biocontrol of meloidogyne incognitaon tomato

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Research ArticleReceived: 16 August 2008 Revised: 28 January 2009 Accepted: 30 January 2009 Published online in Wiley Interscience: 8 May 2009

(www.interscience.wiley.com) DOI 10.1002/ps.1777

Biocontrol of Meloidogyne incognita on tomatousing antagonistic fungi,plant-growth-promoting rhizobacteriaand cattle manureZaki A Siddiquia,b∗ and Kazuyoshi Futaib

Abstract

BACKGROUND: Biocontrol achieved by a single biocontrol agent is generally inconsistent under field conditions. The aim of thepresent study was to increase the competitiveness and efficacy of biocontrol agents by using them together with cattle manure.

RESULTS: The effects of antagonistic fungi [Aspergillus niger v. Teigh., Paecilomyces lilacinus (Thom) Samson and Penicilliumchrysogenum Thom] and plant-growth-promoting rhizobacteria (PGPR) [Azotobacter chroococcum Beijer., Bacillus subtilis(Ehrenberg) Cohn and Pseudomonas putida (Trev.) Mig.] were assessed with cattle manure on the growth of tomato and onthe reproduction of Meloidogyne incognita (Kof. & White) Chitwood. Application of antagonistic fungi and PGPR alone and incombination with cattle manure resulted in a significant increase in the growth of nematode-inoculated plants. The highestincrease (79%) in the growth of nematode-inoculated plants was observed when P. putida was used with cattle manure,followed by use of P. lilacinus plus cattle manure. Paecilomyces lilacinus resulted in a high reduction in galling and nematodemultiplication, followed by P. putida, B. subtilis, A. niger, A. chroococcum and P. chrysogenum. The combined use of P. lilacinuswith cattle manure resulted in a maximum reduction in galling and nematode multiplication.

CONCLUSION: Application of P. lilacinus or P. putida with cattle manure was useful to achieve greater biocontrol of M. incognitaon tomato.c© 2009 Society of Chemical Industry

Keywords: antagonistic fungi; biocontrol; cattle manure; nematode; PGPR; Solanum lycopersicum

1 INTRODUCTIONTomato, Solanum lycopersicum L., is an important vegetable crop,and its cultivation is worldwide. Yield loss due to root-knotnematodes (Meloidogyne spp.) on tomato range from 40 to 46%in India.1,2 Plants infected with Meloidogyne spp. show typicalsymptoms of root galling. Some infected plants exhibit deficiencysymptoms, particularly nitrogen.3 This disease has become a majorconstraint to the successful cultivation of tomato in India.4

Rhizosphere microorganisms provide an initial barrier againstpathogen attack on the root.5 The rhizosphere organisms, antag-onistic fungi, have a great potential against plant pathogens.6 Thefungus Paecilomyces lilacinus (Thom) Samson is primarily a sapro-phyte, infecting the eggs and females of root-knot nematodesand destroying the embryo within 5 days,7 and it is a successfulbiocontrol agent under various conditions.8 The commercial prod-uct MeloCon WG (Prophyta GmbH, Germany) of P. lilacinus strain251 is successfully used for the control of nematodes.9 Aspergillusand Penicillium are common genera occurring in most agricul-tural fields of India. Aspergillus species are known to producea variety of secondary metabolites and have shown biocontrolpotential against plant-parasitic nematodes.10 Similarly, Penicil-lium spp. are also useful as antagonists of nematodes.11 On theother hand, plant-growth-promoting rhizobacteria (PGPR) may

impart beneficial effects on plants. PGPR enhance plant emer-gence, colonise roots and stimulate overall plant growth, andalso improve seed germination, root development, nutrient up-take, water utilisation and plant health.12 The management ofthe crop rhizosphere with PGPR towards enhanced biocontrol ofplant pathogens has shown considerable promise.12,13 Organicmanure may suppress plant-parasitic nematodes and improvecrop tolerance.14 Nematode-infected plants generally show foliarsymptoms of nutrient deficiency,3,15 and application of manuresmay influence nematode development and reproduction.4,16,17

Composted cattle manure is commonly available and used by thefarmers in India. Although this manure has been found to be lesseffective than other manures in reducing galling and nematodemultiplication in chickpea,18 it still provides a good source ofnutrients for rhizosphere microorganisms.

