Ureibacillus aquaedulcis sp. nov., isolated from freshwater well and reclassification of Lysinibacillus yapensis and Lysinibacillus antri as Ureibacillus yapensis comb. nov. and Ureibacillus antri comb. Nov.

A Gram-stain-positive aerobic, rod-shaped, spore-producing bacterium forming colonies with convex elevation and a smooth, intact margin was isolated from a freshwater sample collected from a well situated in an agricultural field. The 16S rRNA gene sequence of the isolated strain BA0131T showed the highest sequence similarity to Lysinibacillus yapensis ylb-03T (99.25%) followed by Ureibacillus chungkukjangi 2RL3-2T (98.91%) and U. sinduriensis BLB-1T (98.65%). The strain BA0131T was oxidase and catalase positive and urease negative. It also tested positive for esculin hydrolysis and reduction of potassium nitrate, unlike its phylogenetically closest relatives. The predominant fatty acids in strain BA0131T included were anteiso-C15:0, iso-C16:0, iso-C15:0, iso-C14:0 and the major polar lipids comprised were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The respiratory quinones identified in strain BA0131T were MK8 (H2) (major) and MK8 (minor). The strain BA0131T shared the lowest dDDH values with L. yapensis ylb-03T (21%) followed by U. chungkukjangi 2RL3-2T (24.2%) and U. sinduriensis BLB-1T (26.4%) suggesting a closer genetic relationship U. sinduriensis BLB-1T. The ANI percentage supported the close relatedness with U. sinduriensis BLB-1T (83.61%) followed by U. chungkukjangi 2RL3-2T (82.03%) and U. yapensis ylb-03T (79.57%). The core genome-based phylogeny constructed using over 13,704 amino acid positions and 92 core genes revealed the distinct phylogenetic position of strain BA0131T among the genus Ureibacillus. The distinct physiological, biochemical characteristics and genotypic relatedness data indicate the strain BA0131T represents a novel species of the genus Ureibacillus for which the name Ureibacillus aquaedulcis sp. nov. (Type strain, BA0131T = MCC 5284 = JCM 36475) is proposed. Additionally, based on extensive genomic and phylogenetic analyses, we propose reclassification of two species, L. yapensis and L. antri, as U. yapensis comb. nov. (Type strain, ylb-03T = JCM 32871T = MCCC 1A12698T) and U. antri (Type strain, SYSU K30002T = CGMCC 1.13504T = KCTC 33955T).

Draft genome sequence of 'Candidatus Phytoplasma asteris,' strain SW86 associated with sandal spike disease (SSD).

The sandal spike disease (SSD), related to 'Ca. Phytoplasma asteris' (Aster Yellows group), poses a significant threat to Indian sandalwood (Santalum album L.), making it the second most expensive wood globally due to declining population density. The epidemiology of SSD and the nature of the pathogen remain poorly understood. The SW86 isolate, collected from the Marayoor Sandalwood Reserve, was chosen for genome sequencing subsequent to confirming its titer and enriching phytoplasma DNA. Genome sequencing, utilizing Illumina and Oxford Nanopore Technology platforms, enabled a targeted hybrid metagenomic assembly resulting in 20 scaffolds totaling 554,025 bp, housing 436 protein-coding genes, 27 tRNA, and 1 rRNA operon. The genome analysis highlighted specific gene distributions, emphasizing translation, ribosomal structure, and biogenesis, with 352 genes assigned to 18 functional categories. Additionally, 322 proteins received functional assignments in the KEGG database, emphasizing 'Genetic Information Processing' and 'Environmental Information Processing'. Key potential pathogenicity factors, including signal peptide proteins and virulence proteins, were identified. Noteworthy findings include homologs of effectors genes like SAP11 and SAP05 and pathogenesis-related proteins, such as hemolysin III and SodA genes, in the SW86 genome. The duplicated cation-transporting P-type ATPase in the SW86 genome suggests a role in enhancing adaptability and contributing to the severity of SSD symptoms. This genome analysis provides crucial insights into the genomic features and potential virulence factors of 'Ca. Phytoplasma asteris' strain SW86, advancing our understanding of pathogenicity mechanisms and offering avenues for future disease management strategies in Indian sandalwood. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03952-5.

