Biological control is a promising approach to reduce plant diseases caused by nematodes to ensure high productivity in agricultural production. Large‐scale analyses of genetic variation in fungal species used for biocontrol can generate knowledge regarding interaction mechanisms that can improve efficacy of biocontrol applications. In this study, we performed a genome‐wide association study (GWAS) for in vitro antagonism against the root lesion nematode Pratylenchus penetrans in 53 previously genome re‐sequenced strains of the biocontrol fungus Clonostachys rosea. Nematode mortality in C. rosea potato dextrose broth (PDB) culture filtrates was highly variable and showed continuous variation (p < .001) between strains, indicating a polygenic inheritance. Twenty‐one strains produced culture filtrates with higher (p ≤ .05) nematode mortality compared with the PDB control treatment, while ten strains lowered (p ≤ .05) the mortality. The difference in in vitro antagonism against P. penetrans correlated with antagonism against the soybean cyst nematode Heterodera glycines, indicating lack of host specificity in C. rosea. An empirical Bayesian multiple hypothesis testing approach identified 279 single nucleotide polymorphism markers significantly (local false sign rate < 10^–10) associated with the trait. Genes present in the genomic regions associated with nematicidal activity included several membrane transporters, a chitinase and genes encoding proteins predicted to biosynthesize secondary metabolites. Gene deletion strains of the predicted nonribosomal peptide synthetase genes nps4 and nps5 were generated and showed increased (p ≤ .001) fungal growth and conidiation rates compared to the wild type. Deletion strains also exhibited reduced (p < .001) nematicidal activity and reduced (p ≤ .05) biocontrol efficacy against nematode root disease and against fusarium foot rot on wheat. In summary, we show that the GWAS approach can be used to identify biocontrol factors in C. rosea, specifically the putative nonribosomal peptide synthetases NPS4 and NPS5.

中文翻译：

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生防真菌红竹根病害线虫拮抗作用的自然变异以及通过全基因组关联图谱鉴定生防因子


生物防治是减少线虫引起的植物病害以确保农业生产高生产力的一种有前途的方法。对用于生物防治的真菌物种的遗传变异进行大规模分析可以产生有关相互作用机制的知识，从而提高生物防治应用的功效。在这项研究中，我们进行了一项全基因组关联研究 (GWAS)，以体外对抗 53 个先前基因组重新测序的生防真菌Clonostachysrosea菌株中的根部病害线虫穿透短体线虫 (Pratylenchus penetrans) 。 C. Rosea马铃薯葡萄糖肉汤 (PDB) 培养物滤液中的线虫死亡率变化很大，并且在菌株之间表现出连续变化 ( p < .001)，表明多基因遗传。与 PDB 对照处理相比，21 个菌株产生的培养滤液具有更高的 ( p ≤ .05) 线虫死亡率，而 10 个菌株降低了 ( p ≤ .05) 死亡率。对P. penetrans 的体外拮抗作用的差异与对大豆胞囊线虫Heterodera Ganes的拮抗作用相关，表明C. Rosea缺乏宿主特异性。经验贝叶斯多重假设检验方法识别出 279 个与该性状显着相关的单核苷酸多态性标记（局部错误符号率 < 10 ^–10 ）。存在于与杀线虫活性相关的基因组区域的基因包括几种膜转运蛋白、几丁质酶和编码预计可生物合成次级代谢物的蛋白质的基因。 生成了预测的非核糖体肽合成酶基因nps4和nps5的基因缺失菌株，与野生型相比，它们显示出增加的 ( p ≤ .001) 真菌生长和分生孢子形成率。缺失菌株还表现出杀线虫活性降低 ( p < .001)，对线虫根病和小麦镰刀菌根腐病的生物防治功效降低 ( p ≤ .05)。总之，我们表明 GWAS 方法可用于鉴定C. Rosea中的生物防治因子，特别是假定的非核糖体肽合成酶 NPS4 和 NPS5。