Detection and identification ofXanthomonaspathotypes associated with citrus diseases using comparative genomics and multiplex PCR

Abstract

BackgroundInCitruscultures, three species ofXanthomonasare known to cause distinct diseases.X. citrisubsp.citripatothype A,X. fuscanssubsp.aurantifoliipathotypes B and C, andX. alfalfaesubsp.citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species ofXanthomonas.ResultsUsing comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µlin vitroand 1.5 × 104CFU ml−1in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes ofXanthomonasnot associated withCitrusand one species ofXylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species ofXanthomonasinvestigated in this study.ConclusionsWe demonstrated that these markers can be used as potential candidates for performingin vivomolecular diagnosis exclusively for citrus-associatedXanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.