To determine the species, the four isolates were molecularly identified by amplifying and sequencing the internal transcribed spacer (ITS) region and the translation elongation factor 1‑α (EF‑1α) using primers ITS1/ITS4 and EF1‑728F/EF1‑986R, respectively (White et al. 1990).
Abstract
In March 2025, leaf spot symptoms were detected in approximately 50% of five‑month‑old Momordica charantia plants in a 1.5‑ha commercial field located in Culiacán, Sinaloa, Mexico (24°14'26.8"N, 107°11'43.2"W). Affected plants exhibited circular to irregular leaf spots lesions with grayish centers and brown to reddish margins, along with chlorosis and in advanced stages, mild defoliation. Forty symptomatic leaf samples were randomly collected for pathogen isolation. Samples were surface‑disinfected with 1% NaClO and rinsed with sterile distilled water. After drying, five tissue fragments were placed per Petri dish containing PDA supplemented with streptomycin sulfate (0.3 g L⁻¹) and incubated at 25 °C for 7 days. Among eight isolates obtained, four isolates were selected for identification: CERCCH1CULSIN, CERCCH2CULSIN, CERCCH3CULSIN, and CERCCH4CULSIN. Microscopic examination showed smooth, branched, brown and hyphae. Conidiophores were fascicles, brown, straight and unbranched, measuring 18.45–295.60 × 3.20–6.55 μm (n = 10). Conidia were hyaline, straight or slightly curved, and acicular to clavate, measuring 20.10–228.75 × 3.05–6.90 μm (n = 10). Based on these traits, the isolates were preliminarily identified as Cercospora spp. (Chupp 1954). To determine the species, the four isolates were molecularly identified by amplifying and sequencing the internal transcribed spacer (ITS) region and the translation elongation factor 1‑α (EF‑1α) using primers ITS1/ITS4 and EF1‑728F/EF1‑986R, respectively (White et al. 1990; Carbone and Kohn 1999). Sequences were deposited in GenBank under codes PQ670974, PV628715, PV628716, and PV628717 for ITS, and PQ671957, PV651779, PV651780, and PV651781 for EF‑1α, corresponding to isolates CERCCH1CULSIN, CERCCH2CULSIN, CERCCH3CULSIN, and CERCCH4CULSIN. BLAST analysis showed that ITS sequences shared about 99.8% identity with the reference C. citrullina sequence ON849061.1 (PQ670974.1, 476/477 pb; PV628715.1, 480/481 pb; PV628716.1, 479/481 pb; PV628717.1, 472/473 pb), while EF‑1α sequences showed 100% with ON890306.1 (PQ671957.1, 246/246 pb; PV651779.1, 249/249 pb; PV651780.1, 253/253 pb; PV651781.1. 253/253 pb). A Phylogenetic analysis using the Neighbor‑Joining method with 1000 bootstrap replicates grouped the isolates within C. citrullina clade. Pathogenicity of each of the four isolates was confirmed through foliar spray inoculation. Spore suspensions (1.0 × 10⁴ conidia mL⁻¹) of each isolate were applied to 15 healthy, 30‑day‑old M. charantia plants. An additional set of 15 plants was sprayed with sterile water to serve as the control. All plants were maintained at 25 ± 2 °C, 90% RH, and a 12:12 h photoperiod. Eight to ten days after inoculation, leaf spot symptoms developed and were like those observed in the field for all four isolates, whereas no symptoms were observed in the control plants. Cercospora citrullina was successfully reisolated and identified from symptomatic foliar tissue of each isolate, thereby fulfilling Koch’s postulates. This fungus has previously been reported as a causal agent of foliar disease in M. charantia (Hsieh and Goh 1990; Gautam et al. 2020); however, no prior reports exist for this pathogen on this host in Mexico. Therefore, this study constitutes the first report of C. citrullina causing leaf spot on M. charantia in Mexico, and this finding will support future research aimed at developing integrated disease management strategies for this disease.
