Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Genetic Monitoring of Plant Growth Promoting Rhizobacterium (PGPR), Bacillus subtilis AH18 using Multiplex PCR in Field Soil

Multiplex PCR을 이용한 생물방제균 Bacillus subtilis AH18의 토양내 Genetic Monitoring

  • Woo, Sang-Min (Department of Applied Microbiology, Yeungnam University) ;
  • Lim, Jong-Hui (Department of Applied Microbiology, Yeungnam University) ;
  • Jeong, Hee-Young (Department of Applied Microbiology, Yeungnam University) ;
  • Kim, Sang-Dal (Department of Applied Microbiology, Yeungnam University)
  • 우상민 (영남대학교 응용미생물학과) ;
  • 임종희 (영남대학교 응용미생물학과) ;
  • 정희영 (영남대학교 응용미생물학과) ;
  • 김상달 (영남대학교 응용미생물학과)
  • Published : 2009.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

The genetic monitoring method was developed for the rapid detection of the PGPR and biocontrol agent, B. subtilis AH18 in red-pepper field soil by multiplex PCR using sid, aec and cel gene primers. The monitoring of B. subtilis AH18 in the soil was carried by amplified a 2,3-dihydro-2,3-dihydroxy benzoate dehydrogenase [EC: 1. 3. 1. 28]gene (sid - 794 bp : EF408238) which is a key enzyme of siderophore synthesis, an auxin efflux carrier gene (aec - 1,052 bp : EF408239) and a cellulase gene (cel - 1,582 bp : EF070194). The natural un sterilized soil was inoculated with B. subtilis AH18 to determine the sensitivity ($1.8\times10^5$ cfu/g) of multiplex PCR for the rapid dectection and then the strain was monitored successfully in rhizosphere or non-rhizosphere soil of red-pepper cultural soil. At 3 weeks after the treatment, density of the strain was monitored more abundantly in rhizosphere soil.

식물성장촉진물질인 auxin을 비롯한 식물병원성진균을 방제하는 siderophore 그리고 식물병원성 진균의 세포벽을 분해하는 cellulase를 동시에 생산하는 생물방제균주 B. subtilis AH18의 토양내 monitoring을 위하여 각 생산물질에 관여하는 유전자를 기초로 primer(sid, aec, cel)를 제작하였다. Single PCR 및 multiplex PCR을 수행하여 800-bp(sid), 1000-bp(air), 1600-bp(cel)의 DNA fragment를 확인하였으며, 각각의 fragment는 siderophore의 생합성 key enzyme인 2,3-dihydro-2,3-dihydroxy benzoate dehydrogenase[EC : 1. 3. 1, 28]gene (sid-794bp)이며, auxin efflux carrier gene (aec - 1052 bp), 그리고 cellulase gene(cel - 1582 bp)임을 확인하고, NCBI Genbank에 등록하였다(Genbank accession sid: No. EF408238, aec: No. EF408239, cel: No. EF070194). 또한 B. subtilis AH18을 처리한 일반 경작지 토양에서 multiplex PCR을 통하여 3종의 유전자에서 증폭된 triple band를 확인하였으며, 고추를 실제 토양을 이용해 Pot에 이식 후 고추의 rhizosphere와 non-rhizosphere에서 B. subtilis AH18의 존재를 확인할 수 있다. 뿐만아니라 본 균주를 고추가 이식된 Pot의 토양에 처리 후 monitoring시 민감도는 $1.8\times10^5$ cfu/g이었고, monitoring 가능한 기간은 3주이상 확인 할 수 있었다.

