Genetic characterization of Raffaelea quercivora isolates collected from areas of oak wilt in Japan

This study was undertaken to improve understanding of the phylogenetic position of pathogenic fungi implicated in the oak wilt in Japan. Sequences were obtained from three regions of partial nuclear ribosomal DNA of 25 isolates of Raffaelea quercivora including an ex-type strain, all of which were collected from seven areas of disease outbreak. All the isolates formed one clear clade with high bootstrap values, distinctly delimited from the closest species, R. montetyi. These results indicate that the R. quercivora is phylogenetically a well-defined taxon.     

Phylogeny of ambrosia beetle symbionts in the genus Raffaelea

The genus Raffaelea was established in 1965 when the type species, Raffaelea ambrosia, a symbiont of Platypus ambrosia beetles was described. Since then, many additional ambrosia beetle symbionts have been added to the genus, including the important tree pathogens Raffaelea quercivora, Raffaelea quercus-mongolicae, and Raffaelea lauricola, causal agents of Japanese and Korean oak wilt and laurel wilt, respectively. The discovery of new and the dispersal of described species of Raffaelea to new areas, where they can become invasive, presents challenges for diagnosticians as well as plant protection and quarantine efforts. In this paper, we present the first comprehensive multigene phylogenetic analysis of Raffaelea. As it is currently defined, the genus was found to not be monophyletic. On the basis of this work, Raffaelea sensu stricto is defined and the affinities of undescribed isolates are considered.     

Microstructure of the prothoracic mycangia in the ambrosia beetle Platypus koryoensis (Coleoptera: Curculionidae: Platypodinae)

Platypus koryoensis (Murayama) (Coleoptera: Curculionidae: Platypodinae) is a minute ambrosia beetle that can cause significant damage to live oak trees (Quercus mongolica) along with its symbiotic pathogenic fungus (Raffaelea sp.). Although both sexes of the beetle have a number of cuticular depressions for carrying microorganisms, only the females have mycangia that are typical in size on the pronotum. The mycangial cavities are filled with a mass of amorphous substances including yeast-like spores. In addition, mycangial cavities are equipped with cuticular pores which release substances to support symbiotic microorganisms during storage. Small depressions of both sexes can be subdivided into two clusters according to their relative sizes and locations. Although they have a typical cuticular peg (basiconic sensillum), they also share a common morphological characteristic with the typical mycangium of female: a round outlet and an invaginated reservoir containing a mass of secretory substances. Accordingly, our fine structural data suggest that both the female mycangia and the clusters of small cuticular depressions in both sexes also have a mycangial function in common regardless of their relative size and location.     

Five new Simplicillium species (Cordycipitaceae) from soils in Tokyo,Japan

Eighteen Simplicillium isolates were discovered from soil samples collected on the Honshu, Bonin, and Izu islands in areas under the jurisdiction of Tokyo, Japan. Using a combination of micro-morphological characteristics and sequences of the ribosomal RNA gene ITS region, the isolates were classified as six Simplicillium species, and five of them were previously undescribed. The five species (Simplicillium aogashimaense, Simplicillium cylindrosporum, Simplicillium obclavatum, Simplicillium subtropicum and Simplicillium sympodiophorum) were discovered from the Chichi-jima, Hachijo, and Aogashima islands, and Simplicillium minatense was discovered from Honshu. The five new species and three known species are distinguished by conidial morphology.     

Ultraviolet Light-Induced Heterokaryon Formation and Parasexuality in Cryphonectria parasitica

Rizwana, R., and Powell, W. A. 1995. Ultraviolet light-induced heterokaryon formation and parasexuality in Cryphonectria parasitica. Experimental Mycology 19, 48-60. The effect of ultraviolet-light on heterokaryon formation, vegetative compatibility, and parasexuality in Cryphonectria parasitica was examined. Heterokaryons of complementary auxotrophic strains could not be made by hyphal anastomosis if the strains belonged to different vegetative compatibility groups. Protoplast fusions overcame incompatibility of strains differing in the alleles of a single but not multiple vegetative incompatibility loci. Fusion of protoplasts from ultraviolet light-treated complementary auxotrophs increased heterokaryon formation by 10⁴ to 10⁵ using the strains differing in alleles of a single vegetative incompatibility gene but had no detectable effect on strains differing in multiple vegetative incompatibility genes. Vegetative compatibility tests of single conidial isolates resolved from these heterokaryons suggest that diploids had formed followed by the loss of one of the VIC alleles. Presence of both auxotrophic markers in some of these single conidial isolates confirms the occurrence of a parasexual cycle. These experiments demonstrate that ultraviolet-light can enhance heterokaryon formation and parasexuality in C. parasitica .     

