In Florida, angular leaf spot, caused by Xanthomonas fragariae, was the only known bacterial disease in strawberry, which is sporadic and affects the foliage and calyx. However, from the 2019-2020 to 2023-2024 Florida strawberry seasons, unusual bacterial-like symptoms were observed in commercial farms, with reports of up to 30 % disease incidence. Typical lesions were water-soaked and angular in early stages that later became necrotic with a circular-ellipsoidal purple halo, and consistently yielded colonies resembling Pseudomonas on culture media. Strains were pathogenic on strawberry, fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco, and lacked pectolytic activity. Although phenotypic assays, such as fatty acid methyl profiles and Biolog protocols, placed the strains into the Pseudomonas group, there was a low similarity at the species level. Further analysis using 16S rRNA genes, housekeeping genes, and whole genome sequencing showed that the strains cluster into the Pseudomonas group but do not share more than 95 % average nucleotide identity compared to representative members. Therefore, the genomic and phenotypic analysis confirm that the strains causing bacterial spot in strawberry represent a new plant pathogenic bacterial species for which we propose the name Pseudomonas fragariae sp. nov. with 20-417T (17T=LMG 32456T=DSM 113340 T) as the type strain, in relation to Fragaria×ananassa, the plant species from which the pathogen was first isolated. Future work is needed to assess the epidemiology, cultivar susceptibility, chemical sensitivity, and disease management of this possible new emerging strawberry pathogen.

Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

BACKGROUND: Reliable species identification of cultured isolates is essential in clinical bacteriology. We established a new study algorithm named NOVA - Novel Organism Verification and Analysis to systematically analyze bacterial isolates that cannot be characterized by conventional identification procedures MALDI-TOF MS and partial 16 S rRNA gene sequencing using Whole Genome Sequencing (WGS).
RESULTS: We identified a total of 35 bacterial strains that represent potentially novel species. Corynebacterium sp. (n = 6) and Schaalia sp. (n = 5) were the predominant genera. Two strains each were identified within the genera Anaerococcus, Clostridium, Desulfovibrio, and Peptoniphilus, and one new species was detected within Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus. Twenty-seven of 35 strains were isolated from deep tissue specimens or blood cultures. Seven out of 35 isolated strains identified were clinically relevant. In addition, 26 bacterial strains that could only be identified at the species level using WGS analysis, were mainly organisms that have been identified/classified very recently.
CONCLUSIONS: Our new algorithm proved to be a powerful tool for detection and identification of novel bacterial organisms. Publicly available clinical and genomic data may help to better understand their clinical and ecological role. Our identification of 35 novel strains, 7 of which appear to be clinically relevant, shows the wide range of undescribed pathogens yet to define.

A single strain from the family Paenibacillaceae was isolated from the wall behind the Waste Hygiene Compartment aboard the International Space Station (ISS) in April 2018, as part of the Microbial Tracking mission series. This strain was identified as a gram-positive, rod-shaped, oxidase-positive, catalase-negative motile bacterium in the genus Cohnella, designated as F6_2S_P_1T. The 16S sequence of the F6_2S_P_1T strain places it in a clade with C. rhizosphaerae and C. ginsengisoli, which were originally isolated from plant tissue or rhizosphere environments. The closest 16S and gyrB matches to strain F6_2S_P_1T are to C. rhizosphaerae with 98.84 and 93.99% sequence similarity, while a core single-copy gene phylogeny from all publicly available Cohnella genomes places it as more closely related to C. ginsengisoli. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values to any described Cohnella species are <89 and <22%, respectively. The major fatty acids for strain F6_2S_P_1T are anteiso-C15:0 (51.7%), iso-C16:0 (23.1%), and iso-C15:0 (10.5%), and it is able to metabolize a wide range of carbon compounds. Given the results of the ANI and dDDH analyses, this ISS strain is a novel species within the genus Cohnella for which we propose the name Cohnella hashimotonis, with the type strain F6_2S_P_1T (=NRRL B-65657T and DSMZ 115098T). Because no closely related Cohnella genomes were available, this study generated the whole-genome sequences (WGSs) of the type strains for C. rhizosphaerae and C. ginsengisoli. Phylogenetic and pangenomic analysis reveals that F6_2S_P_1T, C. rhizosphaerae, and C. ginsengisoli, along with two uncharacterized Cohnella strains, possess a shared set of 332 gene clusters which are not shared with any other WGS of Cohnella species, and form a distinct clade branching off from C. nanjingensis. Functional traits were predicted for the genomes of strain F6_2S_P_1T and other members of this clade.

