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Article

Halomonas kashgarensis sp. nov., a Novel Species Isolated from the Rhizosphere Soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China

by
Zhen-Pu Liang
1,2,*,†,
Yi Wang
2,†,
Xiao-Yue Chen
2,
Rui Wang
2,
Yan Xu
2,
Jin-Ping Dai
1,
Deepali Singh
3 and
Xiao-Xia Zhang
2,*
1
Institute of Microbial Application, Xinjiang Academy of Agricultural Sciences, Urumqi 830000, China
2
College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, China
3
School of Biotechnology, Gautam Buddha University, Greater Noida 201312, India
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2025, 17(2), 98; https://doi.org/10.3390/d17020098
Submission received: 27 December 2024 / Revised: 19 January 2025 / Accepted: 26 January 2025 / Published: 29 January 2025
(This article belongs to the Special Issue 15th Anniversary of Diversity—Biodiversity, Conservation and Ecology of Animals, Plants and Microorganisms)
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Abstract

:
A novel Gram-negative, orange-colored, rod-shaped, oxidase and catalase-positive, non-spore-forming bacterium, designated as zp-37T, was isolated from the rhizosphere soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China. The phylogenetic analysis, based on the 16S rRNA genes, revealed that strain zp-37T belongs to the genus Halomonas. Growth of strain zp-37T was observed at 10–43 °C, pH 6.0–11.0, and 0–20% NaCl (w/v). The principal fatty acids of strain zp-37T were summed feature 8 (C18:1ω7c and/or C18:1ω6c, 55.67%) and summed feature 3 (C16:1ω7c and/or C16:1ω6c, 20.16%). The polar lipid profile contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids (UPL 1–3), unidentified aminophospholipids (UAPL 1–2), and unidentified lipid (UL). Its main respiratory quinone was ubiquinone Q-9 (100%). The genome of strain zp-37T was 3,489,967 bp in size, containing two plasmids with lengths of 18,112 bp and 4364 bp, respectively. The genomic DNA G+C content of strain zp-37T was 59.3%. By the genome annotation, various genes related to the function of saline-alkaline stress tolerance and plant growth promotion were predicted. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain zp-37T and its five closely related strains were 72.64–75.59% and 19.70–20.40%, respectively, which were lower than the threshold for species delineation (ANI: 95–96%, dDDH: 70%). Based on the phylogenetic, phenotypic, and chemotaxonomic analyses and genomic comparisons, strain zp-37T was suggested to represent a novel species within the genus Halomonas, for which the name Halomonas kashgarensis sp. nov. is proposed. The strain type was designated zp-37T (=CGMCC 1.62213T = JCM 37305T).

1. Introduction

Halomonas, classified within the Halomonadaceae family, Oceanospirillales order, Gammaproteobacteria class, Pseudomonadota phylum, was proposed by Vreeland et al. in 1980 [1]. Members of the genus Halomonas are usually rod-shaped, Gram-negative, aerobic, and halophilic bacteria. They are highly halotolerant, and numerous strains are able to survive in an environment with salts that range from 0.1 to 32.5% [2]. Therefore, a lot of members of the genus Halomonas have been isolated in a variety of hypersaline environments, such as seawater, saline soil, saline lakes, and marine sediments [3,4,5,6,7,8,9]. Most strains of Halomonas also have strong alkali tolerance and can grow under pH conditions exceeding 10 [10]. In addition, some of them have the capacity to produce exopolysaccharides, compatible solutes, and degrade aromatic compounds [9,11]. Due to their excellent salt tolerance and plant growth-promoting effect, Halomonas spp. has outstanding performance in improving saline-alkaline soil and promoting plant growth under saline-alkaline stress. In Zhang et al.’s study, Halomonas sp. MC1 significantly increased the growth of cabbage roots under salt stress [12]. Tiwari et al. showed an increase in carotenoid and chlorophyll content in wheat after inoculating with Halomonas sp. SL9 [13]. The study by Bekkaye et al. also showed that under salt stress, inoculation of strain Halomonas sp. BSSM328 could significantly reduce the content of abscisic acid, jasmonic acid, and proline in wheat, which could help wheat alleviate salt stress and promote its growth [14].
Up to now, December 2024, the genus Halomonas includes 144 species with valid publications (https://lpsn.dsmz.de/search?word=Halomonas, accessed on 27 December 2024). A variety of Halomonas were used as microbial inoculants to promote plant growth [15]. During investigations of rhizosphere soil of Phragmites australis (Cav.) Trin. ex Steud in Xinjiang, China, a novel strain belonging to the genus Halomonas with plant growth-promoting ability was isolated and designated as zp-37T. This study aimed to characterize the zp-37T strain via physiological, chemotaxonomic, and genome analyses. The discovery of a novel bacterium of the genus Halomonas expands our understanding of this genus and provides the potential bacterium resource for the future preparation of microbial agents for the improvement of saline-alkaline soil.

