First Report of <i>Cophinforma tumefaciens</i> Causing Dieback of Cashew in Costa Rica

In 2024, cashew trees (Anacardium occidentale) on a 3-hectare commercial farm in Esparza, on the Pacific Coast of Costa Rica, showed dieback symptoms, including shoot and branch dieback, with severe vascular necrosis and defoliation. More than 80% of trees showed symptoms with varying degrees of severity, including some cases of total branch death (Figure 1). Samples were taken from five trees and transported to the laboratory. There, the tissue was cut and disinfected with commercial bleach (1% v/v), followed by 70% ethanol. Rinsing steps with sterile distilled water were performed in between and following disinfection. Sections of tissue taken 3–5 mm from the edge of the lesions were placed on 25% potato dextrose agar (PDA) plates and incubated for 10 days at 26°C. Two representative isolates, 5906-2FI and 6175-OR422, were further purified by transferring to fresh 25% PDA plates, and individual colonies were obtained using the hyphal tip method. Initially, the colonies grew with a white colour transitioning to dark olive green and later black within 10 days. They had a cottony appearance with irregular edges and submerged mycelia (Figure 2). Agar plugs were placed on 2% water agar plates containing pine needles and incubated for 3 weeks at 28°C. The conidia obtained from these cultures were unicellular, hyaline, thin-walled, fusiform with no septa, and measured 25–40 × 7.5–15 µm (mean 33.29 ±4.00 × 11.18 ±1.93 µm, n = 20) (Figure 3). These features are consistent with Cophinforma (Phillips et al. 2013; Zhao et al. 2021). Genomic DNA was obtained from the isolates and used in PCR with primers ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995) and EF1-728F/EF1-986R (Carbone and Kohn 1999), to amplify the internal transcribed spacer (ITS), β-tubulin gene (TUB2) and translation elongation factor 1α gene (EF1α), respectively. The resulting sequences were deposited in Genbank under the following Accession Nos: ITS, PX275515 and PX275516; TUB2, PX273771 and PX273773; and EF1α, PX273770 and PX273772. A BLASTn search revealed that the sequences were greater than 99% identical to Cophinforma tumefaciens (syn. C. atrovirens and C. eucalypti) accessions (ITS: KF531822.1; TUB2: MN539176.1; EF1α: MK340865.1). Based on maximum-likelihood phylogenetic inference of the concatenated ITS and EF1α DNA sequences, the identity of isolates 5906-2FI and 6175-OR422 was confirmed to be C. tumefaciens (Figure 4). A pathogenicity test was performed on 3-month-old, healthy A. occidentale trees. A wound was made with a sterile scalpel at least 5 cm from the shoot. Then, a PDA disc (Ø = 6 mm) with 1-week old mycelium was placed on the exposed tissue and sealed with plastic film. Three additional plants were inoculated with sterile PDA as controls. All plants were kept in a greenhouse at 26°C–30°C for 3 weeks. The stems were then removed and processed as previously described (Figure 5). Only the inoculated stems exhibited fungal colonies with the same morphology as the original isolates, thus fulfilling Koch's postulates. The inoculation was conducted in two separate experiments with similar results. Cophinforma tumefaciens has been confirmed as a pathogen of cashew in Brazil (Cardoso et al. 2019) and of multiple other hosts (Zhao et al. 2021). Phylogenetically, C. eucalypti and C. atrovirens are identical to C. tumefaciens; however, the latter species takes priority in naming (Zhao et al. 2021). The genus also contains C. mamane, which is also closely related to the other species, with only a 3-bp difference in their ITS sequences (Phillips et al. 2013). However, no other genes from the type isolate are currently available for comparison. In Costa Rica, it has been reported that Sphaeropsis tumefaciens (syn. C. tumefaciens) was isolated from diseased Citrus spp. stems and branches, but no further details were provided (Villalobos et al. 2009). To our knowledge, this is the first report of C. tumefaciens causing dieback symptoms in cashew trees in Costa Rica. This work was funded by grants C4263 (Vicerrectoría de Investigación) and ED-313 (Vicerrectoria de Acción Social) from the Universidad de Costa Rica.