International Journal of Food Microbiology 84 (2003) 93 104 www.elsevier.

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Biological control of Monilinia laxa and Rhizopus stolonifer in postharvest of stone fruit by Pantoea agglomerans EPS125 and putative mechanisms of antagonism
Anna Bonaterra a, Marta Mari b, Lucia Casalini b, Emilio Montesinos a,*
a

s Santalo , 17071 Girona, Spain Institute of Food and Agricultural Technology and CeRTA-CIDSAV, University of Girona, Av. Llu b CRIOF, University of Bologna, Via Fanin, 47, 40127 Bologna, Italy Received 20 March 2002; received in revised form 29 June 2002; accepted 5 August 2002

Abstract Treatment of stone fruits (apricot, peach and nectarine) with Pantoea agglomerans strain EPS125 decreased the incidence and diameter of lesions of brown rot caused by Monilinia laxa and soft rot caused by Rhizopus stolonifer. Root control was achieved on fruits either wounded and subsequently inoculated with the pathogens or non-wounded and naturally infected from orchards. The efficacy of biocontrol was dependent on the concentration of the biocontrol agent and pathogen. At medium to low pathogen dose, optimal EPS125 concentrations were above 107 CFU ml 1. The median effective dose (ED50) of EPS125 was 4.5 104 in M. laxa and 2.2 105 CFU ml 1 in R. stolonifer. However, EPS125 was more effective in M. laxa than in R. stolonifer as indicated by the ratio between ED50 of the biocontrol agent and pathogen (Kz/Kx) which was 166 and 1263, respectively. Interactions between the strain EPS125 and the fruit surface, and M. laxa and R. stolonifer, were studied to determine the mechanisms of protection from postharvest rots. The strain EPS125 colonizes, grows and survives on stone fruit wounds. Significant inhibition of conidial germination and hyphal growth of R. stolonifer and M. laxa was achieved when the fungal and EPS125 cells were cocultivated on peel leachate or nectarine juice. However, no effect was observed when the antagonist and the pathogen cells were physically separated by a membrane filter which permits nutrient and metabolite interchange. Therefore, a direct interaction between the strain and the pathogen cells is necessary for antagonism, without a significant contribution of the production of antibiotic substances or nutrient competition. Preemptive exclusion by wound colonization and direct interaction with the pathogen is proposed as the mechanism of biocontrol. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Stone fruit rot; Dose response; Biocontrol agent pathogen interaction; Pantoea agglomerans; Bacterial biocontrol agent; Postharvest disease

1. Introduction Stone fruits are usually marketed immediately after harvest without long-term cold storage. Losses of economic importance are produced by several decays due to fungal rot (Batra, 1991; Ogawa et al., 1995).

* Corresponding author. Tel.: +34-972-418427; fax: +34-972418399. E-mail address: emonte@intea.udg.es. (E. Montesinos).

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(02)00403-8

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Brown rot caused by Monilinia laxa (Aderh. and Ruhl.) and soft rot caused by Rhizopus stolonifer (Ehrenb.: Fr.) Vuill are the most important postharvest stone fruit decays in Europe. Yield losses due to brown rot can be important depending on weather conditions in the orchard, and are especially severe if high humidity, warm temperatures and abundant rainfall prevail prior to harvest. The conidia of Monilinia produced on mummies are dispersed in early spring and infect fruits in the orchard throughout the growing season. Brown rot is controlled by the use of sanitation practices and by fungicide spray programs in the field. In Italy and Spain a fungicide application is recommended during the bloom and pre-harvest phases if conditions are favorable to disease development and cultivars are susceptible to Monilinia. Postharvest treatments are not performed. Control programs are often inefficient and significant levels of brown rot may occur during storage, transport and marketing. Losses due to R. stolonifer (soft rot) appear in storage and in the consumers home. If the temperature is higher than 5 jC soft rot spreads rapidly from the infected to adjacent fruits. Soft rot is not efficiently controlled by registered fungicides and can be economically important especially on processing fruit harvested mature and ripened at room temperature (Ogawa et al., 1995). In the last 15 years, interest in alternative postharvest disease management practices other than chemical pesticides have increased due to the need to eliminate chemical residues on fruit. Several bacteria (Pusey and Wilson, 1984; Pratella et al., 1993; Smilanick et al., 1993) and yeasts (McLauglin et al., 1992; ChandGoyal and Spotts, 1996) have been identified as postharvest biocontrol agents of brown and soft rot of stone fruits. Among bacteria used as biological control agents, strains of Pantoea agglomerans were reported as effective in postharvest against Penicillium expansum on pear and Penicillium digitatum and Penicillium et al., italicum on orange (Nunes et al., 2001; Teixido 2001). However, there are no reports on the efficacy of P. agglomerans for the control of postharvest fruit rot in apricot, peach and nectarine. In a screening program performed in our laboratory to isolate naturally occurring bacteria from plants, with potential application for biocontrol (Montesinos et al., 1996), a strain of P. agglomerans named

EPS125 was selected. The strain was reported to be effective in the control of blue mold of apple and pear s, 2000). (France The present study was conducted to determine: (1) the potential of strain EPS125 for control of postharvest decays caused by M. laxa and R. stolonifer on stone fruits; (2) the influence of antagonist and pathogen concentration on biocontrol efficacy; and (3) the putative mechanism of action.

