Sequences Of Internal Transcribed Spacer Region Research Articles

First Report of Colletotrichum godetiae Causing Anthracnose and Twig Blight on Persian Walnut in Hungary.

Persian walnut (Juglans regia L.) fruit with preharvest anthracnose symptoms, necrotic fruit stalks, and twigs with necrotic buds, and peaks were collected in a Hungarian orchard next to Nágocs, in September 2018. Disease incidence was approximately 15% on a Hungarian bred walnut cultivar 'Milotai 10'. Similar symptoms were found on Persian walnut in other locations (eg. Milota, Érd, Sarród, and Kocs). Acervuli were observed on necrotic lesions on fruit, and twigs with pale orange conidial masses. Conidia were hyaline, unicellular, and fusiform. Morphometric measurements of conidia showed mean length ± SD × width ± SD = 15.9 ± 1.7 × 4.5 ± 0.4 μm, length/width ratio 1:0.3 (n=100). The fungus was isolated from conidial masses on potato dextrose agar (PDA) medium amended with Chlorampenicol (25 mg/L). A total of 12 isolates were obtained as pure cultures by single-spore isolations and incubated at 23°C in dark for 10 days. The colonies were white to gray or grayish-orange on the upper side and with black spots on the reverse side. The isolates showed morphological characteristics of Colletotrichum acutatum in sensu lato (Jayawardena et al. 2016). Molecular analyses were conducted to identify the exact species. Internal transcribed spacer (ITS) region, actin (ACT), and calmodulin (CAL) partial genes were amplified by ITS1F/ITS4R, ACT512F/ACT783R and CAL1/CAL2 primers (White at al. 1990, Carbone and Kohn 1999, O'Donnell et al. 2000). The sequences of ITS region (GenBank Accession Nos: MK367398-99, MK367401-02) showed 100% identity with C. godetiae sequence. Based on ACT gene (GenBank Accession Nos: MK415991-92, MK415994-95) were 100% identity with the deposited C. godetiae type strains from walnut. The obtained sequences of CAL gene (GenBank Accession Nos: MK415998-99, MK416001-02) were same and showed 100% with other C. godetiae sequences from other host plants. The fungus was identified as Colletotrichum godetiae Neerg. Pathogenicity tests were accomplished in the field and under laboratory conditions (25°C on thermostat) on 10 green 'Milotai 10' walnut fruit, and 10 walnut twigs each. Tests were conducted on living trees, collected fruit, and two-year-old twigs by inserting mycelial agar plugs (5 mm in diameter) onto wounded pericarp tissues, which were then wrapped with wet cotton and parafilm. Wounded tissues on 5 fruit and 5 two-year-old twigs were treated with non-colonized PDA plugs as noninoculated controls. After 14 d necrotic lesions 9 to 17 mm in diameter developed on fruit on living trees. Lengths of 12 to 17 mm and width of 7 to 12 mm necrosis was measured on phloem of walnut twigs, and almost two times larger in cambium. No necrosis developed around control wounds. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues, isolates were identical morphologically and by sequence analysis of ITS region, ACT, and CAL partial genes to the original isolates. Damm et al. (2012) described two C. godetiae strains associated with walnut, one isolated in Austria and another one of unknown origin. An epidemic event of walnut anthracnose caused by Colletotrichum species mainly C. godetiae was reported in France (Da Lio et al. 2018). The pathogen was isolated from nuts, buds, insects, and stems. To our knowledge, this is the first report of anthracnose of walnut fruit caused by C. godetiae in Hungary. Anthracnose caused by C. godetiae, and previously reported C. fioriniae (Varjas et al. 2019) is becoming an increasing preharvest problem on Persian walnut in Hungary.

Phenotypic plasticity and taxonomy of Cladophora gracilis (Griffiths) Kützing (Cladophorales, Chlorophyta) in the western Yellow Sea with the implication of its DNA barcode

Due to extensive morphological plasticity, the taxonomy of Cladophora species remains unclear. As one of the widely distributed species, C. gracilis was reported to hold many varieties, which make it difficult to identify the species. This study explored the morphology diversity of nine C. gracilis samples collected along the coast of western Yellow Sea. Some samples showed extremely varied characteristics, by which one cannot classify them correctly. Hence, 18S rDNA and nuclear ribosomal DNA internal transcribed spacer regions (ITS) sequences were employed to delimit species. For 18S rDNA, sequence similarity ranged from 99.6% to 100%. For ITS region, the similarity ranged from 98.7% to 100%. Molecular data strongly suggested that the morphologically heterogeneous samples were actually the same species. Characteristics comparison of the samples revealed that the taxonomy criteria including branching pattern and density, thallus color, height and texture varied widely, influenced by environmental conditions and age of alga. Besides, cell dimensions, as the relatively stable criterion, also exhibited intraspecific variance. Successful application of 18S rDNA and ITS sequences indicated that molecular method can be a powerful assistant as DNA barcodes to traditional morphology taxonomy.