2. Materials and Methods

2.1. Isolation and Culture Conditions

The soil samples were collected from the rhizosphere of Phragmites australis (Cav.) Trin. ex Steud, in Kashgar County, Xinjiang, China (39°79′ N, 78°55′ E) on 20 August 2020. The soil samples were packed in sterile plastic bags and immediately transported to the laboratory in ice-cooled boxes. One gram of the soil sample was added to 9 mL of double-distilled water (ddH2O) and serially diluted, and 100 µL of the soil dilutions were plated on a modified Luria–Bertani (mLB) solid medium (comprising 5 g of yeast extract, 10 g of tryptone, 15 g of agar, and 1000 mL of ddH2O and NaCl supplemented to a final concentration of 7.5% with pH 9.0). Plates were incubated at 30 °C for 7 days, and colony formation was monitored [16]. Colonies of different morphotypes were selected and re-streaked on mLB solid medium until they were purified. A total of 109 colonies were isolated and subsequently identified. One of these isolates, an orange, round, single colony, was grown on mLB plates, designated as zp-37T, and stored in a 25% (v/v) glycerol solution at −80 °C for further use [17]. Samples of strain zp-37T have been deposited in the China General Microbiological Culture Collection Center (CGMCC) as 1.62213 and the Japan Collection of Microorganisms (JCM) as JCM 37305.

2.2. Phylogenetic Analysis

The strain zp-37T was identified by 16S rRNA gene sequencing. The universal PCR primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for 16S rRNA gene amplification [18]. The PCR mixture was set up in a total volume of 25 μL, comprising 10.5 μL of ddH2O, 1 μL of 27F primers (10 μmol/L), 1 μL of 1492R primers (10 μmol/L), and 12.5 μL of 2 × Phanta Max Mix (P515, Novozan, Nanjing, China). Colonies of strain zp-37T approximately 1 mm in diameter were picked up with a sterilized pipette tip and directly transferred to the PCR tube as DNA templates [19]. The amplified products were sent to Sangon Biotech (Shanghai, China) for sequencing. The sequencing results were analyzed and identified using a nucleotide BLAST search in the EzbioCloud database (https://www.ezbiocloud.net/, accessed on 9 December 2024). The phylogenetic analysis was carried out using the MEGA (v.11) [20] software with the maximum-likelihood [21], neighbor-joining [22], and maximum-parsimony [23] algorithms. The phylogenetic trees were evaluated by bootstrap analysis with 1000 replications [24].

2.3. Morphological, Physiological, and Biochemical Characterizations

The zp-37T strain was cultured on mLB solid medium with aerobic condition at 30 °C for the observation of colony morphology. Cell morphology and the presence of spores of this strain were observed by using an optical microscope (CX-22, Olympus, Tokyo, Japan), scanning electron microscope (SEM, Q45, FEI, Hillsboro, OR, USA), and transmission electron microscope (TEM, ht7800, Hitachi, Tokyo, Japan). The Gram-staining reactions were performed by using a Gram-staining kit (Hopebio, Qingdao, China) following the guidelines provided by the manufacturer. The range and optimal NaCl concentration for growth were determined in mLB liquid medium at 30 °C (supplemented NaCl with a final concentration of 0–20%, an interval of 2%, and pH adjusted to 9.0). The range and optimal pH for zp-37T growth were tested in mLB liquid medium (supplemented with final 7.5% NaCl) at 30 °C, and the pH buffers were adjusted from 2 to 12 as described by Qiu et al. [25]. Additionally, the temperature range that the strain could grow (4, 10, 15, 20, 25, 30, 37, 40, 43, and 45 °C) was determined in mLB liquid medium under aerobic conditions. The OD600 values of the bacterial culture medium were measured by spectrophotometer (VIS-7220N, Beifen-Ruili, Beijing, China) every 24 h.
Catalase activity was determined by observing the formation of bubbles when adding 3% (v/v) H2O2 to the liquid culture of strain zp-37T. Oxidase activity was measured through the oxidation of tetramethyl-ρ-phenylenediamine [26]. The microbial biochemical identification kits (Hopebio, Qingdao, China) and Biolog ECO microplates were used to assess the carbon source utilization capacity of strain zp-37T, including L-arabinose, D-glucose, β-Methyl-D-Glucoside, D-Galactonic Acid γ-Lactone, L-Arginine, Pyruvic Acid Methyl Ester, D-Xylose, D-Galacturonic Acid, L-Asparagine, Tween 40, I-Erythritol, 2-Hydroxy Benzoic Acid, L-Phenylalanine, Tween 80, D-Mannitol, 4-Hydroxy Benzoic Acid, L-Serine, α-Cyclodextrin, N-Acetyl-D-Glucosamine, γ-Hydroxybutyric Acid, L-Threonine, Glycogen, D-Glucosaminic Acid, Itaconic Acid, Glycyl-L-Glutamic Acid, D-Cellobiose, Glucose-1-Phosphate, α-Ketobutyric Acid, Phenylethylamine, α-D-Lactose, D L-α-Glycerol Phosphate, D-Malic Acid, Putrescine, D-galactose, D-sucrose, and D-fructose [27]. Seven traits related to plant growth-promoting were determined, including nitrogen fixation, ACC deaminase production, phosphate and potassium solubilization, siderophore production, indole acetic acid (IAA) synthesis, and cellulase production. Nitrogen fixation was tested on the Ashby solid medium [28]. ACC deaminase production was tested using the method described by El-Tarabily [29]. Phosphate and potassium solubilization of the strain zp-37T were tested on PVK solid medium and potassium-dissolving solid medium, respectively, according to the method described by Li et al. [30]. The siderophore production ability was assayed on Chrome azurol S (CAS) blue solid medium described by Schwyn and Neilands [31]. IAA synthesis was tested using the Salkowski method described by Reang et al. [32]. Cellulase production capacity was performed on a carboxy methyl cellulose (CMC) congo red solid medium described by Rasool et al. [33].

