First report of leaf spot caused by Colletotrichum siamense on Bauhinia purpurea from India

The Pharbitis purpurea (L.) Voisgt, a member of the Convolvulaceae, is a graceful plant with an air purifying function and ornamental values. It is often cultivated in parks and roadsides. In April 2021, leaf spots (with approximately 67.9% disease incidence) were observed on P. purpurea grown in Xichang city (27°49'N; 102°16'E). More than 1000 square meters of planting area were investigated. Initially, yellowish-brown spots were of different sizes with a yellow irregular border, and slightly sunken necrotic lesions. Gradually, the necrotic lesions expanded and developed into brown spots that often coalesced and expanded to cover the entire leaves. Finally, the leaves wilted, died and fell off. For fungal isolation, infected tissues from ten samples were cut into small pieces of (2.5 × 2.5 mm) sterilized with 3% NaOCl for 30 s and 75% ethanol for 60 s, rinsed three times with sterilized water, blot-dried and cultured on potato dextrose agar (PDA) at 25°C in dark for 8 days. After culturing for 8 days, the colony diameter reached 75.2 to 79.7 mm. The pure colonies were grayish-white with pale yellowish borders and grayish black and pale yellowish borders on the reverse side. The conidia were hyaline, single-celled, cylindrical, smooth-walled, subcylindrical with obtuse to slightly rounded ends, measuring 11.6 to 17.9 × 3.7 to 5.8 μm (n = 100; average=14.7 × 4.9μm). These morphological characteristics were consistent with the description of Colletotricum siamense (Zhang et al. 2021). For molecular identification, the genomic DNA of the representative isolate LBH202104 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). Partial of internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, and GDF/GDR, respectively (Weir et al. 2012). BLAST results of obtained sequences (ITS: OM948680, ACT: OM959361, CAL: OM959366, and GAPDH: OM959364), showed >99% identity with C. siamense sequences (MN305712, MZ461478, MK141754, and MK361203) in GenBank. Based on morphology and phylogenetic analysis, the representative isolate was identified as Colletotrichum siamense (Fig. S1&S2). For pathogenicity test, the conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old eight potted P. purpurea plants. Fifteen leaves of each plant were inoculated. For negative controls, 8 plants were sprayed with sterilized distilled water. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 68 to 75% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. siamense cultures were re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. siamense on P. purpurea worldwide. The identification of this pathogen provides a foundation for the management of Leaf spot in P. purpurea.

A leaf spot, found on Sophora tonkinensis in Hechi city, Guangxi province, China was identified as Colletotrichum siamense based on morphological and molecular phylogenetic analysis of the internal transcribed spacer (ITS) of ribosomal DNA, β-tublin (TUB2), the translation elongation factor 1-alpha (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase 1 (CHS-1), calmodulin (CAL) and glutamine synthetase (GS) genes. Koch’s postulates were satisfied by successful reisolation of C. siamense only from plants inoculated with the pathogen. This is the first report of leaf spot caused by C. siamense on Sophora tonkinensis.

