Characterization of Sinomonas gamaensis sp. nov., a Novel Soil Bacterium with Antifungal Activity against Exserohilum turcicum

A novel Gram staining positive, aerobic bacterium NEAU-HV1T that exhibits antifungal activity against Exserohilum turcicum was isolated from a soil collected from Gama, Hadjer lamis, Chad. It was grown at 10–45 °C (optimum 30 °C), pH 5–10 (optimum pH 8), and 0–4% (w/v) NaCl (optimum 1%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NEAU-HV1T was closely related to Sinomonas susongensis A31T (99.24% sequence similarity), Sinomonas humi MUSC 117T (98.76%), and Sinomonas albida LC13T (98.68%). The average nucleotide identity values between NEAU-HV1T and its most closely related species were 79.34−85.49%. The digital DNA–DNA hybridization values between NEAU-HV1T and S. susongensis A31T, S. albida LC13T, and S. humi MUSC 117T were 23.20, 23.50, and 22.80%, respectively, again indicating that they belonged to different taxa. The genomic DNA G+C content was 67.64 mol%. The whole cell sugars contained galactose, mannose, and rhamnose. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and four glycolipids. The respiratory quinone system comprised MK-9(H2), MK-10(H2), and MK-8(H2). The major cellular fatty acids (>5%) were anteiso-C15:0, anteiso-C17:0, C16:0, and iso-C15:0. Based on the polyphasic analysis, it is suggested that the strain NEAU-HV1T represents a novel species of the genus Sinomonas, for which the name Sinomonas gamaensis sp. nov. is proposed. The type strain is NEAU-HV1T (= DSM 104514T = CCTCC M 2017246T).


Introduction
The genus Sinomonas, a member of the family Micrococcaceae in the phylum Actinobacteria, was first proposed by Zhou et al. [1] with the description of Sinomonas flava and the reclassification of Arthrobacter atrocyaneus as Sinomonas atrocyaneus. At the time of writing, the genus Sinomonas comprised 10 species (http://www.bacterio.net/sinomonas.html) that were isolated from soil, volcanic rock, and the surface of weathered biotite: Sinomonas flava, Sinomonas atrocyaneus, Sinomonas echigonensis, Sinomonas albida, Sinomonas soli, Sinomonas notoginsengisoli, Sinomonas mesophila, Sinomonas susongensis, Sinomonas humi, and Sinomonas halotolerans [1][2][3][4][5][6][7]. The species of this genus are aerobic and rod-shaped, contain MK-9(H 2 ) as the predominant respiratory quinone, and have iso-C 15:0 , anteiso-C 15:0 , and anteiso-C 17:0 as the major fatty acids. Other typical features are the presence of galactose, mannose, and ribose as the major cell wall sugars; A3α as the peptidoglycan type; and diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol as the major phospholipids. Some members of the genus Sinomonas have been reported to be able to synthesize silver nanoparticles with antimicrobial activity [8], hydrolyze starch [9], biodesulfurize coal [10], and degrade sesamin [11]. Recently, S. atrocyaneus has exhibited plant growth promoting effects and antagonistic activity against a broad-spectrum of root and foliar pathogens, implying its potential use in sustainable agriculture [12]. In this study, a strain NEAU-HV1 T with inhibitory activity against phytopathogenic fungi Exserohilum turcicum was isolated from a soil sample. Based on the polyphasic analysis, this strain was classified as representative of a novel species in the genus Sinomonas.

Isolation of Bacterial Strain
Strain NEAU-HV1 T was isolated from a soil sample collected from a cotton field (13 • 0´N, 15 • 44´E), situated in Gama, Hadjer lamis Region, Chad, which is considered as the principal agricultural region of Chad. The soil sample was suspended in sterile water (2 mL) followed by an ultrasonic treatment (160 w) for 3 min. After the addition of distilled water (43 mL), the soil suspension was incubated at 28 • C and 250 rpm on a rotary shaker for 20 min. Then, 200 µL of the suspension was spread on a plate of humic acid-vitamin (HV) agar [13] supplemented with cycloheximide (50 mg L −1 ) to inhibit any fungal contaminants. After seven days of aerobic incubation at 28 • C, colonies were transferred and purified on nutrition agar (NA; BD Difco, Becton, Dickinson and Company, Sparks, MD, USA) and maintained as glycerol suspensions (20%, v/v) at −80 • C for long term preservation.

