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Crop Protection 27 (2008) 189–197

www.elsevier.com/locate/cropro

Control of damping-off caused by Rhizoctonia solani andFusarium solani using olive mill waste water and some of its

indigenous bacterial strains

Thabet Yanguia, Ali Rhoumab,�, Mohamed Ali Trikib, Kamel Gargouric, Jalel Bouzida

aLaboratoire Eau, Energie et Environnement, Ecole Nationale des Ingenieurs de Sfax, Rte de Sokra Km 4.5, 3038 Sfax, TunisiabUnite de recherche Protection des Plantes Cultivees et Environnement, Institut de l’olivier de Sfax, Rte de Sokra Km 1.5, 3003 Sfax, TunisiacLaboratoire de recherche Amelioration de la Productivite de l’Olivier et des Arbres fruitiers, Institut, de l’Olivier, BP 1087 3000 Sfax, Tunisia

Received 8 February 2007; received in revised form 9 May 2007; accepted 13 May 2007

Abstract

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in vivo for their efficacy against

damping-off caused by two soilborne fungi Rhizoctonia solani and Fusarium solani. OMW and polyphenols displayed a high level of

antifungal activity against R. solani. However, F. solani was more resistant and only the highest dose (2%) prevented its mycelial growth.

In pot experiments, the percentage of tomato plants showing symptoms of damping-off was significantly reduced with different doses of

OMW (0.5%, 1% and 2%) as compared to the control (soil treated with water). Nine indigenous bacterial strains isolated from OMW

exhibited an antagonistic effect against the two fungi. Based on the gene 16S rRNA sequence analysis, four isolates showed 99.2%

similarity to known sequences of Bacillus subtilis, three isolates demonstrated low percentage similarities (94.3–96.5%) to the genera

Bacillus, whereas two isolates were associated with Burkholderia caryophylli and Pseudomonas fluorescens (98.2–99.6% similarities).

Among these bacteria, the strain B1 proved efficient against the two soilborne pathogens in vitro and in pot experiments.

Our study in controlled conditions suggested that addition of OMW to soil exerts significant disease suppressiveness against R. solani

and F. solani.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Olive mill waste water; Polyphenols; Indigenous bacteria; Damping-off; Rhizoctonia solani; Fusarium solani

1. Introduction

Damping-off is a serious disease complex worldwide of awide range of seedlings in nurseries, glasshouses, gardens,crops and forests (Agrios, 2005), and can kill bothgerminating seeds and young seedlings. Several fungi thatare widely distributed in soils can cause this disease,including Rhizoctonia solani, Pythium spp., Phytophthora

spp., Sclerotinia spp. and Fusarium spp. (Stephens et al.,1982). Strategies to control soilborne diseases are limitedbecause cultivars with complete resistance are not available(Li et al., 1995). Control of the soilborne pathogens isdifficult because of their ecological behaviour, their

e front matter r 2007 Elsevier Ltd. All rights reserved.

opro.2007.05.005

ing author. Tel.: +216 74 241 442; fax: +216 74 241 033.

ess: roumaal@yahoo.fr (A. Rhouma).

extremely broad host range and the high survival rate ofresistant forms such as chlamydospores and sclerotia underdifferent environmental conditions. Many research studieshave shown that biological control offers an environmen-tally friendly alternative to protect plants from soilbornepathogens (Emmert and Handelsman, 1999; Whipps, 2001;Weller et al., 2002). Although the number of biocontrolproducts is increasing, these products still represent only avery small proportion of fungicides (Fravel, 2005). Inrecent years, several bacterial and fungal antagonistsagainst soilborne plant pathogenic fungi have beendescribed (Howell, 2003; Faltin et al., 2004). However,many of these showed inconsistent in vitro effects and onlyvery few antagonists were analysed under open fieldconditions (Grosch et al., 2005). Therefore, other alter-natives to control damping-off and root rot are currently of

ARTICLE IN PRESST. Yangui et al. / Crop Protection 27 (2008) 189–197190

great importance. The use of organic amendments plays animportant role in the outcome of the plant–pathogeninteractions (Hoitink and Boehm, 1999; Abawi andWidmer, 2000). The decomposition level of organic matteraffects the composition of bacterial taxa as well as activitiesof biocontrol agents (Hoitink and Boehm, 1999). Thedecomposition of organic matter increases the populationof saprophytic micro-organisms and some of them act asantagonists to plant pathogens (Mazzola, 2002; Maniciet al., 2004).

