Pantoea Agglomerans CPA-2 Research Articles

DNA-based methodologies for the quantification of live and dead cells in formulated biocontrol products based on Pantoea agglomerans CPA-2

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 2.17log10CFU or cellsg/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 3days, spray drying formulation rehydrated with 10% non-fat skimmed milk (NFSM) was as effective as fresh cells to control Penicillium digitatum in oranges.

Environmental monitoring of the biocontrol agentPantoea agglomeransCPA-2 applied to citrus fruit at preharvest

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.

Molecular tools applied to identify and quantify the biocontrol agent Pantoea agglomerans CPA-2 in postharvest treatments on oranges

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.

Development of PMA real-time PCR method to quantify viable cells of Pantoea agglomerans CPA-2, an antagonist to control the major postharvest diseases on oranges

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.

Detection and quantification by PCR assay of the biocontrol agent Pantoea agglomerans CPA-2 on apples

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 103 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.

Influence of diluent and sample processing methods on the recovery of the biocontrol agent Pantoea agglomerans CPA-2 from different fruit surfaces

Determining the populations of biocontrol agents applied as a postharvest treatment on fruit surfaces is fundamental to the assessment of the microorganisms' ability to colonise and persist on fruit. To obtain maximum recovery, we must develop a methodology that involves both diluent and processing methods and that does not affect the viability of the microorganisms. The effect of diluent composition was evaluated using three diluents: phosphate buffer, peptone saline and buffered peptone saline. An additional study was performed to compare three processing methods (shaking plus sonication, stomaching and shaking plus centrifugation) on the recovery efficiency of Pantoea agglomerans strain CPA-2 from apples, oranges, nectarines and peaches treated with this biocontrol agent. Overall, slight differences occurred among diluents, although the phosphate buffer maintained the most ideal pH for CPA-2 growth (between 5.2 and 6.2). Stomaching, using the phosphate buffer as diluent, was the best procedure for recovering and enumerating the biocontrol agent; this fact suggested that no lethal effects from naturally occurring antimicrobial compounds present on the fruit skins and/or produced when the tissues were disrupted affected the recovery of the CPA-2 cells, regardless of fruit type. The growth pattern of CPA-2 on fruits maintained at 20°C and under cold conditions was similar to that obtained in previous studies, which confirms the excellent adaptation of this strain to conditions commonly used for fruit storage.

Endospore production allows using spray-drying as a possible formulation system of the biocontrol agent Bacillus subtilis CPA-8

The role of endospore production by Bacillus subtilis CPA-8 on survival during spray-drying was investigated by comparison with a non-spore-forming biocontrol agent Pantoea agglomerans CPA-2. Endospore formation promoted heat resistance in CPA-8 depending on growth time (72 h cultures were more resistant than 24 h ones). The survival of CPA-8 and CPA-2 after spray-drying was determined after being grown in optimised media for 24 and 72 h. Spray-dried 72 h CPA-8 had the best survival (32%), while CPA-2 viability was less than 2%. CPA-8 survival directly related with its ability to produce endospores. Spray-dried CPA-8 reduced Monilinia fructicola conidia germination similarly to fresh cells, demonstrating that spray-drying did not adversely affect biocontrol efficacy. Endospore production thus improves CPA-8 resistance to spray-drying. These results can provide a reliable basis for optimising of the spray-drying formulation process for CPA-8 and other microorganisms.

Anti-oxidant activity of oranges after infection with the pathogen Penicillium digitatum or treatment with the biocontrol agent Pantoea agglomerans CPA-2

