title: Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California.
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 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-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 beta-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.
authors.affiliation: Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, 9240 South Riverbend Ave, Parlier, CA 93648, USA. zhma@uckac.edu
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Ma
authors.forename: Michael A
authors.initials: MA
authors.lastname: Yoshimura
authors.forename: Brent A
authors.initials: BA
authors.lastname: Holtz
authors.forename: Themis J
authors.initials: TJ
authors.lastname: Michailides
mesh_terms: D001203:Ascomycota / Q000235:genetics; D001483:Base Sequence; D001562:Benzimidazoles; D002140:California; D025141:Drug Resistance, Fungal; D005659:Fungicides, Industrial; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014404:Tubulin / Q000235:genetics
chemical_list: D001562:Benzimidazoles; D005659:Fungicides, Industrial; D014404:Tubulin