Internal Transcribed Spacer Fragments Research Articles

First report of Diplodia quercivora causing branch cankers on declining chestnut oak (Quercus montana) in Virginia.

Since the beginning of the twentieth century oak decline has been documented in central and eastern hardwood forests of the United States as a stress-mediated disease (Oak et al. 2016). Opportunistic canker pathogens, including Diplodia corticola, D. quercivora, D. sapinea, and Botryosphaeria dothidea have been associated with crown dieback of declining oak trees in several mid-Atlantic states (Ferreira et al. 2021). On 02 August 2022, a survey was conducted at two natural hardwood sites in Fredrick and Shenandoah Counties, Virginia that exhibited symptoms of decline (Fig. 1A). At both sites, mature Quercus montana trees were observed with bole and branch cankers, bleeding and sooty lesions, and discolored sapwood. Pycnidia were present on the margin of seven branch cankers from three trees that were felled, with hyaline, elliptical to oblong conidia 19.0 - 26.8 × 8.5 - 11.2 µm (n = 40) in size (Fig. 1C and D). Six cultures were derived from single spores that were placed on PDA medium and incubated for 10 days in the dark at 22 ± 2°C. Additionally, a 4-mm piece of necrotic tissue was selected from the margin of each of the seven cankers, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, seven colonies from each canker assayed were transferred to full-strength PDA plates and incubated for 10 days in the dark at 22 ± 2°C. Colonies derived from spores and the necrotic wood were morphologically identical, with white, aerial, floccose mycelium that turned dark gray to olivaceous after five days (Fig. 1B). DNA was individually extracted from four, 10-day-old cultures (two from spores and two from wood). Mycelia was harvested with a sterile pin and extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A segment of the internal transcribed spacer (ITS), large subunit rRNA (LSU), and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990), LR5/LROR (Vilgalys and Hester 1990), and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY). The nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). The resulting ITS sequences from the four isolates were identical. A 544-bp, 1131-bp, and 273-bp segment of the ITS, LSU, and tef1 loci from isolate GS22-DSB-17 was deposited into the GenBank database (accessions OQ597712, OQ597714, and OR754429, respectively). A Genbank BLAST analysis revealed that the ITS and tef1 fragments shared 510/516 (99%) and 271/273 (99%) nucleotides with the D. quercivora ex-type BL8 (JX894205/JX894229). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. montana trees (average stem caliper 6.5 cm) with D. qercivora isolate GS22-DSB-17, while five plants were used as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 6°C. The same procedure was performed on the control plants using sterile PDA plugs. After six weeks the bark was carefully removed, and all five stems treated with D. quercivora had necrotic lesions with a mean canker linear growth ([length+width]/2) of 15.6 mm from the edge of the wound, which was significantly larger (P = 0.001) than the controls (2.3 mm; Fig. 1E-M). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. quercivora based on morphology and 100% ITS sequence homology to isolate GS22-DSB-17. D. quercivora was not recovered from the control plants. In the United States, D. quercivora has been isolated from declining white oak trees in Maryland, Massachusetts, West Virginia, and Florida (Dreaden et al. 2014; Ferreira et al. 2021; Haines et al. 2019). More surveys are needed to understand the host range and distribution of D. quercivora in the United States, as well as the environmental and site factors that impact oak health and predispose trees to infection from opportunistic cankering pathogens.

Comparison of five DNA extraction methods in three medicinal plants: Peganum harmala L., Tamarix ramosissima Ledeb., and Potentilla reptans L.

Extracting high-yield, high-quality DNA from plant samples is challenging due to the presence of the cell wall, pigments, and some secondary metabolites. The main CTAB method, two of its modified protocols (beta-mercaptoethanol or ammonium acetate were eliminated), the modified Murray and Thompson method, and the Gene All kit were statistically compared based on the quantity and quality of the total DNA (tDNA) extracted from fresh and dried leaves of three medicinal herbs P. harmala, T. ramosissima, and P. reptans. The suitability of the tDNAs for molecular studies was evaluated by polymerase chain reaction (PCR) of the fragments of the internal transcribed spacer (ITS) in nuclear DNA and the trnL-F region in chloroplast DNA. Some significant differences were found between the tDNAs extracted by five extraction methods. With the exception of P. harmala, where the PCR of both the ITS fragments and the trnL-F region worked successfully in all DNA samples, but only the ITS fragments, not the chloroplast trnL-F region, were amplified in the DNA samples of T. ramosissima and P. reptans. The chloroplast trnL-F region was amplified only in DNA samples extracted from fresh and dried leaves of the three studied herbs using the commercial kit. Gene All kit, the main CTAB method, and its modified protocols were the less time-consuming protocols that yielded DNA suitable for downstream PCR vis-a-vis the modified Murray and Thompson method.

Metabarcoding of insect-associated fungal communities: a comparison of internal transcribed spacer (ITS) and large-subunit (LSU) rRNA markers.

Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations and early detection of pathogens and invasive species. Complex communities of fungi are regularly characterised by metabarcoding using the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus, but reliance on a single short sequence fragment limits the confidence of identification. Here we link metabarcoding from the ITS2 and LSU D1-D2 regions to characterise fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type), but identification against the respective reference databases using various taxonomic classifiers revealed poor resolution towards lower taxonomic levels, especially the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, using phylogenetic trees (focused on the epidemiologically important Sordariomycetes) we placed OTUs obtained with either marker relative to reference sequences of the entire rRNA cistron that includes both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. Sensitivity analysis of congruence in both markers suggested the biologically most defensible threshold values for OTU delimitation in Sordariomycetes to be 98% for ITS2 and 99% for LSU D1-D2. Studies of fungal communities using the canonical ITS barcode require corroboration across additional loci. Phylogenetic analysis of OTU sequences aligned to the full rRNA cistron shows higher success rate and greater accuracy of species identification compared to probabilistic taxonomic classifiers.

Konjac anthracnose is caused by Colletotrichum gloeosporioides in Yunnan Province of China.

Konjac (Amorphophallus konjac) is an economically important traditional crop in Fengqing County, Yunnan Province, China. Anthracnose symptoms were observed on this crop in June and July of 2020. The plants developed round, oval, or irregular leaf spots with brown edges and central taupe spots, and exhibited serious defoliation and tree weakness. Disease incidence in affected fields reached up to 35%. Anthracnose caused significant economic losses in konjac production and became a limiting factor of the konjac industry in Fengqing County. To date, no control measures of konjac anthracnose have been reported and tested in China. To determine the causal pathogen, symptomatic leaves were collected and cut into 5 mm2 pieces. The leaf peces were surface sterilized in 70% ethanol for 10 s, followed by treatment with 0.1% mercuric chloride for 3 min and three rinses in sterile distilled water. The tissue pieces were transferred onto potato dextrose agar (PDA) and incubated at 28°C. After 4 days of incubation, hyphal tips from leaf pieces were transferred to new PDA to generate pure cultures. The hyphae were initially white, and then became dark green; red-orange conidial masses were observed on the mycelium plate surface at a growth rate of 13.14 mm/day. The conidia, observed under 400× magnification, were colorless, long-oval to fusiform, one-celled, and 15.4 to 18.2 × 3.3 to 5.9 μm in size. To identify the isolate, the genomic DNA of the pathogen was extracted using the CTAB method. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and β-tubulin (TUB) gene were PCR amplified using the ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b primer pairs, respectively (Lee et al. 2020). BLASTn search of the obtained 536 bp ITS fragment (GenBank accession no. MT785772), 229 bp GAPDH sequence (MW187543), and 717 bp TUB sequence (MW187544) revealed a 99.44% to 99.63% sequence homology (100% query cover) with ITS (JQ005152, 99.44%), GADPH (JQ005239, 99.63%), and TUB (JQ005587, 99.60%) sequences of the C. gloeosporioides type strain CBS 112999, respectively. The highest homology with other Colletotrichum species was only 98.16%, including C. siamense, the causal agent of anthracnose in A. paeoniifolius and A. konjac (Prasad et al. 2017; Wu et al. 2020). To complete Koch's postulates, leaves of 3-month-old konjac plants grown in the field were sprayed with a conidial suspension (106 spores/ml) of the isolate YNFQ-1 (sterile water was used as a negative control). Approximately 5 days after inoculation with YNFQ-1, symptoms similar to those in natural conditions appeared, whereas the negative control plants and fruits inoculated with the sterile water had no disease. The pathogen was re-isolated (strain YNFQ-1) from inoculated leaf tissues, and its identity was confirmed with both morphological and molecular (DNA sequences) tools, thus fulfilling Koch's postulates. The culture properties, morphological characteristics, and molecular identification confirmed the identity of the pathogen as C. gloeosporioides. There have been many reports about anthracnose of Amorphophallus; C. siamense causes anthracnose on A. paeoniifolius in India (Prasad et al. 2017) and A. konjac in Hubei, China (Wu et al. 2020), and C. gloeosporioides causes anthracnose of A. muelleri in Yunnan, China (Yang et al. 2020). To the best of our knowledge, this is the first report of C. gloeosporioides causing anthracnose on A. konjac in Fengqing County, China. The results are expected to have important implications in the diagnosis, control, and future research of anthracnose on A. konjac.

