First report of leaf spot caused by Colletotrichum kahawae on cultivated rocket (Eruca sativa) in Italy

Eruca sativa common name cultivated rocket, is an annual species in the Brassicaceae, grown for fresh consumption. In fall 2014, in a commercial plastic-house in northern Italy, where the crop was grown for 7 years in the same soil, a new leaf spot was observed on rocket plants ('Coltivata') 7 to 35 days after sowing. The incidence ranged from 5 to 15% in the more humid area of the tunnel, with 5 to 75% of the affected leaf area showing white spots. Symptoms first appeared on the tip or edge of leaf as pale white or cream-colored circular spots 5 to 50 mm in diameter. Spots had thin dark brown or black borders. They enlarged, with round or irregular shapes, to form extensive dead areas. The disease developed at 20 to 28°C and 75 to 90% relative humidity. Isolations were carried out from affected leaf tissues (1 mm2) dipped in 1% sodium hypochlorite for 1 min, then rinsed in sterilized water, and placed onto PDA amended with 25 mg/liter of streptomycin sulfate. A Colletotrichum sp. (Bailey and Jeger 1992) was consistently recovered. Hyaline, cylindrical, aseptate, and thin-walled conidia (9.3 to 16.5 × 3.4 to 6.3 μm, avg. 14.4 × 4.7 μm; n = 41) were produced abundantly in acervuli (79.0 to 91.5 μm; n = 15) in gray mycelium. Genomic DNA was extracted from 10 mg of fresh mycelium collected from PDA plates of one representative, single-conidium isolate. The ITS1-5.8S-ITS2 region of ribosomal DNA (rDNA) and a fragment of the beta-tubulin 2 gene (TUB2) between exons 2 and 6 were amplified using 200 ng of genomic DNA as template. Amplicons were analyzed by electrophoresis, purified (ExoSAP-IT), and sequenced in both directions. BLASTn sequence analysis was performed (Morgulis et al. 2008). The 489-and 600-bp fragments of ITS rDNA (GenBank Accession No. KT259854) and TUB2 (KT259853), respectively, showed 100% similarity with ITS and TUB2 sequences of C. kahawae (JN715847.1 and KC425710.1, respectively). Pathogenicity tests were performed on healthy, 25-day-old Coltivata plants by spraying leaves with a conidial and mycelium suspension from PDA plates of one isolate of the pathogen, adjusted at 1 × 105 conidia/ml. Control plants were sprayed with sterilized water. Fifteen plants/treatment were used. The plants were covered with plastic bags for 5 days, and kept in a growth chamber at 25°C under white fluorescent lamps (12-h photoperiod). Spots similar to those on the plants with natural infections were observed 7 to 10 days after inoculation; control plants remained healthy. A Colletotrichum sp. similar to C. kahawae was reisolated consistently from inoculated plants; no fungal colonies were obtained from control plants. C. kahawae has been reported in Italy on olive (Schena et al. 2014). This is, to our knowledge, the first report of C. kahawae on E sativa in Italy and worldwide. Due to the wide host range of C. kahawae and the economic value of cultivated rocket, this disease could be a threat for this crop In other production areas In Italy.