∗ Correspondence to: Zaki A Siddiqui, Department of Botany, Aligarh MuslimUniversity, Aligarh 202002, India. E-mail: zaki [email protected]

a Department of Botany, Aligarh Muslim University, Aligarh 202002, India

b Graduate School of Agriculture, Kyoto University, Kyoto, Sakyo-ku 606-8502,Japan

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www.soci.org ZA Siddiqui, K Futai

In most studies, a single biocontrol agent is used to control asingle pathogen.19 This may sometimes account for inconsistentperformance because a single agent is not active in all soilenvironments or against all pathogens that attack the host plant.On the other hand, combining biocontrol agents with organicmanures might improve biocontrol efficacy because manuresgenerally facilitate multiplication and establishment of biocontrolagents in the rhizosphere.18

The rhizosphere harbours a variety of microorganisms includingbacteria, fungi and arbuscular mycorrhizal (AM) fungi. The aimof the present study was to increase the efficacy of PGPR andantagonistic fungi for the biocontrol of nematodes by usingthem in combination with cattle manure. Three antagonistic fungi(Paecilomyces lilacinus (Thom) Samson, Aspergillus niger v. Teigh.and Penicillium chrysogenum Thom) and the same number ofPGPR species [Azotobacter chroococcum Beijer., Bacillus subtilis(Ehrenberg) cohn and Pseudomonas putida (Trev.) Mig.] weretested alone and together with cattle manure for their effect onthe growth of tomato and on the reproduction of the root-knotnematode Meloidogyne incognita (Kof. & White) Chitwood.

2 MATERIALS AND METHODS2.1 Preparation of soilSandy loam soil (pH 7.2) collected from the field of the Departmentof Botany, AMU Aligarh, India, was added to jute bags. Water waspoured into each bag to wet the soil before transferring themto an autoclave for sterilisation at 137.9 kPa for 20 min. Sterilisedsoil was allowed to cool to room temperature before filling 15 cmdiameter clay pots with 1 kg of sterilised soil.

2.2 Nematode inoculumMeloidogyne incognita was collected from tomato field soil andmultiplied on eggplant (Solanum melongena L.) using a single eggmass. After 3 months, the inoculum was further multiplied on a fewother eggplants. Egg masses from the roots of these plants werehand picked using sterilised forceps and placed in 9 cm diametersieves of 1 mm pore size, which had been lined with cross-layeredtissue paper. The sieves were placed in petri dishes with distilledwater for hatching and incubated at 27 ◦C. Two thousand hatchedsecond-stage juveniles (J2) in 10 mL water were applied to eachplant as described below.

2.3 Biocontrol agent preparationThree fungi, A. niger, P. lilacinus and P. chrysogenum, were isolatedfrom the rhizosphere of tomatoes grown at a field near Chherat,Aligarh, India. The effects of these fungi were tested on thehatching and penetration of M. incognita in a preliminary test in icecream cups. These fungi were found to have antagonistic potentialagainst the tested nematode, and were therefore selected forthe present study. Pure cultures of these fungi were stored inthe Mycology Laboratory, Department of Botany, Aligarh MuslimUniversity, Aligarh, India. These isolates were designated as A. nigerCAI, P. lilacinus CAI and P. chrysogenum CAI (CAI = ChheratAligarh, India). These antagonistic fungi were separately culturedon Richard’s liquid medium for 15 days.20 Mycelia mats of eachwere collected separately on blotting paper to absorb excessof water and nutrients. A quantity of 100 g of mycelium fromeach was macerated in 1000 mL distilled water, and 10 mL of thissuspension containing 1 g fungus was used as inoculum per plant.PGPR (A. chroococcum, B. subtilis and P. putida) were obtained from

Table 1. Effects of biocontrol agents and cattle manure on the length(cm) of tomato plants inoculated with Meloidogyne incognita anduninoculated plantsa

