Water Agar Plates Research Articles

Seedborne Fungal Detection Differs with Seed Assay Method, and Fungal Diversity and Abundance Are Impacted by Fungicide Treatment, Harvest Timing, and Storage Environment

Previous studies have shown that foliar fungicides increase the abundance of Diaporthe spp. in mature soybean seeds, thereby reducing seed quality and potentially increasing seedling, stem, and pod disease in subsequent plantings. To mitigate this, we examined a fungicide containing mefentrifluconazole, fluxapyroxad, and pyraclostrobin applied during the R3 growth stage. We compared the seedborne fungal community from soybeans harvested at full maturity and 2 to 3 weeks later to assess fungal colonization. Six trials were conducted in Iowa and South Dakota in 2021 and 2022. Asymptomatic, recently harvested seed was assayed using either an agar-plate or a seed-blotter test, and the results were compared. The agar-plate method involved disinfesting seeds in sodium hypochlorite and ethanol and then transferring them to acidified water agar plates. Fungal isolates were identified using morphology and DNA sequences. This method detected 562 fungal isolates from 25 species and 14 genera. Dry conditions reduced seed infection, but a 2-week delay in harvest increased the likelihood of Alternaria and Cercospora infection in nontreated controls. The seed-blotter test involved disinfesting seeds, placing them on blotter paper, and incubating them. This assay detected higher numbers of fungal infected seed, with Aspergillus as the dominant genus. Storage conditions significantly impacted fungal presence, with more Aspergillus in seeds stored in a shed than in the cooler. Both assay methods showed that the fungicide treatment reduced the number of seeds infected with Alternaria, Cercospora, and Aspergillus. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Biocontrol of Hemileia vastatrix, the Causal Agent of Coffee Leaf Rust, by Paenibacillus sp. NMA1017.

Coffee leaf rust (CLR), caused by Hemileia vastatrix, is considered a highly important phytosanitary problem in Mexico. Currently, there are few microorganisms used as biocontrol alternatives to chemical control of CLR in organic coffee fields in Mexico. This study evaluates the use of Paenibacillus sp. NMA1017 as a biocontrol agent to inhibit the development of H. vastatrix in in vitro and in vivo (greenhouse) experiments. Hemileia vastatrix urediniospores were placed on water agar plates, and then Paenibacillus sp. NMA1017 was inoculated simultaneously or 8 h later. Urediniospores germination rate was reduced by 94% when the NMA1017 strain was inoculated simultaneously with the urediniospores and reduced by 38% when NMA1017 was inoculated 8 h later. Experiments with 8-month-old Bourbon coffee plants that were infected with H. vastatrix showed that disease incidence was reduced by 38, 90, and 50% when NMA1017 was applied 8 days before, simultaneously, or 8 days after the application of H. vastatrix, respectively. Paenibacillus sp. NMA1017 also reduced the severity of CLR on the leaves by up to 62%. The germination urediniospores of other rust pathogens such as Puccinia sorghi (maize leaf rust), Puccinia triticina (wheat leaf rust), Puccinia graminis f. sp. tritici (black stem rust of wheat), Uromyces striatus (alfalfa leaf rust), and Phragmidium sp. (rosebush leaf rust) were also inhibited. Use of the potential biocontrol agent Paenibacillus sp. NMA1017 might help reduce the application of chemical fungicides for the control of CLR, making coffee a more sustainable crop and providing management options for organic coffee growers.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 μm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 μm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

First Report of Root and Collar Rot caused by Phytophthora nicotianae on ⨯Graptoveria 'Silver Star' in Korea.

⨯Graptoveria 'Silver Star' (a cross between Graptopetalum filiferum and Echeveria agavoides) from the Crassulaceae family, are an evergreen succulent with lotus constellation-shaped flowers, making it consumer favorite ornamental plant in Korea. In 2019, Korea's ornamental production was estimated at KRW 517.4 billion (EUR 382 million), from 4,244 ha of farming area according to the Ministry of Agriculture, Food and Rural Affairs of Korea. In July 2023, ⨯Graptoveria 'Silver Star' plants with chlorotic leaves, root and collar rot were observed in a greenhouse in Yongin (37°14'27.9"N, 127°10'39.19"E), Korea. To isolate the causal agent, small pieces (1 mm2) of symptomatic tissues were surface-sterilized using 1% NaOCl for 1 min, then put onto a water agar (WA) plate and incubated in the dark at 25℃ for five days. Two isolates (FD00202, FD00203) were obtained from diseased leaves, stem and roots by isolating single sporangium. To investigate the morphological characteristics of the isolates, the mycelium from potato dextrose agar (PDA) were transferred to V8 agar (V8A) followed by incubation at 25°C in the dark for 7 days. The isolates produced dense cottony mycelium, with slightly petaloid and light rossette pattern, with coralloid edges measuring 70 to 83 mm diameter. Sporangium were spheroid (30.0-48.0 µm long, 25.0-35.0 µm wide) with globose chlamydospores (17.0-50.0 µm long, 18.0-38.0 µm wide). Oogonia were not observed. Morphological and cultural characteristics of these isolates were phenotypically similar to that of Phytophthora nicotianae (Faedda et al. 2013; Abad et al. 2023). For molecular identification, genomic DNA was extracted from 5 days old cultures using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). Two gene regions, the rDNA-ITS, COX I were amplified and sequenced using primers ITS1/ITS4 and FM83/FM84, respectively (White et al. 1990; Martin and Tooley 2003). The resulting sequences were deposited in GenBank with accession no. LC783858 to LC783861. A BLASTn search of the DNA sequences from ITS, COX I showed 99.81 and 98.94% identity to P. nicotianae isolate IMI 398853, respectively. Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS, COX I using MEGA7 under the Tamura-Nei model (Kumar et al. 2016). The isolates formed a monophyletic group with P. nicotianae isolate IMI 398853, CPHST BL162, and CPHST BL 44. Based on morphological characteristics and molecular analysis, the isolates were identified as P. nicotianae. T confirm their pathogenicity, inoculum was prepared in accordance with Ann (2000). Artificially wounded healthy plant roots were dipped in zoospore suspension (3.0 × 106 zoospore/ml) for 24 hours, with mock-treated plants (control) dipped in sterile distilled water (Ann. 2000). Thereafter, the plants were transplanted into new medium and kept under high humidity. Symptoms were observed after 10 days of incubation. The plants inoculated with P. nicotianae showed similar symptoms of chlorotic leaves with root and collar rot, while control remained symptomless. The pathogen was re-isolated from all inoculated plants and confirmed as P. nicotianae by morphological and molecular analysis. but not from controls, fulfilling Koch's postulates. Phytophthora nicotianae was previously report on Echeveria derenbergii and Kalanchoe blossfeldiana causing brown spot on stems and roots in California and Korea, respectively (French 1989; Oh and Son 2008). To best of our knowledge, this is the first report of P. nicotianae causing root and collar rot on ⨯Graptoveria 'Silver Star' plants in the Korea.

