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.

Molecular characterisation of Histoplasma capsulatum sensu lato from Ethiopian horses reveals two distinct phylogenetic clades.

Epizootic lymphangitis (EL) is a highly prevalent and contagious infectious disease affecting horses in many parts of Ethiopia caused by Histoplasma capsulatum sensu lato ('var. farciminosum'). In this study, 12 suspected isolates of H. capsulatum sensu lato or yeasts unidentified by conventional biochemical tests isolated from Ethiopian horses with EL were characterised by internal transcribed spacer sequencing. Six of the 12 isolates were identified to be members of H. capsulatum sensu lato and the other six were Pichia kudriavzevii (synonym: Candida krusei) (n = 3), Trichosporon asahii (n = 1), Geotrichum silvicola (n = 1) and Moesziomyces aphidis (n = 1), respectively. The six H. capsulatum sensu lato isolates were further characterised by multilocus sequence analysis. Four distinct gene loci (arf [462 bases], H-anti [410 bases], ole1 [338 bases] and tub1 [272 bases]) of these six isolates as well as those of two H. capsulatum sensu lato ('var. farciminosum') reference strains (ATCC 58332 and ATCC 28798) were polymerase chain reaction (PCR)-amplified and sequenced. Phylogenetic analyses of their concatenated nucleotide sequences showed that three of the isolates and the reference strain ATCC 58332 were identical and belonged to the Eurasia clade within Latin American (LAm) A (H. suramericanum), and those of the other three isolates and the reference strain ATCC 28798 were identical and belonged to the Africa clade. At least two distinct phylogenetic clades of H. capsulatum sensu lato were circulating in Ethiopian horses with EL. Advanced molecular technologies and bioinformatics tools are crucial for the accurate identification and typing of pathogens as well as the discovery of novel microorganisms in veterinary microbiology.

Using multilocus sequence analysis with four concatenated housekeeping gene loci, at least two distinct phylogenetic clades, namely Eurasia clade and Africa clade, of Histoplasma capsulatum sensu lato were confirmed to be circulating in Ethiopian horses with epizootic lymphangitis.

Whole genome-based comparative analysis of the genus Streptomyces reveals many misclassifications.

Streptomyces species are experts in the production of bioactive secondary metabolites; however, their taxonomy has fallen victim of the tremendous interest shown by the scientific community, evident in the discovery of numerous synonymous in public repositories. Based on genomic data from NCBI Datasets and nomenclature from the LPSN database, we compiled a dataset of 600 Streptomyces species along with their annotations and metadata. To pinpoint the most suitable taxonomic classification method, we conducted a comprehensive assessment comparing multiple methodologies, including analysis of 16S rRNA, individual housekeeping genes, multilocus sequence analysis (MLSA), and Fast Average Nucleotide Identity (FastANI) on a subset of 409 species with complete data. Due to insufficient resolution of 16S rRNA and inconsistency observed in individual housekeeping genes, we performed a more in-depth analysis, comparing only FastANI and MLSA, which expanded our dataset to include 502 species. With FastANI validated as the preferred method, we conducted pairwise analysis on the entire dataset identifying 59 non-unique species among the 600, and subsequently refined the dataset to 541 unique species. Additionally, we collected data on 724 uncharacterized Streptomyces strains to investigate the uniqueness potential of the unannotated fraction of the Streptomyces genus. Utilizing FastANI, 289 strains could be successfully classified into one of the 541 Streptomyces species. KEY POINTS: • Evaluation of taxonomic classification methods for Streptomyces species. • Whole genome analysis, specifically FastANI, has been chosen as preferred method. • Various reclassifications are proposed within the Streptomyces genus.

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.

Francisella sciaenopsi sp. nov. isolated from diseased red drum Sciaenops ocellatus in Florida, USA.