Treatment No manure Cattle manure

Without nematode Control 65.9 b 68.7 f∗

A. niger 73.4 a 77.4 bc∗

P. lilacinus 72.2 a 79.5 ab∗

P. chrysogenum 71.4 a 75.2 cd∗

B. subtilis 71.7 a 78.4 ab∗

P. putida 73.6 a 80.6 a∗

A. chroococcum 71.5 a 77.1 bc∗

With nematode Control 42.5 e 59.4 g∗

A. niger 56.6c d 70.5 f∗

P. lilacinus 58.7 c 72.7 def∗

P. chrysogenum 54.5 d 68.2 f∗

B. subtilis 56.3 cd 72.2 ef∗

P. putida 58.2 c 73.9 de∗

A. chroococcum 55.9 cd 69.3 f∗

a Data followed by different letters within one column are significantlydifferent at P = 0.05, while a significant difference between rows(between no manure and cattle manure) is shown by ∗ .

the Microbial Type Culture Collection and Gene Bank, Institute ofMicrobial Technology, Chandigarh, India. PGPR were grown onnutrient broth (HiMedia Laboratories, Mumbai, India) incubatedat 37 ± 2 ◦C for 72 h. A quantity of 10 mL of the suspension(1.5 × 107 cells mL−1) was used as inoculum per plant.

2.4 Manure preparationA quantity of 10 g of composted cattle manure was added, asshown in Table 1. Cattle dung was collected from local farmers,and prior to use had been allowed to decompose in a 50 L plasticdrum for 1 year. The manure was mixed at 10 day intervals, and 10 Lwater was added after mixing. A quantity of 10 g of compostedcattle manure was added to the treatments (see Table 1); thiscontained about 30 mg N, 4.4 mg P and 8.4 mg K.

2.5 Experimental set-upSeeds of tomato variety K-25 were surface sterilised in 0.1%sodium hypochlorite for 2 min and then washed 3 times withdistilled water. Seeds were sown in seedling trays and transplanted,one seedling per pot, 2 weeks after germination. Seedlings wereplaced in a greenhouse and watered as needed. Two days aftertransplanting, seedlings were inoculated with the treatments;uninoculated plants served as a control. Treatments were 2000freshly hatched J2 of M. incognita (10 mL nematode suspensionwith 200 juveniles mL−1); PGPR (10 mL at 1.5 × 107 bacterial cellsmL−1); 1 g antagonistic fungus (10 mL at 0.1 g mycelia mL−1);cattle manure (10 g).

Cattle manure is generally added to the soil before plantingthe crop, but here it was added at the time when nematodes,PGPR and antagonistic fungi were inoculated in order to avoidpre- and post-effects with other treatments. For inoculation ofM. incognita, PGPR, antagonistic fungi and cattle manure, the soilaround the root was carefully removed without damaging theroots. The inoculum suspensions were poured or placed aroundthe roots, and the soil was replaced. An equal volume of sterilewater was added to control treatments.

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The experiment was set up in a completely randomised blockdesign with seven experimental variables: (1) control; (2) A. niger;(3) P. lilacinus; (4) P. chrysogenum; (5) A. chroococcum; (6) B. subtilis;(7) P. putida. These seven treatments were tested alone andtogether with cattle manure (7 × 2 = 14 treatments). These 14treatments were tested both in the presence and in the absence ofM. incognita (14 × 2 = 28 treatments). Each of the 28 treatmentswas replicated 5 times (28 × 5 = 140 pots), and the experimentwas repeated once. Data of both experiments were almost similar,and this paper presents pooled data of both experiments.

2.6 Plant parameters and nematode quantificationThe plants were harvested 90 days after inoculation. Data wererecorded on plant length, shoot dry mass, number of galls,percentage root colonisation and nematode population. A 250 gsubsample of well-mixed soil from each treatment was processedby Cobb’s sieving and decanting method, followed by Baermann’sfunnel extraction to determine the nematode population.21 Forestimating the numbers of eggs, juveniles and females inside theroots, roots of different treatments were cut separately into smallpieces. Subsamples (1 g) of roots from the different treatments,in five replicates, were collected from well-mixed homogeneousmixtures and macerated separately in a Waring blender, andcounts were made on the suspensions thus obtained. The numberof nematodes in roots was calculated by multiplying the numberof nematodes in 1 g of root by the total root mass.

