ITS Gene Sequences Research Articles

Isolation and Identification of Candida tropicalis as a Cause of Cutaneous Candidiasis in Kalar District, Iraq.

Fungal infections are currently causing health issues all over the world, among which are Candida species that cause cutaneous infection. Numerous dermatological studies concentrated on a single species. However, the virulence factors and the spread of specific candidiasis in specific areas have remained poorly understood. Therefore, the current study was designed to shed light on Candida tropicalis, which has been identified as the most prevalent yeast among Candida non-albicans species. Forty specimens were collected from patients with cutaneous fungal infection (25 females and 15 males) and underwent examination. According to conventional identification based on macroscopic and microscopic examinations, eight isolates were identified as C. tropicalis from Candida non-albicans. Molecular diagnosis for internal transcribed spacers (ITS1 and ITS4) using conventional polymerase chain reaction (PCR) yielded an amplicon of 520 bp for all isolates. Further investigation of PCR-restriction fragment length using Mitochondrial sorting protein; Msp1 enzyme revealed two bands of 340 and 180 bp. The ITS gene sequence in one isolated species was found to be 98% identical to C. tropicalis strain MYA-3404 chromosome R ATCC CP047875.1. Another isolate shared 98.02% identity with C. tropicalis strain MA6 18S ribosomal RNA gene DQ666188.1, indicating C. tropical species identity, implying that non-Candida species should be considered when diagnosing candidiasis. This study demonstrated the significance of Candida non-albicans, particularly C. tropicalis, in terms of pathogenic potential, the ability to cause potentially fatal systemic infections and candidiasis, and acquired flucozonal resistance with a high mortality rate.

Reliability and Utility of Standard Gene Sequence Barcodes for the Identification and Differentiation of Cyst Nematodes of the Genus Heterodera

Difficulties inherent in the morphological identification of cyst nematodes of the genus Heterodera Schmidt, 1871, an important lineage of plant parasites, has led to broad adoption of molecular methods for diagnosing and differentiating species. The pool of publicly available sequence data has grown significantly over the past few decades, and over half of all known species of Heterodera have been characterized using one or more molecular markers commonly employed in DNA barcoding (18S, internal transcribed spacer [ITS], 28S, coxI). But how reliable are these data and how useful are these four markers for differentiating species? We downloaded all 18S, ITS, 28S, and coxI gene sequences available on the National Center for Biotechnology Information (NCBI) database, GenBank, for all species of Heterodera for which data were available. Using a combination of sequence comparison and tree-based phylogenetic methods, we evaluated this dataset for erroneous or otherwise problematic sequences and examined the utility of each molecular marker for the delineation of species. Although we find the rate of obviously erroneous sequences to be low, all four molecular markers failed to differentiate between at least one species pair. Our results suggest that while a combination of multiple markers is best for species identification, the coxI marker shows the most utility for species differentiation and should be favored over 18S, ITS, and 28S, where resources are limited. Presently, less than half the valid species of Heterodera have a sequence of coxI available, and only a third have more than one sequence of this marker.

Soil Microbial and Organic Carbon Legacies of Pre-Existing Plants Drive Pioneer Tree Growth during Subalpine Forest Succession

Fast-growing pioneer tree species play a crucial role in triggering late successional development in forests. Experimental evidence of the soil legacy effects of pre-existing plants on pioneer tree performance is lacking. We explored the legacy effects of soils conditioned by early successional herbs (Poa poophagorum Bor and Potentilla fragarioides L.) and mid-successional shrubs (Rhododendron fortunei Lindl. and Enkianthus quinqueflorus Lour.) on late-successional ectomycorrhizal (ECM) pioneer tree (Betula platyphylla Sukaczev) seedling growth. The soils were analyzed for soil nutrient status and fungal and bacterial compositions using ITS and 16S rRNA gene sequencing. B. platyphylla seedlings produced higher biomass in soils conditioned by shrubs. Soil organic carbon (SOC) and bacterial and fungal legacies most impacted pioneer tree seedling growth. Additionally, the partial least squares path model revealed that soil nutrients, especially SOC, indirectly affected seedling biomass by their direct effects on the bacterial and fungal communities. The changes in bacterial community composition had a stronger effect on seedling biomass than those of fungi because bacteria with shorter turnover times are generally considered to be more efficient than fungi in enhancing nutrient availability. Our study integrates soil microbial and nutrient legacies to explain the potential mechanisms of pioneer tree regeneration.

