First Report of Leaf Spot of Rocket (Eruca sativa) Caused by Fusarium equiseti in Italy

Abstract

During the summer of 2010, rocket (Eruca sativa) plants grown in an open field and under a plastic tunnel in Piedmont (northern Italy) showed symptoms of a previously reported foliar disease. Symptoms were observed on 30-day-old plants and consisted of small, circular, brown leaf spots (1 to 3 to 10 to 12 mm in diameter), sometimes later becoming elliptical. Necrotic lesions were cracked in the center and showed a well-defined border, frequently surrounded by a violet-brown halo. Approximately 40% of the plants were affected with 30 to 40% of the leaves infected. An orange-brown colony with characteristics of Fusarium was isolated from leaf tissues of 30 infected plants on potato dextrose agar (PDA). Isolates were purified, subcultured on PDA, and single-spore cultures were obtained. On PDA, they produced orange-brown colonies and purple pigments. On Spezieller Nährstoffarmer agar (SNA) (1), the isolates produced hyaline macroconidia with dorsiventral curvature, five to seven septate, and measuring 36.2 to 49.3 × 3.4 to 5.3 (average 41.9 × 4.0) μm. Chlamydospores, solitary but also in short chains (two to three elements), measuring 7.2 to 15.3 (average 10.1) μm were produced on carnation leaf agar (CLA) after 10 days and became verrucose 20 days later. Macroconidia were produced on CLA in orange sporodochia from monophialides on branched conidiophores. Microconidia were not observed. Such characteristics are typical of the genus Fusarium (1). The rDNA ITS region (internal transcribed spacer) was amplified using the primers ITS1/ITS4 (2) and sequenced. BLASTn analysis of the 480-bp product obtained showed an E-value of 0.0 with Fusarium equiseti. The nucleotide sequence has been assigned the GenBank Accession No. JF460797. The translation elongation factor-1α (EF-1α) gene (GenBank Accession No. JN127347) was amplified using primers EF-1/EF-2 and sequenced. The 702-bp fragment showed 99% identity with F. equiseti (GenBank Accession No. FJ939673.1). To confirm pathogenicity, 20-day-old rocket plants were transplanted into 2-liter volume pots, filled with a steamed peat/perlite/sand (60:20:20 vol/vol) substrate and maintained in a growth chamber at 25 ± 1°C. Five pots per treatment were used, each pot containing two plants. The artificial inoculation was carried out either by spraying leaves with a spore suspension prepared from 15-day-old cultures of the pathogen on PDA or by applying CLA agar disks (6 mm in diameter) from 10-day-old cultures onto leaves. Control plants were inoculated with distilled water or with noninoculated agar disks. Plants were covered with plastic bags for 5 days. The first symptoms, consisting of chlorotic leaf halo and leaf spots surrounded by a violet-brown halo, developed 15 days after inoculation by foliar spraying and 5 days after inoculation by disks. Noninoculated plants remained healthy. F. equiseti was consistently isolated from symptomatic plants. The pathogenicity test was conducted twice. To our knowledge, this is the first report of F. equiseti on E. sativa in Italy. Currently, this disease is present in several farms in northern Italy. Its importance might increase because of the widespread cultivation of cultivated rocket in Italy. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.