Phenotypic and Genetic Diversity of Benomyl-Degrading Bacteria Isolated from Agricultural Soils

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부(식물미생물학전공), 2015. 8. 가종억.Benomyl is a benzimidazole family of fungicide which is used for fungi control in agricultural soils. Although benomyl has been used worldwide, a few benomyl-degrading microorganisms have been reported. In this study, nineteen bacterial strains were isolated from agricultural soils across Korea, and their phenotypic and genetic characteristics were investigated. The isolates were able to utilize benomyl as a sole carbon and energy source in mineral medium. Analysis of the 16S rRNA gene sequence revealed that all the isolates were related to members of the genera, Mycobacterium and Rhodococcus. Nine different chromosomal DNA fingerprinting patterns were obtained by polymerase-chain-reaction (PCR)amplification of repetitive extragenic palindromic (REP) sequences. The isolates were classified into three groups according to their growth and degradation properties, taking 48?100 hours to completely degrade 100ppm of benomyl in mineral medium. 2- aminobenzimidazole and 2-hydroxybenzimidazole were identified as intermediate metabolites by HPLC. Degradation experiments with various benzimidazoles showed that they had narrow degradation capabilities. They could degrade carbendazim but hardly utilized other benzimidazoles such as thiabendazole, thophanate-methyl, and fuberidazole. When analyzed with PCR amplification using previously reported carbendazim-hydrolyzing esterase gene, mheI, four isolates produced positive DNA bands. This is the first time that Mycobacterium can degrade benomyl as a sole carbon and energy source. Considering complete degradation capability of these strains, they might be useful for biodegradation in soils contaminated with benomyl.Contents Abstract····························································································································i Contents·························································································································iii List of tables···················································································································v List of figures·················································································································vi I. Introduction·················································································································1 II. Materials and Methods······························································································3 1. Media and culture condition················································································3 2. Chemicals··········································································································3 3. Soil sampling and isolation of bacterial strains····················································7 4. Phylogenetic identification by 16S rDNA sequence analysis·························10 5. Colony REP-PCR···························································································12 6. Axenic culture experiments·············································································14 7. Degradation phenotype analysis········································································15 8. Plasmid profiling experiment············································································15 9. High Performance Liquid Chromatography (HPLC) Analysis······················16 10. PCR amplification of the benzimidazole and carbamate hydrolyzing gene··16 III. Results ·················································································································18 1. Isolation of benomyl-degrading bacteria·······················································18 2. 16S rDNA sequence analyses··········································································19 3. REP-PCR genomic fingerprinting····································································23 iv 4. Growth patterns of isolates on benomyl····························································25 5. Identification of 2-aminobenzimidazole and 2-hydroxybenzimidazole as intermediate metabolites in the bacterial degradation of benomyl·······················28 6. Plasmid detection experiment···········································································30 7. Degradative diversity analysis······································································32 8. Sequence diversity analysis of benzimidazole-degradative genes ·······················35 IV. Discussion··············································································································38 Literature Cited ···········································································································41 Abstract in KoreanMaste