Molecular and phylogenetic analysis of inulinase-producing yeast isolated from nira siwalan (Borassus flabellifer) based on ITS sequences

Inulinase (E.C. 3.2.1.7) is an enzyme capable hydrolyze inulin to be a fructose monomer, which is widely used in various fields, especially the food industry. This enzyme can be found in various plant species and can be produced by microorganisms such as bacteria, yeast, and fungi. Previous research has succeeded in isolating the potential yeast of inulinase from nira siwalan (Borassus flabellifer), named N1. This research purpose to identify molecularly the yeast inulinolytic N1 and its kinship with other species based on internal transcribed spacer (ITS) rDNA sequences. The stages in this research are yeast DNA extraction using the chelex method, DNA amplification with primers ITS 4 and ITS 5, electrophoresis, sequencing analysis, and construction of the phylogenetic tree. Phylogenetic trees are reconstructed using the neighbor-joining method based on their evolutionary relationship. The results of DNA isolation showed that the concentration of yeast DNA was 495.8 ng/ μl and purity of 2,12. The PCR product from the ITS fragment amplification produced a band size of ± 510 bp. The results of molecular identification and phylogenetic analysis showed that the N1 yeast isolate was closely related to Candida parapsilosis (MK638869.1) originating from India with a homology of 99.45%.

Molecular Profile of Trichophyton mentagrophytes and Microsporum canis based on PCR-RFLP of Internal Transcribed Spacer

<p><em>Trichophyton mentagrophytes</em> and <em>Microsporum canis</em> are dermatophytes fungi which commonly infect animal and human. Conventional and molecular methods were used for identification of the fungus. The region of internal transcribed spacer (ITS) has a high probability for fungal identification. PCR-RFLP was reported as a useful method to differentiate dermatophytes fungi. The objective of the study was<em> </em>to compare molecular profile of <em>T. mentagrophytes</em> and <em>M. canis</em> based on the result of ITS fragment digestion using Dde I, Hinf I and Mva I. The molds were isolated from skin scrapping of 18 animals which showed dermatophytosis lesion. The isolated molds were grown on agar plate for 14 days of incubation at 37<sup>o</sup>C and then identified based on macro and microscopic morphologies. Amplification of chitin synthase gene was used for confirmation and separation of dermatophytes from other fungi. ITS fragment was amplified and then digested using restriction enzymes Dde I, Hinf I and Mva I. The result showed that digestion products from ITS fragment of <em>T. mentagrophytes</em> and <em>M. canis </em>were different<em>.</em> The fragment 159 bp from Dde I, 374 bp from Hinf I and 89 bp from Mva I were present in <em>T. mentagrophytes</em> but absent in <em>M. canis</em>. Based on these results, specific RFLP profile of digestion ITS region by Dde I, Hinf I and Mva I can be used as a specific marker for species of dermatophytes fungi.</p>

First Report of Leaf Spot Disease Caused by Stemphylium vesicarium on Fagopyrum esculentum in China.

Common buckwheat (Fagopyrum esculentum) is a widely cultivated non-grass cereal. It has a considerable market value with nutritional qualities and ability to treat or prevent cancers, hypertension and diabetes (Cawoy et al., 2006). In July-August 2018 and 2019, leaves exhibiting unfamiliar symptoms were observed in Huan County and Huachi County, Gansu, China, with 15% incidence and moderate to severe severity across the field. Initial symptoms consisted of small chlorotic, circular to oval leaf spots. As disease progressed, the spots enlarged and turned light brown to brown with chlorotic margins. When the disease was severe, the leaf spots coalesced and the leaves became prematurely chlorotic and senescent. Spots occurred mostly but not exclusively on older foliage. Diseased tissues were surface sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, placed on PDA medium and incubated at 20 °C for 48-72 h. A total 24 isolates were obtained and purified through single-spore cultures, 19 of which were characterized. Colonies on PDA of all 19 isolates were identical, exhibiting a light gray color, with whitish aerial mycelium that later turned light brown on the reverse of the culture plates, and sporulated sparsely. Conidia were brown colored, cylindrical, and borne singly, often had three main transverse septa, at which points there were conspicuous constrictions, and measured (17-37) μm long × (13-21) μm wide (n=30) in V8 juice agar, (22-38) μm long × (11-19) μm wide (n=30) in PCA, (21-41) μm long × (13-20) μm wide (n=30) in SNA, the mean length/width ratio was 1.6 to 2.0 in V8 juice agar, 1.6 to 2.5 in PCA, 1.3 to 2.2 in SNA. Conidiophores were unbranched, 5.1 to 7.9 μm wide. Dictyospores were produced on well-differentiated conidiophores, the apical cell of conidiophore was slightly to distinctly swollen. Based on morphological features, the isolates were tentatively identified as a member of the Stemphylium vesicarium species complex (Koike et al., 2013; Simmons, 1967). Genomic DNA of representative isolate B1 was extracted, and the internal transcribed spacer (ITS) region and calmodulin gene (cmdA) were amplified using ITS1/4, V9G/ITS4, and CALDF1/CALDR1, respectively. The resulting sequences were deposited in GenBank (acc. nos. MT629829, MW406903, MW417122). Nucleotide BLAST similarity analysis of the sequence fragment of ITS and cmdA from isolate B1 resulted in higher than 99% (99.32% for ITS1/4, 100% for V9G/ITS4, and 100% for CALDF1/CALDR1) identity with S. vesicarium strains (GenBank acc. nos. LC512757, MH863402 and MH206181). Based on morphological features and molecular data, the buckwheat isolates were identified as S. vesicarium. To verify pathogenicity, the back side of leaves from six asymptomic plants were inoculated by spraying the spore suspension (104 spores/ml) harvested from isolate B1 grown on V8 juice agar for 20 days. Control plants were sprayed with sterile water. Each plant was covered with a black plastic bag for 48 h and then was kept in a greenhouse. Stemphylium spot symptoms were observed on all inoculated leaves after 14 days, whereas control leaves were symptomless. The pathogen was reisolated from symptomatic leaf spots, micromorphological features and colony characters of the reisolated fungi were identical to the original isolate. To our knowledge, this is the first documentation of leaf spot of buckwheat caused by S. vesicarium in China, and the first characterization of a Stemphylium foliar pathogen on this crop.

