Internal Transcribed Spacer Region Of rDNA Research Articles

Novel species of Botryosphaeriaceae associated with shoot blight of pistachio

Various species of phytopathogenic Botryosphaeriaceae were identified previously from pistachio trees worldwide. Disease symptoms caused by pathogens in Botryosphaeriaceae on pistachio include panicle and shoot blight, leaf defoliation, fruit discoloration and decay. In this study species of Botryosphaeriaceae were collected from blighted pistachio shoots in Arizona, USA, and Greece. The aims of this study were to identify these Botryosphaeriaceae isolates and to test their pathogenicity to pistachio. The fungi were identified based on comparisons of DNA sequence data of the nuclear rDNA internal transcribed spacer region (ITS), a partial translation elongation factor 1-alpha gene (TEF1), a partial β-tubulin gene (TUB2) and morphological characteristics. Results indicated that some isolates collected from pistachio represent two previously undescribed species, which we described here as Lasiodiplodia americana sp. nov. from the United States and Neofusicoccum hellenicum sp. nov. from Greece. Field inoculations of L. americana and N. hellenicum on branches of four pistachio cultivars showed that both L. americana and N. hellenicum are pathogenic on pistachio. The four pistachio cultivars differed in their susceptibility to the Botryosphaeriaceae species. Results of this study suggested that the two new species of Botryosphaeriaceae need to be monitored carefully to determine the distribution of these pathogens and the possible spread to other areas.

Phylogenetic and chemotypic diversity of Periglandula species in eight new morning glory hosts (Convolvulaceae)

Periglandula ipomoeae and P. turbinae (Ascomycota, Clavicipitaceae) are recently described fungi that form symbiotic associations with the morning glories (Convolvulaceae) Ipomoea asarifolia and Turbina corymbosa, respectively. These Periglandula species are vertically transmitted and produce bioactive ergot alkaloids in seeds of infected plants and ephemeral mycelia on the adaxial surface of young leaves. Whether other morning glories that contain ergot alkaloids also are infected by Periglandula fungi is a central question. Here we report on a survey of eight species of Convolvulaceae (Argyreia nervosa, I. amnicola, I. argillicola, I. gracilis, I. hildebrandtii, I. leptophylla, I. muelleri, I. pes-caprae) for ergot alkaloids in seeds and associated clavicipitaceous fungi potentially responsible for their production. All host species contained ergot alkaloids in four distinct chemotypes with concentrations of 15.8–3223.0 μg/g. Each chemotype was a combination of four or five ergot alkaloids out of seven alkaloids detected across all hosts. In addition, each host species exhibited characteristic epiphytic mycelia on adaxial surfaces of young leaves with considerable interspecific differences in mycelial density. We sequenced three loci from fungi infecting each host: the nuclear rDNA internal transcribed spacer region (ITS), introns of the translation factor 1-α gene (tefA) and the dimethylallyl-tryptophan synthase gene (dmaW), which codes for the enzyme that catalyzes the first step in ergot alkaloid biosynthesis. Phylogenetic analyses confirmed that these fungi are in the family Clavicipitaceae and form a monophyletic group with the two described Periglandula species. This study is the first to report Periglandula spp. from Asian, Australian, African and North American species of Convolvulaceae, including host species with a shrub growth form and host species occurring outside of the tropics. This study demonstrates that ergot alkaloids in morning glories always co-occur with Periglandula spp. and that closely related Periglandula spp. produce alkaloid chemotypes more similar than more distantly related species.

A re-evaluation of the genus Myceliophthora (Sordariales, Ascomycota): its segregation into four genera and description of Corynascus fumimontanus sp. nov.

