Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia.

Multi-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations.

Epicoccum spp. Causing Maize Leaf Spot in Heilongjiang Province, China.

Maize leaf spot occurs worldwide and affects maize production. Maize can be infected by several pathogens causing leaf spot, such as Bipolaris zeicola, Bipolaris maydis, Curvularia species, Alternaria species, etc. In the current study, 30 Epicoccum isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of nuLSU, ITS, tub2, and rpb2. These maize isolates were grouped into five Epicoccum species, including E. nigrum, E. layuense, E. sorghinum, E. latusicollum, and E. pneumoniae. Pathogenicity tests showed that all five Epicoccum species could produce small ellipse- and spindle-shaped spots on maize leaves. The lesion center was grayish yellow to dark gray and surrounded by a chlorotic area. Furthermore, the Epicoccum isolates exhibited high pathogenicity to 20 main maize varieties of Heilongjiang Province but showed different sensitivities to the commonly used fungicides carbendazim and tebuconazole. In addition, these Epicoccum isolates showed different production capacity of pectinase, cellulase, protease, amylase, laccase, and gelatinase, but all showed high lipase activity. This is the first report globally of E. layuense, E. latusicollum, and E. pneumoniae as causal agents of maize leaf spot. E. pneumoniae was first reported as a plant pathogen.

First report of Preharvest Decay caused by Colletotrichum chrysophilum on apples in Italy (South Tyrol).

Apple (Malus × domestica Borkh.) is economically the most important fruit crop in South Tyrol (Italy). At the end of the growing season 2020, necrotic lesions and chlorosis developed on leaves and premature leaf dropping was observed on the cultivars Gala, Granny Smith and Cripps Pink(cov)/Rosy Glow(cov) in the Etsch/Adige valley. After the appearance of these symptoms, small circular brownish spots were observed on above 90 % of apples in the respective orchards. Fungal isolates were obtained from symptomatic apples by culturing small portions of fruit flesh from the lesion margin on potato dextrose agar (PDA) at 25°C in the dark. The colonies showed a white to peachy color on the upper surface and were greyish on the reverse side. Conidia were cylindrical, predominantly rounded and averaged 16.39 ± 1.37 µm and 5.75 ± 0.81 µm (n = 33), consistent morphological characteristics as described recently in Fuentes-Aragón et al. (2021). A multi-locus sequence analysis according to Astolfi et al. (2022) and Weir et al. (2022) was conducted based on the internal transcribed spacer (ITS) region and on fragments of actin (ACT), DNA-(apurinic or apyrimidinic site) lyase (APN2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), beta-tubulin (TUB2), and intergenic spacer and partial mating type (Mat1-2) genes. MegaBlast analysis revealed 100 % identity to the reference genome of C. chrysophilum for ITS (NR_160821), for ACT (KX093982), APN2 (KX094018), CAL (KX094063), GAPDH (KX094183), 99.29 % for TUB2 (KX094285), 99.85 % for Mat1-2 (KX094325) and 99.87 % for GS (KX094204). Sequences were deposited in GenBank (ITS: OK485032; ACT: OK539650; APN2: ON624100; CAL: ON624104; GAPDH: OK539654; GS: ON624108; TUB2: OK539658; Mat1-2: ON645973). Pathogenicity assays were performed on wounded and unwounded apples of cvs. Gala and Rosy Glow(cov). Apples of both cvs. were wounded with a sterile needle. For each variant, 8 fruits were inoculated with 10 µL spore suspension (1 × 106 spores mL-1) in 0.05 % Tween®20 or with 10 µL 0.05 % Tween®20 as control. The apples were put into plastic boxes containing moist tissue and incubated at 25°C, 100 % relative humidity and 12 h photoperiod. First lesions appeared on inoculated wounded fruits after 5 days, whereas unwounded and control fruits remained asymptomatic. After 15 days, symptoms could be observed only on inoculated wounded apples with spore suspension: on 95 % of individual wounds of Rosy Glow(cov) and on 77.5 % of Gala. Koch's postulates were fulfilled by re-isolating the fungus and by identifying the re-isolates as C. chrysophilum (ITS: OK485033-OK485035; ACT: OK539651-OK539653; APN2: ON624101-ON624103; CAL: ON624105-ON624107; GAPDH: OK539655-OK539657; GS: ON624109; TUB2: OK539659-OK539661; Mat1-2: ON645974-ON645976). To date, only one study confirmed C. chrysophilum as causal agent of apple bitter rot in Europe (Cabrefiga et al. 2022). Recently, Astolfi et al. (2022) reclassified C. chrysophilum as the main causal agent of GLS on apples in Southern Brazil and Uruguay. The authors stressed that C. chrysophilum might also be the potential agent of GLS in Europe (Astolfi et al. 2022). This confirms the observations made in 2020 in South Tyrol, where massive leaf spots preceded the symptoms on fruit. To the best of our knowledge, this is the first report of a preharvest decay on apples caused by C. chrysophilum in Italy (South Tyrol).

