Internal Transcribed Spacer Region Gene Research Articles

Façade eluates affect active and total soil microbiome

The application of biocides in building materials has become a prevalent practice to mitigate the growth of microorganisms such as algae, fungi, and bacteria on the façades. These can leach out from the material and reach the nearby soil environment. This study aimed to characterize the effect of façade eluates generated within different leaching experiments on total and metabolic active soil microbial community composition and functions. Façade eluates were produced by immersion testing DIN EN 16105 and a natural weathering experiment. Afterward, soil microcosms were treated with the respective façade eluate and incubated for 29 days. Subsequently, the active and total soil microbial community compositions were investigated.Fungal internal transcribed spacer region gene and bacterial 16S rRNA gene were sequenced for active (bromodeoxyuridine labeled DNA) microbial community and total community. Façade eluates reduced total bacterial and fungal gene copy numbers. Overall, active bacterial and fungal richness was reduced and altered in community composition in comparison to the total richness and composition, respectively. Façade eluates retrieved of façade samples without biocides did alter the soil microbial communities to the same extent as façade eluates with biocides. Additionally, members of the active microbiome that benefit from the presence of façade eluates and omitted ones could be identified. Our result demonstrated that façade eluates affect active and total soil microbial community composition and function regardless of the leaching procedure and biocides addition.

Postharvest blown-arc plasma treatment did not reduce Fusarium graminearum or its mycotoxins in field-infested wheat grain

The increasing prevalence of Fusarium graminearum, a major causative pathogen of Fusarium head blight in cereal crops, leads to contamination of grain with mycotoxins, which necessitates the development of effective management strategies. This study investigated whether postharvest Blown-Arc plasma treatment could reduce F. graminearum and associated mycotoxins in field-infested grain. Postharvest Blown-Arc plasma treatment for 60 or 180 s was trialled to reduce Fusarium graminearum and deoxynivalenol (DON) mycotoxins in field-infested wheat grain. Changes in fungal components of the grain microbiome following the treatment were assessed using metabarcoding analysis of the nuclear ribosomal internal transcribed spacer gene region, and liquid chromatography mass spectrophotometry was used to measure changes in concentrations of the mycotoxins deoxynivalenol (DON), DON-3-glucoside (DON3-G) and 3-acetyl-DON (3-ADON). Fusarium spp. were the most common taxa in the grain over two seasons, with the relative abundance being higher in 2020 (72%) compared to 2021 (45%). Postharvest plasma treatment of field-infested grain for 60 or 180 s did not significantly lower Fusarium abundance. Alpha and beta diversity of the natural mycobiota in the grain were also unaffected. The concentrations of mycotoxins were not significantly different after the treatments, except that in the 2021 trial, the 180 s treatment caused a significant increase in DON concentration. We concluded that the postharvest plasma treatment used in this study did not reduce Fusarium contamination or the concentration of mycotoxins, DON, DON3-G and 3-ADON in field-infested grain. The results highlight the importance of using field-infested grain in experiments, as opposed to grain inoculated under laboratory conditions, which provides only superficial infection. This approach is crucial for accurately understanding the dynamics of treatment efficacy.

Recommendations on approving the name " Entomosporium", with a new species, E.dichotomanthes from China (Leotiomycetes, Drepanopezizaceae).

The phytopathogenic genus, Entomosporium can cause serious leaf diseases worldwide. Entomosporium has long been regarded as a synonym of Diplocarpon. However, different morphologies between Entomosporium and Diplocarpon make this doubtful. Based on morpho-phylogenetic analyses, the placement of the genus was re-evaluated in this study. The combined the internal transcribed spacer gene region (ITS) and the 28S large subunit ribosomal RNA gene region (LSU) phylogenetic analysis shows that Entomosporium is an independent clade within Drepanopezizaceae and formed a sister clade to the generic type Diplocarpon. Moreover, Hymenula and Pseudopeziza do not cluster in Drepanopezizaceae. We propose to resurrect the name Entomosporium, and exclude Hymenulacerealis and Pseudopezizamedicaginis from Drepanopezizaceae and propose to treat them under Ploettnerulaceae. A new species, E.dichotomanthes is also introduced from China based on morpho-molecular analyses which is associated with Dichotomanthestristaniicarpa.

