Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California
Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California
复制标题
DOI：
10.1002/ps.982
复制
发表时间：
2005-05-01
影响因子：
4.1
通讯作者：
Michailides, TJ
中科院分区：
文献类型：
--
作者：
Ma, ZH;Yoshimura, MA;Michailides, TJ
松褐腐病菌（Monilinia laxa）是加州核果和扁桃褐腐病的病原菌，引起开花枯萎和果实腐烂。在这项研究中，低水平的抗性苯并咪唑类杀菌剂苯菌灵和甲基托布津检测到田间分离的M laxa从核果和杏仁在加州。低抗性（LR）菌株在分别用苯菌灵和甲基硫菌灵（1 μ g/ml和5 μ g/ml）改良的马铃薯葡萄糖琼脂（PDA）平板上生长，但在用苯菌灵（5 μ g/ml）或甲基硫菌灵（50 μ g/ml）改良的平板上不生长。苯并咪唑LR分离物的特征在于温度敏感性和β-微管蛋白基因的DNA序列。LR分离物表现出高温敏感性，在28 ℃时对1 μ g/ml苯菌灵敏感，但在8 - 24 ℃时具有抗性。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处有一个点突变，导致亮氨酸被苯丙氨酸取代。根据该点突变设计了一对等位基因特异性PCR引物，用于对Mlaxa LR分离株的快速检测。此外，根据M laxa、M fructicola等真菌β-微管蛋白基因内含子6 DNA序列的差异，开发了一对针对M laxa的PCR引物。引物对扩增预期的376 bp的DNA片段，从所有的M laxa菌株测试，但不是从核果和杏仁作物分离的其他14种真菌。限制性内切酶BsmA Ⅰ只识别敏感株（S）PCR产物中的GTCTCC序列，而不识别LR株PCR产物中的GTTTCC序列。该酶切376bp的PCR产物，在琼脂糖凝胶上产生两条带（111和265bp）。因此，等位基因特异性PCR和PCR-限制性片段长度多态性（PCR-RFLP）的方法可以用于快速检测苯并咪唑耐药菌株的M laxa核果和杏仁作物在加州。(c)2004化学工业协会。
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 I and 5 mu g ml(-1), respectively, but not in plates amended with benomyl at 5 mu g ml(-1) or thiophanate-methyl at 50 mu g 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 mu g 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 265bp) 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. (c) 2004 Society of Chemical Industry.