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This study assessed the infection rates of different spore inoculum doses of the grapevine trunk pathogens Diplodia seriata, Phaeomoniella chlamydospora and Eutypa lata following artificial inoculation of pruning wounds. Potted vines of cv. Tempranillo were inoculated with doses ranging from 10 to 4000 conidia per wound of D. seriata and P. chlamydospora and led to recovery percentages of 10–100% for D. seriata and 16–94% for P. chlamydospora. Eutypa lata, when inoculated onto wounds of vines in a mature vineyard (cv. Shiraz) and on detached canes (cv. Cabernet Sauvignon) with a dose range of 10 to 1000 ascospores per wound, led to recovery percentages of 17–95%. In the field assay, there was no difference in recovery from wounds that were exposed to single or double inoculations with the same total spore dose, or between canes that were harvested 7 or 11 months after inoculation. The results obtained in this study showed significant variability in pathogen recovery between trials, comparable with that reported previously, which suggests that factors such as pathogen virulence, environmental parameters and experimental conditions may influence the infection process. According to this study, in order to obtain optimal recovery percentages of 50–70% for robust evaluation of pruning wound treatments, dose ranges of 100-1000 conidia of D. seriata, 100–2000 conidia of P. chlamydospora, and 100–500 ascospores of E. lata per wound would be required.