Morpho-Phylogenetic Evidence Reveals Novel Pleosporalean Taxa from Sichuan Province, China.

Pleosporales is the largest and most morphologically diverse order in Dothideomycetes, including a large proportion of saprobic fungi. During the investigation of microfungi from decaying wood in Sichuan Province, several novel fungal taxa of asexual and sexual morphs were collected, identified, and well-described. Phylogenetic analyses based on SSU, ITS, LSU, RPB2 and TEF1α gene sequences suggested that these new taxa were related to Pleosporales and distributed in five families, viz. Amorosiaceae, Bambusicolaceae, Lophiostomataceae, Occultibambusaceae and Tetraplosphaeriaceae. The morphological comparison and molecular phylogeny evidence justify the establishment of six new taxa, namely Bambusicola guttulata sp. nov., Flabellascoma sichuanense sp. nov., Neoangustimassarina sichuanensis gen. et sp. nov., Occultibambusa sichuanensis sp. nov. and Pseudotetraploa bambusicola sp. nov. Among them, Neoangustimassarina was introduced as the second sexual morph genus in Amorosiaceae; Bambusicola guttulata, O. sichuanensis and P. bambusicola were isolated from bamboos, which contributed to the diversity of bambusicolous fungi. The detailed, illustrated descriptions and notes for each new taxon are provided, as well as a brief note for each family. The potential richness of fungal diversity in Sichuan Province is also discussed.

Species diversity and phylogenetic relationships within the tribe Mentheae (Lamiaceae) in Uzbekistan using ITS sequence data

Regular monitoring and comprehensive study of natural populations rare endangered species, both by classical taxonomy and molecular genetic analysis methods are of considerable importance all over the world. This work has focused at the research of species diversity flora of Uzbekistan using combinations of DNA markers. Currently, there are no universal DNA site for plant species identification. Pairwise sequence alignments and multiple sequence alignments are made using ClustalW program. DNA sequence data were used to verify the taxonomic identity and phylogenetic aspects of the studied representatives the tribe Mentheae (Lamiaceae) collected in Uzbekistan territory. The phylogenetic analyzes of this tribe was reconstructed for 44 species, representing 19 genera using nuclear ribosomal DNA internal transcribed spacer region (nrITS), containing ITS1 and ITS4 adopted as an important basis, since it can provide a high resolution of relationships. 15 consensus ITS gene sequences were obtained for studied species in this work and 29 published molecular sequence data from GenBank were used. Dendrograms were constructed using hierarchical clustering with the software Mega X. The parsimony analyses were resulted in an accurate consensus tree. Three major clades was identified within the tribe Mentheae. So, this paper demonstrates the successfully usefulness of DNA technology as a tool for genetic inventory at the species diversity assessment of rare endangered plants.

New host record, Sclerophrys regularis (Bufonidae), for Rhabdias africanus (Rhabdiasidae, Kuzmin, 2001) based on molecular and morphological evidence

With diversity of hosts range, most identified nematode species still lack the crucial connection between morphological and molecular make-up, which is important for precisely classifying the specimens. The present study provides the complete description of Rhabdias africanus in the new host record, Sclerophrys regularis. Fifty toad specimens were collected, and a high prevalence of R. africanus infection (74%) was observed. Morphology and ultrastructure were observed using light and scanning electron microscopes. Morphological characteristics, including peculiarities of the head, the shape and position of the lips, and the number of labial papillae, were described. The length of the body, the esophageal length, the distance from an anterior end to the nerve ring, and the tail length were reduced in the studied samples relative to previously described specimens. Furthermore, some variable matrices that have not previously been described, e.g., ovarian part widening, the nerve ring and its location, and eggs with different stages of larvae, were included in the present study. Genus and species identification was confirmed by comparing partial 12S (619 bp) and ITS (878 bp) gene sequences to those of Rhabdias species deposited in GenBank. The studied species showed a 99.34% resemblance to R. africanus from South Africa. We assume our findings will aid in the molecular identification of adult and larval stages of this genus in amphibians. We strongly recommend further studies on the environmental factors that promote Rhabdias infection and transmission.

