Pattaraporn Yukphan

Two endophytic bacterial strains, designated as Sx8-8ᵀ and SI8-4ᵀ, were isolated from the stem and leaf tissues, respectively, of Kaempferia marginata Carey collected in Thailand. A comprehensive taxonomic investigation employing a polyphasic approach was conducted to characterize these strains. Strain Sx8-8ᵀ is a Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium that produces yellow pigment on Reasoner’s 2A agar (R2A) medium. Phylogenetic analysis based on 16S rRNA gene sequences placed this strain within the genus Sphingobium , showing the highest similarity (98.5%) to Sphingobium chungbukense KACC 12347ᵀ (=DJ77 ^T ). The draft genome was 4.7 Mb in size with a G+C content of 64 mol%. Genome-wide comparisons with closely related type strains yielded an average nucleotide identity based on blastn (ANIb) and MUMmer (ANIm) values of 82.2–84.0% and 86.7–86.9%, respectively, with digital DNA–DNA hybridization (dDDH) values of 28.4–29.1%. Chemotaxonomic data supported its affiliation to the genus, with Q-10 identified as the major respiratory quinone, along with characteristic fatty acid and polar lipid profiles. These findings support the proposal of a novel species, Sphingobium trunci sp. nov., with Sx8-8ᵀ (=LMG 33805ᵀ=TBRC 17746ᵀ) as the type strain. Strain SI8-4ᵀ is a Gram-positive, aerobic, spore-forming, rod-shaped bacterium, producing pale pink pigment on R2A medium. It was identified as a member of the genus Peribacillus , sharing the highest 16S rRNA gene sequence similarity (99.0%) with Peribacillus frigoritolerans JCM 11681ᵀ and Peribacillus muralis DSM 16288ᵀ. The genome was 4.6 Mb in size, with a G+C content of 43 mol%. Comparative genomic analyses revealed ANIb and ANIm values of 80.0–86.0% and 84.3–87.8%, respectively, while dDDH values were 24.3–32.1%. The major quinone was MK-7, and the fatty acid and polar lipid compositions were consistent with members of the genus Peribacillus . Based on genotypic and phenotypic distinctiveness, strain SI8-4ᵀ is proposed to represent a novel species, Peribacillus folii sp. nov. The type strain is SI8-4ᵀ (=KACC 23903ᵀ=KCTC 43756ᵀ=TBRC 17747ᵀ)

Two Gram-stain-negative, aerobic, non-motile, non-gliding, rod-shaped bacterial strains, designated as TBRC 19031^T and TBRC 19032, were isolated from water samples collected from the Mekong River, Thailand. Strain TBRC 19031^T was obtained from Chiang Saen in the upstream section near the borders with China and Myanmar, while TBRC 19032 originated from Khong Chiam, in the downstream section where the river exits Thailand. Colonies of both strains were circular, smooth and deep yellow on Reasoner’s 2A agar and did not produce flexirubin-type pigments. Phylogenetic analysis with 16S rRNA gene sequences placed both strains within the genus Flavobacterium, showing the highest sequence similarity to Flavobacterium cheonhonense ARSA-15^T (98.29% for TBRC 19031^T and 98.22% for TBRC 19032). However, whole-genome comparisons between the strains and F. cheonhonense ARSA-15^T revealed average nt identity (89.39% and 89.29%), average aa identity (92.84% and 92.95%) and digital DNA–DNA hybridization (35.00% and 34.70%). The predominant fatty acids were iso-C_15:1, iso-C_15:0 and iso-C_15:0 3-OH, and menaquinone MK-6 was the major respiratory quinone. The major polar lipids of both strains included phosphatidylethanolamine, steryl ester and diacylglycerol. The genome sizes were 3.02 and 3.04 Mbp, with G+C contents of 38.3% and 38.2% for TBRC 19031^T and TBRC 19032, respectively. Comparative genomic analyses revealed the absence of genes involved in sulphate reduction and denitrification pathways and the presence of a gene encoding phosphatidylinositol synthase, distinguishing them from other Flavobacterium within the clade. Ecological profiling using public metagenomic datasets showed that both strains were associated with lotic freshwater environments. This study not only introduces Flavobacterium mekongense sp. nov. as a new species but also provides broader insights into the ecology, metabolism and environmental distribution of freshwater Flavobacterium. The genomic features identified here offer promising leads for future studies in microbial ecology, comparative genomics and functional gene mining in aquatic ecosystems. The type strain is TBRC 19031^T (TBRC 19031^T=NBRC 117006^T).