Olive mill waste water (OMW) is a major environmentalproblem owing to its high organic load and antimicrobialproperties, particularly for Mediterranean countries.Many studies established that these wastes have a highfertilizer value when applied to the soil because of thehigh organic matter content and some mineral nutrientcontent (Paredes et al., 1999). However, despite thepotential agronomic value, soil amendment with OMW isalso known for its antimicrobial activity (Capasso et al.,1995; Kistner et al., 2004). The incorporation of freshOMW into the soil increases the number of soil micro-organisms and induces a change in the microbial popula-tion (Tardioli et al., 1997). In this context, Kotsou et al.(2004) reported that soil treatment with OMW created anr-environment that selectively enhanced and sustainedthe bacterial population of r-strategists for a prolongedtime period and consequently induced the soil suppressive-ness against the plant pathogen R. solani and possiblyalso against other telluric pathogens. In the same way,Kistner et al. (2004) found that the addition of OMW tohydroponic nutrient solutions provided an environmentsuppressing plant pathogens while favouring beneficials.In this project, we studied the biofungicide effect ofOMW against two soilborne pathogens R. solani andFusarium. solani. We analysed the direct effect of OMWon mycelial growth of these fungi in Petri dishes. Thiseffect was verified in pot experiments containing sterilesoil inoculated by R. solani and F. solani and amendedwith three dosage rates of OMW. We isolated someOMW indigenous bacteria in order to verify theirpossible antagonism against these two soilborne patho-genic fungi.

2. Materials and methods

2.1. OMW and its characteristics

OMW was taken from a three-phase continuous extrac-tion factory located in the region of Sfax (south-east ofTunisia) and was kept at �20 1C until use. Its physical andchemical characteristics were pH: 4.96, electrical conduc-tivity: 10mS cm�1, chemical of oxygen demand: 100 g l�1,total organic carbon: 38.64 g l�1, fat matter: 10.5 g l�1, totalpolyphenols: 5.8 g l�1, K: 830mg l�1, Fe: 3.04mg l�1, P:4.2mg l�1, NO3: 17mg l�1, SO4: 17mg l�1, Cl: 210mg l�1,Ca: 12.3mg l�1 and Na: 356mg l�1.

2.2. Plant pathogenic fungi

R. solani and F. solani were originally isolated fromtomato plants exhibiting symptoms of tomato damping-off. The isolates were stored at 4 1C in tubes containingpotato–dextrose–agar (PDA, 200 g of potato, 20 ofdextrose and 20 g of agar agar) and at �20 1C in tryptonesalt medium (tryptone: 1 g, NaCl: 8.5 g, Tween 20: 1%,glycerol: 15% and distilled water: 1 l).

2.3. Effect of OMW on mycelial growth of R. solani and

F. solani

To study the activity of OMW against mycelialgrowth of R. solani and F. solani, different concentrationswere prepared (0.5%, 1%, 2%, 4%, 6%, 8%, 10%,15% and 20%) by OMW incorporation in the PDAmedium at approximately 45 1C. After mixing, theamended PDA was dispensed into 9-cm-diameter Petridishes and allowed to cool. Five-millimetre-diameter plugsof agar from young pure cultures of R. solani and F. solani

was placed with the surface mycelium facing down onthe test PDA medium. The plates were incubated at 25 1C,and the radial growth of mycelium was measured at24-h intervals for a week. Controls were run withinoculated PDA without OMW addition. Four replicatesof each concentration were used, and three separate testswere performed. Plates showing contamination werediscarded.