Piscine francisellosis is one of the most important bacterial diseases affecting various fish species worldwide. Francisella orientalis, F. noatunensis, and F. salimarina (F. marina) have been reported as etiological agents of disease in fish. A Francisella sp. was isolated from several diseased red drum Sciaenops ocellatus experiencing morbidity in Florida, USA, in 2008. In this study, molecular and phenotypic characterization of the recovered isolate was conducted. Phenotypically, the isolate showed a biochemical reaction profile distinct from that of F. orientalis and F. salimarina. Although the 16S rRNA sequence of this isolate shared 99.61% identity to the type strain of F. philomiragia O#319LT, whole genome analysis (average nucleotide identity <95%; digital DNA-DNA hybridization <70%) and a multilocus sequence analysis of 8 concatenated housekeeping genes in comparison with other Francisella spp. indicated that this isolate was a novel Francisella species, more closely related to F. orientalis. Immersion, intracoelomic injection, and co-habitation challenges using a Nile tilapia Oreochromis niloticus fingerling model of infection were done to investigate virulence in a piscine model. Variably pigmented granulomas and pigmented macrophage aggregates were observed in the kidneys and spleens of the challenged fish, but no mortality was recorded during the 15 d challenge period, suggesting that this novel Francisella sp. might be an opportunistic pathogen of fish. Based on the phenotypic and genotypic differences from other Francisella spp. observed in this study, we propose the name Francisella sciaenopsi sp. nov. for this novel isolate.

Pantoea tagorei sp. nov., a Rhizospheric Bacteria with Plant Growth-Promoting Activities.

A Gram-negative, short-rod, non-motile, facultatively anaerobic, potassium-solubilizing bacterium MR1 (Mine Rhizosphere) was isolated from rhizospheric soil of an open-cast coal mine of Jharia, Jharkhand, India. Isolate MR1 can grow in a broad range of temperature, pH, and NaCl concentrations. The 16S rRNA gene sequence of the strain showed 99.24% similarity with Pantoea septica LMG 5345T. However, maximum-likelihood tree constructed using 16S rRNA gene sequence, multilocus sequence analysis using concatenated sequences of ten housekeeping genes, whole-genome based phylogenetic reconstruction, digital DNA-DNA hybridization, and average nucleotide identity (ANIm and ANIb) values indicated segregation of MR1 from its closest relatives. Fatty acid profile of MR1 also suggested the same, with clear variation in major and minor fatty acid contents, having C13:0 anteiso (10-Methyldodecanoic acid) as the unique one. Thus, considering all polyphasic data, strain MR1T (= MTCC 13265T, where 'T' stands for Type strain) is presented as a novel species of the genus Pantoea, for which the name Pantoea tagorei sp. nov. is proposed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01147-9.

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.

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.

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.

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.

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.

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).

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.

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.

Genetic and Phenotypic Characterization of Xanthomonas Species Pathogenic in Wheat in Uruguay.

Bacterial diseases affecting wheat production in Uruguay are an issue of growing concern yet remain largely uninvestigated in the region. Surveys of 61 wheat fields carried out from 2017 to 2019 yielded a regional collection of 63 strains identified by 16S rRNA gene analysis as Xanthomonas spp. A real-time PCR protocol with species-specific primers previously reported allowed the identification of 44 strains as X. translucens, the causal agent of bacterial leaf streak (BLS) in wheat and other cereal crops. Multilocus sequence analysis of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD) revealed that these strains were most closely related to X. translucens pv. undulosa, the pathovar that is most commonly associated with BLS of wheat. Multilocus sequence typing was applied to examine the genetic diversity of X. translucens strains. Strains were assigned to four different sequence types, three of which were previously reported globally. Additionally, 17 Xanthomonas strains not belonging to X. translucens were obtained from diseased wheat leaves. Phylogenetic analysis showed that these strains are closely related to X. prunicola and clustered together with previously uncharacterized Xanthomonas strains isolated from wheat in Minnesota. In planta pathogenicity assays carried out on a BLS-susceptible wheat cultivar showed that X. translucens pv. undulosa strains caused brown necrosis symptoms typical of BLS, whereas non-translucens Xanthomonas sp. strains elicited an atypical symptom of dry necrosis. These findings suggest that local wheat fields are affected by X. translucens pv. undulosa and by a new wheat pathogen within the Xanthomonas genus.

Characterization, Pathogenicity, Phylogeny, and Comparative Genomic Analysis of Pseudomonas tolaasii Strains Isolated from Various Mushrooms in China.

