Application of a new designed high resolution melting analysis for mycobacterial species identification.

The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

Geographical and climatic distribution of lentil-nodulating rhizobia in Iran.

Lentil is one of the most important legumes cultivated in various provinces of Iran. However, there is limited information about the symbiotic rhizobia of lentils in this country. In this study, molecular identification of lentil-nodulating rhizobia was performed based on 16S-23S rRNA intergenic spacer (IGS) and recA, atpD, glnII, and nodC gene sequencing. Using PCR-RFLP analysis of 16S-23S rRNA IGS, a total of 116 rhizobia isolates were classified into 20 groups, leaving seven strains unclustered. Phylogenetic analysis of representative isolates revealed that the rhizobia strains belonged to Rhizobium leguminosarum and Rhizobium laguerreae, and the distribution of the species is partially related to geographical location. Rhizobium leguminosarum was the dominant species in North Khorasan and Zanjan, while R. laguerreae prevailed in Ardabil and East Azerbaijan. The distribution of the species was also influenced by agroecological climates; R. leguminosarum thrived in cold semiarid climates, whereas R. laguerreae adapted to humid continental climates. Both species exhibited equal dominance in the Mediterranean climate, characterized by warm, dry summers and mild, wet winters, in Lorestan and Kohgiluyeh-Boyer Ahmad provinces.

Occurrence of Pseudomonas syringae pvs. actinidiae, actinidifoliorum and Other P. syringae Strains on Kiwifruit in Northern Spain.

Pseudomonas syringae pv. actinidiae (Psa), the agent causing bacterial canker of kiwifruit, has been present in the Principality of Asturias (PA), Northern Spain, since 2013, although with restricted distribution. In this study, 53 strains collected in kiwifruit orchards in PA during the period 2014-2020 were characterized by a polyphasic approach including biochemical and phylogenetic analysis. Thirty-three strains, previously identified by PCR as Psa, have been found to be a homogeneous group in phylogenetic analysis, which seems to indicate that there have been few introductions of the pathogen into the region. Two strains were confirmed as P. syringae pv. actinidifoliorum (Pfm), so this is the first report of Pfm in the PA. The remaining 18 strains were found to be close to P. avellanae and P. syringae pv. antirrhini or to strains described as Pfm look-alikes. Pathogenicity tests carried out on peppers with a selection of strains have shown that both Psa and Pfm caused clear damage, while the 18 atypical strains caused variable lesions. It would be necessary to carry out pathogenicity testing of atypical strains on kiwifruit plants to study the role of these strains in the kiwifruit pathosystem to evaluate their pathogenic potential in this crop.

Molecular Analysis for Potential Hospital-Acquired Infection Caused by Aspergillus Tubingensis Through the Environment.

The identification of Aspergillus species has been performed mainly by morphological classification. In recent years, however, the revelation of the existence of cryptic species has required genetic analysis for accurate identification. The purpose of this study was to investigate five Aspergillus section Nigri strains isolated from a patient and the environment in a university hospital. Species identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identified all five black Aspergillus strains as Aspergillus niger. However, calmodulin gene sequence analysis revealed that all five strains were cryptic species, four of which, including the clinical strain, were Aspergillus tubingensis. Hospital-acquired infection of the patient with the A. tubingensis strain introduced from the environment was suspected, but sequencing of six genes from four A. tubingensis strains revealed no environmental strain that completely matched the patient strain. The amount of in vitro biofilm formation of the four examples of the A. tubingensis strain was comparable to that of Aspergillus fumigatus. An extracellular matrix was observed by electron microscopy of the biofilm of the clinical strain. This study suggests that various types of biofilm-forming A. tubingensis exist in the hospital environment and that appropriate environmental management is required.

Diversity of Strains in the Pseudomonas syringae Complex Causing Bacterial Stem Blight of Alfalfa (Medicago sativa) in the United States.