Degradation of crop straw in natural environment has been a bottleneck. There has been a recent increase in the exploration of cold-adapted microorganisms as they can solve the problem of corn straw degradation under low temperatures and offer new alternatives for the sustainable development of agriculture. The study was conducted in low-temperature (10°C) and high-efficiency cellulose-degrading bacteria were screened using carboxymethyl cellulose (CMC) selection medium and subjected to genome sequencing by the third-generation Pacbio Sequl and the second-generation Illumina Novaseq platform, and their cellulase activity was detected by 3,5-dinitrosalicylic acid (DNS) method. The results showed that the low-temperature (10°C) and high-efficiency cellulose-degrading bacterium Bacillus subtilis K1 was 4,060,823 bp in genome size, containing 4,213 genes, with 3,665, 3,656, 2,755, 3,240, 1,261, 3,336 and 4,003 genes annotated in the non-redundant protein sequence database (NR), Pfam, clusters of orthologous groups of proteins (COGs), Genome Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Annotation databases, respectively. In addition, a large number of lignocellulose degradation-related genes were annotated in the genome. The cellulose activity of B. subtilis K1 was higher, exhibiting the highest activity of endo-β-glucanase (24.69 U/ml), exo-β-glucanase (1.72 U/ml) and β-glucosaccharase (1.14 U/ml). It was found that through adding cold-adapted cellulose-degrading bacteriaK1 in the corn straw composting under 6°C (ambient temperature), the average temperature of straw composting was 58.7°C, and higher 86.7% as compared to control. The HA/FA was higher 94.02% than the control and the lignocellulose degradation rate was lower 18.01-41.39% than the control. The results provide a theoretical basis for clarifying the degradation potential of cold-adapted cellulose-degrading bacteria and improving the cellulose degradation efficiency.

Dendrobium plants are members of the family Orchidaceae, many of which are endangered orchids with ornamental and medicinal values. Dendrobium endophytic microbes have attracted attention for the development of strategies for plant protection and utilization of medicinal principles. However, the role of endophytic bacteria is poorly elucidated due to the lack of their successful cultivation. This study obtained a total of 749 endophytic isolates from Dendrobium roots using solid media prepared by simply modified methods (separate sterilization of phosphate and agar [PS] and use of gellan gum as a gelling reagent [GG]) and by a conventional method of autoclaving the phosphate and agar together (PT method). Notably, based on a comparison of 16S rRNA gene sequences between the isolates and the Dendrobium root endophyte community, we successfully retrieved more than 50% (17 out of 30) of the predominant endophytic bacterial operational taxonomic units (OTUs) using PS and GG media, which is a much higher recovery rate than that of PT medium (16.7%). We further found that a number of recalcitrant bacteria, including phylogenetically novel isolates and members of even the rarely cultivated phyla Acidobacteriota and Verrucomicrobiota, were obtained only when using PS and/or GG medium. Intriguingly, the majority of these recalcitrant bacteria formed colonies faster on PS or GG medium than on PT medium, which may have contributed to their successful isolation. Taken together, this study succeeded in isolating a wide variety of Dendrobium endophytic bacteria, including predominant ones using PS and GG media, and enables performance of future studies to clarify their unknown roles associated with the growth of Dendrobium plants. IMPORTANCE Dendrobium endophytic bacteria are of great interest since their functions may contribute to the protection of endangered orchids with ornamental and medicinal values. To understand and reveal the \"true roles\" of the endophytes, obtaining those axenic cultures is necessary even in the metagenomic era. However, no effective methods for isolating a variety of endophytic bacteria have been established. This study first demonstrated that the use of simply modified medium is quite effective and indeed allows the isolation of more than half of the predominant endophytic bacteria inhabiting Dendrobium roots. Besides, even phylogenetically novel and/or recalcitrant endophytic bacteria were successfully obtained by the same strategy. The obtained endophytic bacteria could serve as \"living material\" for elucidating their unprecedented functions related to the conservation of endangered orchid plants. Furthermore, the culture method used in this study may enable the isolation of various endophytic bacteria dominating not only in orchid plants but also in other useful plants.