Pseudomonas tohonis sp. nov., isolated from the skin of a patient with burn wounds in Japan.

Strain TUM18999T was isolated from the skin of a patient with burn wounds in Japan. The strain was successfully cultured at 20-42 °C (optimum, 30-35 °C) in 1.0-4.0% NaCl (w/v) and at pH 5.5-9.5, optimum pH 5.5-8.5. The phylogenetic tree reconstructed using 16S rRNA, gyrB, rpoB and rpoD gene sequences indicated that strain TUM18999T is closely related to Pseudomonas otitidis MCC10330T. Although the partial 16S rRNA gene sequence (1412 bp) of TUM18999T exhibits high similarity to those of Pseudomonas alcaligenes NBRC 14159T (99.08 %) and Pseudomonas otitidis MCC10330T (98.51 %), multi-locus sequence analysis using 16S rRNA, gyrB, rpoB and rpoD genes reveals a clear distinction between TUM18999T and other Pseudomonas species. In addition, an average nucleotide identity >90 % was not observed in the P. aeruginosa group. Moreover, TUM18999T and P. otitidis can be distinguished based on the minimum inhibitory concentration for carbapenem. Meanwhile, the cellular fatty acids are enriched with C18 : 1 ω7c/C18 : 1 ω6c (34.35 %), C16 : 1 ω7c/C16 : 1 ω6c (24.22 %), C16 : 0 (19.79 %) and C12 : 0 (8.25 %). Based on this evidence, strain TUM18999T can be defined as representing a novel Pseudomonas species, with the proposed name Pseudomonas tohonis sp. nov. The type strain is TUM18999T (GTC 22698T=NCTC 14580T).

Pseudomonas piscium sp. nov., Pseudomonas pisciculturae sp. nov., Pseudomonas mucoides sp. nov. and Pseudomonas neuropathica sp. nov. isolated from rainbow trout.

Six Gram negative, motile bacteria were isolated from rainbow trout (Oncorhynchus mykiss). The 16S rRNA sequence similarity values grouped them in the Pseudomonas mandelii (strains P49, P50T, 154aT and P154b), Pseudomonas fluorescens (strain P115T) and Pseudomonas koreensis (strain P155T) phylogenetic subgroups in the genus Pseudomonas. The DNA G+C content ranged from 58.5 to 60 mol%. The strains were characterized phenotypically using API 20NE and Biolog GENIII tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and fatty acid contents. Multi-locus sequence analysis with four housekeeping gene sequences (rpoD, rpoB, gyrB and 16S rRNA) together with genome comparisons by average nucleotide identity and genome-to-genome distance calculations were performed. Results showed that the similarity values of these strains to known species type strains were lower than the thresholds established for species in the genus Pseudomonas. Based on these data, we concluded that strains P49, P50T, P115T, P154aT, P154b and P155T belonged to four novel species. The names proposed are: Pseudomonas piscium sp. nov. for strains P49 and P50T with P50T (=CECT 30175T=CCUG 74871T) as the type strain; Pseudomonas pisciculturae sp. nov. for strain P115T (CECT 30173T=CCUG 74873T); Pseudomonas mucoides sp. nov. for strains P154aT and P154b with P154aT (=CECT 30177T=CCUG 74874T) as the type strain; and Pseudomonas neuropathica sp. nov. for strain P155T (=CECT 30178T=CCUG 74875T).

African histoplasmosis: new clinical and microbiological insights.

