State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, No. 71, East Beijing Road, P.O. Box 821, Nanjing 210008, China; University of Chinese Academy of Sciences, Beijing 100049, China.
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, No. 71, East Beijing Road, P.O. Box 821, Nanjing 210008, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Ecotoxicol Environ Saf. 2021 Feb;209:111749. doi: 10.1016/j.ecoenv.2020.111749. Epub 2020 Dec 18.
Elucidation of the inhibitory effects of humic substances (HSs) on phytopathogenic fungi and the underlying molecular mechanisms are highly important for improved biocontrol. In this study, we investigated the growth suppression, morphological characteristics, transcriptomic sequence, and radical signals of Rhizoctonia solani following HS addition (50 mg/L). Through mycelial cultured experiment, mycelia growth of R. solani had been suppressed with HS addition, and the inhibition rate was 24.88 ± 0.11% compared to the control. Field emission-scanning electron microscopy showed increased and superimposed branching mycelial growth, with a shriveled appearance. RNA samples of R. solani cultured with or without HSs were both extracted to examine the sequence on molecular level by Illumina HiSeq sequencing platform. RNA sequencing analysis revealed 175 differentially expressed genes (DEGs; 111 upregulated and 64 downregulated) between the HSs treatment and control. The upregulated unigenes were annotated and significantly enriched to three molecular processes: vitamin B6 metabolism, ABC transporters, and glutathione metabolism, while the downregulated unigenes were annotated to carbohydrate metabolism, but not significantly enriched. Real time-quantitative polymerase chain reaction analysis showed that the unigenes related to hexokinase, glucose-6-phosphate isomerase, glutathione synthase, and glutathione reductase were significantly decreased (by 60.03%, 70.70%, 60.33%, and 57.59%, respectively), while those related to glutathione S-transferase were significantly increased (2.66-fold). The electron paramagnetic resonance spectra showed that HSs induced increased the intensity of radical signals of R. solani in a cultured system increased by 59.56% compared to CK (without HSs addition). Network analysis based on DEGs expression and the chemical structure of HSs revealed that the carbonyl moiety in HSs formed the most links with nodes of the DEGs (sum of the links of positive and negative effects = 70), implicating this structure as the active fraction responsible for the inhibitory effect. This study provides molecular and chemical evidence of the biofungicidal activity of HSs with the potential for practical application.
阐明腐殖质（HSs）对植物病原菌真菌的抑制作用及其潜在的分子机制，对于提高生物防治效果至关重要。在本研究中，我们研究了 HSs（50mg/L）添加后对 Rhizoctonia solani 的生长抑制、形态特征、转录组序列和自由基信号的影响。通过菌丝培养实验，发现 HSs 添加抑制了 R. solani 的菌丝生长，与对照相比，抑制率为 24.88±0.11%。场发射扫描电子显微镜显示，菌丝生长增加且呈叠加状分枝，外观皱缩。提取添加或不添加 HSs 培养的 R. solani 的 RNA 样本，通过 Illumina HiSeq 测序平台在分子水平上检查序列。RNA 测序分析显示，HSs 处理与对照之间有 175 个差异表达基因（DEGs；111 个上调，64 个下调）。上调的 unigenes 被注释并显著富集到三个分子过程：维生素 B6 代谢、ABC 转运体和谷胱甘肽代谢，而下调的 unigenes 被注释到碳水化合物代谢，但未显著富集。实时定量聚合酶链反应分析显示，与己糖激酶、葡萄糖-6-磷酸异构酶、谷胱甘肽合酶和谷胱甘肽还原酶相关的 unigenes显著降低（分别降低 60.03%、70.70%、60.33%和 57.59%），而与谷胱甘肽 S-转移酶相关的 unigenes显著增加（2.66 倍）。电子顺磁共振波谱显示，与 CK（未添加 HSs）相比，HSs 在培养系统中诱导 R. solani 自由基信号强度增加了 59.56%。基于 DEGs 表达和 HSs 化学结构的网络分析表明，HSs 中的羰基部分与 DEGs 的节点形成了最多的链接（正效应和负效应的链接总和=70），表明该结构是负责抑制作用的活性部分。本研究为 HSs 的生物杀菌活性提供了分子和化学证据，具有实际应用的潜力。
