Internal Transcribed Spacer Gene Research Articles

Diversity trumps acidification: Lack of evidence for carbon dioxide enhancement of Trichodesmium community nitrogen or carbon fixation at Station ALOHA

We conducted 11 independent short‐term carbon dioxide (CO2) manipulation experiments using colonies of the filamentous cyanobacteria Trichodesmium isolated on three cruises in the North Pacific Subtropical Gyre (NPSG). Dinitrogen (N2) and carbon (C) fixation rates of these colonies were compared over CO2 conditions ranging from ∼ 18 Pa (equivalent to last glacial maximum atmospheric ) to ∼ 160 Pa (predicted for ∼ year 2200). Our results indicate that elevated has no consistent significant effect on rates of N2 or C fixation by Trichodesmium colonies in the NPSG under present environmental conditions. Differences between treatments were not modulated by phosphorus amendments, iron amendments, or light level. Sequencing the hetR, nifH, 16S, and internal transcribed spacer genes of Trichodesmium colonies revealed a highly diverse community of Trichodesmium and other N2‐fixing colony‐associated organisms. The species composition of Trichodesmium demonstrated spatiotemporal variability, but over half of total sequences were phylogenetically closely related (> 99% hetR sequence similarity) to isolate H9‐4 of T. erythraeum, which showed no response to elevated in previous laboratory experiments. Our handpicked Trichodesmium colonies included a substantial number of organisms other than Trichodesmium with the metabolic capacity for N2 and C fixation. We suggest that the diverse assemblage of Trichodesmium species and coexisting microorganisms within the colonies can explain the lack of an observed CO2 enhancement of N2 or C fixation rates, because different species are known to have different specific affinities for CO2.

Molecular characterization of Toxocara spp. from soil of public areas in Ahvaz southwestern Iran

In the present study, the microscopy and polymerase chain reaction methods were used for detection and identification of soil contamination by Toxocara eggs in squares, streets, public parks, and rubbish dumps in Ahvaz, southwestern Iran.A total of 210 soil samples were collected from different parts of the city and examined by microscopy and polymerase chain reaction (PCR) methods, following sodium nitrate flotation. Nucleotide sequencing was performed to confirm the results of the PCR method. Toxocara eggs were found in 64 and 71 soil samples using the microscopy and PCR methods, respectively. The highest contamination rate was observed in the central part of Ahvaz (39.5% and 46.5% by the microscopy and PCR methods, respectively). Based on internal transcribed spacer 2 (ITS2) PCR identification, 28% of the samples were diagnosed as Toxocara cati and 5.7% as Toxocara canis; no mixed contamination was observed. DNA sequencing of the ITS2 gene confirmed our findings.Compared to the conventional microscopic detection following by flotation, used as the gold standard, the PCR method appears to be rapid and sensitive as well as allows analysis of Toxocara spp. isolated from soil independent of the stage of egg development. Therefore, the PCR method appears to be a valuable tool for the diagnosis and differentiation of Toxocara spp. from soil samples in epidemiological studies, and will help the local health systems in effective prevention and control of disease.

Analyses of ITS and LSU gene regions provide congruent results on fungal community responses

The Internal Transcribed Spacer (ITS) regions and the Large Subunit (LSU) of the nuclear ribosomal RNA (rRNA) gene complex are commonly used to elucidate questions in fungal community ecology. Here, we compared the congruence across these gene regions using two ecological experiments (primary successional dynamics at a receding glacier forefront and community dynamics in stored Sorghum biomass), in which both ITS1 and LSU were sequenced from the same DNA extracts. We analyzed richness, diversity and evenness estimators along with community shifts inferred from ordination analyses. Our analyses show that ITS and LSU provide similar results and consistent conclusions. Taken together, we conclude that either gene region is appropriate for testing ecological hypotheses as long as there are no a priori hypotheses that preclude the use of one gene region over the other.

