Species-specific isothermal nucleic acid amplification assay targeting Internal Transcribed Spacer (ITS) for rapid authentication of the medicinal crop Cirsium japonicum and Cirsium setosum in herbal markets.

Methodological approaches for identifying rhizosphere bacteria and fungi: A systematic review of comparative insights and best practices

Dry bubble disease, attributed to the filamentous fungus Lecanicillium fungicola (Cordycipitaceae) results in huge yield losses in mushroom (Agaricus bisporus) cultivation worldwide. The possibilities for controlling the disease using commercial fungicides are highly limited, and therefore, there is an increasing demand for novel, alternative means of pest management. Our research objective was the comprehensive examination of viruses in the causal agents of dry bubble disease, which may open up an avenue for its virocontrol in the future. Out of 57 fungal isolates obtained from dry bubble-affected A. bisporus crops in various countries, 47 (82%) were confirmed by ITS (Internal Transcribed Spacer) sequence analysis as L. fungicola. In addition, different members of the genera Akanthomyces and Simplicillium (7 and 3 isolates, respectively), yet unknown to cause dry bubble symptoms, have also been detected. Cellulose column chromatography revealed the presence of double-stranded (ds) RNA in seven L. fungicola and three Akanthomyces sp. isolates, suggesting viral infection. The ten dsRNA-positive and eight randomly selected dsRNA-negative fungal strains were subjected to rRNA-depletion high-throughput RNA-sequencing analysis. The presence of seven new viruses representing four new species in the established families, Partitiviridae, Polymycoviridae, Botourmiaviridae and the narna-like virus group, and three previously established/proposed species in the families Chrysoviridae and "Mycovirgaviridae" were confirmed. The impact of the detected and identified viruses on their host fungi, and their potential applicability for virocontrol purposes will be examined in the future. This study provides the first detailed report on viruses of mushroom pathogenic fungi.

Trichophyton rubrum is the primary causative pathogen of onychomycosis, and terbinafine remains the first-line treatment for both oral and topical therapy. However, increasing reports of terbinafine resistance-associated mutations in T. rubrum from India, Europe, and other parts of Asia have raised global concern. This study aimed to investigate mutations in the squalene epoxidase (SQLE) gene in T. rubrum from onychomycosis samples in South Korea. Between August 2021 and November 2023, toenail specimens from patients with KOH-positive fungal microscopy were collected from six hospitals. Fungal species were identified through internal transcribed spacer (ITS) sequencing, and mutations in the squalene epoxidase (SQLE) gene were analyzed. Among 388 clinical samples, 231 (59.5%) were ITS PCR-positive. Sanger sequencing identified T. rubrum in 185 samples (80.5%), followed by Candida parapsilosis (7), T. mentagrophytes (4), T. violaceum (2), and various other fungi (24). Among 106 SQLE-positive T. rubrum clinical samples, 3.8% (4/106) harbored resistance-associated mutations, including Phe397Leu (n = 3) and Leu393Phe (n = 1), while the remaining 102 samples showed wild-type SQLE sequences at these positions. These findings demonstrate the presence of SQLE mutations in T. rubrum from onychomycosis samples in South Korea. Further studies incorporating fungal isolation and antifungal susceptibility testing are needed to determine their clinical significance.

Plant-fungi interaction serves various insights for research. Therefore, the study aimed to isolate endophytic fungi from Allium cepa (onion) bulbs and Allium sativum (garlic) cloves to evaluate the biological activity of their metabolites. Remarkably, a heteromorphic strain of Fusarium oxysporum was successfully isolated and identified via Internal Transcribed Spacer (ITS) amplicon sequencing: the strain from onion was designated ACE (OR062575), and the one from garlic as ASE (OR062574). Morphologically, ACE spores exhibited no defined shape, whereas ASE produced oval and amygdaloid chlamydospores. The fungal extracts were evaluated using the free radical scavenging DPPH assay, antimicrobial disc diffusion method, and brine shrimp lethality bioassay. Notably, ASE demonstrated approximately 1.5 times higher 50% free radical inhibition compared to ACE. The ACE extract (80 µg/disc) showed significant antibacterial activity against Escherichia coli (18 mm) and Bacillus megaterium (16 mm), whereas ASE exhibited roughly half this activity at the same dose against E. coli and Pseudomonas aeruginosa. Interestingly, the cytotoxicity of ACE had a logarithmic 50% lethal dose of 0.44 µm/ml, and no significant difference was observed from the reference vincristine sulfate. These findings highlight the need for further investigation into how different Allium species influence fungal genetic variation and metabolic activities.

Pterula bispora sp. nov. (Pterulaceae) is described as a new species based on morphological and molecular evidence from two specimens collected from Guizhou and Jilin provinces, China. This species is distinguishable by its slender branches, darkened apices, and 2-spored basidia. Phylogenetic analysis of a concatenated dataset of internal transcribed spacer (ITS), nuclear ribosomal large subunit (LSU), and RNA polymerase II subunit B gene (RPB2) sequences revealed that the two accessions were nested within the Pterula clade and were grouped together in a distinct subclade, thus supporting their identification as an unnamed species of Pterula. A full description, illustrations, and the results of molecular phylogenetic analyses for the new species are provided.

