Table_1_Determining the biocontrol capacities of Trichoderma spp. originating from Turkey on Fusarium culmorum by transcriptional and antagonistic analyses.docx

In this study aiming to investigate potential fungal biocontrol agents for Fusarium culmorum, several isolates of Trichoderma spp. were evaluated for their antagonistic effects by means of transcriptional analyses. At first, 21 monosporic Trichoderma spp. isolates were obtained from natural wood debris and wood area soils in Manisa, Turkey. Trichoderma spp. Isolates were identified as belonging to four different species (T. atroviride, T. harzianum, T. koningii, and T. brevicompactum) by tef1-α sequencing. Then, the linear growth rate (LGR) of each species was calculated and determined to be in a range between 13.22 ± 0.71 mm/day (T. atroviride TR2) and 25.06 ± 1.45 mm/day (T. harzianum K30). Inter-simple sequence repeat (ISSR) genotyping validated the tef1-α sequencing results by presenting two sub-clusters in the dendrogram. We determined the genetically most similar (TR1 & TR2; 97.77%) and dissimilar (K9 & K17; 40.40%) individuals belonging to the same and different species, respectively. Dual sandwich culture tests (which are useful for antagonism studies) revealed that T. harzianum K21 (the least suppressive) and T. brevicompactum K26 (the most suppressive) isolates suppressed F. culmorum with growth rates of 3% and 46%, respectively. Expressions of genes previously associated with mycoparasitism-plant protection-secondary metabolism (nag1, tgf-1, and tmk-1) were tested by quantitative real-time polymerase chain reaction (qRT-PCR) in both those isolates. While there were no significant differences (p>0.05) in expression that were present in the K21 isolate, those three genes were upregulated with fold change values of 2.69 ± 0.26 (p<0.001), 2.23 ± 0.16 (p<0.001), and 5.38 ± 2.01 (p<0.05) in K26, meaning that the presence of significant alteration in the physiological processes of the fungus. Also, its mycoparasitism potential was tested on Triticum aestivum L. cv Basribey in planta, which was infected with the F. culmorum FcUK99 strain. Results of the trials, including specific plant growth parameters (weight or length of plantlets), confirmed the mycoparasitic potential of the isolate. It can be concluded that (i) nag1, tgf-1, and tmk-1 genes could be approved as reliable markers for evaluation of BCA capacities of Trichoderma spp. and (ii) the T. brevicompactum K26 strain can be suggested as a promising candidate for combating in F. culmorum diseases following the necessary procedures to ensure it is non-hazardous and safe.

本研究旨在探究针对禾谷镰孢（Fusarium culmorum）的潜在真菌生防制剂，通过转录组分析评估了木霉属（Trichoderma spp.）多个分离株的拮抗活性。首先从土耳其马尼萨省的天然木本残体及林地土壤中分离得到21个单孢木霉属分离株。通过tef1-α测序鉴定，这些分离株隶属于4个不同物种：深绿木霉（T. atroviride）、哈茨木霉（T. harzianum）、康宁木霉（T. koningii）以及短密木霉（T. brevicompactum）。计算各菌株的线性生长速率（LGR），结果介于13.22±0.71 mm/天（深绿木霉TR2）至25.06±1.45 mm/天（哈茨木霉K30）之间。简单序列重复区间（Inter-simple sequence repeat, ISSR）基因分型通过聚类树生成两个亚簇，验证了tef1-α测序的结果。本研究确定了遗传相似度最高的菌株（TR1与TR2，相似度97.77%）以及遗传差异最大的菌株（K9与K17，相似度40.40%），二者分别隶属于相同及不同物种。采用拮抗研究常用的双重三明治培养试验，结果显示哈茨木霉K21（抑制效果最弱）与短密木霉K26（抑制效果最强）对禾谷镰孢的抑制率分别为3%与46%。通过实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测了此前与重寄生、植物保护及次级代谢相关的基因（nag1、tgf-1及tmk-1）在这两株木霉中的表达情况。K21菌株中这三个基因的表达无显著差异（p>0.05）；而在K26菌株中，这三个基因均出现上调，其折叠变化值分别为2.69±0.26（p<0.001）、2.23±0.16（p<0.001）及5.38±2.01（p<0.05），表明该菌株的生理过程发生了显著改变。此外，在接种禾谷镰孢FcUK99菌株的普通小麦（Triticum aestivum L. cv Basribey）植株上验证了该菌株的重寄生活性。试验结果（包括幼苗的重量、长度等特定植物生长参数）证实了该分离株的生防重寄生潜力。综上可得出两点结论：（1）nag1、tgf-1及tmk-1基因可作为评估木霉属菌株生防制剂潜力的可靠分子标记；（2）短密木霉K26菌株可作为对抗禾谷镰孢病害的潜在候选生防菌株，后续需开展相关安全无毒化验证流程以确保其使用安全性。