Characterization and PCR‐based detection of benzimidazole‐resistant isolates of Monilinia laxa in California

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Abstract
Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low‐level resistance to the benzimidazole fungicides benomyl and thiophanate‐methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low‐resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate‐methyl at 1 and 5 µg ml−1, respectively, but not in plates amended with benomyl at 5 µg ml−1 or thiophanate‐methyl at 50 µg ml−1. The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the β‐tubulin gene. The LR isolates showed high‐temperature sensitivity, being sensitive to 1 µg ml−1 of benomyl at 28 °C but resistant at 8–24 °C. Analysis of the DNA sequence of the β‐tubulin gene showed that the LR isolates had a point mutation at the amino‐acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele‐specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of β‐tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376‐bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376‐bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele‐specific PCR and the PCR‐restriction fragment length polymorphism (PCR‐RFLP) methods could be useful for rapidly detecting benzimidazole‐resistant isolates of M laxa from stone fruit and almond crops in California. Copyright © 2004 Society of Chemical Industry

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