A rare urinary tract infection of multidrug-resistant Chryseobacterium urinae sp. nov. isolated from a diabetic, non-catheterized patient.

Chryseobacterium demonstrates a diverse environmental presence and a significant pathogenic potential across various ecosystems. This clinical case showcases a rare instance of bacterial infection in a 75-year-old male with untreated diabetes and recurrent urinary tract infections (UTIs). The patient presented symptoms of abdominal pain, burning urination, fever, and an elevated eosinophil count. A subsequent urine culture identified a Chryseobacterium-related bacterium as the causative agent, exhibiting sensitivity to piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and nitrofurantoin, which led to successful treatment using oral nitrofurantoin. Analysis of the 16S rRNA gene sequence of APV-1T revealed a close relationship of 98.2% similarity to Chryseobacterium gambrini strain 5-1St1aT (AM232810). Furthermore, comparative genome analysis, incorporating Average Nucleotide Identity (ANI), Digital DNA-DNA Hybridization (dDDH) values, and comprehensive phylogenetic assessments utilizing 16S rRNA gene sequences, core genes, and amino acid sequences of core proteins, highlighted the unique phylogenetic positioning of APV-1T within the Chryseobacterium genus. Distinct carbon utilization and assimilation patterns, along with major fatty acid content, set APV-1T apart from C. gambrini strain 5-1St1aT. These findings, encompassing phenotypic, genotypic, and chemotaxonomic characteristics, strongly support the proposal of a novel species named Chryseobacterium urinae sp. nov., with APV-1T designated as the type strain (= MCC 50690 = JCM 36476). Despite its successful treatment, the strain displayed resistance to multiple antibiotics. Genomic analysis further unveiled core-conserved genes, strain-specific clusters, and genes associated with antibiotic resistance and virulence. This report underscores the vital importance of elucidating susceptibility patterns of rare pathogens like Chryseobacterium, particularly in immunocompromised individuals. It advocates for further analyses to understand the functional significance of identified genes and their implications in treatment and pathogenesis.

Fictibacillus fluitans sp. nov., isolated from freshwater pond.

A Gram-positive, aerobic, rod-shaped, spore-forming bacterium, designated NE201T, was isolated from a freshwater pond in Village Nerur, India. Growth was observed in the range of 15-45 °C temperature with optimum at 30 °C, pH range of 5-9 (optimum at 7.0), and at concentrations of NaCl ranging between 0 and 14% (optimum 0%, w/v). The 16S rRNA gene sequence showed the highest similarity with Fictibacillus enclensis NIO-1003T (JF893461) at 99.01% followed by F. rigui WPCB074T (EU939689) at 98.9% and F. solisalsi CGMCC 1.6854T (EU046268) at 98.66%. The digital DNA-DNA hybridization (dDDH) and orthoANI values for strain NE201T against F. enclensis NIO-1003T (GCA_900094955.1) were 33.7% and 87.68%, respectively. The phylogenetic analysis based on the 16S rRNA gene, 92 core genes derived from the genome, and 20 proteins involving over 20,236 amino acid positions revealed the distinct phylogenetic position of strain NE201T and the formation of a clearly defined monophyletic clade with F. enclensis. The strain NE201T showed a unique carbon utilization and assimilation pattern that differentiated it from F. enclensis NIO-1003T. The major fatty acids were anteiso -C15:0 (51.42%) and iso-C15:0 (18.88%). The major polar lipids were phosphatidylglycerol (PG), phosphatidylethanolamine (PE, and diphosphatidylglycerol (DPG). The antiSMASH analyzed genome of NE201T highlighted its diverse biosynthetic potential, unveiling regions associated with terpene, non-ribosomal peptide synthetases (NRPS), lassopeptides, NI-siderophores, lanthipeptides (LAP), and Type 3 Polyketide Synthases (T3PKS). The overall phenotypic, genotypic, and chemotaxonomic characters strongly suggested that the strain NE201T represents a novel species of genus Fictibacillus for which the name Fictibacillus fluitans sp. nov. is proposed. The type strain is NE201T (= MCC 5285 = JCM 36474).

From sequences to species: Charting the phytoplasma classification and taxonomy in the era of taxogenomics.

