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농 학 석 사 학 위 논 문
Phenotypic and Genetic Diversity of
Benomyl-Degrading Bacteria Isolated from
Agricultural Soils
경작 토양으로부터 분리한 benomyl 분해 세균의
표현형적 유전적 다양성 구명
2015 년 8 월
서울대학교 대학원
농생명공학부 식물미생물학전공
이 지 형
A THESIS FOR THE DEGREE OF MASTER OF SCIENCE
Phenotypic and Genetic Diversity of
Benomyl-Degrading Bacteria Isolated from
Agricultural Soils
By
Ji Hyeong Lee
Major in
Plant Microbiology in Agricultural Biotechnology
The Graduate School of Seoul National University
농학석사학위논문
경작 토양에서 분리한 베노밀 분해 세균의
표현형적 유전적 다양성 구명
지도교수 가 종 억
이 논문을 농학석사학위논문으로 제출함
2015년 8월
서울대학교 대학원
농생명공학부 식물미생물학전공
이 지 형
이지형의 논문을 석사학위논문으로 인준함
2015년 8월
위 원 장
부위원장
위 원
A THESIS FOR THE DEGREE OF MASTER OF SCIENCE
Phenotypic and Genetic Diversity of
Benomyl-Degrading Bacteria Isolated from
Agricultural Soils
UNDER THE DIRETION OF ADVISER
JONG-OK KA
SUBMITTED TO THE FACULTY OF THE GRADUATE
SCHOOL OF SEOUL NATIONAL UNIVERSITY
BY JI HYEONG LEE
MAJOR IN PLANT MICROBIOLOGY
DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY
AUGUST, 2015
APPROVED AS A QUALIFIED THESIS OF JI HYEONG LEE
FOR THE DEGREE OF MASTER OF SCIENCE
BY THE COMMITTEE MEMBERS
C H A I R M A N
VICE CHIRMAN
Abstract
Phenotypic and Genetic Diversity of
Benomyl-Degrading Bacteria Isolated from Agricultural Soils
JI HYEONG LEE
Major in Plant Microbiology in
Agricultural Biotechnology
The Graduate School of
Seoul National University
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.
Key words: Benomyl-degrading bacteria, biodegradation, degradative genes
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
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 Korean
List of tables
Table 1. The composition of bacterial culture media
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Table 2. PCR conditions for 16S rDNA amplification
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙11
Table 3. Colony REP-PCR conditions
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13
Table 4. PCR primers and product sizes
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙17
Table 5. Nearest relatives of the benomyl-degrading isolates based upon 16S rDNA sequence analysis
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
List of figures
Fig. 1. Structures of benomyl and other benzimidazole fungicides
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5
Fig. 2. Locations of sampling sites
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙8
Fig. 3. Isolation of benomyl-degrading bacteria
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙9
Fig. 4. Phylogenetic relationships established by the neighbor-joining method
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙22
Fig. 5. REP-PCR band patterns of the representative isolates
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24
Fig. 6. Degradation of benomyl and growth of the representative isolate, SG-D1, grown benomyl-MMO
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙26
Fig. 7. Degradation of benomyl and growth of the representative isolate, JS-E2, grown benomyl-MMO
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27
Fig. 8. Degradation of benomyl and growth of the representative isolate, JC-E4, grown benomyl-MMO
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27
Fig. 9. Chromatograms (HPLC) obtained from representative isolate incubated with 100 ppm benomyl for 12 hours
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Fig. 10. Plasmid profiles of the isolates
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Fig. 11. PCR-amplification with the primers specific for the mhe1gene
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙36
I. Introduction
Benzimidazole is a chemical fungicide that is characterized by the benzimidazole functional group. Benomyl (methyl1-(butylcarbamoyl) benzimidazol-2-ylcarbamate), carbendazim (methylbenzimidazol-2-ylcarbamate), thiophanate-methyl (dimethyl 4,4’-(o-phenylene)bis(3-thioallophanate), thiabendazole (2-(thiazol-4-yl) benzimidazole), and fuberidazole (2-(2-furyl) benzimidazole) belong to this group. They are widely and abundantly used in agricultural fields to kill or control harmful fungi. They bind to microtubules interfering cell function such as meiosis and intracellular transportation. The hydrolyzed form, carbendazim, is chemically stable and persistent in environment harming the liver and endocrine system. Among benzimidazole fungicides, benomyl is, since 1970, applied mainly in agricultural soils to control fungi, acarids and nematodes in many crop growing countries across the world. The major use of benomyl is to control fungal diseases on fruits, vegetables and ornamentals. Recent studies indicate that considerable fractions of the applied fungicides are transported from agricultural soils to other food stuffs, including surface water and even ground water, posing serious environmental problems (Pandey et al., 2010). Furthermore, carbendazim is a suspected mutagen, carcinogen, and endocrine disruptor, and its use is tightly controlled by regulatory bodies in many countries (Pandey et al., 2010). Therefore, it is very important to assess the persistence and fate of the fungicides and investigate the factors responsible for their degradation, both biotic and abiotic.