High-quality genomic DNA extraction remains a major bottleneck for fungal genomics, particularly for worldwide aerobic and non-photosynthetic mushroom species that rely on their rigid cell walls, interference between metabolites, polysaccharides, etc., and complex genomes. This study systematically compares five DNA extraction protocols involving four distinct sample preparation procedures (fresh (A), filtered (B), frozen (C) and cryogenic mycelium (D)) across mycelial cultures of eight Tunisian fungal strains representing Ascomycota and Basidiomycota to identify the optimal combination for genomic DNA extraction from mycelium. The eight phylogenetically diverse fungal species were analyzed using short-read (MiSeq and NextSeq550) and/or long-read (MinION Mk1C) sequencing technologies, giving a depth coverage between 3.7× and 83×. The generation and quality of the assemblies were assessed within the Galaxy platform, which revealed a gap percentage of 0–0.509%. Taxonomic characterization and phylogenetic inference were performed with SANGER technology using the Internal Transcribed Spacer (ITS) and D1/D2 region of the 26S rRNA gene, assigning the species to our eight different strains: Clitopilus baronii (BS6), Porostereum spadiceum (BS200), Trametes versicolor (BS22-9), Schizophyllum commune (BS23-13), Gloeophyllum abietinum (BS23-14), Irpex laceratus (BS100), Trichoderma asperellum (GC9) and Trichoderma harzianum (S3). The optimized DNeasy Plant Pro Kit protocol with cryogenic biomass treatment presents a safe and cost-effective method for fungal genome sequencing and taxonomic resolution. This integrated comparative evaluation of extraction for sequencing identifies an optimal Qiagen-based extraction strategy combined with cryogenic treatment for eight diverse Tunisian fungal species, guiding method selection based on specific cell wall characteristics rather than proposing a universal protocol limited by unequal replication and strain numbers.

Pneumocystis pneumonia (PcP) caused by Pneumocystis jirovecii is one of the most common and serious opportunistic infections in immunocompromised patients. People at risk for developing Pneumocystis pneumonia include human immunodeficiency virus positive individuals, cancer patients, people receiving immunosuppressive therapy, organ transplant recipients and people with compromised immune systems. Despite effective treatment and prophylaxis, mortality is reported to be between 15-40%, even in people with immunodeficiency other than acquired immunodeficiency syndrome. Although the number of immunosuppressed patients is increasing, diagnostic and epidemiological studies for this agent are still insufficient. This study aimed to investigate the prevalance and epidemiological characteristics of P.jirovecii isolated from various patient groups. Among the 469 bronchoalveolar lavage fluids or sputum samples sent to the medical microbiology laboratory from various units, 114 samples were selected for indirect fluorescent antibody (IFA) testing and molecular studies based on an examination of the patients' files to determine underlying diseases, use of immunosuppressive drugs, corticosteroid treatments received and the presence of patchy or nodular ground-glass opacity on computed tomography scans and the presence of a cyst-like structure on May-Grunwald Giemsa staining. The presence of P.jirovecii was investigated by IFA and polymerase chain reaction methods and genotyping was performed by sequencing of mtLSU rRNA and internal transcribed spacer (ITS) region. The obtained ITS gene region DNA sequences were aligned and TCS network analysis was performed in the aligned region. As a result, positivity was found in eight samples out of 469 samples by using mtLSU rRNA genotyping, Genotype 1 (n= 3; 37.5%) was found to be the most common genotype in our samples. Genotype 2 (n= 2, 25%), genotype 4 (n= 2, 25%) and genotype 3 were the most frequently detected other genotypes, respectively. When the relationships between the sequences were examined, it was observed that our samples were generally related to the samples originating from Iran. In the present study, the genotyping analysis of the ITS region, constructed using the consensus sequence employed in the study by Lee et al., revealed that the most common genotype was Eh (n=3; 42.85%), followed by Bg (n= 2; 28.57%), Bi (n= 1; 14.28%) and Eg (n=1; 14.28%). Our study is the first genotyping study conducted in our country using the ITS gene region. Different epidemiological findings were found in P.jirovecii genotype frequencies in studies conducted in different geographies. This suggests that genetic variations in P.jirovecii have a geographical component and this may affect the distribution of P.jirovecii strains among humans. According to TCS network analysis, our samples are generally associated with samples originating from Iran. None of our samples are found alongside samples originating from India. However, in certain areas of the analysis, New World and Old-World samples exist together. It can be assumed that globalization and thus the increase in human movement over time has led to the spread of different genotypes to different geographical regions and the formation of genotypic mosaics in certain geographical regions. Man-made destruction of nature and the consequent intertwining of urban and rural boundaries, as well as global warming and climate change have undeniably contributed to these movements.