Phytoplasma taxonomy has been a topic of discussion for the last two and half decades. Since the Japanese scientists discovered the phytoplasma bodies in 1967, the phytoplasma taxonomy was limited to disease symptomology for a long time. The advances in DNA-based markers and sequencing improved phytoplasma classification. In 2004, the International Research Programme on Comparative Mycoplasmology (IRPCM)- Phytoplasma/Spiroplasma Working Team - Phytoplasma taxonomy group provided the description of the provisional genus 'Candidatus Phytoplasma' with guidelines to describe the new provisional phytoplasma species. The unintentional consequences of these guidelines led to the description of many phytoplasma species where species characterization was restricted to a partial sequence of the 16S rRNA gene alone. Additionally, the lack of a complete set of housekeeping gene sequences or genome sequences, as well as the heterogeneity among closely related phytoplasmas limited the development of a comprehensive Multi-Locus Sequence Typing (MLST) system. To address these issues, researchers tried deducing the definition of phytoplasma species using phytoplasmas genome sequences and the average nucleotide identity (ANI). In another attempts, a new phytoplasma species were described based on the Overall Genome relatedness Values (OGRI) values fetched from the genome sequences. These studies align with the attempts to standardize the classification and nomenclature of 'Candidatus' bacteria. With a brief historical account of phytoplasma taxonomy and recent developments, this review highlights the current issues and provides recommendations for a comprehensive system for phytoplasma taxonomy until phytoplasma retains 'Candidatus' status.

Nested Real-Time PCR Assessment of Vertical Transmission of Sandalwood Spike Phytoplasma ('Ca. Phytoplasma asteris').

The Sandalwood Spike disease (SSD)-related to 'Ca. Phytoplasma asteris' has threatened the existence of sandalwood in India. The epidemiology of SSD is still poorly understood despite the efforts to understand the involvement of insect vectors in SSD transmission and alternate plant hosts over the last two decades. Apart from the transmission of SSD phytoplasma through insect vectors, the information on vertical transmission is entirely unknown. Over 200 seeds from SSD-affected trees and over 500 seedlings generated using commercially purchased seeds were screened for the presence of SSD phytoplasma to understand the vertical transmission in an insect-free environment. The end-point nested PCR and real-time nested PCR-based screening revealed an alarming rate of 38.66% and 23.23% phytoplasma positivity in one-month and four-month-old seedlings, respectively. These results were further validated by visualizing the phytoplasma bodies in sandalwood tissues using scanning electron microscopy. The presence of phytoplasma DNA in the seeds and seedlings is a concern for the commercial distribution of sandalwood seedlings in the current setup. This also poses a fear of spreading the disease to newer areas and negatively affecting the economy. The seedling mortality was also suspected to be associated with isolated bacterial and fungal isolates such as Erwinia, Curtobacterium, Pseudomonas, Rhodococcus, Aspergillus, Fusarium, and Neofusicoccum isolated using a culture-dependent approach. These findings strongly recommend the accreditation of commercial production of sandalwood seedlings curtailing SSD phytoplasma's menace. Additionally, a new nested end-point and qRT PCR assays developed in this study proved valuable for the rapid screening of phytoplasma in many plant samples to detect phytoplasmas.

Rothia santali sp. nov., endophytic bacteria isolated from sandalwood (Santalum album L.) seedling.

A novel, mustard yellow-pigmented aerobic bacterial strain designated AR01T was isolated from hypocotyl tissue of a sandalwood seedling from Bangalore, India. The 16S rRNA gene of strain AR01T had the highest 98.97% sequence similarity with Rothia halotolerans YIM 90716T (KCTC 19172) followed by Rothia kristinae PM 129T (NBRC 15354T) (97.31%) and Rothia koreensis P31T (JCM 15915) (97.11%), respectively. The strain AR01T was coccoid-shaped, non-motile, non-spore forming, oxidase negative and catalase positive. The strain AR01T has a genome size of 3.31 Mb containing 2993 protein-coding genes including 48 tRNA and 10 rRNAs spread across 84 contigs. The genomic DNA G + C content was 71.77 mol%. The calculated dDDH was 31.10% and the OrthoANI value was 85.27% when compared with its closest related type strain Rothia halotolerans YIM 90716T. The predominant cellular fatty acids were C16:0 iso, C15:0 anteiso and C17:0 anteiso. The strain AR01T contains major polar lipids including diphosphatidylglycerol and phosphatidylglycerol. The distinct physiological, biochemical characteristics and genotypic relatedness indicated that AR01T represents a novel species of the genus Rothia, for which the name Rothia santali sp. nov. (Type strain AR01T = MCC 4800T = JCM 35593T) is proposed.