First Report of Damping-Off Caused by Mucor circinelloides on Aconitum carmichaelii in China.

Aconitum carmichaelii is a typical traditional Chinese medicinal herb that has been grown for more than one thousand years in China (Singhuber et al. 2009). Surveys for damping-off of A. carmichaelii were conducted from 2016 to 2018 in three of China's major planting areas (Shaanxi, Sichuan, and Yunnan Province). Damping-off was observed from April to June with disease incidences ranging from 5% to 11% in ten investigated fields. In the early stage of disease development, the roots were fully covered by white mycelia. When the disease was severe, seedlings were stunted, turned yellow, had withered roots, and some eventually died. One fungus was consistently isolated from the diseased roots in the planting areas of Shaanxi Province (33°7'42″N and 107°20'27″E) on PDA medium. It was identified as Mucor circinelloides, based on morphological characteristics (Schipper 1976). Three isolates from different regions were characterized by yellowish colonies composed of tall and short sporangiophores 6-11 µm in width, terminal and globose sporangia, ellipsoidal sporangiospores 5-8 µm in length and 4-5 µm in width, and obovoid columellae. The internal transcribed spacer regions (ITS) and the large subunit (28S) of ribosomal RNA gene from the representative isolate, MC180610 (GenBank accession no. MK087755, MT043749), were also amplified and sequenced using the universal primer sets, ITS1/ITS4 and NL1/NL4, respectively. BLAST analyses of the ITS sequence showed 99.38% identity with the type strain (CBS 195.68) of M. circinelloides (GenBank accession no. NR_126116), and the 28S sequence showed 99.44% identity with the strain (CBS 274.49) of M. circinelloides f. circinelloides (GenBank accession no. MH868051). The pathogenicity of M. circinelloides isolate (MC180610) to A. carmichaelii was examined by inoculating 150 ml of the hypha suspension of M. circinelloides prepared from a 10-day-old potato dextrose broth culture into the soils of healthy potted A. carmichaelii plants. Uninoculated potted A. carmichaelii plants served as controls. There were three replicates for the inoculated and control plants. All the potted A. carmichaelii plants were incubated at 25°C under 12 h light 12 h dark conditions. The symptoms that developed on M. circinelloides inoculated A. carmichaelii plants were similar to those observed in the field; control plants did not show symptoms. The pathogen was reisolated from symptomatic roots onto the PDA medium and morphologically identified as M. circinelloides. M. circinelloides has been reported as a pathogen of papaya (Carica papaya), Mandarin fruits (Citrus reticulata), and sweet potatoes (Ipomea batatas), respectively (Cruz-Lachica et al. 2018; Saito et al. 2016; Oladoye et al. 2016), but it has never been reported on A. carmichaelii before. This is the first report of damping-off caused by M. circinelloides on A. carmichaelii. This pathogen may present a threat to the production of A. carmichaelii in China.

First report of Athelia rolfsii causing stem rot disease on Vasaka (Justicia adhatoda)

Athelia rolfsii is a facultative plant pathogen which develops characteristic white mycelial mats and sclerotia at the crown infection sites when conditions are favourable. In July 2019, stem rot symptoms such as dark brown necrotic lesions were observed at collar of the stem base. The infected plants showed dark brown symptoms which later turned into light white mycelial growth with immature white sclerotia, while the isolated fungus was initially white in colour and later turned black. The identification of the isolate as Athelia rolfsii was also confirmed by using internal transcribed spacer region sequencing. To the best of our knowledge, this is the first report of stem rot of Justicia adhatoda caused by Athelia rolfsii in the world.

Mycelial Growth and Biological Control Measures of Botrytis cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh

Fruit rot disease of strawberry caused by fungus is most economically important disease in Bangladesh, which reduces the production and quality of strawberry at both pre and post-harvest period. Present study was conducted to identify the causal organism of the fruit rot disease of strawberry through classical and molecular techniques and its eco-friendly control measures. A fungal pathogen causing fruit rot was isolated and identified as Botrytis cinerea through classical fungal taxonomy and molecular characterization based on their internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sequence analysis showed that 5.8S of rDNA sequences were identical. The reciprocal homologies of the ITS region sequences ranged from 98 to 100%. Experimental results suggested that studied fungus (MH371474.1) was genetically similar with MF521935.1 Botrytis cinerea. Isolated fungus was evaluated on six different culture media and at five different temperature conditions. The optimum mycelial growth of B. cinerea was found on Richard agar medium at 25 ℃ temperature at 7days post inoculation (dpi), while mycelial growth was drastically reduced the temperature 30 ℃ and above. Three Trichoderma species viz., T. ressei, T. harzianum and T. asperellum were used to assess the antagonistic effect on isolated pathogenic fungus in which the mycelial growth of it was inhibited mostly by T. ressei. Experimental results revealed that Trichoderma ressei was the effective antagonistic fungus against Botrytis cinerea for the biological control.