Pesticide use is widespread in agricultural systems in Iraq, raising concerns about their potential impact on the gut mycobiome. This study investigated how residues of a fungicide containing metalaxyl-M and azoxystrobin affect the gut fungal microbiome using a mouse model. Thirty 12-week-old male Swiss albino mice (Mus musculus) weighing 30–40 g were divided into two groups: a control group (n=15) fed a regular diet and a treatment group (n=15) fed the same diet supplemented with 10 ppm of fungicide. Due to high inter-individual variation observed in preliminary analysis and cost constraints, fecal samples from mice within each group were pooled (5 pools per group, 3 mice per pool) prior to DNA extraction, effectively resulting in n=5 biological replicates per group for sequencing. Metagenetic libraries of the internal transcribed spacer (ITS) gene were generated and analyzed using QIIME2, DESeq2, DECIPHER, and Phangorn. A total of 191 amplicon sequence variants of fungal taxa were identified across all samples. The bioinformatic analysis revealed that the gut fungal community was significantly altered by fungicide treatment. Some species were enriched, including uncultured Nakazawaea and Neosartorya hiratsukae, and others were reduced, including Trichomonascus ciferrii, Alternaria conjuncta, and Ophiostoma canum. This study reveals that fungicides used in Iraq have profound effects on gut microbiome communities in mice, and it is recommended to pursue these aspects towards the further development of a research agenda focused on the health implications of dietary fungicide exposure and also on the methods of minimization.

Isodon serra is a hepatoprotective herb widely used in traditional Chinese medicine. In July 2024, plants at a cultivation base in Maoming (21°41'11" N, 110°29'12" E), China, exhibited stem rot-like symptoms, with a field incidence rate of 50%. Initial disease presentation involved foliar chlorosis with brown stem lesions, progressing to leaf desiccation, internodal browning, and eventual plant collapse. Pathogen isolation was accomplished by first surface-sterilizing symptomatic stem segments with 75% ethanol for 30 seconds, followed by treatment with 1% NaClO for 2 minutes. The segments were rinsed thoroughly 3 times with sterile water finally and then cultivated on potato dextrose agar (PDA) at 28°C. The experiment was repeated 3 times, with isolations performed from multiple infected stem tissue segments, resulting in a total of 3 pure cultures with consistent morphology. Hyphal growth initiated within 12h, achieving complete medium colonization within 3-4 days while colony morphology transitioned from white to brownish-black after 7 days. The average diameter of pycnidium was 80.283 μm (n=20); spherical or oblate in shape; a hole on the top was for the release of condidium. The condidium were long oval or elliptical in shape with the average of 8.83 ± 1.39 μm long × 5.87 ± 1.17μm wide (n = 20); colorless transparent; warty protuberance on the surface. Chlamydospores are spherical, colorless and transparent. The morphological features consistent with Macrophomina sp. characteristics (Wu et al., 2022). The genomic DNA was extracted from a representative isolate by Fungi Genomic DNA Extraction Kit for molecular identification. The primer sets ITS4/ITS5, LROR/LR5, EF1-728F/EF1-986R and Bt2a/Bt2b (Wu et al. 2022) were used to amplify the internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), translation elongation factor-1α (tef-1α) and β-tubulin regions (β-TUB). The outcome of a blast of sequences showed 100% identity to M. euphorbiicola sequences (MN270855.1, MN270854.1, MH716048.1 and MF457657.1) in β-tubulin regions. Three groups of specific primers (MeTefF/MeTefR, MsTefF/MsTefR and MpTefF/MpTefR) (Santos et al., 2020) corresponding to the translation elongation factor-1 α were used for verification. The result showed that only the primer of M. euphorbiicola (MeTefF/MeTefR) was able to amplify the band. The result of multilocus sequence analysis (MLSA) (Altschul et al. 1990) constructed by ITS, LSU, tef1-α and β-tubulin also showed 100% homology to M. euphorbiicola MFLUCC 23-00057. Both morphological and molecular characteristics confirmed the fungi was M. euphorbiicola. The sequences were submitted in Genbank under accession numbers (PV544976(ITS), PV549410(LSU), PV595098(tef1-α), PV595099(TUB)). Three 25-cm-tall Isodon serra seedlings each were used for the treatment and control. A combined wound and spore suspension method was applied: treated plants were needle-wounded at the stem base, inoculated with M. euphorbiicola plugs, and drenched with 10 mL of spore suspension (1×10 6 spores/mL). Controls received PDA plugs and sterile water. All plants were kept at 30°C, 90% RH, and a 12-h photoperiod. Symptoms appeared around day 4, followed by wilting and death by day 7, while controls stayed healthy. Re-isolation and PCR with specific primers confirmed M. euphorbiicola as the causal agent. This is the first report of Macrophomina euphorbiicola as the causal agent of stem rot on Isodon serra.

