Pseudomonas glycinae sp. nov. isolated from the soybean rhizosphere

Abstract Strains MS586T and MS82, which are aerobic, Gram‐negative, rod‐shaped, and polar‐flagellated bacteria, were isolated from the soybean rhizosphere in Mississippi. Taxonomic positions of MS586T and MS82 were determined using a polyphasic approach. 16S rRNA gene sequence analyses of the two strains showed high pairwise sequence similarities (>98%) to some Pseudomonas species. Analysis of the concatenated 16S rRNA, rpoB, rpoD, and gyrB gene sequences indicated that the strains belonging to the Pseudomonas koreensis subgroup (SG) shared the highest similarity with Pseudomonas kribbensis strain 46‐2T. Analyses of average nucleotide identity (ANI), genome‐to‐genome distance, delineated MS586T and MS82 from other species within the genus Pseudomonas. The predominant quinone system of the strain was ubiquinone 9 (Q‐9), and the DNA G+C content was 60.48 mol%. The major fatty acids were C16:0, C17:0 cyclo, and the summed features 3 and 8 consisting of C16:1ω7c/C16:1ω6c and C18:1ω7c/C18:1ω6c, respectively. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. Based on these data, it is proposed that strains MS586T and MS82 represent a novel species within the genus Pseudomonas. The proposed name for the new species is Pseudomonas glycinae, and the type strain is MS586T (accession NRRL B‐65441 = accession LMG 30275).

ANI evaluates a large number of nucleic acid sequences, including some that evolve quickly and others that evolve slowly, in its calculation and reduces the influence of horizontal gene transfer events or variable evolutionary rates. It has been suggested that species descriptions of bacteria and archaea should include a high-quality genome sequence of at least the type strain as an obligatory requirement (Rosselló-Móra & Amann, 2015). The current metagenome databases have shown evidence for approximately 8000 sequence-discrete natural populations, which is roughly equivalent to species at the 95% ANI level (Rosselló-Móra & Whitman, 2018). Genome-to-genome distance (GGDC 2.0) is another highly effective method for inferring whole-genome distances. GGDC effectively mimics DNA-DNA hybridization for genome-based species delineation and subspecies delineation (Meier-Kolthoff, Auch, Klenk, & Göker, 2013). Therefore, ANI and GGDC are highly effective ways to evaluate the genetic relatedness between genomes. Strains MS586 T and MS82 were isolated from the rhizosphere soybean plants growing in fields where most plants were infected by the charcoal rot pathogen Macrophomina phaseolina.
Plate bioassay indicated both strains MS586 T and MS82 exhibited striking antimicrobial activity (Ma et al., 2017). This research is focused on the characterization of the taxonomic position of the two strains.

| Bacterial strains and growth conditions
MS586 T and MS82 were isolated from a soybean rhizosphere sample by standard dilution plating on nutrient broth yeast extract (NBY) agar medium (Vidaver, 1967) at 28°C. Antimicrobial activity against multiple plant pathogens was detected with an antifungal plate assay as previously described (Gu, Wang, Chaney, Smith, & Lu, 2009). Following purification, the bacterium was preserved in 20% glycerol at −80°C. Pseudomonas spp. type strains and reference strains were provided by the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). All strains used in this study are summarized in Table A1.

| Cell morphology and physiological tests
Colony morphology of the strains MS586 T and MS82 was determined after growth on NBY agar plates. Gram staining was performed as described previously (Murray, Doetsch, & Robinow, 1994); cell morphology and flagellation types were observed with a transmission electron microscope (TEM) using routine negative glutaraldehyde staining; and the production of fluorescent pigments was tested on King B medium (King, Ward, & Raney, 1954).
Optical density (OD600) metrics recorded for NBY liquid cultures were used to evaluate optimal growth and pH, at temperatures from 4°C to 40°C, with an interval of 4°C for 24 hr, and at pH 4.0-10.0.
Cellular fatty acids were identified using the Sherlock 6.1 system (Microbial IDentification Inc.) and the library RTSBA6 (Sasser, 1990).
Biochemical features and enzyme activities were determined using API 20 NE and API 50 CH strips with API 50 CHB/E medium (bio-Merieux), as well as Biology GENIII Microplates (Biolog) as directed in the manufacturer's instructions; results were recorded after incubation for 48 hr at 28°C.
Sequences of type strains used in the MLSA were downloaded from Tolerance of NaCl at

Propionic acid
NCBI (accession numbers in Table A2). The maximum-likelihood method was used to construct the phylogenetic tree with 1000 bootstrap replicates.

| DNA fingerprinting
DNA fingerprinting has been evaluated and proposed as a reliable method for distinguishing different strains in the same taxon, which are not clonal varieties. Thus, the primer sequence corresponding to BOX elements (BoxA1R: 5′-CTACGGCAAGGCGACGCTGACG-3′) was used for DNA fingerprinting (Koeuth, Versalovic, & Lupski, 1995). PCR amplification was conducted as follows: initial denaturation at 94°C for 5 min, followed by 30 cycles (94°C for 1 min, 52°C or 53°C for 1 min, and 72°C for 2 min), and finally 72°C for 8 min. The DNA fragments were analyzed in a 2% agarose gel.

