Effect of small lipophilic molecules in tomato and rice root exudates on the behaviour of Meloidogyne incognita and M. graminicola

Nematology, 2012, Vol. 14(3), 309-320

Effect of small lipophilic molecules in tomato and rice rootexudates on the behaviour of Meloidogyne incognita

and M. graminicola

Tushar K. DUTTA 1,2, Stephen J. POWERS 1, Hari S. GAUR 2,Michael BIRKETT 1 and Rosane H.C. CURTIS 1,∗

1 Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK2 Indian Agricultural Research Institute, New Delhi-110 012, India

Received: 8 February 2011; revised: 15 July 2011Accepted for publication: 21 July 2011; available online: 30 January 2012

Summary – Plant chemicals in the rhizosphere originating from root exudates or sites of previous nematode penetration can influencenematode behaviour, and a number of plant compounds, some present in root exudates, have been shown either to attract nematodes tothe roots, or to result in repellence, motility inhibition, or even death. The present work was conducted to isolate small lipophilicmolecules (SLMs) emitted by root exudates of Solanum lycopersicum and Oryza sativa to investigate their effect on root-knotnematodes. SLMs extracted, through solid phase extraction, from hydroponically collected root exudates of 40 tomato and rice plantshad an inhibitory impact on the motility of second-stage juveniles of Meloidogyne incognita and M. graminicola and showed anematotoxic or nematostatic (upon dilution) effect on both species. The semiochemicals present in the SLMs induced a very small,albeit statistically significant, effect on stylet thrusting. A small quantity of salivary secretion around the stylet tip and a significantdecrease in nematode head movement were observed. The semiochemicals negatively influenced behaviour of M. incognita and M.graminicola by strongly affecting their mobility. Therefore, it is proposed that SLMs present in both tomato and rice root exudates playimportant roles during the interaction of Meloidogyne spp. with their host plant, and that they might exert a repellent, or allellopathic,effect on these nematodes.

Keywords – motility, root-knot nematode, semiochemicals, stylet thrusting.

Root-knot nematodes are major, economically impor-tant, pests of cereals, as well as dicotyledonous crops,in many countries. Meloidogyne graminicola Golden &Birchfield causes widespread yield losses in rice, wheatand several other graminaceous plants (MacGowan, 1989;Gaur et al., 1993) and can also infect certain dicotyle-donous plants (MacGowan & Langdon, 1989; Sabir &Gaur, 2005). Meloidogyne incognita (Kofoid & White)Chitwood is a serious pest of dicotyledonous crops, al-though it occasionally infects cereals. The underlyingmechanism for differences in host preferences of the twogroups of Meloidogyne, i.e., one preferring cereals and theother preferring dicotyledonous plants, has not been elu-cidated.

Parasites suffer greater mortality during the transmis-sion phase of their life cycles. Sedentary plant-parasiticnematodes, such as Meloidogyne spp., have co-evolved

∗ Corresponding author, e-mail: [email protected]

with their hosts to develop mechanisms that optimise thechances of successful root invasion. Infective nematodestages rely on responses to plant signals originating fromroot exudates or sites of previous nematode penetration tofind a host in the soil. When a root is encountered, its sur-face is explored for a suitable penetration site. Nematodeshave the ability to chemo-orientate using a combinationof head and tail chemosensory organs to compare, simul-taneously, the intensities of a stimulus at each end of theirbodies (reviewed in Curtis et al., 2009).

The infective second-stage juveniles (J2) do not feedduring their migration in soil and roots, but rely on storedlipid reserves to provide the energy for their movement.Plant signals are therefore essential for nematodes to lo-cate hosts and feeding sites before these reserves areoverly depleted; nematodes with >60% of their lipid re-serves depleted are no longer capable of directed move-

© Koninklijke Brill NV, Leiden, 2012 DOI:10.1163/156854111X612306Also available online - www.brill.nl/nemy 309

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ment (Robinson et al., 1987). However, the particularplant stimuli involved in key stages of the plant-nematodeinteraction have not yet been clearly identified.