Morphological and Biochemical Characteristics of NEAU-HV1 T
Colony morphology of strain NEAU-HV1 T was observed after two days of incubation at 28 • C on NA. Cell morphology was examined by transmission electron microscopy (Hitachi H-7650, Hitachi Co., Tokyo, Japan). Motility was detected by the presence of turbidity throughout tubes containing semi-solid medium and was further confirmed by the hanging-drop method [14,15]. Gram staining was performed as described by Smibert and Krieg [16]. Growth at different temperatures (0-45 • C, steps of 1 • C below 5 • C, and then intervals of 5 • C) was determined in a tryptic soy broth (TSB; BD Difco, Becton, Dickinson and Company, Sparks, MD, USA) medium after incubation for seven days. Growth tests for pH ranging from 4.0-12.0 in 1.0 pH unit intervals and NaCl tolerance (0-10% (w/v) in 1% intervals) were determined in TSB medium at 28 • C for seven days on a rotary shaker. The buffer systems were: pH 4.0-5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0-8.0, 0.1 M KH 2 PO 4 /0.1 M NaOH; pH 9.0-10.0, 0.1 M NaHCO 3 /0.1 M Na 2 CO 3 ; and pH 11.0-12.0, 0.2 M KH 2 PO 4 /0.1 M NaOH [17]. Growth was determined by monitoring the turbidity at OD 600 by using a spectroscopic method (U-3900; Hitachi Co., Tokyo, Japan) and plate counting. The utilization of organic compounds as the sole carbon and energy source, decomposition of cellulose, hydrolysis of starch and aesculin, reduction of nitrate, peptonization of milk, liquefaction of gelatin, and production of H 2 S were analyzed as recommended by Ventosa et al. [18]. Anaerobic growth was tested on NA for up to two weeks at 28 • C in Bacteron anaerobic chambers (Sheldon Manufacturing, Carson, CA). The methyl red and Voges-Proskauer reactions, hydrolysis of Tweens 20, 40, and 80, production of esterase and urease were tested as described by Smibert and Krieg [16]. Metabolic properties and enzyme activities were tested using API 50CH, API 20NE, and API ZYM strips with API 50CHB suspension medium (bioMérieux, Marcy-I'Etoile, France) according to the manufacturer's protocols.

Phylogenetic Analysis of NEAU-HV1
For DNA extraction, strain NEAU-HV1 T was cultured in TSB to the early stationary phase (~24 hours) and harvested by centrifugation. The genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN Biotech, Co. Ltd., Beijing, China). The 16S rRNA gene was PCR-amplified with the universal primers 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1541R (5´-AAGGAGGTGATCCAGCC-3´) under conditions described previously [19]. The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL, Applied Biosystems Inc., Foster City, California, USA) and software provided by the manufacturer. The almost full-length 16S rRNA gene sequence was compared with the available 16S rRNA gene sequences of validly named species in the EzBioCloud server (https://www.ezbiocloud.net/) [20] to search the related similar species for tree reconstructions and calculate the pairwise 16S rRNA gene sequence similarity. Phylogenetic trees were reconstructed with the neighbor joining [21], maximum likelihood [22], and minimum evolution [23] methods using MEGA version 7.0 after multiple alignments of the sequences using the CLUSTAL W algorithm [24]. For the neighbor joining algorithm, Kimura's 2-parameter model and pairwise deletion were used to reconstruct the phylogenetic tree. The Tamura-Nei model together with Gamma distribution and Invariant sites (TN93+G+I) and Nearest-Neighbor-Interchange (NNI) heuristic method for the maximum likelihood algorithm, and close-neighbor-interchange (CNI) search for the minimum evolution algorithm were applied. Distances were calculated according to Kimura's two-parameter model [25]. The topology of the phylogenetic tree was evaluated by using bootstrap analysis with 1000 replicates [26]. All positions containing gaps and missing data were eliminated from the dataset and the root position of the trees was inferred by using Nocardia inohanensis DSM 44667 T (GenBank accession no. AB092560) as an outgroup. The genome sequences of strain NEAU-HV1 T and its most closely related strains S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T were determined by Novogene Bioinformatics Technology (Beijing, China) using a IlluminaHiSeq PE150 (Illumina) following the manufacturer's suggested protocols. The resulting reads were quality trimmed to the Q20 confidence level. The draft genome was assembled using SOAP de novo version 2.04 [27] with default parameters. The estimated dDDH values were calculated using formula two at the Genome-to-Genome Distance Calculation (GGDC) website (http://ggdc.dsmz.de/distcalc2.php) as described by Meier-Kolthoff et al. [28]. Average nucleotide identity (ANI) calculations were performed using the OrthoANIu tool (https://www.ezbiocloud.net/tools/ani) [29] and JSpecies software (http://jspecies.ribohost.com/jspeciesws/) [30]. For core-genome analysis, cores genes were extracted from the genomes of NEAU-HV1 T and its related strains using the USEARCH program with BPGA (Bacterial Pan Genome Analysis tool), with >50% sequence identity cut-off [31]. The concatenated amino acid sequences of core genes were aligned to generate the phylogenetic tree using the neighbor joining method. Genome mining for bioactive secondary metabolites was performed using "antibiotics and secondary metabolite analysis shell" (antiSMASH) version 4.0 [32].

