ORIGINAL ARTICLE

Biocontrol bacteria selected by a direct plant protectionstrategy against avocado white root rot show antagonismas a prevalent traitM.A. Gonzalez-Sanchez1, R.M. Perez-Jimenez1, C. Pliego2, C. Ramos2, A. de Vicente3

and F.M. Cazorla3

1 IFAPA-CICE-Malaga, CAP-Junta de Andalucıa, Cortijo de la Cruz s ⁄ n, Churriana, Malaga, Spain

2 Area de Genetica, Facultad de Ciencias, Universidad de Malaga, Malaga, Spain

3 Departamento de Microbiologıa, Facultad de Ciencias, Universidad de Malaga, Malaga, Spain

Introduction

Biological control offers an environmentally sound alter-

native to chemical pesticides and is an attractive method

for plant protection against soil-borne diseases. Many

biological control agents are found by screening large

numbers of micro-organisms against plant pathogens

in vitro or in planta (Berg et al. 2001; Anith et al. 2003).

Antagonistic micro-organisms have been selected using

such screening methods (Howell and Stipanovic 1979;

Kloepper 1991; Tjamos et al. 2004). Despite these

successes, the application of biocontrol agents is generally

not yet as reliable as that of their chemical counterparts

(Weller 1988; Gerhardson 2002). One of the biggest

reasons for this is that there is a lack of appropriate

screening procedures to select micro-organisms most suit-

able for disease control in diverse soil environments. Cur-

rent screening procedures may ignore the influence of

Keywords

agriculture, biocontrol, mechanism of action,

plant pathology, plant diseases.

Correspondence

Francisco M. Cazorla, Departamento de

Microbiologıa, Facultad de Ciencias,

Universidad de Malaga, Campus Universitario

de Teatinos s/n, 29071-Malaga, Spain,

E-mail: cazorla@uma.es

2009 ⁄ 1719: received 30 September 2009,

revised 6 November 2009 and accepted 11

November 2009

doi:10.1111/j.1365-2672.2009.04628.x

Abstract

Aim: This study was undertaken to study bacterial strains obtained directly for

their efficient direct control of the avocado white root rot, thus avoiding pre-

screening by any other possible mechanism of biocontrol which could bias the

selection.

Methods and Results: A collection of 330 bacterial isolates was obtained from

the roots and soil of healthy avocado trees. One hundred and forty-three repre-

sentative bacterial isolates were tested in an avocado ⁄ Rosellinia test system,

resulting in 22 presumptive protective strains, all of them identified mainly as

Pseudomonas and Bacillus species. These 22 candidate strains were screened in

a more accurate biocontrol trial, confirming protection of some strains (4 out

of the 22). Analyses of the potential bacterial traits involved in the biocontrol

activity suggest that different traits could act jointly in the final biocontrol

response, but any of these traits were neither sufficient nor generalized for all

the active bacteria. All the protective strains selected were antagonistic against

some fungal root pathogens.

Conclusions: Diverse bacteria with biocontrol activity could be obtained by a

direct plant protection strategy of selection. All the biocontrol strains finally

selected in this work were antagonistic, showing that antagonism is a prevalent

trait in the biocontrol bacteria selected by a direct plant protection strategy.

Significance and Impact of the Study: This is the first report on the isolation

of biocontrol bacterial strains using direct plant protection strategy in the sys-

tem avocado ⁄ Rosellinia. Characterization of selected biocontrol bacterial strains

obtained by a direct plant protection strategy showed that antagonism is a pre-

valent trait in the selected strains in this experimental system. This suggests

that antagonism could be used as useful strategy to select biocontrol strains.

Journal of Applied Microbiology ISSN 1364-5072

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78 65

biotic and abiotic factors in the rhizosphere. For example,

dual-culture with fungal pathogens on agar plates has

often been used as a screening method (Kloepper and

Schroth 1981; Schroth and Hancock 1982). However, the

method may be inappropriate because it excludes host–

antagonist–pathogen interacting factors, and it cannot

select biocontrol agents that provide disease control by

other mechanisms such as root colonization, induction of

systemic resistance and ⁄ or niche competition (Lugtenberg

et al. 2001; Bakker et al. 2003; Kamilova et al. 2005;

Lugtenberg and Kamilova 2009; Pang et al. 2009). Some

of these biocontrol traits have been used individually or

in combination for the isolation and selection of potential

novel biocontrol agents in many experimental biological

systems, such as cucumber (Pang et al. 2009), tomato

(Compant et al. 2005), potato (Bakker et al. 1986) and

other profitable crops (Bakker et al. 2003).

The avocado (Persea americana Mill.) is an important

tropical and subtropical crop in Spain and worldwide.

Phytophthora cinnamomi is an oomycete which causes the

most serious fungal root disease in avocado plants world-

wide (Erwin and Ribeiro 1996). However, in the Mediter-

ranean area, white root rot caused by the fungus

Rosellinia necatrix is the main limiting factor of the

avocado crop (Perez-Jimenez 2006; Pliego et al. 2009).

Several strategies have been reported for control of

R. necatrix, such as soil solarization (Lopez-Herrera et al.

1999) and applications of fungicides such as fluazinam

(Kanadani et al. 1998), but in some cases, these treat-

ments are very expensive or still experimental. Other

approaches include biological control, for example control

of R. necatrix by the fungus Trichoderma harzianum has

been reported (Freeman et al. 1986).

Recently, several studies have reported the isolation of

antagonistic bacteria against R. necatrix for control of this

pathogen (Cazorla et al. 2006, 2007; Ten Hoopen and

Krauss 2006). These studies have focused almost exclu-

sively on understanding how rhizobacterial antagonists

impact fungal pathogen survival and disease-causing

activity (Cazorla et al. 2006, 2007). To avoid selection of

antibiotic-producing micro-organisms that could have

undesirable secondary effects mainly because of unspecific

toxicity of the antimicrobial compounds, and to improve

the selection of potential biocontrol agents, a novel proce-

dure based on the selection of competitive root tip coloniz-

ers (Kamilova et al. 2005) was recently applied to generate

a collection of bacterial isolates that efficiently colonize the

roots of avocado plants (Pliego et al. 2007, 2008). This

strategy yielded a selection of antagonistic strains, but with

a low frequency of antibiotic-producing strains, and con-

tributed to the isolation of rhizobacterial strains with a

higher variety of potential biocontrol traits, such as compe-

tition for niches and nutrients (Pliego et al. 2007, 2008).

Materials and methods

Strains and growth conditions

Bacterial strains used in this study are listed in Table 1.

Pseudomonas spp. were grown on King’s B (KB) medium

(King et al. 1954) at 25�C, and the Bacillus spp. were

grown on nutrient agar (NA; Difco Lab, Detroit, MI,

USA) supplemented with 1% glucose (NAG) at 30�C.

Cycloheximide (100 lg ml)1) was used to prevent the

growth of fungi. Nutrient media were supplemented with

rifampicin (50 lg ml)1) when required.

Fungal and oomycete strains used in this study

(Table 2) were grown at 25�C on potato dextrose agar

(PDA; Difco Lab).