African histoplasmosis is defined as the fungal infection caused by Histoplasma capsulatum var. duboisii (Hcd). Studies focused on distinguishing Hcd and H. capsulatum var. capsulatum (Hcc), which coexist in Africa, are scarce or outdated, and African strains are continuously underrepresented. In this work, 13 cases of African patients with histoplasmosis diagnosed in the Spanish Mycology Reference Laboratory have been reviewed showing that 77% had disseminated disease and AIDS as underlying disease although Hcd infection has been classically considered a rare presentation in AIDS patients. Strains isolated from these patients and other clinical and reference strains were studied by assessing classical identification methods and performing a three-loci multi-locus sequence analysis (MLSA). Classical identification methods based on biochemical tests and measurement of yeast size proved to be useless in distinguishing both varieties. The MLSA defined an African cluster, with a strong statistical support, that included all strains with African origin. Finally, mating type was also determined by using molecular methods revealing an unequal mating type distribution in African strains. In conclusion, historical statements and classical identification methods were useless to distinguish between varieties, whereas molecular analyses revealed that all strains with African origin grouped together suggesting that traditional classification should be revised. Further investigation is required in order to unravel traditional concepts about Hcd infection and support results obtained in this work.

Characterization of three strains of Capnocytophaga canis isolated from patients with sepsis.

Capnocytophaga canimorsus and Capnocytophaga cynodegmi, both commensal bacteria in the oral cavities of dogs and cats, are zoonotic pathogens. In particular, C. canimorsus causes sepsis and fatal septic shock. Recently, a novel Capnocytophaga species, C. canis, was isolated from the oral cavities of healthy dogs. It is reportedly oxidase-negative and therefore considered avirulent in humans. In the present study, three strains of C. canis were isolated from Japanese patients with sepsis. All three strains, HP20001, HP33001 and HP40001, were oxidase-positive. Nucleotide sequence identities of the 16S rRNA gene of the three strains to the C. canimorsus type strain ATCC35979, C. cynodegmi type strain ATCC49044 and C. canis type strain LMG29146 were 96.9-97.0%, 96.9-97.0% and 99.7-99.8%, respectively. Multi-locus sequence analysis based on seven house-keeping genes, dnaJ, fumC, glyA, gyrB, murG, trpB and tuf, revealed that the oxidase-positive C. canis strains isolated in Japan and oxidase-negative strains of C. canis from canine oral cavities in Switzerland were clustered in different genetic subgroups. These results indicate that the virulence of C. canis strains in humans is associated with oxidase activity.

Pseudomonas moraviensis subsp. stanleyae, a bacterial endophyte of hyperaccumulator Stanleya pinnata, is capable of efficient selenite reduction to elemental selenium under aerobic conditions.

AIMS: To identify bacteria with high selenium tolerance and reduction capacity for bioremediation of wastewater and nanoselenium particle production. METHODS AND RESULTS: A bacterial endophyte was isolated from the selenium hyperaccumulator Stanleya pinnata (Brassicaceae) growing on seleniferous soils in Colorado, USA. Based on fatty acid methyl ester analysis and multi-locus sequence analysis (MLSA) using 16S rRNA, gyrB, rpoB and rpoD genes, the isolate was identified as a subspecies of Pseudomonas moraviensis (97.3% nucleotide identity) and named P. moraviensis stanleyae. The isolate exhibited extreme tolerance to SeO3(2-) (up to 120 mmol l(-1)) and SeO4(2-) (>150 mmol l(-1)). Selenium oxyanion removal from growth medium was measured by microchip capillary electrophoresis (detection limit 95 nmol l(-1) for SeO3(2-) and 13 nmol l(-1) for SeO4(2-)). Within 48 h, P. moraviensis stanleyae aerobically reduced SeO3(2-) to red Se(0) from 10 mmol l(-1) to below the detection limit (removal rate 0.27 mmol h(-1) at 30 °C); anaerobic SeO3(2-) removal was slower. No SeO4(2-) removal was observed. Pseudomonas moraviensis stanleyae stimulated the growth of crop species Brassica juncea by 70% with no significant effect on Se accumulation. CONCLUSIONS: Pseudomonas moraviensis stanleyae can tolerate extreme levels of selenate and selenite and can deplete high levels of selenite under aerobic and anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas moraviensis subsp. stanleyae may be useful for stimulating plant growth and for the treatment of Se-laden wastewater.

The genetic diversity and evolution analysis of the Hainan melioidosis outbreak strains.

