Colonization pattern of plant root and leaf surfaces visualized by use of green-fluorescent-marked strain of Methylobacterium suomiense and its persistence in rhizosphere
- 583 Downloads
The localization of bacterial cell, pattern of colonization, and survival of Methylobacterium suomiense CBMB120 in the rhizosphere of rice and tomato plants were followed by confocal laser scanning, scanning electron microscopy, and selective plating. M. suomiense CBMB120 was tagged with green fluorescent protein (gfp), and inoculation was carried out through seed source. The results clearly showed that the gfp marker is stably inherited and is expressed in planta allowing for easy visualization of M. suomiense CBMB120. The colonization differed in rice and tomato—intercellular colonization of surface-sterilized root sections was visible in tomato but not in rice. In both rice and tomato, the cells were visible in the substomatal chambers of leaves. Furthermore, the strain was able to compete with the indigenous microorganisms and persist in the rhizosphere of tomato and rice, assessed through dilution plating on selective media. The detailed ultra-structural study on the rhizosphere colonization by Methylobacterium put forth conclusively that M. suomiense CBMB120 colonize the roots and leaf surfaces of the plants studied and is transmitted to the aerial plant parts from the seed source.
KeywordsMethylobacterium Green fluorescent protein Conjugation Flow cytometry Colonization Microscopy
Financial support was provided by the Korea Research Foundation, Republic of Korea to S. Poonguzhali and M. Madhaiyan and partially funded through Agricultural Research Promotion Center (ARPC) by the Ministry of Agriculture and Forestry, Korea. We thank Dr. Lee Yi (Research Centre for Agricultural and Biotechnology Science, Chungbuk National University, Korea) for kindly providing the plasmid pFAJ1820 and Florence Wisniewski-Dyè (Ecologie Microbienne, Université, France) for providing the helper strain E. coli HB101 (pRK2013). Flow cytometry, confocal laser scanning, and scanning electron microscopy were performed at the Center for Research Instruments and Experimental Facilities, Chungbuk National University with the assistance of Jung-Hee Lee and Jeong-Seon Jeon. The authors wish to thank anonymous referees for critical review of the manuscript.
- Bozzola JJ, Russell LD (1998) Electron microscopy, 2nd edn. Jones & Bartlett, Sudbury, MSGoogle Scholar
- Madhaiyan M, Poonguzhali S, Sa TM (2007b) Influence of plant species and environmental conditions on epiphytic and endophytic pink-pigmented facultative methylotrophic bacterial populations associated with field-grown rice cultivars. J Microbiol Biotechnol 17:1645–1654Google Scholar
- Normander B, Hendriksen NB, Nybore O (1999) Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability and activity in the natural barley rhizosphere. Appl Environ Microbiol 65:4646–4651Google Scholar
- Simons M, van der Bij AJ, Brand I, de Weger LA, Wijffelman CA, Lugtenberg BJJ (1996) Gnotobiotic system for studying rhizosphere colonization by plant growth promoting Pseudomonas bacteria. Mol Plant Microbe Interact 9:600–607Google Scholar
- Tombolini R, Unge A, Davey ME, de Bruijn FJ, Jansson JK (1999) Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria. FEMS Microbiol Ecol 22:17–28Google Scholar
- Unge A, Tombolini R, Davey ME, de Bruijn FJ, Jansson JK (1998) GFP as a marker gene. In: Akkermans AD, van Elsas JD, de Bruijn FJ (eds) Molecular microbial ecology manual. Kluwer, Dordrecht, pp 1–16Google Scholar
- Whittenbury R, Davies SL, Wilkinson JF (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61:205–218Google Scholar