REGULAR ARTICLE

Arbuscular mycorrhizal fungi reduce root-knot nematodepenetration through altered root exudation of their host

Christine Vos & Sofie Claerhout &Rachel Mkandawire & Bart Panis &

Dirk De Waele & Annemie Elsen

Received: 22 September 2011 /Accepted: 11 November 2011 /Published online: 29 November 2011# Springer Science+Business Media B.V. 2011

AbstractAims Arbuscular mycorrhizal fungi (AMF) can con-trol root-knot nematode infection, but the mode ofaction is still unknown. We investigated the effects ofAMF and mycorrhizal root exudates on the initialsteps of Meloidogyne incognita infection, namelymovement towards and penetration of tomato roots.Methods M. incognita soil migration and root pene-tration were evaluated in a twin-chamber set-upconsisting of a control and mycorrhizal (Glomusmosseae) plant compartment (Solanum lycopersicumcv. Marmande) connected by a bridge. Penetrationinto control and mycorrhizal roots was also assessed

when non-mycorrhizal or mycorrhizal root exudateswere applied and nematode motility in the presence ofthe root exudates was tested in vitro.Results M. incognita penetration was significantlyreduced in mycorrhizal roots compared to controlroots. In the twin-chamber set-up, equal numbers ofnematodes moved to both compartments, but themajority accumulated in the soil of the mycorrhizalplant compartment, while for the control plants themajority penetrated the roots. Application of mycor-rhizal root exudates further reduced nematode pene-tration in mycorrhizal plants and temporarilyparalyzed nematodes, compared with application ofwater or non-mycorrhizal root exudates.Conclusions Nematode penetration was reduced inmycorrhizal tomato roots and mycorrhizal root exu-dates probably contributed at least partially byaffecting nematode motility.

Keywords Glomus mosseae .Meloidogyneincognita . Motility test . Mycorrhiza-inducedresistance . Nematode penetration . Root exudates

Introduction

In the light of growing concerns about human andenvironmental safety related to pesticide use, increasingcost of chemical inputs and emergence of pathogenresistance to chemical pesticides, arbuscular mycorrhi-zal fungi (AMF) may provide a more sustainable and

Plant Soil (2012) 354:335–345DOI 10.1007/s11104-011-1070-x

Responsible Editor: Thom W. Kuyper.

C. Vos (*) : S. Claerhout : R. Mkandawire :B. Panis :D. De WaeleLaboratory of Tropical Crop Improvement,Department of Biosystems, Faculty of BioscienceEngineering, University of Leuven (K.U.Leuven),Kasteelpark Arenberg 13,3001 Leuven, Belgiume-mail: christine.vos@biw.kuleuven.be

A. ElsenBodemkundige Dienst van België,Willem de Croylaan 48,3001 Heverlee, Belgium

R. Mkandawire :D. De Waele :A. ElsenDepartment of Biology, Faculty of Sciences,Ghent University,Ledeganckstraat 35,9000 Ghent, Belgium

environmentally sound alternative to pesticide use(Harrier and Watson 2004; Dong and Zhang 2006).AMF are obligate root symbionts of more than 80%of all land plants, including most agricultural crops. Inexchange for photosynthetic carbon from their host,they can not only improve plant growth through anincreased nutrient uptake but also alleviate plant stresscaused by abiotic and biotic factors, in addition toproviding other ecosystem services such as increasedsoil stability (Smith and Read 2008; Gianinazzi et al.2010). The biocontrol effect of AMF has beenobserved in a wide range of plants against differentpathogens. Most reports deal with soil-borne fungalpathogens causing root rot or wilting, like Rhizocto-nia, Fusarium and Phythophthora, but the interactionwith some bacterial and aboveground pathogens hasalso been investigated (Whipps 2004; Pozo andAzcon-Aguilar 2007; St-Arnaud and Vujanovic2007; Akhtar and Siddiqui 2008).

