Isolation and diversity of endophytic actinomycetes from Melia toosendan

Abstract:

[Objective] To investigate the diversity of endophytic actinomycetes isolated from Chinese medicinal 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.

Key words:16S rRNA genes, Melia toosendan, Endophytic actinomycetes, Isolation, Diversity