Internal Transcribed Spacer Gene Research Articles

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.

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 Hymenula cerealis and Pseudopeziza medicaginis 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 Dichotomanthes tristaniicarpa.

Occurrence of Stem Rot on Acacia mangium Caused by Amauroderma subresinosum.

Acacia mangium has the characteristics of developed root system, nitrogen fixation and soil improvement, fast growth and high yield, and improvement of soil fertility. It is often used as a windbreak tree species in rubber plantations, a highway shade tree, for coastal and mountain restoration in Hainan . In October 2021, a stem rot disease with an incidence of 3% was found in Baisha city(19°22'18″N,109°16'58″E), Hainan Province, China. In the early stage of the disease, the crown showed chlorotic leaves, followed by defoliation. In later stages, whole tree dieback was observed. The basal tissue of the stem of the diseased tree had white rot, and black-brown basidiocarps were observed about 1 m away from the ground. The basidiocarps surface of fresh was disinfected with 75 % ethanol, the epidermal tissue was removed, and the inner tissue blocks were transferred to PDA medium. After culturing in dark at 28°C for 3 days, a colony with white aerial mycelium was isolated and designated: HNBSMZXS20211011001. The basidiocarp was dark brown, sessile, mostly one-year-old, and the cap is nearly semi-circular, wooden, slightly shiny, with a size of 14.7 to 18.1cm × 9.5 to 10.1 cm. The base is thick (5 to 6.5 cm) and the edge is thin (0.3 to 0.7 cm). The basidiospores are oval, 10.7 to 13.65μm × 6.7 to 9.06μm in size, with a double-layer wall. The outer wall is transparent and the inner wall is light yellow. The basidiospores contain 1~2 oil droplets. The morphological features are consistent with those of Amauroderma subresinosum (Murrill) Corner (Zhang et al., 2000). The basidiocarps and type strain cultures were stored as accessions in the Laboratory of Plant Pathogen Fungus Biology, Hainan University. For pathogenicity tests, sawdust culture medium was used (soft sawdust 82%, wheat bran 15%, glucose 2%, gypsum 1%, mixed with water in proportion, sterilized at 121°C for 40min). The mycelium plug from a fresh culture (d=5mm) was taken from the edge of the colony of type strain, and transferred to the sterilized sawdust medium. When mycelium has colonized the media, it was used to inoculate plants. Media without mycelium was used as a control. Naturally growing seedlings (three year old) of A.mangium were selected from the teaching nurseries of Hainan University (20°6'25''N,110°32'24''E). First, 75 % alcohol was sprayed on the stem of the base of A.mangium for surface disinfection. After the surface was dried, a slight wound (about 4×2cm) was made on the surface with a sterilized scalpel. A inoculated and control sawdust media rods were tightly attached to the wound, moistened with cotton balls soaked in sterile water, and then fixed with plastic wrap, and the outer layer was wrapped with newsprint. Inoculation and controls were replicated three times. Two months after inoculated, the stems of the plants inoculated with the isolated fungus grew white hyphae and showed white rot symptoms, and the leaves became chlorotic and defoliated with complete tree decline in six months, which was consistent with the original symptoms observed. By comparison, white callus had grown on the edge of the stem wounds of the control plants. The same fungus was re-isolated from the inoculated plants and confirmed as A.subresinosum based on the internal transcribed spacer (ITS), the ribosomal large subunit(LSU), and the translation elongation factor 1-α(EF1-α) gene sequence, the fungus was not isolated from control plants thus fulfilling Koch's postulates. The ITS region of r-DNA, the ribosomal large subunit(LSU), the translation elongation factor 1-α gene(EF1-α) were amplified using ITS1/ITS4(White et al. 1990), LR0R/LR5(Hu et al. 2021), EF1-983F/EF1-1567R(Buckley et al. 2005) primers, respectively. The sequences of ITS (OQ674500), LSU (OQ674502) and EF1-α gene (OQ883944) were submitted to GenBank. Through with BLAST, the identities of the ITS, LSU and EF1-α sequences to A.subresinosum (GenBank Accession no. ITS: LC176755; LSU: MK119903 and EF1-α: MK121572) was 99.82%; 99.15% and 99.82%, respectively, the identities were more than 99 %. It was reported that A.subresinosum could infect Casuarina equisetifolia and Areca catechu(Chen et al., 2016; Cheng. 2017; Wu et al., 2019). However, this is the first report of Amauroderma subresinosum causing stem rot of Acacia mangiumin Hainan, China. This report will facilitate field diagnosis and provide scientific reference for further research on the disease.