2.4. Antifungal activity of total polyphenols extracted from

OMW

2.4.1. Polyphenol extraction and analysis

OMW was centrifuged at 7000 rpm for 20min. Thesupernatant (SP) was extracted three times with ethylacetate. The collected organic fraction was dried andevaporated under vacuum. The residue was extracted twicewith dichloromethane in order to remove the non-phenolicfraction (lipids, aliphatic fractions and sugars). Theliquid phase was discarded, while the washed residue wasweighed and re-suspended in ethyl acetate (4.2mgml�1).The latest compound was analysed by gas chromatographycoupled with mass spectroscopy according to Sampedroet al. (2005). Gas chromatography–mass spectrometric(GC–MS) analyses were performed on OMW ethyl acetateextract derivatized with N,O-bis (trimethylsilyl) trifluoroa-cetamide in pyridine. Mass spectra were recorded by theuse of a Hewlet-Packard 5973 spectrometer equipped witha capillary column HP 5 MS (30� 0.25mm) at 100–280 1Cwith an isothermal programme at 100 1C for 2min, then at5 1C up to 280 1C and finally isothermal at 280 1C for 5min.Identification of aromatic compounds was based oncomparison with retention times and mass spectra of purestandards.

ARTICLE IN PRESST. Yangui et al. / Crop Protection 27 (2008) 189–197 191

2.4.2. Effect of polyphenols on mycelial growth

Spore and mycelium suspensions of F. solani andR. solani (100 ml) were spread on the plates containingPDA medium, and wells of 6-mm diameter were punchedin the agar with a sterile steel borer. Twenty microlitres oftotal polyphenols was filtered through 0.45-mm filters understerile conditions and pipetted into the wells on PDA platesinoculated with the fungi. The plates were incubated for48 h at 25 1C and then examined for haloes of inhibitionaround the well.

2.5. Effect of OMW in suppressing damping-off diseases in

pot experiments

2.5.1. Soil preparation

An agricultural soil sample (clay: 6%, silt: 3%, sand:91%, pH: 8.2, electrical conductivity: 1.1mS cm�1, organicmatter content: 0.17%, Ntotal: 176 mg g

�1, P: 42 mg g�1 andK: 130 mg g�1) was taken from a field located in the regionof Chaal (60 km to the south-west of Sfax). The soil wasmoistened with distilled water to 60% of its water-holdingcapacity and autoclaved for 1 h at 121 1C twice, on 2successive days. The soil was maintained at 60% of itswater-holding capacity for a week under sterile conditions.After that, the soil was again autoclaved twice for 20min at121 1C. It was placed in plastic pots (dimensions:20 cm� 20 cm� 5 cm, 2 kg of soil per pot).

2.5.2. Soil inoculation

R. solani and F. solani were cultured in a liquid mediumof potato dextrose for 7 d on a shaker at 200 rpm.Mycelium was separated from the liquid medium byfiltration using Whatman paper No. 1. Soil was inoculatedwith 25mg of prepared mycelium mixed with 1 kg ofautoclaved soil. Seven days after inoculation, three doses offresh OMW were added (0.5%, 1% and 2% (w/w)) to theinoculated soil. Fifteen days after the addition of OMW, 30tomato seedlings (cv. Riogrande) at the stage of two trueleaves were transplanted into 10 pots (three plants/pot)for each dose. There were three replicates for each dose.Pots were placed under ambient conditions and monitoredfor a growing period of 4 weeks. Plants showing sym-ptoms of damping-off were noted. There were twocontrols, one for non-infested soil with each dose of freshOMW and another for infested soil without amendmentwith OMW.

2.6. Isolation of bacteria from OMW

For isolation of bacterial strains, 10 g of OMW wassuspended in 90ml of sterile distilled water and shaken for10min at 250 rpm. One millilitre of this suspension wasused to prepare serial 10-fold dilutions in 0.9% of NaCl.Aliquots (100 ml) of a dilution of each suspension werespread on Lauria-Bertani agar (LBA: tryptone: 10 g, yeastextract: 5 g, NaCl: 5 g, agar: 18 g and distilled water: 1 l).Some representative colonies, which differed morphologi-

cally, were selected from the countable plates and re-streaked on a new plate of the same media to obtain purecolonies. Purified bacterial isolates were stored in 30%glycerol at �20 1C.