Biodegradation is considered to be primarily responsible for removal of benomyl and carbendazim. Although several bacterial strains that can degrade benzimidazole fungicides have been isolated from soil, only few bacterial strains have been discovered on benomyl, its
degradative metabolites, biodegradation pathways, degradative genes and enzymes. To the best of our knowledge, this is the first report on diversity of bacteria that utilize benomyl as the sole carbon source and identification of an intermediate in the degradation of benomyl by various bacterial strains.
This study aimed to look for strains that were able to degrade benomyl completely as a sole carbon source. Nineteen strains isolated from agricultural soils, which had been routinely treated with the benomyl, were characterized on their metabolic, genetic, and physiological basis.
II. Materials and Methods
1. Media and culture condition
All isolated bacteria were cultured on MMO mineral medium (Stainer et al, 1966) containing benomyl at 100 ppm (μg/ml) as the only carbon source. The solid medium used for strain isolation was peptone-tryptone-yeast extract-glucose medium consisting of 0.25 g of peptone (Difco Laboratories, Detroit, Mich), 0.25 g of tryptone (Difco), 0.5 g of yeast extract (Difco), 0.5 g glucose, 0.03 g of magnesium sulfate, and 0.003 g of calcium chloride (Table 1). All cultures were incubated at 28℃ and liquid culture were aerated by shaking at 150 rpm on a rotary shaker (Vision Co. Korea).
2. Chemicals
Analytical grade benomyl (methyl1-(butylcarbamoyl) benzimidazol-2-ylcarbamate), carbendazim (methylbenzimidazol-2-ylcarbamate), thiophanate-methyl (dimethyl 4,4’-(o-phenylene)bis (3-thioallophanate), thiabendazole (2-(thiazol-4-yl) benzimidazole), fuberidazole (2-(2-furyl) benzimidazole), and carbaryl (2-isopropoxyphenyl N-methylbenzimidazole) were obtained from Sigma Chemical Co., St. Louis, Mo (Fig. 1).
Table 1. The composition of bacterial culture media Medium Composition (g/L) PTYG Peptone 0.25 Tryptone 0.25 Yeast Extract 0.5 Glucose 0.5 MgSO4 0.03 CaCl2 0.003
MMO Sol A Na2HPO4 0.71
KH2PO4 0.68 Sol B (NH4)2SO4 0.3 Sol C MGSO4 ∙ 7H2O 0.05 Sol D CaCl2∙ H2O 0.001 Sol E FeSO4 ∙ 7H2O 0.006 ZnSO4 ∙ 7H2O 0.0028 MnSO4 ∙ 7H2O 0.0012 Co(NO3)2 ∙ 6H2O 0.0017 CuSO4 ∙ 5H2O 0.0004 (NH4)6Mo7O24 ∙ 4H2O 0.0002
(1) (2) (3) (4) (5) (6) (7)
(8)
Fig. 1. Structural formulas of benomyl (1), carbendazim (2), thiophanate-methyl (3), thiabendazole (4), fuberidazole (5), 2-aminobenzimidazole (6), 2-hydroxybenzimidazole (7), carbaryl (8)
3. Soil sampling and isolation of bacterial strains
Soil samples were collected from the 15 cm top layer in agricultural soils across South Korea (Fig. 2). One hundred forty three soil samples were taken and screened through a 2-mm-pore-size sieve, and kept at 4℃ prior to use. For enrichment of benomyl-degrading bacteria from the agricultural soil, 20 g of each soil sample was treated with benomyl dissolved in distilled water to a final concentration of 100 μg/g soil and thoroughly mixed. The treated soil was incubated and periodically stirred with water at room temperature keeping the moisture in the soil. Five weeks after benomyl application, 1 g of soil sample was transferred into a tube containing 3 ml of MMO mineral medium supplemented with benomyl (100 μg/ml) and incubated at 28℃ on a rotary at 150 rpm for 14 days. The enriched culture was serially diluted and spread onto PTYG agar plates and the plates were incubated at 28℃. The strains were then tested for benomyl degradation in fresh benomyl-MMO (100 μg/ml benomyl) medium before strain purification (Fig. 3).
