First Report of Fusarium oxysporum Causing a Vascular Wilt of Statice (Limonium sinuatum) in the U.K.

Abstract

Statice (Limonium sinuatum) is grown commercially in many countries as a cut-flower crop. Fungal and oomycete pathogens reported for this plant include Colletotrichum, Botrytis, Cercospora, Rhizoctonia, and Peronospora (Moorman 2016). In the UK, 80% of all statice production (2.5 ha, value $800,000) is through a specialist grower in Lincolnshire. In 2016, 30% of the 70,000 statice plants raised at this nursery were affected by a vascular wilt. Symptoms observed on approximately 1 m tall flowering plants (cv. Velvet Wings) consisted of wilted leaves and flower stalks, often progressing down one side of the stem, followed by leaf necrosis and plant death. Brown staining of vascular tissue was observed in dissected stems and, in advanced infections, mycelium was evident at the stem base. Isolations were carried out from 18 plants by excising 2 to 3 cm sections of infected stem, surface disinfecting in 5% sodium hypochlorite for 2 min, rinsing in sterile water and plating on potato dextrose agar (PDA) containing 20 µg/ml chlortetracycline. After incubation at 20°C for 4 days under natural light, fungal colonies were sub-cultured onto PDA and grown for a further 10 days. Cultures displayed morphology typical of Fusarium species, with a peach/pale pink color. Macroconidia were 3-septate, straight or slightly curved, 16.1 to 25.7 µm x 2.8 to 5.4 µm. Microconidia were abundant, elliptical or reniform, 6.1 to 12.4 µm x 1.6 to 4.9 µm. DNA was extracted from four isolates, and the translation elongation factor 1-α (EF1-α) gene amplified by PCR and sequenced using exTEF-F/ FUexTEF-R primers (Taylor et al. 2016). All isolates shared identical EF1-α sequences (GenBank accession number KX822794) and were confirmed as Fusarium oxysporum using BLAST analysis. The sequences were 99% identical to the EF1- α sequences of a wide range of F. oxysporum isolates with the greatest homology to F. oxysporum f. sp. freesia (KP964900, 99.7% over 989 bp). To confirm pathogenicity, statice (cv. Velvet Wings) plants produced by tissue culture were raised in pots. Once the first flower stalk reached approximately 30 cm, plants were removed, the bottom third of the root system excised and the remaining roots soaked in a conidial suspension (1 x 106 conidia/ml) for 5 min. As a negative control treatment, F. oxysporum isolate Fo47, a biocontrol strain known to be non-pathogenic on a range of hosts (Aime et al. 2013), was used to inoculate plants in the same way, while noninoculated control plants were soaked in sterile water. All statice (six plants / isolate or control treatment) were then replanted in compost and pots placed in a randomized design in a temperature controlled glasshouse (25°C day, 18°C night, 16 hour photoperiod). After 20 days, typical wilt symptoms were observed in all the plants inoculated with the four F. oxysporum isolates from statice, and after 40 days all the plants were dead (e-Xtra). All noninoculated control plants and those inoculated with F. oxysporum isolate Fo47 remained healthy. The pathogen was successfully re-isolated from infected stems with the fungal cultures exhibiting the same morphology as described previously. Furthermore, following DNA extraction, amplification and sequencing of the EF1-α gene of the re-isolated fungi, all sequences were 100% identical to those obtained for the original isolates, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of Fusarium wilt of statice. Further work is required to establish the host range of this potentially new forma specialis.