Diversity of an uncommon elastic hypersaline microbial mat along a small-scale transect.

We evaluated the microbial diversity and metabolome profile of an uncommon hypersaline elastic microbial mat from Cuatro Ciénegas Basin (CCB) in the Chihuahuan Desert of Coahuila, México. We collected ten samples on a small scale transect (1.5-m) and described its microbial diversity through NGS-based ITS and 16S rDNA gene sequencing. A very low number of taxa comprised a considerable proportion of the mat and were shared across all sampling points, whereas the rare biosphere was more phylogenetically diverse (Faith’s Phylogenetic Diversity (FPD) index) and phylogenetically disperse (using a null model distribution of Phylogenetic Species Clustering (nmdPSC)) than the abundant (high read count) taxa for both analyzed libraries. We also found a distinctive metabolome profile for each sample and were able to tentatively annotate several classes of compounds with relevant biological properties.

First Report of Leaf Spot Caused by Nigrospora hainanensis on Oxalis corymbosa in China.

Oxalis corymbosa DC. introduced into China as an ornamental plant in the mid-19th century is commonly known as an important medicinal and edible perennial herb (Zhou et al. 2021). The plant native to South America is also an invasive and widely distributed weed found in agricultural farms, gardens, and lawns, especially in sugarcane fields of Guangxi province, China. The coverage rate of O. corymbosa in sugarcane fields was normally more than 70%, sometimes up to 100%. In March of 2021, a leaf spot disease of O. corymbosa from sugarcane fields was encountered in Nanning city of Guangxi province, China. Early symptoms appeared as small yellowish round spots. The spots turned to be irregularly, usually exhibiting pale brown necrosis in the center with dark brown necrotic well-defined margins. Severely infected leaves turned to be blighted, then dead. To isolate the pathogen, diseased leave tissue fragments (4 mm × 4 mm) were soaked in 75% ethanol for 10 s followed by 2% sodium hypochlorite for 1 min, and rinsed by sterile water for three times. They were transferred to potato dextrose agar (PDA) medium cultured at 25 °C. Pure cultures were obtained by collected hypha tip from upcoming colonies. The colony features were similar to each other, floccose, white at first, becoming brown, dark brown or black on PDA after 7 days fully covered the 90 mm petri-dishes. Conidial determination were conducted on synthetic nutrient-poor agar medium (SNA) according to Wang et al. (2017). Conidia abundantly dispersed on SNA arising from conidiophores, which normally reduced to conidiogenous cells generated from hyphae. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, 6-8.5 (-12.5) × 5-7.5 (-9) μm in size (n=50). Conidia were solitary, smooth, black, sphaerical or ellipsoidal, (11-) 13-16.5 × (8-) 10-15.5 μm in size (n=100). Setae were not observed during the observation. The fungus was identified as Nigrospora sp. based on the morphology. One of the representative strains (FSC-3) was selected for genomic DNA extraction. The sequences of transcribed spacer region of rDNA (ITS), the partial translation elongation factor (TEF1), and the Beta-tubulin fragment (TUB) were respectively amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F and EF2 (Carbone & Kohn 1999, Crous et al. 2013) and Bt2a and Bt2b (Glass & Donaldson 1995), deposited in the NCBI GenBank with accession numbers of OK083685 (541 bp), OK184809 (481 bp) and OK086377 (421 bp). BLASTn analysis showed that those ITS, TEF1 and TUB gene sequences shared 99%-100% identity with the type strain (CGMCC3.18129) of Nigrospora hainanensis (GenBank accession nos. NR153480, KY019415, KY019464, respectively). In addition, a maximum likelihood analysis using concatenated gene sequences of ITS, TEF1 and TUB was performed in RAxML v.7.2.8 (Stamatakis 2006) implementing the model of GTRCAT with 1,000 bootstrap replicates. The phylogenetic results indicated that the strain FSC-3 was N. hainanensis, which also confirmed after a morphological comparison with N. hainanensis (Wang et al. 2017). Pathogenicity was tested on living Oxalis corymbosa leaves (3 plants for each test) arising from cultivated roots grown for three weeks. It was conducted by dropping 5 μL conidial suspensions (105 conidia / mL) on the living leaves (two sites per leave) incubated in separate containers at 25 °C with 90-100% relative humidity after inoculation. Controls were treated with sterile distilled water. Pale brown small spots came up after 24 h, and then extended to brown larger spots. Symptoms after inoculation were similar to field ones, while the control plants remained healthy. The pathogenicity test was repeated twice with the similar results. Re-isolation of the pathogen from the inoculated leaves was determined based on morphology and sequence analysis to fulfill Koch's postulates. Nigrospora hainanensis had been found from diseased root and leaf tissues of sugarcane in Liuzhou city, Guangxi province (Raza et al. 2019). The results indicated that O. corymbosa was another host in sugarcane fields in Guangxi, China. To our knowledge, this is the first report of Nigrospora hainanensis causing leaf spot on Oxalis corymbosa in China.

