渐绿木霉抑菌物质的分离纯化及其对植物病原菌的抑制作用

Isolation and Purification of Active Compound from Trichoderma viridescens and Its Inhibitory Activities Against Phytopathogens

【目的】从木霉(Trichoderma spp.)代谢产物中获得对疫霉菌(Phytophthora spp.)有抑制作用的活性拮抗化合物,并评价其对其他植物病原菌的生防潜力,为木霉生防菌株及其产生拮抗化合物的利用提供依据。【方法】用玻璃纸筛选法筛选产生拮抗化合物的木霉菌株,这些化合物对疫霉菌有强烈的抑制活性。在PDA上培养获得的木霉菌株作为接种体,进一步接种在稻米培养基上扩大培养,用于拮抗化合物的提取。木霉培养物经乙酸乙酯萃取、过滤和浓缩等程序,获得最初的粗提物。粗提物进一步通过柱层析、薄层层析纯化和生物活性测定,确定活性组分并获得纯的样品。依据样品的化学特性,采用气相色谱-质谱联用(GC-MS)进行化学结构鉴定,确定拮抗化合物的化学分子式和结构。选用不同类群的植物病原菌,包括卵菌门的辣椒疫霉(P.capsici)和黄瓜疫霉(P.melonis)、子囊菌门中的尖镰孢(Fusarium oxysporum)和担子菌门的立枯丝核菌(Rhizoctonia solani),测定拮抗化合物对它们发育不同阶段的拮抗活性。【结果】最初筛选试验结果显示,渐绿木霉(T.viridescens)菌株TS0404能产生对疫霉菌有强烈抑制活性的拮抗化合物。分离、纯化和生物活性试验表明,具有生物活性的活性组分是一种黄色油状液体。质谱图揭示该化合物最大离子峰166,化合物被鉴定为6-戊基-2H-吡喃酮(6-pentyl-2H-pyran-2-one,6-PP)。生物活性测定结果显示,该化合物对辣椒疫霉、黄瓜疫霉、立枯丝核菌、尖镰孢菌丝生长有显著抑制作用(EC50分别为115.26、99.58、126.46和315.75μg·m L-1),其中对黄瓜疫霉抑制效果最好,300μg·m L-1浓度完全抑制黄瓜疫霉菌丝生长。该化合物对辣椒疫霉和黄瓜疫霉游动孢子囊萌发也有显著抑制效果(EC50分别为168.67和111.87μg·m L-1),其中对黄瓜疫霉游动孢子囊萌发抑制效果最好,在400μg·m L-1时,完全抑制其游动孢子囊的萌发。此外,6-戊基-2H-吡喃酮还对尖镰孢分生孢子和立枯丝核菌菌核萌发有显著的抑制效果,其对尖镰孢分生孢子萌发抑制的EC50为151.81μg·m L-1;300μg·m L-1时,完全抑制立枯丝核菌菌核的萌发。【结论】从渐绿木霉筛选分离获得了6-戊基-2H-吡喃酮,其对卵菌中的辣椒疫霉和黄瓜疫霉子实体有强烈的抑制活性,对尖镰孢和立枯丝核菌菌核也有显著的抑制作用,显示该物质是一种广谱性的拮抗化合物,在作物疫病防治中具有潜在的应用价值。

【Objective】 An antagonistic compound against Phytophthora spp. was obtained from Trichoderma spp. and its biocontrol potential was evaluated for controlling phytopathogens, thus providing a scientific basis for utilization of biocontrol strains and their antagonistic compounds. 【Method】 Trichoderma strains producing antagonistic compounds were screened by the cellophane method and grew on PDA as inoculants, and then were transfered into rice media for isolation of antagonistic compounds.The cultures of the Trichoderma strains from the rice media were extracted, filtered and concentrated, and initial crude extract was obtained. The crude extract was further purified by column and thin-layer chromatography and the active fractions were ascertained by the biological activity determination. Based on the chemical property of the active fraction obtained, their chemical structure was identified by analysis of gas chromatography-mass spectrometry（GC-MS）. The biological activity of different types of plant pathogens was determined with the antagonistic compound obtained, including the oomycete P. capsici and P. melonis, ascomycete Fusarium oxysporum and basidiomycete Rhizoctonia solani. 【Result】 The initial screening assays showed that the strain TS0404 of T. viridescens produced an antagonistic compound with strong inhibitory activity against P. capsici. The results of isolation,purification and bioactivity determination displayed that the active fraction obtained was yellow oily liquid. Mass spectra revealed the biggest ion peak of active fraction was 166, which was identified as 6-pentyl-2H-pyran-2-one（6-PP）. The bioactive determination demonstrated that the 6-PP had significant inhibitory activity against hyphal growth of P. capsici, P. melonis, R. solani and F.oxysporum（EC50 115.26, 99.58, 126.46 and 315.75 μg·m L^-1, respectively） but the inhibitory effect on P. melonis was the best among them and hyphal growth was completely inhibited when its concentration reached 300 μg·m L^-1. Similarly, the 6-PP had a conspicuous inhibitory activity against zoosporangial germination of P. capsici and P. melonis but the inhibitory effect on P. melonis was the best and zoosporangial germination of P. melonis was completely inhibited at 400 μg·m L^-1 level. In addition, the 6-PP had significant inhibitory effect on conidial germination of F. oxysporum（EC50 151.81 μg·m L^-1） and sclerotial germination of R. solani（300 μg·m L^-1for complete inhibitory concentration）. 【Conclusion】 The 6-PP is an antagonistic compound isolated from T. viridescens, which showed strong inhibitory activities against the fruiting bodies of P. melonis, P. capsici, F. oxysporum and R. solani. It was concluded that the 6-PP is a type of broad-band antagonistic compound, which has great potential values of application in biocontrol.