Evolutional and Geographical Relationships of <Emphasis Type="Italic">Bartonella grahamii</Emphasis> Isolates from Wild Rodents by Multi-locus Sequencing Analysis

To clarify the relationship between Bartonella grahamii strains and both the rodent host species and the geographic location of the rodent habitat, we have investigated 31 B. grahamii strains from ten rodent host species from Asia (Japan and China), North America (Canada and the USA), and Europe (Russia and the UK). On the basis of multi-locus sequencing analysis of 16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB, the strains were classified into two large groups, an Asian group and an American/European group. In addition, the strains examined were clearly clustered according to the geographic locations where the rodents had been captured. In the phylogenetic analysis based on gltA, the Japanese strains were divided into two subgroups: one close to strains from China, and the other related to strains from Far Eastern Russia. Thus, these observations suggest that the B. grahamii strains distributed in Japanese rodents originated from two different geographic regions. In the American/European group, B. grahamii from the North American continent showed an ancestral lineage and strict host specificity; by contrast, European strains showed low host specificity. The phylogenetic analysis and host specificity of B. grahamii raise the possibility that B. grahamii strains originating in the North American continent were distributed to European countries by adapting to various rodent hosts. An erratum to this article can be found at     

Assessment ofGibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Speciation ofBotryobasidium subcoronatum (Basidiomycota) collected in Taiwan: Morphology, mating tests, and molecular data

Taiwan is a phytogeographically isolated area, with a rate of ca. 40% endemism in higher plants. In this study, the strong tendency towards genetic isolation and speciation ofBotryobasidium subcoronatum (Basidiomycota) from Taiwan is evaluated in comparison with European specimens. Mating tests amongB. subcoronatum specimens collected in Taiwan and Europe were performed. Mon-mon and di-mon matings confirm different intersterility groups or biological species. The biological species concept was compared with results from morphological comparison and molecular data from mtSSU rDNA and the nuclear ITS2 region. Three intersterility groups have been detected comprising European and Taiwanese strains from temperate and subtropical areas. MtSSU rDNA and the ITS2 rDNA sequences confirm consepecifity ofB. subcoronatum.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

Detection and characterization of the African citrus greening Liberobacter by amplification,cloning, and sequencing of the rplKAJL-rpoBC operon

Greening disease of citrus is caused by a phloem-restricted, uncultured bacterium, recently characterized and named Liberobacter. As shown previously, a probe encoding ribosomal protein genes (rplKAJL-rpoBC operon) from an Asian liberobacter could detect all Asian liberobacter strains tested, but not African strains. Using the sequence of the rplKAJL-rpoBC operon of the Asian liberobacter strain from Poona (India), we have defined primers for PCR amplification of the equivalent genes of an African liberobacter strain. The amplified fragment was cloned in pUC18 and successfully used as a probe to detect African liberobacter strains by Southern and dot hybridizations. Sequence comparisons of the African and Asian liberobacter operons indicate that they represent two different species in the proposed genus Liberobacter.     

Protective Activity of Antisera against Isolates of Borrelia burgdorferi from Various Geographical Origins

Antisera from rabbits immunized with two Japanese strains of Borrelia burgdorferi, HP3 an isolate from Ixodes persulcatus and HO14 an isolate from I. ovatus, or the European strain P/Bi isolated from human cerebrospinal fluid (CSF) did not passively protect hamsters from challenge with the infectious strain 297, a North American isolate from patient CSF. Antisera to strains 297 and B31, a North American isolate from I. dammini, however, provided protective effect to challenge with strain 297. Immune mice sera in the presence of homologous B. burgdorferi antigen induced the production of oxygen intermediates from mouse peritoneal exudate cells. Heterologous B. burgdorferi antigen had no effect. These results suggest that antigenic properties of Japanese strains are different from those of North American and European isolates.     

Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae

Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically. These strains belonged to the genus Xenorhabdus. The Newfoundland (NF) strain was shown to be X. bovienii but the Texas (TX) strain was not identified at the species level. Four additional cultures of Xenorhabdus were included in the study. These were a strain of X. bovienii (Ume?), which was from a nematode of European origin, and strains of X. nematophilus, X. beddingii, and X. poinarii. The tests used in this study indicated identical properties for the NF (North American) and Ume? (European) strains of X. bovienii. These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid. The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C. Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.     

New features in the vegetative and reproductive morphology of Cystoclonium purpureum (Cystocloniaceae,Rhodophyta) from the North Atlantic Ocean

The aim of this study was to evaluate whether the success of invasive Caulerpa cylindracea (Caulerpa racemosa var. cylindracea) in Mediterranean habitats may be linked to particular nutritional requirements and/or nutrient accumulation/reallocation dynamics within the algal thallus. To this end, a manipulative field experiment was carried out transplanting C. cylindracea and supplying nutrients to subtidal rocky macroalgal assemblages. Morphological features of C. cylindracea were measured, while percentage cover and tissue nitrogen, carbon and phosphorus were determined for C. cylindracea and two co-occurring species (the introduced Laurencia chondrioides and native Flabellia petiolata) in nutrient-enriched and unenriched conditions. Growth of C. cylindracea was greater in the nutrient-enriched plots while no effect of nutrient enrichment was detected in co-occurring macroalgae. A particularly high requirement for nitrogen has been observed in C. cylindracea, but without severe N-limitation, while a general nutritional state of P-limitation was detected in both invasive and co-occurring macroalgae. The absence of inhibition in hypertrophic conditions and under high levels of ammonia together with a strong uptake and quick reallocation of nutrients within the coenocytic thallus probably represent the main advantages over the other co-occurring species which could explain part of the success of C. cylindracea in the Mediterranean Sea.     

Recent approaches to the taxonomy of the Gracilariaceae (Gracilariales,Rhodophyta) and the Gracilaria verrucosa problem

Recognition of species in the Gracilariaceae, often notoriously difficult, is being aided by a combination of classical and modern techniques. We review some recent findings and present new results that may lead to redefinition of Gracilaria verrucosa, the type species of its genus. Plastid DNA restriction profiles (patterns of banding obtained by electrophoresis of DNA after restriction endonuclease digestion) from eleven strains ascribed to G. verrucosa indicated that the concept of this species in northern Europe includes possibly three taxa, one of which is known now to be a species of Gracilariopsis. In contrast, restriction profiles from Argentinian and Japanese strains were closely similar to the predominant pattern for European G. verrucosa. Profiles of several other strains, from the western Atlantic and eastern Pacific, were dissimilar to the European group and to each other. A chromosome number of n = 24 was determined for a representative of the predominant European group, and preliminary results of hybridization trials suggest that these strains, and others with approximately the same plastid DNA restriction pattern, are interfertile.NRCC 30559     

Virulence and type III effector diversities of Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli in Brazil

Common bacterial blight (CBB) is caused by four genetic lineages belonging to two species of Xanthomonas, namely Xanthomonas citri pv. fuscans (includes fuscans, NF2 and NF3 lineages) and X. phaseoli pv. phaseoli (lineage NF1). A collection of 117 strains of Xanthomonas isolated from common bean plants grown in several producing regions of Brazil, between 2007 and 2016 was established. For species and lineage identification, the following tests were performed: multiplex PCR with a set of four specific primer pairs, pathogenicity tests on susceptible cultivar BRS Artico and phylogenetic analysis based on housekeeping gene sequences. The presence of the two species were confirmed among the 117 strains, being 62 non-fuscans strains (NF1, NF2 and NF3) and 55 fuscans strains of X. citri pv. fuscans. To select a set of representative strains for the virulence assay, a PCR-based analysis of effector diversity was performed with 42 strains belonging to the two species. PCR with primers for xopL, avrBsT, xopE2 and xopE1 genes were positive for all strains, while for the other six effectors there was variation. Six distinct effector profiles were detected, and one strain representing each type was inoculated in 15 common bean cultivars with varying levels of resistance to CBB. The fuscans strains showed uniformity in their effector profiles and were the most virulent. The phylogenetic analyses of our strain collection revealed that all genetic variants of CBB pathogens (NF1, NF2, NF3 and fuscans) are present in Brazil, with significant variability in virulence to common bean cultivars.     

