First report of Pantoea agglomerans causing Walnut Blight in Juglans regia L. in Chile

Walnut (Juglans regia L.) is Chile’s most cultivated nut tree, with 43,236 ha in 2024 and 62.8% of total tree nut export value (ODEPA, 2024). In summer 2024, symptoms were observed on walnut leaves and fruits in several Chilean regions: Metropolitana (Melipilla; 33°41´S, 71°12′W), O'Higgins (Santa Cruz; 34°38´S, 71°21´W), Maule (Linares; 35°50´S, 71°36´W), Ñuble (Chillán; 36°36´S, 71°52´W), and Biobío (Los Ángeles; 37°28´S, 72°13´W). Symptoms included brown leaf spots, irregular necrotic lesions and pericarp necrosis in fruits (23% incidence). Twenty fields were evaluated across these regions. In each field, 10 mixed symptomatic leaf and fruit samples were collected (200 total). Because Pantoea agglomerans may cause symptoms like those of Xanthomonas arboricola pv. juglandis (Xaj) (Yang et al., 2011), samples were surface-disinfected with 0.5% NaClO, macerated in saline solution, and plated onto a semi-selective medium for Pantoea spp. (Kini et al., 2019). After 48 h at 28°C, dominant circular, smooth, convex colonies (blue-violet on the semi-selective medium) were purified by repeated streaking on nutrient agar (NA), turning yellow-pigmented. Gram staining revealed Gram-negative bacilli. A total of 51 presumptive Pantoea spp. isolates were obtained from walnut tissues. Based on colony morphology and geographic origin, one strain per region (BC-0573 to BC-0577) was selected for molecular identification. DNA was extracted from the selected strains, and five housekeeping genes (16S rRNA, gyrB, pyrG, rplB, fusA) were amplified (Delétoile et al., 2009) and sequenced. The results were deposited in GenBank: BC-0573, PX023326 and PX101744 to PX101747; BC-0574, PX023500 and PX101748 to PX101751; BC-0575, PX024028 and PX101752 to PX101755; BC-0576, PX023273 and PX101756 to PX101759; and BC-0577, PX024313 and PX101760 to PX101763. BLASTn searches against NCBI sequences derived from type material identified P. agglomerans as the closest match for all five genes, with identity values ranging from 98.23 to 100%. To confirm identity, multilocus sequence analysis was performed using the concatenated 5-gene sequences. Phylogenetic analysis (Maximum Likelihood, Tamura-Nei model, MEGA v12.0.4) confirmed clustering within the P. agglomerans clade. Pathogenicity tests were performed on five 2-year-old walnut trees for each strain. Disinfected leaves were spray-inoculated with a bacterial suspension adjusted to 1x10⁸ CFU/mL; plants were kept at 24±2°C and 50% relative humidity (RH). Additionally, mature and healthy fruits (n=3) were collected, wounded, and each fruit was inoculated with a single strain by applying 50 µL of the same suspension into each wound; controls were inoculated with sterile water. Fruits were incubated at 28°C and 60% RH. After 5 days, all P. agglomerans strains produced necrotic leaf lesions and brown pericarp spots. Koch’s postulates were confirmed by reisolation and species-specific PCR using pagR primers (Shin et al., 2022). P. agglomerans causes leaf blight and vascular wilt in sorghum and maize in Mexico (Morales-Valenzuela et al., 2007), leaf blight and bulb rot of onion in Chile (Sepúlveda et al., 2023), and brown apical necrosis in walnut trees in China (Yang et al., 2011). This is the first report of P. agglomerans causing walnut blight in Chile. Because symptoms closely resemble those of Xaj, misdiagnosis is possible, highlighting the need for accurate diagnosis and effective management in walnut orchards.