Enrichment for microbes living in association with plant tissues

To investigate a cultivation-independent method of enrichment for microbes living in association with plant tissues. A large quantity of leaves or seeds was enzymatically hydrolyzed, and the pellets were collected by differential centrifugation. Enzyme concentration, buffer and incubation time were optimized for release of plant-associated microbes. The relative abundance of plant nuclear DNA and bacterial DNA in the enriched sample was estimated by PCR amplification of genome-specific marker genes. The efficiency of microbe enrichment was estimated from the proportion of bacterium-derived clones and their restriction fragment length polymorphism (RFLP) types as detected by 16S rRNA gene-based techniques. With a higher ratio of bacterial to plant nuclear DNA, the enriched samples showed a considerably enhanced proportion of bacterium-derived clones and a wider sequence diversity of those clones. The method described here proved to be remarkably effective in enriching for bacteria living in association with plant tissues. The method can be applied to study plant-associated microbes in the field of environmental molecular ecology and environmental metagenomics.