根结线虫拮抗放线菌的筛选及菌株HA10002的鉴定与活性物质分析

为了筛选获得对根结线虫具有拮抗能力的强活性放线菌菌株，并从菌株中发现新的杀线虫活性化合物，从海南东寨港红树林采集海泥样品9份，从五指山原始森林等地采集土壤样品51份，用平板稀释法对海泥和土壤样品进行分离；以南方根结线虫2龄幼虫为靶标线虫，采用24孔细胞培养板液体线虫法进行初筛和复筛；根据形态特征、培养特征、生理生化特征和16S rDNA序列对复筛出的活性菌株进行鉴定；菌株发酵液经溶媒萃取、硅胶柱层析、Sephadex LH 20凝胶过滤和制备薄层板层析，采用活性跟踪法从中分离纯化杀线虫活性化合物；化合物经光谱及波谱分析和文献对照，进行结构鉴定。从60份样品中分离得到356株菌落形态有差异的放线菌。初筛得到16株对根结线虫校正死亡率在80%以上的菌株，复筛获得3株线虫校正死亡率在95%以上的菌株，其中分离自海泥样品中的菌株HA10002抗线虫活性最强，发酵原液的校正死亡率达100%。HA10002经鉴定为白浅灰链霉菌，从其发酵液中分离获得2个活性化合物，其中A26 3为Nocardamine，A22 1的结构正在进一步鉴定中。上述结果表明，筛选出的放线菌HA10002能合成2种以上杀根结线虫的活性代谢产物，是一株遗传稳定且具有开发潜力的活性菌株。

Screening of actinomycetes against root knot nematodes and the active compounds produced by strain HA10002

In order to isolate new actinomycete strains with high nematicidal activity, and to discover new compounds against root knot nematodes, nine mangrove sediments collected from Dongzhaigang mangrove and 51 soil samples from Five finger Mountain,in Hainan Province were isolated using dilution plate method. Strains were selected by using Meloidogyne incognita as target nematodes.The selected antagonistic strains were identified through morphological observation and 16S rDNA sequencing and analyses. Fermentation broth of the strain was separated and purified by means of solvent extraction, column chromatography, Sephadex LH 20 and preparation TLC. Structures of the active compounds were elucidated based on the results of 1H NMR, 13C NMR, HMBC, 1H 1H COSY and ESI MS. From 9 mangrove sediments, 93 actinomycete strains were isolated and from 51 soil samples, 263 actinomycete strains were isolated. Of the 356 strains, 16 antagonistic strains against M. incognita were selected, among which strain HA10002 from mangrove sediment possessed the highest nematicidal activity, with lethal rate as high as 100%, and it was identified as Streptomyces albogriseolus. By adopting the tracing method of bioactivity, two active compounds were separated from the fermentation broth of HA10002, of which the compound A26 3 was identified as nocardamine, and the structure of A22 1 is being elucidating. It is concluded that the selected actinomycete strain HA10002 could produce more than two active metabolites against Meloidogyne spp., and it can be a promising strain in biological control of root knot nematodes.