The mycorrhiza-induced resistance seems to becharacterized by both localized and systemic mecha-nisms (Cordier et al. 1998; Pozo et al. 2002) but theexact modes of action remain to be elucidated.Whipps (2004) classified suggested modes of actionof AMF into different groups. The first groupcomprises indirect effects of AMF through the hostplant, including induced plant defense. Formation ofmycorrhizal symbiosis indeed evokes a weak, tran-sient or extremely localized defense response in thehost (Gianinazzi-Pearson et al. 1996) which is thoughtto predispose the host to react more rapidly tosubsequent pathogen attack (Pozo and Azcon-Aguilar 2007). The second group of suggested modesof action encompasses more direct effects of AMF onthe pathogen, including competition for space ornutrients and the effect of compounds exuded byAMF. Whipps (2004) also included the effect ofmycorrhizal root exudates on pathogens in this group,although this effect can also be considered as anindirect effect through the host and it is also closelyintertwined with the third group, covering effects onpathogens through development of an antagonisticmicrobiota.

Root exudation is an important process throughwhich 5 to 21% of the photosynthesized carbon isreleased into the rhizosphere (Walker et al. 2003; Baiset al. 2004), creating a small zone around the plantroot system differing in physical, biochemical andecological properties (Vivanco et al. 2002). Low-

molecular-weight compounds, including amino acids,organic acids, sugars, phenolics and various second-ary metabolites, constitute the largest portion of theroot exudates (Vivanco et al. 2002) and are altered bymycorrhizal symbiosis (Hodge 2000; Jones et al.2004). Both quantitative and qualitative differences inmycorrhizal root exudates have been reported, includ-ing differences in amino acids (Harrier and Watson2004), flavonoids (Steinkellner et al. 2007), phenoliccompounds (McArthur and Knowles 1992), sugarsand organic acids (Sood 2003; Lioussanne et al.2008), although the root exudates composition alsodepends on the degree of symbiosis (Scheffknecht etal. 2006), plant species or AMF species (Badri andVivanco 2009). The altered root exudation is alsoreflected in the systemic autoregulation of themycorrhizal colonization by the host (Pinior et al.1999; Vierheilig et al. 2003): once roots are colonizedby AMF, plants appear to be able to regulate furthercolonization through the release of root exudates andit has been proposed that plants might use thismechanism as a preventative measure against furthermycorrhizal colonization and defense against patho-gens at the same time (Vierheilig and Piché 2002;Vierheilig et al. 2008). Changes in the mycorrhizalroot exudates composition also lead to alterations inthe microbial rhizosphere populations of facultativeanaerobic bacteria, fluorescent pseudomonads, Strep-tomyces species and chitinase-producing actinomy-cetes (Marschner and Baumann 2003; Wamberg et al.2003; Harrier and Watson 2004) which might in turnhave antagonistic effects on nematodes.

Roots thus have a large impact on their immediaterhizosphere surroundings. The challenge for plant-parasitic nematodes is to locate a suitable host plant inthe soil by detection of the gradients created by theplant. Different types of stimuli exist in the soilenvironment, including for example chemical andthermal signals, which can stimulate nematodes tomove in a specific direction (Curtis et al. 2009). Perry(2005) divided nematode host finding behaviour indifferent phases: long-range host finding, short-rangehost finding and infection site finding by means oflocal or contact cues. According to this classificationthe corresponding long-distance cues, relevant to thescale of several centimeters, serve to direct thenematodes to areas that are likely to contain a possiblehost, while the short-distance cues attract the nemat-odes to the roots themselves. The local cues, finally,

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guide the nematodes to their preferred penetrationsites (Spence et al. 2008).

Nematode host finding in relation to possible stimulihas been examined in many studies, but holisticinvestigations to evaluate the relative importance ofeach stimulus are still lacking. The long-distance cuesare thought to be stimuli of a more general nature, ofwhich CO2 has been studied most extensively. CO2 isthe most abundant volatile component of the rootexudation, and is generally accepted as a long-distance nematode-attractant (Curtis et al. 2009), butthe fact that it cannot be the only factor in hostfinding is illustrated by the report that nematodeswere attracted to living roots, away from a non-biological CO2 source (Bird 1960). Root-knot nemat-odes are also remarkably sensitive to thermal gra-dients and have been shown to respond to minute heatdifferences (Curtis et al. 2009). It has been hypothe-sized that metabolic heat attracts nematodes to a root,but it seems impossible for nematodes to detect such asmall gradient in the presence of fluctuating thermalgradients that are inherent to the soil environment.Perry (2005) instead classified heat as a local cue,orienting the nematodes to their preferred penetrationsite in the metabolically highly active root zoneimmediately behind the root tip. Once the nematodearrives in the root area, other root exudates can serveas a stimulus. Wuyts et al. (2006c) performedchemotaxis studies showing that nematodes wereonly attracted towards plant roots as long as the rootexudates had not been removed. Several phenoliccompounds have been tested for their effect onnematode chemotaxis, motility or survival, but theactual presence and concentration of the specificallytested compounds in root exudates has usually not beeninvestigated (Wuyts et al. 2006b; Curtis et al. 2009).

