【作者】 朱国胜； 刘作易； 喻子牛； 桂阳；

【Author】 Zhu GuoSheng~(1,2,3) LIU ZuoYi~(1,4*) YU ZiNiu~1 GUI Yang~(1,2) (1 State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University, Wuhan,Hubei,430070 2 Guizhou Key Laboratory of Agricultural Biotechnology,Guiyang,Guizhou,550006 3 Institute of Chinese Medical Materials Development,Guizhou Academy of Agricaltural Sciences, Guiyang,Guizhou,550006 4 Guizhou Academy of Agricultural Science,Guiyang,Guizhou,550006)

【机构】 华中农业大学农业微生物国家重点实验室； 贵州省农业生物技术重点实验室； 贵州省现代中药材研究所； 贵州省农业科学院；

【摘要】 对兰科植物菌根真菌进行研究,首要的问题是准确高效地分离出菌根真菌,消除污染菌的干扰,而这些问题解决的关键在于建立一个准确、高效的菌根真菌分离方法。用于兰科植物菌根真菌的分离方法主要包括组织块表面消毒分离法和单菌丝团分离法。但这些方法都无法解决以下问题:1)未能在不毒杀菌丝团的条件,解决细菌和真菌污染问题;2)内生细菌在菌根真菌分离过程中的作用未得到重视和应用;3)分离效率低,无法区分活的菌丝团和死的菌丝团,可培养的菌丝团和不可培养的菌丝团;4)无法将含有多种菌的菌丝团中的各种菌分离纯化出来;5)难将生长慢的种类分离纯化出来。这些问题未解决,致使不能很好地认识菌根真菌的种类和承担的角色,也不能很好地将菌根真菌应用到兰科植物种子萌发,原球茎、幼苗、块茎和成年植株的生长发育中。本研究建立了兰科植物菌根真菌单菌丝团分离方法,涉及根的选择、根的处理、菌丝团的制备、菌丝团的处理、菌丝团的选择及培养、菌株纯化等步骤;该技术解决了:1)细菌和真菌污染问题,2)内生细菌在分离菌根真菌的科学应用问题,3)从成千上万的菌丝团中分离菌根真菌的大海捞针问题,4)具有多种菌的菌丝团中各菌分离纯化问题,5)生长慢的菌根真菌的分离问题。本方法的建立大大提高菌根真菌分离的准确性和高效性,大大降低后续有效菌株的筛选的工作量,促进对兰科植物菌根真菌生物学作用的理解,提高菌根真菌应用的水平。更多还原

【Abstract】 One of the key techniques for studying ochid mycorrhizal fungi is to isolate them from roots accurately.Techniques used for the isolation of orchid mycorrhizal fungi include plating fragments of surface-sterilized roots(PFSSR) on nutrient agar and plating individual carefully separated fungal pelotons(PICSFP) on nutrient agar.There are however,many problems associated with these isolation methods.These include 1) problems of fungal and bacterial contamination that cannot be avoided without destroying the pelotons,2) the unknown role of endophytic bacteria in isolation,3) a low isolation efficiency as dead pelotons or those from which fungi cannot be isolated could not be identified,4) a single peloton may contain several mycorrhizal taxa and all of these cannot usually be isolated and analysed,and 5) it is difficult to isolate slow growing mycorrhizal fungi.These problems have resulted in difficulties in understanding the taxonomy and role of mycorrhizal fungi of orchids and thus make it hard to successfully apply these fungi to promote seed germination,development and growth of protocorms,seedlings,juveniles,tubers and adult plants without chlorophyll.A novel technique for isolating mycorrhizal fungi from roots of orchids was established.This technique involves selection and treatment of roots,preparation of pelotons,treatment of pelotons,culture of pelotons so that fungal hyphae grow out and strain purification.The technique is considered better because 1) problems of fungal and bacterial contamination are resolved,2) endophytic bacteria are suppressed and also used to promote hyphal growth from the pelotons,3) live and dead pelotons, and those from which fungi are culturable or unculturable can easily be identified,providing increased isolation efficiency,4) a single taxon can be isolated from a single peloton containing several mycorrhizal taxa,5) slow-growing mycorrhizal taxa can easily be isolated.The implications and potential use of this technique in future studies is discussed.更多还原