The suppression or generation of hydrogen peroxide (H 2O 2) in fruit as well as the changes in the superoxide dismutase (SOD) and catalase (CAT) activities due to Penicillium digitatum infection were evaluated on oranges. These biochemical parameters were also determined when oranges were treated with the biocontrol agent Pantoea agglomerans CPA-2. After harvest, oranges were divided in two batches of fruit: one was used immediately and another stored at 4 °C for 2 months. In both cases, H 2O 2 content and changes in enzyme activities were determined at specific time intervals in oranges inoculated with P. digitatum or treated with P. agglomerans. Similar levels of H 2O 2 were observed during the first 3 days after inoculation in all treatment (control, infected and treated fruit). A short time after, CPA-2 treated fruit showed an accumulation of H 2O 2 while infected fruit exhibited a sharp decrease in H 2O 2 levels. An increase of SOD and CAT activities was also observed in CPA-2 treated oranges; in contrast, infected fruit showed a significant reduction of both enzymatic activities. This biochemical approach suggests that P. digitatum suppresses H 2O 2 production as well as SOD and CAT activities in orange tissue as response to infection process. In contrast, the biocontrol agent P. agglomerans CPA-2 triggers H 2O 2 production and both enzymatic activities probably as a mechanism of action to prevent citrus fruit against future infections of green mold. Our data suggest that P. agglomerans CPA-2 may act as antagonist against green mold not only by competition for nutrients and space but also by inducing oxidative response in orange tissue.

Preventive and curative activity of combined treatments of sodium carbonates and Pantoea agglomerans CPA-2 to control postharvest green mold of citrus fruit

Preventive and curative activity of 2 min dips in 3% sodium carbonate (SC) or sodium bicarbonate (SBC) aqueous solutions heated to 40 °C, alone or followed by the application of 2 × 10 8 CFU/mL of the biocontrol agent Pantoea agglomerans CPA-2 (BA), were simultaneously evaluated for the control of postharvest green mold, caused by Penicillium digitatum, in artificially inoculated Lanelate and Valencia oranges. Fresh cells of BA proliferated inside rind wounds and their survival was not adversely affected by the presence of residues of SC or SBC. Green mold incidence after 7 d of incubation at 20 °C in rind wounds treated after fungal inoculation (curative activity) was 15%, 40%, or 15% in oranges treated with SC, BA, or SC + BA and 5%, 45% or 0% in oranges treated with SBC, BA, or SBC + BA, respectively, while it was about 90% in untreated control fruit. Green mold incidence in rind wounds treated before inoculation or reinoculation with the pathogen (preventive activity in pre-existing wounds) was 10% and 2%, or 15% and 8%, respectively, in oranges treated with SC and SC + BA, and 3% and 5%, or 20% and 5%, respectively, in oranges treated with SBC and SBC + BA. Green mold incidence in wounds inoculated after treatment (preventive activity in new wounds) was 55% and 25%, and 60% and 40% in oranges treated with SC and SC + BA, or SBC and SBC + BA, respectively. Additionally, the duration of the protective effect of SBC, BA, and SBC + BA was assessed in Eureka lemons and Valencia oranges. In both species, all three treatments effectively protected pre-existing rind wounds during 7 d of storage at 10 °C. After 0, 1, and 2 d, but not after 4 or 7 d, the protective effect of SBC was significantly inferior to that of BA and SBC + BA. The integration of treatments is a promising approach to replace the use of conventional fungicides to control green mold in citrus packinghouses.

Control of postharvest diseases on citrus fruit by preharvest application of the biocontrol agent Pantoea agglomerans CPA-2 : Part I. Study of different formulation strategies to improve survival of cells in unfavourable environmental conditions

The objective in the present study was to investigate the survival and effectiveness of different biological agent Pantoea agglomerans formulations against Penicillium spp. with different preharvest treatments. Results indicated a high sensitivity of non-adapted and osmotic-adapted P. agglomerans cells to environmental conditions in the field, resulting in preharvest treatments which were ineffective against Penicillium spp. In the second part of this study, dry conditions and solar radiation were identified as important environmental conditions that seriously affect populations of P. agglomerans cells. Different formulation strategies were tested in order to improve the resistance of cells to unfavourable environmental conditions. Osmotic-adapted P. agglomerans cells in the presence of 25 g L −1 of NaCl in the production medium [osmotic-adapted treatment (P25)] or at water activities ( a w) of 0.98 [osmotic-adapted treatment (P98)] had higher survival rates than non-adapted cells, when these cells were sprayed on oranges and stored in hermetically sealed chambers at a low RH of 43%. Among seven additives tested, the presence of 5% Fungicover in the bacterial suspension improved adherence and persistence of P. agglomerans cells on oranges exposed to unfavourable conditions. Therefore, while P. agglomerans cells sprayed alone had log values of 0.5 CFU cm −2, in combination with Fungicover the population level of P. agglomerans cells reached log values of 5 and 4.2 CFU cm −2, at 0 and 24 h after application. Lyophilised cells showed greater resistance to unfavourable environmental conditions than fresh cells. The present study has demonstrated that the formulation improvement can provide better performance of biocontrol agents under environmental conditions non-conducive for growth and survival.

