First Report of Peronospora sparsa Causing Downy Mildew (Dryberry) of Rubus fruticosus in Mexico.

Abstract

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.