The modes of action of mycorrhiza-induced resis-tance against plant-parasitic nematodes remain largelyunknown, although recently the systemic nature ofAMF-induced reduction of nematode reproductionand the involvement of a class III chitinase gene havebeen demonstrated (Li et al. 2006; Elsen et al. 2008).No reports exist to date on the involvement of rootexudates in mycorrhiza-induced resistance againstnematodes. Therefore, the aim of this study was toinvestigate the effect of AMF on the two crucialinitial steps of root-knot nematode infection, namelymovement towards a host root and subsequentpenetration into the host root.

Materials and methods

Biological materials

In all experiments, the tomato (Solanum lycopersi-cum) cv. Marmande was grown under greenhouseconditions at an ambient temperature of 20–27°C and75% relative humidity. The AMF Glomus mosseae,originally isolated from banana in Tenerife, Spain,and maintained as a greenhouse stock culture onsorghum, was applied as a layer of 200 ml mycorrhi-zal inoculum per 1 L pot at sowing. The inoculumconsisted of rhizosphere soil from 6-months-oldsorghum pot cultures containing spores, hyphae andheavily colonized root pieces. Plants from the controltreatment received 200 ml of rhizosphere soil and rootpieces from non-colonized sorghum plants, and wererandomly selected and tested for mycorrhizal coloni-zation in every experiment. All plants received 1 g ofa slow release fertilizer at planting (Substral Osmo-cote® controlled release fertilizer, NPK; 14-13-13).Mycorrhizal colonization was determined by stainingthe roots with an ink-vinegar solution (Vierheilig et al.1998). After clearing in 10% KOH, staining in 5%ink-vinegar solution and destaining in water, 20 rootpieces of 1 cm were mounted on slides and observedwith a light microscope (100x magnification). Thefrequency of mycorrhizal colonization (F%) wascalculated as the percentage of root pieces colonizedby hyphae, arbuscules or vesicles. In addition, theabundance of these structures, i.e. the intensity of themycorrhizal colonization (I%), was estimated visuallyusing different classes (1–20, 20–40, 40–60, 60–80,80–100%) that correspond to the area of the cortexcolonized by the mycorrhizal fungus. The results wereexpressed as a percentage by calculating (20x+40y+60z+80t+100v)/(x+y+z+t+v) with x, y, z, t and vthe number of root fragments in each class (Plenchetteand Morel 1996). Before nematode inoculation of thetest plants in every experiment, the degree ofmycorrhizal colonization of plants grown in parallelwas determined.

The Meloidogyne incognita population was origi-nally isolated from banana in Malaysia and main-tained as a greenhouse stock culture on tomato cv.Marmande. A pure culture was initiated in thegreenhouse starting from a single egg mass. Inoculumfor the experiments was obtained after 4 months byextraction of egg masses from heavily infected tomato

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roots, followed by 5-day incubation on modifiedBaermann dishes to obtain freshly hatched secondstage juveniles (J2) (Hooper et al. 2005).

Twin-chamber experiment

Tomato seeds were planted in the greenhouse in 1 Lpots containing a mixture of sand and potting soil(2:1). As described above, 200 ml of rhizosphere soilcolonized by G. mosseae was added for the mycor-rhizal treatment while plants from the control treat-ment received 200 ml of rhizosphere soil from non-colonized sorghum plants. After 6 weeks, eightmycorrhizal plants were uprooted to determine my-corrhizal colonization as described above.

