!!!!!Monilinia laxa

Biological control of Monilinia laxa and Rhizopus stolonifer in

postharvest of stone fruit by Pantoea agglomerans EPS125

and putative mechanisms of antagonism

Anna Bonaterraa, Marta Marib, Lucia Casalinib, Emilio Montesinosa,*

a Institute of Food and Agricultural Technology and CeRTA-CIDSAV, University of Girona, Av. Llus Santalo, 17071 Girona, SpainbCRIOF, 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 effectivein 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 agentpathogen interaction; Pantoea agglomerans; Bacterial biocontrol agent; Post-

harvest 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).

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0168-1605(02)00403-8

* Corresponding author. Tel.: +34-972-418427; fax: +34-972-

418399.

E-mail address: [email protected]. (E. Montesinos).

www.elsevier.com/locate/ijfoodmicro

International Journal of Food Microbiology 84 (2003) 93104

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 temper-

atures 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 temper-

ature is higher than 5 jC soft rot spreads rapidly fromthe infected to adjacent fruits. Soft rot is not effi-

ciently 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 postharv-

est 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; Chand-

Goyal and Spotts, 1996) have been identified as post-

harvest 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 expan-

sum on pear and Penicillium digitatum and Penicillium

italicum on orange (Nunes et al., 2001; Teixido et al.,

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

(Frances, 2000).

The present study was conducted to determine: (1)

the potential of strain EPS125 for control of post-

harvest decays caused by M. laxa and R. stolonifer on

stone fruits; (2) the influence of antagonist and patho-

gen 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 sev-

eral phytopathogenic microorganisms and it was char-

acterized phenotypically and genotypically as a P.

agglomerans according to Bergeys Manual of Sys-

tematic Bacteriology and to several authors (Dye,

1969; Ewing and Fife, 1972; Gavini et al., 1989).

The strain has a characteristic pattern DNA macro-

restriction 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 retainsphenotypical and genotypical characteristics and per-

formance of the parental strain, was used in the

present study. Cultures were grown in LB agar (Mani-

atis et al., 1982) and were stored in 20% glycerol at

80 jC. Isolates of M. laxa and R. stolonifer werefrom 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 potatodextrose agar

(Dhingra and Sinclair, 1985), and both were main-

tained 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 for24 h. Then, it was inoculated into 100 ml of LB broth

A. Bonaterra et al. / International Journal of Food Microbiology 84 (2003) 9310494

in 250-ml flasks. The flasks were incubated on a

rotary shaker at 150 rpm at 25 jC for 16 h, and cellswere then pelleted by centrifugation at 6000 g,resuspended in sterile distilled water and the concen-

tration adjusted to 109 CFU ml 1 as a stock suspen-sion. 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-hdark. Sporangiospores of R. stolonifer were obtained

from 3-day-old potatodextrose agar cultures grown

at 20 jC in the dark. Spores were collected byscraping the culture surface with a wet cotton swab

and resuspending the material in distilled water con-

taining Tween-80 at 0.5 x. The concentration ofspores 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 culti-

vars 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 homoge-

neous in maturity and size. Fruits were stored at 1 jCand 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 surface-

disinfected 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 ofM. 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 1103 spores ml 1 ofM. laxa or R. stolonifer. Then, fruits were placed on

polystyrene tray packs which were placed in boxes. In

R. stolonifer assays, Independence and Venus nectar-

ine 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 incu-

bated 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 treat-ments 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. Theexperimental design was completely randomized for

each treatment replicate and consisted of three repli-

cates of 30 fruits per replicate. Incidence of fruit rot

per replicate was determined after 3, 4, 6 and 7 days.