Oral bacteriome and mycobiome of patients with idiopathic membranous nephropathy with different tongue coatings treated with a Chinese herbal formula

Ethnopharmacological relevanceMoshen Fuyuan Formula (MSFY) is one of the representative Chinese medicine compound for Idiopathic membranous nephropathy (IMN), that originate from Fang Ji Huang Qi decoction in the Han dynasty. IMN is usually accompanied by different tongue coatings in traditional Chinese medicine (TCM), and tongue microorganisms are important factors affecting the formation of the tongue coating. Recently, oral microbiomes, including bacteria and fungi, have been identified as pivotal factors that contribute to disease development. However, the regulation of oral microbiomes by MSFY has not been defined. Aim of the studyIn this work, we explore the characteristics of oral bacteria and fungi in IMN patients with different tongue coatings, and clarify the therapeutic effect of MSFY based on oral microbiome. Materials and methodsWe enrolled 24 patients with IMN, including 11 with white tongue (IMN-W) and 13 with yellow tongue (IMN-Y), and recruited an additional 10 healthy individuals. Patients with IMN were treated with the MSFY. The oral bacteriome and fungi before and after treatment were detected using full-length 16S rRNA and internal transcribed spacer gene sequencing. ResultsThe therapeutic effect of MSFY on patients with yellow tongue coating was more significant than that on patients with white tongue coating. In terms of oral bacteriome, Campylobacter bacteria were enriched in patients with yellow tongue and could be a promising biomarker for yellow coating. Enrichment of Veillonella parvula_A may partially account for the therapeutic effect of MSFY. As for oral fungi, Malassezia globosa was enhanced in patients with IMN-W and reduced in patients with IMN-Y. Notably, it was reduced by MSFY. We also found that mycobiome-bacteriome interactions were highly complex and dynamic in patients with IMN. ConclusionThe regulation of the dynamic balance between oral fungi and bacteria by MSFY contributes to the treatment of IMN. This study determined the oral bacteriome and mycobiome of patients with IMN with different tongue coatings before and after MSFY treatment, which aids in promoting personalized treatment in clinical TCM and provides direction for investigating the mechanism of Chinese herbal medicines.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sphagneticola trilobata in Hainan Province, China.

Sphagneticola trilobata (L.) Pruski is a perennial creeping herb of the Asteraceae family, which is native to South America. It was introduced into Southern China as a groundcover in the 1970s (Zhang et al. 2023). Now it is mainly used for folk medicine to treat various kinds of inflammatory, incuding joint pain, rheumatic diseases, arthritis, in addition to treating persistent wounds, ulcers, and edemas (Gonçalves et al. 2022). In February and November 2023, powdery mildew symptoms were observed on 60% of S. trilobata plants on the Hainan Medical University campus (19° 58' 53″ N; 110° 19' 47″ E) in Haikou, Hainan Province, China. Powdery mildew colonies covered the leaf surfaces and stems of affected plants, causing discoloration and defoliation. Mycelia were superficial and hyphal appressoria were nipple-shaped. Conidiophores (n =30) were unbranched, cylindrical, 74 to 161 × 10 to 14 µm, and produced three to five immature conidia in chains with a crenate outline. Foot cells (n =30) were cylindrical, straight or sometimes curved at the base, and 27 to 56 µm long. Conidia (n =100) were ellipsoid-ovoid to doliiform, 17 to 30 ×14 to 28 µm (length/width ratio = 1.1 to 1.9), with well-developed fibrosin bodies, and produced germ tubes from the lateral position. Based on these morphological characteristics, the pathogen was provisionally identified as Podosphaera xanthii (Braun and Cook 2012). The teleomorph was not observed. A specimen was deposited in the Hainan Medical University Plant Pathology Herbarium as HMST-23. To confirm the genus identification and ascertain a putative species, genomic DNA was extracted from mycelium, conidiophores, and conidia using a fungal DNA kit (Omega Bio-Tek, USA). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990) and sequenced directly. The resulting 577-bp sequence was deposited in GenBank (accession no. OR784549). A BLASTn search in GenBank of this sequence showed 100% similarity with the ITS sequences of P. xanthii isolates from China (MT260063, MN203658, OP765400, and MT739423), Thailand (LC270780), and Vietnam (KM260731, KM260730, and KR779870). Additionally, the 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O´Donnell 1993; accession no. OR784550). This region shared 100% similarity with P. xanthii isolates (LC371334, LC270782, AB936277, and OP765401) as well. Powdery mildew from Hainan sample belonged to the P. xanthii group with strong bootstrap values support 99% in maximum likelihood phylogenetic tree based on ITS and 28S gene sequences. To confirm pathogenicity, five healthy potted plants of S. trilobata were inoculated by gently pressing a powdery mildew-infected leaf onto 15 young leaves. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 24 to 30°C, 70% relative humidity, with a 16-h photoperiod. After 7 days, inoculated leaves showed powdery mildew symptoms whereas no symptoms were observed on control plants. The fungal colonies observed on inoculated plants were morphologically identical to those found on the originally infected leaves collected from Hainan Province. Based on the morphological characteristics and molecular identification, the fungus was identified as P. xanthii. In different countries and regions, P. xanthii has been previously reported on S. trilobata in Taiwan (Yeh et al. 2021). To our knowledge, this is the first record of P. xanthii infecting S. trilobata in Hainan Province, China. S. trilobata is often planted as an ornamental plant on both sides of the road, and we are concerned that it may serve as a new host, spreading this pathogen to other economic crops.