After determination of mycorrhizal colonization,plants were transferred to a twin-chamber set-up(Declerck et al. 1998; Dababat and Sikora 2007),consisting of two 1 L pots connected by a plasticbridge (1×1×5 cm) filled with the same soil mixture,at a soil depth of 3 cm. Two days after transplanting,2,000 freshly hatched M. incognita J2 were inoculatedper twin chamber, onto the middle of the bridge. Thecompartments and bridge were watered to fieldcapacity at time of inoculation, and 2,000 J2 wereinoculated in 2 ml of water into a hole in the soilmixture exactly in the middle of the bridge. Theexperiment consisted of three treatments: (a) controlplants in both compartments (control treatment), (b)mycorrhizal plants in both compartments (AMFtreatment) and (c) a control plant in the leftcompartment and a mycorrhizal plant in the rightcompartment (mixed treatment). All twin-chamberswere placed in a completely randomized design andeach treatment consisted of eight replications. Twelvedays after inoculation, nematode penetration wasassessed in control and mycorrhizal root systems ofall treatments. Plants from both compartments wereuprooted and roots were gently washed to removeadhering soil. Nematodes were extracted from thewhole root sample by maceration-sieving (Speijer andDe Waele 1997). Roots were cut into 1–2-cm seg-ments and macerated in a blender for two times 10 s.The homogenized solution was then passed through aseries of sieves (250–100–25 μm sieve) and thecollected J2 from root samples were counted with alight microscope (40x magnification). Nematodepenetration rate was calculated as the penetratednematode population (Pp) divided by the inoculated

initial nematode population (Pi). A soil subsample of100 ml was taken in both compartments of the mixedtreatment and nematodes were extracted on modifiedBaermann dishes (Hooper et al. 2005) and J2 countedwith a light microscope (40× magnification).

Root exudates experiments

Control and mycorrhizal tomato plants were grown asdescribed above, and the mycorrhizal colonizationwas determined before exudates collection and nem-atode inoculation, as described above. After determi-nation of mycorrhizal colonization, 16 control plantsas well as 16 mycorrhizal plants of 6-weeks old wereused for collection of root exudates according to theprocedure described by Vierheilig et al. (2003).Whole plants were harvested non-destructively andplant roots were gently rinsed to remove the soilmixture. Plants were then placed in a growth chamber(12 h photoperiod, 20–22°C, 50% relative humidity)in flasks with distilled water. The roots were protectedfrom light by covering the flasks in aluminum foil.During 12 days, the root exudates were collected on adaily basis (i.e. every 24 h), and the empty flaskswere daily refilled with distilled water. Every 4 days anew batch of control and mycorrhizal plants was usedfor root exudates collection. After collection, the rootexudates were immediately pooled per treatment and30 ml of each solution was applied to the soil surfaceof individual plants according to the different treat-ments. Control plants as well as mycorrhizal plantsdaily received (a) distilled water, (b) root exudatescollected from control plants or (c) root exudatescollected from mycorrhizal plants, resulting in sixtreatments. One hour after the first application of rootexudates or distilled water to the plants in thegreenhouse, plants were inoculated with 1,000 freshlyhatched J2 per plant, as described above. Twelve daysafter nematode inoculation, the nematodes wereextracted from the roots by the maceration-sievingmethod and the number of J2 that had penetrated theroots was counted using a light microscope (Speijerand De Waele 1997) as described above. Nematodepenetration rate was calculated as the penetratednematode population (Pp) divided by the inoculatedinitial nematode population (Pi). All plants wereplaced in a completely randomized design, eachtreatment consisted of 8 replications, and the exper-iment was repeated.