Molecular detection and genotyping of microsporidia species in chickens in Turkey

Microsporidia are obligate intracellular pathogens that infect various hosts including invertebrates and vertebrates. Despite the importance, knowledge on the prevalence and molecular characteristics of microsporidia in chickens is limited, and no data are available for Turkey. A total of 300 fecal samples from chickens in the Central Anatolia Region of Turkey were analyzed by using a nested polymerase chain reaction assay targeting the rRNA internal transcribed spacer (ITS) region for the common microsporidia species. Corresponding PCR amplicons from the positive samples were sequenced for genotyping. Enterocytozoon bieneusi was identified in 22 (7.3 %) samples, whereas Encephalitozoon spp. was not detected. The prevalence of E. bieneusi was 63.6 % in Kayseri and 36.4 % in Nevsehir provinces, and 8.8 % in soft fecal samples and 9.7 % in diarrhoeic samples. No infections were found in Kirsehir Province. Significant differences were found for the distribution of E. bieneusi among provinces and fecal conditions. Infections were found only in free-range chickens. As a result of ITS region sequencing, two genotypes were characterized. The novel genotype ERUNT1 (n = 21), belonging to zoonotic group 1, was the most common genotype throughout the study area. The other known genotype, ERUSS1 (n = 1), had a restricted distribution and was previously detected in cattle and sheep in the same region. Our study provides the first data on microsporidia species from chickens in Turkey. None of these genotypes have been reported in humans; thus, the risk potential for public health is limited but needs further investigation.

First Report of Alternaria alternata Causing Leaf Blight on Actinidia arguta in Liaoning, China

HomePlant DiseaseVol. 104, No. 10First Report of Alternaria alternata Causing Leaf Blight on Actinidia arguta in Liaoning, China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Alternaria alternata Causing Leaf Blight on Actinidia arguta in Liaoning, ChinaQin Wang, Qing-Shu Luan, Jian-Jun Wang, Xu Jiang, and Gang ChenQin Wanghttp://orcid.org/0000-0003-2932-8512Provincial Key Laboratory of Forest Protection, Liaoning Academy of Forestry, Shenyang 110032, ChinaSearch for more papers by this author, Qing-Shu LuanProvincial Key Laboratory of Forest Protection, Liaoning Academy of Forestry, Shenyang 110032, ChinaSearch for more papers by this author, Jian-Jun WangProvincial Key Laboratory of Forest Protection, Liaoning Academy of Forestry, Shenyang 110032, ChinaSearch for more papers by this author, Xu JiangProvincial Key Laboratory of Forest Protection, Liaoning Academy of Forestry, Shenyang 110032, ChinaSearch for more papers by this author, and Gang Chen†Corresponding author: G. Chen; E-mail Address: chengang1625@163.comProvincial Key Laboratory of Forest Protection, Liaoning Academy of Forestry, Shenyang 110032, ChinaSearch for more papers by this author AffiliationsAuthors and Affiliations Qin Wang Qing-Shu Luan Jian-Jun Wang Xu Jiang Gang Chen † Provincial Key Laboratory of Forest Protection, Liaoning Academy of Forestry, Shenyang 110032, China Published Online:18 Aug 2020https://doi.org/10.1094/PDIS-09-19-1871-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Actinidia arguta is a Chinese native fruiting vine and has been cultivated in several countries for its commercial advantages (Williams et al. 2003). During the 2017 and 2018 growing seasons, leaf spot and blight symptoms were observed on young and mature leaves of A. arguta at four orchards in Dandong, Liaoning Province, China, with the disease incidence ranging from 8 to 23%. To identify the pathogen, three symptomatic leaf samples from each of the plantations were selected for pathogen isolation. Eight isolates similar in appearance were obtained by the hyphal tip method. Colonies on potato carrot agar were cottony and were mouse gray or black olive. Conidiophores were simple or branched, straight or flexuous, brown in color, smooth, with one or more conidial scars. Conidia were borne solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid, with a cylindrical or coniform beak at the tip. Conidia ranged from 13.0 to 50 μm long (mean 31.3 μm) and 9 to 18 μm wide (mean 13.9 μm) with one to five transverse and rarely zero to three longitudinal septa. To make further identification, the rDNA internal transcribed spacer regions (ITS) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of all isolates were amplified using primer pairs ITS5/ITS4 and GDF1/GDR1, respectively. All isolates had identical ITS sequence and identical GAPDH sequence, exhibiting 100% identity with that of Alternaria alternata strain CBS 130265 (KP124391 for ITS, KP124243 for GAPDH). Only one representative sequence of each gene was submitted to GenBank (MN249500 for ITS, MN258117 for GAPDH). Based on morphological characteristics (Simmons 2007) and DNA sequence data, the pathogen was identified as A. alternata. For the pathogenicity test, one isolate was used for artificial inoculation in 3-year-old seedlings of A. arguta (cultivar Longcheng). Ten healthy leaves from three individuals were selected, sprayed with a suspension of 106 conidia/ml, and covered with plastic bags for 4 days. A control treatment with an equal number of leaves was sprayed with distilled water only. Symptoms identical to those observed in the field were observed 7 to 10 days after inoculation, whereas all control leaves were asymptomatic. A. alternata was consistently reisolated from inoculated leaves. A. alternata has previously been reported to cause fruit rot on A. arguta in Korean (Kim et al. 2017). However, to our knowledge, this is the first report of A. alternata causing leaf spots on A. arguta in China.The author(s) declare no conflict of interest.

