Biochemical and molecular characterization of Xanthomonas citri pv. mangiferaeindicae causing bacterial black spot of mango in Pakistan.

Bacterial black spot (BBS) disease of mango (Mangifera indica L.) is associated with Xanthomonas citri pv. mangiferaindicae (Xcm) and can cause substantial production and fruit-quality losses in susceptible orchards. This study was investigated to isolate and characterize Xcm associated with symptomatic mango samples from major mango-growing areas of South Punjab, Pakistan, using phenotypic, biochemical, pathogenicity, PCR, sequencing, and phylogenetic approaches. Symptomatic mango samples were collected from surveyed orchards, and Xcm isolates were obtained, purified, and characterized based on colony morphology and biochemical tests, including gelatin hydrolysis, starch hydrolysis (amylase activity), catalase, and oxidase assay. Pathogenicity assays were performed to evaluate the ability of selected isolates to produce disease symptoms. Molecular identification was performed by PCR amplification with the primers targeting the 16 S rRNA gene (27 F/1492R) and the pthA region (VM3/VM4), followed by sequencing and phylogenetic analysis. The isolates produced yellow or offwhite, mucoid colonies, while biochemical assays provided supporting phenotypic characteristics for their identification. BLAST analysis showed 99.9% identity with previously reported Xcm sequences fromChina. Phylogenetic analysis further demonstrated that the Pakistani isolates clustered closely with Xcm isolates reported from mango in china revealed that the Pakistani isolates are clustered closely with the Xcm isolates reported from mango in China and an isolate associated with Ambarella in France The combined phenotypic, pathogenicity, PCR, sequencing, and phylogenetic evidence supported the association of Xcm with bacterial black spot of mango in the surveyed areas of Pakistan. This study confirmed the association of Xcm with bacterial black spot of mango on molecular basis in Pakistan for the first time and provide baseline information for future additional molecular markers diagnosis such as gyrB, rpoD, dnaK, fyuA, or multilocus sequence analysis, to better understand the genetic diversity of Xcm and facilitate improved disease diagnosis and management.