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In vitro motility assay

Root exudates were collected from eight control andmycorrhizal tomato plants of 6-weeks old, anddistilled water was used as a control, resulting inthree treatments. The root exudates were collected asdescribed above, pooled, filter sterilized (0.2 μm poresize filter, DynaGard syringe filter tips, MicrogonInc., California) and stored in 1 ml aliquots at −20°C.Mycorrhizal colonization of plants from the samebatch was determined before the start of the experi-ment, and mycorrhizal colonization of the plants usedfor exudates collection was determined after exudateshad been collected. In a 24-well plate, 250 μl of eithersolution was applied and 100 freshly hatched J2 wereadded per well. Plates were incubated at 25°C in thedark, with four replications per treatment and theassay was performed twice. The number of immotileJ2, i.e. without active sinusoidal form and movement,was monitored in each well at 1, 2 and 3 days afterapplication of the solutions by using an inverted lightmicroscope (40× magnification) (Wuyts et al. 2006b).In an additional experiment, the immotile nematodesat 1 day after application of each solution werehandpicked with a glass micropipette (Speijer and DeWaele 1997) and transferred to a new well with250 μl of the same solution that had been freshlyadded to the new well. Nematode motility was thenobserved after 4–24–48 h.

Statistical analysis

Nematode infection and plant data of the twin-chamber and root exudates experiments were statisti-cally analyzed by ANOVA when the conditions weremet (i.e., normal distribution and homogeneity ofvariances), using Statistica® (Release 7, Statsoft,Tulsa, USA). Nematode data of each compartmentfrom the twin-chamber experiment were analyzed asproportions, relative to the amount of nematodes inthe other compartment. The twin-chamber experimentwas analyzed as a 2-way ANOVA, with treatment andcompartment side as factors. Data from both repli-cations of the exudates experiment were analyzed as a3-way ANOVA, with mycorrhizal colonization, exu-dates application and experiment repetitions as fac-tors. The Tukey HSD test was applied for multiplecomparisons of group means. Prior to analysis,nematode numbers were log(x+1) transformed, while

nematode proportions and penetration rates werearcsin(x/100) transformed to reduce data variance.Data of the in vitro motility assay were analyzedusing non-parametric Kruskal-Wallis analysis ofvariance by ranks, because of the smaller samplesize. When the Kruskal-Wallis statistic was signifi-cant, comparisons of treatments versus control andmultiple comparisons among treatments were calcu-lated as described by Siegel and Castellan (1988).

Results

Mycorrhizal plants in the twin-chamber experimentdisplayed a well-established and active mycorrhizalcolonization, as confirmed by the observation ofhyphal structures, arbuscules and vesicles inside thestained roots. On average, the colonization frequencyreached 79±3% with an intensity of 25±5%. Mycor-rhizal colonization did not spread to the controlcompartments of the mixed treatment in the twin-chamber experiment and had no significant effect onfresh root and shoot weight (data not shown).Nematode penetration was similar in both compart-ments when plants from the same treatment werepresent on both sides of the bridge (Fig. 1). Penetra-tion of tomato roots by the J2 was significantly (P=

B

B

A

A

Fig. 1 Number of Meloidogyne incognita second stage juve-niles (J2) that penetrated in tomato roots in the left or rightcompartment of the twin-chamber (Pi=2,000 J2), eithercolonized by Glomus mosseae (AMF treatment) or not (controltreatment), 12 days after nematode inoculation. Controltreatment: both compartments contain non-mycorrhizal tomatoplants; AMF treatment: both compartments contain mycorrhizaltomato plants. Bars represent standard error, based on eightreplications.Different letters indicate significant differences (P<0.05) between the treatments according to the 2-way ANOVATukey HSD test on arcsin(x/100) transformed proportion data

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0.002; F=7.3; df=1) higher in non-mycorrhizal plantsof the control treatment compared to penetration inmycorrhizal plants of the AMF treatment. Penetrationrate per compartment reached 23% on average in thecontrol treatment vs 14% in the AMF treatment. Thesame effect was observed in the mixed treatment, witha control plant in the left compartment and amycorrhizal plant in the right compartment. Nema-tode penetration in this case was significantly (P=0.006; F=10.7; df=1) higher in the control compart-ment with a penetration rate of 19% vs a penetrationrate of 7% in the mycorrhizal compartment (Fig. 2).When considering the number of nematodes in thesoil in the compartments of the mixed treatment,significantly (P=0.010; F=8.6; df=1) more nemat-odes were extracted from the mycorrhizal compart-ment (Fig. 2). Combined with the number ofnematodes that penetrated the roots, this provided anestimation of the total number of nematodes thatmoved away from the inoculation point on the bridge,towards either compartment. No significant differencewas observed between the total number of nematodes(data from roots and soil) between the control andmycorrhizal compartment of the mixed treatment(Fig. 2).

