First report of Colletotrichum karstii causing anthracnose on soybean in China.

Soybean (Glycine max) is a significant grain and oil crop. Among the various challenges faced by soybean cultivation, anthracnose stands out as one of the most prevalent diseases. In June 2023, anthracnose symptoms on leaves characterized by irregular disease spots featuring gray-white centers and brown edges, along with many small black dots on their surface, were observed in a 20-hectare soybean (variety "Liu Yuehuang") field located in Luodian County (25°40'20″ N, 106°53'50″ E, 575 m), Guizhou Province, China. Around 30% of the 300 soybean plants examined were symptomatic, and a total of ten leaves were collected. Fragments (5×5 mm) from the edge of disease spots were sheared and surface-sterilized with 3% sodium hypochlorite and 75% ethanol for 60 s and 30 s, respectively. They were then flushed twice with sterile water, dried using sterile filter papers, finally placed on potato dextrose agar (PDA) and incubated at 28°C for two days. In total, 11 isolates with identical morphological characteristics were obtained. The colonies grown with white aerial mycelia on their surface; conidia were cylindrical, both ends are rounded, aseptate, hyaline, 11.0-14.0 (12.5) × 4.5-6.0 (5.0) µm (n = 30); appressoria were nearly ovoid, brown to black, 8.5-10.5 (9.5) × 5.5-7.5 (6.0) µm (n = 30). The morphological characteristics closely resembled the description of C. karstii (Damm et al., 2012). To further identify the isolates, chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the internal transcribed spacer (ITS) loci were amplified by using CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), Bt2F/Bt2R (Woudenberg et al., 2009), GDF/GDR (Guerber et al., 2003) and ITS1/ITS4 (White et al., 1990) PCR primers, respectively. The BLAST results showed that the sequences of two representative strains, LD 2023048-1 and LD 2023048-2, were highly similar to those of strain C. karstii CGMCC3.14194 (ITS: OR342620 (99%) and OR342621 (99%) with HM585409, ACT: OR412337 (97%) and OR423341 (100%) with HM581995, CHS-1: OR423344 (99%,) and OR423345 (100%) with HM582023, GAPDH: OR423348 (98%) and OR423349 (98%) with HM585391, and TUB: OR423352 (99%) and OR423353 (99%) with HM585428). The phylogenetic tree combined five sequences showed that the two strains clustered into a branch of C. karstii CGMCC3.14194 with high support values. Thirty-day-old soybean plants (n = 10) (variety Liu Yuehuang) were separately sprayed with 1 × 105 spore suspensions/mL of the two strains by spray method, and plants sprayed with sterile distilled water were used as the negative control (n = 5). All the plants were then covered with plastic bags and cultured in the greenhouse (28℃, 80% humidity, 12 h light dark cycle). After ten days of inoculation, plants inoculated with C. karstii began to produce typical anthracnose symptoms, while the control remained asymptomatic. The confirmation of the reisolated pathogen as C. karstii was established through a comprehensive analysis of morphology and five sequencing loci. Pathogenicity tests were repeated three times. Anthracnose on soybean is caused by Colletotrichum spp. reported in China including C. truncatum (Hu et al., 2015), C. brevisporum (Shi et al., 2021) and C. fructicola (Xu et al., 2023). As far as we know, this study is the initial report of C. karstii inducing anthracnose on soybean to date, which establishes a fundamental reference for preventing and controlling this disease.

Taxonomy, biological characterization and fungicide sensitivity assays of Hypomyces cornea sp. nov. causing cobweb disease on Auricularia cornea.

