Brenneria Yuansilingia Sp. Nov., Isolated From Symptomatic Bark of Willow Canker

Two Gram-negative, facultative anaerobic strains are isolated from the symptomatic tissue of willow bark canker. The cells grow at 4–41 ℃ , pH 5.0–10.0, with optimal growth occuring at 30 °C and pH 7.0. Two novel strains share the highest 16S rRNA gene sequence similarity with Brenneria nigriuens LMG 2694 T (98.8%). In the phylogenetic trees based on sequences of 16S rRNA gene and four housekeeping genes (gyrB, rpoB, atpD and infB), two novel strains form a distinct branch from Brenneria species, indicating that two novel strains should belong to a novel species in the genus Brenneria, which is conrmed by the results of average nucleotide identity analysis and Digital DNA–DNA hybridization. Two novel strains have 81.9–85.0 % average nucleotide identity values with its closely relatives, B. nigriuens LMG 2694 T , B. alni DSM 11811 T , B.corticis CFCC11842 T , B. populi CFCC 11963 T and B. corticis gBX10-1-2 T , respectively, lower than the proposed species boundary cut-off for ANI (95-96 %). The main fatty acids are C 16:0, C 16:1 ω7c and C 18:1 ω7c. The DNA G+C content is 53.5 -53.7 %. Based on these data, two novel strains represent a novel species within the genus Brenneria. The name Brenneria yuansilingia sp. nov. is proposed. The type strain is hezel4-2-4 T (=CFCC 15597 T = LMG 31719 T ). Cells are Gram-negative, facultative anaerobic, motile with agella, 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 H 2 S 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-ranose, 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 C 16:0, C 16:1 ω7c, and C 18:1 ω7c. The G+C content is G+C 53.5-53.7 %. Type strain hezel4-2-4 T (=CFCC 15597 T = LMG 31719 T ) 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 hezel4-2-4 T MT036110,

Recently, two novel strains named hezel4-2-4 T and L3-3C-1 are isolated from samples of willow (Salix matsudana) bark canker colletected from Heze City, Shandong Province and Zhongwei city, Ningxia Hui Autonomous Region, China, respectively. This disease caused by Lonsdalea populi is often found in the branches and trunks of willow trees leading to tree or branch death (Li et al. 2019b). In the present study, the taxonomic status of two novel strains will be determined by using 16S rRNA gene analysis, multilocus sequence analysis (MLSA), genome sequencing, average nuclide identity (ANI), digital DNA:DNA hybridization (dDDH), fatty acid analysis, and physiological and biochemical characterization.

Materials And Methods
Isolation and cultivation of bacteria Bark canker samples of willows were collected from Heze City, Shandong Province, China, in September 2018. The bacterial strains were isolated according to the method described by Li et al. (2014). In brief, the collected bark was sterilized in 4% sodium hypochlorite solution for 3 minutes. After washing three times in sterile water, the bark tissues were cut into small pieces and ground using sterile mortar by the pestle. Then the ground tissue was transferred into a conical ask with 10 ml sterile water and shaking 10 mins at 30 ℃. The resulting suspension was serially diluted to 10 -1 -10 -4 with sterile water and spread onto nutrient agar plates. After incubation at 30 ℃ for 48 h, single colonies were streaked out on nutrient agar plate. The puri ed strains were preserved at -80 ℃.
Anaerobic growth was observed after incubation of the novel strain on the same medium at 30 ℃ for one week in anaerobic jars (candle-jar method; Gerhardt et al. 1981). The catalase and oxidase activities were determined according to the method described by Smibert & Krieg (Smibert & Krieg 1994). Other physiological and biochemical characteristics were tested by using API 20E, API 50CHB/E test kits (bioMérieux) and Biolog Gen III according to the manufacturer's instructions.

Phylogenetic analysis
The 16SrRNA gene was ampli ed and sequenced by using primers 27F/1492R (5'-AGAGTTTGATCCTGGCTCAG-3'/5'-GGTTACCTTGTTACGACTT-3') (Lane 1991). An almost complete 16S rRNA gene sequence (1443 bp) was obtained for subsequent similarity analysis. The 16S rRNA gene sequence similarities were carried out by EzTaxon-e (http://eztaxon-e.ezbiocloud.net; Kim et al. 2012). After alignment and trimming, a 1302 bp-long sequence (corresponding to nucleotide positions 81-1382 in the Escherichia coli) was used for phylogenetic tree reconstruction by using Mage 7.0 Kumar et al. 2016 based on maximum likelihood (Kimura 2-parameter model with Gamma distributed with invariant sites G+I). Cronobactersakazakii was used as the out group, and the resulting trees were evaluated using 1000 resamplings.
MLSA based on partial sequences of four housekeeping genes (gyrB, 743 bp; rpoB, 637 bp; infB, 615 bp; atpD, 638bp) was performed on two novel and reference strains (Brady et al. 2008). Sequences of the four housekeeping genes for two novel strains were retrieved from its genomic sequence. Sequences of the reference strains were downloaded from GeneBank and the accession numbers were listed in table S1. The concatenated data of four housekeeping genes were aligned and used to reconstruct maximum likelihood method tree using Mage7.0 (Kumar et al. 2016). The general time reversible model with gamma distributed with invariant sites (G+I) was used and the resulting trees were evaluated using 1000 resamplings.

Genome analysis
Genome sequencing of two novel strains were carried out as described by Li et al. (2016). In brief, the library was constructed by the 300 to 500-bp DNA fragmentation performed using a TruSeq™ DNA Sample Prep Kit. Draft genomes were assembled using SOAPdenovo v2.04. The Glimmer 3.02 software were used to coding sequences prediction (Lagesen et al. 2007). The G+C contents were deduced from the genomic data. Virulence genes of hezel4-2-4 T were predicted using the Pathogen Host Interactions

Fatty acid analysis
The cellular fatty acid analysis was carried out for two novel and reference strains. After culturing on TSA at 30 °C for 24 h, the cells were harvested during the exponential phase. The fatty acids were extracted and analyzed according to the protocol of the Sherlock Microbial Identi cation System (MIDI, version 6.0) (Sasser M. 1990).

Results And Discussion
Physiological and biochemicalcharacteristics Cells are Gram-negative, facultative anaerobic, motile with agella, 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.
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 3934 T , which next to the branch formed by B. corticis, B. nigri uens, 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 hezel4-2-4 T 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.

Fatty acid analysis
The main fatty acids of two novel strains were C 16:0, C 16:1 ω7c, C 18:1 ω7c, similar to closely related reference strains (Table2). The amount of C 16:1 ω7c of the novel strains was higher than their closely related reference strains, which could be used to differentiate them from its close relatives.

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