Isolation and In Vitro/In vivo evaluation of epiphytic bacterial strains against walnut bacterial blight pathogen Xanthomonas arboricola pv. juglandis

Abstract

Bacterial blight of walnut (Juglans regia), caused by Xanthomonas arboricola pv. Juglandis (Xaj) leads to substantial economic losses in walnut-producing regions globally. In this study, epiphytic bacteria isolated from the walnut phyllosphere in Kohgiluyeh and Boyer-Ahmad Province, southwest Iran (2018–2019), were evaluated as potential biocontrol agents against Xaj. Eighty-five epiphytic bacterial isolates from symptomatic and asymptomatic walnut leaves were screened for antagonistic activity against Xaj. Six isolates exhibited inhibitory effects in both ex-planta and in-planta experiments. Among them, D5 and D4 showed the strongest inhibition, with halo diameters of 28 ± 0.06 mm and 27 ± 0.08 mm, respectively, in well diffusion assays. Molecular methods and dendrogram analysis confirmed that D4 was the most similar to Bacillus amyloliquefaciens, and D5 was the most similar to Bacillus subtilis. The other four isolates, K2, H11, B3, and B11, exhibited weaker antagonistic activity compared to D4 and D5 and were identified as Pseudomonas hibiscicola, Pseudomonas putida, Pseudomonas geniculata, and Pantoea agglomerans, respectively. Greenhouse experiments using foliar spray and needle-pricking methods demonstrated that Bacillus amyloliquefaciens and Bacillus subtilis effectively reduced disease severity, necrotic spots on leaves, and the percentage of infected leaf area compared to untreated controls. Importantly, pretreatment with D4 and D5 significantly inhibited the growth of Xaj bacteria in plants. These findings suggest that B. amyloliquefaciens and B. subtilis have potential as biocontrol agents against walnut bacterial blight, providing a more environmentally friendly option than chemical pesticides. The use of phyllosphere-derived Bacillus and Pseudomonas strains represents a sustainable approach that capitalizes on the natural microbiome's inherent advantages over introduced biocontrol organisms. Further research in field conditions is needed to confirm their effectiveness.