Auricularia cornea is an important edible mushroom crop in China but the occurrence of cobweb disease has cause significance economic loss in its production. The rate of disease occurrence is 16.65% all over the country. In the present study, a new pathogen Hypomyces cornea sp. nov. was found to cause the cobweb disease. In July 2021, three strains of fungal pathogen were isolated from infected fruiting bodies and identified as H. cornea based on morphological studies and molecular phylogenetic analysis of internal transcribed spacer (ITS) of nuclear ribosomal DNA, mitochondrial large subunit (LSU) of rRNA and the partial translation elongation factor 1-alpha genes. The representative isolates of the pathogenic Hypomyces species used to perform pathogenicity test with spore suspension that caused similar symptoms as those observed in the cultivated field, and same pathogens could be re-isolated, which fulfill Koch's postulates. The typical biological characterization was examined of the serious pathogen to determine its favorable growth conditions, including suitable temperature, pH, carbon, nitrogen sources and light conditions. The findings revealed an optimum temperature of 25 °C, pH of 6, and soluble starch and peptone as the preferred carbon and nitrogen sources, respectively. The hyphal growth inhibition method was used for primary in vitro screening test of seven common fungicides, and the most suitable fungicide is Prochloraz manganese chloride complex, the EC50 values of cobweb pathogen and mushrooms were 0.085 µg/mL and 2.452 µg/mL, respectively. The results of our research provide an evidence-based basis for the effective prevention and treatment of A. cornea cobweb disease.

First report of Powdery Mildew Caused by Podosphaera xanthii on Siraitia grosvenorii in Guizhou, China.

Swingle (Siraitia grosvenorii), a member of the Cucurbitaceae family, stands out as a distinctive plant with both economic and medicinal significance. In October 2023, severe powdery mildew were observed on S. grosvenorii in Guiyang City (26.50°N; 106.66°E), Guizhou Province, China. About 80 % of the plants in the greenhouse showed powdery mildew symptoms. Three infected plant samples were selected for morphological and molecular analysis (GZAAS 23-0801, GZAAS 23-0802 and GZAAS 23-0803). The voucher specimens are deposited in the Key Laboratory of Agricultural Biotechnology of Guizhou Province. The symptoms initially manifested as irregular to nearly circular, small yellow spots, with distinct depressions as well as surfaces covered in white mycelium. Over time, these spots gradually expanded and merged patches. In the final stages, the entire leaves turned into yellow and withered. Microscopic observations showed that fungal hyphae were septate, branched, and flexuous to straight and 5 to 9 µm wide, and appressoria were indistinct to slightly nipple-shaped. Conidia were hyaline and ellipsoid to oval with fibrosin bodies and measured 31 to 43 × 18 to 24 µm (n = 50) with a length/width ratio of 1.3 to 2.3. Conidiophores were unbranched, straight, 120 to 268 × 14 to 22 µm (n = 30), producing two to five immature conidia in chains. Foot cells of conidiophores were cylindrical, 39 to 84 × 8 to 14 µm (n = 30), followed by one to three short cells. Short cells were cylindrical, 12 to 32 × 8 to 15 µm (n = 50). The morphological characteristics were identical with the previous description of Podosphaera xanthii (Braun and Cook, 2012). Total DNA was extracted from conidia and mycelia by the Chelex method (Walsh et al., 1991). The ribosomal DNA internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) were amplified by using the primers ITS1/ITS4 (White et al., 1990) and LSU1/LSU2 (Scholin et al., 1994), respectively. The ITS (OR825802, OR825803 and OR825804, respectively) and LSU (OR825805, OR825806 and OR825807, respectively) sequences of three isolates, were deposited in GenBank. The BLAST results revealed that both the ITS and LSU region sequence were 100% identical to those of P. xanthii (ITS: MF043939, MG754404 and KJ698669; LSU: OQ061319, AB936277and OP218411). Phylogenetic analyses of ITS and LSU sequences showed that our three isolates were clustered with P. xanthii (KX842351, LC270782 and LC270779) with high statistical support (ML/MP/BI: 100%/97%/1.00). Combined with their morphological characteristics, these three isolates were identified as P. xanthii. Pathogenicity tests were performed by gently brushing conidia onto the leaves of five healthy S. grosvenorii plants. Five non-inoculated plants were used as the control. All plants were maintained in a greenhouse at 25 ± 2°C. One week after inoculation, similar symptoms were observed in the inoculated plants, whereas no symptoms occurred on the control plants. By microscopic observation, the fungus on the inoculated plants was morphologically identical to those on originally diseased plants. Powdery mildew caused by P. xanthii has been reported on Vernonia cinerea (Wu et al., 2023), Vigna unguiculata (Zhang et al., 2023), Cucumis melo (Meesam et al., 2023). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. grosvenorii in Guizhou, China. The occurrence of powdery mildew on S. grosvenorii may pose a potential threat to its large-scale cultivation. The pathogen could become a threat to other Cucurbitaceae members in the future.