Chemotaxonomic Analysis of NEAU-HV1 T
Biomass for chemical studies was prepared by growing the strains in TSB medium in shake flasks at 28 • C until the exponential phase of growth was reached. Cells were harvested by centrifugation, washed with distilled water, and freeze-dried. The cell-wall diamino acid was determined from whole-cell hydrolysates, as described by McKerrow et al. [33] and analyzed by a HPLC method using an Agilent TC-C 18 column (250 × 4.6 mm, i.d. 5 µm) (Agilent Technologies, Santa Clara, CA, USA). Procedures for identification of cell-wall amino acids and whole-cell sugars were performed as described by Tang et al. [34]. Polar lipids were extracted and examined by two-dimensional TLC and identified according to the method of Minnikin et al. [35]. Menaquinones were extracted from freeze-dried biomass and purified according to Collins [36]. Extracts were analyzed using an HPLC-UV method [37]. Fatty acid methyl esters were extracted and analyzed using the microbial identification system (MIDI) and the RTSBA6 database (Microbial Identification Inc., Newark, DE, USA) [38].

In Vitro Antifungal Activity Test
The antagonistic activity of NEAU-HV1 T against six phytopathogenic fungi (Fusarium oxysporum, Fusarium orthoceras, Sclerotinia sclerotiorum, Alternaria solani, Corynespora cassiicola, and Exserohilum turcicum) was evaluated using petri dish assays [39]. To further investigate the antifungal components produced by NEAU-HV1 T , this strain was cultured in GYM medium [40] and the inhibitory activity of the supernatant was tested. Briefly, strain NEAU-HV1 T was inoculated into GYM medium and incubated at 28 • C for seven days on a rotary shaker. The supernatant was obtained by centrifugation at 8000 rpm and 4 • C for 10 min and subsequently filtrated with a 0.2 µm membrane filter. The cell precipitate was extracted with an equal volume of methanol. Then, the antifungal activity was evaluated using the well diffusion method [41], and each well contained 150 µL of the supernatant or methanol extract. In order to test the effect of pH on the antifungal stability, pH values of the supernatant was adjusted to pH 3.0, 5.0, 7.0, and 11.0 by 1 mol/L hydrochloric acid or 1 mol/L NaOH, and the adjusted supernatant was kept at 4 • C for 24 h. Afterward, the pH values were readjusted to 7.0. To examine the effect of temperature on antifungal activity, the supernatant was placed in a water bath at 40, 60, 80, and 100 • C for 30 min, and then cooled to room temperature. A total of 200 µL of the treated supernatant was added into 25 mL of sterile potato dextrose agar (PDA), and the mixture was poured into 9 cm plates. The PDA containing 200 µL of sterilized water was used as the control. Then, 5 mm plugs of E. turcicum were placed at the center of each plate, and all plates were placed at 28 • C for seven days. The antifungal activity was evaluated when the fungal mycelium reached the edges of the control plates.

