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Soil Biology & Biochemistry 47 (2012) 60e66

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Soil Biology & Biochemistry

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Mycorrhiza-induced resistance in banana acts on nematode host locationand penetration

Christine Vos a,*, Daphné Van Den Broucke a, Franklin Mbongo Lombi c, Dirk De Waele a,b,Annemie Elsen b,d

a Laboratory of Tropical Crop Improvement, Department of Biosystems, University of Leuven, Kasteelpark Arenberg 13, 3001 Leuven, BelgiumbDepartment of Biology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, 9000 Gent, BelgiumcCentre Africain de Recherches sur Bananier et Plantains (CARBAP). B.P. 832 Douala-Cameroun, BelgiumdBodemkundige Dienst van België, Willem de Croylaan 48, 3001 Heverlee, Belgium

a r t i c l e i n f o

Article history:Received 9 June 2011Received in revised form20 December 2011Accepted 23 December 2011Available online 5 January 2012

Keywords:Mycorrhiza-induced resistanceBiological controlMode of actionBurrowing nematode (Radopholus similis)PenetrationRoot exudatesBanana

* Corresponding author. Tel.: þ32 16 329603; fax:E-mail address: christine.vos@biw.kuleuven.be (C.

0038-0717/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.soilbio.2011.12.027

a b s t r a c t

Mycorrhiza-induced resistance has been observed against a broad range of mainly soil-borne pathogens,including plant-parasitic nematodes, but the modes of action involved remain unclear. In this study therole of mycorrhiza-induced resistance was investigated during the pre-infectional phase of nematodehost finding and penetration. Banana plants were colonized by Glomus mosseae or Glomus intraradices,two arbuscular mycorrhizal fungi. The plant-parasitic nematode Radopholus similis was inoculated afterestablishment of the mycorrhizal colonization. Nematode attraction and penetration were assessedwithin a 12-day period. In root exudate experiments, root exudates collected from both control andmycorrhizal plants were added both to control and mycorrhizal plants to assess their direct impact on thenematode penetration. In an in vitro chemotaxis bio-assay, the chemotactic behavior of R. similis wasdetermined towards isolated root exudates of control and mycorrhizal plants. The penetration experi-ments clearly showed lower nematode penetration in mycorrhizal plants and the important contributionof differential root exudation by mycorrhizal plants was demonstrated in the exudate experiments aswell as in the in vitro chemotaxis bio-assay, with the largest impact on juveniles. The root exudateexperiments and in vitro chemotaxis bio-assay point towards a reduced attraction of the nematodes tothe mycorrhizal plant roots. The results demonstrate that a water-soluble compound in mycorrhizal rootexudates is at least partially responsible for the mycorrhiza-induced resistance at the pre-infectionallevel of R. similis infection.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The worldwide restriction on the use of nematicides has forcedproducers to look for alternative nematode management strategies.Within an integrated and more sustainable management approachthe use of biocontrol organisms, like arbuscular mycorrhizal (AM)fungi, seems to be a promising alternative (Whipps, 2004). AMfungi are obligate biotrophic fungi colonizing the roots of approx-imately 80% of all terrestrial plants. It is known that symbiosis byAM fungi can confer resistance or tolerance to biotic stresses, inparticular soil-borne fungi and plant-parasitic nematodes (Whipps,2004). This activity is also referred to as mycorrhiza-induced

þ32 16 321993.Vos).

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resistance by Pozo and Azcon-Aguilar (2007). In banana, one ofthe world’s major food crops (De Waele and Elsen, 2007), AM fungican reduce the severity of diseases caused by the soil-borne fungiFusarium oxysporum f. sp. cubense and Cylindrocladium spathiphylli(Jaizme-Vega et al., 1998; Declerck et al., 2002). AM fungi were alsoreported to suppress population build-up of the plant-parasiticburrowing nematode Radopholus similis in the roots of variousbanana genotypes (Elsen et al., 2003a,b).

Yield losses caused by R. similis infection can go up to 50% (Sarahet al., 1996; Speijer and Kajumba, 2000). After host finding, thisnematode penetrates the roots preferably near the root apexthough any portion of the entire root lengthmay be invaded (i.e. thepre-infectional phase). Following successful root penetration, thenematodes migrate intercellularly in the cortical parenchyma,feeding on the cytoplasm of surrounding cells. This feedingbehavior creates dead cells and cavities, visually observed as

C. Vos et al. / Soil Biology & Biochemistry 47 (2012) 60e66 61

necrosis. During this post-infectional phase, nematode develop-ment and reproduction takes place.

Knowledge on the underlying mechanisms of mycorrhiza-induced resistance against plant-parasitic nematodes could leadto improvement of the use of AM fungi as biocontrol organisms.However, so far only limited efforts have been made in this respect.In 2008, Elsen et al. demonstrated for the first time thatmycorrhiza-induced resistance against plant-parasitic nematodesis systemically induced in banana roots. The next step is to deter-mine when and where during the nematode infection cycle the AMfungi interfere. For example, mycorrhizal plants could producesubstances that hamper nematode host finding, resulting ina reduction of nematode penetration.

In this study we demonstrate for the first time that mycorrhiza-induced resistance acts at the pre-infectional phase of a nematodeinfection. The results of the penetration experiment clearly showlower penetration in mycorrhizal plants and the important role ofroot exudates is also demonstrated in root exudate experiments aswell as in the in vitro chemotaxis bio-assay with root exudates.

