Bacterial Leaf Spot Susceptibility Screening of Chili Pepper Cultivars Using qPCR Determination of Xanthomonas euvesicatoria pv. euvesicatoria titers

Abstract

Bacterial leaf spot (BLS) is a serious disease of chili pepper (Capsicum spp.) caused by Xanthomonas euvesicatoria pv. euvesicatoria. Conventional resistance screening is time and resource intensive. It was considered that a quick and simple determination of cultivar susceptibility could be achieved through estimating bacterial titers of inoculated plants. A SYBR quantitative polymerase chain reaction (qPCR)-based assay was compared to conventional PCR, then used to detect and enumerate pathogen titers in serial dilutions and DNA extracted from infected plant leaves. The qPCR detection limit was approximately 1 CFU µl-1, ten times more sensitive than conventional PCR. A linear correlation (R2 = 0.994) was obtained from the standard curve comparing plate-truthed serial dilutions of the pathogen with qPCR cycle threshold. Six strains were used to inoculate cultivars Hugo and Warlock. One strain, X. euvesicatoria pv. euvesicatoria BRIP62403, was consistently the most virulent based on visual symptoms and pathogen titers in planta inferred by qPCR performed on DNA extracted from infected leaves 2 and 6 weeks post-inoculation. Visual observations 6-weeks after inoculation were highly correlated (R2 = 0.8254) to pathogen titers. The qPCR method was used to categorize 20 chili pepper cultivars two weeks after inoculation. A high positive correlation (R2 = 0.6826) was observed between visual scoring and pathogen titers from 20 chili pepper cultivars, facilitating categorization of susceptible, intermediate and resistant cultivars. The qPCR approach developed here facilitates susceptibility-screening of chili pepper cultivars at an early stage of selection and could be readily adapted to a range of other pathosystems.