Nageia nagi (Thunb.) Kuntze is widely cultivated in China for its ornamental and economic value. In August 2019, a leaf spot was observed on N. nagi plants at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). Disease incidence was about 35%, and the diseased leaf rate was above 40%. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became reddish-brown, eventually developing large irregular lesions. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C (Zhang et al. 2021). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-six isolates of Colletotrichum ssp. were obtained (isolation frequency about 82%). Three representative single-spore isolates (ZB-1, ZB-3, and ZB-7) were used for morphological studies and phylogenetic analyses. Colonies on PDA medium of the three isolates were white to gray in color with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.1-17.9 ×4.4-6.8 µm (15.6 ± 1.2 × 5.4 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.7-9.3 × 4.6-6.9 µm (7.8 ± 0.2 × 5.6 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum siamense complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), β-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. Sequences of them deposited in GenBank under nos. OL826760 - OL826762 (ITS), OL830205 - OL830207 (ACT), OL830196 - OL830198 (GAPDH), OL830193 - OL830195 (TUB2), OL830199 - OL830201 (CHS-1), and OL830202 - OL830204 (CAL). A Blast search of GenBank showed that ITS, ACT, GAPDH, TUB2, CHS-1, and CAL sequences of the three isolates were identical to Colletotrichum siamense at a high level (Table 1). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed ZB-1, ZB-3, and ZB-7 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on six N. nagi plants (three for inoculation, three for controls), which were grown in the field. Six healthy leaves were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (1 × 106 conidia/mL) per plant. Healthy leaves were inoculated with ddH2O as a control by the same method. All the inoculated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiment was repeated three times. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Up to now, Cephleuros virescens, Pestalotiopsis longisetula, Alternaria tenuissima, A. alternate, and Phoma glomerata could infect N. nagi (Zhou et al. 2015; Zhang et al. 2016), and cause leaf spots in China. To our knowledge, this is the first report of C. siamense causing leaf spots on N. nagi worldwide. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Philodendron bipinnatifidum belongs to the Araceae family, as its graceful plant architecture and the function of purifying air and water, which has important ornamental value (Yu et al. 2019). P. bipinnatifidum is often cultivated in parks and along roadsides. In August 2019 and June 2020, leaf spot disease was observed on over 80% of P. bipinnatifidum plants in Qingxiushan Park (N22°47'23.35″, E108°23'4.26″), Nanning, Guangxi, China. The disease was also frequently observed on P. bipinnatifidum in some other places in Nanning. Symptoms began as small, round, brown spots. As the disease developed, the center of the lesions was sunken with a dark brown border (Fig. 1). Under severe conditions, some spots were joined into larger irregular spots, and even whole leaves died. For fungal isolation, small pieces (5 × 5 mm) were cut from the margin between lesion and healthy tissues of symptomatic leaves, disinfected with 75% ethanol for 10 s, 1% sodium hypochlorite solution for 1 min and washed by sterile water for three times. Over one hundred morphologically similar colonies with white mycelia and a dark green pigment were obtained after 5 days incubation on potato dextrose agar (PDA) at 25°C. Isolates GBZ6-1, GBZ9-1 and GBZ9-2 were selected for intensive study, and the mycelial growth rates of them averaged 14.5 mm/day, 14.6 mm/day and 13.2 mm/day, respectively. Isolates could produce orange conidia on PDA for 7 days. Conidia were elliptical, aseptate and colourless, with sizes of 15.4 ± 0.14 µm × 5.8 ± 0.1 µm, 15.6 ± 0.14 µm × 5.3 ± 0.1 µm and 15.0 ± 0.16 µm × 6.3 ± 0.1 µm for GBZ6-1, GBZ9-1 and GBZ9-2, respectively. Appressoria were mostly brown ovoid, conidial appressoria averaged 7.62-9.31 μm × 5.84-742 μm, and mycelial appressoria were 8.06-9.22 μm × 6.28-6.92 μm. Genomic DNA was extracted by the DNAsecure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd] and the rDNA internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012) were sequenced. Phylogenetic analysis was performed using RAXML (Version 2.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, and GAPDH). The results showed that three isolates were identified as C. siamense (accessions MW116805-MW116816, for ACT, CAL, CHS-1 and GAPDH of GBZ6-1, GBZ9-1 and GBZ9-2; MW073391-MW073393 for ITS of GBZ6-1, GBZ9-1 and GBZ9-2). According to the cultural and conidial morphology, as well as phylogenetic analysis, these isolates were identified as Colletotrichum siamense. Pathogenicity tests were conducted on one-year-old plants. Young healthy leaves of three plants were artificially wounded by gently scratching with a sterile needle and 10 µl of conidial suspension (106 spores/ml) were applied to per wound site for each isolate. Wounded leaves were inoculated with 10 µl of water as controls. All plants were sprayed with water and covered with plastic bags to maintain high humidity. Symptomatic lesions were observed on the inoculated leaves after 15 days at 28 °C, whereas no symptoms were observed on the control leaves. To fulfill Koch's postulates, fungi were reisolated from symptomatic leaves and morphologically identical to the inoculated isolates (Xue et al. 2020). To our knowledge, this is the first report of leaf spot caused by C. siamense on P. bipinnatifidum worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. siamense on P. bipinnatifidum.