Phylogeny of the land snails Bradybaena and Phaeohelix (Pulmonata: Bradybaenidae) in Japan

The Japanese Archipelago harbours high diversity of endemic bradybaenid land snails. However, there have been few systematic studies of these snails. The resolution of the taxonomy and phylogenetic relationships of these bradybaenid land snail taxa is important both for describing species diversity and for promoting the conservation of these land snails. We investigated the molecular phylogeny of Bradybaena and Phaeohelix using the CO1 and internal transcribed spacer genes, to clarify whether morphological traits and the current species taxonomy of these genera reflect their phylogenetic relationships. Our results show that the Japanese species in these genera are genetically divided into three clades, and the geographical distribution pattern of the lineages tends to reflect phylogenetic relationships. Although the nominal species taxonomy of these genera was not consistent with their molecular phylogenetic relationships, their shell and genital morphology reflected phylogenetic relationships to some extent. Inferred phylogeny and observed genital morphology showed that Phaeohelix submandarina, P. miyakejimana and Bradybaena circulus oceanica from Hachijo-kojima Island belong to P. phaeogramma. In addition, the distinction between Bradybaena and Phaeohelix was not supported by molecular phylogeny, showing instead that Phaeohelix should be synonymized with Bradybaena. This study suggests that a further taxonomic revision of Japanese Bradybaenidae is needed and, to address this issue, genital anatomy is useful in addition to molecular phylogenetic analyses.

Prevalence, Isolation and Molecular Characterization ofBartonellaSpecies in Republic of Korea

To determine the prevalence of Bartonella species and identify which species of Bartonella naturally infects the striped field mouse (Apodemus agrarius) in the Republic of Korea (ROK), spleens from 200 mice were assayed by nested polymerase chain reaction (nPCR) targeting the RNA polymerase subunit beta (rpoB) gene and the 16S-23S internal transcribed spacer (ITS) region for members of the genus Bartonella. Utilizing PCR techniques, the prevalence of Bartonella spp. ranged from 31.5% (63/200) to 62.0% (124/200) for the rpoB and ITS gene fragments, respectively. The most prevalent species, Bartonella grahamii, was assigned to 17 genotypes and closely related to the zoonotic pathogens, B.taylorii, B.tribocorum, B.phoceensis and B.henselae, which also were detected. Two Bartonella isolates (KRBG28 and KRBG32) were recovered from blood of A.agrarius captured in Gyeonggi Province, ROK. Comparison of the 16S rRNA, hemin-binding protein E (hbpE), glutamate dehydrogenase 1 (gdh1), invasion-associated protein B (ialB), cell division protein (ftsZ), citrate synthase (gltA), 60 kDa heat shock protein (groEL), rpoB gene fragments and the ITS region sequences from the isolates with GenBank was confirmed as B.grahamii. Phylogenetic analysis based on the alignment of concatenated sequences (4933 bp) of KRBG28 and KRBG32 clustered with B.grahamii, forming an independent clade between Asian and American/European B.grahamii genogroups.

Phylogenetic relationship between different race representative populations of Fusarium oxysporum f. sp. ciceris in respect of translation elongation factor-1α, β-tubulin, and internal transcribed spacer region genes

Genetic diversity of 70 isolates of Fusarium oxysporum f. sp. ciceris originated from various states of India representing eight races causing wilt in chickpea (Cicer arietinum) was analyzed using translation elongation factor-1α (TEF-1α), β-tubulin, and internal transcribed spacer (ITS) gene regions. TEF-1α, β-tubulin, and ITS gene-specific markers produced ~720-, ~500-, and ~550-bp amplicons, respectively, in all the isolates of the pathogen. A phylogenetic tree constructed from the sequences generated in the present study along with the sequences of foreign isolates of Fusarium species available in NCBI database sharing more than 90 % nucleotide sequence similarity grouped the isolates into two major clusters. Most of the isolates of the present study showed more or less similar grouping pattern in case of the three gene sequences. Each group had the isolates representing different races as well as place of origin indicating low level of diversity among the isolates in respect of these gene sequences. Except TEF-1α, the groups generated by β-tubulin and ITS gene sequences did not correspond to the state of origin and races of the pathogen. However, the groups of TEF-1α partially corresponded to the place of origin as well as races of the pathogen. The isolates did not show any race-specific grouping patterns; however, most of the isolates representing race 1 clustered separately.