2. Materials and methods

2.1. Plant growth, inoculation of AM fungi and nematodes

Tissue-cultured plants of banana cv. Grande Naine (Musa acu-minata AAA, Cavendish subgroup, ITC 1256) were provided by theInternational Musa Germplasm Collection at the InternationalTransit Center, University of Leuven, Belgium. Cultivar GrandeNaine was selected for its high mycorrhizal dependency (Elsenet al., 2003a) and for its high susceptibility to R. similis (Daniellset al., 2001). The plants were proliferated and regenerated onMurashige and Skoog medium including vitamins, 30 g/l sugar,10 mg/l ascorbic acid and 2 g/l gelrite with pH 6.2 (Banerjee and DeLanghe, 1985). Well-rooted plantlets were transferred to thegreenhouse.

Two different AM fungi isolates were maintained and propa-gated in sorghum pot cultures in the greenhouse at an ambienttemperature of 20e27 �C, with a 12-h photoperiod (170e190 PAR),and a relative humidity of 70%. The Glomus mosseae isolate wasoriginally isolated from Pome banana (Musa AAB, Pome-group)grown on a biological farm in Los Realejos, Tenerife, Spain. TheGlomus intraradices isolate (MUCL 41833) originated from bananaroots in Guadeloupe, France. The G. intraradices was used as inoc-ulum for the penetration experiment, while G. mosseae was usedfor the root exudate experiments and for the in vitro chemotaxisbio-assay. Mycorrhizal inoculum consisted of rhizosphere soil from6-month old sorghum pot cultures containing spores, hyphae andheavily colonized root pieces.

The burrowing nematode R. similis was maintained and multi-plied on in vitro carrot disc cultures (Pinochet et al., 1995) at 26 �C.The isolate used in all experiments was originally collected frombanana roots in Uganda and has been characterized as highlypathogenic (Fallas et al., 1995). Before inoculation, nematodes werecollected from the carrot discs by the maceration-sieving method(Speijer and De Waele, 1997).

2.2. Penetration experiment

8-week old rooted in vitro banana plants were deflasked andplanted in 200-ml pots containing a substrate mixture of sand andpotting soil (2:1). For the mycorrhizal treatment, a 1-cm layer ofrhizosphere soil colonized by AM fungi was added to each pot,between two layers of substrate. Plants from the control treatmentreceived a layer of rhizosphere soil from non-colonized sorghumplants. The plants were grown in the greenhouse at an ambient

temperature of 20e27 �C, with a 12-h photoperiod (170e190 PAR),and a relative humidity of 70% during 6 weeks to allow goodcolonization of the roots in the mycorrhizal treatment. After 6weeks, eight mycorrhizal plants were harvested to determinemycorrhizal colonization. Secondary and tertiary roots werestained with ink-vinegar (Vierheilig et al., 1998). After clearing in10% KOH, staining in 5% ink-vinegar solution and destaining inwater, 20 1-cm root pieces weremounted on slides and observed bybright field microscopy. The frequency (F%) of the mycorrhizalcolonization was calculated as the percentage of root segmentscolonized by either hyphae, arbuscules or vesicles, while theintensity (I%) was estimated as the abundance of hyphae, arbus-cules and vesicles in each mycorrhizal root segment (Plenchetteand Morel, 1996). Plants were inoculated with 200 vermiformR. similis, consisting mainly of juveniles. Pots were watered to fieldcapacity at time of inoculation, and nematodes were inoculated in2 ml of water into two 1-cm deep holes in the substrate around thestem base. At 1e2e4e6e10e12 days after inoculation, the nema-todes were extracted from the roots by the maceration-sievingtechnique (Speijer and De Waele, 1997). To determine how manynematodes had penetrated the roots, the number of juveniles,females and males was counted using bright field microscopy. Eachtreatment consisted of eight replicates.

2.3. Root exudate experiments

Two separate root exudate experiments were carried out. Thefirst experiment consisted of four treatments: (1) control plantsreceiving distilled water (Control plants/H2O), (2) mycorrhizalplants receiving distilledwater (Mycorrhizal plants/H2O), (3) controlplants receiving root exudates collected from control plants (Controlplants/ex �AM) and (4) control plants receiving root exudatescollected from mycorrhizal plants (Control plants/ex þAM). In thesecond experiment more treatments were included: (1) controlplants receiving distilled water (Control plants/H2O), (2) mycor-rhizal plants receiving distilled water (Mycorrhizal plants/H2O),(3) control plants receiving root exudates collected from controlplants (Control plants/ex �AM), (4) mycorrhizal plants receivingroot exudates collected from control plants (Mycorrhizal plants/ex �AM), (5) control plants receiving root exudates collected frommycorrhizal plants (Control plants/ex þAM) and (6) mycorrhizalplants receiving root exudates collected from mycorrhizal plants(Mycorrhizal plants/ex þAM). In both experiments each treatmentconsisted of eight replicates.

As described for the penetration experiment, 8-week old rootedin vitro banana plants were deflasked and transplanted in 200-mlpots containing a substrate mixture of sand and potting soil (2:1).For the mycorrhizal treatment the plants were inoculated with AMfungi as described before, and plants from the control treatmentreceived a layer of rhizosphere soil from non-colonized sorghumplants. Six weeks after planting, eight (experiment 1) or 16(experiment 2) control plants as well as eight or 16 mycorrhizalplants were used for root exudates collection according to theprocedure described by Vierheilig et al. (2003). The whole plantswere harvested non-destructively and plant roots were gentlyrinsed to remove the substrate and placed in a growth chamber(12 h photoperiod, 20e22 �C, 50% relative humidity) in flasks withdistilled water. The roots were protected from light by covering theflasks in aluminum foil. During 12 days, the exudates were collectedon a daily basis (i.e. every 24 h), and the empty flasks were dailyrefilled with distilled water. Every four days a new batch of controland mycorrhizal plants was used for exudates collection. Nobacterial growth was observed inside the flasks. After collection,the exudates were immediately pooled per treatment and after pHcheck 30 ml of each solutionwas applied to the soil substrate of the

Table 1Penetration of Radopholus similis nematodes up to 12 days after inoculation(Pi¼ 200), in control and mycorrhizal roots of banana (Musa Grande Naine).

