An outbreak of Neopestalotiopsis sp. causing red leaf spot of sapota in Bangladesh

In June 2017, severe leaf spots symptoms were observed by growers on pineapple leaves of Josapine variety in in Alor Pongsu (5°01'60.00" N, 100°34' 59.99" E), Perak,northwest of Peninsular Malaysia. The early infection stage shows that several brown spots could be observed, which then would merge to form large brown to creamy white lesions that cover all the leaf surface. This infection finally caused the plant to die after a while. Disease observations conducted from 2018 - 2023 showed that 10-15% incidences of the disease were observed in several pineapple farms located in Johor, Kedah, and Sarawak. The aim of this study to confirm the causal pathogen of the disease by performing isolation, pathogenicity testing, and identification of the primary causal pathogen from 20 samples of infected leaves collected from Alor Pongsu. The leaf tissues between infected and healthy were cut into small pieces (0.5 cm 0.5 cm), and surface sterilized with 1% sodium hypochlorite for 30 seconds, followed by 70% ethanol for 30 seconds, and rinsed thrice with sterilized water before placing on Potato Dextrose Agar (PDA). The PDA plates were incubated at room temperature (28 ± 2℃) in natural light. After five days of incubation, the potential causal pathogen was purified using a single conidial isolation technique for morphological and molecular characterizations. All 32 isolates displayed similar phenotypes. Based on morphological observation on PDA, the colonies were initially white of aerial mycelia but gradually darkened as the culture aged. Microscopic features of the 14-day-old fungal culture showed that the mycelia were branched with 0- 1 septa, pigmented, and brown. Arthroconidia were ellipsoid to ovoid or round shaped, hyaline, with rounded apex, truncate base, and occurring singly or in chains averaging 9 ± 3 × 5 ± 2 μm (n = 20). Based on the morphological characteristics, the fungal isolates were tentatively identified as Neoscytalidium species. A representative isolate of Neoscytalidium coded as UiTMPMD2 was further identified through PCR implication of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers and BLAST homology search as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on 100% similarity (575 bp out of 575 bp) to a reference sequence (accession no. KU204558.1). The sequence was deposited in Gen Bank (accession no. OR366479) with reference sequence code of INBio:30A. Pathogenicity tests were performed on 10 whole plants of Josapine pineapple (4 months old) using a leaf inoculating method (Wu et al. 2022) in a glasshouse (25-32°C) and repeated twice. Four mature leaves per each plant were wounded at two points and inoculated with mycelium PDA plugs from 7-days-old cultures of N. dimidiatum. Control plants were wounded in the same manner but inoculated with sterilized PDA plugs. Seven days post inoculation, leaf spot symptoms were observed on treated plants with the pathogen, while the control plants remained symptomless. Pathogen was successfully reisolated from brown leaf spot symptoms in which the cultural and morphological characteristics were identical to those of the originals. Neoscytalidium dimidiatum has a wide range of hosts and it has been reported in Malaysia to cause stem canker on pitahaya (Mohd et al. 2003; Khoo et al. 2023 ) and fruit rot of guava (Ismail et al. 2021). To the best of our knowledge this is the first report of N. dimidiatum causing leaf spots on pineapples in Malaysia. This report establishes a foundation for further study ofN. dimidiatum that can effectively address the disease in pineapple.

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, leaf spot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 2 ha). Leaf spots were round or irregular with a clear yellow halo around a brown, necrotic lesion. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C in the darkfor 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (CLCT-1, CLCT-2, and CLCT-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. The colonies on PDA were gray white at first, subsequently turning grayish to dark gray, with numerous black microsclerotia and setae. Conidia were hyaline, aseptate, falcate with pointed ends, and 16.5 to 22.3 × 2.5 to 3.2 μm (n = 30). Morphological characteristics of the isolates were consistent with the description of Colletotrichum truncatum (Schwein.) Andrus & W. D. Moore (Sawant et al. 2012). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequences were submitted to GenBank under accession numbers OP740964 to OP740966 (ITS), OP800837 to OP800839 (GAPDH), and OP800843 to OP800845 (ACT). The sequences of the three isolates were 100% identical (ITS, 547/547 bp; GAPDH, 290/290 bp; and ACT, 266/266 bp) with those of C.truncatum (accession nos. GU227869, GU228261, and GU227967) through BLAST analysis.. In addition, a phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that nested within the clade containing C. truncatum (the type strain CBS 112998) by the maximum likelihood method. From the combination of the morphological and molecular characteristics, the isolates were determined to be C.truncatum. A pathogenicity test was performed in a greenhouse at 24 to 30°C with 80% relative humidity. Wampee plants (cv. JIXIN, n =5, 1-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water. Leaf spots were observed on the inoculated plants after 10 days while the control ones remained healthy. The pathogen re-isolated from all the symptomatic leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C.truncatumhas also beenreportedto be thecausalagent of anthracnose disease in multiple crops (Diao et al. 2014;Villafana et al. 2018; Stella de et al. 2021), thus, this is the first to report C.truncatum causing leaf spot on C. lansium in China. This study provides an important reference for the control of the disease due to the high host range ofC.truncatum.