Fig. 3. Isolation of benomyl-degrading bacteria. Enrichment with benomyl
(0.1ul/g soil, 5 weeks)
Mineral medium with benomyl (100 μg/ml) Streaking on a PTYG agar medium Confirmation of pure culture UV/VIS spectrophotometer HPLC analysis
4. Phylogenetic identification by 16S rDNA sequence analysis
Total genomic DNA was extracted from the isolates and PCR amplification of 16S rRNA genes was performed with forward primer 27mf and reverse primer 1492r as previously described (Kim et al., 2005; Lane, 1991). The amplified 16S rRNA genes were sequenced using a ABI Prism BigDye Terminator Cycle Sequencing Ready Kit according to the manufacturer’s instruction (Perkin-Elmer) with the sequencing primers 515r, 926f and 1055r (Chapalamadugu et al., 1992; Lane, 1991). Approximately 1400 unambiguous nucleotide positions were used for comparison to the data in GenBank using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). Sequences from nearest relatives were identified from the Ribosome Database Project (RDP) using the SIMILARITY-RANK program of the RDP (Maidak et al., 2000).
Table 2. PCR conditions for 16S rDNA amplification PCR reaction mixture 10x buffer 5.0 ㎕ dNTP (2.5 mM) 5.0 ㎕ 27mf primer (20 pmole/㎕) 5.0 ㎕ 1492r primer (20 pmole/㎕) 5.0 ㎕
Taq polymerse (5 U/㎕) 0.5 ㎕
Distilled Water 28.5 ㎕ Template DNA 1.0 ㎕ PCR reaction condition Step 1 94℃ 5.0min Step 2 94℃ 1.0min Step 3 55℃ 1.0min Step 4 72℃ 1.0min Step 2, 3, 4: 29 cycles Step 5 72℃ 10min Step 6 4℃
5. Colony REP-PCR
Aliquots of genomic DNA from isolates were used as templates to generate repetitive extragenic palindromic-PCR (REP-PCR) genomic fingerprints with the BOXA1R primer (5’-CTACGGCAAGGCGACGCTCACG-3’), as described previously (de Bruijn, 1992). PCR amplification were carried out in a model PTC 100 cycler (MJ Research, Waltham, U.S.A) in 25 μl of PCR mixtures containing Gitschier buffer [1 M (NH4)2SO4, 1 M
Tris-HCL (pH 8.8), 1 M MgCI2, 0.5 M EDTA (pH 8.8), 14.4 M B-mercaptoethanol], 0.1%
bovine serum albumin, 100% dimethyl sulfoxide, each deoxynucleoside triphosphate at a concentration of 2.5 mM, 50 pmol/ul BOXA1R primer, 5 U of Tap DNA polymerase per ml, and 1 μl of DNA. The cycle used were as follows: 1 cycle as 95℃ for 7 min, 35 cycles at 92℃ for 1 min, 52℃ for 1 min and at 65℃ for 8 min; 1 cycle at 65℃ for 16min; and a final soak at 4℃. After the reactions, PCR products were separated by electrophoresis on 1.2% agarose gels. After electrophoresis, the image was photographed with UV transillumination (306 nm).
Table 3. Colony REP-PCR conditions PCR reaction mixture Gitschier buffer 5.0 ㎕ BSA (0.1%) 0.4 ㎕ DMSO (100%) 2.5 ㎕ dNTP (2.5 mM) 5.0 ㎕
BOX1R primer (50 pmole/㎕) 5.0 ㎕
Taq polymerase (5 U/㎕) 0.8 ㎕
Distilled Water 1.8 ㎕ Template DNA 1.0 ㎕ PCR reaction condition Step 1 93℃ 7.0min Step 2 92℃ 1.0min Step 3 52℃ 1.0min Step 4 65℃ 8.0min Step 2, 3, 4: 35 cycles Step 5 65℃ 16min Step 6 4℃
6. Axenic culture experiment
After growth in benomyl-MMO medium (100 μg/ml), cells were harvested by centrifugation at 5,000 rpm for 1 min at 4℃, washed twice with MMO medium, and resuspended in mineral medium. Aliquots of resuspended cells were inoculated into flasks containing 100 ml of mineral medium supplemented with 100 ppm (100 μg/ml) of benomyl as the sole carbon source at a final density of OD600 = 0.004. All cultures were incubated at
28℃ in the dark and were aerated by shaking on a rotary shaker (150 rpm). The experiments for each were conducted in triplicate. At specific intervals, aliquots of the cultures were taken out and used to determine cell growth and the remaining concentrations of benomyl. Cell growths were determined with optical density at 600 nm. For the quantification of benomyl, 3 ml culture of the flasks was filtered by Minisart® 0.2 μm Syringe Filter. After filtration, the culture was used for the measurement of optical densities at 230 nm for benomyl. The concentration of benomyl was calculated using standard curves prepared from the known concentrations of benomyl in the same medium.
