Abstract: 【Objective】The objective of this study is to develop a nested-PCR method to rapidly and accurately detect Aac (Acidovorax avenae subsp. citrulli) in watermelon seeds, and to provide technique support for prevention and control bacterial fruit blotch of watermelon (WFB).【Method】Two pairs of specific primer sets (BX-L1/BX-R5 and BX-L1/BX-S-R2) derived from BOX short repeated sequence of Aac were selected to establish the nested-PCR. 5 µL of suspension were added in the tubes, boiled at 99℃ for 10 min, and then chilled on ice for 5 min, the released pathogen DNA was used as template for PCR reaction. PCR reaction system was performed with 5 μL of 10× reaction buffer (25 mmol?L-1 MgCl2), 0.4 μL of Taq DNA polymerase (DR001B, 5 U?μL-1), 3 μL of each primer (5 μmol?L-1), 4 μL of dNTP（D4030RA, 2.5 mmol?L-1）, ddH2O was added to the final reaction volume of 50 μL. DNA amplification was performed with 2 min at 95℃, followed by 35 cycles consisting of denaturation (30 s at 95℃), annealing (45 s at 65℃) extension (1 min at 72℃), and a final extension at 72℃ for 7 min. The nested-PCR was performed using (BX-L1/BX-R5 primers for the first run and the BX-L1/BX-S-R2 sets for the second run by using 1 μL of the first PCR product as the template applying the same thermal profile and number of cycles and annealing (45 s at 66℃). Analytical sensitivity, specificity and reproducibility were assessed, respectively. The nested-PCR method was used to detect a series of dilution of Aac suspension and different bacteria-carrying rates of seeds. 【Result】Aac in different strains was amplified by the specific primer sets BX-L1/ BX-R5 and BX-L1/BX-S-R2, respectively. A target band was amplified from Aac strains but not from A. avenae subsp. cattleyae, A. avenae subsp. konjaci and other bacteria. The nested-PCR assay had a lowest detection limit of 4.7×101 cfu/mL, the sensitivity was 1 000 times higher than the conventional direct-PCR. When the carrier rate of infected seed was 0.1%-0.5% , the positive detection rate was 66.7%. When the carrier rate of infected seed was 1%-10% , the positive detection rate was 83%-100%.【Conclusion】The present study demonstrated that the procedure of nested-PCR is a sensitive, specific, rapid, reproducible method to detect Aac in watermelon seeds.

Key words: bacterial fruit blotch of watermelon , watermelon seed , PCR , detection