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Microvirga Amygdalina sp. nov., and Microvirga Alba sp. nov., Two New Species Isolated from Soil in South Korea


 Two novel Gram-stain-negative, aerobic, rod-shaped, circular, convex, light-pink and white-coloured bacterial strains BT291T and BT350T were isolated from soil collected in Uijeongbu city (37° 44′ 55″ N, 127° 2′ 20″ E) and Jeju island (33° 22′ 48″ N, 126° 31′ 48″ E), respectively, South Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains BT291T and BT350T belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alpha Proteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rRNA gene sequence similarity between the two strains BT291T and BT350T was 97.4 %. The two strains were found to have the same quinone system, with Q-10 as the major respiratory quinone. The major polar lipids of strains BT291T and BT350T were phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT291T were C18:1 ω7c (58.2 %) and cyclo-C19:0 ω8c (25.7 %). The major cellular fatty acids of strain BT350T were C18:1 ω7c (38.5 %) and cyclo-C19:0 ω8c (27.7 %). Based on the polyphasic analysis (phylogenetic, chemotaxonomic and biochemical), strains BT291T and BT350T can be suggested as two novel bacterial species within the genus Microvirga and the proposed names are Microvirga amygdalina and Microvirga alba, respectively. The type strain of Microvirga amygdalina is BT291T (= KCTC 72368T = NBRC 114845T) and the type strain of Microvirga alba is BT350T (= KCTC 72385T = NBRC 114848T).


Introduction
The genus Microvirga was rst described by Kanso and Patel (2003) allocated to the family Methylobacteriaceae order Rhizobiales. At the time of writing (January 2021), the genus comprises 23 published and not validly published species (http://www.bacterio.net/Microvirga.html). Microvirga species have been retrieved from various polar environments in the last years, e.g. from regoliths from Tibet hot spring sediments (Liu et al. 2020  ) and root nodule (Msaddak et al. 2020). In genus Microvirga, cells are Gram-strain-negative, C 18: 1 ω7c and cyclo-C 19: 0 ω8c was contained in the major fatty acids, and the genome size and DNA G + C content were 3.53-9.63 Mb and 61.1-65.1 %, respectively (Zhang et al. 2019).
In this study, both strains BT291 T and BT350 T were newly isolated from a soil sample Uijeongbu city (37°4 4′ 55″ N, 127° 2′ 20″ E) and Jeju island (33° 22′ 48″ N, 126° 31′ 48″ E), respectively, South Korea. In the present study, we conducted a phylogenetic analysis based on the 16S rRNA gene sequences and phenotypic, genotypic and chemotaxonomic characteristics to determine the taxonomic position of strains BT291 T and BT350 T . The results suggested that strains BT291 T (Felsenstein 1981) and maximum-parsimony algorithms (Fitch 1971). The stability of the tree topologies was evaluated by bootstrap analysis based on 1,000 resampling method (Felsenstein 1985). Evolutionary distances were calculated according to the Kimura two-parameter model (Kimura 1983).

Genome sequencing
The genomic DNA was extracted using a genomic DNA extraction kit according to the manufacturer's instruction (Solgent

Chemotaxonomic characteristics
For analysis of cellular fatty acid, polar lipid and quinone strains BT291 T and BT350 T were grown on R2A agar at 25°C for three days and cells were freeze-dried. Polar lipids of strains BT291 T and BT350 T were extracted as described previously. The total lipids, glycolipids, phosphatidylcholine and amino groups were separated using two-dimensional thin-layer chromatography (TLC). The polar lipid spots were detected by spraying the proper detection reagents (Komagata and Suzuki 1987;Minnikin et al. 1984).
The fatty acids were puri ed by saponi cation, methylation and extraction procedures (Sasser 1990). The quinones were extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) based on the previous methods (Hiraishi et al. 1996). The fatty acid methyl esters (FAME) were identi ed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc).

Results And Discussion
Morphology, Physiology and Biochemical analysis Strains BT291 T and BT350 T were Gram-staining-negative bacteria and they showed rod-shaped morphology ( Fig. S1). Colonies of strains BT291 T and BT350 T were circular, convex and smooth after incubation for three days at 25°C. Colonies color of strains BT291 T and BT350 T were light-pink and white, respectively. Cells of strain BT291 T could survive at 10 to 30°C (optimum 25°C) and pH 6.0-9.0 (optimum 8.0) in R2A medium and the cells of strain BT350 T could survive at 10 to 30°C (optimum 25°C) and pH 5.0-9.0 (optimum 8.0) in R2A medium. Differential features between the new strains and reference strains were provided in Table 1. In addition, the negative reaction of strains BT291 T and BT350 T on API kits were given as supplementary tables (Table S2 and S3).

Phylogenetic and Genome sequence analysis and analysis
Based on the 16S rRNA gene sequence similarities, strains BT291 T and BT350 T were a liated with the family Methylobacteriaceae and showed high sequence similarities with the genus Microvirga. The strain BT291 T was closely related to Microvirga aerophila 5420S-12 T (97.5 % 16S rRNA gene similarity) and Microvirga subterranean DSM 14364 T (97.2 %). The strain BT350 T was closely related to Microvirga aerophila 5420S-12 T (97.6 %) and Microvirga brassicacearum CDVBN77 T (96.8 %). The 16S rRNA gene sequence similarities of strains BT291 T and BT350 T with the closely related type strains were less than 97.5 % and 97.6 %, respectively and with other Microvirga species were less than 96.9 %. These values were around or below the 98.7 % 16S rRNA gene sequence similarity recently used as the threshold for differentiating among bacterial species (Chun et al. 2018). The remaining Microvirga species exhibited sequence similarities lower than 97.0 %. After the reconstruction of neighbor-joining, maximum-likelihood ( Fig. S2) and maximum-parsimony (Fig. S3) trees strain BT350 T clustered with M. brassicacearum CDVBN77 T and M. avescens c27j1 T and strain BT291 T clustered independently (Fig. 1)

Chemotaxonomic characterization
The fatty acid pro les of strains BT291 T and BT350 T and three reference strains of genus Microvirga were presented in Table 2. The major fatty acids of strain BT291 T C 18:1 ω7c (58.2 %) and cyclo-C 19:0 ω8c    Supplementary Files