Genetic Relationships of Some Samples of Ginger (Zingiber officinale (Wild) Roscoe) as Medicinal Herbs in the Cuu Long River Delta, Vietnam.

<b>Background and Objective:</b> Ginger (<i>Zingiber officinale</i> (Wild). Roscoe) is a plant that has long been used as a medicinal herb for humans, so it has been grown and popularized in the Mekong Delta Provinces, but the research systematic genetics has not been given much attention. The current study evaluated the genetic characteristics of some ginger samples collected in eight provinces in the Mekong Delta based on morphological characteristics and ITS gene sequence regions. <b>Materials and Methods:</b> Samples of Ginger varieties were collected in eight provinces, fresh leaf samples were collected and stored frozen at -20°C. Ginger morphology observation and description based on improved plant research methods. Total DNA extraction of ginger leaf samples was extracted from fresh leaf samples according to the extraction procedure by the modified CTAB method. <b>Results:</b> The phenotypes of the samples like length, leaf width, flower length and fruit diameter were significantly different between growing regions due to different environmental and farming conditions. Genetic relationship analysis showed that there are two distinct groups. Group I has 5 ginger samples from G8-Vinh Long, G4-Ben Tre, G2-Ca Mau, G5-Dong Thap and G6-Hau Giang provinces and group II includes ginger varieties belonging to G7-Ho Chi Minh City, G1-An Giang Province and G3-City. Can Tho. <b>Conclusion:</b> Samples/cultivars collected from eight provinces in the Mekong Delta showed variation in agronomic characteristics, but only stem height and length changes were statistically significantly different. Genotyping most of the samples belonged to the species <i>Zingiber officinale</i> (Wild) Roscoe.

Enenterum kyphosi Yamaguti, 1970 and Enenterum petrae n. sp. (Digenea: Enenteridae) from kyphosid fishes (Centrarchiformes: Kyphosidae) collected in marine waters off eastern Australia.

Species of the digenean genus Enenterum Linton, 1910 (Lepocreadioidea: Enenteridae) are characterised primarily by their elaborate oral suckers, which are divided into varying numbers of anteriorly directed lobes, and their host-restriction to herbivorous marine fishes of the family Kyphosidae. We describe Enenterum petrae n. sp. from the brassy chub Kyphosus vaigiensis (Quoy Gaimard) collected off Lizard Island, Great Barrier Reef, Queensland, Australia. Enenterum petrae n. sp. is readily differentiated from congeners by its unique oral sucker morphology, in having a minute pharynx, and the combination of a genital cap and accessory sucker. We also provide the first record of Enenterum kyphosi Yamaguti, 1970 from Australia based on material obtained from the blue sea chub Kyphosus cinerascens (Forsskl) collected off Lizard Island and North Stradbroke Island, Queensland. Morphologically, our specimens of E. kyphosi agree closely with descriptions of this species from Hawaii and South Africa, and despite lack of molecular data from outside of Australian waters, we consider all three reports to represent a single, widespread species. The first ITS2 and COI mtDNA gene sequences for species of Enenterum are provided and molecular phylogenetic analyses of 28S rDNA gene sequences place these species in a strongly-supported clade with the type-species of the genus, Enenterum aureum Linton, 1910. The oral suckers of both E. kyphosi and E. petrae n. sp. can be interpreted as having varying numbers of lobes depending on the particular specimen and how the division between lobes is defined. Scanning electron microscopical images improves understanding of the morphology of the enenterid oral sucker, and permits speculation regarding the evolutionary history leading to its specialisation in this lineage.