Keywords

References

  1. Boldt, T. S., J. Sorensen, U. Karlson, S. Molin, and C. Ramos. 2004. Combined use of different gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa. FEMS Microbiol. Ecol. 48: 139-148 https://doi.org/10.1016/j.femsec.2004.01.002
  2. Bourque, S. N., J. R. Valero, J. Mercier, M. C. Lavoie, and R. C. Levesque. 1993. Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis. Appl. Environ. Microb. 59: 523-527
  3. Chεun， H. I., S. I. Makino, M. Watarai, J. Erdenεbaatar, K. Kawamoto, and I. Uchida. 2003. Rapid and effective detection of anthrax spores in soil by PCR. J. Appl. Microbiol. 95: 728-733 https://doi.org/10.1046/j.1365-2672.2003.02038.x
  4. Demoz, B. T. and L. Korsten. 2006. Bacillus subtilis attachment, colonization, and survival on avocado flowers and its mode of action on stem-end rot pathogens. Biol. Control. 37: 68-74 https://doi.org/10.1016/j.biocontrol.2005.11.010
  5. Gera, R., D. V. Pathak, and P. K. Sharma. 2003. Monitoring rhizosphesre colonization by Azospirillum using constitutively exprlεssed gfp gene. lnd. J. Microbiol. 43: 219-222
  6. Glick, B. R., C. L. Patten, G. Patten, and D. M. Penrose. 1999. Biochemical and genetic mechanisms used by plant growth promoting bacteria. Imperial College Press. Canada
  7. Hui, C. K., S. Bowden, H. Y. Bowden, A. Wong, S. Lewin, F. Rousseau, H. Mommeja-Marin, N. P. Lee, J. M. Luk, and S. Locarnini. 2006. Comparison of Real-Time PCR Assays for Monitoring Serum Hepatitis B Virus DNA Levels during Antiviral Therapy. J Clin. Microbiol. 44: 2983-2987 https://doi.org/10.1128/JCM.02544-05
  8. Jung, H. K., J. C. Ryoo, and S. D. Kim. 2005. A muti microbial biofungicide for the biological control against several important plant pathogenic fungi. J. Kor. Soc. Appl. Biol. Chem. 48: 40-47
  9. Jung, H. K., J. R. Kim, S. M. Woo and S. D. Kim. 2006. An auxin producing plant growth promoting rhizobacterium Bacillus subtilis AH18 which has siderophore-Producing biocontrol activity. Kor. J. Microbiol. Biotechnol. 34: 94-100
  10. Kang, S. J., J. H. Kim, and G J. Joo. 2005. Isolation of antagonistic bacteria against Fusarium oxysporum and physicochemical properties of compost mixed with microbial formulation. Kor. J. Hort. Sci. Technol. 23: 342-350
  11. Kim, M. K., H. S. Kim, B. O. Kim, S. Y. Yoo, J. H. Seong, D. K. Kim, S. E. Lee, S. J. Choe, J. C. Park, B. M. Min, M. j. Jeong, D. K. Kim, Y. K. Shin, and J. K. Kook. 2004. Mu1tiplex PCR using conserved and species-specific 16s rDNA primers for simu1taneous detection of Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans. J. Microbiol. Biotechnol. 14: 110-115
  12. Kim, M. Y., J. H. Kim, H. J. Kim, S. H. Park, G J. Woo, and H. Y. Kim. 2003. Monitoring of genetically modified soybean and processed foods in korean market using PCR. J. Kor. Soc. Agric. Chem. Biotechn. 46: 344-347
  13. Kim, Y. S., J. B. Kim, and Y. B. Eom. 2004. Detection of pathogenic Yersinia enterocolitica strains by a rapid and spedific multiplex PCR assay. J. Exp. Biomed. Sci. 10: 333-339
  14. Kunst F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero et al. 1997. The complete genome sequence of thε gram-positive bacterium Bacillus subtilis. Nature. 390: 249-256 https://doi.org/10.1038/36786
  15. Kwon, D. H., J. H. Choe, H. K. Jeong, J. H. Lim, G J. Ju, and S. D. Kim. 2004. Selection and identification of auxinproducing plant growth promoting rhizobacteria having phytopathogen antagonistic activity. J. Kor. Soc. Appl. Biol. Chem. 47: 17-21
  16. Lee, E. T., and S. D. Kim. 2000. Selection and antifungal activity of antagonistic bacterium pseudomonas sp. 2112 against red-pepper rotting Phytophthora capsici. Kor. J. Appl. Microbiol. Biotechn. 28: 334-340
  17. Lee, I. K., C. I. Kim, S. D. Kim, and I. D. Yoo. 1990. Antifungal antibiotic against fruit rot disease of red pepper form Streptomyces parvullus. Kor. J. Appl. Microbiol. Biotechnol. 18: 142-147
  18. Lee, J. M., E. S. Do, S. B. Baik, and S. C. Chun. 2003. Effect of organic amendments on efficacy of biological control of seedling damping-off of cucumber with severa1 microbial products. Kor. J. Mycology. 31: 44-49 https://doi.org/10.4489/KJM.2003.31.1.044
  19. Lee, J. M., H. S. Lim, T. H. Chang, and S. D. Kim. 1999. Isolation of siderophore-producing Pseudomonas fluorescens GL7 and its biocontrol activity against root-rot disease. Kor. J. Appl. Microbiol. Biotechn. 27: 427-432