Treatment Time Juveniles Females Males Total

Control plants 1 DAI 6� 4 ab 4� 4 a 0 10� 4 a2 DAI 5� 4 ab 6� 3 ab 1 12� 5 ab4 DAI 10� 3 abc 18� 7 abc 1 29� 6 bc6 DAI 11� 4 abc 15� 2 bc 1 27� 6 c10 DAI 9� 5 ab 22� 6 bc 1 31� 9 c12 DAI 159� 23 c 37� 13 c 5 200� 28 d

Mycorrhizal plants 1 DAI 2� 2 a 2� 2 a 0 4� 3 a2 DAI 2� 2 a 4� 3 ab 0 6� 4 ab4 DAI 5� 3 ab 7� 5 abc 6 14� 5 bc6 DAI 4� 3 ab 12� 4 bc 0 16� 7 c10 DAI 20� 8 bc 17� 8 bc 5 41� 10 c12 DAI 86� 17 c 29� 11 c 5 116� 19 d

P-value (treatment) 0.020 0.029 n.s. 0.001P-value (time) <0.001 <0.001 n.s. <0.001P-value (treatment� time) 0.034 n.s. n.s. n.s.

DAI¼ days after inoculation. n.s.¼ not significant.Data are represented as mean� standard error. A two-way ANOVA was carried outon log(xþ 1) transformed data to identify significant main (treatment, time) and/orinteraction (treatment� time) effects. Within each column, values followed bydifferent letters are significantly different (P� 0.05) according to Tukey HonestSignificant difference test.

C. Vos et al. / Soil Biology & Biochemistry 47 (2012) 60e6662

individual plants according to the different treatments. One hourafter the first application of root exudates or distilled water tothe plants in the greenhouse, they were inoculated with 1000(first experiment) or 200 (second experiment) vermiform R. similis,according to the procedure described above. A higher inoculumdensity was used in the first experiment to compensate for inoc-ulum loss because of possible nematode leaching due to the dailyexudate application. Twelve days after nematode inoculation, thenematodes were extracted from the roots by the maceration-sieving method (Speijer and De Waele, 1997) and the number ofjuveniles, females and males was counted using bright fieldmicroscopy. Frequency (F%) and intensity (I%) of the colonizationin the mycorrhizal plants were estimated as described above(Plenchette and Morel, 1996).

2.4. In vitro chemotaxis bio-assay

The in vitro chemotaxis bio-assay was based on the method-ology described by Wuyts et al. (2006), which was modified fromHewlett et al. (1997). Petri dishes (5-cm diameter) were filled with5 ml modified Strullu Romand medium (Diop, 1995). At oppositesides of the plate 1-cm diameter wells were made using a corkborer. These wells were filled with 100 ml root exudate, rootextract or control solution 3 h prior to the onset of the experimentand incubated at 26 �C in the dark. The root exudates werecollected as described before, and filter sterilized (0.2 mmpore sizefilter, DynaGard syringe filter tips, Microgon Inc., California). Theroot extracts were collected by crushing frozen roots, followed bydissolving the resulting powder in distilled water. Banana plantsgrown for 6 weeks in the greenhouse, with and withoutG. mosseae, were used for the collection of root exudates and rootextracts. Control treatments consisted of distilled water (neutralcontrol), 1% acetic acid (repellent control) and 0.5 M CaCl2(attractive control) added to both holes. Both control and mycor-rhizal root exudates and extracts were tested. Ten R. similisfemales were pipetted onto the medium in the middle of theplate in a minimal volume of water. The nematodes were allowedto move on the medium surface for 1 h while incubated at 26 �Cin the dark. After 1 h, the nematode movement was stoppedby spraying the plate surface with 70% ethanol. Movementtracks were recorded on photographic film as described byWard (1973). A visual assessment of the nematode chemotacticbehavior was made with the three control treatments asreferences.

2.5. Statistical analyses

Nematode infection data of all experiments were statisticallyanalyzed by analysis of variance (ANOVA) when the conditions forANOVA (i.e., normal distribution and homogeneity of variances)were met, using Statistica� (Release 7, Statsoft, Tulsa, USA). TheTukey HSD test was applied for multiple comparisons of groupmeans. Prior to analysis, nematode numbers and penetration rateswere log(xþ 1) or arcsin(x/100) transformed respectively, to reducethe variance in the data (Gomez and Gomez, 1984).

3. Results

3.1. Penetration experiment

A well-established and active mycorrhizal colonization wasobserved, as hyphal structures, arbuscules and vesicles werepresent in the stained roots. Colonization frequency was higherthan 90%, with an average intensity of 31%.

Nematode penetration increased over time, with the largestincrease towards the end of the 12-day period. The nematodepenetration rate (defined as the ratio between the amount ofpenetrated nematodes and the amount of inoculated nematodes)reached 1 in the control treatment 12 days after nematode inocu-lation while it reached only 0.58 in the mycorrhizal roots. Onlya few male R. similis were recovered, while the majority of thepenetrated nematodes consisted of juveniles and to a lesser extentfemales (Table 1).

