First Report of Fusarium oxysporum and Fusarium redolens Causing Wilting and Yellowing of Wild Rocket (Diplotaxis tenuifolia) in the United Kingdom

Abstract

In summer 2015, a UK grower reported crop losses of 20% (>£170k) in glasshouse-grown wild rocket (Diplotaxis tenuifolia, cvs. Voyager and Napoli) with disease symptoms initially seen as chlorosis of leaf veins followed by wilting, yellowing and plant death. Diseased plants were collected from three areas of a glasshouse with further samples taken from two areas of another glasshouse in 2017. Roots were washed, surface sterilized in 70% ethanol for 10s, rinsed twice in sterile water and 5mm sections placed on potato dextrose agar (PDA) containing 20 µg/ml chlorotetracycline. Following incubation at 20°C for 4 days, fungal colonies were sub-cultured onto PDA and examined after 14 days. All colonies resembled Fusarium with four morphotypes identified. Morphotype 1: flat, white colonies, abundant oval to cylindrical microconidia (9.4 x 4.6 µm), no macroconidia. Morphotype 2: pale yellow colonies which darkened with age to orange/brown, no microconidia, abundant macroconidia (33.3 x 5.7 µm), 3-7 septate, dorsiventral curvature, hooked basal cell and papillate apical cell. Morphotype 3: white colonies, cotton-like aerial mycelium with pink pigmentation on the underside. Abundant cylindrical (occasionally oval) microconidia (8.9 x 3.6 µm), sparse macroconidia (15.2 x 4.1 µm), slightly curved and slender, 3 septate with hooked basal cell and pointed apical cell. Morphotype 4: white colonies, cotton like aerial mycelium with orange/red pigmentation on the underside, abundant cylindrical microconidia (8.2 x 3.3 µm), macroconidia extremely sparse (15.4 x 3.9 µm), slightly curved and slender, 3-4 septate with hooked basal cell and pointed apical cell. Morphotype 1 was most frequently isolated from diseased plants. DNA was extracted from cultures of each morphotype (19 x type 1, 4 x type 2, 1 x type 3 and 4) and PCR carried out to amplify and sequence part of the translation elongation factor 1-α (EF1-α) gene (Taylor et al., 2016). Following BLAST searches and phylogenetic reconstruction (Fig. S2), isolates were identified as Fusarium redolens (morphotype 1, GenBank accession no. MK172061), Fusarium equiseti (morphotype 2, MK172060) and Fusarium oxysporum (morphotype 3, MK172059 and type 4, MK172058). F. oxysporum isolates were only identified in 2015. The pathogenicity of each Fusarium sp. was determined on six wild rocket cultivars. Roots of five week-old plants (eight plants per isolate/cultivar combination) were trimmed by 2-3mm and soaked in 100 ml of conidial suspension (1 x 106 cfu/ml) for 10 minutes before transplanting into compost in 9 cm pots. Control plants were dipped in sterile water. Pots were placed in a glasshouse (27°C day, 20°C night) and disease symptoms recorded twice weekly. Typical symptoms of wilting and yellowing (particularly in leaf veins) were observed 12 days after inoculation with F. oxysporum and F. redolens, while uninoculated control plants and those inoculated with F. equiseti remained healthy. Fusarium was consistently re-isolated from symptomatic plants, and following DNA extraction, PCR and sequencing of the EF1-α gene, sequences matched the identity of the inoculated isolate, thus fulfilling Koch’s postulates. This is the first report of Fusarium wilt of rocket in the UK and to our knowledge the first ever report of F. redolens causing wilt of wild rocket. F. oxysporum has been reported to cause wilt of wild rocket in Italy (Garibaldi et al. 2003) whilst F. redolens is known to cause wilt of chickpea (Jimenez-Fernandez et al. 2011).