  20. Lee, T. S., B. K. Park, and D. W. Oh. 2003. Detection of pathogenic Yersinia enterocolitica in drinking water and vegetables by multiplex-PCR. J. Kor. Soc. Food. Sci. Nutr. 32: 35-41 https://doi.org/10.3746/jkfn.2003.32.1.035
  21. Leonid, N. T., M. J. Lee, H. Park, and J. H. Yoon. 2000. Production of auxin - like compounds by ginseng growth - promotiog Bacterium Pseudomonas fluorescens KGPP207. Agric. Chem. Biotechnol. 43: 264-268
  22. Lindstrom, M., M. Nevas, and H. Korkeala. 2006. Detection df Clostridium botulinum by multiplex PCR in food and feces. Method. Biotechnol. 21: 37-45 https://doi.org/10.1385/1-59259-990-7:037
  23. Liu, L., J. W. Kloepper, and S. Tuzun. 1995. Induction of systemic resistance in cucumber by p1ant growth-promoting rhizobacteria: duration of protection and effect of host resistance on protection and root colonization. Phytopathology 85: 1064-1068 https://doi.org/10.1094/Phyto-85-1064
  24. Mahadi, N. M., S. Hastowo, B. Lay, and D. H. Dean. 1998. Application of multiplex PCR for rapid determination of cryI gene profIles of new Bacillus thuringiensis isolates. J. Microbiol. Biotechnol. 8: 517-522
  25. Martinez, I., E. Mateo, E. Churruca, C. Girbau, R. A1onso, and A. Femandez-Astorga. 2005. Detection of cdtA, cdtB, and cdtC genes in Campylobacter jejuni by multiplex PCR. Int. J. Med. Microbiol. 296: 45-48 https://doi.org/10.1016/j.ijmm.2005.08.003
  26. Oh, Y. J. 2000. Cultural conditions for the improvement in gibberellic acid productivity by a mutant of Gibberella fujikuroi ATCC 12616 - Gibberella fujikuroi G-36. Kor. J. Appl. Microbiol. Biotechnol. 28: 152-155
  27. Panicker, G , D. R. Cal1, M. J. Kmg, and A. K. Bej. 2004. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays. Appl. Environ. Microbiol. 70: 7436-7444 https://doi.org/10.1128/AEM.70.12.7436-7444.2004
  28. Ping, L. and W. Boland. 2004. Signals from the underground: bacrerial volatiles promote growth in Arabidopsis. Trends Pla. Sic. 9: 263-266 https://doi.org/10.1016/j.tplants.2004.04.008
  29. Reva, O. N., C. Dixelius, J. Meijer, and F. G Priest. 2004. Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiol. Ecol. 48: 249-259 https://doi.org/10.1016/j.femsec.2004.02.003
  30. RεY M. w., P. Ramaiya, B. A. Nelson, S. D. Brody-Karpin, E. J. Zaretsky, M. Tang et al. 2004. Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol. 5: R77 https://doi.org/10.1186/gb-2004-5-10-r77
  31. Rhoads,J. A., K. S. Rhoads, J. A. Riesing, K. S. Pabich, E. K Thamm, S. Zoulim, F. Thibault, V. Mullen, C. R. Pabich, E. K. Thamm, S. Zoulim, F. Thibault, V. and C. R. Mullen. 2006. Characteristics of a new automated real time PCR assay for monitoring HBV patients. J. Clin. Virol. 36: S6
  32. Sergeev, N., M. Distler, M. Vatgas, V. Chizhikov, K. E. Hero1d, and A. Rasoo1y. 2006. Microarry ana1ysis of Bacillus cereus group virulence factors. J. Microbiol. Meth. 65: 488-502 https://doi.org/10.1016/j.mimet.2005.09.013
  33. Sturtevant, D. B., and B. J. Taller. 1989. Cytokinin production by Rhizobium japonicum. Plant physiology. 39: 1247-1452
  34. van Loon, L. C., P. A. H. M. Bakkεr, and C. M. Pieterse 1998. Systemic resistance induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36: 453-483 https://doi.org/10.1146/annurev.phyto.36.1.453
  35. Wang, S. H., J. K. Wen, Y. F. Zhou, Z. P. Zhang, R. F. Yang, J. B. Zhang, J. Chen, and X. E. Zhang. 2004. Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip. Biosens. Bioelectron. 20: 807-813 https://doi.org/10.1016/j.bios.2004.03.019
  36. Woo, S. M., H. K. Jung, and S. D. Kim. 2006. Cloning and characterization df a celluase gene from a plant growth promoting rhizobacterium, Bacillus subtilis AH18 against phytophthora blight disease in red-pepper. Kor. J. Microbiol. Biotechnol. 34: 311-317
  37. Woo, S. M., J. U. Woo, and S. D. Kim. 2007. Purification and characterization of the sidrophore from Bacillus licheniformis Kl1, a multi-functional plant growth promoting rhizobacterium. Kor. J. Microbial. Biotechnol. 35: 128-134
  38. Woo, S. M. and S. D. Kim. 2007. Confmnation of nonsiderophore antifungal substance and cellulase from Bacillus licheniformis Kl1 containing antagonistic ability and plant growth promoting activity. J. Life. Science. 17: 983-989