Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms.

BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Microbacterium humicola sp. nov., Microbacterium terrisoli sp. nov., Paenibacillus pedocola sp. nov., Paenibacillus silviterrae sp. nov., Flavobacterium terrisoli sp. nov., and Aquabacterium humicola sp. nov., isolated from soil.

Four Gram-stain-positive and two Gram-stain-negative bacterial strains, designated as W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T, were isolated from soil samples collected from the Republic of Korea. The 16S rRNA gene sequence analysis showed that strains W4T and FW7T belonged to the genus Microbacterium, strains TW48T and UW52T were affiliated to the genus Paenibacillus, strain PT-3T was related to the genus Flavobacterium, and strain RJY3T was associated with the genus Aquabacterium. The closest phylogenetic taxa to W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T were Microbacterium bovistercoris NEAU-LLET (97.7 %), Microbacterium protaetiae DFW100M-13T (97.9 %), Paenibacillus auburnensis JJ-7T (99.6 %), Paenibacillus allorhizosphaerae JJ-447T (95.7 %), Flavobacterium buctense T7T (97.1 %), and Aquabacterium terrae S2T (99.5 %), respectively. Average nucleotide identity and digital DNA-DNA hybridization values between the novel strains and related reference type strains were <95.0 % and <70.0 %, respectively. The major cellular fatty acid in strains W4T, FW7T TW48T, and UW52T was antiso-C15 : 0. Similarly, strain PT-3T revealed iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, and iso-C15 : 0 3-OH as its principal fatty acids. On the other hand, RJY3T exhibited summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), and C12 : 0 as its predominant fatty acids. Overall, the polyphasic taxonomic data indicated that strains W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T represent novel species within the genera Microbacterium, Paenibacillus, Flavobacterium, and Aquabacterium. Accordingly, we propose the names Microbacterium humicola sp. nov., with the type strain W4T (=KCTC 49888T=NBRC 116001T), Microbacterium terrisoli sp. nov., with the type strain FW7T (=KCTC 49859T=NBRC 116000T), Paenibacillus pedocola sp. nov., with the type strain TW48T (=KCTC 43470T=NBRC 116017T), Paenibacillus silviterrae sp. nov., with the type strain UW52T (=KCTC 43477T=NBRC 116018T), Flavobacterium terrisoli sp. nov., with the type strain PT-3T (=KCTC 92106T=NBRC 116012T), and Aquabacterium humicola sp. nov., with the type strain RJY3T (=KCTC 92105T=NBRC 115831T).

Flavobacterium facile sp. nov., isolated from water system of Atlantic salmon (Salmo salar) fry cultured in Chile.

Strain T-12T, an orange, Gram-stain-negative, non-motile, rod-shaped strain, was isolated in November 2013 from water samples collected from an Atlantic salmon (Salmo salar) fry culturing system at a fish farm in Chile. Phylogenetic analysis based on 16S rRNA sequences (1394 bp) revealed that strain T-12T belonged to the genus Flavobacterium, showing close relationships to Flavobacterium bernardetii F-372T (99.48 %) and Flavobacterium terrigena DS-20T (98.50 %). The genome size of strain T-12T was 3.28 Mb, with a G+C content of 31.1 mol%. Genome comparisons aligned strain T-12T with Flavobacterium bernardetii F-372T (GCA_011305415) and Flavobacterium terrigena DSM 17934T (GCA_900108955). The highest digital DNA-DNA hybridization (dDDH) values were 42.6 % with F. bernardetii F-372T (GCA_011305415) and 33.9 % with F. terrigena DSM 17934T (GCA_900108955). Pairwise average nucleotide identity (ANI) calculations were below the species cutoff, with the best results with F. bernardetii F-372T being: ANIb, 90.33 %; ANIm, 91.85 %; and TETRA, 0.997 %. These dDDH and ANI results confirm that strain T-12T represents a new species. The major fatty acids were iso-C15 : 0 and C15 : 1ω6с. Detected polar lipids included phospholipids (n=2), aminophospholipid (n=1), aminolipid (n=1) and unidentified lipids (n=2). The predominant respiratory quinone was menaquinone MK7 (80 %) followed by MK-6 (20 %). Phenotypic, chemotaxonomic, and genomic data support the classification of strain T-12T (=CECT 30410T=RGM 3222T) as representing a novel species of Flavobacterium, for which the name Flavobacterium facile sp. nov. is proposed.

