Ma Zhonghua, Yoshimura Michael A, Holtz Brent A, Michailides Themis J
Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, 9240 South Riverbend Ave, Parlier, CA 93648, USA.
Pest Manag Sci. 2005 May;61(5):449-57. doi: 10.1002/ps.982.
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.
核果链核盘菌是加利福尼亚州核果和杏仁褐腐病的病原菌，可引起花腐和果腐。在本研究中，从加利福尼亚州的核果和杏仁中采集的核果链核盘菌田间分离株对苯并咪唑类杀菌剂苯菌灵和甲基托布津表现出低水平抗性。低抗（LR）分离株分别在添加了1和5微克/毫升苯菌灵以及甲基托布津的马铃薯葡萄糖琼脂（PDA）平板上生长，但在添加了5微克/毫升苯菌灵或50微克/毫升甲基托布津的平板上不生长。通过温度敏感性和β-微管蛋白基因的DNA序列对苯并咪唑LR分离株进行了鉴定。LR分离株表现出高温敏感性，在28℃时对1微克/毫升苯菌灵敏感，但在8-24℃时具有抗性。对β-微管蛋白基因的DNA序列分析表明，LR分离株在氨基酸位置240处存在点突变，导致亮氨酸被苯丙氨酸取代。基于该点突变，开发了一对等位基因特异性PCR引物用于快速检测核果链核盘菌的LR分离株。此外，根据核果链核盘菌、果生链核盘菌和其他真菌物种β-微管蛋白基因内含子6的DNA序列差异，开发了一对核果链核盘菌特异性PCR引物。该引物对从所有测试的核果链核盘菌分离株中扩增出预期的376 bp DNA片段，但未从从核果和杏仁作物中分离的其他14种真菌物种中扩增出该片段。限制性内切酶BsmA I仅识别敏感（S）分离株PCR产物中的GTCTCC序列，而不识别LR分离株PCR产物中的GTTTCC序列。该内切酶消化S分离株的376 bp PCR产物，在琼脂糖凝胶上产生两条带（111和265 bp）。因此，等位基因特异性PCR和PCR-限制性片段长度多态性（PCR-RFLP）方法都可用于快速检测加利福尼亚州核果和杏仁作物中对苯并咪唑类抗性的核果链核盘菌分离株。