Rose of Sharon, Hibiscus syriacus L., is a flowering shrub in the family Malvaceae planted as the national flower of South Korea. In September 2012, previously unknown leaf spots with premature defoliation were observed on dozens of Rose of Sharon plants growing in the shaded area in a park of Dongducheon, Korea. The same symptoms were found on Rose of Sharon in several localities of Korea in 2012. The symptoms usually started as small, dark brown to grayish leaf spots, eventually causing leaf yellowing with significant premature defoliation. The diseased leaves retained for a while green color at the margin of the spots. Representative samples (n = 5) were deposited in the Korea University Herbarium (KUS). Conidiophores of the fungus observed microscopically on the leaf spots were erect, brown to dark brown, single or in clusters, amphigenous but mostly hypophyllous, and measured 80 to 400 × 5 to 10 μm. Conidia were borne singly or in short chains, ranging from cylindrical to broadest at the base and tapering apically, straight to slightly curved, pale olivaceous brown, 2 to 16 pseudoseptate, 50 to 260 × 9 to 20 μm, each with a conspicuous thickened hilum. On potato dextrose agar, single-spore cultures of two isolates were identified as Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei on the basis of morphological and cultural characteristics (1,2). Two monoconidial isolates were preserved at the Korean Agricultural Culture Collection (KACC46956 and KACC46957). Genomic DNA was extracted using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequences of 520 bp were deposited in GenBank (Accession Nos. KC193256, KC193257). A BLAST search in GenBank revealed that the sequences showed 100% identity with those of numerous C. cassiicola isolates from diverse substrates. To conduct a pathogenicity test, a conidial suspension (ca. 2 × 104 conidia/ml) was prepared in sterile water by harvesting conidia from 2-week-old cultures of KACC46956, and the suspension was sprayed onto the leaves of three healthy 2-year-old plants. Inoculated plants were kept in humid chambers for the first 48 h and thereafter placed in the glasshouse. After 10 days, typical leaf spot symptoms developed on the leaves of all three inoculated plants. C. cassiicola was reisolated from the lesions, confirming Koch's postulates. Control plants treated with sterile water remained symptomless. C. cassiicola is cosmopolitan with a very wide host range (1,2). Though Corynespora hibisci Goto was recorded to be associated with brown spot disease of H. syriacus in Japan (4), there is no previous record of C. cassiicola on H. syriacus (3). To our knowledge, this is the first report of Corynespora leaf spot on Rose of Sharon in Korea. According to our field observations in Korea, this disease was found in August and September, following a prolonged period of moist weather. Severe infection resulted in leaf yellowing and premature defoliation, reducing tree vigor and detracting the beauty of green leaves.

Coreopsis lanceolata L. (Compositae), an ornamental species grown in parks and gardens, is very much appreciated for its long-lasting flowering period. In August of 2008, pot-grown plants with necrotic leaf lesions were observed in a commercial nursery located near Biella (northern Italy). Lesions were present, especially along the margin of basal leaves, and sometimes had a chlorotic halo. On infected leaves, dark brown necrosis developed. Leaf stalks were sometimes affected. In many cases, the leaves, especially those at collar level, were withered. Of 1,500 plants, 15% were infected by the disease. Microscopic examination did not reveal any fungal structures within the lesions. Small fragments of tissue from 30 affected leaves were macerated for 15 min in casein hydrolysate and 0.1-ml aliquots of the resulting suspension were spread onto Luria Bertani agar (LB) and potato dextrose agar (PDA). Plates were maintained at 22 ± 1°C for 48 h. No fungi were isolated from the leaf spots on LB or PDA. Colonies similar to those of Pseudomonas spp. were consistently isolated on LB. Colonies were fluorescent on King's medium B, levan negative, oxidase positive, potato soft rot negative, arginine dihydrolase negative, and tobacco hypersensitivity positive (LOPAT test). The bacterial colonies were identified as Pseudomonas cichorii (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers 27F and 1492R and sequenced (GenBank Accession No. FJ534557). BLAST analysis (1) of the 998-bp segment showed a 98% homology with the sequence of P. cichorii. The pathogenicity of one isolate was tested twice by growing the bacterium in nutrient broth shake cultures for 48 h at 