Euonymus fortunei (Turcz.) Hand.-Maz., an evergreen vine shrub, commonly used for landscaping and herbaceous medicines in China. In December 2024, leaf spot symptoms were noticed on E. fortunei plants in three parks with a 15-25% disease incidence out of 160 trees in Weifang City, Shandong Province in China (118°44'34.9''E, 36°52'17.3''N). Irregularly shaped lesions with a grayish-white center and brownish margins surrounded by a yellow halo developed on infected leaves and coalesced at later stages. To isolate the causal agent, 14 diseased leaves were randomly collected from 10 trees in different parks and a small piece (2 x 2 mm) was sliced from each lesion at the edge, surface-disinfected with 75% ethanol for 30 s, rinsed 3 times in sterilized water, placed on a potato dextrose agar (PDA), and incubated at 27°C in the dark for 3 days. Botryosphaeria sp.-like fungal colonies were isolated from 93% of the sampling plates and 3 representative fungal isolates (FFT01 to FFT03) were selected randomly for morphological and molecular characterization and hyphal-tip purified. Fungal colonies initially appeared to be white with fluffy mycelia and gradually developed a grayish-black pigment. The hyaline, unicellular, and oblong-fusiform conidia were 20.1 - 25.7 μm (mean = 23.2 ± 1.4) long and 6.3 - 9.1 μm (mean = 7.2 ± 0.8) in diameter (n = 30), similar to the morphological features of Botryosphaeria dothidea (Zhang et al. 2021). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1) gene, and beta-tubulin (tub2) gene were amplified and sequenced using primer pairs ITS1/ITS4, EF1- 728F/EF1-986R, and Bt2a/Bt2b, respectively (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999) and desposited in GenBank (Accession Nos. PV422744 to PV422746 for ITS, PV441492 to PV441494 for tef1, and PV4414485 to PV441487 for tub2). Sequences had > 99% identity with their corresponding sequences from the ex-type B. dothidea strain CMW 8000. In the maximum-likelihood phylogeny derived from the concatenated alignments, all three isolates were grouped in the B. dothidea clade. To validate the pathogenicity of FFT01 isolate, five leaves from each of three one-year-old E. fortunei seedlings were scratched with a sterilized syringe needle and then a 5 mm2 mycelial PDA plug of 6-day-old culture was placed on each wounded site. Three E. fortunei seedlings inoculated with sterile PDA plugs served as controls. Each wound-inoculated seedling was enclosed in a transparent plastic bag for 3 days then kept in a growth chamber under controlled conditions (27°C, 14/10-h light/dark cycle, and 75% relative humidity). Seven days post-inoculation, grayish-white lesions surrounded by yellow halo were observed on leaves with black pycnidia developing in lesions, similar to the symptoms on the naturally infected leaves, while no lesions appeared on mock-inoculated leaves. The identity of the fungus re-isolated from symptomatic leaves was confirmed as B. dothidea based on morphology and DNA sequence. The pathogenicity test was repeated three times with similar results. Although B. dothidea has been reported as a common fungal pathogen causing leaf spot, fruit rot or stem canker on a variety of plants including plum, Kadsura coccinea, and soybean (Chen et al. 2021; Su et al. 2024; Yuan et al. 2024), this is the first report on its occurrence on E. fortunei in China. This study will provide a basis for the prevention and control of leaf spot on E. fortunei in the future.

Pomegranate (Punica granatum L.), which is native to central Asia, is considered as one of the most renowned commercial fruit trees in the world. The planting area in China is roughly 120 thousand hectares. In June 2020, symptoms of leaf spot on P. granatum appeared in Nanyang City (32º40´34˝N, 111º44´20˝E), Henan Province, with an incidence rate of 35% in several 3.3-hectare orchards. Initially, the lesions showed as round or subrounded brown spots on affected leaves. The spots then progressively developed into irregular lesions with distinct yellow halos surrounding them. Parts of the lesions were weakly zonate, which finally led to leaf withering and falling off. Diseased tissues were cut into 5×5 mm2 pieces, which were surface sterilized with 75% ethanol solution for 30 s, washed 3 times in sterilized water, and put on potato dextrose agar (PDA) plates supplemented with 50 μg ml-1 streptomycin. A total of 16 purified fungal isolates with similar phenotypic features were obtained. Three randomly chosen isolates SLY11, SLY24, and SLY25 were utilized for the investigation. Fungal colonies on PDA were first white to gray and later mycelium became olive green to blackish brown. To examine the morphological properties of conidia, we utilized potato carrot agar (PCA) culture medium and incubated it at 23°C under a 12-hour light/dark alternation. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa and 1 to 4 longitudinal septa. Conidiophores were septate, solitary, and measured 22.7 (±4.64) × 10.6 (±2.15) μm (n=50), with a conical beak length of 0 to 5.5 μm. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (TEF1), β-tubulin gene (TUB), and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and GDF1/GDF2 from genomic DNA. Sequences were submitted to GenBank with accession numbers OL840230, OL840231, OL840232 for ITS, OL982540, OL982541, OL982542 for TEF1, OL982543, OL982544, OL982545 for TUB, OL862608, OL862609, OL862610 for GAPDH sequences of isolates SLY11, SLY24, and SLY25, respectively. BLASTn analysis of ITS (OL840230), TEF1 (OL982540), TUB (OL982543), GAPDH (OL862608) sequences indicated 100, 99.59, 99.68, and 100% similarity to the sequences of Alternaria alternata strain HC-2 (MT644140), BJFA-1 (MK895958), CS36-5 (KY814630), and ag1 (KP057228) in GenBank. Isolates SLY11, SLY24, and SLY25 formed a clade with the type strains CBS 130265 and CBS 130258 of A. alternata in phylogenetic trees established, clearly seperating from other Alternaria spp. The morphological features and molecular analyses supported the isolates as members of A. alternata. To validate the pathogenicity of the isolates, ten healthy leaves of 3-year-old potted pomegranate trees were utilized for testing and inoculated with conidial suspension (106 conidia ml-1), 20 µl each leaf. Control plants were inoculated with sterilized water. An additional pathogenicity test was repeated on wounded leaves. The inoculated plants were placed at 28°C in a greenhouse (12 h light per day and 90% relative humidity) for 5 days. The pathogenicity testing was conducted three times. Distinct lesions were found on the nonwounded and wounded leaves of inoculated plants after 3 to 5 days. The morphology and ITS sequences of the fungi that were reisolated from each of the inoculated plants were similar to that of the inocula, fulfilling Koch's postulates. Fruit rot of pomegranate induced by A. alternata was not identified in our investigation. A. alternata is reported to induce leaf spot disease on P. granatum in India (Zakir et al. 2009), Israel (Ezra et al. 2010), Spain (Garcia-Jimenez et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot disease on P. granatum in China. Severe leaf disease caused by A. alternata can lead to reduced pomegranate yields in the harvest stages. This note will aid in pathogen identification and disease control.