Clinical and laboratory characterization of cutaneous leishmaniasis in Chinese migrant workers returned from Iraq.

Imported cutaneous leishmaniasis (CL) is a growing problem with increasing global travel to endemic areas. Returned travelers with CL are easy to be misdiagnosed and mistreated due to the lack of awareness for the disease to the physicians in non-endemic region that may lead to unfavorable outcome. Our study intends to summarize the characteristics of Leishmania infection imported from Iraq, so as to help Chinese physicians diagnose and treat the disease. All CL patients were treated with intralesional injection of antimony. The definitive diagnosis of CL is based on the parasite identification by microscopic examination directly on lesion smear or parasite culture, PCR amplification of Leishmania-specific internal transcribed spacer 1 (ITS-1). The phylogenetic analysis, the immunopathological examination and the cytokine detection were proceeded after the diagnosis. We have identified 25 CL cases in migrant Chinese workers returned from Iraq for the first time with L. major as the major species of infected Leishmania parasite. Clinical features of the Iraq-imported CL include the history of skin exposure to sandflies bite and the lesions mostly on the exposed limbs. More ulcerative wet lesion was observed than nodular dry lesion. PCR is not only used to detect Leishmania parasite with high sensitivity, but also to identify the species of infected parasite through sequencing the amplified Leishmania-specific ITS-1 gene. The phylogenetic analysis based on the amplified ITS-1 sequences revealed that the infected Leishmania was closed related to the species and strains endemic in Iraq. The immunopathological examination revealed the T-cell filtrated cellular immune response with less B cells and NK cells involved. The cytokine profile measured in the skin lesion also confirmed the Th1 cellular response with higher expression levels of IFN-γ, IL-6 and IL-8. The skin lesions in CL patients were healed after being treated locally with antimony. The clinical and parasitological features of these Chinese CL cases imported from Iraq provide useful information for the diagnosis and treatment of CL that is not commonly seen in Chinese local population.

First Report of Fusarium proliferatum Causing Bulb Rot on Lilium davidii var. willmottiae in China.

Lilium davidii var. willmottiae, known as Lanzhou lily, is widely cultivated in China for its edible bulbs. In July 2023, symptoms of bulb rot were observed on Lanzhou lilies harvested from Lanzhou, Gansu Province, during storage at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China), at an incidence of nearly 70%. The surface of the lily scales had dark water-stained spots, after the development of which the color gradually darkened, the bulbs became soft, accompanied by a pungent smell. Finally, the whole bulb became ruined and rotten, and there were thick mycelium layers on the bulbs. The infected bulbs were washed with clean water, sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 5 min, and then rinsed three times with sterile distilled water. The 5 mm×5 mm tissue pieces from the junction of the diseased part and the healthy part were clipped, placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 fungal isolates were obtained, all of which exhibited typical Fusarium characteristics. The colonies were white to pink with white to cream-colored aerial mycelia. After 10 to 15 days of incubation, the macroconidia (n = 50) were hyaline, relatively slender with a curve, three to five septate, and 8.73 to 33.24 × 2.16 to 4.19 μm in length. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 4.04 to 8.48 × 1.24 to 2.65 μm. These morphological characteristics were similar to those described for Fusarium proliferatum (Leslie and Summerell 2006). For molecular identification, a cetyltrimethylammonium bromide (CTAB) protocol was used to extract total genomic DNA (O'Donnell et al., 1998), after which the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymerase Ⅱ subunit 2 (RPB2) genes were amplified using the universal primers ITS1/ITS4, EF1/EF2 and RPB2-5f2/fRPB2-7cr, respectively, and subsequently sequenced (White et al., 1990; O'Donnell et al., 1998; Liu et al., 1999; Reeb et al., 2004; O'Donnell et al., 2007; Jiang et al., 2018). The sequences of a representative isolate (CAAS01) were analyzed and submitted to GenBank under accession numbers OR554007 (ITS), OR594233 (TEF1-α) and OR603932 (RPB2). A BLAST analysis revealed that the sequences of the ITS, TEF1-α, and RPB2 genes shared 100%, 100%, and 100% identity, respectively, with those of Fusarium proliferatum (MT466521.1, MK952792.1, and LT841266.1) in GenBank. In addition, the ITS, TEF1-α and RPB2 sequences shared 100%, 100%, and 100% identity with those of Fusarium annulatum (LC13675, the Fusarium fujikuroi species complex; previously known as the Gibberella fujikuroi species complex) in the Fusarium-ID database. Fusarium proliferatum, whose common synonyms are Gibberella fujikuroi mating population D and Gibberella fujikuroi var. intermedia, is the anamorphic form of the Gibberella fujikuroi complex that belongs to the Nectriaceae family. A phylogenetic tree was constructed based on the combined TEF1-α and RPB2 sequences of CAAS01 and other Fusarium isolates, revealing that CAAS01 was grouped with Fusarium proliferatum. Based on sequence alignment and phylogenetic analysis, the isolate was identified as Fusarium proliferatum. To determine the pathogenicity of the isolated fungi, healthy bulbs were punctured with disposable sterilized needles and soaked in equal amounts of sterile water and conidial suspension (1×107 conidia/mL) for 30 min respectively. The pathogenicity experiment was repeated three times. After 7 days of inoculation at 25 °C and 80% relative humidity, the surface of the inoculated bulbs produced water-stained spots and mycelium layers consistent with the symptoms exhibited by Lilium davidii var. willmottiae bulbs during storage, while the uninoculated lily bulbs remained symptomless. Fusarium proliferatum was reisolated from the infected bulbs and identified based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of bulb rot on Lilium davidii var. willmottiae caused by Fusarium proliferatum in China. This study will contribute to the development of management strategies for this postharvest disease in Lilium davidii var. willmottiae.