Alfalfa growers in the Intermountain West of the United States have recently seen an increased incidence in bacterial stem blight (BSB), which can result in significant herbage yield losses from the first harvest. BSB has been attributed to Pseudomonas syringae pv. syringae and P. viridiflava; however, little is known about the genetic diversity and pathogenicity of these bacteria or their interaction with alfalfa plants. Here, we present a comprehensive phylogenetic and phenotypic analysis of P. syringae and P. viridiflava strains causing BSB on alfalfa. A multilocus sequence analysis found that they grouped exclusively with P. syringae PG2b and P. viridiflava PG7a. Alfalfa symptoms caused by both bacterial groups were indistinguishable, although there was a large range in mean disease scores for individual strains. Overall, PG2b strains incited significantly greater disease scores than those caused by PG7a strains. Inoculated plants showed browning in the xylem and collapse of epidermal and pith parenchyma cells. Inoculation with a mixture of PG2b and PG7a strains did not result in synergistic activity. The populations of PG2b and PG7a strains were genetically diverse within their clades and did not group by location or haplotype. The PG2b strains had genes for production of the phytotoxin coronatine, which is unusual in PG2b strains. The results indicate that both pathogens are well established on alfalfa across a wide geographic range and that a recent introduction or evolution of more aggressive strains as the basis for emergence of the disease is unlikely.

Pectobacterium carotovorum Subsp. brasiliense Causing Soft Rot in Eggplant in Xinjiang, China.

An outbreak of stem rot in eggplants was observed in Heshuo County, Xinjiang, during winter 2021-2022 in about 12-35% of the eggplants in the region (about 40 hm2). The infected tissues yielded a total of four bacterial strains, which were subsequently subjected to physiological and biochemical assays as well as molecular identification. Based on these analyses, the pathogen was identified as Pectobacterium carotovorum subsp. brasiliense. The pathogenicity was confirmed through the fulfillment of Koch's postulates. The host range test confirmed the broad spectrum of species susceptible to infection by the strains. This study represents the first case of infection caused by P. carotovorum subsp. brasiliense resulting in stem rot in eggplant.

Siderophore-producing Pantoea ferrattrahens sp. nov. isolated from a clinical specimen and Pantoea ferramans sp. nov. isolated from soil at the bottom of a pond.

Two Gram-negative facultative anaerobes were isolated from a sepsis patient with pancreatic cancer (strain PAGU 2156T ) and soil at the bottom of a pond (strain PAGU 2198T ), respectively. These two strains formed haloes around the colonies on chrome azurol S agar plates, indicating the production of siderophores. Two isolates assigned to the genus Pantoea based on the 16S rRNA gene were differentiated from established species by using polymorphic taxonomies. Phylogenetic analysis using four housekeeping genes (gyrB, rpoB, atpD, and infB) showed that strain PAGU 2156T is closely related to Pantoea cypripedii LMG 2657T (89.9%) or Pantoea septica LMG 5345T (95.7%). Meanwhile, strain PAGU 2198T formed a single clade with Pantoea rodasii DSM 26611T (93.6%) and Pantoea rwandensis DSM 105076T (93.3%). The average nucleotide identity values obtained from the draft genome assembly showed ≤90.2% between strain PAGU 2156T and closely related species and ≤81.5% between strain PAGU 2198T and closely related species. Based on various phenotypes, biochemical properties, and whole-cell fatty acid composition compared with related species, it was concluded that each strain should be classified as a new species of the genus Pantoea. In this manuscript, Pantoea ferrattrahens sp. nov. and Pantoea ferramans sp. nov. with strain PAGU 2156T (=NBRC 115930T = CCUG 76757T ) and strain PAGU 2198T (=NBRC 114265T = CCUG 75151T ) are proposed as each type strain.

Genomic Analysis of the Carrot Bacterial Blight Pathogen Xanthomonas hortorum pv. carotae in Korea.

