, Soybean (Glycine max L.) is an important oil, food, and economic crop worldwide (Mishra et al., 2024). Due to its high protein and fat content, soybean serves as a raw material for food, feed, and various industrial sectors. Soybean originated from China, which ranks fourth in terms of planting area and total production, following rice, maize, and wheat in China (Di et al., 2023). In soybean production, biological and abiotic diseases are critical factors limiting both yield and quality, especially pests and diseases (Rupe and Luttrell, 2008). In recent years, soybean root diseases, seed-borne diseases, and leaf related diseases have garnered increasing global attention, particularly in regions with intensive soybean cultivation (Lin et al., 2022). Therefore, effectively controlling technologies to pests and diseases is an urgent issue to reduce yield losses in soybean production, particularly in light of the severe diseases occurs or breaks out on a large scale in recent years.
Paramyrothecium spp. is predominantly isolated from soil, where it acts primarily as a saprophyte, decomposing organic plant materials (Krisai-Greilhuber et al., 2017; Lombard et al., 2016). Notably, it often emerges as an important plant pathogen and infects root of diverse plant species (Aumentado and Balendres, 2022; Moumni et al., 2020; Rennberger and Keinath, 2020). The life cycle of Paramyrothecium is restricted to an asexual stage during pathogenic development, with no sexual morphs identified to date (Krisai-Greilhuber et al., 2017; Lombard et al., 2016). The genus is characterized by the production of septated, branched, and filamentous hyphae, which facilitate nutrient uptake and promote colonization on plant tissues. Its sporodochial conidiomata produced masses of conidia that range in pigmentation from olivaceous green to dark green (Aumentado and Balendres, 2022; Lombard et al., 2016). Conidial production occurs on conidiophores and serves a pivotal role in dispersal and survival in diverse environmental conditions. Morphologically, the conidia can be single-celled or septate (typically with one septum), with shapes ranging from cylindrical to oval. These structures are generally smooth, exhibit straight to slightly curved forms, and are transparent to light green in appearance (Lombard et al., 2016). A distinguishing trait of Paramyrothecium is the presence of thin-walled setae enclosing the sporodochia, typically bearing 1-3 septa, which differentiates it from closely related Myrothecium genera (Lombard et al., 2016). These physiological and structural adaptations enable Paramyrothecium to occupy a broad range of ecological niches spanning soil ecosystems and plant hosts (Lombard et al., 2016; Matić et al., 2019).
Paramyrothecium (Stachybotryaceae, Hypocreales, Sordariomycetes) was established to reclassify taxa previously assigned to Myrothecium sensu lato (Lombard et al., 2016; Wijayawardene et al., 2020). Morphological characteristics combined with molecular analyses demonstrated that this group fungi were distinct from other Myrothecium species, leading to the formal recognition of the genus Paramyrothecium by Lombard et al. (2016). This genus comprises primarily plant-pathogenic fungi. Among the 20 species currently identified in Paramyrothecium genus, P. foliicola and P. roridum are the most widely reported as causal agents of leaf spot diseases in a diverse range of plant hosts (Ben et al., 2017; Chen et al., 2016; MycoBank, 2025; Withee et al., 2022). P. foliicola has been recorded on various economically significant crops, including melon (Cucumis melo) (Sabahi et al., 2022), tomato (Solanum lycopersicum) (Huo et al., 2023), and eggplant (Solanum melongena) (Aumentado and Balendres, 2022). It has also been referred as stem canker of cucumber (Cucumis sativus) (Huo et al., 2021) and apple (Malus domestica) (Azizi et al., 2020). Similarly, P. roridum and P. breviseta have been reported on coffee (Coffea sp.), causing leaf spots and stem rot (Huaman-Pilco et al., 2023; Wu et al., 2021). Additionally, P. roridum has been identified as the causal agent of crown canker on false dragonhead (Physostegia virginiana) (Wang et al., 2021b), mulberry (Morus sp.), and muskmelon (Cucumis melo). Recent taxonomic advancements reported the genus Paramyrothecium was added new members, P. eichhorniae and P. vignicola. P. eichhorniae was isolated from Aristolochia sp., Centrosema sp., Eichhornia crassipes, Oroxylum indicum, Psophocarpus sp., Spilanthes sp., and P. vignicola was isolated from Coccinia grandis, Commelina benghalensis, Lablab purpureus, Solanum virginianum, Vigna mungo, Vigna sp., and V. unguiculata, which indicated the genus Paramyrothecium was important related to ecological diversity and agricultural significance (Pinruan et al., 2022; Withee et al., 2022).
