16S-23S rRNA ITS Research Articles

Molecular characterization uncovering a novel genus of tapering-filamentous cyanobacteria from Thailand: Phayaothrix lacustris gen. & sp. nov. (Nostocales, Cyanophyta)

In this study, novel Calothrix-like strains NUACC09 and NUACC10 were isolated from the surfaces of rocks in Phayao Lake, Thailand. Morphological, molecular and ecological comparisons were investigated to characterize the taxonomic status of these novel strains. Under the light microscope, morphological studies indicated that these two strains were morphologically similar to Calothrix but could be differentiated by production of a non-hyaline hair at the terminal end, a low degree of tapering, twisted or loop-forming filaments, a knotted growth form, intertwined trichomes and presenting more than one trichome within the single sheath. In the 16S rRNA and rbcLX gene phylogenetic tree analyses, our strains formed a monophyletic clade with former freshwater/terrestrial Calothrix-like taxa separating distantly from other Calothrix-like genera. Furthermore, low 16S rRNA gene sequence similarity (<94.9%) to the closely related genera and species delimitation analyses (PTP/bPTP, GMYC, ABGD and ASAP methods) indicated that this clade should be considered as a different cyanobacterial lineage. The phylogenetic tree and secondary structures (D1–D1′, V2, Box-B and V3 helix) based on 16S–23S rRNA ITS regions suggested that multiple species might be contained in this clade. Therefore, here, Phayaothrix was proposed as a novel cyanobacterial genus with Phayaothrix lacustris sp. nov. as the type species following the International Code of Nomenclature for algae, fungi and plants.

Diversity of Flea Species (Siphonaptera) and Their Vector-Borne Pathogens from Bats (Chiroptera) in Lithuania

Bat fleas (Insecta: Siphonaptera: Ischnopsyllidae) are highly host-specific ectoparasites distributed worldwide (except Antarctica). The identification of flea species and the detection of flea-borne pathogens plays a significant role not only in veterinary and public health, but also in providing insights into the ecology, behaviour, and geographical distribution of fleas and flea-associated pathogens. The present study aimed to conduct a morphological and molecular identification of fleas collected from Lithuanian bats, as well as to screen for the presence of vector-borne pathogens. A total of 58 flea specimens were collected from bats in Lithuania. Based on morphological analysis, seven flea species were identified (Ischnopsyllus variabilis, I. octactenus, I. simplex, I. elongatus, I. hexactenus, Nycteridopsylla pentactena, and N. eusarca) and screened for the presence of Anaplasma spp., Borrelia spp., Babesia spp., Bartonella spp., Rickettsia spp., and Mycoplasma spp. Molecular analysis of fleas’ COI and 18S rRNA genes showed that the flea species collected from bats are closely genetically related. Bartonella DNA was detected in bat fleas (41.4%). No DNA of Anaplasma, Borrelia, Babesia, Rickettsia, and Mycoplasma was amplified in the tested samples. The Wolbachia sp. was accidentally found in I. elongatus. The phylogenetic analysis of four Bartonella genes (16S-23S rRNA ITS, gltA, rpoB, and groEL) revealed specificity to bats or bats’ ectoparasites. This is the first report about bat flea species and the pathogens in bat fleas in Lithuania. New host records for bat fleas were also reported. This study extends the knowledge about bat fleas and their role as vectors of different pathogens.

Metabolomic and taxonomic characterization of Haloleptolyngbya lusitanica sp. nov. (Cyanobacteria, Synechococcales)

ABSTRACT The morphological plasticity of cyanobacteria and their widespread ecological dominance in a wide range of habitats highlights the need for in-depth taxonomic studies. This work focused on the taxonomical revision of Leptolyngbya (Cyanophyceae) strains deposited in the ESSACC culture collection and their metabolomic characterization. Although the studied ESSACC strains were morphologically identified as Leptolyngbya sp., the 16S rRNA gene and 16S–23S rRNA ITS analysis revealed that two strains (LMECYA 079 and LMECYA 173) belong to Haloleptolyngbya and represent a new taxonomical unit, genetically unique, ecologically plastic and adapted to both freshwater and thermal habitats, here described as Haloleptolyngbya lusitanica sp. nov. To perform a suspect screening of cyanometabolites in these strains, we used a non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomic approach. Several metabolites were identified in Haloleptolyngbya lusitanica: micropeptin MM978, spumigin 640, oscillatoxin A and anabaenopeptin D. Strains were maintained and grown under the same conditions, revealing the common production of oscillatoxin A by both H. lusitanica strains. Other identified metabolites, however, were strain-specific, such as anabaenoptin D, which was only detected in LMECYA 173. The different cyanometabolite profiles reinforce the notion that cyanobacteria have the ability to adapt to different habitats, which is maintained under long-term culturing conditions.