20 ± 1°C. The suspension was centrifuged, the cell pellet resuspended in sterile water to a concentration of 107 CFU/ml, and 30 4-month-old healthy coreopsis plants were sprayed with the inoculum. The same number of plants was sprayed with sterile nutrient broth as a control. After inoculation, plants were covered with plastic bags for 48 h and placed in a growth chamber at 20 ± 1°C. Five days after inoculation, lesions similar to those seen in the field were observed on all plants inoculated with the bacterium, but not on the controls. Ten days later, 40% of the leaves were withered. Isolations were made from the lesion margins on LB and the resulting bacterial colonies were again identified as P. cichorii. The pathogen caused the same symptoms also on plants of Dendranthema frutescens (cv. Camilla), Chrysanthemum morifolium (cvs. Eleonora and Captiva), and an Osteospermum sp. (cv. Wild side) when artificially inoculated with the pathogen with the same methodology. The same bacterial leaf spot caused by P. cichorii was observed in 2005 in other nurseries in the same area on Phlox paniculata (3). To our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on C. lanceolata in Italy.

First report of leaf spot caused by Colletotrichum siamense on Ocimum tenuiflorum in India

Kiwifruit (Actinidia spp.) is an important fruit with high nutritional and economic value, which is widely cultivated in China. In April 2021, leaf spots were observed on the leaves of 'Xuxiang' (A. deliciosa) in a kiwifruit plantation of Hefei city, Anhui province, China (117°26'E, 31°85'N). Disease incidence was about 10% of the observed plants. Small yellow spots initially developed on the leaves and gradually expanded into irregular dark brown spots, and eventually the diseased leaves curled and withered. Leaf tissues (n=10, 5×5 mm) were collected from five infected plants, sterilized in 75% ethanol solution for 30 s and 1% NaOCl for 5 min, washed, dried and plated on PDA at 25°C. In total, ten isolates were obtained, including two previously reported Botryosphaeria dothidea (Zhou et al. 2015) and Diaporthe actinidiae strains (Bai et al. 2017) and eight unknown isolates with similar morphology. All unknown isolates initially appeared white with many aerial hyphae, and at the later stage, the center of all colonies turned gray. Colonies were transferred to new PDA with 0.1% yeast extract for three days. Then, aerial hyphae were scraped with sterile cotton swabs, and continued to grow for four days. Orange conidial masses were produced. Conidia were hyaline, smooth-walled, single-celled, cylindrical with broadly rounded ends, with average size around 4.1-5.5×13.2-18.2 µm (n=100). Appressoria (n=50) were ovoid in shape with average size around 4.9-6.7×8.6-11.8 µm. Morphological features were similar to Colletotrichum. gloeosporioides species complex (Weir et al. 2012). To confirm their species identification, internal transcribed spacers (ITS), β-tubulin (TUB2), glyceraldehydec-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS) were amplified by PCR using the primer pairs ITS1/ITS4, Bt2a/Bt2b, GDF/GDR, ACT-512F/ACT-783R CHS-79F/CHS-234R, respectively (Weir et al. 2012). Based on alignment analysis, sequences of the eight unknown isolates were 100% homologous. The representative isolate LSD3-1 was selected for further study. BLAST analysis showed that the ITS (OM033371), TUB2 (OM044376), GAPDH (OM044377), ACT (OM044379) and CHS (OM044378) sequences of isolate LSD3-1 were 98.7%-100% identical with the collected sequences of C. fructicola strain ICMP:18581 (NR_144783, JX010405, JX010033, JX009866, JX009501). Phylogenetic analysis of multiple genes was conducted with the Maximum likelihood method using MEGA 7. Based on morphological and molecular characteristics, the LSD3-1 was identified as Colletotrichum fructicola (Prihastuti et al., 2009). Koch's postulates were performed on six one-year-old 'Xuxiang' plants, which were used to test pathogenicity in the greenhouse (at 28℃, relative humidity 80%, 16/8 h light/dark). Surface-sterilized leaves were sprayed with a conidial suspension (107 conidia/mL). Yellow and brown lesions were formed 14 to 21 days after inoculation, whereas the mock-inoculated controls remained asymptomatic. The experiment was performed three times. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Although C. fructicola has been reported as a leaf spot disease on many plants (Shi et al. 2018), this is the first report of leaf spot caused by C. fructicola on kiwifruit in China. This result is helpful to better understanding the pathogen of kiwifruit leaf spot diseases in China and formulate effective control strategies.