Plant chemicals originating from root exudates orsites of previous penetration can influence nematodebehaviour, and a number of plant compounds, somepresent in root exudates, have been shown either toattract nematodes to the roots or to result in repellence,motility inhibition, or even death (Rao et al., 1996; Zhao,1999; Zhao et al., 2000; Wuyts et al., 2006; Curtis etal., 2009). A combination of signals emanating fromdifferent areas of the roots affects nematode behaviourin a given plant-nematode interaction (Prot, 1980). J2 ofMeloidogyne spp. are attracted to the zone of elongationin growing root tips and display characteristic nematodeexploratory behaviour at the root surface, including styletthrusting, release of secretions in preparation for rootpenetration, aggregation and an increase in mobility (vonMende, 1997). This exploratory behaviour was inducedin vitro by compounds present in root exudates, and anumber of plant compounds, such as catechol and caffeicacid, induced nematode stylet thrusting and production ofsecretions (McClure & von Mende, 1987; Grundler et al.,1991; Robinson, 2004; Curtis, 2007). Neurotransmitters,such as serotonin and resorcinol, have also been shown toinduce stylet thrusting and an increase in motility of J2 ofMeloidogyne spp. (McClure & Von Mende, 1987). Indoleacetic acid, in vitro, induced the production of secretionsfrom Globodera pallida and M. incognita (Duncan et al.,1995; Curtis et al., 2006). Host cues from root exudatesof solanaceous plants have been shown to be responsiblefor increasing the lipophilicity of the cuticle surface ofJ2 of Globodera spp., leading to increased attractiontowards those plants (Akhkha et al., 2004). Quercetin,a flavonoid that inhibits PI3-kinase, blocks the effect ofphytohormones on the nematode cuticle (Akhkha et al.,2002), this compound acting as a repellent and nematodemotility inhibitor (Wuyts et al., 2006).

In general, very little information is available aboutthe behaviour of plant-parasitic nematodes in response tocompounds in roots and the rhizosphere. Plant volatilesare lipophilic molecules that serve various ecologicalroles, and J2 of Meloidogyne spp. could follow the gra-dient formed by different volatile chemical stimuli ema-nating from the host root during penetration (Reynolds etal., 2011), and/or their migration inside the root tissue us-ing amphidial and phasmidial receptors.

Preliminary attraction bioassay studies in our labora-tory have shown that Meloidogyne spp. are attracted dif-

ferentially to good and poor hosts, while no attraction wasobserved for non-host plants (Dutta et al., 2010). We hy-pothesised that either: i) the blend of attractants and repel-lents are different in good and poor hosts, or ii) relativelylong-range attractants, together with shorter-range repel-lents, might affect nematode movement patterns. Witha view to elucidating the chemical ecology of plant-nematode interactions, we chose to analyse the effect oflipophilic low molecular weight compounds present inthe root exudates on nematode behaviour, as these com-pounds have been shown to act as host signals for bene-ficial organisms (Giovannetti et al., 1996; Nagahashi &Douds, 1999; Akiyama & Hayashi, 2006). The aim ofthis study was to provide evidence for a role played bychemical signals in the mediation of these interactions,using; i) solid phase extraction (SPE) designed to iso-late small lipophilic molecules (SLMs) from root exudates(RE) of tomato, Solanum lycopersicum, and rice, Oryzasativa; and ii) stylet thrusting, mobility and motility bioas-says to investigate the impact of SLMs on behaviour ofJ2 of M. incognita and M. graminicola. Identification ofhost-derived semiochemicals that can affect nematode be-haviour could provide a new strategy for nematode con-trol.

Materials and methods

NEMATODES

Meloidogyne incognita (race 1 NCSU) and M. gramini-cola (from Bangladesh) were maintained on tomato(S. lycopersicum cv. Tiny Tim) and rice (Oryza sativa cv.Ballila) in 15 cm diam. pots containing a mix of equalparts of sterilised sand : peat : top soil, held at 27°C, 30-65% relative humidity (RH) and 16 h : 8 h light : dark pho-toperiod in a glasshouse. Egg masses were handpickedusing sterilised forceps from carefully washed roots of8-week-old plants and kept in a piece of 10 μm porouscloth supported on Miracloth (Calbiochem, Nottingham,UK) held by two plastic rings in a flat-bottomed evapo-rating dish containing distilled water (Hooper, 1986), andincubated at 27°C. Freshly hatched J2 were used for allthe experiments.

GERMINATION OF SEEDS

Seeds of tomato (S. lycopersicum cv. Tiny Tim) andrice (O. sativa cv. Ballila) were surface-sterilised in 0.1%sodium hypochlorite for 2 min, then washed three times

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with distilled water and placed in wet filter paper inPetri dishes wrapped in aluminum foil. Five- to 6-day-oldgerminated seedlings were used for hydroponic culture.