Bacterial leaf blight of carrots caused by Xanthomonas hortorum pv. carotae (Xhc) is an important worldwide seed-borne disease. In 2012 and 2013, symptoms similar to bacterial leaf blight were found in carrot farms in Jeju Island, Korea. The phenotypic characteristics of the Korean isolation strains were similar to the type strain of Xhc. Pathogenicity showed symptoms on the 14th day after inoculation on carrot plants. Identification by genetic method was multi-position sequencing of the isolated strain JJ2001 was performed using four genes (danK, gyrB, fyuA, and rpoD). The isolated strain was confirmed to be most similar to Xhc M081. Furthermore, in order to analyze the genetic characteristics of the isolated strain, whole genome analysis was performed through the next-generation sequencing method. The draft genome size of JJ2001 is 5,443,372 bp, which contains 63.57% of G + C and has 4,547 open reading frames. Specifically, the classification of pathovar can be confirmed to be similar to that of the host lineage. Plant pathogenic factors and determinants of the majority of the secretion system are conserved in strain JJ2001. This genetic information enables detailed comparative analysis in the pathovar stage of pathogenic bacteria. Furthermore, these findings provide basic data for the distribution and diagnosis of Xanthomonas hortorum pv. carotae, a major plant pathogen that infects carrots in Korea.

Identification and Genetic Characterization of Pseudomonas syringae pv. actinidiae from Kiwifruit in Sichuan, China.

Pseudomonas syringae pv. actinidiae causes kiwifruit bacterial canker and poses a major threat to the kiwifruit industry. This study aimed to investigate the genetic characteristics of the P. syringae pv. actinidiae population from kiwifruit in Sichuan, China. Sixty-seven isolates obtained from diseased plants were characterized using morphological features, multiplex-PCR, and multilocus sequence analysis (MLSA). The isolates exhibited the typical colony morphology of P. syringae pv. actinidiae. Multiplex PCR amplification identified every isolate as P. syringae pv. actinidiae biovar 3. MLSA of the three housekeeping genes gapA, gyrB, and pfk, revealed that the reference strains of the five described biovars were clearly distinguished by a combined phylogenetic tree, and all of the tested isolates clustered with the reference strains of P. syringae pv. actinidiae biovar 3. Through a phylogenetic tree constructed from a single gene, it was found that pkf gene alone could distinguish biovar 3 from the other biovars. Furthermore, all P. syringae pv. actinidiae isolates analyzed by BOX-A1R-based repetitive extragenic palindromic (BOX)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR clustered into four groups. The clustering results of BOX- and ERIC-PCR indicated that group III had the largest number of isolates, accounting for 56.72 and 61.19% of all 67 isolates, respectively, and the two characterization methods were similar and complementary. The results of this study revealed that the genomes of P. syringae pv. actinidiae isolates from Sichuan had rich genetic diversity but no obvious correlation was found between clustering and geographical region. This research provides novel methodologies for rapidly detecting kiwifruit bacterial canker pathogen and a molecular differentiation at genetic level of P. syringae pv. actinidiae biovar diversity in China.

Streptomyces longhuiensis sp. nov, a novel species isolated from soil of Lilium brownii in Hunan Province, PR China.

An actinobacterium strain, designated BH-MK-02T, was isolated from the soil of Lilium brownii. The taxonomic position was determined using a polyphasic approach. Strain BH-MK-02T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into straight spore chains with a wrinkled surface. The diagnostic diamino acid was ll-diaminopimelic acid. The major menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and unidentified lipid spots. The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. The phenotypic characteristics of strain BH-MK-02T indicated that it belonged to the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain BH-MK-02T was most closely related to Streptomyces aureus CGMCC 4.1833T (99.7 %). However, the average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of strain BH-MK-02T and S. aureus CGMCC 4.1833T were 78.1 and 23.2 %, respectively, below the 96.7 and 70 % cut-off points respectively recommended for delineating Streptomyces species. Furthermore, the novel isolate could be distinguished from S. aureus CGMCC 4.1833T by morphological, physiological and biochemical characteristics. Based on all these data, strain BH-MK-02T (=MCCC 1K06237T=JCM 34789T) clearly represents a novel species within the genus Streptomyces, for which the name Streptomyces longhuiensis sp. nov. is proposed.