Description of Pseudocalidococcus azoricus gen. sp. nov. (Thermosynechococcaceae, Cyanobacteria), a Rare but Widely Distributed Coccoid Cyanobacteria

Coccoid cyanobacteria represent an important part of cyanobacterial freshwater diversity, with many studied strains in public databases identified as Synechococcus. This is a diverse genus, both morphologically and ecologically, with a global distribution. However, many of the so-called Synechococcus-like cyanobacteria strains could represent several independent genera that require further studies. In this work, four strains of a Synechococcus-like cyanobacteria isolated from freshwater lakes and terrestrial atmophytic habitats on São Miguel and Flores Islands (Azores archipelago) were studied genetically using the 16S rRNA and 16S–23S rRNA ITS, morphologically with light and transmission electron microscopy, and ecologically. A draft genome was produced from the reference strain by Illumina sequencing, which allowed a more complete phylogenetic study and a deeper taxonomic analysis, revealing a divergent phylogenetic evolution and low ANI and AAI values (69.4% and 66.3%, respectively) to Thermosynechococcus, the closest phylogenetic genus. Although morphologically similar to Synechococcus, the 16S rRNA and genome phylogenetic analysis placed the studied strains in a clade sister to Thermosynechococcus, inside the Thermosynechococcaceae. Thus, Pseudocalidococcus azoricus gen. sp. nov. is described as a new coccoid freshwater genus and species from the Azores archipelago. A detailed comparison with similar morphological taxa is provided, supporting the separation of the new genus. The 16S rRNA with a high genetic similarity to other strains from several continents identified as Synechococcus sp. suggests that the new genus probably has a worldwide distribution. Future studies should be performed to clarify the taxonomic identity of those strains.

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

TaqMan Quantitative PCR Detection of Xylella taiwanensis in Taiwan.

Xylella taiwanensis (Xt) is a nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, loss of tree vigor, and reduction in fruit yield and quality. No cure for PLSD is available. The only option for growers to control the disease is to use pathogen-free propagation material, which requires early and accurate detection of Xt. Currently, only one simplex PCR method is available for the diagnosis of PLSD. We developed five Xt-specific TaqMan quantitative PCR (TaqMan qPCR) systems (primers-probe sets) for the detection of Xt. The PCR systems target three conserved genomic loci commonly used in bacterial pathogen detection: the 16S rRNA gene (rrs), the 16S-23S rRNA intergenic transcribed sequence (16S-23S rRNA ITS), and the DNA gyrase gene (gyrB). BLAST analysis using the GenBank nr sequence database, including whole genome sequences of 88 Xanthomonas campestris pv. campestris (Xcc) strains, 147 X. fastidiosa (Xf) strains, and 32 Xt strains, showed that all primer and probe sequences were specific only to Xt. Single nucleotide polymorphisms (SNPs) provided the primer/probe specificity to Xt. The PCR systems were evaluated by using DNA samples from pure cultures of two Xt strains, one Xf strain, one Xcc strain, and 140 plant samples collected from 23 pear orchards in four counties in Taiwan. The two-copy rrs and 16S-23S rRNA ITS-based PCR systems (Xt803-F/R, Xt731-F/R, and Xt16S-F/R) showed higher detection sensitivity than the two single-copy gyrB-based systems (XtgB1-F/R and XtgB2-F/R). A metagenomic analysis of a representative PLSD leaf sample detected the presence of non-Xt proteobacteria and fungal pathogens that should be taken into consideration in PLSD, as they might interfere with diagnosis.

Khargia gen. nov., a new genus of simple trichal Cyanobacteria from the Persian Gulf

Information regarding the diversity of Cyanobacteria in many parts of the world is still minimal. One example of a region that has not yet been widely studied is south-western Asia, including the region of the Persian Gulf. A culture-dependent study of cyanobacterial diversity in a rainwater basin on Kharg Island enabled the isolation of a novel taxon, previously unnamed, from a simple trichal cyanobacterium. Further comparisons showed the existence of closely related strains/taxa from other parts of the world, namely the strains CCALA 945 isolated from South Italy and "<i>Leptolyngbya india" </i>from India. Herein, we have thus described the new genus <i>Khargia</i> and the new species <i>Khargia iranica. </i>Other strains, isolated by other authors, were included in the <i>Khargia</i> genus as additional species: <i>Khargia italica</i> and <i>Khargia indica</i>. The recognition of the new genus was based on morphological evaluations (identification by both light and electron microscopy), the phylogenetic analyses based on the 16S rRNA gene, and species delimitation based on Automatic Barcode Gap Definition (ABGD), Generalized Mixed Yule Coalescent (GMYC), bayesian version of Poisson Tree Processes (bPTP), and the secondary structure of 16S-23S rRNA ITS region of the studied strains.

