First report of Sclerotinia sclerotiorum associated with olive fruit rot in Italy

Picholine in an orchard of 2 ha located in the countryside of the city of Monopoli (southern Italy). Symptomatic olives represented ∼10% of drupes on each tree, exhibiting dehydrated and sunken areas but without observable mycelium. Symptoms were not ascribable to any known causal agent of olive rots such as Colletotrichum or Fusarium spp. (Schena et al. 2011) and drupes were not over-ripe or damaged by insects. Affected drupes were surface-disinfected by dipping for 2 min in a hypochlorite solution (40 g/liter) and marginal rotted tissues were seeded onto potato dextrose agar (PDA) amended with ampicillin (0.25 mg/liter) and streptomycin (0.25 mg/liter). Plates were incubated in the dark for 4 days at 23°C and individual colonies were transferred to new plates of PDA to obtain pure cultures. After 14 days of incubation, white mycelium and irregularly shaped black sclerotia, measuring 1.9 to 4.7 mm in length and 1.6 to 3.5 mm in diameter, were produced. Five representative isolates were identified as Sclerotinia sclerotiorum (Lib.) de Bary based on morphology of the colony, sclerotia, and microscopic observations. Furthermore, the ITS region of the rDNA was amplified and sequenced using the universal primers ITS5 and ITS4. All isolates showed identical ITS sequences and a representative sequence was deposited in GenBank (accession no. KX424963). The BLAST search on GenBank and on the Fungal Barcoding Database (http://www.fungalbarcoding.org) of this sequence resulted in a 100% match with several sequences of S. sclerotiorum with a query cover also at 100% (e.g., KP340898, JN012606, and HM769812), enabling discrimination from other fungal species. To confirm Koch’s postulates, and as healthy olive drupes of the cv. Frantoio were not available anymore when the putative pathogen was isolated and identified, 20 healthy olive fruit from both cvs. Leccino and Picholine were surface sterilized as described above, washed twice with sterile distilled water, air-dried, and wounded in the equatorial zone with a pin (0.5 mm in diameter). Olives were inoculated by placing a PDA agar plug (5 mm in diameter) of actively growing mycelium from one of the isolates on each wound. A sterile agar plug was placed on the same number of olive drupes as a control. After 7 days of incubation at 20°C in a moist chamber, rot symptoms were observed on 75 and 60% of the inoculated fruit of cvs. Leccino and Picholine, respectively. The pathogen was consistently reisolated from inoculated fruit and reidentified morphologically and molecularly as S. sclerotiorum, as described above. Control drupes remained symptomless and the pathogen was not recovered. Olive is one of the most important crops in Italy, the second largest producer in the world (FAOSTAT 2016). S. sclerotiorum is a necrotrophic fungal pathogen with a wide host range comprising many economically important plant species (Saharan and Mehta 2008). It is controlled mainly by using fungicides and crop rotation for annual crops, but biological control strategies have also been reported (Bolton et al. 2006). To our knowledge, this is the first report of S. sclerotiorum on olive fruit worldwide.