Single Spore Research Articles

First report of Diaporthe hongkongensis causing leaf spot on Rhododendron latoucheae in China.

During a field study in the Baili Azalea Forest Area in Guizhou Province, China (27°12'N, 105°48'E) between May and July 2023, symptoms of leaf spot were observed on Rhododendron latoucheae Franch. The incidence of leaf spot on R. latoucheae leaves was about 12% in a field of 1 hm2, significantly reducing their ornamental and economic value. The affected leaves bore irregular, grey-white lesions with distinct dark brown borders, accompanied by black conidiomata. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. Conidiomata present on the lesions were transferred onto water agar and incubated at 25℃ for 24 hours. Germinated spores were then transferred onto potato dextrose agar (PDA) to generate single spore cultures for morphological analysis. This process yielded three single-spore isolates: GZULJ 3-6.1, GZULJ 3-6.2, and GZULJ 3-6.3, all displaying identical morphological characteristics. Representative isolate GZULJ 3-6.1 was used for further study. The colonies, cultivated on PDA at 25°C in the dark, appeared pale gray with white aerial mycelium. Alpha conidia were single celled, translucent and fusiform, measuring 6.2 to 8.1 × 2.6 to 3.0 μm (n=50). Beta conidia were filamentous and hook-shaped, measuring 17.0 to 23.0 × 0.9 to 1.5 μm (n=50). The morphological features were consistent with the established description of Diaporthe hongkongensis (Zhang et al. 2021). For molecular identification, total genomic DNA was extracted. The complete internal transcribed spacer region (ITS), partial sequence of beta-tubulin (TUB), translation elongation factor 1-alpha (TEF1), and calmodulin (CAL) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson. 1995), EF1-728F/EF1-986R and CAL-228F/CAL-737R (Carbone and Kohn. 1999), respectively. The ITS, TUB, TEF1, and CAL sequences (GenBank accession nos. OR807761, OR825453, OR825450, and OR825447, respectively) were obtained, and BLASTn analysis against sequences in GenBank showed 97.37 to 99.30% identity with Diaporthe hongkongensis CBS 115448. Phylogenetic analysis reaffirmed the isolate's placement within a well-supported cluster alongside D. hongkongensis. The pathogenicity of GZULJ 3-6.1 was evaluated using three 2-year-old R. latoucheae plants inoculated with spore suspension (106 spores per ml). The inoculation targeted three leaves per plant, while an additional three plants, serving as controls, were sprayed with sterile water. The plants were maintained in the growth chamber at 25°C, with a 12-hour photoperiod and 75% relative humidity. The pathogenicity test was repeated three times. After 14 days, the inoculated leaves developed brown lesions similar to those observed in the field, whereas the control leaves remained symptom-free. The fungus was successfully reisolated from the infected leaves and identified through morphological characterization and molecular analyses, confirming its identity. D. eres, a closely related species to D. hongkongensis, has been reported to induce similar disease symptoms on R. latoucheae (Wu et al. 2024). However, to our knowledge, this is the first report of D. hongkongensis causing leaf spot disease on R. latoucheae in China. This finding will facilitate the development of effective management strategies to control the complex leaf spot disease affecting R. latoucheae and provide critical insights into its broader implications for the health of this species.

Co-encapsulation of Creatininase, Creatinase, and Sarcosine Oxidase in Yeast Spore for Creatinine Degradation.

Creatinine clearance is used to reflect the glomerular filtration rate to assess kidney function. Creatinine degradation-related enzymes have been used for creatinine detection in clinical medicine. The mixture of spores encapsulating either creatininase or creatinase or sarcosine oxidase could mediate a three-step reaction to produce hydrogen peroxide from creatinine. In this study, to achieve consecutive and efficient creatinine detection, degradation enzymes creatininase, creatinase, and sarcosine oxidase were co-encapsulated in a single Saccharomyces cerevisiae spore. The co-encapsulation spores performed high specific activity and enzymatic properties and converted creatinine to H2O2, which was 160% higher than the mixture of spores that individually expressed these three enzymes. The detection condition of co-encapsulation was optimized for the store at room temperature and resistance to environmental stresses. The S. cerevisiae spores can co-encapsulate enzyme families and catalyze consecutive reactions in the spore wall, having potential application prospects.

Lasiodiplodia theobromae causes rachis blight on coconut palms (Cocos nucifera L.) in Florida.

Lasiodiplodia, a genus in the family Botryosphaeriaceae, has a broad host range and causes dieback, root rot, fruit rot, leaf rot, and blights in many plant species across sub-tropical and tropical geographical areas (Alves et al., 2008). In palms, this fungal pathogen is known to cause fruit and heart rot, wood decay and leaf blight around the globe (Atallah et al., 2024; Santos et al., 2020). In field-grown coconut palms symptoms usually start on older fronds progressing to younger fronds. Symptoms including discoloration of internal tissue, profuse pathogen sporulation, fruiting body formation, and death of entire fronds, universally characterize rachis blight caused by Lasiodiplodia. In summer 2023, a disease incidence of 30% and severity ranging from 20% to 100% of the canopy was observed in Florida. Three diseased coconut palms were sampled from a five-acre plot and the symptomatic tissue (internal rachis discoloration) was excised at the margin of the advancing lesion, and surface sterilized (30s 75% EtOH, 1 min 3% sodium hypochlorite, 1 min rinse in sterile water). Six pieces were plated on one Potato Dextrose Agar (PDA, Difco) plate supplemented with three antibiotics, penicillin, streptomycin, and neomycin, each at 5mg/L (Gibco PSN), and a total of three PDA plates per sample were processed. Plates were incubated at 25°C under darkness and Lasiodiplodia-like colonies were consistently recovered from all 54 rachis pieces. Initially white in color, the fungal colony grew radially, turned gray, and eventually took on a darker shade, with an abundance of aerial mycelium and rare occurrence of conidiomata and conidia in older plates. Two morphologically similar isolates Las1A and Las1B were obtained from single spores and grown at 28°C in the dark for 7 days. DNA was extracted using the quick DNA extraction protocol (Liu et al., 2000) and regions from two fungal barcoding genes, nuclear ribosomal internal transcribed spacer (nrITS) and translation elongation factor 1-α (TEF1-α) were amplified using primers ITS1/ITS4 (White et al., 1990) and EF1-728F/EF1-986R (Carbone & Kohn, 1999), respectively, and sequenced. The ITS (PQ282128 and PQ282129) and TEF1-α sequences from both isolates (PQ278132 and PQ278133) were 100% identical to Lasiodiplodia theobromae isolate G112-8 (OR453211 and OR482955, respectively). One-year-old seedlings of coconut palm (Cocos nucifera L.) were used for pathogenicity tests. Agar plugs from margins of actively growing cultures of Las1A and Las1B were placed on cut end of petioles from fronds close to the spear leaf and covered with parafilm. The control palms were treated with distilled water. The onset of disease symptoms was observed about 14 days post inoculation. The inoculated petioles were soft, listless, lost turgidity, and could be easily bent without snapping about 1 month following inoculation. In contrast, the palm control seedlings that were inoculated with water remained turgid, healthy and alive. Fruiting body development was observed on inoculated petioles, the pathogen was reisolated, and examination of its colony and spore morphology as well as presence of paraphyses was used to verify its identity. This is the first report of Lasiodiplodia theobromae causing disease on Cocos nucifera in Florida. This pathogen is a potential threat to coconut palm production in Florida and further research on pathogen diversity, pathogenicity, and distribution is needed to develop options for disease management.