Melioidosis is a zoonotic disease, with its outbreaks being rare and indicative of an unusual concurrence of extreme climate and natural environmental factors. An outbreak of melioidosis cases emerged in Hainan following Typhoon "Dianmu" from October to December 2021, presenting an opportunity to identify the environmental sources of infection for these cases due to its nature as a well-defined point-source cluster. To investigate the relationship between the occurrence of these melioidosis cases and the environment, we extracted the entire genome of 25 clinical strains and conducted MLST typing, followed by whole genome sequencing and analysis of molecular genetic information for four ST46 genotypes from these strains. Phylogenetic and evolutionary relationships between Hainan sequence types (STs) and those found in other endemic regions were analyzed using IslandPath-DIMO, PHASTER, e-BURST, PHYLOViZ, and the maximum likelihood method. Notably, a total of 25 clinical strains were identified, encompassing 12 STs (ST46, ST1105, ST1991, ST30, ST1992, ST50, ST164, ST55, ST70, ST1993, ST1545, and ST58), with ST1991, ST1992, and ST1993 being newly discovered subtypes. PHYLOViZ clustering analysis divided the strains into two groups (A and B), both closely related to the Asian region. Phylogenetic tree analysis further revealed that most of the strains in this study were closely related to those found in Australia and Thailand. Analysis of patient information and visits to their residences suggested that contaminated water sources might be the primary source of infection during this outbreak. Our findings underscore that extreme weather events, such as typhoons, significantly increase the infection rate of B. pseudomallei, along with its genetic diversity, necessitating additional prevention strategies to control these B. pseudomallei infections.

Redescription, molecular characterisation and Wolbachia endosymbionts of Mansonella (Tupainema) dunni (Mullin & Orihel, 1972) (Spirurida: Onchocercidae) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia.

The genus Mansonella Faust, 1929 includes 29 species, mainly parasites of platyrrhine monkeys in South America and anthropoid apes in Africa. In Malaysia, Mansonella (Tupainema) dunni (Mullin & Orihel, 1972) was described from the common treeshrew Tupaia glis Diard & Duvaucel (Scandentia). In a recent classification of the genus Mansonella, seven subgenera were proposed, with M. (Tup.) dunni as a monotypic species in the subgenus Tupainema. In this study, we collected new material of M. (Tup.) dunni from common treeshrews in Peninsular Malaysia and redescribed the morphological features of this species. We found that M. (Tup.) dunni differs from M. (Cutifilaria) perforata Uni et al., 2004 from sika deer Cervus nippon (Cetartiodactyla) in Japan, with regards to morphological features and predilection sites in their respective hosts. Based on multi-locus sequence analyses, we examined the molecular phylogeny of M. (Tup.) dunni and its Wolbachia genotype. Species of the genus Mansonella grouped monophyletically in clade ONC5 and M. (Tup.) dunni was placed in the most derived position within this genus. Mansonella (Tup.) dunni was closely related to M. (M.) ozzardi (Manson, 1897) from humans in Central and South America, and most distant from M. (C.) perforata. The calculated p-distances between the cox1 gene sequences for M. (Tup.) dunni and its congeners were 13.09% for M. (M.) ozzardi and 15.6-16.15% for M. (C.) perforata. The molecular phylogeny of Mansonella spp. thus corroborates their morphological differences. We determined that M. (Tup.) dunni harbours Wolbachia endosymbionts of the supergroup F genotype, in keeping with all other Mansonella species screened to date.

Phylogenetic diversity of Rhizobium species recovered from nodules of common beans (Phaseolus vulgaris L.) in fields in Uganda: R. phaseoli, R. etli, and R. hidalgonense.

A total of 75 bacterial isolates were obtained from nodules of beans cultivated across 10 sites in six agro-ecological zones in Uganda. Using recA gene sequence analysis, 66 isolates were identified as members of the genus Rhizobium, while nine were related to Agrobacterium species. In the recA gene tree, most Rhizobium strains were classified into five recognized species. Phylogenetic analysis based on six concatenated sequences (recA-rpoB-dnaK-glnII-gyrB-atpD) placed 32 representative strains into five distinct Rhizobium species, consistent with the species groups observed in the recA gene tree: R. phaseoli, R. etli, R. hidalgonense, R. ecuadorense, and R. sophoriradicis, with the first three being the predominant. The rhizobial strains grouped into three nodC subclades within the symbiovar phaseoli clade, encompassing strains from distinct phylogenetic groups. This pattern reflects the conservation of symbiotic genes, likely acquired through horizontal gene transfer among diverse rhizobial species. The 32 representative strains formed symbiotic relationships with host beans, while the Agrobacterium strains did not form nodules and lacked symbiotic genes. Multivariate analysis revealed that species distribution was influenced by the environmental factors of the sampling sites, emphasizing the need to consider these factors in future effectiveness studies to identify effective nitrogen-fixing strains for specific locations.