Identification of the Causal Agent of Bacterial Leaf Spot on Watermelon in China.

Watermelon diseases caused by pathogenic bacteria were endemic in Liaoning and Jilin Provinces from 2019 to 2020 in China, resulting in serious economic losses to the watermelon industry. This study characterized 56 strains isolated from symptomatic watermelon leaves collected from Liaoning and Jilin Provinces. Through morphological observation, 16S rRNA and gyrB sequence analysis, and BIOLOG profiles, the pathogen was identified as Pseudomonas syringae. In China, the watermelon disease caused by P. syringae was reported for the first time. The multilocus sequence analysis showed that the isolated strains belonged to three different clades within P. syringae phylogroup 2. Interestingly, most of them (79%) belonged to clade 2a, 14% were clade 2b, and 7% were clade 2d. This indicates that bacterial leaf spot outbreaks of watermelon in China were caused by multiple sources and mainly by P. syringae clade 2a.

Characterization of Pseudomonas syringae Strains Associated with Shoot Blight of Raspberry and Blackberry in Serbia.

During May 2016, severe blight symptoms were observed in several raspberry and blackberry fields in Serbia. In total, 22 strains were isolated: 16 from symptomatic raspberry shoots, 2 from asymptomatic raspberry leaves, and 4 from symptomatic blackberry shoots. Additionally, eight raspberry strains, isolated earlier from two similar outbreaks, were included in the study. Pathogenicity of the strains was confirmed on detached raspberry and blackberry shoots by reproducing the symptoms of natural infection. The strains were Gram-negative, fluorescent on King's medium B, ice nucleation positive, and utilized glucose oxidatively. All strains were levan positive, oxidase negative, nonpectolytic, arginine dihydrolase negative, and induced hypersensitivity in tobacco leaves (LOPAT + - - - +, Pseudomonas group Ia). Furthermore, all strains liquefied gelatin and hydrolyzed aesculin but did not show tyrosinase activity or utilize tartrate (GATTa + + - -). Tentative identification using morphology, LOPAT, GATTa, and ice-nucleating ability tests suggested that isolated strains belong to Pseudomonas syringae. The syrB gene associated with syringomycin production was detected in all strains. DNA fingerprints with REP, ERIC, and BOX primers generated identical profiles for 29 strains, except for strain KBI 222, which showed a unique genomic fingerprint. In all, 9 of 10 selected strains exhibited identical sequences of four housekeeping genes: gyrB, rpoD, gapA, and gltA. Five nucleotide polymorphisms were found in strain KBI 222 at the rpoD gene locus only. In the phylogenetic tree based on a concatenated sequence of all four housekeeping genes, strains clustered within phylogroup 2 (i.e., genomospecies 1) of the P. syringae species complex, with pathotype strains of P. syringae pv. aceris and P. syringae pv. solidagae as their closest relatives. There was no correlation between genotype and geographic origin, particular outbreak, host, or cultivar.

Tomato and Jute Mallow are Two New Hosts of Papaya Bunchy Top Phytoplasma, a 16SrXII-O Subgroup Strain in Nigeria.

