Strain MC02^T, a Gram-stain-negative, rod-shaped bacterium, was isolated from field soil collected from California, USA. To examine if MC02^Trepresents a novel species, we compared its colony morphology, 16S rRNA gene and whole genome sequence, and its metabolic phenotype using Biolog GenIII and MALDI-TOF analyses compared to reference strains. Based on 16S rRNA gene and whole genome sequencing, MC02^Tbelongs to the genusMassiliaandMassilia agriK-3–1^Tis the most similar strain with 96.97 % 16S rRNA gene sequence identity. MALDI-TOF analysis revealed thatMassilia aerilataDSM19289^Tis the closest match, but the similarity score was much lower than the ≥1.7 threshold for a reliable identification at the genus level. The predominant fatty acids were summed feature 3 (C_16 : 1⍵7cand/or C_16 : 1⍵6c; 49.07 %) and C_16 : 0(30.01 %). The genome is 5.02 Mbp and the G+C content is 66.2 mol%. Whole genome comparisons to the closest related strains revealed an average amino acid identity value of 67.4 %, an OrthoANI similarity of 77.1 %, and a DNA–DNA-hybridization probability ≥70 %, confirming that MC02^Trepresents a novel species. Strain MC02^Tcan grow at pH 6 but not at pH 5, and a salt concentration of ≥1 % inhibits its growth. In contrast to otherMassiliastrains, MC02^Tcan utilize turanose, inosine andl-serine. The genome of MC02^Tshows putative endophyte genes such as a nitrate reductase, several phosphatases, and biotin biosynthesis genes, 26 flagellar motility genes and 14 invasion and intracellular resistance genes. Based on its metabolic, physiological and genomic characteristics, we propose that strain MC02^T(NRRL B-65554^T=ATCC TSD-200^T=LMG 31737^T) represents a novel species of the genusMassiliawith the nameMassilia arenosasp. nov.

We isolated two new soil bacteria: ONC3^T(from garden soil in NC, USA; LMG 31738^T=NRRL B-65553^T) and M1^T(from farmed soil in MI, USA; NRRL B-65551^T=ATCC TSD-197^T=LMG 31739^T) and characterized their metabolic phenotype based on Biolog, MALDI-TOF MS and fatty acid analyses, and compared 16S rRNA and whole genome sequences to other members of theOxalobacteraceaeafter sequencing on an Illumina Nextera platform. Based on the results of 16S rRNA sequence analysis, ONC3^Tshows the highest sequence similarity toMassilia solisilvaeJ18^T(97.8 %),Massilia terraeJ11^T(97.7 %) andMassilia agilisJ9^T(97.3 %). Strain M1^Tis most closely related toNoviherbaspirillum denitrificansTSA40^T,Noviherbaspirillum agriK-1-15^TandNoviherbaspirillum autotrophicumTSA66^T(sequence identity of 98.2, 98.0 and 97.8 %, respectively). The whole genome of ONC3^Thas an assembled size of 5.62 Mbp, a G+C content of 63.8 mol% and contains 5104 protein-coding sequences, 56 tRNA genes and two rRNA operons. The genome of M1^Thas a length of 4.71 MBp, a G+C content of 63.81 mol% and includes 4967 protein-coding genes, two rRNA operons and 44 tRNA genes. Whole genome comparisons identifiedMassiliasp. WG5 with a 79.3 % average nucleotide identity (ANI) and 22.6 % digital DNA–DNA hybridization (dDDH), andMassiliasp. UBA11196 with 78.2 % average amino acid identity (AAI) as the most closely related species to ONC3^T. M1^Tis most closely related toN. autotrophicumTSA66^Twith an ANI of 80.27 %, orN. denitrificansTSA40^Twith a dDDH of 22.3 %. The application of community-accepted standards such as <98.7 % in 16S sequence similarity and <95–96 % ANI or 70 % DDH support the classification ofMassilia hortiONC3^TandNoviherbaspirillum arenaeM1^Tas novel species within theOxalobacteraceae.