First report of Fusarium asiaticum and F. meridionale causing leaf spot on Hemerocallis citrina in Sichuan province, China.

Daylily (Hemerocallis citrina) is widely cultivated in China as vegetable and medicine. Its flower buds are nutritious vegetables rich in ascorbic acid, mineral elements and et al (Wang et al. 2024). Whereas, little is known about the pathogen identification in daylily. In April 2023, leaf spot symptoms were observed on 20% daylily seedlings in Dazhou city (30.91° N, 106.87° E), Sichuan province, China. The symptomatic leaves were superficially disinfected with 70% ethanol for 20 s, rinsed twice in sterile dH2O and subsequently disinfected with 1% NaClO for 40 s, and rinsed twice in sterile dH2O again. The disinfected leaves were cut into pieces (5 × 5 mm), placed on PDA amended with streptomycin sulfate (50 mg/L), and incubated in dark at 25 ℃ for two days. The fungal isolates, displaying morphological features of Fusarium species (Leslie and Summerell 2006), were purified through transferring single spores. Consequently, two distinct type fungal isolates were obtained. Cultured on PDA, Type Ⅰ (Q22, Q26, Q27) and Type Ⅱ (Q29, Q29-1) isolates showed similar growth phenotypes. Type Ⅰ and Ⅱ isolates' cultures were initially white but gradually became yellow, and scarlet diffusible pigments were produced with time (Fig. S1). On SNA medium, Type Ⅰ isolates produced macroconidia with 3 to 6 septa, which measured 5.56±0.54 × 48.65±6.12 µm (n = 50). While, Type Ⅱ isolates' macroconidia contained 3 to 11 septa that measured 4.08±0.93 × 51.40±12.83 µm (n = 50) (Fig. S1). All the isolates were properly preserved in our lab. To further identify these isolates, DNA fragments of Beta-tubulin (TUB2) (Glass and Donaldson 1995), translation elongation factor-1 alpha (TEF1) (Rehner and Buckley 2005), and DNA-directed RNA polymerase II largest (RPB1) (Hofstetter et al. 2007) and second largest subunit (RPB2) (O'Donnell et al. 2012) were amplified and sequenced. BLASTN analyses of TUB2 (PP266396 ~ PP266398), TEF1 (PP404039 ~ PP404041), RPB1 (PP404042 ~ PP404044) and RPB2 (PP408000 ~ PP408002) of Type Ⅰ isolates showed 99.35 ~ 100% identity to those of F. asiaticum NRRL 13818 (AF212745.1, MW233069.1, MW233240.1, JX171573.1). Type Ⅱ isolates' sequences (PP408003 ~ PP408010) showed their homology with those of F. meridionale NRRL 28436 (AF212730.1, MW233092.1, MW233263.1, MW233435.1) at 100% identity. The phylogenetic tree based on combined datasets of TUB2, TEF1, RPB1 and RPB2 of Fusarium species confirmed that Type Ⅰ and Ⅱ isolates were F. asiaticum and F. meridionale, respectively (Fig. S2). Seedlings of cultivar "chuanhuanghua No.1" (n = 5) in a greenhouse (25°C, relative humidity 90%) were inoculated with conidial suspension (3 × 105 conidia / mL). Controls were treated with sterile dH2O. Fifteen days post-inoculation, natural symptoms appeared on inoculated leaves (Fig. S3). Whereas, non-inoculated controls were disease-free. The pathogenicity assay was repeated three times. F. asiaticum and F. meridionale were successfully re-isolated from diseased leaves and verified using morphological and molecular methods described above, fulfilling Koch's postulates. F. ussurianum, a Fusarium graminearum species complex (FGSC) member, and F. proliferatum were identified as causal agents of leaf spot on daylily in China (Chen et al. 2024; Li et al. 2018). To our knowledge, this is the first report of F. asiaticum and F. meridionale causing leaf spot on daylily worldwide. Our studies demonstrate that FGSC members are the common pathogens causing leaf spot on daylily in Sichuan, China.

First Report of Colletotrichum siamense Causing Leaf Anthracnose on Jackfruit in Thailand.

Jackfruit (Artocarpus heterophyllus Lam.) is commonly grown in Thailand. In June 2023, leaf anthracnose on this plant was observed at a field in Chai Prakan District (19°42'24"N, 99°01'59"E), Chiang Mai Province, Thailand, with ~25% disease incidence in a 1000-m2 plantation area. The initial symptom had brown spots with a yellow halo, enlarged, elongated, 0.2 to 2 cm in diameter, irregular, sunken, brown, with a dark brown halo, and leaves withered and dried. Pale yellow conidiomata developed on the lesions in high humidity. Ten symptomatic leaves were used to isolate the fungal causal agents through a single spore isolation method (Tovar-Pedraza et al. 2020). Four fungal isolates (SDBR-CMU492 to SDBR-CMU495) with similar morphology were obtained. Colonies on potato dextrose agar (PDA) were 70 to 85 mm in diameter, white to grayish white with cottony mycelia, the reverse pale yellow after incubation at 25°C for 1 week. All isolates produced asexual structures. Setae were brown with 1 to 3 septa, 40 to 100 × 2.2 to 4.0 µm, a cylindrical base, and acuminate tip. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 7.4 to 27.2 × 2.0 to 4.5 µm. Conidia were one celled, hyaline, smooth walled, cylindrical, ends rounded, guttulate, 11.1 to 15.7 × 3.4 to 6.1 µm. Appressoria were dark brown to black, oval to irregular, 8.8 to 24.9 × 3.6 to 10 µm. Morphologically, all isolates resembled the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) region, actin (act), β-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using primer pairs ITS5/ITS4, ACT-512F/ACT-783R, T1/T22, CL1C/CL2C, and GDF1/GDR1, respectively (White et al. 1990; Weir et al. 2012). Sequences were deposited in GenBank (ITS: PP068858, PP068859, PP446789, PP446790; act: PP079636, PP079637, PP460760, PP460761; tub2: PP079638, PP079639, PP460762, PP460763; CAL: PP079634, PP079635, PP460758, PP460759; GAPDH: PP079632, PP079633, PP460756, PP460757). Maximum likelihood phylogenetic analyses of the concatenated five genes identified all isolates as C. siamense. To pathogenicity test, the mature leaves of a healthy plant were surface disinfested using 0.1% NaClO for 3 min, rinsed three times with sterile water, and wounded. Conidia suspensions (15 µl of 1 × 106 conidia/ml) of each isolate grown on PDA at 25°C for 2 weeks were used to inoculate wounded and unwounded samples by the attached method. Control leaves were mock inoculated with sterile distilled water. Ten replications were conducted for each treatment and repeated twice. Plants were placed in a greenhouse at 25 to 30°C and 80 to 90% relative humidity. After 7 days, all inoculated leaves displayed brown lesions, while control leaves had no symptoms. Colletotrichum siamense was reisolated from inoculated tissues on PDA to complete Koch's postulates. Prior to this study, C. fructicola and C. gloeosporioides caused leaf anthracnose on jackfruit worldwide (Sangchote et al. 2003; Chitambar 2016). Leaf anthracnose on jackfruit caused by C. siamense has been reported from Australia (James et al. 2014) and Bazil (Borges et al. 2023). In Thailand, Bhunjun et al. (2019) reported that C. artocarpicola causes leaf anthracnose in jackfruit. Therefore, this is first report of C. siamense causing leaf anthracnose on jackfruit in Thailand. The finding will inform epidemiological investigations and future approaches to managing this disease.