Watermelon (Citrullus lanatus L.) is widely cultivated and consumed in Malaysia for its nutritional value. In June 2018, nearly 40% of the 'Red Rocky' watermelon plants in experimental plots of the research farm at Faculty of Agriculture, UPM Serdang, Selangor, Malaysia had leaf spot symptoms. Leaf spots were small, ranging 5 to 30 mm, yellow to brown, and circular to irregular in shape. With ages, the leafspots gradually enlarged and coalesced. To investigate the disease, ten symptomatic leaves were collected from the experimental plots. Diseased tissues (5 x 5 mm) were excied and surface sterilized with 0.5% sodium hypochlorite (NaOCl) for 2 min, rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C for 5 days. A total of ten isolates with similar colony morphologies were obtained from tissue samples. A single representative isolate "F" was further characterized by molecular analysis. All colonies were initially white in color, but later turned gray to black upon sporulation after 7 days. Conidia were produced in culture and were single-celled, black, smooth-walled, spherical in shape measuring 11.4 to 14 μm x 13.8 to 19 μm in diameter (n=40). These were borne on hyaline vesicles at the tip of a conidiophore. For molecular identification, genomic DNA was extracted from fresh mycelium of isolate F using DNeasy Plant Mini kit (Qiagen, Germantown, MD, USA). The internal transcribed spacer (ITS) region of rDNA and the translation elongation factor 1-alpha (TEF1-α) gene were amplified using the ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. BLASTn analysis of the ITS sequence revealed 100% identity (526 bp out of 526 bp) to Nigrospora sphaerica (GenBank Accession no. HQ608063). TEF1-α sequence had 100% identity (494 bp out 494 bp) with N. sphaerica (GenBank Accession no. MN995332). The resulting sequences were deposited in GenBank (ITS: Accession no. MK544066; TEF1-α Accession no. MT708197). Based on morphological and molecular characteristics, isolate "F" was identified as Nigrospora sphaerica (Sacc.) Mason (Chen et al. 2018). A pathogenicity test was conducted on five healthy leaves of five one-month-old watermelon 'Red Rocky' plants grown in a greenhouse. Leaves were wounded using a 34-mm-diameter florist pin frog and spray-inoculated until runoff with a conidial suspension (1 × 106 conidia/ml) of a 7-day-old culture. Five leaves from additional 2 plants were sprayed with sterile distilled water to serve as controls. Inoculated plants were covered with polyethylene bags for 48 h to maintain high humidity. Ten days post-inoculation, symptoms on inoculated leaves developed brown-to-black lesions similar to those observed in the field, while control leaves remained asymptomatic. N. sphaerica was re-isolated from all symptomatic tissues confirming Koch's postulates. N. sphaerica is distributed on a wide range of hosts and has been reported from 40 different host genera including monocotyledonous and dicotyledonous hosts (Wang et al. 2017). N. sphaerica has been reported to cause leaf spot of date palm in Pakistan (Alam et al. 2020) and kiwifruit in China (Chen et al. 2016). To our knowledge, this is the first report of N. sphaerica causing leaf spot of watermelon in Malaysia. This new disease could reduce fruit quality since sweetness and ripening are dependent on healthy foliage. Additionally, this disease can cause premature defoliation which would also reduce watermelon productivity.

Coconut (Cocos nucifera) is a high economic value cash crop in Malaysia. In December 2021, irregular spots with dotted rust-like appearance were observed mainly on the tip of the leaves of MATAG variety coconut seedlings at the nursery in Perak state. More than 90% of the coconut seedlings surveyed were infected with leaf spot symptoms. These symptoms could bring huge economic losses due to the downgrade value of the seedlings. 15 symptomatic leaves were obtained from the nursery, 10 mm2 of cut leaves were disinfected with 10% sodium hypochlorite for 10 minutes and rinsed with sterile distilled water before plated on potato dextrose agar (PDA). A total of 4 single-spore isolates were obtained and were observed morphologically. The isolates had white cotton-like appearance with undulate edge. Black acervuli were seen after 7 days of incubation at 26 °C. The conidia were fusiform and contained five cells with four septate and three versicolor cells in between the apical and basal cell. The conidia were 17.2 µm long and 5.9 µm wide (n=30). Conidia consisted of two to three apical appendages and one basal appendage. These morphological characters were consistent with the original description of Neopestalotiopsis clavispora (Santos et al., 2019; Abbas et al., 2022). Species identification was done by amplifying internal transcribed spacer (ITS) region using primers ITS 4 and ITS 5 (White et al., 1990) and beta-tubulin (TUB2) using primers Bt2a and Bt2b (Glass & Donaldson et al., 1995) of the representative isolate LKR1, then sequenced. The 488 bp ITS and 409 bp TUB2 sequences were deposited in GenBank under the accession numbers ON844193 and OP004810, respectively. Isolate LKR1 shares 99.8% identity with the ITS sequence (MH860736.1) of the reference pathogenic N. clavispora strain CBS:447.73 and 100% identity with the TUB2 sequence (KM199443.1) of the reference pathogenic N. clavispora strain CBS 447.73. The phylogenetic analysis confirmed that the isolate LKR1 belonged to N. clavispora when a supported clade is formed with 98% and 94% bootstrap support for ITS and TUB2 respectively with other related N. clavispora. Pathogenicity test was conducted by using five replicates of 8 month old seedlings, they were incubated under greenhouse condition and were watered daily. The leaves of the seedlings were injured with sterile needles and were sprayed with conidial suspension (1 x 10^6 conidia/ml). The control plants were also injured but sprayed with sterile distilled water. After a month, signature symptoms of spots on the leaves appear but none on the control seedling. N. clavispora was successfully re-isolated only from the inoculated symptomatic leaves and identified morphologically. No fungus was re-isolated from the control seedlings. The result was consistent even after repeating the test one more time. N. clavispora has been reported causing leaf spot on Macadamia integrifolia (Santos et al., 2019), Phoenix dactylifera L. (Basavand et al., 2020) and Musa acuminata (Qi et al., 2022). N. clavispora has also been reported causing rust-like appearance of leaves on strawberry (Fragaria × ananassa Duch.) (Obregón et al., 2018). To our knowledge, this is the first report of N. clavispora causing leaf spot disease on coconut seedlings in Malaysia. Through the identification of N. clavispora as the causal agent of leaf spot on coconut, this can help coconut growers to tackle the disease problem earlier thus, preventing the disease from spreading until the adult phase.