Construction and preliminary characterization of a

bacterial strain collection from avocado roots and soils

Bacterial strains were isolated in 2004 from root and soil

samples from 20 healthy avocado trees located in 12 avo-

cado orchards affected by root pathogens in Malaga and

Granada (Spain). Root and soil samples were collected

from avocado trees at a distance of 1 m from the trunk

and 10 cm from the soil surface. Samples were main-

tained in sterile bags, stored at 4�C and transported to

the laboratory where they were processed. Roots from

each sample were separated from the soil. Root samples

were washed twice in tap water and homogenized in a

laboratory blender for 3 min with 10 ml of sterile phos-

phate-buffered saline (PBS) (pH 7Æ2, 0Æ1 mol l)1) per

gram of fresh weight of root material. The remaining soil

samples were sifted through a mesh of 0Æ2 cm, and subse-

quently, 25 g of sifted soil was shaken in an Erlenmeyer

flask with 250 ml of sterile PBS for 1 h. To isolate and

estimate the bacterial counts in each sample, the subse-

quent suspensions obtained from root or soil samples

were serially diluted and plated on KB and NAG media

amended with cycloheximide (100 lg ml)1). After 2 days

of incubation at 25�C, colony counts were recorded, and

representative isolates (based on abundance and colony

morphology) were transferred to fresh KB and NAG-agar

plates to grow as pure cultures, which were included in

the bacterial collection and used for further experiments.

The strains in the bacterial collection were preliminarily

characterized by a few general tests, such as Gram staining

(Gerhardt et al. 1994), production of diffusible fluorescent

pigments under UV light on KB agar at 25�C, and antago-

nistic activity against R. necatrix. To study antagonism, a

dual-plate assay was used (Kloepper and Schroth 1981).

Briefly, screening for in vitro antifungal activity on KB and

PDA agar plates against R. necatrix CH53 (Table 2) was

performed by placing in the centre of a Petri dish a

Antagonism is a prevalent trait M.A. Gonzalez-Sanchez et al.

66 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78

ª 2009 The Authors

0Æ6-cm diameter mycelium disk from a 7-day-old fungal

culture grown at 24�C, followed by inoculation of the bac-

terial strains at a distance of about 3 cm from the fungus.

Bacterial strains inhibiting mycelial growth after 5–7 days

were reported as R. necatrix antagonists.

Selection of bacterial candidates for potential biocontrol

Among the bacterial collection, bacterial isolates obtained

from different samples were chosen for further experi-

mentation based on differences in abundance, colony

morphology, Gram staining, fluorescence and R. necatrix

antagonism.

These bacterial isolates from avocado soils and roots

were screened as potential biocontrol candidates against

white root rot using a biocontrol assay including avocado

plantlets (cvs Reed and Topa-Topa) with a well-developed

root system and homogeneous characteristics. These

plants were obtained by in vitro culture from germinated

embryos as previously described with slight modifications

(Pliego-Alfaro 1988; Cazorla et al. 2006). Plantlets were

used in these assays after 8 weeks of ex vitro development

on perlite to harden the roots (Pliego-Alfaro 1988). Roots

of avocado plantlets were surface disinfected by immer-

sion in 0Æ06% NaOCl for 10 min and then washed twice

in tap water. Subsequently, disinfected avocado roots were

Table 1 Bacterial strains used in this study

Strain Relevant characteristics* Reference or source

Bacillus

Bacillus sp. CB153 Isolated from avocado rhizoplane This study

Bacillus cereus CB12 Isolated from avocado rhizoplane This study

Bacillus mycoides CB135 Isolated from avocado soil This study

Bacillus subtilis CB43 Isolated from avocado soil This study

B. subtilis CB100 Isolated from avocado soil This study

B. subtilis CB115 Isolated from avocado soil This study

B. subtilis CB301 Isolated from avocado soil This study

Enterobacter

Enterobacter sp. CB240 Isolated from avocado rhizoplane This study

Pseudomonas

Pseudomonas sp. CB217 Isolated from avocado rhizoplane This study

Pseudomonas sp. CB225 Isolated from avocado rhizoplane This study

Pseudomonas chlororaphis CB58 Isolated from avocado rhizoplane This study

Ps. chlororaphis CB254 Isolated from avocado soil This study

Ps. chlororaphis CB303 Isolated from avocado soil This study

Ps. fluorescens CB21 Isolated from avocado rhizoplane This study

Ps. fluorescens CB32 Isolated from avocado rhizoplane This study

Ps. fluorescens CB78 Isolated from avocado rhizoplane This study

Ps. fluorescens CB306 Isolated from avocado rhizoplane This study

Pseudomonas putida CB6 Isolated from avocado rhizoplane This study

Ps. putida CB95 Isolated from avocado soil This study

Ps. putida CB104 Isolated from avocado soil This study

Ps. putida CB286 Isolated from avocado soil This study

Ps. putida CB320 Isolated from avocado rhizoplane This study

Reference strains

B. subtilis UMAF6614 Produce FEN, BAC and SUR, biocontrol of Podosphaera fusca Romero et al. 2007

B. subtilis UMAF6639 Produce FEN, ITU and SUR, biocontrol of P. fusca Romero et al. 2007

Escherichia coli DH5a General-purpose host strain Boyer and Roulland-Dussoix 1969

Pseudomonas chlororaphis PCL1391 Produce PCA and PCN, biocontrol of Fusarium oxysporum

f. sp. radicis-lycopersici.

Chin-A-Woeng et al. 1998

Pseudomonas fluorescens PCL1606 Isolated from avocado roots, producing HPR, biocontrol of

avocado white root rot caused by Rosellinia necatrix

Cazorla et al. 2006

Ps. fluorescens Pf5 Produce DAPG, PLT and PRN, biocontrol of Rhizoctonia solani Howell and Stipanovic 1979

Ps. putida WCS358-Rif Spontaneus RIF-resistant mutant derived from Ps. putida

WCS358, plant-growth-promoting strain isolated from

the rhizosphere of potato plants

Geels and Schippers 1983

*DAPG, 2, 4-diacetyl phloroglucinol; PRN, pyrrolnitrin; PLT, pyoluteorin; HPR, 2-hexyl-5 propyl resorcinol; PCA, phenazine-1-carboxilyc acid; PCN,

phenazine-1-carboxamide; FEN, fengycin; ITU, iturin; SUR, surfactin; BAC, bacillomycin; RIF, rifampicin.

M.A. Gonzalez-Sanchez et al. Antagonism is a prevalent trait

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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78 67

dipped for 20 min into a suspension of the bacterial iso-

late to be tested in PBS (ranging between 108 and

109 CFU ml)1) or into sterile PBS as a control. Treated

plants were placed into 250-ml pots containing 30 g of

wet potting soil and inoculated with the virulent strain

R. necatrix CH53, using four R. necatrix-infected wheat

grains per pot (Freeman et al. 1986; Cazorla et al. 2006).

Because of the high number of bacterial strains to be

tested and space requirements, only five plantlets were

used per strain in this initial direct selection process.