2.7 Biocontrol agent quantificationA separate experiment to determine root colonisation by PGRP wasconducted, and plants were grown as described in Section 2.5. Theexperiment was set up in a completely randomised blocked designwith four experimental variables: (1) control; (2) A. chroococcum;(3) B. subtilis; (4) P. putida. These four treatments were tested aloneand together with cattle manure (4 × 2 = 8 treatments). Theseeight treatments were tested both in the presence and in theabsence of M. incognita (8 × 2 = 16 treatments). Each of the 16treatments was replicated 5 times (16 × 5 = 80 pots). Tomatoroots inoculated with PGPR were collected 1 month after sowing.Roots were surface sterilised in 0.1% sodium hypochlorite andwashed with sterilised water. The roots of one treatment werecut into small pieces. Subsamples (1 g), with five replicates, weretaken from the homogeneous mixtures and crushed separatelyin sterile normal saline solution (NSS), and 0.1 mL serially dilutedextracts were plated on nutrient agar plates and incubated at 37 ◦Cfor 24 h. The plates were placed on a Quebec colony counter forcounting the bacterial colonies. Colonies of rhizobacteria fallingwithin 30–300 colony-forming units (CFU) on a petri dish wereselected and multiplied by a reciprocal dilution factor to obtainthe bacterial colony number22 and represented as CFU g−1 root.

Antagonistic fungi were re-isolated from nematode eggs andfemales to determine percentage infection in the remainingpopulation. The nematode eggs and females were collected fromroots obtained by macerating the latter in a Waring blender asdescribed above. For re-isolation, eggs and females were surfacesterilised with 0.1% mercuric chloride for 2 min, washed 3 times indistilled water and placed on potato dextrose agar medium. After1 week of incubation at 25 ◦C, fungus growth was identified andcounted.

2.8 Statistical analysisThe biocontrol agents were tested in a 2 × 2 factorial design.Data obtained were analysed statistically by analysis of variance

Table 2. Effects of biocontrol agents and cattle manure on the shootdry weight (g) of tomato plants inoculated with Meloidogyne incognitaand uninoculated plantsa


Without nematode Control 17.93 d 20.56 f∗

A. niger 20.28 ab 21.32 cd∗

P. lilacinus 19.93 bc 21.93 ab∗

P. chrysogenum 19.75 c 20.96 e∗

B. subtilis 19.85 c 21.65 bc∗

P. putida 20.34 a 22.22 a∗

A. chroococcum 19.78 c 21.24 d∗

With nematode Control 11.58 g 16.42 j∗

A. niger 15.42 f 19.44 h∗

P. lilacinus 15.96 e 20.02 g∗

P. chrysogenum 14.93 e 18.86 i∗

B. subtilis 15.45 f 19.92 g∗

P. putida 15.88 e 20.76 ef∗

A. chroococcum 15.20 f 19.12 hi∗


(P = 0.05). Tukey’s test (P = 0.05) was then used to distinguish dif-ferences between treatments. All these analyses were performedby computer software Stat View 5.0 (SAS Institute, Cary, NC).Graphs for galling and nematode population were prepared usingSigmaPlot and error bars showing standard error.

3 RESULTSEffects of nematodes, biocontrol agents and cattle manureindividually and their interactions were significant at P = 0.05 bothon plant length and shoot dry weight, except for the interactions ofall three factors on plant length. Application of antagonistic fungiand PGPR alone or in combination with cattle manure to plantswith and without nematodes resulted in a significant increasein the growth of tomato over the uninoculated and nematode-inoculated controls (Tables 1 and 2). Application of A. niger toplants without nematodes resulted in a 13.1% increase in shootdry weight, followed by P. lilacinus (11.2%) and P. chrysogenum(10.2%), compared with the control (Table 2). Similarly, applicationof P. putida to plants without nematodes resulted in a 13.4%increase in shoot dry weight, followed by B. subtilis (10.7%) andA. chroococcum (10.3%), compared with the control. Use of cattlemanure on plants without nematodes resulted in a 14.7% increasein shoot dry weight over the uninoculated control. The combineduse of P. putida with cattle manure resulted in the highest increase(23.9%) in shoot dry weight of plants without nematodes (Table 2).

On the other hand, use of P. lilacinus resulted in a 37.8% increasein shoot dry weight of nematode-inoculated plants, followed byP. putida (37.1%), B. subtilis (33.4%), A. niger (33.2%), A. chroococcum(31.3%) and P. chrysogenum (28.9%). Moreover, addition of cattlemanure resulted in a 42% increase in the growth of nematode-inoculated plants. Integration of P. putida with cattle manureresulted in the highest increase (79.3%) in shoot dry weight ofnematode-inoculated plants, followed by P. lilacinus plus cattlemanure (72.9%). Use of P. chrysogenum with cattle manure wasthe least effective in increasing growth (62.9%) of nematode-inoculated plants among combined treatments (Table 2).