Evolutionary Relationships between Rhynchosporium lolii sp. nov. and Other Rhynchosporium Species on Grasses

The fungal genus Rhynchosporium (causative agent of leaf blotch) contains several host-specialised species, including R. commune (colonising barley and brome-grass), R. agropyri (couch-grass), R. secalis (rye and triticale) and the more distantly related R. orthosporum (cocksfoot). This study used molecular fingerprinting, multilocus DNA sequence data, conidial morphology, host range tests and scanning electron microscopy to investigate the relationship between Rhynchosporium species on ryegrasses, both economically important forage grasses and common wild grasses in many cereal growing areas, and other plant species. Two different types of Rhynchosporium were found on ryegrasses in the UK. Firstly, there were isolates of R. commune that were pathogenic to both barley and Italian ryegrass. Secondly, there were isolates of a new species, here named R. lolii, that were pathogenic only to ryegrass species. R. lolii was most closely related to R. orthosporum, but exhibited clear molecular, morphological and host range differences. The species was estimated to have diverged from R. orthosporum ca. 5735 years before the present. The colonisation strategy of all of the different Rhynchosporium species involved extensive hyphal growth in the sub-cuticular regions of the leaves. Finally, new species-specific PCR diagnostic tests were developed that could distinguish between these five closely related Rhynchosporium species.     

Neotyphodium sinicum, from several Roegneria species throughout China, provides insights into the evolution of asexual endophytes

The fungus Neotyphodium sinicum forms an asymptomatic symbiosis with Roegneria grass species which are widely distributed in China. We collected 1,809 asymptomatic specimens of Roegneria, belonging to six species, from around 14 cities in nine provinces. 45.7% of the 1,809 asymptomatic Roegneria plants were endophyte-infected. Of 100 Neotyphodium isolates subsequently obtained, 31 were tested for cultural characteristics. These isolates showed morphological diversity and varying growth rates as well as differences in colony appearance and in conidial lengths. While conidial length of most of the isolates was typical of interspecific hybrid Neotyphodium species, conidial length of some isolates was smaller, similar to that of haploid species. A phylogenetic study was conducted of the tubB and tefA genes in 21 Neotyphodium isolates. The isolates were phylogenetically clustered into three types: 15/21 isolates had two divergent alleles of both tubB and tefA; 3/21 had two alleles of only tubB and 3/21 had two alleles of only tefA. In the tubB NJ tree, alleles-1 formed two distinct sub-clades in the Epichlo? bromicola/E. yangzii clade (EBY); allele-2 was located in three sub-clades widely distributed within the E. typhina clade (ETC). The allele-1 of the tefA gene was also located within the EBY clade and formed two sub-clades; allele-2 was located in 3 sub-clades within the ETC clade. No relationship was observed between phylogenetic relationships and morphology in the N. sinicum isolates. The phylogenetic characteristics had little relationship to the host species or site of origin of the isolates. An explanation consistent with the phylogenetic findings is that N. sinicum is a complex of species that has resulted from multiple independent hybridization events.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

Metapochonia hahajimaensis (Clavicipitaceae,Hypocreales), a new species from soil in Hahajima Island,Tokyo, Japan

A new species, Metapochonia hahajimaensis is proposed. Three isolates were obtained from soil samples collected from Hahajima, Ogasawara Islands, Tokyo, Japan. Using a combination of morphological characteristics and multigene (ITS, SSU, LSU, TEF) phylogenics, the isolates were identified as a Metapochonia species which were previously undescribed. The new species is distinguished from the closely-related M. bulbillosa and M. goniodes or other Metapochonia taxa by conidial size and colonial characteristics.     

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.     