In September 2025, a total of 15 soybean fields were surveyed in Muan and Gongju, Korea. Irregular leaf blight symptoms were observed in five fields, with approximately 1–10% of soybean plants showing symptoms in each affected field. To isolate the causal pathogen, symptomatic leaf tissue was surface-disinfected with 1% NaOCl for 1 min, rinsed with sterile distilled water, and placed on potato dextrose agar (PDA). The plates were incubated at 20℃ for 7 days, and olive-brown hyphae growing from the tissues were subcultured twice on PDA. Five isolates with similar morphology were obtained from symptomatic leaves, and one representative isolate, DA17-37, was selected for further morphological, molecular, and pathogenicity analyses. The conidia were predominantly oval to ellipsoidal and aseptate, measuring 3.71-6.45 x 1.77-2.83 μm, with a mean size of 5.11 x 2.14 μm (n=30). The ascospores were mostly oval and 1-septate, measuring 12.49-18.25 x 4.13-5.41 μm with a mean size of 15.08 x 4.48 μm (n=30). Additionally, multicellular chlamydospores were observed. Overall, these morphological characteristics were consistent with those of the genus Didymella. To genetically characterize isolate DA17-37, genomic DNA was extracted, and the internal transcribed spacer (ITS), beta-tubulin (tub2), and RNA polymerase II second largest subunit (rpb2) genes were amplified by PCR using the respective primer sets described previously (Gorny et al. 2016; Liu et al. 1999; Woudenberg et al. 2009). The resulting sequences were deposited in GenBank under accession numbers PZ274701, PZ433240, and PZ439292. Maximum likelihood phylogenetic analysis of the concatenated dataset showed that isolate DA17-37 clustered with the reference strains of Didymella americana. To confirm the pathogenicity of isolate DA17-37, a conidial suspension (1 x 10
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conidia/mL) prepared from 30-day-old PDA cultures grown at 20℃ was sprayed onto 2-week-old potted soybean plants. Control plants were sprayed with sterile distilled water. The inoculated plants were incubated in a dew chamber at 26℃ for 3 days and then transferred to a growth chamber maintained at 26℃ and 70% relative humidity under a 16-h photoperiod. Symptoms first appeared at 7 days post-inoculation (dpi), and representative symptoms showing disease progression were photographed at 14 dpi, whereas the uninoculated control plants remained healthy. To satisfy Koch’s postulates, the same pathogen was re-isolated from symptomatic leaves and identified based on morphology and DNA sequence analysis. Didymella americana has been reported as a causal pathogen of leaf blight on lima bean in the United States. To the best of our knowledge, this is the first report of leaf blight caused by D. americana on soybean in Korea. Continued monitoring is needed to assess the potential spread of this disease in Korea and to support the development of appropriate disease management strategies.
S. Choi, Eun Young Kim, Shinhwa Kim et al.· Plant Disease· 0 citations
In 2024 fall, symptoms of leaf blade and petiole spots were observed on strawberry plants 'Rociera FNM' and 'Marisma FNM'. In 450 randomly selected plants from 3 nurseries of Segovia province (Spain) 3.5% of incidence was recorded. Lesions on the leaf were reddish-brown with yellowish halos and became necrotic. Dark brown lesions with defined edges were noted on petioles. Affected tissue sections were excised, rinsed with 0.1% Tween 20, submerged in 70% ethanol (20 s), followed by 1% NaOCl (60 s), and then placed onto potato dextrose agar (Condalab, Madrid, Spain) with 50 mg/l of streptomycin sulphate. Plates were incubated at 28°C under darkness. From 6 crowns and 2 petioles 8 isolates (Di 3 to Di 10) were obtained from different pycnidia via single conidia isolation. Colonies had petal-shaped margins. Hyphae were initially hyaline, later developing an orange coloration in the center of the colony, and black conidiomata were visible two weeks post-incubation. Conidiomata were ellipsoidal to globose. Single-celled conidia were brown, ellipsoidal with slightly mucronate apices, narrowly truncated bases and smooth walls. This morphology was consistent with Coniella sp. as described by Álvarez et al. (2016). Isolates were stored in 50% glycerol solution at -80ºC. Genomic DNA was extracted from the 8 selected isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1), and large subunit ribosomal RNA (LSU) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF728/EF986 (Carbone and Kohn 1999), and LR0R/LR7 (Chethana et al. 2017), respectively. Sequence alignments performed in MEGA