濒危兰科植物三蕊兰根内生真菌的分离鉴定及促生作用

Identification and growth promotion of endophytic fungi from endangered Neuwiedia singapureana(Orchidaceae)

【目的】开展三蕊兰(Neuwiedia singapureana)可培养内生真菌多样性及其促生潜能研究,为发掘我国海南地区三蕊兰内生真菌资源提供参考依据。【方法】以分布于海南吊罗山自然保护区野生三蕊兰为材料,通过分离培养,结合ITS测序及分析开展三蕊兰根内可培养真菌分离鉴定,并对其溶磷能力进行检测。将分离获得的内生真菌与铁皮石斛(Dendrobium officinale)和多花脆兰(Acampe rigida)原球茎进行共培养,检测其对2种兰科植物生长的影响。【结果】在光学显微镜下可明显观察到三蕊兰根细胞内分布的真菌菌丝。共获得三蕊兰根内生真菌24个OTUs,分属于9科14属,木霉属(Trichoderma)真菌分离概率最高;Nectriaceae科5个属(如Cylindrocarpon和Gliocephalotrichum等)真菌分离率次之。其中5株内生真菌具有不同程度的溶磷能力,为Apiotrichum sp.(Neu242),Cylindrocarpon spp.(Neu83和Neu154),Gliocladiopsis sp.(Neu245)和Mucor sp.(Neu123)属真菌。菌株Mucor sp.Neu123和Apiotrichum sp.Neu242对铁皮石斛和多花脆兰幼苗生长具有显著促进作用。【结论】三蕊兰根内具有丰富的可培养内生真菌,筛选鉴定出具溶磷能力的兰科促生真菌,可为兰科植物的人工繁育提供促生菌株资源,也为兰科菌根共生磷营养机制研究奠定基础。

【Objective】The purpose of the paper was to study the diversity and growth promoting potential of culturable endophytic fungi in Neuwiedia singapureana,and provide a reference for the exploration of endophytic fungi resources in Hainan of China.【Method】The endophytic fungi strains of N.singapureana rooted from the Dialuoshan Nature Reserve in Hainan province were identified by culture method,combined with ITS sequencing,and the phosphorus dissolving and growth promotion ability of some fungi was tested.The endophytic fungi was co-cultured with the protocorms of D.officinale and A.rigida,to test their effects on the growth of the two orchids.【Result】By freehand sections and microscopic observation,the fungal hypha in the cortex of root of the N.singapureana could be clearly observed.24 OTUs were obtained,belonging to nine families and 14 genera,among which Trichoderma in Moniliaceae family had the highest isolation rate,with five genera(eg.Cylindrocarpon,Gliocephalotrichum)strains in Nectriaceae followed.Among them,five strains showed different degrees of phosphorus-decompressing ability,contaning Apiotrichum sp.(Neu242),Cylindrocarpon spp.(Neu83 and Neu154),Gliocladiopsis sp.(Neu245)and Mucor sp.(Neu123).Morover,Mucor sp.Neu123 and Apiotrichum sp.Neu242,showed significantly promoting effects on the growth of D.officinale and A.rigida seedlings.【Conclusion】There are abundant culturable fungi in the roots of N.singapureana.The endophytic fungi with potential phosphorus-soluble ability are screened from N.singapureana roots,which can provide growth-promoting strain resources for the artificial breeding of orchids,and also lay a foundation for the research on the mechanism of orchid mycorrhiza symbiotic phosphorus nutrition.