First Report of Leaf Spot of Wild (Diplotaxis tenuifolia) and Cultivated (Eruca vesicaria) Rocket Caused by Alternaria japonica in Italy

Abstract

Wild (Diplotaxis tenuifolia) and cultivated (Eruca vesicaria) rocket, popular crops in Italy as well as in many Mediterranean areas, are grown for fresh consumption as well as for dish decoration. During fall and winter of 2010 to 2011, extensive necroses were observed on leaves of D. tenuifolia and E. vesicaria that were grown in commercial greenhouses in Piedmont and Liguria (northern Italy). The disease affected 30 to 40% of 60-day-old plants. First symptoms were usually black-brown lesions, 1 to 30 mm in diameter, which progressively turned black. Lesions usually started on the upper side of older leaves at the leaf margins and tips and developed a yellow halo. Eventually, lesions also affected leaf veins and stems. A fungus was consistently isolated from infected leaves on potato dextrose agar and was grown on water agar (15 g/liter) amended with autoclaved rocket tissues (100 g/liter). After 12 days of growth at 22°C and 12-h dark/12-h light, conidia that were produced were dark brown, obclavate, obpyriform, ovoid or ellipsoid, with beaks. Round conidia without beaks were also present. Conidia showed two to seven (average three to four) transverse and one to three longitudinal septa, and measured 17.7 to 56.2 (average 30.9) × 6.6 to 17.8 (average 10.8) μm. Conidia were produced singly or in short chains (two to three elements) and mostly presented a conical or cylindrical beak, 1.8 to 7.3 (average 3.6) μm, pale light brown to brown. On the basis of its morphological characteristics, the pathogen was identified as an Alternaria sp. (3). DNA was extracted with Terra PCR Direct Polymerase Mix (Clontech, Mountain View, CA) and PCR was carried out with ITS 1/ ITS 4 primer (4). A 553-bp PCR product was sequenced and a BLASTn search (1) confirmed that the sequence corresponded to Alternaria japonica. The nucleotide sequence has been assigned the GenBank Accession No. JP 742643. Pathogenicity tests were performed by spraying leaves of healthy 30-day-old wild and cultivated rocket plants with an aqueous 1 × 105 spore/ml suspension. The inoculum was obtained from cultures of the fungus grown on sterilized host leaves placed on water agar for 20 days in light/dark at 22 ± 1°C. Plants sprayed only with water served as controls. Three pots (four plants per pot) were used for each treatment. Plants were covered with plastic bags for 4 days after inoculation and maintained in a glasshouse at 22 ± 1°C. Lesions developed on leaves 7 days after inoculation with the spore suspension, whereas control plants remained healthy. A. japonica was consistently reisolated from these lesions. The pathogenicity test was carried out twice. The presence of A. japonica has been reported on several brassica hosts, such as Brassica napus, B. nigra, B. oleracea, and B. rapa (2). This is, to our knowledge, the first report of A. japonica on wild and cultivated rocket in Italy as well as in Europe. Because of the importance of rocket in many countries, the potential impact of this disease is high. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) J. C. David, IMI Description of Fungi and Bacteria. 144:1432, 2000. (3) E. G. Simmons. Alternaria. An Identification Manual. CBS Biodiversity Series 6, Utrecht, The Netherlands, 2007. (4) T. J. White et al. In: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.