Draft genome sequence of 'Candidatus Phytoplasma australasia', strain SS02 associated with sesame phyllody disease.

'Candidatus Phytoplasma' is an uncultivated, intracellular bacterial plant pathogen transmitted by phloem-feeding insect vectors. Among the group of phytoplasmas, the Peanut Witches' Broom or 16SrII group of phytoplasmas associated with various diseases cause severe crop losses every year in India. The 'Ca. Phytoplasma sp.' strain SS02 was associated with phyllody disease of sesame plants collected from New Delhi. The genome sequence of strain SS02 was obtained using its genomic DNA enrichment and hybrid assembly of sequences generated on Illumina and Oxford Nanopore Technologies MinION platforms. The hybrid assembly strategy generated a draft genome with 60 contigs totaling 553,228 bp of length with more than 400 × depth coverage and 95.21% of the estimated completeness. The SS02 genome draft sequence contains 465 protein-coding genes, 17 tRNA genes, and 3 rRNA genes. The availability of this draft genome also provided a foundation for genome-scale genotypic analyses.

Hydrogenophaga crocea sp. nov. associated with cyanobacterial mat isolated from farmland mud.

A catalase and oxidase-positive strain BA0156T was isolated from a cyanobacterial mat collected from the farmland mud cultivated with sugarcane from Ahmednagar, India. The 16S rRNA gene of strain BA0156T showed the highest percent sequence similarity with Hydrogenophaga borbori LMG 30805T (98.5%), followed by H. flava DSM 619T (98.3%) and H. intermedia DSM 5680T (98.2%). The strain BA0156T contained the major fatty acids, C16:0 (25.1%) and C17:0 cyclo (3.9%), whereas phosphatidylethanolamine and diphosphatidylglycerol were the major polar lipids. The OrthoANI and dDDH values between strain BA0156T and its closest relative H. borbori LMG 30805T were 84.6% and 28.3%, respectively. The DNA G+C content of strain BA0156T was 69.4 mol %. Furthermore, the biochemical and physiological features of strain BA0156T showed a distinct pattern from their closest phylogenetic neighbours. The phenotypic, genotypic and chemotaxonomic characteristics indicated that the strain BA0156T represents a new species for which the name Hydrogenophaga crocea (type strain BA0156T = MCC 3062T = KCTC 72452T = JCM 34507T) is proposed.

Paenibacillus albicereus sp. nov. and Niallia alba sp. nov., isolated from digestive syrup.

Two aerobic, Gram-stain variable, catalase-positive and oxidase-negative rods named strain UniB2T and UniB3T, were isolated from digestive syrup containing fungal diastase (10 mg/ml), pepsin (2 mg/ml) and sugar base containing polyethylene glycol. Based on 16S rRNA gene sequence analysis, strain UniB2T has the highest sequence similarity with Paenibacillus humicus NBRC 102415T (98.3%) and strain UniB3T showed the highest sequence similarity with Niallia circulans DSM 11T (98.9%). The DNA G + C content of UniB2T was 63.7 mol %. The dDDH and ANI values between the strain UniB2T and its phylogenetically close relative were < 38.3% and < 89.5%, respectively. The major fatty acids of the strain UniB2T were C16:0 (13.9%), C15:0 anteiso (39.7%), C17:0 anteiso (15.5%). The DNA G + C content of UniB3T was 35.6 mol %. The dDDH and ANI values between the strain UniB3T and its close relatives were < 29.1% and 84.6%, respectively. The major fatty acids of strain UniB3T were C16:0 (13.5%), C15:0 anteiso (40.1%) and C17:0 anteiso (16.0%). Major polar lipids for both strains were Diphosphatidylglycerol and phosphatidylethanolamine. Both strains showed unique carbon utilization and assimilation pattern that differentiated them from their phylogenetically related neighbours. These phenotypic, genotypic and chemotaxonomic characters indicated the strains UniB2T and UniB3T represent two novel species for which the names Paenibacillus albicereus sp. nov. (Type strain UniB2T = MCC 3997T = KCTC 43095T = JCM34513T) and Niallia alba sp. nov. (Type strain UniB3T = MCC 3998T = KCTC 43235T = JCM 34492T) are proposed.

