Isolation and Identification of Actinomycetes from Soil for Controlling Aedes aegypti Larvae
Keywords:
Actinomycetes, Aedes aegypti, 16S rDNA, phylogeneticAbstract
Aedes aegypti is not only an annoying insect but also a carrier of many diseases to humans. Many control methods have been applied continuously. Microbial control using microorganism or its product is becoming outstanding because of highly efficiency, easily to apply and normally safe to human and environment. The objective of this research was to isolate and identify actinomycetes from soil for controlling Aedes aegypti larvae. Soil samples were collected from 2 collecting sites in Mae Hong Son Province with 20 soil samples/site. Actinomyces were isolated using serial dilution technique from soil suspension and spread on starch casein agar (SCA) plates. The actinomycetes colonies were picked up and cultured in glucose yeast malt (GYM) liquid media for 21 days and extracted using high speed centrifuge at 10,000 rpm for 10 min. The supernatant of culture medium was collected, adjusted to 5% concentration and used for efficacy test on 10 mosquito larvae (3rd stage). The isolate with the highest efficacy of crude extract to control mosquito larvae was selected and processed for 2 steps: first, to determine the efficacy of their crude extracts on mosquito larvae as above after being cultured in 5 liquid media: GYM, GYB, SY, ISP1 and ISP3 and second, to search for the related species using phylogenetic analysis based on ITS of 16S rDNA sequence. The result revealed that the total of 195 isolates of Actinomyces were screened from all soil samples. Only was the crude extract of MSsm 007-6 the highest efficacy to control mosquito larvae with 50% mortality at 48 hours after application. The crude extracts of MSsm 007-6 cultured in GYM, Glucose Yeast extract Broth (GYB) and SY liquid media had high efficacy to control mosquitos at 73.3, 96.7 and 63.3 % mortality, respectively. The Neighbor-Joining phylogenetic analysis represented that ITS of 16S rDNA sequence of MSsm 007-6 was very closely related to Streptomyces kasugaensis strain M338-M1 with supporting bootstrap value at 75%
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