2. Materials and methods 2.1. Bacterial antagonist and fungal pathogen strains Strain EPS125 was isolated from the surface of a pear fruit and is deposited in the Spanish Type Culture Collection with the referential code CECT 5392. Strain EPS125 has biofungicide activity against several phytopathogenic microorganisms and it was characterized phenotypically and genotypically as a P. agglomerans according to Bergeys Manual of Systematic Bacteriology and to several authors (Dye, 1969; Ewing and Fife, 1972; Gavini et al., 1989). The strain has a characteristic pattern DNA macrorestriction fragment length polymorphysm (MRFLP) which differs from other strains of P. agglomerans (Montesinos et al., 2001). A spontaneous mutant resistant to 100 Ag ml 1 of rifampicin, which retains phenotypical and genotypical characteristics and performance of the parental strain, was used in the present study. Cultures were grown in LB agar (Maniatis et al., 1982) and were stored in 20% glycerol at 80 jC. Isolates of M. laxa and R. stolonifer were from the collection of CRIOF (University DeGli Studi di Bologna, Bologna, Italy). The pathogens were isolated from stored nectarines showing the typical brown and soft rots, produced by M. laxa and R. stolonifer, respectively. M. laxa was grown on V8 agar and R. stolonifer on potato dextrose agar (Dhingra and Sinclair, 1985), and both were maintained in agar slants at 4 jC. 2.2. Preparation of bacteria and pathogen spore suspensions EPS125 was grown on LB agar plates at 25 jC for 24 h. Then, it was inoculated into 100 ml of LB broth

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in 250-ml flasks. The flasks were incubated on a rotary shaker at 150 rpm at 25 jC for 16 h, and cells were then pelleted by centrifugation at 6000 g, resuspended in sterile distilled water and the concentration adjusted to 109 CFU ml 1 as a stock suspension. Conidia of the fungal pathogens were obtained from the pure cultures using aseptic procedures to avoid contamination. Conidia of M. laxa were obtained from 7-day-old V8 agar cultures incubated at 25 jC under a photoperiod of 12-h light and 12-h dark. Sporangiospores of R. stolonifer were obtained from 3-day-old potato dextrose agar cultures grown at 20 jC in the dark. Spores were collected by scraping the culture surface with a wet cotton swab and resuspending the material in distilled water containing Tween-80 at 0.5 x . The concentration of spores was adjusted with a hemacytometer at 106 conidia ml 1 as a stock suspension. 2.3. Source of fruit Nectarine, apricot and peach fruit of cultivars used in the experiments were obtained from commercial orchards in Emilia-Romagna near Bologna (Italy). The nectarine cultivars were Independence, Venus, Fantasia, Vega and Stark Red Gold. The peach cultivars used were May Crest and Flavor Crest and the apricot cultivars were Tyrinthos, and Reale dImola. Fruits were free of wounds and rot and were homogeneous in maturity and size. Fruits were stored at 1 jC and used within 5 days of harvesting. 2.4. Assays of biological control of brown and soft rot with pathogen inoculated wounded fruits Peach, apricot and nectarine fruit were surfacedisinfected by immersion for 1 min in a dilute solution of sodium hypochlorite (1% active chlorine), washed two times by immersion in distilled water, and let dry. Then, fruits were wounded in the equatorial zone (one wound per fruit) with a flame sterilized nail to a uniform depth of 3 mm. In the biological control treatment the fruits were treated with a 108 CFU ml 1 suspension of P. agglomerans EPS125. In some cases a fungicide treatment was included consisting of tebuconazol (Folicur, Bayer) at 0.125 mg a.i. ml 1 for control of M. laxa, and iprodione (Rovral, Aventis) at 1.0 mg a.i. ml 1 for control of R. stolonifer. A

nontreated control with water was done. All the treatments were applied by immersion of fruits for 1 min into the treatment solutions. Two hours after the treatments the fruits were inoculated by immersion for 1 min in a spore suspension of 1 103 spores ml 1 of M. laxa or R. stolonifer. Then, fruits were placed on polystyrene tray packs which were placed in boxes. In R. stolonifer assays, Independence and Venus nectarine and Reale dImola apricot cultivars were used. In M. laxa assays Tyrinthos apricot, May Crest and Flavor Crest peach, and Venus nectarine cultivars were used. The boxes were covered with plastic bags to maintain high humidity conditions, and were incubated at 20 jC. The incidence of infected wounds (%) and the lesion diameter were determined after 7 days of incubation. All treatments (biological, chemical and nontreated control) consisted of four replicates of 25 fruits per replicate. 2.5. Assays of biological control with unwounded fruits Nectarine fruits of Fantasia, Vega and Stark Red Gold cultivars were directly collected from orchards which have been affected by brown and soft rot during the previous year and having high humidity and warm temperatures during bloom and preharvest. These conditions were conducive to Monilinia and Rhizopus rot. Treatments consisted of 108 CFU ml 1 suspension of EPS125, tebuconazol at 0.125 mg a.i. ml 1, or water as a nontreated control. All the treatments were done by immersion, and fruits were placed on packing trays in plastic boxes which were covered with plastic bags and incubated at 20 jC. The experimental design was completely randomized for each treatment replicate and consisted of three replicates of 30 fruits per replicate. Incidence of fruit rot per replicate was determined after 3, 4, 6 and 7 days. 2.6. Dose response experiments The effect of pathogen and biological control agent concentrations on the incidence and severity of fruit rot was assessed at several concentrations of spores of M. laxa and R. stolonifer (5 102, 1 103, 5 103, 1 104 spore ml 1) and cells of P. agglomerans EPS125 (106, 107, 108, 109 CFU ml 1). The dose response assay was done on Flavor Crest peach using