Since 2016, devastating bacterial blotch affecting the fruiting bodies of Agaricus bisporus, Cordyceps militaris, Flammulina filiformis, and Pleurotus ostreatus in China has caused severe economic losses. We isolated 102 bacterial strains and characterized them polyphasically. We identified the causal agent as Pseudomonas tolaasii and confirmed the pathogenicity of the strains. A host range test further confirmed the pathogen's ability to infect multiple hosts. This is the first report in China of bacterial blotch in C. militaris caused by P. tolaasii. Whole-genome sequences were generated for three strains: Pt11 (6.48 Mb), Pt51 (6.63 Mb), and Pt53 (6.80 Mb), and pangenome analysis was performed with 13 other publicly accessible P. tolaasii genomes to determine their genetic diversity, virulence, antibiotic resistance, and mobile genetic elements. The pangenome of P. tolaasii is open, and many more gene families are likely to emerge with further genome sequencing. Multilocus sequence analysis using the sequences of four common housekeeping genes (glns, gyrB, rpoB, and rpoD) showed high genetic variability among the P. tolaasii strains, with 115 strains clustered into a monophyletic group. The P. tolaasii strains possess various genes for secretion systems, virulence factors, carbohydrate-active enzymes, toxins, secondary metabolites, and antimicrobial resistance genes that are associated with pathogenesis and adapted to different environments. The myriad of insertion sequences, integrons, prophages, and genome islands encoded in the strains may contribute to genome plasticity, virulence, and antibiotic resistance. These findings advance understanding of the determinants of virulence, which can be targeted for the effective control of bacterial blotch disease.

Genetic diversity of Lactobacillus delbrueckii isolated from raw milk in Hokkaido, Japan.

Lactic acid bacteria (LAB) play important roles in acid production and flavor formation in fermented dairy products. Lactic acid bacteria strains with distinct characteristics confer unique features to products. Diverse LAB have been identified in raw milk and traditional fermented milk prepared from raw milk. However, little is known about LAB in raw milk in Japan. To preserve diverse LAB as potential starters or probiotics for future use, we have isolated and identified various kinds of LAB from raw milk produced in Japan. In this study, we focused on Lactobacillus delbrueckii, one of the most important species in the dairy industry. We identified L. delbrueckii subspecies isolated from raw milk in Hokkaido, Japan, by analyzing intraspecific diversity using 4 distinct methods, hsp60 cluster analysis, multilocus sequence analysis, core-genome analysis, and whole-genome analysis based on average nucleotide identity. The subspecies distribution and a new dominant subset of L. delbrueckii from raw milk in Japan were revealed. The discovery of new strains with different genotypes is important for understanding the geographic distribution and characteristics of the bacteria and further their use as a microbial resource with the potential to express unconventional flavors and functionalities. The strains identified in this study may have practical applications in the development of fermented dairy products.

Cruciferous Weeds Do Not Act as Major Reservoirs of Inoculum for Black Rot Outbreaks in New York State.

Cruciferous weeds have been shown to harbor diverse Xanthomonas campestris pathovars, including the agronomically damaging black rot of cabbage pathogen, X. campestris pv. campestris. However, the importance of weeds as inoculum sources for X. campestris pv. campestris outbreaks in New York remains unknown. To determine if cruciferous weeds act as primary reservoirs for X. campestris pv. campestris, fields that were rotating between cabbage or had severe black rot outbreaks were chosen for evaluation. Over a consecutive 3-year period, 148 cruciferous and noncruciferous weed samples were collected at 34 unique sites located across five New York counties. Of the 148 weed samples analyzed, 48 X. campestris isolates were identified, with a subset characterized using multilocus sequence analysis. All X. campestris isolates originated from weeds belonging to the Brassicaceae family, with predominant weed hosts being shepherd's purse (Capsella bursa-pastoris), wild mustard (Sinapis arvensis), yellow rocket (Barbarea vulgaris), and pennycress (Thlaspi arvense). Identifying pathogenic X. campestris weed isolates was rare, with only eight isolates causing brown necrotic leaf spots or typical V-shaped lesions on cabbage. There was no evidence of cabbage-infecting weed isolates persisting in an infected field by overwintering in weed hosts; however, similar cabbage and weed X. campestris haplotypes were identified in the same field during an active black rot outbreak. X. campestris weed isolates are genetically diverse both within and between fields, but our findings indicate that X. campestris weed isolates do not appear to act as primary sources of inoculum for B. oleracea fields in New York.