The Botryosphaeriaceae: genera and species known from culture.

In this paper we give an account of the genera and species in the Botryosphaeriaceae. We consider morphological characters alone as inadequate to define genera or identify species, given the confusion it has repeatedly introduced in the past, their variation during development, and inevitable overlap as representation grows. Thus it seems likely that all of the older taxa linked to the Botryosphaeriaceae, and for which cultures or DNA sequence data are not available, cannot be linked to the species in this family that are known from culture. Such older taxa will have to be disregarded for future use unless they are epitypified. We therefore focus this paper on the 17 genera that can now be recognised phylogenetically, which concentrates on the species that are presently known from culture. Included is a historical overview of the family, the morphological features that define the genera and species and detailed descriptions of the 17 genera and 110 species. Keys to the genera and species are also provided. Phylogenetic relationships of the genera are given in a multi-locus tree based on combined SSU, ITS, LSU, EF1-α and ß-tubulin sequences. The morphological descriptions are supplemented by phylogenetic trees (ITS alone or ITS + EF1-α) for the species in each genus. TAXONOMIC NOVELTIES: New species - Neofusicoccum batangarum Begoude, Jol. Roux & Slippers. New combinations - Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves, Botryosphaeria ramosa (Pavlic, T.I. Burgess, M.J. Wingf.) A.J.L. Phillips & A. Alves, Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips, Cophinforma mamane (D.E. Gardner) A.J.L. Phillips & A. Alves, Dothiorella pretoriensis (Jami, Gryzenh., Slippers & M.J. Wingf.) Abdollahz. & A.J.L. Phillips, Dothiorella thailandica (D.Q. Dai., J.K. Liu & K.D. Hyde) Abdollahz., A.J.L. Phillips & A. Alves, Dothiorella uruguayensis (C.A. Pérez, Blanchette, Slippers & M.J. Wingf.) Abdollahz. & A.J.L. Phillips, Lasiodiplodia lignicola (Ariyawansa, J.K. Liu & K.D. Hyde) A.J.L. Phillips, A. Alves & Abdollahz., Neoscytalidium hyalinum (C.K. Campb. & J.L. Mulder) A.J.L. Phillips, Groenewald & Crous, Sphaeropsis citrigena (A.J.L. Phillips, P.R. Johnst. & Pennycook) A.J.L. Phillips & A. Alves, Sphaeropsis eucalypticola (Doilom, J.K. Liu, & K.D. Hyde) A.J.L. Phillips, Sphaeropsis porosa (Van Niekerk & Crous) A.J.L. Phillips & A. Alves. Epitypification (basionym) - Sphaeria sapinea Fries. Neotypifications (basionyms) - Botryodiplodia theobromae Pat., Physalospora agaves Henn, Sphaeria atrovirens var. visci Alb. & Schwein.

Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species.

Xylophilus ampelinus is the causal agent of blight and canker on grapevine. Only a few data are available on this species implying that the occurrence of this pathogen may be underestimated, and its actual ecological niche may not be understood. Moreover, its genetic diversity is not well known. To improve our knowledge of this species, an analysis of the complete genome sequences available in NCBI was performed. It appeared that several sequences are misidentified. The complete genome sequence of the type strain was obtained and primers designed in order to sequence gyrB and rpoD genes for the strains held in CIRM-CFBP. The genetic barcoding data were obtained for 93 strains, isolated over 35 years and from several geographical origins. The species revealed to be strongly homogenous, displaying nearly identical sequences for all strains. However, the oldest strains of this collection were isolated in 2001 therefore, a new isolation campaign and epidemiological surveys are necessary, along with the obtention of new complete genome sequences for this species.

Taxonomy and Phylogenetic Appraisal of Dothideomycetous Fungi Associated with Magnolia, Lilium longiflorum and Hedychium coronarium.