In both repetitions of the exudates experiment,mycorrhizal colonization of the plants used fornematode inoculation and for root exudates collection

had an average frequency of 83±4%, with anintensity of 27±7%. There were no significant differ-ences between both repetitions of the exudatesexperiment. Mycorrhizal colonization resulted in asignificantly (P<0.001; F=27.2; df=1) lower pene-tration of the J2 (Fig. 3). In addition, daily applicationof root exudates originating from mycorrhizal plantssignificantly (P=0.001; F=7.4; df=2) reduced nema-tode penetration compared to the application ofdistilled water or root exudates originating fromcontrol plants. A significant (P=0.002; F=6.7; df=2) interaction effect was observed between mycorrhi-zal colonization and root exudates application, withnematode penetration being highest when non-mycorrhizal root exudates were applied to controlplants and lowest when mycorrhizal root exudateswere applied to mycorrhizal plants (Fig. 3).

The mycorrhizal plants providing root exudates forthe in vitro motility assays had an average mycorrhi-zal colonization frequency of 89±3%, with anaverage intensity of 31±5%. A significantly (P=0.033) higher percentage (21% more) of the J2 wasimmobilized 1 day after application of root exudatesoriginating from mycorrhizal plants, compared to thepercentage of immobilized J2 after application ofdistilled water or control root exudates. Nematodesregained motility at day 2, with no significant differ-ences among the treatments at day 2 or 3 (Fig. 4). Out

A

A B

B

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Fig. 2 Number of Meloidogyne incognita second stagejuveniles (J2) in soil and/or tomato roots of the mixedtreatment, consisting of a left compartment with a controltomato plant (L) and a right compartment with a tomato plantcolonized by Glomus mosseae (R) (Pi=2,000 J2), 12 days afternematode inoculation. Bars represent standard error, based oneight replications. Different letters indicate significant differ-ences (P<0.05) between the compartments according to the 1-way ANOVA Tukey HSD test on arcsin(x/100) transformedproportion data

A

B B

C C

D

Fig. 3 Number of Meloidogyne incognita second stagejuveniles (J2) that had penetrated tomato roots colonized byGlomus mosseae or not, to which mycorrhizal exudates,distilled water or non-mycorrhizal exudates were applied daily,12 days after inoculation (Pi=1,000 J2). Bars representstandard error, based on eight replications, and two repetitionsof the experiment. Different letters indicate a significant (P<0.05) interaction effect between exudates application andmycorrhizal colonization, according to the 2-way ANOVATukey HSD test on log (x+1) transformed data

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of the immotile nematodes that had been transferredto a fresh solution of the same treatment 1 day afterthe onset of the assay (data not shown), none regainedmotility at either time point when non-mycorrhizalroot exudates or water had been applied, while 72%of the immotile nematodes in the mycorrhizal rootexudates treatment regained motility 48 h after thetransfer to a fresh solution of mycorrhizal rootexudates.

Discussion

This study demonstrated that root penetration of M.incognita J2 was significantly reduced in G. mosseae-colonized tomato plants compared to control plants,as observed in the twin-chamber and exudates experi-ments. Reports about mycorrhiza-induced resistanceagainst plant-parasitic nematodes have been reviewedby Pinochet et al. (1996), Hol and Cook (2005) andAkhtar and Siddiqui (2008) but the contribution ofreduced nematode penetration to the overall biocon-trol effect has rarely been described. The twin-chamber experiment provided a better insight. J2migrated equally towards both compartments whenplants from the same treatment were present on bothsides, thus validating the set-up. In the mixedtreatment, overall nematode penetration was lowerthan in the other treatments. We have made similarobservations in in vitro chemotaxis choice-experiments. It seems that, when two differentcompounds are present, nematodes stay longer onthe inoculation spot before starting their migration toeither side, compared to when the same compound ispresent on both sides, although they might eventuallymove equally to both compounds. Significantly fewer

