naturally-infected vines

Grapevine trunk diseases (GTDs) caused by pathogenic fungi are considered a serious problem to worldwide. Some GTD pathogens produce phytotoxic metabolites (PMs) that play an important role in their virulence. It is hypothesized that these PMs are translocated into the leaves causing foliar symptoms. However, foliar symp- toms in vines affected with Botryosphaeria dieback have never been reported in Australian vineyards. A previous investigation demonstrated that different PMs, including R-(-)-mellein, were produced in vitro by Australian Bot- ryosphaeriaceae isolates.




molecular and analytical chemistry techniques. Wood samples from vines naturally-infected with Botryospha- eria dieback were collected from three vineyards in New South Wales, Australia. For artificially-inoculated vines, one-year-old rootlings (cv. Chardonnay and Cab- ernet Sauvignon) were inoculated with Diplodia seriata, Spencermartinsia viticola and Dothiorella vidmadera. All symptomatic and asymptomatic wood samples were analysed by cultural isolations and qPCR to detect and quantify the pathogens. An LC-MS/MS protocol was optimized for the detection of spencertoxin, protocat- echuic alcohol and R-(-)-mellein from infected wood. R-(-)-mellein was detected by LC-MS/MS but was only present in symptomatic wood and its amount correlated with the amount of pathogen DNA detected by qPCR. These results suggest that this PM was not translocated as previously hypothesized. The inability to detect the other two PMs could be explained by the following: they could be under the detection limit, they could be pro- duced during the later stages of infection, they could be broken down into other compounds or detoxified by the plant. Our findings suggest that R-(-)-mellein may not be directly involved in the development of foliar symptoms and these symptoms may be caused by a combination of diverse factors which require more in-depth studies.