Identification, characterization, and sensitivity to phytochemicals of a novel Curvularia species associated with leaf spot disease on Curcuma kwangsiensis

A leaf spot disease affecting Curcuma kwangsiensis (Zingiberaceae) has been observed in Qinzhou City, Guangxi Province. Infected leaves exhibit yellow-brown spots that progressively expand and eventually lead to leaf death. Curvularia isolates were obtained from the diseased leaves with tissue isolation and single spore purification methods. To accurately identify these isolates, we analyzed their morphological characteristics and phylogenetic relationships using combinations of ITS, GAPDH, and EF-1α gene sequences. Phylogenetic analysis showed that the investigated strains formed a distinct clade separate from other recognized Curvularia species. Furthermore, the strains exhibited differences in conidiophore size and conidia shape/size. Based on phylogenetic studies, morphology, and pathogenicity tests, the pathogen was identified as a new species named Curvularia qinzhouensis. Optimal conditions for mycelial growth were observed at 30 °C and pH 8. The sensitivity of the pathogen to various phytochemicals was also examined. Honokiol, thymol, and citral demonstrated effective antifungal effects, with EC50 values of 6.72 ± 1.75, 25.74 ± 4.30, and 54.24 ± 4.69 µg/ml, respectively. The present investigation provides the first report of leaf spot disease on C. kwangsiensis caused by C. qinzhouensis, and valuable insights for the prevention and control of this disease.

First Report of Basal Stem Rot of Achnatherum inebrians Caused by Fusarium pseudograminearum in China.

Drunken horse grass (Achnatherum inebrians) is a perennial bunchgrass that is widely distributed in arid and semi-arid grasslands in northwest China (Zhang et al., 2021). In July 2023, Basal stem rot was found in artificially grown drunken horse grass plots in Yuzhong County (35.85° N, 104.12° E), Gansu Province, China, with an average incidence of 5.2%. Diseased plants showed crown and basal stem rot with chocolate brown discoloration at the base of the stem and slight constriction of some basal stems. Five field's foci were surveyed and at least 6 basal stems per focus were collected. Infected basal stems were surface-sterilized (75% ethanol for 30 s and 1% NaClO for 90 s), rinsed three times with sterilized water, placed on potato dextrose agar (PDA), and incubated at 22°C in the dark for 3 days. Isolates were purified by single spore cultures (Leslie and Summerell, 2006). The average mycelial growth rate was 4.8 to 7.5 mm/day at 25°C on PDA, and the colonies produced aerial mycelium varying from rose to yellow white, and rose to burgundy pigment diffused into the agar. Macroconidia of the isolates were produced on carnation leaf agar (CLA) incubated under black light and observed to be abundant, but no microconidia were found. Macroconidia were relatively slender, curved to almost straight, commonly 3-6 septate, averaging 30.1 × 3.8 μm (n=50). The morphological characteristics of this fungus fully fit the description of F. pseudograminearum (Aoki and O'Donnell, 1999). To obtain the phylogenetic support, DNA of three representative isolates YZ-Y-1, YZ-Y-2 and YZ-Y-3 was extracted by using an HP Fungal DNA Kit (D3195), and a portion of the RNA polymerase II second largest subunit (RPB2) gene and elongation factor 1 alpha (EF1-α) gene were amplified using primers RPB2-5f2 and RPB2-7cr (O'Donnell et al. 2010) and EF1 and EF2 (O'Donnell et al. 1998), respectively. Results of sequences were deposited in GenBank (accession nos. PP179044 to PP179049). A nucleotide BLAST search revealed RPB2 and EF1-α sequences to be 99.8 and 100% similar to the corresponding sequences of the ex-type strain NRRL 28062 F. pseudograminearum accessions numbers MW233433 and MW233090, respectively. For pathogenicity tests, 15 μl of conidia suspension (1×106 conidia/ml) was inoculated into the stem bases of 10 healthy drunken horse grass seedlings (around 3 weeks old) using a sterile syringe, then wrapped with moistened sterile gauze, while the other 10 drunken horse grass seedlings were injected with sterile water as a control. All seedlings were placed in a greenhouse with a plastic cover at 15-22°C and 90-100% relative humidity. All inoculated drunken horse grass seedlings showed symptoms similar to those of natural infection with stem basal rot, whereas uninoculated drunken horse grass seedlings remained healthy after 15 days. Fungi re-isolated from the basal stems of inoculated plants were confirmed phenotypically and molecularly as F. pseudograminearum. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of drunken horse grass in China. The disease has become a potential threat to the growth of drunken horse grass in China.

First report of leaf spot on Gleditsia sinensis caused by Colletotrichum gloeosporioides in China.

