Detection The Pathogen Of Bacterial Fruit Blotch Of Watermelon By Polymerase Chain Reaction

Bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli (A.a.c) is an important quarantine disease. The pathogen can infect watermelon, cantaloupe, melon and other cucurbits, thus the production of cucurbits and related industries confronted with a significant threat. The bacteria are carried by seeds. It is necessary to establish a method of fast, accurate and convenient detection to the pathogen.In this study, the 16S rDNA and ITS sequence of A.a.c and its related species were amplified by polymerase chain reaction. According to the differences of ITS sequences, the primers were designed and screened which could specifically detect the A.a.c. The specific primers were TIF2: 5-GCT GGA TCA CCT CCT TTC GT-3 and TIR3: 5-TGA CGC AAT CAA ATT TTT GTCA-3. The procedure of PCR was optimized. The result showed that a 462 bp target band of PCR product was generated from the A.a.c, but no amplification product was generated from the related species of the A.a.c and other bacterial strains used in this study. The detection sensitivity of bacterial liquid by PCR was 105 CFU/mL and the whole process took about 90 min.What is more, the gyrB sequences of A.a.c and its related species were amplified by touchdown PCR. According to the differences of gyrB sequences, the nested primers were designed and screened which could specifically detect the A.a.c. The outer primers were GTF: 5-GTG CTC TGC TTC ACC AAC AA CA-3, GTR: 5-TCA GGG TCT CTC CGT CCA GG-3, and the inner primers were GTF2: 5-ATC GG CAA GTA CAT CGAGCA GAA T-3, GTR2: 5-TCG ACG ATG TAG ATC TCG CTC AGG-3. The procedure of PCR was optimized. The result indicated that a 395 bp target band of PCR product was generated from the A.a.c, but no amplification product was generated from the related species of the A.a.c and other bacterial strains used in this study. The detection sensitivity of bacterial suspension by nested PCR was 103 CFU / mL, and the whole process took about 2 h.Due to the persistence of DNA in the environment after bacterial cells death, DNA-based detection methods cannot differentiate whether positive signals originate from living or dead bacterial targets, so that it was a hot research subject how to eliminate the influence of dead cells. In this study, we investigated that whether the DNA dye (Ethidium Monoazide) could differentiate the living or dead cells of A.a.c. The result indicated that when the concentration of dead cells was less than 107 CFU / mL, the EMA could inhibit the DNA amplification of dead cells when the nested PCR was carried out.