Ligustrum japonicum (Japanese privet) is an evergreen shrub native to East Asia. It is widely cultivated as an ornamental plant. Since September 2023, a necrotic leaf spot outbreak on L. japonicum was observed in Baihe Wetland Park (32°5'42"N, 112°28'13"E) in Nanyang, China. The disease caused sporadic and irregularly shaped brown lesions, with chlorotic halo and dark margin on leaves, and disease incidence was up to 29% among 120 plants surveyed. As the disease progressed, leaf lesions enlarged, became slightly sunken and dark brown, and sometimes coalesced. In severe cases, necrosis extended from leaves into adjacent shoots. To isolate the pathogen, small tissue pieces excised from the margin of necrotic lesions on symptomatic leaves were surface sterilized by immersing in 75% ethanol for 30 s followed by 1% NaClO for 1 min, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 3 days. A total of 22 fungal isolates were obtained. Three representative isolates (Bd1, Bd6, and Bd17) sampled at separate sites were selected for further investigation. On PDA, the colonies were white at first and gradually turned dark gray with irregular margins within 7 days at 25°C. Conidia were hyaline, aseptate, fusiform, measuring 19.6-24.4 × 4.4-8.6 μm (n = 100). These morphological characteristics were connected to Botryosphaeriaceae fungi (Slippers et al. 2014). For molecular identification, the rDNA internal transcribed spacer (ITS), 28S large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha gene (tef1-α), and β-tubulin genes (tub) were amplified using primers ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), EF1-688F/EF1-1251R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were deposited in GenBank under accession numbers OR922737, OR976075, OR976076 (ITS), OR977584, OR977713, OR978107 (LSU), OR993993 to OR993995 (tef1-α), OR947471, OR993991, OR993992 (tub). A phylogenetic tree was constructed using neighbor-joining method based on concatenated ITS, LSU, tef1-α, and tub sequences, together with sequences from type or reference strains. The three isolates clustered closely with the reference strains CBS 115476 and CBS 110302 of Botryosphaeria dothidea in phylogenetic analysis and were clearly separated from other Botryosphaeria species. The molecular analyses confirmed that the isolates are B. dothidea (Slippers et al. 2014). Pathogenicity experiments were performed on healthy L. japonicum seedlings (three-month-old, 60 to 80 cm tall) using conidial suspensions (106 conidia ml-1) as inoculum. For each isolate (Bd1, Bd6, and Bd17), the conidial suspension was sprayed onto the aerial parts (leaves and young shoots) of 10 seedlings. Control plants were sprayed with sterile distilled water. All plants were maintained in controlled-environment chambers at 25°C and 90% relative humidity under a 12 h light/12 h dark photoperiod. Initial symptoms on leaves were first observed at 5 days post inoculation. At 10 dpi, inoculated seedlings developed necrotic brown lesions mainly at the petiole bases and leaf margins. By 21 dpi most affected leaves had abscised, and some lesions occurred on young shoots leading to partial shoot dieback. All inoculated seedlings developed symptoms (30/30 total), whereas control plants remained symptomless. The three isolates caused similar symptoms with comparable disease incidence. B. dothidea was consistently reisolated from symptomatic leaves and shoots, thereby fulfilling Koch’s postulates. B. dothidea is a well-known pathogen that causes diseases in various plants, including Camellia oleifera (Hao et al. 2023), Quercus dentata (Liu et al. 2023), and Cornus officinalis (Zhang et al. 2023). This is the first report of B. dothidea as the foliar disease causal agent on L. japonicum in China. Identifying this pathogen contributes to a better understanding of the disease affecting L. japonicum in this region and provides a basis for future disease management strategies.

Chinese horse chestnut (Aesculus chinensis Bunge) is an important medicinal and ornamental tree species that is widely planted throughout China (Sun et al. 2023). In June 2024, A. chinensis plants in and around Dushan Forest Park (33°04′13″N, 112°35′11″E) in Nanyang exhibited numerous foliar lesions. Across an 8.1-ha area, 100 A. chinensis trees were inspected, and leaf spot symptoms were observed on 59 trees. Early symptoms appeared as small black spots on the leaf margin, which gradually expanded toward the center. Dark halos subsequently developed around the lesions. Five diseased leaves were collected from each of ten individual plants (n = 50). Pieces were excised from the lesion margin and placed on potato dextrose agar. In total, 33 purified fungal strains were obtained from symptomatic leaves. Three isolates (Q-1, Q-2, and Q-5) from different sampling sites were selected for additional analysis. On PDA, colonies produced abundant aerial mycelia. The colony surface was olivaceous to dark green, and the reverse was greyish brown to dark brown (Figure S1c, d). Conidia were produced in unbranched chains of 5 to 11, obclavate or spheroidal, dark brown, with 2 to 5 transverse septa and 0 to 3 longitudinal septa, measuring 11.5 to 37.2 × 7.3 to 11.8 μm (n = 100) (Figure S1e, f). These morphological characteristics are consistent with Alternaria spp. (Simmons 2007). Genomic DNA was extracted from fresh mycelia using the CTAB method. The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and the second largest subunit of RNA polymerase II (rpb2) were amplified by PCR using primer pairs ITS1/ITS4, EF1-728F/EF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively, following the PCR protocols described in the corresponding references. Sequences were deposited in GenBank: ITS (PV342512, PV342513, PV412997), tef1 (PV417283–PV417285), gapdh (PV528059, PV528060, PV543653), and rpb2 (PV528056–PV528058). A maximum-likelihood phylogeny based on the concatenated ITS-tef1-gapdh-rpb2 dataset was inferred in MEGA12. The Kimura 2-parameter model with gamma-distributed rate variation and a proportion of invariant sites (K2+G+I) was selected as the best-fit nucleotide substitution model. It placed all three isolates within the Alternaria alternata clade (Figure S2), clearly separated from other congeners (Woudenberg et al. 2015). Together, morphological characteristics and multilocus evidence identified the causal agents as A. alternata. Pathogenicity tests were conducted using three representative isolates. For each isolate, five healthy 6-month-old potted A. chinensis seedlings (~30 cm tall) were inoculated (15 seeding in total), and five seedlings sprayed with sterile distilled water served as controls. Inoculation was performed by spraying 50 μL of a conidial suspension onto the leaf surfaces, and the inoculated plants were maintained in a greenhouse at 25 °C and 90% relative humidity. Typical symptoms developed on incubated leaves within 7 days and were consistent with field observations, whereas negative controls remained symptomless (Figure S1g,h). The inoculated fungus was reisolated from symptomatic tissues and confirmed as A. alternata based on morphology, thereby fulfilling Koch’s postulates. Previously reported diseases of A. chinensis in China include leaf blotch caused by Colletotrichum sp. (Sun et al. 2020) and leaf blight caused by Botryosphaeria dothidea (Wang et al. 2025). Our current results constitute the first report of A. alternata as the causal agent of leaf spot on A. chinensis in China. Given the high ornamental value, leaf spot symptoms can reduce aesthetic quality and may lead to premature defoliation under favorable conditions, highlighting the need for timely diagnosis and management. At present, there is no direct evidence demonstrating a host jump from another Aesculus plant. Resolving this question will require broader sampling across hosts and locations, coupled with cross-inoculation assays.

