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

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria 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 Alkanna tinctoria 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 Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. 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 A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora 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

Phylogenomics reveals insights into the functional evolution of the genus Agrobacterium and enables the description of Agrobacterium divergens sp. nov

The genus Agrobacterium was initially described as mainly phytopathogenic strains. Nowadays, the genus includes phytopathogenic and non-phytopathogenic bacteria that are distinctive among the Rhizobiaceae family. Recently we have isolated two closely related strains, LMG 31531T and LMG 31532, from soil and plant roots, respectively. Both strains differ from previously reported species based on the genomic and phenotypic data. A. arsenijevicii KFB 330T and A. fabacearum LMG 31642T showed the highest 16S rRNA similarity (98.9 %), followed by A. nepotum LMG 26435T (98.7 %). A clear genomic feature that distinguishes LMG 31531T and LMG 31532 from other Agrobacterium species is the absence of a linear chromid. Nevertheless, typical values of the core-proteome Average Amino Acid Identity (cpAAI > 85 %) and 16S rRNA gene sequence similarity (>96 %) when compared to other members of the genus confirm the position of these two strains as part of the Agrobacterium genus. They are therefore described as Agrobacterium divergens sp. nov. Besides, our comparative genomic study and survey for clade-specific markers resulted in the discovery of conserved proteins that provide insights into the functional evolution of this genus

Roseomonas hellenica sp. nov., isolated from roots of wild-growing Alkanna tinctoria

Two Gram-negative, aerobic, rod-shaped and yellow-orange pigmented bacterial strains (LMG 31523(T) and LMG 31524) were isolated from roots of wild-growing Alkanna tinctoria plants collected near Thessaloniki, Greece. Analysis of their 16S rRNA gene sequences revealed that they form a separate cluster related to the genus Roseomonas. A comparative whole genome analysis of the two strains and the type strains of related Roseomonas species revealed average nucleotide identity values from 78.84 and 80.32%. The G + C contents of the genomic DNA of strains LMG 31523(T) and LMG 31524 were 69.69% and 69.74%, respectively. Combined data from phenotypic, phylogenetic and chemotaxonomic studies indicated that the strains LMG 31523(T) and LMG 31524 represent a novel species of the genus Roseomonas. Genome analysis of the new strains showed a number of genes involved in survival in the rhizosphere environment and in plant colonization and confirmed the endophytic characteristics of LMG 31523(T) and LMG 31524. Since the strains LMG 31523(T) and LMG 31524 were isolated from a plant collected in Greece the name Roseomonas hellenica sp. nov. is proposed. The type strain is LMG 31523(T) (=CECT 30032(T)). (C) 2021 The Author(s). Published by Elsevier GmbH

Root-associated bacteria modulate the specialised metabolome of Lithospermum officinale L.

Bacteria influence plant growth and development and therefore are attractive resources for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of bacteria on the modulation of SM production in the medicinal plant Lithospermum officinale (Boraginaceae family) with a focus on the naphthoquinones alkannin/shikonin and their derivatives (A/Sd). The study was conducted in an in vitro cultivation system developed for that purpose, as well as in a greenhouse. Targeted and non-targeted metabolomics were performed, and expression of the gene PGT encoding for a key enzyme in the A/S biosynthesis pathway was evaluated with qPCR. Three strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of A/Sd in L. officinale in both systems, demonstrating the strength of our approach for screening A/Sd-inducing bacteria. The bacterial treatments altered other plant metabolites derived from the shikimate pathway as well. Our results demonstrate that bacteria influence the biosynthesis of A/Sd and interact with different metabolic pathways. This work highlights the potential of bacteria to increase the production of SM in medicinal plants and reveals new patterns in the metabolome regulation of L. officinale