2.4. Chemotaxonomic Characteristics

The zp-37T strain was incubated in mLB liquid medium at 180 r/min 30 °C for 48 h to determine its cellular fatty acids, polar lipids, respiratory quinones, and chemical composition of the cell wall. Fatty acids were saponified, extracted, methylated, and analyzed using the Sherlock® Microbial Identification System (MIDI Inc, Newark, NJ, USA) according to the manufacturer’s instructions. Gas chromatography (GC) was used for the identification of cellular fatty acids [25]. Chloroform-methanol filtration was used to extract the cellular polar lipids, and the examination of polar lipids was performed by two-dimensional thin layer chromatography (TLC) on silica gel (Kieselgel 60 F254; Merck Inc., Darmstadt, Germany). A high-performance liquid chromatography (HPLC) system (Shimadzu Inc., Kyoto, Japan) was utilized to analyze respiratory quinones [34].

2.5. Genome Sequencing and Analysis

The E.Z.N.A® Bacteria DNA kit (Omega, Norcross, GA, USA) was utilized to extract and purify the genome DNA of strain zp-37T according to the instructions. A highly qualified DNA sample (OD260/280 = 1.8–2.0, >6 μg), which was quantified by using a TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA, USA), was used to construct a fragment library. The complete genome of strain zp-37T was sequenced by combining the Illumina NovaSeq 6000 system and Pacific Biosciences Sequel IIe technology (PacBio) (Shanghai Biozeron Biotechnology Co., Ltd., Shanghai, China). The assembly of the whole genome was conducted using the ABySS software (v2.2.0) (http://www.bcgsc.ca/platform/bioinfo/software/abyss, accessed on 20 July 2023). The CheckM (v1.2.2) was used to assess genome completeness and contamination [35].
Gene analysis was carried out using GeneMarkS (v4.17). The genome annotation was performed using multiple databases, including Non-Redundant Protein Database (NR; https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/, accessed on 20 July 2023), SwissProt (http://uniprot.org, accessed on 20 July 2023), Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/, accessed on 20 July 2023), Clusters of Orthologous Groups of proteins (COG; https://www.ncbi.nlm.nih.gov/research/cog/, accessed on 20 July 2023), Gene Ontology (GO; http://www.geneontology.org/, accessed on 20 July 2023), and Carbohydrate-Active Enzymes Database (CAZy; http://www.cazy.org/, accessed on 20 July 2023). Secondary metabolites biosynthesis gene clusters (BGCs) were predicted by using the antiSMASH (v.7.1.0) platform [36].
Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values were analyzed by ANI Calculator (https://www.ezbiocloud.net, accessed on 14 December 2024) [37] and the Genome-to-Genome Distance Calculator (GGDC) 3.0 (https://ggdc.dsmz.de, accessed on 14 December 2024) [38]. The phylogenomic tree, including 24 type strains of the genus Halomonas was constructed through the EasyCGTree pipeline [39]. The analysis of orthologous homologous gene clusters was using the OrthoVenn 3.0 online platform [40].

2.6. Accession Numbers

The sequence of the 16S rRNA gene and the whole genome of strain zp-37T has been deposited in the GenBank database under the accession numbers OQ996844 and CP137552, respectively.

3. Results

3.1. Phylogenomic Analysis Based on 16S rRNA Gene Sequence

The 16S rRNA gene sequence of strain zp-37T was 1433 bp in length (GenBank accession number OQ996844). Its 16S rRNA gene sequence was analyzed to determine the phylogenetic relationships using the EzBioCloud database [41].
Phylogenetic analysis showed that the 16S rRNA gene of strain zp-37T exhibited the highest similarities with two strain types, Halomonas maris QX-1T and Halomonas zhaodongensis NEAU-ST10-25T. The 16S rRNA gene sequence similarities between strain zp-37T and these two closely related type strains were 97.67% and 97.65%, respectively, which were lower than the species identification threshold (98.65%) for prokaryotic species [42].
To determine the phylogenetic position of strain zp-37T, phylogenetic trees based on the 16S rRNA sequence of strain zp-37T and its closely related type strains were constructed. In the phylogenetic tree constructed using the maximum-likelihood (ML) algorithm (Figure 1), strain zp-37T was closely associated with the genus Halomonas and formed a separate branch. Phylogenetic trees based on the neighbor-joining (NJ) and maximum-parsimony (MP) methods also corroborated this relationship (Figures S1 and S2). Thus, the lower sequence similarity and distinctive branching pattern in the phylogenetic tree indicated that the strain zp-37T was a potential novel species in the genus Halomonas.