Control of postharvest diseases on citrus fruit by preharvest applications of biocontrol agent Pantoea agglomerans CPA-2 : Part II. Effectiveness of different cell formulations

Infection of citrus fruit by postharvest pathogens often occurs in the field prior to harvest; therefore, it could be advantageous to apply biocontrol agents before harvest, which would reduce initial infection and then remain active and control pathogens in storage and under commercial conditions. The objective of the present work was to evaluate the effectiveness of different formulations of Pantoea agglomerans applied preharvest for controlling postharvest diseases on citrus. Results confirmed the protective effect of the additive Fungicover (FC) on populations of P. agglomerans exposed to non-conducive field conditions. In general, when osmotic-adapted and lyophilised P. agglomerans cells were used in bacterial treatments, these treatments showed greater survival rates than treatments with non-osmotic-adapted or fresh cells under field conditions. However, this superiority was only found when Fungicover was also added to suspensions of bacterial treatments. Therefore, bacterial treatments with Fungicover had population levels of P. agglomerans cells 1.2 and 2.8 log CFU cm −2 higher than bacterial treatments without Fungicover during field experiments. These results allowed us to conclude that it is possible to improve environmental stress tolerance and ecological competence of P. agglomerans cells by integrating certain formulation strategies. Consequently, the improved formulation of P. agglomerans provided an effective control for orange fruit against natural postharvest pathogen infections and artificial infections of Penicillium digitatum with values of decay reduction higher than 50%. These latter results also demonstrated that it is possible to control postharvest pathogens using bacterial preharvest treatments.

Development and application of a SCAR marker to monitor and quantify populations of the postharvest biocontrol agent Pantoea agglomerans CPA-2

Pantoea agglomerans CPA-2 is an effective biocontrol agent of postharvest diseases of citrus and pome fruit. A monitoring technique was developed for its identification and to quantify its populations. The methodology used consisted of (i) searching for a semi-selective medium, (ii) identification of molecular markers and (iii) monitoring population dynamics in a commercial trial. As a semi-selective medium, Malonate Broth Agar supplemented with tetracycline hydroxychloride and incubation at high temperature (max. of 40 °C) facilitated the selective recovery of P. agglomerans CPA-2 colonies. The RAPD technique was applied to a collection of 13 strains of P. agglomerans, including CPA-2. Among the 12 primers tested, OPL-11 amplified a fragment (about 720 bp) specific to strain CPA-2. On the basis of this fragment, two SCAR markers were amplified using a primer pair derived from OPL-11 elongation. A first SCAR marker of 720 bp was specifically amplified for the strain CPA-2 and a second one of 270 bp was obtained for all P. agglomerans strains tested, including CPA-2. Commercial trials demonstrated a significant reduction of decay with the treatment of formulated cells of P. agglomerans CPA-2. Population dynamics of CPA-2 in commercial trials were determined on fruit surfaces and in the environment using both the classical plating technique and PCR with SCAR primers. In general, no significant differences were observed between results obtained from the two methods. On fruit surfaces, 1 day after CPA-2 applied its population by classical methods was 4.37 × 10 6 cfu wound −1 and at the end of the experiment the population increased to 5.8 × 10 5 cfu wound −1. The percentages of colonies identified as P. agglomerans CPA-2 at these sampling times using SCAR primers were 90 and 95%, respectively. Population dynamics in the environment to evaluate the environmental fate of P. agglomerans CPA-2 showed that it has a limited persistence and limited capacity for dispersion.