Flavobacterium acetivorans sp. nov. and Flavobacterium galactosidilyticum sp. nov., psychrotolerant flavobacteria isolated from cultivated fish.

Two yellow-coloured strains, F-29T and F-340T, were isolated from fish farms in Antalya and Mugla in 2015 and 2017 during surveillance studies. The 16S rRNA gene sequence analysis showed that both strains belong to the genus Flavobacterium. A polyphasic approach involving a comprehensive genome analysis was employed to ascertain the taxonomic provenance of the strains. The overall genome-relatedness indices of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) between the strains and the other members of the genus Flavobacterium were found to be well below the established thresholds of 70 and 95 %, respectively. The whole-genome-based phylogenetic analysis revealed that strain F-29T is closely related to Flavobacterium granuli (dDDH 39.3 % and ANI 89.4 %), while strain F-340T has a close relationship with the type strain of Flavobacterium pygoscelis (dDDH 25.6 % and ANI 81.5 %). Both strains were psychrotolerant with an optimum growth temperature of 25 °C. The chemotaxonomic characteristics of the strains were typical of the genus Flavobacterium. Both strains had phosphatidylethanolamine, aminolipids and unidentified lipids in their polar lipid profile and MK-6 as the isoprenoid quinone. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The genome size of the strains was 3.5 Mb, while G+C contents were 35.3 mol% for strain F-29T and 33.4 mol% for strain F-340T. Overall, the characterizations confirmed that both strains are representatives of two novel species within the genus Flavobacterium, for which the names Flavobacterium acetivorans sp. nov. and Flavobacterium galactosidilyticum sp. nov. are proposed, with F-29T (JCM 34193T=KCTC 82253T) and F-340T (JCM 34203T=KCTC 82263T) as the type strains, respectively.

Flavobacterium adhaerens sp. nov. and Flavobacterium maritimum sp. nov., two novel flavobacteria isolated from the Pearl River Estuary.

Two strains, designated as SYSU M80004T and SYSU M80005T, were isolated from water sampled in the Pearl River Estuary, Guangzhou, Guangdong, PR China. The strains were Gram-stain-negative and aerobic. Strain SYSU M80004T could grow at pH 6.0-8.0 (optimum, pH 7.0), 22-30 °C (optimum, 28 °C) and in the presence of 0-1 % NaCl (w/v; optimum 0 %). Strain SYSU M80005T could grow at pH 6.0-8.0 (optimum, pH 7.0), 4-37 °C (optimum, 28 °C) and in the presence of 0-1 % NaCl (w/v; optimum 0%). Both strains contained MK-6 as the predominant menaquinone. C16 : 0 and iso-C15 : 0 were identified as the major fatty acids (>10 %) of strain SYSU M80004T while strain SYSU M80005T contained iso-C15 : 0 and iso-C17 : 0 3-OH as major fatty acids. Phosphatidylethanolamine was present as the major polar lipid in both strains. The average nucleotide identity and digital DNA-DNA hybridization values between these two strains and their closest relatives were 73.5-79.3 % and 19.6-23.2 %, respectively. Phylogenetic analysis based on the 16S rRNA gene and genomic sequences indicated they belonged to the genus Flavobacterium. Therefore, on the basis of phenotypic, physiological, chemotaxonomic and genomic evidence, two novel species, Flavobacterium adhaerens sp. nov. (type strain=SYSU M80004T=CDMCC 1.4522T=KCTC 102268T) and Flavobacterium maritimum sp. nov. (type strain=SYSU M80005T=CGMCC 1.4523T= KCTC 102269T) are proposed.

Flavobacterium poyangense sp. nov., an ammonifying bacterium isolated from a freshwater lake.

A Gram-stain-negative, aerobic, non-spore-forming, nonmotile, rod-shaped, and yellow-pigmented bacterium, designated strain JXAS1T, was isolated from a freshwater sample collected from Poyang Lake in China. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the isolate belonged to the genus Flavobacterium, being closest to Flavobacterium pectinovorum DSM 6368T (98.61 %). The genome size of strain JXAS1T was 4.66 Mb with DNA G+C content 35.7 mol%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain JXAS1T and its closest relatives were below the threshold values of 95 and 70 %, respectively. The strain contained menaquinone 6 (MK-6) as the predominant menaquinone and the major polar lipids were phosphatidylethanolamine, one unidentified glycolipid, and one unidentified polar lipid. The major fatty acids (>5 %) were iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C15 : 0, iso-C17 : 0 3OH, iso-C15 : 0 3OH, and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). Based on phylogenetic, genotypic, and phenotypic evidence, the isolated strain represents a new species in the genus Flavobacterium, and the name Flavobacterium poyangense is proposed. The type strain is JXAS1T (=GDMCC 1.1378T=KCTC 62719T).

Flavobacterium capsici sp. nov., isolated from the rhizospheric soils of bell pepper (Capsicum annuum L.).

Two separate bacterial strains, PMTSA4T and PMR2A8, were isolated from the rhizospheric soils of bell pepper plants grown in a plant nursery. These strains are Gram-negative, non-motile and rod-shaped and grow in aerobic conditions. They exhibit a positive reaction for catalase activity but negative results for oxidase activity. Phylogenetic analysis of the 16S rRNA gene sequences revealed that the strains PMTSA4T and PMR2A8 are closely related to Flavobacterium piscinae ICH-30T (95.6%, respectively), Flavobacterium ahnfeltiae 10Alg 130T (95.5%) and Flavobacterium maris KMM 9535T (95.3%), aligning them within the genus Flavobacterium. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identities (ANIs) of the whole-genome sequences for the two strains and related Flavobacterium species were significantly below the established thresholds for prokaryotic species delineation (<70% for dDDH and <95% for ANI). The observed values were as follows: Flavobacterium aquatile LMG 4008T (dDDH: 19.8% and ANI: 75.5%), F. piscinae ICH-30T (dDDH: 18.6% and ANI: 73.3%) and F. stagni WWJ 16T (dDDH: 18.5% and ANI: 72.0%). The strains have genome sizes of 3 068 185 bp and 3 068 330 bp, with a G+C content of 32.5 mol%. In phenotypic characterization, the new strains grew at 10-35 °C and tolerated up to 4% NaCl at pH 5-9 (optimum pH 8). The predominant cellular fatty acids were observed to be iso-C15:0, iso-C17:0 3-OH and iso-C15:0 3-OH. Menaquinone-6 was the predominant quinone. Considering the results from phenotypic, chemotaxonomic, phylogenetic and genomic analyses, it is proposed that the strains PMTSA4T and PMR2A8 represent a novel species within the genus Flavobacterium.

Flavobacterium rivulicola sp. nov., Isolated from a Freshwater Stream.

A Gram-strain-negative, aerobic, yellow-colored, non-motile, and rod-shaped bacterial strain, designated IMCC34852T, was isolated from a freshwater stream in the Republic of Korea. Cellular growth occurred at 10-37 °C, pH 6.0-9.0, and with 0-0.5% (w/v) NaCl. The 16S rRNA gene sequence analysis showed that strain IMCC34852T belonged to the genus Flavobacterium and that the strain was most closely related to F. cheonhonense ARSA-15 T (97.6%), F. buctense T7T (96.7%), F. silvisoli RD-2-33 T (96.1%), and F. paronense KNUS1T (96.1%). The whole-genome sequence of strain IMCC34852T was 3.2 Mbp in size, with a DNA G + C content 37.3%. The average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) values between strain IMCC34852T and its related species were all below 79.8% and 22.7%, respectively, which are significantly lower than the thresholds of 95% for ANI and 70% for DDH for species delineation. The major respiratory quinone of strain IMCC34852T was menaquinone-6 (MK-6) and the predominant cellular fatty acids were iso-C15:0 (32.6%), iso-C16:0 (11.7%), iso-C15:1 G (10.3%), and iso-C14:0 (6.7%). The major polar lipids of the strain were phosphatidylethanolamine, two unidentified aminolipids and six unidentified lipids. Based on these results, it was concluded that strain IMCC34852T represents a novel species in the genus Flavobacterium, for which the name Flavobacterium rivulicola sp. nov is proposed. The type strain of the proposed novel species is IMCC34852T (= KACC 23133 T = KCTC 82066 T = NBRC 114419 T).

Flavobacterium coralii sp. nov., a marine bacterium isolated from coral culture seawater.

A Gram-stain-negative, non-motile, strictly aerobic, rod-shaped bacterium, with one polar flagellum and named D11R37T, was isolated from coral culture seawater of Acropora digitifera. Strain D11R37T grew with 0-6 % (w/v) NaCl (optimum, 0.5%), at 10-41 °C (optimum, 28 °C) and at pH 6.0-7.0 (optimum, 7.0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain D11R37T formed a lineage within the genus Flavobacterium, and it was distinct from the most closely related species Flavobacterium suzhouense XIN-1T and Flavobacterium suaedae G16-7T with 16S rRNA gene sequences similarities of 95.97% and 95.48 %. The major respiratory quinone was menaquinone-6. The polar lipids comprised one phosphatidylethanolamine, two aminolipids and one unknown polar lipid. The predominant fatty acids (more than 10 % of total fatty acids) were iso-C15 : 0 (18.0%), iso-C17 : 0 3-OH (11.9 %) and summed feature 3 (10.9 %). The DNA G+C content was 41.3 mol%. Based on polyphasic taxonomic data, strain D11R37T is considered to represent a novel species within the genus Flavobacterium, for which the name Flavobacterium coralii sp. nov. is proposed. The type strain is D11R37T (=KCTC 82968T=MCCC 1K06440T).

Flavobacterium endoglycinae sp. nov., an endophytic bacterium isolated from soybean (Glycine max L. cv. Gwangan) stems.

A Gram-stain-negative, facultatively anaerobic, motile by gliding, rod-shaped, oxidase- and catalase-positive bacterial strain, designated BB8T, was isolated from the stems of a Korean soybean cultivar (Glycine max L. cv. Gwangan). The strain produced a yellow pigment on tryptic soy agar. Growth of strain BB8T occurred at pH 5.0-8.0 (optimum, pH 7.0), at 10-35 °C (optimum, 25-30 °C) and in the presence of 0-1 % (w/v) NaCl (optimum, 0.5%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BB8T formed a lineage within the genus Flavobacterium and was most closely related to Flavobacterium artemisiae SYP-B1015T (96.9 % 16S rRNA gene sequence similarity) and Flavobacterium ustbae T13T (96.8%). The complete genome sequence of strain BB8T was 5 513 159 bp long with a G+C content of 34.1 mol%. The major fatty acids (>10 %) of strain BB8T were iso-C15 : 0 (21 %), summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c, 20.3%) and iso-C16 : 0 3-OH (13.7%). The predominant polar lipids were phosphatidylethanolamine and unidentified aminolipids, and the major respiratory quinone was menaquinone-6. Based on these phenotypic, genotypic and chemotaxonomic characteristics, strain BB8T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium endoglycinae sp. nov. is proposed. The type strain is BB8T (=KCTC 82167T=CCTCC AB 2020070T).

Flavobacterium agrisoli sp. nov., a novel bacterium isolated from soil.

A Gram-stain negative, rod shaped, motile by gliding, yellow-pigmented, aerobic bacterium, designated SE-1-eT, was isolated from a soil sample collected on Dokdo Island, South Korea. The isolate was characterized taxonomically using a polyphasic approach based on the phenotypic and genomic analyses. The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SE-1-eT belonged to the genus Flavobacterium in the family Flavobacteriaceae and had the highest sequence similarity with Flavobacterium cheongpyeongense IMCC34759T (97.5%), Flavobacterium arsenitoxidans S2-3HT (97.4%), Flavobacterium resistens BD-b365T (97.4%), and Flavobacterium chungangense CJ7T (97.4%). The predominant respiratory quinone of the isolate was found to be MK-6; the main polar lipid was phosphatidylethanolamine; and the major fatty acids were identified as summed feature 3 (C16:1 ω7c/C16:1 ω6c), C15:0 iso, and C16:0. The draft genome of strain SE-1-eT had a length of 3,715,609 bp and a DNA G + C content of 34.8 mol%. The nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the novel isolate and F. cheongpyeongense IMCC34759T, F. resistens BD-b365T, and Flavobacterium chungangense CJ7T ranged from 74.9 to 75.3% and from 20.2 to 20.9%, respectively. On the basis of its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain SE-1-eT represents a novel species in the genus Flavobacterium, for which the name Flavobacterium agrisoli sp. nov. is proposed. The type strain is SE-1-eT (= KCTC 82352 T = JCM 34302 T).

Flavobacterium difficile sp. nov., isolated from a freshwater waterfall.

A bacterial strain designated KDG-16 T is isolated from a freshwater waterfall in Taiwan and characterized to determine its taxonomic affiliation. Cells of strain KDG-16 T are Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and form light yellow colonies. Optimal growth occurs at 20-25 °C, pH 6-7, and with 0% NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set reveal that strain KDG-16 T is affiliated with species in the genus Flavobacterium. Analysis of 16S rRNA gene sequences shows that strain KDG-16 T shares the highest similarity with Flavobacterium terrigena DSM 17934 T (97.7%). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain KDG-16 T and the closely related Flavobacterium species are below the cut-off values of 95-96, 90 and 70%, respectively, used for species demarcation. Strain KDG-16 T contains iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH as the predominant fatty acids. The polar lipid profile consists of phosphatidylethanolamine, one uncharacterized aminophospholipid, one uncharacterized phospholipid, two uncharacterized aminolipids and two uncharacterized lipids. The major polyamine is homospermidine. The major isoprenoid quinone is MK-6. Genomic DNA G + C content of strain KDG-16 T is 31.6%. Based on the polyphasic taxonomic data obtained, strain KDG-16 T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium difficile sp. nov. is proposed. The type strain is KDG-16 T (= BCRC 81194 T = LMG 31332 T).

Flavobacterium inviolabile sp. nov. isolated from stream water.

A Gram-stain-negative, rod-shaped, aerobic and non-motile bacterium, designated P2-65T, was isolated from Moonsan stream water in the Republic of Korea. The temperature, NaCl concentration and pH ranges for growth of strain P2-65T were 10-37 °C, 0.0-3.0% (w/v) and 6.5-8.5 with optimum growth at 25-30 °C, 0.0-1.0% and 7.0-7.5, respectively. Comparison of 16S rRNA gene sequence showed that strain P2-65T was closely related to Flavobacterium cauense (95.4%) and Flavobacterium cheniae (95.3%). The major fatty acids were iso-C15:0, iso C17:0 3-OH, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0) and iso-C15:0 3-OH. The predominant respiratory quinone was menaquinone-6 (MK-6). The major polar lipids detected in the strain were phosphatidylethanolamine, one aminophospholipid, one unidentified aminolipid and one unidentified polar lipid. The G + C content of the genomic DNA was 39.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values for strain P2-65T with closely related Flavobacterium species were below 74.8% and 20%, respectively. Based on polyphasic features, strain P2-65T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium inviolabile sp. nov. is proposed. The type strain is P2-65T (= KCTC 62055T = NBRC 112953T).

Flavobacterium agri sp. nov., a novel bacterial species isolated from rhizospheric soil of Coriandrum sativum.

A Gram-stain-negative, strictly aerobic, cream yellow colored, non-motile, rod-shaped bacterial strain, designated MAH-1T was isolated from rhizospheric soil of Coriandrum sativum. A polyphasic taxonomic study was performed on the isolated strain. Optimal growth occurred at 28-30 °C, pH 6.5 and 0% NaCl. The strain showed activity for both catalase and oxidase tests. Cell growth occurs on R2A agar, nutrient agar and Luria-Bertani agar. Cells were able to hydrolyze starch, aesculin, gelatin, and Tween 20. Alignment of 16S rRNA gene sequences indicated that strain MAH-1T was associated with the genus Flavobacterium and was most closely related to Flavobacterium longum YIT 12745T (94.5% sequence similarity) and Flavobacterium caeni LM5T (93.0%). Strain MAH-1T had a genome size of 3,975,600 bp. Genome contained 67 contigs encoded by 3,522 protein-coding genes with 38 tRNA and 6 rRNA genes. The genomic DNA G + C contents of strain MAH-1T was 47.1 mol %. The genomic ANI and dDDH values between strain MAH-1T and one of the close relatives F. caeni LM5T were 72.2 and 18.8%, respectively. The major fatty acids were C15:0 iso, C16:0 iso and C15:0 anteiso. The predominant respiratory quinone was menaquinone 6 (MK-6). Based on physiological, biochemical and phylogenetic data for this isolate, it was confirmed that strain MAH-1T was affiliated to the genus Flavobacterium and represented a novel species, for which the name Flavobacterium agri sp. nov. is proposed. The type strain is MAH-1T (= KACC 19300T = CGMCC 1.16617T).

Flavobacterium undicola sp. nov., isolated from a freshwater lake.