61 Determining in vivo and in vitro predatory activity of Duddingtonia flagrans and interaction with dietary coccidiostat

Abstract A nematode trapping fungus, Duddingtonia flagrans (Df), fed to sheep is useful for biocontrol of gastrointestinal nematodes on pasture. The objectives were: 1) validate methods to determine viability of a commercial source of Df before beginning research studies, and 2) examine the interaction between Df and dietary coccidiostat (as decoquinate). Methods: Study 1: In Jan 2023, weaned Katahdin lambs (97.3 ± 1.2 d of age; 19.5 ± 1.3 kg BW; packed cell volume of 26.5 ± 1.4 %), naturally infected with predominantly Haemonchus contortus (89%) and mean fecal egg count (FEC) of 6,860 ± 1,535 eggs/g, were randomly assigned to: 1) no Df (-g)/(FEC-g)×100]. Study 2: In Aug 2023, 7 mo old anthelmintic-treated ram lambs (FEC < 50 egg/s; 51.6 ± 3.9 kg in BW) were fed grain supplement daily with decoquinate (Deccox; +C) or without (-C; n = 3/diet) on d -7 to 1, and, within diet, administered 3 g BioWorma/d in 6 gel capsules (+DF; n = 2/diet) or not (-DF; n = 1) on d -1 and -2. Fecal samples were collected on d 0 (2 g/plate + 300 L3 of H. contortus) and d 1 (6 g/plate + 1000 L3) and placed on water agar plates (n = 3/lamb) at 25°C. Trapping structures were visually assessed after 24, 48, and 144 h (6 d), and cultures Baermannized on d 7 for L3 recovery. Relative reduction was (x̅-DF-C-x̅+DF+C/ x̅-DF-C)×100%. Data for variables collected one time were analyzed by Proc GLM (SAS), and for those collected serially, a Proc Mixed with repeated measures. A 2×2 factorial was used in Study 2 and included treatments, day and interaction in model. Results: Study 1: As expected, L3 recovery was reduced (P = 0.005) in DF lambs by 67-78% relative to control. Study 2: DF formed loops and traps around L3 by d 6 and reduced L3 recovery on both days (P < 0.001) without an interaction by decoquinate (P = 0.69). The L3 reduction relative to -DF-C was between 60.2-73.5%. The coccidiostat, decoquinate, can be added to the diet in the presence of BioWorma without antagonism. In conclusion, the in vitro studies allow detection of adverse issues with viability of Df and can reduce the number of animals needed for research studies.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

Occurence of leaf spot on Narrow-head ragwort (Ligularia stenocephala) caused by Alternaria cinerariae in Korea.

Narrow-head ragwort (Ligularia stenocephala (Maxim.) Matsum. & Koidz.) has been used for medicinal purposes and a leafy vegetable in South Korea (Choi et al., 2007; Debnath et al., 2017). In May 2022, brown spots and blight were observed on the leaves in a farmer's field in Namwon (35°26'58.9"N, 127°28'55.9"E), Korea. About 80% of the plants in a 3000 m2 cultivation area were infected. To isolate the causal agent, small pieces (1 mm2) surface-sterilized (1% NaOCl for 1 min) symptomatic leaf tissues were put onto a water agar (WA) plate and incubated in the dark at 25℃. After five days of incubation, two isolates (FD00021, FD00022) were obtained from diseased leaves using a single spore isolation technique. Morphological characteristics were examined after seven days of incubation at 25℃ in the dark. Colonies were 51.6 to 65.3 mm in diameter, gray-green in the center, and ivory at the edge. Conidiophores were straight or curved and 10 - 34 × 4 - 5 μm. Conidia were solitary or two to four in a chain, long ellipsoid to obclavate, one to thirteen transverse septa, 52 - 169 ×14 - 34 μm, blunt-tapered beak variable in size 4 - 56 × 3 - 10 μm (n=75). The morphological and cultural characteristics of the isolates were consistent with that of Alternaria cinerariae (Nishikawa and Nakashima, 2015; Simmons, 2007). For molecular identification, genomic DNA was extracted from 5-day-old cultures using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). Five gene regions, including rDNA ITS, GPD, Alt a, RPB2, and EF1-α were amplified and sequenced using ITS1/ITS4, gpd1/gpd2, Alt-a1-for/ Alt-a1-rev, RPB2-5F2/RPB2-7cR, and EF1-728F/EF1-986R primer sets respectively (Wang et al., 2022; Garibaldi et al., 2022). The resulting sequences were deposited in GenBank with accession no. OP785152, OP785153 and OP832000 to OP832007). The concatenated genes (rDNA ITS, GPD, Alt a, RPB2, and EF1-α) sequence identity of the FD00021 and FD00022 against the reference strain A. cinerariae CBS 116495 is 99.92% (2572/2574) and 99.84% (2570/2574), respectively. Maximum Likelihood tree was inferred based on the concatenated sequences of the five gene regions using the Kimura 2-parameter model with 1,000 bootstrap replications. The phylogenetic tree showed that the present strains and A. cinerariae CBS 116495 fell into the same clade with high bootstrap support (100%). Based on morphological characteristics and molecular analysis, the isolates were identified as A. cinerariae. To confirm their pathogenicity, drops (70 μl) of conidial suspension (1×104 spores/ml) were applied on intact healthy leaves (3 leaves/plant) of plants (3 plants/isolate) that had been cultivated for one month after transplantation as seedlings. Controls were treated with sterile distilled water. The treated plants were covered with plastic boxes to maintain humidity around 90% and were maintained in an incubator at 25℃ in a 12-hour light-dark cycle. Symptoms appeared only on inoculated leaves after four days of inoculation, while controls remained asymptomatic. The A. cinerariae isolates were re-isolated from infected tissue of the inoculated leaves, thus fulfilling Koch's postulates. Alternaria cinerariae is an important plant pathogen that can cause leaf spot and blight on a variety of host plants including Cineraria spp. and Tussilago farfara (He et al. 2020). This is the first report on leaf spot on Narrow-head ragwort caused by A. cinerariae in the world. Leaf spot disease caused by Alternaria cinerariae is a significant threat to narrow-head ragwort agriculture in South Korea. Therefore, its control strategies are necessary for increasing productivity.

First Report of Leaf Spot Caused by Arthrinium arundinis on Amaranthus hybridus in Korea.