Characteristics of Ralstonia solanacearum Biovar N2 Strains in Asia

Ralstonia solanacearum biovar N2 strains isolated in Asia were compared by biochemical tests with biovar N2 strains from South America and biovar 2 (race 3) strains from Africa, America, Asia and Europe. Distinct differences were found between Asian and South American strains of biovar N2, and between Asian biovar N2 and biovar 2 strains with respect to their ability to utilize several carbon sources. Using cluster analysis based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) genomic fingerprints, the Asian biovar N2 strains were divided into two groups, group 1 containing Japanese strains and group 2 containing Indonesian and Philippine strains. The fingerprints showed the genetic diversity of biovar N2 strains in Asia.     

Introduced American eels Anguilla rostrata in European waters: life‐history traits in a non‐native environment

This study investigated growth, condition and development of American eels Anguilla rostrata that were introduced into a European river to estimate their competitive potential in a non‐native habitat. Results demonstrate that A. rostrata develops normally in European waters and successfully competes with the native European eel Anguilla anguilla. In addition, A. rostrata appears to be more susceptible to the Asian swimbladder nematode Anguillicola crassus than A. anguilla and could support the further propagation of this parasite. Detected differences in fat content and gonad mass between Anguilla species are assumed to reflect species‐specific adaptations to spawning migration distances. This study indicates that A. rostrata is a potential competitor for the native fauna in European fresh waters and suggests strict import regulations to prevent additional pressure on A. anguilla and a potential further deterioration of its stock situation.     

Microbotryozyma collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria oleosa (Miridae)

Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666^T (= PRA303-1S = CBS 12537) designated as the type strain.     

Phylogeny of Castanea (Fagaceae) based on chloroplast trnT-L-F sequence data

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.     

Isolation and identification of mucinolytic actinomycetes

Biochemical and physiological tests, and 16S rRNA gene sequences, were used to classify nine Actinomycete strains isolated from soil and sand samples in Thailand. These strains were isolated based on their ability to readily degrade mucin glycoproteins. A turbidometric based mucinolytic assay was developed to confirm this. In addition all strains showed significant production of proteases. Phylogenetic analysis of the strains revealed that from the nine isolated Actinomycete strains eight were closely related to Streptomyces species and one was identified as belonging to the genus Kitasatospora. The biochemical and physiological tests performed identified two strain pairs that were similar (with only 3.9% difference observed) and this was in accordance with the phylogenetic results obtained. The remaining strains were distinct from each other, with the soil-isolated strains forming a separate clade to the sand-isolated strains in the inferred phylogenetic trees. The isolated mucinolytic Actinomycete strains will be the subject of further investigations into their proteolytic and glycosidic activity. Mucin degrading enzymes such as these are studied for their potential to be used for the development of a drug delivery system.     

Hybridization and sexual stimulation inTrichophyton mentagrophytes

Mating and sexual stimulation tests applied to 132 strains of this dermatophyte isolated in Czechoslovakia revealed among them strains ofArthroderma benhamiae (40 strains of the+mating type, one of the—mating type) andA. vanbreuseghemii (three strains of the+type, seven of the-type). No dependence was found concerning the anamorphic variety (T. mentagrophytes var.granulosum, var.interdigitale, var.mentagrophytes, var.quinckeanum), teleomorphic species, mating type and the clinical localization of dermatophytosis. Plausible reasons of different frequencies of the mating types are discussed.