The application of the biocontrol agent Pantoea agglomerans EPS125 to unwounded fruits was practically ineffective for control of postharvest blue mould caused by Penicillium expansum when the treatment and subsequent wounding and pathogen inoculation were separated by periods of unfavourable conditions. This was due to a rapid decrease in viability of the alocthonous introduced biocontrol agent in the intact peel surface. A system for osmoadaptation of the biocontrol agent was developed by combining saline osmotic stress and osmolyte amendment to the growth medium. Osmoadapted cells accumulated trehalose and glycine betaine (GB) intracellularly and showed a higher tolerance to desiccation than non-osmoadapted cells. Osmoadaptation in NaCl plus GB during inoculum preparation increased considerably survival on the peel surface of apple fruits. This effect was significant under low relative humidity (RH) and fluctuating RH conditions, but was not significant at high RH. Osmoadaptation significantly improved blue mould control under conditions where the standard biological control treatments were ineffective. The rot diameter was significantly reduced in apple fruits which were treated with EPS125 and incubated for several days under low, high or fluctuating RH, followed by wounding and inoculation of P. expansum. Growth of EPS125 with NaCl, either with or without the addition of GB, was an effective osmoadaptation treatment for improving blue mould rot control. However, the addition of GB to the NaCl amended growth medium increased 4–5-fold growth rate and OD of the cultures. This is an advantage for mass production of P. agglomerans EPS125 in a NaCl amended growth medium.
Osmotic stress tolerance
Blue mould control
Biological control of postharvest rot of fruits and vegetables has been the focus of considerable research . Several bacteria and fungi have been isolated and some are developed commercially as biopesticides . However, one of the main limitations of this technology is that upon introduction of the antagonists in intact plant surfaces, survival is limited and frequently population levels decrease . If mechanical damage of the epidermal tissues occurs too late after biofungicide treatment, the remaining population levels of the antagonist may not colonize, grow and protect these sites from pathogen entrance and infection . Moreover, strategies of postharvest fruit rot control have been developed based on preharvest treatment in the field . However, preharvest application of the biological control agent may reduce its survival due to unfavourable conditions in the field and may render ineffective fungal rot control during handling and storage. Also, the development of commercial products requires mass production and the dehydration for formulation to get a stable product through time for delivery . Unfortunately, losses of viability can be as high as several orders of magnitude due to cells viability damage during the process of dehydration . Therefore, survival of the biocontrol agent under different stages of its development and use is one of the critical points for implementation of biofungicides in postharvest fruit control.
Several microorganisms survive under limited availability of water by means of a physiological process of osmoadaptation that consists of intracellular accumulation of compatible solutes or membrane stabilisation [6–8]. Sugars, polyols, heterosids, aminoacids or aminoacid derivatives, and diverse substances are synthesized or taken from the growth medium, and accumulated intracellularly to counterbalance the osmotic pressure of the environment, and maintain cell turgor [9–11]. Osmoprotectants as glycine betaine (GB) and trehalose have been detected in Gram-negative bacteria exhibiting an increased tolerance to desiccation and other beneficial effects under stress conditions .
Pantoea agglomerans is one of the abundant components of the normal bacterial microbiota of plants . Strains have been isolated that exert beneficial effects on plants as biological control agents of certain aerial plant pathogens or in frost damage control  or are active in plant growth promotion . Two strains of P. agglomerans exhibit a high capacity to colonize fruit wounds and are interesting as biocontrol agents of postharvest diseases of fruits. Strain EPS125 control stone fruit diseases caused by Rhizopus stolonifer or Monilinia laxa  and strain CPA-2 that control fruit rot diseases on apple, pear and citrus [17,18]. However, the limited survival of P. agglomerans under osmotic stress conditions is one important limitation affecting inoculum preparation and efficacy. Procedures for freeze-drying and spray-drying of P. agglomerans to preserve cells in a dehydrated form have been reported for strain CPA-2 . However, physiological studies and methods of osmoadaptation to improve environmental stress tolerance and ecological competence during mass production have not been previously reported.
The aim of the present work was to study the effect of osmoadaptation treatments on improvement of: (i) growth under osmotic stress conditions, (ii) survival to desiccation or low relative humidity on the fruit surface, and (iii) efficacy of control of blue mould rot under low humidity conditions and delayed pathogen inoculation.
