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The 16S rRNA gene is a primary marker used to study bacterial taxonomy, particularly for bacteria with ‘Candidatus’ status, where it serves as a primary reference sequence for identification. Despite its usefulness, ensuring the authenticity of publicly available sequences has always posed a challenge. To address this issue, multiple tools and databases with curated collections of reference 16S rRNA gene sequences were established. However, even with these efforts, many sequence chimeras still exist. These chimeric gene sequences are formed by the fusion of sequence parts from multiple organisms and can arise from errors during PCR amplification or sequencing. The objective of this study was to identify chimeric sequences from a dataset of over 4500 phytoplasma 16S rRNA gene sequences using multiple tools. The tools ChimeraSlayer and UCHIME identified 12 and 11 chimeric sequences, respectively. Notably, these sequences included the reference 16S rRNA gene sequence of ‘Ca. Phytoplasma wodyetiae’ strain Bangi-2 (KC844879) and ‘Ca. Phytoplasma allocasuarinae’ (AY135523). The study’s outcomes indicated the existence of chimeric 16S rRNA gene sequences, emphasizing the threat posed by such sequences in correctly assigning taxonomic status to phytoplasma strains. These findings underscore the importance of rigorously verifying the authenticity of 16S rRNA gene sequences to ensure their accuracy in identifying and classifying bacterial species.

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).

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 (98.55 %), followed by H. flava DSM 619T (98.35 %) and H. intermedia DSM 5680T (98.21 %). The strain BA0156T consisted of 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 84.59 % and 28.3 %, respectively. The DNA G+C content of strain BA0156T was 69.42 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 BA0156 T = MCC 3062T = KCTC 72452T = JCM 34507T) is proposed.

A catalase and oxidase-positive strain BA0156

Two aerobic, Gram-stain variable, catalase and oxidase-positive, spore-forming, motile rods of strain UniB2T and UniB3T, were isolated from digestive syrup containing fungal diastase (10 mg/ml), pepsin (2 mg/ml) and sugar base containing polyethene glycol. Based on 16S rRNA gene sequence analysis, strain UniB2T has the highest sequence similarity with Paenibacillus humicus NBRC 102415T (98.30 %) and strain UniB3T showed the highest sequence similarity with Niallia circulans DSM 11T (98.95 %). The DNA G+C content of UniB2T was 63.74 mol %. The dDDH and ANI values between the strain UniB2T and its phylogenetically close relative were T were C16:00 (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 T were C16:0 (13.54 %), C15:0 anteiso (40.09 %) and C17:0 anteiso (16.03 %). Major polar lipids for both strains were Diphosphotidylglycerol and Phosphatidylethanolamine. Additionally, both the 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 albicerus 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.

Two aerobic, Gram-stain variable, catalase-positive and oxidase-negative rods named strain UniB2

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.

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.

Marigold plants with suspected symptoms of phytoplasma infection were collected during field surveys from three different districts of Karnataka where the incidence of these symptoms was ranging from 1 to 11%. The presence of phytoplasmas in the samples showing phyllody and virescence symptoms was confirmed by nested PCR assays using P1/P7 and R16F2n/R16R2 phytoplasma specific primer pairs. The nucleotide sequence identity and phylogenetic analyses of the 16S rRNA gene sequence indicated the presence of a ‘Candidatus Phytoplasma australasia’ -related strain in the diseased marigold samples. The phylogenetic analysis of secA gene sequences of this phytoplasma strain showed its clustering with strains enclosed in the 16SrII-D subgroup, confirming the phytoplasma identification. To the best of our knowledge, this constitutes the first report of 16SrII phytoplasmas associated with marigold phyllody from the Karnataka state. As the most widely cultivated commercial flower of Karnataka, marigold has a significant role in the national and international flower trade. Phyllody and associated symptoms have a direct negative impact on the quality and market value of these flowers.