More than 30 species of the genus Myrothecium have been reported worldwide (Seifert et al., 2011), and listed in Index Fungorum (2025). It is difficult to confirm Myrothecium species due to the similarity of morphological features and the absence of molecular data (Chen et al., 2016, Lombard et al., 2016). Additionally, numerous studies emphasize the pivotal role of the phylogenetic relationships for phytopathogens using multi-gene phylogenetic analysis, particularly for Myrothecium species (Jayawardena et al., 2016, 2019; Phoulivong et al., 2010). In order to increase the accuracy of Paramyrothecium spp. identification, molecular methods were applied for rapid identification of Paramyrothecium species, particularly using multi-locus combined phylogenetic analysis (Bhunjun et al., 2021; Cai et al., 2009; Hyde et al., 2018). The related loci for identification of Paramyrothecium species were mainly composed of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cmdA), RNA polymerase II second largest subunit (rpb2), and β-tubulin (tub2) gene regions, and any combination of them can be used to analyze and identify Paramyrothecium species (Lombard et al., 2016).
During the survey of soybean diseases in late August, 2024, typical symptoms of leaf spots were observed on the soybean plants growing in Changping district, Beijing, China. The infected leaves became brown to black and produce black stroma arranged in a circular pattern on necrotic areas. To confirm the causal agent, the diseased leaves of soybean plants were collected for isolating the pathogen. The objective of the present study was to identify the pathogen species causing the soybean leaf spots by using morphological and molecular analysis.
Materials and Methods
Disease survey and sample collection
In late August 2024, a field disease survey of soybean was conducted by our research team in Beijing, China. The new symptoms on soybean leaves were observed resembling with leaf spot caused by Paramyrothecium spp., in a field located in Changping District (40°13′N, 116°14′E), Beijing, China. Diseased leaves were characterized by circular, brown necrotic lesions with tiny black particles. The incidence of diseased plants with these symptoms was approximately 20%, with affected plants scattered across several areas within the field. To confirm the causal agents, we collected diseased leaves showing typical symptoms of circular, brown necrotic lesions, primarily characterized by round or oval spots varying in color from light to dark brown. These samples were subsequently used for pathogen isolation and further studies.
Pathogen isolation
The fresh leaf lesions were cut into several 2-3 mm sections from the margins of diseased areas (1/3) and healthy parts (2/3). After being sterilized in 2% NaClO for 2 min, the tissues were subsequently rinsed three times with sterile distilled water and dried on sterilized filter paper. Every three to four pieces were cultivated on a potato dextrose agar (PDA; AoBoXing Biotech, Beijing, China). Plates were incubated at 25°C for 2-3 days. Fungal isolates were purified by transferring single hyphal tips to a fresh medium. The plates were incubated at 25°C for 7-10 days. All isolates obtained were stored in 30% glycerol at −80°C for long-term preservation.
Morphological characteristics
The 5-mm-diameter mycelial plugs of each isolate were cut from the edge of an active colony by a puncher. The morphological characteristics of these isolates were observed by sub-culturing at 25°C for 21 days on PDA. The colony produced a lot of stroma and released conidia. The morphology of isolates were observed and measured under a light microscope (Olympus CX31, Tokyo, Japan).