Differentiation Among Rodentibacter Species Based on 16S-23S rRNA Internal Transcribed Spacer Analysis.

The internal transcribed spacer (ITS) regions of Rodentibacter pneumotropicus, R. heylii, R. rarus, R. ratti, and R. heidelbergensis and of a Rodentibacter- related β-hemolytic Pasteurellaceae taxon isolated from laboratory rodents were studied for their feasibility to discriminate among these species. The 6 species analyzed showed species-specific ITS patterns that were shared by the type strains and clinical isolates and that allowed their identification. Nevertheless, differentiating between the ITS band patterns of R. pneumotropicus and R. ratti is visually challenging. In all species tested, sequence analysis of the ITS fragments revealed a larger ITSile+ala, which contained the genes for tRNAIle(GAU) and tRNA Ala(UGC), and a smaller ITSglu with the tRNAGlu(UUC) gene. The ITS sequences varied among the 6 species evaluated, displaying identity levels ranging from 62% to 86% for ITSile+ala and 68% to 90% for ITSglu. Overall, ITS amplification proved to be a reliable method to differentiate among these important Pasteurellaceae species of laboratory rodents. Moreover, the ITS sequence variations recorded here might facilitate the design of probes for specific identification of these species. The ability to diagnose these organisms to the species level could increase our understanding of their clinical significance.

First Report of Verticillium Wilt Caused by Verticillium dahliae Infection on Chinese Cabbage in Korea.

Chinese cabbage (Brassica rapa L.) is one of the most important vegetables in Korea due to its role as the main ingredient for the making of Kimchi. In June 2014, disease symptoms of leaves wilt, dry, and drop off on Chinese cabbage were observed in a Chinese cabbage farm located at Taebeak (37°26'50.7"N 128°95'50.0"E), Gangwon province, Korea. This disease was observed on approximately 35% of the plants in the field, causing an almost 10% decrease in total production. At the early stage of infection, the color at the edge of the plant foliage changed from green to yellow. As the disease progressed, infected leaves wilted, dried off, and detached from the plant. Soft rot that occurred at the base of the leaf stem and root tissues caused the infected leaves to dry and fell off the plant. To identify the causal agent, a small piece of infected leaf tissues was sterilized with 1% sodium hypochlorite solution for 1 min and rinsed with sterile water before it was transferred onto potato dextrose agar (PDA) media. The plates were then incubated at 25°C for 10 days in the dark. Fungal colonies grown on PDA media were of white-creamy in color with an abundance of mycelia and later develop into black color due to the formation of microsclerotia embedded in the media. Microscopic examination showed conidiophores and phialides were both appeared in a verticillate arrangement, whereas conidia were hyaline, smooth-walled, and ellipsoidal to oval with average size 5.4×2.5 µm (n=100). Microsclerotia appeared in elongate to an irregularly spherical shape and greatly variable in size. The morphological attributes of the fungal isolate described above were comparable to the characteristics of Verticillium dahliae Kleb. (V. dahliae) described by Hawksworth and Talboys (1970), and V. dahliae isolated from Chinese cabbage in Japan reported in Kishi (1998). Pathogenicity test was performed by soaking twelve individual Chinese cabbage seedlings for 15 min into fungal pathogen conidial suspension (1x106 conidium/ml) before transferred into soil tray. The same number of non-inoculated seedlings on the soil tray was used as a control. Inoculated and control plants were then covered with a plastic bag for 24 hours to maintain high humidity before transferred into the greenhouse (25°C). Seven days post-inoculation (dpi), treated plant leaves turned yellow, and soft rot was observed. At 10-dpi, plant leaf tissues dried off and severe soft rot occurred. Pathogenicity test was repeated three times and consistent results were obtained. The re-isolated fungal pathogen from the inoculated plants showed identical morphological characteristics to the original isolates, thus fulfilling Koch's postulates. For further identification, PCR amplification targeting Internal Transcribed Spacer (ITS) and RNA polymerase II gene (RPB2) regions were performed (Liu et al., 1999; White et al., 1990). Each PCR product was sequenced and deposited in the GenBank under the accession LC549667 and LC061275, respectively. Sequence analysis using BLAST showed that the nucleotide sequences of ITS and RPB2 DNA fragments are 99-100% identical to the reference strain of V. dahliae available in the NCBI database (MG585719, HE972023, XM_009652520 and DQ522468, respectively). Therefore, based on the results of morphological and molecular analyses, the fungal pathogen isolated from Chinese cabbage in this study was identified as V. dahliae and deposited in the National Institute of Horticultural and Herbal Science germplasm collection (NIHHS 13-252). Recently, due to high demand and a more competitive price, more Chrysanthemum farmers in Korea switch their crops to Chinese cabbage. Interestingly, the occurrence of V. dahliae infection was also reported to occur in Chrysanthemum plants in Korea (Han et al. 2007), which indicates a serious problem ahead to these farmers. Therefore, in this current study, the identification of V. dahliae pathogenic to Chinese cabbage will provide vital knowledge for the development of disease management strategies to minimize the loss of crop production. To our knowledge, this is the first report that V. dahliae causes Verticillium wilt disease on Chinese cabbage in Korea.