v11.0 (Tamura et al. 2021) showed that all isolates were identical across the three analyzed loci. Sequences of Di6 and Di10 isolates were deposited in GenBank. BLASTn analyses revealed that ITS (PX663172, PX663173), TEF1 (PX666006, PX666007), and LSU (PX663628, PX663629) sequences showed 100% identity with C. fragariae strain CBS 198.18 for ITS and 99% and 100% identity with strain CBS 167.84 for TEF1 and LSU, respectively. A concatenated phylogenetic tree was constructed with representative Coniella species and outgroup sequences from NCBI GenBank (Chethana et al., 2017) and confirmed the identity. Pathogenicity test was conducted using isolates Di6 and Di10. Inoculation with each isolate was performed by spraying 25 mL of a conidial suspension (5 x 104 conidia/mL) onto 5 plants of 'Rociera FNM'. Control plants were sprayed with sterile water. The assay was carried out twice. After inoculation, plants were covered with a plastic bag and kept in darkness for two days. Growth chamber was maintained at 25±2°C with 12-h photoperiod. Identical symptoms to the initially observed appeared on inoculated plants 6 days post-inoculation. Both isolates exhibited comparable severity (proportion of symptomatic leaf area) 20 days after inoculation. An assay was also performed on 'Marismas FNM', giving the same results. Fungal colonies re-isolated from the lesions had the same morphology and ITS, TEF-1 and LSU sequences of the inoculated isolates, thereby fulfilling Koch's postulates. In Switzerland and Great Britain C. fragariae has previously been isolated from strawberry plants (Rigotti et al. 2003; Jones and Baker, 2007). To our knowledge, this is the first report of C. fragariae on strawberry in Spain. Monitoring of the pathogen is essential since it can spread through asymptomatic plants into strawberry’s nurseries and fields.
In September 25, 2025, numerous symptoms resembling leaf spot were observed on the leaves of Welsh onions planted at the Beijing Academy of Agricultural and Forestry Sciences (39°57′N, 116°18′E). Samples were taken from the diseased leaves. The lesions mostly appeared at the leaf tips and middle parts of the leaves, exhibiting a yellowish-white discoloration, circular or oval shapes, slightly sunken, and in severe cases. The samples were isolated in a hood, soaked in 75% alcohol for 30 s, rinsed twice with sterile water for 5 s each time, and finally cut into leaf pieces（5 × 5 mm）. These pieces were inoculated onto Potato Dextrose Agar (PDA) medium and cultured in a dark incubator at 25°C. Two isolates (designated BJST4 and BJST6) were isolated. Mycelia were white and irregularly floccular at the initial stage. The medium gradually turned yellow, later, they turned cottony-woolly. The medium gradually turned reddish-brown. Conidia are pale yellowish-brown, elliptical, and muriform with transverse septa, constricted at the central septum. After 10 d, the mycelia were picked and DNA was extracted using the CTAB method. Primers ITS1/ITS4 were used to amplify the Internal Transcribed Spacer (ITS) regions (White et al., 1990), and primers gpd1/gpd2 were used to amplify the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (BerBee et al., 1999). The amplification products were sent to Tsingke BiotechnologyCo., Ltd. for sequencing. BLASTn analysis revealed that the ITS sequences of both isolates were 100% homology to Stemphylium vesicarium (YLXXC10-21 and YLXXC08-9), and the gpd sequences showed over 99% homology to multiple S. vesicarium. MEGA7 was used to align and trim the obtained sequences, and phylogenetic trees were constructed for the ITS and gpd sequences separately. The ITS and gpd sequences have been submitted to GenBank (GenBank accession nos. PZ169639, PZ169640, PZ179494, and PZ179495, respectively). To verify Koch's postulates, inoculation was performed on detached Welsh onion leaves. The leaves were cleaned with 75% alcohol and rinsed with sterile water. The edge hyphae of a 5-day-old strain were inoculated onto the pricked and wounded leaves. The control group used PDA medium without inoculation. The inoculated leaves were placed in a light incubator (humidity 99%, temperature 25°C with a 12-h photoperiod). On the third day of inoculation, disease spots appeared in the treatment, while the control group remained healthy. The strain re-isolated from the lesions was identical to the original inoculated strain. To our knowledge, this is the first report of leaf spot disease caused by S. vesicarium on Welsh onions in Beijing, China. This discovery provides an effective direction for the identification and treatment of Welsh onion diseases in production.