Iris domestica, commonly known as “she gan”, is an herbaceous plant known for its medicinal properties and is distributed throughout China (Tang et al. 2025). In August 2025, leaf spot were observed on I. domestica plants in Handan (36.55°N, 113.26°E), Hebei Province, China (incidence rate 80%), plants in 150 ha were observed to be severely affected by the disease, causing a yield loss of 70%. Initial symptoms appeared on the leaves as circular yellow or brown lesions that expanded over time; later, the leaves turned yellow; and about 1 month later, the whole plant died. To isolate the causal agent, tissues (5×5 mm) from four symptomatic plants were removed from the border of lesions, surface sterilized in 75% ethanol for 30 s and 0.1% mercury chloride (HgCl2) for 1 min, then rinsed three times with sterile distilled water, plated on potato dextrose agar (PDA) at 25℃, and incubated in the dark for 7 days, two consistent surface morphology strains (202520 and 202521) were prepared based on single spore isolates. All isolates surface were gray-green and the reverse were dark green, the conidia were obclavate and light brown, 5 to 9 transverse and longitudinal septate, moderate in size (29.5 to 65.4 μm in length and 15.1 to 18.2 μm in width), the conidiophores were produced in clusters, erect or flexuous, according to the colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF gene), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and major allergen Alt a 1 (Alta1) regions were amplified (accession nos. PX588428 for ITS, PX549681 for tef1, PX549689 for GAPDH, and PX549690 for Alta1) with ITS1/ITS4 (Zhang et al. 2022), EF1-728F/EF1-986R (Glass and Donaldson 1995), GDF/GDR (Templeton et al. 1992), and Alt-F/Alt-R (Hong et al. 2005) primers. When compared with other Alternaria species in GenBank, the isolate exhibited 100% (ITS, LC269975), 100% (TEF-1α, OP374452), 100% (GAPDH, OP352299), and 100% (Alta1, OR486298) similarity with Alternaria iridicola (query coverage=100%, per-centage identity=100%). A phylogenetic tree was constructed in MEGA software (version 11.0.10) (Tamura et al. 2021) using the partial concatenated gene sequences. Neighbor-joining analysis revealed that the isolates were closely related to A. iridicola (100% bootstrap). Pathogenicity tests were performed on 5- to 6-month healthy seedlings of I. domestica, the culture disks of 202520 and 202521 was inoculated via leaf wounding on 36 plants per isolate. For the control treatment, 36 plants were treated with an equivalent area of PDA disks. The experiment was repeated three times. The plants were placed in a greenhouse from 26 to 30℃ and 95% relative humidity. The typical symptom were observed 10 days after inoculation, except on the control samples where no symptoms were observed. The same fungus was successfully reisolated from the symptomatic plant tissue and was reidentified as A. iridicola through morphological characteristics, ITS, TEF, GAPDH and Alta1 sequencing analysis (accession nos. ITS: PX925249, TEF: PX926523, GAPDH: PX926521 and Alta1: PX926522), thereby fulfilling Koch’s postulates. This study represents the first documented occurrence of A. iridicola induced leaf spot on I. domestica in China. The finding is greatly significant for preventing and controlling root rot in I. domestica and reducing economic losses for farmers.

