Rebollar-Alviter A, Silva-Rojas H V, Zelaya-Molina L X, Ellis M A
Universidad Autónoma Chapingo, Centro Regional Morelia, Morelia, Michoacán, 58170, México.
Colegio de Postgraduados, Programa de Semillas, Campus Montecillo, Texcoco, Edo. de México, 56230, México.
Plant Dis. 2009 Jun;93(6):674. doi: 10.1094/PDIS-93-6-0674B.
Michoacan State is the largest producer of blackberries (Rubus fruticosus) in Mexico with more than 4,000 ha in production. During the rainy season of 2007 (July to September), purple, angular, vein-delimited leaf spots along the midrib and major veins were observed. Affected young fruit lost their shine, became shriveled, and later dried. Some fruit split. Symptomatic leaflets from cv. Tupy were collected from the field in Tangancicuaro and Los Reyes counties. In the laboratory, 20 detached leaflets were washed with 10% bleach for 2 min, rinsed with sterile distilled water, and placed into sterile petri dishes containing 0.5% water agar. To promote sporulation of fungi, leaflets were incubated at 17°C with a 12-h photoperiod in a growth chamber. Sporangia and sporangiophores, which developed 20 days later on the underside of the leaves, were transferred to the underside of detached healthy leaves of the same cultivar with a sterile needle, and incubated as previously described. A set of noninoculated leaves were included as controls. Sporangiophores and sporangia developed on the underside of angular purple lesions on leaves 15 to 22 days after inoculation. Symptoms were identical to those observed on leaves in the field. The pathogenicity test was repeated twice with the same results. Sporangia were light brown, ovoid to elliptical, and measured 14 to 22 × 11 to 18 μm. Sporangiophores were dichotomously branched with slender curved ends. Symptoms on the leaves and fruit and oomycete morphology were similar to those described for downy mildew (2). To confirm pathogen identity, a product of ~500 bp of the nuclear ITS-rRNA was amplified from total DNA from symptomatic and asymptomatic leaves and fruit by nested PCR. The primers sets PS3/PS1 and PR3/PR4 were used for the first and second reaction, respectively (1,3). PCR products were sequenced in both directions and sequences were deposited in GenBank under Accession Nos. EU601168, EU601169, EU601170, and EU601171. Consensus sequences obtained in this study were compared with the same region of Peronospora sparsa (GenBank Accession Nos. EU391654 from Denmark and AF266783 from the UK). Similarity among these sequences varied between 99 and 100%. To our knowledge, this is the first report of downy mildew (dryberry) of blackberry caused by P. sparsa in Mexico. References: (1) B. J. Aegerter et al. Plant Dis. 86:1363, 2002. (2) W. D. Gubler. Page 15 in: Compendium of Raspberry and Blackberry Diseases and Insects. M. A. Ellis et al., eds. The American Phytopathological Society, St Paul MN, 1998. (3) H. Lindqvist et al. Plant Dis. 82:1304, 1998.
米却肯州是墨西哥最大的黑莓（悬钩子属）生产地，种植面积超过4000公顷。在2007年雨季（7月至9月）期间，沿着中脉和主要叶脉观察到紫色、有角、脉限清晰的叶斑。受影响的幼果失去光泽，变得干瘪，随后干枯。一些果实开裂。从坦甘西夸罗和洛斯雷耶斯县的田间采集了来自“图皮”品种的有症状小叶。在实验室中，将20片离体小叶用10%漂白剂洗涤2分钟，用无菌蒸馏水冲洗，然后放入含有0.5%水琼脂的无菌培养皿中。为促进真菌产孢，将小叶在生长室中于17°C、12小时光周期下培养。20天后在叶片下表面形成的孢子囊和孢囊梗，用无菌针转移到同一品种的离体健康叶片下表面，并按前述方法培养。设置一组未接种的叶片作为对照。接种后15至22天，在叶片有角的紫色病斑下表面形成孢囊梗和孢子囊。症状与在田间叶片上观察到的相同。致病性测试重复了两次，结果相同。孢子囊浅褐色，卵形至椭圆形，大小为14至22×11至18微米。孢囊梗二叉分枝，末端细长弯曲。叶片和果实上的症状以及卵菌形态与霜霉病（2）描述的相似。为确认病原菌身份，通过巢式PCR从有症状和无症状的叶片及果实的总DNA中扩增出约500 bp的核糖体DNA内转录间隔区（ITS-rRNA）产物。引物对PS3/PS1和PR3/PR4分别用于第一轮和第二轮反应（1,3）。PCR产物进行双向测序，序列存入GenBank，登录号为EU601168、EU601169、EU601170和EU601171。将本研究获得的一致序列与稀疏霜霉（Peronospora sparsa）的相同区域（来自丹麦的GenBank登录号EU391654和来自英国的AF266783）进行比较。这些序列之间的相似性在99%至100%之间。据我们所知，这是墨西哥首次报道由稀疏霜霉引起的黑莓霜霉病（干腐病）。参考文献：（1）B. J. Aegerter等人，《植物病害》86:1363，2002年。（2）W. D. Gubler，载于《树莓和黑莓病害及昆虫防治手册》，M. A. Ellis等人编，美国植物病理学会，明尼苏达州圣保罗，1998年，第15页。（3）H. Lindqvist等人，《植物病害》82:1304，1998年。