Optimum vegetative growth conditions and genetic diversity in different strains of Pleurotus salmoneastramineus L.J.N. Vassilzeva

This experiment was undertaken to depict the favourable condition for mycelial growth, molecular identification and phylogenetic relationship of the selected strains of Pleurotus salmoneostramineus. Suitable temperature and pH were obtained at 25ºC and 6, respectively. Mushroom complete, glucose peptone and yeast malt extract culture media were favorable, while Hennerberg and Hoppkins were unfavorable. Dextrin was the best and xylose was the less effective carbon sources. Inorganic nitrogen sources were less effective for the mycelial growth of P. salmoneostramineus. The sequences of internal transcribed spacer (ITS) region of selected strains revealed that the total length ranged from 614 to 663 bp. The size of the ITS1 and ITS2 regions varied among the strains. Sequence analysis showed that 5.8S of rDNA sequences were identical. Phylogenetic tree of the ITS region sequences indicated that strains of P. salmoneostramineus belong to same cluster. The reciprocal homologies of the ITS region sequences ranged from 98 to 100%. The strains of P. salmoneostramineus were also analyzed by random amplification of polymorphic DNA (RAPD) with 20 arbitrary primers. RAPD results suggested that tested strains of P. salmoneostramineus were genetically similar with some variations, thus it could be concluded that RAPD and ITS techniques were well competent for detecting the genetic diversity of all tested strains of P. salmoneostramineus.

Isolation and molecular identification of a strain belonging to the new species Zalaria obscura from a deteriorated wooden artwork.

We report the case of an outdoor deteriorated wooden sculpture of Madonna, completely blackened in the face, and thus suspected of fungal attack. A multi-disciplinary approach, including microbiological analysis, molecular biology, and Fourier transform infrared (FT-IR) spectroscopy, was applied to understand the real nature of the observed alteration. FT-IR showed that the blackening was due to the application of a natural terpene resin subjected to alteration over time. The microbiological assay allowed to isolate a particular black fungus that has been recovered in the vegetative phase, growing as the only species adapted to the examined substrate. Basic Local Alignment Search Tool (BLAST) analysis of the ITS (internal transcribed spacer) region sequence identified the fungus (LS31012019) as Zalaria obscura, a black yeast belonging to the new genus Zalaria, family Dothideales. Overall, this study evidenced the importance of a multi-disciplinary approach to understand the real causes of observed deterioration of artworks. More interestingly, the recovery of a strain identified as Z. obscura from this type of substrate is never reported in the literature and this finding could offer the possibility to investigate the role of this microorganism in the deterioration process of cultural heritage.

First Report of Sclerotinia sclerotiorum Causing Sclerotinia Rot on Codonopsis lanceolata in Korea