3.2. Morphological, Physiological, and Biochemical Characteristics

The morphological characteristics of strain zp-37T were observed on mLB plates. Its colonies were orange, round, smooth, and shiny in appearance, with an average diameter of 1.44 mm. Strain zp-37T was identified as Gram-negative and non-spore-forming. The morphology of cells was analyzed under a scanning electron microscope and transmission electron microscope, revealing it was a rod-shaped structure with dimensions of 0.3–0.5 μm in width and 2.5–3.0 μm in length (Figure 2).
Physiologically, the vigorous growth of strain zp-37T was observed in mLB liquid medium at 10–43 °C (optimum: 37 °C) under aerobic conditions. Its pH range for growth was 6.0–11.0 (optimum: 7.0–8.0). The strain zp-37T could grow in mLB liquid medium with 0 to 20% (w/v) NaCl, and the optimal NaCl concentration was 12%. Some differences were observed in morphological, physiological, and biochemical characteristics when they were compared between strain zp-37T and its closely related strains (Table 1).
The cells of strain zp-37T were catalase and oxidase-positive. In addition, to analyze its carbon source utilization, thirty-six various carbon sources were examined, proving that zp-37T could utilize thirty-three of those as the sole carbon source: L-arabinose, D-glucose, β-Methyl-D-Glucoside, D-Galactonic Acid γ-Lactone, L-Arginine, Pyruvic Acid Methyl Ester, D-Xylose, D-Galacturonic Acid, L-Asparagine, Tween 40, I-Erythritol, 2-Hydroxy Benzoic Acid, L-Phenylalanine, Tween 80, D-Mannitol, 4-Hydroxy Benzoic Acid, L-Serine, α-Cyclodextrin, N-Acetyl-D-Glucosamine, γ-Hydroxybutyric Acid, L-Threonine, Glycogen, D-Glucosaminic Acid, Itaconic Acid, Glycyl-L-Glutamic Acid, D-Cellobiose, Glucose-1-Phosphate, α-Ketobutyric Acid, Phenylethylamine, α-D-Lactose, D L-α-Glycerol Phosphate, D-Malic Acid, and Putrescine. It could not utilize D-galactose, D-sucrose, and D-fructose as carbon sources (Table S1).
IAA production levels were checked continuously for 14 days, and a maximum level of 57.15 μg/mL was detected on the 11th day. The strain zp-37T had good growth on the Ashby solid medium, proving it can produce nitrogenase and its ability to fix nitrogen. In addition, strain zp-37T had a halo zone around its colonies on PVK, CAS, and CMC congo red solid medium, indicating its ability for phosphate solubilization, siderophore production, and cellulolytic activity (Figure 3).

3.3. Chemotaxonomic Characterization

The primary fatty acids of strain zp-37T were summed feature 8 (C18:1ω7c and/or C18:1ω6c, 55.67%) and summed feature 3 (C16:1ω7c and/or C16:1ω6c, 20.16%), which were consistent with the characteristics of the genus Halomonas described by Franzmann [47]. The polar lipids of strain zp-37T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids (UPL 1–3), unidentified aminophospholipids (UAPL 1–2), and unidentified lipid (UL). The type of respiratory quinone of strain zp-37T was ubiquinone Q-9 (100%), which was consistent with other members of the genus Halomonas [48]. The cell wall of strain zp-37T was found to lack detectable DPA components. The primary sugars presented in the cell wall were identified as ribose, glucose, and galactose. Strain zp-37T and its closest related stains exhibited similar chemical characteristics (Table 2).