2 Materials and methods
2.1 Bacterial strain and growth conditions
P. agglomerans EPS125 was grown in a glucose minimal medium (GMM) containing per liter 5 g of glucose, 1 g of NH4Cl, 3 g of KH2PO4, 2.4 g of Na2HPO4, 0.5 g of NaCl and 0.2 g of MgSO4 at pH 7. The medium was supplemented with NaCl prior to autoclaving to give final concentrations of 0.1, 0.3, 0.5 or 0.7 M depending on the experiment. GB or trehalose (T) were also added in some experiments upon sterilization by filtration through 0.45 μm pore diameter filters, to a final concentration of 0.1 or 1 mM. For inoculation, a preculture was first prepared with the same medium composition of the characteristics of the medium used in the experiment: GMM alone or amended with NaCl, GB or T. Cultures were grown in an orbital shaker at 150 rpm and 25 °C for 24 h.
2.2 Effect of timing of treatment with EPS125, wounding of fruits and pathogen inoculation on efficacy of control of blue mould rot
Liquid cultures of P. agglomerans EPS125 were prepared in LB broth and incubated at 25 °C for 24 h. Then cells were harvested by centrifugation at 3000g for 30 min and resuspended in sterile distilled water to a concentration of 108 cfu ml−1. Golden apples were collected from a commercial orchard near Girona, and were washed, surface-disinfected by immersion for 1 min in a dilute solution of hypochlorite (1% active chlorine), washed two times by immersion in distilled water, and dried in air until they were no longer wet. The fruits were treated with EPS125 in two different ways. In the standard inoculation fruits were wounded with a cork borer making nine wounds per fruit of approximately 9 mm2 and 5 mm depth of each wound, treated by immersion in the suspension of EPS125 and inoculated immediately with the pathogen P. expansum with 10 μl of a pathogen suspension of 104 conidia ml−1 into wounds. In the delayed inoculation fruits were treated with EPS125 and maintained for 15 days at 20 °C. Then wounds were made and the pathogen was inoculated. Inoculated apples were placed in polystyrene tray packs which were placed into boxes and maintained at 20 °C. Three replicates of six fruits per replicate were prepared for each treatment. Appropriate controls consisting of non-treated fruits inoculated with the pathogen were done. The lesion diameter was measured at 5, 7, 10 and 13 days after pathogen inoculation. The experiment was repeated two times.
2.3 Effect of osmoadaptation on growth in liquid culture under osmotic stress conditions
Cultures were grown in GMM supplemented with NaCl and the osmoprotectants GB or T (0, 0.1 or 1 mM). Triplicates of each treatment were done on each of the two experiments performed. The experiment 1 was performed at 0.5 M NaCl and experiment 2 at 0.7 M NaCl. The medium was inoculated with a 1% v/v stationary phase preculture grown in the equivalent growth medium. Growth was monitored at 25 °C for 48 h using a Bioscreen C microplate reader (Labsystems) by means of measurements of OD at 600 nm. A vibration shaking of 2 min was programmed before OD reading to prevent cell sedimentation and favour aeration. Maximum OD and growth rate (μ) were determined for each medium composition condition and replicate.
2.4 Analysis of intracellular trehalose and glycine betaine contents
EPS125 was grown in 500 ml Erlenmeyer flasks containing 200 ml GMM supplemented with either 0.01, 0.1, 0.3, 0.5 or 0.7 M NaCl and the corresponding osmoprotectant treatment at 0, 0.1 or 1 mM GB. A triplicate of each NaCl-GB combination was done. Cultures were grown as described above and viable cell concentrations were determined by means of 10-fold serial dilution and plating onto LB agar Petri plates. Then cells were harvested by centrifugation at 3000g after 30 min and the pellet was extracted with 20 ml of ethanol under vigorous shaking at room temperature for 10 h. The ethanol extracts were rotary evaporated to dryness at 40 °C and the dry material was redissolved in 1 ml of acetonitrile–water (1/1, v/v) before analysis. Trehalose and GB were quantified by high-pressure liquid chromatography (HPLC) upon injection of 20 μl in a Waters HPLC (model 610, Waters, Mildford, Madison, USA). The apparatus was equipped with a Zorbax carbohydrate analytical column (Agilent Technologies) attached to a precolumn and operating at a flow rate of 1.4 ml min−1 with a mobile phase of acetonitrile–water (75:25, v/v). Absorbance measurements were performed with a refractive index detector (RID) (model 990, Waters). Under these conditions GB and trehalose were detected at 4.7 and 11.3 min retention time, respectively. The intracellular osmolyte contents was expressed as specific contents in fg cfu−1.