3.2. Root exudate experiments

As in the penetration experiments, a well-established and activemycorrhizal colonization was observed in both root exudateexperiments, with clear observations of hyphal structures, arbus-cules and vesicles in the roots. Colonization frequency was above90% with an average intensity of 34%. Application of root exudatesto the soil substrate of the plants had no effect on mycorrhizalcolonization.

In the first root exudate experiment, a significantly lowernematode penetration rate (P� 0.05) was observed in the mycor-rhizal roots than in the control roots when both treatments hadreceived distilled water (mycorrhizal plants/H2O vs. control plants/H2O) (Table 2). The highest nematode penetration rate wasobserved in the control roots to which root exudates from controlplants (control plants/ex �AM) had been applied. In none of thetreatments male R. similis were recovered from the roots 12 daysafter inoculation, while in all treatments a small number of femaleswas observed (Table 2). The majority of the penetrated nematodesconsisted of juveniles.

In the second root exudate experiment, the nematode pene-tration rate in the mycorrhizal plants that had received mycorrhizalroot exudates (mycorrhizal plants/ex þAM) was significantly lower(P� 0.05) than in all the other treatments (Table 3). Based on thetwo significant main effects for the two-way ANOVA of the totalnematode population, significantly less R. similis penetratedmycorrhizal roots, or roots that had received mycorrhizal rootexudates (ex þAM) (P� 0.05). In this experiment the majority ofthe R. similis nematodes that penetrated the roots consisted ofjuveniles and females (Table 3). Significantly (P� 0.05) fewerjuveniles had penetrated the roots of plants to which mycorrhizal

Table 2Penetration of Radopholus similis nematodes 12 days after inoculation (Pi¼ 1000) incontrol and mycorrhizal roots of banana (Musa Grande Naine), receiving distilledwater or root exudates from control plants (ex -AM) or from mycorrhizal plants(ex þAM). Experiment 1.

Treatment Juveniles Females Males Total

Control plants/H2O 214� 23 b 11� 4 a 0 225� 22 bMycorrhizal plants/H2O 100� 13 a 6� 2 a 0 106� 13 aControl plants/ex �AM 343� 39 c 17� 4 a 0 360� 37 cControl plants/ex þAM 203� 12 b 13� 3 a 0 216� 12 b

P-value (treatment) 0.000 n.s. n.a. 0.000

n.s.¼ not significant. n.a.¼ not applicable.Data are represented as mean� standard error. A one-way ANOVA was carried outon log(xþ 1) transformed data. Within each column, values followed by differentletters are significantly different (P� 0.05) according to Tukey Honest Significantdifference test.

C. Vos et al. / Soil Biology & Biochemistry 47 (2012) 60e66 63

root exudates (ex þAM) had been added, while significantly lessfemales had penetrated mycorrhizal roots.

3.3. In vitro chemotaxis bio-assay

In the in vitro chemotaxis bio-assay, the nematode tracks showedthat the presence of 1% acetic acid in both wells on the side of thepetri dish had a repellent effect on the nematode. Nematode trackscould only be observed around the inoculation zone in the middle ofthe petri dish, and the nematodes clearly did not approach the aceticacid, since no tracks were observed in the zones near the wells(Fig. 1E). The addition of 0.5 M CaCl2 to the wells resulted inattraction of the nematodes, as can be seen by the nematode tracksfrom the inoculation spot towards the wells (Fig. 1F). Randomnematode movement was observed in the distilled water treatment(Fig. 1G). On the agar plates containing root exudates or extractsfrom mycorrhizal roots a repellent effect on the nematodes wasobserved, similar to the behavior in the presence of acetic acid(Fig. 1A and C). When root exudates or root extracts from controlroots were applied, the nematodes showed a neutral to attractivemovement towards the root exudates and extracts (Fig. 1B and D).

4. Discussion

In the present study we demonstrate that mycorrhiza-inducedresistance in banana acts already at the pre-infectional level ofR. similis infection, and comprises a water soluble compound of themycorrhizal root exudates that negatively impacts nematode host

Table 3Penetration of Radopholus similis nematodes 12 days after inoculation (Pi¼ 200) incontrol and mycorrhizal roots of banana (Musa Grande Naine), receiving distilledwater or root exudates from control plants (ex �AM) or from mycorrhizal plants(ex þAM). Experiment 2.

Treatment Juveniles Females Males Total

Control plants/H2O 36� 10 b 16� 8 b 0 52� 11 bMycorrhizal plants/H2O 20� 8 b 8� 3 a 0 28� 5 b

Control plants/ex �AM 21� 5 b 27� 5 b 0 48� 8 bMycorrhizal plants/ex �AM 18� 5 b 5� 3 a 3 23� 5 b

Control plants/ex þAM 16� 6 a 23� 10 b 0 39� 8 bMycorrhizal plants/ex þAM 3� 3 a 2� 2 a 0 5� 3 a

P-value (plant) n.s. 0.014 n.a. <0.001P-value (exudates) 0.006 n.s. n.a. <0.001P-value (plant� exudates) n.s. n.s. n.a. 0.001

n.s.¼ not significant. n.a.¼ not applicable.Data are represented as mean� standard error. A two-way ANOVA was carried outon log(xþ 1) transformed data to identify significant main (treatment, time) and/orinteraction (treatment� time) effects. Within each column, values followed bydifferent letters are significantly different (P� 0.05) according to Tukey HonestSignificant difference test.

