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Identification of plant species is a crucial process in natural products. Ocimum, often referred to as the queen of herbs, is one of the most versatile and globally used medicinal herbs for various health benefits due to it having a wide variety of pharmacological activities. Despite there being significant global demand for this medicinal herb, rapid and comprehensive metabolomic fingerprinting approaches for species- and variety-specific classification are limited. In this study, metabolomic fingerprinting of five Ocimum species (Ocimum basilicum L., Ocimum sanctum L., Ocimum africanum Lour., Ocimum kilimandscharicum Gurke., and Hybrid Tulsi) and their varieties was performed using LC-MS, GC-MS, and the rapid fingerprinting approach FT-NIR combined with chemometrics. The aim was to distinguish the species- and variety-specific variation with a view toward developing a quality assessment of Ocimum species. Discrimination of species and varieties was achieved using principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA), data-driven soft independent modelling of class analogy (DD-SIMCA), random forest, and K-nearest neighbours with specificity of 98% and sensitivity of 99%. Phenolics and flavonoids were found to be major contributing markers for species-specific variation. The present study established comprehensive metabolomic fingerprinting consisting of rapid screening and confirmatory approaches as a highly efficient means to identify the species and variety of Ocimum, being able to be applied for the quality assessment of other natural medicinal herbs.

Azospirillum brasilense is a plant growth–promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA, however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C4-dicarboxylates in A. brasilense, respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana, with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth.

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Azospirillum brasilense is a plant growth promoting rhizobacterium which is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can co-metabolize ethanol in media having fructose or glycerol as carbon source, and contribute to its growth. In minimal medium containing fructose or glycerol as carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense . But, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense led to the loss of the AldA protein and ethanol utilization ability in fructose or glycerol supplemented media. Furthermore, ethanol inhibited the growth of the aldA::km mutant. The exaA::km mutant also lost the ability to utilize ethanol in fructose supplemented medium. But, in glycerol supplemented media, it utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced only by glycerol, not by fructose. Unlike exaA , expression of aldA and exaA1 were not dependent on σ 54 . Instead, they were negatively regulated by RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as carbon source without or with malate overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating co-metabolism of ethanol by inducing the expression of ethanol oxidizing enzymes and of RpoH2 in repressing them. IMPORTANCE This study has unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol is used as primary growth substrate. It opens the possibility of studying the regulation of expression of ethanol oxidation pathway for generating high yielding strains, which can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.

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