Longitudinal Investigation of the Native Grass Hay from Storage to Market Reveals Mycotoxin-Associated Fungi.

This study aimed to characterize the fungal diversity and mycotoxin concentrations of native grass hay in various storage periods. In the present study, the native grass hay samples were collected when stored for 0 d (D0 group), 30 d (D30 group), and 150 d (D150 group), respectively. Here, mycotoxin analyses combined with ITS gene sequence were performed to reveal the changes in response to the storage period. There were notable differences in deoxynivalenol and aflatoxin B1 concentrations among the three groups. Compared to the D150 group, the diversity of the fungal community was higher in the D0 and D30 groups, which indicating the diversity was significantly influenced by the storage period. No significant (p > 0.05) difference was observed among the three groups on the dominant phyla. Interestingly, a significant (p < 0.05) difference was also observed in Chactomella and Aspergillus among the three groups, the abundance of the Chactomella was significantly (p < 0.05) decreased and the abundance of Aspergillus was statistically (p < 0.05) increased in the D150 group. Correlation analysis of the association of fungi with mycotoxin could provide a comprehensive understanding of the structure and function of the fungal community. These results indicated that the good practices of storage are essential for the prevention of mycotoxin. The information contained in the present study is vital for the further development of strategies for hay storage with high quality in the harsh Mongolian Plateau ecosystem.

Molecular phylogeny of Ruppia maritima L. from Chilika Lake, Odhisa using nuclear and plastid genes

Aim: The present study aims to confirm the molecular identification of Ruppia maritima L. from Chilika Lake, Odhisa using different molecular markers and find out the evolutionary lineage of Ruppia species. Methodology: For molecular identification, R. maritima was collected from Chilika Lake and the rbcL, matK, and ITS gene sequences were investigated and analyzed using BLAST to verify the gene fragment. Phylogenetic tree was constructed using MEGA X software following Neighbor joining (NJ) and Maximum parsimony (MP) methods. Results: The nucleotide (ITS) and plastid gene (rbcL and matK) sequences of Ruppia maritima were examined. The tree topology of R. maritima rbcL and matK gene sequences revealed a paraphyletic cluster at 99 and 100% bootstrap. The conserved region was high in matK (74.8%), followed by rbcL (45.2%) and ITS (19.1%) with seagrass and freshwater species. ITS had the most significant proportion of variable sites (79.7%), followed by rbcL (52.4%) and matK (25.2%). The ITS region showed less parsimonious-informative character (66.1) than rbcL and matK region. Interpretation: The study concludes that the R. maritima was identified through molecular markers, which was a closed lineage with seagrasses than freshwater species.

Diversity and Pathogenicity of Pythium Species Associated with Reduced Yields of Processing Tomatoes (Solanum lycopersicum) in Victoria, Australia.

Yield decline associated with poor crop establishment, stunting, wilting, and diminished root systems was reported in processing tomato crops in Victoria, Australia. During surveys between 2016 and 2018 Pythium species were isolated by soil baiting and by culturing from the diseased roots and collars of plants exhibiting these symptoms. Eleven species of Pythium were identified based on cultural characteristics and phylogenetic analysis with ITS, Cox-1, and Cox-2 gene sequences. None of the 11 Pythium species had been reported previously from processing or fresh tomatoes in Australia. Pythium dissotocum was the most abundant and widespread species isolated during surveys in each of two growing seasons. In pathogenicity tests, these Pythium species ranged from nonpathogenic to highly aggressive. P. aphanidermatum, P. ultimum, and P. irregulare were consistently the most aggressive species, causing serious damage or death at the pregermination, postgermination, and later stages of plant growth. Five processing tomato cultivars varied significantly in their susceptibility to Pythium disease. These results suggest that Pythium species could be contributing to yield loss in processing tomatoes in Victoria both in the crop establishment phase and through the season.

