Li Yang, Ma Xiao, Gai Wenxian, Xiao Luodan, Gong Zhenhui
Northwest A&F University, 12469, College of Horticulture, Yangling, China;
Institute of Fruit, Vegetable, Tea and Flower, Yibin Academy of Agricultural Sciences, Yibin 644600, P. R. China, Yibin, China;
Plant Dis. 2021 Mar 23. doi: 10.1094/PDIS-01-21-0123-PDN.
Pepper (Capsicum annuum L.) is an important solanaceous vegetable crop, with high nutritional and economic value. However, it is susceptible to Colletotrichum spp. infection during its growth and development, which seriously affects production yield and quality. Chili anthracnose, caused by Colletotrichum spp., is one of the most destructive diseases of pepper. In August 2020, chili anthracnose was observed with widespread distribution in the horticulture field of Northwest A&F University (34.16° N, 108.04° E) in Shaanxi Province, China. Approximately 60% of the pepper plants had disease symptoms typical of anthracnose. Lesions on pepper fruits were dark, circular, sunken, and necrotic, with the presence of orange to pink conidial masses (Figure S1A). To perform fungal isolation, the tissue at the lesion margin was cut from eight symptomatic fruits, surface disinfested with 75% ethanol for 30 s, and 2% NaClO for 1 min, then rinsed three times with sterile distilled water and dried on sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 28 ºC in the dark. After 3 days, hyphae growing from tissue of each lesion were recultured on PDA (Liu et al. 2016). A representative single-spore isolate (NWAFU2) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. NWAFU2 colonies had gray-white aerial mycelium, and the reverse side of the colonies was dark gray to light yellow after 10-days growth on PDA (Figure S1B-C). Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, and measured 10.1 to 16.9 (length) × 4.7 to 7.0 (width) μm (n=50) (Figure S1D). Based on morphological features, the isolate was consistent with the description of C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted using a CTAB method and the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial sequences of actin (ACT) genes were amplified and sequenced using primers ITS1F/ITS4, GDF1/GDR1 and ACT-512F/ACT-783R, respectively (Dowling et al. 2020). Using the BLAST, ITS, ACT, GAPDH gene sequences (GenBank accession nos. MW258690, MW258691 and MW258692, respectively) were 100%, 100% and 98.19% identical to ZJL-4 of C. gloeosporioides (GenBank accession nos. MN075757, MN058142 and MN075666, respectively). Phylogenetic analysis was conducted using MEGA-X (Version 10.0) based on the concatenated sequences of published ITS, ACT and GAPDH for Colletotrichum species using Neighbor-Joining algorithm. The identified isolate (NWAFU2) was closely related to C. gloeosporioides (Figure S2). To confirm the pathogenicity, ten healthy pepper fruits were surface-sterilized and 2 μL of conidial suspension (1×106 conidia/mL) was injected the surface of pepper. Five fruits were inoculated with 2μL sterile distilled water as controls. After inoculation, the fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. Anthracnose symptoms similar to those observed in the field, were observed 7 days after inoculation (Figure S1F) and control fruits remained healthy. A similarly inoculated detached leaf assay resulted in water-soaked lesions 3 days after inoculation. C. gloeosporioides was reisolated from the infected pepper fruits, fulfilling Koch's postulates. C. gloeosporioides has been reported to cause chili anthracnose in Sichuan Province, China (de Silva et al. 2019; Liu et al. 2016). However, Shaanxi is one of the main pepper producing areas in china and it is geographically distinct from Sichuan; its climate and environmental conditions are different from Sichuan. Knowledge that C. gloeosporioides causes chili anthracnose of pepper in Shaanxi province, China may aid in the selection of appropriate management tactics for this disease. Reference: de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301-2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180.
辣椒（Capsicum annuum L.）是一种重要的茄科蔬菜作物，具有很高的营养价值和经济价值。然而，它在生长发育过程中易受炭疽菌属（Colletotrichum spp.）感染，这严重影响了产量和品质。由炭疽菌属引起的辣椒炭疽病是辣椒最具毁灭性的病害之一。2020年8月，在中国陕西省西北农林科技大学（北纬34.16°，东经108.04°）的园艺场观察到辣椒炭疽病广泛分布。约60%的辣椒植株出现典型的炭疽病症状。辣椒果实上的病斑呈黑色、圆形、凹陷且坏死，有橙色至粉红色的分生孢子团（图S1A）。为进行真菌分离，从8个有症状的果实上切取病斑边缘组织，用75%乙醇表面消毒30秒，再用2%次氯酸钠消毒1分钟，然后用无菌蒸馏水冲洗3次，并在无菌滤纸上干燥。将组织置于马铃薯葡萄糖琼脂（PDA）上，于28℃黑暗中培养。3天后，将每个病斑组织上长出的菌丝在PDA上再次培养（Liu等人，2016）。一个具有代表性的单孢分离株（NWAFU2）用于形态学特征分析、分子分析、系统发育分析和致病性测试。NWAFU2菌落有灰白色气生菌丝，在PDA上生长10天后，菌落背面为深灰色至浅黄色（图S1B - C）。分生孢子呈圆柱形，无隔膜，两端钝圆至稍圆，大小为10.1至16.9（长）×4.7至7.0（宽）μm（n = 50）（图S1D）。根据形态特征，该分离株与胶孢炭疽菌复合种（C. gloeosporioides species complex）的描述一致（Weir等人，2012）。为进行分子鉴定，采用CTAB法提取基因组DNA，并分别使用引物ITS1F/ITS4、GDF1/GDR1和ACT - 512F/ACT - 783R扩增和测序内转录间隔区（ITS）、甘油醛 - 3 - 磷酸脱氢酶（GAPDH）和肌动蛋白（ACT）基因的部分序列（Dowling等人，2020）。使用BLAST分析，ITS、ACT、GAPDH基因序列（GenBank登录号分别为MW258690、MW258691和MW258692）与胶孢炭疽菌的ZJL - 4（GenBank登录号分别为MN075757、MN058142和MN075666）的序列一致性分别为100%、100%和98.19%。基于已发表的炭疽菌属ITS、ACT和GAPDH的串联序列，使用邻接法，通过MEGA - X（版本10.0）进行系统发育分析。鉴定出的分离株（NWAFU2）与胶孢炭疽菌密切相关（图S2）。为确认致病性，对10个健康辣椒果实进行表面消毒，向辣椒表面注射2μL分生孢子悬浮液（1×106个分生孢子/mL）。5个果实接种2μL无菌蒸馏水作为对照。接种后，将果实置于28℃黑暗的保湿箱中。该实验重复3次。接种7天后观察到与田间观察到的类似的炭疽病症状（图S1F），对照果实保持健康。类似接种的离体叶片试验在接种3天后出现水渍状病斑。从感染的辣椒果实中重新分离出胶孢炭疽菌，符合柯赫氏法则。在中国四川省，胶孢炭疽菌已被报道可引起辣椒炭疽病（de Silva等人，2019；Liu等人，2016）。然而，陕西是中国主要的辣椒产区之一，在地理上与四川不同；其气候和环境条件与四川不同。了解胶孢炭疽菌在中国陕西省引起辣椒炭疽病，可能有助于为该病选择合适的管理策略。参考文献：de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301 - 2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115 - 180.