Gleditsia sinensis Lam (Lamarck et al., 1788) is an endemic species widely distributed in China. In Sep. 2022, leaf spot symptoms were observed on G. sinensis in Xuhui district (31◦9'16''N, 121◦26'36''E), Shanghai, China, with an incidence rate of 55% in the examination of 9 trees. The leaves showed typical symptoms of anthracnose with irregular gray-brown spots and sunken areas. For isolation, 5 × 5 mm sections were cut from the lesion edge of 20 infected leaves collected from 2 trees. The surface of the sections was sterilized by immersion in 75% ethanol for 30 s, followed by 5% NaClO for 1 min, rinsed three times with sterile water, and dried on sterile filter paper. These sections were placed on PDA plates incubated at 25°C in darkness. Eighteen isolates with similar colony morphology were obtained and purified by single spore culturing. Two isolates (YKY2301, 2302) from separate trees were further tested. On the 6th day, the colonies had a diameter of 7.6 to 8.4 cm and appeared white to gray-white with aerial hyphae. The colony's central part exhibited an orange hue due to the conidia accumulation, while the undersides displayed an orange-yellow color. The hyphae were hyaline and smooth, with septa and branches, and the conidia were cylindrical with blunt to slightly rounded ends, measuring 13.1 to 18.8 (average 15.9) μm× 4.0 to 6.6 (average 5.4) μm (n=184). From conidia germinated on glass slides, the appressoria measured 5.5 to 6.3 μm ×4.9 to 5.1 μm (n=50) and were nearly spherical or elliptical in shape. These characteristics matched those of the Colletotrichum gloeosporioides species complex (Cannon et al., 2012; Weir et al., 2012). For molecular identification, the genomic DNA was extracted using a modified CTAB method (Luo et al., 2012). Gene fragments including ITS (PP125667, PP125668), GAPDH (PP153428, PP153429), ACT (PP153424, PP153425), TUB2(PP153917, PP190256), and ApMAT (PP153426, PP153427) were obtained by PCR using universal primers (Huang et al., 2022) and sequenced. The sequences exhibited 98.19% to 99.82% identity with the corresponding gene of the type strain C. gloeosporioides IMI356878 (JX010152, JX010056, JX009531, JX010445, JQ807843) in NCBI BLAST. A multilocus Maximum likelihood phylogenetic tree was constructed based on concatenated the five genes by PhyloSuite. It showed that YKY2301, 2302 were on the same branch with C. gloeosporioides. Based on these results, the isolates were identified as C. gloeosporioides. Pathogenicity tests were conducted by mycelial and conidia inoculation. 5 mm mycelial or blank agar plugs were inoculated onto the leaves of 2 healthy trees in a garden (25 to 30 °C), with and without wounds made by toothpick pricking (n≥3 per group). All mycelial inoculated leaves showed leaf spots on the 6th day. Three healthy 2-year-old seedlings were inoculated with either conidia (108 conidia/ml) or water by leaf spray, and maintained in a climate chamber (27 °C, 80% humidity). Inoculated seedlings showed necrotic leaf spots on day 14, and wilted within 3 weeks. The controls in all tests remained asymptomatic. The pathogen has been re-isolated and confirmed by sequencing, thus fulfilling Koch's postulates. This is the first report of leaf spots caused by C. gloeosporioides on G. sinensis in the world. As illustrated by the example of legume pod infection (Gerusa et al., 2019), it poses a potential threat to the fruits of G. sinensis, despite currently only affecting their ornamental value. This report provides basic information for future research.

Foliar blight, leaf spot, and stem blackening diseases caused by Stemphylium beticola, Alternaria alternata, and Alternaria atra on dragon's head (Lallemantia iberica) in Iran

Dragon's head, Lallemantia iberica, is an oilseed crop adapted to be grown in cold-dry areas. In the growing season of 2022–23 a severe foliar blight, leaf spot, and stem blacking symptoms were observed in commercial fields in northern and northwestern Iran, leading to more than 50 % yield loss. Infected samples were collected and transferred to the laboratory. Following the isolation of fungal agents, five representative isolates were selected for further studies. Morphological studies, based on conidial shape, conidiation, and other mycological characteristics, were carried out on a pure culture established from a single spore. Furthermore, multigene phylogenetic analyses were performed using sequences of translation elongation factor 1-α (TEF1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Internal Transcribed Spacer (ITS) rDNA genes for Alternaria isolates, along with calmodulin (cmdA) for the Stemphylium isolate. The morphological and molecular studies identified the fungal isolates as Alternaria atra, Alternaria alternata, and Stemphyliium beticola. Pathogenicity tests on the commercial cultivar ‘Sara’ indicated that all isolates were pathogenic to dragon's head, but they differed significantly in aggressiveness. To the best of our knowledge, this is the first report of the association of these fungal species with an important foliar disease on this crop in Iran and worldwide.

First Report of Didymella pomorum Causing Leaf Blight on Angelica sinensis in China.

Angelica sinensis (Oliv.) Diels, is a perennial herbaceous plant of the Umbelliferae family. It has a long history of cultivation and is highly valued as a traditional Chinese medicine in China (Zhang et al. 2012). In September 2023, leaf blight on A. sinensis with an average disease incidence of 56% was recorded in an approximately 6.7-ha production field in Lijiang, Yunnan province, China (26.8215°N, 100.2369°E). At first, small, chlorotic lesions appeared on the leaves. They subsequently increased in density and gradually merged, causing leaves to yellow and wither. Ultimately the blight casused death of the entire foliage. In order to identify the causal agent, cross-sectional segments (5×5 mm2) were cut from the edge of leaf lesions, surface disinfected with a 1% sodium hypochlorite solution for 3 min and rinsed three times with sterile distilled water. They were subsequently placed on potato dextrose agar (PDA) plates and incubated for 3 days under a 12-h photoperiod at 28℃. A total of ten isolates with similar morphological characteristics were obtained by single spore isolation. After 10 days of incubation on PDA, the colony morphology of these isolates was characterized by a brownish central area with a white edge. Aged colonies became wrinkled in the center of the colony. Conidia (n = 30) were elliptical and brown, with a size range of 4.11 to 6.55 μm (average 5.37±0.74 μm) × 3.17 to 4.62 μm (average 3.92±0.43 μm). Chlamydospores (n = 30) formed chains in series, spherical or elliptical in shape, ranging from yellow-brown to dark brown, with a size range of 12.30 to 13.70 μm (average 12.98±0.46 μm) × 4.20 to 5.30 μm (average 4.63±0.26 μm). The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of RNA polymerase II (RPB2), and the 28S nuclear ribosomal large subunit rRNA (LSU) region of two isolates were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), and LR0R/LR5 (Schoch et al. 2012), respectively. These amplicons were sequenced bidirectionally and assembled. The two isolates produced the same nucleotide sequences, and the sequences of a representative isolate (AsDp1) were deposited in GenBank. BLASTn analyses showed that the ITS (PP510616), RPB2 (PP526010), and LSU (PP550143) sequences of isolate AsDp1 were 100%, 99.66%, and 100% identical with those of Didymella pomorum ex-type isolate CBS 354.52 (MH857081, KT389616, and MH868616), respectively. A phylogenetic tree was constructed based on the ITS, RPB2, and LSU concatenated nucleotide sequences using the maximum likelihood method in MEGAX. Isolate AsDp1 was clustered with four D. pomorum isolates. According to the morphological and nucleotide sequences analyses, isolate AsDp1 was identified as D. pomorum (Chen et al. 2015). To determine pathogenicity, 1-year-old A. sinensis plants (approximately 20 cm tall) grown in 7-liter pots filled with sterilized field soil were sprayed until runoff with a 1×106 conidia/ml suspension of isolate AsDp1 onto the foliage, while control plants were sprayed with sterile water. All plants were cultivated under a 12-h photoperiod at 25℃. The pathogenicity tests were performed in triplicate with ten plants in each treatment. After fifteen days, numerous chlorotic lesions appeared on the leaves of all inoculated plants. The symptoms were similar to those found on naturally infected plants in the field, while the control plants remained asymptomatic. Subsequently, D. pomorum was reisolated from the diseased leaves, and the identity was confirmed based on its ITS sequence and morphological characteristics. D. pomorum causing stem canker on Rosa spp. was reported in Canada (Ilyukhin 2022). To our knowledge, this is the first report of D. pomorum causing leaf blight on A. sinensis in China. This etiological finding will potentially pave the way for the development of control strategies of this disease.