Polyphasic Taxonomic Characterization of NEAU-HV1 T
Strain NEAU-HV1 T was aerobic, Gram staining positive, and non-motile. Light microscopy at 1000× magnification showed that the cells formed bent rods after cultivation for 12 h and then fragmented into a coccoid shape at 24 h, and the changed shape was also observed by transmission electron microscopy ( Figure 1). This metamorphic feature of rod-coccus growth cycle was also observed for other species of Sinomonas such as S. susongensis A31 T , S. halotolerans CFH S0499 T , S. mesophila MPKL 26 T , and S. echigonensis CW59 T [2,[4][5][6]. Neither flagella nor spore were found. Colonies on NA were circular, convex, and pale yellow, with a smooth surface and diameter of 0.5-1.5 mm after incubation for 48 h at 28 • C. Strain NEAU-HV1 T was able to grow at 10-45 • C (optimum, 30 • C), at pH 5-10 (optimum, pH 8), and with 0-4% (w/v) NaCl (optimum, 1%). Phenotypic properties useful for distinguishing NEAU-HV1 T from its three phylogenetic neighbors are shown in Table 1 and Table S1. The detailed characteristics of strain NEAU-HV1 T are given in the species description.    BLAST sequence analysis of the 16S rRNA gene sequence (1518 bp; GenBank/EMBL/DDBJ accession number MF418645) indicated that strain NEAU-HV1 T was related to members of the genus Sinomonas. The EzBioCloud analysis showed that strain NEAU-HV1 T was most closely related to S. susongensis A31 T , S. humi MUSC 117 T , and S. albida LC13 T with a gene sequence similarity of 99.24, 98.76, and 98.68%, respectively. In the neighbor joining tree (Figure 2), strain NEAU-HV1 T clustered with members of the genus Sinomonas and formed an independent subclade with the most closely related strains of S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T . The topology of the neighbor joining tree was further confirmed using the maximum likelihood and minimum evolution methods ( Figures S1 and S2). De novo assembly of the genome sequencing data of strain NEAU-HV1 T resulted in 39 contigs, an N50 of 233,829 bp, and an average genome coverage of 441.0× ( Table 2). The draft genome size of strain NEAU-HV1 T was 4,320,429 bp and its DNA G+C content was 67.64 mol% for strain NEAU-HV1 T , which is within the range (66.6-71.8 mol%) previously obtained for species of the genus Sinomonas [5]. A phylogenetic tree (Figure 3) based on the conserved core genome showed that NEAU-HV1 T was most related to S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T , supporting the phylogenetic analysis based on 16S rRNA gene sequences ( Figure 2). The dDDH values ( Table 2) between strain NEAU-HV1 T (NCBI accession number NZ_SSCL00000000) and S. susongensis A31 T (NZ_SSNH00000000), S. albida LC13 T (NZ_SWDW00000000), and S. humi MUSC 117 T (NZ_JTDL00000000) were 23.20, 23.50, and 22.80%, respectively, which were below the 70% DDH species boundary as recommended by Meier-Kolthoff et al. [28]. The values of OrthoANIu, ANI-MUMmer (ANIm), and ANI-Blast (ANIb) of strain NEAU-HV1 T with S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T (Table 3) were below the 94-96% cut-off value previously proposed for species delimitation [42], indicating that strain NEAU-HV1 T represents a putative novel species of the genus Sinomonas.  All data were obtained from this study. +, positive; -, negative. All strains were positive for the utilization of glucose, sucrose, ribose, maltose, mannose, sorbose, L-aspartic acid, L-proline, L-glutamine, L-serine, L-asparagine, L-glutamic acid, decomposition of cellulose, and the hydrolysis of aesculin. All strains were negative for H 2 S production, liquefaction of gelatin, hydrolysis of Tweens 20, 40, and 80, and the methyl red test.
Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 12 All data were obtained from this study. +, positive; -, negative. All strains were positive for the utilization of glucose, sucrose, ribose, maltose, mannose, sorbose, L-aspartic acid, L-proline, Lglutamine, L-serine, L-asparagine, L-glutamic acid, decomposition of cellulose, and the hydrolysis of aesculin. All strains were negative for H2S production, liquefaction of gelatin, hydrolysis of Tweens 20, 40, and 80, and the methyl red test.
BLAST sequence analysis of the 16S rRNA gene sequence (1518 bp; GenBank/EMBL/DDBJ accession number MF418645) indicated that strain NEAU-HV1 T was related to members of the genus Sinomonas. The EzBioCloud analysis showed that strain NEAU-HV1 T was most closely related to S. susongensis A31 T , S. humi MUSC 117 T , and S. albida LC13 T with a gene sequence similarity of 99.24, 98.76, and 98.68%, respectively. In the neighbor joining tree (Figure 2), strain NEAU-HV1 T clustered with members of the genus Sinomonas and formed an independent subclade with the most closely related strains of S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T . The topology of the neighbor joining tree was further confirmed using the maximum likelihood and minimum evolution methods (Figures S1 and S2). De novo assembly of the genome sequencing data of strain NEAU-HV1 T resulted in 39 contigs, an N50 of 233,829 bp, and an average genome coverage of 441.0× ( Table  2). The draft genome size of