Amaranthus hybridus (=A. patulus), often called green amaranth, is an annual herbaceous plant of the Amaranthaceae. This plant is considered a harmful weed in the agricultural context of North America and has expanded its distribution to Asia and Europe. In Korea, it has become a problematic invasive issue, leading to economic losses due to reduced crop yields and rising weed management costs (Park et al., 2014), although its seeds and young leaves are edible and frequently consumed. In October 2020, we observed leaf spot symptoms on A. hybridus plants that were growing within perilla farms (Perilla frutescens var. japonica) located in Damyang (35°14'07"N, 126°59'40"E), Korea, with a disease incidence of 20 to 30% of the inspected plants. The initial signs appeared as grey to brown dots on the leaves, which gradually expanded into irregular, brown patches with a diameter of 2-3 cm. A single spore was isolated from the diseased leaf under a dissecting microscope, placed onto a 2% water agar plate, and incubated in darkness at 25°C for three days. Pure cultures were obtained by transferring single hyphal tips onto potato dextrose agar (PDA) plates. Five single-spore isolates were the same in the cultural and morphological examination, and a representative isolate (P309) was preserved at the Korean Agricultural Culture Collection (KACC49813), Korea. Colonies appeared light gray to white with regular margins and reached 4 to 5 cm in diameter after a week. After two weeks, black patches of spores were often visible in the aerial mycelia. Conidiophores were brown to pale brown, often branched, thick-walled, and measured 6.8 × 2.7 µm (n = 30). Conidia were single-celled, dark brown, globose to ellipsoid, and measured 6.8 × 5.0 µm (n = 50), with a ratio of length/width of 1.1 to 1.6 (n = 50). These morphological characteristics matched those of Arthrinium arundinis (Crous et al., 2013). For molecular identification, genomic DNA was extracted from conidia and mycelia on two-week-old PDA culture of the KACC49813. PCR was performed for the internal transcribed spacer (ITS) (primers ITS1/ITS4, White et al. 1990), the large subunit (LSU) rDNA (primers LROR/LR5, Vilgalys et al. 1990), the beta-tubulin gene (TUB) (primers T1/Bt-2b, O'Donnell and Cigelnik 1997), and the translation elongation factor 1-alpha (TEF) (primers EF1-728F/EF-2, Crous et al. 2013). A BLASTn search of the resulting sequences of ITS (560 bp; OL744431), LSU (881 bp; OL744432), TUB (790 bp; PP084934), and TEF (445 bp; PP084935) revealed 100 % similarity (e-value=0.0, coverage=100%) to previously reported sequences of Arthrinium arundinis (e.g. MF627422 for ITS, KF144930 for LSU, KF144973 for TUB, and KY705146 for TEF), confirming the identity of the Korean isolate. Pathogenicity assays were performed twice by spraying 1 ml of a conidial suspension (1.1 × 104 conidia per mL) onto the leaf surface of sixteen healthy A. hybridus plants. Sixteen control plants were sprayed with sterile water. All plants were kept in a growth chamber at 80% relative humidity and 23 °C with a 12-h light/dark cycle. Three weeks after the inoculation, initial symptoms mirroring the aforementioned ones appeared, while the control plants remained symptomless. Fungal isolates were successfully re-isolated from the inoculated leaves, and their identity as A. arundinis was confirmed by DNA sequencing, thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot caused by A. arundinis on Amaranthus hybridus, not only in Korea but globally. Arthrinium arundinis has also been reported as a plant pathogen on some agricultural crops (Ji et al. 2020; Liao et al. 2022; Farr and Rossman 2023), suggesting its polyphagous behavior. Then, this pathogen could represent a potential risk to the cultivation of edible amaranth in Korea and other crops where Amaranthus species are spread as weeds. For this reason, continuous monitoring is necessary to assess the impact of this fungus on Amaranthus and other crops.

First report of leaf spot disease caused by Alternaria alternata on Polygonatum cyrtonema Hua in Hunan Province of China.

Polygonatum cyrtonema Hua, a perennial plant of the Asparagaceae family, is an important herb in Chinese medicine and is mainly grown in the Chinese provinces of Guizhou, Hunan, Yunnan, Anhui, and Zhejiang (Chen et al. 2021). In June 2021, a new case of leaf spot disease was detected in an 80 m2 plantation of P. cyrtonema on Xuefeng Mountain, Huaihua City, Hunan Province (27°17'30″N, 110°24'20″E). It infected almost 40% of the total planted area. Initially, irregular light brown spots appeared on the leaves, gradually turning dark brown and coalescing to form large necrotic areas, after which the affected plant turned yellow and eventually died. Ten disease samples were collected from ten plants in the plantation area. The leading edge of necrotic tissues were rinsed with sterile water and then disinfected with 3% hydrogen peroxide for 30 s, followed by 75% ethanol for 90 s, and rinsed three times with sterile water. Samples were then placed on water agar plates and incubated in the dark in a constant temperature incubator at 28 ℃ for 3-5 days. After mycelial growth was observed in the media, the hyphae were transferred to potato dextrose agar plates and incubated for 3-5 days at 28 ℃ in the dark. Ultimately, 12 purified fungal isolates were obtained, some of which were morphologically similar, including 10 that were Alternaria (83.3% isolation rate). Three representative isolates (HJYB1, HJYB2, and HJYB3) were selected for further study. The initial colonies were grayish green with white fluffy mycelia on the surface and a prominent white rim, which became brown with dense, cottony aerial mycelia as the colonies matured. The conidia were obpyriform or ellipsoidal, pale to dark brown, with 0-4 transverse and 0-3 longitudinal septa, some with a short cylindrical beak at the tip. They measured 11.826-28.873 × 6.231-26.018 μm (n = 100). To further confirm the identity of the isolates, their rDNA internal transcribed spacer region (ITS), β-microtubulin (TUB2) and translation elongation factor-1 (TEF-1) genes were amplified and sequenced using the ITS4/ITS5, TUB2F/R and EF-526F/1567R primers, respectively (Hong et al. 2006). The sequences were submitted to GenBank (ITS: OR513924, OR513964, OR519874; TUB2: OR526928, OR533421, OR526929; TEF: OR526926, OR533420, OR526927). A concatenated phylogenetic tree of the three genes showed that the isolate clustered significantly with Alternaria alternata. Based on morphological identification and phylogenetic tree analysis, the isolate was identified as A. alternata. We carried out pathogenicity tests on four uniformly growing P. cyrtonema plants. Three of these plants were used as experimental plants and one as a control. For each plant, three young leaves were selected and inoculated with 6 × 6 mm PDA blocks, while sterile PDA blocks were used as controls. The treated plants were subjected to 10 days of stable temperature in a climatic chamber set at 28°C, 80% constant relative humidity and 12 hours of light per day. The pathogenic lesions appeared and the pathogens re-isolated from the diseased leaves showed similar morphological characteristics to representative isolates and were confirmed as A. alternata by DNA sequencing, thus fulfilling Koch's postulates. A. alternata is the major causal agent of leaf spot on P. sibiricum (Zou et al. 2023) and Agrimonia pilosa (Jiang et al. 2023). As far as we know, leaf necrosis caused by A. tenuissima has been found on P. cyrtonema (Li et al. 2020). To our knowledge, this is the first report of A. alternata causing leaf spot disease in P. cyrtonema. These findings form the basis for the management of this leaf spot disease.

First report of Fusarium tricinctum Causing Leaf Blight on Amorphophallus konjac in Hunan Province of China.

