First report of Neofusicoccum batangarum as causal agent of scabby cankers of cactus pear (Opuntia ficus-indica) in minor islands of Sicily

Cactus pear (Opuntia ficus-indica, Cactaceae), native to Mexico, is a multipurpose crop. About 90% of Italian production of cactus pear fruit is from Sicily. In 2013, a disease of cactus pear was noticed in minor islands of Sicily, Lampedusa and Linosa (Pelagie archipelago), Favignana (Aegadian archipelago), and Ustica, where cactus pear is grown as living fences. Symptoms were on flattened stems functioning as leaves (cladodes) and included radially expanding cankers, up to 20 cm in diameter, concentric, crusty, silvery areas, with minute, black dots (pycnidia erumpent from epidermis) and a leathery, brown halo. A milky to buff colored exudate, caking on contact with air, oozed from active cankers. Cankers coalesced and the cladode wrinkled. Infected plants declined and took a gray, ghostly appearance. The disease was found on 100% of plants in Lampedusa and Linosa, whereas it occurred sporadically in Favignana and Ustica but with an incidence of 100% of infected plants in some sites. It was not found in Sicily. Ten plants per island were randomly chosen and pieces (5 mm) of diseased tissue or single pycnidia were plated onto potato dextrose agar supplemented with 1 mg/ml of streptomycin and incubated at 20°C. A fast-growing fungus with a gray, aerial mycelium was consistently recovered. Optimum temperature for growth was around 25°C. Conidia from pycnidia produced on 1.5% water agar and sterilized pine needles were unicellular, hyaline, smooth, fusoid to ovoid, thin-walled,15.0 ± 1.5 × 5.5 ± 0.6 μm (length/width ratio = 2.9). Four representative isolates, one from each island, were identified according to internal transcribed spacer (ITS), EF1-α, and β-tubulin genes (Dissanayake et al. 2016; Lopes et al. 2016; Phillips et al. 2013). All isolates had identical ITS (GenBank accession nos. MF414730, MF414738, MF414747, and MF414748), β-tubulin (MF414749, MF414757, MF414766, and MF414767), and EF1-α (MF414768, MF414776, MF414785, and MF414786) sequences and 100% identity with the corresponding sequences of a reference isolate (CBS124923) of Neofusicoccum batangarum from Indian almond (Terminalia catappa; Lopes et al. 2016). Two polymorphic bases were identified in ITS and TEF sequences as compared with another reference isolate (ex-type isolate CBS124924) of N. batangarum from the same host (Lopes et al. 2016). Isolates were identified as N. batangarum and deposited at CBS-KNAW Biodiversity Centre (CBS143023, CBS143024, CBS143025, and CBS143026). On May 2015, field-grown cactus pear plants were wound-inoculated with these isolates (3 plants/isolate, 6 cladodes/plant). On each cladode, two holes (5-mm diameter) were made 20 cm apart with a cork-borer, and an agar plug from a 5-day-old colony was inserted into the hole. Three plants inoculated with sterile agar served as a control. Wounds were sealed with the excised tissues. Four days after inoculation (a.i.), a brown halo appeared around the wound and a buff-colored exudate oozed from the hole. Cankers were identical to those on naturally infected plants. Pycnidia emerged 20 days a.i. N. batangarum was reisolated from cankers and identified by sequencing the ITS, EF-α, and β-tubulin regions. Controls were asymptomatic. This disease is most likely the same reported as gummy canker caused by Dothiorella ribis (Somma et al. 1973). A similar disease of a distinct cactus species (Nopalea cochenillifera) incited by N. batangarum has been reported in Brazil (Conforto et al. 2016). This is the first report of N. batangarum in Europe and as a pathogen of cactus pear worldwide.