Genetic basis and phenotypic characterization of copper resistance in Xanthomonas citri from citrus orchards in Pakistan

Safi Ullah, Iqbal Munir, Musharaf Ahmed, Sajid Ali

Abstract


Copper resistance in citrus orchards is an emerging concern, requiring effective strategies to manage the spread of bacterial citrus canker. During a two-year surveillance (2020-2021), 5,490 citrus canker lesion samples were collected from major citrus-growing regions of Pakistan, including Punjab, Khyber Pakhtunkhwa, and the Federal area. Of these, 3,160 isolates were successfully cultured, giving an overall recovery rate of 58%, with variation across regions, districts, and cultivation zones. Sargodha district, the primary citrus-producing area of Punjab, was the main focus for copper-resistance assessment. A total of 120 bacterial isolates, along with a copper-resistant reference strain (LH-201), were evaluated on nutrient glucose agar amended with copper sulfate at 0.5 g, 1 g, and 2 g concentrations. Phenotypic characterization showed that most isolates tolerated 0.5 g CuSO₄ (60% recovery), fewer survived at 1 g (24%), and none grew at 2 g. In contrast, the reference strain (LH-201) exhibited complete resistance at all tested levels. Copper-tolerance patterns also varied among Sargodha sub-locations: isolates from Chak 48 and Choranga (2020) and Chak 82 (2021) showed higher recovery at 1 g CuSO₄. Overall, resistance frequency was greater in 2020 than in 2021. Molecular screening using primers targeting copper-resistance genes (CopLAB) detected no such genes in any isolates, despite their phenotypic tolerance at lower copper concentrations. Overall, Pakistani citrus canker populations were largely copper-sensitive, particularly at higher concentrations, with no genetic evidence of stable copper-resistance mechanisms. These findings highlight spatial and temporal variation in pathogen recovery and provide important understandings for copper-based management practices in Pakistan.


Keywords


Copper resistance, Xanthomonas citri, Citrus canker, Phenotypic characterization, Molecular detection

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References


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DOI: https://doi.org/10.33804/pp.009.04.5933

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