HYDROPONIC CULTURES OF TOMATO AND RICE

Murashige and Skoog (MS) basal salt medium (SigmaLife Science, St Louis, MO, USA) was prepared with4.3 g l−1, pH 6, autoclaved, and with Gamborg’s vitaminadded at 1 ml l−1 of medium. Metal sheets (15 × 10 cm)having 40 circular holes and glass dishes (14 × 9 × 4 cm)were autoclaved separately. Forty 5-day-old seedlingsof tomato or rice were put through the holes of eachmetal sheet using sterilised forceps and mounted onto aglass dish (containing 250 ml of the medium) in sucha way that roots were submerged in the medium. Theplants were supported on the metal sheet with adhesivetape. This assembly was maintained in a glasshousechamber at 27°C, 30-65% RH and with a 16 h : 8 hlight : dark photoperiod. Medium containing root exudateswas collected every 3-4 days (on days 4, 7, 10 and 14after the seedlings had been placed into the hydroponicculture), with two pools, one each for tomato and rice,being made. After each collection of root exudates, freshMS medium was added; this frequent change of mediumkept all plants very healthy and no contamination wasobserved. The root exudates were stored at 4°C untilneeded. The 14-day-old plants were frozen in liquidnitrogen and kept at −20°C.

SOLID PHASE EXTRACTION (SPE) OF ROOT

EXUDATES

Tomato and rice root exudates (a total of 900 mlof each, obtained from 40 plants) were collected andfiltered through Whatman No. 1 filter paper to removeparticulate matter. The filtrates were subjected to solidphase extraction (SPE) using octadecasilyl (C18) glassSPE cartridges (Kinesis, St Neots, UK), which werepre-conditioned and washed with HPLC-grade methanol(2 ml) and water (2 ml) respectively. After completionof the SPE, interefence analytes that potentially interferewith the derivatisation process were removed by washingwith HPLC-grade water (2 ml). The SPE extracts obtainedwere eluted with distilled diethyl ether (2 ml) and residualwater was removed from the extracts by adding a smallquantity of anhydrous magnesium sulphate (MgSO4) tothem. The SPE extracts of tomato and rice were then keptin tightly capped vials and stored at −20°C until required.For bioassays using SPE extracts of the root exudates,

half of the extracts (roughly equivalent to 450 ml of rootexudates from 20 plants) were removed and evaporatedcarefully under a gentle stream of nitrogen to dryness,then re-dissolved in 1 ml of 0.01% aqueous ethanolicsolution per sample. In addition to the preparation of SPEextracts of tomato and rice root exudates, an SPE extractof MS hydroponic medium (900 ml) and named MSfraction was also prepared in a similar manner and usedas a control for the bioassays. The respective SPE extractscontaining SLM from approximately 20 plants werenamed tomato SLM (ToSLM) and rice SLM (RiSLM).These samples were used in the bioassays either neat,or diluted using distilled water 1 : 1 (ToSLM and RiSLMsingle dilution), or 1 : 2 (ToSLM and RiSLM doubledilution). J2 were incubated with 10 μl root exudate (RE)(equivalent to root exudates of 0.2 plants) of each solutionfor the stylet thrusting and motility bioassays and J2 wereincubated with 50 μl RE (equivalent to root exudatesof one plant) of each solution for the immobility andmortality bioassays.

STYLET THRUSTING BIOASSAY

The bioassay for monitoring nematode stylet thrustingwas adapted from methodology described by McClureand von Mende (1987), and the neurotransmitters resor-cinol and octopamine were used as controls for this bioas-say. When J2 of Meloidogyne are incubated with oc-topamine, it induces an increase in nematode body move-ment but does not stimulate stylet thrusting. By contrast,resorcinol induces nematode stylet thrusting and produc-tion of secretions but does not affect nematode bodymovement (McClure & von Mende, 1987). Therefore,these neurotransmitters were used as positive and nega-tive controls, respectively, for the stylet thrusting bioas-say. Aliquots (2 μl) of concentrated suspension contain-ing approximately 50 J2 of M. incognita or M. gramini-cola were treated with 10 μl RE of: i) tomato root exu-date SPE extract (ToSLM); ii) rice root exudate SPE ex-tract (RiSLM); iii) 0.01% ethanol (control); iv) distilledwater (control); v) MS medium SPE extract; vi) 0.1% re-sorcinol; or vii) 0.1% octopamine (both Sigma), in 0.5 mlpolyethylene microcentrifuge tubes. In each tube, 10 μlof 0.2% (w/v) Coomassie Brilliant Blue R250 was addedto give a final volume of 22 μl. From each tube, a 10 μlaliquot was then pipetted into a previously formed ringof petroleum jelly (no chemicals added) on a microscopeslide. A coverslip was then quickly applied with slightpressure from forceps to make an airtight seal and tospread the nematode suspension until it made contact with

