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

Grapevine trunk diseases (GTDs) represent a serious threat to vineyards worldwide causing substantial economic and yield losses. To date, no curative methods are available and the connection between pathogen and symptom expression is not completely understood. Among the GTDs, Botryosphaeria dieback (BD) is considered a serious problem worldwide causing cankers, dieback and eventually death of vines. At least 27 species of the Botryosphaeriaceae are associated with BD worldwide, although only 11 species have been reported in Australian vineyards to date. In Europe, the disease is also associated with foliar symptoms. However, these symptoms have not been reported in Australian vineyards to date. The expression of foliar symptoms is usually associated with phytotoxic metabolites (PMs) produced by the pathogen that are translocated into the leaves. Prior to this investigation, limited information was available on the PMs produced by Australian isolates of Botryosphaeriaceae spp. The most widespread and most virulent species of Botryosphaeriaceae involved in BD in South Australia (SA) and New South Wales (NSW) vineyards were selected. The chromatographic profiles
of organic extracts from culture filtrates of eight species of Botryosphaeriaceae showed the ability of all isolates to produce several and different metabolites. The phytotoxicity of the organic extracts varied among isolates and species when tested on grapevine leaves and tomato cuttings. The PMs produced by Diplodia seriata H141a, Dothiorella mutila DAR79137, Neofusicoccum australe DAR79506, N. parvum B19, and for the first time, those produced by N. luteum DAR81016,
Spencermartinsia viticola DAR78870 and Do. vidmadera DAR78993 were isolated and fully characterised by spectroscopic method (1D and 2D NMR and HR ESIMS). Furthermore, the exopolysaccharides (EPSs) produced by three Neofusicoccum spp. were isolated and assayed on grapevine leaves in vitro and all the EPSs were shown to be phytotoxic at different concentrations. The in planta production of PMs was investigated by molecular and analytical chemistry techniques. Wood samples from vines naturally-infected with BD were collected from three vineyards in NSW. In addition, one-year-old rootlings (cvs. Chardonnay and Cabernet Sauvignon) were inoculated with D. seriata H141, S. viticola DAR78870 and Do. vidmadera DAR78993. All symptomatic and asymptomatic wood samples were analysed by cultural isolations, quantitative PCR (qPCR) and LCQqQ in MRM mode. (R)-mellein was only detected in symptomatic wood samples and its amount was correlated with the amount of pathogen DNA detected by qPCR. These results suggest that (R)-mellein was not translocated throughout the wood as previously hypothesised, and foliar symptoms may be caused by a combination of diverse factors which require further in-depth studies. Genetic analysis of the pathogen genome was also attempted to identify pathogenicity and virulence genes involved in the production of (R)-mellein. The production of (R)-mellein by D. seriata A141a and N. parvum DAR78998 was confirmed by HPLC quantification. Several sets of primers previously developed for the amplification of the polyketide synthase (PKS) gene of Parastagonospora nodorum were optimised and tested on isolates of D. seriata and N. parvum. Only one set of primers was suitable for N. parvum. The BLASTn analysis for resulting sequences showed the highest similarity with the putative gene involved in the expression of the 6-methyl-salycilic acid (6-MSA) synthase protein in D. corticola and N. parvum. This protein is a PKS and it is likely that the amplified sequence encodes for the biosynthesis of a partially reduced polyketide (PK) compound similar to 6-MSA such as (R)-mellein. Based on these results, novel primers were designed, optimised and tested on the genomic DNA of D. mutila, D. seriata, Lasiodiplodia theobromae, Do. vidmadera, N. australe, N. luteum and N. parvum. The resulting PCR products were sequenced and had the highest homology to D. corticola PKSs involved in the synthesis of 6-MSA. However, these preliminary results should be confirmed by further investigations. To our knowledge, this was the first study to investigate the PMs produced by Botryosphaeriaceae spp. isolated from grapevines in Australia. Insights from this multidisciplinary research will provide knowledge that may assist in elucidating the role of PMs in the pathogenicity, symptom development and plant-pathogen interaction. Future investigation may result in methods being developed to better assist in field diagnosis and control of Botryosphaeria dieback in Australian vineyards.