Based on a number of isolates of Myceliophthora (Chaetomiaceae, Sordariales, Ascomycota) recently isolated from soil samples collected in USA, the taxonomy of the genus was re-evaluated through phylogenetic analyses of sequences from the nuc rDNA internal transcribed spacer region and genes for the second largest subunit of RNA polymerase II and translation elongation factor 1α. Members of Myceliophthora were split into four monophyletic clades strongly supported by molecular and phenotypic data. Such clades correspond with Myceliophthora, now restricted only to the type species of the genus Corynascus, which is re-established with five species, the new monotypic genus Crassicarpon and also the new genus Thermothelomyces (comprising four species). Myceliophthora lutea is mesophilic and a permanently asexual morph compared to the members of the other three mentioned genera, which also are able to sexually reproduce morphs with experimentally proven links to their asexual morphs. The asexual morph of M. lutea is characterized by broadly ellipsoidal, smooth-walled conidia with a wide, truncate base. Crassicarpon thermophilum is thermophilic and heterothallic and produces spherical to cuneiform, smooth-walled conidia and cleistothecial ascomata of smooth-walled, angular cells and ascospores with a germ pore at each end. Corynascus spp. are homothallic and mesophilic and produce spherical, mostly ornamented conidia and cleistothecial ascomata with textura epidermoidea composed of ornamented wall cells, and ascospores with one germ pore at each end. Thermothelomyces spp. are thermophilic, heterothallic and characterized by similar ascomata and conidia as Corynascus spp., but its ascospores exhibit only a single germ pore. A dichotomous key to distinguish Myceliophthora from the other mentioned genera are provided, as well as dichotomous keys to identify the species of Corynascus and Thermothelomyces. A new species, namely Corynascus fumimontanus, characterized by verrucose ascomatal wall cells and irregularly shaped ascospores, is described and illustrated.

Sarcodon in the Neotropics I: new species from Guyana, Puerto Rico and Belize

Four species of the ectomycorrhizal (ECM) genus Sarcodon (Bankeraceae, Thelephorales, Basidiomycota) are described as new to science. Sarcodon pakaraimensis sp. nov. is described from forests dominated by the ECM trees Pakaraimaea dipterocarpacea (Dipterocarpaceae) and Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae) in the Pakaraima Mountains of Guyana. Sarcodon portoricensis sp. nov. is described from lower montane wet forest within the El Yunque National Forest of Puerto Rico. Sarcodon quercophilus sp. nov. and Sarcodon umbilicatus sp. nov. are described from Quercus (Fagaceae) cloud forests within the Maya Mountains of Belize. The discovery of these species is significant given that the majority of the approximately 87 described Sarcodon species are north temperate or boreal in distribution and frequently associate with coniferous host plants; these constitute the most recent records for Sarcodon from the greater Neotropics. Each of the new species is morphologically consistent with accepted diagnostic characters for Sarcodon: pileate-stipitate stature, a dentate hymenophore, determinate basidiomatal development, fleshy, non-zonate context and brown, tuberculate basidiospores. DNA (ITS) sequence analysis corroborated the generic placement of S. pakaraimensis, S. portoricensis, S. quercophilus and S. umbilicatus and, along with morphological differences, supported their recognition as distinct species. Macromorphological, micromorphological, habitat and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) are provided for each of the new species. A key to Neotropical Sarcodon species and similar extralimital taxa is provided.

Characterization of Trichoderma species isolated in Ecuador and their antagonistic activities against phytopathogenic fungi from Ecuador and Japan

Native Trichoderma spp. were isolated from agricultural fields in several regions of Ecuador. These isolates were characterized via morphological observation as well as molecular phylogenetic analysis based on DNA sequences of the rDNA internal transcribed spacer region, elongation factor-1α gene and RNA polymerase subunit II gene. Fifteen native Trichoderma spp. were identified as T. harzianum, T. asperellum, T. virens and T. reesei. Some of these strains showed strong antagonistic activities against several important pathogens in Ecuador, such as Fusarium oxysporum f. sp. cubense (Panama disease) and Mycosphaerella fijiensis (black Sigatoka) on banana, as well as Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease) on cacao. The isolates also showed inhibitory effects on in vitro colony growth tests against Japanese isolates of Fusarium oxysporum f. sp. lycopersici, Alternaria alternata and Rosellinia necatrix. The native Trichoderma strains characterized here are potential biocontrol agents against important pathogens of banana and cacao in Ecuador.

Acaulospora ignota and Claroideoglomus hanlinii, two new species of arbuscular mycorrhizal fungi (Glomeromycota) from Brazil and Cuba