Heliconia subulata, a New Host of Colletotrichum tropicale Causing Anthracnose in China.

Heliconia subulata is a common ornamental plant, it has been widely planted in southern China for greening parks, roads, and residential areas. H. subulata plants with spots on their leaves were observed in East Coast Wetland Park (18°16'53.37″N, 109°30'19.36″E), Sanya City, Hainan Province, China on Aug. 31, 2023. The symptoms of the leaves are irregular gray-white, spots, that develop into brown and black, with yellow halos at the disease-health junction. Following an on-the-spot investigation, it was found that the incidence of the disease was 40 to 50%. The leaves were disinfected with 70% ethanol for 1 min, rinsed with sterile water 3 times, disinfected for 1 min with 0.1% HgCl2, rinsed with sterile water 3 times, dried, put on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. The red conidia pile was selected from the culture, dispersed in sterile water and diluted to 20 μL containing 1 to 2 conidia. After absorbing 20 μL spore suspension for many times and inoculating it on the new PDA plate, five pure cultures of single spore, J-1-1 to J-1-5, were obtained. After 7 days of growth, the colonies were grayish aerial mycelium on the front and light orange conidia on the reverse. The white aerial mycelia, conidia, acervulus, and appressorium were observed (Supplementary Fig. S1). The morphological characteristics showed that the isolate had the same characteristics as the previously described Colletotrichum spp. (Wang et al. 2021). The genomic DNA of isolates J-1-1 and J-1-5 were extracted by Fungal DNA Kit (OMEGA bio-tek, Guangzhou, China). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and β-tubulin 2 genes (TUB2) were amplified by primers ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b, respectively (Weir et al. 2012). Based on sequencing and gene sequence alignment analysis, it was found that the consistency between the ITS sequences of isolates J-1-1 and J-1-5 was 99.82%. The consistency between GADPH and TUB2 sequences was 100%. The gene sequences of isolates J-1-1 and J-1-5 were submitted to GenBank with accession numbers PP455510/PP455511 (ITS), PP510210/PP510211 (GADPH) and PP510212/PP510213 (TUB2) respectively. Based on the BLAST analysis, the three sequences were more than 99% identical to those of theC.tropicalestrain FC1 (ITS: MT192648, GAPDH: MT155819, TUB2: MT199874; Duan et al. 2022). A phylogenetic tree was constructed by MEGA 11 based on the ITS, GADPH, and TUB2 gene sequence by the maximum-likelihood method. The results showed that the isolates J-1-1 and J-1-5 were clustered with C. tropicale CBS:124949 (Supplementary Fig. S2). Based on morphological and molecular biological analysis, two isolates were identified as C. tropicale. To further test the pathogenicity of isolates J-1-1 and J-1-5, spore suspensions (1×106conidia/mL) were prepared and 20 μL spore suspensions were inoculated on the leaves of healthy H. subulata potted plants stabbed with sterile toothpicks. Three leaves were inoculated in each treatment, and sterile water was inoculated as a control. The treated plants were placed in an incubator with a temperature of 28℃, relative humidity of 90%, and light/dark (12h/12h). After 15 days, the spore suspension treatment showed the same symptoms as the naturally diseased H. subulata plants in the field, but the leaves treated with sterile water were not infected (Supplementary Fig. S1). The morphology of the isolates obtained from diseased leaves was the same as that of isolates J-1-1 and J-1-5 on the PDA plate. To our knowledge, this is the first report of H. subulata, a new host of C. tropicale causing anthracnose in China.

First Report of Colletotrichum queenslandicum Causing Leaf Anthracnose on Jatropha curcas L in China.

Jatropha curcas L. (Euphorbiaceae), is a valuable multi-purpose crop, previously used for disease treatment and environmental restoration, recently is attention to use jatropha oil for produce biodiese. In June 2023, the leaf disease of approximately 60% of J. curcas was observed in Mazhang, Zhanjiang, Guangdong Province (E110°27'26.8'' N22°6'14.7''). Diseased leaves showed typical anthracnose symptoms of chlorotic regions with brownish sunken necrotic lesions (Figure 1). Sections from the junction of disease were surface disinfected in 75% ethanol and 3% hydrogen peroxide solution for 1 minute each. Four small pieces of infected tissue were removed from the lesion and placed on potato dextrose agar (PDA), and incubated at 25 to 28℃ in the dark. Hyphal tips from the inoculated tissues were subcultured on PDA and two isolates were purified by single spore method. The colonies on PDA were 7.8 cm diam after 10 d at 25 to 28 ℃, covered with dense, cottony, grayish-white aerial mycelium and small dark-based acervuli with orange ooze and dark brown straight setae. Conidia were hyaline, smooth-walled, aseptate, the apex and base rounded, slightly constricted near centre, 12.9 - 13.8 × 3.9 - 4.6 um (av.13.6 × 4.3 μm, n = 50). Appressoria were variable in shape, mostly simple, subglobose or irregular lobes, 5.8-9.6 × 5.7-11.2 um (Figure 2). Perithecia were not observed. These characteristics were consistent with Colletotrichum sp. (Weir, B. S., et al. 2012). Sequences of isolates ACCC 35630 and ACCC 35631 stored in Agricultural Culture Collection of China including internal transcribed spacer (ITS), actin (ACT), beta-tubulin (tub2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (chs). were amplified (Weir et al, 2012), sequenced and submitted to GenBank (ITS: PP474979 and PP474984; ACT: PP505487 and PP505488; TUB: PP505493 and PP505494; GAPDH: PP505491 and PP505492; CHS: PP505489 and PP505490). The amplicon sizes of ITS, ACT, TUB, GAPDH, and CHS were 550, 652, 500, 264, and 301 bp, respectively. Phylogenetic analyses showed that Isolates 35630 & 35631 were clustered closely association with RHCOL1 and RHCOL3. Phylogenetic analysis with MEGA 7 using the combined ITS-ACT-CHS-GAPDH-TUB2 sequences showed that the two isolates clustered with C. queenslandicum (Figure 3). To test the pathogenicity, ten healthy leaves on plants in the field were wiped with 75% alcohol and sterile water, punctured with a sterile needle and inoculated by adding 10 uL of spore suspension (1 × 105 conidia/ml) to the wounded sites. And two other leaves were added sterile water as controls. Symptoms of anthracnose were observed on leaves similar to the disease described above after 7 days of inoculation, whereas the leaves from the controls remained asymptomatic. C. queenslandicum was reisolated from the inoculated leaves. C. queenslandicum has been reported as a pathogen causing leaf and fruit anthracnose on papaya, coffee, rambutan, avocado and Persian lime etc. in tropical and temperate regions (Kunta, M., et al. 2018), and this is the first report on J. curcas in China as well as worldwide. This disease may have a significant negative impact on J. curcas cultivation.

Population distribution characteristics of mating type genes and genetic stability in Morchella sextelata.