Water convolvulus (Ipomoea aquatica Forsk.), a member of the Convolvulaceae family, is an important tropical vegetable cultivated in China (Liu et al. 2017). From 2016 to 2020, dark-brown leaf spots were observed in major water convolvulus (cv. Large leaf) growing areas (2 ha) in Honghe City (24°12' N, 103°6' E), Yunnan Province, China. Field investigations showed that a leaf spot disease occurred on water convolvulus in four fields with 15% incidence (50 plants in each field were investigated) and resulted in up to a 10% decrease in its total production. Symptoms on water convolvulus plants appeared as small lesions, yellowish-green and circular on the leaves. Ten plants were selected randomly from the growing area, with three diseased leaves collected from each plant. Symptomatic tissues were excised, surface sterilized with 75% ethanol for 30 s, washed in sterile-distilled water three times, and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 7 days. Colonies on PDA were gray to green in color and fuzzy in the middle, with irregular borders. Conidiophore morphology showed single, yellowish-brown or brown structures with 1~6 septa, and long 22~125 µm, wide 3.5~5.5 µm. Conidia were elliptical, black-brow, solitary, with a smooth surface, 1~6 longitudinal septa and 1~3 transverse septa, 20~30 µm in length, and 15~22 µm in width. The morphological characteristics of the fungus were consistent with the description of Stemphylium solani (Chai et al. 2014; Weber, 1930). To further confirm the identity of the 30 isolates, the partial gapdh (glyceraldehyde-3-phosphate dehydrogenase), tef1 (translation elongation factor 1-alpha), cmdA (Calmodulin) and ITS (intemal transcribed spacers) sequences were amplified by PCR with the primer pairs of gpd1/gpd2, EF1-728F/EF1-986R, CALDF1/CALDR2 and ITS1/ITS4, respectively (Berbee et al. 1999; Carbone & Kohn. 1999; Lawrence et al. 2013; White et al. 1990). Multiple sequence alignments were generated using MEGA7, and phylogenetic analysis was conducted with the neighbor-joining (NJ) method (Tamura et al. 2007), the results indicated that all sequences from the 30 isolates were identical. Thus, one representative isolate, KXC11033003 was chosen for further analysis. The ITS, gapdh, cmdA and tef1 sequences of this isolate were submitted to the NCBI GenBank database (accession nos. OL444947~OL444950). The strain KXC11033003 and S. solani (CBS-408.54) formed a clade with 82% bootstrap value (Figure S2). To fulfill Koch's postulates, 30 plants were inoculated for each of the thirty isolates. Conidia were sprayed on leaves of water convolvulus (8-true-leaf stage) in a suspension of 107 conidia/mL or water as a healthy control in a greenhouse at 15~18℃ (night) / 25~28℃ (day) with 95% humidity. Symptoms of dark brown spots appeared on the leaves after 7 days, whereas controls remained healthy The pathogens were reisolated from the lesions and confirmed identical to the original isolate by gene sequences. No pathogens were isolated from the control plants. To our knowledge, this is the first report of leaf spot caused by S. solani on water convolvulus in Yunnan Province, China. Further, Stemphylium leaf spot caused by S. solani has been reported previously on tomato, garlic, pepper (Zheng et al.2008; Nasehi et al.2018). This study stresses the need to identify appropriate management strategies for S. solani that help prevent quality and yield losses in water convolvulus in China.