Crassiparies yunnanensis sp. nov. (Neohendersoniaceae, Pleosporales) from dead twigs of Coffea arabica in China

Arabica coffee (Coffea arabica) accounting for about 60% of the world’s coffee is popular all over the world as a refreshing beverage and food additive. During surveys of fungi occurring on coffee plants, we collected a species of Neohendersoniaceae on dead twigs of an Arabica coffee plant in Pu’er, Yunnan Province, China. Phylogenetic analyses of combined SSU, LSU, ITS, rpb2, and tef gene sequences confirmed that our fungus is a distinct new species within Crassiparies. Morphological characteristics also support this taxon to be a novel species in Crassiparies. Full description, photo plate, and a phylogenetic tree to show the placement of the new species in Crassiparies are provided.

Fungal Richness of Cytospora Species Associated with Willow Canker Disease in China.

Species of Cytospora are considered important plant pathogens of a wide range of plant hosts, especially Salicaceae plants. Salix (Salicaceae, Malpighiales) has been widely cultivated in China because of its strong ecological adaptability, fast growth, and easy reproduction. In this study, a total of eight species of Cytospora were discovered on Salix in China, including C. ailanthicola, C. alba, C. chrysosperma, C. gigaspora, C. nivea, C. paracinnamomea, C. rostrata, and C. sophoriopsis. Among them, C. alba and C. paracinnamomea were identified as novel species based on morphology and phylogenetic analyses of ITS, act, rpb2, tef1-α, and tub2 gene sequences and were confirmed as pathogens of willow canker disease by pathogenicity tests. The mycelial growth rates of strains from these two novel species (C. alba and C. paracinnamomea) had optimum temperatures of 21 to 22 °C and an optimum pH value of 5 to 6. The effectiveness of six carbon sources on the mycelial growth showed that fructose and maltose had the highest influence. Cytospora species richness was significantly positively correlated with dry and wet areas. This study represents a significant evaluation of Cytospora associated with willow canker disease in China and provides a theoretical basis for predicting the potential risk of willow canker disease.

Asexual morph and molecular phylogeny of Phyllactinia rhoina on Choerospondias axillaris

AbstractChoerospondias axillaris (Anacardiaceae) is a fast‐growing tree species with edible wild fruits, used as pioneer tree for reforestation in south China. Fruits, leaves and bark are utilized for diverse culinary and pharmaceutical purposes. In December 2019, C. axillaris infected by powdery mildew was collected in Lianhuashan Park (117.01°E, 25.10°N), Fujian, China. The disease affected approximately 50‒60%, which severely influenced the yield and quality of the infected trees. Based on morphological and molecular analyses, the causal agent was identified as Phyllactinia rhoina. As far as we are aware, this is the first detailed description of the asexual morph for this species and the first complete molecular phylogeny inferred from ITS and 28S rDNA gene sequences. The information may be helpful for disease monitoring purposes of C. axillaris for growers.

Structural, metabolic, and functional characteristics of soil microbial communities in response to benzo[a]pyrene stress

