Genomic DNA extracted from a representative colony (strain SPIN2) confirmed the identification of SPIN2 as C. spinaciae, and maximum parsimony analysis of combined sequences confirmed the identification of SPIN2 as C. dematium species complex.
Abstract
Spinacia oleracea L. is an important leafy vegetable crop in Italy, with the Apulia region (Capitanata area) cropping about 800 ha with an annual production of 15.339 tons. During the 2024–2025 growing season, anthracnose-like symptoms were observed on spinach cv. ‘Kangaroo’ in a field in the Capitanata area (41.3501° N, 15.6025° E). Disease incidence ranged 23 to 36%, based on visual assessment of 200 random plants. Initial symptoms on leaves consisted of small circular water-soaked lesions surrounded by a chlorotic halo. Lesions enlarged, becoming tan to dark brown and coalescing into larger necrotic areas with thin papery tissue; acervuli bearing dark setae were visible on mature lesions. Symptomatic leaves were surface sterilized (70% ethanol 20 s, 20% NaOCl 20 s, 70% ethanol 20 s), rinsed three times in sterile distilled water and dried on sterile filter paper. Leaf pieces (0.5–0.8 cm diameter) were placed on potato dextrose agar (PDA; 39 g L⁻¹, Oxoid) amended with streptomycin sulphate (500 ppm). Plates were incubated at 23 ± 2°C in the dark. After 14 days, colonies resembling Colletotrichum sp. were transferred to fresh PDA. Hyphal tips from the colonies, were spread on water agar (20 g L-1). After 24-36 h, hyphal tips were transferred to fresh PDA plates for 7-10 days to obtain pure cultures. Colonies showed irregular margins with dense mycelium, dark olivaceous-grey at the center and cream toward the periphery. Microscopic observation revealed hyaline, aseptate, falcate conidia (16.01–19.7 × 2.9–3.8 µm) produced on conidiophores within acervuli on oatmeal agar (OA; 72.5 g L⁻¹, Fluka). Genomic DNA was extracted from a representative colony (strain SPIN2) following Carlucci et al. (2013). The internal transcribed spacer (ITS) region and partial fragments of actin (act), beta-tubulin (tub2), chitin synthase (chs-1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and histone H3 (his3) genes were amplified using primers ITS1/ITS4, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-354R, GDF1/GDR1, and CYLH3F/CYLH3R, respectively (Damm et al. 2009). Sequences of strain SPIN2 (GenBank acc. nos. PZ091375 (ITS); PZ118972 (act); PZ118971 (tub2); PZ118973 (chs-1); PZ118974 (gapdh); PZ118975 (his3) showed 100% identity with Colletotrichum spinaciae strain CBS 128.57. Maximum parsimony analysis of combined sequences, including reference taxa from the C. dematium species complex (Damm et al. 2009; Liu et al. 2022), confirmed the identification of SPIN2 as C. spinaciae. Pathogenicity was assessed on ten healthy 30-day-old spinach seedlings (cv. ‘Kangaroo’) grown in pots (soil:sand:peat 2:1:1). Plants were sprayed with a suspension of propagules (4 × 10⁵ mL⁻¹) of the SPIN2 strain (collected on OA plates for 14 d) until runoff; ten control plants received sterile distilled water. Plants were maintained in a greenhouse at 25 ± 3°C, 70–80% relative humidity. The experiment was conducted three times. Twelve days after inoculation, dark brown to black circular lesions developed on inoculated plants, whereas control plants remained symptomless. Colletotrichum spinaciae was consistently re-isolated from symptomatic tissues (95%), fulfilling Koch’s postulates. Colletotrichum spinaciae has been previously reported on spinach in Canada (Cerkauskas et al. 1991), North America (Correll et al. 1994), Australia (Washington et al. 2006), and Turkey (Kurt et al. 2016). To our knowledge, this is the first report of C. spinaciae causing anthracnose on S. oleracea in Italy, and can thus form the basis for improved disease scouting and management in spinach crops.