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Table 3. Colonisation of tomato roots by rhizobacteria in thepresence and absence of cattle manure in nematode inoculated anduninoculated plantsa


Without nematode Control – –

B. subtilis 2.3 × 105 cd 2.8 × 105 c∗

P. putida 2.9 × 105 e 3.4 × 105 e∗

A. chroococcum 2.0 × 105 b 2.5 × 105 b∗

With nematode Control – –

B. subtilis 2.1 × 105 bc 2.6 × 105 bc∗

P. putida 2.5 × 105 d 3.1 × 105 d∗

A. chroococcum 1.6 × 105 a 2.1 × 105 a∗


Table 4. Re-isolation of fungal biocontrol agents from females andeggs of Meloidogyne incognita obtained from plants inoculated withcattle manure and uninoculated plantsa

Re-isolation of antagonistic fungi

Treatment females eggs

No manure Control – –

A. niger 3 c 1 b

P. lilacinus 42 b 75 a

P. chrysogenum 4 c 2 b

Cattle manure Control – –

A. niger 5 c 2 b

P. lilacinus 47 a 79 a

P. chrysogenum 6 c 4 b

a Data followed by different letters within one column are significantlydifferent at P = 0.05.

The effects of nematodes and cattle manure individually oncolonisation of roots by PGPR were significant at P = 0.05, whileinteraction of nematodes and cattle manure was non-significant.P. putida resulted in a greater colonisation of roots, followedby B. subtilis and A. chroococcum (Table 3). Colonisation of rootsby PGPR was increased in the presence of cattle manure butreduced in the presence of nematodes. The effect of cattle manureon the re-isolation of antagonistic fungi from females and eggsof nematodes was non-significant. Only parasitism of nematodefemales by P. lilacinus was increased in the presence of cattlemanure (Table 4).

The effects of biocontrol agents and cattle manure individuallyand their interactions on root galling and nematode multiplicationwere significant at P = 0.05. Inoculation of antagonistic fungiand PGPR alone and together with cattle manure resulted ina significant reduction in galling and nematode multiplication(Fig. 1). Application of P. lilacinus resulted in high reduction ingalling and nematode multiplication, followed by P. putida, B.subtilis, A. niger, A. chroococcum and P. chrysogenum. In combinedtreatments, maximum reduction (73%) in galling and nematodemultiplication was observed when P. lilacinus was used withcattle manure, and minimum reduction when P. chrysogenum wasapplied with cattle manure (Fig. 1).

4 DISCUSSIONThe parasitism on M. incognita eggs and females by P. lilacinusresulted in reduced nematode multiplication, and thereby anincrease in the growth of nematode-infected plants. Paecilomyceslilacinus was isolated from a larger number of nematode femalesand improved plant growth to a greater extent than otherantagonistic fungi used. Paecilomyces lilacinus parasitised eggsof M. incognita more frequently and destroyed the embryo,while females were parasitised at the anus. Similar parasitismby P. lilanicus has been reported earlier.7,23 Aspergillus speciescommonly occur in soils in warmer climates and in compost anddecaying plant material, and are known to produce a varietyof secondary metabolites.24 Some Aspergillus species have alsobeen reported for their biocontrol potential against root-knotnematodes.25 The biocontrol potential of A.niger may be attributedto its ability to produce a variety of secondary metabolites.Moreover, Aspergillus and Penicillium inhibit egg hatch, whichsuggests the involvement of mechanisms other than parasitism,11

as neither species was isolated from eggs or females of nematodes,indicating their inability to parasitise the nematodes, and hencetheir exogenous effect. Moreover, the enzymatic disintegration ofvitelline and chitin layers might have increased the permeability ofeggshell and enhanced the mycelial penetration, leading to totaldisintegration of the egg contents.11