First Report of Aspergillus niger causing rot of Morchella sextelata in China.

As an important edible mushroom, morel mushroom (Morchella spp.) has been widely spread and cultivated in China. However, between 2022 and 2023, a rot disease with a natural incidence of 28% occurred in morel mushroom farms in the Qingpu district of Shanghai (N30°97', E121°06'), China. High temperatures (>20℃) and high humidity (>70%) provide conditions conducive to the spread of this disease. First, a small white mold-like symptoms appeared on the surface or the pinnacle of pileus. The tissues in the infected parts stop growing and developing.Then the lesion developed to encircle the pileus and spread gradually to the stipe, seriously affecting its yield and quality. The infected tissue of morel fruiting body at the edge of the lesions was isolated and cultivated on potato dextrose agar (PDA) at 28℃ in the dark. After 3 days, monospore cultures formed black cottony colonies. In order to reliably identify, isolates were transferred to Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. Scanning electron microscope analysis revealed that mature mycelia produced conidiophores ended with numerous metula and phialides. The phialides showed the number of conidia bearing rounded spores, which coincides with previous research(Silva et al, 2020). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), beta-microtubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) gene of the isolate were amplified by PCR (White et al. 1990; Glass et al. 1995; Hong et al. 2005; Liu et al. 1999). The resulting sequence was deposited in GenBank with accession OQ931346.1, OR393310, OR393311, and OR393312, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as MOR02. Comparison results indicated that the sequences with accession numbers KF305756.1, MK450794.1, HQ285594.1, and HQ285594.1 have high identity with the molecular sequences of A. niger MOR02, which is 99%, 98%, 98%, 99%, respectively. Phylogenetic analysis with ITS and RBP2 genes of the isolated strain and 9 Aapergillus spp. strains were performed using MEGAX software with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as A. niger. A spore suspension of the A. niger strain MOR02 (1 x107 conidia/mL) was inoculated back to healthy morel mushrooms. Five healthy fruit bodies of M. sextelata were injected, and another five healthy morels were treated with potato dextrose broth（PDB） medium as controls. Morels were incubated for 7 days at 20℃ and 85% to 90% relative humidity. The pathogen successfully infected the morel showing a similar white mold-like lesion as the natural occurrence disease. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as A. niger MOR02 based on its morphological characteristics and phylogenetic marker genes. To our knowledge, this is the first report of A. niger causing rot disease of M. sextelata. This study confirms that A. niger is the pathogenic fungus causing morel rot on the Qingpu farm in Shanghai. The disease occurs under conditions of high humidity and high-temperature conditions. Better production management is the most important to prevent the disease.

First Report of Leaf Spot on Cucumis melo L. Caused by Arcopilus aureus in China.

Cucumis melo L. is an important fruit with widespread consumption and commercial value. However, an undescribed disease affecting Hami melon (Cucumis melo L. var. Luhoutian) plants has consistently emerged in the Qihe region of Dezhou, Shandong Province of China since 2021. The disease can occur in both seedling and mature stages of Hami melon plants, and in severely diseased areas, the incidence rate was seen as 40 to 80%. During the seedling stage, the initial symptom is the appearance of water-soaked spots on the leaves. As the disease progresses, the leaves develop necrotic spots, and severely affected plants may exhibit stem rot and decay. In the mature stage, the disease primarily affects the leaves, causing necrotic spots and chlorosis. Under conditions of high humidity, black mold can be observed in the affected areas. Small pieces of symptomatic leaves from six different infected plants were collected and surface-sterilized with 5% NaClO for 3 min and 75% alcohol for 30 s for pathogen isolation (Wang et al., 2020). After rinsing with sterile water and blotted on sterile filter paper, the tissues were established on potato dextrose agar (PDA) media and incubated at 28℃ for 3-4 days. Pure isolates showed up at PDA were obtained through single-spore isolation. Colonies of all 16 isolates obtained by single-spore isolation had similar morphological characteristics on the PDA medium, the mycelium of the isolate appears dense and yellowish-brown on the PDA medium, and also secretes a brownish-red pigment on PDA. Under the opticalmicroscope, the perithecia from PDA media are subglobose spherical in shape, 80-100 μm in diameter, brownish by reflected light, wholly and densely hairy. Terminal hairs are very dense, greyish by reflected light, olive brown to reddish brown by transmitted light, thick-walled, arcuate, circinate, or spirally coiled at the apex. The ascospores within the perithecia are elliptical or droplet-shaped, initially colorless hyaline but later becoming subhyaline slightly gray, with dimensions of 7-9 μm × 4-5 μm. The morphological characteristics of the isolates were consistent with the description of Arcopilus aureus (Wang et.al. 2016). The internal transcribed spacer (ITS) region and β-tubulin genes of three randomly selected isolates were PCR amplified and sequenced using primers ITS4/ITS5 and Bt2a/Bt2b. The sequences of ITS and β-tubulin genes were submitted to NCBI with GenBank Accession No. OR539527 and OR640972, respectively. Based on morphological features and phylogenetic analysis, we concluded that the isolates belonged to A. aureus. Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of Hami melon seedlings (n=12) grown at 28°C with 60% humidity in a greenhouse, the assay was repeated three times. Symptoms appeared on the pathogen-inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as A. aureus based on morphological, and molecular phylogenetic analysis, while Koch'sostulate was used to confirm its life mode. To the best of our knowledge, this is the first report of leaf spot caused by A. aureus on Cucumis melo L. in China.