Sequence analysis in partial genes of five isolates of Angiostrongylus cantonensis from Taiwan and biological comparison in infectivity and pathogenicity between two strains

Angiostrongylus cantonensis is the most common infectious agent causing eosinophilic meningitis and is present in Taiwan, Thailand and the Pacific islands. Clinical symptoms vary within different endemic regions, and their severity is probably dependent on the number of ingested parasites and the diversity among strains. The experimentally definitive host is the rat, and non-permissive hosts are certain mammals such as humans and mice. In this study, the partial gene sequences of two A. cantonensis strains isolated from five different regions in Taiwan were selected and molecularly analyzed. The internal transcribed spacer gene and cytochrome-c oxidase subunit I gene sequences of the Hualien (H) strain of A. cantonensis differed from those of the Pingtung (P) strain and the other three strains by 19% and 11%, respectively. We analyzed the infectivity, fecundity, and development of the H and P strain in rats and host pathogenicity in mice inoculated with both strains. The number of the emerged first-stage larvae, adult recovery, and average length of adults in Sprague–Dawley rats significantly differed between rats inoculated with the H and P strain. Young adult recovery, average length of young adults, eosinophil counts in the cerebrospinal fluid (CSF), glutathione peroxidase concentration, levels of reactive oxygen species as well as malondialdehyde concentration in the CSF, and the survival of mice significantly differed between BALB/c mice inoculated with the H and P strain. The H strain of A. cantonensis had lower infectivity, delayed fecundity, and poor development in rats, and caused milder pathology and lower mortality in mice than the P strain. These data clearly indicate that the H strain of A. cantonensis is a pathogenically distinct strain with lower infectivity to its definitive host, and causing mild pathogenic symptoms to its non-permissive host.

DNA barcoding assessment of green macroalgae in coastal zone around Qingdao, China

An assessment with assistance of DNA barcoding was conducted on green macroalgae in coastal zone around Qingdao, China, during the period of April–December, 2011. Three markers were applied in molecular discrimination, including the plastid elongation factor tufA gene, the internal transcribed spacer (ITS) region of the ribosomal cistron and rubisco large subunit gene 3′ regions (rbcL-3P). DNA barcoding discriminated 8 species, excluding species of genus Cladophora and Bryopsis due to failures in amplification. We ascertained and corrected 4 species identified by morphological methods for effectively assisting the classification. The gene tufA presented more advantages as an appropriate DNA marker with the strongest amplification success rate and species discrimination power than the other two genes. The poorest sequencing success largely handicapped the application of ITS. Samples identified by tufA and rbcL as Ulva flexuosa were clustered into the clade of U. prolifera by ITS in the neighbor-joining tree. Confusion with discrimination of the complex of U. linza, U. procera and U. prolifera (as the LPP complex) still existed for the three DNA markers. Based on our results, rbcL is recommended as a preferred marker for assisting tufA to discriminate green macroalgae. In distinguishing green-tide-forming Ulva species, the free-floating sample collected from the green tide in 2011 was proved to be identical with U. prolifera in Yellow Sea for ITS and rbcL genes. This study presents a preliminary survey of green macroalgae distributed in the coastal area around Qingdao, and proves that DNA barcoding is a powerful tool for taxonomy of green macroalgae.