Honeysuckle (Lonicera japonica Thunb, LJ) is a common medicinal and edible plant (He et al. 2022). It has been utilized in various industries such as biomedicine, animal husbandry, and food production (Li et al. 2014; Su et al. 2020). In June 2023, a significant leaf lesion was observed on approximately 20% of honeysuckle "Juhua No.1" leaves in a 3.33-ha field at the base of Julu County, Hebei province, China. Almost all leaves were infected. Leaf spot disease occurred in the field honeysuckle throughout the flowering period, especially after picking. The disease mainly infected the leaves of honeysuckle, forming irregular spots on the edge of the leaf surface with black-brown edges, the midrib and lateral veins were affected (Figure S1A). In advanced stages, the entire leaf would become necrotic. For pathogen isolation, small pieces (4×4 mm) of the infected tissue from diseased leaves were surface sterilized with 75% ethanol and 5% sodium hypochlorite, rinsed with the sterile water, incubated on PDA. Finally, six isolated pathogens were obtained. Hyphae were white. The mycelium was multicellular, had diaphragm. Conidiophores protruded from the stroma, started as spherical structures and gradually developed into radial, black-brown formations. Spore acrosome was subglobose, bilayered pedicels covering acrosome, 40-60 µm in diameter, yellowish brown (Figures S1B, S1C). Based on morphological and cultural characteristics, the leaf spot disease fungus was tentatively identified as Aspergillus spp. (Wei 1979). To test the pathogenicity of pathogen, leaves of three healthy potted honeysuckle "Juhua No.1" plants were inoculated by sprayed with conidial suspensions (106 spores/ml) (Figure S1D). Negative controls were established by inoculating leaf with sterile distilled water. All plants were incubated in a greenhouse at 28 ± 2℃. The experiment was replicated three times. After 10 days, typical leaf spot symptoms were observed on inoculated leaves, whereas no symptoms were found on the control groups. The re-isolated fungus from the inoculated leaves displayed the same morphological traits (Figures S1E-S1H), again identified as Aspergillus spp., confirming Koch's postulates, designated as H2. To confirm the pathogen's identity, genomic DNA was extracted from the pathogenicity isolate H2. The 18S rDNA and the ITS genes were amplified and sequenced using primer pairs S1/S2 (Zhang et al. 2018) and ITS1/ITS4 (Zhang et al. 2023), respectively. Results of BLAST searches showed that the 18s rDNA and ITS sequences of H2 were highly homologous (>99%) with Aspergillus niger. The close genetic relationship indicated that H2 belonged to the genus Aspergillus (Figure S2a). We further sequenced the whole genome of H2. The sequence data were available in the NCBI GenBank (Accession number: PRJNA1117256). We also analyzed the ANI (Yoon et al. 2017) and digital DNA-DNA blotting (dDDH) (Figures S2b, S2c). The ANI values of H2 compared to Aspergillus niger CBS 554.65 and Aspergillus niger KJC3 were higher than 95%. The dDDH values of H2 compared to Aspergillus niger CBS 554.65 and Aspergillus niger KJC3 were higher than 70%. Above all results, honeysuckle leaf spot disease was identified as Aspergillus niger. This is the first report of leaf spot on Lonicera japonica caused by Aspergillus niger in China. Our findings expand the geographical range of A.niger-infected plants, also provide reference for scientific prevention and control of honeysuckle leaf spot disease.

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.