First Report of Anthracnose on Ilex cornuta Caused by Colletotrichum aenigma in China

Ilex cornuta (Aquifoliaceae) is a dark green evergreen shrub with glossy leaves that is widely distributed in China and East Asia and used as an ornamental and medicinal plant. In March 2022, typical symptoms of anthracnose were observed on I. cornuta leaves (with approximately 30% of leaves affected) in Jiangxi Academy of Forestry, Nanchang city, Jiangxi Province, China. The early symptoms were light brown spots on the edge or tip of the leaves. The spots gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a dark brown margins. To isolate the pathogen, ten symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface sterilized by dipping in 70% ethanol for 30 s and 1% sodium hypochlorite (NaClO) for 30 s, and then washed three times with sterile distilled water. Leaf pieces were then placed on potato dextrose agar (PDA) plates at 28℃ in the dark. Subsequently, six isolates were obtained using the single-spore method, five of them were similar in morphological characteristics. Colonies grown on PDA for 7 days were 75-85 mm in diameter, and were cottony, dense, and pale white on the surface and white to grayish-green on the reverse side. Conidia were single-celled, transparent and subcylindrical to clavate. The contents of the conidia were granular and 15.63–20.63 × 5.63–7.50 μm in size (=17.78 ± 1.41× 6.50 ± 0.55 μm, n = 40). The species was also identified by analysis of the internal transcribed spacer (ITS) region, actin (ACT), glyceradehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUB2), calmodulin (CAL), glutamine gynthetase (GS), DNA Lyase (APN2), intergenic spacer and partial mating type (ApMat) genes using ITS4/ITS5, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, CL1C/CL2C, GSF/GSR, ColDL-F3/CgDL-R1 and CgDL-F6/CgMAT1F2 primers, respectively (Weir, et al. 2012; Maharachchikumbura, et al. 2014; Khodadadi, et al. 2020). The sequences were deposited in GenBank (Accession Nos. OQ600619, OQ603370, OQ603373, OQ603379, OQ974177, OQ974176, OQ974178, and OQ974175). BLASTN analysis in GenBank showed that these genes exhibited 100% similarity to the sequences of Colletotrichum aenigma (MT476812, MN525817, MN525878, MN525904, MN525836, KX620296, and MN338281) and 99% similarity to the sequence of Colletotrichum sp. strain (MT071110). Concatenated sequences of these eight genes and Colletotrichum species sequences from GenBank were then used to construct a phylogenetic tree by using the maximum likelihood method in IQtree V1.5.6. Isolate JFRL 03-1005 was grouped into a clade with C. aenigma with a high bootstrap value. Thus, the isolate was identified as C. aenigma based on morphological and molecular data. To verify Koch's postulates, pathogenicity was tested on 2-year-old healthy potted plants of I. cornuta. Ten disinfected leaves were wounded with a sterile scalpel, and then inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) from isolate JFRL 03-1005. The control leaves were inoculated with 10 μl of sterile water. Then, the potted plants were incubated at 28°C with a 12 h photoperiod and 80% humidity. After 10 days, distinct spots appeared on all inoculated leaves, whereas control leaves remained asymptotic. C. aenigma was reisolated from the spots and identified by sequencing the ITS, ACT, GAPDH, TUB2, CAL, GS, APN2 and ApMat genes. Previous studies reported that C. aenigma can caused anthracnose on the leaves of various cash crops in China, such as apple, tree peonies, mulberry, and walnut (Wang et al. 2020; Zhang et al. 2021; Wang et al. 2022; Zhu et al. 2022). To the best of our knowledge, this is the first report of C. aenigma causing anthracnose on I. cornuta in China, this report further confirmed that C. aenigma has a wide range of hosts in nature. the anthracnose on I. cornuta caused by C. aenigma has seriously affected its ornamental value. Therefore, more attention should be paid to this disease and appropriate control strategies should be formulated.