Treated plantlets were grown in a chamber at 25�C with

70% relative humidity, and 16 h at 40 lE m)2 s)1 daily

light exposures. Plants were watered from the bottom

twice per week (25 ml each time). Aerial symptoms,

resulting from root infection, were evaluated on a scale of

values (0, healthy plant; 1, wilting of leaves; 2, overall

wilting and dryness of leaves and 3, dead plant), and a

disease index (DI) percentage was recorded by applying

the previously described formula (Cazorla et al. 2006).

The previously mentioned 143 strains were tested in 29

independent experiments. In each experiment, five plants

were used for each bacterial isolate, and five to seven iso-

lates were tested per independent experiment. The bio-

control strain Pseudomonas fluorescens PCL1606 (Table 1)

was included as a positive control in all the experiments,

and plants with noninoculated roots were used as a nega-

tive control. To normalize the results, the experiments

were terminated when the untreated plantlets (negative

control) reached a DI value of 50%.

Biological control of selected protective candidates

Bacterial isolates displaying protection and reducing the

DI below 30% in the initial biocontrol trials were selected

for further testing in a second trial of complete biocontrol

experiments. Thirty plants per treatment in three inde-

pendent experiments of ten plants each were used for

each selected bacterial isolate. In these assays, clonal avo-

cado plantlets were used in order to reduce the variability

because of plant genotypes. Clonal avocado plants were

obtained following a previously described method

(Pliego-Alfaro and Murashige 1987), with slight modifica-

tions. Briefly, shoots of avocado, belonging to clone TT53

(rootstock cv. Topa Topa), were grown in the active pro-

liferation medium Murashige & Skoog (MS; Sigma-

Aldrich, St Louis, MO, USA) modified with macronutri-

ents N45K (Barcelo-Munoz et al. 1990) supplemented

with benziladenine (0Æ3 mg l)1). To induce the appear-

ance of roots, terminal shoots 1Æ5 cm in length from these

cultures were transferred into tubes (25 · 150 mm)

containing 25 ml of a modified MS medium (MS) with

macronutrients at one-third of standard concentration

and indole-3-butyric acid (1 mg l)1). The tubes were sha-

ken at 5 rev min)1 in a roller drum for 3 days, at 25�C,

70% relative humidity, and 16 h at 40 lE m)2 s)1 of daily

light exposure. Subsequently, to promote root growth,

shoots were transferred to similar tubes with modified MS

medium with active charcoal (1 mg l)1) and agar (6 g l)1).

Tubes were incubated for 6 weeks in the same conditions

without shaking. Then, avocado plantlets were placed into

pots of 250 ml with a vermiculite: perlite mix (50%) and

0Æ1% fertilizer (Osmocote Exact Mini; Scotts O.M. Espana,

Tarragona, Spain). Every 2 weeks for a period of 8 weeks,

the seedlings were fertilized with 20 ml of 0Æ2% Bayfolan S

(Bayer, Barcelona, Spain). Plantlets were maintained in a

growth chamber under the conditions described earlier for

10–12 weeks after which 15–20-cm seedlings were used in

biocontrol experiments.

The moderately virulent strain R. necatrix CH33

(Table 2) was used instead of the highly virulent R. neca-

trix strain CH53 in order to slightly reduce the inoculum

pressure. Root bacterization, inoculum size of pathogen,

Table 2 Fungal and oomycete strains used in this study

Strains Relevant characteristics Reference or source*

Fusarium oxysporum f. sp.

radicis-lycopersici ZUM2407

Causes crown and foot rot of tomato IPO-DLO

Phytophthora cactorum CH462 Causes cankers on avocado trunks; pathogen to many plants INIA

Phytophthora cinnamomi CH14 Isolated from avocado root rot; high virulence Perez-Jimenez (1997)

P. cinnamomi CH23 Isolated from avocado root rot; medium virulence Perez-Jimenez (1997)

P. cinnamomi CH24 Isolated from avocado root rot; low virulence Perez-Jimenez (1997)

Rhizoctonia solani CH559 Causes Rhizoctonia seed and root rot on avocado; pathogen to many plants INIA

Rosellinia necatrix CH10 Isolated from white root rot of avocado; medium virulence Perez-Jimenez (1997)

R. necatrix CH33 Isolated from white root rot of avocado; low virulence Perez-Jimenez (1997)

R. necatrix CH53 Isolated from white root rot of avocado (former Rn 400); high virulence Perez-Jimenez (1997)

Verticillium dahliae CH460 Causes Verticillium wilt of avocado; pathogen to many plants INIA

*IPO-DLO, Institute for Plant Protection-Agriculture Research Department, Wageningen, The Netherlands; INIA, Instituto Nacional de Investigacion

y Tecnologıa Agraria y Alimentaria, Madrid, Spain.

Antagonism is a prevalent trait M.A. Gonzalez-Sanchez et al.

68 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78

ª 2009 The Authors

growth conditions of the plants and evaluation of aerial

symptoms were performed following the same methods

described in the previous biocontrol assays.

Identification of selected bacterial isolates

A number of phenotypic and physiological tests to iden-

tify and characterize the selected biocontrol bacterial iso-

lates were performed by the conventional tests of glucose

metabolism, oxidase and arginine dihydrolase reaction

(Gerhardt et al. 1994) and by using the API 20NE� sys-

tem (BioMerieux, Mercy L’Etoile, France). To avoid selec-

tion of potential phytopathogenic bacteria, bacterial

isolates were tested for elicitation of the hypersensitive

response (HR) on tobacco leaves var. Newdel (Lelliott

and Stead 1987). Bacterial characterization was comple-

mented by analysis of the 16S rDNA sequence. For this,

colony PCR on the 22 selected strains was performed,

using the primers 41F and 1486-N for Gram-negative

isolates, and primers 41F and 1468r-P for Gram-positive

isolates (Cazorla et al. 2006, 2007). The resulting PCR

fragment was purified (QIAquick PCR purification kit 50;

Westburg, Leusden, The Netherlands) and used directly

for sequencing (Macrogen Inc., Seoul, Korea). The

sequences were analysed using dnaman software (Lynnon

Biosoft, Quebec, QC, Canada). Homology studies were

carried out using the National Center for Biotechnology

Information GenBank Blast Software (NCBI, Bethesda,

MD, USA).

Antagonism against soil-borne fungal and oomycete

plant pathogens

For the selected bacterial isolates, a more complete study

of their in vitro spectra of fungal inhibition was carried out

against a number of fungi and oomycetes pathogenic to

avocado and other plants, including additional strains of

R. necatrix (Table 2) via a dual-plate assay, performed as

described earlier. All assays were repeated at least twice.

Production of antifungal metabolites

Selected bacterial isolates were tested for production of

hydrogen cyanide (HCN), proteases, lipases, b-glucanases

and cellulases by general procedures (Gerhardt et al.

1994). Siderophore production was assayed according to

the method of Schwyn and Neilands (1987) using chrome

azurol S (CAS) medium.

For detection of the antibiotics produced by antagonis-

tic Pseudomonas spp., extractions of cell-free supernatants

from 5-day-old KB cultures of the corresponding strains

were carried out with chloroform ⁄ methanol (2 : 1 v ⁄ v).

