First Report of Neofusicoccum nonquaesitum Causing Branch Dieback of English Walnut (Juglans regia) in the Maule Region, Chile

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English walnut (Juglans regia) cultivation has been expanding in recent years in Chile, with a surface of 9,000 ha planted in the Maule Region (35° 26' S, 71° 40' W), central Chile. In a field survey conducted between August (2022) and January (2023) in three localities of the Maule Region, several declined trees were observed. English walnuts exhibited small chlorotic leaves, cankers, dead twigs, and dieback of branches, with an incidence of 10 to 35% of trees affected per orchard. Internally, diseased branches exhibit brown to dark brown wood cankers. Symptomatic branches (n = 30) were collected and surface disinfected with 96% ethanol, and flamed. Pieces of wood (approximately 5 mm) were placed on potato dextrose agar (PDA), supplemented with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (Díaz et al. 2018), and incubated at 22°C for 5 days in darkness. Four isolates (VLC-1-6-20, VLC-1-10-20, VLC-1-12-20, and VLC-1-15-20) exhibited moderate growth rates (16.4 mm/day), developing a white hue to olivaceous black colonies after 7 days at 22°C on PDA. Chlamydospores were absent. Black pycnidia were formed individually or in clusters. The isolates produced aseptate, hyaline, and fusiform, with base truncate conidia measuring 24.8 ± 1.6 x 6.8 ± 0.63 µm (l/w = 3.1; n = 50). The four isolates characterized were amplified using of the internal transcribed spacer (ITS1/ITS4), a portion of beta-tubulin (Bt2a/Bt2b), and part of the translation elongation factor 1- α (EF1-728/EF1-968) genes (Phillips et al. 2013). Blast analyses showed 100, 99 and 100 % identity with ex-type isolate PD-484 of Neofusicoccum nonquaesitum for ITS (deposited as GenBank accession no. PP697845 to PP697848), Bt (PP789576 to PP789579) and EF1 (PP830824 to PP830827) regions, respectively. Combined phylogenetic analysis using MEGA 7 software and the maximum likelihood test clustered the four isolates with ex-type of N. nonquaesitum. Pathogenicity was performed using two isolates (VLC-1-6-20 and VLC-1-10-20), which were inoculated in healthy tree walnuts cv. Chandler (8 years old), using 40 μl of mycelial suspension (105 fragments of mycelium/ml) on rounded wounds in the middle of each attached young branch (n=30 branches) (Twizeyimana et al. 2013). Sterile distilled water was used as a control treatment. Pathogenicity tests were repeated twice. After 6 months, necrotic streaks with mean lengths of 13 mm and 28 mm were observed in the middle of wounded young branches. No necrotic lesions were observed in the control treatments. Reisolation of N. nonquaesitum was positive (100%) only from inoculated branches and molecularly identified (EF1-a), fulfilling Koch’s postulates. Previously, N. nonquaesitum has been reported causing dieback on English walnut in California (Chen et al. 2014). To our knowledge, this is the first report of N. nonquaesitum causing canker and branch dieback of English walnut in central Chile. Previously, Diplodia mutila, Dothiorella sarmentorum, and N. austral have been associated with walnut dieback in Chile (Díaz et al. 2018; Iqbal et al. 2023; Barcos et al. 2023). In conclusion, it is imperative to implement epidemiological studies to avoid the spread and severity of branch dieback of walnuts in the Maule region, central Chile.