Testing two types of molecular methods for the detection of Candidatus Liberibacter, the causative agent of huanglongbing (HLB) in the greening of citrus fruits in Ecuador.

Abstract

Citrus greening disease, caused by the pathogenic bacterium Candidatus Liberibacter, is a looming problem in Ecuador, as it is present in neighbouring countries. The damage caused by this disease disrupts phloem function and severely impairs the translocation of assimilates in the host plant, often leading to death. To accurately identify the causative bacterium of citrus greening using molecular techniques and a rapid-action protocol, clustered sampling was conducted on symptomatic plants in four provinces of Ecuador, where the vector is already present and citrus-producing areas exist. Two methodologies, CTAB DNA extraction and the DNA-easy plant mini kit, were employed to extract DNA for subsequent verification of the presence/absence of this microorganism through PCR, followed by electrophoresis and cost analysis. Results showed similar DNA extractions using both methods, with quantification through electrophoresis and spectrophotometry indicating a positive correlation of r2=0.9402. PCR reaction with the 16S rRNA molecular markers of the 22 samples did not detect the presence of the bacterium in any of the areas. The total diagnostic cost for PCR of 18 samples was determined to be $84.93, with a unit value of $4.71. Using the Qiagen® DNA-easy plant mini method, the unit value per sample was $3.36, resulting in a total cost of $60.65 for the CTAB method.