2.5 Effect of osmoprotection treatments in the viability of EPS125 under dry conditions
Osmoadapted cells were obtained by growth in GMM amended with 0.5 M NaCl alone or plus 0.1 mM GB. Non-osmoadapted cells were cultured in unamended GMM. Culture aliquots of 1 ml were distributed into 1.5 ml Eppendorf tubes and harvested by centrifugation at 3000g for 5 min. The supernatant was carefully discarded and the pellet containing cells was dried in a vacuum desiccator at 25 °C using silica gel as desiccant. The tubes were maintained in the desiccator and samples were taken for cell viability analysis at different times during a period of three months. The experimental design consisted of three replicates of each treatment and date of assessment. For analysis, the dry pellets were resuspended in 1 ml of sterile 0.9% w/v NaCl and maintained under shaking for 1 h in the refrigerator. Suspensions were 10-fold serially diluted in water and appropriate dilutions were plated onto LB agar plates and incubated for 24 h at 25 °C. Colonies were counted and counts were transformed to cfu ml−1 of culture. Data were transformed to percentage of surviving cells.
2.6 Effect of osmoadaptation on survival in the peel apple surface under different storage conditions
Golden apples were collected and prepared as described above. Then fruits were treated by immersion in a 108 cfu ml−1 suspension of EPS125 Rif+ (resistant to rifampycine). The biological control agent was prepared under non-osmoadapted or osmoadapted conditions as previously described, either in GMM 0.5 M NaCl alone or amended with 0.1 mM GB. After the corresponding treatment, fruits were placed in polystyrene tray packs which were placed into non-hermetic boxes and maintained at 20 °C under three different conditions: high (90% RH), low (45% RH) and fluctuating (24 h low, 24 high RH) relative humidity. Incubations at the proper RH conditions were performed in controlled environment chambers (PGR-15, Conviron, Canada; SGC097.PFX.F, Fitotron, Sanyo Gallenkamp PLC, UK). RH was measured by the control system of the corresponding equipment either using wet-dry bulb temperature (Conviron) or electronic sensor (Fitotron). The variability of the actual values of RH during experiments was always less than 5% around the selected values. Low relative humidity (45%) was obtained by including into the chamber CaCl2 as humidity absorber. Low, high and fluctuating conditions were used to simulate three usual exposure situations of fruits since storage to market and consumer conditions. Three replicates of six fruits each were prepared for each osmoadaptation treatment and storage condition. Samples of two apples were taken immediately or after 14 days in experiment 1 and after 21 days in experiment 2. Then 8 subsamples were taken from the two apples consisting of intact peel surface using a sterilized 10 mm cork borer. The tissue cylinders were trimmed to about 5 mm in length discarding part of the mesocarp tissue. Then the tissue plug was placed in a sterile plastic bag with 20 ml of 0.05 M phosphate buffer (pH 7) and 0.1% peptone, and ground with a pestle. The clear supernatant was serially diluted and appropriate dilutions were seeded onto LB agar plates supplemented with 100 μg ml−1 of rifampicin to counterselect the strain inoculated. Plates were incubated at 25 °C and the colonies counted after 24 h. The population levels of P. agglomerans were expressed as log10cfu cm−2 of fruit peel.
2.7 Effect of osmoadaptation on efficacy of control of blue mould rot
The treatments consisted of immersion of Golden apples in water (non-treated control), non-osmoadapted, osmoadapted in 0.5 M NaCl, and osmoadapted in 0.5 M NaCl plus 0.1 mM GB. The concentration of EPS125 was 108 cfu ml−1. After the treatment, fruits were placed in polystyrene tray packs which were placed in boxes and maintained at 20 °C under three different conditions of incubation: high (90% RH), low (45% RH) and fluctuating (24 h low, 24 high RH) relative humidity. After 15 days of treatment, the fruits were wounded with a flame sterilized cork borer (nine wounds per fruit) (3 mm depth, 5 mm diameter) and were incubated again at 20 °C for 24 h. Then each wound was inoculated with 10 μl of a suspension of Penicillium expansum at 104 conidia ml−1 and incubated at the same temperature. The lesion diameter was assessed at 3 and 7 days after pathogen inoculation. The experimental design consisted of three replicates of three fruits per replicate and nine wounds per fruit, for each treatment (three osmoadaptation treatments, three incubation conditions). The experiment was performed two times.
