Resistance of Monilinia fructicola to thiophanate-methyl in Croatia

Abstract

Croatian isolates of Monilinia fructicola, M. laxa and M. fructigena have been collected from peach, nectarine, sweet cherry, plum and apricot fruits and assessed for their sensitivity to thiophanate-methyl. Out of 66 isolates collected, 34 were identified as M. fructicola (52%), 22 as M. fructigena (33%) and 10 as M. laxa (15%). Mycelial radial growth on water agar amended with thiophanate-methyl was measured to determine half maximal effective concentration values (EC50). All isolates of M. laxa and M. fructigena showed as sensitive, with EC50 values lower than 1 µg ml− 1 in 31 from 32 isolates. In M. fructicola, 19 isolates (56%) were sensitive, nine (26%) were resistant (EC50 2–30 µg ml− 1), and six (18%) were highly resistant (EC50 > 30 µg ml− 1). Positive allele-specific PCR with primer pair HRR/HRF, detecting resistant point mutations in β-tubulin gene, was recorded in 15 out of 16 highly resistant, resistant and less sensitive M. fructicola isolates. Among all species, PCR assay coincided with agar assay in 95% of cases. Highly resistant M. fructicola isolates AL 24/19 and VR 8/18 caused typical brown rot on nectarine fruits inoculated with conidia suspended in 5 or 10 µg ml− 1 of thiophanate-methyl. Four sensitive isolates of all three species did not develop on inoculated fruits. Besides implications for brown rot management, the finding of M. fructicola strains resistant to thiophanate-methyl may indicate that this species could be adapting to fungicides more rapidly than M. laxa or M. fructigena. As resistant M. fructicola isolates were also found in orchards where benzimidazoles have never been used, there is a possibility they originate from already benzimidazole-resistant strains which entered and established in Croatia.