Morphological studies of spores and mycorrhizal structures and phylogenetic analyses of sequences of the partial small subunit rRNA gene, the whole internal transcribed spacer rDNA region, including the 5.8S rRNA gene, and the partial large subunit rRNA gene of two arbuscular mycorrhizal fungi (Glomeromycota) have indicated that they are undescribed species, one of the genus Acaulospora and the second of Claroideoglomus. The first species, here named A. ignota, is distinguished morphologically by yellow-coloured, relatively small (65–80 μm diam when globose) spores, whose upper surface of the structural laminate spore wall layer is ornamented with inconspicuous warts or flattened elevations, usually deteriorating and disappearing with age. The most distinguishing features of the second species, C. hanlinii, are its relatively small (45–90 μm diam when globose) and dark-coloured (olive yellow) spores, with a simple two-layered spore wall, of which the inner structural laminate layer 2 may be much thicker than that in other known Claroideoglomus species. The occasional pale staining of spore wall layer 1 in Melzer’s reagent is also distinctive in the species. Both species were grown in single-species pot cultures established from spores extracted from pot trap cultures with mixtures of rhizosphere soils and roots of plants colonizing maritime sand dunes near Natal, Brazil (A. ignota), and Varadero, Cuba (C. hanlinii). To date, these are the only sites in the world where these two fungi are known to occur, as confirmed by comparisons of their sequences with those deposited in available databases.

The epiphytic orchids Ionopsis utricularioides and Psygmorchis pusilla associate with different Ceratobasidium lineages at Valle del Cauca, Colombia

In Orchidaceae, association with symbiotic fungi is required for seed germination and seedling development, thereby being the main energy source during the first steps of germination. Colombia is one of the countries with the greatest biodiversity of orchids, with an estimated 3,200 species, but few studies on orchid mycorrhiza have been conducted. In our study, we isolated and sequenced the internal transcribed spacer rDNA region of fungi from two co-occurring Colombian epiphytic orchids, I. utricularioides and P. pusilla, both belonging to the subtribe Oncidiinae. All sequences were recognized as belonging to the genus Ceratobasidium, known to be a common orchid mycorrhizal fungus in both tropical and temperate orchids. One sequence was 100% similar to fungi isolated from I. utricularioides in Costa Rica in a previous study. I. utricularioides was confirmed to be a specialist, associating with only one clade of mycorrhizal fungi. However, P. pusilla was shown to be a generalist, associating with three clades. This finding indicates that the variation in mycorrhizal specificity could be an important factor in the co-existence of orchids. The high affinity between the subtribe Oncidiinae and Ceratobasidium was also reinforced.

Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia.

Development of a PCR assay based on the 16S–23S rDNA internal transcribed spacer for identification of strictly anaerobic bacterium Zymophilus

PCR-primers were designed for identification of strictly anaerobic bacteria of the genus Zymophilus based on genus-specific sequences of the 16S–23S rDNA internal transcribed spacer region. The specificity of the primers was tested against 37 brewery-related non-target microorganisms that could potentially occur in the same brewery specimens. None DNA was amplified from any of the non-Zymophilus strains tested including genera from the same family (Pectinatus, Megasphaera, Selenomonas), showing thus 100% specificity. PCR assay developed in this study allows an extension of the spectra of detected beer spoilage microorganisms in brewery laboratories.

Genetic diversity of medically important and emerging Candida species causing invasive infection.

BackgroundGenetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.MethodsWe selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C® system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.ResultsAmong 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C® profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.ConclusionsMolecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0793-3) contains supplementary material, which is available to authorized users.

Detection of damping-off of Cape gooseberry caused by Pythium aphanidermatum and its suppression with phosphonate

Cape gooseberry cultivated in commercial greenhouses developed wilt and root rot during the summer growing season of 2013 in Aichi Prefecture, Japan. A Pythium species was isolated from affected roots and identified as P. aphanidermatum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, this isolate caused severe wilt and root rot. P. aphanidermatum, which previously was not known to cause damping-off in Cape gooseberry, was isolated by a bait method from soil samples collected from within and outside of the greenhouses. Pre- and post-inoculation activity of phosphonate and two fungicides, cyazofamid and metalaxyl, was evaluated with in vivo experiments. The three compounds were applied 3 days before inoculation, and phosphonate and cyazofamid also were applied at 24 h after inoculation. All three substances provided similar protectant activity. Phosphonate and cyazofamid also exhibited curative activity. In vitro activity of phosphonate on mycelial growth, sporulation, and zoospore cyst germination was evaluated; the EC50 concentration for the inhibition of mycelial growth was low. Zoospore formation was sensitive to phosphonate, while the germination of encysted zoospores was not.

A Multiplex Real-Time PCR Assay for the Detection of Puccinia horiana and P. chrysanthemi on Chrysanthemum.

