摘要
五指山位于海南岛中部,北纬18°45'20"-18°58'54",东经109°39'38"-109°47'50",主峰位于五指山市境内,最高峰海拔高度1867米,属国家自然保护区的面积134.4平方公里,为海南最大的自然保护区,是世界上现存地势最高的原始热带雨林之一,蕴藏着极其丰富的稀有放线菌资源,至今仍然未开发利用。我们对五指山原始林区不同海拔高度和不同植被生境的土壤样品的稀有放线菌进行了分离、初步鉴定及以香蕉枯萎镰刀菌4号小种(Fusaium oxysporum f.sp.cubense race 4)为对象进行了抑菌效果初步评价,旨在发现稀有放线菌新属、新种,获得对香蕉枯萎镰刀菌病害具良好抑菌效果的稀有放线菌菌株。初步研究结果如下：
一、采用稀释平板法,主要选取HV、葡萄糖天门冬素、甘油天门冬素和土壤浸汁四种选择性培养基(每种培养基上均含有20mg/L的制霉菌素、20mg/L放线菌酮及50mg/L的重铬酸钾),结合苯酚、SDS、干热和超声波四种不同的预处理方法,共从五指山原始林区不同海拔高度不同生境的116份土样中分离得到702株稀有放线菌,其中以苯酚预处理后再涂布于HV琼脂上分离得到的稀有放线菌最多。
二、对不同海拔高度分离到的稀有放线菌数量进行统计分析,发现稀有放线菌的数量随海拔高度的升高而降低；同时我们还发现不同生境样品中分离得到的稀有放线菌的数量也是不一样的。
三、根据气生菌丝有无、菌落的颜色、生长及形态的观察,产水溶性色素的情况等,将702株稀有放线菌划分为14个类群。
四、从上述14个类群中挑选26株稀有放线菌进行了形态观察和16S rRNA基因序列分析,结果显示此26株菌分别为小单孢菌属,冢村菌属,小双孢菌属,指孢囊菌属,野村菌属,游动四孢属,Luedemannella及Streptosporangiaceae str.,其中以小单孢菌属为主。
五、以香蕉枯萎病菌4号小种为抑制对象,对702株稀有放线菌进行抑菌活性初步测定和筛选,发现菌株210-1-61具有较强的拮抗活性,抑菌距离达8.2mm。
六、对拮抗菌210-1-61进行鉴定,通过形态特征及16S rDNA序列分析,再通过生理生化等多项特征与相关近缘种属比较,我们将其鉴定为小单孢菌属,与Micromonospora pattaloongensis JCM12833T亲缘关系最近。
七、对菌株119-1-07的进行初步鉴定,发现其形态特征与Luedemannella flava7-40(26)T较为相似；且其16SrDNA序列相似性与Luedemannella flava 7-40(26)T最高,为97.2%,并其能在进化树上与Luedemannella形成一个稳定独立的分枝；但其培养特征及生理生化特性与Luedemannella flava 7-40(26)T则有一定的差异。因此,119-1-07极为可能是Luedemannella属的一个新种。
Wuzhishan is located at 18°45'20"-18°58'54"N,109°39'38"-109°47'50"E, in the central part of Hainan island. Its main peaks are located at the Wuzhishan City and the highest altitude is 1867 m. It is the largest nature reserve in Hainan with an area of 134.4 km2 belonging to the national government. Wuzhishan has also one of the highest virgin tropical rain forests in the world. Wuzhishan contains abundant rare actinomycetes resources which have not been exploited so far. In order to obtain new, highly efficient antibiotic-producing isolates of actinomycetes that have good biocontrol potentials against Fusarium oxysporum f.sp. cubense race 4 (Focr4) we carried out soil sample collection from different altitudes and vegetation habitats of Wuzhishan forest, isolation and identification of the actinomycetes, as well as evaluating their antagonistic effects against Fusarium oxysporum f.sp. cubense race 4 (Focr4), The preliminary results are as follows:
1. A total of 702 actinomycetes isolates were isolated from 116 soil samples collected from Wuzhishan and grown on various agar media including mainly HV, glucose asparagine, soil extract and glycerol-asparagine, all supplemented with 20mg/l nystatin,20mg/l nalidixic acid and 50mg/l potassium dichromate. Soil dilution plate method with pretreatment using phenol, SDS, dry heat or ultrasonic was used. Pretreatment with phenol followed by spreading the samples onto HV agar yielded the maximum number of actinomycete isolates.
2. Statistical analysis on the number of isolates from soil samples collected at different altitudes indicated that the quantity of actinomycetes decreased with the increase of the altitude of sampling sites. We also found the number of actinomycetes isolated varied with different habitats.
3.702 isolates of actinomycetes were divided into 14 groups based on the characteristics of the colonies on culture plates, including the presence/absence of aerial mycelia, growth pattern, color of the colony and the production or absence of soluble pigments.
4.26 actinomycetes isolates were selected for detailed morphological studies and 16S rDNA sequences analysis. The results indicated that they belonged to eight genera: Micromonospora, Tsukamurella, Microbispora, Dactylosporangium, Nonomuraea, Planotetraspor, Luedemannella and Streptosporangiaceae.
5.702 isolates were tested for antifungal activity assay by pairing each with Fusarium oxyporm f.sp. cubense race 4 on PDA plates. The results showed that 4 isolates were equally most effective against Fusarium oxyporm f.sp. cubense race 4 but after further screening, the strain 210-1-61 proved to have the highest antagonistic activity and was selected for further studies.
6. To indentify isolate 210-1-61, the phenotypic and physiological characteristics as well as the 16S rDNA sequences were further studied. Based on the results, strain 210-1-61 should be classified as a member of the genus Micromonospora and the closest relative was Micromonospora pattaloongensis JCM12833T (98.89% gene sequence similarity). Although the strain was also very close to Micromonospora eburnea JCM 12345T (98.33% gene sequence similarity) they could be readily differentiated based on some cultural and physiological properties..According to the cultural and physiological characteristics as well as phylogenetic analysis, strain 210-1-61 was identified of Micromonospora,and it's closest to Micromonospora pattaloongensis JCM12833T.
7. As for the identification of strain 119-1-07, its phenotypic characteristics are close to those of Luedemannella flava 7-40(26)T and the 16S rDNA sequences of both had 97.2% similarity. Additionally, the strain was very close to Luedemannella constituting a separate phylogenetic tree branch with the nearest evolutionary distance. However, the strain could be readily distinguished from Luedemannella flava 7-40(26)T based on some cultural and physiological properties. Therefore, strain 119-1-07 should be a novel species belonging to the genus of Luedemannella.
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