De Miccolis Angelini Rita M, Masiello Mario, Rotolo Caterina, Pollastro Stefania, Faretra Francesco
Plant Pathology Section, Department of Soil, Plant and Food Sciences, University of Bari, Bari, Italy.
Pest Manag Sci. 2014 Dec;70(12):1884-93. doi: 10.1002/ps.3748. Epub 2014 Mar 24.
Succinate dehydrogenase inhibitors (SDHIs), interfering with fungal respiration, are considered to be fungicides at medium to high risk of resistance. Boscalid was the first molecule belonging to the SDHIs that was introduced for the control of Botryotinia fuckeliana. A range of different target-site mutations leading to boscalid resistance have been found in field populations of the fungus. The different types of mutation confer different cross-resistance profiles towards novel SDHIs, such as the recently introduced fungicide fluopyram. This study combines the determination of cross-resistance profiles and the setting-up of methods for fast molecular detection of the mutations.
By means of in vitro tests, a range of SdhB mutations were characterised for resistance levels towards boscalid and fluopyram. SdhB mutations conferring P225L and P225F substitutions conferred high resistance to boscalid and high or moderate resistance to fluopyram respectively. Mutants carrying the N230I replacement were moderately resistant to both SDHIs. Substitutions at position H272 responsible for a high level of resistance to boscalid conferred sensitivity (H272R), hypersensitivity (H272Y) or moderate resistance (H272V) to fluopyram. Allele-specific (AS) PCR was developed and used for genotyping 135 B. fuckeliana isolates. The assay confirmed the strict association between resistance profiles and allelic variants of the SdhB gene. Real-time AS-PCR proved to be sensitive and specific for quantitative detection of different SDHI-resistant genotypes.
Fluopyram-resistant mutants are currently rarely detected in the field sprayed with boscalid, but this may change with intensive exposure of the fungal population to fluopyram. PCR assays/methods developed in the study provide tools for fast monitoring of field populations and observing possible changes in population composition following fluopyram introduction, useful for the setting-up of appropriate preventive measures.
琥珀酸脱氢酶抑制剂（SDHIs）通过干扰真菌呼吸作用，被认为是具有中等至高抗药性风险的杀菌剂。啶酰菌胺是首个引入用于防治灰葡萄孢的属于SDHIs的分子。在该真菌的田间种群中已发现一系列导致啶酰菌胺抗性的不同靶位点突变。不同类型的突变赋予对新型SDHIs（如最近引入的杀菌剂氟吡菌酰胺）不同的交叉抗性谱。本研究结合了交叉抗性谱的测定以及快速分子检测突变方法的建立。
通过体外试验，对一系列SdhB突变体针对啶酰菌胺和氟吡菌酰胺的抗性水平进行了表征。赋予P225L和P225F替换的SdhB突变分别赋予对啶酰菌胺的高抗性和对氟吡菌酰胺的高抗性或中等抗性。携带N230I替换的突变体对两种SDHIs均具有中等抗性。导致对啶酰菌胺高水平抗性的H272位点的替换赋予对氟吡菌酰胺的敏感性（H272R）、超敏感性（H272Y）或中等抗性（H272V）。开发了等位基因特异性（AS）PCR并用于对135株灰葡萄孢分离株进行基因分型。该检测证实了抗性谱与SdhB基因等位变异之间的严格关联。实时AS-PCR被证明对不同SDHI抗性基因型的定量检测具有敏感性和特异性。
目前在喷施啶酰菌胺的田间很少检测到对氟吡菌酰胺的抗性突变体，但随着真菌群体大量接触氟吡菌酰胺，这种情况可能会改变。本研究中开发的PCR检测方法为快速监测田间种群以及观察氟吡菌酰胺引入后种群组成的可能变化提供了工具，有助于制定适当的预防措施。
