Antibiosis has been shown to be an important mode of action by Trichoderma species used in the protection of grapevine pruning wounds from infection by trunk pathogens. The major active compound from Trichoderma isolates known to protect grapevine pruning wounds from trunk pathogen infection was isolated and identified. The compound, a 6-pentyl-a-pyrone (6PP), was found to be the major secondary metabolite, by quantity, which accumulated in the culture filtrate of T. harzianum isolate T77 and the two T. atroviride isolates UST1 and UST2. Benzimidazole resistant mutants generated from these isolates also produced 6PP as their main secondary metabolite, except for a mutant of T77 that had lost its ability to produce 6PP. The isolates UST1 and UST2 were co-cultured with the grapevine trunk pathogens Eutypa lata and Neofusicoccum parvum in a minimal defined medium and a grapevine cane-based medium (GCBM). Co-culturing UST1 with N. parvum induced 6PP production in the minimal defined medium and the GCBM. The production of 6PP by UST2 was induced in the GCBM, while co-culturing with the two trunk pathogens either reduced or had no effect on 6PP production. Mycelial growth and ascospore/conidia germination of E. lata, N. australe, N. parvum and Phaeomoniella chlamydospora were inhibited by 6PP in a concentration-dependent manner. The results show that the presence of N. parvum and grapevine wood elicits the production of 6PP, suggesting that this metabolite is involved in Trichoderma-pathogen interactions on grapevine pruning wounds.

Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

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Background and Aims The application of Trichoderma species on grapevine pruning wound surfaces reduces wound infection from trunk pathogens that cause grapevine decline. The aim of this study was to determine the efficacy of pruning wound treatments containing Trichoderma as influenced by the time of pruning and time of application following pruning. Methods and Results Cultivars Chenin Blanc and Cabernet Sauvignon were pruned early and late in the winter season, respectively, corresponding to the break from the winter dormancy period and the normal pruning time for these cultivars. After pruning, the wounds were treated immediately, 6, 24, 48 or 96 h after pruning with either T. atroviride or T. harzianum. Colonisation of grapevine pruning wounds by the Trichoderma spp. was dependent on the physiological state of the vines as well as the weather conditions at pruning. In dormant vines, colonisation remained high from immediate application up to 48 h after pruning. In vines at break of dormancy, colonisation was highest at 6 and 24 h after application. Natural wound infection was higher in wounds pruned in late winter compared with that in early winter. Conclusions Applying the biocontrol agent 6 h after pruning consistently resulted in a high incidence of Trichoderma spp. in both cultivars at either early or late pruning regardless of vine physiological state or the weather conditions. Significance of Study Pruning early in the season in combination with the application of Trichoderma spp. pruning‐wound agents approximately 6 h after pruning can significantly reduce wound infection by trunk pathogens.

Grapevine trunk disease fungi infect vines through openings, primarily pruning wounds. The main objective of this study was to understand the role of sucker wounds and wounds made by the removal of green shoots from the stems of potted grapevines as potential points of infection for grapevine trunk disease pathogens. Six wine and four table grape vineyards of different ages were sampled in different production areas in the Western Cape grape region of South Africa. Isolations were made from 161 sucker wounds, and fungal pathogens were identified using morphology and DNA sequence analysis of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal RNA gene, the translation elongation factor 1alpha or the partial β-tubulin gene. The results show that 62% of the sucker wounds were infected by trunk disease pathogens, including Diaporthe ampelina, Diplodia seriata, Phaeomoniella chlamydospora, Phaeoacremonium minimum, Eutypella microtheca, Cryptovalsa ampelina and Neofusicoccum australe. Diaporthe ampelina was the most common, followed by D. seriata and P. chlamydospora, in both the wine and table grape sucker wounds. Under glasshouse conditions, wounds made by the removal of young green shoots on one-year-old potted grapevine plants were inoculated with spore suspensions of D. ampelina, E. lata, N. parvum, P. minimum and P. chlamydospora. After four months, all the inoculated pathogens could be re-isolated. This study shows that grapevine sucker and green shoot wounds are susceptible to different grapevine trunk disease pathogens and may therefore play a role in the epidemiology of trunk diseases.