First Molecular Detection of Bartonella bovis and Bartonella schoenbuchensis in European Bison (Bison bonasus)

Bartonella bacteria infect the erythrocytes and endothelial cells of mammalians. The spread of the Bartonella infection occurs mainly via bloodsucking arthropod vectors. Studies on Bartonella infection in European bison, the largest wild ruminant in Europe, are lacking. They are needed to clarify their role in the maintenance and transmission of Bartonella spp. The aim of this study was to investigate the presence of the Bartonella pathogen in European bison and their ticks in Lithuania. A total of 38 spleen samples from bison and 258 ticks belonging to the Ixodes ricinus and Dermacentor reticulatus species were examined. The bison and tick samples were subjected to ssrA, 16S-23S rRNA ITS, gltA, and rpoB partial gene fragment amplification using various variants of PCR. Bartonella DNA was detected in 7.9% of the tissue samples of European bison. All tick samples were negative for Bartonella spp. The phylogenetic analysis of 16S-23S rRNA ITS, gltA, and rpoB partial gene fragment revealed that European bison were infected by B. bovis (2.6%) and B. schoenbuchensis (5.3%). This is the first report addressing the occurrence of B. bovis and B. schoenbuchensis in European bison in Europe.

Neocylindrospermum variakineticum gen. & sp. nov. (Nostocales, Cyanobacteria), a novel genus separated from Cylindrospermum using a polyphasic method

ABSTRACT The large genus Cylindrospermum is polyphyletic as currently circumscribed, and its cryptic diversity requires detailed taxonomic evaluation. Two novel cyanobacterial strains were isolated by scraping the surface of wet wood above the water level, in the stream below Mae Phun Waterfall in the northern part of Thailand, and investigated with a polyphasic study combining morphological and molecular analyses. The morphological features of these two novel strains fitted the original description of the genus Cylindrospermum but they differed from known species in cell size, exospore features and akinete formation. Phylogenetic analyses based on 16S rRNA, 16S–23S rRNA ITS region, rpoC1 and nifH genes indicated that the novel strains formed a unique clade with strongly supported values from BI and ML methods. This unique clade was distantly separated from the Cylindrospermum sensu stricto clade and other Cylindrospermum-like clades. Molecular analyses showed that the two novel strains had <96.3% 16S rRNA gene sequence similarity and <83.2% 16S–23S rRNA ITS region sequence similarity to known cyanobacterial genera and other related taxa. Furthermore, ITS secondary structures between the 16S–23S rRNA gene in both strains also exhibited singular folding patterns of D1–D1', Box-B and V3 helix, thereby distinguishing them from the closely related taxa. The establishment of Neocylindrospermum gen. nov. with the type species as N. variakineticum sp. nov. is proposed according to the International Code of Nomenclature for Algae, Fungi, and Plants.

Bartonella spp. detection in laelapid (Mesostigmata: Laelapidae) mites collected from small rodents in Lithuania.

The genus Bartonella contains facultative Gram-negative intracellular bacteria from the family Bartonellaceae that can cause diseases in humans and animals. Various Bartonella species have been detected in rodents' ectoparasites, such as fleas, ticks, mites, and lice. However, the role of laelapid mites (Mesostigmata: Laelapidae) as carriers of Bartonella spp. needs to be confirmed. We aimed to investigate the presence of Bartonella spp. in laelapid mites collected from small rodents in Lithuania using real-time PCR targeting the transfer-messenger RNA/tmRNA (ssrA) gene and to characterize Bartonella strains using nested PCR and sequence analysis of the 16S-23S rRNA intergenic transcribed spacer region (ITS). A total of 271 laelapid mites of five species (Laelaps agilis, Haemogamasus nidi, Eulaelaps stabularis, Myonyssus gigas, and Hyperlaelaps microti) were collected from five rodent species (Apodemus flavicollis, Apodemus agrarius, Clethrionomys glareolus, Micromys minutus, and Microtus oeconomus) during 2015-2016. Bartonella DNA was detected in three mite species L. agilis, M. gigas, and Hg. nidi with an overall prevalence of 11.4%. Sequence analysis of the 16S-23S rRNA ITS region revealed the presence of Bartonella taylorii in L. agilis, Hg. nidi, and M. gigas, and Bartonella grahamii in L. agilis. Our results suggest that laelapid mites are involved in the maintenance of rodent-associated Bartonella spp. in nature. To the best of the authors' knowledge, this is the first study to demonstrate the presence of Bartonella spp. DNA in laelapid mites from small rodents.

