RESUMO
Melon (Cucumis melo L.) is an economically important crop in Brazil, with an annual production of 699.281 tons (FAO 2024). Fungal diseases are one of the biggest problems in melon production, and melon growers in northeastern Brazil have reported over 80% of plants showing anthracnose symptoms in the fields during rainy seasons. Plants were wilted, displaying brown necrotic lesions and water-soaked spots with yellowish edges on the leaves and vines. Melon fruits displayed necrotic lesions on the outside. From June 2022 to June 2023, melon leaves (varieties Yellow, Galia, and Cantaloupe) from anthracnose-symptomatic plants were collected in four melon farms located in the municipalities of Afonso Bezerra, Mossoró, Tibau, and Upanema in the state of Rio Grande do Norte. Small fragments of symptomatic leaves were disinfected in 70% ethanol (30 sec) and 2.5 % sodium hypochlorite (1 min), rinsed in sterile distilled water, and plated on PDA Petri dishes with tetracycline (0.05g/liter). Plates were maintained in a bio-oxygen demand incubator (BOD) for 3 days at 28 ± 2 °C, under a 12 hr photoperiod. Eleven representative fungal colonies resembling Colletotrichum spp. were selected and monosporically grown on PDA for seven days for morphology, pathogenicity, and molecular analyses.ight colonies showed pinkish-dark brown with acervuli in the center and cottony mycelium, and showing black edges in some isolates, resembling C. plurivorum (Zhang et al. 2023). Conidia from those colonies were hyaline, cylindrical with obtuse ends, and 17.76 x 7.06 µm, n= 50. Three colonies developed pinkish-gray mycelia with numerous black microsclerotia, and the conidia were hyaline, falcate, and 27.38 x 4.10 µm, n= 50, resembling C. truncatum (Yu et al. 2023). The total DNA of the eleven isolates was extracted, and the internal transcribed space (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB), and chitin synthase 1 (CHS-1) regions were partially amplified by PCR. Amplicons were sequenced and deposited to Genbank (Table eXtra1). A phylogenetic tree was built with the Maximum likelihood method with the concatenated sequences of the five partial gene sequences on Software MEGA (Version 11.0.10) (Tamura et al. 2021). The isolates CML5, CML8, CML9, CML10, CML11, CML14, CML15, and CML25 were grouped with Colletotrichum plurivorum CBS 125474 (orchidearum complex), and the isolates CML26, CML27 and CML28 with Colletotrichum truncatum CBS 15:35 (truncatum complex) with 87 % e 97 % of Bootstrap support, respectively. C. plurivorum was detected in four farms visited (we selected two representative isolates per farm), while C. truncatum isolates were all from the farm in Afonso Bezerra municipality. A pathogenicity test was performed following the method of Baishuan et al. (2023), micro-injuries were made in leaves of melon seedlings 'Goldex Yellow' and inoculated with a spore suspension of colonies with seven days of growth (106 spore/mL) of each isolate and sprayed to the point of dripping. Sterile water was used as mock. After nine days, anthracnose symptoms similar to those observed in the field were seen in all inoculated leaves, while no symptom was observed in the leaves of the mock plants. The pathogens were reisolated and their identification was confirmed by morphology and sequencing. Five seedlings were inoculated per isolate and mock, the assay was repeated, and the same results were observed. The species C. plurivorum has already been reported to cause disease in Cucumbers in Brazil (Silva et al. 2023) and C. plurivorum and C. truncatum in Citrullus lanatus in China (Guo et al. 2022). To the best of our knowledge, this is the first report of C. plurivorum and C. truncatum causing anthracnose in melon plants in Brazil.
RESUMO
Melon (Cucumis melo L.) is the second most exported fruit in Brazil with an annual production of 27.5 million tons (FAO 2023). From September 2019 through February 2020, 50-day-old melon plants started showing root rot symptoms (dark-brow necrotic zones in their roots that extended to the collar zone) in northeastern Brazil, 30% of the plants in the fields were affected by the disease. The fields are in clay soil where melon, in monoculture, is produced all year long with three cycles of the culture per year. A total of 132 samples from "Yellow" and "Cantaloupe" cultivars were collected from four melon fields (4°59'45.3"S, 37°33'39.7"W; 4°57'10.2"S, 37°31'37.1"W; 5°38'17.9"S, 37°56'27.7"W; and 5°00'25.5"S, 37°23'55.3"W). Small pieces of diseased tissues were surface disinfested in 70% ethanol for 30 sec, in 2% sodium hypochlorite for 1 min, washed in sterilized distilled water, plated on a PDA Petri dishes with tetracycline (0.05g/L), and incubated for seven days at 28 ± 2 ºC. Nine representative isolates were selected for downstream analysis. Colonies were white and later became dark gray, pycnidia and conidia were produced after 30 days ofncubation at 25°C under near-UV light in water-agar medium. Conidia were hyaline when immature and dark brown when mature, ranging from cylindrical subovoid to ellipsoidal and septate to non-septate, and with an average size of 12.54 to 21.97 µm. The colonies were morphologically identified as Lasiodiplodia sp. (Phillips et al. 2013). Total DNA from the isolates was extracted and the ITS, TUB, and TEF-1α genes (Jayawardena et al. 2019) were partially amplified by PCR, Sanger sequenced, and deposited in Genbank: ITS (OM102511 to OM102520), TUB (OR062087 to OR062094 and OR062095), and TEF-1α (OP536826 to OP536835). Blastn analysis of the partial sequences ITS (519bp), TUB (388bp), and TEF-1α (315bp) showed 100% nucleotide similarity of the isolates with sequences of L. brasiliensis and L. theobromae from the GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis method. All nine isolates were grouped into the L. brasiliensis clade with 71% bootstrap support, confirming the isolates's identity. Pathogenicity assays were conducted in a greenhouse using the wooden toothpick inoculation method (Nogueira et al. 2019). "Goldex" Yellow melon seedlings were used in a completely randomized experimental design, with 10 treatments (9 isolates + Mock) and six replicates, with one plant per pot. Plants were inoculated 15 days after sowing, and disease severity was evaluated 50 days after inoculation. All nine isolates caused symptoms in the assessed melon plants. The fungus was reisolated from the lesions and looked morphologically identical to the inoculated fungus, fulfilling Koch's postulates. The pathogenicity test was repeated and yielded similar results. All samples in this study were provided by melon growers who were concerned about the high incidence of root rot disease in their plantations. More research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to melons to develop strategies for disease control to properly assist the growers's concerns. This pathogen has been reported to cause disease in other crops in Brazil, e.g., watermelon (Alves et al. 2023) and apples (Martins et al. 2018). However, to the best of our knowledge, this is the first report of L. brasiliensis causing root rot in melons in Brazil.