First report of 16SrII group (Peanut witches' Broom) Phytoplasmas associated with the Leucas aspera Phyllody in India.

Leucas aspera (Wild.) Linn. (Family: Lamiaceae) is a commonly found weed throughout India, known for its pharmacological properties. Its white flowers and leaves are used in many Ayurvedic formulations for the treatment of chronic rheumatism, psoriasis, snake bites and skin eruptions (Prajapathi et al., 2010). During a survey of commercial flower crop fields in May 2018, a few L. aspera plants, growing as unwanted weeds in the fields and surrounding agricultural wastelands with the symptoms of phyllody, virescence and little leaves were observed in Emmekoppalu (12.2106, 76.2511; n= 1/26 plants) and Beerihundi (12.1630, 76.3225; n= 2/59 plants) localities of Mysuru district, and Srirangapatna in Mandya district (12.2541, 76.411; 1/67 plants), Karnataka- India(Figure 1). 'n' denotes the symptomatic/ asymptomatic samples observed. The disease incidence in the surveyed localities ranged less than four per cent. The total genomic DNA was extracted from the leaf midrib tissues of three representative symptomatic and two asymptomatic samples using the CTAB method. The phytoplasma 16S rRNA gene was amplified in nested PCR assay by P1/P7 followed by R16F2n/R16R2 primers using Long Amplification (LA) Taq polymerase (Takara, Japan). Additionally, the PCR assays were performed for the amplification of phytoplasma secA gene using the primers SecAfor1/SecArev3 and SecAfor2/SecArev3 (Hodgetts et al., 2008). The DNA templates from all the symptomatic samples generated amplicons of approximately 1.25kb (16S rRNA gene) and 480 bp (secA gene) revealing the association of phytoplasma strains. No amplifications were observed for the asymptomatic L. aspera samples. The obtained 16S rRNA gene sequences (MN223676, MT807111 and MZ093053) showed 97.96, 98.37 and 98.18 % sequence identity, respectively; with the 'Candidatus Phytoplasma aurantifolia', strain 'WBDL (U15442) using EzBiocloud database. The NCBI-BLAST analysis revealed maximum identity to various Peanut witches' Broom (PWB) phytoplasma strains. The virtual RFLP tool, iPhyClassifier delineated the Leucas phyllody phytoplasma strains (MN223676, MT807111 and MZ093053) to group 16SrII (PWB, Peanut Witches' broom group) subgroup D with the similarity coefficient 1.0 (Zhao et al. 2009). The obtained secA gene sequences (MZ151944, MZ151945 and MZ151946) were 98.15 to 100 % similar to the strain sequences of PWB phytoplasma strains. Further, the clustering pattern in the phylogenetic trees (16S rRNA and secA genes) constructed using MEGA 7 confirmed that the Leucas phyllody phytoplasma sequences were closely related to PWB strains. To the best of our knowledge, this is the first report on the association of 16SrII-D subgroup phytoplasma with the phyllody disease of L. aspera. In India, many weeds and wild plants serve as alternative hosts of PWB phytoplasmas and aid in the emergence of related diseases in economically important crops (Thorat et al., 2016; Thorat et al., 2017). The close genetic association of phytoplasma strains found in L. aspera and many other crops indicates the presence of common insect vector(s) transmitting these phytoplasmas (Yadav et al. 2015). This report is an addition to the catalogue of the weed species harboring phytoplasma strains associated with economically important crop plants (Rao et al., 2017). The screening of phytoplasma strains in weeds, alternate hosts and known/ unknown insect vectors is therefore essential to develop management strategies and effective management of phytophagous insect vectors feeding on both weeds and crop plants.

'Candidatus Phytoplasma sacchari', a novel taxon - associated with Sugarcane Grassy Shoot (SCGS) disease.