Soursop (Annona muricata L: Annonaceae) is a small tropical fruit tree native to South America (Pinto, 2005). The flesh of its fruits is widely used as a main ingredient of pastries, even young fruits are used as a vegetable. In June 2022, leaf spots symptoms were observed on fifty soursop plants in a commercial nursery located in Juan José Ríos (25°45'20"N 108°50'21"W), Ahome, Sinaloa State. The incidence of the disease was 75%, while the severity was 12%. Symptoms were round, small black necrotic spots, that grew up to 6 mm in diameter with brown or gray color at the center. Fungal isolation was done on potato dextrose agar (PDA) and Colletotrichum-like colonies were obtained. Five isolates were recovered and purified by single spore culture and only a single morphotype was observed. One random isolate was selected for pathogenicity tests, morphological and molecular characterization. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Biotic Products Development Center at the National Polytechnic Institute under accession no. IPN 13.0102. Colonies in PDA at 25°C grow at a rate of 9.0-14.0 mm/d. After 14 days, the colony was olive to gray with orange conidial masses, and conidia (n =100) were hyaline, aseptate, cylindrical, and straight with rounded ends, measuring 11.5 to 18.5 and 3.5 to 5.5 μm. Appressoria were melanized and circular or oval in shape, measuring 6.0 to 4.0 μm (n=20). According to the morphological characteristics observed, the isolate was placed tentatively within the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular confirmation, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), partial sequences of actin (ACT) (Weir et al. 2012) and span style="font-family:'Times New Roman'">glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced. Sequences were deposited in GenBank under the accession numbers: ITS, OQ606966; ACT, OQ617292 and GAPDH, OQ617293. A phylogenetic tree including published sequences of the C. gloeosporiodes species complex was constructed according to Talhinhas and Baroncelli (2021) and the isolate IPN 13.0102 was grouped in a clade with the ex-type culture of C. siamense (ICMP18578) and C. pandanicola. However, C. pandanicola was recorded only as an epiphytic fungus occurring on leaves of Pandanus sp. (Pandanaceae) (Tibpromma et al. 2018) and there are no additional reports of this fungus as a plant pathogen on Pandanus or any other plant. Therefore, the isolate IPN 13.0102 corresponds to C. siamense. Pathogenicity was demonstrated by spraying a conidial suspension (1 × 105 conidia/ml) onto four healthy soursop plants, while two control plants were sprayed using sterile distilled water. All plants were kept in a wet chamber for 48 h at 28  2°C and 85% RH. The characteristic symptoms of the disease were observed 14 days after inoculation, while control plants remained healthy. The pathogenicity test was repeated twice obtaining the same results. The morphology of the recovered fungus was consistently identical to that originally isolated from diseased leaves, fulfilling Koch's postulates. Colletotrichum siamense has been previously reported on Anona spp. in Brazil (Costa et al. 2019). To our knowledge, this is the first report of Colletotrichum siamense causing leaf spots on Annona muricata in Mexico. Further studies for monitoring and control strategies of leaf spots on soursop are required.

Cowpea (Vigna unguiculata, Fabaceae) is an annual herbaceous legume cultivated for seed and fodder. During a survey in May 2022, cowpea leaves with leaf spot symptoms were collected from a field in which c. 40% of the plants were diseased (Karaj City, Alborz province, Iran). Leaf spots began as small circular water-soaked lesions at the leaf centre and margins which expanded and on occasion, coalesced. The infected tissue became pale brown and necrotic. Isolation of the causal pathogen was performed using the high humidity blotter method. After five days, growing fungi were transferred with a fine sterile needle to 2% water agar. Pure cultures were obtained using the hyphal tip method on synthetic nutrient-poor agar (SNA) and potato carrot agar culture media. Fungal colonies were incubated at 25°C with an 8 hour light/16 hour dark photoperiod for seven days. A typical colony (ALT1) reached 58 mm in diameter after seven days incubation on SNA culture medium. The colony was olive-brown to dark brown at the centre, with a white margin. Aerial mycelia were sparse. Mycelia were septate, pale brown and smooth–walled. Sporulation was abundant on SNA, mostly from the aerial hyphae. Conidiophores arose singly from the aerial mycelia, and were brown, erect, simple, short, smooth, frequently reduced to conidiogenous cells with a slight swelling at the apices, 7–30 × 3–5 μm. Conidiogenous loci were 1–3 per conidiogenous cell, and brown. Ramoconidia were 0–3 septate, ellipsoid to cylindrical, smooth, brown, 15–32 × 3–5 μm, with a short beak (n = 50). Conidia were subcylindrical, ellipsoid or fusiform, brown, smooth, in long acropetal chains, simple or with lateral branches and 8–11 × 3–3.5 μm (n = 50) (Figure 1). Morphological and cultural characteristics of the recovered isolate (ALT1) were similar to the description of Alternaria malorum provided by Goetz & Dugan (2006) and Crous et al. (2009). Molecular characterisation of isolate ALT1 was done by PCR amplification and sequencing of the internal transcribed spacer (ITS) region using the ITS1 and ITS4 primers (White et al., 1990). The obtained sequence was deposited in GenBank (Accession No. OP006224) and a BLAST search showed it had 100% identity to isolates of A. malorum (MW335156, LR134074, FJ839611 and KY077556). Maximum likelihood analysis was performed by heuristic search with Mega X software. In the generated phylogenetic tree (Figure 2), isolate ALT1 was separated from closely related Alternaria species and other fungal genera, and clustered in the same group as A. malorum isolates. The molecular analysis confirmed the morphological identification of the pathogen. Pathogenicity testing was performed by spraying a conidial suspension (106 conidia/ml) of isolate ALT1 on healthy and surface-sterilised cowpea leaves without wounding, and was replicated three times. Sterile water was sprayed on the leaves of control plants. All plants were covered with plastic bags to maintain high humidity and placed on a glasshouse bench at 25°C with a 12 hour photoperiod until the development of disease symptoms. Leaf spots appeared twelve days after inoculation (Figure 3) and resembled those observed in the field. The pathogen was re-isolated from the newly generated leaf spots, fulfilling Koch's postulates. In Iran, Alternaria malorum was first reported by Asgari et al. (2004) as Cladosporium malorum from the leaves of the Hordeum vulgare in East Azarbaijan province. Alternaria malorum has also been reported as a fungus associated with declining Persian oak trees (Alidadi et al., 2018). Bagherabadi & Zafari (2022) also reported A. malorum as the causal agent of bark canker in walnut trees. Several diseases are known to infect cowpea, including anthracnose, Cercospora leaf spot, damping off, Fusarium wilt, Sclerotium stem, root and crown rot, Septoria leaf spot, and scab (Dugje et al., 2009). This is the first report of Alternaria malorum as a causal agent of cowpea leaf spot disease in Iran and globally. We are thankful to the University of Tehran for supporting this research.