Isolation and Characterization of Rhizospheric Bacteria From Vuralia turcica Rhizospheric Soil

Plant growth-promoting rhizobacteria are highly active in soil ecosystems for legumes due to their biotic activities. Vuralia turcica (Kit Tan, Vural &amp; Kucukoduk) Uysal &amp; Ertugrul is a Turkish endemic legume plant with potential value for ornamental and food crops. However, reports of plant growth-promoting rhizobacteria in V. turcica rhizosphere are lacking in the literature. This study includes endophytic bacterial isolation from V. turcica rhizospheric soil. The objective of this study was to isolate and characterize growth-promoting bacteria. V. turcica rhizospheric soils from four different locations of Central Anatolia of Turkey, namely L1 (Gölçayır, 38° 28´ 10.5´´ N/31° 21´ 04.4´´ E), L2 (Akşehir, 38° 28´ 17.328´´ N/31° 20´ 52.468´´ E), L3 (Dereçine, 38° 30´ 36.702´´ N/31° 17´ 56.702´´ E), L4 (Sultandağı, 38° 32´ 43.2168´´ N/31° 16´ 54.4728´´ E), were selected for bacterial isolation. Ten bacterial strains were isolated and identified through the comparison of the 16S rRNA and 16S-23S rRNA ITS region. Among them, 4 isolates belonged to Bacillus megaterium, 3 strains belonged to Stenotrophomonas rhizophila, 1 strain belonged to Rhodococcus erythropolis, 1 strain belonged to Xanthomonas albilineans, and the remaining 1 strain belonged to Lysobacter enzymogenes, respectively.

Mycoplasma bovis-pneumonia and polyarthritis in feedlot calves in Argentina: First local isolation

Bovine respiratory disease (BRD) is one of the most frequent clinical concerns in weaned calves after their arrival at the feedlot. This work reports the first local isolation of Mycoplasma bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Twenty four out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing, and fever. Thirty percent of the affected calves also showed lameness and swelling of elbow or carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal to coalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibrous tissue, and fibrinonecrotic arthritis and tenosynovitis were detected. Mycoplasma was isolated from lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from these isolates showed 100% similarity with the same region of M. bovis strains. Since there are no commercially available vaccines in the region for the prevention and control of M. bovis pneumonia and arthritis, surveillance is a priority to reduce the source of disease to naïve animals.

High rate of reinfection and possible transmission of Mycobacterium avium complex in Northeast Thailand

The Mycobacterium avium complex (MAC) includes two main species of non-tuberculous mycobacteria (NTM), M. avium and Mycobacterium intracellulare. These can cause serious disease, especially in immunocompromised patients. Little information is available concerning genetic diversity of NTM. We used multilocus sequence typing (MLST) based on a highly discriminative gene set to analyze MAC serially isolated from patients to determine the rate of MAC reinfection. Genomic DNA was sequenced from 49 MAC isolates (15 cases comprised of 11 true infections and 4 instances of colonization). More than half of the MAC isolates tested were found to be multidrug resistant. The discriminatory power was assessed of 24 house-keeping genes (fusA, atpD, pheT, glnA, topA, secA, argH, glpK, murC, cya, pta, rrl, rrs, hsp65, rpoB, 16S-23S rRNA ITS, recF, lipT, pepB, gnd, aspB, groEL, sodA and est) previously used for genotyping of MAC and other NTM. Seven genes (fusA, secA, rpoB, hsp65, 16S rRNA, 23S rRNA, 16S-23S rRNA ITS) had a discriminatory power index higher than 0.9 and were included in the optimized set that we used. This set was significantly better for genotyping and diagnosis of MAC than previously used 4-gene, 5-gene and 9-gene sets. MLST using our 7-gene set indicated that the rate of reinfection was 54.55% (6/11 cases). Persistent infections (n = 5 cases, 45.45%) were found. A changing of clone in the same patient was found in 1/4 (25%) of the colonization cases. Two small clusters of possible MAC transmission between humans were found. Our study demonstrated that the high frequency of apparent treatment failure of MAC might be artefactual, as a consequence of a high rate of MAC reinfection in Thai population. Our useful highly discriminative gene set for MAC species and clonal strain analysis could be further applied for the diagnosis and patient management.