Coffea canephora (conilon coffee) represents approximately 30% of the coffee marketed worldwide. The state of Espírito Santo is the largest conilon coffee-producing state in Brazil. In 2013 and 2014, leaves with a leaf spot were observed on most of the conilon coffee seedlings in a commercial nursery in Laranja da Terra, Espírito Santo, Brazil. The infected leaves were deposited in the VIC Herbarium (VIC 42482) and a pure single-spore culture of the pathogen was deposited in the culture collection of the Universidade Federal de Viçosa (Accession No. COAD 1729). The initial symptoms were circular, brown to dark brown lesions with yellow margins occurring on both leaf surfaces. In high humidity, concentric rings formed and the lesions expanded rapidly to reach up to 30 mm in diameter, and later became dark brown with a grayish center. Black sporodochia with white, and marginal mycelial tuffs bearing black spore masses were observed in the older lesions. These symptoms were consistent with those of Myrothecium leaf spot reported on Coffea spp. (3). Microscopic observation revealed aseptate, hyaline, and cylindrical conidia, rounded at both ends, greenish to black in mass, and 5 to 6 μm long and 1 to 2 μm wide. The symptoms and morphological characteristics described above matched the description of Myrothecium roridum Tode (4). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and the sequence of an internal transcribed spacer (ITS) region was obtained and deposited in GenBank (Accession No. KJ815095). The sequence of the ITS region exhibited 100% identity over 561 bp with another M. roridum sequence in GenBank (JF343832). To verify the pathogenicity of the fungus, healthy leaves of the C. canephora clones 12v and 14 (four seedlings each) were wounded superficially with a sterilized needle and inoculated by spraying them with a suspension of M. roridum conidia (106 conidia ml-1). The seedlings were covered with plastic bags and incubated in a growth chamber at 25°C under a photoperiod of 12 h light/12 h dark for 5 days. The control seedlings were sprayed with distilled water and incubated similarly. Fifteen days after inoculation, symptoms in all inoculated seedlings were consistent with those initially observed on the naturally infected seedlings, whereas the controls remained healthy. Re-isolation and identification confirmed Koch's postulates. M. roridum has a wide host range, and symptoms were similar to those reported in other hosts of the pathogen in Brazil (2,3). There is only one report of M. roridum on C. canephora in Colombia (1); however, this pathogen was previously reported on C. arabica in Brazil, Colombia, Costa Rica, Guatemala, India, Indonesia, Puerto Rico, and the Virgin Islands (1,3). To our knowledge, this is the first report of a leaf spot caused by M. roridum on conilon coffee in Brazil. The cultivation of conilon coffee is increasing and the reported leaf spot disease affects the quality of the seedlings in nurseries. It is therefore important to conduct a thorough study of management strategies for this disease.

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.

Oat (Avena sativa) is an annual gramineous crop, which contains a source of soluble dietary fiber, β-glucan, unsaturated fatty acids, vitamins, minerals, phenolic acids and avenanthramides. It widely cultivated in cool and semi-arid areas in northern China (Li et al, 2017). In July 2018, a severe leaf spot infection was observed in the Forage Germplasm Nursery (31°17'22″N, 103°40'15″E, 2885 m elevation) in Tianzhu County, Wuwei City of Gansu Province in China. Disease incidence (total number of diseased leaves / total number of surveyed leaves X 100%) was 93% over 300 m2 planting area. Symptoms initially appeared as small circular to irregular, gray-green, water-soaked spots on the leaves in the middle or along the margin of leaves, that enlarged and coalesced. The center of the leaf spots turned brown to reddish-brown. Infected tissues from symptomatic leaves were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 60 s, soaked in 5% commercial bleach (~0.275% NaClO) for 5 min (Xue et al, 2018), rinsed five times with distilled water, plated on potato dextrose agar (PDA) medium, and incubated for 3 days in the dark at 25°C (Zhang, 2003; Blagojević et al, 2020; Humpherson et al, 1989). Hyphae emerging from the tissue were subcultured on fresh PDA medium for purification. All colonies were light brown with intensive sporulation in rings that was grayish white, and later became grayish brown. The back of the colony was dark brown. Conidiophores were light brown, unbranched, grew vertically on hyphae, and each conidiophore produced 3 to 7 conidia (mostly 6). Conidia