Screening of Endophytic Bacteria of Leucojum aestivum 'Gravety Giant' as a Potential Source of Alkaloids and as Antagonist to Some Plant Fungal Pathogens.

Leucojum aestivum is a medicinal plant belonging to the Amaryllidaceae family well known as a producer of alkaloids such as galanthamine and lycorine. However, the endophytic microbes that colonize different plant tissues without causing any damage have not been reported in this plant. Here, we explored the different endophytic bacterial communities isolated from different surface disinfected tissues of L. aestivum 'Gravety giant' and screened bacterial isolates producing alkaloids and their potential use as biocontrol agent against wheat pathogens. For that purpose, endophytic bacteria were isolated from bulbs, roots and shoots of L. aestivum. After taxonomical characterization, these microorganisms were screened for their ability to produce alkaloids using high-performance thin-layer chromatography (HPTLC) and untargeted liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) strategies. We isolated 138 bacteria belonging to four phyla and 42 genera, mainly from roots and shoots. The most abundant genera were Rahnella in shoot, Patulibacter in bulb and Bacillus in roots. Among the different bacterial isolates, the methanolic extracts of Luteibacter rhizovicinus (LaBFB3301) and Commamonas denitrificans (LaBFS2103) slightly delayed the growth of F. graminearum colonies in in vitro dual tests against F. graminearum and M. nivale strains with 15.5% and 19.9% inhibition rates, respectively. These isolates are able to produce an indolic alkaloid tryptophol (C10H11NO, [M + H]+ 162.0913). These endophytic bacteria might be investigated to characterize the plant protection effect and the plant growth promotion effect.

Screening of Antimicrobial Activities and Lipopeptide Production of Endophytic Bacteria Isolated from Vetiver Roots.

The exploration of certain microbial resources such as beneficial endophytic microorganisms is considered a promising strategy for the discovery of new antimicrobial compounds for the pharmaceutical industries and agriculture. Thirty-one endophytic bacterial strains affiliated with Bacillus, Janthinobacterium, Yokenella, Enterobacter, Pseudomonas, Serratia, and Microbacterium were previously isolated from vetiver (Chrysopogon zizanioides (L.) Roberty) roots. These endophytes showed antifungal activity against Fusarium graminearum and could be a source of antimicrobial metabolites. In this study, in particular, using high-throughput screening, we analyzed their antagonistic activities and those of their cell-free culture supernatants against three species of Fusarium plant pathogens, a bacterial strain of Escherichia coli, and a yeast strain of Saccharomyces cerevisiae, as well as their capacity to produce lipopeptides. The results showed that the culture supernatants of four strains close to B. subtilis species exhibited antimicrobial activities against Fusarium species and E. coli. Using mass spectrometry analyses, we identified two groups of lipopeptides (surfactins and plipastatins) in their culture supernatants. Whole-genome sequencing confirmed that these bacteria possess NRPS gene clusters for surfactin and plipastatin. In vitro tests confirmed the inhibitory effect of plipastatin alone or in combination with surfactin against the three Fusarium species.

Composition and functional comparison of vetiver root endophytic microbiota originating from different geographic locations that show antagonistic activity towards Fusarium graminearum.

Given the current trend towards reducing the use of chemical controls in agriculture, microbial resources such as plant endophytes are being intensively investigated for traits that are conducive to plant protection. Among the various important target pathogens, Fusarium graminearum is a fungal pathogen of cereal crops that is responsible for severe yield losses and mycotoxin contamination in grains. In the present study, we investigated the bacterial endophytic communities from vetiver (Chrysopogon zizanioides (L.) Roberty) roots originating from 5 different geographic locations across Europe and Africa. This study relies on a global 16S metabarcoding approach and the isolation/functional characterization of bacterial isolates. The results we obtained showed that geographical location is a factor that influences the composition and relative abundance of root endophyte communities in vetiver. Three hundred eighty-one bacterial endophytes were isolated and assessed for their in vitro antagonistic activities towards F. graminearum mycelium growth. In total, 46 % of the isolates showed at least 50 % inhibitory activity against F. graminearum. The taxonomic identification of the bioactive isolates revealed that the composition of these functional culturable endophytic communities was influenced by the geographic origins of the roots. The selected communities consisted of 15 genera. Some endophytes in Bacillus, Janthinobacterium, Kosakonia, Microbacterium, Pseudomonas, and Serratia showed strong growth inhibition activity (≥70 %) against F. graminearum and could be candidates for further development as biocontrol agents.

Comparative Proteomic Analysis of Lithospermum erythrorhizon Reveals Regulation of a Variety of Metabolic Enzymes Leading to Comprehensive Understanding of the Shikonin Biosynthetic Pathway.

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.