Yexuan Zheng, Mifeng Bai, Yujie Yang et al.· Plant Disease· 0 citations
Snap bean (Phaseolus vulgaris L.) is an important crop in Sinaloa, Mexico where 2,076 ha were cultivated in 2024, representing 21.88% of the national production area (DGSIAP 2026). In December 2024, root rot was observed in ~ 40% 4‑month‑old P. vulgaris plants grown in a 5‑ha outdoor commercial field in Culiacán, Sinaloa (24°14'44.7"N 107°10'59.6"W). Plants also exhibited crown rot, stunted growth, wilting, leaf yellowing, and partial foliage necrosis and defoliation, and plant death. Thirty pieces (1‑cm²) of rotted tissue were collected from the roots of 15 randomly selected diseased plants. The fragments were surface‑sterilized in 1% NaClO, rinsed in sterile distilled water and dried on paper. Five pieces were placed equidistantly per Petri dish containing PDA supplemented with streptomycin sulfate (0.3 g L⁻¹) and incubated at 30 °C for 5 days. Out of the 6 isolates generated, 3 were selected for identification. The isolates were plated on V8 medium (800 mL distilled water, 200 mL V8 juice, 2 g CaCO₃, and 15 g agar) and incubated for 7 days at 30 °C. Cultures developed white, dense, cottony, aerial, and well‑branched mycelia; the hyphae were hyaline and coenocytic, and the sporangia were filamentous, lobulate, and irregular in shape. These traits were consistent with Pythium aphanidermatum (Watanabe 2002; Castro-Diego et al. 2025). PCR analysis was performed with the internal transcribed spacers ITS1 and ITS4 (White et al. 1990) and the cytochrome oxidase c subunit 1 (COI) (Robideau et al. 2011). The sequences were deposited in GenBank under PX671483, PX671484, and PX671485 for ITS, PX677389, PX677390, and PX677391 for COI, corresponding to isolates PYTHFRI3CULSIN, PYTHFRI1CULSIN, and PYTHFRI8CULSIN, respectively. BLAST analysis showed that PX671483, PX671484, and PX671485 shared 100% identity with P. aphanidermatum sequence HQ643439 (555/555 bp, 664/664 bp, and 739/739 bp, respectively), and that PX677389, PX677390, and PX677391 also exhibited 100% identity with sequence HQ708486 (596/596 bp, 628/628 bp, and 626/626 bp, respectively) (Robideau et al. 2011). Phylogenetic analyses using the Neighbor‑Joining method with COI sequences and 1,000 bootstrap replications in MEGA 11 placed the three isolates within the P. aphanidermatum clade. To verify the pathogenicity of the isolates, 2 pathogenicity assays were conducted. In each assay, 10 P. vulgaris seedlings were individually inoculated at the stem base with 5 mL of inoculum (0.25 g of macerated mycelium in 100 mL of sterile water) 15 days after emergence. An additional 10 seedlings were inoculated with sterile water to serve as controls. All plants were maintained in a growth chamber at 28 ± 2 °C, 70 ± 5% RH, and a 12:12 h photoperiod for 30 days. Inoculated plants developed symptoms similar to those observed in the field, whereas control plants remained asymptomatic. Pythium aphanidermatum was successfully reisolated and identified from symptomatic root tissues, fulfilling Koch’s postulates, while no isolates were obtained from control plants. Although P. aphanidermatum was previously reported on P. vulgaris in Mexico (McGuire and Crandall 1967), details on methodology and isolation site were not provided. This study represents the first confirmed occurrence of P. aphanidermatum causing root rot in P. vulgaris in Mexico supported by both morphological and molecular evidence and the first documented case in the state of Sinaloa.