Extracted material was fractionated in chloroform ⁄ ace-

tone 9 : 1 (v ⁄ v) via thin-layer chromatography (TLC)

using silica RP-18F254s TLC plates (Merck AG, Darmstadt,

Germany). After drying, the chromatograms were

observed under UV light of 254 nm. The presence of spots

with Rf values of 0Æ50 and 0Æ87 indicate production of

phenazine-1-carboxylic acid (PCA) and phenazine-1-carb-

oxamide (PCN), respectively. Spots of other antibiotics

were also reported after spraying with diazotized sulfanilic

acid (Whistler et al. 2000). Antibiotics were identified via

their characteristic colours and Rf values, corresponding to

those produced by the reference strains: pyoluteorin (PLT),

Rf = 0Æ68 brown; pyrrolnitrin (PRN), Rf = 0Æ81 maroon; 2,

4 diacetyl phloroglucinol (DAPG), Rf = 0Æ74 yellow; and 2-

hexyl 5-propyl resorcinol (HPR), Rf = 0Æ93 dark green.

Strains Pseudomonas chlororaphis PCL1391, Ps. fluorescens

Pf5 and Ps. fluorescens PCL1606 were used as references to

compare their production of antibiotics to that of the tested

strains (Table 1).

The presence of antifungal lipopeptides in bacterial cul-

ture supernatants of Bacillus spp. was analysed via TLC as

described previously (Romero et al. 2007). Briefly, bacte-

rial cultures were grown on nutrient broth (NB) supple-

mented with 1% glucose for 4 days at 28�C. Cell-free

supernatants were obtained by centrifugation at 2500 g

for 15 min and then extracted with n-butanol. Once the

n-butanol layers were evaporated to dryness under a

vacuum, the residues were dissolved in methanol and

fractionated by TLC using silica gel plates (HPTLC

60-F254; Merck AG) with chloroform–methanol–water

(65 : 25 : 10, v ⁄ v ⁄ v). For identification of these antifungal

compounds, methanolic extracts of the Bacillus subtilis

strains UMAF6614 and UMAF6639 (Table 1) were

obtained following the procedure described previously,

and used as controls (Romero et al. 2007). Spots of lipo-

peptides were detected after spraying with water and

identified by Rf values corresponding to those of refer-

ence strains: fengycines, Rf = 0Æ1; iturin A Rf = 0Æ3;

bacillomycines Rf = 0Æ3–0Æ6; and surfactines, Rf = 0Æ7. All

experiments were repeated at least twice.

Motility assays

Swimming and swarming abilities were tested in KB med-

ium diluted 1 ⁄ 20 in milliQ water with agar concentra-

tions of 0Æ3 and 0Æ5%, respectively. Twitching motility

was assayed on 1% agar plates as previously described

(Pliego et al. 2007). For all motility tests, the strains to be

tested were stab-inoculated through agar plates. Swim-

ming and swarming motility was recorded after 48 h of

incubation. Twitching motility was recorded after

3–4 days of growth at 25�C, staining the zone of the agar

and Petri dish interface with 1% crystal violet (CV). All

assays were repeated at least twice.

M.A. Gonzalez-Sanchez et al. Antagonism is a prevalent trait

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78 69

Bacterial persistence and avocado root colonization

assays

For these assays, avocado plants from in vitro germinated

embryos with 12 weeks of ex vitro growth were used.

Spontaneous rifampicin-resistant (50 mg ml)1) strains

were obtained for the bacterial isolates to be tested in

these experiments and their growth characteristics in

liquid media were compared with those of the wild type

strains. Plant roots were disinfected, inoculated with bac-

terial suspensions of the rifampicin-resistant strains con-

taining about 108–109 CFU ml)1 and grown in

vermiculite under the same conditions described for the

biocontrol assays. Bacteria were recovered from different

parts of the roots; those bacteria located within 1 cm of

the root tips were considered colonizers, and those

located 2–6 cm from the root tips were considered persis-

tent forms of the assayed bacteria.

Bacterial recovery from the roots was performed as fol-

lows. One hour after inoculation (time 0), and at 7, 14

and 21 days of plant growth, three plants per bacterial

strain inoculated were removed from the vermiculite and

their roots were processed independently. Two root sam-

ples were collected: the root tip (1 cm to the tip), and a

segment 2–6 cm from the root tip. For both types of

samples, bacterial population sizes were determined as

follows. Root samples were weighed and homogenized in

a laboratory blender for 2 min with 10 ml of sterile PBS

per gram of fresh root material. The resulting suspensions

were serially diluted and plated on KB or NAG medium,

for Pseudomonas spp. or Bacillus spp. strains respectively,

and supplemented with rifampicin (50 lg ml)1) and

cycloheximide (100 lg ml)1). To select Bacillus spp.

spores, the resulting suspension was also heated to 80�C

for 10 min. Then, rifampicin-resistant colony counts were

determined after 48 h of incubation at 25�C.

Biofilm formation: attachment assay

Biofilm formation was assessed using the microtitre plate

assay (O’Toole et al. 2000). Selected bacterial strains were

grown and shaken in Luria-Bertani (LB) medium at 25�C

to mid-exponential growth and cells diluted to a theoretical

OD595 of 0Æ0025 in fresh LB medium. Samples of 150 ll of

the diluted cells were allocated to each well of 96-well poly-

styrene microtitre plates. The microtitre plates were incu-

bated at stationary conditions at 25�C for a 48 h. Cells that

had adhered to each well were stained with 175 ll of a

0Æ1% (w ⁄ v) CV solution at room temperature for 20 min.

Excess CV was then removed, and the wells were rinsed

with water. CV stain was measured after the addition of

175 ll of dimethyl sulfoxide for each dry well. The samples

then sat for c. 20 min, and their OD595 were measured on

a plate reader. Pseudomonas putida WCS358-Rif (Geels and

Schippers 1983; Bakker et al. 1986) and B. subtilis

UMAF6639 (Romero et al. 2007) were included as refer-

ence strains. Escherichia coli DH5a was included as negative

control (Tomaras et al. 2003). This experiment was

repeated at least three times with eight independent wells

per sample.

Growth promotion assay

For this assay, the method described previously by Cham-

bel et al. (1994) was employed with minor modifications.

Briefly, seeds of lettuce cv. Maravilla de verano (Fito,

Barcelona, Spain) were dipped into a bacterial suspension

(109 CFU ml)1), for 1 h at 25�C. The treated seeds were

placed between two sterile wet filter paper sheets in a

plate. Three plates with seven seeds each were used per

treatment. Ten days after incubation (25�C, 16 h artificial

light and 8 h dark) in a growth chamber, the fresh

weights of seedlings were recorded. The strain Ps. fluores-

cens WCS358-Rif was used as a positive control.

Statistical methods

Data obtained from different experiments were statisti-

cally analysed via analysis of variance followed by Fisher’s

least significant difference test (P = 0Æ05) using spss

software (SPSS Inc., Chicago, IL, USA).