7. Degradation phenotype analysis
Each strain was grown in benomyl-MMO mineral medium (100 μg/ml) to produce cells induced for benomyl metabolism. Cells were harvested, washed, and prepared in the same way as described above. Aliquots of suspended cells were inoculated into culture tubes, each of which contained 3 ml mineral medium supplemented with 2-aminobenzimidazole and 2-hydroxybenzimidazole, thiophanate-methyl, thiabendazole, and fuberidazole at a concentration of 100μg/ml. The tubes were cultured by shaking 150 rpm at 28℃ for 4 weeks, after which the optical density at 600 nm was determined. To determine the degradation of fungicides, the cultures were centrifuged to remove the cellular material, and UV absorption was measured to monitor degradation of the substrates.
8. Plasmid profiling experiment
For detection of plasmid DNA from isolates, cells were lysed using the procedure described by Kado et al. (Kado and Liu, 1981) and alkaline method (Bimboim and Doly, 1979).
9. High Performance Liquid Chromatography (HPLC) Analysis
The growth condition was the same as described above. The 1 ml of culture samples were centrifuged with swing rotor at 340 g for 10 min (combi-514R, Hanil, Korea). The supernatant was dissolved in 1 ml acetonitrile and then filtered with PTFE syringe filters with a pore size of 02.um (Pall Corporation, USA). Filtered samples were analyzed by HPLC on a Luna 5u 18 column (4.6mmVx250mm). Carbendazim, 2-aminobenzimidazole, 2-hydroxybenzimidazole were detected at 286nm. The mobile phase was acetonitrile/water (70:30, v/v) and the flow rate was 1ml/min. Metabolites of benomyl were determined by comparison with the authentic compound based on the retention time in the HPLC analysis.
10. PCR amplification of the carbendazim hydrolyzing esterase
gene and carbamate hydrolase gene
The partial gene sequences specific to the benomyl degradation pathway were amplified by PCR with specific primers targeting for carbendazim degradative genes involved in the initial pathways (Pandey et al., 2010; Wang et al., 2010; Pang et al., 2010; Xu et al., 2006). The amplification of the carbendazim degradative gene with the corresponding primers is expected to produce 726bp (Pandey et al., 2010) and the amplifications of the carbamate degradative gene with the corresponding primers is expected to produce 1498bp (Hashiomoto et al., 2006) (Table 4).
Table 4. PCR primers and product sizes
Primer name Target Sequences Size Source of reference
mhe1-F mhe1-R
Carbendazim hydrolyzing
esterase gene (mhe1) 5‘-GCATGGCCAACTTCGTCCTCG5‘-GCGCCCAGCGCCGCCAGC- 3-3‘ ‘ 726bp (Pandey et al., 2010)
cahA-F cahA-R
Carbaryl hydrolase gene
III. Results
1. Isolation of benomyl-degrading bacteria
Nineteen benomyl degrading bacteria were isolated through enrichment process from different agricultural soils. Among 143 agricultural soil samples, 127 soil samples apparently did not show any detectable degradation of benomyl during 5 weeks of the enrichment period, but benomyl completely disappeared from 16 enrichment cultures of soil samples. Through repeated enrichment and purification steps, nineteen benomyl-degrading bacteria were isolated from the 16 enrichment cultures. Soil samples were collected throughout South Korea areas, and the most benomyl-degrading bacteria were found from variousprovinces, where the crop is most largely and widely grown. It seems to be related to the fact that benomyl is mainly used for control of fungi in agricultural soils. The isolates were able to aerobically grow on benomyl as a sole carbon and energy source.
2. 16S rDNA sequence analysis
Analysis of 16S rRNA gene sequences indicated that all of the benomyl-degrading bacteria aligned within the members of the actinobacteria group, Mycobacterium and Rhodococcus (Table 5), having >98% sequence similarity to previously reported species. Althoughthey were isolated from different agricultural soil, most isolates were closely related to the same genus Rhodococcus. The result is not surprising in that many previous studies reported that Rhodococcus show broad catabolic diversity and enzymatic capabilities of environmental and biotechnological importance (Warhurst & Fewson, 1994; Bell et al., 1998).
Furthermore, several strains belonging to Mycobacterium, known for utilizing a wide range of PAH substrates, were isolated. (Lease et al., 2011; Badejo et al., 2013; Pontiroli et al., 2013; Chun Zhang and Anne J. Anderson, 2012). They have not been reported as benomyl-degrading bacteria until now. The neighbor-joining method was used to establish the phylogenetic relationships amongst these isolates on the basis of their 16S rRNA gene sequences and also their relationships between closely related type strains (Fig. 4).
