First report of Macrophomina phaseolina causing crown and root rot on strawberry in Italy

Strawberry (Fragaria × ananassa) is an important annual crop in southern Italy (about 2,800 ha in 2016, http://www.istat.it). In June 2017, about 20% of the plants in three strawberry fields of cultivar Melissa located in Basilicata (40°10′49.1″N, 16°38′11.3″E) showed wilting and chlorosis of the leaves. Crown and root sections of symptomatic plants displayed the presence of necrotic tissues and brown-red to dark brown discoloration of the vascular ring. Symptomatic tissue fragments were exposed to 2% sodium hypochlorite for 2 min, rinsed twice with sterile distilled water, and then placed on potato dextrose agar (PDA) plates. Dark gray Macrophomina-like colonies were observed after 7 days incubation at 24°C. Abundant dark oblong-shaped sclerotia were present in infected crown tissues as well as in pure cultures. Fifty sclerotia from each of three representative isolates were measured averaging 121 (68 to 174) μm × 174 (58 to 290) μm in size. Molecular identification of 10 fungal isolates using the species-specific primers MpKFI and MpKRI (Babu et al. 2007) yielded, in all cases, the expected 350-bp fragment. DNA of one representative isolate was amplified by polymerase chain reaction using the universal ITS5/ITS4 primers, and the 501-bp product was custom sequenced (Genewiz, Takeley, UK). BLASTn analysis of this sequence (GenBank accession no. MG836711) showed 99% identity (E value = 0.0, coverage = 100%) with Macrophomina phaseolina (Tassi) Goidanich (GenBank accession nos. KF951698.1 and KC357271.1). Pathogenicity assays were performed as described by Mertely et al. (2005) with slight modifications, using three representative isolates to inoculate 1-year-old strawberry plants (cv. Melissa). Briefly, toothpicks, sterilized by autoclaving twice in deionized water and finally in malt extract, were placed on the surface of M. phaseolina colonies grown on malt extract agar, and 7-days-colonized toothpicks were inserted into the crowns of six replicated strawberry plants for each isolate. Malt-extract-infused toothpicks were inserted in six replicated control plants. Plants were singularly placed in pots (10 × 12 cm), maintained in greenhouse under natural light, at 24°C and watered every 5 days with 150 ml of water per pot. Necrosis at the base of petioles and chlorosis of the leaves, followed by wilting, were observed after 3 weeks only on inoculated plants. About 6 weeks postinoculation, all plants collapsed or died, whereas the controls remained healthy. M. phaseolina was readily reisolated on PDA from all the symptomatic plants and identified based on both morphological and molecular features, hence fulfilling Koch’s postulates. M. phaseolina-induced crown rot of strawberry has recently been reported from Chile (Sánchez et al. 2013) and Tunisia (Hajlaoui et al. 2015), whereas in Italy there was only a report on sunflower (Manici et al. 1995). Because of the favorable climatic conditions, M. phaseolina represents a potential threat to southern Italy strawberry production in coming years.