First Report of Fusarium clavum Causing Leaf Spot and Fruit Rot on Tomato in Italy

During July to October 2019, an unknown leaf spot and fruit rot disease was observed on 90- to 120-day-old Solanum lycopersicum L. plants in plastic tunnels (0.5 ha) in Piedmont, Italy. Small circular, brown leaves spots (1 to 5 mm in diameter), with a well-defined border, surrounded by a yellow halo, were observed on 20 to 35% plants. Lesions expanded up to 30 mm in diameter, becoming necrotic and broken in the center, and eventually dried out. In September, 1 to 5% of fruit showed dry rot (up to 40 mm in diameter) and fruit mummification. Approximately 1-mm2 tissue pieces of 50 symptomatic leaves and 20 fruits were dipped in 1% sodium hypochlorite for 1 min, rinsed in sterilized water, and placed on potato dextrose agar (PDA) with 25 mg/liter of streptomycin sulfate. Fusarium was isolated at 80% frequency from both tissues incubated at 22°C for 5 days. Single-spore cultures of two selected isolates, isolates 8-19.1 and 8-19.3, respectively, from the leaves and fruits, were subcultured on carnation leaf agar (CLA) (Leslie and Summerell 2006). Microconidia, although rare, were single-celled, hyaline, nonseptate, ovoid, and 10.1 to 14.5 × 3.2 to 3.9 µm. Macroconidia were hyaline one to five septate, with dorsiventral curvature, and measured 19.9 to 41.6 × 2.3 to 5.6 (mean 30.4 × 4.0, N = 40) µm. Chlamydospores developed in a 21-day-old culture grown on CLA, either terminally or intercalary, of 6.7 to 10.9 (mean 9.0, N = 40) µm. The DNA extraction was performed (E.Z.N.A. Fungal DNA Mini Kit, Omega Bio-Tek, Darmstadt, Germany). Subsequently, the partial calmodulin gene (cmdA; Carbone and Kohn 1999; Groenewald et al. 2013) and partial RNA polymerase II beta subunit (rpb2; Xia et al. 2019) were amplified and sequenced, producing 579 to 580 bp (cmdA) and 874 to 876 bp (rpb2) (GenBank nos. MT182816 and MT182814 for isolate 8-19.1, and MT182817 and MT182815 for isolate 8-19.3). BLASTn analysis exhibited 100% identity with the reference strains of Fusarium clavum known as FIESC 5 (Villani et al. 2016; Xia et al. 2019) in rpb2 portion (GenBank nos. MN170395 and LN901605) and 99% identity in cmdA portion (GenBank no. MN170328). Both isolates clustered in the same clade as F. clavum following the maximum parsimony algorithm on the concatenated alignment of cmdA and rpb2 sequences (supplementary material). The suspension (1 × 105 conidia/ml) from 15-day-old PDA cultures of both isolates was sprayed onto five 30-day-old plants (cv. Rugantino) covered with plastic bags for 7 days in a greenhouse at 20 to 26°C. Fruits were surface disinfected, wounded with a 5-mm borer (four fruits, five wounds/fruit), and inoculated (104 conidia/ml of both isolates). Negative control leaves and fruits were treated with sterilized distilled water. Inoculated and negative control fruits were kept in a chamber at 22°C for 7 days. Both tests were repeated. Symptoms were observed 8 to 10 days after inoculation of leaves and 5 days after inoculation of fruit and progressed as observed in the field. All water-inoculated controls remained asymptomatic. Fusarium colonies were recovered from the inoculated leaves and wounded fruits. F. equiseti was found to cause tomato crown and root rot (Yezli et al. 2019) and fruit rot (Khokhar et al. 2019). This is the first report of F. clavum, a member of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019), causing fruit rot and leaf spot on tomato in Italy. The new disease poses a threat to tomato cultivation in Italy and needs to be properly monitored and managed to prevent severe yield loss.