Table5. Nearest relatives of the benomyl-degrading isolates based upon 16S rDNA sequence analysis
Isolate REP
pattern Sample site Nearest relative
a Similarity(%)a
CN-T1 1 Taean, Chumgcheongnam-do Mycobacterium obuense 98.7
CN-S1 4 Seosan, Chumgcheongnam-do Rhodococcus qingshengii 98.6
BR-A3 8 Boryeong, Chumgcheongnam-do Rhodococcus qingshengii 100
JN-J1 3 Jangseong, Jeollanam-do Mycobacterium gilvum 99.06
JS-E2 4 Jangseong, Jeollanam-do Rhodococcus qingshengii 99.71
JS-G2 4 Jangseong, Jeollanam-do Rhodococcus qingshengii 98.7
KJ-S1 4 Jeongseon, Kangwon-do Rhodococcus qingshengii 98.6
KJ-N1 3 Najeonri, Kangwon-do Mycobacterium gilvum 99.06
KJ-N2 4 Najeonri, Kangwon-do Rhodococcus qingshengii 98.6
SG-E1 4 Seoguipo, Jeju-do Rhodococcus qingshengii 99.71
SG-D1 7 Pyoseonri, Jeju-do Mycobacterium chlorophenolicum 99.72
JC-F1A 4 Euntanri, chungcheongbuk-do Rhodococcus qingshengii 98.6
CW-B1 5 Kagokri, chungcheongbuk-do Rhodococcus qingshengii 100
JC-E4 2 Euntanri, chungcheongbuk-do Mycobacterium wolinskyi 98.07
KY-S3 9 Seori, Gyeonggi-do Mycobacterium conceptionense 99.58
KY-S1 4 Seori, Gyeonggi-do Rhodococcus qingshengii 98.6
KP-H1 4 Hoehwari, Gyeonggi-do Rhodococcus qingshengii 99.58
a
Fig.4. Phylogenetic relationships established by the neighbor-joining method, based on 16S rRNA gene sequences of REP representative strains, closely related type strains (*), and previously isolated pesticide degrading bacterial strains. Numbers on braches indicate bootsrap confidence estimates obtained with 1,000 replicates. Scale bar represents an evolutionary (Knuc) of 0.01. JN-J1 JC-E4 Mycobacterium sphagni DSM 44076T (FR733719)* Mycobacterium pallens czh-8T (DQ370008) Mycobacterium crocinum czh-42T (DQ534008) Mycobacterium psychrotolerans WA101T (AJ534886)
SG-D1
Mycobacterium chlorophenolicum PCP-1T(X79094)* Mycobacyerium chubuense ATCC27278T(X55596) Mycobacterium gilvum ATCC 43909T(X81996)
Mycobacterium iranicum M05T (HQ009482) CN-T1
Mycobacterium obuense ATCC 27023T(X55597)* Mycobacterium senegalense CIP 104941T (AY457081) Mycobacterium houstonense ATCC 49403T (AY457067 ) Mycobacterium conceptionense CIP 108544T (AY859684)* KY-S3
Mycobacterium peregrinum ATCC 14467T (AF058712)
Rhodococcus baikonurensis GTC 1041T (AB071951)
ES-A3 CW-B1
Rhdococcus erythropolis DSM 43066 T(X79289)* Rhodococcus jialingiae djl-6-2T (DQ185597)* Rhodococcus erythropolis NBRC 100887(AP008957) BR-A3 Rhodococcus qingshengii djl-6T (DQ090961 )* JS-E2 Nocardioides sp. SG-4G 62 100 100 76 100 97 98 49 80 79 46 36 100 17 32 50 21 26 35 0.01
3. REP-PCR genomic fingerprinting
To identify identical strains and investigate genomic relatedness among the closely related isolates by 16S rDNA sequences analysis, REP-PCR experiment was carried out by PCR amplification with the BOXA1R primer (de Bruijn, 1992). It was revealed that the 19 isolates were grouped into nine distinct DNA fingerprint patterns (Fig. 5). Some isolates showed unique REP-patterns, but many isolates showed identical REP-pattern regardless of their regions of origin. For example, as seen in Table 5, an isolate from Jeju province could be the same as one from Gyeonggi, Chungchung, Kangwon or Jeolla province. Their detection frequencies in the soils reflect their ubiquity in South Korea.
M 1 2 3 4 5 6 7 8 M 9 M
Fig.5. REP-PCR band patterns of the representative isolates. Lanes: 1, CN-T1; 2, JC-E4; 3, JN-J1, 4; JS-E2; 5, CW-B1; 6, ES-A3; 7, SG-D1; 8, BR-A3; 9, KY-S3; M, DNA size marker
4. Growth patterns of the isolates on benomyl
Growth curves of the nine representative isolates of REP-PCR patterns were analyzed by growing them in mineral medium containing benomyl (100μg/ml). During the course of the experiment, optical densities of the cultures were measured regularly. All the representative 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. Strain SG-D1, CN-T1, and KY-S3 grown on benomyl-MMO medium totally degraded 100 ppm benomyl within about 48 hours, and the bacterial cell density (OD600) increased in proportion
to benomyl degradation (Fig. 6). Strain JS-E2, ES-A3, CW-B1, and BR-A3 grown on benomyl-MMO medium completely degraded 100 ppm benomyl within about 100 hours and strain JC-E4, JN-J1 within about 80 hours with concomitant cell growth (Fig.7, Fig.8). There was no significant initial lag period and each strain showed a sharp log growth with benomyl degradation.