finding behavior and is at least partially responsible for thesubsequent suppression of nematode penetration. The effect wasmost pronounced on juveniles, since they constituted the mostabundant life stage in the inoculum and are known to be the mostactive food seekers, resulting in a higher penetration rate comparedto females (Wyss, 2002). Fewer females will develop inside theroots as a consequence of the lower juvenile penetration. Due totheir lower penetration, the impact of mycorrhizal colonization andexudation on the female nematodes could be observed morereadily in the in vitro chemotaxis bio-assay, with the females beingattracted to non-mycorrhizal exudates, but not to mycorrhizalexudates. The males are present only as a minority in the inoculumand possess a degenerative stylet, resulting in sporadic penetrationonly, probably through penetration sites created by the other lifestages (Hunt et al., 2005).

Harrier andWatson (2004) and Gianinazzi et al. (2010) reviewedreports about the protection against nematodes that AM fungi canoffer their host plants. In banana, the bioprotective effect of AMfungi has been reported previously for migratory endoparasiticnematodes such as P. coffeae (Umesh et al., 1988), Pratylenchusgoodeyi (Jaizme-Vega and Pinochet, 1997) and R. similis (Elsen et al.,2003a,b), in which case it was shown to be systemically induced(Elsen et al., 2008).

But so far, few attempts had been made towards explaining themode of action of the observed mycorrhiza-induced resistanceagainst plant-parasitic nematodes. The influence of AMF on the otherfacets of migratory nematode behavior like migration, feeding andreproduction has not yet been investigated. Hol and Cook (2005)hypothesized some mechanisms and discussed their plausibility.AM fungi can enhance nutrient uptake, especially of phosphorous,and thus increase vitality of theplant. Increased root growth is indeedoften observed for mycorrhizal roots and might increase planttolerance towards nematode infection (Pinochet et al., 1996).However, extra phosphorous addition to plants did not result in thesame nematode suppressive effect as colonization by AM fungi(Azcon-Aguilar and Barea, 1996), indicating that other mechanismsplay a role. Mycorrhizal colonization can also lead to increased rootbranching, which is suggested to negatively impact nematodeinfection and in addition counterbalances the suppressed rootbranching caused by the nematodes (Stoffelen et al., 2000). Since thebioprotective effect has also been reported to be systemicallyinduced, a direct impact of the AM fungi on the nematodes can beexcluded (Elsen et al., 2008). To our knowledge, so far only one studylooked at the possible molecular basis of the mycorrhiza-inducedresistance against plant-parasitic nematodes. Li et al. (2006) re-ported the primed transcriptional activation of a class III chitinasegene in mycorrhizal grapevine roots upon infection by sedentaryendoparasitic root-knot nematodes. However, in viewof the differentinfection cycles of sedentary and migratory nematodes, it would notbe surprising that differentmechanismsmight be responsible for thesameeffect, asWhipps (2004) already suggested for other pathogens.

The hypothesis that nematodes respond differentially towardsroot exudates from mycorrhizal plants has been investigated herefor the first time. Application of mycorrhizal exudates led to thesuppression of nematode penetration, especially of the juveniles, inboth exudate experiments. A higher nematode inoculum densitywas used in the first experiment to counteract possible nematodeleaching due to daily exudate application, but this seemed unnec-essary and penetration rates were similar in both exudate experi-ments. The symbiosis with AM fungi influences host metabolism,and leads to both quantitative and qualitative differences in rootexudation (Jones et al., 2004), which might in turn lead to differ-ential attraction of nematodes that rely on root exudates for hostrecognition (Wuyts et al., 2006). Differences in root exudatecomposition reported previously include amino acids and organic

Fig. 1. In vitro chemotaxis bio-assay with visualization of nematode tracks on agar medium. Pictures were taken 1 h after inoculation with 10 female Radopholus similis nematodesin the middle of the plate. Three hours before nematode inoculation the opposite wells in the agar medium were filled with exudates of mycorrhizal roots (A), exudates ofcontrol roots (B), extracts of mycorrhizal roots (C), extracts of control roots (D), 1% acetic acid as repellent control (E), 0.5M CaCl2 as attractive control (F) or distilled water as neutralcontrol (G).

C. Vos et al. / Soil Biology & Biochemistry 47 (2012) 60e6664

acids (Sood, 2003; Harrier and Watson, 2004, Lioussanne et al.,2008), and increased concentrations of phenolic compounds havealso been reported (McArthur and Knowles, 1992). It has also beensuggested that the change in root exudate composition of mycor-rhizal plants has an effect on the microbial rhizosphere population(Marschner and Baumann, 2003) which might in turn have aneffect on the soil nematode population. However, mycorrhiza-induced resistance against R. similis was also observed in a dix-enic in vitro culture (Elsen et al., 2001) which indicates at least thatthere are other mechanisms at work as well. Lioussanne et al.(2010) stated that not the root exudates per se are responsible foran alteration in the mycorrhizosphere, but rather physical orchemical factors associated with the mycelium. The altered rootexudation is also reflected in the systemic autoregulation of themycorrhizal colonization (Pinior et al., 1999; Vierheilig et al., 2003),and it has been proposed that plants might use this mechanism asa preventative measure against further mycorrhizal colonizationand defense against pathogens at the same time (Vierheilig andPiché, 2002; Vierheilig et al., 2008).