New species of Chalciporus and Tylopilus from India, with keys to the known species

ABSTRACT Two new species, Chalciporus rubrostipitatus and Tylopilus purpureus, are proposed from India based on morphological and molecular data. Chalciporus rubrostipitatus is characterized by basidiomata having purplish red to reddish pileus with subtomentose to rugose surface, whitish pileal context, round to angular pores, and reddish orange to red stipe, which is pruinose toward the apex. Tylopilus purpureus produces basidiomata having a purple to vinaceous purple pileus, whitish pore surface that changes to reddish brown on bruising, and a minutely pubescent purplish stipe. Morphological descriptions and comparisons, taxonomic keys, and results of phylogenetic analyses using sequences of the ITS (internal transcribed spacer), 28S (28S rRNA), and RPB2 (second largest subunit of RNA polymerase II) gene regions are presented.

Hidden diversity of Pestalotiopsis and Neopestalotiopsis (Amphisphaeriales, Sporocadaceae) species allied with the stromata of entomopathogenic fungi in Taiwan

Pestalotiopsissensu lato, commonly referred to as pestalotiopsis-like fungi, exhibit a broad distribution and are frequently found as endophytes, saprobes and pathogens across various plant hosts. The taxa within pestalotiopsis-like fungi are classified into three genera viz. Pestalotiopsis, Pseudopestalotiopsis and Neopestalotiopsis, based on the conidial colour of their median cells and multi-locus molecular phylogenies. In the course of a biodiversity investigation focusing on pestalotiopsis-like fungi, a total of 12 fungal strains were identified. These strains were found to be associated with stromata of Beauveria, Ophiocordyceps and Tolypocladium in various regions of Taiwan from 2018 to 2021. These strains were evaluated morphologically and multi-locus phylogenetic analyses of the ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α) and tub2 (beta-tubulin) gene regions were conducted for genotyping. The results revealed seven well-classified taxa and one tentative clade in Pestalotiopsis and Neopestalotiopsis. One novel species, Pestalotiopsis manyueyuanani and four new records, N. camelliae-oleiferae, N. haikouensis, P. chamaeropis and P. hispanica, were reported for the first time in Taiwan. In addition, P. formosana and an unclassified strain of Neopestalotiopsis were identified, based on similarities of phylogeny and morphology. However, the data obtained in the present study suggest that the currently recommended loci for species delimitation of pestalotiopsis-like fungi do not deliver reliable or adequate resolution of tree topologies. The in-vitro mycelial growth rates of selected strains from these taxa had an optimum temperature of 25 °C, but growth ceased at 5 °C and 35 °C, while all the strains grew faster under alkaline than acidic or neutral pH conditions. This study provides the first assessment of pestalotiopsis-like fungi, associated with entomopathogenic taxa.

Research advances of deciphering Shalgam microbiota profile and dynamics

The relationship between the microbiota and their functions in the quality and characteristic flavors of the fermented foods that provide them autochthonous attributes has been remained elusive, so far. With the demand in elucidating the microbiota of the autochthonous fermented foods, the characterization of the shalgam microbiota via culture-dependent and culture-independent methods has been carried out. To shed light on shalgam microbiota harboring Lactic acid bacteria (LAB) and yeasts, microorganisms isolated from shalgam have been identified by culture-dependent methods including 16S rRNA and ITS (Internal Transcribed Spacer) gene regions sequencing, RAPD-PCR, Rep-PCR, and API CHL50. Culture-independent characterization methods such as 16S rRNA and ITS meta-barcoding sequencing were performed to pinpoint the microbial diversity within shalgam. More recently, bioinformatics and in vitro analysis of bacteria and yeast isolated from shalgam to find prospective probiotics and elucidate shalgam microbiota dynamics due to the types of salts used in shalgam production have been reported. In this review, we intend to collate the data on microorganisms identified via culture-dependent and culture-independent methods. Taken together, we presented a broad perspective on the shalgam microbiota and how future endeavors in shalgam microbiota research can move forward.