J2 in the mixed treatment were recovered from themycorrhizal roots in the right compartment than fromthe control roots in the left compartment. However,the total amount of nematodes that moved away fromthe inoculation point on the bridge towards eithercompartment was equal, irrespective of the plantbeing mycorrhizal or not. The AMF thus did notseem to act through a long-distance cue (Spence et al.2008) but rather at the moment the nematodes cameinto proximity of the roots, resulting in a significantlyhigher penetration of the control roots and anaccumulation of non-penetrating nematodes near themycorrhizal roots. Using a twin-chamber set-up,Dababat and Sikora (2007) also concluded that M.incognita J2 were more prone to penetrate non-treatedtomato roots than roots colonized by a non-pathogenic endophytic Fusarium oxysporum strain,although no data were reported concerning nematodeaccumulation in the soil. Two factors could explainour observations: since the action radius of the rootexudates is small (Bais et al. 2006), nematodebehavior in soil might be affected once they are closeto mycorrhizal roots (Wuyts et al. 2006c), or theactual nematode penetration of mycorrhizal rootsmight be hampered.

Plant defense in mycorrhizal roots is accompaniedby biochemical changes, including the formation ofstructural barriers like lignins, callose andhydroxyproline-rich glycoproteins and the activationof the phenylpropanoid metabolism and defense-related proteins like chitinases and β-1,3-glucanases(see for example reviews by Azcon-Aguilar and Barea1996; Morandi 1996; Whipps 2004; St-Arnaud andVujanovic 2007). Evidence for the importance ofthese compounds in nematode infection has beenprovided in other reports. Resistance against M.

Fig. 4 Percentage of immo-bilizedMeloidogyne incog-nita second stage juvenilesat 1, 2 and 3 days afterapplication of mycorrhizalroot exudates, distilled wa-ter or non-mycorrhizal rootexudates. * indicate signifi-cant differences (P<0.05)according to the Kruskal-Wallis test. Bars representstandard error, n=4

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incognita in the pepper cultivar CM334, for example,is characterized by limited nematode penetration,accompanied by accumulation of phenolic com-pounds, chlorogenic acid in particular (Pegard et al.2005). In resistant soybean cultivars, reduction of M.incognita penetration appeared concomitantly withincreased transcriptional activation and enzyme activ-ity of phenylalanine ammonia lyase and 4-coumarylCoA ligase, two major enzymes of the centralphenylpropanoid pathway (Edens et al. 1995). In-creased cell wall lignifications have also beenassociated with mycorrhizal symbiosis (Harrier andWatson 2004), and might contribute to the lowernematode penetration observed. In the case of tomatoinfection by the fungal pathogen Phytophthora para-sitica, the role of cell wall modifications inmycorrhiza-induced resistance was demonstrated(Cordier et al. 1998), and modifications in the ligninbiosynthetic pathway have also been shown to affectM. incognita reproduction (Wuyts et al. 2006a).

Our results clearly point to a role for mycorrhizalroot exudates in the reduction of M. incognitapenetration. Mycorrhizal symbiosis indeed not onlyalters the root metabolism, it also leads to an alteredroot exudation composition (Hodge 2000; Jones et al.2004). Root exudates play an important role indefending the rhizosphere against pathogenic micro-organisms (Bais et al. 2006) and the ability of apathogen to infect a root system might depend on itsability to overcome the plants antimicrobial rootexudates (Bais et al. 2005). Plant-parasitic nematodesneed root exudates to guide themselves towards asuitable penetration site (Curtis et al. 2009) and canthus be attracted, repelled or paralyzed by theirpresence (Koltai et al. 2002). The results of theexudates experiments show that mycorrhizal rootexudation contributed to a reduced nematode pene-tration, since application of mycorrhizal root exudatesdecreased nematode penetration compared to applica-tion of distilled water or control root exudates. Thedifference between nematode penetration in controland mycorrhizal plants was smallest when distilledwater was applied and was increased when mycorrhi-zal or non-mycorrhizal root exudates were applied. Inthe case of control plants, application of non-mycorrhizal root exudates resulted in the highestnematode penetration, indicating the presence ofcertain stimulating factors absent in the water controland mycorrhizal root exudates. But this is probably

not the only difference between mycorrhizal and non-mycorrhizal root exudates, since application of my-corrhizal root exudates to mycorrhizal plants reducednematode penetration beyond the level of the watercontrol and non-mycorrhizal root exudates, implyingnematode inhibiting activity of the mycorrhizal rootexudates. When non-mycorrhizal root exudates wereapplied to mycorrhizal plants, or when mycorrhizalroot exudates were applied to control plants, nodifferences were observed compared to the watercontrol, probably due to nullification by the plant’sown root exudation.