The reproductive mode of morels (Morchella spp.) is governed by mating type genes, specifically MAT1-1 and MAT1-2. This study investigated the presence of mating type genes at various growth stages and in different parts of cultivated Morchella sextelata. This study revealed significant fluctuations in the detection ratio of the two mating type genes during ascocarps growth. Single ascospore strains with MAT1-1, MAT1-2 and both mating types were selected for experimentations. Stress stimuli including H2O2, Congo red and NaCl were introduced into the medium. Differences in the cultural and physiological characteristics of single spore strains were analyzed, and mating type genes were identified after subculturing to assess their stability. The results indicated that a total of 297 samples with a single mating type gene were detected in 480 samples selected from the five stages of fruiting body growth, accounting for 61.9%. Stress exposure influenced colony morphology, mycelial growth rate, and biomass, leading to significant increases in malondialdehyde content and osmotic adjustment compounds, including soluble protein and proline. Physiological and biochemical parameters varied among the three mating type strains under different stress conditions. Principal component analysis was used to calculate the weight values, which showed that the MAT1-2 strain exhibited the highest tolerance to chemical stresses, particularly oxidative stress. Subculturing under stress revealed that single mating type strains ceased growth by the 8th generation, whereas both mating type strains could continue to the 15th generation without loss of mating type genes, indicating broader environmental adaptability and higher viability. These findings offer novel insights into mating type gene function and serve as a scientific foundation for the development of high-yield, stress-resistant morel varieties.

First Report of Neopestalotiopsis clavispora Causing Fruit Brown Spot of Pomegranate in China.

Pomegranate (Punica granatum) is an important fruit crop for therapeutic and food applications. In June 2022, brown spots were observed on the fruit surface of pomegranate cultivar named Guangyan in Mengzi (23°20'6''N,103°25'5''E), Yunnan, China. The early spots appeared as circular or irregular lesions, measuring 1~1.5 mm in diameter. They were light brown with a clear boundary between disease and healthy tissues. Over time, these spots developed into polygonal lesions covering the entire fruit surface. Eventually, the diseased fruits decayed, and more than 50% of fruits were infected in pomegranate orchards. The tissues from the interface between health and disease were cut down, immersed in 75% ethanol for 15 s, then 5% NaOCl disinfecting for 2 min, washed three times with sterile water, and the PDA cultured at 26 °C in an incubator under dark conditions. Twenty-five samples were collected for pathogen isolation, ten fungal isolates were obtained by single spore germination, and these isolates had similar morphological characters. The colonies were white with 81 mm diameter at 7 days of incubation, containing undulate edges with dense aerial mycelium. After 14 days, the black conidiomata formed superficially, gathering into black droplets. Conidiogenous cells were hyaline, short, and filiform. Conidia were fusiform, straight or slightly curved, and comprised five cells, 24.12 to 34.53 (x̄=29.78) μm × 4.21 to 12.15 (x̄=8.68) μm (n=50). The three median cells were 13.13 to 25.22 μm (x̄=18.54), dark brown, whose septa and periclinal walls were darker than the other two cells. The apical cells showed two to four appendages, 12.31 to 29.15 (x̄=21.56) μm. Only a single appendage was found on the basal cell, 2.34 to 7.16 μm. Based on morphological features, these isolates were identified as Neopestalotiopsis clavispora (Maharachchikumbura et al., 2012, 2014). Molecular identification of isolate YNSL-3 was performed by amplification and sequencing of ITS4/ITS5, BT2A/ BT2B and EF1-728F/EF-2, respectively (White et al. 1990, Glass et al.1995, Carbone et al. 1999, O'Donnell et al. 1998). These base sequences were deposited in GenBank with accession numbers OQ891378 (ITS), OR088917 (Tef) and OR513439(Tub), respectively. BLAST searches of the sequences revealed 100% (478/478 bp), 100% (484/484 bp), and 94.67% (426/450 bp) homology with those of N. clavispora NM16311a from GenBank (LC209216, LC209220, and LC209221), respectively. Phylogenetic analysis (IQ-TREE) by maximum likelihood method showed that the isolate YNSL-3 was clustered with N. clavispora. The pathogenicity was tested with the isolate of YNSL-3, YNSL-5 and YNSL-8 by detached assay. The fruit surface of pomegranate cultivar Guangyan was wounded with a sterilized needle. The mycelial blocks (5mm2) of isolates cultured on PDA for 7 days were attached to the points of inoculation. Controls were inoculated with sterile PDA agar. All inoculated fruits were maintained in a growth chamber at 26°C with 75% relative humidity. The test was performed thrice. The brown lesions were observed after 7 days, whereas the controls showed no symptoms. The same pathogens reisolated were identical to the original isolates based on morphological characterization and molecular analyses. N. clavispora could cause different diseases in many plants (Rajashekara et al. 2023, Loredana et al. 2020). To our knowledge, this is the first report of fruit brown spot on Punica granatum caused by N. clavispora in China. This finding will help improve management strategies against the fruit brown spots on P. granatum in China.

A simplified laboratory approach for isolating M. oryzae spores from rice samples infected with multiple pathogens

A method for separating M. oryzae from rice samples infected with multiple pathogens using basic laboratory equipment is described. We conducted a series of experiments to obtain a single spore of M. oryzae. This method can also be used to isolate spores from other fungal species.

First Report of Alternaria alternata causing leaf spot on Chinese quince (Chaenomeles sinensis (Thouin) Koehne) in Korea.

