First report of root and crown rot caused by Fusarium oxysporum Schltdl. on Prunus cerasifera L. in Spain.

Abstract

In 2021, Spain was the largest producer of cherries in Europe with a production of 125810 tons (FAOSTAT, 2021). In May 2022, in several production fields in Huelva (Spain), wilting was noted in 4-year-old cherry trees cv. Crystal Champaign grafted on rootstock cv. Adara (Prunus cerasifera L.). Gumming and wilting affected approx. 1% of the production area, leading to the eventual collapse and death of most affected trees within 2-3 years. Discoloration of the vascular system of the crown and roots was also noted. Symptomatic crown and root pieces (0.5 cm) were subjected to surface sterilization: immersed in 1% NaClO for 2 min, rinsed in sterile distilled water, and air-dried in a laminar flow cabinet. Then, plant tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated in a lab bench at room temperature for a week. Cottony and pink colonies were observed growing from the tissues. Two single strains (F175 and F176) were obtained from each tree by excising single spores (Gordon and Okamoto 1991). Isolates produced sparse aerial mycelia with white to pinkish-orange pigmentation on Spezieller Nährstoffarmer Agar (SNA). Both isolates produced microconidia in false heads on short monophialides. Microconidia were hyaline and measured in the range of 5.0-17.5 × 2.5-3.8 µm for both isolates (n = 50). Macroconidia were less abundant, falciform, and hyaline. Morphological characteristics were consistent with identification as Fusarium spp. (Leslie and Summerell 2006). A portion of the translation elongation factor-1 alpha (EF-1α) gene was sequenced using EF1/2 primers (O'Donnell et al. 1998) (GenBank Accession Nos. OR733348 and OR733349). Based on a comparison of 619 base pairs (bp), both isolates exhibited different sequences, with a 99.5% similarity (616/619 bp). A comparison with previously described isolates revealed a 100% match with published F. oxysporum sequences in the GenBank database (KT323846 and MZ404079, respectively). Isolates were used to conduct pathogenicity tests on 1-year-old plants cv. Adara growing in 512 cm3 pots. Using a scalpel, a 6-7 mm-length wound (2-3 mm deep) was made 5 cm above the soil line in all plants. For each isolate, 5 plants were inoculated by placing a 5 mm plug containing 10-day-old mycelia grown in AMAP medium (Borrero et al. 2009) at the incision point. Non-colonized AMAP plugs were used to inoculate 5 control plants. The inoculated sites were sealed with parafilm. Plants were randomly placed in a growth chamber with a temperature of 28/24ºC and a 12-hour photoperiod. A reddish-brown vascular stem discoloration was noticed in all the inoculated plants 73 days after inoculation. On average, the length of the necrotic area was 12.73 cm for F175, 20.12 cm for F176, and 4.59 cm for the control plants. Fusarium oxysporum was successfully re-isolated from all the inoculated plants. Recovered isolates were confirmed to be the same as the inoculated ones by sequencing the EF-1α gene. A one-way ANOVA indicates that plants cv. Adara were susceptible to both F. oxysporum isolates (P < 0.05). This is particularly noteworthy as cherries are predominantly planted on rootstocks and cv. Adara is a widely used rootstock in Spain. While F. oxysporum has been reported as the cause of root and crown rot in sweet cherry (P. avium L.) in British Columbia (Úrbez-Torres et al. 2016), this is the first report of F. oxysporum causing root and crown rot in cherry rootstocks (P. cerasifera L.) in Spain.