First Report of Fusarium solani Causing Stem Canker in English Walnut in Spain

Abstract

In March 2017, stem canker was observed in 1-year-old English walnut (Juglans regia L.) cultivars Chandler and Howard from a nursery (Valencia, eastern Spain) and in a recently established orchard (Granada, southern Spain) using plants from the same nursery. The complete lot from the nursery was composed of 3,500 plants, of which 33% were affected. In general, plants showed lack of sprouting after the transplantation in the field, abundant wounds in roots and rootstock, internal wood discoloration, and sectorial crown and root necrosis. Necrotic tissues were washed with tap water, surface disinfected in a sodium hypochlorite solution (Cl at 5 g/liter) in sterile distilled water (SDW) for 1 min, and washed with SDW. Small wood pieces were plated on potato dextrose agar (PDA) with 0.06% lactic acid and incubated at 23°C for 14 days. Fungal isolates morphologically similar to Fusarium were consistently obtained. A representative one, ColPat-359, was placed on PDA and carnation leaf agar (CLA) and incubated as described above. On PDA, it produced whitish-beige floccose mycelium, brownish-violet in the center. On CLA, microconidia were oval to cylindrical, 5.2 to 10.5 × 1.9 to 4.3 µm; macroconidia were 0- to 2-septate, cylindrical and curved, 12.2 to 20.4 × 3.6 to 5.7 μm. First, it was identified as Fusarium solani species complex (Leslie and Summerell 2006). ITS, EF-1α, and RPB2 genomic areas were amplified with primers ITS4/ITS5, EF1/EF2, and fRPB2-5f2/fRPB2-7cr/fRPB2-7cf/fRPB2-11ar, respectively. ITS (MK968891), EF-1α (MK984235), and RPB2 (MK984234) sequences were deposited in GenBank. They showed 100% similarity with the reference sequences of F. solani AF14 (ITS: JX173101), NRRL 52705 (EF-1α: JF740787), and dH22442 (RPB2: KF255534). Pathogenicity was assessed on 1-year-old plants of walnut cultivar Chandler grown on plastic pots (20 liter, one plant per pot) filled with sterilized peat. Inoculations were done by irrigating (1 liter/pot) with inoculum of ColPat-359 (1.4 × 10⁷ conidia/ml) on potato dextrose broth (PDB) incubated at 25°C for 14 days, shaken at 90 rpm. Pots treated with sterile PDB were used as control (eight pots per treatment). Prior to inoculation, the crown and roots of the walnut plants were wounded to simulate the transplanting. Plants grew for 1 year in a greenhouse and were irrigated twice a week. Finally, detached shoots of walnut cultivar Chandler were wounded, inoculated with mycelial PDA plugs of ColPat-359 or with sterile PDA plugs (control), and incubated at 100% relative humidity and 25°C for 21 days (15 shoots per treatment). Each test was conducted twice. Inoculated walnut plants did not show aerial symptoms. Internal wood discoloration and sectorial necrosis were observed in crown and roots. Detached shoots showed internal wood discoloration (average lesion length = 56.4 mm). No symptoms were observed in the controls. F. solani was consistently reisolated (50 to 100%) from affected tissues on Komada medium. F. solani was described causing stem canker of J. regia and J. nigra in the United States (Tisserat 1987) and South Africa (Chen and Swart 2000), as well as root rot and wilting of Juglans spp. in China (Zheng et al. 2015), and it was associated with Geosmithia morbida causing thousand cankers disease of J. nigra and J. regia in Italy (Montecchio et al. 2015). To our knowledge, this is the first report of F. solani alone causing a stem canker syndrome in J. regia in Spain. This study highlights the pathogenic capacity of F. solani in English walnut, which should be considered during the propagation process to prevent the dispersal of this pathogen to the orchards.