Results

Isolation and preliminary characterization of bacteria

from avocado roots and soils

The numbers of culturable bacterial cells present on the

avocado root and soil samples were estimated after isola-

tion and growth on NAG and KB agar. Avocado roots

contained between 5Æ7 and 7Æ4 log CFU of aerobic cultur-

able bacteria per gram of fresh weight, while soil samples

contained between 6Æ3 and 7Æ3 log CFU g)1 of soil.

From avocado soil and root samples, a collection of

330 bacterial isolates was obtained by selecting isolated

colonies, based on abundance and differences in colony

morphology. All strains in the bacterial collection were

characterized based on Gram staining assay, production

of diffusible fluorescent pigments under ultraviolet light

on KB plates and antagonistic ability against the fungal

strain R. necatrix CH53 using a dual plate assay.

From the bacterial collection, 110 isolates (33%) were

Gram-positive, and 220 isolates (67%) were Gram-

negative. Among the Gram-negative isolates, 84 (38%)

were fluorescent on KB plates. Antagonistic assays revealed

26 isolates (8%) with antifungal activity against R. necatrix

Antagonism is a prevalent trait M.A. Gonzalez-Sanchez et al.

70 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78

ª 2009 The Authors

CH53; among them, 22 were Gram-negative, of which 14

were fluorescent, and four isolates were Gram-positive.

Selection of bacterial candidates for Rosellinia necatrix

biocontrol by a direct avocado-protection method

Among the collection of 330 bacterial isolates, a total of

143 isolates were chosen to be screened as biocontrol can-

didates. For this, a simplified biocontrol assay comprising

a single trial that included plantlets obtained by in vitro

germinated embryos were used. The 143 screened isolates

included the 26 antagonistic against R. necatrix CH53, as

well as other 117 isolates, all selected based on the sample

source and differences in colony morphology and abun-

dance.

These biocontrol experiments yielded a total of 22 iso-

lates showing a DI below 30% when the nonbacterized

control DI was 50% (Table 1 and Fig. 1). The other 121

assayed isolates had DI higher than 30% (data not

shown), and were thus discarded. Pseudomonas fluorescens

PCL1606, a biocontrol strain used as a reference, showed

an average DI of 28% in these biocontrol experiments.

Among the 22 selected isolates, 11 isolates had been pre-

viously characterized as antagonistic to R. necatrix CH53.

Twelve of the strains had been isolated from root samples

and ten from soil samples (Fig. 1).

Identification of these 22 selected bacterial isolates was

performed by some conventional tests, such as Gram

staining, glucose metabolism, fluorescence, and the API

20NE� miniaturized gallery, combined with homology

analyses of nucleotide sequences of their PCR-amplified

16S rDNA. These studies revealed that fourteen isolates

could be assigned to the genus Pseudomonas, one to the

genus Enterobacter and seven to the genus Bacillus

(Table 1). None of them elicited the HR on tobacco

leaves. The strains antagonistic to R. necatrix CH53 were

identified as the Pseudomonas spp., including Ps. chlorora-

phis and Ps. fluorescens, and B. subtilis (Fig. 1).

In a second set of biocontrol trials, the 22 selected can-

didate strains were tested in a more accurate biocontrol

test including three repetitions per strain with ten plants

each. Bacillus subtilis CB115, Ps. chlororaphis CB254 and

80

60

70

50

40

30

20

10

0Inoculant strain

Source* R

B. s

p. C

B15

3

B.c

. CB

12

B.m

. CB

135

B.s

. CB

43

B.s

. CB

100

B.s

. CB

115

B.s

. CB

301

Ent

. sp.

CB

240

P. s

p. C

B21

7

P. s

p. C

B22

5

P.c

. CB

58

P.c

. CB

254

P.c.

CB

303

P.f.

CB

21

P.f.

CB

32

P.f.

CB

78

P.f.

CB

306

P.p.

CB

6

P.p.

CB

95

P.p.

CB

104

P.p.

CB

286

P.p.

CB

320

Non

-bac

teriz

ed

P.f.

PC

L160

6

+

+

+ + + +

+ + + + + + +

+ + + + + + +

++++++++++++++–

–

–

– – – – – –

–

– – – – – – – –

– – – – – –

–––––––

–

–––

R R R R R R R R R R R RSSSSSSSSSS

Gram stain

Antagonism Rn†

Fluroscence‡

Dis

ease

in in

dex

(%)

Figure 1 Biocontrol shown by candidates of potential biocontrol agents after selection against avocado white root rot. Twenty-two isolates

among the 143 tested showed protection [a disease index (DI) below 30% when the DI of the nonbacterized control was 50%] against avocado

white root rot. Five avocado seedlings were used per strain. Strain Pseudomonas fluorescens PCL1606 was used as a control. The table below the

figure shows the characteristics of the strains after prescreening. Table below: (*) sample source: R, roots; S, soil; (�) antagonism against Rosellinia

necatrix CH53: +, fungal inhibition; ), no fungal inhibition; (�) production of fluorescent pigments under ultraviolet light (254 nm) on King’s B

medium.

M.A. Gonzalez-Sanchez et al. Antagonism is a prevalent trait

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78 71

Ps. fluorescens CB32, as well as the reference biocontrol

strain Ps. fluorescens PCL1606, showed significant reduc-

tions of DI in the experiments at two different disease

pressure, when the DI of the nonbacterized avocado con-

trol plants was 50 and 90% (Fig. 2). Strains Ps. fluorescens

CB306 and Enterobacter sp. CB240 showed a significant

reduction of the DI only when nonbacterized control DI

was 50% (Fig. 2). The remaining strains were not able to

reduce the DI significantly. Interestingly, the three strains

showing broader biocontrol ability, B. subtilis CB115,

Ps. chlororaphis CB254 and Ps. fluorescens CB32, were

antagonistic to R. necatrix CH53 (Fig. 1).

Bacterial traits correlated with the biocontrol activity

All 22 bacterial strains selected in the initial biocontrol

assay were tested for several traits potentially involved in

biological control of R. necatrix, in order to detect possi-

ble differences among bacteria selected using each of the

selection strategies. The 22 bacterial strains selected in the

80

*

*

*

*

* *

* *

* *

60

70

50

40

30

20

10

100

90

80

70

60

50

40

Dis

ease

in in

dex

(%)

B. s

p. C

B15

3

B.c

. CB

12

B.m

. CB

135

B.s

. CB

43

B.s

. CB

100

B.s

. CB

115

B.s

. CB

301

Ent

. sp.

CB

240

P.s

p. C

B21

7

P.s

p. C

B22

5

P.c

. CB

58

P.c

. CB

254

P.c.

CB

303

P.f.

CB

21

P.f.

CB

32

P.f.

CB

78

P.f.

CB

306

P.p.

CB

6

P.p.

CB

95

P.p.

CB

104

P.p.

CB

286

P.p.

CB

320

Non

-bac

teriz

ed

P.f.