Decoding seasonal changes: soil parameters and microbial communities in tropical dry deciduous forests.

In dry deciduous tropical forests, both seasons (winter and summer) offer habitats that are essential ecologically. How these seasonal changes affect soil properties and microbial communities is not yet fully understood. This study aimed to investigate the influence of seasonal fluctuations on soil characteristics and microbial populations. The soil moisture content dramatically increases in the summer. However, the soil pH only gradually shifts from acidic to slightly neutral. During the summer, electrical conductivity (EC) values range from 0.62 to 1.03 ds m-1, in contrast to their decline in the winter. The levels of soil macronutrients and micronutrients increase during the summer, as does the quantity of soil organic carbon (SOC). A two-way ANOVA analysis reveals limited impacts of seasonal fluctuations and specific geographic locations on the amounts of accessible nitrogen (N) and phosphorus (P). Moreover, dehydrogenase, nitrate reductase, and urease activities rise in the summer, while chitinase, protease, and acid phosphatase activities are more pronounced in the winter. The soil microbes were identified in both seasons through 16S rRNA and ITS (Internal Transcribed Spacer) gene sequencing. Results revealed Proteobacteria and Ascomycota as predominant bacterial and fungal phyla. However, Bacillus, Pseudomonas, and Burkholderia are dominant bacterial genera, and Aspergillus, Alternaria, and Trichoderma are dominant fungal genera in the forest soil samples. Dominant bacterial and fungal genera may play a role in essential ecosystem services such as soil health management and nutrient cycling. In both seasons, clear relationships exist between soil properties, including pH, moisture, iron (Fe), zinc (Zn), and microbial diversity. Enzymatic activities and microbial shift relate positively with soil parameters. This study highlights robust soil-microbial interactions that persist mainly in the top layers of tropical dry deciduous forests in the summer and winter seasons. It provides insights into the responses of soil-microbial communities to seasonal changes, advancing our understanding of ecosystem dynamics and biodiversity preservation.

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.

Revealing the Diversity of the Mycobiome in Different Phases of Ticks: ITS Gene-Based Analysis

Ticks are obligate ectoparasites and vectors of a variety of pathogens in humans and animals. Certain tick-borne pathogens (TBPs) have been identified as the cause of zoonoses, posing potentially significant threats to the human health and livestock industries. Fungi are one of the major TBPs that can affect ticks and cause disease in humans. At present, there are few studies on the diversity of fungal microbial communities carried by Ixodes. Therefore, profiling tick-borne fungi will contribute to understanding the tick-fungal interaction. This study evaluated the community profile and differences in the fungal microbiome in Ixodidae collected on parasitic ticks or nonparasitic ticks in Wuwei, Gansu Province, China. The Shannon index, Simpson index, and Richness index were used to evaluate the diversity of mycobiome. Principle coordinates analysis (PCoA) was conducted to determine patterns of diversity in mycobiome. Using correlation analysis to determine the correlation of mycobiome. The results show that the high-throughput sequencing of the internal transcribed spacer gene generated 3,634,943 raw reads and 7,482 amplicon sequence variants. The dominant tick species in this region was Dermacentor nuttalli (Ixodidae). The mycobiome belonged to four classes—Dothideomycetes, Sordariomycetes, Ustilaginomycetes, and Tremellomycetes—and more than 261 genera, the most abundant genera were Cladosporium, Purpureocillium, Aureobasidium, Tranzscheliella, and Sporormiella. Alpha diversity indicated that the abundance and evenness of mycobiome were marginally higher in nonparasitic ticks than in parasitic ticks. PCoA showed that the community structures of parasitic ticks vary from nonparasitic ticks, samples from nonparasitic ticks tended to cluster more closely than those from the parasitic ticks. Correlation analysis indicated that there was a significant positive correlation or negative correlation between the mycobiome. Our results indicate that the mycobiome carried by Dermacentor nuttalli had rich diversity, and there was a significant difference in mycobiome between parasitic ticks and nonparasitic ticks. These findings may conducive to understand the complex interaction between ticks and commensal fungi and provide help for the further exploration of the behavioral characteristics of ticks and formulation of effective biological control measures.

First Report of Root Rot on Hydroponically Grown Romaine Lettuce Caused by Pythium dissotocum in Korea.