Molecular Identification and Genetic Diversity of Thalassia hemprichii Through DNA Barcoding Using Internal Transcribed Spacer gene (ITS) from Awur Bay Jepara, Indonesia

Thalassia hemprichii is a type of seagrass that dominates in Awur Bay of Jepara. Conventional identification has many obstacles and the environmental pressure causes seagrass stand damage and incomplete. This leads to difficulty in morphological identification. Therefore, it is necessary to do molecular identification. DNA barcode using ITS gene is one method of molecular identification using short sequences that are efficient and have a high level of accuracy. The purposes of this research were to identify Thalassia hemprichii using DNA barcode of the Internal Transcribed Spacer (ITS). The research methods includeds DNA extraction using CTAB, PCR amplification of ITS fragment, electrophoresis, DNA sequencing, and analysis with MEGA 6 and BLAST. The result of this research showed that all of the samples studied were Thalassia hemprichii. DNA barcode of ITS is a gene marker which capable of identifying seagrass species of Thalassia hemprichii.

First Report of Clonostachys rhizophaga as a Pathogen of Water Chestnut (Eleocharis dulcis) in Anhui Province of China

HomePlant DiseaseVol. 103, No. 1First Report of Clonostachys rhizophaga as a Pathogen of Water Chestnut (Eleocharis dulcis) in Anhui Province of China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Clonostachys rhizophaga as a Pathogen of Water Chestnut (Eleocharis dulcis) in Anhui Province of ChinaX. Yang, A.-F. Zhang, C.-Y. Gu, H.-Y. Zang, and Y. ChenX. YangSearch for more papers by this author, A.-F. ZhangSearch for more papers by this author, C.-Y. GuSearch for more papers by this author, H.-Y. ZangSearch for more papers by this author, and Y. Chen†Corresponding author: Y. Chen; E-mail: E-mail Address: chenyu66891@sina.comhttp://orcid.org/0000-0001-9767-5255Search for more papers by this authorAffiliationsAuthors and Affiliations X. Yang A.-F. Zhang C.-Y. Gu H.-Y. Zang Y. Chen † , Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China; Scientific Observing and Experimental Station of Crop Pests in Hefei, Ministry of Agriculture, China; Laboratory of Quality & Safety Risk Assessment for Agro-Products, Hefei, Ministry of Agriculture, China; and Key Laboratory of Anhui Agricultural Sciences (18S1103), Hefei 230031, China. Published Online:26 Oct 2018https://doi.org/10.1094/PDIS-05-18-0840-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Water chestnut, Eleocharis dulcis (Burm. f.) Trin., is a sedge native to China and is widely grown in the world, especially in the tropical and subtropical regions. In September 2017, black spots were observed on about 80% of the water chestnuts in several villages of Wuwei County, Anhui Province. The diseased water chestnuts showed longitudinal chlorosis streaks with black spots on the stem surface and internal vascular necrosis. To isolate the pathogen, the diseased stem segments (2 mm in length) were surface sterilized with 1% NaClO for 1min, rinsed twice for 30 s with sterile deionized water, and then incubated on water agar medium at 25°C in a growth chamber for 3 days. Then a mycelial plug was transferred to potato dextrose agar (PDA) medium and incubated for 2 weeks in the growth chamber at 25°C. Representative isolates formed off-white mycelia, dimorphic, Verticillium-like (primary) or penicillate (secondary) conidiophores, and ovoidal to elongated, slightly curved or asymmetrical, hyaline conidia about 5 to 8.8 μm long and 2.5 to 3.9 μm wide (more than 50 conidia were measured),with laterally displaced hilum. To identify the eight isolates, partial fragments of internal transcribed spacer (ITS) and β-tubulin (tub2) gene were amplified according to the method of Abang et al. (2009). A 525-bp ITS fragment (GenBank accession no. MG872331) was amplified from the ITS for all eight isolates with universal primers ITS1/ITS4, showing 100% similarity to the Clonostachys rhizophaga strain CML2312 (GenBank accession no. KC806275) and CML2310 (GenBank accession no. KC806272). A 592-bp fragment of tub2 (GenBank accession no. MH325937) was amplified with the primers bt2a (5′-AACATGCGTGAGATTGTAAGT-3′) and bt2b (5′-TCTGGATGTTGTTGGGAATCC-3′) and showed 100% similarity to the C. rhizophaga strain 1019 (GenBank accession no. FJ593883). To verify the pathogenicity of these isolates, a 5-mm-diameter mycelial plug was cut from 2-week-old colonies grown on PDA for each of the isolates and inoculated on the punctured stem of healthy water chestnuts. The inoculation site was covered with wet cotton and wrapped with Parafilm. The control plants were treated by the same method but using sterile PDA plugs of the same size. All the treatments were incubated in a plant growth chamber at 25°C with a 12-h photoperiod. Two weeks after inoculation, symptoms on all inoculated stems were identical to those described above with a dark area surrounded by a yellow halo on the stem surface and internal browning, whereas the control plants did not show any symptoms. Furthermore, the fungus was reisolated from all the inoculated stems and identified to be C. rhizophaga as described above, fulfilling Koch’s postulates. This pathogen has been reported to infect other plants, such as chickpea (Abang et al. 2009), giant bamboo (Dendrocalamus giganteus) (Zazzerini and Quaglia 2010), and Canary lsland pine (Pinus canariensis) (Piontelli and Giusiano 2003); however, to our knowledge, this is the first report of C. rhizophaga infecting water chestnut plants in China. This disease is an emerging problem in Anhui Province, and it is necessary to develop new disease management practices to sustain commercial production in this region.