2.7. In vitro antagonistic activity of bacteria isolated from

OMW

2.7.1. Dual culture

The in vitro inhibition of mycelial growth of R. solani

and F. solani by the bacterial isolates was tested using thedual culture method as described by Landa et al. (1997).Three 50 ml drops from a suspension of antagonisticbacteria (108 cfuml�1) were equidistantly placed on themargins of PDA medium and incubated at 25 1C for 24 h.A 4-mm agar disc from fresh PDA cultures of R. solani andF. solani was placed at the centre of the PDA plate for eachbacterial isolate tested and incubated at 25 1C for 7 d.Growth diameter of the pathogen mycelium was measuredand compared with the control growth where the bacterialsuspension was replaced by sterile distilled water. Eachexperiment was run in triplicate and was repeated at leastthree times. The percentage growth inhibition was calcu-lated using the following formula: % inhibition ¼(1�(fungal growth/control growth))� 100.

2.7.2. Production of diffusible metabolites

The ability of the OMW indigenous bacterial strains toproduce diffusible metabolites was tested according to theagar well diffusion assay (AWDA) as reported by Tagg andGiven (1971). All bacterial isolates were transferredindividually to 50ml of Luria-Bertani broth medium(LB broth) in 250-ml Erlenmeyer flasks and incubated byshaking each culture at 200 rpm for 4 d under ambientconditions. LBA medium (20ml) was poured into eachsterile Petri dish (90mm diam.). Spore and myceliumsuspensions of F. solani and R. solani (100 ml) werespread on the plates containing PDA medium, and wellsof 6-mm diameter were punched in the agar with a sterilesteel borer. The bacterial cultures were centrifuged at15 000 rpm for 30min to remove cell debris. Aftercentrifugation, 20 ml of each sample was filtered through0.45 mm filters under sterile conditions and pipetted intowells on PDA plates inoculated with the fungi. The plateswere incubated for 48 h at 25 1C and then examined forhaloes of inhibition around the well. The diameter (di) ofthe zone of inhibition was calculated using the followingformula: di ¼ d�Dw, where di is the diameter of theinhibition zone, d is the diameter of the halo and Dw

represents the diameter of the well (6mm). Four replicatesof each bacterial isolate were used, and three separate testswere performed.

2.8. Identification of the potential antagonistic bacteria

The identification of bacterial strains was achieved bysequencing the 16S rRNA gene (rrs). Amplification was

ARTICLE IN PRESST. Yangui et al. / Crop Protection 27 (2008) 189–197192

carried out by PCR with primers F667-pA-rrs AGAGTTT-GATCCTGG CTCAG and F668-pH-rrs AAGGAGGT-GATCCAGCCGCA designed by Bruce et al. (1992).Standard PCR conditions were 1min DNA denaturationat 94 1C, 1min annealing at 57 1C and 1min extension at72 1C for 35 cycles. The 16S rDNA sequences werecompared with sequences in the GenBank database withthe Basic Alignment Search Tool (Altschul et al., 1990).

2.9. Effect of potential antagonistic bacteria against

R. solani in pot experiments

To investigate the effect of suppression of damping-offcaused by R. solani, 25mg of hyphae separated from PDAliquid medium as mentioned above was mixed with 1 kg ofthe autoclaved soil with 2% cornflour. Each pot (300mlvolume) was inoculated 7 days before seeding. Twentymillilitres of SP or cell suspension (108 cfuml�1 bacteriapelleted, washed twice and resuspended in distilled sterilewater) was added to pathogen-infested pots 3 days beforeseeding. One additional treatment with pathogen inoculumonly served as the inoculated control (C). Thirty sterilizedtomato seeds (Lycopersicon esculentum, CV. Riogrande)

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0.0 0.5 1.0 2.0

OMW doses

Mycelial g

row

th (

cm

)

R. solani

F. solani

Fig. 1. Effect of OMW doses on mycelial growth of R. solani and

F. solani. Different letters denote significant difference according to

Duncan’s multiple range test at po0.05.

Fig. 2. Mycelium deformation and vacuolization of Fusarium solani treated by

mycelia.

were sown in 10 pots and then placed under fieldconditions. There were three replicates for each treatment.Disease incidence was expressed as the percentage of thenumber of plants showing typical symptoms caused by R.

solani.