Two novel Gram-negative, aerobic, rod-shaped, non-motile bacteria, strains TBRC 10068T and TBRC 16381T, were isolated from a fluid sample from a close-pitcher cup (Nepenthes gracilis) and an insect sample (Junonia lemonias), respectively. Comparing the 16S rRNA gene sequences with those found in EzBioCloud’s publicly available databases revealed that the two strains exhibited a close genetic relationship with Commensalibacter intestini A911T; the calculated sequence similarities were 98.56 and 97.70 %, respectively. The average nucleotide identity and digital DNA–DNA hybridization values of the two Commensalibacter strains, as well as those of their closely related type strains, were found to be lower than the species demarcation threshold of 95 and 70 %, respectively. The phylogenomic analysis of strains TBRC 10068T and TBRC 16381T showed that they belong to the genus Commensalibacter. However, they formed distinct lineages separate from all other strains of Commensalibacter by use of 81 bacterial core genes. In addition, the comparative genomic analysis revealed that the core orthologues of strains TBRC 10068T and TBRC 16381T, compared to the closely related type strains of Commensalibacter species, had distinct genetic profiles. Strain TBRC 10068T contained 163 unique genes, whereas strain TBRC 16381T contained 83. The three Commensalibacter species possessed Q-9 as the primary isoprenoid quinone homologue. The results of a polyphasic taxonomic investigation indicated that strains TBRC 10068T and TBRC 16381T represent two separate new species within the genus Commensalibacter. The species were designated as Commensalibacter nepenthis sp. nov. with the type strain TBRC 10068T (=KCTC 92798T) and Commensalibacter oyaizuii sp. nov. with the type strain TBRC 16381T (=KCTC 92799T).

Four newly discovered Gram-stain-negative bacteria, designated as BL00010^T, BL00058, D8-11^T and BL00200, were isolated from water samples collected at three hydrological monitoring stations (namely Chiang Saen, Chiang Khan and Nong Khai) located along the Mekong River in Thailand. An investigation encompassing phenotypic, chemotaxonomic and genomic traits was conducted. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that all four isolates represented members of the genus Rhodoferax. These isolates were closely related to Rhodoferax bucti KCTC 62564^T with a similarity of 99.59%. The major fatty acids of the four novel isolates included C_16:0 and C_16:1ω7c and/or C_16 : 1ω6c, whereas the major respiratory quinone was identified as ubiquinone Q-8. In addition, phosphatidylethanolamine was identified as a major polar lipid in these bacteria. The genomes of BL00010^T, BL00058, D8-11^T and BL00200 were similar in size (3.88–4.01 Mbp) and DNA G+C contents (59.5, 59.3, 59.5 and 59.3 mol%, respectively). In contrast to R. bucti KCTC 62564^T and Rhodoferax aquaticus KCTC 32394^T, the newly discovered species possessed several genes involved in nitrite and nitrile metabolism, which may be related to their unique adaptation to nitrile-rich environments. From the results of the pairwise analysis of average nucleotide identity of the whole genome and digital DNA–DNA hybridisation, it was evident that BL00010^T and D8-11^T represented two novel species, for which we propose the nomenclature Rhodoferax potami sp. nov., with the type strain BL00010^T (TBRC 17198^T = NBRC 116413^T), and Rhodoferax mekongensis sp. nov., with the type strain D8-11^T (TBRC 17307^T = NBRC 116415^T).

A novel Gram-stain-negative, obligately aerobic, short rod-shaped and motile bacterium, designated strain BC00092^T, was isolated from brackish ground water collected in Stegodon Sea Cave located at Satun UNESCO Global Geopark, Satun Province, Thailand. The phylogenetic analysis of BC00092^T based on 16S rRNA gene sequences revealed that the strain represented a member of the genus Leeia and was closely related to Leeia oryzae DSM 17879^T (96.68 %) and Leeia aquatica IMCC25680^T (94.89 %). The average nucleotide identity and digital DNA–DNA hybridization values calculated from the whole-genome sequences between BC00092^T and closely related type strains of species within the family Leeiaceae were lower than the species demarcation threshold values of 95 and 70 %, respectively. Moreover, five conserved signature indels of members of the family Leeiaceae were found in the protein sequences from the annotated assembled genome of BC00092^T. According to the results of the polyphasic taxonomic study, strain BC00092^T represents a novel species within the genus Leeia , for which the name Leeia speluncae sp. nov. is proposed. The type strain is BC00092^T (TBRC 13508^T = KCTC 92111^T).

Culture-independent and culture-dependent approaches were employed to investigate the taxonomic diversity and biosynthetic gene cluster potential of Actinomycetota in the Mekong River. Through 16S rRNA gene metabarcoding, 21,103 OTUs were revealed to represent 190 genera and at least 595 species of Actinomycetota, including putatively novel taxa. Conventional and in situ cultivation (IC) methods provided 75 Actinomycetota isolates representing 72 species from 21 genera. Of these, 45 species in 4 genera were new to the Thailand Bioresource Research Center (TBRC), a collection of 20,079 Actinomycetota strains from 660 species. Applying both culture-independent and culture-dependent approaches to the same sample revealed greater diversity among the Actinomycetota in the Mekong River than one approach alone.