Puccinia horiana, the cause of chrysanthemum white rust, is a regulated fungal plant pathogen in the United States, while P. chrysanthemi, the cause of chrysanthemum brown rust, is a widespread but less destructive pathogen. Accurate identification is essential to enforce quarantine measures, but the two species cannot be differentiated visually in the absence of mature spores or symptoms. A multiplex real-time PCR assay was developed to detect and discriminate between P. chrysanthemi and P. horiana. Species-specific hydrolysis probes labeled with different fluorescent dyes were designed based on the rDNA internal transcribed spacer region. Seven fresh samples and 270 herbarium specimens of chrysanthemum rust were tested with the assay with results confirmed using spore morphology. P. horiana and P. chrysanthemi were accurately detected from all fresh samples, and as little as 1 pg of template DNA was reproducibly detected. Of the herbarium specimens, 99% were positive for at least one species using the multiplex assay with 7% positive for both species. This multiplex assay can discriminate between P. chrysanthemi and P. horiana and provides an additional tool for identification of P. horiana to ensure appropriate application of quarantine measures.

Comparison of the contamination rates of culture media used for isolation and identification of dermatophytes

Mycological media that promote spore production are essential for the diagnosis of dermatophytosis. However, these culture media frequently become contaminated by multiple fungal or bacterial species during culture. The aim of this study was to compare the contamination rates of 6 culture media used for the isolation and identification of dermatophytes, including Borelli's lactritmel agar (BLA), brain-heart infusion agar (BHIA), Lowenstein-Jensen agar (LJA), malt extract agar (MEA), potato dextrose agar (PDA), and Sabouraud glucose agar (SGA). Agar plates were inoculated with 43 well-characterized dermatophyte strains, belonging to the genera Arthroderma, Epidermophyton, Microsporum, or Trichophyton. The agar plates were incubated at 26 °C and examined every 5 days for 1 month. By the end of the incubation period, 97 of the 258 plates (37.6%) were contaminated by fungi. No bacteria were detected. Overall, BLA demonstrated the lowest rate of contamination, followed by SGA, MEA, BHIA, PDA, and LJA. Sequencing of the internal transcribed spacer region rDNA of the contaminant fungi revealed that Aspergillus and Penicillium species were the most common contaminants. These results suggest that nonenriched culture media types, such as BLA or SGA, reduced contamination during dermatophyte subculture.

FIRST REPORT OF GUMMOSIS OF MANGO TREES CAUSED BY NEOFUSICOCCUM PARVUM IN SICHUAN, SOUTHWEST CHINA

Mango (Mangifera indica) is an important fruit crop in southern China, and gummosis is one of the most important diseases. In September 2012, symptomatic branches were collected in Panzhihua, Sichuan province. On potato dextrose agar, isolates of similar morphological characteristics were consistently recovered from surface-sterilized branch pieces. The fungus was identified as Neofusicoccum parvum based on morphology and DNA sequence comparisons (Costa et al., 2010). Conidia were hyaline, thin walled, spindle-shaped to ellipsoid, nonseptate, and ranged from 15 to 23.5 × 4 to 7 μm (average 19.5 × 5.6 μm). The rDNA internal transcribed spacer region, a partial sequence of the β-tubulin gene, and the translation elongation factor 1-α gene of one isolate (L13), showed 100% identity to N. parvum (GenBank accession Nos GU997685, HM480386 and HQ859955). Pathogenicity of three isolates was tested in three green twigs and three 3-year-old branches in mango orchards of Guangxi Academy of Agricultural Sciences, Guangxi, China.Five wounds were made for each location with a sterilized needle. Mycelial plugs were placed at wounds and then covered with parafilm. Control twigs were inoculated with uncolonized PDA plugs. Two weeks later, typical brown lesions were observed on inoculated branches, and gum exuded from infected tissues wound. No symptoms were seen on the controls. Koch's postulates were fulfilled by reisolation of N. parvum from diseased branches. In China, the disease was mainly associated with Lasiodiplodia theobromae (Li et al., 2013) and Botryosphaeria dothidea (Mo et al., 2013). To our knowledge, this is the first report of N. parvum causing mango gummosis in China.