Mytilaria laosensis is a fast–growing and high–yielding timber tree species widely grown in the southern subtropical region of China (Yu et al. 2023). In September 2024, a survey was conducted in Yingde City, Guangdong Province, southern China. Sapling stem canker symptoms were found on M. laosensis, and the incidence rate was 33.12% (investigated 1561 trees and infected 517 trees). The symptoms included dark brown necrotic spots that were sunken and shrunken, with cracked cortex and yellowing leaves. The epidermis was stripped from four stem pices (∼0.5 cm) between the healthy and infected tissues, the pieces were disinfected with 75% alcohol for two minutes, rinsed three times with sterile water, and dried for 10 minutes. They were then placed in Petri dishes on potato dextrose agar (PDA) medium and cultured at 28°C in the dark for 2–3 days to obtain pure cultures. Then, monosporic cultures were obtained (isolates MLP112, MLP414, MLP424, and MLP511). The fungal colonies were initially composed of white hyphae, after 5 days became gray and after 10 days turned black. Immature conidia were hyaline, aseptate, ellipsoid, thick–walled, and mature conidia elliptic or oblong, simple septate, dark brown in color, 19.3–31.3 μm in length and 9.2–15.7 μm in width, (mean length=23.9 μm, width average=11.9 μm, N=40), length–to–width ratio was 2.0. Based on morphological characteristics the fungus was temptatively identified as Lasiodiplodia theobromae (Alves et al. 2008). To confirm the indentity of the isolates, the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1 (tef1) and beta tubulin genes were amplified with the universal ITS (White et al. 1990), EF1–α (Stielow et al. 2015) and β–tubulin (Glass & Donaldson 1995) primers, respectively, and sequenced. The obtained sequences were deposited in Gen Bank (ITS: PV855669, PV855670, PV855671, PV855672; EF1–α: PV903164, PV903165, PV903166, PV903167; β–tubulin: PX564918, PX564919, PX564920, PX564921). BLAST results showed 100% similarity with reliable references of L. theobromae (ITS, OR116087.1; EF1–α, OR536247.1; β–tubulin, MT212400.1). To fulfill Koch’s postulates, 5 mm diameter mycelial plugs of PDA cultures were inoculated into eight M. laosensis stem segments, while four stem segments were inoculated with sterile PDA plugs and served as a control. Both ends of stem segments were sealed with paraffin, Segments were incubated at 28°C and 80% relative humidity. Within 5 to 7 days, inoculation sites exhibited obvious dark brown to black lesions and epidermal necrosis, consistent with symptoms of natural infections. The control stems remained asymptomatic. The fungus was isolated from the tissue and identified as L. theobromae based on morphology and DNA sequencing (ITS, tef1 and β–tubulin). The host range of L. theobromae include more than 500 plants species (Hattori Y. et al. 2023). To the best of our knowledge, this is the first report of L. theobromae causing sapling stem canker on M. laosensis in China. The results of this study will help to develop management strategies of M. laosensis sapling stem canker.

Sugarcane (Saccharum officinarum L.) is an important sugar and economic crop, mainly cultivated in tropical and subtropical regions, where it plays a key role in the local economy. Its stems can be processed into sugar, ethanol, and feed. In October 2025, a severe leaf spot outbreak (disease incidence > 50%) was observed at the sugarcane research institute of Guangxi academy of agricultural sciences of China (22°50′58″N, 108°14′47″E), covering an area of approximately 150 m2. Initially, small brown or reddish-brown spots appeared on the leaves, which later expanded into elliptical or irregular lesions. In severe infections, these lesions coalesced, causing the leaves to wither, yellow, and senesce prematurely. Leaf samples with leaf spot symptoms were collected from infected sugarcane plants for pathogen isolation. Leaf tissues (5 mm²) were excised from the lesion margins, surface-sterilized (75% ethanol, 30 s; 2% NaClO, 2 min), rinsed five times in sterile water, and placed on potato dextrose agar (PDA), followed by incubation in the dark at 28°C. Three morphologically distinct isolates were obtained by transferring hyphal tips to fresh PDA medium. One isolate was preliminarily identified as Colletotrichum siamense (designated Jiang3.2, a new host record) based on morphology and ITS sequencing, and was selected for further characterization. The isolate on PDA formed white to grayish-white colonies with dense aerial mycelium and dark brown to black pigmentation on the reverse. Conidia were unicellular, hyaline, straight, cylindrical with bluntly rounded ends, measuring (12.6-17.8) × (3.4-6.2) μm (n = 100). Hyphae were hyaline and septate. Appressoria were melanized, oval to clavate in shape, irregular, measuring (10.1-15.0) × (5.1-7.4) μm (n = 20). These morphological characteristics are consistent with the genus Colletotrichum. For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and β-tubulin 2 (TUB2) were amplified using the corresponding primer pairs (Weir et al. 2012). All sequences were submitted to GenBank under accession numbers PX851062 (ITS), PX915664 (GAPDH), PX915662 (ACT), and PX915665 (TUB2). A phylogenetic tree based on the concatenated dataset of all gene sequences showed that the isolate clustered with C. siamense. Pathogenicity of the isolate was verified in a potted experiment at 28 ± 2°C, 70–85% relative humidity, and a 12 h light/dark photoperiod. Five sugarcane seedlings were spray-inoculated with a conidial suspension (1×10⁷ conidia/mL), while five control plants were sprayed with sterile water. Typical leaf spot symptoms developed on inoculated leaves within 10 days, while controls remained asymptomatic. Colletotrichum siamense was re-isolated from symptomatic tissues and confirmed by morphology and molecular analysis, fulfilling Koch's postulates. This species has been reported to cause leaf diseases on various hosts, including Capsicum annuum, Macropanax rosthornii, and Annona muricata (de Silva et al. 2019; Beltrán-Peña et al. 2023; Wang et al. 2024). To our knowledge, this is the first report of C. siamense causing ring spot on sugarcane in China. This pathogen poses a threat to production by potentially reducing yield, which could result in significant economic losses for the region. These findings provide a key basis for epidemiological surveillance and sustainable disease management to minimize economic losses.

