Molecular identification and pathogenesis of Xanthomonas arboricola pv. juglandis isolates from walnut in Azad Jammu and Kashmir, Pakistan

Abstract

Xanthomonas arboricola pv. juglandis causes bacterial blight of walnut, a highly destructive disease of walnut (Juglans regia) in the temperate climates of Pakistan. Although the geographical distribution and biochemical characterization of the pathogen in Azad Jammu and Kashmir (AJK) have been reported previously, its molecular characterization and pathogenicity on local cultivars remain largely unexplored. In the present study, we provided the first comprehensive molecular characterization and phylogenetic analysis of 20 X. arboricola pv. juglandis isolates recovered from infected walnut trees across multiple locations in AJK, along with one reference isolate from the University of Punjab, Lahore. The bacterial isolates were cultured, and genomic DNA was extracted. The 16S ribosomal RNA (rRNA) gene was amplified using universal bacterial primers, sequenced, and analyzed alongside reference sequences retrieved from GenBank. Phylogenetic analysis was performed using the maximum likelihood method with appropriate outgroup sequences. The 16S rRNA sequences exhibited high nucleotide identity (98.5-100%) with previously reported sequences of X. arboricola pv. juglandis, confirming the species and pathovar identity of the isolates. Phylogenetic analysis revealed two major genetically distinct clades among the studied isolates, indicating both evolutionary divergence and close relatedness to reference strains. Pathogenicity assays on walnut seedlings demonstrated that all isolates were virulent, producing disease severity scores ranging from 1.0 to 3.5, indicating moderate to high pathogenic potential. These molecular and pathogenicity findings enhance the understanding of the epidemiology of walnut bacterial blight in AJK and are expected to support the development of region-specific disease management strategies and breeding programs for sustainable walnut production.