Purification and Identification of Paenibacillus sp., Isolated from Diseased Larvae of Allomyrina dichotoma (Linné, 1771)(Coleoptera: Scarabaeidae) in Insect Farms
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In reared populations of Allomyrina dichotoma, commercial insects, the skin of last instar larvae was changed softer with opaque white, and infested grubs eventually died. To clarify the cause of the symptom,we collected the larvae of A. dichotoma from five farms and examined their intestinal bacterial florae using pyrosequencing technique. From those results, a member of Paenibacillus was found only in the larvae showing the symptom of disease. Through PCR analysis using a Paenibacillus specific primer set, we obtained the partial 16S rRNA gene sequence and confirmed the microbe as Paenibacillus sp. For clear identification,a whole guts was extracted from each larva showing the sign of the disease and incubated at 70oC for 15 min to isolate spore forming bacteria. After then,each content of guts was cultured on MYPGPNAL agar medium(12.5 μg/ml of nalidixic acid) at 30oC. The 16S rRNA gene sequence analysis for the isolated bacteria showed that they were closely related to P. rigui(97.9%similarity), to P. chinjuensis(96.1% similarity), and to P. soli(95.3% similarity). Additional tests including API test and cellular fatty acid composition analysis were performed, but the strain couldn't be identified at species level, suggesting it may represent novel species of the genus Paenibacillus.
Abstract
In reared populations of Allomyrina dichotoma, commercial insects, the skin of last instar larvae was changed softer with opaque white, and infested grubs eventually died. To clarify the cause of the symptom,we collected the larvae of A. dichotoma from five farms and examined their intestinal bacterial florae using pyrosequencing technique. From those results, a member of Paenibacillus was found only in the larvae showing the symptom of disease. Through PCR analysis using a Paenibacillus specific primer set, we obtained the partial 16S rRNA gene sequence and confirmed the microbe as Paenibacillus sp. For clear identification,a whole guts was extracted from each larva showing the sign of the disease and incubated at 70oC for 15 min to isolate spore forming bacteria. After then,each content of guts was cultured on MYPGPNAL agar medium(12.5 μg/ml of nalidixic acid) at 30oC. The 16S rRNA gene sequence analysis for the isolated bacteria showed that they were closely related to P. rigui(97.9%similarity), to P. chinjuensis(96.1% similarity), and to P. soli(95.3% similarity). Additional tests including API test and cellular fatty acid composition analysis were performed, but the strain couldn't be identified at species level, suggesting it may represent novel species of the genus Paenibacillus.
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