2.6. Doseresponse 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, 1103, 5 103,1104 spore ml 1) and cells of P. agglomeransEPS125 (106, 107, 108, 109 CFU ml 1). The doseresponse assay was done on Flavor Crest peach using

A. Bonaterra et al. / International Journal of Food Microbiology 84 (2003) 93104 95

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 Alof the antagonist suspension, let stand for 2 h for

complete water absorption by the wound, and inocu-

lated with 50 Al of the spore suspension of thepathogen. 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 cham-

ber. 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 calcu-

lated 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 tonectarines (cultivar Independence) which were previ-

ously wounded with a cork borer (as above). The

wounds were treated with 50 Al of the correspondingbacterial antagonist suspension and incubated for 7

days at 20 jC. Three replicates of three fruits for eachtreatment were periodically sampled and the fruit

tissue containing the wound was removed with a cork

borer (10 mm diameter 2 cm depth), placed in asterile 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 supple-

mented with 100 Ag ml 1 of rifampicin. Plates wereincubated at 25 jC and the colonies counted after 24h. 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 ofEPS125 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 andincubated on a rotary shaker at 25 jC for 48 h. Then,the culture was centrifuged and filtered through a 0.2-

Am pore filter and the filtrate was inoculated with 100Al 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 experi-

ment was repeated twice. Conidium germination was

assessed after incubation at 25 jC by means ofmicroscopy at 200 , and for each of the three treat-ment replications (coinoculation, spent medium and

nontreated). Also, dry weight of the fungus was

determined after 7 days of incubation at 25 jCfollowing 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 nec-

tarines 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 Alof 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 condi-

tions at 20 jC. Treatments consisted of four replicatesof 25 fruits per replicate, and the experiment was

performed twice.

The competition for nutrients between the strain

EPS125 andM. laxa or R. stolonifer was studied using

the method developed by Janisiewicz et al. (2000). This


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 treatmentswere 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 sporesml 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 0.2ml of the spore suspension of 1104 spore ml 1 in thecylinder insert. The third treatment consisted of 0.6 ml

of nectarine juice and EPS125 (1108 CFU ml 1) inthe well and 0.4 ml of the fungal spore suspension

(5 103 spores ml 1) inside the cylinder insert. Eachtreatment 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 jCfor 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 PV 0.05. Theanalysis was performed with the GLM procedure of

the PC-Statistical Analysis System version 6 (SAS

Institute, Cary, NC).

Disease severity data of the doseresponse experi-

ments 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 biocon-

trol agent and pathogen is as follows:

y Ymax x1 Ix1 I Kx

where Ymax is the maximum disease proportion the

pathogen can produce, Kx is a half-saturation constant

corresponding to the pathogen concentration produc-

ing half the maximum disease proportion, x is the

pathogen density, and I is the proportion of the

pathogen inactivated by the biocontrol agent concen-

tration. The proportion of pathogen inactivated (I)

depends on the biocontrol agent concentration (z)

according to the following equation:

I Imax zz 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 com-

paring doseresponse 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 inocu-

lated 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 ofbrown rot was between 70% and 100% on nontreated

fruits and the preventive treatment with EPS125

decreased incidence to 2030% (efficacy of 48% to

87%). Incidence of soft rot was between 75% and


100% on nontreated fruits and the preventive treat-

ment with EPS125 reduced disease levels to 020%

(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 fungi-

cide 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).

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 reducedbrown rot decay incidence significantly (PV 0.05)with an efficiency of 49% to 61%, and did not differ

significantly from the tebuconazole fungicide treat-

ments. Soft rot and blue mold occurred only in Stark

Red Gold nectarines. In both cultivars the treatment

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 (%) Rot diameter (mm)

Nontreated EPS125 Nontreated EPS125

Nectarine Independence R. stolonifer 100.0 a 0.0 b 59.3 a 0.0 b

Venus R. stolonifer 100.0 a 20.0 b 58.0 a 6.7 b

Apricot Reale dImola R. stolonifer 81.0 a 8.0 b 34.5 a 3.0 b

Reale dImola R. stolonifer 75.0 a 3.0 b 25.2 a 0.5 b

Tyrinthos M. laxa 71.0 a 27.3 b 22.0 a 9.5 b

Tyrinthos M. laxa 71.5 a 37.0 b 33.8 a 13.0 b

Peach May Crest M. laxa 85.4 a 26.6 b 22.3 a 4.0 b

May Crest M. laxa 81.1 a 23.7 b 31.2 a 4.0 b

Flavor Crest M. laxa 100.0 a 13.3 b 67.7 a 7.3 b

x EPS125 was applied by immersion of wounded fruits in a suspension of 108 CFU ml 1.y 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 ( PV 0.05) according to the Tukey test.

