Characterization of fungicide resistant isolates of Phaeoacremonium aleophilum infecting grapevines in Spain

•This is the first report of carbendazim and flusilazole resistance in Phaeoacremonium aleophilum isolated from Spanish vineyards.•No AFLP markers were associated with resistant P. aleophilum single-spore cultures.•Two different point mutations at codon 198 of the beta-tubulin gene were found in some resistant single-spore cultures.•All P. aleophilum single-spore cultures were able to infect wood of Vitis vinifera without fungicide.•Only resistant single-spore cultures showed high severity on wood segments previously impregnated with Escudo®.

Fungicide resistance in Phaeoacremonium aleophilum, one of the most frequent fungal pathogens associated with grapevine trunk diseases, was investigated and found to exist in some isolates of the pathogen against a commercial formulation, Escudo®. The effect of this compound and its two active substances, carbendazim and flusilazole, was first evaluated on the mycelial growth of P. aleophilum. Escudo®-resistant isolates were estimated at a frequency of 24% in Spanish vineyards. Then, the two active substances were used individually to test their effect on mycelia growth of twelve single-spore cultures originating from six Escudo®-resistant isolates. Flusilazole (DMI-triazole) did not inhibit mycelia growth of any single-spore cultures of P. aleophilum. Carbendazim (benzimidazole) used alone allowed the growth of the same single-spore cultures that were also resistant to Escudo®. AFLP characterization of sensitive and resistant single-spore cultures showed genetic diversity within P. aleophilum isolates but no AFLP markers were associated with resistance. New primers set (L2/R1) were designed to partially amplify the exon 6 of the beta-tubulin gene of P. aleophilum. Two different point mutations resulted in glycine (GGC) or lysine (AAA) replacing the glutamic acid (GAG) at codon 198 of the beta-tubulin gene in some of the resistant single-spore cultures studied. Resistant single-spore cultures of P. aleophilum were shown to have different aggressiveness levels as sensitive single-spore cultures by inoculation of wood segments of Vitis vinifera in the presence and absence of fungicide.