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
Michelia × alba (M. alba), an evergreen magnolia, is widely planted as courtyard and street ornamental tree across subtropical South China. It grows in open ground in southern provinces and pots in the Yangtze River basin (Liang and Nooteboom 1993). In February 2026, severe anthracnose was observed on M. alba in the Qujing community courtyard, Qujing City (103.783028 E, 25.472859 N), Yunnan Province, China. Disease symptoms were observed on 60 - 80% of leaves in planting plots (~ 0.015 - 0.020 ha) with 15 - 20 mature M. alba trees. The disease forms small water-soaked leaf spots expanding into irregular brown lesions with clear yellow halos. Lesions range 1.2 × 1.0–4.5 × 3.8 cm, with sunken necrotic centers and concentric rings. Under humid conditions, lesions coalesce to trigger widespread leaf blight and early leaf drop, with dark fungal fruiting bodies occasionally present on dead tissue. To isolate the fungus, 9 symptomatic leaves were randomly collected from three trees and rinsed with sterile water. Small, infected tissue segments (~ 5 mm²) were surface sterilized with 75% ethanol for 30 s, then 3% hypochlorous acid for 3 min, rinsed thrice with sterile water, and inoculated on potato dextrose agar (PDA). Plates were incubated at 28°C with a photoperiod of 12 h. 9 strains with similar morphology characterizations were isolated, and two representative isolates (BLYB1 and BLYB2) were purified. Purified colonies initially had sparse edges on PDA, becoming dense after 3 days, covering the dish by 12 days, with round, flat edges. Microscopically, the conidia were observed to be hyaline, cylindrical, and obtuse at the apex, measuring (12.30 - 16.90) × (5.56 - 8.36) μm, avg. (14.85 ± 2.30) × (6.96 ± 1.40) μm (n = 50). These morphological characteristics resembled Colletotrichum siamense. To confirm identification, genomic DNA from mycelium of these two isolates was extracted, and the sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS1), and calmodulin (CAL) were amplified (Zhang et al. 2020), with GenBank accessions: ITS (PZ537092, PZ537093), GAPDH (PZ584306, PZ584307), ACT (PZ584308, PZ584309), CHS1 (PZ584310,PZ584311), CAL (PZ584312, PZ584313). Pathogenicity of isolates BLYB1 and BLYB2 was assayed on healthy one-year-old leaves with needle wounding, using 5 mm mycelial plugs in three biological replicates. Inoculated leaves were cultured at 25 - 30℃, 85 - 95% relative humidity and a 12 h photoperiod. Typical leaf spots formed on all inoculated leaves at 5 days post-inoculation, whereas control leaves treated with blank PDA plugs or sterile water showed no lesions. The target fungus re-isolated from diseased tissues was identified as C. siamense, which fulfilled Koch’s postulates. The fungus was re-isolated and identified as C. siamense, fullfilling Koch's postulates. C. siamense infects at least 60 plant species across the world (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. alba in Yunnan, China via multilocus phylogenetic analysis. This finding aids targeted disease management to protect M. alba plantations, as C. siamense anthracnose reduces the ornamental value of M. alba, and supports the need for research on host-pathogen interactions and resistance breeding.
Anthracnose of lima beans (Phaseolus lunatus) in South Carolina (SC) has been exclusively attributed to Colletotrichum truncatum, which causes reddish brown lesions on aerial plant tissues. Here we report C. magnum as an additional causal agent of this disease. In October 2023, anthracnose symptoms were observed on the pods and leaves of lima beans in a ~1000 sq. ft plot at a market farm in Charleston County, SC, with all foliage and ~10% of pods affected. Pod lesion margins were excised and surface disinfested by briefly agitating in 0.1% Tween, soaking in 70% ethanol for 30 s and 1% sodium hypochlorite for 1 min, and blotting dry. The lesions were incubated on 1:10 potato dextrose agar (PDA) amended with tetracycline (10 µg/mL) for 4 days at room temperature. Putative Colletotrichum spp. were plated onto full-strength PDA and incubated for 5 days at room temperature. Three isolates were obtained via single hyphal tipping. Isolate PL7 exhibited growth and sporulation on PDA at 20°C under 1550 lm LED lights. Colonies exhibited cottony, filamentous growth that was slightly raised from the colony surface. Mycelia were white in appearance and septate. Acervuli were dark brown to black and produced salmon mucilage when sporulating. Conidia were cylindrical with an average length and width of 19.21 µm and 5.52 µm, respectively (n = 10). Species-level identification was performed by amplifying the internal transcribed spacer (ITS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions using primer pairs ITS1F/ITS4 (Glass and Donaldson 1995; White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), and GDF/GDR (Templeton et al. 1992), respectively. These loci were selected based on their established utility for species-level identification within the Colletotrichum genus (Talhinhas and Baroncelli 2021). The sequences shared 99.83% (ITS), 99.62% (ACT), and 100% (GAPDH) similarities with Colletotrichum magnum (CBS 519.97) when analyzed with BLASTn. The resulting sequences were deposited into GenBank (ITS, PZ433468; ACT, PZ445365; GAPDH, PZ445309) and aligned to 214 Colletotrichum species reference sequences (Talhinhas and Baroncelli 2021) using MAFFT. A maximum-likelihood phylogenetic tree was constructed in IQtree (Minh et al. 2020) and visualized with iTOL (Letunic and Bork 2024). PL7 clustered with Colletotrichum magnum (CBS 519.97) within the magnum species complex. Koch’s postulates were repeated twice by spray inoculating three 2-week-old lima bean plants (cv. Jackson Wonder) with a PL7 conidial suspension of 1.0 x 10
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conidia/mL; three control plants received sterile deionized (DI) water. High humidity was maintained by daily misting with DI water. Characteristic anthracnose symptoms appeared on the abaxial side of leaves after 7 days. Control plants did not develop symptoms in any experiment. C. magnum was reisolated from the leaves of each inoculated plant. The reisolates had identical morphology and ITS, GAPDH, and ACT sequences to the initial isolate, fulfilling Koch’s postulates. Disease symptoms were notably milder than those of C. truncatum. C. magnum is known to cause disease in Cucurbitaceae (Damm et al. 2019). To our knowledge, this is the first report of Colletotrichum magnum causing anthracnose of lima bean in SC. The presence of an additional Colletotrichum species causing lima bean anthracnose in SC underscores the importance of accurate species-level identification for effective disease management.
