川楝内生放线菌的分离及多样性研究

Isolation and diversity of endophytic actinomycetes from Melia toosendan

【目的】分离四川省各个地区川楝内生放线菌并研究其物种多样性。【方法】应用7种选择性分离培养基分离样品根、茎、叶、树皮和果实中的内生放线菌,采用16SrRNA基因RFLP分析代表菌株多样性。【结果】研究共获得403株内生放线菌。不同地点、不同植株部位、不同培养基分离得到的内生放线菌数目均有差异。广元采集的样品分离得到的数目最多,为86株;最少的是绵阳,仅有12株。从植物表皮中分离到148株放线菌,占获得菌株总数的36.7%;而从果中分离到31株,仅占获得菌株总数的7.6%;虽然从根部分离到的数量也很少,但是其出菌率却是最高的。5号和3号培养基的分离效果最为理想。16S rRNA基因RFLP分析结果显示所有供试菌株在68%的相似性上聚在一起,在84%的相似水平上分成了10个遗传类型。代表菌株的16SrRNA基因序列测定及系统发育分析结果表明:分离得到的放线菌包括4个属,分别是链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。其中,链霉菌是优势类群,占代表菌株数目的比例高达91%,而稀有放线菌的比例只有9%。【结论】研究发现的川楝内生放线菌主要属于链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。

[Objective] To investigate the diversity of endophytic actinomycetes isolated from Chinesemedicinal plant Melia toosendan from different regions of Sichuan province. [Methods] Seven kinds of selective isolation media were used to isolate endophytic actinomycetes from the roots, stems, leaves, bark, and fruits of Melia toosendan. 16S rRNA gene-RFLP was used to analysis the diversity and community structure of endophytic actinomycetes. [Results] A total of 403 strains of endophytic actinomycetes were isolated and purified. The number of isolates was varied with different sampling sites, media and plant tissues. Eighty-six strains were obtained from the samples in Guangyuan, which was the highest. The least was the samples from Mianyang, only 12 strains. One hundred and forty-eight strains of actinomycetes were isolated from the bark, accounting for 36.7% of the total strains. Only 31 strains were isolated from the fruits, accounting for 7.6% of the total strains. Although the quantity of isolates from the roots was very low, the isolation frequency was the highest. The most ideal isolation media were numbers 5 and 3, indicating that this two media were most suitable for the growth of endophytic actinomycetes. The results of 16S rRNA gene PCR-RFLP analysis indicated that all of the strains were clustered together at the 68% similarity level and divided into 10 genetic types at the 84% similarity level. Thirty-seven representative strains were chosen for 16S rRNA sequencing. The results of 16S rRNA sequencing and phylogenetic tree ananlysis of 16S rRNA of endophytic actinomycetes showed that the tested strains belonged to Streptomyces, Kitasatospora, Arthrobacter, and Kribbella, among which Streptomyces was dominant, accounting for 91% of the representative strains. [Conclusion] The results presented above indicate that the main endophytic actinomycetes belong to Streptomyces, Kitasatospora, Arthrobacter, and Kribbella in Chinese medicinal plant Melia toosendan in Sichuan province.