were light brown, septate, straight to slightly curved, single or in chains, oval or obclavate, measured 17 to 32 µm wide and 63 to 106 µm long with the conical beak cell, 7 to 12 transverse septa, 0 to 5 longitudinal septa. These morphological characteristics were similar to the descriptions of Alternaria spp. (Simmons, 2008). A single isolate, YMZZ1, was selected for molecular identification. The ITS region of rDNA, partial GAPDH and Tef1-α gene sequences were amplified by PCR with the primer pairs of ITS1/ITS4, gpd1/gpd2 and EF1-728F/EF1-986R, respectively (Woudenberg et al, 2013). Sequences were deposited in GeneBank under accessions MN446739 (ITS), MN481462 (GAPDH), and MN464104 (Tef1-α). A nucleotide BLAST search revealed ITS, GAPDH and Tef1-α sequences to be 99% similar to accessions numbers MN856410 (565/573 bp), MK026431 (575/575 bp), and MH754531 (211/211 bp), respectively) of A. brassicae. Neighbor-joining (NJ) and Maximum Likelihood (ML) phylogenetic analysis were conducted based on ITS, GAPDH and TEF-1α sequences using MEGA7.0 under Kimura 2-parameter model. The isolate YMZZ1 clustered with a representative strain A. brassicae LGBA22 with 100% bootstrap support. To test its pathogenicity, six healthy 3 week old plants were spray-inoculated with a suspension of 3×105 conidia/mL of YMZZ1. The same number of plants were sprayed with sterilized water as control. All plants were covered with transparent plastic bags for 48 h to maintain high relative humidity and incubated in a 25°C growth chamber (16/8 h light/dark) for observation. Ten days after inoculation, leaf spot symptoms were observed on leaves similar to those previously observed in nursery; no symptoms were observed on the control. The pathogenicity test was repeated twice under the same conditions and A. brassicae was re-isolated from inoculated plants each time fulfilling Koch's postulates. A. brassicae has not been previously reported as a pathogen of A. sativa in the world, but has been mentioned as a pathogen of horse radish (Armoracia rusticana) in Serbia (Blagojevic et al, 2015). To our knowledge, this is the first report of leaf spot caused by A. brassicae on A. sativa in China. This study stresses an urgent need to identify appropriate management strategies of A. brassicae that help in preventing losses in quality and yield of oats in northern China.

Japanese plum (Prunus salicina Lindley) is a deciduous tree in the family Rosaceae. In Korea, this plant is widely distributed in orchards as an important stone fruit as well as in gardens as an ornamental tree because of their abundant white blossoms. Every September to November since 2001, leaf spots were observed on Japanese plum in a garden in Cheongyang, Chungnam District, Korea. Early symptoms consisted of small, brown spots that were 2 to 5 mm in diameter. Later, the leaf lesions became circular or irregular, dark brown, expanded to 15 mm in diameter, and resulted in discoloration with necrosis on twisted leaves that was followed by defoliation. In November, older lesions sometimes appeared blackish brown as sporulation occurred on the lesions. The causal fungus was isolated from diseased leaves and cultured on potato dextrose agar. A culture has been placed in the CABI Herbarium (IMI Accession No. 387139). Conidial dimension averaged 34 × 12 μm. On the basis of morphological characteristics of conidia and conidiophores, the causal fungus was identified as a small-spored species of Alternaria as described by E. G. Simmons (1). Pathogenicity tests were conducted by inoculating slightly wounded and nonwounded leaves with a conidial suspension adjusted to 1 × 106 conidia/ml. Four leaves per each experiment were either wounded or not and inoculated with a spore suspension. The eight leaves were placed in a moist chamber at 25°C. After 6 to 10 days, small brown spots appeared on 87% of the wounded and nonwounded leaves. Control leaves sprayed with distilled water did not develop any symptoms. The causal fungus was consistently reisolated from the leaf spots. Results from pathogenicity tests were similar in a repeated test. It is possible that small-spored Alternaria spp. isolates are host specific (2). Eight Alternaria spp., including A. alternata, A. tenuis, A. tenuissima, and A. citri, have been found to cause black spot on fifteen Prunus spp. in China, Japan, Hong Kong, Libya, Mexico, Australia, and the United States (2). Further studies on the host-specific toxin production, geographical distribution, and host ranges for the species of Alternaria isolated from Japanese plum are in progress. To our knowledge, this is the first report of leaf spot on Japanese plum (P. salicina) caused by a small-spored Alternaria sp. in Korea.