Strain GY_HT was isolated from an artificial wetland in Okcheon, Chungcheongbuk-do Province, Republic of Korea. Strain GY_HT was closely related to Pseudolabrys taiwanensis CC-BB4T based on 16S rRNA gene sequences (94.7 % similarity) and clustered within the family Nitrobacteraceae. Cells of the isolate were Gram-stain-negative, catalase-negative and oxidase-positive, and colonies were white or pale transparent. A flagellum was observed, and the isolate could respire both aerobically and anaerobically. Growth of GY_ HT was observed in the following conditions: 10-45 °C, pH 5-11 and 0-4 % NaCl. The optimal conditions for growth were 25 °C, pH 6.5-7.5 and 0.5-1.5 % NaCl. The major fatty acids were C19 : 0 cyclo ω8c (35.8 %) and summed feature 8 (C18 : 1  ω7c/C18 : 1  ω6c; 27.4 %). The major quinone was found to be ubiquinone-10. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine were the major polar lipids. The G+C content of the genome of GY_HT was 63.3 mol%. Based on its phylogenomic, physiological and biochemical attributes, strain GY_HT represents a novel species of a novel genus of the family Nitrobacteraceae. We propose the name as Undibacter mobilis gen. nov., sp. nov. The type strain is GY_HT (=KCTC 62792T=JCM 32856T).

Recent metagenomic analysis has revealed that our gut microbiota plays an important role in not only the maintenance of our health but also various diseases such as obesity, diabetes, inflammatory bowel disease, and allergy. However, most intestinal bacteria are considered \'unculturable\' bacteria, and their functions remain unknown. Although culture-independent genomic approaches have enabled us to gain insight into their potential roles, culture-based approaches are still required to understand their characteristic features and phenotypes. To date, various culturing methods have been attempted to obtain these \'unculturable\' bacteria, but most such methods require advanced techniques. Here, we have tried to isolate possible unculturable bacteria from a healthy Japanese individual by using commercially available media. A 16S rRNA (ribosomal RNA) gene metagenomic analysis revealed that each culture medium showed bacterial growth depending on its selective features and a possibility of the presence of novel bacterial species. Whole genome sequencing of these candidate strains suggested the isolation of 8 novel bacterial species classified in the Actinobacteria and Firmicutes phyla. Our approach indicates that a number of intestinal bacteria hitherto considered unculturable are potentially culturable and can be cultured on commercially available media. We have obtained novel gut bacteria from a healthy Japanese individual using a combination of comprehensive genomics and conventional culturing methods. We would expect that the discovery of such novel bacteria could illuminate pivotal roles for the gut microbiota in association with human health.

A yellow-pigmented strain, designated Y4AR-5T, was characterized by using a polyphasic approach. The strain was isolated from a tundra soil from near Longyearbyen, Svalbard Islands, Norway. The cells were Gram-stain-negative, aerobic, rod-shaped and non-motile. Growth occurred at 4-28 °C (optimum 20 °C) and pH 6.0-9.0 (optimum pH 8.0) and with 0-0.5 % (w/v) NaCl (optimum 0 %). The major respiratory quinone was MK-7. The polar lipids were phosphatidylethanolamine (PE), an aminophospholipid (APL), a phospholipid (PL), an unidentified aminolipid (AL) and two unidentified lipids. The results of analysis of the 16S rRNA gene indicated that the novel strain was most closely related to members of the genus Spirosoma (96.2 % sequence similarity with Spirosoma endophyticum). The genomic DNA G+C content was 45.9 mol%. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0. On the basis of its phenotypic and genotypic properties, strain Y4AR-5T should be classified as representing a novel species of the genus Spirosoma, for which the name Spirosomaflavum sp. nov. is proposed. The type strain is Y4AR-5T (=CCTCC AB 2015352T=KCTC 52490T).