Macleaya cordata is a perennial herb that belongs to the Papaveraceae and is typically prescribed as a traditional antibacterial medicine in China (Kosina et al. 2010). The extract from M. cordata has been widely used in the manufacturing of natural growth promoters as an alternative to antibiotic growth promoters in the livestock industry (Liu et al. 2017), and the products are marketed in 70 countries such as Germany, China, etc (Ikezawa et al. 2009). During the summer of 2019, symptoms of leaf spot were observed on M. cordata (cv. HNXN-001) in two commercial fields (approximately 1, 300 m2 and 2, 100 m2) of Xinning county, Shaoyang City, Hunan Province, China, where approximately 2 to 3% of the plants were affected. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to leaf blight. Six symptomatic basal leaf sections from six plants from two fields were surface disinfested in 0.5% NaClO for 1 min, then 75% ethanol for 20 s, rinsed in sterile water three times, air dried, and placed onto potato dextrose agar (PDA), one dish for samples from a single leaf. Plates were incubated at 26°C in darkness. Nine strains with similar morphological characters were isolated, and one representative isolate ( BLH-YB-08) was used for morphological and molecular characterization. The colonies on PDA were grayish-green with white round margins. Conidia were typically obclavate to obpyriform, brown to dark brown, and 12.0 to 35.0 × 6.0 to 15.0 μm, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa (n=50). Isolates were identified as Alternaria sp. on the basis of mycelial characteristics, color, and conidial morphology. To confirm identity of the pathogen, DNA was extracted from isolate BLH-YB-08 with the DNAsecure Plant Kit (TIANGEN, Biotech, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), actin (ACT), 28S nrDNA (LSU), 18S nuclear ribosomal DNA (SSU), histone 3 (HIS3), internal transcribed spacer (ITS) region of ribosomal DNA, and translation elongation factor 1-α (TEF) genes ( Berbee et al. 1999; Carbone and Kohn. 1999; Glass and Donaldson. 1995; White et al. 1990.) were amplified and sequenced. Sequences were deposited into the GenBank database. They were 100% sequence identity of GAPDH (OQ224996) with A. alternata strain AA2-8 (MH65578; 578/578bp), 100% sequence identity of RPB2 (OQ190460) with A. alternata strain SAX-WN-30-2 ( MK605877; 933/933bp), 100% sequence identity of ACT (OQ923292) with A. alternata strain FCBP0352 (OL830257; 939/939 bp), 100% sequence identity of LSU (OQ891167) with A. alternata XL14 (MG839509 ; 908/908 bp), 100% sequence identity of SSU (OQ139544) with A. alternata strain BJ19.4.1(OM736063; 1,067/1,067 bp), 100% sequence identity of HIS3 (MT454856) with A. alternata YJ-CYC-HC2 (OQ116440 ; 442/442 bp), 100% sequence identity of ITS (MT212225) with A. alternata CS-1-3 (OQ947366; 543/543bp), and 100% sequence identity of TEF (OQ190461) with A. alternata strain YZU 221185 (OQ512730; 252/252 bp). To test pathogenicity, the isolate BLH-YB-08 was cultured on PDA for 7 days to prepare conidial suspensions and the spore concentration adjusted to a final concentration of 1×106 spores/ml. The leaves of five potted 45-day-old M. cordata (cv. HNXN-001) plants were sprayed with conidial suspensions, and five control potted plants were wiped with 75% alcohol and washed five times with sterile distilled water. They were then sprayed with sterile distilled water. Plants were placed in a greenhouse at 25 to 30°C with 90% relative humidity. Pathogenicity tests were conducted twice. Fifteen days after inoculation, lesions were found on inoculated leaves, and the symptoms were the same as those in the field, whereas the controls were healthy. A fungus was consistently isolated from the inoculated leaves and identified as A. alternata by DNA sequencing of the GAPDH, ITS, and HIS3 genes, fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on M. cordata caused by A. alternata in China. Understanding its etiology may help to control this fungal pathogen, thus reducing economic losses. Funding: Hunan Provincial Natural Science Foundation General Project (2023JJ30341) Hunan Provincial Natural Science Foundation Youth Fund (2023JJ40367) Seed Industry Innovation Project of Hunan Provincial Science and Technology Department Special project for the construction of Chinese herbal medicine industry technology system in Hunan Province "Xiangjiuwei" Industrial Cluster Project of the Ministry of Agriculture and Rural Affairs.