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the petroleum jelly. There were three replicates for eachtreatment. Observations of frequency of head movementmin−1 along with stylet thrusting were made for ten J2chosen at random per microscope slide (replicate). Thenumber of thrusts in a single period of 60 s for each J2 wascounted (Rolfe & Perry, 2001; Masler et al., 2007), andobservations of the formation of blue stains around thestylet were made after 20 min, 1, 2, 4 and 16 h. The LeicaM205FA (Leica Microsystems, Wetzlar, Germany) micro-scope used was set at 400× magnification with bright-field optics. Slides with Coomassie Blue alone were keptas another control, to check for any effect of this chemi-cal on nematode movement. Photographs were taken us-ing the Leica M205FA microscope and Leica ApplicationSuite software.

MOTILITY BIOASSAY

Assays were developed to assess nematode movementin Petri dishes (55 mm diam.) containing 4 ml of auto-claved agar substitute medium that contained 0.8% Phy-tagel (Sigma) and 0.1% MgSO4 · 7H2O. Aliquots (2 μl)of concentrated suspension containing approximately 100J2 of M. incognita or M. graminicola were incubated with10 μl of: i) ToSLM; ii) RiSLM; iii) 0.01% ethanol (con-trol); iv) distilled water (control); or v) MS fraction (con-trol), in 0.5 ml polyethylene microcentrifuge tubes for 1 h.A 5 μl aliquot containing about 50 J2 was then placed inthe centre of the dish to monitor nematode movement. Thenumbers of J2 were counted in three circular areas of thePetri dish: area 1 (radius 0.5 cm), area 2 (radius 1.5 cmexcluding area 1) and area 3 (radius 2.5 cm excluding ar-eas 1 and 2) (see Fig. 2) at three time points (30, 60 and90 min). There were three replicates for each treatment.Photographs were again taken using a Leica M205FA mi-croscope and Leica Application Suite software.

IMMOBILITY AND MORTALITY BIOASSAY

Assays were developed to assess nematode immobilityand mortality using Costar® 96-well cell culture clusters(Corning Inc., New York, USA). Aliquots of 50 μl of:i) neat ToSLM; ii) single dilution (1 : 1 RE to distilledwater) ToSLM; iii) double dilution (1 : 2 RE to distilledwater) ToSLM; iv) neat RiSLM; v) single dilution (1 : 1)RiSLM; vi) double dilution (1 : 2) RiSLM; vii) 0.01%ethanol (control); viii) distilled water (control); ix) MSfraction (control); or x) 0.2% eugenol (used as the positivecontrol due to its nematotoxic effect on Meloidogyne;R. Curtis, unpubl.) were added to the wells with 5 μlof concentrated suspension containing approximately 50

J2 of M. incognita or M. graminicola. The numbers ofimmobile J2 were counted after 1, 2, 4 and 24 h. After24 h, 150 μl of distilled water was added to each well andthe number of immobile J2 counted after another 24 h;these were considered as dead nematodes. There werethree replicates for each treatment.

STATISTICAL ANALYSIS

For each nematode species separately, data from thestylet thrusting bioassay (means of the ten J2 per replicatefor each treatment) were subjected to analysis of variance(ANOVA) for a completely randomised design to test theeffect of treatments. Similarly, data from immobility as-says (i.e., percentage of immobile J2 after 1, 2, 4 and 24 h)were distributed as Normal, and so were subjected to asplit-plot in time ANOVA, taking account of the replicatesas statistical blocks, and testing the effect of treatments,dilutions, time points and interactions between these fac-tors. Again for each nematode species separately, datafrom the motility bioassay (i.e., counts of nematodes inthe different areas and over time) and the mortality bioas-say (i.e., proportion of immobile J2 after another 24 hfollowing addition of water) were log transformed for re-gression analysis to fit generalized linear models (GLM;McCullagh & Nelder, 1989) assuming a Poisson distri-bution for the motility data and a Binomial distributionfor the mortality data. Change in deviance, which is dis-tributed as Chi-squared, was used to assess the effects oftime, area (i.e., distance as 0, 0.5, 1.5 or 2.5 cm), treatmentand the interactions between these variates and factors forthe motility bioassay, and the effects of treatment, dilutionand their interaction for the mortality bioassay. All theanalyses were done using GenStat® (2009) (12th Edition,Lawes Agricultural Trust (Rothamsted Research), VSNInternational, Hemel Hempstead, UK). Means in relevantmodel terms from ANOVA, and predicted means in rele-vant model terms from GLM, were compared using leastsignificant difference (LSD) values at the 5% (P = 0.05)level of significance.

