Analysis of endophytic bacterial communities of potato by plating anddenaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCRfragments

摘要

The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desiree) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable endophytic bacterial communities detected in potato stem bases as well as in roots were in most cases on the order 10(3) to 10(5) CFU g(-1) of fresh plant tissue. Dilution plating revealed that a range of bacterial types dominated these populations. Dominant isolates fell into the alpha and gamma subgroups of the Proteobacteria, as well as in the Flavobacterium/Cytophaga group. Different representatives of the Firmicutes were also found. The most frequently isolated strains (>5% of the total) were characterized as different Pseudomonas spp. (including P. aureofaciens, P. corrugata, and P, putida), Agrobacterium radiobacter, Stenotrophomonas malruphilia, and FIuvobacterium resinovorans, using fatty acid methyl ester (FAME) analysis and/or sequencing of their partial 16S ribosomal RNA genes. Other Proteobacteria or Firmicutes were also found, albeit infrequently, and mainly in potato stem tissue. The fate of three putative potato endophytes, Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis, was monitored following their release into potato plants via injection, via root dipping, or via the soil. Following stem injection, the S, maltophilia and Bacillus inoculants could be tracked over time periods of, respectively, 22 and 1 day(s) by dilution plating as well as via PCR-DGGE. However, only S. maltophilia was able to colonize, and persist in, plant tissue from soil or dipped roots. S. paucimobilis was never recovered from the plant irrespective of the mode of introduction. The diversity of the indigenous bacterial flora associated with potato was then monitored via PCR-DGGE. The patterns obtained revealed the existence of bacterial communities of limited complexity, with communities from potato stems typically differing from those from stem peel and roots. Evidence was obtained for the endophytic occurrence of a range of organisms falling into the alpha, beta, and gamma subgroups of the Proteobacteria as well as in the Firmicutes. Several of the sequences found matched those from isolates, suggesting that the molecular evidence reported culturable organisms. However, a number of sequences did not have matching sequences from isolates, suggesting that non-culturable or as-yet-uncultured endophytic organisms were being detected.