Perilla (Perilla frutescens var. japonica), a member of the family Labiatae, is an annual herbaceous plant native to Asia. Its fresh leaves are directly consumed and its seeds are used for cooking oil. In July 2018, leaf spots symptoms were observed in an experimental field at Gangneung-Wonju National University, Gangneung, Gangwon province, Korea. Approximately 30% of the perilla plants growing in an area of about 0.1 ha were affected. Small, circular to oval, necrotic spots with yellow borders were scattered across upper leaves. Masses of white spores were observed on the leaf underside. Ten small pieces of tissue were removed from the lesion margins of the lesions, surface disinfected with NaOCl (1% v/v) for 30 s, and then rinsed three times with distilled water for 60 s. The tissue pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Five single spore isolates were obtained and cultured on PDA. The fungus was slow-growing and produced 30-50 mm diameter, whitish colonies on PDA when incubated at 25ºC for 15 days. Conidia (n= 50) ranged from 5.5 to 21.3 × 3.5 to 5.8 μm, were catenate, in simple or branched chains, ellipsoid-ovoid, fusiform, and old conidia sometimes had 1 to 3 conspicuous hila. Conidiophores (n= 10) were 21.3 to 125.8 × 1.3 to 3.6 μm in size, unbranched, straight or flexuous, and hyaline. The morphological characteristics of five isolates were similar. Morphological characteristics were consistent with those described for Ramularia coleosporii (Braun, 1998). Two representative isolates (PLS 001 & PLS003) were deposited in the Korean Agricultural Culture Collection (KACC48670 & KACC 48671). For molecular identification, a multi-locus sequence analysis was conducted. The internal transcribed spacer (ITS) regions of the rDNA, partial actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using primer sets ITS1/4, ACT-512F/ACT-783R and gpd1/gpd2, respectively (Videira et al. 2016). Sequences obtained from each of the three loci for isolate PLS001 and PLS003 were deposited in GenBank with accession numbers MH974744, MW470869 (ITS); MW470867, MW470870 (ACT); and MW470868, MW470871 (GAPDH), respectively. Sequences for all three genes exhibited 100% identity with R. coleosporii, GenBank accession nos. GU214692 (ITS), KX287643 (ACT), and 288200 (GAPDH) for both isolates. A multi-locus phylogenetic tree, constructed by the neighbor-joining method with closely related reference sequences downloaded from the GenBank database and these two isolates demonstrated alignment with R. coleosporii. To confirm pathogenicity, 150 mL of a conidial suspension (2 × 105 spores per mL) was sprayed on five, 45 days old perilla plants. An additional five plants, to serve as controls, were sprayed with sterile water. All plants were placed in a humidity chamber (>90% relative humidity) at 25°C for 48 h after inoculation and then placed in a greenhouse at 22/28°C (night/day). After 15 days leaf spot symptoms, similar to the original symptoms, developed on the leaves of the inoculated plants, whereas the control plants remained symptomless. The pathogenicity test was repeated twice with similar results. A fungus was re-isolated from the leaf lesions on the inoculated plants which exhibited the same morphological characteristics as the original isolates, fulfilling Koch's postulates. R. coleosporii has been reported as a hyperparasite on the rust fungus Coleosporium plumeriae in India & Thailand and also as a pathogen infecting leaves of Campanula rapunculoides in Armenia, Clematis gouriana in Taiwan, Ipomoea batatas in Puerto Rico, and Perilla frutescens var. acuta in China (Baiswar et al. 2015; Farr and Rossman 2021). To the best of our knowledge, this is the first report of R. coleosporii causing leaf spot on P. frutescens var. japonica in Korea. This disease poses a threat to production and management strategies to minimize leaf spot should be developed.

The areca palm (Areca catechu L.), belonging to the Palmaceae family, is an important economic crop in Hainan. In June 2018, symptoms of leaf spot were observed on nearly 20% of A. catechu in Qionghai, Hainan (19°40′N; 110°33′E). At first, leaves exhibited small yellow spots; as symptoms progressed, the middle of the lesions appeared black with distinct yellow halos. Lesions were long oval, sometimes irregular with black to brown, small black spots and distinct yellow halos. As the lesions continued to expand, necrotic spots enlarged, gradually gray, and then combined to form larger necrotic areas. Ten leaves with typical symptoms were randomly collected. Small tissue sections (5 × 5 mm) were excised from the margins of lesions, disinfected with 75% ethanol for 10 s and 1% sodium hypochlorite for 2 min, followed by a triple wash with sterile water, plated on PDA, and incubated at 28°C. Three days after incubation, fast-growing fungal colonies with white mycelia appeared. Incubation continued for 3 weeks and few pycnidia developed. Alpha conidia were 5.9 to 8.5 × 2.3 to 3.0 µm (avg. 7.3 × 2.7 µm, n = 100), aseptate, hyaline, fusiform, and acute at both ends. Beta conidia were 14.8 to 29.6 × 1.3 to 2.1 µm (avg. 22.5 × 1.7 µm, n = 100), hyaline, filiform, curved, and tapering toward both ends. These morphological characteristics were similar to Diaporthe spp. (Guarnaccia and Crous 2017). To further confirm the identity of the isolate, single-spore isolates were cultured on PDA and selected for DNA extraction. The internal transcribed spacer (ITS) gene was amplified using primers ITS1 and ITS4 (White et al. 1990), a partial sequence of β-tubulin (TUB) gene by T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995), translation elongation factor 1-α (TEF1) gene by EF1-728F/EF1-986R, and calmodulin (CAL) by CAL-228F/CAL-737R (Carbone and Kohn 1999); sequence data were deposited in GenBank (MN424525, MN424539, MN424567, MN424581). BLAST analysis demonstrated that these sequences were 99% similar to the ITS (MF418423), TUB (MF418583), TEF1 (MF418502), and CAL (MF418257) of D. limonicola. Moreover, a phylogenetic tree was constructed using the method of maximum likelihood (MEGA7.0 (Kumar et al. 2016)) with a combined dataset of ITS, TUB, TEF1, and CAL sequences, which clustered the isolate with D. limonicola (CPC 31137) with high bootstrap support (100%). Based on the morphology, sequence data, and phylogenetic analysis, these isolates were determined as D. limonicola. To validate the results, a pathogenicity test was performed. Ten healthy leaves were washed with sterile water, and each leaf was slightly punctured eight times by a sterile pin and divided into two groups. The first group was placed on a mycelial disk taken from the edges of 4-day-old PDA cultures on each wounded leaf, whereas the other group was placed on PDA agar as a control. All plants were incubated at 28 ± 2°C and 100% relative humidity. Seven days after inoculation, leaves placed on the mycelial disk exhibited the typical symptoms (i.e., small black spots and gray to brown lesions surrounded by yellow halos), whereas controls showed no symptoms. Further, D. limonicola was reisolated from the leaf spot. Pathogenicity tests were repeated thrice with the same results. Koch’s postulate was achieved by the reisolation of D. limonicola from the inoculated leaves. D. limonicola has previously been reported to cause trunk canker of Citrus limon and C. sinensis in Italy (Guarnaccia and Crous 2017). To our knowledge, this is the first report of D. limonicola causing leaf spot on A. catechu in China.