Nigerian papaya bunchy top (NGPBT) phytoplasma was first identified in diseased papaya plants growing in Ibadan, Oyo State, Nigeria (Kazeem et al. 2021). The NGPBT phytoplasma is a 'Candidatus Phytoplasma convolvuli'-related strain and represents a subgroup lineage, 16SrXII-O (the accession number of the reference strain is MW530522, Kazeem et al. 2021). The present communication reports that NGPBT phytoplasma can also infect tomato (Solanum lycopersicum) and jute mallow (Corchorus olitorius). Since May 2020, tomato and jute mallow grwn in Ibadan have been observed to develop yellowing, little leaf, and stunting symptoms (Fig. 1). Because the symptomatic plants occurred in the region approximately 1 km adjacent to where the NGPBT disease was reported, and the symptoms of infected plants resembled those of phytoplasma infection, molecular diagnostic assays for phytoplasma detection were deployed. Total DNAs were extracted from symptomatic plants, including four tomato plants and three jute mallows, as well as from asymptomatic two tomato and two jute mallow plants. The DNA samples were subjected to semi-nested PCR using phytoplasma 16S rRNA gene-specific primers P1A and P7A, followed by P1A and 16S-SR (Lee et al. 2004). An amplicon of 1.5 kb was obtained from each of the symptomatic plants, while no amplicon resulted from DNA samples of asymptomatic plants or negative controls without DNA templates (water and PCR reagents only). PCR products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), and three clones were chosen for each sample for Sanger sequencing (Psomagen Inc., Rockville, MD, USA). The nearly full-length 16S rRNA gene sequences (1.53kb) derived from tomato (OP123558) and jute mallow (OP123559) samples were identical. Based on the iPhyClassifier phytoplasma classification web tool (Zhao et al. 2009) and the BLAST search against the NCBI nucleotide database, these phytoplasma strains showed 100% sequence identity in 16S rRNA gene with the NGPBT phytoplasma (16SrXII-O, MW530522). Moreover, two additional genetic loci including ribosomal protein genes rplV-rpsC, and rplO-secY-adk were also amplified by nested PCR or semi-nested PCR with specific primers rpStolF/rpStolR followed by rpStolF2/rpStolR (Martini et al. 2007), and SecYF1a (Xll)/MapR-703-a, followed by SecYF2a (Xll)/MapR-703-a (Lee et al. 2010). Gene fragments of rplV-rpsC (1238bp) and rplO-secY-adk (2064bp) were amplified from DNAs of diseased papaya, tomato, and jute mallow plants. The obtained sequences were deposited into GenBank, respectively: rplV-rpsC (OP123560, OP123562, and OP123563) and rplO-secY-adk (OP123565, OP123567, and OP123568). Multilocus sequence analysis (MLSA) indicated that the sequences of phytoplasmas amplified from three different plant hosts were also identical in rp, secY, and adk genes. The MLSA results demonstrate that tomato and jute mallow are two new hosts of NGPBT phytoplasmas. This also marks the first time that phytoplasma diseases are associated with tomato and jute mallow in Nigeria, as prior to this study, phytoplasma diseases were only reported in coconut palm and papaya in the country (Osagie et al. 2016; Kazeem et al. 2021). Results from the present study suggest that insect vector(s) for the transmission of the NGPBT phytoplasma are present in the region. Since both tomato and jute mallow are important vegetable crops in Nigeria, timely dissemination of emerging disease information is needed to alert growers and extension personnel in the region. In addition, ongoing incidence, and prevalence surveys of NGPBT disease indicate that more infected papaya and tomato plants have been observed in the region than in previous years. A better understanding of the NGPBT phytoplasma disease epidemiology will help devise strategies to control the diseases associated with the NGPBT phytoplasma.

Pseudomonas fitomaticsae sp. nov., isolated at Marimurtra Botanical Garden in Blanes, Catalonia, Spain.

