Analysis of rhizosphere bacteria of rice cultivated in Andosol lowland and upland fields using molecular biological methods

Abstract

Bacteria in the rhizosphere influence plant growth and interact with plant roots. Microscopy and culture method have been used for studies of microorganisms of the rhizosphere, but these methods are insufficient for evaluation because most rhizosphere bacteria are viable but non-culturable (VBNC). Bacteria in the rhizosphere of rice cultivated in Andosol lowland and upland fields were analyzed in this study using PCR-DGGE and FISH, in combination with modified pretreatments. Results show that the two methods with the pretreatments, more than conventional methods, provided a rapid and simple analysis of rhizosphere bacteria. The 16S rDNA band pattern of bacteria in the rhizosphere obtained using PCR-DGGE indicated different species composition of bacterial community in the two ecosystems and greater diversity of bacteria in the rhizosphere in upland field. Sequencing of major 16S rDNA bands identified Bacterium A35 and Clostridium bifermentans as dominant bacteria in the rhizosphere of rice in lowland fields and Klebsiella planticola and Bacillus fusiformis in upland fields. Furthermore, FISH observation indicated the predominance of gram-positive low GC bacteria in both rhizospheres and a higher proportion of Clostridium spp. in lowland fields, which is consistent with results of PCR-DGGE analysis. The results suggest that the bacteria in the rice rhizosphere can be changed depending on aerobic and anaerobic conditions of fields. It is expected to apply the PCR-DGGE and FISH to agricultural field experiments as reliable methods to evaluate the rhizosphere bacteria.