Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites

Abstract

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as <i>Lithospermum</i> spp. and <i>Alkanna</i> spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by <i>Alkanna tinctoria</i> might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing <i>Alkanna tinctoria</i> collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were <i>Pseudomonas</i>, <i>Xanthomonas</i>, <i>Variovorax</i>, <i>Bacillus</i>, <i>Inquilinus</i>, <i>Pantoea</i>, and <i>Stenotrophomonas</i>. Several bacteria were then tested <i>in vitro</i> for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of <i>Pseudomonas</i>, <i>Pantoea</i>, <i>Bacillus</i> and <i>Inquilinus</i> showed positive plant growth properties whereas those of Bacteroidetes and <i>Rhizobiaceae</i> showed pectinase and cellulase activity <i>in vitro</i>. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of <i>A. tinctoria</i>. Four strains belonging to <i>Chitinophaga</i> sp., <i>Allorhizobium</i> sp., <i>Duganella</i> sp., and <i>Micromonospora</i> sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization.