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 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 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 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.

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).

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.

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 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 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 turcicum sp. nov. and Flavobacterium kayseriense sp. nov. isolated from farmed rainbow trout in Turkey.

During a study on culturable microorganisms from fish farms, four yellow-pigmented gram negative, rod shaped isolates, F-47T, F-339T, F-380 and F-400, were recovered from rainbow trout samples exhibiting clinical signs. Based on 16S rRNA gene sequence analysis, the strains were identified as members of the genus Flavobacterium. Strains F-47T and F-380 shared the highest 16S rRNA gene sequence identity level of 97.6% with the type strain of Flavobacterium frigoris DSM 15719T while strains F-339T and F-400 shared the highest identity level of 97.6% with the type strain of F. caseinilyticum AT-3-2T. A polyphasic taxonomic approach including phenotypic and genomic characterization as well as whole-cell MALDI-TOF mass spectrometry analyses was employed to ascertain the taxonomic position of the strains within the genus Flavobacterium. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses between strains F-47T, F-339T and their close neighbours F. frigoris DSM 15719T and F. caseinilyticum AT-3-2T, respectively, confirmed that both strains represent novel species in the genus Flavobacterium. The DNA G+C contents of the strains F-47T and F-339T are 34.3% and 35.3%, respectively. It can be concluded on the basis of polyphasic characterization as well as pairwise genome comparisons that the strains F-47T and F-339T represent two novel species within the genus Flavobacterium, for which Flavobacterium kayseriense sp. nov. F-47T (=JCM 34195T=KCTC 82255T) and Flavobacterium turcicum sp. nov. F-339T (=JCM 34202T=KCTC 82262T) are proposed, respectively.

Absolute quantification of priority bacteria in aquaculture using digital PCR.

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.

Flavobacterium bizetiae sp. nov., isolated from diseased freshwater fish in Canada at the end of the 1970s.

Genome sequence analysis of two strains collected in Canada at the end of the 1970s and deposited in 1998 at the Collection de l'Institut Pasteur has led to the taxonomic description of a novel fish-associated species in the genus Flavobacterium. Both strains, CIP 105534T and CIP 105535, were yellow-pigmented, Gram-stain-negative, non-spore-forming rod-shaped bacteria that exhibited gliding motility. They grew aerobically in a temperature range from 5 to 30 °C with optimal growth at 25 °C on trypticase soy or Reasoner's 2A agar but they did not grow on marine agar. Their major fatty acid profiles were similar, consisting of iso-C15 : 0, C16 : 1 ω7c and/or iso-C15 : 0 2-OH (shown as summed feature 3), C16 : 0 3-OH, iso-C17 : 0 3-OH and C16 : 0. The major polyamine was sym-homospermidine. Phosphatidylethanolamine and, most notably, ornithine-containing lipid OL2 and unidentified aminophospholipid APL1 were major polar lipids. A yellow pigment spot was visible after chromatographic analysis. The predominant respiratory quinone was MK-6. The G+C content of the two genomes was 34 mol% and their size was around 5.8 Mb. Comparison of the 16S rRNA gene sequences with those of the closely related type strains showed high levels of relatedness with Flavobacterium collinsii and Flavobacterium pectinovorum. All average nucleotide identity (ANI) and digital DNA-DNA hybridization values estimated against publicly available Flavobacterium genome assemblies were lower than 90 and 30 %, respectively. Phylogenetic, phenotypic and chemotaxonomic data indicated that the two strains represent a novel species of the genus Flavobacterium, for which the name Flavobacterium bizetiae sp. nov. is proposed. The type strain is CIP 105534T (=LMG 1342T). The unique ability of F. bizetiae to use melibiose as a sole source of carbon could provide a simple phenotypic test to discriminate F. bizetiae from its closest relatives.

High Genetic Diversity in Flavobacterium psychrophilum Isolates from Healthy Rainbow Trout (Oncorhynchus mykiss) Farmed in the Same Watershed, Revealed by Two Typing Methods.

Flavobacterium psychrophilum affects salmonid health worldwide and causes economic losses. The genetic diversity of the pathogen must be considered to develop control methods. However, previous studies have reported both high and low levels of genetic diversity. The present longitudinal study aimed at assessing the genetic diversity of F. psychrophilum at a small temporal and geographic scale. Four farms located on the same watershed in France were studied. Rainbow trout (Oncorhynchus mykiss) batches were monitored, and apparently healthy individuals were sampled over 1 year. A total of 288 isolates were recovered from fish organs (gills and spleen) and eggs. Pulsed field gel electrophoresis revealed high genetic diversity. Multilocus sequence typing performed on a selection of 31 isolates provided congruent results, as follows: 18 sequence types (STs) were found, of which 13 were novel. The mean gene diversity (H = 0.8413) was much higher than that previously reported for this host species, although the sampling was restricted to a single watershed and 1 year. Seven isolates out of 31 were assigned to clonal complex ST10 (CC-ST10), which is the predominant clonal complex in the main salmonid production areas. A split decomposition tree reflected a panmictic population. This finding is important for aquaculture veterinarians in their diagnostic procedure, as the choice of adequate antibiotic treatment is conditioned by the correct identification of the causative agent. Furthermore, this study expands our knowledge on genetic diversity required for the development of an effective vaccine against F. psychrophilumIMPORTANCE The bacterium Flavobacterium psychrophilum is a serious pathogen in many fish species, especially salmonids, that is responsible for considerable economic losses worldwide. In order to treat infections and to develop vaccines, the genetic diversity of this bacterium needs to be known. We assessed the genetic diversity of F. psychrophilum isolates from apparently healthy rainbow trout raised in several fish farms in the same watershed in France. Two different genotyping methods revealed high diversity. The majority of isolates were unrelated to clonal complex sequence type 10 (CC-ST10), the clonal complex that is predominant worldwide and associated with disease in rainbow trout. In addition, we found 13 novel sequence types. These results suggest that a diverse subpopulation of F. psychrophilum may be harbored by rainbow trout.

Flavobacterium panici sp. nov. isolated from the rhizosphere of the switchgrass Panicum virgatum.

A Gram-staining-negative non endospore-forming strain, PXU-55T, was isolated from the rhizosphere of the switchgrass Panicum virgatum and studied in detail to determine its taxonomic position. The results of 16S rRNA gene sequence analysis indicated that the isolate represented a member of the genus Flavobacterium. The isolate shared highest 16S rRNA gene sequence similarities with the type strains of Flavobacterium chungangense (98.78 %) and Flavobacterium chilense (98.64 %). The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between the PXU-55T genome assembly and the ones of the most closely related type strains of species of the genus Flavobacterium were 87.3 and 31.9% (Flavobacterium defluvii), and 86.1 and 29.9% (Flavobacterium johnsoniae). Menaquinone MK-6 was the major respiratory quinone. As major polar lipids, phosphatidylethanolamine, an ornithine lipid and the unidentified polar lipids L2, L3 and L4 lacking a functional group were found. Moderate to minor amounts of another ornithine lipid, the unidentified lipid L1 and a glycolipid were present, as well. The major polyamine is sym-homospermidine. The fatty acid profiles contained major amounts of iso-C15:0, iso-C15:0 3-OH, iso-C17:0 3-OH, C15:0, summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH) and various hydroxylated fatty acids in smaller amounts, among them iso C16:0 3-OH, C16:0 3-OH and C15:0 3-OH, which supported the classification of the isolate as a member of the genus Flavobacterium. Physiological and biochemical characterisation and ANI calculations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation of the strain. For this reason, we propose that strain PXU-55T (=CIP 111646T=CCM 8914T) represents a novel species with the name Flavobacterium panici sp. nov.

Flavobacterium salmonis sp. nov. isolated from Atlantic salmon (Salmo salar) and formal proposal to reclassify Flavobacterium spartansii as a later heterotypic synonym of Flavobacterium tructae.

A Gram-staining-negative non endospore-forming strain, T13(2019)T was isolated from water samples from Atlantic salmon (Salmo salar) fry culture in Chile and studied in detail for its taxonomic position. The isolate shared highest 16S rRNA gene sequence similarities with the type strains of Flavobacterium chungangense (98.44 %) followed by Flavobacterium tructae and Flavobacterium spartansii (both 98.22 %). Menaquinone MK-6 was the predominant respiratory quinone in T13(2019)T. Major polar lipids were phosphatidylethanolamine, an ornithine lipid and the unidentified polar lipids L1, L3 and L4 lacking a functional group. The major polyamine was sym-homospermidine. The fatty acid profile contained major amounts of iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH, C15 : 0, summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH) and various hydroxylated fatty acids in smaller amounts, among them iso-C16 : 0 3-OH, and C15 : 0 3-OH, which supported the grouping of the isolate into the genus Flavobacterium. Physiological/biochemical characterisation and ANI calculations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation. In addition it became obvious, that the type strains of F. tructae and F. spartansii showed 100 % 16S rRNA gene sequence similarities and ANI values of 97.21%/ 97.59 % and DDH values of 80.40 % [77.5 and 83%]. These data indicate that F. tructae and F. spartansii belong to the same species and it is proposed that F. spartansii is a later heterotypic synonym of F. tructae. For strain T13(2019)T (=CIP 111411T=LMG 30298T=CCM 8798T) a new species with the name Flavobacterium salmonis sp. nov. is proposed.

Gemmobacter aquarius sp. nov., Runella rosea sp. nov. and Flavobacterium fluviale sp. nov., isolated from the Namhangang River system.

Three bacterial strains, namely HYN0069T, HYN0085T and HYN0086T, were isolated from freshwater samples taken from the Namhangan River system in Korea. 16S rRNA gene sequence similarities and phylogenetic tree topologies indicated that the three strains belonged to the genera Gemmobacter, Runella and Flavobacterium by showing the highest sequence similarities with Gemmobacter straminiformis (98.4 %), Runella aurantiaca (98.3 %) and Flavobacterium chungangense (98.1 %). No bacterial species with validly published names showed 98.7 % or higher sequence similarity with the novel isolates. The average nucleotide identities between the genome sequences of the three new isolates and the three closest neighbours were 80.2-92.0 %, all below the threshold for bacterial species delineation (95-96 %). Many biochemical and physiological features also discriminated the isolates from previously known species of the genera Gemmobacter, Runella and Flavobacterium. Based on the phylogenetic and phenotypic data presented in this study, we suggest three novel species with the following names: Gemmobacter aquarius sp. nov. (type strain HYN0069T=KACC 19488T=NBRC 113115T), Runella rosea sp. nov. (type strain HYN0085T=KACC 19490T=NBRC 113116T) and Flavobacterium fluviale sp. nov. (type strain HYN0086T=KACC 19489T=NBRC 113117T).

Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture.

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production.

Flavobacterium pokkalii sp. nov., a novel plant growth promoting native rhizobacteria isolated from pokkali rice grown in coastal saline affected agricultural regions of southern India, Kerala.

Nine plant-associated bacterial strains designated as L1I52T, NRK F1, NRK F15, NRK F16, NRK F41, NRK F42, NRK F47, NRK F49, and NRK F50 originating from the roots and rhizosphere region of a coastal saline tolerant pokkali rice were taxonomically characterized in this study. Genomic fingerprinting using Enterobacterial Repetitive Intergenic Consensus (ERIC) primers discriminated the nine strains based on the DNA fingerprint patterns indicating that they were not clonal in origin. Phylogenetic analysis using 16S rRNA and other five housekeeping genes (gyrB, glyA, atpA, dnaK and murG) revealed that the novel strains constituted a single novel species within the genus Flavobacterium. In all tree construction methods, the novel strains formed a distinct phylogenetic branch, with Flavobacterium daejeonense GH1-10T, F. sufflavum BBQ-12T, and F. glycines Gm-149T as their nearest phylogenetic neighbours. However, average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) comparison between the draft genomes of L1I52T (representative isolate) and it's nearest phylogenetic neighbours were well below the proposed threshold values (<95 % and <70 %) used for species discrimination. Thus, based on the phenotypic, genotypic and chemotaxonomic data obtained in this study, we describe a novel Flavobacterium species for which we propose the name Flavobacterium pokkalii sp.nov., with strain L1I52T (=MTCC 12454T=KCTC 42429T) as the type strain. In addition, L1I52T is a potential plant growth promoting rhizobacteria as they can promote pokkali rice growth and we identified several plant associated gene features in the genome of L1I52T that are potentially involved in plant microbe interactions.

Flavobacterium ajazii sp. nov., Isolated from Seaweed of Gouqi Island, China.

A novel bacterium designated SSM4.2T was isolated from seaweed of Gouqi Island, which is the center of the Zhoushan fishing ground in the East China Sea. Strain SSM4.2T was Gram-stain-negative, bright yellow-pigmented, short rod-shaped, non-flagellated, non-spore forming, aerobic and motile by gliding. Growth was observed at 4-37 °C (optimum 25-30 °C), pH 6.0-8.0 (optimum pH 7.0) and 0-2.0% (w/v) NaCl (optimum 0%) concentration. The strain was catalase- and oxidase-positive. Menaquinone-6 (MK-6) was found as the sole respiratory quinone and zeaxanthin as the main carotenoid pigment. The predominant fatty acids (≥ 10%) were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1 ω7c /C16:1 ω6c). The major polar lipid was phosphatidylethanolamine (PE). The genome size was 5.7 Mbp. The DNA G + C content was 34.1 mol%. 16S rRNA gene sequence revealed that strain SSM4.2T belongs to the genus Flavobacterium and shares high-sequence similarity with F. limi KACC 18851T (98.1%), F. hydrophilum KACC 19591T (97.6%), F. defluvii KCTC 12612T (97.1%), F. cheongpyeongense KACC 19592T (97.0%) and F. fluviatile KCTC 52446T (96.9%). Strain SSM4.2T had 73.2-84.6% average nucleotide identity and 19.1-29.4% digital DNA-DNA hybridization values with its closest type strains. Based on its phenotypic, chemotaxonomic, phylogenetic and genomic features, strain SSM4.2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ajazii sp. nov. is proposed. The type strain is SSM4.2T (= KCTC 72807T = MCCC 1K04370T).

Flavobacterium restrictum sp. nov., Flavobacterium rhamnosiphilum sp. nov., and Flavobacterium zepuense sp. nov. isolated from glaciers.

Three strains, designated as LB1R34T, LB3P52T and ZT4R6T, were isolated from glaciers located on the Tibetan Plateau, PR China. The strains were Gram-stain-negative, aerobic, rod-shaped, non-motile and yellow. Phylogenetic analysis based on the 16S rRNA gene and genomic sequences indicated that they were related to the members of the genus Flavobacterium. The 16S rRNA gene sequences similarities between the three strains were 92.31-96.93 %. The average nucleotide identity values and digital DNA-DNA hybridization values between these three strains and their closest relatives were 76.80-91.33 % and 21.3-44.2 %, respectively. LB1R34T, LB3P52T and ZT4R6T contained MK-6 as the major menaquinone, summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and iso-C15 : 0 as the major fatty acids. Phosphatidylethanolamine was present in their polar lipids profiles. On the basis of the phenotypic characteristics, he results of phylogenetic analysis and genotypic data, three novel species, Flavobacterium restrictum sp. nov. (type strain=LB1 R34T=CGMCC 1.11493T=NBRC 113650T), Flavobacterium rhamnosiphilum sp. nov. (type strain=LB3 P52T=CGMCC 1.11446T=NBRC 113776T) and Flavobacterium zepuense sp. nov. (type strain=ZT4 R6T=CGMCC 1.11919T=NBRC 113653T) are proposed.