Wilt and root rot of poinsettia caused by three high-temperature-tolerant Pythium species in ebb-and-flow irrigation systems

Poinsettia plants growing in ebb-and-flow irrigation systems developed wilting and root rot during the summer growing seasons of 2010 in Gifu Prefecture and 2011 in Aichi Prefecture. Pythium species were isolated from roots with rot symptoms. The isolates were identified as P. helicoides and P. myriotylum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, these isolates caused severe wilting and root rot. This is the first report of poinsettia root rot disease caused by P. helicoides and P. myriotylum, although P. aphanidermatum was reported as a pathogen of poinsettia root rot. To better understand these diseases, we performed an epidemiological study of three high-temperature-tolerant Pythium species, P. aphanidermatum, P. helicoides and P. myriotylum. Disease incidence as a percentage of diseased plants was greatest at 35 °C for all three species. Disease severity using the rating scale of root rot was also highest at 35 °C, particularly with high zoospore inoculum densities (100.0 zoospores/mL). Although the disease incidence and severity were reduced at lower temperatures, the three Pythium species were able to cause disease at temperatures as low as 20 °C.

Characterization of Alternaria species associated with leaf blight of sunflower in China

Nine Alternaria species have been reported to be associated with sunflower leaf blight worldwide, and A. helianthi has been recognized as the most prevalent and damaging species. However, the population structure of Alternaria species causing leaf blight of sunflower in China had not been examined thoroughly prior to this study. During 2010 to 2013, a total of 272 Alternaria isolates were obtained from infected sunflower leaves in 11 provinces, autonomous regions, and municipalities of China. Based on morphological traits and sequence analyses of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) and the partial coding sequences of the histone 3 gene, 227 (83.5 %) isolates were identified as belonging to Alternaria tenuissima, the remainder 45 isolates were grouped to A. alternata (16.5 %). Compared with the ITS regions of rDNA, sequence analyses of the partial coding sequences of histone 3 gene displayed a critical role in discrimination of the small-spored Alternaria species. Phylogenetic analysis of the partial coding sequence of histone 3 gene clearly divided the representative Alternaria isolates into two main clades, A. tenuissima and A. alternata. The pathogenicity of A. tenuissima and A. alternata on detached leaves of sunflower cv. Gankui No.2 did not significantly differ between the two species or among isolates from different geographical origins. Our results indicate that the population structure of Alternaria species associated with sunflower leaf blight differed from that reported previously in China since A. helianthi was not found in this study. In addition, this is the first report about A. tenuissima causing leaf blight on sunflower in China.

Prevalence and genetic diversity of Oesophagostomum stephanostomum in wild lowland gorillas at Moukalaba-Doudou National Park, Gabon

Abstract Using a sedimentation method, the prevalence of the nodular worm Oesophagostomum stephanostomum (Nematoda: Strongylida) in western lowland gorillas at Moukalaba-Doudou National Park (MDNP), Gabon, was determined in fecal samples collected between January 2007 and October 2011, along with their coprocultures. Concurrently, possible zoonotic Oesophagostomum infections in villagers living near MDNP were assessed from their fecal samples collected during October and November of 2011. In the gorillas, strongylid (Oesophagostomum and/or hookworm) eggs were found in 47 of 235 fecal samples (20.0 %) and Oesophagostomum larvae were detected in 101 of 229 coprocultures (44.1 %). In the villagers, strongylid eggs were found in 9 of 71 fecal samples (12.7 %), but no Oesophagostomum larvae were detected in coprocultures. The internal transcribed spacer (ITS) region of ribosomal RNA gene (rDNA) and cytochrome c oxidase subunit-1 (cox-1) region of mitochondrial DNA (mtDNA) of coprocultured Oesophagostomum larvae were amplified using parasite DNA extracted from 7–25 larvae/sample, cloned into Escherichia coli, and sequenced. Sequenced rDNA contained 353/354-bp long ITS1, 151-bp long 5.8S rDNA, and 227-bp long ITS2. Parts of clones showed variations at 1–3 bases in the ITS1 region at a frequency of 24/68 (35.3 %) and at 1–2 bases in the ITS2 region at a frequency of 7/68 (10.3 %), whereas the 5.8S rDNA was essentially identical. Sequenced cox-1 gene of the parasites, 849 bp in length, showed a higher number of nucleotide variations, mainly at the third nucleotide position of the codon. The majority of clones (27/41 (65.9 %)) had an identical amino acid sequence. These results suggest that at MDNP, Gabon, only a single population of O. stephanostomum with a degree of genetic diversity is prevalent in western lowland gorillas, without zoonotic complication in local inhabitants. The possible genetic variations in the ITS region of rDNA and cox-1gene of mtDNA presented here may be valuable when only a limited amount of material is available for the molecular species diagnosis of O. stephanostomum.

