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The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3'-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TagMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 103 cells per reaction, and the standard curve was linear within a range of 5 log units. Results from semi-commercial trials showed that CPA-2 had a low impact The maximum persistence of P. agglomerans CPA-2 was not longer than 5 days in plastic boxes stored at 0 degrees C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2 populations on apple surfaces during large-scale experiments that could ensure efficient and successful treatments
This research was supported by the national project RTA2009-00053-00-00 (Plan Nacional de I + D Ministerio de Ciencia e Innovación, Spanish Government), the National Council of Science and Technology of México (CONACYT) for scholarship 198363 (L. Soto) and the Catalonian Government, AGAUR, for the grant for research stays outside Spain BE-DRG 2009 BE1 00367 (R. Torres). We want to thank Dr. Jean-Claude Pech and Dr. Alain Latché (GBF Laboratory, UMR 990 INRA/INP-ENSAT, Castanet Tolosan, France) for sharing with us all their molecular expertise. We also thank Dr. Ramona N. Pena (Department of Animal Production, University of Lleida) for help in designing primers and the TaqMan MGB probe. Finally, we are also grateful to Cristina Solsona for her excellent technical assistance. |
