Physiological and biochemicalcharacteristics
Cells are Gram-negative, facultative anaerobic, motile with flagella, short rods, approximately 0.6-0.7 × 1.5-2.0 µm (Fig S1). The physiological and biochemical results were listed in Table 1 and the species description given below.
Phylogenetic analysis
Two novel strains have 99.8% 16S rRNA gene sequence similarity, and show highest similarity with Brenneria nigrifluens LMG 2694 (98.8 %), followed by B. corticis gBX10-1-2 (97.8 %) and B. roseae (96.9 %). In the ML phylogenetic tree based on 16S rRNA gene (Fig.1), two novel strains form a distinct branch from the reference strains within Brenneria clade, and cluster together with B. nigrifluens ATCC 13208T with 91 % bootstrap, and next to the branch of B. corticis CFCC 11842T. Two novel strains and its closely relatives, B. nigrifluens ATCC 13208T, B. corticis CFCC 11842T, B. alni NCPPB 3934T and B. populi strains formed one big cluster within Brenneria clade.
In the ML phylogenetic tree based on MLSA data (Fig.2), two novel strains form a separate branch, and cluster together with B. alni NCPPB 3934T, which next to the branch formed by B. corticis, B. nigrifluens, B. goodwinii and B. populi. Phylogenetic trees based on 16S rRNA gene sequences and MLSA data suggest that two novel strains should belong to a novel species within the genus Brenneria.
Genomic analysis
The genome length of strain hezel4-2-4T is 4.54 Mb size across 32 contigs (N50= 479,859 bp), including 3,936 coding genes, 66 pseudogenes, 17 rRNAs, 69 tRNAs and 8 other RNAs. The max length of those contigs is 889,800 bp, the DNA G+C content is 53.48 %. 547 genes are found to be associated with pathogen and host interactions according to database of the Pathogen Host Interactions. 269 genes of those genes are reduced virulence, and 118 genes do not affect pathogenicity of the trains (detailed in Fig S 2). To detect potential contamination of the novel strain genome, the 16S rRNA gene sequence determined by conventional Sanger sequencing is compared with the sequence retrieved from its genome. They share 100% sequence similarity.
Five closely related species, B. nigrifluens LMG 2694T, B.alni DSM 11811T and B. corticis gBX10-1-2T, are selected for ANI analysis and dDDH with two novel strains. Two novel strains showed 81.9-85% ANI values with closely relatives (Table S2), which are lower than the proposed species boundary cut-off for ANI (95-96 %) (Richter M, Rosselló-Móra R, 2009), revealed that they represent a novel species within genus Brenneria. Two novel strains have 26.0-29.5 dDDH values with their closely related referenc strains (Table S2), lower than the species boundary DDH value (70 %) (Wayne et al. 1987).
Fatty acid analysis
The main fatty acids of two novel strains were C16:0, C16:1 ω7c, C18:1 ω7c, similar to closely related reference strains (Table2). The amount of C16:1 ω7c of the novel strains was higher than their closely related reference strains, which could be used to differentiate them from its close relatives.
Taxonomic conclusion
The phylogenetic trees based on 16S rRNA gene and MLSA data suggest that two novel strains should represent a novel species within the genus Brenneria. The results of fatty acids, ANI, dDDH are most useful data to demonstrate that two novel strains belong to a novel species of Brenneria. Moreover, there are several physiological and biochemical characteristics which can differentiate two novel strains from their Brenneria reference strains. For example, the disability of utilization of dextrin, d-glucuronic acid, α-keto butyric acid, L-glutamic acid can distinguish two novel strains from their closely reference strains Brenneria alni DSM 11811T, B. corticis CFCC 11842T, B. goodwinni LMG 26270T, B. nigrifluens LMG 2694T and B. populi CFCC 11963T. Based on those data, we propose that the two novel stains should be assigned to a novel species within genus Brenneria. The name proposed for them are Brenneriayuansilingia sp. nov.
Description of Brenneria yuansilingiasp. nov.
Brenneria yuansilingia (Yuan.si.lin.gia N.L. gen. neut. n. Yuansiling named in honour of researcher Siling Yuan (1919–2017), a well-known forest pathologist in China)
Cells are Gram-negative, facultative anaerobic, motile with flagella, short rods, approximately 0.6-0.7 × 1.5-2.0 µm. Colonies are circular, light cream, smooth with entire margin, and approximately 1.3-1.5 mm growth at 30 °C on TSA for 48 h. The cells grow at 4-41 ℃, pH 5-10 and optimal growth 30 °C and pH 7.0. Growth occurs in conditions of 0–7 % (w/v) salinity. Positive for oxidase and negative for catalase. Nitrite not produced by reduction of nitrate. Negative for the activities of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, β-galactosidase, tryptophane deaminase, gelatinase, and production of H2S and indole. Citrate is not utilized. It is positive for acid production from glycerol, l-arabinose, d-ribose, d-glucose, d-fructose, d-mannose, l-rhamnose, inositol, d-mannitol, methyl-αd-glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, aesculin, salicin, d-maltose, d-saccharose, d-trehalose, d-raffinose, gentiobiose, d-turanose, potassium gluconate, potassium 2-ketogluconate and potassium 5-keto-gluconate (API 50CHB/E). Cells are positive for assimilation of acetic acid, d-aspartic acid, d-fructose, d-fructose-6-PO4, d-galactose, d-gluconic acid, d-glucose-6-PO4, d-mannitol, d-mannose, d-saccharic acid, D-salicin, d-turanose, formic acid, glycerol, l-aspartic acid, l-rhamnose, l-serine, methyl pyruvate, mucic acid, myo-inositol, N-acetyl-d-glucosamine, pectin, β-methyl d-glucoside, quinic acid, sucrose, and variable for assimilation of gentiobiose (Biolog Gen III). The main fatty acids are C16:0, C16:1 ω7c, and C18:1 ω7c. The G+C content is G+C 53.5-53.7 %. Type strain hezel4-2-4T (=CFCC 15597T= LMG 31719T) is isolated from bark tissue of a willow bark canker. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genome sequence of strain hezel4-2-4T is MT036110, JABJXS000000000.