Karen Wakeley, Suman Sharma, K. Paugh et al.· Plant Disease· 0 citations
Soybean (Glycine max L.) is an economically important legume crop in South Korea (Lee and Ahn 2017; Chae et al. 2025; Cho and Yoon 2025). In October 2021, anthracnose symptoms were observed on soybean plants in two commercial (1 ha each) fields in Gimje, Jeollabuk-do, South Korea (35°80′N, 126°88′E), with disease incidence exceeding 30%. Symptoms consisted of irregular, dark brown necrotic lesions surrounded by yellow chlorotic halos on leaves. Six symptomatic leaves were collected from the two fields for pathogen isolation. Small pieces (approximately 5 × 5 mm), surface-disinfested tissues from lesion margins were plated on water agar amended with streptomycin. Five isolates (FD0006, FD0007, FD8, FD0033, and FD34) were obtained and purified on potato dextrose agar (PDA). Colonies were pale gray with entire margins after 7 days at 25°C. Conidia (n = 50) were hyaline, aseptate, cylindrical with rounded ends, measuring 14.4–21.0 × 4.1–6.2 μm (mean 16.4 × 5.3 μm). Appressoria were dark brown, irregular to strongly lobed, measuring 7.4–15.9 × 4.7–11.3 μm (mean 11.7 × 8.2 μm). Asci were cylindrical to clavate, 38.0–70.4 × 6.5–14.2 μm, and ascospores were hyaline, aseptate, and 13.5–22.1 × 4.6–6.8 μm. These characteristics were consistent with species of the Colletotrichum orchidearum species complex, including C. reniforme (Liu et al. 2022; Zhao et al. 2026). For molecular identification, partial sequences of gapdh, chs-1, act, tub2, and his3 were amplified and sequenced using primers described by Damm et al. (2019). Representative sequences were deposited in GenBank (gapdh: LC797748–LC797752; tub2: LC797788–LC797792; chs-1: LC797828–LC797832; act: LC797868–LC797872; his3: LC797906–LC797910). Multilocus phylogenetic analysis (Bayesian and Maximum Likelihood) of the combined dataset placed all five isolates within the C. reniforme clade with strong support. Pathogenicity was assessed using isolates FD0007 and FD00034 on four soybean plants (cv. Daewon and Cheongja 5) at the V2–V3 growth stage. Plants were inoculated by spraying approximately 10 mL of a conidial suspension (1 × 10
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conidia/mL) containing 0.02% Tween 20 onto unwounded leaves until runoff. Control plants (four) were sprayed with sterile distilled water containing 0.02% Tween 20. After inoculation, plants were maintained in a dew chamber at 24°C in the dark for 24 h and then transferred to a greenhouse, where they were kept at approximately 95% relative humidity by enclosing the plants with plastic film. Necrotic lesions similar to those observed in the field developed within six days, with an 80% disease incidence. The fungus was reisolated from symptomatic tissues and identified as C. reniforme based on morphological and molecular characteristics described above, thereby fulfilling Koch’s postulates. The experiment was repeated twice with similar results. Colletotrichum reniforme was originally described from China as a member of the C. orchidearum species complex (Liu et al. 2022). To our knowledge, this is the first report of soybean anthracnose caused by C. reniforme and the first record of this species in South Korea. This finding expands the known geographic distribution and host range of C. reniforme and indicates that the pathogen may represent an emerging threat to soybean production in Korea. Accurate identification of Colletotrichum species associated with soybean anthracnose will be important for disease surveillance, epidemiological studies, and the development of effective management strategies.
Woohyung Lee, O. Hassan, Eunchong Jeong et al.· Plant Disease· 0 citations
Based on morphology and multilocus analyses, the pathogen was identified as C. eragrostidis, and the obtained sequences were deposited in GenBank placed the three isolates within the C. eragrostidis clade.
Xiang Lu, Chumiao Chen, Yunzhi Bai 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.
M. Avilés, J. Ordóñez, A. M. Pastrana et al.· Plant Disease· 0 citations
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