DNA-based methodologies to detect and quantify the postharvest biocontrol agent Pantoea agglomerans CPA-2 applied on oranges

Abstract

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) for postharvest diseases of citrus and pome fruits. However, for registration purposes and to implement their use as effective control strategy, it is necessary to study the traceability and survival of BCAs in their target application sites. The main objective of this work was to evaluate the persistence and quantify the population of CPA-2 after its postharvest application on orange cultivar 'Valencia Late' by molecular techniques. After application, the persistence of CPA-2 was evaluated by sampling the packing line and storage chambers, as well as on clothing of the workers by conventional PCR. The results showed that the maximum persistence of CPA-2 was lower than 3 days in surfaces of packing line. Furthermore, CPA-2 did not survive more than 1 day on working clothes, while in the environment or on different storage chamber surfaces it was not detected. In addition, the CPA-2 populations were quantified by quantitative PCR (qPCR) combined with a DNA intercalating reagent, propidium monoazide dye (qPCR-PMA) to quantify the CPA-2 viable cells on fruit surface. The qPCR-PMA method was compared with qPCR and dilution plating method. Results showed that CPA-2 populations quantified by qPCR-PMA were significantly different compared with those obtained by qPCR during the time-course of the assay; however, no significant differences were observed between qPCR-PMA and dilution plating. In conclusion, the persistence of CPA-2 was low at different sampling areas, suggesting that it cannot grow and survive on the surfaced sampled. Furthermore, qPCR-PMA method can be a quick and specific tool to monitor the viable population of CPA-2 on fruit surface.

The authors are grateful to the Spanish Government for financial support by national project RTA2009-00053-00-00, and CONACyT for L. Soto Ph.D. grant 198363