PC

L160

6

Inoculant strain

Antagonism† +

+

+

6·6 3·5

2·7

1·6 4·5 6·0

5·33·6 6·2 6·7

7·06·3 6·3 6·8

6·8 4·6

5·9

6·3

6·6

6·1

6·1

5·7 5·5

6·06·37·0NT NT

NT

NT NT

NT

NT

NTNTNTNTNT

NTNT

NTNTNTNTNTNTNT

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

NTNT5·85·3<1·0 <1·0

<1·0

6·3

+

+ + + +

+ + +

+ + + + + + +

+ + + + + + + + + + + + + + +

– – –

––

–

– – –

– – – – –

– – –

– – – – – – –

– – – – –

– – – – –

– –+ + + + + + + + + + + + + + – – –

– –+++++ +

Biofilm (adhesion)††

PGPR activity‡‡

Antif. metabolities‡

Siderophores

Root persistence§

Root colonization**

Figure 2 Protection conferred by 22 selected bacterial isolates against avocado white root rot when the disease index of the nonbacterized

control reaches 50 and 90%. Thirty avocado seedlings were used per strain. Strain Pseudomonas fluorescens PCL1606 was used as a control.

Asterisks denote statistically significant differences with the nonbacterized control (P = 0Æ05). Below each strain, a summary of the biocontrol traits

tested. NT, Not tested. �Antagonism against different fungi and oomycetes; see details in Table 3. �See details in Table 4. §Log CFU g)1 fresh

root sample taken around 6 cm from the root tip 21 days postinoculation. See Table 5. **Log CFU g)1 fresh root tip sample 21 days postinocula-

tion. See Table 5. ��Adhesion to polystyrene. ��Plant-growth promoting activity.

Antagonism is a prevalent trait M.A. Gonzalez-Sanchez et al.

72 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78

ª 2009 The Authors

initial biocontrol assays were tested for antagonistic activ-

ity against some different soil-borne pathogens affecting

avocado and other plants, using the dual culture tech-

nique on KB agar and PDA plates (Table 3). All Pseudo-

monas strains antagonistic to R. necatrix CH53 also

showed activity against the two other tested strains of

R. necatrix and against other soil-borne fungal and

oomycete pathogens tested (Table 3). However, the

strains Bacillus cereus CB12, Ps. fluorescens CB306 and

Ps. putida CB95 that had not previously shown antifungal

activity to R. necatrix did so against Verticillium dahliae

and ⁄ or Phytophthora spp. The three Ps. chlororaphis

strains exhibited the strongest antifungal activity against

all the pathogens tested (Table 3).

The selected strains were also analysed for exoenzyme

production, HCN and antibiotics. The results obtained

for these bacteria with potential biocontrol activity are

summarized in Table 4. Protease activity was detected for

most antagonistic strains. b-glucanase or cellulase activi-

ties were present only in some Bacillus strains. Lipase

activity was present only in the Ps. chlororaphis strains,

and HCN production was detected in several isolates of

Pseudomonas (Table 4). On the other hand, all Pseudomo-

nas strains produced siderophores in CAS agar medium

(Fig. 2).

The identification of common antibiotics of Pseudomo-

nas was carried out by organic extractions and fraction-

ation using TLC. This analysis revealed that the three

strains of Ps. chlororaphis produced compounds with the

same Rf values as PCA (Rf = 0Æ50) and PCN (Rf = 0Æ87),

when compared to the reference strain Ps. chlororaphis

PCL1391. No compounds with similar Rf values to

DAPG, HPR, PLT or PRN were detected for any of the

tested strains (Table 4).

To detect lipopeptidic antibiotics in the Bacillus strains,

methanolic fractions from butanolic extracts of cell-free

culture filtrates of the strains were separated in TLC

sheets using the strains B. subtilis UMAF6639 and

Table 3 Identification and antagonistic ability against soil-borne fungal and oomycete phytopathogens via dual plate assay

Strain

Antagonistic activity*

Rosellinia necatrix

(n = 3)�

Phytophthora

cinnamomi (n = 3)�

Phytophthora

cactorum

Fusarium

oxysporum

Rhizoctonia

solani

Verticillium

dahliae

Bacillus

Bacillus sp. CB153 ) ) ) ) ) )Bacillus cereus CB12 ) ) ) + ) ++

Bacillus mycoides CB135 ) ) ) ) ) )Bacillus subtilis CB43 ++ ++ ++ ++ ++ ++

B. subtilis CB100 ++ + + ++ + ++

B. subtilis CB115§ ++ + + + + ++

B. subtilis CB301 ++ + + ++ + +

Enterobacter

Enterobacter sp. CB240§ ) ) ) ) ) )Pseudomonas

Pseudomonas sp. CB217 + + + ) ) +

Pseudomonas sp. CB225 + + + ) ) +

Pseudomonas chlororaphis CB58 ++ ++ ++ ++ ++ ++

Ps. chlororaphis CB254§ ++ ++ ++ ++ ++ ++

Ps. chlororaphis CB303 ++ ++ ++ ++ ++ ++

Pseudomonas fluorescens CB21 ) ) ) ) ) )Ps. fluorescens CB32§ + + + ) ) ++

Ps. fluorescens CB78 + + + ) + ++

Ps. fluorescens CB306§ ) + + ) ) +

Pseudomonas putida CB6 ) ) ) ) ) )Ps. putida CB95 ) + + ) ) ++

Ps. putida CB104 ) ) ) ) ) )Ps. putida CB286 ) ) ) ) ) )Ps. putida CB320 ) ) ) ) ) )

*Pseudomonas spp. and Enterobacter sp. were tested on King’s B agar, and Bacillus spp. on PDA; +, diameter of the growth inhibition zone

between 1 and 5 mm; ++, diameter of the growth inhibition zone >5 mm; ), no antifungal activity observed.

�Three strains of R. necatrix (CH53, CH10, and CH33) were tested and all produced similar results.

�Three strains of P. cinnamomi (CH14, CH23 and CH24) were tested and all produced similar results.

§Bacterial strains displaying the most efficient biocontrol activity.

M.A. Gonzalez-Sanchez et al. Antagonism is a prevalent trait

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78 73

UMAF6614 as references. The B. subtilis strains CB43,

CB100 and CB115 produced spots with similar Rf values

to fengycin (Rf = 0Æ09) and surfactin (Rf = 0Æ7), and

B. subtilis CB43 also produced bacillomycin (Rf = 0Æ3–

0Æ6).

Characterization of motility types showed swimming in

all the tested strains except Ps. fluorescens CB78. The

lowest diameter in swimming assays was displayed by

B. subtilis CB115 (diameter below than 1Æ5 cm), while the

rest of the assayed strains always showed higher diameter

values, reaching values above 3 cm of swimming diameter

for Ps. chlororaphis CB58 or Ps. fluorescens CB306.

Swarming motility was detected only in Ps. chlororaphis

and B. subtilis strains. None of the tested strains exhibited

twitching motility under the conditions tested.

