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J Appl Microbiol. 2006 Apr;100(4):830-7.

Enrichment for microbes living in association with plant tissues.

Author information

1
Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan, China.

Abstract

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.

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