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• Biological control agents were tested in the field in almond, cherry, and grapevine. • Trichoderma atroviride SC1 protected efficiently grapevine pruning wounds. • Clonostachys rosea J1446 protected efficiently almond and cherry pruning wounds. The ascomycete fungi that cause wood canker diseases in perennial, horticultural crops are often generalist pathogens able to infect multiple host plants. These pathogens frequently infect pruning wounds, and the protection of such wounds with chemicals or biological control agents (BCAs) is an essential component of disease management. This study aimed at assessing four commercial BCAs for their ability to prevent almond, cherry, and grapevine pruning wound infections by three canker pathogens under field conditions for two growing seasons: Calosphaeria pulchella in cherry, Neofusicoccum parvum in almond and grapevine, and Eutypa lata in all three hosts. The two bacterial BCAs tested, Bacillus amyloliquefasciens MBI 600 and Streptomyces lydicus WYEC 108, performed poorly overall as infection rates in treated wounds were not significantly different than in the water-treated wounds for most pathogens in all crops. Trichoderma atroviride SC1 provided the best protection against E. lata and N. parvum in grapevine, achieving superior or equivalent protection than the standard conventional chemical thiophanate-methyl. Field trials also demonstrated that Clonostachys rosea J1446 provided satisfactory protection of pruning wounds against E. lata and C. pulchella in cherry and against E. lata and N. parvum in almond trees. The identification of effective BCAs for use in multiple crops and against multiple pathogens is a great step towards a sustainable management of fungal canker diseases by mitigating the negative impacts of chemical fungicides. [ABSTRACT FROM AUTHOR]