Four novel Pleurocordyceps (Polycephalomycetaceae) species from China.

Entomopathogenic fungi comprise an ecologically important group of specialized pathogens infecting other fungi, invertebrates, and plants. These fungi are species-rich with high diversity and broad distribution worldwide. The majority of entomopathogenic fungi belong to clavicipitoids, which consist of the hypocrealean families, Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae. The latter is a newly established entomopathogenic family that recently separated from the family Ophiocordycipitaceae to accommodate the genera, Perennicordyceps, Pleurocordyceps, and Polycephalomyces. In recent years, Polycephalomycetaceae has been enriched with parasitic and hyperparasitic fungi. With 16 species spread across China, Ecuador, Japan, and Thailand, Pleurocordyceps is the most speciose genus in the family. In this study, we expand the number of taxa in the genus by introducing four new Pleurocordyceps species from China, namely, P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. We provide detailed descriptions and illustrations and infer genus-level phylogenies based on a combined 6-loci gene sequence dataset comprising the internal transcribed spacer gene region (ITS), small subunit ribosomal RNA gene region (SSU), large subunit rRNA gene region (LSU), translation elongation factor 1-alpha gene region (TEF-1α), RNA polymerase II largest subunit gene region (RPB1), and RNA polymerase II second largest subunit (RPB2). This study contributes to knowledge with regard to the diversity of Pleurocordyceps specifically and entomopathogenic Hypocreales more broadly.

Identification of four autochthonous yeasts from the intestines of goldfish, Carassius auratus with potential probiotic properties and their effects on the most common fish bacterial pathogens

In aquaculture, probiotic yeasts have gained particular interest because of their numerous health benefits for farmed fish. Many autochthonous yeasts have been isolated and identified from fish species with potential probiotic characteristics. In the present study, four autochthonous yeast strains were identified and characterized from the intestinal tracts of 16 healthy goldfish, Carassius auratus. Their in vitro probiotic properties were examined in terms of cell surface hydrophobicity, co-aggregation, and tolerability to different pH values and bile salt concentrations. These strains were identified by culture characters and sequence analysis of ITS (Internal Transcribed Spacer) gene regions. Four strains, namely Cutaneotrichosporon jirovecii isolate jpn01, Debaryomyces nepalensis isolate jpn02, Blastobotrys proliferans isolate jpn05, and Diutina catenulata isolate jpn06, were identified and added to the NCBI GenBank with accession numbers defined as MT584874.1, MT584873.1, MT649918.1, and MT501155.1, respectively. Results demonstrated the capability of these strains to co-aggregate with several fish-associated bacterial pathogens such as Lactococcus garvieae, Vagococcus salmoninarum, Vibrio anguillarum, Yersinia ruckeri, and Aeromonas hydrophila. Only the jpn05 strain did not co-aggregate with A. hydrophila. All identified yeast isolates could grow and tolerate low pH conditions (pH 2.0) and bile salt concentrations (up to 1.5%). Of interest, the hydrophobicity (%) of the yeast isolates was 80%, 94.0%, 80.6%, and 66.4% for jpn01, jpn02, jpn05, and jpn06 isolates, respectively. In this context, our data provide important in vitro evidence for the potential probiotic features of the yeast isolates. These strains could be considered candidate probiotic yeasts; however, their application in aquaculture nutrition necessitates further in vivo assays.

Calcarisporium yuanyangense sp. nov. (Calcarisporiaceae, Hypocreales), a fungal pathogen isolated from Ophiocordyceps nutans

In this study, a new species, Calcarisporium yuanyangense, was isolated from Ophiocordyceps nutans collected from Yuanyang County, Yunnan Province, China, and described by morphology and molecular phylogeny. Based on the combined dataset of five genes including the internal transcribed spacer gene region (ITS), the nuclear ribosomal small and large subunit gene regions (nrSSU and nrLSU), the gene encoding translation elongation factor 1-α (tef-1α), and the RNA polymerase II second largest subunit gene region (rpb2), phylogenetic analyses were performed by the maximum likelihood (ML) and the Bayesian inference (BI) methods to determine the phylogenetic position of C. yuanyangense in Calcarisporium. This species was clustered with C. cordycipiticola, C. xylariicola, and C. arbuscula to form a separate branch. Calcarisporium yuanyangense had the basic morphological characteristics of Calcarisporium, but still differed morphologically from the other species in the genus. This species was similar to C. arbuscula in conidia shape, but the conidia of C. yuanyangense were larger and had a higher length/width ratio than those of C. arbuscula. This study deepens the understanding of the host diversity and the distribution of this taxonomic unit, as well as provides important taxonomic data for further exploration of the genus Calcarisporium.