Pineapple (Ananas comosus (L.) Merr.) is an important perennial monocotyledonous plant that serves as an important fruit crop globally and is also produced in the Hainan Province of China where production in 2009 was 296,600 t. In July 2009, atypical symptoms of a leaf spot disease were observed on mature pineapple leaves in Chengmai County; approximately 15% of plants propagated from suckers became symptomatic after 150 to 300 days, eventually causing a 3 to 10% yield loss. In the initial infection stage, grayish white-to-yellowish white spots emerged on the leaf surfaces that ranged from 1.0 to 2.4 × 0.3 to 0.7 cm; black specks were not always present in the spots. Leaf spots also had distinctive light brown-to-reddish brown banding pattern on the edges. Several spots would often merge to form large lesions, 6.5 to 15.4 × 2.5 to 5.6 cm, covering more than 67% of the leaf surface, which can lead to death of the plant. Infected pineapple leaves collected from an orchard of Chengmai County were surface sterilized (75% ethanol for 30 s, 0.1% HgCl2 for 2 min, and rinsed three times in sterile distilled water). Leaf pieces were placed on potato dextrose agar medium and then incubated at 25°C. The emerging fungal colonies were grayish white to brown. Similar strains were obtained from Qionghai City and Wanning City subsequently. Two isolates, ITF0706-1 and ITF0706-2, were used in confirmation of the identity of the pathogen and in pathogenicity tests. Colonies were fast growing (more than 15 mm per day at 25 to 30°C) with dense aerial mycelia. Conidia were fusiform, pyriform to oval or cylindrical, olive brown to dark brown, 3 to 10 septate (typically 5 to 8), 33.2 to 102.5 × 9.0 to 21.3 μm, with a strongly protruding hilum bulged from the basal cell, which were similar to the Type A conidia described by Lin et al. (3). The strains were subjected to PCR amplification of the internal transcribed spacer (ITS)1-5.8S-ITS2 regions with universal primer pair ITS1/ITS4. The ITS sequence comparisons (GenBank Accession Nos. JN711431 and JN711432) shared between 99.60 and 99.83% identity with the isolate CATAS-ER01 (GenBank Accession No. GQ169762). According to morphological and molecular analysis, the two strains were identified as Exserohilum rostratum (Drechs.) Leonard & Suggs. Pathogenicity experiments were conducted five times and carried out by spraying a conidial suspension (105 CFU/ml) on newly matured leaves of healthy pineapple plants; plants sprayed with sterile water served as the negative control. Plants were incubated in the growth chamber at 20 to 25°C. Symptoms of leaf spot developed on test plants 7 days after inoculation while the control plants remained asymptomatic. Koch's postulates were fulfilled with the reisolation of the two fungal strains. Currently, E. rostratum is one of the most common pathogens on Bromeliads in Florida (2) and has been reported on Zea mays (4), Musa paradisiacal (3), and Calathea picturata (1) in China, but to our knowledge, this is the first report of leaf spot disease caused by E. rostratum on pineapple in Hainan Province of P.R. China.

In August 2020-2021, symptoms of leaf spot were observed in luffa (Luffa cylindrical) fields in Qingdao city, Shandong Province. In all the 10 fields investigated, leaf spot occurred. The incidence (% luffa plants with symptoms from a defined number of plants assessed) was 35 to 60%. Early symptoms of infected leaves were small and irregular chlorotic lesions which later became irregular brown spots. As the disease progressed, the lesions gradually spread from the edge to the center of leaves to the middle, and became dark brown. The enlarged spots coalesced and eventually led to the withering and death of the leaves. In order to isolate the pathogen, 30 symptomatic leaves were collected from different planting fields. Small pieces of leaf tissues (5×5 mm) were cut from the junction of healthy and diseased tissues, sanitized with 2% NaClO for 1 min, rinsed three times with sterile distilled water. The tissue samples were then placed on potato dextrose agar (PDA) amended with 50 mg/L streptomycin sulfate, and incubated at 28℃ for 5 days in the dark. Ten purified fungal isolates were obtained by single spore isolation method. Colonies of these fungal isolates on the PDA medium were initially grayish-white, and then turned olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these fungi produced light brown and solitary conidiophore with septum. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 13.2 to 49.5 × 9.5 to 21.6 µm (n=50). The morphological characteristics of these isolates were consistent with that of Alternaria spp. (Simmons 2007). The representative isolate NEAU-SG-1 was selected for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α gene (TEF), histone 3 (HIS3), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), H3-1a/H3-1b (Glass and Donaldson 1995), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007), respectively. Sequences of these genes of isolate NEAU-SG-1 were deposited into GenBank database with the accession numbers of OL307719, OL415166, OL415169, OL415167, and OL415168. BLAST analysis of these sequences showed 99-100% homology with sequence homology with Alternaria tenuissima strains (ITS, MH824269; GAPDH, MK683783; TEF, MN056178; HIS3, MH824371; RPB2, LC621694). To fulfill Koch's postulates, ten surface disinfected 30-day-old luffa seedlings were inoculated by spraying conidia suspension (106 conidia/ml) of isolate NEAU-SG-1. The other ten surface disinfected seedlings inoculated with sterile distilled water served as the control group. After inoculation, each plant was covered with plastic bags for three days and cultured in greenhouse at 25℃. One week later, leaves inoculated with conidia suspension were observed with the same symptoms as described above, while the leaves of the control group were asymptomatic. Pathogenicity test was repeated twice. The Alternaria isolates were successfully re-isolated from those infected leaves and identified using the morphological and molecular methods described above. A. tenuissima has a wide host range in the world, and is the pathogen of leaf spot of many crops (Ma et al. 2021). To our knowledge, this is the first report of A. tenuissima causing leaf spot on luffa in China. This report will provide basic information for the diagnosis and prevention and control strategies of luffa leaf spot.