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 -20 0 20 40 60 80 100 120 0h 6h 12h 18h 24h 30h 36h 42h C el l d en si ty a t O D 6 0 0 b en o m y l 1 0 0 p p m Time(hr)
benomyl con O.D
-0.05 0 0.05 0.1 0.15 0.2 0.25 -20 0 20 40 60 80 100 120 0h 12h 24h 36h 48h 60h 72h 84h 96h ce ll d en si ty a t O D 6 0 0 b en o m y l 1 0 0 p p m Time (hr)
benomyl con O.D
0 0.05 0.1 0.15 0.2 0.25 -20 0 20 40 60 80 100 120 0h 12h 24h 36h 48h 60h 72h 84h ce ll d en si ty a t O D 6 0 0 b en o m y l 1 0 0 p p m Time (hr)
benomyl con O.D
Fig.7. Degradation of benomyl and growth of the representative isolate JS-E2 grown on benomyl-MMO
5. Identification of aminobenzimidazole and
2-hydroxybenzimidazole as intermediates in the bacterial
degradation of benomyl
In benomyl degradation experiments, two metabolite peaks were detected by HPLC analysis at retention time (RT) of 2.5 min and 2.6 min after 3-12 hours incubation (Fig. 9). The intermediates were observed to be accumulated in the biodegradation of benomyl by the isolates after 3-12 hours of incubation. The metabolite peaks were identified as 2-aminobenzimidazole and 2-hydroxybenzimidazole by comparison with the authentic compounds based on the retention time in the HPLC analysis.
Fig.9. Chromatograms (HPLC) obtained from cultures incubated with 100 ppm of benomyl for 12 hours. Minutes 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 V o lt s 0 .0 0 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0 .0 2 0 0 .0 2 5 0 .0 3 0 V o lt s 0.000 0.005 0.010 0.015 0.020 0.025 0.030 1 .8 9 5 2 .5 2 5 2 .6 5 7 3 .1 4 5 Detector A (286nm) D5B T1B6/18 20150628-0009 Retention Time
6. Plasmid detection experiment
When the isolates were subjected to Kado’s plasmid detection procedure (Kado and Liu, 1981), all the nine representative isolates degrading benomyl exhibited no plasmid DNA bands (Fig. 10). They are all gram positive strains, Rhodococcus and Mycobacterium species, and appeared to have no plasmid DNAs.
Fig.10. Plasmid profiles of the isolates. Lanes: 1, CN-T1; 2, JC-E4; 3, JN-J1, 4; JS-E2; 5, CW-B1; 6, ES-A3; 7, SG-D1; 8, BR-A3; 9, KY-S3; M, DNA size marker
7. Degradative diversity analysis
The ability of the isolates to degrade other compounds structurally-related with benomyl was examined by cultivating each isolate grown in the mineral medium supplemented with the respective compound. The substrate degradation capabilities of the representative isolates of each REP-PCR group were shown in Table 6. All isolates could utilize benomyl, carbendazim, 2-aminobenzimidazole and 2-hydroxybenzimidazole. Furthermore, all isolates could degrade carbaryl which belongs to carbamate fungicide. However, the isolates could not degrade other representative benzimidazoles including thiabendazole, thiophanate-methyl, and fuberidazole.
Table 6. Substrate utilization patterns by the benomyl-degrading isolates Isolatesa Substrateb Benomyl 2-aminobenzi midazole 2-hydroxybenz imidazole Carbendazm Thiaben dazole Thiophanate
-methyl Fuberidazole Carbaryl
CN-T1 ++ ++ ++ ++ - - - ++ CN-S1 ++ ++ ++ ++ - - - ++ BR-A3 ++ ++ ++ ++ - - - ++ JN-J1 ++ ++ ++ ++ - - - + JS-E2 ++ ++ ++ ++ - - - ++ JS-G2 ++ ++ ++ ++ - - - ++ KJ-S1 ++ ++ ++ ++ - - - ++ KJ-N1 ++ ++ ++ ++ - - - ++ KJ-N2 ++ ++ ++ ++ - - - ++ SG-E1 ++ ++ ++ ++ - - - ++ SG-D1 ++ ++ ++ ++ - - - ++ SG-D4 ++ ++ ++ ++ - - - ++ ES-A3 ++ ++ ++ ++ - - - ++
a
The isolates were grown on R2Abefore the test of substrate utilization
b
++: Over 95% reduction in peak height as determined by UV scanning and substantial growth (OD600 >0.05), '+: 40 to 60% reduction in peak height as determined by UV scanning and substantial growth (OD600 >0.025), '-: below 10% reduction in peak height and scant growth (OD600<0.1)
JC-F1A ++ ++ ++ ++ - - - ++ CW-B1 ++ ++ ++ ++ - - - ++ JC-E4 ++ ++ ++ ++ - - - ++ KY-S3 ++ ++ ++ ++ - - - ++ KY-S1 ++ ++ ++ ++ - - - ++ KP-H1 ++ ++ ++ ++ - - - ++
8. Genetic diversity analysis by PCR amplification
To determine whether the isolates had any sequence homology with carbendazim hydrolyzing esterase gene which was previously reported in other bacterial strains, PCR amplification was performed using PCR primers targeting for previously-reported degradative genes, such as mhe1 gene (Pandey et al., 2010) and cahA gene (Hashimoto et al., 2006) (Fig. 11-12). Four isolates showed positive DNA bands of carbendazim hydrolyzing esterase gene (mhe1), but no isolates showed any positive DNA bands in PCR experiments with cahA gene (Fig. 11-12).