strain NEAU-HV1 T was 4,320,429 bp and its DNA G+C content was 67.64 mol% for strain NEAU-HV1 T , which is within the range (66.6-71.8 mol%) previously obtained for species of the genus Sinomonas [5]. A phylogenetic tree (Figure 3) based on the conserved core genome showed that NEAU-HV1 T was most related to S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T , supporting the phylogenetic analysis based on 16S rRNA gene sequences (Figure 2). The dDDH values ( Table 2) between strain NEAU-HV1 T (NCBI accession number NZ_SSCL00000000) and S. susongensis A31 T (NZ_SSNH00000000), S. albida LC13 T (NZ_SWDW00000000), and S. humi MUSC 117 T (NZ_JTDL00000000) were 23.20, 23.50, and 22.80%, respectively, which were below the 70% DDH species boundary as recommended by Meier-Kolthoff et al. [28]. The values of OrthoANIu, ANI-MUMmer (ANIm), and ANI-Blast (ANIb) of strain NEAU-HV1 T with S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T (Table 3) were below the 94-96% cut-off value previously proposed for species delimitation [42], indicating that strain NEAU-HV1 T represents a putative novel species of the genus Sinomonas.      The whole cell sugars of strain NEAU-HV1 T were galactose, mannose, and rhamnose, which were consistent with type strains of Sinomonas [6]. The major cellular fatty acids (>5% of the total fatty acids) of strain NEAU-HV1 T were anteiso-C15:0 (44.6%), anteiso-C17:0 (27.0%), C16:0 (20.5%), and iso-C15:0 (7.9%). The fatty acid profile of strain NEAU-HV1 T was similar to those of closely related strains S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T , which support the placement of strain NEAU-HV1 T within the genus Sinomonas. However, some quantitative differences clearly distinguished strain NEAU-HV1 T from the other reference strains (Table 4). For example, C16:0 (20.5%) was detected as a major component in strain NEAU-HV1 T , while it was a minor component (<5%) in the related strains. Additionally, iso-C16:0 was not detected in strain NEAU-HV1 T , but the three reference strains had this component at 3.8, 6.8, and 11.4%, respectively. The polar lipids of strain NEAU-HV1 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and four glycolipids ( Figure S3). This pattern was similar to the three related strains, which contained diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol as the major components [2,5,7]. The respiratory quinone system of strain NEAU-HV1 T consisted of MK-9(H2), MK-10(H2), and MK-8(H2) (approx. 89, 10, and 1%, respectively). The menaquinone composition was in agreement with the previous descriptions that MK-9(H2) was the predominant menaquinone of members of Sinomonas [2,5,7]. A comparison of the menaquinones in strain NEAU-HV1 T and three other related  The whole cell sugars of strain NEAU-HV1 T were galactose, mannose, and rhamnose, which were consistent with type strains of Sinomonas [6]. The major cellular fatty acids (>5% of the total fatty acids) of strain NEAU-HV1 T were anteiso-C 15:0 (44.6%), anteiso-C 17:0 (27.0%), C 16:0 (20.5%), and iso-C 15:0 (7.9%). The fatty acid profile of strain NEAU-HV1 T was similar to those of closely related strains S. susongensis A31 T , S. albida LC13 T , and S. humi MUSC 117 T , which support the placement of strain NEAU-HV1 T within the genus Sinomonas. However, some quantitative differences clearly distinguished strain NEAU-HV1 T from the other reference strains (Table 4). For example, C 16:0 (20.5%) was detected as a major component in strain NEAU-HV1 T , while it was a minor component (<5%) in the related strains. Additionally, iso-C 16:0 was not detected in strain NEAU-HV1 T , but the three reference strains had this component at 3.8, 6.8, and 11.4%, respectively. The polar lipids of strain NEAU-HV1 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and four glycolipids ( Figure  S3). This pattern was similar to the three related strains, which contained diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol as the major components [2,5,7]. The respiratory quinone system of strain NEAU-HV1 T consisted of MK-9(H 2 ), MK-10(H 2 ), and MK-8(H 2 ) (approx. 89, 10, and 1%, respectively). The menaquinone composition was in agreement with the previous descriptions that MK-9(H 2 ) was the predominant menaquinone of members of Sinomonas [2,5,7]. A comparison of the menaquinones in strain NEAU-HV1 T and three other related strains demonstrated that the menaquinone composition of NEAU-HV1 T was very similar to that of S. susongensis A31 T [5], however, the minor component MK-8(H 2 ) in NEAU-HV1 T became the major component in S. albida LC13 T and no MK-8(H 2 ) was detected in S. humi MUSC 117 T [2,7]. The DNA G+C content of strain NEAU-HV1 T was 67.64 mol%, which was within the range (66.6-71.8 mol%) previously obtained for species of the genus Sinomonas [5]. Table 4.
Cellular fatty acid compositions (%) of strain NEAU-HV1 T and its closest phylogenetic neighbors. On the basis of morphological, physiological, chemotaxonomic, and phylogenetic results, strain NEAU-HV1 T is considered to represent a novel species within the genus Sinomonas, for which the name Sinomonas gamaensis sp. nov. is proposed.