Lance asiabell or deodeok (Codonopsis lanceolata L.) is native to East Asia such as Korea, China, Japan, and Russia. In Korea, roots of the plant are widely used as a food and medicine because of its high content of beneficial ingredients such as saponin. In May 2018, Sclerotinia rot symptoms on 2-year-old lance asiabell plants were observed in a field in Jeju (33.51° N, 126.84° E), Korea. Symptoms affecting the crown were water-soaked lesions associated with characteristic white cottony mycelia. Secondary symptoms such as wilting and bleaching were also observed on above-ground tissues. The incidence of diseased plants was approximately 10% in the field. Ten sclerotia were collected from the soil around the diseased plants, surface sterilized with 1% NaOCl for 1 min, and then rinsed twice in sterilized distilled water. The sclerotia were placed on water agar and incubated at 20°C for 5 days in the dark. Four isolates were obtained from mycelium grown from each sclerotium and were cultured on potato dextrose agar (PDA) at 20°C for 3 weeks in the dark. All isolates produced irregular and black sclerotia (1.8 to 4.5 mm in size) on a PDA plate. Based on morphological characteristics of mycelia and sclerotia, all isolates were tentatively identified as Sclerotinia sclerotiorum (Kohn 1979). To confirm the morphological identification of all isolates, DNA sequencing of the internal transcribed spacer (ITS) region of rDNA was carried out using ITS1 and ITS4 primer pair (White et al. 1990). The 505-bp products of nucleotide sequences obtained from four isolates (NC18657 to NC18660) were deposited in GenBank with accession numbers MK418968 to MK418971. The ITS region sequences of the four isolates were 100% identical with GenBank reference accession number KF859933 of S. sclerotiorum CBS 499.50. Pathogenicity assays were conducted using two isolates (NC18657 and NC18659) on the lance asiabell seedlings. Five healthy 2-week-old seedlings were inoculated by placing a sclerotium with PDA plug (3 × 3 mm) around the stem. The same number of seedlings were noninoculated controls and received noncolonized PDA plugs. Plants were maintained in the growth chamber at 20 ± 3°C with a 14-h photoperiod. After 4 weeks, all inoculated seedlings wilted. Black irregular sclerotia with white mycelium were observed on the soil surface. No symptoms were observed in the control plants. The fungus was consistently reisolated from the sclerotia formed on the soil surface, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of S. sclerotiorum causing Sclerotinia rot on lance asiabell in Korea. Further studies are needed to develop integrated management of the disease on the host in Korea.

Review of NIOSH Cannabis-Related Health Hazard Evaluations and Research.

Since 2004, the National Institute for Occupational Safety and Health (NIOSH) has received 10 cannabis-related health hazard evaluation (HHE) investigation requests from law enforcement agencies (n = 5), state-approved cannabis grow operations (n = 4), and a coroner's office (n = 1). Earlier requests concerned potential illicit drug exposures (including cannabis) during law enforcement activities and criminal investigations. Most recently HHE requests have involved state-approved grow operations with potential occupational exposures during commercial cannabis production for medicinal and non-medical (recreational) use. As of 2019, the United States Drug Enforcement Administration has banned cannabis as a Schedule I substance on the federal level. However, cannabis legalization at the state level has become more common in the USA. In two completed cannabis grow operation HHE investigations (two investigations are still ongoing as of 2019), potential dermal exposures were evaluated using two distinct surface wipe sample analytical methods. The first analyzed for delta-9-tetrahydrocannabinol (Δ9-THC) using a liquid chromatography and tandem mass spectrometry (LC-MS-MS) method with a limit of detection (LOD) of 4 nanograms (ng) per sample. A second method utilized high performance liquid chromatography with diode-array detection to analyze for four phytocannabinoids (Δ9-THC, Δ9-THC acid, cannabidiol, and cannabinol) with a LOD (2000 ng per sample) which, when comparing Δ9-THC limits, was orders of magnitude higher than the LC-MS-MS method. Surface wipe sampling results for both methods illustrated widespread contamination of all phytocannabinoids throughout the tested occupational environments, highlighting the need to consider THC form (Δ9-THC or Δ9-THC acid) as well as other biologically active phytocannabinoids in exposure assessments. In addition to potential cannabis-related dermal exposures, ergonomic stressors, and psychosocial issues, the studies found employees in cultivation, harvesting, and processing facilities could potentially be exposed to allergens and respiratory hazards through inhalation of organic dusts (including fungus, bacteria, and endotoxin) and volatile organic compounds (VOCs) such as diacetyl and 2,3-pentanedione. These hazards were most evident during the decarboxylation and grinding of dried cannabis material, where elevated job-specific concentrations of VOCs and endotoxin were generated. Additionally, utilization of contemporary gene sequencing methods in NIOSH HHEs provided a more comprehensive characterization of microbial communities sourced during cannabis cultivation and processing. Internal Transcribed Spacer region sequencing revealed over 200 fungal operational taxonomic units and breathing zone air samples were predominantly composed of Botrytis cinerea, a cannabis plant pathogen. B. cinerea, commonly known as gray mold within the industry, has been previously associated with hypersensitivity pneumonitis. This work elucidates new occupational hazards related to cannabis production and the evolving occupational safety and health landscape of an emerging industry, provides a summary of cannabis-related HHEs, and discusses critical lessons learned from these previous HHEs.