Recognizing novel cyanobacterial diversity in marine benthic mats, with the description of Sirenicapillariaceae fam. nov., two new genera, Sirenicapillaria gen. nov. and Tigrinifilum gen. nov., and seven new species

ABSTRACT Marine benthic proliferations are increasing in occurrence, range and duration by way of anthropogenic nutrient loading and climate change. The spread of these cyanobacteria within a variety of regions has made it fundamental to recognize the diversity and the species involved in these blooms. To expand knowledge on marine cyanobacterial diversity and reveal their phylogenetic relationships, sampling and isolation of benthic proliferative events from underexplored regions were conducted in the USA (Florida) and France (Loire-Atlantique). Cyanobacteria were described with the polyphasic approach using morphology, 16S rRNA gene phylogeny, 16S–23S rRNA ITS secondary structures and pairwise distances. Sampling of marine cyanobacteria from seagrasses, lagoons and marine coastal waters from nine Florida localities, in addition to Florida freshwater canals and French salt flats, revealed floating, epipsammic and epiphytic mats. A total of 30 cyanobacteria were isolated of which 21 represented two novel genera, Sirenicapillaria and Tigrinifilum. Sirenicapillaria is a genus that is found in massive benthic blooms throughout the western and southern Florida Coast.

Isolation and characterization of the most abundant rhizobacterial species associated with Vuralia turcica (Fabaceae: Papilionoideae)

Vurali turcica is naturally grown in a limited area in Central Anatolia in Turkey and was categorized as a critically endangered plant in the Red Data Book of Turkish Plants. This study aimed to analyze whether the symbiotic and mutualistic relation between V. turcica rhizomes and present microflora in the habitat can be active on its distribution. Plant growth-promoting rhizobacteria (PGPRs) colonize the rhizosphere and promote plant growth and physiology. In this paper, the diversity of PGPRs of rhizomes of V. turcica was analyzed. Rhizome samples were obtained from the natural habitats of V. turcica by the workers of Nezahat Gökyiğit Botanical Garden, and bacterial isolation was conducted on the collected samples. MIS analysis, 16S rRNA, and 16S-23S rRNA ITS region sequencing were implemented, and as a result, Bacillus megaterium was found to be one of the most abundant bacterial species of the rhizomes of V. turcica based on nucleotide homology. This study is the first report on the identification of rhizobacterial species in V. turcica.

Molecular epidemiology of certain vector-borne bacterial microorganisms in domestic animals and their ectoparasites in Egypt.

Vector-borne bacterial diseases (VBBD) are a diverse group of tropical and subtropical zoonotic diseases. This study investigated the possibility of domestic animals to carry certain vector-borne bacterial microorganisms (VBBMs), as well as the presence of these targeted DNAs in their ectoparasites in different localities of Egypt using molecular analyses. For this study, 234 animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) in addition to 115 ectoparasites (95 ticks and 20 lice) were investigated for the molecular detection of Bartonella spp., Borrelia spp., and Rickettsia spp., targeting 16S-23S rRNAITS, 16S rRNA, and gltA genes, respectively. The results indicated that the overall prevalence of VBBD was observed in 17 animals (7.26%), of which 16 (6.84%) were positive for Bartonella spp. and one (0.43%) was positive for Borrelia theileri. All blood samples were negative for the DNA of Rickettsia spp. In addition, the results demonstrated that all ectoparasites were free from VBBDNA. Furthermore, of the animals examined for ectoparasite infestation, 28 (11.97%) and 5 (2.14%) represented Rhipicephalus annulatus ticks and Haematopinus tuberculatus lice, respectively, which infested animals. Analysis of epidemiological factors revealed that gender, age, and ectoparasitic infestation of animals had a significant effect on Bartonella infection, whereas no significant difference between animal species was observed. Hence, we report a potential novel Bartonella sp. from cattle and buffaloes, including a new genotype of Bo. theileri from cattle, in Egypt.

Diversity of the genus Brasilonema (Nostocales, Cyanobacteria) in plant nurseries of central Florida (USA) with the description of three new species: B. fioreae sp. nov., B. santannae sp. nov. and B. wernerae sp. nov.