The role of root exudates from mycorrhizal plants has beeninvestigated in several plantefungal pathogen interactions withdifferent outcomes. Norman and Hooker (2000) reported dimin-ished Phytophthora fragariae sporulation in the presence ofroot exudates from mycorrhizal strawberries. Fillion et al. (1999)showed the reduction of Fusarium oxysporum conidia germina-tion when subjected to crude extracts from G. intraradices myce-lium, while in tomato Scheffknecht et al. (2006) observeda stimulatory effect on the microconidia germination of F. oxy-sporum. Interestingly, the latter authors noticed that the effectdepended on the degree of mycorrhizal colonization. Lioussanne

et al. (2008) also nuanced that the attraction of Phytophthoranicotianae zoospores towards mycorrhizal root exudates couldshift to repellency, depending on the maturity of the mycorrhizalcolonization. In our experiments we found that mycorrhizal rootexudates, when applied daily onto the soil substrate of controlplants, significantly lowered nematode penetration. For the fungalpathogen P. nicotianae on the other hand, no effect on infection oftomato roots could be found (Lioussanne et al., 2009). This mightbe explained by the fact that nematodes and fungi are verydifferent pathogens, but might also be attributed to differences inthe exudates collection procedure, with root exudates in the lattercase possibly being collected before mycorrhizal colonizationmaturity, or due to the use of membrane filtering which can affectthe exudates composition and bioactivity (Steinkellner et al.,2008).

So far, bioprotection during early nematode infection, as weobserved in our experiments, had only been demonstrated forother biological control agents. For R. similis, systemically reducedpenetration has been reported in banana colonized by the mutu-alistic endophyte F. oxysporum (Vu et al., 2006), and Sikora et al.(2008) demonstrated that the attractiveness of the roots contain-ing the endophyte seemed significantly reduced. However, otherF. oxysporum isolates did not affect the R. similis pre-infectionalphase (Athman et al., 2006, 2007). In the case of root-knot nema-todes, nematode penetration rate was reduced in tomato colonizedby F. oxysporum (Dababat and Sikora, 2007). As in our experiments,the authors observed that root-knot nematodes were less attractedtowards the F. oxysporum colonized roots and also towards theisolated root exudates, supporting the hypothesis that rootexudates can play an important role in nematode bioprotection.

C. Vos et al. / Soil Biology & Biochemistry 47 (2012) 60e66 65

In conclusion, our study demonstrates that mycorrhiza-inducedresistance acts already at the pre-infectional phase of R. similisinfection, reporting for the first time that root exudates originatingfrom a mycorrhizal plant negatively impact nematode host findingbehavior and subsequent root penetration. The root exudateexperiments and the in vitro chemotaxis bio-assay point towardsa reduced attraction of the nematodes to the host root. Identifica-tion of the active exudate components could potentially contributeto more sustainable management of plant-parasitic nematodes.

Acknowledgements

In commemoration of Daphné Van Den Broucke, for herenthusiastic contribution to this work. The present work wassupported by a Postdoctoral Fellowship of the Research Foundatione Flanders (FWO-Vlaanderen) to A. Elsen, a specialization grantfrom the Institute for the Promotion of Innovation through Scienceand Technology in Flanders (IWT-Vlaanderen) to C. Vos and bya VLIR-UDC grant from the Belgian government to F. Mbongo.

References

Athman, S.Y., Dubois, T., Coyne, D., Gold, C.S., Labuschagne, N., Viljoen, A.,2006. Effect of endophytic Fusarium oxysporum on host preference ofRadopholus similis to tissue culture banana plants. Journal of Nematology38, 455e460.

Athman, S.Y., Dubois, T., Coyne, D., Gold, C.S., Labuschagne, N., Viljoen, A., 2007.Effect of endophytic Fusarium oxysporum on root penetration and reproductionof Radopholus similis in tissue culture-derived banana (Musa spp.) plants.Nematology 9, 599e607.

Azcon-Aguilar, C., Barea, J.M., 1996. Arbuscular mycorrhizas and biological control ofsoil-borne plant pathogens - an overview of the mechanisms involved.Mycorrhiza 6, 457e464.

Banerjee, N., De Langhe, E., 1985. A tissue culture technique for rapid clonal prop-agation and storage under minimal growth conditions of Musa (banana andplantain). Plant Cell Reports 4, 351e354.

Dababat, A.A., Sikora, R.A., 2007. Influence of the mutualistic endophyte Fusariumoxysporum 162 onMeloidogyne incognita attraction and invasion. Nematology 9,771e776.

Daniells, J., Jenny, C., Karamura, D., Tomekpe, K., 2001. Musalogue: a catalogue ofMusa germplasm. Diversity in the genus Musa. The International Network forthe Improvement of Banana and Plantain, Montpellier, France.

Declerck, S., Risède, J.M., Rufyikiri, G., Delvaux, B., 2002. Effects of arbuscularmycorrhizal fungi on severity of root rot of bananas caused by Cylindrocladiumspathiphylli. Plant Pathology 51, 109e115.

De Waele, D., Elsen, A., 2007. Challenges in tropical plant nematology. AnnualReview of Phytopathology 45, 457e485.

Diop, T.A., 1995. Ecophysiologie des champignons mycorhiziens à arbuscules ass-ociés à Acacia albida dans les zones Sahéliennes et Soudano-Guinéennes duSénégal. PhD thesis, University of Angers, France.

Elsen, A., Baimey, H., Swennen, R., De Waele, D., 2003a. Relative mycorrhizaldependency and mycorrhiza-nematode interaction in banana cultivars (Musaspp.) differing in nematode susceptibility. Plant and Soil 256, 303e313.