A Gram-negative, rod-shaped bacterium, strainDuganella callidaDN04^T, was isolated from the soil of a maize field in North Carolina, USA. Based on the 16S rRNA gene sequence, the most similarDuganellaspecies areD. sacchariSac-22^T,D. ginsengisoliDCY83^T, andD. radicisSac-41^Twith a 97.8, 97.6, or 96.9 % sequence similarity, respectively. We compared the biochemical phenotype of DN04^TtoD. sacchariSac-22^TandD. zoogloeoides115^Tand other reference strains from different genera within the Oxalobacteraceae and while the biochemical profile of DN04^Tis most similar toD. sacchariSac-22^Tand otherDuganellaandMassiliastrains, there are also distinct differences. DN04^Tcan for example utilize turanose, N-acetyl-d-glucosamine, inosine, andl-pyroglutamic acid. The four fatty acids found in the highest percentages were C_15 : 0iso (24.6 %), C_15 : 1isoG (19.4 %), C_17 : 0iso3-OH (16.8 %), and summed feature 3 (C_16:1⍵7c and/or C_16:1⍵6c) (12.5 %). We also applied whole genome sequencing to determine if DN04^Tis a novel species. The most similar AAI (average amino acid identity) score was 70.8 % (Massilia plicataNZ CP038026^T), and the most similar ANI (average nucleotide identity) score was 84.8 % (D. radicisKCTC 22382^T), which indicates that DN04^Tis a novel species. The genome-to-genome-distance calculation (GGDC) revealed a DDH of 28.3 % toD. radicisKCTC 22382^T, which is much lower than the new species threshold. Based on the morphological, phenotypic, and genomic differences, we proposeDuganella callidasp. nov. as a novel species within theDuganellagenus (type strain DN04^T=NRRL B-65552^T=LMG 31736^T).

From garden soil, we isolated and sequenced Massilia sp. strain ONC3, a new member of the Oxalobacteraceae within the Massilia genus. Sequence analysis showed an assembled genome size of 5,622,601 bp, with a predicted total of 5,104 protein-coding sequences, 3,194 functionally assigned genes, 2 rRNA operons, and 56 tRNAs.

Endophytic bacteria are known to influence vital activities of host plants. Endophytes can promote plant growth and provide a defense response against pathogens. The use of endophytes in crop production has the potential to reduce the application of fertilizer and pesticide input and thus improve the sustainability of crop production. In this study, we investigated the effects of seed inoculation with non-native endophytic bacteria, harvested from Brassica carinata, on oat (Avena sativa L.) growth with root vigor assays and greenhouse experiments. For root vigor assay experiments, seeds of two different oat cultivars were treated with 16 endophytic bacteria previously shown to promote growth benefits on multiple crop species. For the greenhouse experiments, the effect of seed inoculation with bacterial isolates was evaluated on ten oat cultivars at two fertilization levels. The root vigor assay showed that multiple isolates, including Bacillus licheniformis, Enterobacter kobei, B. halotolerans, B. cereus, B. aryabhattai, and Lysinibacillus fusiformis, had a positive effect on seedling root growth in one of the two oat cultivars. In the other cultivar, the bacterial isolates had either no effect or a negative effect on root growth. Greenhouse studies showed that the magnitude and direction of the effect of bacterial inoculation on oat growth varied with fertilization levels, bacterial strain, and oat cultivar. However, we identified two cultivars that were more responsive to bacterial inoculation than the others and for which bacterial inoculation of seed resulted in enhanced growth in several traits under both reduced and full nitrogen levels, and this response was observed for the two isolates tested. Our results show that inoculating oat seeds with non-native bacterial endophytes can promote root and shoot growth in oats. Developing biofertilizers that are effective across crop species, crop cultivars, and environmental conditions may be possible if cultivars are selected for their responsiveness across multiple bacterial isolates and in multiple growing environments. Overall, this study indicates that non-native endophytes could be considered for the development of biofertilizers with effectiveness across crop species.