Understanding the characteristics of soil microbes responding to benzo[a]pyrene (BaP) helps to deepen the knowledge of the risks of BaP to soil ecosystem. In this study, the structural, metabolic, and functional responses of soil microbial communities to BaP (8.11 mg kg−1) were investigated. Analysis of microbial community structure based on 16 S rRNA and ITS gene sequencing indicated that BaP addition enriched microbes associated with aromatic compound degradation (Sphingomonas, Bacilli, Fusarium) and oligotrophs (Blastocatellaceae, Rokubacteriales), but inhibited Cyanobacteria involved in nitrogen-fixing process. Network analysis showed that the bacterial community enhanced intraspecific cooperation, while fungal community mainly altered the keystone taxa under BaP stress. Biolog EcoPlate assay demonstrated that microbial metabolism of carbon sources, especially nitrogen-containing sources, was stimulated by BaP addition. Functional analysis based on enzyme activity tests, functional gene quantification, and function annotation showed that nitrogen-cycling processes, especially nitrogen fixation, were significantly inhibited. These results suggest that BaP-tolerant microbes may establish cooperative relationships and compete for resources and ecological niches with sensitive microbes, especially those associated with nitrogen cycling, ultimately leading to enhanced carbon source utilization and restricted nitrogen cycling. This study clearly elucidates the adaptation strategies and functional shifts of soil microbial communities to BaP contamination.

Trichoderma hamatum Strain Th23 Promotes Tomato Growth and Induces Systemic Resistance against Tobacco Mosaic Virus.

Trichoderma hamatum strain Th23, isolated from tomato roots, was molecularly identified using phylogenetic analysis based on ITS, tef1, and rpb2 gene sequences and evaluated for its efficiency in suppressing tobacco mosaic virus (TMV) infection for the first time. Under greenhouse conditions, the application of Th23 promoted tomato growth with significant increases in shoot and root parameters as well as improved total chlorophyll content. Compared to the nontreated tomato plants, the soil pretreatment of tomato plants 48 h before TMV inoculation produced a significant reduction in the TMV accumulation level by 84.69% and enhanced different growth parameters. In contrast, TMV had a deleterious impact on fresh and dry matter accumulation and inhibited photosynthetic capacity. Furthermore, the protective activity of Th23 was associated with a significant increase in reactive oxygen species scavenging enzymes (PPO, CAT, and SOD) as well as decreased nonenzymatic oxidative stress markers (H2O2 and MDA) compared to the TMV treatment at 15 days post-viral inoculation (dpi). In addition, considerable increases in the transcriptional levels of polyphenolic genes (HQT and CHS) and pathogenesis-related proteins (PR-1 and PR-7) were shown to induce systemic resistance against TMV. Consequently, the ability of T. hamatum strain Th23 to promote plant growth, induce systemic resistance, and boost innate immunity against TMV infestation supported the incorporation of Th23 as a potential biocontrol agent for managing plant viral infections. To the best of our knowledge, this is the first report of the antiviral activity of T. hamatum against plant viral infection.

Morphological and molecular identification of trematode isloates from laying ducks in Nanchang City

To identify the species of trematodes isolated from laying ducks in Nanchang City using morphological and molecular approaches. Trematodes were isolated from the hepatobiliary duct, gallbladder and large intestine of market-sold laying ducks in Nanchang City. Following morphological characterization, total DNA was extracted from all trematode specimens, and internal transcribed spacer region (ITS) and cytochrome C oxidase subunit 1 (Cox1) genes were amplified using PCR assay and sequenced. Sequence alignment was performed using the Blast software, and homology and phylogenetic analyses were done in the trematode isolates based on ITS and Cox1 gene sequences. The morphological characteristics of two trematode isolates from the large intestine of laying ducks were similar to those of Echinostoma revolutum and E. miyagawai, and the morphological characteristics of eight trematode samples isolated from the hepatobiliary duct and gallbladder of laying ducks were similar to those of Amphimerus anatis. The ITS and Cox1 gene sequences of the two trematode isolates from the large intestine of laying ducks had 99.3% and 98.9%-99.4% homology with E. miyagawai, and the phylogenetic analysis showed that two trematode isolates had the closest genetic relationship with E. miyagawai based on ITS and Cox1 gene sequences. The ITS gene sequences of eight trematode isolates from the hepatobiliary duct and gallbladder of laying ducks shared 95.1%-95.5% with Opisthorchis sudarikovi and Clonorchis sinensis, while the Cox1 gene sequences of eight trematode isolates from the hepatobiliary duct and gallbladder of laying ducks shared 86.3%-86.4% and 85.5%-85.7% with O. viverrini and O. sudarikovi. ITS gene sequence-based phylogenetic analysis showed that the duck-derived trematode isolates had the closest genetic relationship with C. sinensis, and Cox1 gene sequence-based phylogenetic analysis showed that the duck-derived trematode isolates had the closest genetic relationship with Metorchis orientalis and O. viverrini. The trematode isolates from the large intestine of laying ducts in Nanchang City may be E. miyagawai, and the trematode isolates from the hepatobiliary duct and gallbladder may be an unidentified trematode species of the family Opisthorchiidae.