Molecular characteristics and phylogenetic analysis
To identify the Paramyrothecium isolate at a molecular level, those were cultured on cellophane-covered PDA for 10 days at 25°C. The mycelia of isolate were scraped from the cellophanes with a sterilized blade. Genomic DNA was extracted by using the Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. Purified DNA was suspended in 1× TE buffer and stored at −20°C for later use. Partial sequences of the four gene regions of ITS, RNA polymerase II second largest subunit (rpb2), β-tubulin (tub2) and calmodulin (cmdA) gene were amplified on three representative isolates (DQ1, DQ2, and DQ3) by using the universal fungal primers ITS4/ITS5 for ITS (White et al., 1990), RPB2-5F2/RPB2-7cR (O’Donnell et al., 2007) for rpb2, Bt2a/Bt2b (Glass and Donaldson, 1995) for tub2 and CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al., 2013) for cmdA, respectively (Table 1). PCR reactions were carried out using a Gene Amp 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) in 50 μL reaction mixtures containing 25 ng of DNA, 2 μL of each primer, 25 μL of 2×Taq PCR Mastermix (TIANGEN, Beijing, China), and 17 μL ddH2O. The cycling protocol was as follows: an initial denaturation for 5 min at 95°C, followed by 35 cycles of denaturation at 94°C for 45 s, 45 s at 55-65°C (depending on the primer-specific annealing temperature), and extension at 72°C for 60 s, with a final extension of 72°C for 10 min. All PCR products were analyzed using 1.5% agarose gel with the Gelgreen Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). Then, a cloning method was applied to generate a sequence from the obtained amplicons by the four primer pairs, respectively (Sangon Biotech, Shanghai, China). The resulting sequences for each isolate were aligned with Multalin (http://multalin.toulouse.inra.fr/multalin; accessed on 25 October 2024). Phylogenetic analysis was performed based on the concatenated multiple gene regions of the nuclear sequence dataset (ITS-cmdA-rpb2-tub2) using the MEGA X with maximum likelihood analysis and Tamura-Nei distance model with 1000 bootstrap replicates (Kimura, 1980; Kumar et al., 2018). And BLAST online tool in the GenBank database (National Center for Biotechnology Information, NCBI) to compare sequence similarities.
Pathogenicity and host range tests
Firstly, pathogenicity tests were performed on original host seedlings of soybean. Afterwards other several legume crops hyacinth bean (Lablab purpureus), common bean (Phaseolus vulgaris), faba bean (Vicia faba), pea (Pisum sativum), mung bean (Vigna radiata), adzuki bean (Vigna angularis), lentil (Lens culinaris), cowpea (Vigna unguiculata) and peanut (Arachis hypogaea) were inoculated for host range test of the pathogen (Table 2). The ten tested crops were sown in 500 mL paper cups filled with a mix of equal volumes of vermiculite and peat. The planted cups were randomly distributed on a greenhouse bench and incubated at 25°C under a 12 h photoperiod and watered regularly every 3 to 4 days. After the crops reached the stage of fully expanded true leaves, the seedlings were performed by spraying spore suspension (1.0 × 106/mL) of isolates, whereas the controls were inoculated with sterile water. The treated plants were incubated in a misting room at 25°C under high humidity (>90%) for 48 h. Misting room was controlled by an automatically centrifugal humidifier for spraying 30 min every 2 h, which maintained leaf wetness without excessive runoff. Then, the plants were removed to a greenhouse maintained at 25°C until the plants were rated for disease symptoms. Plants were monitored every day to detect symptoms. Pathogen was re-isolated from inoculated plants to verify Koch’s postulates. All tests were conducted twice.
Inoculum concentration screening
To screen inoculation concentration of P. vignicola isolate suspension, this study was performed inoculation concentration screening experiment. This will provide important information for screening resistant germplasms. The experiment was designed six treatments, including sterile water (CK), and conidial suspension at a concentration of 1 × 102 spores/mL, 1 × 103 spores/mL, 1 × 104 spores/mL, 1 × 105 spores/mL, 1 × 106 spores/mL, respectively.