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.

First Report of Bacterial Pineapple Heart Rot Caused by Dickeya zeae in Puerto Rico.

In Puerto Rico, the agricultural production of pineapple (Ananas comosus (L.) Merr.) comprises nearly 5,000 tons harvested annually from over 250 ha (USDA 2018). With an annual income of approximately $3 million USD, pineapple ranks fourth in importance among Puerto Rican crops (USDA 2018). Recently, the pineapple industry on the island underwent a change from growing a local cultivar known as "Cabezona" to cultivar MD2, introduced from Hawaii around 1996 (SEA 2015), because this cultivar produces fruit more than once during a single growing season. In August 2018 (when the rainy season normally starts in Puerto Rico), soft rot symptoms appeared at commercial fields in Manatí (WGS 84 Lat 18.42694, Lng -66.44779) and persisted through 2019. Symptoms observed in the field included foliar water-soaked lesions with gas-filled blisters, especially at the base of the leaf. Leaves exhibited brown discoloration and a fetid odor (rot) at the basal portion of the plant. Finally, leaves collapsed at the center of the pineapple crown, effectively killing the apex and preventing the fruit from developing. Disease incidence ranged from 25% to 40% depending on the weather and season; when there was more rain, there was higher disease incidence. Symptomatic leaves were collected in February 2019, disinfected with 70% ethanol, and rinsed with sterile distilled water. Tissue sections (5mm2) were placed in nutrient agar. Bacterial colony-forming units (CFU) were a translucent cream color, circular, with a flat convex surface and wavy edge. Biochemical analysis showed that bacteria were Gram-negative, oxidase positive, catalase positive, and facultatively anaerobic. Pathogenicity was tested on leaves of one-and-a-half-year-old pineapple seedlings in humid chambers. Bacteria were grown on sterile nutrient agar for 3 days at 25 ± 2°C. Inoculation assays (three replications) were performed using 1X108 CFU/ml of bacteria suspended in sterile water and applied with a cotton swab to leaves wounded with a needle. The inoculated tissue was incubated at 28°C and kept in a dark environment. Negative controls were inoculated with sterile water. Five days after inoculation, foliar water-soaked lesions were observed, followed by the formation of brown leaf tissue and gas-filled blisters, the same symptoms observed in the field. A partial DNA sequence of the 16S rRNA gene of the bacterial isolate and the re-isolated bacteria were amplified using primers 27F and 1492R (Lane et al. 1985) and sequenced. The isolate was determined to the genus Dickeya through a BLAST® search against sequences available in the database of the National Center for Biotechnology Information (NCBI). This partial 16S rRNA sequence of the bacterial isolate was deposited in GenBank® at NCBI (Accession no. MT672704). To determine the identity of the Dickeya species, we sequenced the genes dnaA, gyrB, dnaX, and recN (Marrero et al. 2013) for the bacterial isolate (GenBank accession nos. OM276852, OM276853, OM276854, and OM276855) and conducted a Multilocus Sequence Analysis including reference Dickeya sequences of Marrero et al., 2013. The Phylogenetic analysis (using WinClada) resolved the Puerto Rican isolate as belonging to a clade broadly ascribable to D. zeae, most closely related to strains isolated from earlier Hawaiian pineapple bacterial heart rot outbreaks. Dickeya zeae was responsible for bacterial heart rot of pineapple in Malaysia and was later reported as the causal agent for outbreaks in Costa Rica and Hawaii (Kaneshiro et al. 2008; Sueno et al. 2014; Ramachandran et al. 2015). D. zeae had not previously been reported as causing bacterial heart rot in pineapples in Puerto Rico and this study points to a close relationship with strains first detected in Hawaii and which should further be explored to determine the precise nature of this relationship. This information should facilitate the adoption of effective control measures for this disease on the island, promote more effective methods of preventing future introductions of pathogens, and encourage further investigations into the occurrence of D. zeae on the island.