Study On The Detection Method Of Psa-V In Kiwifruit

Kiwifruit canker is a kind of remarkable bacterial disease.At present,kiwifruit canker has become the key quarantine object of national forest plant.Pseudomonas syringae pv.Actinidiae(Psa)is the pathogenic bacterium of kiwifruit canker.The virulent strain of Psa(Psa-V)has high pathogenicity.It is the main pathogen causing the outbreak of kiwifruit canker in China.Kiwifruit pollen carrying Psa-V are significant factors which lead to its long-distance transmission.Therefore,it is of great significance to strengthen the detection of Psa-V in kiwifruit pollen to prevent the occurrence of kiwifruit canker.In this study,common PCR was used to detect the Psa-V in Shaanxi pollen samples,and then the detection method of active Psa-V was established which utilized PMA-qPCR and RT-PCR.This technology would provide technical support for the disinfection of pollen and for reducing the transmission of Psa-V by pollen.1.The PCR detection of Psa-V in commodity pollen in kiwifruit producing areas of Shaanxi provinceA total of 36 commodity pollen samples were selected and tested by using PCR.The results showed that,through PCR detection,a total of 27 samples were positive,with a positive probability of 75%.Sequencing analysis of the positive samples showed that all the 27 samples with positive results were Psa-V.2.The Psa-V test based on PMA-qPCRThe Propyl azide bromide(PMA)and real-time fluorescence quantitative analysis(q-PCR)were combined to quantitative detect the number of living ulcer dominant pathogenic bacteria(Psa-v)of Shaanxi kiwifruit.By optimizing the optimal concentration of PMA,dark incubation time and exposure time,the optimal conditions for PMA-PCR to distinguish ulcer bacteria from live and dead were determined.Results show that the Psa can be completely dead in 98.3℃ boiling water bath treatment after 13 min.When the death concentration of Psa was 1×107 CFU/mL,the best concentration for covalent cross-linking between PMA and dead bacteria was 105μg/mL.The best dark incubation time was 8 min and the best exposure time was 20 min.Under such a condition,there was no DNA amplification of dead bacteria,without affecting the DNA amplification of living bacterium.The linear regression equation established by Psa standard plasmid was Y=-3.2204x+37.73,R2=0.9955.This equation can detect 6.39×102 copy/reaction system of ulcer bacteria at the minimum.A minimum of 2.38×102 copies/L of ulcer bacteria can be detected.If the artificially infected branches samples-were used to detect,the minimum detection limit would be 6.30×104CFU/mL,which was consistent with the detection results of colony-counting method.3.The Psa-V test based on RT-PCRRT-PCR technology was applied to establish a Psa-V test method for kiwifruit in Shaanxi province.The results showed that the RT-PCR method could effectively detect the living Psa-v.In the pure culture,the minimum detection limit was 1.26×104 CFU/ml.For the sample of infected pollen,the sensitivity can reach 1.39×101 CFU/g after enriching culture medium for 12h.