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M. laxa as pathogen and on Venus nectarine using R. stolonifer. The fruits were surface-disinfected as described above and wounded with a cork borer making a single well per fruit of approximately 9 mm2 and 5-mm depth in the middle of the equatorial zone of each fruit. Wounds were inoculated with 50 Al of the antagonist suspension, let stand for 2 h for complete water absorption by the wound, and inoculated with 50 Al of the spore suspension of the pathogen. Then, the fruits were placed in polystyrene tray packs in boxes that were sealed with plastic bags to maintain high humidity and incubated at 20 jC. The experimental design consisted of three replicates of five fruits per replicate for each pathogen and biocontrol agent concentration. The replicates were completely randomized within the incubation chamber. Percent infected wounds and the diameter of each lesion were determined 3, 4, 6 and 7 days after inoculation. Disease severity for each fruit was calculated as the diameter of the lesion expressed as a proportion of the highest diameter value obtained in the nontreated inoculated controls. 2.7. Interaction experiments between antagonist, pathogen and host In order to study the ability of EPS125 to survive and multiply in wounds, several cell concentrations (106, 107, 108 and 109 CFU ml 1) were applied to nectarines (cultivar Independence) which were previously wounded with a cork borer (as above). The wounds were treated with 50 Al of the corresponding bacterial antagonist suspension and incubated for 7 days at 20 jC. Three replicates of three fruits for each treatment were periodically sampled and the fruit tissue containing the wound was removed with a cork borer (10 mm diameter 2 cm depth), placed in a sterile plastic bag with 20 ml of 0.05 M phosphate buffer (pH 7) and peptone 0.1%, and ground with a pestle. The clear supernatant was serially diluted and the dilutions were seeded on LB agar plates supplemented with 100 Ag ml 1 of rifampicin. Plates were incubated at 25 jC and the colonies counted after 24 h. The population levels were expressed as CFU per wound. The effect of EPS125 on spore germination and mycelial growth of M. laxa was determined on peel leachate which was prepared from Fantasia nectarine

(Droby et al., 1989). Surface-disinfected fruits were wounded with a dissecting needle (100 wounds per fruit), and each set of three wounded fruits was shaken for 15 min at 120 rpm in 100 ml of distilled water. The washing liquid obtained was filter-sterilized through a 0.2-Am pore filter. The effect of coinoculation of EPS125 was determined on spore germination and hyphal growth. Erlenmeyer flasks (100 ml) containing 20 ml of the peel leachate were coinoculated with 1 ml of a suspension of P. agglomerans EPS125 at 109 CFU ml 1 in water, and 100 Al of a 106 spore ml 1 conidial suspension of M. laxa in water. The effect of the culture suspernatant of strain EPS125 grown on peel leachate medium was also assessed. Twenty milliliters of peel leachate was inoculated with 1 ml of a bacterial suspension of 109 CFU ml 1 and incubated on a rotary shaker at 25 jC for 48 h. Then, the culture was centrifuged and filtered through a 0.2Am pore filter and the filtrate was inoculated with 100 Al of a conidial suspension of 106 conidia ml 1 of M. laxa in water. A peel leachate inoculated with a spore suspension was used as a nontreated control. Each treatment was replicated three times and the experiment was repeated twice. Conidium germination was assessed after incubation at 25 jC by means of microscopy at 200 , and for each of the three treatment replications (coinoculation, spent medium and nontreated). Also, dry weight of the fungus was determined after 7 days of incubation at 25 jC following filtration through Whatman no. 1 filter paper and drying overnight at 80 jC. The effect of LB culture filtrates of EPS125 on decay caused by M. laxa and R. stolonifer was studied on nectarine fruits. Surface-disinfected nectarines were wounded in the equatorial zone with a cork borer making a well of approximately 9 mm2 and 5-mm depth. The wounds were treated with 50 Al of the culture filtrate of EPS125. Two hours later, 50 Al of a spore suspension of 103 spores ml 1 of M. laxa or R. stolonifer was inoculated into each treated wound. Percent infection and lesion diameter were determined after 7 days incubation in humid conditions at 20 jC. Treatments consisted of four replicates of 25 fruits per replicate, and the experiment was performed twice. The competition for nutrients between the strain EPS125 and M. laxa or R. stolonifer was studied using the method developed by Janisiewicz et al. (2000). This