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 μm in length, 7.1-17.2 μm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

Identification and pathogenicity analysis of leaf brown spot of Juglans regia in China

English walnut (Juglans regia), has high economic and ecological value. As an important tree species for eliminating poverty, it is planted in many Provinces of China. In 2021, new pathogenic fungi were observed in English walnut in Guangyuan City, Sichuan Province, China. The initial symptom of leaf infection is that the leaves are covered with small black spots, which gradually expand into larger brown spots. Most of the spots appeared at the edges of the leaves, and yellow whorls were observed at the junction between the spots and the healthy leaves. The pathogenic fungi were isoalted form collecting disease samples and purified by single-spore culturing. In vitro and field experiments showed that the pathogen could cause brown spots on walnut leaves. The inoculation experiment showed that the symptoms in the field experiment were the same as those observed on the spot; however, slight differences were observed in the in vitro experiment. Ten isolates were obtained from walnut leaves with brown spot symptoms, and these were further characterized based on morphology and DNA sequencing. ITS (internal transcribed spacer), LSU (large sub-unit rDNA), rpb2 (second largest subunit of RNA polymerase) and tub2 (beta-tubulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. The isolate was identified as Nothophoma quercina by morphological and polygene analyses. As far as we are aware, the brown spots on walnut leaves caused by N. quercina is the first report of its kind.

First Report of Lasiodiplodia brasiliensis Causing Root Rot on Watermelon in Brazil.

Watermelon (Citrullus lanatus (Thumb.) Matsum. & Nakai) is an important crop in Brazil both for export and domestic consumption. In October 2019, watermelon plants showing decline and root rot symptoms were surveyed in 16 commercial fields in Baraúna's municipality (Rio Grande do Norte, Brazil). The disease prevalence was 12.5%, and the average incidence was 5%. Affected root segments were cut into small pieces and surface-disinfected with 70% ethyl alcohol and 1.5 % NaOCl for 1 and 2 min, respectively. Tissues were plated onto potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fungal colonies developed from the infected tissues were dark or greyish, and pure cultures were obtained by hyphal tip isolation technique. Six fungal isolates with the same morphology were obtained. Two of them were selected for morphological and molecular characterization (CFC-1123 and CFC-1124). Isolates grew rapidly in PDA, covering the entire surface of the Petri dishes within 3 days. The aerial mycelium was initially white, turning dark greenish-gray after 4 to 5 days of incubation at 25°C in the dark. Isolates produced pycnidia and conidia in water-agar medium with sterilized pine needles after 30 days of incubation at 25°C under near-UV light. The conidia were initially hyaline and brown with central transverse septum and longitudinal streaks when mature. Conidia were ellipsoid to oval (22.83 ± 3.1 µm long and 11.58 ± 1.5 µm wide). Based on morphological features, the isolates were initially identified as Lasiodiplodia sp. (Phillips et al. 2013). To confirm the identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region as well as fragments of the translation elongation factor 1-α (TEF) and β-tubulin 2 (TUB) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank under accession numbers OL841380, OL865376 and OL890691 for CFC-1123, and OL841381, OL865377 and OL890692 for CFC-1124. Maximum likelihood phylogenetic analysis of the concatenated sequences of ITS, TEF and TUB gene regions of some reference sequences and ex-types of Lasiodiplodia spp. was performed. Phylogenetic analysis revealed that the isolates grouped in the L. brasiliensis clade (Netto et al. 2014) with 80/79% of bootstrap. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi from Cariri (CFC) at the Universidade Federal do Cariri (Crato, Brazil). Pathogenicity of the two isolates was determined using colonized wheat grains as inoculum source. One watermelon seed (cv. Crimson Sweet) was placed in a sterile plastic pot (500-mL) filled with 6 cm layer of a substrate composed of soil and Tropstrato® (5:1 w/w). Three wheat grains (50 mg) colonized with each isolate were placed 10 mm above the seed and covered with the substrate. Control pots were inoculated only with sterile wheat grains. There were five replicates for each isolate. The pots with seedlings were maintained in a greenhouse at 28 ± 2°C under natural light conditions. The inoculated seedlings showed poor growth, withering and drying leaves 45 days after inoculation (DAI), and subsequently root rot symptoms and death at 60 DAI. Control seedlings remained asymptomatic. The pathogen was re-isolated from all inoculated seedlings and identified by conidia morphology to fulfill Koch's postulates. Lasiodiplodia brasiliensis has been reported to cause postharvest rot and gummosis of watermelon (Farr and Rossman 2022). However, to our knowledge, this is the first report of watermelon decline caused by this fungus in Brazil and worldwide. This finding must be considered for developing efficient control strategies for the disease.