The Chinese quince (Chaenomeles sinensis (Thouin) Koehne), belongs to the Rosaceae family, is widely distributed throughout Asia, including Republic of Korea. It is used as a traditional treatment for asthma, common cold, and dry pharynx. Numerous recent pharmacological studies on antiinfluenza, antioxidant, and antidiabetic properties have confirmed the medicinal properties of the Chinese quince fruit (Chun et al., 2012). In March 2022, leaf spots on Chinese quince, resulting in defoliation, were observed in Andong, Gyeongsangbuk Province, Korea (Fig. 1A). The disease symptoms are dark brown spots on leaves. Later, the chlorophyll is lost, causing the entire leaf to become wilted and fell off (Fig. 1B). To identify the pathogen, symptomatic leaves were brought to the laboratory, cut into small pieces, and surface-disinfected in 70% ethanol for 15 s and rinsed with sterile distilled water (SDW). The specimens were then treated with 1% NaOCl for 15 s, followed by rinsing with SDW. Thus, surface-disinfected tissues were placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 7 d. A total of four isolates were obtained from the infected leaves. The colonies were transferred onto freshly prepared PDA plates by the single spore method for further purification. GYUN-10746 isolate was selected as the representative strain among the four isolates and deposited in the Korean Agricultural Culture Collection (KACC 410367). They initially produced white mycelia, which turned dark brown or pale brown at the center and beige at the periphery after 7 d (Fig. 1C and D). Conidiophores were pyriform, sometimes ovoid, or ellipsoidal and brown, measuring 30.8 ± 0.49 × 12.9 ± 0.26 µm (length × width) (n=100) (Fig. 1E). The morphological characteristics were consistent with those of Alternaria alternata (Woudenberg et al. 2015). For molecular identification, DNA was amplified using the following primers: ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone et al. 1999), Gpd-R/Gpd-F (Berbee et al. 1999), Alt a1-F/Alt a1-R (Hong et al. 2005) and rpb2F/rpb2R (Liu et al. 1999) by PCR. DNA sequences from all 4 isolates (GYUN-10746, GYUN-11193, GYUN-11194 and GYUN-11195) were identical. The ITS (OP594615), TEF1-α (OR327062), GAPDH (OR372157), Alt a 1 (OR327061), and RPB2 (OR352741) sequences from the representative isolate GYUN-10746 were 100% identical to those of previously identified A. alternate isolates. A phylogenetic tree was constructed using sequences of ITS, TEF1-α, GAPDH, Alt a l, and RPB2 to illustrate their relationship with A. alternata and related Alternaria species (Fig. 2). For the pathogenicity test, healthy Chinese quince branch containing leaves were inoculated with 7-day-old mycelial plugs of A. alternata, while leaves on a branch inoculated with PDA plugs alone served as a control group. Thus inoculated branches were incubated at 25°C for 7 d. Disease symptoms were developed on leaves of the branches inoculated with mycelial plugs of the fungal pathogen (Fig. 1F), while no symptoms developed on control group. The resulting leaf spots resembled those on the original infected plants. To confirm Koch's postulates, the pathogen was re-isolated from inoculated leaves with identical morphological and molecular characteristics. To the best of our knowledge, this is the first report of leaf spot caused by A. alternata in C. sinensis in Korea. The identification of the pathogen may provide pertinent information for the development of disease controlling strategies.

Leaf grey spot caused by Arcopilus aureus on tobacco in China.

Tobacco (Nicotiana tabacum L.) is one of the most widely cultivated industrial crops worldwide. From April to July 2023, about 40% of tobacco seedlings in the greenhouse exhibited irregular taupe lesions in Zhengzhou, Henan Province, China. At an early stage of the lesion development, light grey spots with the diameter of 1-2 mm were observed, these spots gradually expanded and connected into large irregular lesions causing leaf wrinkling or withered. A total of 12 infected leaf tissues were sterilized with 75% ethanol for 45 s, rinsed three times in sterilized water and then plated on potato dextrose agar (PDA) medium for 10 days at 28°C in darkness. Seven fungal colonies that show the similar appearance were isolated and three of them (MB-1, MB-2 and MB-3) were used for subsequent identification. Colonies of these strains on PDA with loose mycelium and orange-red pigment on the underside, white aerial in the center and light yellow hyphae near the periphery, formed in the shape of a concentric ring pattern. Ascomata appeared from the 14th day, were black, spherical or ellipsoid with walls of textura angularis, and size was 53.8-101.1 μm × 50.3-104.3 μm (n=30). Terminal hairs were brown and straight, gradually tapering toward the tips. Asci clavate or fusiform, spore bearing part 16.2-29.2 × 7.3-11.4 μm (n=21), with 8 irregularly arranged ascospores, evanescent. Ascospores are brown at maturity, biapiculate, navicular or fusiform shapes with size of 8.7-12.8 μm × 4.8-6.9 μm (n=100), and more or less inaequilateral. Single spore strains derived from these strains exhibited the morphological features consistent with the original strains. The morphological characteristics of the fungus were consistent with the description of Arcopilus aureus (Chivers) X.W. Wang & Samson (= Chaetomium aureum Chivers) (Lee et al. 2019). Furthermore, the sequences of RPB2 region were amplified from these strains and the result sequences (GenBank accession no. OR513105-OR513108) all showed a 100.00% identity with A. aureus strain CBS 538.73 (GenBank accession no. KX976807.1). It was reported that the RPB2 gene was efficient in discriminating Arcopilus species (Tavares et al. 2022), thus a maximum likelihood (ML) phylogenetic tree based on the RPB2 gene sequences were constructed using MEGA 7.0 with 1000 replications of bootstrapping (Kumar et al. 2016), which revealed that these strains formed a well-supported clade with A. aureus strains of (CBS 153.52 and CBS538.73) (Wang et al. 2022). Pathogenicity analysis were performed on healthy flue-cured tobacco seedlings leaves (cv Y85) by using mycelial agar plugs (5 mm in diameter) and spore suspension (1×106 spores/mL), and the PDA plugs and sterile water were used for control group, respectively. Tobacco seedlings were incubated in a 25°C and 70% RH growth chamber. After seven days, the leaves showed obvious symptoms, with taupe lesions and yellow halos on the periphery, whereas no symptoms were found on the control leaves. The A. aureu was then reisolated from inoculated diseased leaves. Previously, A. aureus has been only reported to cause leaf black disease on Pseudostellaria heterophylla in China (Yuan et al. 2021). To our knowledge, this is the first reported of A. aureus causing tobacco leaf grey spot worldwide. Arcopilus aureus has been reported as a plant biocontrol fungus (Wang et al. 2013). However, due to the potential serious damage in tobacco seedlings caused by this fungus, the use of A. aureus as a plant biocontrol agent needs to be given more attention, and disease control measures of this pathogen should be developed.

Exploring the influence of rapeseed cultivar and pathogen isolate on Acremonium alternatum's efficacy in clubroot disease control

Clubroot disease, caused by Plasmodiophora brassicae, ranks among the most significant diseases affecting rapeseed cultivars, leading to substantial annual yield losses. Current control methods are limited to a small selection of chemical or biological treatments. Using biocontrol organisms presents a promising strategy for reducing disease severity and promoting plant vigour. However, their efficacy is strongly dependent on biotic and abiotic factors during the growing season, as well as the specific application conditions. In the present study, we evaluated the efficacy of the biocontrol fungus Acremonium alternatum in reducing clubroot disease symptoms across different susceptible and resistant rapeseed cultivars (Brassica napus) under various experimental greenhouse settings employing different types of P. brassicae inoculum: a uniform single spore isolate e3 and two German field isolates P1 and P1 ( +). We found that A. alternatum can reduce clubroot disease symptoms in susceptible rapeseed cultivars Visby, Ability and Jenifer, but not cv. Jumbo, when inoculated with the aggressive single spore isolate P. brassicae e3 at moderate (106 spores mL−1) and high (107 spores mL−1) densities. A. alternatum enhanced plant vitality and shoot biomass in cv. Visby inoculated with field isolates P1 or P1 ( +) but did not considerably reduce clubroot severity there. The clubroot-resistant cv. Mentor displayed a reduction in clubroot symptoms after A. alternatum treatment. In conclusion, A. alternatum holds some promise in managing moderate P. brassicae levels in the soil and could serve as an option in integrated pest management of clubroot disease when combined with resistant cultivars.

First Report of Aspergillus westerdijkiae Causing Leaf Blight on Rehmannia glutinosa in China.

