Italy is the largest tomato (Solanum lycopersicum)-producing country in Europe with a cultivated area of 97,092 ha and a production of 5,798,103 tons/year in 2018 (FAOSTAT, 2020). During July 2020, a postharvest rot occurred in fresh tomatoes ‘Piccadilly’ cultivated in Sicily (Pachino, RG) and commercialized in Northern Italy (Torino, TO). Affected fruit showed circular black rot on the blossom end. The rot had an average incidence of 7% of the fruits, in three batches of 100 tomatoes each. Isolation was carried out by cutting pieces of symptomatic rotten fruits. The fragments were surface-disinfected with 1% sodium hypochlorite for 30 s, rinsed in sterile water and air-dried. Five fragments were cut and plated onto Potato Dextrose Agar (PDA) supplemented with streptomycin, and incubated at 24±1°C in the dark for 5 days. Representative colonies were transferred onto PCA and morphological observations were performed as described by Woudenberg et al. (2017) after 7 and 14 days. Colonies were olive-green, flat with regular margins, while conidia were mid to deep brown, solitary, ovoid or ellipsoid (17.39 µm ± 2.04 × 10.59 ± 3.30 µm) with transverse and longitudinal septa. Based on morphological observations the isolates were identified as Stemphylium eturmiunum (Simmons, 2001). Species identification was confirmed by sequencing rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990), cmdA gene region using primers CALDF1/CALDR2 (Lawrence et al. 2013) and gapdh gene region with primers gpd1/gpd2 (Berbee et al. 1999). Six amplified sequences per region (ANos. from MW158387 to MW158398 and from MW159746 to MW159751) were BLAST-searched in GenBank, obtaining >99 % identity with ex-type strain of S. eturmiunum strain CBS 109845 (AN° KU850541) for ITS, and 100% identity (ANos. KU850831 and KU850689) for cmdA and gapdh, respectively. To confirm the species, DNA sequences were aligned with CLUSTAL W with closely related species of Stemphylium reported in the last revision of the genus (Woudenberg et al., 2017), and a phylogenetic analysis with the Neighbor Joining method based on Tamura Nei model + Gamma distribution (bootstrap 1,000) was performed. The phylogenetic tree confirmed the identity of the isolates as S. eturmiunum (Suppl. Fig. 1). To fulfil Koch’s postulates, pathogenicity tests were conducted on S. lycopersicum cv. Piccadilly fruits. Tomatoes were surface sterilized with 1% sodium hypochlorite and air-dried. Fruits (5 fruits per isolates) were wounded (two injuries of 3 mm each) and inoculated with a spore suspension of 1x105 cell/mL obtained from 15 days-old PCA cultures, as in Spadoni et al. (2020. Negative controls were wounded and inoculated with sterile deionized water. Symptoms occurred on all fruits inoculated after 12 days at 24±1°C and S. eturmiunum was re-isolated from inoculated fruits on PCA (Suppl. Fig. 2), control remained symptomless. Re-isolated colonies were molecularly identified as S. eturmiunum. In Italy a different species, S. vesicarium, was reported on tomato (Porta-Puglia, 1981), while S. eturmiunum was described as a postharvest pathogen of tomato in China, Greece, New Zealand and the United States (Woudenberg et al., 2017; Vaghefi et al., 2020), and from fruits commercialized in Danish and Spanish markets (Andersen and Frisvad, 2004). To the best of our knowledge, this is the first report of S. eturmiunum causing postharvest rot on tomato in Italy. The occurrence of this pathogen further stresses the importance of careful handling to prevent fruit crackings and of preharvest control strategies.
Molecular screening of Bartonella in free-ranging capybaras (Hydrochoerus hydrochaeris) from Paraná State, Southern Brazil