Phylogenetic analysis of black piper (Piper spp.) population collected in different locations of Viet Nam based on the ITSU1-4 gene region

The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses and it has been recommended as a core plant DNA barcode. To compare and find out the genetic diversity difference, some individuals Peper were collected in different localities in Vietnam when using the ITS of nuclear ribosomal DNA. The ITS gene region from the nuclear genomes was tested for suitability as DNA barcoding regions of thirty-nine Peper individuals. Universal primers were used and sequenced products were analyzed using the Maximum Likelihood method and Tamura-Nei model in the MEGA X program. We did not observe high variability in intraspecific distance within the ITSu1-4 gene region between individuals ranging from 0.000 to 0.155. The size of the gene region has fluctuated from 667 to 685 bp between different individuals with the percentage (G + C) contained in the ITSu1-4 gene region which ranged from 54.776% to 60.805%, mean = 60.174%. The values of Fu’s Fs, D, Fu and Li’s D* and F* were negative as well (Fs = -0.209, D = -1.824; p < 0.05, D* = -1.205; not significant, p > 0.10 and F* = -1.699; not significant, 0.10 > p > 0.05) indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred. The value of Strobeck’s S is high (S = 0.684). The results of evolutionary relationships of taxa obtained 3 groups with the highest value of Fst are shown in the pairs of groups II and III (Fst = 0.151) and the lowest is in groups II and I (Fst = 0.015). All of the new sequences have been deposited in GeneBank under the following accession numbers MZ636718 to MZ636756. This database is an important resource for researchers working on Species of Peper in Vietnam and also provides a tool to create ITSu1-4 databases for any given taxonomy.

Characterization and Pathogenicity of Pythium-Like Species Associated with Root and Collar Rot of Kiwifruit in Turkey.

During the period of June to October in 2018, a widespread decline was observed on kiwifruit vines in the vineyards located in the Altınordu, Fatsa, and Perşembe districts of Ordu province in Turkey. The symptoms were associated with reddish-brown rots expanding from the root to the collar with sparse off-color foliage. Based on the percentage of the total infected samples across 18 vineyards, the most common oomycete species were Globisporangium intermedium (37.1%), Phytopythium vexans (34.3%), Globisporangium sylvaticum (14.3%), Globisporangium heterothallicum (11.4%), and Pythium dissotocum (2.9%). The morphological identification of isolates was confirmed based on partial DNA sequences containing the nuclear rDNA internal transcribed spacer region gene and the mitochondrial cytochrome c oxidase subunit II gene. The optimum growth temperature and the optimum pH values of the five species ranged from 22.98 to 28.25°C and 5.67 to 8.51, respectively. Pathogenicity tests on the seedlings of kiwifruit cv. Hayward revealed significant differences in virulence among isolates. Phytopythium vexans and Globisporangium sylvaticum isolates caused severe root and collar rot resulting in seedling death, while Globisporangium heterothallicum and Globisporangium intermedium isolates had relatively lower virulence. All Globisporangium spp. and Phytopythium vexans isolates significantly decreased plant growth parameters (plant height, shoot and root dry weights, and root length); however, Pythium dissotocum caused very mild symptoms and did not affect these parameters of growth. To our knowledge, this is the first study reporting Globisporangium sylvaticum, Globisporangium heterothallicum, and Globisporangium intermedium causing root and collar rot on kiwifruit not only in Turkey but also in the world.