Elsen, A., Beeterens, R., Swennen, R., 2003b. Effects of an arbuscular mycorrhizalfungus and two plant-parasitic nematodes on Musa genotypes differing in rootmorphology. Biology and Fertility of Soils 38, 367e376.

Elsen, A., Declerck, S., De Waele, D., 2001. Effects of Glomus intraradices on thereproduction of the burrowing nematode (Radopholus similis) in dixenicculture. Mycorrhiza 11, 49e51.

Elsen, A., Gervacio, D., Swennen, R., De Waele, D., 2008. AMF-induced biocontrolagainst plant parasitic nematodes in Musa sp.: a systemic effect. Mycorrhiza 18,251e256.

Fallas, G.A., Sarah, J.L., Fargette, M., 1995. Reproductive fitness and pathogenicity ofeight Radopholus similis isolates on banana plants (Musa AAA cv. Poyo). Nem-atropica 25, 135e141.

Fillion, M., St-Arnaud, M., Fortin, J.A., 1999. Direct interaction between the arbus-cular mycorrhizal fungus Glomus intraradices and different rhizosphere micro-organisms. New Phytologist 141, 525e533.

Gianinazzi, S., Gollotte, A., Binet, M.N., van Tuinen, D., Redecker, D., Wipf, D., 2010.Agroecology: the key role of arbuscular mycorrhizas in ecosystem services.Mycorrhiza 20, 519e530.

Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research,second ed. John Wiley and sons, New York.

Harrier, L.A., Watson, C.A., 2004. The potential role of arbuscular mycorrhizal (AM)fungi in the bioprotection of plants against soil-borne pathogens in organicand/or other sustainable farming systems. Pest Management Science 60,149e157.

Hewlett, T.E., Hewlett, E.M., Dickson, D.W., 1997. Response of Meloidogyne spp.,Heterodera glycines, and Radopholus similis to tannic acid. Journal of Nematology29, 737e741.

Hol, W.H.G., Cook, R., 2005. An overview of arbuscular mycorrhizal fungi-nematodeinteractions. Basic and Applied Ecology 6, 489e503.

Hunt, D.J., Luc, M., Manzanilla-Lopez, R.H., 2005. Identification, morphology andbiology of plant parasitic nematodes. In: Luc, M., Sikora, R.A., Bridge, J. (Eds.),Plant Parasitic Nematodes in Tropical and Subtropical Agriculture, second ed.CAB International, Wallingford, pp. 11e51.

Jaizme-Vega, M.C., Hernandez, B.S., Hernandez, J.M.H., 1998. Interaction of arbus-cular mycorrhizal fungi and the soil pathogen Fusarium oxysporum f. sp. cubenseon the first stages of micropropagated Grande Naine banana. Acta Horticulturae490, 285e295.

Jaizme-Vega, M.C., Pinochet, J., 1997. Growth reponse of banana to three mycor-rhizal fungi in Pratylenchus goodeyi infested soil. Nematropica 5, 213e217.

Jones, D.L., Hodge, A., Kuzyakov, Y., 2004. Plant and mycorrhizal regulation ofrhizodeposition. New Phytologist 163, 459e480.

Li, H.Y., Yang, G.D., Shu, H.R., Yang, Y.T., Ye, B.X., Nishida, I., Zheng, C.C., 2006.Colonization by the arbuscular mycorrhizal fungus Glomus versiforme inducesa defense response against the root-knot nematode Meloidogyne incognita ingrapevine (Vitis amurensis Rupr.), which includes transcriptional activation ofthe class III chitinase gene VCH3. Plant and Cell Physiology 47, 154e163.

Lioussanne, L., Jolicoeur, M., St-Arnaud, M., 2008. Mycorrhizal colonization withGlomus intraradices and development stage of transformed tomato rootssignificantly modify the chemotactic response of the pathogen Phytophthoranicotianae. Soil Biology and Biochemistry 40, 2217e2224.

Lioussanne, L., Jolicoeur, M., St-Arnaud, M., 2009. Role of the modification in rootexudation induced by arbuscular mycorrhizal colonization on the intraradicalgrowth of Phytophthora nicotianae in tomato. Mycorrhiza 19, 443e448.

Lioussanne, L., Perreault, F., Jolicoeur, M., St-Arnaud, M., 2010. The bacterialcommunity of tomato rhizosphere is modified by inoculation with arbuscularmycorrhizal fungi but unaffected by soil enrichment with mycorrhizalroot exudates or inoculation with Phytophthora nicotianae. Soil Biology andBiochemistry 42, 473e483.

Marschner, P., Baumann, K., 2003. Changes in bacterial community structureinduced by mycorrhizal colonization in split-root maize. Plant and Soil 251,279e289.

McArthur, D.A.J., Knowles, N.R., 1992. Resistance responses of potato to vesicular-arbuscular mycorrhizal fungi under varying abiotic phosphorus levels. PlantPhysiology 100, 341e351.

Norman, J.R., Hooker, J.E., 2000. Sporulation of Phytophthora fragariae shows greaterstimulation by exudates of non-mycorrhizal than by mycorrhizal strawberryroots. Mycological Research 104, 1069e1073.

Pinior, A., Wyss, U., Piché, Y., Vierheilig, H., 1999. Plants colonized by AM fungiregulate further root colonization by AM fungi through altered root exudation.Canadian Journal of Botany 77, 891e897.