Bacterial persistence and colonization of avocado roots

was studied in a 21-day experiment, in which two differ-

ent root sections of 3-month-old avocado plants were

analysed. Colonization was analysed at root tips (sections

of 0–1 cm at the tip), and bacterial persistence was stud-

ied on root sections 2–6 cm from the root tips. The bac-

terial counts for the different strains in the two types of

sections after 21 days are summarized in Fig. 2. Detailed

results of the four most protective strains are shown in

Table 5. In general, the Pseudomonas spp. tested were able

to colonize both root sections. After 21 days, bacterial

count levels in sections 2–6 cm from the root tips were in

the range of 106–107 CFU g)1 of root; however, in root

tip sections, bacterial counts were more variable among

the tested strains (105–107 CFU g)1 of root). The amount

of bacteria tends to be higher on the root sections

2–6 cm from the tips than in the sections 0–1 cm from

the root tips (Table 5 and Fig. 2). The bacterial levels

reached by the strains of B. subtilis were clearly lower,

especially in samples from the root tips (Fig. 2) and

mainly spores were found, because the bacterial counts

observed from heated and nonheated root samples were

identical. The levels of persistence and colonization

achieved by B. subtilis CB115 (Table 5) were the lowest.

The ability to initiate biofilm formation was tested by

measuring the attachment of the bacteria to the wells of a

polystyrene microtitre plate. Only the reference strains,

Ps. putida WCS358-Rif and Ps. fluorescens PCL1606 and

the assayed strains of B. subtilis, except CB115, showed

Table 4 Production of antifungal metabolites by the studied bacterial

strains

Strain

Production of antifungal

metabolites*

HCN Exoenzymes Antibiotics

Bacillus

Bacillus sp. CB153 ) ) )Bacillus cereus CB12 ) P, C, G )Bacillus mycoides CB135 ) P )Bacillus subtilis CB43 ) P, C, G FEN, SUR, BAC

B. subtilis CB100 ) P, C, G FEN, SUR

B. subtilis CB115� ) P, C, G FEN, SUR

B. subtilis CB301 ) ) )Enterobacter

Enterobacter sp. CB240� ) G )Pseudomonas

Pseudomonas sp. CB217 + P )Pseudomonas sp. CB225 ) ) )Pseudomonas chlororaphis CB58 + P, L PCA, PCN

Ps. chlororaphis CB254� + P, L PCA, PCN

Ps. chlororaphis CB303 + P, L PCA, PCN

Pseudomonas fluorescens CB21 ) ) )Ps. fluorescens CB32� + P )

Ps. fluorescens CB78 ) P )Ps. fluorescens CB306� ) ) )Pseudomonas putida CB6 ) ) )Ps. putida CB95 + P )Ps. putida CB104 ) ) )Ps. putida CB286 ) ) )Ps. putida CB320 ) ) )

*), not detected; +, presence; P, protease; C, cellulase; G, b-glucan-

ase; L, Lipase; PCA, phenazine-1-carboxilyc acid; PCN, phenazine-

1-carboxamide; FEN, fengycin; SUR, surfactin; BAC, bacillomycin.

�Bacterial strains displaying the most efficient biocontrol activity.

Table 5 Bacterial counts (log CFU g)1 root) in different sections of avocado roots bacterized with selected strains to determine the bacterial

colonization and persistence on avocado roots

Root inoculated with

Log 10 (CFU g)1 root)

Sections 0–1 cm from tips (colonization) Sections 2–6 cm from tips (persistence)

0 days 7 days 14 days 21 days 7 days 14 days 21 days

Bacillus subtilis CB115 2Æ74 ± 0Æ17 0Æ57 ± 0Æ47 ND* ND 1Æ73 ± 0Æ25 0Æ50 ± 0Æ50 ND

Pseudomonas chlororaphis CB254 7Æ67 ± 0Æ07 7Æ62 ± 0Æ23 7Æ20 ± 0Æ32 6Æ27 ± 0Æ22 7Æ99 ± 0Æ19 7Æ70 ± 0Æ35 6Æ79 ± 0Æ70

Pseudomonas fluorescens CB32 7Æ44 ± 0Æ04 7Æ69 ± 0Æ12 7Æ28 ± 0Æ36 6Æ82 ± 0Æ23 7Æ59 ± 0Æ15 7Æ66 ± 0Æ20 7Æ01 ± 0Æ22

Ps. fluorescens CB306 7Æ70 ± 0Æ26 7Æ24 ± 0Æ15 7Æ15 ± 0Æ12 6Æ10 ± 0Æ38 7Æ31 ± 0Æ10 7Æ10 ± 0Æ25 6Æ32 ± 0Æ44

Ps. fluorescens PCL1606 7Æ91 ± 0Æ18 7Æ64 ± 0Æ10 6Æ92 ± 0Æ48 6Æ26 ± 0Æ29 7Æ80 ± 0Æ05 7Æ13 ± 0Æ28 6Æ56 ± 0Æ04

*ND, lower than 1Æ4 log 10 CFU g)1 roots.

Antagonism is a prevalent trait M.A. Gonzalez-Sanchez et al.

74 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78

ª 2009 The Authors

significant adhesion values under the conditions tested

(Fig. 2).

Finally, to test whether some of the studied strains pro-

mote plant growth, lettuce seeds were inoculated with

these strains and grown in plates for 10 days. Measure-

ments from fresh lettuce seedlings showed that B. subtilis

CB115, Ps. chlororaphis CB58 and Ps. fluorescens CB306

exert a significant effect on plant growth (Fig. 2), as did

the reference strain Ps. putida WCS358-Rif (data not

shown).

Discussion

The rhizosphere of healthy avocado trees located in areas

affected by soil-borne phytopathogenic fungi represents a

feasible and useful source for isolation of micro-organ-

isms with promising biocontrol traits (Cazorla et al. 2006,

2007; Pliego et al. 2007). The broad presence of bacteria

belonging to the genera Pseudomonas and Bacillus suggest

that representatives of both genera could be habitually

associated with avocado roots, as was previously shown

(Cazorla et al. 2006, 2007; Pliego et al. 2007). This fact is

consistent with the well-established ability of these groups

of bacteria to live in the root and soil environments

(Dekkers et al. 2000b; Lugtenberg et al. 2001).

In previous work, the search for biocontrol microbial

agents against the avocado white root rot causal agent

R. necatrix was based on antagonism (Cazorla et al. 2006,

2007) or colonization (Pliego et al. 2007) assays, the most

frequently used strategies to select bacterial candidates for

biocontrol (Kloepper 1991; Anith et al. 2003; de Weert

and Bloemberg 2006). However, these previous strategies

did not take into account the influence of biotic and abi-

otic factors in the rhizosphere of Rosellinia-infected

plants, and they introduce a bias, selecting mainly those

strains with biocontrol activity based on the mechanism

used in the selection method, such as antifungal produc-

tion (Cazorla et al. 2006; Romero et al. 2007), root colo-

nization (Kamilova et al. 2005) or both root colonization

and antagonism (Pliego et al. 2007). For this reason, a

direct plant-protection strategy was developed to screen

for candidates of biocontrol agents, considering all the

potential biocontrol traits. Twenty-two strains with pre-

sumed protective activity were discovered from 143

assayed, independently of the biocontrol mechanism

involved (Fig. 1). These 22 preliminarily selected bacteria

were mainly members of Pseudomonas and Bacillus, gen-

era that have been extensively reported as exerting biolog-

ical control of soil-borne pathogens (Weller 1988;

Whipps 2001).

