| 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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