Conidial suspensions were made in the same process with above pathogenicity tests. The prepared conidial suspension is quantified using the hemocytometer and then diluted with sterile water to obtain suspensions of different concentrations. The leaves were sprayed with spore suspension till run-off and the plants were maintained with regular watering and observed for the development of leaf spot. All tests were conducted twice.
Data analysis
Pathogenicity was evaluated by the percentage (%) of diseased areas, which was assessed following the method described by Tsushima et al. (2019). The disease severity included five rating scales: −, not susceptible (0, no infection or lesion observed); +, moderately susceptible (<1-30% diseased area relative to the healthy portion); ++, susceptible (31-50% diseased area relative to the healthy portion); and +++, highly susceptible (>51% diseased area relative to the healthy portion). Data were analyzed using one-way analysis of variance. Furthermore, multiple mean comparisons were conducted using Tukey’s honest significant difference test at a 95% significance level (Tukey, 1951). Statistical analyses were performed using SPSS statistical software (version 26; IBM Corp., Armonk, NY, USA).
Results
Disease symptoms
During the disease surveys, a new disease of leaf spot was found in soybean growing in the fields. It was observed by the naked eyes that about 20% infected plants were distributed sporadically in a few spots in the field. The affected plants exhibited typical Paramyrothecium leaf spot symptoms. showing brown to black lesions with crater-like shapes from shallow to deep, and brown to black sporodochia were arranged in a circular pattern on the lesions (Fig. 1A, 1B).
Morphological characteristics
Colony of isolates (DQ1, DQ2, and DQ3) on PDA medium was flat, with an ‘entire’ margin. Aerial mycelium was abundant, white and woolly after 7 days of incubation. Concentric rings with abundant sporodochia were produced after 3 weeks of incubation, and the colony undersides were yellowish (Fig. 1C). In addition, prominent and bright, slimy black masses with greenish-olivaceous sporodochia were observed on seven-day-old culture on PDA (Fig. 1D). Conidiophores branched repeatedly (Fig. 1E). The conidia were a septate, hyaline, smooth, cylindrical, to ellipsoidal-shaped. The conidia were rounded at both ends and measured an average of 5.83 × 1.93 μm (n = 30), and ranged from 5.25-6.51 × 1.49-2.22 μm (Fig. 1F). All morphological characteristics of our isolates were consistent with the previous description of P. vignicola (Lombard et al., 2016).
Molecular characteristics and phylogenetic analysis
To confirm morphology identification, Molecular sequencing and phylogenetic analysis of multiple gene regions were conducted. The ITS, cmdA, rpb2, and tub2 gene regions amplified from the three isolates were sequenced using their respective common primers. Sequences of the ITS, cmdA, rpb2, and tub2 genes obtained from the three isolates showed 99-100% identity with numerous reported strains of P. vignicola in GenBank by BLAST analysis. The aligned sequences of the four loci (ITS, cmdA, rpb2, and tub2) were cascaded, and 3410 bp (ITS: 1-569, cmdA: 570-1355, rpb2: 1356-2425, tub2: 2426-3411) sequences of the three isolates and those from reported Paramyrothecium spp. strains were used to phylogenetic analysis in the dataset (Supplementary Table 1).
The phylogenetic tree was constructed using a concatenated sequence dataset of the four gene regions (Fig. 2). In the phylogenetic tree, the three isolates were grouped with those of eight reference strains of P. vignicola from Thailand in GenBank. The phylogenetic analysis revealed that the three isolates could be distinguished from other species of the Paramyrothecium spp. Altogether, the results of the molecular characteristics and phylogenetic analysis strongly support that the three isolates clearly belong to P. vignicola. This is consistent with the result of morphology identification.