| Genome sequencing and analysis
Genomic DNA of strain MS586 T was extracted using the Wizard  Table   A3. The clustering of the type-based species using a 70% dDDH radius around each type strain was conducted as previously described

| Chemotaxonomic analysis
As important chemical characteristics for bacterial identification, the cellular fatty acid profile of the strain MS586 T was analyzed.
Cellular fatty acids were harvested after 2 days of growth at 28°C on TSA. Fatty acids extracted from the bacteria were methylated and analyzed following the protocol of the Sherlock 6.1 Microbial Identification (MIDI) system (Microbial IDentification Inc.) using the library RTSBA6 (Sasser, 1990). Analyses of respiratory quinones and polar lipids were carried out by the Identification Service of the DSMZ (Braunschweig, Germany).

TA B L E 1 (Continued)
and d-serine. In contrast, all these reactions were not negative for

| Phylogenetic analysis
Sequence analysis revealed that the 16S rRNA genes of MS586 T and MS82 shared significant identities (>98%) to some Pseudomonas Therefore, further extensive research is needed to update the Pseudomonas taxonomy.

| DNA fingerprinting
DNA fingerprinting by BOX-PCR revealed that strains MS586 T and MS82 were different representatives of the proposed novel species.
As shown in Figure A2, two strains have the two common bands (490 bp and 900 bp) in the BOX-PCR profiles; however, each of them produced unique bands (125 bp, 300 bp, 750 bp, and 1350 bp for MS586 T ; 700 bp, 750 bp, 1100 bp, and 1350 bp for MS82), which suggests the two strains are not identical isolates.

| General taxonomic genome features of strain MS586 T
The main characteristics of the whole-genome sequence of strain MS586 T are depicted in Pf0-1, which is the closest relative outside to the novel species.
As reported by Lopes et al. (Lopes et al., 2018), three strains isolated from tropical soils, which share ≥95% ANI values with strain MS586 T , are the potential strains for the novel species. As shown in Figure 2, the whole-genome-based phylogenetic tree obtained with TYGS automated pipeline shows that both MS586 T and MS82 were grouped into the same species cluster and confirmed that P. kribbensis 46-2 T is the closely related type strain.
P. fluorescens Pf0-1 was clustered to independent branch, which indicates its distinct phylogenetic position and potential as a separate species. Collectively, the ANI, GGDC, and whole-genome phylogenetic tree data support that strains MS586 T and MS82 represent a unique species.
Furthermore, strains MS586 T and MS82 were noteworthy, which were isolated from the rhizosphere of soybean plants associated with fungal pathogen infections. Strain MS586 T has shown remarkable antifungal activities against a broad range of plant fungal pathogens (Jia and Lu, unpublished). Similarly, our study has demonstrated that strain MS82 possesses antifungal activities against the mushroom fungal pathogen Mycogone perniciosa, but not the mushroom fungus (Ma et al., 2019). Furthermore, it has been reported that PafR gene confers resistance to the mushroom pathogenic fungus (Ma et al., 2017). As expected, the PafR gene was also found in strains MS586 T . Therefore, it is not surprising that multiple nonribosomal peptide synthetase gene clusters, which are frequently associated with the production of antimicrobial compounds (Mootz & Marahiel, 1997), have been predicted from the genomes of the bacterial strains.
Strain MS586 T also contains the highest amounts of C 10:0 3-OH (6.6%) when compared to the reference strains (2.2%-5.4%). The detailed fatty acid profiles of strain MS586 T and the type strains of closely related species are provided in Table 4. Two-dimensional TLC analysis revealed that the polar lipids of strain MS586 T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), three unidentified phospholipids (PL), and one unidentified lipid (L) ( Figure A3). Strain MS586 T contains higher amounts of PL and L as compared with those of the closest relative of P. kribbensis 46-2 T . As expected, the major polar lipid components of strain MS586 T were PE, DPG, and PG, which agrees with data published previously for the genus Pseudomonas (Moore et al., 2006). Also, the major respiratory quinone of strain

F I G U R E 2
Whole-genome sequence tree generated with TYGS for strain MS586 T and its closely related species of the genus Pseudomonas. Tree inferred with FastME from GBDP distances was calculated from genome sequences. Branch lengths are scaled in terms of GBDP distance formula d5; numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. The colored squares designate species cluster. Accession numbers of sequences used in this study are summarized in Table A3 TA B L E 4 Cellular fatty acid profiles of strain MS586 T and strains of closely related species  (Chang et al., 2016); b, (Pascual et al., 2015); c, (Tvrzova et al., 2006);d, (Lopez et al., 2012); and e, (Camara et al., 2007). Values are percentages of total fatty acids.
Summed features represent groups of two or three fatty acids that cannot be separated by GC with the MIDI system. Summed feature 3 consists of C16:1ω7c/C16:1ω6c; summed feature 8 consists of C18:1ω7c/C18:1ω6c.

ACK N OWLED G M ENTS
This study was supported by the National Institute of Food and Agriculture, United States Department of Agriculture (MIS-401200).
We appreciate Kate Phillips for English proofreading.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
The GenBank accession numbers for the complete genome of