3.1 Influence of timing treatments with EPS125 and inoculation of pathogen on blue mould rot control
Treatment of wounded apples with the biocontrol agent followed by inoculation of the pathogen delayed significantly the progression of infections in the two experiments performed (Fig. 1). However, there was no disease control in fruits in which wounds and inoculation of the pathogen were delayed 15 days from the EPS125 treatment.
Time course of blue mould rot on Golden apples treated with P. agglomerans EPS125 and inoculated with P. expansum according to different strategies of treatment. Fruits were wounded, treated with the biocontrol agent and immediately inoculated with P. expansum (□) or treated with the biocontrol agent incubated for 15 days at 20 °C and then wounded and inoculated with P. expansum (O). Wounded fruits without treatment and inoculated with P. expansum (♦) and non-wounded fruits without treatment maintained for 15 days at 20 °C and then wounded and inoculated with P. expansum (Δ) were used as controls. Data correspond to the mean rot diameter of three repetitions of five fruits per repetition. The experiment was performed two times (panels A and B). The bars represent the mean confidence interval for each experiment.
3.2 Effect of osmoprotectant treatments in liquid culture on growth under osmotic stress conditions induced by NaCl
The growth of P. agglomerans was significantly affected by NaCl and osmolyte treatments. Growth was slight or absent in GMM amended with high concentrations of NaCl in comparison to the non-amended control (Table 1). In the two experiments performed the addition of the osmoprotectant GB restored growth rate and OD values to levels not significantly different than the control, especially at a concentration of 1 mM. However, the effect of trehalose was less important and was only observed in experiment 1 performed at a lower NaCl concentration (0.5 M) than experiment 2.
Effect of addition of the osmolytes glycine betaine and trehalose on growthaof cultures of Pantoea agglomerans EPS125 under saline osmotic stress induced by sodium chloride treatment
Growth rate (h−1)b
aStationary phase cultures previously grown in the equivalent medium were inoculated at 1% v/v in the corresponding medium.
bMaximum OD, Maximum optical density obtained on the culture. Data are the mean of three replicates. Means in the same column followed by the same letter are not significantly different according to the Fisher's LSD test (P= 0.05).
c+, NaCl added; −, NaCl not added. NaCl concentration was 0.5 M in experiment 1 and 0.7 M in experiment 2.
dGB 0.1, glycine betaine 0.1 mM; GB 1, glycine betaine 1 mM; T 0.1, trehalose 0.1 mM; T 1, trehalose 1 mM.
eThe experiment was performed twice.
3.3 Storage of osmolytes during growth under osmotic stress
Trehalose cell contents increased upon increasing NaCl added to the culture medium in the two experiments performed at both GB concentrations (0.1 and 1 mM) (Fig. 2). Trehalose was also accumulated in treatments with NaCl alone at 0.3 and 0.5 M in the second experiment. GB contents increased significantly only at the high GB concentration in the culture medium.
Trehalose (left panels) and glycine betaine (GB) (right panels) cell contents of P. agglomerans EPS125 upon growth in glucose minimal medium amended with GB at 0.1 (), 1 mM (Δ) or non-treated (♦) and at different NaCl concentrations. The results are the mean of three replicates. In experiment 1 (A and B) the preculture was inoculated at 1% (v/v) whereas in experiment 2 (C and D) the inoculation was performed at 10%(v/v). The bars represent the mean confidence interval for each experiment.
3.4 Effect of osmoadaptation in survival under dry conditions
Exponential phase cells grown in standard culture medium were highly sensitive to desiccation and their viability declined rapidly with time (Fig. 3). In contrast, cells cultured in the standard medium either supplemented with NaCl to induce trehalose accumulation or with NaCl plus GB to induce trehalose and GB accumulation, showed a high tolerance to desiccation. After 100 days of incubation under dry conditions, untreated controls had viable cell concentrations of 1 × 106 for experiment 1 and 2 × 103 cfu ml−1 for experiment 2, which correspond to 0.1% and 0.0001% survival. Osmoadapted cells had significantly higher tolerance to dryness than non-osmoadapted controls. In the first experiment both osmoadaptation treatments had a final viable cell concentration of 5 × 107 cfu ml−1 representing 3% of survival. In the second experiment the final viable cell concentrations were 1 × 107 cfu ml−1 in the NaCl osmoadapted (2% survival) and 1 × 106 cfu ml−1 (0.2% survival) in the NaCl plus GB osmoadapted cultures.
