Grapevine trunk diseases (GTDs) are a serious problem of grapevines worldwide. The microbiota of the grapevine endosphere comprises prokaryotic and eukaryotic endophytes, which may form varied relationships with the host plant from symbiotic to pathogenic. To explore the interaction between grapevine endophytic bacteria and GTDs, the endomicrobiome associated with grapevine wood was characterized using next-generation Illumina sequencing. Wood samples were collected from grapevine trunks with and without external symptoms of GTD (cankers) from two vineyards in the Hunter Valley and Hilltops, NSW, Australia and metagenomic characterization of the endophytic community was conducted using the 16S rRNA gene (341F/806R) and ITS (1F/2R) sequences. Among the important GTD pathogens, Phaeomoniella, Phaeoacremonium, Diplodia and Cryptovalsa species were found to be abundant in both symptomatic and asymptomatic grapevines from both vineyards. Eutypa lata and Neofusicoccum parvum, two important GTD pathogens, were detected in low numbers in Hilltops and the Hunter Valley, respectively. Interestingly, Pseudomonas dominated the bacterial community in canker-free grapevine tissues in both locations, comprising 56-74% of the total bacterial population. In contrast, the Pseudomonas population in grapevines with cankers was significantly lower, representing 29 and 2% of the bacterial community in Hilltops and the Hunter Valley, respectively. The presence of Pseudomonas in healthy grapevine tissues indicates its ability to colonize and survive in the grapevine. The potential of Pseudomonas spp. as biocontrol agents against GTD pathogens was also explored. Dual culture tests with isolated fluorescent Pseudomonas against mycelial discs of nine Botryosphaeria dieback, three Eutypa dieback, and two Esca/Petri disease pathogens, revealed antagonistic activity for 10 Pseudomonas strains. These results suggest the potential of Pseudomonas species from grapevine wood to be used as biocontrol agents to manage certain GTD pathogens.

Alternaria alternata is the causal organism of core rot decay symptoms in susceptible cv. Red Delicious but not in resistant cv. Golden Delicious. The two cultivars did not differ in natural colonization of the style and ovary during the first week after full bloom; colonization of the ovary in the susceptible cultivar subsequently decreased with increasing distance from the calycine tube. By 30 days after full bloom, Alternaria recovery from ovary 1, adjacent to the end of the calycine tube, was 100 and 40% in the susceptible and resistant cultivars, respectively. In the susceptible cultivar, Alternaria recovery decreased from 75 to 20% in ovaries 2, 3, and 4, while there was only minor incidence in the resistant cultivar. Inoculation of the mesoderms of the two cultivars induced similar decay symptoms, but inoculated locules of Red Delicious were more susceptible than those of Golden Delicious. Increased inoculum concentration or isolate virulence enhanced the difference in locule susceptibility between the cultivars. Inoculation on isolated seed locules or on media amended with susceptible locule tissue as a carbon source induced greater transcript levels of several genes than the inoculation on resistant tissue. Endo- and exoglucanase activity levels were higher at pH 4.8 than at 4.2, conditions typical of the mesoderm adjacent to the seed locules of the susceptible and resistant cultivars, respectively. Current results suggest that susceptibility of Red Delicious apples to core rot decay is dependent on the sensitivity to locule colonization and on mesoderm pH, a factor that enhances fungal virulence.

Grapevine trunk diseases (GTDs) are considered a serious problem to viticulture worldwide. Several GTD fungal pathogens produce phytotoxic metabolites (PMs) that were hypothesized to migrate to the foliage where they cause distinct symptoms. The role of PMs in the expression of Botryosphaeria dieback (BD) symptoms in naturally infected and artificially inoculated wood using molecular and analytical chemistry techniques was investigated. Wood samples from field vines naturally infected with BD and one-year-old vines inoculated with Diplodia seriata, Spencermartinsia viticola and Dothiorella vidmadera were analysed by cultural isolations, quantitative PCR (qPCR) and targeted LC-MS/MS to detect three PMs: (R)-mellein, protocatechuic acid and spencertoxin. (R)-mellein was detected in symptomatic naturally infected wood and vines artificially inoculated with D. seriata but was absent in all non-symptomatic wood. The amount of (R)-mellein detected was correlated with the amount of pathogen DNA detected by qPCR. Protocatechuic acid and spencertoxin were absent in all inoculated wood samples. (R)-mellein may be produced by the pathogen during infection to break down the wood, however it was not translocated into other parts of the vine. The foliar symptoms previously reported in vineyards may be due to a combination of PMs produced and climatic and physiological factors that require further investigation.

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