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【目的】草酸(Oxalic acid,OA)是灰霉菌(Botrytis cinerea)等植物致病菌的致病因子,一些木霉(Trichoderma spp.)生防菌可消除草酸并降低植物发病率,但其消除草酸防治病害的途径和机制尚未研究透彻。【方法】首先从42株木霉菌株中筛选出一株在30 mmol/L草酸胁迫下耐受性最强的哈茨木霉T.harzianum LTR-2,通过形态学方法观察和原位分析了不同浓度草酸胁迫下LTR-2的发育特征变化,并测定了草酸消除水平、滤液p H和菌丝干重,分析了LTR-2在草酸为唯一碳源的生长情况。通过Real-time PCR分析了OXDC基因LTR_4445在草酸处理下的表达水平。【结果】在PDA固体培养基中,25°C半光照条件下培养5 d,在草酸浓度为50-80 mmol/L时,LTR-2可存活,但无正常菌落形态;在30-50 mmol/L浓度下,LTR-2先萌发厚垣孢子,而后再次萌发菌丝形成菌落;在<30 mmol/L浓度下,LTR-2发育正常,仅生长速度减缓。25°C、160 r/min振荡培养5 d,LTR-2可消除草酸。在20 mmol/L浓度时,草酸消除率最高,为66.50%;10 mmol/L浓度中的消除率次之,为55.06%。草酸浓度>50 mmol/L时,消除能力下降为6.75%-38.94%。相应地,培养液p H被不同程度地提高,在10、20 mmol/L草酸浓度下提高的幅度更大。当草酸浓度<20 mmol/L时,木霉的菌丝干重有不同程度的提高。将草酸作为唯一碳源进行液体培养5 d时,在10、20 mmol/L浓度下,LTR-2可形成肉眼可见的绿色菌丝球,但高于50 mmol/L条件下无法生长。草酸处理下,LTR_4445表达水平上调。【结论】通过分析LTR-2在草酸胁迫条件下的形态特征变化及消除草酸的特性,暗示在木霉消除草酸作用中除了已知的草酸降解代谢途径,还存在响应草酸胁迫模式的转变、将草酸作为前体转化为营养物质的途径等其他消除途径。
【OBJECTIVE】 Oxalic acid (OA) is a causative agent of plant pathogenic bacteria such as Botrytis cinerea. Trichoderma spp. Biocontrol bacteria can eliminate oxalic acid and reduce the incidence of plant disease, but its elimination The pathways and mechanisms of oxalic acid prevention and treatment have not been studied thoroughly. 【Method】 A strain of Trichoderma harzianum LTR-2, which was the most tolerant to 30 mmol / L oxalic acid, was screened from 42 Trichoderma strains. Morphological observation and in situ analysis of the differences were made The development of LTR-2 under oxalic acid stress was studied. The removal of oxalic acid, the pH of filtrate and the dry weight of mycelium were measured. The growth of LTR-2 was analyzed with oxalic acid as sole carbon source. The expression level of OXDC gene LTR_4445 under oxalic acid treatment was analyzed by Real-time PCR. 【Result】 LTR-2 could survive in PDA solid medium for 5 days under the condition of 25 ° C semi-light and 50-50 mmol / L oxalic acid, but no normal colony morphology was observed. When the concentration was 30-50 mmol / L concentration, LTR-2 first germinated chlamydospores, and then re-germinated mycelium colonies; <30 mmol / L concentration, LTR-2 normal growth, slow growth only. 25 ° C, 160 r / min shaking culture 5 d, LTR-2 eliminate oxalic acid. At the concentration of 20 mmol / L, oxalic acid had the highest elimination rate of 66.50%, followed by the elimination rate of 10 mmol / L at 55.06%. When the concentration of oxalic acid was above 50 mmol / L, the elimination ability decreased from 6.75% to 38.94%. Correspondingly, the culture fluid p H was increased to some extent, and the amplitude of the increase was larger at the 10,20 mmol / L oxalic acid concentration. When the concentration of oxalic acid was less than 20 mmol / L, Trichoderma mycelial dry weight increased to some extent. When cultured with liquid oxalic acid as sole carbon source for 5 days, LTR-2 could form green mycelium visible at 10,20 mmol / L, but could not grow above 50 mmol / L. Oxalic acid treatment, LTR_4445 expression increased. 【Conclusion】 The analysis of morphological changes of LTR-2 under oxalic acid stress and the elimination of oxalic acid imply that in addition to the known pathways of oxalate degradation and metabolism, Oxalic acid as a precursor into nutrients and other means of elimination.