Effect of osmoadaptation on the viability of Pantoea agglomerans EPS125 during storage under dry conditions. Bacteria were osmoadapted by amendment of the growth medium with 0.5 M NaCl (□) or 0.5 M NaCl plus at 0.1 mM (Δ) or at 1 mM (Δ). Non-osmoadapted cells were cultured in the absence of NaCl and osmolytes (♦). The results are the mean of three replicates. Error bars represent the confidence interval of the mean. The experiment was performed two times (panels A and B). The initial viable cell amount for each tube was 1.7 × 109 cfu in experiment A and 8.0 × 108 cfu in experiment B.
3.5 Survival on the fruit surface under different humidity conditions depending on osmoadaptation treatment
Cell survival was dependent on the osmoadaptation during cultivation and the environment conditions of fruits after treatment (Table 2). Upon incubation at high RH for 15 days, population levels ranged from 3.8 to 4.1 in the experiment 1 and from 2.8 to 3.8 in experiment 2, but the values were not significantly different between osmoadaptative and non-osmoadaptative treatments. However, at low or fluctuating RH the population levels were lower than at high RH and there were significant differences between treatments. The most consistent effect of increase in viability was found in cells osmoadapted with NaCl plus GB and maintained under fluctuating RH. However, this effect was found in experiment 1 and only for the low RH conditions.
Effect of environmental conditions during storage of Golden apples in population levels of Pantoea agglomerans EPS125 (log10 cfu cm−2) on the fruit surface depending on osmoadaptation treatment during inoculum preparation
Relative humidity (%)a
NaCl + GB
NaCl + GB
NaCl + GB
aApples were stored at 20 °C and 90, 45 or fluctuating (flu) RH (24 h at 90% and 24 h at 45% RH).
bFruits were treated with EPS125 grown under different osmoadaptation treatments: non-treated (NT), plus NaCl (NaCl) and plus NaCl and glycine betaine (NaCl + GB).
cIn the experiment 1 the population level was assessed at 14 days and in the experiment 2 at 21 days from application.
dValues are means of 3 repetitions of 8 plugs per repetition obtained from 2 fruits. Means within the same column and in the same environment that are followed by different letters are significantly different (P < 0.05) according to the Fisher's LSD test.
3.6 Effect of osmoadaptation on efficacy of blue mould control under different relative humidity environments
In experiment 2 osmoadaptative treatments (NaCl alone or plus GB) decreased significantly lesion diameter compared to non-osmoadapted cultures at all incubation conditions except for NaCl plus GB at fluctuating RH (Fig. 4). In experiment 1 the effect was less consistent, but contrast analysis of variance for comparison of non-osmoadapted with osmoadapted treatments gave NaCl plus GB as slightly effective at high RH (NO vs. GB, P= 0.109) and significant at low RH (NO vs. GB, P= 0.023). However, the effect was not significant at fluctuating RH. Osmoadaptation treatment with NaCl alone was not significantly different from NaCl plus GB (P= 0.350). Overall, the osmoadaptation treatment produced reductions in disease severity (lesion diameter) from 35% to 53% with respect to the NO or NT controls.
Effect of osmoadaptation of Pantoea agglomerans EPS125 on blue mould rot control of Golden apples incubated at different relative humidity conditions. P. expansum was inoculated after 15 days of treatment with EPS125 prepared as follows: NO, non-osmoadapted; () NaCl, osmoadapted in NaCl 0.5 M (); NaCl-GB, osmoadapted in NaCl 0.5 M plus glycine betaine at 0.1 mM (□). NT, fruits non-treated and inoculated with the pathogen (█). Incubations at 90% RH (A and B), fluctuating 90–45% RH (C and D), and 45% RH (E and F). Bars with the same letter within the same panel do not differ significantly (P < 0.05) according to Fisher's LSD test. The experiment was performed two times (Left panels, experiment 1; right panels, experiment 2).