Results

STYLET THRUSTING BIOASSAY

Depending on the type of test compounds applied, sev-eral reactions were observed for both M. incognita andM. graminicola J2 (Table 1A, B). Nematodes exposedto the neurotransmitter resorcinol started to thrust theirstylets with very slow body movement within 20 min

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Table 1. Mean, n = 3, head movement and stylet movement (± S.E.) induced by various compounds at 4 h, together with standarderror of difference (SED), degrees of freedom (df), and least significant difference (LSD) at the 5% level between means.

Treatment Head movement min−1 Stylet movement min−1

A: Meloidogyne incognitaToSLM 7 ± 0.58 4.67 ± 0.33RiSLM 6.33 ± 0.33 5 ± 0.580.01% ethanol 15 ± 0.58 0distilled water 15.67 ± 0.33 0MS fraction 16.67 ± 0.33 00.1% resorcinol 5.33 ± 0.33 59 ± 0.580.1% octopamine 30 ± 0.58 0SED (14 df), LSD (5%) 0.642, 1.378 0.471, 1.011

B: M. graminicolaToSLM 6.33 ± 0.33 5 ± 0.58RiSLM 7 ± 0.58 5.33 ± 0.330.01% ethanol 16 ± 0.58 0distilled water 15.67 ± 0.33 0MS fraction 15.33 ± 0.33 00.1% resorcinol 6 ± 0.58 59.33 ± 0.670.1% octopamine 30.33 ± 0.88 0SED (14 df), LSD (5%) 0.777, 1.666 0.504, 1.081

of incubation. Stylet thrusting became more regular andcontrolled with a frequency of about 1 thrust s−1. Occa-sionally, massive accumulation of stylet exudates was ob-served around the heads of the treated nematodes (Fig. 1),and after 16 h all the nematodes were dead. By contrast,nematodes treated with octopamine showed faster bodymovement but did not show any sign of stylet thrustingor stylet exudates around the nematode mouth tip. Thesemiochemicals present in the SLMs induced very lit-tle, but statistically significant (P < 0.05; LSD), styletthrusting compared with 0.01% ethanol, water and MSfraction. Compared with resorcinol, ToSLM or RiSLMextracts showed a significant decrease in stylet thrusting(P < 0.05; LSD) for M. incognita and M. graminicolaJ2, and in comparison with 0.01% ethanol, water and MS-fraction there was a significant decrease in the nematodehead movement (P < 0.05; LSD).

All J2 became quiescent or moved slowly within 1 hof exposure to ToSLM or RiSLM extracts, both of whichinduced a weaker, sporadic stylet movement in a fewJ2, with small quantities of salivary secretion around thestylet tip being observed after 16 h (Fig. 1). MS fraction,0.01% ethanol, Coomassie blue and distilled water wereused as controls and did not induce any stylet movementor salivary secretion. For these treatments all J2 movednormally even after 16 h of exposure.

MOTILITY BIOASSAY

The GLM showed that there was a significant inter-action between treatment, distance moved and time forboth M. incognita (χ2 = 37.8; df = 4; P < 0.001)and M. graminicola (χ2 = 28.9; df = 4; P = 0.002).Comparison of predicted means showed that significantly(P < 0.05; LSD) greater number of M. incognita orM. graminicola J2 remained at the centre of Petri dishespre-incubated with ToSLM or RiSLM compared withthose with 0.01% ethanol, water or MS fraction, at 30,60 and 90 min after initiation of the bioassay. Signifi-cantly (P < 0.05; LSD) greater number of J2 of bothMeloidogyne species incubated with ethanol, water or MSfraction had moved to area 2 and area 3 at 60 min com-pared with 30 min, and at 90 min compared with 60 min,while no significant difference (P > 0.05; LSD) amongthe number of J2 remaining at the centre over time was ob-served when incubated with undiluted ToSLM or RiSLM.Although there had been some significant movement toarea 1 by 60 min (P < 0.05; LSD), very few nematodesincubated with either of these two treatments had movedto area 2 at 60 min and none, or hardly any, had movedto area 3 at 90 min. By contrast, approximately 50% ofnematodes incubated with control treatments had movedout of the centre and were distributed in areas 1, 2 or 3 at90 min (Figs 2, 3, 4). Thus, 0.01% ethanol, water or MS