In the framework of the research project called fitomatics, we have isolated and characterized a bacterial plant-endophyte from the rhizomes of Iris germanica, hereafter referred to as strain FIT81T. The bacterium is Gram negative, rod-shaped with lophotrichous flagella, and catalase- and oxidase-positive. The optimal growth temperature of strain FIT81T is 28 °C, although it can grow within a temperature range of 4-32 °C. The pH growth tolerance ranges between pH 5 and 10, and it tolerates 4% (w/v) NaCl. A 16S rRNA phylogenetic analysis positioned strain FIT81T within the genus Pseudomonas, and multilocus sequence analysis revealed that Pseudomonas gozinkensis IzPS32dT, Pseudomonas glycinae MS586T, Pseudomonas allokribbensis IzPS23T, 'Pseudomonas kribbensis' 46-2 and Pseudomonas koreensis PS9-14T are the top five most closely related species, which were selected for further genome-to-genome comparisons, as well as for physiological and chemotaxonomic characterization. The genome size of strain FIT81T is 6 492 796 base-pairs long, with 60.6 mol% of G+C content. Average nucleotide identity and digital DNA-DNA hybridization analyses yielded values of 93.6 and 56.1%, respectively, when the FIT81T genome was compared to that of the closest type strain P. gozinkensis IzPS32dT. Taken together, the obtained genomic, physiologic and chemotaxonomic data indicate that strain FIT81T is different from its closest relative species, which lead us to suggest that it is a novel species to be included in the list of type strains with the name Pseudomonas fitomaticsae sp. nov. (FIT81T=CECT 30374T=DSM 112699T).

Vibrio salinus sp. nov., a marine nitrogen-fixing bacterium isolated from the lagoon sediment of an islet inside an atoll in the western Pacific Ocean.

A marine, facultatively anaerobic, nitrogen-fixing bacterium, designated strain DNF-1T, was isolated from the lagoon sediment of Dongsha Island, Taiwan. Cells grown in broth cultures were Gram-negative rods that were motile by means of monotrichous flagella. Cells grown on plate medium produced prosthecae and vesicle-like structures. NaCl was required and optimal growth occurred at about 2-3% NaCl, 25-30 °C and pH 7-8. The strain grew aerobically and was capable of anaerobic growth by fermenting D-glucose or other carbohydrates as substrate. Both the aerobic and anaerobic growth could be achieved with NH4Cl as a sole nitrogen source. When N2 served as the sole nitrogen source only anaerobic growth was observed. Major cellular fatty acids were C14:0, C16:0 and C16:1 ω7c, while major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was 42.2 mol% based on the genomic DNA data. Phylogenetic analyses based on 16S rRNA genes and the housekeeping genes, gapA, pyrH, recA and gyrB, revealed that the strain formed a distinct lineage at species level in the genus Vibrio of the family Vibrionaceae. These results and those from genomic, chemotaxonomic and physiological studies strongly support the assignment of a novel Vibrio species. The name Vibrio salinus sp. nov. is proposed for the novel species, with DNF-1T (= BCRC 81209T = JCM 33626T) as the type strain. This newly proposed species represents the second example of the genus Vibrio that has been demonstrated to be capable of anaerobic growth by fixing N2 as the sole nitrogen source.

Phylogenetic Characterization and Genome Sequence Analysis of Burkholderia glumae Strains Isolated in Thailand as the Causal Agent of Rice Bacterial Panicle Blight.

Burkholderia glumae is one of the most critical rice-pathogenic bacteria, and it causes bacterial panicle blight (BPB) in rice plants. In 2017, BPB symptoms were observed from rice fields in Chiang Rai, Northern Thailand. Sixty-one isolates obtained from the symptomatic panicles of rice were initially identified as B. glumae by polymerase chain reaction (PCR) using species-specific primers. Among them, six selected strains isolated from the susceptible japonica rice cultivar DOA2 were characterized in terms of morpho-physiology, pathology, phylogenetics, and genomics. Our genome sequence analysis of the six selected strains revealed the presence of multiple prophages, which may reflect the high level of diversity in this bacterial species through dynamic horizontal gene transfer processes, including phage infection. This notion was supported by the results of phylogenetic and phylogenomic analyses, which showed the formation of several subgroups not related to the years of isolation or the geographical origins. This study reports the isolation of B. glumae as the causal pathogen of BPB disease in japonica rice in Thailand and provides genomic resources to better understand the biology and diversity of this plant pathogenic bacterium. Further studies with a vast collection of B. glumae strains from various rice-growing regions around the world are needed to elucidate the evolution, variability, and lifestyle of the pathogen.

