College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Pestic Biochem Physiol. 2022 Jun;184:105130. doi: 10.1016/j.pestbp.2022.105130. Epub 2022 May 22.
The succinate dehydrogenase inhibitor (SDHI) fungicide boscalid is an excellent broad-spectrum fungicide but has not been registered in China to control Penicillium digitatum, the causal agent of green mold of citrus. The present study evaluated the risk and molecular mechanisms for boscalid resistance in P. digitatum. Resistance induction with four arbitrarily selected sensitive isolates of P. digitatum by ultraviolet (UV) irradiation on conidia plated on boscalid-amended potato dextrose agar (PDA) and consecutive growing on boscalid-amended PDA produced five highly resistant isolates with EC values greater than 1000 μg/mL and two resistant isolates with EC lower than 200 μg/mL. Boscalid resistance of the five mutants with EC values above 1000 μg/mL was stable after successive transfers on PDA for 16 generations. However, for the other two mutants with EC lower than 200 μg/mL, the EC values decreased significantly after successive transfers. There was significant cross-resistance between boscalid and carboxin (r = 0.925, P < 0.001), but no significant cross-resistance was detected between boscalid and fludioxonil (r = 0.533，P = 0.095) or between boscalid and prochloraz (r = -0.543，P = 0.088). The seven resistant mutants varied greatly in the mycelia growth, sporulation, pathogenicity, and sensitivities to exogenous stresses including NaCl, salicylhydroxamic acid (SHAM), and HO. Alignment of the deduced amino acid sequence showed that there was no point mutation in the target enzyme succinate dehydrogenase (Sdh) subunits SdhA, SdhC, or SdhD in each of the seven resistant mutants, and the mutation of a conserved histidine residue to tyrosine (H243Y) in the subunit SdhB (i.e., iron‑sulfur protein) occurred in only three highly resistant isolates. Molecular docking indicated that mutation H243Y could not prevent the binding of boscalid into the quinone-binding site of SDH in the presence of the heme moiety. However, for SDH without the heme moiety, boscalid could bind into a deeper site with a much higher affinity, and the mutation H243Y spatially blocked the docking of boscalid into the deeper site. This may be the molecular mechanism for boscalid resistance caused by SdhB-H243Y mutation.
琥珀酸脱氢酶抑制剂（SDHI）杀菌剂啶酰菌胺是一种优秀的广谱杀菌剂，但尚未在中国注册用于防治柑橘绿霉病的病原菌指状青霉。本研究评估了啶酰菌胺对指状青霉的抗性风险和分子机制。用紫外线（UV）照射孢子平板上的啶酰菌胺处理过的土豆葡萄糖琼脂（PDA）上的 4 个任意选择的敏感青霉菌株，连续在啶酰菌胺处理过的 PDA 上培养，产生了 5 个 EC 值大于 1000μg/mL 的高度抗性分离株和 2 个 EC 值低于 200μg/mL 的抗性分离株。在 PDA 上连续转接 16 代后，5 个 EC 值大于 1000μg/mL 的突变体的啶酰菌胺抗性稳定。然而，对于 EC 值低于 200μg/mL 的另外两个突变体，EC 值在连续转接后显著下降。啶酰菌胺与咯菌腈（r=0.925，P<0.001）之间存在显著的交叉抗性，但啶酰菌胺与氟啶胺（r=0.533，P=0.095）或啶酰菌胺与丙环唑（r=-0.543，P=0.088）之间没有显著的交叉抗性。7 个抗性突变体在菌丝生长、产孢、致病性以及对 NaCl、水杨羟肟酸（SHAM）和 HO 等外源胁迫的敏感性方面差异很大。推导的氨基酸序列比对表明，7 个抗性突变体中的靶酶琥珀酸脱氢酶（SDH）亚基 SdhA、SdhC 或 SdhD 没有发生点突变，只有 3 个高度抗性分离株中的亚基 SdhB（即铁硫蛋白）中的保守组氨酸残基突变为酪氨酸（H243Y）。分子对接表明，在血红素存在的情况下，突变 H243Y 不能阻止啶酰菌胺结合到 SDH 的醌结合位点。然而，对于没有血红素的 SDH，啶酰菌胺可以结合到更深的位点，并且具有更高的亲和力，突变 H243Y 空间上阻止了啶酰菌胺结合到更深的位点。这可能是 SdhB-H243Y 突变引起啶酰菌胺抗性的分子机制。