3.4. Genomic Analyses

The genome of strain zp-37T (GenBank accession number CP137552) was 3,489,967 bp in size, containing two plasmids with lengths of 18,112 bp and 4364 bp, respectively. The genomic DNA G+C content of strains zp-37T was 59.3%. Its genome consisted of three contigs, and the sequencing depth coverage was approximately 88.0X. Assessment of genome assembly showed high completeness (99.21%) and low contamination (3.08%). We confirmed that the 16S rRNA gene sequence obtained from PCR matched that derived from the genome, ensuring the authenticity of its genomic data. The features of the genome were displayed in the genome map (Figure 4). In addition, phylogenetic trees based on the genome sequence of strain zp-37T and its 24 closely related type strains were constructed by using the EasyCGTree pipeline. Carnimonas nigrificans ATCC BAA-78T was used as the outgroup. Like the previous phylogenetic tree based on the 16S rRNA sequence, in the phylogenomic tree, strain zp-37T was closely associated with the genus Halomonas and formed a separated branch, which supported the proposal that strain zp-37T represented a novel species of the genus Halomonas (Figure 5).
The genome of strain zp-37T contains 3249 protein-coding sequences, 60 tRNAs, and 18 rRNAs (6 16S rRNAs, 6 23S rRNAs, and 6 5S rRNAs). The COG database annotated about 2798 protein-coding sequences, which accounted for 86.12% of all protein-coding sequences. These protein-coding sequences were classified into 21 types in the COG database (Figure S3A). Among the 21 functional annotation classifications of strain zp-37T, 1222 (43.67%) protein-coding sequences were related to metabolism: amino acid transport and metabolism (9.04%), energy production and conversion (7.61%), inorganic ion transport and metabolism (7.33%), coenzyme transport and metabolism (4.54%), lipid transport and metabolism (4.54%), carbohydrate transport and metabolism (4.50%), secondary metabolites biosynthesis, transport and catabolism (3.07%), and nucleotide transport and metabolism (3.04%). A total of 2108 protein-coding sequences of strain zp-37T were functionally annotated in the KEGG database (Figure S3B). These coding sequences were divided into 23 pathways of metabolism, including metabolism (12), genetic information processing (4), environmental information processing (2), cellular processes (3), and organismal systems (2).
According to the annotation results, 37 genes related to plant growth-promoting functions were found in the genome, including 7 nitrogen fixation genes (such as Fe-S cluster assembly protein sufB, sufD, and Fe-S cluster assembly transcriptional regulator iscR), 10 phosphate solubilization genes (such as phosphate ABC transporter complex pst A, pstB, and Pho regulon phoU), 9 IAA synthesis genes (such as tryptophan synthase genes trpA, aldB, and aspartate aminotransferase aspC) [49], 8 siderophore production genes (such as iron-siderophore transport system permease protein fepD, fepG, and ironIII transport system substrate-binding protein afuA), 1 tRNA recycling genes involved in cytokinin synthesis (tRNA dimethylallyltransferase miaA) [50], 1 rhizosphere colonization gene (tyrosine recombinase XerC), and 1 chemotaxis ability gene (chemotaxis protein cheW) (Table S2) [51]. Moreover, various saline-alkaline stress tolerance-related genes were predicted by the genome annotation (Table S3), such as 3 K+ transporter genes (potassium transporter ktrD, trk system potassium transporter trkA, and ktrC), 6 Na+/H+ antiporter genes (such as Na+/H+ antiporter subunit mrpD, mrpE, and mrpG), 4 proline synthesis genes (such as glutamate 5-kinase proB, glutamate-5-semialdehyde dehydrogenase proC, and prolyl-tRNA synthetase proS) and 3 antioxidant enzymes genes (catalase-peroxidase katG, vitamin B12 transporter btuB, and glutathione peroxidase bsaA) [50].
The comparative analysis of the genomes of strain zp-37T and its five closely related strains, H. maris QX-1T, H. zhaodongensis NEAU-ST10-25T, H. sulfidaeris ATCC BAA-803T, H. songnenensis NEAU-ST10-39T, and H. hydrothermalis Slthf2T, used OrthoVenn 3.0. The result revealed that their genes had a significant gene overlap (Figure 6). The strain zp-37T had a total of 2662 genes, while the other five closely related strains had 3471, 2920, 3328, 2929, and 3137 genes, respectively. Strain zp-37T shared 1914 common clusters of orthologous genes with other five strains and possessed 14 unique genes. And these unique genes of strain zp-37T were gpt (xanthine-guanine phosphoribosyltransferase), prpE (bis(5′-nucleosyl)-tetraphosphatase PrpE), aer (aerotaxis receptor), etc. (Table S4). Additionally, antiSMASH was used to predict clusters of secondary metabolite genes of strain zp-37T. Just like the type strains H. maris QX-1T, H. zhaodongensis NEAU-ST10-25T, H. sulfidaeris ATCC BAA-803T, H. songnenensis NEAU-ST10-39T, and H. hydrothermalis Slthf2T, zp-37T contained a gene cluster associated with ectoine, indicating a conserved metabolic pathway present in these strains.
The ANI values between strain zp-37T and other five closely related strains, H. maris QX-1T, H. zhaodongensis NEAU-ST10-25T, H. sulfidaeris ATCC BAA-803T, H. songnenensis NEAU-ST10-39T, and H. hydrothermalis Slthf2T, were calculated using OrthoANI (https://www.ezbiocloud.net/tools/ani, accessed on 19 January 2025), which ranged from 72.64% to 75.59%. These ANI values were below the threshold for the prokaryotic species (95–96%). The dDDH calculation results demonstrated that the values of strain zp-37T and the other five closely related strains ranged from 19.70% to 20.40%, which were lower than the boundary of 70% cutoff for species differentiation (Table 3). These results suggest strain zp-37T was considered a novel species of the genus Halomonas and formally named Halomonas kashgarensis sp. nov., subsequently.

4. Taxonomic Conclusions

The morphological, biochemical, chemotaxonomic, physiological, and genomic analyses of the strain zp-37T revealed that it is classified within the genus Halomonas. However, there are notable differences when compared to the closely related type strains. Strain zp-37T also showed low ANI and dDDH values with other closely related type strains. These results confirmed that strain zp-37T is a novel species of the genus Halomonas, for which the name Halomonas kashgarensis sp. nov. is proposed.