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method tests the effect of nutrient depletion by an antagonist on germination and growth of the pathogen. Tissue culture plates and cylinder inserts provided with a membrane filter of 0.45-Am pore size (Millicell-CM, Millipore, Bedford, MA) attached to the inside bottom part of the cylinder were used. Each well of the culture plate was filled with 5% nectarine juice and a cylinder insert containing the spore suspensions. To prepare the nectarine juice, 50 g of fruit homogenate prepared using a waring blender was diluted with distilled water to 1 l, allowed to settle, and the clear supernatant was filtered, first through a Whatman no. 1 filter and after through a 0.2-Am pore filter. Three different treatments were done. The first treatment consisted of 0.6 ml of nectarine juice alone in the well and 0.4 ml of the spore suspension of M. laxa or R. stolonifer (5 103 spores ml 1) inside the cylinder insert as a nontreated control. The second treatment consisted of nectarine juice alone in the well and a mixture of 0.2 ml of a bacterial suspension of EPS125 at 2 108 CFU ml 1 and 0.2 ml of the spore suspension of 1 104 spore ml 1 in the cylinder insert. The third treatment consisted of 0.6 ml of nectarine juice and EPS125 (1 108 CFU ml 1) in the well and 0.4 ml of the fungal spore suspension (5 103 spores ml 1) inside the cylinder insert. Each treatment was replicated three times and the experiment was performed twice. Once the cylinders were placed into the wells, the whole device was incubated at 25 jC for 24 h. Then, the cylinder inserts were removed and the membrane was blotted by the bottom side with tissue paper until all the liquid from the inside of the cylinder was absorbed. Thereafter, a portion of the membrane was cut with a sharp scalpel, transferred to a glass slide and observed under the microscope at 200 to determine conidium germination. 2.8. Data analysis To test the significance of the effect of treatments, a one-way analysis of variance was performed. Means were separated using the Tukey test at P V 0.05. The analysis was performed with the GLM procedure of the PC-Statistical Analysis System version 6 (SAS Institute, Cary, NC). Disease severity data of the dose response experiments were used to estimate efficiency parameters for the biocontrol agent and pathogen using a hyperbolic saturation model (Montesinos and Bonaterra, 1996).

The equation of the hyperbolic saturation model which relates disease to concentrations of the biocontrol agent and pathogen is as follows: y Ymax x 1 I x1 I Kx

where Ymax is the maximum disease proportion the pathogen can produce, Kx is a half-saturation constant corresponding to the pathogen concentration producing half the maximum disease proportion, x is the pathogen density, and I is the proportion of the pathogen inactivated by the biocontrol agent concentration. The proportion of pathogen inactivated (I ) depends on the biocontrol agent concentration (z) according to the following equation: z I Imax z Kz where Imax is the maximum proportion of pathogen the biocontrol agent can inactivate and Kz is the concentration of biocontrol agent that produces an inactivation of Imax/2. This model provides valuable parameters for both the pathogen and the biocontrol agent, such as the median effective dose (ED50) of the pathogen (Kx) and the biocontrol agent (Kz) and the efficiency of the biocontrol agent calculated as the ED50 biocontrol agent/pathogen ratio (Kz/Kx), which are useful in comparing dose response relationships (Montesinos and Bonaterra, 1996). Regression and parameter estimation were performed by a non-linear-least-squares method using the NLIN procedure of the SAS.

3. Results 3.1. Bioassays with wounded and pathogen inoculated fruits Treatment with EPS125 reduced significantly the incidence and lesion diameter of brown rot and soft rot in wounded fruits of several cultivars of nectarine, apricot and peach inoculated with R. stolonifer or M. laxa and stored at 20 jC (Table 1). The incidence of brown rot was between 70% and 100% on nontreated fruits and the preventive treatment with EPS125 decreased incidence to 20 30% (efficacy of 48% to 87%). Incidence of soft rot was between 75% and

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Table 1 Incidence and severity of brown and soft rot in wounded fruits of several cultivars of nectarine, apricot and peach treated with P. agglomerans EPS125x and inoculated with the fungal pathogens M. laxa or R. stolonifer Stone fruit Cultivar Pathogeny Incidence of fruit rotz (%) Nontreated Nectarine Apricot Independence Venus Reale dImola Reale dImola Tyrinthos Tyrinthos May Crest May Crest Flavor Crest R. stolonifer R. stolonifer R. stolonifer R. stolonifer M. laxa M. laxa M. laxa M. laxa M. laxa 100.0 100.0 81.0 75.0 71.0 71.5 85.4 81.1 100.0 a a a a a a a a a EPS125 0.0 20.0 8.0 3.0 27.3 37.0 26.6 23.7 13.3 b b b b b b b b b Rot diameter (mm) Nontreated 59.3 58.0 34.5 25.2 22.0 33.8 22.3 31.2 67.7 a a a a a a a a a EPS125 0.0 6.7 3.0 0.5 9.5 13.0 4.0 4.0 7.3 b b b b b b b b b

Peach

EPS125 was applied by immersion of wounded fruits in a suspension of 108 CFU ml 1. Pathogens were inoculated after EPS125 treatment by immersion in a suspension of 103 spores ml 1. z The trials were performed at 20 jC and disease was assessed 7 days after pathogen inoculation. Values are the mean of four repetitions of 25 fruits per repetition. Treatment means within the same row for incidence or rot diameter that are followed by different letters are significantly different ( P V 0.05) according to the Tukey test.
x y

