Avenot H F, Sellam A, Karaoglanidis G, Michailides T J
Department of Plant Pathology, University of California Davis, Kearney Agricultural Center, Parlier 93648, USA.
Phytopathology. 2008 Jun;98(6):736-42. doi: 10.1094/PHYTO-98-6-0736.
Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.
通过菌丝体生长试验，从曾使用过百菌清（唑菌胺酯+啶酰菌胺）且对啶酰菌胺杀菌剂表现出高抗性水平（平均EC(50)值>500微克/毫升）的开心果果园中鉴定出38株链格孢菌分离株。一项交叉抗性研究表明，相同的分离株对羧菌胺（一种已知的琥珀酸脱氢酶（Sdh）抑制剂）也具有抗性。为了确定链格孢菌对啶酰菌胺抗性的遗传基础，从一个对杀菌剂敏感的分离株中分离出了完整的铁硫基因（AaSdhB）。推导的氨基酸序列与其他生物的铁硫蛋白（Ip）具有高度相似性。对敏感和抗性分离株的AaSdhB全序列进行比较发现，一个高度保守的组氨酸残基（敏感分离株中的密码子CAC）在第277位被转换为酪氨酸（密码子TAC，I型突变体）或精氨酸（密码子CGC，II型突变体）。在其他真菌物种中，该残基与羧酰胺抗性有关。在本研究中，分别有10个和5个突变体为I型和II型，而其他23个抗性分离株（III型突变体）在组氨酸密码子处没有突变。利用I型突变体中检测到的点突变设计了一对等位基因特异性聚合酶链反应（PCR）引物，以促进快速检测。一种PCR-限制性片段长度多态性（RFLP）分析方法，即扩增的基因片段用AciI酶切，已成功用于诊断II型突变体。本文讨论了链格孢菌AaSdhB序列中的这些修饰与赋予啶酰菌胺抗性的相关性。