Konjac (Amorphophallus konjac) is a perennial herbaceous plant of the Araceae family, cultivated mainly in south-western China and used extensively for weight loss (Chua et al. 2010). In June 2022, leaf blight was detected on a 2,00 ha A. konjac plantation in Chenxi County, Huaihua City, Hunan Province. It infected almost 20% of the area under cultivation and tends to occur each year during warm, humid weather from May to July, causing significant economic losses to A. konjac production. There were small brown spots on the leaves which gradually spread to form irregular brown lesions. In severe cases the entire plant turned yellow and died. Nine samples were collected randomly from different plants in three plantation forests to isolate the pathogens. They were washed with sterile water and the lesions were excised. They were subsequently disinfected with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s and rinsed three times with sterile water. The cut sections were then placed on water agar plates and grown in the dark in a constant temperature incubator at 28℃ for 3-5 days, when mycelia grew they were transferred to potato dextrose agar medium and grown in the dark at 28℃ for 3-5 days. Eleven purified fungal isolates were obtained, ten of which looked like Fusarium (90.9% isolation rate), and three representative isolates (MY5, MY7 and MY9) were chosen for further study. The fungal colonies initially appeared white and gradually turnned dark red. Macroconidia were crescent-shaped, elongated, slightly curved and had 2 to 4 septations, with a predominance of 3 septations. They measured 15.540 to 42.083 × 2.760 to 4.558 μm (n=100). Microconidia were oval or pyriform, with a maximum of one septum and measured 6.135 to 24.990 × 2.158 to 4.412 μm (n=100). Two genetic regions, the translation elongation factor-1 (TEF1-α) and RNA polymerase II largest subunit (RPB1) genes, were amplified and sequenced to verify the identity of the fungus (Qiu et al. 2023). The universal primers TEF1-F/R, G2R/Fa were used for amplification and sequencing, and the sequences were submitted to GenBank (TEF1-α: OR545395, OR545397, OR545399; RPB1: OR545394, OR545396, OR545398). A joint phylogenetic tree of the two genes was constructed and analysis showed that the three isolates were significantly clustered with Fusarium tricinctum. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as F. tricinctum. Pathogenicity tests were carried out on 12 uniformly growing leaf expansion stages of konjac plants，each inoculated with five young leaves. Mycelial blocks of 6 × 6 mm grown on PDA media for 5 days were placed on the surface of the leaves, while sterile PDA blocks were placed on the control plant. After 10 days of rearing the treated plants in a constant temperature chamber at 28°C and 90% relative humidity, the lesions appeared and the pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. F. tricinctum has been shown to be the major pathogenic fungus causing leaf blight in wheat (Triticum aestivum L.) (Castañares et al. 2011) and orchardgrass (Dactylis glomerata L.) (Wu et al. 2020). To our knowledge, this is the first time in the world that F. tricinctum has been reported to cause leaf blight in A. konjac. This research could provide a foundation for future control of leaf blight disease.

First Report of Anthracnose Caused by Colletotrichum camelliae on Amorphophallus konjac in Hunan Province of China.

Amorphophallus konjac is a crop in the family Araceae, which is widely cultivated in Hunan, Yunnan, and Guizhou in China. A. konjac flour is highly valuable economically as a product for weight reduction. In June 2022, a new leaf disease was discovered in an understory A. konjac plantation in Xupu County, Hunan Province, China, where 2,000 hm2 of A. konjac had been planted. Approximately 40% of the total cultivated area exhibited symptoms. The disease outbreaks occurred during warm and wet weather (May to June). In the early stages of infection, small brown spots appeared on the leaves and then gradually spread into irregular lesions. There was a light yellow halo around the brown lesions. In severe cases, the whole plant gradually turned yellow and died. Six symptomatic leaf samples were collected from three different fields in Xupu County to isolate the causal agent. They were rinsed with sterile water, and the lesions were cut off. The lesions were rinsed in 3% hydrogen peroxide for 30 s followed by a 90 s treatment in 75% alcohol. They were then rinsed five times in sterile water, placed on water agar plates and incubated for 2-3 days at 28°C. After the mycelium had grown, they were transferred to potato dextrose agar (PDA) plates and incubated for 3-5 days at 28°C. In total, ten isolates were obtained, and seven of them were Colletotrichum (isolation frequency 70%). Three representative isolates (HY1, HY2, and HY3) were selected for further study. This fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were cylindrical, lacked a septum, and were thin-walled. They measured 14.04 to 21.58 × 5.89 to 10.40 μm (n=100). To further confirm its fungal identity, the fungus was amplified and sequenced using six genetic regions, including β-tubulin (TUB2), actin (ACT), the internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), calmodulin (CAL) and chitin synthase (CHS). The universal primers BT2a/TUB2R, ACT512F/ACT783R, ITS4/ITS5, GDF/GDR, CL1C/CL2C, CHS79F/CHS345R were used for amplification (Weir et al. 2012), sequenced by the Sanger chain termination method, and submitted to GenBank (TUB2: OQ506549, OQ506544, OP604480; ACT: OQ506551, OQ506546, OP604482; ITS: OQ457036, OQ457498, OP458555; GAPDH: OQ506553, OQ506548, OP604484; CAL: OQ506552, OQ506547, OP604483; CHS: OQ506550, OQ506545, OP604481). An analysis of a joint phylogenetic tree that was constructed using the six genes showed that the three isolates clearly clustered with Colletotrichum camelliae (syn. Glomerella cingulata f. sp. camelliae) strain ICMP 10646 (GenBank: JX010437.1, JX009563.1, JX010225.1, JX009993.1, JX009629.1, JX009892.1) and HUN1A4 (GenBank: KU252173.1, KU251646.1, KU251565.1, KU252019.1, KU251838.1, KU251913.1). HY3 was used as a representative strain for the pathogenicity test on the leaves of A. konjac from the whole plant. PDA blocks that were 6 × 6 mm and had been cultured for 5 d were placed on the leaf surface, and sterile PDA blocks were used as the control group. The climate chamber was maintained at 28°C at all times, and 90% relative humidity was maintained. The pathogenic lesions appeared after 10 days of inoculation. The pathogen that was re-isolated from the diseased tissues had the same morphological characteristics as HY3. Thus, Koch's postulates were fulfilled. C. camelliae has been shown to be the primary pathogenic fungus responsible for anthracnose in tea (Ca. sinensis (L.) O. Kuntze) (Wang et al. 2016) and camellia oleifera (Ca. oleifera Abel.) (Li et al. 2016). Anthracnose caused by Colletotrichum gloeosporioides has been reported on A. konjac (Li. 2021). However, to our knowledge, this is the first report in China and worldwide that C. camelliae causes anthracnose on A. konjac. This research lays the foundation for future research to control this disease.

First Report of Scab Disease Caused by Venturia effusa on Pecan in Anhui Province of China.