Identification and pathogenicity ofPratylenchus scribnerion tomato in Sichuan Province of People's Republic of China

AbstractRoot-lesion nematodes (Pratylenchusspp.) are a group of economically important pathogens that have caused serious economic losses in many crops. In 2019, root-lesion nematodes were recovered from tomato (Solanum lycopersicum) root samples collected from Sichuan Province, People's Republic of China (PRC). Extracted nematodes were disinfected, and one individual female was cultured on a carrot disc for propagation at 25 °C by parthenogenesis and designated the SC isolate. Afterwards, the isolate was identified on the basis of morphometric and molecular markers. Both morphometric characters and molecular analysis of the internal transcribed spacer region gene (ITS) of ribosomal DNA, the D2-D3 expansion region of the 28S rDNA gene and the mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed that the species of root-lesion nematode wasPratylenchus scribneri. The Bayesian tree inferred from the ITS rDNA, 28S rDNA and mtDNA-COI gene sequences also showed that this isolate formed a highly supported clade with otherP. scribneriisolates. The pathogenicity of the root-lesion nematode SC isolate on tomato was assessed, showing that tomato was a suitable host forP. scribneri. To the best of our knowledge, this is the first report ofP. scribnerion tomato in Sichuan Province, PRC. These are also the first molecular data obtained fromP. scribnerion tomato in the PRC, and the pathogenicity ofP. scribnerito tomato was studied for the first time. This study provides scientific data for the detection, identification and control of tomato root-lesion nematode disease.

Genetic Diversity and Population Structure of Didymellarabiei Affecting Chickpea in Ethiopia.

Ascochyta blight, also known as chickpea blight, which is caused by the fungal pathogen, Didymella rabiei, is an important disease affecting chickpea (Cicer arietinum L.) in many countries. We studied the genetic diversity and population structure of 96 D. rabiei isolates collected from three geographic populations in Ethiopia using simple sequence repeat (SSR) markers. We confirmed the genetic identity of 89 of the D. rabiei isolates by sequencing their rRNA internal transcribed spacer region genes. The chickpea blight pathogen isolates were genetically diverse, with a total of 51 alleles identified across 6 polymorphic SSR loci, which varied from 3 to 18 (average 8.5) alleles per SSR marker. The observed heterozygosity and expected heterozygosity ranged from 0.01 to 0.92 and 0.19 to 0.86, respectively. The mean polymorphic information content value of the D. rabiei populations was 0.58, with a mean gene diversity of 0.61 among loci. Gene flow (Nm = number of migrants) for the three populations of D. rabiei isolates ranged from 1.51 to 24.10 (average 6.2) migrants/cluster. However, the genetic variation between the D. rabiei populations was small (8%), with most of the variation occurring within populations (92%). Principal component analysis to visualize genetic variation showed that the D. rabiei isolates obtained from most of the chickpea samples formed roughly three groups on a two-dimensional coordinate plane. Similarly, the clustering of individuals into populations based on multi-locus genotypes (using Clumpak) grouped isolates into three clusters but with individual isolate admixtures. Hence, no clear geographic origin-based structuring of populations could be identified. To our knowledge, this is the first report of D. rabiei diversity in Ethiopia. Virulence studies should be conducted to develop chickpea varieties that are resistant to more aggressive pathogen populations.

Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans.

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

Molecular Phylogenetic Diversity and Biological Characterization of Diaporthe Species Associated with Leaf Spots of Camellia sinensis in Taiwan.