5. Description of Halomonas kashgarensis sp. nov.

Halomonas kashgarensis (ka.shgar.en’sis. N.L. fem. adj. Kashgarensis, pertaining to Kashgar, from Xinjiang, China, where the bacterium was originally isolated).
The cells are Gram-negative, aerobic, rod-shaped, about 0.3–0.5 μm in width and 2.5–3.0 μm in length. Colonies on mLB are orange, round, and smooth, with an average diameter of 1.44 mm. Growth was observed at 10–43 °C (optimum: 37 °C), pH 6.0–11.0 (optimum: 7.0–8.0), and 0–20% NaCl (w/v; optimum: 12%) on mLB liquid medium. Catalase and oxidase were positive. Thirty-three various carbon sources (L-arabinose, D-glucose, β-Methyl-D-Glucoside, D-Galactonic Acid γ-Lactone, L-Arginine, Pyruvic Acid Methyl Ester, D-Xylose, D-Galacturonic Acid, L-Asparagine, Tween 40, I-Erythritol, 2-Hydroxy Benzoic Acid, L-Phenylalanine, Tween 80, D-Mannitol, 4-Hydroxy Benzoic Acid, L-Serine, α-Cyclodextrin, N-Acetyl-D-Glucosamine, γ-Hydroxybutyric Acid, L-Threonine, Glycogen, D-Glucosaminic Acid, Itaconic Acid, Glycyl-L-Glutamic Acid, D-Cellobiose, Glucose-1-Phosphate, α-Ketobutyric Acid, Phenylethylamine, α-D-Lactose, D L-α-Glycerol Phosphate, D-Malic Acid, and Putrescine) were utilized as the sole carbon source. Principal fatty acids are summed as feature 8 (C18:1ω7c and/or C18:1ω6c) and summed as feature 3 (C16:1ω7c and/or C16:1ω6c). The polar lipids profile contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids (UPL 1–3), unidentified aminophospholipids (UAPL 1–2), and unidentified lipid (UL). The respiratory quinone is ubiquinone Q-9 (100%). The genome of strain zp-37T is 3,489,967 bp in length, with a G+C content of 59.3%.
The strain type zp-37T (=CGMCC 1.62213T = JCM 37305T) was isolated from the rhizosphere soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China. The 16S rRNA genes sequence and genome sequence of strain zp-37T have been deposited in the NCBI database under the accession numbers OQ996844 and CP137552, respectively.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d17020098/s1, Figure S1: The NJ phylogenetic tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of strain zp-37T and other closely related members of the genus Halomonas. Bootstrap support values were calculated from 1000 replicates (only values ≥ 70% were shown). The bold font represents the novel species identified in this study. GenBank accession numbers were provided in parentheses for reference. Carnimonas nigrificans CTCBS1T was used as the outgroup; Figure S2: The MP phylogenetic tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of strain zp-37T and other closely related members of the genus Halomonas. Bootstrap support values were calculated from 1000 replicates (only values ≥ 70% were shown). The bold font represents the novel species identified in this study. GenBank accession numbers were provided in parentheses for reference. Carnimonas nigrificans CTCBS1T was used as the outgroup; Table S1: Carbon sources utilization characteristics of strain zp-37T; Figure S3: Functional analysis of gene and protein sequence annotations from strain zp-37T A: GOC function classification of genes in strain zp-37T B: KEGG function classification of genes in strain zp-37T; Table S2: Potential genes related to plant growth-promoting in zp-37T Genome; Table S3: Potential genes related to saline-alkaline stress tolerance in zp-37T Genome; Table S4: The non-overlapped unique genes in strain zp-37T.

Author Contributions

Conceptualization, Z.-P.L. and X.-X.Z.; methodology, X.-X.Z. and J.-P.D.; software, Y.W., X.-Y.C. and R.W.; validation, Z.-P.L., Y.W. and X.-X.Z.; formal analysis, X.-Y.C. and Y.W.; investigation, X.-Y.C., Y.W. and Y.X.; resources, Z.-P.L., X.-X.Z. and J.-P.D.; data curation, X.-X.Z.; writing—original draft preparation, Z.-P.L. and X.-Y.C.; writing—review and editing, X.-X.Z. and D.S.; visualization, Y.W. and R.W.; project administration, Z.-P.L.; funding acquisition, Z.-P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Special Project on Key Research and Development Tasks of Xinjiang Uygur Autonomous Region, grant number No. 2022B02019, and Support Program for Innovative Development of Nanjiang Key Industries, grant number No. 2022DB026.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Strain zp-37T has been preserved in the China General Microbiological Culture Collection Center (CGMCC) and Japan Collection of Microorganisms (JCM) under the depository number of CGMCC NO. 1.62213 and JCM NO. 37305, respectively. The GenBank accession number for the 16S rRNA gene sequence of strain zp-37T is OQ996844. The GenBank accession number for the whole genome sequences is CP137552.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
mLBModified Luria-Bertani
ANIAverage nucleotide identity
dDDHDigital DNA-DNA hybridization
PGPhosphatidylglycerol
DPGDiphosphatidylglycerol
PEPhosphatidylethanolamine
UPL 1–3Unidentified phospholipids
UAPL 1–2Unidentified aminophospholipids
ULUnidentified lipids
ddH2ODouble-distilled water
CGMCCChina General Microbiological Culture Collection Center
JCMJapan Collection of Microorganisms
SEMScanning electron microscope
TEMTransmission electronic microscope
IAAIndole acetic acid
CASChrome azurol S
CMCCarboxy methyl cellulose
TLCThin layer chromatography
HPLCHigh-performance liquid chromatography
NCBINational Center for Biotechnology Information
PacBioPacific biosciences sequel IIe
KEGGKyoto encyclopedia of genes and genomes
COGClusters of orthologous groups of proteins
GOGene ontology
CAZyCarbohydrate-active enzymes database
BGCsBiosynthesis gene clusters
GGDCGenome-to-genome distance calculator
CDSsCoding sequences

