Enrichment for microbes living in association with plant tissues

Aims: To investigate a cultivation-independent method of enrichment for microbes living in association with plant tissues. Methods and Results: 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. Conclusions: The method described here proved to be remarkably effective in enriching for bacteria living in association with plant tissues. Significance and Impact of the Study: The method can be applied to study plant-associated microbes in the field of environmental molecular ecology and environmental metagenomics.