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 Blue mold Soft rot

Fantasia Stark Red

Gold

Vega Stark Red

Gold

Stark Red

Gold

Water 60.0 a 41.1 a 36.7 a 45.3 a 21.0 a

EPS125 26.7 b 21.1 b 14.4 b 25.3 b 1.0 b

Fungicide 16.7 b 10.0 b 8.9 b 19.0 b 0.0 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 samecolumn that are followed by different letters are significantly

different ( PV 0.05) according to the Tukey test.

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 R. stolonifer

Incidence

(%)zDiameter

(mm)

Incidence

(%)

Diameter

(mm)

Nontreated 99.9 a 57.9 a 98.7 a 70.7 a

EPS125 22.7 b 7.2 b 6.7 b 4.2 b

Fungicide 2.7 c 2.3 b 1.3 b 1.4 b

x All treatments were applied by immersion of fruits. Non-

treated, water; EPS125, a suspension in water of 108 CFU ml 1 ofEPS125; fungicide, tebuconazol at 0.125 mg a.i. ml 1 for M. laxaand iprodione at 1 mg a.i. ml 1 for R. stolonifer.

y Pathogens were inoculated 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 correspond to the mean of

four replicates of 25 fruits per replicate. Means within the same

column followed by different letters are significantly different

( PV 0.05) according to the Tukey test.


with EPS125 reduced soft rot and blue mold inci-

dence significantly.

3.3. Estimation of biocontrol efficiency parameters

from doseresponse 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 con-

centrations 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, respec-

tively) and the asymptotic standard errors for the

estimated parameters. The maximum disease propor-

tion and the maximum proportion of pathogen inac-

tivated reached values of almost 1 in both pathogens.

ED50 of the two pathogens were similar, 2.7 102for M. laxa and 1.7 102 spores ml 1 for R.stolonifer. ED50 of the biocontrol agent was

4.5 104 in M. laxa and 2.2 105 CFU ml 1 inR. 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, respec-

tively. High concentrations of EPS125 (109 CFU

ml 1) produced a full inhibition of disease produc-tion by R. stolonifer at the maximal spore concen-

trations, 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, respec-tively, 2.7 105, 2.8 106, 1.6 107 and 3.3 108CFU per wound, and increased to 5 107 to 6.2 108CFU 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 perwound.

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 CFUml 1 of the bacterial antagonist and 5 103 sporesml 1 of the pathogen). However, no inhibition of

Fig. 1. Infectivity titration ofM. 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), 1103 (.), 5 103 (o), and 1104 (5)spores ml 1. The lines represent predictions of disease proportion atthe 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 thepathogen inoculation.


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 inci-

dence when applying cells at 108 CFU ml 1 (datanot shown).

Spores ofM. laxa and R. stolonifer fully germinated

within 24 h in filter inserts submerged in wells con-

taining 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 CFUml 1 of the bacterial antagonist and 5 103 sporesml 1 of the pathogen). Observation of the filtermembrane 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 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 MSE

Ymax (maximum

disease proportion)

Kx (ED50pathogen)y

Imax (maximum pathogen

proportion inactivated)

Kz (ED50biocontrol agent)

M. laxa 56 0.99 (0.047) 2.69 102 (0.79 102) 0.99 (0.002) 4.48 104 (1.37 104) 0.0098R. stolonifer 56 0.97 (0.007) 1.71102 (1.00 102) 0.99 (0.001) 2.16 105 (1.27 105) 0.0300Data 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 .

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 1and incubated at 20 jC. Data points correspond to the meanpopulation levels of three replicates of three fruits, and bars indicate

the confidence interval for the mean.

Table 5

Inhibition of conidial germination and mycelial growth of M. laxa

by P. agglomerans EPS125 on nectarine peel leachate medium

Treatmentx Conidia

germination (%)yMycelial

dry weight (mg)

Nontreated 99.1 a 7.0 a

EPS125 cells 50.5 b 2.5 c

EPS125 culture

filtrate

95.4 a 4.5 b

x Nontreated, peel leachate; EPS125 cells, 5 107 CFU ml 1 ofEPS125 inoculated in peel leachate; EPS125 culture filtrate, 5 107CFU ml 1 of EPS125 inoculated in peel leachate, incubated during48 h, centrifuged and filtered through a 0.2 Am pore filter. Alltreatments were inoculated with conidia at a final concentration of

5 103 conidiaml 1. Conidia germinationwas determined after 24 hof incubation at 25 jC and mycelial growth was determined after 7days.