Pecan (Carya illinoinensis) is a world-famous nut tree which widely cultivated in China. Quanjiao County, located in Anhui province, is reputed to be the capital of pecan production in China. Since 2019, typical scab symptoms were observed on most pecan cultivars in orchards located in the regions of Quanjiao (32°5'7.08″ N, 118°16'2.91″ E). In April, dark brown to black lesions of scab could be observed on both the abaxial and adaxial surface of the lamina, and were often associated with the veins or midrib. In July, small, brownish, and circular lesions ranging from 1 to 2 mm in diameter were observed at the end of stems and shoulder of the fruit. In the surveyed orchards, disease incidence on the leaves reached more than 35%. While, according to the number of infected nut clusters, disease incidence ranged from 40 to 60% on the infected fruits. Using a sterilized scalpel, conidia were scraped from the surface of a single lesion from the infected leaves or fruits, and a dilute spore suspension was prepared in sterile distilled water, of which 100 microliters was spread on 1% water-agar plate (Bock et al. 2014). The conidia were incubated at 25°C for 48 h under fluorescent lights with a 12-hphotoperiod. Single germinated conidia were selected and transferred into potato dextrose agar (PDA) plate to obtain monospore isolates. From 2019 to 2020, more than 20 isolates were obtained from the infected leaves and fruits. Incubated at 24°C for 6 weeks in darkness on PDA, the colonies were gray-black with circular morphology and floccose texture, which were consistent with the characteristics of Venturia effusa described previously (Gottwald 1982). The conidia were pyriform to ellipsoid, zero to one septate, smooth, attenuated towards apex and base, base truncate, pale brown and 10.08 to 18.14 × 4.86 to 9.56 μm (n = 50) in size. To further identify the isolates, the regions of internal transcribed spacer (ITS), beta-tubulin 2 (TUB2) and translation elongation factor 1 alpha (EF1-a) were amplified and sequenced from genomic DNA for the three representative isolates (AH-81 and AH-82 from the infected leaves, and AH-41 from the infected fruits), respectively (White et al. 1990; Young et al. 2018; Bensch et al. 2006). Sequences of them were deposited in GenBank under nos. OP199056 to OP199058 (ITS), OP566581 to OP566583 (TUB2) and OP566578 to OP566580 (EF1-a). Multilocus phylogenetic analysis revealed that three isolates and V. effusa were clustered in the same clade, indicating high genetic similarity between these organisms. Their morphological and molecular characteristics were consistent with those for V. effusa. The pathogenicity of three isolates were tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 105 conidia/ml was sprayed evenly on the surface of leaves of a healthy pecan seedling, and each isolate inoculated four pecan seedlings. The pathogenicity experiment was repeated three times. The plants inoculated with sterile water were used a negative control. The inoculated plants were enclosed in plastic bags for 2 days, and kept in the nursery greenhouse. Four weeks after inoculation, a similar symptom of scab was observed on leaves of cultivar Mahan, and V. effusa was isolated again from inoculated leaves with the frequency of 100% by the single-spore isolation, whereas no symptoms were observed on the control plants. To our knowledge, this is the first report of V. effusa as a scab pathogen on pecan in Anhui Province of China and underscores the need for monitoring this disease and developing disease control strategies to prevent severe reduction in the value of fruit.

First Report of Leaf Spot Caused by Septoria erigerontis on Conyza sumatrensis in Korea.

Conyza sumatrensis (Retz.) E. Walker (syn. Erigeron sumatrensis) or commonly known as fleabane, is an annual herbaceous plant native to South America. It/span>was accidentally introduced to Korea in the 1930s and became invasive in natural and managed ecosystems of the country (Kim et al. 2008). Leaf spots on this plant were first observed in March 2013 in Jeju (33°29'45"N; 126°26'26"E), and then in February 2017 in Seoguipo (33°14'30"N; 126°32'58"E), Korea. The severity of the disease was estimated to be greater than 50%. Initial symptoms were small, distinct, reddish-brown, then turned into brown spots with dark purplish-brown margins. Conidiomata were pycnidial, epigenous, occasionally hypogenous, scattered, dark brown to rusty brown, globose, embedded in host tissue or partly erumpent, 70 to 165 µm in diameter, with ostioles measuring 10 to 26 µm in diameter. Conidia were straight to flexuous, 24 to 65 × 1.5 to 2.0 µm, hyaline, and 1 to 5 septate. Morphological circumscriptions were consistent with Septoria erigerontis Peck (Verkley et al. 2013). To obtain a monoconidial isolate, conidia were collected from lesions, placed in an Eppendorf tube containing sterile water and streaked onto the surface of 2% water agar (WA) plates supplemented with 100 mg/L of streptomycin sulfate, and after five days transferred onto potato dextrose agar (PDA). The two-week-old colonies incubated at 25°C on PDA had a slightly ruffled, but mostly colorless margin; colonies were 5 to 7 mm in diameter, slightly elevated in the center, surface black, covered with a diffuse to a dense mat of grey aerial mycelium. Voucher specimens were housed in the Korea University Herbarium (KUS-F27274 and F29725), and two cultures were deposited in the Korea Agricultural Culture Collection (Accession Nos: KACC47219 and KACC48297). Nucleotide sequences of the internal transcribed spacer (ITS), translation elongation factor 1-α (EF), actin (ACT), 28S rDNA (LSU), and RNA polymerase II second largest subunit (RPB2) genes obtained from KACC47219 were determined (Verkley et al. 2013) and deposited in GenBank (Accession Nos: OM909018, OM908934, OM974318, OM974319, OM974320). Results of BLASTn search for ITS and LSU were 99-100% identical with reference sequences of Septoria erigerontis (MH865036, MH876473), S. lactucae (MK617321), and S. phlogis (MH876550) in GenBank. Whereas it showed 97% similarity for ACT, 93-94% for EF and RPB2 genes with sequences of S. erigerontis (JQ325031, KF253363, KF252411). Pathogenicity was tested by spraying 20 leaves of four-month-old three potted plants with a conidial suspension (1×104 propagules/mL) harvested from a four-week-old culture (span style="font-family:'Times New Roman'">KACC47219). Ten leaves were used as controls. The plants were placed in a dew chamber at 26°C for 24 h, then moved to a greenhouse. Inoculated leaves developed typical symptoms after seven days, whilst no symptoms were observed on control ones. S. erigerontis was re-isolated from symptomatic lesion and its identity was confirmed by microscopic studies, fulfilling Koch's postulates. Although S. erigerontis has been recorded to be associated with leaf spots of Erigeron spp. and Conyza spp. (Farr and Rossman 2022), there is no previous record of this fungus on C. sumatrensis. To our knowledge, this is the first report of leaf spot caused by S. erigerontis on C. sumatrensis worldwide as well as in Korea. We presume that this fungus may have potential as a biocontrol agent on fleabanes, particularly glyphosate-resistant Conyza species (Sansom et al. 2013).

Arthrobotrys musiformis (Orbiliales) Kills Haemonchus contortus Infective Larvae (Trichostronylidae) through Its Predatory Activity and Its Fungal Culture Filtrates.