Both quantitative and qualitative differences inmycorrhizal root exudation have been reported (Hodge2000; Jones et al. 2004), which might affect pathogeninfection. Norman and Hooker (2000) for examplereported diminished Phytophthora fragariae sporula-tion in the presence of root exudates from mycorrhizalstrawberries. Fillion et al. (1999) showed the reduc-tion of Fusarium oxysporum conidia germinationwhen subjected to crude extracts from Glomus intra-radices mycelium. But the effect of mycorrhizal rootexudates on fungal pathogens is not always unequiv-ocal. In tomato for example, a stimulatory effect onthe microconidia germination of F. oxysporum wasobserved by Scheffknecht et al. (2006), and Phytoph-thora nicotiana infection of tomato was not affectedby mycorrhizal root exudates (Lioussanne et al.2009). Lioussanne et al. (2008) also nuanced thatthe attraction of Phytophthora nicotianae zoosporestowards mycorrhizal root exudates shifted to repel-lency, depending on the maturity of the mycorrhizalcolonization. The only reported effect of mycorrhizalexudates on nematodes deals with improved hatchingof cyst nematodes in the presence of root exudatesfrom mycorrhizal potato (Ryan et al. 2000). Changesin the mycorrhizal root exudation composition alsolead to alterations in the microbial rhizosphere whichmight in turn have antagonistic effects on nematodes.Root exudates originating from mycorrhizal plantsattracted plant growth promoting bacteria like Pseu-domonas fluorescens (Sood 2003) and stimulatedpopulations of beneficial soil micro-organisms likeTrichoderma (Fillion et al. 1999), organisms knownto exert a biocontrol effect on plant-parasitic nemat-odes (Dong and Zhang 2006; Sikora et al. 2008). Itshould be noted that exudate compounds can also bemycelium derived. Hyphae from the mycorrhizalfungus itself also release compounds in the so-called

342 Plant Soil (2012) 354:335–345

hyphosphere soil, thus influencing microbial activityand local soil characteristics (Jones et al. 2004).

No reports exist to date on the effects of mycor-rhizal root exudates on nematode behavior in the soil.The in vitro motility assay performed in this studyrevealed the motility-inhibiting effect of mycorrhizalroot exudates on M. incognita J2, though onlytransient since nematodes resumed motility at 2 daysafter root exudates application. This might beexplained by the degradation of active compoundsduring the assay or the lack of inflow of new rootexudates, since the motility-inhibiting effect could beprolonged by transferring the nematodes to a freshsolution of mycorrhizal root exudates. Nematodemotility has been tested in the presence of non-mycorrhizal root exudates from several plant species,and can depend on the plant species involved: M.incognita juveniles have been reported to temporarilylose motility in presence of root exudates fromlegumes but not from solanaceous species includingtomato (Zhao et al. 2000; Hubbard et al. 2005). In ourmotility assay, using tomato root exudates we did notobserve significant differences between the applica-tions of water or control root exudates.

To summarize, this study revealed that penetrationof M. incognita J2 was reduced in mycorrhizal tomatoroots and that mycorrhizal root exudates contributedto this process, due to a negative effect on nematodemotility in the soil. Molecular and histologicalobservations could greatly contribute to more insightinto the observed effect on penetration, while it wouldalso be interesting to study the profile of mycorrhizaltomato root exudates to further elucidate the effects ofAMF on nematode behavior.

Acknowledgements The authors wish to thank Nicole Viaenefrom the Institute for Agricultural and Fisheries Research(ILVO), Belgium, for valuable comments on the manuscript.This work was supported by a specialization grant from theInstitute for the Promotion of Innovation through Science andTechnology in Flanders (IWT-Vlaanderen) to C. Vos and aVLIR-UDC grant from the Belgian Government to R.Mkandawire.

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