Pinochet, J., Calvet, C., Camprubi, A., Fernandez, C., 1996. Interactions betweenmigratory endoparasitic nematodes and arbuscular mycorrhizal fungi inperennial crops: a review. Plant and Soil 185, 183e190.

Pinochet, J., Fernandez, C., Sarah, J.L., 1995. Influence of temperature on in vitroreproduction of Pratylenchus coffeae, P. goodeyi and Radopholus similis. Funda-mental and Applied Nematology 18, 391e392.

Plenchette, C., Morel, C., 1996. External phosphorus requirements of mycorrhizaland non-mycorrhizal barley and soybean plants. Biology and Fertility of Soils 21,303e308.

Pozo, M., Azcon-Aguilar, C., 2007. Unravelling mycorrhiza-induced resistance.Current Opinion in Plant Biology 10, 393e398.

Sarah, J.L., Pinochet, J., Stanton, J., 1996. The burrowing nematode of bananas,Radopholus similis Cobb, 1913. Musa pest fact sheet no. 1, INIBAP, Montpellier.

Scheffknecht, S., Mammerler, R., Steinkellner, S., Vierheilig, H., 2006. Root exudatesof mycorrhizal tomato plants exhibit a different effect on microconidia germi-nation of Fusarium oxysporum f. sp. lycopersici than root exudates from non-mycorrhizal tomato plants. Mycorrhiza 16, 365e370.

Sikora, R.A., Pocasangre, L., Zum Felde, A., Niere, B., Vu, T., Dababat, A.A., 2008.Mutualistic endophytic fungi and in-planta suppressiveness to plant parasiticnematodes. Biological Control 46, 15e23.

Sood, S.G., 2003. Chemotactic response of plant-growth-promoting rhizobacteriatowards roots of vesicular-arbuscular mycorrhizal tomato plants. FEMS Micro-biology and Ecology 45, 219e227.

Speijer, P.R., De Waele, D., 1997. Screening of Musa germplasm for resistanceand tolerance to nematodes. INIBAP Technical guidelines, nr. 1. INIBAP,Montpellier.

Speijer, P.R., Kajumba, C., 2000. Yield loss from plant parasitic nematodes in EastAfrican highland banana (Musa spp., AAA). In: Craenen, K., Ortiz, R.,Karamura, E.B., Vuylsteke, D.R. (Eds.), Proceedings of the First InternationalConference on Banana and Plantain for Africa. Kampala, Uganda, 14e18 October,1996. Acta Horticulturae 540, 453e459.

Steinkellner, S., Mammerler, R., Vierheilig, H., 2008. Effects of membrane filteringof tomato root exudates on conidial germination of Fusarium oxysporumf. sp. lycopersici. Journal of Phytopathology 156, 489e492.

Stoffelen, R., Verlinden, R., Xuyen, N.T., Swennen, R., De Waele, D., 2000. Host plantresponse of Eumusa and Australimusa bananas (Musa spp.) to migratory endo-parasitic and root-knot nematodes. Nematology 2, 896e907.

Umesh, K.C., Krishnappa, K., Bagyaraj, D.J., 1988. Interaction of burrowing nematode,Radopholus similis (Cobb, 1893) Thorne 1949, and VA mycorrhiza, Glomus

C. Vos et al. / Soil Biology & Biochemistry 47 (2012) 60e6666

fasciculatum (Thaxt.) Gerd. and Trappe in banana (Musa acuminata colla.).Indian Journal of Nematology 18, 6e11.

Vierheilig, H., Coughlan, A.P., Wyss, U., Piché, Y., 1998. Ink and vinegar, a simplestaining technique for arbuscular mycorrhizal fungi. Applied and Environ-mental Microbiology 64, 5004e5007.

Vierheilig, H., Lerat, S., Piché, Y., 2003. Systemic inhibition of arbuscular mycorrhizadevelopment by root exudates of cucumber plants colonized by Glomus mos-seae. Mycorrhiza 13, 167e170.

Vierheilig, H., Piché, Y., 2002. Signalling in arbuscular mycorrhiza: facts andhypotheses. Advances in Experimental Medicine and Biology 505, 23e29.

Vierheilig, H., Steinkellner, S., Khaosaad, T., Garcia-Garrido, G.M., 2008. The biocontroleffect of mycorrhization on soilborne fungal pathogens and the autoregulation ofAM symbiosis: one mechanism, two effects? In: Varma, A. (Ed.), Mycorrhiza:Genetics and Molecular Biology e Eco-Function e Biotechnology e Eco-physiology e Structure and Systematics. Springer-Verlag, Berlin, pp. 307e320.

Vu, T., Hauschild, R., Sikora, R.A., 2006. Fusarium oxysporum endophytes inducedsystemic resistance against Radopholus similis on banana. Nematology 8,847e852.

Ward, S., 1973. Chemotaxis by the nematode Caenorhabditis elegans: identificationof attractants and analysis of the response by use of mutants. Proceedingsof the National Academy of Sciences of the United States of America 70,817e821.

Whipps, J.M., 2004. Prospects and limitations for mycorrhizas in biocontrol of rootpathogens. Canadian Journal of Botany 82, 1198e1227.

Wuyts, N., Zin Thu Zar, M., Swennen, R., De Waele, D., 2006. Banana rhizodeposi-tion: characterization of root border cell production and effects on chemotaxisand motility of the parasitic nematode Radopholus similis. Plant and Soil 283,217e228.

Wyss, U., 2002. Feeding behaviour of plant-parasitic nematodes. In: Lee, D.L. (Ed.),The Biology of Nematodes. Taylor and Francis, London, pp. 233e259.