Description of Sinomonas gamaensis sp. nov.
Sinomonas gamaensis sp. nov. (ga.ma.en'sis N.L. fem. adj. gamaensis of or belonging to Gama, the location of the soil sample from which the type strain was isolated).
The type strain, NEAU-HV1 T (= DSM 104514 T = CCTCC M 2017246 T ), was isolated from a soil sample collected from Gama, Hadjer lamis, Chad. The G+C content of the type strain is 67.64 mol%, calculated from the assembly for the draft genome sequence.

Antifungal Activity of NEAU-HV1 T against E. turcicum
Strain NEAU-HV1 T possessed antagonic activity against E. turcicum with an inhibitory ratio of 56% to the mycelia growth, however, no inhibitory effect on the growth of five other phytopathogenic fungi was observed (Figure 4a). Comparison of the antifungal activity of the supernatant with that of the cell pellet suggested that strain NEAU-HV1 T could produce proteins or lipopeptides as the antifungal substances, because only the supernatant could inhibit the growth of E. turcicum (Figure 4b).
The antifungal substances were sensitive to pH and temperature ( Figure 5), which further confirmed that they were proteins. The antiSMASH analysis led to the identification of seven gene clusters, however, they showed low similarity to the known gene clusters of bottromycin, type III polyketide, two nonribosomal peptides, terpene, bacteriocin, and microansamycin. Therefore, the relationships between the corresponding secondary metabolites produced by NEAU-HV1 T and the antifungal activity are still ambiguous. To date, only 10 species of Sinomonas have been described, and S. atrocyaneus exhibited antimicrobial activity against Acidovorax citrulli, which is the pathogen of bacterial fruit blotch [12]. To the best of our knowledge, this is the first report on the antifungal activity of Sinomonas against plant pathogenic fungi.
Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 12 Strain NEAU-HV1 T possessed antagonic activity against E. turcicum with an inhibitory ratio of 56% to the mycelia growth, however, no inhibitory effect on the growth of five other phytopathogenic fungi was observed (Figure 4a). Comparison of the antifungal activity of the supernatant with that of the cell pellet suggested that strain NEAU-HV1 T could produce proteins or lipopeptides as the antifungal substances, because only the supernatant could inhibit the growth of E. turcicum ( Figure  4b). The antifungal substances were sensitive to pH and temperature ( Figure 5), which further confirmed that they were proteins. The antiSMASH analysis led to the identification of seven gene clusters, however, they showed low similarity to the known gene clusters of bottromycin, type III polyketide, two nonribosomal peptides, terpene, bacteriocin, and microansamycin. Therefore, the relationships between the corresponding secondary metabolites produced by NEAU-HV1 T and the antifungal activity are still ambiguous. To date, only 10 species of Sinomonas have been described, and S. atrocyaneus exhibited antimicrobial activity against Acidovorax citrulli, which is the pathogen of bacterial fruit blotch [12]. To the best of our knowledge, this is the first report on the antifungal activity of Sinomonas against plant pathogenic fungi.