Haemonchus contortus (Hc) is a parasite affecting small ruminants worldwide. Arthrobotrys musiformis (Am) is a nematode-trapping fungi that captures, destroys and feeds on nematodes. This study assessed the predatory activity (PA) and nematocidal activity (NA) of liquid culture filtrates (LCF) of Am against Hc infective larvae (L3), and additionally, the mycochemical profile (MP) was performed. Fungal identification was achieved by traditional and molecular procedures. The PA of Am against HcL3 was performed in water agar plates. Means of non-predated larvae were recorded and compared with a control group without fungi. LCF/HcL3 interaction was performed using micro-tittering plates. Two media, Czapek–Dox broth (CDB) and sweet potato dextrose broth (SPDB) and three concentrations, were assessed. Lectures were performed after 48 h interaction. The means of alive and dead larvae were recorded and compared with proper negative controls. The PA assessment revealed 71.54% larval reduction (p < 0.01). The highest NA of LCF was found in CDB: 93.42, 73.02 and 51.61%, at 100, 50 and 25 mg/mL, respectively (p < 0.05). Alkaloids and saponins were identified in both media; meanwhile, coumarins were only identified in CDB. The NA was only found in CDB, but not in SPDB. Coumarins could be responsible for the NA.

First Report of Chinese Cabbage Wilt Caused by Plectosphaerella cucumerina in Inner Mongolia of China.

Chinese cabbage [Brassica rapa L. subsp. pekinensis (Lour.) Hanelt] has been grown commercially for many decades in Huade County, Inner Mongolia. In 2018 and 2019, an unusual stem and leaf wilt disease with an average disease incidence of approximately 3% was observed. Diseased plants with spindle-shaped stem lesions were collected and small pieces (0.3 × 0.3 cm) of the diseased tissues were cut from the margins of stem lesions, surface sterilized with 75% alcohol for 3 to 5 s, 0.1% NaClO for 2 to 3 min, and washed three times with sterilized water. The treated tissues were placed on 1.5% (w/v) water agar plates and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for culture purification. Three isolates with similar morphology were obtained and named as BC-2, BC3-2 and BG2. To confirm their pathogenicity, Chinese cabbage (cv. Chunqiuhuang) seed was planted into plugs. After 30 days, the fibrous roots were wounded with a fruit knife and root-dipped in the conidium suspension (1 × 106 conidia/ml) for 20 min. Inoculated seedlings were transplanted in pots (30 × 25 cm) with sterilized nursery soil, with one seeding per pot. The roots of control plants were also wounded and dipped in sterilized water. Five seedlings were inoculated with each isolate and the experiment was repeated three times. Treated seedlings were maintained in a greenhouse at 25 to 28°C under a 12-h photoperiod. Chlorosis and wilting were observed approximately 4 weeks after inoculation, and the outer layer of leaves of the inoculated seedlings developed discoloration and wilted symptoms after 50 days. The symptoms induced by all three isolates were the same as the symptoms observed in the field, whereas no symptoms developed on the control plants. To confirm the Koch's postulates, the fungus was successfully re-isolated from the infected leaves and had similar growth and morphology as the original isolates. The three isolates were cultured for both morphology and molecular identification. The 14-day-old colonies on PDA were buff or salmon pink with few aerial hyphae, and slimy surfaces. Aerial hyphae were sparse with simple or branched conidiophores. Conidia were ellipsoidal, hyaline, surface smooth, septate or aseptate, and (4.0 to 9.7 μm × 2.0 to 3.9 μm). Such characteristics are typical of Plectosphaerella spp. (Palm et al. 1995). For molecular identification, the internal transcribed spacer (ITS) region of rDNA was amplified using the primer pair ITS1/ITS4 (White et al. 1990), and the products were directly sequenced. BLAST analysis showed that the sequences of Isolates BC-2 (511 bp out of 515 bp), BC3-2 (512 bp out of 516 bp) and BG2 (503 bp out of 505 bp) showed 99% identity to an isolate of P. cucumerina (acc. no. KT826571.1) from Bottle gourd [Lagenaria siceraria (Molina) Standl.] (Yan et al. 2016). The sequences of Isolates BC-2, BC3-2 and BG2 were deposited in GenBank (acc. nos. MW320463, MW320462 and MW320464). Although P. cucumerina was reported causing root rot of cabbage (B. oleracea) in Gansu, China (Li et al. 2017), to our knowledge, this is the first report of P. cucumerina causing Chinese cabbage wilt in Inner Mongolia, China. The presence of the disease could cause significant economic losses in Chinese cabbage production. For this reason, strategies for the management and control of this disease should be implemented.

First Report Disease of Clonostachys rosea Causing Root Rot on Astragalus membranaceus in China.

Astragalus membranaceus is a perennial plant in the family of Leguminosae. It is one of the most important traditional Chinese medicines. Root rot has become a serious constraint to commercial production of this medicinal plant in northeastern, northern and northwestern China. In June 2020, A. membranaceus plants in a field at the village of Sada, Minhe County, Haidong City, Qinghai Province (102°51'30.8″E, 36°19'8.58″N, H:2437m), showed symptoms in the form of a large number of brown longitudinal cracks or turtle cracks in the root, and weak growth potential of the aboveground part, with yellowing and falling off of the leaves in severe cases. Root rot seriously affects the quality and yield of A. membranaceus. The investigation was performed in the area of field was 3,996 m2, and the incidence of root rot reached 10%. Five A. membranaceus root rot samples were collected. To isolate the fungi, rotted root pieces (about 0.5 cm) were obtained with the sterile scissors from the junction of infected and healthy tissues. The symptomatic roots were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plate in incubator for 2 days at 25°C. After that the surface of root was moldy, abundant conidiophores and conidia formed. Ten isolates were obtained by single spore isolation technique. The mycelia were white or light yellow on PDA. Primary conidiophores were Verticillium-like with two to three levels. The stipe length measured 41.91 to 97.62 μm. Secondary conidiophores were penicillate with two or three whorls of phialides. The stipe length measured 36.67 to 65.98 μm. Conidiogenous cells are slender cylindrical. Conidia were colorless, round or oval, and aseptate, with an average length and width of 6.5 to 8.5×2.5 to 3.5 μm. Chlamydospores were round or oval, with an average length and width of 9.91 to 16.21×8.07 to 13.64 μm. The identity of ten isolates was confirmed by sequencing the internal transcribed spacer (ITS) locus using the primers ITS1F/ITS4 (sequence length was 526 bp), the translation elongation factor 1-alpha (EF-1α) gene using the primers EF-1H/EF-2T (sequence length was 567 bp) (O'Donnell et al. 1998), and the sequences included ITS1, 5.8S ribosomal RNA gene, ITS2, and large ribosomal RNA gene using the primers F1/F2 (sequence length was 1010 bp) (Cao et al, 2020). BLAST analysis of all the three sequences of isolates, HQ-1, HQ-1-1, HQ-2-1, HQ-3-1, HQ-4, HQ-6, HQ-6-1, HQ-7-1, HQ-9-1, and HQ-10-1, in the Genebank database resulted in matches of 99.2% or more for Clonostachys rosea (NCBI accession Nos. MZ433169-MZ433178 (ITS), MZ451390-MZ451399 (TEF1-α), and MZ433195-MZ433204 (ITS 1, 5.8 S rRNA, ITS 2, and LSU gene). Ten isolates were inoculated on their host for pathogenicity test. The isolates were grown on PDA six days for sporulation. The healthy semi-annual seeding stage root were inoculated by treating them with 10 mL of conidia suspension (1 × 105 conidial/mL). A group of 10 seedlings of the same age was treated with sterile water to serve as the control. Inoculated root were moved into artificial climatic box at 25℃, relative humidity was 70%, with 12 h light / 12 h dark condition. Typical symptoms first appeared as brown tiny spot about 3 days, extended and cracked for 5-7 days, and mycelia were observed on the surfaces of the lesions. Uninoculated plants remained healthy. Isolates were re-isolated from the symptomatic root, and the colonial morphology of the recovered isolates were the same with the original isolate. The assays were conducted three time (Fig S1). These results showed that these isolates maintained their pathogenicity to their own hosts. To our knowledge, this is the first report of C. rosea causing root rot of A. membranaceus in China. Clonostachys rosea has been previously reported to cause root rot of soybeans in the United States (Bienapfl et al, 2012). The disease must be considered in existing management practices, and our finding provided foundation for preventing or curing this disease.

