Identification and detection of Acidovorax citrulli (Schaad vd. 2008), a causal agent of watermelon bacterial fruit blotch disease by quantitative real-time PCR, and investigation of susceptibility reactions of watermelon and melon varieties against the disease pathogen

on from both the diseased plant tissue and cell of Acidovorax citrulli (Acidovorax avenae subsp. citrulli), a causal agent of bacterial fruit blotch disease by Real-Time PCR. The susceptibility reactions of the watermelon and melon varieties to the bacterial pathogen have been investigated. The specific primer pairs for identification and detection of Acidovorax citrulli using Real-Time PCR were designed from both regions of the internal sequence of hrpB2 and the Internal Transcribed Spacer (ITS) region. A LNA (Locked Nucleic Acid) probe with 8 bases was developed. The specificity of the primer and probe sets was determined by testing different plant pathogenic bacteria, different Acidovorax citrulli isolates and watermelon genomic DNA. No amplification was detected in the genomes of different plant pathogenic bacteria and watermelon genomic DNA while both primer-probe sets showed a 88 bp- PCR product in all the tested national and foreign Acidovorax citrulli isolates. In this study, bacterial cell sensitivity limit was 2 bacterial cell, and DNA sensitivity limit was 12 fg. Quantitative Real-Time PCR was performed to determine the number of diseaseaffecting bacterial populations and for detection of living bacteria on the disease plant material using Bio-PCR. Quantitative Real-Time PCR resulted a standard curve for both the developed primer-probe sets in this study. The efficiency of the quantitative Real- Time PCR using primer-probe sets for hrpB2 and ITS was found to be highly reliable with 97.941% and 98.215%, respectively. These Real-Time PCR methods developed in this study may help to screening of watermelon and melon seeds in terms of the presence of the bacterial pathogen. The method may also help to detection and identification of the bacterial pathogen from directly the diseased plant materials and bacterial cells. This developed dependable, sensitive and repeatable Real-Time PCR method can help to controlling potential sources of inoculum and prevent to spreading of bacterial pathogen in field. The susceptibility reactions of watermelon and melon varieties to Acidovorax citrulli, were evaluated based on the nine different scales. The susceptibility reactions of tested plants were not statistically important.