M 1 2 3 4 5 6 7 8 9 M
Fig. 11. PCR amplification with the primers specific for the mhe1 gene. Lanes: 1, JN-J1; 2, SG-D1; 3, JC-E4; 4, CW-B1; 5, BR-A3; 6, JS-E2; 7, ES-A3; 8, KY-S3; 9, CN-T1; M, DNA size marker
M 1 2 3 4 5 6 7 8 9 M
1498bp →
Fig. 12. PCR amplification with the primers specific for the cahA gene. Lanes: 1, JN-J1; 2, SG-D1; 3, JC-E4; 4, CW-B1; 5, BR-A3; 6, JS-E2; 7, ES-A3; 8, KY-S3; 9, CN-T1; M, DNA size marker
IV. Discussion
Nineteen bacterial strains capable of utilizing benomyl as the sole carbon and energy source were isolated from agricultural soils in South Korea. Analysis of 16S rDNA sequence showed that all the isolates were phylogenetically related to Mycobacterium and Rhodococcus (Table 4). The bacterial strains belonging to genus Rhodococcus seemed to be ubiquitous, with frequently being isolated from various agricultural soils taken from various locations in South Korea. Nine representative chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from nineteen isolates (Fig. 5).
Members of Rhodococcus are common in nature, possess a wide spectrum of catabolic activities and are able to survive under extremely harsh conditions, which makes them potentially useful in environmental and industrial biotechnology (Shao et al., 1995) Mycobacterium is of particular interest because it is able to utilize a wide range of PAH substrates as sole sources of carbon and energy, including naphthalene, acenaphthene, anthracene, fluoranthene, and pyrene (Lease et al., 2011; Badejo et al., 2013; Pontiroli et al., 2013; Chun Zhang and Anne J. Anderson 2012). These reports on Rhodococcus and Mycobacterium suggest that the members of this group possess the broad range of degradation ability to degrade new compounds in the environment. Previous studies of the biodegradation of benzimidazole fungicides also have demonstrated that benomyl and carbendazim which are the representative benzimidazole fungicides could be degraded by Rhodococcus strains djl-6 (Xu et al., 2007), djl-6-2 (Wang et al., 2010), and SG-4G (Pandey et al., 2010) by hydrolysis. Although benomyl is a major fungicide which have been used abundantly worldwide and so of environmental concerns (Panddey et al., 2010; Wang et al.,
benomyl and catabolic pathways of benomyl biodegradation. To my knowledge, this study is the first report on diversity of benomyl-degrading bacteria from soils.
All the isolated bacteria in this study were grouped into three different growth patterns based on their benomyl degradation and growth characteristics (Fig. 6 and 8). When each isolates were inoculated on benomyl mineral medium (100ug/ml), benomyl began to be quickly degraded without a significant initial lag period, and then was completely mineralized within 100 hours, which could be important for rapid biodegradation of benomyl in contaminated environments.
Our experiment on benomyl degradation revealed the accumulation of an intermediate during its biodegradation. It has been reported that benomyl or carbendazim were converted to the 2-aminobenzimidazole and 2-hydroxybenzimidazole in their degradations (Pandey et al., 2010; Wang et al., 2010; Zhang et al., 2013; Fang et al., 2010;Xu et al., 2006). As expected, the intermediates detected after 3-12 hours of incubation were 2-aminobenzimidazole and 2-hydroxybenzimidazole (Fig. 8). The benomyl-degrading isolates were also able to utilize them as the sole source of carbon and energy. As specific PCR amplification products were detected from four isolates when PCR-amplification with the primers targeting for carbendaim hydrolyzing esterase, mhe1, it appears that carbendazim degrading enzyme in these isolates is an esterase just like mhe1-encoding carbendazim hydrolase.
