Characterization of the plant-associated bacterial microbiota of the Mexican medicinal species Bouvardia ternifolia.

Introduction. Bouvardia ternifolia is a Mexican endemic plant used in the past as a general curative source and contemporaneously as a remedy for dysentery, poison therapy, pain relief, and other afflictions. The hydroalcoholic extract shows acetylcholinesterase inhibition activity, as well as anti-inflammatory and antioxidant effects. “Bouvardin” is a cyclic hexapeptide found in B. ternifolia with in vitro cytotoxicity against B16 melanoma melanotic and P388 lymphocytic leukemia cell lines. These findings support the idea that this plant has potential as a source of therapeutic bioactive compounds. It is well known that many therapeutic secondary metabolites can be produced by plant-associated microorganisms and in the search of these, the characterization of species that live in conjunction with the plant becomes an important research activity. In this study, we report the characterization of the bacterial community associated with the Mexican medicinal plant B. ternifolia.

Material and methods. Five specimens and plant-soil samples were collected from different regions from the central part of Mexico and fractioned in flower, stem, leaf, and root. To establish the total and endophytic bacterial communities, each plant fraction was divided into two subfractions; the first one was processed directly and the second one was carefully tissue-surface sterilized before processing with chemical agents. Bacteria present in subfractions were collected by crushing down the plant tissue in a mortar followed by centrifugation steps. FavorPrep stool DNA isolation kit # FASTI001-1 and QIAGEN Dneasy PowerSoil kit were used to purify bacterial DNA which was employed to sequence a DNA library prepared from the V3 region of the 16S ribosomal gene by Ion Torrent PGM NGS system technology.

Results. We find ~1,000 OTUs shared among total plant, endophytic, and soil samples, ~2,000 OTUs exclusively for soil bacteria, ~1,500 OTU’s for total plant bacteria, and ~250 OTUs for endophytic bacteria. Proteobacteria (46-79%) was the most representative phylum followed by Actinobacteria and Acidobacteria. The phylum Cyanobacteria (8.5%) was more representative in endophytes samples than in total plant samples (1%). Bacterial richness and diversity by plant fractions and soil, from highest to lowest was observed in the following order: Soil > Root > Leaf > Stem > Flower. Linear discriminant effect size analysis showed the order Streptophyta, the families Oxalobacteraceae, Ruminococcaceae, Lachnospiraceae, and the genera Propionilbacterium, Paracoccus, Lactobacillus as the most representative taxa in endophyte bacterial samples.

Conclusions. This study provides evidence for the composition and diversity of the bacterial communities present in soil and tissues of the Mexican medicinal plant B. ternifolia which can be considered for subsequent analysis to profiling therapeutic and bioactive compounds that give this species its medicinal properties.

Acknowledgments. This work was financed by Cinvestav, FONCICYT 2 267416, CONACYT-BMBF-267416, and CONACyT-163235 INFR-2011-01.