Pathogenicity and host range tests
Firstly, the pathogenicity of the three P. vignicola isolates was tested on the original host, soybean. The results showed that all isolates were strongly pathogenic to soybean. All inoculated plants showed typical symptoms similar to those observed in the field, they showed brown water-soaked spots initially, and then gradually become darker. As the disease developed, smaller spots coalesced to form blighted areas on the leaves, small dark olive to black drops can be noticed on the spots, which produced lots of sporodochia (Fig. 3A). In advanced stages black sporodochia were observed with irregularly shapes surrounded by white mycelia. The stroma structures appeared in concentric rings arrangement surrounded by the necrotic areas on the diseased leaves. Seriously infected leaves turned yellowish and defoliated. The pathogens re-isolated were performed with the brown leaf spot of infected leaf on PDA medium. After a week, white floccose colonies produced circular growth of sporodochia in dark green to black concentric with rings pattern and bearing masses of conidia. Pathogenicity test was confirmed, following Koch’s postulates. There were no disease symptoms in the control plants.
After completion of pathogenicity test, the host range of the P. vignicola isolate DQ1 was tested on nine additional legume crops. The isolate DQ1 showed different pathogenicity on all tested legume crops. The isolate DQ1 showed strong virulence and ultimately led to plant death on original host soybean with reaction from susceptible to highly susceptible. And it also showed strong virulence with highly susceptible to other six legume crops, including hyacinth bean, common bean, faba bean, pea, mung bean and lentil, which caused symptoms similar to those observed on soybean (Fig. 3B-3F, 3H). It showed moderate virulence with susceptible to adzuki bean and causing symptoms characterized by small brown spots or necrotic lesions with dark borders and blight (Fig. 3G). By contrast, the isolate DQ1 displayed only mild symptoms with moderately susceptible or without pathogenicity on cowpea and peanut (Fig. 3I, 3J). It is interesting that different cultivar of cowpea and peanut appeared varied reactions to P. vignicola isolate. Thus, these results indicated that P. vignicola from soybean may infect other legume crops under conducive conditions, especially for hyacinth bean, common bean, faba bean, pea, mung bean and lentil (Table 2).
Inoculum concentration screening
To accurately evaluate resistance phenotype of soybean germplasms, this study conducted inoculum concentration screening tests. Seventeen soybean varieties were used and inoculated with five different inoculation concentrations of P. vignicola. The results showed that the reactions were significant different inoculated with five different inoculation concentrations of P. vignicola (Table 3). As the spore concentration increased, the symptoms of soybean plants gradually become more and more seriously. When inoculated with lower spore concentration, the plants only showed mild symptoms. At a low inoculum concentration of 1 × 102 spores/mL, most of the soybean cultivars displayed only mild symptoms with moderately susceptible, only 2 of 17 soybean cultivars showed susceptible. When treated with 1 × 103 spores/mL, the soybean cultivars showed from moderately susceptible to susceptible. Notably, at 1 × 104 and 1 × 105 spores/mL, the soybean cultivars showed from susceptible to highly susceptible. But the reaction of 12 soybean cultivar changed from susceptible to highly susceptible with the spores concentration increasing. And the symptoms increased significantly, treated from 1 × 104 to 1 × 105 spores/mL, it indicated that symptoms of soybean cultivars couldn’t develop fully under the insufficient pressure treated with 1 × 104 spores/mL. In contrast, treatment with 1 × 106 spores/mL, all soybean cultivars showed highly susceptible and all plants became wilting dead finally, it means this treat couldn’t distinguish the resistance of soybean cultivars under excessive inoculation pressure. Therefore, these results indicates the concentration of 1 × 105 spores/mL is the most suitable to use for P. vignicola resistance screening.
