First report of Ilyonectria liriodendri causing black foot on walnut in Chile.

Walnut (Juglans regia L.) is the primary nut tree cultivated in Chile, covering 44.626 ha. In autumn 2021, walnut decline and tree mortality were observed in young and old orchards (two and 18-years-old, respectively) in the O´Higgins Region in the Chilean Central Valley. In the surveyed walnut orchards cv. Chandler (n = 2), the incidence of symptomatic plants ranged from 35 to more than 90 %. Affected trees showed poor growth, twig and branch death, early defoliation, and shoot dieback. In addition, collar base necrosis and root rot were observed in symptomatic trees, resembling the symptoms of black foot disease, with blackening of complete roots and even the entire root system. Symptomatic root tissues from 5 independent trees were cut into pieces of about 25 mm2. The samples were sterilized for three minutes in a sodium hypochlorite solution (1%), rinsed in sterile distilled water, and plated onto Potato Dextrose Agar (PDA) medium supplemented with lactic acid (10%) and 0.5 g/L of streptomycin sulfate. Mycelium grew from the samples after seven days of incubation at 24°C. The characteristics of the culture and morphology of conidiophores and conidia indicated a Cylindrocarpon-like fungus. Then, 5 single-conidial isolates were obtained and subsequently grown on a PDA medium. Genomic DNA was extracted from cultures of three representative isolates (CNCO5; CNCO6; CNSF2) and used for amplification and sequencing of the Internal Transcribed Spacer of rDNA (ITS), β-tubulin (TUB2), Histone3 (HIS3), and Translation Elongation Factor 1-alpha (TEF1-α) partial gene regions using the primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (O'Donnell and Cigelnik 1997; Glass and Donaldson 1995), CYLH3F/CYLH3R (Crous et al. 2004), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. Isolates clustered consistently with Ilyonectria liriodendri after a multilocus molecular phylogenetic analysis. Sequences were deposited in at NCBI Genbank data base (ITS: OR871536-OR871537-OR871538, TUB2: PP780828-PP780829-PP780830, HIS3: PP780822-PP780823-PP780824, and TEF1-α: PP780825-PP780826-PP780827). A pathogenicity test was conducted by inoculating three walnut Vlach rootstocks (six months old) with one representative I. liriodendri isolate (CNCO6). Plant roots were cut 5 cm from the root tip and immersed in a 105/mL conidial suspension for 30 min; control plants were immersed in sterile distilled water. Then, the plants were transplanted to a sterile substrate. After six weeks of growing in a greenhouse, necrosis characterized by brown-black coloration of the roots was observed developing upwards from the wounded roots. Necrotic lesions were found even in secondary roots of infected plants; however, control plants remained asymptomatic. The fungus was re-isolated, completing Koch's postulates. Black foot disease caused by Ilyonectria spp. has been reported worldwide on grapevines (Ye et al., 2021) and causing root rot of other fruit trees such as olive (Úrbez-Torres et al., 2012) and kiwifruit (Erper et al., 2011). Ilyonectria liriodendri was recently identified as the cause of walnuts root rot in California (Lawrence et al. 2019). To our knowledge, this is the first detection of I. liriodendri causing black foot of walnut in Chile and worldwide. The symptoms of this disease closely resemble those of Phytophthora spp. in walnuts. This similarity could lead to misdiagnosis, highlighting the necessity for specific diagnostic tools to accurately identify the pathogen.