Results revealed that P. putida resulted in greater rootcolonisation than the other two PGPR tested. Pseudomonadscan indirectly protect plants by inducing systemic resistanceagainst various pests and diseases.26 – 28 They play a critical rolein naturally occurring soil that is suppressive to Fusarium wilt29

and produce a wide variety of antibiotics, growth-promotinghormones, siderophores and HCN. They can also solubilisephosphorus.30 – 32 Pseudomonads may also improve plant growthby suppressing parasitic and non-parasitic root pathogens33

through the production of biologically active substances34 or theconversion of unavailable minerals and organic compounds intoforms available to plants.35,36 The plant growth promotion abilityof Pseudomonas is a function of good colonisation of roots andproduction of growth hormones.5,37 In a previous study, P. putidaperformed better than the other two species of PGPR because of itsability to produce greater HCN, IAA and siderophores.38 Generally,various secondary metabolites secreted by Pseudomonas spp.,including HCN and siderophores, have inhibitory effects againstdifferent phytopathogens.12,32,39 Bacillus subtilis also reducednematode galling and multiplication, resulting in improved growthof nematode-inoculated plants. Improvement in growth maybe attributed to the inhibitory effect of B. subtilis against plantpathogens.40,41 Previous studies indicated that treatments withB. subtilis increased the yields of several crops.42,43 Additionally,this bacterium improved plant growth by inhibiting non-parasiticroot pathogens, producing biologically active substances, or bytransforming unavailable mineral and organic compounds intoforms available to plants.35 Moreover, a non-cellular extract ofB. subtilis was also reported to have a high degree of larvicidalproperties against root-knot and cyst nematodes.44 Similarly,A. chroococcum forms considerable quantities of biologicallyactive substances such as vitamins of the B group, nicotinicacid, pantothenic acid, biotin, heteroauxin and gibberellins,45

and has an ability to produce antipathogenic substances.46,47

PGPR were cultured in nutrient broth, and the inoculum addedcontained bacteria plus broth. Nutrient broth itself does havesome nutrients, and, although most of these are utilised bygrowing bacteria, some may be left in the broth unconsumed.


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Figure 1. Effects of antagonistic fungi, plant-growth-promoting rhizobacteria and cattle manure on the galling and nematode numbers of tomato.Nematode numbers represent the total number of nematodes (eggs, females and juveniles) in 1 kg of soil and root. Error bars represent the standarderror. Different letters within one parameter are significantly different at P = 0.05. C = control; An = Aspergillus niger; Pl = Paecilomyces lilacinus; Pc =Penicillium chrysogenum; Bs = Bacillus subtilis; Pp = Pseudomonas putida; Ac = Azotobacter chroococcum.

A control with nutrient broth alone was not included because itmay have greater nutrients than the nutrient broth containingbacteria. Organic manure can provide a food base and haslong been recognised as facilitating biological control.48 Edaphicmicroorganisms stimulated by these amendments contributeto the suppressive activity of the amended soils through allfour principal mechanisms of biological control: (a) competition,(b) antibiosis, (c) parasitism/predation and (d) systemic inducedresistance.49 Cattle manure results in several benefits such as bettersoil structure, build-up of antagonistic organisms and supply ofnutrients.14 Increase in plant growth and reduction in nematodepopulation by the use of cattle manure may be attributed to theabove-mentioned factors. Antagonistic fungi and PGPR were usedindividually and concomitantly with cattle manure to control theroot-knot disease of tomato with the aim of establishing thatcombined use may be exploited for biocontrol. The use of theantagonistic fungus P. lilacinus and the plant-growth-promotingrhizobacterium P. putida with cattle manure was more useful forthe management of root-knot nematodes on tomato, which isevident by their re-isolation from a greater number of nematodesand colonisation of roots, respectively, when used with cattlemanure. This may be due to higher multiplication of antagonisticfungi and PGPR in soil augmented with cattle manure, favouringnatural enemies.

The present experiments were performed in pots and withsterilised soil. When added to field soil, these microorganismsmay face competition from other soil microorganisms. Moreover,environmental conditions will also influence the survival andefficacy of these biocontrol agents under field conditions.However, the authors feel that the use of cattle manure withP. putida or P. lilacinus can provide successful biocontrol underfield conditions because cattle manure may be helpful for theirestablishment in the field. The present study suggests that P. putida

or P. lilacinus with cattle manure may be used for the biocontrol ofM. incognita on tomato, but studies under different field conditionsare required to confirm these results.

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