Specific detection of benzimidazole resistance in Colletotrichum gloeosporioides from fruit crops by PCR-RFLP.
Anthracnose diseases, caused by Colletotrichum gloeosporioides, are a worldwide problem and are especially important in Taiwan owing to the severe economic damage they cause to tropical fruits that are grown for local consumption and export. Benzimidazoles are systemic fungicides widely used for controlling these diseases in Taiwan. Thirty-one isolates of C. gloeosporioides from mango and strawberry grown in Taiwan were examined for their sensitivity to benzimidazole fungicides. The responses of the isolates grown on benzimidazole-amended culture media were characterized as sensitive, moderately resistant, resistant or highly resistant. Analysis of point mutations in the beta-tubulin gene by DNA sequencing of PCR-amplified fragments revealed a substitution of GCG for GAG at codon 198 in resistant and highly resistant isolates and a substitution of TAC for TTC at codon 200 in moderately resistant isolates. A set of specific primers, TubGF1 and TubGR, was designed to amplify a portion of the beta-tubulin gene for the detection of benzimidazole-resistant C. gloeosporioides. Bsh1236I restriction maps of the amplified beta-tubulin gene showed that the resistant isolate sequence, but not the sensitive isolate sequence, was cut. The PCR restriction fragment length polymorphism (PCR-RFLP) was validated to detect benzimidazole-resistant and benzimidazole-sensitive C. gloeosporioides isolates recovered from avocado, banana, carambola, dragon fruit, grape, guava, jujube, lychee, papaya, passion fruit and wax apple. This method has the potential to become a valuable tool for monitoring the occurrence of benzimidazole-resistant C. gloeosporioides and for assessment of the need for alternative management practices.
Department of Plant Pathology, National Chung Hsing University, 250, Kuo Kuang Road, Taichung 402, Taiwan, ROC.
Wen-Hsin
WH
Chung
Wen-Chuan
WC
Chung
Mun-Tsu
MT
Peng
Hong-Ren
HR
Yang
Jenn-Wen
JW
Huang
D001562:Benzimidazoles / Q000494:pharmacology*; D020231:Colletotrichum / Q000187:drug effects / Q000235:genetics; D018556:Crops, Agricultural / Q000382:microbiology*; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005638:Fruit / Q000382:microbiology*; D005659:Fungicides, Industrial / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013624:Taiwan; D014404:Tubulin / Q000235:genetics
D001562:Benzimidazoles; D005659:Fungicides, Industrial; D014404:Tubulin