Neorhizobium tomejilense sp. nov., first non-symbiotic Neorhizobium species isolated from a dryland agricultural soil in southern Spain.

We describe for the first time a non-symbiotic species of the recently described genus Neorhizobium, lacking nodulation or nitrogen fixation genes. The strains were isolated from a dryland agricultural soil in southern Spain where no record of legume cultivation is available, thus we propose the name Neorhizobium tomejilense sp. nov. (type strain T17_20T, LMG 30623T and CECT 9621T). N. tomejilense exhibit a clear distinct lineage from the other Neorhizobium species, Neorhizobium galegae, Neorhizobium alkalisoli and Neorhizobium huautlense, based on polyphasic evidence. Phylogenetic marker analysis of 16S rDNA, atpD, glnII, recA, rpoB and thrC genes and genomic identity data derived from the draft genomic sequences showed that N. tomejilense strains clearly separated from the other Neorhizobium species and that N. galegae represents the closest species, with Average Nucleotide Identities (ANIb) ranging from 90% (for type strain HAMBI 540T) to just under 95.0% (for two N. galegae sv. officinalis strains). Genomes from N. galegae and N. tomejilense, however, clearly differed in important traits, such as the number of rRNA operon copies or the number of tRNAs. Phenotypic characterisation of N. tomejilense also displayed differences with the other Neorhizobium species. Whole-cell matrix-assisted laser-desorption time-of-flight mass spectrometry (WC MALDI-TOF-MS) fingerprint analysis and the dendrogram derived from the fingerprint profiles, showed a clearly distinct group formed by the three N. tomejilense isolates (T17_20T, T20_22 and T11_12) from the other Neorhizobium especies.

A chemical genetic screen reveals that iminosugar inhibitors of plant glucosylceramide synthase inhibit root growth in Arabidopsis and cereals.

Iminosugars are carbohydrate mimics that are useful as molecular probes to dissect metabolism in plants. To analyse the effects of iminosugar derivatives on germination and seedling growth, we screened a library of 390 N-substituted iminosugar analogues against Arabidopsis and the small cereal Eragrostis tef (Tef). The most potent compound identified in both systems, N-5-(adamantane-1-yl-ethoxy)pentyl- L-ido-deoxynojirimycin (L-ido-AEP-DNJ), inhibited root growth in agar plate assays by 92% and 96% in Arabidopsis and Tef respectively, at 10 µM concentration. Phenocopying the effect of L-ido-AEP-DNJ with the commercial inhibitor (PDMP) implicated glucosylceramide synthase as the target responsible for root growth inhibition. L-ido-AEP-DNJ was twenty-fold more potent than PDMP. Liquid chromatography-mass spectrometry (LC-MS) analysis of ceramide:glucosylceramide ratios in inhibitor-treated Arabidopsis seedlings showed a decrease in the relative quantity of the latter, confirming that glucosylceramide synthesis is perturbed in inhibitor-treated plants. Bioinformatic analysis of glucosylceramide synthase indicates gene conservation across higher plants. Previous T-DNA insertional inactivation of glucosylceramide synthase in Arabidopsis caused seedling lethality, indicating a role in growth and development. The compounds identified herein represent chemical alternatives that can overcome issues caused by genetic intervention. These inhibitors offer the potential to dissect the roles of glucosylceramides in polyploid crop species.

Novel, non-symbiotic isolates of Neorhizobium from a dryland agricultural soil.

Semi-selective enrichment, followed by PCR screening, resulted in the successful direct isolation of fast-growing Rhizobia from a dryland agricultural soil. Over 50% of these isolates belong to the genus Neorhizobium, as concluded from partial rpoB and near-complete 16S rDNA sequence analysis. Further genotypic and genomic analysis of five representative isolates confirmed that they form a coherent group within Neorhizobium, closer to N. galegae than to the remaining Neorhizobium species, but clearly differentiated from the former, and constituting at least one new genomospecies within Neorhizobium. All the isolates lacked nod and nif symbiotic genes but contained a repABC replication/maintenance region, characteristic of rhizobial plasmids, within large contigs from their draft genome sequences. These repABC sequences were related, but not identical, to repABC sequences found in symbiotic plasmids from N. galegae, suggesting that the non-symbiotic isolates have the potential to harbor symbiotic plasmids. This is the first report of non-symbiotic members of Neorhizobium from soil.