There are several reports which demonstrate the importance of large plasmids in Rhodococcus for the degradation of various toxic compounds. However, there are no plasmids detected in these strains, suggesting that these strains would have benomyl degradative genes in their chromosomes.
The benzimidazole fungicides are of environmental concern because it was reported that benomyl significantly negatively influenced the numbers of two physiological groups of
bacteria: spores of bacteria utilizing organic nitrogen and bacteria and their spores utilizing inorganic nitrogen (SONA et al., 2010). Furthermore, carbendazim is known for contaminants of food stuffs and quite stable in soil and water and repeated applications of carbendazim could reduce soil microbial diversity and alter the bacterial community structure temporarily (WANG et al., 2009). Bioremediation has been recognized as a major method to restore these fungicide-contaminated environments and protect non-target organisms (Xu et al., 2006). The isolation of single bacterial strains being able to rapidly and completely degrade benomyl and carbendazim under aerobic conditions can be potentially useful for removal of benomyl and carbendazim from the contaminated environments. With respect to this perspective, various benomyl-degrading bacteria isolated in this study could be very useful for bioremediation.
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요약 (국문초록)
Benomyl은 벤지미다졸계 살균제로서 주로 밭 토양에서 균과 애멸구, 선충 등 을 방제하기 위해 전 세계적으로 널리 이용되어 왔다. benomyl은 세포의 미세소 관에 붙어 세포의 기능을 저해하며 특히 베노밀의 수화된 형태인 carbendazim 의 경우 화학적으로 매우 안정하며 환경에 잔류하여 간과 내분비계에 악영향을 끼칠 뿐 아니라 아주 적은 양으로도 돌연변이원, 발암물질 등으로 작용할 가능 성이 있다고 알려져 있다. 또한 이 농약을 계속 토양에 처리시 토양미생물의 다 양성을 감소 시키며 미생물 군집구조를 일시적으로나마 변경시킨다는 연구 결 과가 알려져 있다. benomyl은 물리적 혹은 화학적 방법으로도 제거시킬 수 있지 만 미생물을 활용한 biodegradation이 이 농약의 분해에 가장 중요한 역할을 한 다고 알려져 있다. 본 연구에서는 benomyl을 분해하는 세균을 다양한 지역에서 분리 동정하고 분리된 균의 대사 특성과 표현형적, 유전적 특성을 규명하였다. 전국 각지의 밭토양을 중심으로 채취한 토양에 benomyl을 넣어 증폭 배양을 실험한 결과 benomyl을 성장의 유일한 탄소원과 에너지원으로 사용할 수 있는 19개의 분해균을 분리하였다. REP-PCR과 16s rDNA 염기서열 분석 방법을 통해 이 균들이 Mycobacterium 속과 Rhodococcus속에 속하는 것으로 판명되었다. 이 들 균들은 세 가지의 분해 성장 양상을 보이는 것으로 관찰되었고 분해 대사경 로는 HPLC를 통해 확인한 결과 2-aminobenzimidazole과 2-hydroxybenzimidazole을 거쳐 분해됨을 확인할 수 있었다. 기존에 밝혀진 carbendazim hydrolyzing esterase gene (mhe1) PCR을 수행한 결과 19개의 균 중에서 4개의 균들이 mhe1 gene을 가지고 있음을 확인하였다. 이 연구에서 분리된 균들은 모두 그람 양성균으로 모 두가 plasmid DNA를 가지고 있지 않은 것으로 관찰되어 이 균들의 분해 유전자 가 genomic DNA상에 있을 것으로 추정된다. benomyl에 속한 다른 benzimidazole 계의 농약에 대한 분해 가능성 실험을 진행한 결과, 분리된 균들은 carbendazim 을 제외한 나머지 농약인 thiophanate-methyl, thiabendazole, fuberidazole 등을 분해 할 수 없는 것을 확인하였다. 본 논문의 연구에서 살균제 benomyl을 분해시킬 수 있는 균들을 다양한 지역에서 다양하게 분리하여 분해 특성을 규명한 바, 이 균들을 환경과 사람에게 직간접적으로 악영향을 주는 benomyl 을 분해시키는데 활용할 수 있고, 본 연구에서 분리된 benomyl 분해 균들 중 일부는 종래에 밝혀 진 carbendazim 분해 유전자와는 다른 가수분해 유전자를 가진 것으로 보여, 향 후 benomyl 분해경로와 분해 유전자에 대한 연구를 통해 benomyl 분해 균의 다 양성을 밝히는데 기여할 수 있을 것으로 기대된다.
주요어: 벤지미다졸계 살균제, benomyl, benomyl 가수분해, mhe1 gene 학번: 2013-23276