First Report of Diplodia seriata Causing Pear Branch Canker Dieback in California

California produces 26% of the United States pear crop on approximately 5,600 ha. A survey of seven northern California pear orchards (Pyrus communis cv. Bartlett) in summer 2010 revealed the presence of wedge-shaped cankers on 2- to 5-cm diameter branches, equating to 1- to 3-year-old wood. Many of the observed cankers occurred near pruning wounds, and there was decreased foliation on infected branches. Infected wood was surface disinfected with 95% ethanol and briefly flamed. After removing bark, small sections of diseased tissue were plated onto 4% potato dextrose agar (PDA) amended with 0.01% tetracycline and placed on the lab bench at 22°C until fungal growth emerged. Fungal colonies that were consistently isolated were transferred to fresh PDA using hyphal tip isolation. Fungal colonies were dark brown to gray with aerial mycelium and formed pycnidia after 15 days of incubation at 22°C. Conidia were brown, oval to oblong, and measured (16.5-) 20 to 24 (-26) × (7.5) 8.75 to 11 (-12.5) μm (n = 50). DNA from 14- to 21-day-old colonies was extracted and sequences of the rDNA internal transcribed spacer region and part of the β-tubulin gene were amplified using primers ITS4/ITS5 and Bt2a/Bt2b, respectively (2). The DNA sequences of fungal isolates from California showed 99 to 100% homology with the ex-type Diplodia seriata De Not. (1) CBS112555 deposited in GenBank. DNA sequences from three California isolates were submitted to GenBank with accession numbers KC937062, KC937065, KF481957, KF481598, KF481959, and KF481960. Pathogenicity tests were performed in March 2011 on 3-year-old Bartlett pear trees planted at an experimental farm in Davis, CA. A single, circular, 2-cm pruning wound at the top of the trunk was inoculated on each of three single-tree replications using 2-cm mycelial plugs from 14-day-old colonies growing on PDA. After inoculation, mycelial plugs were covered and sealed with Parafilm and aluminum foil for the duration of the trial. Three control trees were inoculated using sterile PDA plugs. Twelve months after inoculation, UCD103 and UCD105 were consistently re-isolated from the margin between necrotic and healthy tissue using the same methods described for the original isolation, and UCD102 was re-isolated in two out of three plants. The average lesion lengths of UCD102, UCD103, UCD105, and control plants were 12.5, 17.3, 23, and 1 mm, respectively. Control lesions were short and sterile, and seemed to be a physiological reaction from the plant. A second pathogenicity test was completed in 5 months beginning in June 2012. UCD105 was consistently re-isolated, and UCD102 and UCD103 were re-isolated in two out of three plants. The average lesion lengths for UCD102, UCD103, UCD105, and control plants were 2, 3, 5, and 1 mm, respectively. Compared to grapevine (Vitis vinifera), the pathogen grows more slowly in pear tissue under natural conditions. To our knowledge, this is the first report describing D. seriata as a causal agent of pear branch canker in California. Canker diseases can reduce the lifespan of perennial plants, ultimately leading to long term economic losses for growers (3).

First Report of Leaf Blight of Rice Caused by Cochliobolus lunatus in China.