Multiple lines of evidence on the genetic relatedness of the parthenogenetic and bisexual Haemaphysalis longicornis (Acari: Ixodidae)

As an obligate hematophagous ectoparasite, the hard tick Haemaphysalis longicornis exhibits two reproductive strategies, bisexual reproduction, and obligate parthenogenesis, which have attracted a widespread attention. However, the speciation of parthenogenetic population remained ambiguous due to its similarity in morphology but the remarkable differences in cytogenetics as compared with those of the bisexual ones. In the present study, we explored several new lines of genetic evidence to resolve this controversial issue. The number of the chromosomes in two lineages was checked by classical methods and their total DNA levels were determined utilizing flowcytometry. In addition, the sequences of 12S rDNA, 16S rDNA, cytochrome c oxidase I and II (COI, COII) and internal transcribed spacer-2 (ITS-2) genes were used to assess their phylogenetic relationship. We observed that the chromosome ploidy of bisexual and parthenogenetic H. longicornis collected by our laboratory was diploid and triploid, respectively. Flowcytometry analysis indicated a ratio close to 2:3 in the DNA contents of bisexual to parthenogenetic H. longicornis. Although the chromosome ploidy is different, their gene sequences are extremely similar. Analogous to the intra-species genetic difference of other invertebrates, sequence differences of all loci examined are below 2%. Phylogenetic trees constructed from 12S rDNA, 16S rDNA, COI, and ITS-2 genes revealed that they were all in the same monophyletic clade instead of splitting independently into evolutional branches. Moreover, according to 4× Rule, the K/θ ratio of two reproductive populations calculated based on COI was much smaller than four, strongly supporting that they belong to the same species. Therefore, we conclude that the evolutionary process just disturbs the chromosome ploidy and the sexual determination of parthenogenetic population and that it would be better to consider parthenogenetic H. longicornis as a metapopulation rather than a cryptic species.

Characterization of ITS1 gene of Leishmania infantum isolated from Iraqi patients with visceral leishmaniasis by PCR- RFLP and sequencing methods.

For the best of our knowledge, there is no information about molecular characterization of Iraqiisolates of visceral leishmaniasis, the present work aimed to characterize three different Iraqiisolates of Leishmania infantum by polymerase chain reaction (PCR), restriction fragmentlength polymorphism (RFLP) and sequencing methods.Three isolates from bone marrow of Iraqi patients infected with kala azar were used in thisstudy. The isolates were already diagnosed by isoenzyme as Leishmania infantum. Patients wereinhabiting different parts of Baghdad. The samples after microscopic examination were culturedon modified NNN media. Then DNA was extracted for amplifying ITS1 (internal transcribedspacer 1) gene by PCR. Identification of samples was studied using RFLP (digestion with Apo1restriction enzyme) and sequencing of PCR products.The PCR of all samples showed a band under about 500 bp. The results by using PCR-RFLPmethod showed no restriction with digestion with Apo1 restriction enzyme. The results ofsequencing showed differences with all separated gene from Leishmania infantum in the genebank.In this study we found that the sequences of ITS1 gene of Leishmania infantum separated fromIraqi patients are different from other samples, as there is no similarity with Leishmaniainfantum (MHOM/TN/80/IPI1). The more similarity is with the Iranian isolate of Leishmaniainfantum (MCAN/IR/97/LON) with 41%, whereas the similarity with Crithidia luciliae internaltranscribed spacer 1, ITS1 is 96%.

Development of molecular markers and probes based on TEF-1α, β-tubulin and ITS gene sequences for quantitative detection of Fusarium oxysporum f. sp. ciceris by using real-time PCR

