DataSheet_1_A Secondary Metabolite Secreted by Penicillium citrinum Is Able to Enhance Parastagonospora nodorum Sensitivity to Tebuconazole and Azoxystrobin.zip

Parastagonospora nodorum causes glume and leaf blotch of wheat, a harmful disease resulting in serious losses in grain yield. In many countries including Russia, fungicidal formulations based on triazoles and on triazoles combined with strobilurins are used to control this fungus. However, their prolonged application may promote the selection of fungicide-resistant strains of P. nodorum leading to significant attenuation or even loss of fungicidal effect. Chemosensitization of plant pathogenic fungi with natural compounds represents a promising strategy for mitigating fungicide resistance and other negative impacts of fungicides. In this work, we applied a chemosensitization approach towards P. nodorum strains non-resistant or resistant to tebuconazole or azoxystrobin using 6-demethylmevinolin (6-DMM), a metabolite of Penicillium citrinum. The resistant strains were obtained by the mutagenesis and subsequent culturing on agar media incorporated with increasing doses of Folicur® EC 250 (i.e., tebuconazole) or Quadris® SC 250 (i.e., azoxystrobin). Test strains m8-4 and kd-18, most resistant to tebuconazole and azoxystrobin, respectively, were selected for sensitization experiments. These experiments demonstrated that combining 6-DMM with Folicur® enhanced fungicidal effectiveness in vitro and in vivo in addition to attenuating the resistance of P. nodorum to tebuconazole in vitro. 6-DMM was also found to augment Quadris® efficacy towards kd-18 when applied on detached wheat leaves inoculated with this strain. Experiments on P. nodorum sensitization under greenhouse conditions included preventive (applying test compounds simultaneously with inoculation) or post-inoculation spraying of wheat seedlings with 6-DMM together with Folicur® at dose rates (DR) amounting to 10% and 20% of DR recommended for field application (RDR). Combined treatments were run in parallel with using the same DR of the fungicide and sensitizer, alone. A synergistic effect was observed in both preventive and post-inoculation treatments, when the sensitizer was co-applied with the fungicide at 10% of the RDR. In this case, disease reduction significantly exceeded the protective effect of Folicur® at 10% or 20% of the RDR, alone, and also a calculated additive effect. Collectively, our findings suggest that 6-DMM is promising as a putative component for formulations with triazole and strobilurin fungicides. Such new formulations would improve fungicide efficacy and, potentially, lower rates of fungicides needed for plant pathogen control.

诺尔姆平脐蠕孢菌（Parastagonospora nodorum）可引发小麦颖枯病与叶斑病，该病害会造成严重的谷物产量损失。包括俄罗斯在内的诸多国家，均采用基于三唑类（triazoles）杀菌剂以及三唑类与甲氧基丙烯酸酯类（strobilurins）杀菌剂复配的制剂来防治该病原菌。但长期施用这类药剂可能会筛选出抗药性诺尔姆平脐蠕孢菌菌株，导致杀菌效果显著减弱甚至完全失效。利用天然化合物对植物病原真菌实施化学增敏，是缓解杀菌剂抗药性及其他杀菌剂负面效应的极具前景的研究策略。
本研究以桔青霉（Penicillium citrinum）的代谢产物6-脱甲基美维诺林（6-demethylmevinolin, 6-DMM）为增敏剂，针对对戊唑醇或嘧菌酯敏感或抗药的诺尔姆平脐蠕孢菌菌株开展增敏试验。抗药性菌株通过诱变处理，并后续在添加递增剂量福连®乳油250（有效成分为戊唑醇，tebuconazole）或阿米西达®悬浮剂250（有效成分为嘧菌酯，azoxystrobin）的琼脂培养基上继代培养获得。研究选取分别对戊唑醇和嘧菌酯抗性最强的供试菌株m8-4与kd-18，用于后续增敏试验。
试验结果显示，将6-DMM与福连®复配后，不仅可在体外削弱诺尔姆平脐蠕孢菌对戊唑醇的抗药性，还能提升其体内外的杀菌活性。此外，在接种kd-18菌株的离体小麦叶片上施用6-DMM时，其对阿米西达®的增效作用同样得到验证。
温室条件下的诺尔姆平脐蠕孢菌增敏试验设置了两种施药模式：预防型施药（供试化合物与菌液同时接种）和接种后施药，将6-DMM与福连®以相当于田间推荐剂量（RDR）10%和20%的剂量复配施用至小麦幼苗。同时设置单独施用对应剂量杀菌剂、单独施用增敏剂的平行对照组。当增敏剂与相当于田间推荐剂量10%的杀菌剂复配时，预防型与接种后施药两种模式下均观察到协同增效作用。在此条件下，病害抑制率显著高于单独施用10%或20%田间推荐剂量福连®所产生的防护效果，同时也高于理论计算的相加效应。
综上，本研究结果表明，6-DMM可作为三唑类与甲氧基丙烯酸酯类杀菌剂复配制剂的潜在增效组分。这类新型制剂不仅可提升杀菌剂的防治效果，还有望降低植物病害防控所需的杀菌剂用量。