Discussion
Soybean is a crucial crop for protein and oil production, widely grown globally for food, animal feed, and industrial purposes (Han et al., 2021; Wilson, 2008). China, faces challenges in domestic production due to being the largest importer (Si and Han, 2021). To reduce reliance on imports, China launched the Soybean Industry Revitalization Plan to boost local production and improve yields (Xu et al., 2008). However, pests and diseases remain a major challenge (Li and Zhang, 2018). Approximately 500 known pests and diseases affect soybean production in China, with more than 50 causing significant yield losses (Ye et al., 2023). These include long-standing issues, such as root rot, cyst nematode disease, viral diseases, gray spot, anthracnose, grubs, Leguminivora glycinivorella, Etiella zinckenella, Ergania doriae yunnanus, Aphis glycines, Bemisia tabaci, Riptortus pedestris, and Spodoptera litura (Chen, 1995; Ministry of Agriculture and Rural Affairs, 2022). In recent years, new or increasingly severe outbreaks of pests have occurred due to changes of climate and planting pattern, including “symptomatic green” disease (Gao et al., 2022; Wang et al., 2021a), rust (Shan and Zhou, 2007), stem rot (stem and pod blight) disease (Zhao et al., 2022), and Beet armyworm (Wang and Fan, 2019).
Plant diseases significantly impact food security (Ristaino et al., 2021), and fungi are major contributors of crop losses (Summuna et al., 2015). Foliar fungal pathogens, in particular, can severely reduce the yield and quality of crops (Iqbal et al., 2018). In this study, we discovered an emerging disease showing leaf spot in soybean from Changping District, Beijing. Based disease symptoms in the field, we suggested the pathogen might be Paramyrothecium spp. Paramyrothecium is widely distributed across diverse geographic regions, with species identified on various hosts and substrates throughout Asia, the Americas, and Europe (Farr and Rossman, 2024). Paramyrothecium species have frequently been identified as the causative agents of leaf spot and blight diseases on a wide range of vegetables, ornamental plants, and economically significant crops (Farr and Rossman, 2024; Lombard et al., 2016; Matić et al., 2019; Soliman, 2020). In addition to leaf damage, these fungi may also cause stem and crown cankers, as well as fruit rot (Azizi et al., 2020; Chen et al., 2018; Haudenshield et al., 2018). Lombard et al. (2016) designated an epitope for the type species, P. roridum (Myrothecium roridum). Paramyrothecium species are distinguished from closely related Myrothecium by the 1-3 septate of conidia, thin-walled setae surrounding the sporodochia. Recently, 19 species of Paramyrothecium have been listed in Index Fungorum (http://www.indexfungorum.org/; accessed on 20 April 2025).
In our previous study, a leaf spot of mung bean caused by P. foliicola was discovered for the first time during the disease investigating of the legume crops in China, and its pathogenicity was confirmed to original host mung bean (Sun et al., 2021). In the present study, pathogenicity and host range of two isolates respective from soybean and mung bean, P. vignicola and P. foliicola, there were also performed. In this study, a comparison of the morphological characteristics of the P. foliicola and P. vignicola revealed notable differences. P. foliicola colonies on PDA were characterized by abundant white aerial mycelium with sporodochia forming on both the aerial mycelium and the surface of the medium, covered by slimy olivaceous green to black conidial masses. By contrast, morphological identification of our isolates indicated that the colony and conidial morphology were the same as those of P. vignicola (Withee et al., 2022). P. vignicola colonies on PDA exhibited dense, circular, flattened, slightly raised, floccose white aerial mycelium, with radiating concentric rings of sporodochia and slimy olivaceous green to black conidial masses. Recently, Armand et al. (2023) revealed a new species P. amorphophalli from symptomatic leaves of elephant foot yam (Amorphophallus sp.), collected from Chiang Rai, Thailand based on distinct morphological characteristics and phylogenetic analyses. Comprehensive sampling across a variety of hosts, employing both morphological and molecular techniques, is critical for elucidating the ecological dynamics and pathogenic potential of this genus. Therefore, in this study, we also conducted phylogenetic analysis combined with multiple gene sequence dataset. Molecular identification showed that the ITS, cmdA, rpb2, and tub2 gene regions of the three isolates were highly similar to P. vignicola strains available in Genbank. The multi-locus phylogenetic analysis based on combining the four genes clustered the three isolates into the P. vignicola phylogenetic group (Fig. 2). Therefore, based on the morphological characteristics, molecular characteristics, and multi-locus phylogenetic analysis, the three isolates DQ1, DQ2, and DQ3 were identified as P. vignicola.