This paper highlights the taxonomy of some interesting saprobic microfungi associated with dead plant materials of Hedychium coronarium, Lilium longiflorum, and Magnolia species. The taxa reported in this study belong to the orders Pleosporales and Kirschsteiniotheliales (Dothideomycetes). These taxa were identified based on multi-locus phylogeny of nuclear ribosomal DNA (rDNA) (LSU, SSU, and ITS) and protein-coding genes (tef1-α and rpb2), together with comprehensive morphological characterization. Two novel saprobic species, Leptoparies magnoliae sp. nov. and Neobambusicola magnoliae sp. nov., are introduced from Magnolia species in Thailand. Another new species, Asymmetrispora zingiberacearum sp. nov., is also described from dead stems of H. coronarium, which is the first asexual morph species of the genus Asymmetrispora. In addition, Ramusculicola thailandica and Kirschsteiniothelia thailandica are reported as new host records from dead twigs of Magnolia species. Sphaerellopsis paraphysata is reported as a new host record from L. longiflorum. Newly described taxa are compared with other similar species and detailed descriptions, micrographs, and phylogenetic trees to show the positions are provided.

Epidemiology of Nocardia Species at a Tertiary Hospital in Southern Taiwan, 2012 to 2020: MLSA Phylogeny and Antimicrobial Susceptibility.

The identification and antimicrobial susceptibility of Nocardia spp. are essential for guiding antibiotic treatment. We investigated the species distribution and evaluated the antimicrobial susceptibility of Nocardia species collected in southern Taiwan from 2012 to 2020. A total of 77 Nocardia isolates were collected and identified to the species level using multi-locus sequence analysis (MLSA). The susceptibilities to 15 antibiotics for Nocardia isolates were determined by the broth microdilution method, and the MIC50 and MIC90 for each antibiotic against different species were analyzed. N. cyriacigeorgica was the leading isolate, accounting for 32.5% of all Nocardia isolates, and the prevalence of Nocardia isolates decreased in summer. All of the isolates were susceptible to trimethoprim/sulfamethoxazole, amikacin, and linezolid, whereas 90.9% were non-susceptible to cefepime and imipenem. The phylogenic tree by MLSA showed that the similarity between N. beijingensis and N. asiatica was as high as 99%, 73% between N. niigatensis and N. crassostreae, and 86% between N. cerradoensis and N. cyriacigeorgica. While trimethoprim/sulfamethoxazole, amikacin, and linezolid remained fully active against all of the Nocardia isolates tested, 90.9% of the isolates were non-susceptible to cefepime and imipenem.

Phylogenetically diverse Bradyrhizobium genospecies nodulate Bambara groundnut (Vigna subterranea L. Verdc) and soybean (Glycine max L. Merril) in the northern savanna zones of Ghana.

A total of 102 bacterial strains isolated from nodules of three Bambara groundnut and one soybean cultivars grown in nineteen soil samples collected from northern Ghana were characterized using multilocus gene sequence analysis. Based on a concatenated sequence analysis (glnII-rpoB-recA-gyrB-atpD-dnaK), 54 representative strains were distributed in 12 distinct lineages, many of which were placed mainly in the Bradyrhizobium japonicum and Bradyrhizobium elkanii supergroups. Twenty-four of the 54 representative strains belonged to seven putative novel species, while 30 were conspecific with four recognized Bradyrhizobium species. The nodA phylogeny placed all the representative strains in the cosmopolitan nodA clade III. The strains were further separated in seven nodA subclusters with reference strains mainly of African origin. The nifH phylogeny was somewhat congruent with the nodA phylogeny, but both symbiotic genes were mostly incongruent with the core housekeeping gene phylogeny indicating that the strains acquired their symbiotic genes horizontally from distantly related Bradyrhizobium species. Using redundancy analysis, the distribution of genospecies was found to be influenced by the edaphic factors of the respective sampling sites. In general, these results mainly underscore the high genetic diversity of Bambara groundnut-nodulating bradyrhizobia in Ghanaian soils and suggest a possible vast resource of adapted inoculant strains.