Establishment and validation of ARMS (amplification-refractory mutation system) for identification of Anisakis species collected from Korean waters

The identification of Anisakis species in Korean waters was performed using an amplification-refractory mutation system (ARMS). ARMS is typically used to detect single nucleotide polymorphisms or allele types in the same species. However, the application of ARMS for species identification has not been reported. We designed a tetra-primer binding the internal transcribed spacer (ITS) region of 76 samples of Anisakis species and obtained reproducible results. ARMS revealed 380 bp and 130 bp ITS fragments in A. pegreffii, 380 bp and 280 bp fragments in A. simplex, a 130 bp fragments in A. typica and 380 bp, 280 bp and 130 bp fragments in an A. simplex – A. pegreffii hybrid. ARMS using a tetra-primer could be a more rapid, efficient, and reliable tool for monitoring Anisakis species more rapidly than restriction fragment length polymorphism.

Phylogenetic relationship and genetic differentiation of Populus caspica and Populus alba using cpDNA and ITS noncoding sequences

Populus caspica Bornm. (section Leuce and subsection Albida), one of the most endangered endemic tree species in the Hyrcanian Forest in Iran, has numerous morphological characteristics that are closely similar to Populus alba; to clarify their taxonomic relatedness and genetic differentiation and thus inform conservation strategies, we used the noncoding regions of chloroplast DNA (cpDNA; trnL-F and trnH-psbA) and the internal transcribed spacer (ITS). Leaf samples were collected from six populations across northern Iran. cpDNA and ITS fragments were amplified by universal primers using the PCR technique and directed sequencing. The results showed that P. caspica is genetically differentiated from P. alba, and two ITS variants were detected within some P. caspica individuals. Conflicts between topologies from ITS and plastid genomes were observed. High differentiation of P. caspica from the other Populus species shown in this study confirmed the diverging taxonomic status of this endangered species. We recommend in situ conservation measures (e.g., protected areas) for at least several populations of this species, especially in the plain regions of the Hyrcanian forest.

Assessing S. mansoni prevalence in Biomphalaria snails in the Gombe ecosystem of western Tanzania: the importance of DNA sequence data for clarifying species identification

