Department of Mycology & Microbiology, Tea Research Association, North Bengal Regional R & D Centre, Nagrakata 735225, Jalpaiguri, West Bengal, India.
Department of Mycology & Microbiology, Tea Research Association, North Bengal Regional R & D Centre, Nagrakata 735225, Jalpaiguri, West Bengal, India.
Pestic Biochem Physiol. 2024 Nov;205:106115. doi: 10.1016/j.pestbp.2024.106115. Epub 2024 Sep 2.
The gray blight incited by Pestalotiopsis and allied genera is a prevalent disease affecting tea cultivation, and managing it with Trichoderma spp. is an alternative to synthetic fungicides. Plants modify their arsenal system against pathogens when they are exposed to Trichoderma spp., which produces proteins and enzymes associated with pathogenesis. Understanding the expression pattern of defense-related markers will help in developing gray blight resistance tea cultivars. Thus, this study intended to induce resistance against gray blight in tea by Trichoderma harzianum TIND02. For this, a total of eight Trichoderma isolates originated from organic tea rhizospheres were characterized and evaluated for their efficacy. Dual culture test revealed isolate TIND02 as the most potential candidate with 74.6% inhibitory activity against gray blight pathogen Pseudopestalotiopsis theae. Molecular characterization based on ITS and tef-1 alpha genes confirmed isolate TIND02 as T. harzianum. Scanning electron microscopic study showed the mycoparasitic nature of T. harzianum TIND02 (TH-TIND02) to Ps. theae. The ethyl acetate extract of TH-TIND02 at 100 and 200 μg mL showed potential inhibitory activity (>69.9%) against Ps. theae which confirmed the presence of higher volatile metabolites. Gas chromatography-Mass spectrometry study revealed that ethyl acetate extract of TH-TIND02 was composed of 21 major and minor volatile organic compounds with acetamide, 2, 2, 2-trifluoro-N, N-bis trimethyIsilyl-C (94.74%) as a major component. The isolate also produced chitinase, cellulase, β-1, 3 glucanase, and protease hydrolytic enzymes. Nursery experiments revealed that 2% and 5% doses (2 × 10 CFU mL) of TH-TIND02 significantly reduced respective 65.0% and 70.0% disease severity over control with improved plant growth. Besides, expressions of defense-related enzymes (chitinase, pHenolics, peroxidase, phenylalanine ammonia lyase, β-1, 3-glucanase, and polyphenol oxidase) and pathogenesis-related genes (chitinase and β-1, 3-glucanase) due to TH-TIND02 were determined. The secretion of defense-related enzymes was highly upregulated in plants applied with TH-TIND02 followed by Ps. theae inoculation compared to controls. The RT-qPCR analysis showed that the expression of both genes in co-inoculated plants was two-fold higher than in control after 21-day post incubation. These results suggest that TH-TIND02 application reduced gray blight severity by elevated enzyme activity and overexpressed pathogenesis-related genes in tea plants which offer for its eco-friendly and sustainable use as a bio-fungicide in tea gardens.
木霉及其相关属引起的灰霉病是一种普遍影响茶树种植的疾病，用木霉属（Trichoderma spp.）来管理它是一种替代合成杀菌剂的方法。当植物暴露于木霉属时，它们会修改针对病原体的防御系统，木霉属会产生与发病相关的蛋白质和酶。了解防御相关标记物的表达模式将有助于开发抗灰霉病的茶树品种。因此，本研究旨在通过哈茨木霉（Trichoderma harzianum）TIND02 诱导茶树对灰霉病产生抗性。为此，共对来自有机茶园根际的 8 种木霉属分离物进行了特征描述和功效评估。双层培养试验表明，分离物 TIND02 对灰霉病病原菌拟盘多毛孢（Pseudopestalotiopsis theae）具有 74.6%的抑制活性，是最有潜力的候选物。基于 ITS 和 tef-1 alpha 基因的分子特征鉴定分离物 TIND02 为哈茨木霉（T. harzianum）。扫描电子显微镜研究表明，哈茨木霉 TIND02（TH-TIND02）对拟盘多毛孢（Ps. theae）具有寄生性。TH-TIND02 的乙酸乙酯提取物在 100 和 200 μg mL 时表现出对 Ps. theae 的潜在抑制活性（>69.9%），这证实了存在更高的挥发性代谢物。气相色谱-质谱联用研究表明，TH-TIND02 的乙酸乙酯提取物由 21 种主要和次要挥发性有机化合物组成，其中乙酰胺、2,2,2-三氟-N, N-双三甲基硅基-C（94.74%）为主要成分。该分离物还产生几丁质酶、纤维素酶、β-1,3 葡聚糖酶和蛋白酶水解酶。苗圃试验表明，TH-TIND02 的 2%和 5%剂量（2×10 CFU mL）分别显著降低了对照的 65.0%和 70.0%的疾病严重程度，并改善了植物生长。此外，还测定了 TH-TIND02 诱导的茶树防御相关酶（几丁质酶、总酚、过氧化物酶、苯丙氨酸解氨酶、β-1,3-葡聚糖酶和多酚氧化酶）和与发病相关的基因（几丁质酶和β-1,3-葡聚糖酶）的表达。与对照相比，TH-TIND02 处理的植物在接种拟盘多毛孢后，防御相关酶的分泌量显著上调。RT-qPCR 分析显示，在接种 21 天后，共接种植物中这两个基因的表达量是对照的两倍。这些结果表明，TH-TIND02 的应用通过提高茶树的酶活性和过度表达与发病相关的基因来降低灰霉病的严重程度，这为其在茶园中的生态友好和可持续使用提供了依据。
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