Acid tolerance response induced in the biocontrol agent Pantoea agglomerans CPA-2 and effect on its survival ability in acidic environments

The aim of this work was to optimize acid stress conditions for induction of acid tolerance response (ATR) in the biocontrol agent Pantoea agglomerans and study the effect of ATR induced on the ability to survive under acidic conditions. Initially, Pantoea agglomerans was grown in mild acidic conditions (pH 6.0, 5.5, 5.0 and 4.0) in order to induce ATR. The highest ATR was induced at initial pH of 5 using malic or citric acid. A first in vitro experiment was carried out. Thus, basal liquid medium at different pHs (3.0, 3.5, 4.0 and non-acidified) were then inoculated with acid-adapted and non-adapted inocula of P. agglomerans and survivals were examined during incubation at 25 or 4 degrees C. It was found that acid adaptation enhanced the survivals of Pantoea agglomerans CPA-2 cells at pH levels at which the cells were unable to grow (<3.5 and 4.0, at 25 and 4 degrees C, respectively). In contrast, in pH levels at which the cells were able to grow (pH 4.0 at 25 degrees C and non-acidified medium at 25 and 4 degrees C) no-differences were found between adapted and non-adapted cells. In in vivo tests, adapted and non-adapted cells were inoculated in wounds on mandarins and pome fruits. No differences were found between adapted and non-adapted cells and biocontrol efficacy was maintained. The present study demonstrated that exposure of Pantoea agglomerans to mild acidic conditions could induce acid resistance in this biocontrol agent.

Relative importance of amino acids, glycine?betaine and ectoine synthesis in the biocontrol agent Pantoea agglomerans CPA-2 in response to osmotic, acidic and heat stress

The objective of this work was to determine the role of different compatible solutes in adaptation of Pantoea agglomerans CPA-2 at different stages of growth to solute (0.98, 0.97, 0.96 aw), heat (35 and 40 degrees C) and acidic (pH 4.0, 5.0, 6.0) stress. Solute stress was imposed by using NaCl, glucose or glycerol, and pH was imposed with malic and citric acids. The accumulation of glycine-betaine, ectoine and amino acids in bacterial cells was quantified using high performance liquid chromathography (HPLC). There was a significant (P<0.05) accumulation of glycine-betaine (NaCl modified, 100-150 micromol g(-1) dry weight of cells) and ectoine (glucose modified media, >340 micromol g(-1) dry weight of cells) in the cells over a 48 h incubation period when compared with controls (<10 micromol g(-1) dry weight of cells). Chromatographic profile of amino acids was different with respect to control when NaCl or glucose was used as osmolyte. Pantoea agglomerans CPA-2 cells synthesised significant amounts of glycine-betaine and ectoine in response to imposed solute stress. However, these compounds and tested amino acids were not involved in cellular adaptation to either heat or pH stress. This type of information can be effectively applied to improve ecophysiological quality of cells of bacterial biocontrol agents for better survival and biocontrol efficacy in the phyllosphere of plants.

Application of Pantoea agglomerans CPA-2 in combination with heated sodium bicarbonate solutions to control the major postharvest diseases affecting citrus fruit at several mediterranean locations

We determined the potential of using a formulated product based on Pantoea agglomerans CPA-2, either alone or in combination with heated sodium bicarbonate (SBC) solutions, to control the major postharvest diseases affecting citrus crops in the Mediterranean region. Treatments applied either individually or in combination were tested in semi-commercial and commercial trials carried out with oranges and mandarins from the Algarve, Andalusia and Catalonia. Firstly, several formulations of the biocontrol agents were tested in laboratory trials; one of them, a freeze-dried formulation of P. agglomerans strain CPA-2 called FD10-3, was chosen for combined with SBC. This formulation, applied at 2 × 108cfu ml−1 and the SBC treatment, applied at 3% 50°C for 20–40 s, demonstrated that it was possible to reduce decay development in laboratory trials. Semi-commercial applications of FD10-3 and 3% SBC solution at 50°C for 40 s showed excellent control of decay in unwounded mandarins and oranges artificially inoculated with both Penicillium digitatum and P. italicum. No rind injuries or residues attributable to hot water or SBC were observed on treated fruits. Combined treatment provided better control than the two treatments applied separately. Commercial trials demonstrated an important reduction in natural decay with the treatment of SBC 3% at 50°C for 40 s. Furthermore, bacterial-product formulation treatment significantly reduced the percentage of infected fruit and in some cases this reduction was equal to chemical treatments. Even so, no improvement in efficacy was observed with the combination of FD10-3 and SBC in the commercial test. We also assessed the ability of FD10-3 to grow at the wound site in oranges, whether alone or in the presence of SBC, and also its compatibility with standard citrus packinghouse practices.

Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments

To study the improvement of tolerance to low water activity (aw) and desiccation during spray drying in Pantoea agglomerans cells subjected to mild osmotic stress during growth. The micro-organism was cultured in an unmodified liquid (control) or in aw-modified media, and viability of these cells was evaluated on unstressed (0.995) and 0.96 aw stressed solid media, in order to check total viability and aw stress tolerance respectively. Significant improvements in viability on unmodified medium were observed with cells grown for 24 h in NaCl 0.98 aw, glycerol 0.98 aw and 0.97 aw and for 48 h in NaCl 0.98 aw and 0.97 aw modified media. Both yield improvements and water stress tolerance were achieved with low aw media. Cells grown for 24 h in NaCl 0.98 aw or for 48 h in NaCl 0.98 aw, 0.97 aw and 0.96 aw, glucose 0.97 aw and glycerol 0.97 aw showed improved aw stress tolerance in comparison with control cells. The best results were obtained with NaCl treatments (0.98 aw and 0.97 aw) which also exhibited better survival rates than control cells during spray-drying process and maintained their efficacy against postharvest fungal pathogens in apples and oranges. NaCl treatments are very appropriate for improving P. agglomerans low aw tolerance obtaining high production levels and maintaining biocontrol efficacy. Improving stress tolerance of biocontrol agents could be an efficient way to obtain consistency and maintain efficacy of biological control under practical conditions.

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.

Modes of Action ofPantoea agglomeransCPA-2, an Antagonist of Postharvest Pathogens on Fruits

Pantoea agglomerans CPA-2 is an effective antagonist against the postharvest pathogens Penicillium digitatum and Penicillium italicum on citrus fruits but its mode of action is unknown. Possible mechanisms studied in this work were antibiosis, induced resistance, competition and production of chitinolytic enzymes. P. agglomerans CPA-2 was unable to produce antibiotics or chitinolytic enzymes under the conditions tested. Induction of resistance by P. agglomerans CPA-2 was studied in oranges by measuring phenylalanine ammonia lyase and peroxidase enzyme activity in the orange peel at different time points after inoculation with the antagonist and/or the pathogen. No significant augmentation of enzyme activity after inoculation of oranges with P. agglomerans CPA-2 in the presence or absence of the pathogen was observed. P. agglomerans was effective only when it is in close contact with the pathogens. Competition for nutrients was studied using tissue culture plates with cylinder inserts, which allowed competition for nutrients to be studied without competition for space since physical contact between pathogen and antagonist was avoided. The presence of P. agglomerans in the tissue culture wells clearly decreased the germination of Penicillium conidia present in the cylinder when diluted orange peel extract or diluted potato dextrose broth was the nutrient source. Germination of Penicillium conidia, however, was almost completely inhibited when pathogen and antagonist were in physical contact. These results indicate that competition for nutrients is one of the modes of action of P. agglomerans CPA-2, but that physical contact between pathogen and antagonist is important for effective control.

Water activity, temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2.

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P. agglomerans did not change at varying pH levels (4.5-8.6). The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes. This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.

Survival of Pantoea agglomerans Strain CPA-2 in a Spray-Drying Process

Spray drying could be a suitable method for preserving microorganisms, as it allows large quantities of cultures to be dried at low cost. The aims of this paper were to evaluate the effects of spray-drying conditions on survival of the biocontrol agent Pantoea agglomerans CPA-2, which has shown antifungal activity against Penicillium expansum and Penicillium digitatum on citrus fruits. Various compounds cited in the bibliography as carriers were tested in our spray drying, and some salts (MgSO4, K2SO4, and Na2CO3) and dairy products (lactoserum or nonfat skimmed milk [NFSM]) showed the best results in terms of recovered powder. Outlet temperature had more influence on the death of bacteria than inlet temperature. P. agglomerans was heat sensitive, and the activation energy was around 6 kcal/mol K when MgSO4 (10%) or NFSM (10%) were used as carriers and only 3 kcal/mol K when the combination of MgSO4 (10%) and NFSM (10%) was used. The highest powder recovery was obtained when NFSM was used as the rehydration medium. Although the percentage of powder recovery was not high (around 50%) and viability was low, the results suggest that with bigger spray dryers, we could expect a lower outlet temperature and probably an increased viability. Further research into spray-dryer design is needed in order to demonstrate this.