Nasehi A, Kadir J B, Abidin M A Zainal, Wong M Y, Ashtiani F Abed
Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.
Plant Dis. 2012 Aug;96(8):1227. doi: 10.1094/PDIS-03-12-0262-PDN.
Symptoms of gray leaf spot were first observed in June 2011 on pepper (Capsicum annuum) plants cultivated in the Cameron Highlands and Johor State, the two main regions of pepper production in Malaysia (about 1,000 ha). Disease incidence exceeded 70% in severely infected fields and greenhouses. Symptoms initially appeared as tiny (average 1.3 mm in diameter), round, orange-brown spots on the leaves, with the center of each spot turning gray to white as the disease developed, and the margin of each spot remaining dark brown. A fungus was isolated consistently from the lesions using sections of symptomatic leaf tissue surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto PDA and V8 agar media (3). After 7 days, the fungal colonies were gray, dematiaceous conidia had formed at the end of long conidiophores (19.2 to 33.6 × 12.0 to 21.6 μm), and the conidia typically had two to six transverse and one to four longitudinal septa. Fifteen isolates were identified as Stemphylium solani on the basis of morphological criteria described by Kim et al. (3). The universal primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region (ITS1, 5.8, and ITS2) of ribosomal DNA (rDNA) of a representative isolate (2). A 570 bp fragment was amplified, purified, sequenced, and identified as S. solani using a BLAST search with 100% identity to the published ITS sequence of an S. solani isolate in GenBank (1). The sequence was deposited in GenBank (Accession No. JQ736024). Pathogenicity of the fungal isolate was tested by inoculating healthy pepper leaves of cv. 152177-A. A 20-μl drop of conidial suspension (10 spores/ml) was used to inoculate each of four detached, 45-day-old pepper leaves placed on moist filter papers in petri dishes (4). Four control leaves were inoculated similarly with sterilized, distilled water. The leaves were incubated at 25°C at 95% relative humidity for 7 days. Gray leaf spot symptoms similar to those observed on the original pepper plants began to develop on leaves inoculated with the fungus after 3 days, and S. solani was consistently reisolated from the leaves. Control leaves did not develop symptoms and the fungus was not reisolated from these leaves. Pathogenicity testing was repeated with the same results. To our knowledge, this is the first report of S. solani causing gray leaf spot on pepper in Malaysia. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. P. S. Camara et al. Mycologia 94:660, 2002. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.
2011年6月，在马来西亚主要辣椒产区（约1000公顷）的金马仑高原和柔佛州种植的辣椒（辣椒属）植株上首次观察到灰斑病症状。在感染严重的田地和温室中，发病率超过70%。症状最初表现为叶片上微小（平均直径1.3毫米）、圆形、橙褐色斑点，随着病情发展，每个斑点的中心变为灰色至白色，每个斑点的边缘仍为深褐色。使用在1%次氯酸钠中表面消毒2分钟的有症状叶片组织切片，冲洗、干燥后接种到PDA和V8琼脂培养基上，从病斑中持续分离出一种真菌（3）。7天后，真菌菌落呈灰色，在长分生孢子梗末端形成暗褐色分生孢子（19.2至33.6×12.0至21.6微米），分生孢子通常有两到六个横向隔膜和一到四个纵向隔膜。根据Kim等人描述（3）的形态学标准，15个分离株被鉴定为茄链格孢。使用通用引物ITS5和ITS4扩增代表性分离株核糖体DNA（rDNA）内部转录间隔区（ITS1、5.8和ITS2）（2）。扩增出一个570bp片段，纯化、测序，通过BLAST搜索鉴定为茄链格孢，与GenBank中已发表的茄链格孢分离株ITS序列100%同源（1）。该序列已存入GenBank（登录号JQ736024）。通过接种cv. 152177 - A健康辣椒叶片来测试真菌分离株的致病性。用20微升分生孢子悬浮液（10个孢子/毫升）接种四个置于培养皿湿滤纸上的45日龄离体辣椒叶片（4）。四个对照叶片用灭菌蒸馏水同样接种。叶片在25°C、95%相对湿度下培养7天。接种真菌后3天，接种叶片开始出现与原辣椒植株上观察到的类似的灰斑病症状，并且从叶片中持续重新分离出茄链格孢。对照叶片未出现症状，且未从这些叶片中重新分离出真菌。致病性测试重复进行，结果相同。据我们所知，这是茄链格孢在马来西亚引起辣椒灰斑病的首次报道。参考文献：（1）S. F. Altschul等人，《核酸研究》25:3389，1997年。（2）M. P. S. Camara等人，《真菌学》94:660，2002年。（3）B. S. Kim等人，《植物病理学杂志》15:348，1999年。（4）B. M. Pryor和T. J. Michailides，《植物病理学》92:406，2002年。