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Figure 1. The ML phylogenetic tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of strain zp-37T and other closely related members of the genus Halomonas. Bootstrap support values were calculated from 1000 replicates (only values ≥ 70% were shown). The bold font represents the novel species identified in this study. GenBank accession numbers were provided in parentheses for reference. Carnimonas nigrificans CTCBS1T was used as the outgroup.
Figure 1. The ML phylogenetic tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of strain zp-37T and other closely related members of the genus Halomonas. Bootstrap support values were calculated from 1000 replicates (only values ≥ 70% were shown). The bold font represents the novel species identified in this study. GenBank accession numbers were provided in parentheses for reference. Carnimonas nigrificans CTCBS1T was used as the outgroup.
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Figure 2. Morphological characteristics of strain zp-37T. (A,B): Analysis under the FEI Q45 scanning electron microscope. (C,D): Analysis under the Hitachi ht7800 transmission electron microscope.
Figure 2. Morphological characteristics of strain zp-37T. (A,B): Analysis under the FEI Q45 scanning electron microscope. (C,D): Analysis under the Hitachi ht7800 transmission electron microscope.
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Figure 3. Plant growth-promoting trait of strain zp-37T (A). Nitrogen fixation (B). Phosphate solubilization (C). Siderophore production (D). Cellulolytic activity.
Figure 3. Plant growth-promoting trait of strain zp-37T (A). Nitrogen fixation (B). Phosphate solubilization (C). Siderophore production (D). Cellulolytic activity.
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Figure 4. Genome map of strain zp-37T. Each of the rings represents the following features labeled from outside to inside: ring 1, scale marks; rings 2 and 3, the coding sequences (CDSs) on the positive and negative chains, respectively (color-coded by the functional categories); ring 4, rRNA and tRNA; ring 5, G+C content (above average: red; below average: blue); and ring 6, G+C skew (positive: orange; negative: green).
Figure 4. Genome map of strain zp-37T. Each of the rings represents the following features labeled from outside to inside: ring 1, scale marks; rings 2 and 3, the coding sequences (CDSs) on the positive and negative chains, respectively (color-coded by the functional categories); ring 4, rRNA and tRNA; ring 5, G+C content (above average: red; below average: blue); and ring 6, G+C skew (positive: orange; negative: green).
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Figure 5. Phylogenomic tree of the strain zp-37T constructed using the EasyCGTree pipeline. Support values were calculated by using the Shimodaira-Hasegawa test with FastTree and indicated in the middle of branches or near the nodes. Bar, 0.03 substitutions per amino acid position. The bold font represents the novel species identified in this study. GenBank accession numbers were provided in parentheses for reference. Carnimonas nigrificans ATCC BAA-78T was used as the outgroup.
Figure 5. Phylogenomic tree of the strain zp-37T constructed using the EasyCGTree pipeline. Support values were calculated by using the Shimodaira-Hasegawa test with FastTree and indicated in the middle of branches or near the nodes. Bar, 0.03 substitutions per amino acid position. The bold font represents the novel species identified in this study. GenBank accession numbers were provided in parentheses for reference. Carnimonas nigrificans ATCC BAA-78T was used as the outgroup.
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Figure 6. A Venn diagram of whole-genome orthologous genes in strain zp-37T and its five closely related strains. The numbers in the diagram indicate overlapped conserved genes or non-overlapped unique genes in each species. The numbers below the strain names identify the total number of protein-coding genes within each genome.
