Caires N P, Pinho D B, Souza J S C, Silva M A, Lisboa D O, Pereira O L, Furtado G Q
Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570-900, Minas Gerais, Brazil.
Plant Dis. 2014 Oct;98(10):1437. doi: 10.1094/PDIS-04-14-0426-PDN.
Anthracnose is major disease of pepper (Capsicum annum) in the tropics and causes severe damage both in the field and postharvest. In Brazil, this disease is caused by Colletotrichum acutatum, C. boninense, C. capsici, C. coccodes, and C. gloeosporioides, where the first species is responsible for 70% of all occurrences (3). Recently, C. acutatum has been considered a species complex (1); thus, the aim of this study was to verify the etiology of anthracnose on peppers using a morphological and molecular approaches. In 2011, pepper fruits with typical symptoms of anthracnose (dark, sunken spots with concentric rings of orange conidial masses) were collected in Viçosa, Minas Gerais, Brazil. A single spore isolate was obtained on potato dextrose agar (PDA), and the derived culture was deposited in the Coleção de Culturas de Fungos Fitopatogênicos "Prof. Maria Menezes" (code CMM-4200). The upper side colonies on PDA were gray, cotton-like, and pale gray to pale orange. Conidia were hyaline, aseptate, smooth, straight, cylindrical with round ends or occasionally with end ± acute, 12.5 to 17 μm long and 3.5 to 4 μm wide on synthetic nutrient deficient agar. The isolate was morphologically typical of species belonging to the C. acutatum complex. Molecular identification of the pathogen was carried out and sequences of the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (βt) were obtained and deposited in GenBank (Accession Nos. KJ541821 to KJ541823). A search in the Q-bank fungi database using the ITS, βt, and GAPDH sequences retrieved C. scovillei with 100% identity for all three genes. This pathogen was previously reported in Capsicum spp. only in Thailand, Indonesia, and Japan (1,2). To confirm pathogenicity, drops with 10 spores/ml were deposited in 10 artificially wounded fruits (cv. Itapuã 501 and Melina). In control fruits, drops of sterilized water were deposited onto wounds. The fruits were covered for one day with a transparent plastic bag with moisture supplied by a wet filter paper. The fruits were detached and mature. The bags were removed, and the fruits were incubated for 10 days in a growth chamber at 25°C with a photoperiod of 12 h. After 4 days, gray-brown to black sunken spots with concentric rings were observed on 100% of the wounded fruits that had been inoculated. No disease was observed on the control fruits. The fungus C. scovillei was successfully re-isolated from symptomatic fruits to fulfill Koch's postulates. To our knowledge, this is the first report of anthracnose on pepper fruit caused by C. scovillei in Brazil. Due to the diversity of species that cause anthracnose in Capsicum, future studies using morphological and molecular tools are essential for the correct identification of Colletotrichum spp. on pepper in Brazil. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) T. Kanto et al. J. Gen. Plant. Pathol. 80:73, 2014. (3) M. J. Z. Pereira et al. Hortic. Bras. 29:569, 2011.
炭疽病是热带地区辣椒(辣椒属)的主要病害,在田间和收获后都会造成严重损害。在巴西,这种病害由尖孢炭疽菌、香蕉炭疽菌、辣椒炭疽菌、薯蓣炭疽菌和胶孢炭疽菌引起,其中第一种病原菌导致了70%的发病情况(3)。最近,尖孢炭疽菌被认为是一个复合种(1);因此,本研究的目的是采用形态学和分子方法来验证辣椒炭疽病的病因。2011年,在巴西米纳斯吉拉斯州维索萨收集了具有典型炭疽病症状(深色、凹陷斑点,带有橙色分生孢子团的同心环)的辣椒果实。在马铃薯葡萄糖琼脂(PDA)上获得了单孢分离物,并将得到的培养物保藏于植物病原真菌培养物保藏中心“玛丽亚·梅内塞斯教授”(编号CMM - 4200)。PDA上的正面菌落呈灰色、棉絮状,浅灰色至浅橙色。在合成营养缺陷型琼脂上,分生孢子透明、无隔膜、表面光滑、直、圆柱形,两端圆形,偶尔一端 ± 尖锐,长12.5至17μm,宽3.5至4μm。该分离物在形态上是尖孢炭疽菌复合种的典型种。对病原菌进行了分子鉴定,获得了内部转录间隔区(ITS)、甘油醛 - 3 - 磷酸脱氢酶(GAPDH)和β - 微管蛋白(βt)区域的序列,并保藏于GenBank(登录号KJ541821至KJ541823)。使用ITS、βt和GAPDH序列在Q - bank真菌数据库中搜索,检索到斯氏炭疽菌,这三个基因的序列一致性均为100%。这种病原菌此前仅在泰国、印度尼西亚和日本的辣椒属植物中报道过(1,2)。为了确认致病性,将含10个孢子/ml的菌悬液滴在10个人工创伤的果实(品种Itapuã 501和Melina)上。在对照果实的伤口上滴加无菌水。果实采下且已成熟,用透明塑料袋覆盖一天,袋内通过湿滤纸提供湿度。去除塑料袋后,将果实置于生长箱中,在25°C、光周期12小时的条件下培养10天。4天后,在接种处理过且有创伤的果实上100%观察到了灰褐色至黑色的凹陷斑点,带有同心环。对照果实未观察到病害。从有症状的果实中成功重新分离出斯氏炭疽菌,从而完成了柯赫氏法则验证。据我们所知,这是巴西首次报道由斯氏炭疽菌引起的辣椒果实炭疽病。由于引起辣椒炭疽病的物种具有多样性,未来使用形态学和分子工具进行研究对于正确鉴定巴西辣椒上的炭疽菌属物种至关重要。参考文献:(1)U. Damm等人,《真菌学研究》73:37,2012年。(2)T. Kanto等人,《植物病理学报》80:73,2014年。(3)M.J.Z Pereira等人,《巴西园艺》29:569,2011年。