Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria, fungi and protozoa in Eleutherococcus sp plant tissue cultures

In vitro cultures of Eleutherococcus sieboldianus originating from surface sterilized leaf explants were found to be associated with several microorganisms. The associations included bacteria, fungi and protozoa within the rhizosphere and inside root hairs. To determine if this phenomenon is unique to this species, plant tissue cultures of E. gracilistylus and E. senticosus were included in our studies for comparison. A methodology consisting of isothermal amplification, cloning and sequencing was established for analysing 16S ribosomal DNA of cultivated and non-cultivated bacteria from different tissue types. The same methodology was used to obtain internal transcribed spacer regions and 18S regions of fungal and protozoan rDNA. Comparative analyses of sequencing data resulted in the identification of various genera within the Firmicutes and gamma-proteobacteria kingdoms and a broad spectrum of fungal genera related to several uncultured fungi. In addition, amoebal and chrysophyte species were detected. Most of the species were identified in different plant organs and in in vitro culture cell types indicating the microorganisms are systemically distributed. The presence of identical microorganisms in different plant species argues for an evolutionary long-lasting and stable association between the plant genus and the microinhabitants.