BackgroundSnails are essential for the transmission and maintenance of schistosomiasis in endemic areas, as they serve as intermediate hosts for schistosome parasites. A clear understanding of the snail species present, their local distribution and infection status is therefore a prerequisite for effective control of schistosomiasis. The purpose of this study was to establish the infection status and distribution of Schistosoma mansoni in snails in the Gombe area along the shores of Lake Tanganyika in western Tanzania, using both detection of cercarial shedding and molecular approaches.MethodsSnails were collected from streams located close to human settlements in Gombe National Park, as well as from nearby villages (Kiziba, Mtanga, Mwamgongo and Bugamba) and the largest town in the region (Kigoma). Snails were individually exposed to light to induce shedding of schistosome larvae, which were examined using a compound light microscope. Additionally, the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster was simultaneously amplified in both snails and their trematodes using a single polymerase chain reaction (PCR) and sequenced to confirm species identification.ResultsSnails morphologically identified as Biomphalaria pfeifferi were present in all streams except at Mtanga but their distribution was patchy in both time and space. Sequencing of PCR products indicated that not all snails were B. pfeifferi. None of the snails from Gombe or Bugamba shed schistosome larvae, while larvae were shed at all other sites. Overall, an infection prevalence of only 12% was observed in snails based on cercarial shedding. While 47% of the snails were PCR-positive for the 500 bp ITS fragment, which was predicted to indicate infection with S. mansoni, sequence data demonstrated that these bands are not species-specific and can be amplified from other trematode infections. In addition, a 1000 bp band was amplified in 14% of samples, which was identified as a trematode in the family Derogenidae.ConclusionsThe results support the previous assumption that B. pfeifferi snails may be involved in transmitting schistosomiasis in the area but suggest that the community structure of both snails and trematodes may be more complicated than previously thought. This emphasises the importance of confirming species identifications using sequencing, rather than relying only on PCR-based diagnostics or cercarial shedding.

Diversity and community structure of wood-inhabiting fungi found in Japanese wooden houses analyzed by the next-generation sequencing

The diversity and community structures of wood-inhabiting fungi in 16 decayed wood samples from ten wooden houses in Japan were analyzed using a next-generation sequencing (NGS) to determine the fungi responsible for wood decay. DNA of fungi in decayed wood was extracted directly, the internal transcribed spacer (ITS) region in ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR), and then, sequences of tagged ITS fragments were analyzed by NGS. Results of sequencing indicated that 68 species of ascomycetes, 37 species of basidiomycetes, and one fungus each from Mortierellales and Mucoromycetes were detected. The fungal community structures showed diversity and included various species of ascomycetes. A microscopic examination of cell wall structure in decayed wood samples suggested that some ascomycetes were soft-rot fungi. Heat map analysis indicated that the similarity in the structures of fungal communities was influenced to a greater extent by the wood species of samples than where they were used as a component.

Cyanobacterial Contribution to Travertine Deposition in the Hoyoux River System, Belgium.

Travertine deposition is a landscape-forming process, usually building a series of calcareous barriers differentiating the river flow into a series of cascades and ponds. The process of carbonate precipitation is a complex relationship between biogenic and abiotic causative agents, involving adapted microbial assemblages but also requiring high levels of carbonate saturation, spontaneous degassing of carbon dioxide and slightly alkaline pH. We have analysed calcareous crusts and water chemistry from four sampling sites along the Hoyoux River and its Triffoy tributary (Belgium) in winter, spring, summer and autumn 2014. Different surface textures of travertine deposits correlated with particular microenvironments and were influenced by the local water flow. In all microenvironments, we have identified the cyanobacterium Phormidium incrustatum (Nägeli) Gomont as the organism primarily responsible for carbonate precipitation and travertine fabric by combining morphological analysis with molecular sequencing (16S rRNA gene and ITS, the Internal Transcribed Spacer fragments), targeting both field populations and cultures to exclude opportunistic microorganisms responding favourably to culture conditions. Several closely related cyanobacterial strains were cultured; however, only one proved identical with the sequences obtained from the field population by direct PCR. This strain was the dominant primary producer in the calcareous deposits under study and in similar streams in Europe. The dominance of one organism that had a demonstrated association with carbonate precipitation presented a valuable opportunity to study its function in construction, preservation and fossilisation potential of ambient temperature travertine deposits. These relationships were examined using scanning electron microscopy and Raman microspectroscopy.

Bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae): obligate symbionts in mycelial cultures of the black truffles Tuber melanosporum and Tuber brumale.

BackgroundThis work aimed at characterizing 12 isolates of the genus Tuber including Tuber melanosporum (11 isolates) and Tuber brumale (one isolate). This was done using internal transcribed spacer (ITS) sequences, confirming their origin.ResultsAnalysis of their mating type revealed that both MAT1-1 and MAT1-2 exist within these isolates (with 3 and 8 of each, respectively). We observed that each of these cultures was consistently associated with one bacterium that was intimately linked to fungal growth. These bacterial associates failed to grow in the absence of fungus. We extracted DNA from bacterial colonies in the margin of mycelium and sequenced a nearly complete 16S rDNA gene and a partial ITS fragment. We found they all belonged to the genus Rhodopseudomonas, fitting within different phylogenetic clusters. No relationships were evidenced between bacterial and fungal strains or mating types. Rhodopseudomonas being a sister genus to Bradyrhizobium, we tested the nodulation ability of these bacteria on a promiscuously nodulating legume (Acacia mangium), without success. We failed to identify any nifH genes among these isolates, using two different sets of primers.ConclusionsWhile the mechanisms of interaction between Tuber and Rhodopseudomonas remain to be elucidated, their interdependency for in vitro growth seems a novel feature of this fungus.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2756-6) contains supplementary material, which is available to authorized users.