Leaf-streak symptoms were observed on rice (Oryza sativa L.) starting at the booting stage through harvest in Zhejiang Province, China, in 2012. Based on Fuyang County, only 15% of the rice fields were estimated to show these symptoms. However, incidence could be 40 to 80% when the rice got infected. Typical symptoms started as green water-soaked streaks from the tip or edge of leaf blades, similar to bacterial leaf blight caused by Xanthomonas oryzae. Infected leaves turned yellow, then eventually became wilted and dry. No bacterial streaming was observed and no bacteria were isolated. Pieces of infected leaf tissue were surface sterilized using 0.1% (v/v) mercuric chloride, rinsed with sterilized water, then placed on water agar (WA). After 2 or 3 days on WA at 28°C, only fungal growth was observed from surface sterilized tissues. Fungi were isolated, purified by single spore separation process, and subcultured to potato dextrose agar (PDA) plates. Growing on PDA, the surface of the colony was circular, fluffy, and shiny velvety-black, whereas the under surface was dark Prussian blue. Conidiophores were single or fascicled, brown to dark brown, rarely branched, multiseptate, and straight or often geniculate near the apex. Conidia were brown, smooth, fusiform, geniculate or hook-shaped, 17.5 to 28.5 × 8.5 to 14.0 μm, and 3-septate, with the third cell from the base larger and darker than the others. Molecular identification was performed by analysis of the rDNA internal transcribed spacer region (ITS1-5.8S-ITS2). The rDNA-ITS region was amplified with primer pair ITS1 and ITS4 (5), sequenced, and deposited in GenBank (Accession No. KC462186). The sequence of rDNA-ITS (KC462186) showed 100% identity with Cochliobolus lunatus R.R. Nelson & Haasis (JN943422) after BLAST. Based on the results of morphological and molecular analyses, the fungus isolated from infected leaves was identified as C. lunatus (anamorph: Curvularia lunata (Wakk.) Boedijn) (3). Pathogenicity tests were conducted three times by spraying a conidial suspension (1 × 105 spores/ml) with 0.1% (v/v) Tween 20 on 12 healthy rice plants at late tillering stage. The same number of the healthy rice plants sprayed with sterilized water with 0.1% (v/v) Tween 20 were used as control. All plants were kept at 30°C and 75 to 85% relative humidity (RH) under a 12-h light/dark rotation. About 5 to 7 days after inoculation, green water-soaked streaks began to appear on inoculated plants. From 7 to 14 days after inoculation, the lesions developed quickly and the leaves began to wilt. After 14 days, inoculated plants showed symptoms similar to those originally observed in the field, while control plants (sprayed with sterilized water) remained healthy. C. lunatus was re-isolated from all inoculated plants, and re-identified by the same methods (morphological and molecular methods) as described above, thereby satisfying Koch's postulates, and confirming C. lunatus as the cause of the disease. C. lunatus is a pathogen of a wide range of plants and is common in paddy environments. It was reported as one of the causal agents of black kernel of rice (4) and rice spikelet rot disease (SRD) (1,2). The level of incidence observed in the affected fields suggest that this disease could potentially cause major losses under favorable weather conditions if susceptible cultivars are grown. To our knowledge, this is the first report of C. lunatus causing leaf blight of rice in China.

First Report of Diplodia pinea on Pseudotsuga menziesii in Turkey

Diplodia pinea is a latent, opportunistic pathogen of Pinus and other coniferous species, including Pseudotsuga menziesii (3). The fungus causes twig blight, branch cankers, tree and seedling collar rot, root rot, and can also infect cones (1). D. pinea has often been reported causing tip and shoot blight on various Pinus spp. in different parts of Turkey. During disease surveys on Pinus spp. carried out in May 2012 in Izmit in the Marmara Region (37°36'54″N, 31°20'00″E), typical shoot blight symptoms of D. pinea infection were also observed on the neighboring P. menziesii trees. Shoots and cones of P. menziesii were investigated for the presence of D. pinea pycnidia. Pycnidia from cones and shoots were placed on potato dextrose agar (PDA) and incubated at 23°C. Three isolates were obtained from shoot and cone samples. Identification of the pathogen was based on morphological characteristics of the conidia and by PCR of the ITS region of nuclear rDNA. Colonies on PDA were woolly, whitish at first turning black, sometimes partly or entirely turning light gray. Micromorphological characteristics of the Diplodia isolates were similar to those described in (2): conidia width 18.4 μm (SD ± 2.8) (range 11 to 22 μm) × length 34.0 μm (SD ± 5.3) (range 20 to 41 μm) (n = 100). Conidia were at first hyaline, later becoming brown to dark brown, oblong ellipsoid, bicellular with a distinct septum. To confirm the identity of the isolates, genomic DNA was extracted and the internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS4 (4). Amplicons were 483 bp in length (GenBank Accession No. KF372874) and shared 98% nucleotide identity with HM100285.1 and 97% nucleotide identity with JX981458.1 of D. pinea. Inoculation tests were performed on 2-year-old P. menziesii seedlings by placing mycelial plugs of three isolates obtained from pycnidia on the main stem after wounding with a cork borer. Control seedlings were inoculated with PDA plugs without mycelium. All seedlings were incubated at 24°C for 3 weeks in a climate chamber. Following incubation, the seedlings displayed dark brown-to-black discoloration, measuring on average 10.7 ± 0.6 cm, of the bark and wood tissues around the inoculation points on the stems. The pathogen was successfully re-isolated from symptomatic stem tissues, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. menziesii as a host of D. pinea in Turkey. P. menziesii is not endemic to Turkey and to date has a limited distribution (approximately 140 ha), but it is an important fast growing tree species for new industrial plantations.