A Fatal Case of Bloodstream Infection by Fusarium Solani in a Patient with Adrenocortical Carcinoma From Isfahan, Iran

Introduction: Fusarium species are hyaline saprophytic fungi that are frequently found in the soil, air, and water. They can cause severe systemic infections in immunocompromised patients. Clinical manifestations depend on the way of entry of the mold and host immune system status. The main ways of entrance are the airways, skin, and mucosal membranes. Disseminated fusariosis often occurs in patients with hematological disorders, patients with cancer, and solid organ transplant recipients. Case Presentation: Herein we report a case of Fusarium fungemia in a patient with adrenocortical carcinoma from Isfahan, Iran. The patient was a 41-year-old female with stage III adrenal cortical carcinoma. Despite antifungal therapy with liposomal amphotericin B, the patient passed away 6 days after admission. Internal transcribed spacer region sequencing applied for species identification and its sequence deposited in the GenBank (accession number: MK880379). Conclusions: Since the ideal strategies against invasive fungal infections remain uncertain and the mortality rate is high, we recommend primary prophylaxis with a broad-spectrum antifungal agent for vulnerable patients particularly those admitted to high-risk units such as oncology, hematology, and transplant units.

Species discrimination of mountain garlic based on the nrDNA ITS region sequence

Mountain garlic is an edible perennial herb widely distributed in Asian, European, and Siberian regions, referring to a class of Allium species. In South Korea, mountain garlic mainly consists of two Allium species, Allium ochotense Prokh. and A. microdictyon Prokh. Due to having spicy garlic odor and nutrition contents, mountain garlic is used as a vegetable and traditional medicine in South Korea, Japan, and China. However, the genetic relationship between A. ochotense and A. microdictyon belonging to the subgenus Anguinum is still unclear. To understand the genetic diversity and phylogenetic relationships of mountain garlic populations in South Korea, we collected 25 mountain garlic materials from 8 geographical areas and analyzed their genetic diversity and relationships based on sequence variations of the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region. The DNA alignment result showed that A. ochotense and A. microdictyon were clearly divided into two groups, sharing 11 respective, particular nucleotide variable sites. It suggested that the nrDNA ITS region could be used to discriminate among Allium intraspecies. This species identification method would be utilized to distinguish unclear mountain garlic materials. This work not only provides more sequence resources of mountain garlic, but also validates the efficiency of the molecular discrimination method based on the nrDNA ITS region sequence.

Morpho-molecular characterization of <i>Ceratobasidium</i> sp.: A mycorrhizal fungi isolated from a rare epiphytic orchid <i>Gastrochilus calceolaris</i> (J. E. Sm.) D. Don

A mycorrhizal fungus, Ceratobasidium sp. GC (NCBI Gene Bank Accession no GQ369961) associated with the roots of an epiphytic orchid Gastrochilus calceolaris was investigated by cultural morphology, microscopic features and molecular analysis of Internal Transcribed Spacer (ITS) regions sequences of nuclear ribosomal DNA. The colony appearance of the fungal endophyte was fluffy growth pattern and the colour of the young colony was milky white on both surfaces that turned in to brown at maturity on the upper surface and deep brown on reverse surface. The microscopic features of the fungus i.e. hyphal diameter, multi-nucleate vegetative cells, right angle branching pattern with slight constriction at branching point and a dolipore septum near the branching point were observed. All the characters corroborated the identity with anamorphic Rhizoctonia like fungi. The ITS regions sequences of nrDNA and phylogenetic analysis based on the neighbor-joining method showed clustered with Rhizoctonia like fungi, and the maximum identity (98.28%) with Ceratobasidium RR and Ceratobasidium FPUB isolated from Rhynchostylis retusa and Dactylorrhiza hetagera, respectively. Thus, the ITS of nrDNA sequences validated the morphological data. This is the first report of orchid mycorrhizal fungi from Bangladesh.

Takashi Nakase's last tweet: what is the current direction of microbial taxonomy research?

During the last few decades, type strains of most yeast species have been barcoded using the D1/D2 domain of their LSU rRNA gene and internal transcribed spacer (ITS) region. Species identification using DNA sequences regarding conspecificity in yeasts has also been studied. Most yeast species can be identified according to the sequence divergence of their ITS region or a combination of the D1/D2 and ITS regions. Studies that have examined intraspecific diversity have used multilocus sequence analyses, whereas the marker regions used in this analysis vary depending upon taxa. D1/D2 domain and ITS region sequences have been used as barcodes to develop primers suitable for the detection of the biological diversity of environmental DNA and the microbiome. Using these barcode sequences, it is possible to identify relative lineages and infer their gene products and function, and how they adapt to their environment. If barcode sequence was not variable enough to identify a described species, one could investigate the other biological traits of these yeasts, considering geological distance, environmental circumstances and isolation of reproduction. This article is dedicated to late Dr Takashi Nakase (1939-2018).

Sympatric western lowland gorillas, central chimpanzees and humans are infected with different trichomonads.

