Search

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.

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) against the major postharvest pathogens present on pome and citrus fruits. Dehydration, such as freeze-drying, spray-drying and fluidized bed drying is one of the best ways to formulate BCAs. In this work, the survival of CPA-2 cells after formulation was determined by dilution plating and molecular methods as qPCR alone and combined with a sample pretreatment with a propidium monoazide dye (PMA-qPCR) and they were used to calculate treatment concentrations in efficacy trials on postharvest oranges. Furthermore, no significant differences in CPA-2 survival were observed as determined by dilution plating and PMA-qPCR after both the freeze drying and fluidized bed drying processes; however, an interesting significant difference was observed in the spray dried product comparing all quantitative methods. A difference of 0.48 and 2.17 log10 CFU or cells g/dw was observed among PMA-qPCR with qPCR and dilution plating, respectively. According to our study, dilution plating was shown to be an unreliable tool for monitoring the survival of CPA-2 after spray drying. In contrast, the combination of PMA and qPCR enabled a quick and unequivocal methodology to enumerate viable and VBNC CPA-2 cells under stress-dried conditions. Efficacy trials showed that, after 3 days, spray drying formulation rehydrated with 10% non-fat skimmed milk (NFSM) was as effective as fresh cells to control Penicillium digitatum in oranges.

The aim of this study was to evaluate the environmental fate and behaviour of the biocontrol product based on Pantoea agglomerans CPA-2 cells after its release into the field. Three different methods, dilution plating, quantitative real-time PCR (qPCR) and propidium monoazide combined with qPCR (PMA-qPCR) were used to track the CPA-2 population. Populations quantified by qPCR were statistically different compared with those by the PMA-qPCR and dilution plating methods. In addition, the spray dispersion of the CPA-2 treatment was evaluated using water-sensitive papers. A lack of dispersion of the CPA-2 treatment was observed at distances of 2.5 ± 0.5 m. The presence and persistence of CPA-2 in the environment were monitored in different areas, by dilution plating and confirmed by conventional PCR. The results showed that CPA-2 can survive but not proliferate on weed and leaves. The persistence on inert surfaces, such as motorised backpack sprayer, gloves and working clothes, was less than 7 days. In conclusion, PMA-qPCR was a potential tool for fast and specific monitoring of viable populations of CPA-2 and it gave valuable information on population behaviour. Moreover, the results demonstrate that CPA-2 would not present a risk for the environment because of its low establishment, survival and dispersion.

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) for postharvest diseases of citrus and pome fruit. To implement their use as a control strategy is necessary to study the traceability of BCAs in the environment during application, for registration issues. In this study, the presence and persistence of CPA-2 was monitored in the packing line, storage chambers and on working clothes by conventional PCR. After postharvest application, the presence of CPA-2 was not detectable in the environment and storage chambers, whereas on working clothes and the packing line its persistence was less than 1 and 3 days, respectively. Additionally, the CPA-2 population was quantified on oranges stored at two different temperatures (20 °C and 4 °C) by quantitative PCR (qPCR), sample pretreatment with a propidium monizade dye (PMA–qPCR) and the dilution plating method. At the initial time of the assay, no differences were observed in CPA-2 populations quantified by qPCR, PMA–qPCR, and dilution plating, at both storage temperatures. However, CPA-2 populations quantified by PMA–qPCR were significantly different compared with those obtained by qPCR during the time-course of the assay; no significant differences were observed between PMA–qPCR and dilution plating. In conclusion, the persistence of P. agglomerans CPA-2 at different sampling areas after postharvest application was low. Furthermore, PMA–qPCR gave valuable information on viable population behavior and the presence of residual DNA from dead cells. In general, these studies help to understand the persistence of antagonists when applied under postharvest conditions and will lead to optimization of time and mode of application.

Dilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface.

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 TaqMan 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 10(3) cells per reaction, and the standard curve was linear within a range of 5log 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 5days in plastic boxes stored at 0°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.