As mentioned earlier, we have selected 22 potentially

protective strains; interestingly, 11 of them were antago-

nistic towards R. necatrix CH53. On the other hand, from

the total number of bacterial isolates selected from soil

and root samples in this work, the percentage of bacteria

antagonistic against R. necatrix CH53 in vitro was only

8Æ0%, a similar value to that obtained by Cazorla et al.

(2006). These results pointed to antagonism as a relevant

trait in the biocontrol activity of Pseudomonas and Bacil-

lus strains against the white root rot of avocado.

A second and more accurate round of biocontrol tests

was performed using clonal avocado plants. Biocontrol

ability was recorded at two different disease pressure

(when ID was 50 and 90%, respectively), because higher

pressure can lead to different biocontrol abilities of the

bacterial strains (Fig. 2). Five strains imparting significant

protection against R. necatrix were finally selected

(Fig. 2). This revealed the lack of accuracy of the first

biocontrol experiment. Out of 22 strains tested positive,

only three finally were selected as robust biocontrol

strains, possibly because of the low number of plants

assayed and the natural variability of the plants obtained

from germinated embryos. Among the most efficient

strains including CB32, CB115, CB254 and CB306, three

had previously shown antifungal activity towards R. neca-

trix; however, Ps. fluorescens CB306 does not show this

antagonistic activity (Table 3). This strain would not have

been selected by antagonism against R. necatrix. However,

this strain showed antagonistic activity against Phytoph-

thora spp. and V. dahliae (Table 3). Enterobacter sp.

CB240 was not considered for further experiments,

because it could be a potential opportunistic human

pathogen (Berg et al. 2005).

This screening method, without a dominant selection

pressure (e.g. colonization abilities), expands the variety

of strains selected and the different mechanisms of bio-

control against avocado root rot. However, this strategy

was expensive, difficult, time-consuming, and only

allowed the study of a limited number of bacterial strains.

Taking into account the findings of this work, the four

efficient biocontrol strains selected using a direct plant-

protection strategy were all antagonistic to some fungi or

oomycetes, and all of them could have also been isolated

via an easier dual-plate screening of antagonism against a

few soil-borne fungal pathogens (such as R. necatrix and

P. cinnamomi). This antagonism-based selection strategy

is more reliable, fast, and economic and would have pro-

duced the selection of the same selected bacterial strains.

All of the more efficient biocontrol strains showed

antagonistic activity against different target fungi and

oomycetes (Table 3). Thus, Ps. chlororaphis CB254

showed a very strong antagonistic activity against all

assayed fungi and Phytophthora, and B. subtilis CB115

also inhibits all of them. Pseudomonas fluorescens CB32

and CB306 showed specific inhibition to some of the

assayed fungal and oomycete targets, suggesting a specific

M.A. Gonzalez-Sanchez et al. Antagonism is a prevalent trait

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 65–78 75

mode of action of these strains against the soil-borne

phytopathogens assayed. However, other strains of B. sub-

tilis and Pseudomonas, especially of Ps. chlororaphis, dis-

played a strong antagonism but did not exhibit

biocontrol activity (Table 3, Fig. 2).

Analysis of antifungal compound production showed

that all of the strains with antifungal activity produced

some type of antifungal metabolite. Well-known antifungal

antibiotics were only detected in Ps. chlororaphis strains

(PCA, PCN) and some B. subtilis strains (fengycines, sur-

factines and bacillomycin, Table 4). Phenazines produced

by Pseudomonas strains have been demonstrated to be

involved in the suppression of different fungal-based plant

diseases (Thomashow and Weller 1988; Chin-A-Woeng

et al. 1998), and the production of lipopeptidic antibiotics

by B. subtilis strains have been demonstrated to have a role

in biocontrol (Ongena et al. 2005; Romero et al. 2007). In

fact, strains including antibiotic-producing Pseudomonas

spp. and lipopeptidic antibiotics-producing B. subtilis,

have previously been reported to have biocontrol activity

against white root rot in avocado (Cazorla et al. 2006,

2007). However, the production of antifungal metabolites

appeared to be associated with strains displaying efficient

biocontrol activity, such as B. subtilis CB115 or Ps. chloro-

raphis CB254; and, it also appeared in strains that did not

exhibit biocontrol activity (Table 4, Fig. 2). In this sense, a

modulation of the production of antifungal compounds in

the rhizosphere environment by bacteria because of their

interactions with plants has been described (Fiddaman and

Rossall 1994; de Werra et al. 2008), thus explaining the

failure of antibiotic-producing strains to protect plants

when they were applied to the rhizosphere.

There is evidence correlating the efficacy of biocontrol

micro-organisms against soil-borne pathogens with their

ability to colonize the root system of the plant to be pro-

tected, especially when the mode of action used by these

bacterial strains is antibiosis (Chin-A-Woeng et al. 1998)

or competition for space and nutrients (Kamilova et al.

2005; de Weert and Bloemberg 2006; Lugtenberg and

Kamilova 2009). We observed that Pseudomonas spp.

strains were able to colonize avocado roots and to persist

for 21 days (Table 5), also correlating with higher values

for swimming motility. Bacillus subtilis CB115, originally

isolated from a soil sample, persisted mainly as spores

and showed lower colonizing ability, suggesting that root

colonization could not be relevant for its biocontrol abil-

ity. This hypothesis is supported by mutations in the col-

onization genes of Ps. fluorescens WCS365, which did not

consistently decrease the biocontrol activity of the strain

(Dekkers et al. 2000a). Moreover, CB115 showed growth

promoting activity (PGPR) in lettuce (Fig. 2). In most

cases, bacterial strains that promote plant growth also eli-

cit induced systemic resistance (van Loon et al. 1998;

Kloepper et al. 2004). Pseudomonas fluorescens CB306 also

has this PGPR activity, as does Ps. chlororaphis CB58;

however, the latter does not show efficient biocontrol

activity. Nevertheless, the mode of action observed in

Ps. fluorescens CB306, which does not produce antibiotics,

could be related to PGPR activity, its colonization and

persistence abilities.

In summary, all the studied mechanisms (antifungal

production, root colonization, PGPR activity) could be

involved in the efficient biocontrol activity of some of

these bacteria against R. necatrix (Fig. 2), but each one of

these traits would be absent in at least one of the efficient

biocontrol strains. Because of this, any studied mecha-

nism appeared to be an essential or generalized trait

involved in R. necatrix biocontrol. Furthermore, each of

the studied traits is also present in some strains that do

not show biocontrol activity; for this, none of these

mechanisms could be considered sufficient to explain effi-

cient biocontrol activity. In conclusion, the observed bio-

control appears to be a polyphasic activity with different

traits involved, and so the four strains considered showed

several of the studied mechanisms related to biocontrol.

These findings suggest that different traits could act

jointly in biocontrol, and these traits were neither suffi-

cient nor generalized for all the efficient bacteria.

Acknowledgements

This research has been supported by the Spanish Plan

Nacional I+D+I Grants AGL 2005-06347-C03-01 and

AGL 2008-05453-C02-01 and by the Junta de Andalucıa

Grant AGR-169. The authors want to express our sincere

gratitude to A. Barcelo for technical assistance in growing

the avocado plants in vitro.

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