Bacterial strain BBQ-18T, isolated from a freshwater lake in Taiwan, is characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicate that strain BBQ-18T forms a phylogenetic lineage in the genus Flavobacterium. Strain BBQ-18T is most closely related to Flavobacterium alvei HR-AYT with 98.5% 16S rRNA gene sequence similarity. Strain BBQ-18T shows 70.5-89.5% average nucleotide identity and 13.7-38.2% digital DNA-DNA hybridization identity with the type strains of other closely related Flavobacterium species. The strain is Gram-stain negative, strictly aerobic, motile by gliding, rod shaped and formed yellow colonies. Optimal growth occurs at 25 °C, pH 6, and in the absence of NaCl. Strain BBQ-18T contains iso-C15:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c) and anteiso-C15:0 as the predominant fatty acids. The polar lipid profile consists of phosphatidylethanolamine, four uncharacterized aminophospholipids and two uncharacterized phospholipids. The major polyamine is homospermidine. The major isoprenoid quinone is MK-6. The DNA G+C content of the genomic DNA is 33.8%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain BBQ-18T should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacterium undicola sp. nov. is proposed. The type strain is BBQ-18T (= BCRC 81050T = LMG 30052T = KCTC 52810T).

Flavobacterium muglaense sp. nov. isolated from internal organs of apparently healthy rainbow trout.

Two yellow-pigmented isolates, F-60T and F-392, were isolated from the internal organs of an apparently healthy rainbow trout (Oncorhynchus mykiss). The strains were identified as members of the genus Flavobacterium based on the results of 16S rRNA gene sequence analysis. Strains F-60T and F-392 had the highest 16S rRNA gene sequence identity level of 97.4 % to the type strain of Flavobacterium crassostreae LPB0076T. A polyphasic taxonomic approach including phenotypic, chemotaxonomic and genomic characterization was employed to ascertain the taxonomic position of the strains within the genus Flavobacterium. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity based on blast (ANIb) values for strains F-60T and F-392 were calculated as 100 %. However, dDDH and ANI analyses between the strains and their close neighbours confirmed that both strains represent a novel species in the genus Flavobacterium. The strains shared the highest dDDH and ANIb levels of 23.3 and 77.9%, respectively, with the type strain of Flavobacterium frigidarium DSM 17623T while those values for F. crassostreae LPB0076T were obtained as 21.4-21.5 % and 76.3 %. The DNA G+C content of the strains was 34.5 mol%. Chemotaxonomic and phylogenomic analyses of these isolates confirmed that both strains are representatives of a novel species for which the name Flavobacterium muglaense sp. nov. is proposed, with F-60T as the type strain (=JCM 34196T=KCTC 82256T).

Flavobacterium franklandianum sp. nov. and Flavobacterium gawalongense sp. nov., isolated from glaciers on the Tibetan Plateau.

The genus Flavobacterium (family Flavobacteriaceae) can be found in diverse environments. In this study, seven novel strains were isolated from glaciers in PR China and subjected to taxonomic research. Phylogenetic analyses based on the 16S rRNA gene revealed that the strains belonged to the genus Flavobacterium. None of the seven strains grew at temperatures above 22 °C, indicating that they are psychrophilic. Furthermore, the average nucleotide identity (ANI) values of the seven strains were calculated and indicated that they represented two novel species in Flavobacterium. Strain LB3P56T was most closely related to Flavobacterium soyangense IMCC26223T (97.70 %) and strain GSP16T was most closely related to Flavobacterium sinopsychrotolerans 0533T (98.03 %). The ANI values between the two Flavobacterium strains and their closest relatives were less than 83.47 %, which was much lower than the threshold for species delineation of 95-96 %. Therefore, we propose two novel species, Flavobacterium franklandianum sp. nov. (LB3P56T=CGMCC 1.11934T=NBRC 113651T) and Flavobacterium gawalongense sp. nov. (GSP16T=CGMCC 1.24642T=NBRC 113664T).

Flavobacterium solisilvae sp. nov. and Flavobacterium silvaticum sp. nov., isolated from forest soil.