100% on nontreated fruits and the preventive treatment with EPS125 reduced disease levels to 0 20% (efficacy of 80 to 100%). Lesion diameter of wounds on fruits was also reduced significantly by EPS125 in all cases. When comparing the EPS125 with a fungicide treatment no significant differences were observed in control levels of soft rot, while the incidence of brown rot (23%) was different from the fungicide treatment (3%) (Table 2).
Table 2 Incidence and severity of brown and soft rot in wounded fruits of nectarine cv Venus treated with P. agglomerans EPS125, in comparison to a fungicide and a nontreated control and inoculated with the fungal pathogens M. laxa or R. stolonifer Treatmentx M. laxay Incidence (%)z Nontreated EPS125 Fungicide
x

3.2. Efficacy assays with unwounded fruits collected from orchards Brown rot was the most common type of decay found in nectarine fruits in this trial (Table 3). Brown rot accounted for a 37% incidence on Vega and 60% on Fantasia nectarines in the untreated controls. EPS 125 treatments performed at 108 CFU ml 1 reduced brown rot decay incidence significantly ( P V 0.05) with an efficiency of 49% to 61%, and did not differ significantly from the tebuconazole fungicide treatments. Soft rot and blue mold occurred only in Stark Red Gold nectarines. In both cultivars the treatment
Table 3 Incidence of fruit rot (%) on non-wounded Vega, Stark Red Gold and Fantasia nectarines upon treatment with P. agglomerans EPS125 in comparison to a fungicide and a nontreated control Treatmentx Fungal decayy Brown rot Fantasia Stark Red Vega Gold Water EPS125 Fungicide 60.0 a 26.7 b 16.7 b 41.1 a 21.1 b 10.0 b Blue mold Soft rot Stark Red Gold Stark Red Gold 21.0 a 1.0 b 0.0 b

R. stolonifer Diameter (mm) 57.9 a 7.2 b 2.3 b Incidence (%) 98.7 a 6.7 b 1.3 b Diameter (mm) 70.7 a 4.2 b 1.4 b

99.9 a 22.7 b 2.7 c

All treatments were applied by immersion of fruits. Nontreated, water; EPS125, a suspension in water of 108 CFU ml 1 of EPS125; fungicide, tebuconazol at 0.125 mg a.i. ml 1 for M. laxa and iprodione at 1 mg a.i. ml 1 for R. stolonifer. y Pathogens were inoculated by immersion in a suspension of 3 10 spores ml 1. z The trials were performed at 20 jC and disease was assessed 7 days after pathogen inoculation. Values correspond to the mean of four replicates of 25 fruits per replicate. Means within the same column followed by different letters are significantly different ( P V 0.05) according to the Tukey test.

36.7 a 45.3 a 14.4 b 25.3 b 8.9 b 19.0 b

x The treatments were done by immersion. EPS125 was applied at 108 CFU ml 1 and tebuconazol at 0.125 mg a.i. ml 1. y Values are the means of incidence of three replicates of 30 fruits per replicate after 7 days at 20 jC. Means within the same column that are followed by different letters are significantly different ( P V 0.05) according to the Tukey test.

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with EPS125 reduced soft rot and blue mold incidence significantly. 3.3. Estimation of biocontrol efficiency parameters from dose response experiments Fig. 1 shows the effect on disease levels of the treatment of wounded nectarine and peach fruits with several concentrations of EPS125 at different concentrations of M. laxa and R. stolonifer. Table 4 shows the estimated parameters of efficiency of the

pathogens and the biocontrol agent and the goodness of fit to the hyperbolic saturation model. The model adequately fit the data sets for both fungal pathogens M. laxa and R. stolonifer on the basis of the mean square error (MSE) (0.0098 and 0.0300, respectively) and the asymptotic standard errors for the estimated parameters. The maximum disease proportion and the maximum proportion of pathogen inactivated reached values of almost 1 in both pathogens. ED50 of the two pathogens were similar, 2.7 102 for M. laxa and 1.7 102 spores ml 1 for R. stolonifer. ED 50 of the biocontrol agent was 4.5 104 in M. laxa and 2.2 105 CFU ml 1 in R. stolonifer. However, EPS125 was more effective in M. laxa than in R. stolonifer as indicated by the ratio between ED50 of the biocontrol agent and pathogen (Kz/Kx) which was 166 and 1263, respectively. High concentrations of EPS125 (109 CFU ml 1) produced a full inhibition of disease production by R. stolonifer at the maximal spore concentrations, but 20% of residual infections were not controlled in M. laxa when inoculated at high spore concentrations (above 5 103 spore ml 1) (Fig. 1). 3.4. Colonization and competitive fitness Strain EPS125 colonized and grew rapidly in stone fruit wounds. The population levels of EPS125 in wounds, immediately after application at 5 106, 5 107, 5 108 and 5 109 CFU ml 1 were, respectively, 2.7 105, 2.8 106, 1.6 107 and 3.3 108 CFU per wound, and increased to 5 107 to 6.2 108 CFU per wound within 24 h after application (Fig. 2). These population levels remained stable during the following 7 days of incubation, and wounds appeared healed without symptoms of necrosis or rot. No significant differences in population levels were observed among the initial concentrations inoculated except for the lowest concentration applied (106 CFU ml 1) which attained levels of only 5 107 CFU per wound. Spore germination and hyphal growth of M. laxa were inhibited in peel leachate medium when cells of EPS125 were added (Table 5). The germination was 100% in the absence and 50% in the presence of EPS125 at a ratio of 104 CFU per spore (5 107 CFU ml 1 of the bacterial antagonist and 5 103 spores ml 1 of the pathogen). However, no inhibition of

