板栗内生木霉及其挥发性代谢产物的鉴定

Identification of Endophytic Trichoderma of Chestnut and Its Volatile Metabolites

【目的】筛选具有生物熏蒸抑制板栗果腐病病原菌作用的有益内生性木霉菌,用于研发板栗贮藏期病害的绿色防控技术。【方法】表面消毒法分离培养板栗果实组织内生真菌,依据形态学和ITS、rpb2、tef1-α三序列系统发育分析鉴定木霉菌,通过不接触共培养方法,明确木霉菌挥发性代谢产物对多种病菌的抑制作用,并利用顶空固相微萃取和GC-MS技术(HS-SPME-GCMS)分析挥发性代谢产物组成。【结果】从板栗果实中分离获得了4株木霉菌株,从中发现了对板栗果腐病4种病原菌表现广谱熏蒸抑制作用的菌株FPYF6037,经鉴定该菌株为木霉属新种,命名为栗木霉Trichoderma castaneae。该菌株挥发性代谢产物优势组成为萜烯化合物,约占55%。抑菌试验表明,α-水芹烯、β-水芹烯、1,3,5,7-环辛四烯表现出明显抑制病原菌生长的作用,浓度0.4μL/mL的α-水芹烯的抑制率为31.6%,1.0μL/mL的β-水芹烯的抑制率为42.67%,0.8μL/mL的1,3,5,7-环辛四烯抑制率为41%。【结论】首次报道了板栗果实中的内生性木霉菌株,发现了新种栗木霉及其挥发性代谢产物抑制病原菌生长的作用,为利用生物熏蒸作用绿色防控板栗果实腐烂病提供了资源和途径。

【Objective】The study aims to screen beneficial endophytic Trichoderma species with the ability of biological fumigation to suppress the pathogenic fungi causing chestnut fruit rot,for the development of green prevention and control technologies for diseases during the chestnut storage period.【Method】The endophytes in chestnut fruit tissues were isolated and cultivated through the surface disinfection method.Trichoderma species were identified based on morphology and phylogenetic analyses of the ITS,rpb2,and tef1-αsequences.The inhibitory effect of the volatile metabolites of Trichoderma on various pathogenic fungi was clarified by non-contact co-culture,and the composition of its volatiles was analyzed using headspace solid-phase microextraction and GC-MS technology(HS-SPME-GC-MS).【Result】Among the four Trichoderma strains isolated from chestnut fruits,strain FPYF6037 was discovered,which exhibited broad-spectrum fumigation inhibitory effects on the four pathogenic fungi causing chestnut fruit rot.This strain was identified as a new species of the Trichoderma genus and named Trichoderma castaneae.The dominant components of the volatile metabolites of this strain were terpene compounds,accounting for approximately 55%.Component bacteriostasis tests indicated thatα,β-phellandrene and 1,3,5,7-cyclooctatetraene manifested obvious inhibitory effects on the growth of pathogenic fungi.The inhibition rate ofα-phellandrene at a concentration of 0.4μL/mL was approximately 31.6%,that of 1.0μL/mLβ-phellandrene was 42.67%,and that of 0.8μL/mL 1,3,5,7-cyclooctatetraene was approximately 41%.【Conclusion】The endophytic Trichoderma strains within chestnut fruits were reported for the first time,and the new species Trichoderma castaneae and the inhibitory effects of its volatiles on the growth of pathogenic fungi were identified,providing resources and approaches for the green prevention and control of chestnut fruit rot through biological fumigation.