Two Gram-stain-negative, yellow-pigmented and strictly aerobic bacteria, designated strains SE-s27T and SE-s28T, were isolated from forest soil. Both strains were non-motile rods that were catalase-positive and oxidase-negative and grew optimally at 25-30 °C, pH 8.0 and with 0 % (w/v) NaCl. Strain SE-s28T produced flexirubin-type pigments, but strain SE-s27T did not produce them. Both strains contained menaquinone-6 as the sole respiratory quinone and phosphatidylethanolamine as a major polar lipid. As the major cellular fatty acids (>10 %), SE-s27T contained iso-C15 : 1 and iso-C15 : 1G, whereas SE-s28T contained iso-C15 : 0 and summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH. The DNA G+C contents of strains SE-s27T and SE-s28T were 33.1 and 44.3 mol%, respectively. The results of phylogenetic analysis based on 16S rRNA gene sequences revealed that SE-s27T and SE-s28T formed respective distinct phylogenetic lineages within the genus Flavobacterium. Strains SE-s27T and SE-s28T were most closely related to Flavobacterium macrobrachii an-8T and Flavobacterium piscinae ICH-30T with 98.0 and 94.5 % 16S rRNA gene sequence similarities, respectively. In conclusion, strains SE-s27T and SE-s28T represent novel species of the genus Flavobacterium, for which the names Flavobacterium solisilvae sp. nov. and Flavobacterium silvaticum sp. nov. are proposed. The type strains of F. solisilvae and F. silvaticum are SE-s27T (=KACC 18802T=JCM 31544T) and SE-s28T (=KACC 18803T=JCM 31545T), respectively.

Flavobacterium hydrocarbonoxydans sp. nov., isolated from polluted soil.

This paper presents a polyphasic taxonomic study of a Gram-stain-negative bacterium designated GA093T, a soil isolate capable of benzo(α)pyrene degradation. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain GA093T is a member of the genus Flavobacterium, and formed an independent phylogenetic line while clustering with the type strains of Flavobacterium hibernum, Flavobacterium branchiarum and Flavobacterium hydatis. Strain GA093T was facultatively anaerobic, and could grow at 4-33 °C (optimum, 30 °C), at pH 6-11 (optimum, pH 7) and in the presence of 0-2 % (w/v) NaCl (optimum, 0 %). Strain GA093T was capable of producing acid from various carbon sources, which was comparable to other related species of Flavobacterium. The strain contained MK-6 as the only isoprenoid quinone, iso-C15 : 0 as the major cellular fatty acid, phosphatidylethanolamine and phosphatidylinositol as diagnostic polar lipids, and sym-homospermidine as the major polyamine. The chemotaxonomic properties of strain GA093T were consistent with the general properties of Flavobacterium except the presence of phosphatidylinositol, which distinguished it from other related species. The total stretch of the obtained genome of GA093T was 5.05 Mbp, and the DNA G+C content was 34.79 mol%. The genome contained genes potentially related to the degradation of aromatic hydrocarbons. On the basis of the present polyphasic analysis, strain GA093T was found to have properties that distunguished it as representing a novel species of the genus Flavobacterium, for which the name Flavobacterium hydrocarbonoxydans sp. nov. is proposed. The type strain is GA093T (=KCTC 72594T=LMG 31760T).

Flavobacterium dauae sp. nov., isolated from rhizosphere soil of a tomato plant.

A bacterial strain, designated TCH3-2T, was isolated from the rhizosphere of tomato plant grown at Dong-A University Agricultural Experiment Station, Republic of Korea. The strain was Gram-stain-negative, obligate aerobic, orange yellow-coloured, motile by gliding and short rod-shaped. Strain TCH3-2 T only grew on 1/2 tryptic soy agar and Luria-Bertani agar among the media tested, with optimum growth at 28 °C and pH 7. Salt of 1 % NaCl was necessary to support the growth of TCH3-2T. Strain TCH3-2T produced flexirubin-type pigments. The predominant cellular fatty acids were iso-C15 : 0 (55.6 %), iso-C17 : 0 3-OH (17.9 %), summed feature 9 (comprising C16 : 0 10-methyl and/or iso-C17 : 1 ω9c; 10.5 %), iso-C15 : 0 3-OH (4.8 %) and anteiso-C15 : 0 (2.3 %). The major menaquinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, five unknown aminolipids and three unknown lipids. Phylogenetic analysis based on 16S rRNA sequences indicated that TCH3-2T was closely related to Flavobacterium ummariense DS-12T (95.16 %), Flavobacterium marinum SW105T (95.14 %) and Flavobacterium viscosus YIM 102796T (94.54 %). The draft genome of TCH3-2T comprised ca. 2.8 Mb with a G+C content of 34.61 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between TCH3-2T and closely related Flavobacterium species showed that it belongs to a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of TCH3-2T from its closest relatives. Thus, chemotaxonomic characteristics together with phylogenetic affiliation illustrate that TCH3-2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium dauae sp. nov. (type strain TCH3-2T=KACC 19054T=JCM 34025T) is proposed.