Florida is a diverse region that supports abundant cyanobacterial diversity, especially in terrestrial environments. To exploit this environment for cyanobacterial diversity, several greenhouses from central Florida were sampled to identify common nuisance and contaminating algae. Most of the algae observed were mat forming, covering nursery pots, plants, and equipment which were macro- and microscopically morphologically analogous to Brasilonema. Although macroscopic thallus morphology was similar among the samples, microscopic morphological characteristics such as size, color, and sheath formation were disparate. To uncover the cryptic diversity, mats were processed for species isolation, culture, and molecular taxonomic identification. A total of eleven Brasilonema strains were isolated into culture and systematically identified using 16S rRNA and 16S-23S rRNA ITS sequences. Based on morphology and molecular data, five species of Brasilonema were found and three are new to science: B. fioreae, B. santannae, and B. wernerae.

Latitudinal Characteristic Nodule Composition of Soybean-Nodulating Bradyrhizobia: Temperature-Dependent Proliferation in Soil or Infection?

A species-specific latitudinal distribution of soybean rhizobia has been reported; Bradyrhizobium japonicum and B. elkanii dominate in nodules in northern and southern areas, respectively. The aim of this study was to elucidate whether temperature-dependent proliferation in soil or infection is more reliable for determining the latitudinal characteristic distribution of soybean-nodulating rhizobia under local climate conditions. Three study locations, Fukagawa (temperate continental climate), Matsue and Miyazaki (humid sub-tropical climate), were selected in Japan. Each soil sample was transported to the other study locations, and soybean cv. Orihime (non-Rj) was pot-cultivated using three soils at three study locations for two successive years. Species composition of Bradyrhizobium in the nodules was analyzed based on the partial 16S rRNA and 16S–23S rRNA ITS gene sequences. Two Bradyrhizobium japonicum (Bj11 and BjS10J) clusters and one B. elkanii (BeL7) cluster were phylogenetically sub-grouped into two (Bj11-1-2) and four clusters (BjS10J-1-4) based on the ITS sequence. In the Fukagawa soil, Bj11-1 dominated (80–87%) in all study locations. In the Matsue soil, the composition was similar in the Matsue and Miyazaki locations, in which BeL7 dominated (70–73%), while in the Fukagawa location, BeL7 decreased to 53% and Bj11-1 and BjS10J-3 increased. In the Miyazaki soil, BeL7 dominated at 77%, and BeL7 decreased to 13% and 33% in the Fukagawa and Matsue locations, respectively, while BjS10J-2 and BjS10J-4 increased. It was supposed that the B. japonicum strain preferably proliferated in the Fukagawa location, leading to its nodule dominancy, while in the Miyazaki location, temperature-dependent infection would lead to the nodule dominancy of B. elkanii, and both factors would be involved in the Matsue location.

The occurrence of Affixifilum gen. nov. and Neolyngbya (Oscillatoriaceae) in South Florida (USA), with the description of A. floridanum sp. nov. and N. biscaynensis sp. nov.

South Florida (USA) has a subtropical to tropical climate with an extensive and diverse coastline that supports the growth of benthic cyanobacterial mats (BCMs). These BCMs are widespread and potentially house numerous bioactive compounds; however, the extent of the cyanobacterial diversity within these mats remains largely unknown. To elucidate this diversity, BCMs from select locations in South Florida were sampled and isolated into unicyanobacterial cultures for morphological and molecular studies. Phylogenetic relationships of isolated taxa were assessed using the markers 16S rRNA and 16S-23S rRNA ITS by both maximum likelihood and Bayesian inference. We propose Affixifilum gen. nov. based on morphological characteristics and the 16S rRNA phylogeny. Two species are included: Affixifilum granulosum comb nov. (=Neolyngbya granulosa) found in Brazil and Florida (USA) and A. floridanum sp. nov. Several other features, including pair-wise distance of 16S rRNA and 16S-23S rRNA ITS, 16S-23S rRNA ITS secondary structure, morphology, and ecology, provide support for Affixifilum. We also propose the transfer of Lyngbya regalis to Neolyngbya as N. regalis comb. nov. and include the description of one novel species, N. biscaynensis sp. nov.

Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology.

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S-23S rRNA ITS, rpoB, rpoC, rpoD, uvrA, parC, topA, dnaE, fusA and pyk. The genome was sequenced for type strain 1220T. The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02-99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00-96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma. Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis. Based on the genetic data, we propose a novel species of the genus Mycoplasma, for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220T (=ATCC BAA-2147T=NCTC 13513T=DSM 23982T). The G+C content is 26.70 mol%, genome size is 959110 bp.