First Report of Dragon Fruit (Hylocereus undatus) Stem Rot Caused by Diaporthe ueckerae in Taiwan.

Dragon fruit (Hylocereus polyrhizus & H. undatus) is a rapidly growing commodity in Taiwan. The production acreage has been tripled since 2011, with an estimation of over 2,800 ha in 2019. From disease survey conducted in July 2020, reddish orange to blackish brown lesions similar to stem canker caused by Neoscytalidium dimidiatum on dragon fruit cladodes (Supplementary Fig. S1, Q) were observed from two orchards in Central Taiwan. Diseased cladodes were brought back to the lab, surface disinfested with 70% ethanol for 15 to 30 sec, and then blotted dried with a paper towel. Small pieces (about 3x3 mm) of necrotic spots were excised, placed on 2% water agar (WA) plates, and incubated with 12 h photoperiod at 28 ± 2 ℃ for 3 days. Among the necrotic spots that were used for fungal isolation, some were detected to have N. dimidiatum accounting for 21 isolates, while three isolates detected in other spots were unknown. Single hyphal tips of the three unknown fungal colonies with similar morphology were transferred on potato dextrose agar (PDA). Brownish- to grayish-white colonies with fluffy aerial mycelium were observed on PDA (Supplementary Fig. S1, A, B, E, F, I and J) after 8 days of incubation. To induce the sporulation, all the fungal isolates were cultivated on autoclaved cowpea pods on 2% WA plates with 12 h photoperiod at 25 ± 2 ℃ for 3 weeks. Black pycnidia embedded in cowpea tissues and creamy yellowish exudates with pycnidiospores extruding from the ostiole were observed (Supplementary Fig. S1, C, G and K). Alpha-conidia were characterized as aseptate, hyaline, smooth, ellipsoidal or fusiform, often bi-guttulate and measured about 6.0 to 6.5 μm × 2.0 to 2.3 μm (n = 50 for each isolate) (Supplementary Fig. S1, D, H and L). Beta-conidia were not observed. Morphological characteristics of these isolates were similar to Diaporthe spp. described by Udayanga et al. (2015). To further identify the fungal isolates, the internal transcribed spacer (ITS), β-tubulin (TUB) and translation elongation factor 1-α (EF1-α) regions were amplified using primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass & Donaldson 1995) and EF1-728F/EF1-986R (Carbone & Kohn 1999), respectively. BLAST analysis of isolates CH0720-010 (ITS: OK067377; TUB: OK149767; EF1-α: OK149764), CH0720-013 (ITS: OK067378; TUB: OK149768; EF1-α: OK149765) and TC0720-016 (ITS: OK067379; TUB: OK149769; EF1-α: OK149766) showed 99.78 to 100% of ITS identity, 98.8 to 99.2% of TUB identity, and 100% of EF1-α identity with Diaporthe ueckerae (ITS: KY565426; TUB: KY569384; EF1-α: KY569388). Phylogenetic trees were constructed using concatenated ITS, TUB, and EF1-α sequences based on maximum likelihood with HKY+G model, maximum parsimony, and Bayesian inference method in MEGA X and Geneious Prime 2020.2.4. All isolates were clustered in D. ueckerae with similar topology based on aforementioned methods, hence the phylogram of maximum likelihood was presented (Supplementary Fig. S2). To confirm the pathogenicity, detached dragon fruit (H. polyrhizus and H. undatus) cladodes (20 to 30 cm in length) were surface disinfested, wounded with sterilized syringe (about 2 mm in depth), and inoculated with mycelial plugs (6 mm in diam.) from 5-day-old colonies on PDA. Each isolate had three mycelial plugs and the PDA plugs without mycelium were inoculated as negative control. Inoculated cladodes were placed in a moisture chamber and incubated at 30 ± 2 ℃ with 12 h photoperiod. Two days after inoculation (DAI), the agar plugs were removed and symptom development on the cladodes was photo recorded every other day. The inoculation experiment was repeated twice. At 6 DAI, round to irregular, dark-brown, and water-soaking lesions were observed on the cladodes of both species inoculated with the three D. ueckerae isolates whereas all negative controls remained asymptomatic (Supplementary Fig. S1, M-P). Morphologically identical fungi were re-isolated from inoculated cladodes, fulfilling Koch's postulates. Several Diaporthe species have been reported infecting dragon fruit in the southeastern Asian countries such as Thailand, Bangladesh and Malaysia (Udayanga et al. 2012; Karim et al. 2019; Huda-Shakirah et al. 2021). To our knowledge, this is the first report of stem rot caused by D. ueckerae in Taiwan. Since the field symptoms may be easily confused with those caused by N. dimidiatum, the potential threat of Diaporthe species complex on dragon fruit should be aware and may warrant further study.

First Report of Root Rot Caused by Fusarium armeniacum on Soybean in Korea.