Taxonomy and phylogeny of <Emphasis Type="Italic">Cryptocoryneum</Emphasis> (Pleosporales,Dothideomycetes)

Species of Cryptocoryneum were taxonomically reassessed on the basis of morphological observations and the results of molecular phylogenetic analysis. Eighteen isolates of Cryptocoryneum species, namely two strains from Africa, three from Europe, and 13 from Japan, were phylogenetically analysed using sequences of nuclear rDNA internal transcribed spacers (ITS) and the partial sequence of the translation elongation factor 1α gene (TEF1). The phylogenetic analysis indicated that Cryptocoryneum species formed a monophyletic clade and were closely related to Lophiotrema (Lophiotremataceae) and Aquasubmersa (incertae sedis) in the Pleosporales (Dothideomycetes). We examined holotype specimens of C. fasciculatum, C. hysterioides, and Torula uniformis and concluded that these species are conspecific, with C. hysterioides having priority. Although C. hysterioides has long been regarded as a synonym of C. condensatum, we consider C. hysterioides to be a distinct species within the genus. We found several cryptic species that were morphologically similar to C. condensatum sensu lato, but that could be separated on the basis of conidial size and the number of conidial arms and conidial septa, characters that seem to be informative for species delimitation within Cryptocoryneum. A total of seven new species, namely C. akitaense, C. brevicondensatum, C. congregatum, C. japonicum, C. longicondensatum, C. paracondensatum, and C. pseudorilstonei, are described and illustrated. A key to species accepted in Cryptocoryneum is provided.     

Mating Type Gene (MAT1-2) of Trichophyton verrucosum

Trichophyton verrucosum is a zoophilic dermatophyte species that is the most frequent etiologic agent of bovine dermatophytosis throughout the world. Since no teleomorph of T. verrucosum has been found, polymerase chain reaction (PCR) analysis on the genome of T. verrucosum isolated from the Czech Republic and Japan was performed to confirm the presence of a mating type locus in the genome of the fungus and to clarify its classification and ecological characteristics. The mating type gene (MAT1-2) allele was detected by PCR analysis in all 22 isolates (four isolates from the Czech Republic and 18 isolates from Japan). The nucleotide sequence of the region exhibited 99–100 % identity among all isolates, including the reference strain of T. verrucosum. Phylogenetic analysis revealed that the sequences of the internal transcribed spacer region at the MAT1-2 locus clustered together in the isolates examined, forming a branch distinct from that of the other dermatophyte species. These results suggest that T. verrucosum is a clonal offshoot that has drifted away from Arthroderma benhamiae.     

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.     

Investigation of variation in phenotype and DNA content between single-conidium isolates of single Penicillium strains

Variation in phenotypic properties was examined in three strains of closely related fasciculate species of Penicillium using 114 morphological, physiological, and biochemical characters. Thirty-six of these characters showed variation within single-conidium isolates of the same strain. Conidial sizes and nuclear DNA contents were compared using flow microfluorimetry; these results suggested that significant differences in conidial DNA content are associated with phenotypic variation. The taxonomic significance of the results is discussed.     

Molecular phylogenetic analyses based on the nuclear rRNA genes and the intron–exon structures of the nuSSU rRNA gene in Dictyocatenulata alba (anamorphic Ascomycota)

Molecular phylogenies inferred from the nuclear small subunit rRNA gene (nuSSU), nuclear large subunit rRNA gene D1/D2 region (nuLSU), and ITS-5.8S rRNA gene (ITS) indicated that five cultures of the lichenized hyphomycete Dictyocatenulata alba isolated from Japan form a monophyletic clade with high bootstrap support, and a close relationship to the Ostropomycetidae (Lecanoromycetes, Pezizomycotina, Ascomycota). Insertion sequences were found in the nuSSU of all isolates [e.g., nine insertions in the strain JCM 5358 (Japan Collection of Microorganisms)], some of which were group I introns. Five new insertion positions were found among the D. alba isolates. Using BLAST, none of the insertion sequences of D. alba were closely related to those of fungi or other organisms deposited in public DNA databases. Insertion positions were similar in some isolates, and two positions were common to all isolates. Although all phylogenetic analyses based on nuSSU, nuLSU, and ITS revealed the monophyly of D. alba, the isolates were divided into two (in the nuSSU tree) or three (in the nuLSU and ITS trees) groups. Based on the phylogenetic analyses and the intron–exon structures, the five isolates identified as D. alba belong to three cryptic species and therefore D. alba should be considered a species complex. The very slow-growing, tough agar colonies of the isolates, the occurrence of the species on both slightly lichenized and nonlichenized surfaces of trees, or pebbles (rarely on soil), suggest that the members of the D. alba complex may be lichenized. The photobiont was not clearly identified in this study.     