The application of the biocontrol agent P. agglomerans to unwounded fruits was practically ineffective in control of blue mould rot when the treatment and subsequent wounding and pathogen inoculation were separated by long periods of unfavourable relative humidity conditions. The lack of efficacy can be attributed to difficulties in colonization and survival of the biocontrol agent in the intact peel surface compared to the rapid growth observed in fresh wounds [2,16–18]. The results also agree with a reduced growth potential of Candida oleophila and Pseudomonas syringae in old or air dried wounds of apple [3,20]. The great differences in supportiveness between intact peel surface and old wounds compared to fresh wounds can be due to a decreased availability of water causing some kind of osmotic stress in the biological control agent.
Several studies have reported an improvement of resistance of microorganisms to physical–chemical stress by means of adaptative growth to unfavourable osmotic conditions [6,7,10,21–23]. We were interested in physiological methods for improvement of tolerance to environmental stresses that may favour the establishment of P. agglomerans in the fruit surface. Therefore, in the present work a system for osmoadaptation of strain EPS125 was developed by combining saline osmotic stress and GB osmolyte amendment to relieve inhibition of growth. As has been described in other microorganisms [10,24] osmoprotectant accumulation is triggered by osmotic upshifts at sodium chloride concentrations around 0.5 M. However, in P. agglomerans the osmotic upshock observed at NaCl concentrations above 0.5 M induces intracellular accumulation of trehalose and in a lesser extent of GB. We do not detected compounds like sacarose, glycerol, proline or ectoine that have been described in other microorganisms [22,23]. Trehalose accumulation by de novo synthesis has been reported in several Gram negative bacteria, mainly Enterobacteriaceae [6–8,25] and fungi . It is interesting to notice that saline stress alone or combined with GB have induced trehalose synthesis in P. agglomerans, contrarily to that happens in other Gram negative bacteria .
The osmoadapted cells of P. agglomerans showed an improved tolerance to desiccation and increased survival on the surface of apple fruits. This is in agreement with the reported relationship between accumulation of trehalose and increased tolerance to desiccation in E. coli , Saccharomyces cerevisiae  and in the biocontrol agents Trichoderma harzianum  and Candida sake . However, increased levels of trehalose, although it increases shelf-life in several microorganisms, not always improve stress tolerance to water availability . The mechanism of increasing tolerance to desiccation seems to operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state  and stabilising proteins by water replacement via hydrogen bounding [6,9]. Other types of osmotic adaptations not involving compatible solute accumulation have been characterized such as membrane phospholipid composition readjustment or synthesis of membrane derived oligosaccharides .
The present work illustrates the applicability of osmoadaptation to increase survival and fitness, as well as to improve blue mould control efficacy under conditions at which the standard biocontrol treatments may be ineffective, such as delayed inoculation of the pathogen after the biocontrol agent under unfavourable relative humidity conditions. Also, the role of trehalose and GB accumulation observed in P. agglomerans agree with the report of a direct relationship between trehalose cell contents induced by different growth media composition and blue mould rot reductions in apples by Candida sake CPA-1 . Such an improvement of survival by osmotic upshift during mass production of the biocontrol agent can explain the increase in efficacy observed in P. agglomerans under unfavourable conditions at low or fluctuating relative humidity, but not under conditions of moisture availability. This unexpected result can be interpreted because osmotic stress can be produced by the fruit tissue juice release upon wound formation and drying, and therefore osmoadapted cells are better prepared to colonize and growth in wounds. In addition, osmolyte accumulation may not be the only factor responsible of the beneficial effects observed, because osmotic stress adaptation also can have additional advantages in the biological control agent. For example, osmotic stress induced cross-protection against acid shock conditions in Listeria monocytogenes  and has been implicated as a factor contributing to the virulence potential of certain pathogenic bacteria .
Finally, the present study demonstrates that physiological manipulation of the biocontrol agent oriented to force osmoadaptation render beneficial effects such as better survival on the fruit surface under unfavourable conditions and enhancement of preemptive exclusion of the pathogen with improved efficacy of rot control.
This research was supported by grants AGL2003-03354 from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and CAL03-084 from INIA of Spain, and 2001SGR00293 from CIRIT-Generalitat de Catalunya. We thank E. Recas for assistance in HPLC analysis.
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