Rapid Detection Of Acidovorax Avenae Subsp. Citrulli And Xanthomonas Campestris PV. Vesicatoria

Phytobacteria is one of the most damaging plant pathogens, which can make the yields lost in crops and deterioration in quality in flowers and woods seriously. Bacterial fruit blotch (BFB), one of a bacterial deisease in cucurbits, is a seed borne disease caused by Acidovorax. avenae subsp. citrulli (Aac). This species of bacteria can infect melon, watermelon, cantaloupe and other cucurbits and cause devastating lost in fruits. It has been holding as a quarantine pest in China because of its distribution in some regions of China in the recent years.X. campestris pv. vesicatoria (Xcv), the causal agent of the bacterial spot disease on pepper and tomato, is another damaging bacteria in China and other countries in the world. Bacteria spot disease can cause serious damage in tomato especially in areas with high temperature and humidity. Xcv has been listed as an A2 quarantine pest by European Plant Protection Organization (EPPO). More specific and sensitive techniques for the identification of the bacteria are important to the protection of the import and export trade of pepper. So it is necessary to explore one or more testing methods rapidly and accurately for the detection of these two bacteria.Here in this study, we analyzed the effects of the detection methods of GICA (Gold Immunochromatography Assay)-PCR and TaqMan real-time PCR in testing A. avenae subsp. citrulli (Aac) and X. campestris pv. vesicatoria (Xcv) respectively. The results are as follows.(1) Gold Immunochromatography Assay (GICA) Strip is widely used in bacteria diagnosis because of its convenience in performing and visual judgment easily. However, GICA can be used only in the preliminary test because it may show many false positive results in detection. In this study, we developed a method named GICA-PCR to detect the Aac which combined the GICA and PCR assay. The test line of the GICA strip was cut down and soaked in 20μL double distilled water as the template of PCR reaction. Using GICA-PCR, Aac can be detected not only in bacteria cultured in medium but also in leaves and stems of watermelon infected by Aac. This method is more sensitive than that of GICA and can effectively reduce the false positive of the strip. During the assay, PCR showed a clear band even when the test line of the strip that could not be observed clearly by visual. Therefore, this method is worth of being applied for the detection of bacterial fruit blotch.(2) TaqMan real-time PCR has been widely used for the detection of bacteria because it's more specific and sensitive than traditional PCR assay. This technique can be used to measure PCR product accumulation through a dual-labelled probe. In this research, we designed a pair of primers XCV-F-F/R and a probe XCV-F-P based on the rhs family gene for the identification of the Xcv which infects pepper only using TaqMan real-time PCR. The result showed that the TaqMan real-time PCR was 10×more sensitive and specific than that of traditional PCR assay. We also detected Xcv in leaves of inoculated pepper and washing solution of the seeds soaked in Xcv suspension. Therefore, the real-time PCR method in this study is rapid and reliable for the detection of Xcv on pepper in the field.The GICA-PCR and TaqMan real-time PCR in this research were sensitive and specific for the rapid and accurate detection of the target bacteria. These two methods solved the problems of the GICA and traditional PCR assay respectively. Both assays are significant for the safety of the environment and the import and export trade in our country.