Journal of Plant Pathology & Microbiology

Abstract

Formulation of Novel Surface Sterilization Method and Culture Media for the Isolation of Endophytic Actinomycetes from Medicinal Plants and its Antibacterial Activity

Waheeda K and Shyam KV

Endophytic actinomycetes from medicinal plants were rarely reported as compared to those from soil and marine sources. The present results correlate the presence of endophytic actinomycetes in medicinal plants by isolating them from three medicinal plants i.e. Ocimum basillicum, Withania somnifera and Rauvolfia tetraphylla. In the present work surface sterilization method and media was standardized and 32 endophytic actinomycetes were isolated from three medicinal plants. We assessed the efficiency of four different surface sterilization methods and four medium for isolation of endophytic actinomycetes. The method with calcium hypochlorite, sodium hydrogen carbonate and sodium azide (New method) used was more effective in eliminating epiphytic microorganisms. Among four tested media starch casein agar (SCA) found to be best media for the isolation of endophytic actinomycetes. We succeeded in obtaining fast and luxurious growth of pure culture of endophytic actinomycetes on modified ISP-4 (M) (inorganic salt solution agar) in comparison with standard ISP-4(S). Preliminary antibacterial assay of all isolates were tested by confrontation test. The secondary screening of selected isolates were tested by disc diffusion test by using ethyl acetate extract which showed broad spectrum antibacterial activity against test human pathogens. Based on the morphological and phenotypic characters 12 isolates were identified as Streptomyces spp. Among 12 isolates A3 as a representative was characterized by SEM and identified by 16SrRNA analysis as Streptomyces flavoviridis A3WK, it showed significant antibacterial activity against test human pathogen. This is the first report of successful isolation of endophytic actinomycetes from the said medicinal plants, for using newly formulated surface sterilization method and new comparative study on ISP-4 medium.