Fusarium wilt (Fusarium oxysporum f. sp. ciceris) causes significant yield losses in chickpea worldwide. Faster, reliable and more specific molecular detection techniques were developed for the detection of Fusarium oxysporum f. sp. ciceris (Foc). The sequences obtained from multiple alignments of target genes, namely, translation elongation factor-1α (TEF-1α), β-tubulin, and internal transcribed spacer (ITS), were used to design Foc-specific markers/probes. One set of TEF-1α-based molecular marker, namely, SPα-F and R, two sets of β-tubulin-based markers, namely, SPβ1-F and R, and SPβ2-F and R, and one set of ITS gene, namely, SPT-F and R, were developed for the detection and quantification of Foc from diverse samples. The specificity and sensitivity of the designed molecular markers were evaluated through conventional and real-time PCR assays which differentiated the Foc from closely related species of Fusarium and other plant pathogens. In conventional PCR, the minimum detection limits of the markers ranged from 12.5 pg to 100 pg for genomic DNA of Foc and 0.5 ng to 10 ng for infected plant samples. In real-time PCR assay, the minimum detection limits of the markers ranged from 0.001 pg to 0.25 pg for genomic DNA of Foc and from 0.04 pg to 1.5 pg for the infected plant samples. Thus, the markers designed in the present study were found to be specific for Foc and can be used consistently for the detection and identification of Foc isolates. The probes developed from the two sets of markers, namely, SPα and SPβ2, also showed specificity with Foc.

Characterization of Diaporthe australafricana and Diaporthe spp. Associated with Stem Canker of Blueberry in Chile.

Stem canker and dieback are important factors that limit the longevity and reduce the yield of blueberry (Vaccinium spp.) in Chile. In this study, species of Diaporthe associated with blueberry were isolated and identified. The internal transcribed spacer (ITS) regions of ribosomal DNA of 30 isolates and the translation elongation factor 1-α (EF1-α) of 14 isolates were sequenced, analyzed, and compared with their morphological and pathological characteristics. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Diaporthe ambigua, D. australafricana, D. neotheicola, D. passiflorae, and Diaporthe sp. 1. However, morphology alone was insufficient to identify these species. The combined analysis of ITS and EF1-α gene sequences grouped the Chilean blueberry isolates in the same five groups obtained in the ITS analysis. Pathogenicity tests conducted with attached and detached blueberry shoots (<1 year old) and stems (1 to 2 years old) confirmed that isolates of these Diaporthe spp. were pathogenic. The symptoms were reproducible and consisted of necrotic reddish-brown cankers on blueberry shoots and stems. These isolates were capable of infecting blueberry fruit, causing a soft decay, suggesting that they were tissue nonspecific and were also pathogenic on shoots of apple, grapevine, and pear. D. australafricana was the most frequently isolated species and D. ambigua, D. australafricana, and D. passiflorae were highly virulent in shoots, stems, and fruit of blueberry. This study showed that at least four species of Diaporthe are primary pathogens, capable of causing stem canker symptoms on blueberry, and this is the first report of D. ambigua, D. neotheicola, and D. passiflorae attacking this host.

What are the common anthracnose pathogens of tropical fruits?

Species of Colletotrichum are associated with anthracnose of a wide range of host plants including cultivated and wild tropical fruits. The genetic and ecological diversity of species associated with wild fruits are poorly explored, as compared to those associated with pre and postharvest diseases of cultivated fruits. In the present study, isolates of Colletotrichum were obtained from commercially available cultivated fruits, wild fruits (from native trees in natural habitats) and a few herbaceous hosts collected in northern Thailand. These isolates were initially characterized based on analysis of complete sequences of nuclear ribosomal internal transcribed spacer (ITS), into the genetically defined species complexes of Colletotrichum gloeosporioides, C. acutatum, C. boninense and C. truncatum. The isolates were primarily identified in the C. gloeosporioides species complex, based on a strongly supported clade within the ITS gene tree and were further characterized using multi-gene phylogenetic analyses and morphology. Phylogenetic analyses of ITS, partial sequences of actin (ACT), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS) and β-tubulin (TUB2) genetic markers were performed individually and in combination. Colletotrichum gloeosporioides sensu stricto was identified from lime (Citrus aurantifolia) and rose apple (Syzygium samarangense). Colletotrichum fructicola was isolated from dragon fruit (Hylocerous undatus) and jujube (Ziziphus sp.). Colletotrichum endophytica was found only from an unknown wild fruit. We observed a considerable genetic and host diversity of species occurring on tropical fruits within the clade previously known as Colletotrichum siamense sensu lato. The clade consists of isolates identified as pre and postharvest pathogens on a wide range of fruits, including coffee (Coffea arabica), custard apple (Annona reticulata), Cerbera sp., figs (Ficus racemosa) mango (Mangifera indica), neem (Azadirachta indica) and papaya (Carica papaya) and was the dominant group of species among most wild fruits studied. With the exception of one isolate from banana, which grouped in the C. siamense clade, all the other isolates were identified as Colletotrichum musae. A new species, Colletotrichum syzygicola, associated with Syzygium samarangense in Thailand, is introduced with descriptions and illustrations. This study highlights the need to re-assess the evolutionary relationships of Colletotrichum species occurring on cultivated and wild fruits with emphasis on their ecology and cryptic diversification including sampling at regional and global scales.