Taxonomy and control of Trichoderma hymenopellicola sp. nov. responsible for the first green mold disease on Hymenopellis raphanipes.

Trichoderma spp. are a group of widespread fungi with important applications in many aspects of human life, but they are also pathogens that cause green mold disease on mushrooms. During a survey of mushroom cultivation in Guizhou, China, five strains of Trichoderma from three different localities were isolated from soil in mushroom bags of Hymenopellis raphanipes. The typical morphology of having gregarious, reddish stromata and gregarious phialides and the results of phylogenetic analyses based on a combined dataset of RPB2, TEF, and ITS gene sequences demonstrated that these green-spored Trichoderma belong to a new taxon, Trichoderma hymenopellicola. Pathogenicity tests by covering fungal mycelial blocks or soil mixed with spore suspension in mushroom bags showed similar symptoms to those in the field, and the same fungal pathogen had been observed and re-isolated from these symptoms, which fulfill Koch's postulates. A primary screening test of nine common fungicides indicated that prochloraz-manganese chloride complex and propiconazole are the top two effective fungicides inhibiting the pathogen, whereas the former was further indicated as a suitable fungicide to control Trichoderma hymenopellicola, with a high inhibition ratio to the pathogen and low toxicity to the mushroom.

Characterization and fungicide sensitivity of Colletotrichum godetiae causing sweet cherry fruit anthracnose in Guizhou, China.

Sweet cherry is an important fruit crop with high economic and ornamental value in China. However, cherry fruit anthracnose, caused by Colletotrichum species, greatly impacts cherry yield and quality. Here, we surveyed cherry anthracnose in Guizhou, China from 2019-2020. Necrotic sweet cherry fruits were collected from different areas in Guizhou and examined. A total of 116 Colletotrichum strains were isolated from these symptomatic fruits. Based on the morphological characteristics of the isolates and phylogenetic analyses of concatenate internal transcribed spacer (ITS) region and ACT, CHS-1, GAPDH, TUB2, and HIS3 genes, the pathogen responsible for causing sweet cherry anthracnose was identified as Colletotrichum godetiae. Pathogenicity tests were conducted by inoculating healthy sweet cherry fruits with spore suspensions of the fungal pathogen, and Koch's postulates were confirmed by pathogen re-isolation and identification. The Q-1 isolate showed different sensitivities to 13 fungicides, exhibiting seven different modes of action, and its EC50 values ranged from 0.04 to 91.26 µg ml-1. According to that, the sensitivity of 20 isolates from different samples to ten fungicides with better performance, were measured. The results showed that 6 of the 10 fungicides (difenoconazole, propiconazole, prochloraz-manganese, pyraclostrobin, trifloxystrobin-tebuconazole, and difenoconazole-azoxystrobin) all showed higher sensitive to the 20\u00B0C. godetiae isolates, and no resistance groups appeared. Its EC50 values ranged from 0.013 to 1.563 µg ml-1. In summary, this is the first report demonstrating that C. godetiae causes sweet cherry anthracnose and the results of this study provide insights into how sweet cherry anthracnose could be effectively controlled in China.