Fusarium wilt samples were collected in 2017 and 2019 from two soybean (Glycine max) fields, Yesan (36°73′N, 126°81′E) and Gimje (35°76′N, 126°80′E), in Korea. The disease incidence rate in each field was approximately 1%. For fungal isolation, root lesion fragments were surface-sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and then incubated on water agar (WA) plates at 28 °C in an incubator for 5 days. Two isolates (YS37231 and GJ3050) were obtained using the hyphal tip method. Colonies of the isolates on potato dextrose agar (PDA) produced white aerial mycelia, which later turned pinkish yellow. The isolates on PDA formed abundant chlamydospores and macroconidia, but microconidia were absent. Macroconidia were 3–5 septate and prominently curved, measuring 12.4 to 41.2 × 3.3 to 4.3 µm (Leslie and Summerell, 2006). For the identification of the isolates, translation elongation factor 1 alpha (EF-1α) and RNA polymerase second largest subunit (RPB2) regions were amplified and sequenced using EF1, EF2, RPB2-5f2, and RPB2-7cr primers, respectively (O’Donnell et al. 2010). EF-1α sequences of YS37231 (MT445439) and GJ3050 (MT445440) showed 99.01 and 99.67% identity with F. armeniacum (FD_01843 and FD_01305; FUSARIUM-ID database), respectively. The RPB sequences of YS37231 (MT445442) and GJ3050 (MT445441) showed 100 and 98.48% identity with that of F. armeniacum (FD_01869; FUSARIUM-ID database), respectively. The sequences MT445439, MT445440, MT445441, and MT445442 were deposited in NCBI GenBank. The pathogenicity of the two isolates on the soybean cultivar Daewonkong was determined using two inoculation methods. In the first method, a pathogenicity assay was performed on seedlings using WA plates (Cruz Jimenez et al. 2018). Eight surface-sterilized seeds were transferred to WA plates, with or without actively growing cultures, for 3 days; and then incubated at 25 °C in a growth chamber (12 h photoperiod) for 7 days. After 7 days, brown lesions were observed on the roots in inoculated plates; however, no symptoms were observed in the control. -In the second method, 10-day old soybean seedlings were inoculated by cutting and soaking the roots in the spore suspension (1 × 106 conidia/mL) for 2 h. The inoculum was prepared by incubating isolates on PDA for 10 days, then adding sterile distilled water, scraping the growth medium, and filtering the suspension. The seedlings were then transplanted into 18 cm plastic pots (20 cm height) and grown under greenhouse conditions (26 °C ± 3 °C, 13 h photoperiod) for 2 weeks. After 7 days, the inoculated plants showed wilting symptoms, developed brown lesions in the roots, and eventually died within 2 weeks after inoculation. No such symptoms were observed in the control (inoculated with sterile distilled water). The isolates were re-isolated from the inoculated seedlings for confirmation. Although the fungus and associated mycotoxins have been reported in the rice produced in Korea (Hong et al. 2015), to the best of our knowledge, this is the first report of F. armeniacum causing Fusarium wilt on soybean in Korea. In the US, it was first reported by Ellis et al. (2012). Fusarium wilt is a soilborne disease of growing concern in soybean cultivation worldwide. Our findings will help increase awareness about the global spread of this disease.

First Report of Soybean Seedling Disease caused by Rhizoctonia solani AG-7 in Taiwan.

From August to November 2020, reduced emergence and damping-off of soybean seedlings were observed in two fields (Benzhou and Wandan) in Taiwan. Disease incidence was approximately 40% in Benzhou by field scouting. The roots of damping-off seedlings were brown. Affected seedlings could be easily pulled out from the soil and the lesions on the roots/stem were generally dry and sunken. These symptoms suggested the possibility of Rhizoctonia infection. Soil surrounding symptomatic seedlings were collected to bait the potential pathogen and symptomatic plants were used for pathogen isolation. The diseased tissues were washed with tap water and surface-disinfected with 1% bleach before placing on the Dexon selection medium at 26°C for 2 days (Ko and Hora 1971). Hyphae were transferred to potato dextrose agar (PDA), and a brown colony with brown and irregular-shaped sclerotia grew from 90 out of 99 isolates. The hyphae exhibited typical characteristics of Rhizoctonia solani, including a constriction and a septum near the end of branching hyphae (Ajayi-Oyetunde and Bradely, 2018). Two isolates from Benzhou and two isolates from Wandan were tested for their pathogenicity, and eight surface-disinfected seeds were distributed evenly on the water agar plates covered by 2-day-old mycelia at 25°C in dark for 7 days. All isolates caused cotyledon rot and reduced germination. To verify their pathogenicity in pots, double-sterilized sorghum seeds were inoculated with two strains and incubated at 25°C for 2 weeks to be used as fungal inoculum (Ajayi-Oyetunde and Bradely, 2017). A layer of 15 ml of fungal inoculum was placed 5 cm beneath the soil surface in pots. Four soybean seeds were planted approximately 3 cm above the inoculum in each pot. After two weeks, reddish lesions on the hypocotyls or taproots of all seedlings in the inoculated pots were observed, while seedlings in the control pots inoculated with sterile sorghum seeds remained healthy. The pathogen was re-isolated from lesions and had identical morphology to the original isolates. To characterize the fungal identity, the internal transcribed spacer (ITS) was sequenced using the primers ITS1/ITS4 (Sharon et al., 2006). Using BLASTN in the NCBI database, the sequence (GenBank no. MW410857 and MW410858) showed 100% (639/639 bp) similarity to KF907734 and 99.83% (635/636 bp) similarity to AF354099, both belong to R. solani anastomosis group 7 (AG-7) (Hua et al. 2014; Gonzalez et al. 2001). Phylogenetic analysis comparing sequences with different AGs (Ajayi-Oyetunde and Bradely, 2017) grouped our isolates within the AG-7 clade with a 100% bootstrap confidence. In the anastomosis test, an incompatible zonation and unequal mycelial growth rates were observed when AG-7 isolates were paired with an AG-1 IA isolate. On the other hand, the compatible tuft reaction was observed when two AG-7 isolates were paired, and the compatible merge reaction was observed in the self-pairing tests (Macnish et al. 1997). Accordingly, the molecular and morphological characterizations confirmed the causal pathogen as R. solani AG-7. R. solani AG-7 was first reported on radishes in Japan (Homma et al., 1983), first found on carnation in Taiwan (Lo et al., 1990), and in field soils of various crops but not soybean (Chuang, 1997). It was suggested that Rhizoctonia diseases of soybean may be present in Taiwan, but molecular confirmation was lacking (Anonymus, 1979). As R. solani AG-7 causes diseases of soybean in the US and Japan (Baird et al., 1996), the importance of AG-7 as an endemic pathogen of soybean in Taiwan should be recognized and its prevalence determined as a first step to managing this disease.

Droplet-based microfluidics platform for antifungal analysis against filamentous fungi

Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. The conventional antifungal screening techniques, such as water agar and 96-well plates, are based on laborious protocols and bulk analysis, restricting the analysis at the single spore level and are time consuming. In this study, we present a droplet-based microfluidic platform that enables antifungal analysis of single spores of filamentous fungus Alternaria alternata. A droplet-based viability assay was developed, allowing the germination and hyphal growth of single A. alternata spores within droplets. The viability was demonstrated over a period of 24 h and the antifungal screening was achieved using Kunshi/Tezuma as antifungal agent. The efficacy results of the droplet-based antifungal analysis were compared and validated with the results obtained from conventional protocols. The percentage inhibitions assessed by the droplet-based platform were equivalent with those obtained by the other two methods, and the Pearson correlation analysis showed high correlation between the three assays. Taken together, this droplet-based microfluidic platform provides a wide range of potential applications for the analysis of fungicide resistance development as well as combinatorial screening of other antimicrobial agents and even antagonistic fungi.