Genetic characterization and phylogenetic analysis of Eimeria arloingi in Iranian native kids

Among the 16 species of Eimeria from goats, Eimeria arloingi and Eimeria ninakohlyakimovae are regarded as the most pathogenic species in the world and cause clinical caprine coccidiosis. E. arloingi is known to be an important cause of coccidiosis in Iranian kids. Molecular analyses of two portions of nuclear ribosomal DNA (internal transcribed spacer1 (ITS1) and 18S rDNA) were used for the genetic characterization of the E. arloingi. Comparison of the sequencing data of E. arloingi obtained in the present study (ITS1: KC507793 and 18S rDNA: KC507792) with other Eimeria species in the GenBank database revealed a particularly close relationship between E. arloingi and Eimeria spp. from the cattle and sheep. The phylogram based on the ITS1 sequences shows that the E. arloingi, Eimeria bovis, and Eimeria zuernii formed a distinct group separate from the other remaining Eimeria spp. in cattle and poultry. In pairwise alignment, 18S rDNA sequence derived from E. arloingi showed 99% similarity to Eimeria ahsata with differences observed at only three nucleotides. This study showed that the ITS1 and 18S rDNA gene are useful genetic markers for the specific identification and differentiation of Eimeria spp. in ruminants.

Multiplex PCR assay for the detection of common dermatophyte nail infections

Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. Multiplex (MX) PCR was reported as a reliable alternative. Dermatophyte gene sequence records were used to design a MX PCR for detection and identification of dermatophytes in nail specimens. A MX PCR method based on the amplification of the chitin synthase 1 and internal transcribed spacer genes was developed. The study included 93 strains of dermatophytes and non-dermatophytic fungi, six dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction directly from nail samples was carried out by using the QIAamp DNA extraction kit (Quiagen). A set of primers was designed and their specificity was assessed. MX PCR detected the causal agent in specimens from which Trichophyton rubrum and T. interdigitale grew in culture and also identified a dermatophyte species in an additional 32 specimens that were negative in microscopy and culture. None of the investigated non-dermatophytic strains was positive. Sensitivity of MX PCR was higher as compared to mycological examination (97% vs. 81.1%). MX PCR for direct detection of dermatophytes from nail samples yielded mixed flora in 32.8% of samples. MX PCR proved sensitive and adequate for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing moulds, which makes the identification of the causal agent problematic.