The host ranges of P. vignicola and P. foliicola are also significant different. The representative P. foliicola isolate QB1 from mung bean demonstrated high pathogenicity to a wide range of legume crops, including mung bean, soybean, hyacinth bean, common bean, faba bean, pea, adzuki bean, lentil, cowpea, and peanut. By contrast, the representative P. vignicola isolate DQ1 from soybean showed high pathogenicity to other legume crops, including soybean, mung bean, hyacinth bean, common bean, faba bean, pea and lentil. The pathogenicity of the two isolates from mung bean and soybean are significant different on three of ten inoculated crops, adzuki bean, cowpea, and peanut. As for adzuki bean, P. vignicola isolate DQ1 and P. foliicola isolate QB1 showed moderated and strong pathogenicity, respectively. As for cowpea and peanut, P. vignicola isolate DQ1 and P. foliicola isolate QB1 showed mild and strong pathogenicity. Paramyrothecium species are known to infect both their original hosts and novel hosts, exhibiting host shifts within the same plant family and host jumps across different families (Aumentado and Balendres, 2022; Rennberger and Keinath, 2020). The expansion of host range may be associated with changes of planting pattern and climatic conditions (Matić et al., 2019).
Since disease symptoms is influenced by inoculation quantity of pathogen, such as spore suspension concentration. Thus, pathogenicity or disease resistance tests should select an appropriate spore concentration, which is crucial for disease resistance evaluation. Therefore, this study was conducted to investigate the effect of spore suspension concentration on disease severity using the spore suspension spraying method. Five different spore suspensions were designed in order to determine the appropriate concentration for resistance identification experiments. The results showed that the disease symptoms became more and more serious with the spore suspension concentration increasing, indicating a positive correlation between inoculum concentration and disease severity. At lower concentrations (1 × 102-1 × 104 spores/mL), plants exhibited only mild to moderate symptoms, which were insufficient to fully reveal the variation in susceptibility among cultivars. At the highest concentration (1 × 106 spores/mL), all cultivars exhibited severe wilting and death, indicating that excessive inoculum pressure can make loss of the host resistance. In contrast, a concentration of 1 × 105 spores/mL, the inoculated plants developed full disease symptoms, which can distinguish highly susceptible and susceptible varieties. Therefore, we selected it as the appropriate inoculation concentration. P. vignicola was first isolated from leaf spots on Vigna sp. in northern Thailand by Patchareeya Withee et al. (2022). Further cross-pathogenicity tests revealed that these pathogens exhibited pathogenicity on several plants, including Coffea arabica, Commelina benghalensis, Glycine max, and Dieffenbachia seguine. Recently, P. vignicola has been reported causing leaf spot on B. chinensis var. parachinensis in Thailand (Haituk et al., 2024). In this study, an emerging disease with typical symptoms of Paramyrothecium leaf spot was discovered during the disease investigation. We performed a detailed identification of the causal agent of leaf spot from soybean by morphology, molecular characteristics, multi-gene phylogenetic analysis, pathogenicity, and host range tests. Our results were sufficient to demonstrate that the three isolates from soybean were P. vignicola. In soybean, the other species of Paramyrothecium have been identified as pathogens causing related disease. Paramyrothecium spp. inducing myrothecium leaf-spot disease was reported significantly reduce yield of soybean in the field in Indian (Srivastava and Khan, 1997). Moreover, Myrothecium roridum (syn. P. roridum) inducing leaf-spot disease was proved leading to severe tissue necrosis and seedling mortality (Talukdar and Dantre, 2014). P. roridum was also reported inducing leaf spot on soybean in Africa (Haudenshield et al., 2018). To date, this is the first report that P. vignicola naturally causes leaf spot on soybean in China and worldwide. Our results indicate that P. vignicola might pose a potential risk to several legume crops in the future.
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