Camellia sinensis is one of the major crops grown in Taiwan and has been widely cultivated around the island. Tea leaves are prone to various fungal infections, and leaf spot is considered one of the major diseases in Taiwan tea fields. As part of a survey on fungal species causing leaf spots on tea leaves in Taiwan, 19 fungal strains morphologically similar to the genus Diaporthe were collected. ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α), tub2 (beta-tubulin), and cal (calmodulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. In total, six Diaporthe species, including one new species, Diaporthe hsinchuensis, were identified as linked with leaf spot of C. sinensis in Taiwan based on both phenotypic characters and phylogeny. These species were further characterized in terms of their pathogenicity, temperature, and pH requirements under laboratory conditions. Diaporthe tulliensis, D. passiflorae, and D. perseae were isolated from C. sinensis for the first time. Furthermore, pathogenicity tests revealed that, with wound inoculation, only D. hongkongensis was pathogenic on tea leaves. This investigation delivers the first assessment of Diaporthe taxa related to leaf spots on tea in Taiwan.

MOLECULAR IDENTIFICATION OF Diplodia seriata De Not. CAUSING DIEBACK EFFECT ON GRAPEVINES AND EVALUATION OF in vitro EFFICACY OF FIVE DIFFERENT SYNTHETIC FUNGICIDES AGAINST THIS DISEASE

The aim of this study was to realize the molecular identification of Diplodia seriata De Not., a member of the Botryosphaericea family, isolated from 2-10 years old vines in vineyards showing symptoms of dieback disease. The susceptibility of the pathogen against the fungicides with the fosetyl-Al+triadimenol, azoxystorbin+difecanozole, fludioxanil+cyprodinil, metrafenone, fluopyram+tebuconazole combinations were evaluated. The isolates obtained from the root and crown parts of the vine samples were identified as D. seriata according to the morphological and molecular methods. In molecular identification, the ITS (Internal Transcribed Spacer) and TUB2 (β-tubulin) gene regions of the isolates were amplified by Real-Time PCR and the nucleotide sequences were obtained in these gene regions. After using the MEGA 7 software, ITS and TUB2 sequences were aligned and a combined phylogenetic tree was made. It has been molecularly confirmed that the D. seriata isolate has a 100% similarity index with Diplodia species according to the phylogenetic analyses. The mean effective concentration (EC50) values of fungicides used with different concentrations (0, 1, 3, 10, 30, 50, 100 μL mL-1) were determined by Probit analysis. Cyprodinil + fludioxonil showed the highest efficacy (100%) at a concentration of 1 μL mL-1. According to EC50 values, cyprodinil + fludioxonil (0.001 μL mL-1) was recorded as the most effective fungicide followed by fluopyram + tebuconazole (0.520 μL mL-1) and, azoxystrobin + difenoconazole (2.958 μL mL-1), respectively.

Combining natural language processing and metabarcoding to reveal pathogen-environment associations.

Cryptococcus neoformans is responsible for life-threatening infections that primarily affect immunocompromised individuals and has an estimated worldwide burden of 220,000 new cases each year-with 180,000 resulting deaths-mostly in sub-Saharan Africa. Surprisingly, little is known about the ecological niches occupied by C. neoformans in nature. To expand our understanding of the distribution and ecological associations of this pathogen we implement a Natural Language Processing approach to better describe the niche of C. neoformans. We use a Latent Dirichlet Allocation model to de novo topic model sets of metagenetic research articles written about varied subjects which either explicitly mention, inadvertently find, or fail to find C. neoformans. These articles are all linked to NCBI Sequence Read Archive datasets of 18S ribosomal RNA and/or Internal Transcribed Spacer gene-regions. The number of topics was determined based on the model coherence score, and articles were assigned to the created topics via a Machine Learning approach with a Random Forest algorithm. Our analysis provides support for a previously suggested linkage between C. neoformans and soils associated with decomposing wood. Our approach, using a search of single-locus metagenetic data, gathering papers connected to the datasets, de novo determination of topics, the number of topics, and assignment of articles to the topics, illustrates how such an analysis pipeline can harness large-scale datasets that are published/available but not necessarily fully analyzed, or whose metadata is not harmonized with other studies. Our approach can be applied to a variety of systems to assert potential evidence of environmental associations.