Molecular Detection ofAnaplasma,Bartonella, andBorreliaSpecies in Ticks Collected from Migratory Birds from Hong-do Island, Republic of Korea

Bird migration is a recurring annual and seasonal event undertaken by more than 100 species of birds in the southeast Asian and northeast Palearctic regions that pass through or remain for short periods from April to May and September to November at Hong-do Island, Republic of Korea (ROK). A total of 212 ticks (40 Haemaphysalis flava, 12 H. longicornis, 146 Ixodes turdus, 13 I. nipponensis, and 1 I. ornithophila) were collected from 65/2,161 (3.0%) migratory birds consisting of 21 species that were captured from January, 2008, through December, 2009, as part of the Migratory Birds Center, Hong-do bird banding program for studying bird migration patterns. Adult ticks were assayed individually while larvae and nymphs were pooled (1-22 and 1-6 ticks per pool, respectively) into 31 and 65 pools, respectively. Ticks were assayed for zoonotic pathogens by PCR using 16S rRNA, heat shock protein (groEL), and internal transcribed spacer (ITS) gene primers to amplify genera specific for Anapalsma, Bartonella, and Borrelia PCR amplicons. Using the 16S rRNA-based nested PCR, A. phagocytophilum (n=1) was detected in I. nipponensis collected from Zoothera sibirica and A. bovis (n=1) was detected in I. turdus collected from Emberiza chrysophrys. Borrelia turdi 16S rRNA genes (n=3) were detected in I. turdus and I. nipponensis collected from Turdus pallidus and Zoothera aurea. Borrelia spp. 16S rRNA genes (n=4) were detected in Ixodes ticks collected from Emberiza tristrami, T. pallidus, and Z. aurea. The Bartonella grahamii ITS gene (n=1) was detected by nested PCR assay in I. turdus collected from Z. aurea. These results provide insight into the potential role of migratory birds in the dispersal of ticks and associated tick-borne pathogens throughout their ranges in Asia.

Soil Suppressiveness to Fusarium Disease: Shifts in Root Microbiome Associated with Reduction of Pathogen Root Colonization

Soil suppressiveness to Fusarium disease was induced by incubating sandy soil with debris of wild rocket (WR; Diplotaxis tenuifolia) under field conditions. We studied microbial dynamics in the roots of cucumber seedlings following transplantation into WR-amended or nonamended soil, as influenced by inoculation with Fusarium oxysporum f. sp. radicis-cucumerinum. Disease symptoms initiated in nonamended soil 6 days after inoculation, compared with 14 days in WR-amended soil. Root infection by F. oxysporum f. sp. radicis-cucumerinum was quantified using real-time polymerase chain reaction (PCR). Target numbers were similar 3 days after inoculation for both WR-amended and nonamended soils, and were significantly lower (66%) 6 days after inoculation and transplanting into the suppressive (WR-amended) soil. This decrease in root colonization was correlated with a reduction in disease (60%) 21 days after inoculation and transplanting into the suppressive soil. Fungal community composition on cucumber roots was assessed using mass sequencing of fungal internal transcribed spacer gene fragments. Sequences related to F. oxysporum, Fusarium sp. 14005, Chaetomium sp. 15003, and an unclassified Ascomycota composed 96% of the total fungal sequences in all samples. The relative abundances of these major groups were highly affected by root inoculation with F. oxysporum f. sp. radicis-cucumerinum, with a 10-fold increase in F. oxysporum sequences, but were not affected by the WR amendment. Quantitative analysis and mass-sequencing methods indicated a qualitative shift in the root's bacterial community composition in suppressive soil, rather than a change in bacterial numbers. A sharp reduction in the size and root dominance of the Massilia population in suppressive soil was accompanied by a significant increase in the relative abundance of specific populations; namely, Rhizobium, Bacillus, Paenibacillus, and Streptomyces spp. Composition of the Streptomyces community shifted significantly, as determined by PCR denaturing gradient gel electrophoresis, resulting in an increase in the dominance of a specific population in suppressive soils after only 3 days. This shift was related mainly to the increase in Streptomyces humidus, a group previously described as antagonistic to phytopathogenic fungi. Thus, suitable soil amendment resulted in a shift in the root's bacterial communities, and infection by a virulent pathogen was contained by the root microbiome, leading to a reduced disease rate.

