木霉对草酸耐受和消除作用的初步分析

Tolerance and elimination of oxolic acid by Trichoderma spp.

摘要:

【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pH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pH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。

Abstract:

[Objective] Oxalic acid (OA) is a virulence factor of several plant pathogens, e.g. Sclerotinia sclerotiorum and Botrytis cinerea. Some strains of Trichoderma spp. have the ability to eliminate OA and thereby reduce disease incidence, while the pathways and mechanism of OA elimination have not been well discovered. [Methods] In this study, T. harzianum LTR-2, performed the best capacity of OA elimination among 42 Trichoderma strains under the stress of 30 mmol/L OA. The developmental features of LTR-2 under OA stresses were analyzed using morphology and in situ observational methods. OA elimination rates, ambient pH and biomass under different OA stresses were analyzed. Then, the growing states of LTR-2 were described under the condition of OA as the sole carbon source. In the end, Real-time PCR was used to analyze expression level of OXDC coding gene LTR_4445. [Results] LTR-2 was cultured on PDA plate containing different concentrations of OA. With 50?80 mmol/L of OA, LTR-2 could survive, but didn’t form normal colon. With 30?50 mmol/L of OA, LTR-2 developed chlamydospores firstly, which then germinated hypha and formed normal colon. LTR-2 could eliminate OA in liquid culture. LTR-2 showed the highest OA elimination rate of 66.50% under 20 mmol/L OA, and the second highest of 55.06% under 10 mmol/L. When OA concentration was enhanced to 50?80 mmol/L, the OA elimination rate was dropped down to 6.75%?38.94%. pH of culture filtrate was enhanced to varying extents along with OA elimination under different OA stresses, and the highest increase occurred under the concentration of 10 and 20 mmol/L. Interestingly, OA showed growth-promoting effect when concentration was under 20 mmol/L. Moreover, LTR-2 could develop macroscopic mycelium pellet when OA (<20 mmol/L) was the sole carbon source in media. Transcriptional level of LTR_4445 was up-regulated under OA stress. [Conclusion] Our study implied that Trichoderma spp. may employ several strategies under OA stress besides degradation, e.g. transformation on stress-resistance mode, converting OA into a precursor of nutrients, etc.