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 ( PV 0.05) accordingto the Tukey test.


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., 1998; Nunes et al., 2001; Teixido et al., 2001).

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

treatments (Frances, 2000).

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 agentpathogenhost plant

systems, the activity of EPS125 depends on the

concentration of both the pathogen and the antagonist

cells. Knowledge of antagonistpathogen density

relationships provides data on the population levels

of the antagonist required to achieve adequate disease

control (Johnson, 1994). Doseresponse models have

been used as tools to determine quantitative parame-

ters 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 patho-

gen, Kz/Kx in the hyperbolic saturation model, meas-

ures the efficiency of the biocontrol agent in terms of

cells needed to inhibit a pathogen cell (Montesinos

and Bonaterra, 1996). According to the results pre-

sented here in stone fruits, EPS125 was highly effec-

tive against M. laxa and R. stolonifer (median

effective dose ED50 from 0.5 105 to 2 105 CFUml 1), especially when considering the high virulenceof both pathogens (median effective dose from

1.7 102 to 2.7 102 spores ml 1). EPS125 wasmore efficient in controlling M. laxa than R. stoloni-

fer; 166 EPS125 cells were needed to inactivate one

conidium of M. laxa while 1263 EPS125 cells were

required to inactivate one sporangiospore of R. stolo-

nifer. 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 conidiaml 1) and of the biocontrol agent (6.3 106 CFUml 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

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 M. laxa R. stolonifer

Well Membrane cylinder

Fungal spores 95.7y a 98.0 a

EPS125+ fungal spores 1.3 b 2.0 b

EPS125 Fungal spores 92.0 a 96.0 a

x The experiment was performed with 5% (w/v) nectarine juice.

Cylinders were separated from wells by a 0.45-Am pore sizemembrane filter. The concentration of fungal spores in the cylinders

was 5 103 spores ml 1. The concentration of EPS125 was 1108CFU 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 ( PV 0.05) accordingto the Tukey test. Spore germination was determined after 24 h of

incubation at 25 jC.


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 107108 CFU ml 1,since a concentration of 108 CFU ml 1 is enough toinhibit the infection by 5 103 spores of R. stoloniferor M. laxa in wounded fruit. This efficiency is higher

than that reported for other bacteria. 2 108 CFU ofEnterobacter cloacae were needed to control 2 102sporangiospores of R. stolonifer (Wilson et al., 1987),

and 108 CFU ml 1 of Bacillus subtilis were needed tocontrol 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 controlledbrown 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 membranefacienscompletely inhibited R. stolonifer inoculated at

5 104 spores ml 1 on nectarine fruit (Qing andShiping, 2000). The efficiency was lower for Kloeck-

era apiculata in peach, where 5 108 CFU ml 1were 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 sug-

gested to operate on postharvest biocontrol, including

antibiosis, parasitism, induced resistance, and compe-

tition 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 Aureoba-

sidium 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 andWilson,

1995). Induction of host defense reactions was pro-

posed 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 coloniza-

tion (Wilson et al., 1992; Kearns and Hale, 1996),

competition for nutrients (Goodman, 1967), produc-

tion 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. agglom-

erans CPA-2 was not known (Nunes et al., 2001;

Teixido et al., 2001).

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 interac-

tion. It is unlikely that production of antibiotic sub-

stances 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


culture filtrates could be due to nutrient depletion from

themedium 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, andsustain populations long enough time to permit effi-

cient 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 mecha-

nisms 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

Ciencia y Tecnologa of Spain, and from the CIRIT

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.

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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 antagonismIntroductionMaterials and methodsBacterial antagonist and fungal pathogen strainsPreparation of bacteria and pathogen spore suspensionsSource of fruitAssays of biological control of brown and soft rot with pathogen inoculated wounded fruitsAssays of biological control with unwounded fruitsDose-response experimentsInteraction experiments between antagonist, pathogen and hostData analysis

ResultsBioassays with wounded and pathogen inoculated fruitsEfficacy assays with unwounded fruits collected from orchardsEstimation of biocontrol efficiency parameters from dose-response experimentsColonization and competitive fitness

DiscussionAcknowledgementsReferences

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