Fig. 1. Infectivity titration of M. laxa on Flavor Crest peach (A) and of R. stolonifer on Venus nectarine (B) wounded and treated with increasing concentrations of P. agglomerans EPS125. The pathogen densities were 5 102 (n), 1 103 ( ), 5 103 (o), and 1 104 (5) spores ml 1. The lines represent predictions of disease proportion at the different pathogen concentrations according to the hyperbolic saturation model, using estimated parameters shown in Table 2. Disease severity values were assessed at 20 jC after 7 days from the pathogen inoculation.

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Table 4 Estimated parameters and goodness-of-fit for the hyperbolic saturation model that relates the disease severity of brown or soft rot to the biocontrol agent and pathogen concentrations Pathogen DFEx Parameter Ymax (maximum disease proportion) M. laxa R. stolonifer 56 56 0.99 (0.047) 0.97 (0.007) Kx (ED50 pathogen)y 2.69 102 (0.79 102) 1.71 102 (1.00 102) Imax (maximum pathogen proportion inactivated) 0.99 (0.002) 0.99 (0.001) Kz (ED50 biocontrol agent) 4.48 104 (1.37 104) 2.16 105 (1.27 105) 0.0098 0.0300 MSE

Data correspond to the infectivity titration of M. laxa on peach and R. stolonifer on nectarine wounded fruits after treatment with increasing concentrations of P. agglomerans EPS125 shown in Fig. 1. x DFE = degrees of freedom for the error; MSE = mean square error. The asymptotic standard errors for the parameter estimates are given in parentheses. y Densities for M. laxa and R. stolonifer are spore ml 1 and for P. agglomerans EPS125 are CFU ml1 .

germination was observed with cell-free peel leachate culture filtrate of EPS125. The mixed culture of EPS125 and M. laxa in peel leachate medium caused a nearly threefold reduction in mycelial development compared to the control. Peel leachate cell-free culture filtrate of EPS125 also produced a reduction of mycelial growth, but less than in the presence of EPS125 cells. Culture filtrate from EPS125 grown in LB broth was not effective in protecting nectarine surface wounds from infection by M. laxa and R. stolonifer compared to the highly effective reduction of incidence when applying cells at 108 CFU ml 1 (data not shown).

Spores of M. laxa and R. stolonifer fully germinated within 24 h in filter inserts submerged in wells containing 5% w/v nectarine juice. However, germination of both pathogens was prevented by the addition of EPS125 at a ratio of 2 104 CFU per spore (108 CFU ml 1 of the bacterial antagonist and 5 103 spores ml 1 of the pathogen). Observation of the filter membrane insert under the microscope revealed cells of EPS125 closely interacting with spores and with the few germ tubes which germinated from fungal spores. However, complete germination was observed when EPS125 cells were separated from spores by a mem-

Table 5 Inhibition of conidial germination and mycelial growth of M. laxa by P. agglomerans EPS125 on nectarine peel leachate medium Treatmentx Nontreated EPS125 cells EPS125 culture filtrate Conidia germination (%)y 99.1 a 50.5 b 95.4 a Mycelial dry weight (mg) 7.0 a 2.5 c 4.5 b

Fig. 2. Time-course of the population levels of P. agglomerans EPS125 on wounds of nectarine fruit inoculated at concentrations of 5 106 (5), 5 107 (w ), 5 108 ( ) or 5 109 (D) CFU ml 1 and incubated at 20 jC. Data points correspond to the mean population levels of three replicates of three fruits, and bars indicate the confidence interval for the mean.

x Nontreated, peel leachate; EPS125 cells, 5 107 CFU ml 1 of EPS125 inoculated in peel leachate; EPS125 culture filtrate, 5 107 CFU ml 1 of EPS125 inoculated in peel leachate, incubated during 48 h, centrifuged and filtered through a 0.2 Am pore filter. All treatments were inoculated with conidia at a final concentration of 5 103 conidia ml 1. Conidia germination was determined after 24 h of incubation at 25 jC and mycelial growth was determined after 7 days. y Values are means of two experiments consisting of three repetitions per treatment. Means within the same column followed by different letters are significantly different ( P V 0.05) according to the Tukey test.