Molecular Characterization of a Novel Relapsing Fever Borrelia Species from the Desert Cottontail (Sylvilagus audubonii) in New Mexico, USA.

The Borrelia genus comprises vector-borne, spirochete bacteria infecting vertebrates worldwide. We characterized a novel relapsing fever Borrelia species from a desert cottontail (Syvilagus audubonii) from New Mexico, US, using an established multilocus sequence analysis approach. Phylogenetic analysis of the flagellin gene (flaB) and four other protein-coding loci (clpX, pepX, recG, rplB) grouped the novel Borrelia species with hard tick relapsing fever borreliae Borrelia lonestari, Borrelia theileri, and Borrelia miyamotoi. The identity of the vectors and other vertebrate hosts, geographic distribution, and zoonotic potential of this novel Borrelia species deserve further investigation.

New design of multilocus sequence analysis of rpoB, ssrA, tuf, atpE, ku, and dnaK for identification of Mycobacterium species.

BACKGROUND: Differentiating Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is very important in the treatment process of patients. According to the American Thoracic Society guideline (ATS), NTM clinical isolates should be identified at the species level proper treatment and patient management. This study aimed to identify NTM clinical isolates by evaluationg rpoB, ssrA, tuf, atpE, ku, and dnaK genes, and use multilocus sequence analysis (MLSA) to concatenate the six genes. METHODS: Ninety-six Mycobacterium isolates, including 86 NTM and 10 MTB isolates, from all the patients referred to the certain TB Reference Centres were included. All isolates were evaluated by PCR amplification of rpoB, ssrA, tuf, ku, atpE, and dnaK genes and MLSA. RESULTS: Out of 96 isolates, 91 (94.8%), 87 (90.6%), 72 (75%), 84 (87.5%) and 79 (82.3%) were differentiated to the species level by rpoB, tuf, ssrA, dnaK and atpE genes, respectively. The ku gene was able to identify 69 (80.2%) isolates of the 86 NTM isolates to the species level. We could identify 100% of the isolates to the species level by MLSA. CONCLUSIONS: None of the PCR targets used in this study were able to completely differentiate all species. The MLSA technique used to concatenate the six genes could increase the identification of clinical Mycobacterium isolates and all 16 species were well-differentiated.

Characterization and Genetic Diversity of Pseudomonas syringae pv. syringae Isolates Associated with Rice Bacterial Leaf Spot in Heilongjiang, China.

In China, rice is one of the most important cereal crops. Rice bacterial brown leaf spot caused by P. s. pv. syringae is among the most damaging rice diseases in the Heilongjiang Province of China and results in substantial yield losses. In this study, a comprehensive analysis of the pathogen, population structure, and genetic diversity within the species was performed. For this purpose, 176 bacterial isolates of P. s. pv. syringae collected from 15 locations were characterized by using biochemical tests such as the LOPAT test, and genetic characterizations such as multilocus sequence analysis (MLSA) and repetitive PCR, using BOX, REP and ERIC primers. Biochemical testing and detection of syrB genes confirm the presence of P. s. pv. syringae, genetic characterization by MLSA and genetic fingerprinting by repetitive PCR confirmed that high genetic heterogeneity exists in the P. s. pv. syringae isolates, and clustering of the tested isolates and reference strains are related with the same genomospecies 1. This work contributes to the physiological classification of the P. s. pv. syringae isolated from Heilongjiang Province, China, and the results present new data concerning the phylogeny and genetic diversity. This type of study about P. s. pv. syringae has been not reported from this region until now.

Corrigendum: Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.

[This corrects the article DOI: 10.3389/fmicb.2021.666689.].