2.10. Microscopic observations

Morphological aspects of the mycelia of R. solani and F.

solani treated by either OMW or the antagonists includingfragmentation, vacuolization and lyses were examinedmicroscopically at 40� magnification.

2.11. Data analysis

Data were subjected to analysis of variance using SPSSsoftware (version 11). Means values among treatmentswere compared by Duncan’s multiple range test at the 5%(p ¼ 0.05) level of significance.

3. Results

3.1. In vitro effect of OMW and polyphenols against R.

solani and F. solani

The incorporation of OMW in the culture mediumshowed a potent antifungal activity against R. solani and acomplete inhibition of mycelium growth was observed forall the tested doses (Fig. 1). However, for F. solani, only thehigh dose (2%) was efficient in preventing myceliumgrowth. The microscopic observation of mycelia showedsliming of cell walls, deformation and vacuolization(Fig. 2). These results indicated that R. solani was probablymore sensitive to the OMW than F. solani.The results with OMW were confirmed by the effect of

polyphenols. Indeed, F. solani was found to be moreresistant to polyphenols than R. solani. This resistance isexpressed by the very small inhibition zone recordedaround the well and by the intensive formation ofchlamydospores (data not shown). GC–MS analysis ofpolyphenols revealed the presence of vanilline, M-hydroxy-phenylethanol, 4-hydroxyphenylethanol, 1,2-dihydroxyl-4-(1-propyl) benzene, 4-hydroxyphenyl-propionic acid,

OMW. (a) Control intact mycelia. (b) Deformation and vacuolization of

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Fig. 3. GC–MS chromotogram (total ion current) of phenolic compounds extracted from olive mill waste water. The following identified compounds have

been numbered according to their retention times: vanilline [1], M-hydroxyphenylethanol [2], 4-hydroxyphenylethanol [3], 1,2-dihydroxyl-4-(1-propyl)

benzene [4], 4-hydroxyphenyl-propionic acid [5], vanillethanediol [6], 2-hydroxyphenyl acetic acid [7], 3,4-dihydroxyphenylglycol [8].

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1

OMW doses

Perc

en

tag

e o

f d

am

pin

g-o

ff o

f to

mato F. solani

R. solani

a

d d

c

a

b

c

b

2

Fig. 4. Percentage of tomato damping-off incidence by Rhizoctonia solani and Fusarium solani after incorporation of OMW in soil at different doses. Bars

topped by a different letter denote significant reduction of damping-off according to Duncan’s multiple range test at po0.05.

T. Yangui et al. / Crop Protection 27 (2008) 189–197 193

vanillethanediol, 2-hydroxyphenyl acetic acid and3,4-dihydroxyphenylglycol (Fig. 3).

3.2. Suppression of damping-off disease by OMW in pot

experiments

The percentage of tomato plants showing symptoms ofdamping-off was significantly reduced by all doses ofOMW (0.5%, 1% and 2%) (Fig. 4), and the suppressiveeffect was observed against both R. solani and F. solani.However, the highest dose (2%) of OMW significantlyreduced the percentage of infected tomato seedlings

compared with the lower doses. This could be related tothe high toxicity of OMW to the tomato seedlings. Thisfinding was confirmed by results of seed germination oftomato in soil free from the soilborne pathogen butcontaining different doses of OMW (Fig. 5).

3.3. In vitro effect of the indigenous bacterial strains of

OMW

Nine bacterial strains isolated from OMW exhibitedantifungal activity towards R. solani and F. solani in agarwell diffusion assays and in dual culture (Table 1). Based

ARTICLE IN PRESST. Yangui et al. / Crop Protection 27 (2008) 189–197194

on 16S rRNA sequences analysis, the strains B2, BM2,BT211 and BM3 were identified as Bacillus subtilis, BM16 asBurkholderia caryophylli, PsM2 as Pseudomonas fluorescens

and the other isolates B1, BM8 and BM14 were associatedwith the genus Bacillus.