A type II variant of Toxoplasma gondii infects the Eurasian otter (Lutra lutra) in southern Italy.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is a widespread zoonosis capable to affect a wide range of warm-blooded vertebrates. In the past two decades, T. gondii emerged as a significant aquatic pathogen with some pathogenic atypical genotypes isolated and characterized from stranded marine mammals. In contrast, no information is available for mammals in freshwater environment. Although otters are considered highly susceptible to T. gondii infection, to date molecular evidence of T. gondii in Eurasian otter (Lutra lutra) does not exist. We report the first molecular evidence of T. gondii in a free-ranging Eurasian otter from southern Italy and characterized the present strain as a genotype type II variant, with all loci type II except PK1 (locus sequence corresponding to type II variant B), B1 (locus sequence corresponding to type II/X A) and C29-2 (locus with SNPs). Our results indicate circulation of a type II variant in freshwater environment which suggests potential risk of transmission to animals and humans. The finding of a potential pathogenic strain is of great concern for future conservation programmes of the critically endangered Eurasian otter in southern Italy.

First Isolation of Virulent Tenacibaculum maritimum Isolates from Diseased Orbicular Batfish (Platax orbicularis) Farmed in Tahiti Island.

The orbicular batfish (Platax orbicularis), also called 'Paraha peue' in Tahitian, is the most important marine fish species reared in French Polynesia. Sudden and widespread outbreaks of severe 'white-patch disease' have occurred since 2011 in batfish farms one to three weeks after the transfer of juveniles from bio-secured hatcheries to lagoon cages. With cumulative mortality ranging from 20 to 90%, the sustainability of aquaculture of this species is severely threatened. In this study, we report for the first time the isolation from diseased batfish of several isolates belonging to the species Tenacibaculum maritimum, a major pathogen of many marine fish species. Histopathological analysis, an experimental bath challenge and a field monitoring study showed that T. maritimum is associated with 'white-patch disease'. Moreover, molecular and serological analyses performed on representative isolates revealed some degree of genetic diversity among the isolates, a finding of primary importance for epidemiological studies and the development of management and control strategies such as vaccination.

Origin of Pathogens of Grapevine Crown Gall Disease in Hokkaido in Japan as Characterized by Molecular Epidemiology of Allorhizobium vitis Strains.

Crown gall is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of grapevine crown gall is tumorigenic Allorhizobium vitis (Ti) strains that harbor a tumor-inducing plasmid (pTi). The epidemic of grapevine crown gall has not been widely elucidated. In this study, we investigated the genetic diversity of 89 strains of Ti and nonpathogenic A. vitis to clarify their molecular epidemiology. Multi-locus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD was performed for molecular typing of A. vitis strains isolated from grapevines with crown gall symptoms grown in 30 different vineyards, five different countries, mainly in Japan, and seven genomic groups A to F were obtained. The results of MLSA and logistic regression indicated that the population of genetic group A was significantly related to a range of prefectures and that the epidemic of group A strains originated mainly in Hokkaido in Japan through soil infection. Moreover, group E strains could have been transported by infected nursery stocks. In conclusion, this study indicates that both soil infection and transporting of infected nursery stocks are working as infection source in Hokkaido.

Identification and Characterization of Xanthomonas arboricola pv. juglandis Causing Bacterial Blight of Walnuts in Korea.

The present study describes the bacterial blight of walnut, caused by Xanthomonas arboricola pv. juglandis (Xaj) in the northern Gyeongbuk province, Korea. Disease symptoms that appear very similar to anthracnose symptoms were observed in walnut trees in June 2016. Pathogens were isolated from disease infected leaves, fruits, shoots, bud, flower bud of walnut, and cultured onto yeast dextrose carbonate agar plates. Isolated bacteria with bacterial blight symptoms were characterized for their nutrient utilization profiles using Biolog GN2 and Vitek 2. In addition, isolates were subjected to physiological, biochemical, and morphological characterizations. Furthermore, isolates were identified using 16S rDNA sequence analysis, and multi-locus sequence analysis using atpD, dnaK, efp, and rpoD. To confirm pathogenicity, leaves, fruits, and stems of 3-year-old walnut plants were inoculated with bacterial pathogen suspensions as a foliar spray. One week after inoculation, the gray spots on leaves and yellow halos around the spots were developed. Fruits and stems showed browning symptoms. The pathogen Xaj was re-isolated from all symptomatic tissues to fulfill Koch's postulates, while symptoms were not appeared on control plants. On the other hand, the symptoms were very similar to the symptoms of anthracnose caused by Colletotrichum gloeosporioides. When walnut plants were inoculated with combined pathogens of Xaj and C. gloeosporioides, disease symptoms were greater in comparison with when inoculated alone. Xaj population size was more in the month of April than March due to their dormancy in March, and sensitive to antibiotics such as oxytetracycline and streptomycin, while resistant to copper sulfate.