We investigated intestinal trichomonads in western lowland gorillas, central chimpanzees and humans cohabiting the forest ecosystem of Dzanga-Sangha Protected Area in Central African Republic, using the internal transcribed spacer (ITS) region and SSU rRNA gene sequences. Trichomonads belonging to the genus Tetratrichomonas were detected in 23% of the faecal samples and in all host species. Different hosts were infected with different genotypes of Tetratrichomonas. In chimpanzees, we detected tetratrichomonads from 'novel lineage 2', which was previously reported mostly in captive and wild chimpanzees. In gorillas, we found two different genotypes of Tetratrichomonas. The ITS region sequences of the more frequent genotype were identical to the sequence found in a faecal sample of a wild western lowland gorilla from Cameroon. Sequences of the second genotype from gorillas were almost identical to sequences previously obtained from an anorexic French woman. We provide the first report of the presence of intestinal tetratrichomonads in asymptomatic, apparently healthy humans. Human tetratrichomonads belonged to the lineage 7, which was previously reported in domestic and wild pigs and a domestic horse. Our findings suggest that the ecology and spatial overlap among hominids in the tropical forest ecosystem has not resulted in exchange of intestinal trichomonads among these hosts.

Trichophyton mentagrophytes and Tinterdigitale genotypes are associated with particular geographic areas and clinical manifestations.

The fungi Trichophyton mentagrophytes and Tinterdigitale account for significant amount of dermatophytosis cases worldwide. These two dermatophytes form a species complex and have a number of ribosomal internal transcribed spacer (ITS) region genotypes, allowing simultaneous species identification and strain typing. Our aim was to describe the geographic distribution of Tmentagrophytes/Tinterdigitale ITS region genotypes and find an association between the genotypes and clinical presentations of respective infections. We performed rDNA ITS region sequencing in 397 Iranian Tmentagrophytes/Tinterdigitale isolates and analysed all available in GenBank entries with sequences of this kind. For the study, 515 clinical annotations were available. Statistical analysis was performed by chi-squared test and Spearman rank correlation analysis. A total of 971 sequences belonged to genotypes with at least 10 geographic annotations and were classified on the basis of exclusive occurrence in a particular region or high relative contribution to a regional sample. We discerned Asian and Oceanian ("KU496915" Type V, "KT192500" Type VIII, "KU315316"), European ("FM986750" Type III, "MF926358" Type III*, "KT285210" Type VI) and cosmopolitan ("FM986691" Type I, "JX122216" Type II, "KP132819" Type II* and "AF170453" Type XXIV) genotypes. There was statistically significant difference in the ITS genotype distribution between different affected body sites. Trichophyton mentagrophytes "KT192500" Type VIII correlated with tinea cruris, Tmentagrophytes "KU496915" Type V correlated with tinea corporis, Tinterdigitale "JX122216" Type II correlated with tinea pedis and onychomycosis. Trichophyton mentagrophytes and Tinterdigitale genotypes can be associated with distinct geographic locations and particular clinical presentations.

MOLECULAR DIVERSITY OF nrDNA ITS REGION AFFECTED HETEROSIS IN SUNFLOWER (Helianthus annuus)

A study was conducted to specify mutations of the internal transcribed spacer (ITS) in the nucleotides of nuclear ribosomal DNA (nrDNA) and their molecular relationship with heterosis exhibited by the six A- lines and three R-testers of sunflower. The trail was conducted at the fields of the College of Agriculture, University of Anbar, Abu Ghraib during two seasons (spring and fall 2016). Parental lines were crossed according to lineΧtester crossing scheme to produce 18 single hybrids, genotypes were sown to assess their phenotypic performance in the fall season. Results of the molecular analysis of ITS sequencing showed that the rearrangement of single nucleotides had accumulated in a higher rate in the F1 hybrids compared with their ancestor inbreds. The total number of mutations was 268, and deletion mutations accounted for the largest proportion with 209 mutation (71 in lines and 138 in hybrids). In contrast, the transition mutations were 25 all occurred in the hybrids, however the number of transversions recorded 17only. Based on DNA sequence of the nrDNA ITS region the total genotypes were separated into two main groups in cluster analysis following the nearest neighbor method. There was a significant convergence between R2 and both A4ΧR2 and A6ΧR3 hybrids scoring higher values of similarity in context ITS sequence. To be specific, the hybrid A2ΧR3 exhibited the best desirable heterosis for days to 75% flowering and leaf area, while hybrid A5ΧR3 had the highest heterosis for head traits, height and area.

Potential occupational and respiratory hazards in a Minnesota cannabis cultivation and processing facility.