Cantaloupe (Cucumis melo L.) is a commercially important fruit crop that is widely cultivated in Thailand. In June 2025, anthracnose disease was observed on cantaloupe during postharvest storage at 25–32°C and 70–75% relative humidity over a period of 3 to 7 days in Chiang Mai Province, Thailand. The disease incidence was 15% to 20% among 100 fruits per pallet box. The symptoms appeared as small, water-soaked lesions on the fruit surface that expanded over time, becoming depressed and dark brown to black spots. In advanced stages, the lesions coalesced, leading to extensive fruit rot, and pink to orange spore masses developed on the lesions under humid conditions. Two fungal isolates (SDBR-CMU752 and SDBR-CMU753) with similar morphology were obtained from lesions using the single conidial isolation method (Choi et al. 1999). Colonies on potato dextrose agar (PDA) reached 80–85 mm in diameter after 1 week of incubation at 25 °C. The colonies were white to pale gray, cottony in texture, with the reverse side appearing pale orange to pale brown. Both isolates produced asexual structures after 1 week at 25 °C on PDA. Light conidial masses were observed. Conidiophores were hyaline, septate, and clavate to cylindrical in shape. Conidiogenous cells were also hyaline, clavate to cylindrical, measuring 15–20 × 3–5 μm (n = 50). Conidia were subcylindrical to oblong, rounded tips, guttulate, and sized 10–20 × 4–5 μm (n = 50). The morphological characteristics of the present isolates align with those of Colletotrichum species within the Colletotrichum gloeosporioides species complex. This species complex contains plant pathogens associated with anthracnose diseases and other diseases afflicting various crops (Jayawardena et al. 2021; Suwannarach et al. 2025). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB), actin (ACT), and calmodulin (CAL) regions were amplified using the primer pairs ITS1/ITS4, GDF1/GDR1, T1/Bt2b, ACT-512F/ACT-783R, and CL1C/CL2C, respectively (Weir et al. 2012; Jayawardena et al. 2021). The resulting sequences were deposited in GenBank under accession numbers PX533053 and PX533054 for ITS, and PX582285 to PX582292 for the other four gene regions. A maximum likelihood tree constructed from concatenated ITS, GAPDH, TUB, ACT, and CAL sequences placed the two isolates within the C. siamense clade. To confirm pathogenicity, healthy commercial cantaloupe fruits (Cu. melo) from the market were surface disinfected by 0.1% NaClO for 5 min, rinsed three times with sterile distilled water, and wounded (Nuangmek et al. 2019). Conidia were collected from 2-week-old cultures on PDA and suspended in sterile distilled water. Fifteen microliters of a conidial suspension (1 × 106 conidia/ml) were dropped onto the wounded fruits. Mock inoculations were performed using sterile distilled water as a control. Each treatment was conducted with ten replications and repeated twice. The inoculated fruits were stored individually in sterile plastic boxes at 25°C with 70 to 80% relative humidity. After 7 days, all inoculated fruits exhibited brown to dark brown lesions, while control fruits remained asymptomatic. Colletotrichum siamense was consistently reisolated from the inoculated tissues on PDA and identified to complete Koch's postulates. Cantaloupe fruit is commonly affected by fungal pathogens such as Alternaria, Colletotrichum, Didymella, and Fusarium species (Nuangmek et al. 2019; Giménez-Santamarina et al. 2025). Prior to this study, C. siamense was known to cause anthracnose on several fruit crops worldwide, including avocado, culinary melon, mango, papaya, and strawberry among others (Talhinhas & Baroncelli 2023; Suwannarach et al. 2025), but has not been reported in cantaloupe fruit. To our knowledge, this is the first global report of C. siamense causing postharvest anthracnose on cantaloupe fruit. The outcomes will contribute to guiding epidemiological studies and further developing improved management approaches for the disease.

Pluteus sinensis , a new species of Pluteus sect. Celluloderma from China, is described and illustrated based on morphological and molecular data. It is mainly characterized by terrestrial habitat, very small to small basidiomata, slightly umbonate at disk, a cracking pileus surface, broadly ellipsoid basidiospores measuring 5.5–6.5 × 4.5–5.5 µm, fusoid pleurocystidia, an epithelioid hymeniderm pileipellis with a predominantly mucronate apex, and the common presence of narrowly cylindro‐clavate caulocystidia. Phylogenetic analysis based on internal transcribed spacer (ITS) region indicated that P. sinensis forms a well‐supported, independent lineage corresponding to cinereofuscus clade and distinct from previously known Pluteus species. A morphological description and illustration of the new species, as well as comparison with closely related taxa of Pluteus, are provided.