Molecular diagnosis of the brown root rot disease agent Phellinus noxius on trees and in soil by rDNA ITS analysis

Phellinus noxius (P. noxius) is an important pathogen that causes brown root rot of trees in tropical and sub-tropical areas and has led to severe damage to trees. A quick and accurate diagnostic technique is essential to the timely confirmation of the pathogen and possible treatment. In this study, a fast, sensitive and accurate approach of molecular technique was used to diagnose the brown root rot pathogen on trees and in soils of subtropical Hong Kong. Two pairs of specific PCR primers were used to amplify the target rDNA internal transcribed spacer (ITS) region through polymerase chain reaction (PCR) for both tree tissues and soil samples. The amplified ITS fragments were then sequenced and analyzed phylogenetically for the diagnostic identification of the pathogen P. noxius. The results showed that 13 of 38 suspected trees in Hong Kong were infected with P. noxius through molecular detection. The pathogen showed no specific preference to any particular tree species. Quantitative PCR was applied to soils grown with trees identified both positive and negative for P. noxius, but the soils with healthy trees were also found positive for P. noxius. For the first time, P. noxius was reported to infect a wide range of tree species in Hong Kong and widely presented in soils, probably serving as a reservoir for the pathogen. Through this study, it is proposed that P. noxius is a soil-borne pathogen, which increases its infectivity when trees start to grow in the soil as a means in addition to the previously proposed root-to-root contact.

Isolation and characterization of yeasts associated with plants growing in heavy-metal- and arsenic-contaminated soils.

Yeasts were quantified and isolated from the rhizospheres of 5 plant species grown at 2 sites of a Mexican region contaminated with arsenic, lead, and other heavy metals. Yeast abundance was about 10(2) CFU/g of soil and 31 isolates were obtained. On the basis of the phylogenetic analysis of 26S rRNA and internal transcribed spacer fragment, 6 species were identified within the following 5 genera: Cryptococcus (80.64%), Rhodotorula (6.45%), Exophiala (6.45%), Trichosporon (3.22%), and Cystobasidium (3.22%). Cryptococcus spp. was the predominant group. Pectinases (51.6%), proteases (51.6%), and xylanases (41.9%) were the enzymes most common, while poor production of siderophores (16.1%) and indole acetic acid (9.67%) was detected. Isolates of Rhodotorula mucilaginosa and Cystobasidium sloffiae could promote plant growth and seed germination in a bioassay using Brassica juncea. Resistance of isolates by arsenic and heavy metals was as follows: As(3+) ≥ 100 mmol/L, As(5+) ≥ 30 mmol/L, Zn(2+) ≥ 2 mmol/L, Pb(2+) ≥ 1.2 mmol/L, and Cu(2+) ≥ 0.5 mmol/L. Strains of Cryptococcus albidus were able to reduce arsenate (As(5+)) into arsenite (As(3+)), but no isolate was capable of oxidizing As(3+). This is the first study on the abundance and identification of rhizosphere yeasts in a heavy-metal- and arsenic-contaminated soil, and of the reduction of arsenate by the species C. albidus.

The predatory mite Neoseiulus paspalivorus (Phytoseiidae) in Brazil: taxonomic status, reproductive compatibility and morphological and molecular variability.

The predatory mite Neoseiulus paspalivorus (De Leon) is often found in association with the coconut mite, Aceria guerreronis Keifer. The identification of natural enemies is essential for the definition of biological control strategies. Therefore, the present study aimed to confirm whether the mite populations from different Northeastern Brazilian states identified as N. paspalivorus belong to the same species. This determination was accomplished through the study of morphometric variability in 33 anatomical characters and of molecular variability in two DNA fragments: Internal Transcribed Spacer (ITS) rDNA and cytochrome c oxidase subunit I (COI) mtDNA. This study also determined whether there is reproductive isolation between the two most morphologically distinct populations (Rio Grande do Norte and Paraíba). Intraspecific morphometric variability was observed among the five populations of N. paspalivorus. Despite this variability, the crosses and backcrosses of the most morphologically distinct populations did not show reproductive incompatibility. The molecular analysis indicated the absence of genetic differences among the N. paspalivorus populations for the ITS fragment. Three haplotypes were identified for the COI fragment, and the genetic distance ranged from 0 to 0.2 %. Despite the morphometric differences, the results of the molecular and biological analysis corroborate the previous identification of N. paspalivorus for all of the studied populations. The present study contributes to the systematics of Phytoseiidae predatory mites and to the biological control of A. guerreronis by the accurate identification and characterization of one of its main natural enemies along extensive areas in Brazil.