The romaine lettuce (Lactuca sativa L.) is one of the most frequently consumed vegetables in Korea. In January 2023, the romaine lettuce cultured within an indoor hydroponic farm in South Korea displayed severe disease, with an incidence of approximately 13.7% of 300 plants. The diseased plants showed symptoms of stunted growth, lower leaf yellowing, and brown or black-colored soft and mushy root rot, in which the outer layer of root was sloughed off, leaving a thread-like appearance. These symptoms were similar to those of Pythium root rot previously reported to occur on lettuce (McGehee et al., 2018; Stanghellini and Kronland, 1986). Samples of romaine lettuce with rot symptoms were collected from the hydroponic farm. The infected roots were rinsed three times with sterilized distilled water (SDW), dried on sterilized filter paper, and sliced into segments (about 0.5 cm in length), which were placed into Petri dishes (9 cm in diameter) containing V8 juice agar (V8A: 8% V8 juice and 1.5% agar powder) and cultured at 25°C for 2 days. The emerging hyphae were transferred to new Petri dishes containing V8A. After four rounds of sub-culturing, a total of 11 strains were isolated and all of them exhibited the same morphology. Strain KNU2301TP was purified via isolation of a single zoospore and stored at -80°C. The mycelia were non-pigmented. The hyphae obtained from a three-day-old culture grown in V8A were aseptate and the diameter of major hyphae was up to 7 µm. The filamentous sporangia were not to slightly inflated and formed dendroid-like branches. Vesicles containing zoospores were formed on the filamentous sporangia. The encysted zoospores were spherical with a diameter of 8.1 to10 µm (average 8.9 ± 0.6 µm; n = 50). These morphological characteristics of KNU2301TP were similar to those of a previously reported oomycete, Pythium dissotocum (Van der Plaats-Niterink 1981). The genomic DNA was extracted from mycelia cultured on V8A by a previously described extraction method (Chi et al. 2009). Sequences of internal transcribed spacer (ITS) region and cytochrome c oxidase subunit II (cox2) gene were amplified with paired primers ITS1/ITS4 and cox2_F/R, respectively (Callaghan et al., 2022; Hudspeth et al. 2000; White et al. 1990). The resulting sequences were deposited in GenBank (GenBank Accession Nos.; ITS: OQ683867; cox2: OQ700848). The sequences of ITS and cox2 gene of strain KNU2301TP were 99.61% and 100% identical to the sequences of ITS (MG719858) and cox2 gene (MG719859) of P. dissotocum strain YNP-3, respectively. Based on the result of the morphological characterization and sequence analysis, the strain KNU2301TP was identified as P. dissotocum. For Koch's postulates, 15 lettuce seedlings (eighteen-day-old) were inoculated by immersing the roots in a spore suspension (1 × 105 zoospores/ mL) and incubated at 25°C under 16/8 h light/dark cycles for 8 days. Fifteen plants of same age were treated with SDW in the same manner as a control The symptoms resembling those originally found at the farm were developed on the inoculated plants, but not on controls. The strain reisolated from the inoculated plants by same method mentioned above was confirmed as strain KNU2301TP by analysis of morphology and ITS and cox2 sequences. To our knowledge, this is the first report of root rot on hydroponically grown lettuce caused by P. dissotocum in Korea. Root rot on lettuce caused by P. dissotocum has been previously reported in USA, Canada, and Finland (McGehee et al. 2018; Stanghellini and Rasmussen 1994). Since lettuce is an important and popular leafy vegetable around the world, further work would focus on developing efficient strategies to manage this Pythium root rot disease.

First Report of Colletotrichum chrysophilum Causing Anthracnose on Nance (Byrsonima crassifolia) in Mexico.

Nance fruit [Byrsonima crassifolia (L.) HBK] is a native crop widely distributed in Mexico and South America (Medina-Torres et al. 2018). It has been reported that nance is a good source of active compounds with anti-inflammatory, neuropharmacological and antioxidant effects. In 2022, the annual production of nance fruit in Mexico was of 7,713.13 tons and average yield of 5.64 t/ha with economic value of 51,952.66 million pesos (SIAP, 2022). This production generated significant economic income for the communities at a local, regional, and national level. In January 2023, irregular necrotic spots were observed on leaves and fruit of nance in an orchard of 50 nance trees located in San Sebastián Nopalera (16°54'52.73"N; 97°47'50.35"W), Oaxaca, Mexico. The incidence of the disease ranged from 50 to 60% of the trees. Infected fruit first showed dark-brown lesions with defined borders that coalesced to form large necrotic area. Isolates were purified by single spore isolation method (Choi et al. 1999).Colletotrichum strains were grown in PDA medium and five monoconidial isolates were obtained. A representative sample was selected (CNC-NP3) and deposited in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. Colony on PDA was white with sparse aerial mycelium, and the center was dark grey with abundant acervuli. Conidia (n = 100) were hyaline, aseptate, cylindrical with rounded apex, 13.5 to 15.2 × 4.3 to 5.1 μm. Appressoria (n = 20) were terminal or lateral, obovoid to clavate and some with slightly lobed, 9.9 to 11.6 × 5.3 to 6.6 μm. Based on the morphology, the isolate was identified as belonging to the Colletotrichum gloeosporioides species complex (Jayawardena et al. 2016). The representative isolate CNC-NP3 was identified by multilocus phylogenetic analysis using nucleotide sequences of internal transcribed spacer (ITS), actin (ACT), β-tubulin (TUB2), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Jayawardena et al. 2016). The sequences were deposited in GenBank (accessions nos. OQ861102 (ITS), OQ870548 (ACT), OQ870549 (TUB2), OQ870550 (GAPDH). The phylogenetic analysis was carried out by Maximum likelihood method using concatenated sequences of ITS, ACT, TUB2 and GAPDH genes (Kozlov et al. 2019). The multilocus phylogenetic analysis revelated clearly the isolate CNC-NP3 as Colletotrichum chrysophilum. To confirm pathogenicity of CNC-NP3, 30 healthy fruits were inoculated. Fifteen disinfected nance with wounds and fifteen nance without wounds were inoculate with 10 µL of conidial suspension (1×105 spores/mL) from 7-day old culture. And controls were inoculated using sterile distilled water. Fruits were placed in a moist chamber covered with plastic bag at 25 °C for 48 h to maintain high humidity. After 4 days the inoculation sites development symptoms that were identical to those initially observed in the field, whereas the control group remained symptomless. The pathogenicity test was performed twice, with the same results. The pathogen was re-isolated from the lesion to fulfill Koch's postulates. Currently, Colletotrichum chrysophilum has been reported causing anthracnose disease in several crops: apple in New York (Khodadadi et al. 2020), papaya in Mexico (Pacheco- Esteva et al.2022), Blueberry (Soares et al. 2022) and banana in Brazil (Astolfi et al. 2022). To our knowledge, this is the first report of anthracnose in Byrsonima crassifolia caused by Colletotrichum chrysophilum in Oaxaca, Mexico.

