Lasiodiplodia theobromae is a fungal pathogen associated with perennial tropical fruit plants worldwide. In citrus, L. theobromae causes stem-end rot (Diplodia stem-end rot), a damaging postharvest disease that is aggravated when trees are also infected with the citrus greening bacteria ‘Candidatus Liberibacter asiaticus’. Due to the latent infection of L. theobromae during the preharvest stage, it becomes difficult to control the disease by chemical or physical treatment. In the current study, we sequenced and assembled strain CITRA15, the first genome of L. theobromae obtained from diseased Citrus paradise ‘Flame’ grapefruit in Florida, and thereby provided a genomic resource for future research on diagnostics, and postharvest and preharvest disease management of citrus and other fruit crops.

Nitrogen gas (N ₂ ) fixation driven by diazotrophs is a crucial process for supplying nitrogen to paddy soil ecosystems. The genus Geomonas has been considered to be an important potential diazotroph in paddy soils, but direct experimental evidence of the nitrogen-fixing ability of Geomonas in pure culture is still lacking. Hence, we aimed to demonstrate this nitrogen-fixing capability and shed light on how this process was regulated in response to ammonium (NH ₄ ⁺ ) in Geomonas . In this study, we determined that a key nitrogenase gene ( nifH ) was present in 50 isolates from paddy soils. Members of Geomonas contained the minimum nitrogen fixation gene cluster ( nifBHDKEN ) based on genomic analysis, implying Geomonas species had the potential to fix nitrogen. Acetylene reduction assay (ARA), ¹⁵ N ₂ isotope labeling, and total nitrogen accumulation assays validated that Geomonas was, indeed, able to fix nitrogen in pure culture. Under nitrogen-fixing conditions, the cell morphology of Geomonas changed from short rod-shaped (with NH ₄ ⁺ ) to long rod-shaped and flagella became longer and thicker. The expression of genes correlated to nitrogen fixation in the Geomonas transcriptome was quantified in response to NH ₄ ⁺ . Expression of genes associated with nitrogenase, flavin-based electron bifurcation complexes (such as the FixAB system), NH ₄ ⁺ uptake, and transformation (e.g., glutamine and glutamate synthetases) were significantly upregulated under nitrogen-fixing conditions, suggesting these mechanisms might be involved in N ₂ fixation in Geomonas . These results were verified by RT-qPCR. Taken together, our results demonstrate that Geomonas species possess the ability to fix N ₂ and expand our understanding on the ecological significance and potential applications of Geomonas in paddy soil ecosystems. IMPORTANCE: The ability of Geomonas species to fix nitrogen gas (N ₂ ) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria . We demonstrated N ₂ -dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas .

The present study was carried out to clarify the taxonomic relationship between two closely related Bacillus species, Bacillus okuhidensis Li et al. 2002 and Bacillus halodurans (ex Boyer 1973) Nielsen et al. 1995. The maximum-likelihood tree based on the 16S rRNA gene sequence and the phylogenomic tree based on concatenation of 16 protein-marker genes showed that these species were similar. Average nucleotide identity (ANIm 99.25 %, ANIb 98.2 %) and digital DNA–DNA hybridization values (93.5 %) between B. okuhidensis DSM 13666T and B. halodurans DSM 497T were greater than the threshold values for bacterial species delineation, indicating that they belong to the same species. Therefore, B. okuhidensis Li et al. 2002 should be reclassified as a later heterotypic synonym of B. halodurans (ex Boyer 1973) Nielsen et al. 1995.