Cannabis has been legalized in some form for much of the United States. The National Institute for Occupational Safety and Health (NIOSH) received a health hazard evaluation request from a Minnesota cannabis facility and their union to undertake an evaluation. NIOSH representatives visited the facility in August 2016 and April 2017. Surface wipe samples were collected for analysis of delta-9 tetrahydrocannabinol (Δ9-THC), delta-9 tetrahydrocannabinol acid (Δ9-THCA), cannabidiol, and cannabinol. Environmental air samples were collected for volatile organic compounds (VOCs), endotoxins (limulus amebocyte lysate assay), and fungal diversity (NIOSH two-stage BC251 bioaerosol sampler with internal transcribed spacer region sequencing analysis). Surface wipe samples identified Δ9-THC throughout the facility. Diacetyl and 2,3-pentanedione were measured in initial VOC screening and subsequent sampling during tasks where heat transference was greatest, though levels were well below the NIOSH recommended exposure limits. Endotoxin concentrations were highest during processing activities, while internal transcribed spacer region sequencing revealed that the Basidiomycota genus, Wallemia, had the highest relative abundance. To the authors' knowledge, this is the first published report of potential diacetyl and 2,3-pentanedione exposure in the cannabis industry, most notably during cannabis decarboxylation. Endotoxin exposure was elevated during grinding, indicating that this is a potentially high-risk task. The findings indicate that potential health hazards of significance are present during cannabis processing, and employers should be aware of potential exposures to VOCs, endotoxin, and fungi. Further research into the degree of respiratory and dermal hazards and resulting health effects in this industry is recommended.

First Report of Powdery Mildew Caused by Podosphaera elsholtziae on Dracocephalum tanguticum in China

Dracocephalum tanguticum Maxim. is endemic to the Qinghai Tibetan Plateau. It has been widely used in Tibetan traditional medicine to treat pneumonia and respiratory inflammation, but powdery mildew has seriously affected the quality of the plants and reduced the economic value. In August 2017, severe powdery mildew was observed on D. tanguticum in Huangnan (35.31°N, 101.93°E, altitude 3,300 m), China. Voucher specimens were deposited in the Herbarium of Plant Pathology at Qinghai University under accession number QHU2017043. This powdery mildew fungus is morphologically characterized as follows. Mycelium was observed on the entire surface of the leaves and stem, and more than 70% of leaf areas were affected; hyphae hyaline, thin-walled, smooth, 3 to 6 µm wide; hyphal appressoria nipple shaped; conidia ellipsoid-ovoid to doliiform, 22 to 30 × 12 to 17µm; chasmothecia amphigenous, scattered to gregarious, dark brown, globose or subglobose, 67 to 83 µm in diameter; peridium cells irregular shaped, 8 to 37 µm in diameter; appendages usually few, mostly fewer than six, arising from the lower half, myceloid, simple, usually unbranched, 0.3 to 2 times as long as the chasmothecial diameter, 3 to 7 µm wide, brown below and paler toward the tip, septate, wall thin, smooth; ascus broadly ellipsoid-ovoid, sessile or with a very short stalk, 60 to 71 × 50 to 58 µm, six- to eight-spored; ascospores broad ellipsoid-ovoid or subglobose, 11 to 18 × 8 to 15 µm, colorless. This fungus was identified as Podosphaera elsholtziae based on the morphology of its asexual and sexual morphs (Braun and Cook 2012). To confirm the identification, the internal transcribed spacer (ITS) region were amplified. Genomic DNA was extracted from chasmothecia using Chelex-100 (Hirata and Takamatsu 1996; Walsh et al. 1991). The ITS5/P3 and PM5/ITS4 primers were used to amplify the ITS region by nested polymerase chain reaction, and the cloned fragments were sequenced. The ITS region sequences were deposited in GenBank (accession no. MK402076). A BLAST search analysis of the two sequences revealed 99% identity with P. elsholtziae infecting Ajuga reptans Linn. in Japan. Based on the ITS rDNA phylogenetic tree, the sequences retrieved from the specimen clustered within the same clade (bootstrap value 97%) with P. elsholtziae (AB026142). Cladistic trees were constructed using the maximum parsimony method with the Kimura two-parameter substitution model in MEGA 5.0. Branch robustness was assessed via bootstrap analysis with 1,000 replicates. Phylogenetic analysis data agreed with morphological characteristics (Takamatsu et al. 1999, 2009). Pathogenicity tests were performed by rubbing infected leaves onto healthy leaves and stems of four D. tanguticum seedlings. Four healthy seedlings were kept separately as a noninoculated control. Inoculated and noninoculated plants were maintained in a greenhouse at 22 to 25°C. Inoculated plants developed signs and symptoms after 17 days, whereas control plants remained symptomless. The morphology of the fungus on inoculated leaves was identical to that originally observed on diseased plants. To our knowledge, this is the first report of powdery mildew caused by P. elsholtziae on D. tanguticum worldwide.