Oscar Alejandro Sánchez-Gómez, Juan Antonio Castro-Diego, W. Rubio-Aragón et al.· Plant Disease· 0 citations
To the authors' knowledge, this is the first report of P. ramorum occurring on C. siliqua globally and the first confirmation of Koch’s postulates for this host-pathogen combination.
Dinh-Hieu Pham, T. Pastalka, Huiying Liu et al.· Plant Disease· 0 citations
Pepper (Capsicum annuum L.) is one of the most economically important cash crops in China. In July 2024, leaf spot was observed on pepper cv. ‘Sujiao 5’ in a 1-ha pepper plantation in Hangzhou (30°17′N, 118°52′E), Zhejiang Province. Among eight greenhouses surveyed, 30 plants per greenhouse were inspected. Disease incidence ranged from 10% to 20%. Early symptoms were round, grayish-white lesions. As the disease advanced, lesions expanded into circular spots with grayish-white centers and brown margins, and a grayish-brown mold layer emerged on the lesions. Thirty diseased leaves were randomly sampled from twenty infected plants for pathogen isolation. Under a stereomicroscope, a single conidium from the mold layer on diseased leaves was transferred onto potato dextrose agar (PDA) medium using a sterilized inoculating needle and incubated at 26°C for 7 days. Twenty single-conidium isolates were successfully obtained. After 15 days on PDA, colonies were grayish-green, velvety, with grayish-white margins. On synthetic nutrient-poor agar (SNA), colonies were light grayish-green with sparse mycelium. The conidiophores were solitary, erect, light brown, and septate. Ramoconidia were light brown, cylindrical to obovoid, aseptate, and measured 6.0-15.3 × 2.5-4.3 μm (n = 30). Conidia were light brown, obovoid to limoniform, forming branched chains with one to four conidia per branch, and measured 3.3-5.7 × 2.3-3.3 μm (n = 30). The morphological features were consistent with Cladosporium sp. (Bensch et al. 2015). All 20 single-conidium isolates shared identical colony morphology and conidial characteristics, so three representative isolates were randomly selected for sequencing. The primer pairs ITS1/4 (White et al., 1990), EF728/EF986 (Carbone and Kohn, 1999), and ACT512/ACT783 (Carbone and Kohn, 1999) were used to amplify the partial fragments of the internal transcribed spacers (ITS, GenBank: PZ437871-PZ437873), translation elongation factor 1-α gene (tef1, GenBank: PZ445478-PZ445480), and actin gene (act, GenBank: PZ445475-PZ445477). The sequences of the three isolates were identical at all three loci. BLASTn analysis showed 99% identity with Cladosporium sp. (ITS: OQ629129; tef1: HM148442; act: MT154165). The phylogeny suggests that three isolates were integrated into the Cladosporium tenuissimum clade (ML bootstrap support values/Bayesian posterior probabilities = 93/1). Based on morphology and phylogeny, the isolates were identified as C. tenuissimum. Since all isolates were morphologically and molecularly identical, isolate LJYB-LA1 was selected as a representative strain for pathogenicity testing. The isolate was cultured on PDA at 26°C in continuous darkness for 14 days. Conidia were rinsed with sterile water containing 0.1% Tween-80 and adjusted to 2 × 10
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conidia/mL using a hemocytometer. Two-month-old pepper plants cv. ‘Sujiao 5’ were each sprayed with 200 mL of conidial suspension; the negative control plants were sprayed with 200 mL of sterile water. The plants were incubated in a greenhouse at 26°C and 85% relative humidity for 48 h. The experiment was repeated three times with five plants per replicate. After 10 days, inoculated plants developed symptoms similar to those observed in the field, whereas control plants remained asymptomatic. The species C. tenuissimum was reisolated from symptomatic tissues and confirmed by morphology and molecular data, confirming Koch’s postulates. The species C. tenuissimum has been reported to infect Hydrangea paniculata in China (Li et al. 2021) and Phaseolus vulgaris in China (Gao et al. 2024). To our knowledge, this is the first report of C. tenuissimum causing leaf spot on C. annuum in China.
