甜瓜(CucumismeloL.)是巴西的重要经济作物,年产量为699.281吨(粮农组织2024年)。真菌病是甜瓜生产中最大的问题之一,巴西东北部的甜瓜种植者报告说,在雨季期间,超过80%的植物在田间出现炭疽病症状。植物枯萎了,显示棕色坏死病变和浸水斑点,叶子和藤本植物边缘淡黄色。甜瓜果实在外部显示坏死病变。从2022年6月到2023年6月,甜瓜叶(品种黄色,Galia,和哈密瓜)在位于阿方索·贝塞拉市的四个甜瓜农场中收集了有炭疽病症状的植物,Mossoró,Tibau,和北里奥格兰德州的乌帕内玛。将有症状的叶子的小碎片在70%乙醇(30秒)和2.5%次氯酸钠(1分钟)中消毒,在无菌蒸馏水中冲洗,并涂布在含有四环素(0.05g/升)的PDA培养皿上。将平板在28±2°C的生物需氧量培养箱(BOD)中保持3天,在12小时光周期下。11个代表性真菌菌落,类似于炭疽菌。选择并在PDA上单生生长7天用于形态学,致病性,和分子分析。光落呈粉红色-深棕色,中心有针状突起和棉质菌丝,在一些分离物中显示黑色边缘,类似于C.plurivorum(Zhang等人。2023年)。那些殖民地的分生孢子是透明的,两端呈钝角圆柱形,和17.76x7.06μm,n=50。三个菌落发育出粉红色灰色菌丝体,带有许多黑色微菌核,分生孢子是透明的,镰刀状,和27.38x4.10μm,n=50,类似于C.truncum(Yu等人。2023年)。提取十一个分离株的总DNA,和内部转录空间(ITS),甘油醛3-磷酸脱氢酶(GAPDH),肌动蛋白(ACT),β-微管蛋白(TUB),和几丁质合酶1(CHS-1)区域通过PCR部分扩增。对扩增子进行测序并保存至Genbank(表eXtra1)。在软件MEGA(版本11.0.10)(Tamuraetal。2021)。分离株CML5,CML8,CML9,CML10,CML11,CML14,CML15和CML25与多炭疽病CBS125474(兰花复合体)分组,和分离CML26,CML27和CML28与炭疽病CBS15:35(芦竹复合体),87%和97%的Bootstrap支持,分别。在访问的四个农场中检测到了多食梭菌(我们每个农场选择了两个代表性的分离株),而C.truncum分离株都来自AfonsoBezerra市的农场。按照Baisuan等人的方法进行致病性测试。(2023),在甜瓜幼苗\“GoldexYellow\”的叶子上进行微伤害,并用菌落的孢子悬浮液接种,每种分离物生长7天(106孢子/mL),并喷洒至滴落点。无菌水用作模拟物。九天后,炭疽病症状与在田间观察到的相似,在所有接种的叶子中都可以看到,而在模拟植物的叶子中没有观察到症状。重新分离病原体,并通过形态学和测序确认其鉴定。每个分离株接种五株幼苗并模拟,重复试验,并观察到相同的结果。已经报道了C.plurivorum在巴西的黄瓜中引起疾病(Silva等人。2023年)和中国枸杞的多利伏香梭菌和元旦梭菌(郭等人。2022年)。据我们所知,这是在巴西甜瓜植物中引起炭疽病的第一个报告。
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), β-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.