Table 1

Diameters of the inhibition zones and percentage of growth inhibition observed

of OMW

Diameter of inhibition zones in agar well diffusion agar test (cm

Rhizoctonia solani Fusarium solani

B1 1.570.16 1.970.51

BT211 1.870.12 1.070.14

BM2 0.070.00 1.770.11

B2 1.870.18 2.070.21

BM14 1.570.18 0.070.00

BM8 1.270.22 0.070.00

BM16 2.070.11 2.570.31

BM3 1.470.15 0.070.21

PsM2 2.570.16 1.570.11

7 Indicate standard error of the mean.

Fig. 6. Mycelium aspect of Rhizoctonia solani after dual culture with the antag

wall, rupture and collapse of mycelium of R. solani.

50

55

60

65

70

75

80

85

90

Seed

germ

inati

on

(%

)

0.0 0.5 1.0 2.0

OMW doses

a

aa

b

Fig. 5. Percentage of seed germination of tomato plants in soil free from

the soilborne pathogens but added with different doses of OMW.

Different letters denote significant difference according to Duncan’s

multiple range test at po0.05.

There was an absence of physical contact between theantagonistic bacteria and the pathogenic fungi in directantagonism through dual cultures. The presence of a wideinhibition zone with the AWDA test indicated that theantagonism could be related to extra-cellular metabolitesreleased in the culture medium. Mycelium of R. solani wasdarker brown after a dual culture with strain B1 (data notshown). Microscopic observation showed an abnormalleakage, collapse and rupture of mycelium (Fig. 6). Similarresults were found by Fiddaman and Rossall, 1993 (cited byMontealegre et al., 2003), who observed hyphal vacuoliza-tion and deformation in R. solani and Pythium ultimum as aconsequence of treatment with a B. subtilis strain thatsecreted a volatile metabolite with fungicidal properties.

3.4. Effect of OMW indigenous bacteria in pot experiments

Among the antagonistic bacterial isolates, the supernatantand washed cells of strain B1 reduced significantly the incidenceof damping-off of tomato caused by R. solani (Fig. 7).

4. Discussion

The results obtained demonstrate that suppression ofdamping-off of tomato using OMW is due to chemical

with Rhizoctonia solani and Fusarium solani treated with bacterial isolates

) Percentage of growth inhibition in dual culture (%)

Rhizoctonia solani Fusarium solani

46.570.70 45.070.00

43.570.70 4270.70

40.070.00 42.570.00

4271.15 4271.73

43.070.05 40.070.00

41.570.70 40.070.57

42.571.00 38.571.15

41.570.70 42.570.00

40.570.57 38.571.41

onistic bacteria strain B1. (a) Control hyphae of R. solani. (b) Sliming cell

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0

10

20

30

40

50

60

70

80

90

100

Rz SC_Rz SP_Rz

Traitements

Perc

en

tag

e o

f to

mato

dam

pin

g-o

ff

incid

en

ce

a

b

c

Fig. 7. Effect of treatments by strain B1 on the percentage of damping-off incidence of tomato plants caused by Rhizoctonia solani. Bars with different

letters are significantly different according to the test of Duncan (to p ¼ 0.05) RZ: Rhizoctonia solani, SC: suspension cell-free bacteria, SP: supernatant.

T. Yangui et al. / Crop Protection 27 (2008) 189–197 195

compounds such as polyphenols, and probably to some ofits indigenous bacterial strains that act as antagonists.

The complete growth inhibition of R. solani demon-strated that this fungus cannot grow on media containingOMW. This result was not consistent with that reported byKotsou et al. (2004), who demonstrated that R. solani isable to grow on solid media containing OMW as the onlycarbon source. The total inhibition could be due to thedirect effect of the acid pH of OMW and to chelatingtransition metals by polyphenols. Wong and Kitts (2006)reported that phenolic compounds are able to chelatetransition metals and also lower the reactivity of metal ironby forming an inert metal–ligand complex. Chelating oftransition metals, such as iron and copper, reducesbioavaibility for fungal growth. The growth inhibition ofmycelia could be due to phenolic compounds that canpotentially impair cellular function and membrane integ-rity (Ciafardini and Zullo, 2003). It has been demonstratedthat phenolic compounds bond tightly on the cell walls,thus damaging them, and are characterized by a verystrong protein-cross-linking and protein-denaturizing ac-tivity (Bais et al., 2002; Ciafardini and Zullo, 2003).Besides, the complete inhibition of growth of R. solani

indicated the possible presence of fungicidal compounds inthe volatile fraction of the extract. Volatile polyphenolscould be stronger and react with nucleotides and protei-naceous materials. Among the polyphenols, flavonoidsinhibited pathogenic growth by reacting with DNA anddisrupting DNA replication, thus explaining the observedgrowth inhibition of soilborne pathogens in this study(Wong and Kitts, 2006).