Endophytic and epiphytic metabarcoding reveals fungal communities on cashew phyllosphere in Kenya.

Plants intimately coexist with diverse taxonomically structured microbial communities that influence host health and productivity. The coexistence of plant microbes in the phyllosphere benefits biodiversity maintenance, ecosystem function, and community stability. However, differences in community composition and network structures of phyllosphere epiphytic and endophytic fungi are widely unknown. Using Illumina Miseq sequencing of internal transcribed spacer (ITS) and 28S rRNA gene amplicons, we characterised the epiphytic and endophytic fungal communities associated with cashew phyllosphere (leaf, flower and fruit) from Kwale, Kilifi and Lamu counties in Kenya. The ITS and 28S rRNA gene sequences were clustered into 267 and 108 operational taxonomic units (OTUs) at 97% sequence similarity for both the epiphytes and endophytes. Phylum Ascomycota was abundant followed by Basidiomycota, while class Saccharomycetes was most dominant followed by Dothideomycetes. The major non-ascomycete fungi were associated only with class Tremellales. The fungal communities detected had notable ecological functions as saprotrophs and pathotrophs in class Saccharomyectes and Dothideomycetes. The community composition of epiphytic and endophytic fungi significantly differed between the phyllosphere organs which was statistically confirmed by the Analysis of Similarity test (ANOSIM Statistic R: 0.3273, for 28S rRNA gene and ANOSIM Statistic R: 0.3034 for ITS). The network analysis revealed that epiphytic and endophytic structures were more specialized, modular and had less connectance. Our results comprehensively describe the phyllosphere cashew-associated fungal community and serve as a foundation for understanding the host-specific microbial community structures among cashew trees.

Assessment of genetic diversity of Trypanosoma evansi in the domestic animal populations through ITS-1 gene sequence analysis.

Trypanosoma evansi infects domestic animals, causing a debilitating and occasionally fatal disease. The disease leads to significant economic losses to farmers and poses a substantial impediment to the growth of livestock production in developing nations, including India. Considering the challenges associated with managing this infection, there is an urgent need to enhance our understanding of the molecular and genetic diversity of T. evansi. Therefore, this study was planned to analyze the genetic diversity of T. evansi using available internal transcribed spacer-1 (ITS-1) gene sequences from India and compare them with sequences from around the globe. Blood samples used in this study were collected from naturally infected animals including dogs, cattle, and buffaloes in the Indian state of Madhya Pradesh. Using the ITS-1 gene, we amplified a 540 base pairs (bp) segment using polymerase chain reaction (PCR), sequenced it, and identified intra-specific variations. Phylogenetic analysis of 90 sequences, including 27 from India, revealed three distinct clusters with high bootstrap support values. A haplotype network analysis identified 34 haplotypes, with H7 being the most prevalent, indicating a complex evolutionary history involving multiple countries. The genetic analysis of the Indian population revealed distinct characteristics. Despite low nucleotide diversity, there was high haplotype diversity in comparison to other populations. Tajima's D, Fu and Li's D, and Fu and Li's F exhibited non-significant negative values, indicating potential stability. Additionally, the slightly positive values in Fu's Fs, Raggedness (r), and Ramos-Onsins and Rozas (R2) statistics suggested a lack of recent significant selective pressures or population expansions. Furthermore, the presence of genetic differentiation and gene flow among T. evansi populations highlighted ongoing evolutionary processes. These findings collectively depicted a complex genetic landscape, suggesting both stability and ongoing evolutionary dynamics within the Indian population of T. evansi. The findings of this study are important for understanding the evolutionary history and population dynamics of T. evansi, and they may help us develop effective control strategies.

Sequence analysis of Paragonimus internal transcribed spacer 2 and cyclooxygenase 1 genes in freshwater crabs in Henan Province

To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China. Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup. The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1). Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.