A. Bonaterra et al. / International Journal of Food Microbiology 84 (2003) 93104 Table 6 Germination (%) of conidia of M. laxa and R. stolonifer in nectarine juice medium on PTFE membrane cylinders upon interaction with P. agglomerans EPS125 Treatmentx Well EPS125
x

101

M. laxa Membrane cylinder Fungal spores EPS125+ fungal spores Fungal spores 95.7y a 1.3 b 92.0 a

R. stolonifer

98.0 a 2.0 b 96.0 a

The experiment was performed with 5% (w/v) nectarine juice. Cylinders were separated from wells by a 0.45-Am pore size membrane filter. The concentration of fungal spores in the cylinders was 5 103 spores ml 1. The concentration of EPS125 was 1 108 CFU ml 1 either in the well or in the cylinder. y Values are means of two experiments composed of three repetitions per treatment. Means within the same column followed by different letters are significantly different ( P V 0.05) according to the Tukey test. Spore germination was determined after 24 h of incubation at 25 jC.

brane filter which permits medium nutrients and metabolite interchange (Table 6).

4. Discussion P. agglomerans is a common epiphytic bacterium of aerial plant parts (Cook and Baker, 1983) and has been reported to control bacterial and fungal diseases of several plants (Vanneste et al., 1992; Yuen et al., 1994; Montesinos et al., 1996; Zhang and Birch, 1997; Stockwell et al., 1998) and postharvest fruit rot (Bryk et al., 2001). et al., 1998; Nunes et al., 2001; Teixido P. agglomerans EPS125 was isolated from the surface of a pear fruit and does not produce primary dermal or eye irritation on rabbit, and the acute oral toxicity on rats was higher than 1010 CFU kg 1 (Montesinos et al., 2001). In the present work, strain EPS125 significantly reduce brown rot (M. laxa) and soft rot (R. stolonifer) on stone fruits that were wounded and inoculated with the pathogens. The strain also exhibits a high efficacy in reduction of blue mold caused by P. expansum on pome fruits under several cold storage conditions, and the efficacy does not differ significantly from reference fungicide s, 2000). treatments (France EPS125 gave consistent control of brown and soft rot on naturally infected nectarine collected from orchards, at high and medium disease pressure, with

efficacy ranging from 49% to 61%. The efficacy of disease control under these conditions was lower than in wounded inoculated fruits probably because fruits from the orchards harbored latent infections or because some pathogen colonization sites were nonaccessible to EPS125. The fact that no artificial wounds were performed in this trial suggest that EPS125 may be able to protect against infections that develop on new wounds produced during harvest or can possibly be curative against some latent infections. As in other biocontrol agent pathogen host plant systems, the activity of EPS125 depends on the concentration of both the pathogen and the antagonist cells. Knowledge of antagonist pathogen density relationships provides data on the population levels of the antagonist required to achieve adequate disease control (Johnson, 1994). Dose response models have been used as tools to determine quantitative parameters describing the efficiency of the biocontrol agents which permit comparison of different biocontrol agents and pathosystems (Johnson, 1994; Raaijmakers et al., 1995; Montesinos and Bonaterra, 1996; Smith et al., 1997; Larkin and Fravel, 1999). One of the most useful parameters, the ratio between the median effective dose of the biocontrol agent and the pathogen, Kz/Kx in the hyperbolic saturation model, measures the efficiency of the biocontrol agent in terms of cells needed to inhibit a pathogen cell (Montesinos and Bonaterra, 1996). According to the results presented here in stone fruits, EPS125 was highly effective against M. laxa and R. stolonifer (median effective dose ED50 from 0.5 105 to 2 105 CFU ml 1), especially when considering the high virulence of both pathogens (median effective dose from 1.7 102 to 2.7 102 spores ml 1). EPS125 was more efficient in controlling M. laxa than R. stolonifer; 166 EPS125 cells were needed to inactivate one conidium of M. laxa while 1263 EPS125 cells were required to inactivate one sporangiospore of R. stolonifer. These values are similar to the median effective dose ratio found in strain EPS5001 of P. agglomerans against the fungus Stemphylium vesicarium on pear, although the ED50 of the pathogen (3.7 104 conidia ml 1) and of the biocontrol agent (6.3 106 CFU ml 1) were different (Montesinos and Bonaterra, 1996). A very high efficiency was found in the case of Bacillus cereus UW85 for control of Pythium on tomato cultivars in which the median effective dose

