MORPHOLOGICAL, BIOCHEMICAL, AND MOLECULAR CHARACTERIZATION OF XANTHOMONAS CITRI SUBSP. CITRI, CAUSE OF CITRUS CANKER DISEASE IN PAKISTAN

Citrus is being cultivated throughout the globe and its production is increasing every year. Pakistan is ranked at 13thnumber for citrus production. Worldwide, Pakistan is largest exporter and producer of Kinnow. Current study includes the survey for collection of citrus canker infected samples from different citrus orchards of Punjab. The pathogen Xanthomonas citri subsp. citri (Xcc) was isolated and purified on yeast dextrose calcium carbonate agar medium. Pathogenicity was confirmed by both attached and detached leaf assays. On water agar media, Xcc exhibited canker like symptoms on leaves of Crizo citrange, Duncan pumelo and Mexican lime whereas attached leaf assay was done on rough lemon plant and pathogenicity was confirmed after development of symptoms i.e. lesions surrounded by yellow halo. Hypersensitive response of bacteria was confirmed by application of bacterial inoculum on tobacco plant and Xcc. produced necrotic spots at the point of inoculums application. Isolated pathogen X. citri was characterized on biochemical and molecular basis. For molecular detection, DNA of isolated Xcc. was performed by SDS method and also through commercial Kit. Extracted DNA was subjected to polymerase chain reaction (PCR) where six set of primers based on 16S rRNA, pathogenicity and internal transcribed spacer were used for its characterization. Most of isolates showed positive results by all primers while few isolates from Sweet Orange gave no result by primers designed on pathogenicity region. Bacterial isolates produced amplification products by PCR were also subjected to RFLP by EcoR1 and Xbal restriction enzymes. RFLP analysis depicted significant variation in two isolates. Phylogenetic analysis revealed that the Pakistani isolates are closely related to the isolates from Japan, India, Spain and distantly related to the isolates reported from Brazil and USA.

Automated summary

This study collected citrus canker infected samples from different citrus orchards in Punjab and isolated the pathogen Xanthomonas citri subsp. citri (Xcc). The pathogenicity of Xcc was confirmed through attached and detached leaf assays, and its hypersensitive response was confirmed on tobacco plants. The isolated Xcc was characterized using molecular methods and revealed significant variation. Phylogenetic analysis showed that the Pakistani isolates are closely related to isolates from Japan, India, and Spain, but distantly related to isolates from Brazil and USA.