Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production

Abstract

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (1H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by 1H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L.aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

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

“…The extracts were analyzed for the identification of molecules using untargeted LC-MS/MS analysis. Liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) analysis was performed on methanolic extracts of the three endophytic bacteria strains displaying signals on HPTLC for Dragendorff’s reagent (LaBFS1102, LaBFB3301, LaBFS2103) using the same analytical conditions as previously published [ 26 ]. LC-MS analysis performed in positive ion mode confirmed the absence of lycorine and galantamine in the methanolic extracts, as previously observed with the HPTLC analysis ( Figure S3 ).…”

“…The plants that have been investigated are Narcissus tazetta [ 25 ], Crinum macowanii [ 30 , 31 , 32 ] and Lycoris radiata [ 33 ]. The latest example was reported by Spina et al [ 26 ], with in vitro bulblets of L. aestivum . In our study, the bulbs harbor a low number of cultivable bacterial strains, which is consistent with the observation of the few endophytic bacteria isolated from in vitro bulblets of L. aestivum [ 26 ].…”

“…The latest example was reported by Spina et al [ 26 ], with in vitro bulblets of L. aestivum . In our study, the bulbs harbor a low number of cultivable bacterial strains, which is consistent with the observation of the few endophytic bacteria isolated from in vitro bulblets of L. aestivum [ 26 ]. This result could be associated with the accumulation of alkaloids such as galantamine, which is highest in the bulbs of Leucojum plants [ 34 ].…”

“…Hexacyclopeptides have been identified in the endophytic fungus Fusarium salani from Narcissus tazetta [ 25 ]. From Leucojum aestivum ’s in vitro bulblets, five Amaryllidaceae alkaloids, four amino acids and fatty acids were identified in the endophytic bacteria belonging to the Bacillus genus [ 26 ]. In this study, we analyze endophytic bacteria from bulbs, roots and shoots of L. aestivum ‘Gravety giant’.…”

Endophytic Bacteria in Banana In Vitro Cultures: Molecular Identification, Antibiotic Susceptibility, and Plant Survival

“…It includes both conserved and variable regions of about 1500 bp encoding the 30S ribosomal protein subunit, which can be used to identify bacteria at the genus level [11,12]. The analysis of the 16S rDNA, GyrA and GyrB gene sequences has been widely used for bacterial taxonomy and identification in different contaminated samples [13][14][15][16][17]. However, the expanded use of 16S rDNA gene-based analysis for the detection and identification of bacteria in many environments, and the application of this approach to identify bacterial contaminants in in vitro plant cultures, is limited.…”