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ratio Kz/Kx was between 1 and 5 CFU per oospore (Smith et al., 1997). Although full inhibition of disease production by R. stolonifer by EPS125 was obtained at 109 CFU ml 1 at high fungal spore concentrations, 20% of M. laxa infections were not controlled at this dosage. This may indicate that some spores of M. laxa are inaccessible to the antagonist in the fruit wounds, or a lack of coincidence in the spatial distributions of the pathogen and the biocontrol agent (Johnson, 1994; Montesinos and Bonaterra, 1996). Compared to other biocontrol agents used against of postharvest rot, EPS125 is highly efficient, with optimal activity in the range of 107 108 CFU ml 1, since a concentration of 108 CFU ml 1 is enough to inhibit the infection by 5 103 spores of R. stolonifer or M. laxa in wounded fruit. This efficiency is higher than that reported for other bacteria. 2 108 CFU of Enterobacter cloacae were needed to control 2 102 sporangiospores of R. stolonifer (Wilson et al., 1987), and 108 CFU ml 1 of Bacillus subtilis were needed to control Monilinia fructicola infection on peach at 103 spores ml 1 (Pusey et al., 1988). In terms of cell concentration of the biocontrol agent needed to control infection by fungal spores, yeast and fungus have variable efficiency depending on the biocontrol agent and pathosystem. Only 106 spores ml 1 of Trichoderma satisfactory controlled brown rot on plum inoculated with M. fructicola at 8 104 spores ml 1 (Hong and Michailides, 1998), and 5 108 CFU ml 1 of Pichia membranefaciens completely inhibited R. stolonifer inoculated at 5 104 spores ml 1 on nectarine fruit (Qing and Shiping, 2000). The efficiency was lower for Kloeckera apiculata in peach, where 5 108 CFU ml 1 were needed to inhibit infection by 103 spores ml 1 of R. stolonifer (McLauglin et al., 1992). Knowledge of the mechanism of action involved in the biocontrol process can permit establishment of optimum conditions for the interaction between the pathogen and the biological control agent and is important for implementing biological control in a given pathosystem (Cook, 1993; Handelsman and Stabb, 1996). Several mechanisms have been suggested to operate on postharvest biocontrol, including antibiosis, parasitism, induced resistance, and competition for space and limited resources. Antibiosis due to production of pyrrolnitrin is the main mode of action of

Pseudomonas cepacia in controlling Botrytis cinerea and P. expansum on apples and pears (Janisiewicz and Roitman, 1988) and in B. subtilis which controls M. fructicola by the production of iturine (Pusey and Wilson, 1984). Attachment alone or in combination with secretion of cell wall degrading enzymes was proposed as the mechanism operating in the biocontrol of B. cinerea by Pichia guilliermondii (Winiewski et al., 1991), or of several fungal pathogens by Aureobasidium pullulans (Castoia et al., 2001). Competition for nutrients was suggested to play a role in the biocontrol of P. digitatum by Debaryomyces hansenii (Droby et al., 1989), and of B. cinerea by Cryptococcus spp. (Filonow et al., 1996). Preemptive exclusion of fungal infection sites by the antagonist was observed in Candida oleophila and Cryptoccocus laurentii which control B. cinerea (Roberts, 1990; Mercier and Wilson, 1995). Induction of host defense reactions was proposed as mechanism in the biocontrol of P. digitatum by Verticillium lecanii (Benhamou and Brodeur, 2000) and of B. cinerea by Candida saitoana (El-Ghaouth et al., 1998). Inhibition of plant pathogens by P. agglomerans is dependent on the strain and has been attributed to production of an acidic environment (Riggle and Klos, 1972; Beer et al., 1984), preemptive colonization (Wilson et al., 1992; Kearns and Hale, 1996), competition for nutrients (Goodman, 1967), production of herbicolin (Ishimaru et al., 1988) or other antibiotics (Vanneste et al., 1992; Kearns and Hale, 1996), parasitism of the pathogen (Bryk et al., 1998) and induction of plant defense response (Slade and Tiffin, 1984). However, the mechanism of biocontrol of postharvest rot in orange and pear by P. agglomerans CPA-2 was not known (Nunes et al., 2001; et al., 2001). Teixido In the study of interaction between EPS125 and the fungal pathogens M. laxa and R. stolonifer, inhibition of germination of conidia or hyphal growth were only observed when there was a direct cell-to-cell interaction. It is unlikely that production of antibiotic substances is important because neither the cell-free culture filtrate of EPS125 nor physical separation by a membrane filter produced inhibition of pathogen spore germination. Moreover, the application of EPS125 culture filtrates in wounds does not prevent brown or soft rot. However, the observation of a slight inhibition of fungus hyphal growth by EPS125 cell-free

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culture filtrates could be due to nutrient depletion from the medium by EPS125 after long periods of incubation (48 h), because antibiotic production has not been detected in previous studies (data not shown). EPS125 bacteria colonize and quickly multiply on stone fruit wounds within 24 h at room temperature, reaching approximately 5 108 CFU per wound, and sustain populations long enough time to permit efficient control of the pathogens on fruits. The fact that the population level of the strain increased to the carrying capacity in only 24 h is an important trait because it is the time required to germinate for many fungal postharvest pathogens. Thus, preemptive exclusion of the pathogen by wound colonization and cell-to-cell interaction with the fungal pathogen appear to be the main mechanisms of biocontrol of brown and soft rot by P. agglomerans EPS125. In conclusion, EPS125 is effective at moderately concentrations in preventive treatments for control of stone fruit rot on several stone fruit cultivars. Its ability to colonize, rapidly grow and survive in wounds, the fact that the main mechanisms of action is mediated by cell-to-cell interaction, and the absence of major toxicological problems, constitute interesting traits for an effective use as a biofungicide under fully commercial conditions. Acknowledgements This study was supported in part by project PETRI 95-0306-OP from the CICYT of the Ministerio de a of Spain, and from the CIRIT Ciencia y Tecnolog of the Generalitat de Catalunya to CeRTA. We also acknowledge the University of Girona for financial support for a study leave in the CRIOF to A. Bonaterra. References
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