A novel Gram-stain-negative, rod-shaped, non-motile, aerobic bacterium isolated from a sea bean flower [Canavalia rosea (Sw.) DC.] collected in Surat Thani Province, Thailand, and designated as AH18^T was characterized on the basis of polyphasic taxonomy. The phylogenetic analysis of 16S rRNA gene revealed that strain AH18^T represented a member of the genus Neokomagataea . In the 16S rRNA gene sequence analysis, the strain's closest phylogenetic neighbour was Neokomagataea thailandica TBRC 376^T. The draft genome size of strain AH18^T was 2613495 bp, and its DNA G+C content was 52.0 mol%. The strain showed 90.3 and 76.3% pairwise-determined whole-genome average nucleotide identity and 39.8 and 19.6% digital DNA–DNA hybridization values with N. thailandica TBRC 376^T and N. tanensis TBRC 7768^T, respectively. The 16S rRNA gene sequences and phylogenomic analysis revealed that the strain clustered with the members of the genus Neokomagataea but was located in a distinct branch closely related to N. thailandica TBRC 376^T. The predominant cellular fatty acids of the strain were summed feature 8 (C_18:1 ω6c and/or C_18:1 ω7c), C_16:0 and C_18:1 2OH (>5%). The major respiratory ubiquinone was Q-10. In addition, strain AH18^T was substantiated by differences in several physiological characteristics and by MALDI-TOF profiling. On the basis of the results obtained from phenotypic, chemotaxonomic, phylogenetic and genomic analyses, the strain clearly represented a novel species within the genus Neokomagataea , for which the name Neokomagataea anthophila sp. nov. (AH18^T=TBRC 2177^T=NBRC 115156^T) is proposed. An emended description of the genus Neokomagataea is also given.

Two isolates, MS16-SU-2T and MS18-SU-3, obtained from fermented mangosteen peel in vinegar were suggested to constitute a new species assignable to the genus Acetobacter based on the results of 16S rRNA gene sequencing. The two isolates showed the highest sequence similarity (98.58%) to Acetobacter tropicalis NBRC 16470T and Acetobacter senegalensis LMG 23690T. However, the calculated similarity values were lower than the threshold for species demarcation. The phylogenetic analysis showed that the branches of the two isolates were separated from other Acetobacter species, and the two isolates constituted a new species in the genus Acetobacter . The genomic DNA of isolate MS16-SU-2T was sequenced. The assembled genome of the isolate was analysed, and the results showed that the highest average nucleotide identity value of 75.9 % was with Acetobacter papayae JCM 25143T and the highest digital DNA–DNA hybridization value of 25.1 % was with Acetobacter fallax LMG 1636T, which were lower than the cutoff values for species delineation. The phylogenetic tree based on the genome sequences showed that the lineage of isolate MS16-SU-2T was most closely related to A. papayae JCM 25143T and Acetobacter suratthaniensis TBRC 1719T, but separated from the branches of these two species. In addition, the two isolates could be distinguished from the type strains of closely related species by their phenotypic characteristics and MALDI-TOF profiles. Therefore, the two isolates, MS16-SU-2T (=TBRC 12339T=LMG 32243T) and MS18-SU-3 (=TBRC 12305), can be assigned to an independent species within the genus Acetobacter , and the name of Acetobacter garciniae sp. nov. is proposed for the two isolates.

Two novel Gram-stain-negative, rod-shaped and non-motile bacterial strains, designated B5-SW-15^T and C2-DW-16, were isolated from water collected in mangrove forests in Ranong Province, Thailand. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains B5-SW-15^T and C2-DW-16 belonged to the genus Donghicola and were most closely related to Donghicola tyrosinivorans DSM 100212^T (98.2 and 98.1 %, respectively) and Donghicola eburneus DSM 29127^T (97.7 and 97.6 %, respectively). The average nucleotide identity and digital DNA–DNA hybridization values between strain B5-SW-15^T, strain C2-DW-16 and related species were 95.8 and 71.6 % (to strain C2-DW-16), 76.8 and 21.3 % (to D. tyrosinivorans DSM 100212^T) and 80.3 and 24.2 % (to D. eburneus DSM 29127^T), respectively. The predominant cellular fatty acids (>5 %) were summed feature 8 (C_18 : 1  ω6c and/or C_18 : 1  ω7c), C_16 : 0 and C_12 : 1 3-OH. Ubiquinone Q-10 was the sole respiratory quinone. DNA G+C contents of the isolates were 61.0 and 61.2 mol% based on whole genome sequences. Strains B5-SW-15^T and C2-DW-16 contained aminolipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. On the basis of the results from phenotypic, chemotaxonomic and phylogenetic analyses, strains B5-SW-15^T and C2-DW-16 constitute a novel species of the genus Donghicola in the family Rhodobacteraceae for which the name Donghicola mangrovi sp. nov. is proposed. The type strain is B5-SW-15^T (=BCC 56522^T=TBRC 9562^T=KCTC 72743^T).