The inhibition of F. solani occurred only at the highestdose of OMW. This result was confirmed by the micro-scopic observation showing a deformation and a vacuoli-zation of the fungal mycelium. Celar (2003) reported thatphytopathogenic Fusarium sp. used a higher rate of

nitrogen in the nitrate form (concentration of NO3:115.75mg l�1). In addition, Sampedro et al. (2005) foundthat Fusarium lateritium had an extracellular enzymeactivity in solid-state cultures on OMW with a dilution ofupto 25%.The significant reduction of damping-off incidence on

tomato plants using the OMW amendment was attributedto the effect of polyphenols and other chemical com-pounds. Several researchers have demonstrated that onlya few micro-organisms are able to survive in this by-product, because it contains various simple and complexphenolic compounds characterized by high antimicrobialactivity (Capasso et al., 1995; Kistner et al., 2004). Somephytopathogenic bacteria like Pseudomonas syringae pv.savastanoi, Corynebacterium michiganense and Xanthomonas

campestris are inhibited by polyphenols present in OMWin their original concentration (Ciafardini and Zullo,2003).With regard to the effect induced by the microbiological

component of OMW, it is possible that indigenousbacterial strains were also involved. The present workshowed that some OMW indigenous bacteria played amajor role against soilborne plant pathogenic fungi. Thestudy conducted by Kotsou et al. (2004) demonstrated thatthe significant disease suppressiveness against R. solani thatwas induced in the OMW-treated soil was mainlyattributed to the shift in the soil microbial communityfrom K- to r-strategy. Our results showed that species ofBacillus, Burkholderia and Pseudomonas were isolated fromOMW and exhibited antimicrobial activity against the twosoilborne plant pathogenic fungi F. solani and R. solani.These antagonists may have different mechanisms of actionincluding interference with spore germination or germ tubeelongation inhibition through abnormal hyphal swelling(Jung et al., 2003). They can also be responsible for lysesand complete degradation of the fungal hyphae (Wang

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et al., 1999). A suppression by competition for nutrientscould occur (Yoshida et al., 2001; Bailey and Lazarovits,2003). The colour change of mycelium observed for R.

solani and hyphal deformation of F. solani could be due tothe antibiotics secreted by these bacteria, which may havefungicidal properties. This finding confirm the hypothesisof Mazzola (2002), who considered that the control ofsoilborne plant pathogenic fungi by using organic amend-ments is due to a specific suppression, which is related to anincrease in the population of specific or groups of micro-organisms that act as antagonists to the plant pathogens.

The suppressive effect of the antagonist B1 against thesoilborne plant pathogen R. solani is likely to be due tocompetition for nutrients and an antibiosis effect. Anti-biosis by strain B1 would play a major role in thesuppression of damping-off caused by R. solani since thenumber of plants exhibiting characteristic symptoms wassignificantly less than the number of plants treated with abacterial cell-free suspension. In addition, inhibition ofdamping-off disease on tomato plants by the bacterialsupernatant of B1 was observed when it was applied afterfungal inoculation, suggesting that the filtrate had atherapeutic effect. Hydrolytic enzymes may also play animportant role in the control of disease caused by soilborneplant pathogens (Jung et al., 2003). Altogether, it isreasonable to suggest that suppression of damping-offincidence in tomato plants caused by R. solani is associatedwith hydrolytic enzymes that act alone or synergisticallycausing hyphal lyses and deformation of fungal cell walls.

Further studies with other crops and soilborne patho-gens of interest are required to extend our knowledge aboutthe management of OMW for soil sanitation.

Acknowledgements

This work was supported by the funds of the ProjectCFC/IOOC/04. We would like to thank Dr. Xavier Nesmefrom Laboratoire d’Ecologie Microbienne (UniversiteClaude Bernard, France) for his contribution in theidentification of the bacterial strains.

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