Pennycress (Thlaspi arvense L.), a Eurasian annual herb widespread in China, is a promising winter oilseed cover crop. It controls soil erosion, runoff, nutrient leaching and weeds, supports insects and pollinators, and its oil is ideal for renewable jet fuel production (McGinn et al. 2019). In 2023 and 2024, a 30%–40% incidence of stem rot in pennycress was observed in Minzhu Town (45°49' N, 126°48' E), Harbin City, China. Initially, the leaves of affected plants exhibited yellowing during the flowering stage. Then, bottom part of stem turned brown and the whole plant lodging, and then died. 5 randomly selected typical disease plants were collected. Diseased tissue at the stem bottom was cut into small pieces, surface sterilized with 70% ethanol for 30 s and 1% NaClO for 5 min, and rinsed three times in sterile H2O. Subsequently, the pieces were plated onto the potato dextrose agar (PDA) medium at 28℃ for 5 days. A single-spore culture was obtained by monosporic isolation. After incubation on PDA at 28℃ for 7 days, the single-spore isolates initially formed abundant white aerial mycelium, then gradually developed a rose pigmentation with a brownish color in the center and grayish rose at the periphery of the colony (Li et al. 2019). The macroconidia of the isolates were slender, falcate, distinctively curved in the bottom half of the apical cell, and had 3 to 5 septa. Their size ranged from 29.8–49.5 µm × 4.9–7.1 µm (n = 50). Microconidia were irregularly oval and with zero to one septa. The morphological characteristics of the isolates were consistent with Fusarium acuminatum (Leslie and Summerell 2006). One representative isolates (MZT-2) identified putatively as F. acuminatum based on morphology. To confirm the species identity, the partial sequence of translation elongation factor 1 alpha (TEF1-a) gene, rDNA internal transcribed spacer (ITS) and RNA polymerase II second largest subunit (RPB2) region were amplified and sequenced (O’Donnell et al. 2015; White et al. 1990; Ponts et al. 2020). The sequences were deposited in GenBank under accession numbers PV067929, PP794646, and PQ656806, showing 100%, 97%, and 98% identity with F. acuminatum isolates MW423623, OQ704301, and OP838086, respectively. For pathogenicity test, a conidial suspension (1 × 106 conidia per mL) was prepared from a 7-day-old culture of the representative isolate MZT-2 grown in potato dextrose broth. Ten plants were drenched with 200 mL of conidial suspension every two days. As control plants were drenched with 200 mL of sterile water every two days until disease symptoms appeared. All pots were maintained in a greenhouse (26℃ ± 2; 60%–70%; 16 h light/8 h dark). The experiment was repeated three times with three replicates in each. The leaves of the inoculated plants gradually turned yellow and wilted within 10–15 days; the epidermal tissue of the stem showed brown discoloration. Eventually, the plants died within 28–35 days. The control plants exhibited no symptoms. The F. acuminatum pathogen was re-isolated from the diseased stem bottom of inoculated plants, and its morphology and molecular characteristics were identical to those of the original isolate, thus fulfilling Koch's postulates. Previous studies reported that F. acuminatum causes root rot in Schisandra chinensis , leaf spot in Atractylodes lancea, and white fruit disease in Morus alba (Chen et al. 2022; Zheng et al. 2023; He et al. 2025). However, to our knowledge, this is the first report of F. acuminatum causing stem rot in pennycress in China.

We report a rapid increase in dermatophyte infections in New York City and surrounding counties from 2022 to 2024. Reported cases include 135 of Trichophyton indotineae (Ti), 39 of Trichophyton mentagrophytes genotype VII (TmVII), and 14 of T. mentagrophytes genotype II* (TmII*). These numbers highlight the urgent need for rapid, high-throughput identification, drug-resistance testing, and surveillance of these pathogens. To address these challenges, an automated deep learning model was developed using internal transcribed spacer (ITS) sequences from 28 well-defined genotypes of Trichophyton interdigitale/T. mentagrophytes Species Complex (TiTmSC). Ti, previously classified as T. mentagrophytes genotype VIII, is now recognized as a distinct species. The model enabled rapid and accurate identification of all 28 genotypes directly from Sanger sequencing output files. In vitro antifungal susceptibility testing (AFST) demonstrated that 57% of Ti isolates exhibited high minimum inhibitory concentrations (MICs) to the first-line drug terbinafine, and 38%-52% showed elevated MICs to azoles. In contrast, all TmVII and TmII* isolates had low MICs to both terbinafine and azoles. Whole-genome sequencing (WGS) of a subset of Ti, TmVII, and TmII* isolates revealed evidence of both clonal transmission and independent introductions. This integrated approach, combining rapid identification, drug resistance testing, and transmission analysis, is expected to improve patient care and infection control measures.IMPORTANCEThis report presents a comprehensive laboratory investigation into the emergence and rapid spread of Trichophyton indotineae (Ti), Trichophyton mentagrophytes genotype VII (TmVII), and T. mentagrophytes genotype II* (TmII*) in New York City and its surrounding counties. A high-throughput pipeline for the identification of Trichophyton species and genotypes is described. Approximately 57% of Ti isolates exhibited elevated minimum inhibitory concentrations (MICs) to terbinafine, while 38%-52% demonstrated elevated MICs to azoles, raising significant concern. Whole-genome sequencing revealed evidence of both clonal spread and independent introductions of Ti, TmVII, and TmII*. These findings have important implications for patient care and infection control measures.

Hydnellum is an ectomycorrhizal fungus with important ecological and medicinal value. However, the species diversity of Hydnellum in China remains poorly understood. To deepen the understanding of the diversity of Hydnellum species in China, this study, based on a combination of morphological observations and molecular phylogenetic analysis of the internal transcribed spacer (ITS) and nuclear ribosomal large subunit (nrLSU) regions, identified and described four new species: H. aureoluteum sp. nov., H. aureotomentosum sp. nov., H. fuscozonatum sp. nov., and H. pileospinosum sp. nov. For each new species, we provided detailed morphological descriptions, hand-drawn illustrations, and comparisons with closely related taxa. In addition, this study systematically compiled key morphological characteristics and ecological distribution data for all known Hydnellum species in China and constructed a dichotomous identification key. This work provides an important basis for taxonomic research on the genus Hydnellum and enhances our understanding of its ecological distribution patterns in China.