Sugarcane Grassy Shoot (SCGS) disease is known to be related to Rice Yellow Dwarf (RYD) phytoplasmas (16SrXI-B group) which are found predominantly in sugarcane growing areas of the Indian subcontinent and South-East Asia. The 16S rRNA gene sequences of SCGS phytoplasma strains belonging to the 16SrXI-B group share 98.07 % similarity with 'Ca. Phytoplasma cynodontis' strain BGWL-C1 followed by 97.65 % similarity with 'Ca. P. oryzae' strain RYD-J. Being placed distinctly away from both the phylogenetically related species, the taxonomic identity of SCGS phytoplasma is unclear and confusing. We attempted to resolve the phylogenetic positions of SCGS phytoplasma based on the phylogenetic analysis of 16S rRNA gene (>1500 bp), nine housekeeping genes (>3500 aa), core genome phylogeny (>10 000 aa) and OGRI values. The draft genome sequences of SCGS phytoplasma (strain SCGS) and Bermuda Grass White leaf (BGWL) phytoplasma (strain LW01), closely related to 'Ca. P. cynodontis', were obtained. The SCGS genome was comprised of 29 scaffolds corresponding to 505 173 bp while LW01 assembly contained 21 scaffolds corresponding to 483 935 bp with the fold coverages over 330× and completeness over 90 % for both the genomes. The G+C content of SCGS was 19.86 % while that of LW01 was 20.46 %. The orthoANI values for the strain SCGS against strains LW01 was 79.42 %, and dDDH values were 22. Overall analysis reveals that SCGS phytoplasma forms a distant clade in RYD group of phytoplasmas. Based on phylogenetic analyses and OGRI values obtained from the genome sequences, a novel taxon 'Candidatus Phytoplasma sacchari' is proposed.

Pseudomonas lalkuanensis sp. nov., isolated from a bacterial consortia of contaminated soil enriched for the remediation of e-waste.

A novel e-waste-degrading strain, PE08T, was isolated from contaminated soil collected from a paper mill yard in Lalkuan, Uttarakhand, India. Strain PE08T was Gram-stain-negative, rod-shaped, aerobic, oxidase-positive and catalase-positive. Optimum growth was observed at 30 °C (range, 5-40 °C), with 1-2 % NaCl (range, 0-3 %) and at pH 7 (range 6-11). The phylogeny based on 16S rRNA gene sequences delineated strain PE08T to the genus Pseudomonas and showed highest sequence similarity to Pseudomonas furukawaii KF707T (98.70 %), followed by Pseudomonas aeruginosa DSM 50071T (98.62 %) and Pseudomonas resinovorans DSM 21078T (97.93 %). The genome of strain PE08T was sequenced and had one scaffold of 6056953 bp, 99.84 % completeness and 182× coverage were obtained. The G+C content in the genome was 64.24 mol%. The DNA-DNA hybridization and average nucleotide identity values between strain PE08T and its closely related type strain, P. resinovorans DSM 21078T were below 34.8 % and 87.96 %, respectively. The phylogenetic analysis based on whole-genome sequence and concatenated GyrB and RpoB proteins revealed that strain PE08T forms a district clade in the family Pseudomonadaceae. The predominant fatty acids were summed feature 8 (C18 :  1ω7c and/or C18 :1 ω6c), summed feature 3 (C16 :  1ω7c and/or C16 :  1ω6c), C16 : 0 and C12 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The phenotypic, chemotaxonomic and genetic analysis, including overall genome relatedness index values, indicated that strain PE08T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas lalkuanensis sp. nov. is proposed. The type strain is PE08T (=MCC 3792=KCTC 72454=CCUG 73691).

Draft Genome Sequences of Two Phytoplasma Strains Associated with Sugarcane Grassy Shoot (SCGS) and Bermuda Grass White Leaf (BGWL) Diseases.

We performed whole-genome sequencing of two phytoplasmas associated with sugarcane grassy shoot (SCGS) and Bermuda grass white leaf diseases. These are the first draft genomes of SCGS phytoplasma (strain SCGS) and 'Candidatus Phytoplasma cynodontis' (strain LW01) and may help to delineate these phytoplasmas at a finer taxonomic level.