Conclusions
A novel strain NEAU-HV1 T that exhibits antifungal activity against E. turcicum was isolated from a soil sample. Morphological features and phylogenetic analysis based on 16S rRNA gene sequences and genomes suggested that strain NEAU-HV1 T belonged to the genus Sinomonas. Strain NEAU-HV1 T possessed antagonic activity against E. turcicum with an inhibitory ratio of 56% to the mycelia growth, however, no inhibitory effect on the growth of five other phytopathogenic fungi was observed (Figure 4a). Comparison of the antifungal activity of the supernatant with that of the cell pellet suggested that strain NEAU-HV1 T could produce proteins or lipopeptides as the antifungal substances, because only the supernatant could inhibit the growth of E. turcicum ( Figure  4b). The antifungal substances were sensitive to pH and temperature ( Figure 5), which further confirmed that they were proteins. The antiSMASH analysis led to the identification of seven gene clusters, however, they showed low similarity to the known gene clusters of bottromycin, type III polyketide, two nonribosomal peptides, terpene, bacteriocin, and microansamycin. Therefore, the relationships between the corresponding secondary metabolites produced by NEAU-HV1 T and the antifungal activity are still ambiguous. To date, only 10 species of Sinomonas have been described, and S. atrocyaneus exhibited antimicrobial activity against Acidovorax citrulli, which is the pathogen of bacterial fruit blotch [12]. To the best of our knowledge, this is the first report on the antifungal activity of Sinomonas against plant pathogenic fungi.

Conclusions
A novel strain NEAU-HV1 T that exhibits antifungal activity against E. turcicum was isolated from a soil sample. Morphological features and phylogenetic analysis based on 16S rRNA gene sequences and genomes suggested that strain NEAU-HV1 T belonged to the genus Sinomonas.

Conclusions
A novel strain NEAU-HV1 T that exhibits antifungal activity against E. turcicum was isolated from a soil sample. Morphological features and phylogenetic analysis based on 16S rRNA gene sequences and genomes suggested that strain NEAU-HV1 T belonged to the genus Sinomonas. Chemotaxonomic and biochemical characteristics together with DDH relatedness values and ANI values clearly indicated that strain NEAU-HV1 T could be differentiated from the closely related strains S. susongensis A31 T , S. humi MUSC 117 T , and S. albida LC13 T . Based on the polyphasic analysis, it is suggested that the strain NEAU-HV1 T represents a novel species of the genus Sinomonas, for which the name Sinomonas gamaensis sp. nov. is proposed. The type strain is NEAU-HV1 T (= DSM 104514 T = CCTCC M 2017246 T ).

Conflicts of Interest:
The authors declare no conflict of interest.