Conidial development in selected ambrosial species of the genus <Emphasis Type="Italic">Raffaelea</Emphasis>

Conidiogenesis of the type species and two additional species of the genus Raffaelea Arx & Hennebert are described. In contrast to previous conclusions based on light microscopy that indicate sympodial production of the conidia, we found that conidium development was by annellidic percurrent proliferation in the type species Raffaelea ambrosiae, as well as in Raffaelea arxii and Raffaelea albimanens. Consequently, this mode of conidium development is similar to the conidiogenesis of anamorphs within the Ophiostomatales, but distinguished by sporodochia formed within the genus Raffaelea. These findings reduce the differences between Raffaelea and other anamorphs with annellidic percurrent proliferation of the conidiogenous cells within the Ophiostomatales to different conidiomata and conidiophores, and they support previous reports of the close phylogenetic relationship between Raffaelea and Ophiostoma.     

Drug Resistance of Enteric Bacteria IX. Distribution of R Factors in Gram-negative Bacteria from Clinical Sources

Many isolates belonging to the Enterobacteriaceae were collected in 1965 from the inpatients at geographically scattered hospitals in Japan. Among 2,650 Shigella strains examined, 58.4% were found to be drug-resistant; 95.0% of these resistant strains were multiply resistant. Among 434 resistant strains examined, 81% carried R factors that were transferable by cell-to-cell contact. Of 160 isolates of other enteric bacteria, drug-resistant strains included 84.2% of the Escherichia coli, 93.0% of the Klebsiella, and 90.0% of the Proteus cultures. Among these resistant strains, 70.3% of the E. coli, 66.7% of the Klebsiella, and 52.0% of the Proteus were multiply resistant. Of these resistant strains, 84.0% of the E. coli, 88.0% of the Klebsiella, and 50.0% of the Proteus strains carried R factors. These results indicate that R factors are widespread among gram-negative bacteria of clinical significance.     

Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms

Background

Vibrio Parahaemolyticus is an aquatic, halophilic, Gram-negative bacterium, first discovered in 1950 in Japan during a food-poisoning outbreak. Infections resulting from consumption of V. Parahaemolyticus have increased globally in the last 10 years leading to the bacterium's classification as a newly emerging pathogen. In 1996 the first appearance of a pandemic V. Parahaemolyticus clone occurred, a new O3:K6 serotype strain that has now been identified worldwide as a major cause of seafood-borne gastroenteritis.

Results

We examined the sequenced genome of V. Parahaemolyticus RIMD2210633, an O3:K6 serotype strain isolated in Japan in 1996, by bioinformatic analyses to uncover genomic islands (GIs) that may play a role in the emergence and pathogenesis of pandemic strains. We identified 7 regions ranging in size from 10 kb to 81 kb that had the characteristics of GIs such as aberrant base composition compared to the core genome, presence of phage-like integrases, flanked by direct repeats and the absence of these regions from closely related species. Molecular analysis of worldwide clinical isolates of V. Parahaemolyticus recovered over the last 33 years demonstrated that a 24 kb region named V. Parahaemolyticus island-1 (VPaI-1) encompassing ORFs VP0380 to VP0403 is only present in new O3:K6 and related strains recovered after 1995. We investigated the presence of 3 additional regions, VPaI-4 (VP2131 to VP2144), VPaI-5 (VP2900 to VP2910) and VPaI-6 (VPA1254 to VPA1270) by PCR assays and Southern blot analyses among the same set of V. Parahaemolyticus isolates. These 3 VPaI regions also gave similar distribution patterns amongst the 41 strains examined.

Conclusion

The 4 VPaI regions examined may represent DNA acquired by the pandemic group of V. Parahaemolyticus isolates that increased their fitness either in the aquatic environment or in their ability to infect humans.