First Report of Leaf Spot Caused by Corynespora cassiicola on Viburnum odoratissimum var. awabuki (Sweet Viburnum) in China.

Sweet viburnum [Viburnum odoratissimum Ker-Gawl. var. awabuki (K. Koch) Zabel ex Rumpl.] belonging to the family Adoxaceae, is a medical and landscape plant, native to Korea (Jeju Island), Taiwan, and Japan (Edita 1988). In June and September 2019, leaf spots were observed on approximately 65% to 80% of sweet viburnum plants in a hedgerow located in Fenghe Xincheng District (28°41'52.9"N 115°52'14.3"E) in Nanchang, China. Initial symptoms of disease appeared as dark brown spots surrounded by red halos (Figure 1 A), which expanded irregularly. Finally, the center of the lesions desiccated and became light-brown, surrounded by a deep-red halos (Figure 1 B). Ten leaf samples with typical symptoms were collected and washed with tap water for about 15 min. The tissue between the healthy and necrotic area (ca. 4 mm × 4 mm) was cut with a sterile scalpel and surface sterilized with 70% alcohol for 45 s, 2% NaClO for 2 min, washed in sterile deionized water three times, dried on sterilized filter paper, then placed in Petri dishes and incubated at 25℃ in the dark. After 3 to 5 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA dishes. Eventually, 54 fungal isolates were obtained and, of these, 39 isolates were identical in their morphological characteristics. Morphological analysis was performed according with Ellis (1971). The isolate S18, chosen as representative, formed a gray to grayish brown colony with concentric circleson PDA, and a diameter of 8.5 to 9 cm after 7 days incubation at 25℃ (Figure 1 G). Conidia were hyaline, straight or slightly curved, needle shaped, truncate at the base, and acuminate at the tip, with 2 to 6 pseudosepta, 18.90 to 38.38 µm (avg. = 27.51 µm) × 1.64 to 4.50 µm (avg. = 2.60 µm) (n = 36) (Figure 1 H). The genes of fungal isolates (i.e., ITS, tub2 and ACT) were amplified with ITS4/ITS5 for ITS (White, Bruns et al. 1990), Bt2a/Bt2b for tub2 (Glass and Donaldson 1995) and ACT783R/ACT512F for ACT (Carbone and Kohn 1999) and sequenced. The sequences were deposited in GenBank (MW165772 for ITS, MW175900 for ACT and MW168659 for tub2), which showing greater than 99.1% similarity to multiple C. cassiicola accessions, respectively. Pathogenicity tests were performed on healthy leaves in field by inoculating surface-sterilized mature leaves with puncture wound (Figure C) and non-wounded young leaves with 20 µL of a conidial suspension (105 conidia ml-1) (Figure F and G) at 26℃. After 4 to 7 days, all inoculated leaves reproduced similar symptoms as observed initially in the field (Figure 1 C, E and F). To fulfill Koch's postulates, the fungus was isolated on PDA from the margins of leaf spots on inoculated leaves and confirmed as C. cassiicola by morphological characters and ITS gene sequencing. Previously, C. cassiicola was reported as an endophyte on Viburnum spp. and Viburnum odoratissimum (Alfieri et al. 1994). More recently, C. cassiicola has been reported as a pathogen of many plant species in China, such as kiwifruit (Cui, Gong et al. 2015), American sweetgum (Mao, Zheng et al. 2021), castor bean (Tang, Liu et al. 2020), and holly mangrove (Xie, He et al. 2020). To our knowledge, this is the first report of leaf spot disease on sweet viburnum caused by C. cassiicola in China and the precise identification of the causal agent will be useful for its management.