1株孜然内生真菌的分离鉴定及代谢产物的GC分析

Isolation and Identification of an Endophytic Fungus from Cumin and GC Analysis for Its Metabolites

采用组织分离法从孜然种子中分离纯化出内生真菌ZR-003菌株,并通过形态学观察和ITS序列分析方法确定其种属。采用平板测定法研究了该菌株对19种常见农业致病真菌的抑制作用,ZR-003菌株发酵液经过滤、离心和萃取,旋转蒸干之后利用GC方法对其代谢产物进行分析。研究结果表明:ZR-003菌落絮状、白色、具不明显的放射状沟纹,分生孢子结构稀少,其菌丝帚状结构明显散开,瓶梗形状和菌落特征应属于散枝亚组微紫青霉系中颜色较淡的类群;经分子生物学鉴定,ZR-003菌株的ITS序列与微紫青霉(Penicillium janthinellum)的相似性为99%,属微紫青霉菌;同时确定其对香蕉灰纹病菌[Cordana musae (Zimm.)Hohn.]、稻曲病菌(Ustilaginoidea virens)、苹果斑点落叶病菌(Alternaria alternaria f.sp mali)、小麦根腐病菌[Bipolaris sorokiniana(Sacc.)Shoemakey]、黄瓜黑星病菌(Cladosporium cucumerinum)都具有非常良好的抑制作用,其抑菌率分别为80.00%、81.82%、80.95%、80.00%和80.49%,其中对稻曲病菌的抑菌作用最好。经GC、UV分析可知,ZR-003菌株发酵液提取物在保留时间17.1789min处有明显出峰,其在328nm(λ),20(ε);280nm(λ),1500(ε);244nm(λ),15000(ε)下有明显吸收,与标准样品枯茗醛的色谱光谱特征一致,经计算得到其在发酵液中含量为28.7μg·mL-1,综上可见,孜然内生真菌ZR-003菌株发酵液中含有与孜然相同的药用成分—枯茗醛,从而为枯茗醛的获得开辟了新的微生物来源。

An endophytic fungi,ZR-003,from the seeds of cumin(Cuminum cyminum L)were isolated and purified by using tissue separation method,and its species was determined by morphological observation and rDNA ITS method.Also,its inhibitions to the 19 kinds of common agricultural pathogenic fungi were conducted with plate method.The metabolites of ZR-003 were analyzed by GC after filtrating,centrifuging,extracting and rotary drying.The results showed that the colony of ZR-003 was white,flocculation with obscure radial grooves.Its conidium structure was scarce;the mycelial broom structure was obviously scattered.The shape of the flask and the characteristics of the colony should belong to the lighter groups of the purple Penicillium of the subgroup.Identified by molecular biology,the ITS sequence similarity of ZR-003 and Penicillium janthinellum was 99%,belonging to Penicillium janthinellum.And it had inhibitions to Cordana musae(Zimm.)Hohn.,Ustilaginoidea virens,Alternaria alternaria f.sp mali,Bipolaris sorokiniana(Sacc.)Shoemakey,and Cladosporium cucumerinum with the inhibition rates of 80%,81.82%,80.95%,80%and 80.49%,respectively.GC and UV analysis showed that the retention time of fermentation products for ZR-003 was 17.1789 min and its peak appeared at 328nm(λ),20(ε);280nm(λ),1500(ε);244nm(λ),15000(ε),which was in accordance with the chromatographic characteristics of standard sample of 4-propan-2-ylbenzaldehyde.And its content was 28.7μg·mL^-1.The results verified that the fermentation of endophytic fungus ZR-003 contains the same medicinal component as cumin.It provided a new source of microorganisms for the acquisition of cuminaldehyde.