Figure 6. A Venn diagram of whole-genome orthologous genes in strain zp-37T and its five closely related strains. The numbers in the diagram indicate overlapped conserved genes or non-overlapped unique genes in each species. The numbers below the strain names identify the total number of protein-coding genes within each genome.
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Table 1. Characteristics that distinguish strain zp-37T from the related species of the genus Halomonas.
Table 1. Characteristics that distinguish strain zp-37T from the related species of the genus Halomonas.
Characteristics123456
Cell size (μm)0.3–0.5 × 2.5–3.0NDND1.0 × 2.0–3.0 c0.6–0.9 × 1.3–2.7 d1.5 × 2.0–3.0 c
Temperature for growth (°C)
Range10–434–50 a4–60 b−1–35 c4–40 d2–40 c
Optimum3737 a35 b20–35 c35 d30 c
NaCl concentration for growth (%, w/v)
Range0–203–25 a0–15 b0.5–24 c0.2–15 d0.5–22 c
Optimum127 a3 b2–3 c4 d4–7 c
pH for growth
Range6.0–11.05.0–11.0 a6.0–12.0 b5.0–10.0 c5.0–10.0 d5.0–12.0 c
Optimum7.0–8.07.0 a9.0 bND7.0 dND
Hydrolysis of:
Tween 40+NDNDNDNDND
Tween 80+ND− b− c− d− c
Acid production from:
L-arabinose++ a− b− c− d− c
D-galactose−ND− b+ c− d− c
D-xylose+− a+ b+ c+ d− c
D-glucose+− a+ b+ c− d+ c
D-sucrose−− a+ b− c− d− c
D-fructose−ND+ b− c− d− c
DNA G+C content (mol%)59.354.4 a53.8 b56.0 c57.4 d56.3 c
Strains 1: zp-37T; 2: H. maris QX-1T; 3: H. zhaodongensis NEAU-ST10-25T; 4: H. sulfidaeris ATCC BAA-803T; 5: H. songnenensis NEAU-ST10-39T; 6: H. hydrothermalis Slthf2T; a Data from Qiu et al., (2021) [43]; b Data from Jiang (2013) [44]; c Data from Kaye et al., (2004) [45]; d Data from Jiang et al., (2014) [46]; Other data from this study; Positive reaction (+), negative reaction (−), no data available (ND).
Table 2. The chemotaxonomic characteristics of strain zp-37T and its closest related stains.
Table 2. The chemotaxonomic characteristics of strain zp-37T and its closest related stains.
Characteristics1234
Fatty acid (>10% in content)C18:1ω7c and/or C18:1ω6c (55.67%), C16:1ω7c and/or C16:1ω6c (20.16%)C16:0 (25.5%), C17:0 cyclo (14.0%), C19:0 cyclo ω8c (18.7%), C18:1ω7c and/or C18:1ω6c (18.1%) aC18:1ω7c (62.3%), C16:0 (17.6%) bC18:1ω7c (47.2%), C16:1ω7c and/or C16:1ω6c (18.9%) and C16:0 (16.3%) c
Major polar lipidsDPG, PG, PE, UPL 1–3, UAPL 1–2, ULDPG, PG, PE, UPL, UAPL, UL aNDDPG, PE, PG, UPL, UL c
QuinoneQ-9Q-9 aQ-9 bQ-9 c
Strains 1: zp-37T; 2: H. maris QX-1T; 3: H. zhaodongensis NEAU-ST10-25T; 4: H. songnenensis NEAU-ST10-39T; a Data from Qiu et al., (2021) [43]; b Data from Jiang (2013) [44]; c Data Jiang et al., (2014) [46]; Other data from this study; No data available (ND).
Table 3. ANI and dDDH analysis of strain zp-37T and its closely related strains.
Table 3. ANI and dDDH analysis of strain zp-37T and its closely related strains.
Starin12345
ANI (%)74.0472.6474.0975.5973.30
dDDH (%)19.8019.7020.4020.2020.40
Strains 1: H. maris QX-1T; 2: H. zhaodongensis NEAU-ST10-25T; 3: H. sulfidaeris ATCC BAA-803T; 4: H. songnenensis NEAU-ST10-39T; 5: H. hydrothermalis Slthf2T.
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Liang, Z.-P.; Wang, Y.; Chen, X.-Y.; Wang, R.; Xu, Y.; Dai, J.-P.; Singh, D.; Zhang, X.-X. Halomonas kashgarensis sp. nov., a Novel Species Isolated from the Rhizosphere Soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China. Diversity 2025, 17, 98. https://doi.org/10.3390/d17020098

AMA Style

Liang Z-P, Wang Y, Chen X-Y, Wang R, Xu Y, Dai J-P, Singh D, Zhang X-X. Halomonas kashgarensis sp. nov., a Novel Species Isolated from the Rhizosphere Soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China. Diversity. 2025; 17(2):98. https://doi.org/10.3390/d17020098

Chicago/Turabian Style

Liang, Zhen-Pu, Yi Wang, Xiao-Yue Chen, Rui Wang, Yan Xu, Jin-Ping Dai, Deepali Singh, and Xiao-Xia Zhang. 2025. "Halomonas kashgarensis sp. nov., a Novel Species Isolated from the Rhizosphere Soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China" Diversity 17, no. 2: 98. https://doi.org/10.3390/d17020098

APA Style

Liang, Z.-P., Wang, Y., Chen, X.-Y., Wang, R., Xu, Y., Dai, J.-P., Singh, D., & Zhang, X.-X. (2025). Halomonas kashgarensis sp. nov., a Novel Species Isolated from the Rhizosphere Soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China. Diversity, 17(2), 98. https://doi.org/10.3390/d17020098

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