Molecular Evidence Shows Low Species Diversity of Coral-Associated Hydroids in Acropora Corals

A novel symbiosis between scleractinians and hydroids (Zanclea spp.) was recently discovered using taxonomic approaches for hydroid species identification. In this study, we address the question whether this is a species-specific symbiosis or a cosmopolitan association between Zanclea and its coral hosts. Three molecular markers, including mitochondrial 16S and nuclear 28S ribosomal genes, and internal transcribed spacer (ITS), were utilized to examine the existence of Zanclea species from 14 Acropora species and 4 other Acroporidae genera including 142 coral samples collected from reefs in Kenting and the Penghu Islands, Taiwan, Togian Island, Indonesia, and Osprey Reef and Orpheus Island on the Great Barrier Reef, Australia. Molecular phylogenetic analyses of the 16S and 28S genes showed that Acropora-associated Zanclea was monophyletic, but the genus Zanclea was not. Analysis of the ITS, and 16S and 28S genes showed either identical or extremely low genetic diversity (with mean pairwise distances of 0.009 and 0.006 base substitutions per site for the 16S and 28S genes, respectively) among Zanclea spp. collected from diverse Acropora hosts in different geographic locations, suggesting that a cosmopolitan and probably genus-specific association occurs between Zanclea hydroids and their coral hosts.

Clinical Spectrum of Exophiala Infections and a Novel Exophiala Species, Exophiala hongkongensis

We characterized 12 Exophiala strains isolated from patients over a 15-year period to the species level using phenotypic tests and internal transcribed spacer (ITS) and Rpb1 sequencing and described the clinical spectrum of the 12 patients. Eight patients had nail or skin infections, two had invasive infections, and two had colonization of the gastrointestinal tract. ITS and Rpb1 sequencing showed that 11 of the 12 strains were known Exophiala species (E. oligosperma [n = 3], E. jeanselmei [n = 2], E. lecanii-corni [n = 2], E. bergeri [n = 1], E. cancerae [n = 1], E. dermatitidis [n = 1], and E. xenobiotica [n = 1]), which included the first reported cases of onychomycosis caused by E. bergeri and E. oligosperma. The 12th strain (HKU32(T)), isolated from the nail clipping of the right big toe of a 68-year-old female patient with onychomycosis, possessed unique morphological characteristics distinct from other Exophiala species. It grew very slowly and had a velvety colony texture after 28 days, short conidiophores of the same olivaceous color as the supporting hyphae, numerous spores, and no chlamydospore-like cells. ITS, Rpb1, β-tubulin, and β-actin gene sequencing unambiguously showed that HKU32(T) was clustered with but formed branches distinct from other Exophiala species in phylogenetic trees. We propose the new species Exophiala hongkongensis to describe this novel fungus.

Morphological and genetic characterization of Hysterothylacium zhoushanensis sp. nov. (Ascaridida: Anisakidae) from the flatfish Pseudorhombus oligodon (Bleeker) (Pleuronectiformes: Paralichthyidae) in the East China Sea

Hysterothylacium zhoushanensis sp. nov. collected from the intestine of the flatfish Pseudorhombus oligodon (Bleeker) (Pleuronectiformes: Paralichthyidae) in the East China Sea is described and illustrated by light and scanning electron microscopy. The new species can be easily distinguished from its congeners by the presence of remarkable lateral alae, the very short intestinal caecum, the unusually long ventricular appendix (ratio of intestinal caecum to ventricular appendix 1:8.74-23.8), the short spicules (0.58-0.81 mm long, representing 1.70-2.08 % of body length) and the number and arrangement of male caudal papillae (35-42 pairs in total, arranged as 26-32 pairs of precloacal, two pairs of paracloacal and six to eight pairs of postcloacal). In addition, the adults and the putative third-stage larvae identified morphologically of the new species are characterised by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA. The result reveals that they are homogeneous genetically, and all belong to the same species. Molecular analysis by comparing the ITS gene of H. zhoushanensis sp. nov. with these species of Hysterothylacium available in GenBank also seem to support the validity of the new species based on the morphological observation.