Rehmannia glutinosa (also known as Chinese foxglove) is a perennial dicotyledonous herb, which plays an important role in traditional Chinese medicine. Its active ingredients have a wide range of pharmacological effects on the blood system, endocrine system, immune system, cardiovascular system, and nervous system (Zhang et al. 2008). In May 2022, leaf blight was observed on 45-day-old R. glutinosa in a seedling nursery in Jiaozuo City (35°01'44.20″N, 113°05'30.63″E), Henan Province, China with an approximate disease incidence up to 54% (~1,300 plants). Irregular brown lesion initially appeared on the tips of basal leaves, then progressed to the entire leaf causing leaf drying out (Supple. Fig. 1-A, B, C). The same symptoms appeared successively in the leaves from the base to the top of the plant, which eventually caused the whole plant to die. To identify the pathogen, eight symptomatic leaves were randomly collected from eight individual plants, and cut into small pieces (5 × 5 mm) at the border of lesions. The pieces were surface disinfected in 75% ethanol for 15 s, followed by 1% NaClO for 1 min, rinsed in sterile water three times, and placed on potato dextrose agar (PDA) medium in the dark for 3 days at 25℃. Finally, 12 purified isolates (DHY1-DHY12) were obtained by using single spore method. Leaves of R. glutinosa seedlings were inoculated with conidial suspension (106 conidia/ml), three plants were inoculated per isolate. Controls were treated with sterilized water. All inoculated and control plants were incubated in a greenhouse at 25℃ under 80 ± 10% humidity and a 8-h/16-h dark/light cycle. This experiment was repeated three times. After 5 days, similar symptoms to those of diseased leaves in the seedling nursery appeared on leaves inoculated with DHY4-DHY10, while plants inoculated with DHY1-DHY3, DHY11-DHY12, and the controls remained asymptomatic (Supple. Fig.1-D, E). The same fungi were re-isolated from diseased leaves, fulfilling Koch's postulates. The causal agents DHY4 to DHY10, showed similar morphology, which were morphologically identified as Aspergillus sp. (Visagie et al. 2014). Isolate DHY5 was selected for further study. On PDA plates, the colonies were covered with white velutinous mycelia (Supple. Fig.1-F). Conidia were ochre yellow and outwards concentric circles. Vesicles were globose, and about 20.1-26.6 μm in diameter (Supple. Fig.1-G). Conidiophore stipes were smooth walled and hyaline, with conidial heads radiating. The conidia were light yellow to orange, exudate clear to orange droplets. The conidia were (2.53-3.25) μm × (2.58-3.47) μm in diameter (n=50) (Supple. Fig.1-H). For further molecular identification, the ITS and TUB gene sequences were amplified with primer pairs ITS1/ITS4 and BT2a/BT2b (Glass and Donaldson. 1995), respectively. BLASTn searches of the ITS (PP355445) and TUB (PP382788) sequences showed 100% and 98.42% similarity to those of A. westerdijkiae (OP237108 and OP700424), respectively. Phylogenetic analysis based on the concatenated sequences of ITS and TUB confirmed that the fungus was A. westerdijkiae, (Supple. Fig.2). A. westerdijkiae was mainly reported on its secondary metabolite ochratoxin A contamination of agricultural products, fruits, and various food products, such as coffee beans (Alvindia et al 2016), grapes (Díaz et al. 2009), oranges and fruit juice (Marino et al. 2009), etc. To our knowledge, this is the first report of A. westerdijkiae causing leaf blight on R. glutinosa in China.

First report of leaf spot caused by Colletotrichum camelliae in mung beans (Vigna radiata (L.) R. Wilczek) in South Korea.

Mung bean (Vigna radiata (L.) R. Wilczek) is a legume with high nutritional and economic value that is cultivated widely across Asia (Kang et al. 2014). In March 2022, a leaf spot disease in mung bean was observed at the Gangneung-Wonju National University Experimental farm (Gangneung, South Korea, 37.77°N, 128.86°E). The affected plants had irregular brown-gray leaf spots, and the bottom of the leaves showed concentric brown-gray rings that eventually progressed to necrotic lesions. Regardless of the cultivar, approximately 30% of the plants in the field were infected. To isolate the pathogen, the affected leaves were surface-sterilized by washing with 70% ethanol for 1 min, followed by washing with 2% NaClO for 2 min, then rinsing with sterile distilled water. We placed 3-mm sized diseased lesions on potato-dextrose agar (PDA), then incubated them at 27 ± 1 °C in the dark for 7 days and we obtained 1 isolate (CC1). The fungus on PDA had white aerial mycelia that became gray toward the center. Single spore cultures were obtained from fungal isolate. Isolated conidia were single celled, hyaline, cylindrical, and measured between 20.75 to 22.07 μm × 5.85 to 6.92 μm (n = 20), similar to other reports of C. camelliae(Wang et al. 2016). Mycelium from the single spore isolate was used for DNA extraction using Exgene™ Plant SV / (GeneAll®, Cat.No. 117-152), and we amplified the ITS region with primers ITS1 + ITS2 and ITS3 + ITS4, a portion of the actin gene with primers ACT-512F + 738R, and a portion of the beta-tubulin gene with primers BT2aF + BT2bR (Carbone et al. 1999; Glass et al. 1995; White et al. 1990). The amplified regions were sequenced by by Macrogen® (Seoul, South Korea). Sequences were deposited under GenBank accession numbers OR523262 (ITS), OR582483 (Actin), and OR566953 (beta-tubulin). MegaBLAST analysis of the ITS1, ITS2, ACT, and TUB regions showed 99% (216/217 bp) similarity with C. camelliae strain HNCS-26 (MK041440.1), 99% (303/305 bp) similarity with C. camelliae strain G3 (ON025203.1), 99% (242/244 bp) similarity with C. camelliae strain FWT41 (MN525820.1), and 99% (456/460 bp) with C. camelliae strain LF152 (KJ955239.1), respectively. To fulfill Koch's postulates, we conducted a pathogenicity teston the mung bean cultivar VC1973A (Seonhwanokdu) grown for four weeks at 25 °C with a 16-h day/8-h night cycle, simulating the field conditions when the symptoms were observed. We tested the pathogenicity on six plants , three control and three infected plants. Using three leaf replicates per plant resulting in total of nine leaves per group. Leaves were first injured using a sterile needle then either sterile 5 mm PDA plugs or plugs with C. camelliae were placed on the leaf for control and infected conditions, respectively. Irregular gray leaf spots were observed on the abaxial and adaxial of the infected leaf after 2 weeks, like the symptoms observed in the field. This was observed only on infected leaves and nowhere else on the plant and the control plants had no infection. We re-isolated the pathogen from diseased leaves and identified it as C. camelliae using the same molecular markers described previously, completing Koch's postulate. To the best of our knowledge, this is the first report of leaf spot caused by C. camelliae in mung bean plants in Korea, previously the fungi was reported to infect tea plants in Korea (Hassan et al. 2023). More studies are required to investigate potentially resistant mung bean varieties to minimize future damage caused by this fungus.