Improved Primers for the Specific Detection of subsp. in Sugarcane Using a Conventional PCR Assay.

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium subsp. (). infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel -specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published -specific primers and one universal generic primer pair LayF/LayR. Sugarcane leaf samples were collected from spp. hybrids in commercial fields (315 samples) and from germplasm clones of five species and (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural -infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and -pathogen detection with satisfactory sensitivity and specificity.