Ginger (Zingiber officinale Rosc.) is an herbal crop widely grown in China for its medicinal and savory qualities of rhizomes. In August 2018, leaf spot symptoms were observed on ginger plants grown in a field in Nanning, Guangxi Province (E108°3'54", N23°14'48"). Disease incidence was above 50%, and in a Nanning field, rhizome yield loss was almost 30%. Early symptoms appeared as circular, necrotic areas that later developed into circular or irregular spots. The centers of the lesions were white and often surrounded by chlorotic halos (Figure S1A). In severe infections, the spots frequently coalesced, causing the entire leaf to become withered and curved. Small pieces (3 to 4 mm2) from the margin of infected lesions were surface sterilized in 75% ethanol for 40 s followed by 1% NaOCl for 90 s, placed on potato dextrose agar (PDA) and incubated at 28°C in the dark for 4 days. Hyphal tips from the leading edge of colonies were transferred to fresh PDA plates to obtain pure cultures. Fungal colonies were initially white, then turned black/grayish brown when maintained in the dark at 28°C after 5 days (Figure S1B). Conidia were single-celled, brown, or black, smooth, spherical, or subspherical with diameters varying from 9.5 to 15 μm (mean = 13.5 ± 0.72 µm, n = 50) (Figure S1C). Based on these morphological characteristics, the isolates were provisionally identified as Nigrospora oryzae (Ellis 1971; Hudson 1963). Genomic DNA was extracted from a representative isolate Sjb-2. The internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The obtained ITS sequence (GenBank accession no. MW555242), TUB2 sequence (MZ048644), and TEF1-α sequence (MZ048645) showed >99% similarity with several GenBank sequences of N. oryzae (KF516962 for ITS; MK550707 for TUB2; and KY019425 for TEF1-α, respectively). Based on the combined sequences of ITS, TUB2 and TEF1-α sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that the isolates were N. oryzae (Figure S2). Pathogenicity of the isolate was confirmed by fulfilling Koch's postulates. Agar blocks (3 mm diameter) containing a fungal mycelium were placed on detached healthy leaves of ginger. The leaves were then wrapped with sterile polyethylene and incubated in a greenhouse at 25°C with 60% RH. Within 7 days, symptoms appeared on inoculated leaves similar to spots observed in the field, whereas controls remained symptomless. The same pathogen was reisolated from the spots. Pathogenicity tests were performed twice with three replications, indicating that N. oryzae is responsible for leaf spot disease on ginger. The disease in ginger caused by N. oryzae had been reported in Southern Africa (Grech et al. 1989). To our knowledge, this is the first report of N. oryzae causing leaf spot of ginger in China. In the field, this pathogen can substantially affect ginger's health and rhizome yield if no effective control measures are implemented. Therefore, management of the disease should be further investigated to avoid major economic losses.

Cinnamomum migao, a plant in the Lauraceae, is a rare and endangered species endemic to China (Yan et al. 2022). In February 2025, symptoms of leaf spot were observed on C. migao plants growing in Pingtang County, Guizhou Province, China (25°29′55″N, 106°40′29″E). Disease incidence reached 100% (n=200). Foliage exhibited ash-white necrotic lesions that were elliptical or irregular in shape, surrounded by a brown halo at the margin. Six symptomatic leaf samples (5 × 5 mm) were surface-sterilized with 75% ethanol for 30 s, followed by 2.5% NaClO for 1 min. After air-drying, the tissues were placed on potato dextrose agar (PDA) and incubated at 28°C for 7 days. Hyphal tips from emerging colonies were transferred to fresh PDA plates to obtain pure cultures. A total of 16 purified isolates were obtained, 12 of which shared similar morphological characteristics. One representative isolate (designated ZYX) was selected for further study. Colonies on PDA were villous, with dense aerial mycelia, initially white and turning grayish over time; the reverse side was white with a yellow gradient and a red concentric ring. Conidia were ellipsoidal, aseptate, and 3.7 - 6.4 x 1.4 - 4.0 μm (average 5.04 × 2.61 μm; n= 60). Morphological characteristics were consistent with those described for Epicoccum latusicollum, a fungal pathogen causing leaf spot on tobacco in China (Guo et al. 2021). For molecular identification, the internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990), the large subunit ribosomal RNA gene (LSU) with LROR/LR5 (Vilgalys and Hester 1990), the beta-tubulin gene (TUB2) with Btb2Fd/Btub4Rd (Woudenberg et al. 2009), and the RNA polymerase II second largest subunit gene (RPB2) with RPB2-5F2/fRPB2-7cR (Liu et al. 1999). Resulting sequences were deposited in GenBank with accession numbers PV946034 (ITS), PV946035 (LSU), PV976759 (TUB2), and PV976758 (RPB2). BLASTn analysis showed isolate ZYX shared 99% to 100% sequence identity with the E. latusicollum type strain CGMCC 3.18346, ITS (KY742101; 478/479 bp), LSU (KY742255, 859/863 bp), TUB2 (KY742343, 333/333 bp), and RPB2 (KY742174, 595/596 bp) sequences in the GenBank database. Furthermore, the amplified sequences were used to construct a phylogenetic tree in PhyloSuite 1.2.3 which showed that ZYX clustered with E. latusicollum. Thus, both morphological and molecular data support the identification of the isolate as E. latusicollum. To fulfill Koch’s postulates, pathogenicity tests were conducted using conidial suspensions (1×106 conidia/mL) sprayed onto three healthy C. migao plants. Three additional plants were treated with sterile distilled water as controls, and plants were maintained at 28°C with 85% relative humidity and a 12-hour photoperiod. After 7 days, the inoculated plants developed symptoms identical to those observed in the field, while the control plants remained asymptomatic. The pathogen was successfully re-isolated from symptomatic leaves and confirmed based on morphological features and DNA sequences, fulfilling Koch’s postulates. No pathogens were isolated from the control plants. E. latusicollum has been reported to cause disease in various medicinal plants (Yan et al. 2024, Wang et al. 2023). To our knowledge, this is the first report of E. latusicollum causing leaf spot on C. migao in China. This report contributes to the development of targeted and efficient disease management strategies for growers.