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Fig. 1. Responses of J2 of Meloidogyne graminicola to A: RiSLM at 4 h; B: RiSLM at 16 h; C: 0.1% resorcinol at 4 h; D: 0.1%resorcinol at 16 h. Nematodes treated with RiSLM showed trivial amounts of stylet exudates around stylet tips at 16 h. Occasionally,massive accumulation of stylet exudate was observed at 4 and 16 h for nematodes subjected to resorcinol. S = secretion. This figure ispublished in colour in the online edition of this journal, which can be accessed via http://www.brill.nl/nemy

fraction did not have any effect on the motility of J2 of M.incognita or M. graminicola, while undiluted ToSLM orRiSLM slowed down their movement.

IMMOBILITY AND MORTALITY BIOASSAYS

ANOVA of the percentage data for immobility showedthat there was a significant interaction between treatment(including ToSLM and RiSLM dilutions) and time forboth M. incognita (F = 5.55; df = 6, 60; P < 0.001)and M. graminicola (F = 9.95; df = 6, 60; P <

0.001). Comparison of means showed that percentageimmobile J2 of M. incognita or M. graminicola decreasedsignificantly (P < 0.05; LSD) with each dilution, for bothToSLM and RiSLM, and at each time point (i.e., 1, 2, 4,24 h). Percentage immobility increased over time for bothspecies with undiluted (neat), single dilution (1 : 1) anddouble dilution (1 : 2) of ToSLM and RiSLM. Immobilitywas again assessed after another 24 h, following theaddition of distilled water, at which time immobile J2were termed dead. The GLM of these mortality data

revealed significant main effects of treatment (χ2 =831.6 (M. incognita), 846.3 (M. graminicola); df = 5;P < 0.001) and dilution (χ2 = 674.9 (M. incognita),640.7 (M. graminicola); df = 2; P < 0.001) for bothspecies, indicating that the rate of decrease in mortalitywith increasing dilution was much the same for ToSLMand RiSLM. No change in the number of immobile J2was observed with undiluted ToSLM or RiSLM followingthe additional 24 h, while a proportion of J2 revivedthat had been exposed to single and double dilution ofToSLM and RiSLM (Figs 5, 6). Thus, both ToSLM andRiSLM showed nematotoxic effects when used undiluted,and nematostatic effects when diluted 1 : 1 or 1 : 2. Not asingle J2 was immobilised at any time point when exposedto 0.01% ethanol, distilled water or MS fraction, whilepercentage immobility of J2 treated with 0.2% eugenolreached 100% at only 1 h into the bioassay and nonerevived after another 24 h, following the addition ofdistilled water (data not shown).

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Fig. 2. Meloidogyne incognita J2 pre-incubated with A: ToSLM; B: 0.01% ethanol in 55 mm diam. Petri dishes containing Phytagel90 min after initiation of the bioassay. J2 incubated with ToSLM are confined to the centre and area 1, whilst J2 incubated with0.01% ethanol have moved to areas 1, 2 and 3. Diagram of 55 mm diam. Petri dish divided into different areas, used in the motilitybioassay, is shown in the figures. This figure is published in colour in the online edition of this journal, which can be accessed viahttp://www.brill.nl/nemy

Fig. 3. Results of the motility bioassay using J2 of Meloidogyne incognita in 55 mm diam. Petri dishes containing Phytagel. Predictedmeans are presented, n = 3, given a GLM fitted to observed counts. Significant differences between different treatments within eacharea and time point combination are marked with a different letter, while significant differences within each treatment across time,within each area, are indicated as * (using LSD, P < 0.05). Error bars indicate prediction standard error.

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Fig. 4. Results of the motility bioassay using Meloidogyne graminicola in 55 mm diam. Petri dishes containing Phytagel. Predictedmeans are presented, n = 3, given a GLM fitted to observed counts. Significant differences between different treatments within eacharea and time point combination are marked with a different letter, while significant differences within each treatment across time,within each area, are indicated as * (using LSD, P < 0.05). Error bars indicate prediction standard error.

Fig. 5. Results of the immobility and mortality assay for Meloidogyne incognita. Means, n = 3, are presented following ANOVA forimmobility, and following GLM for mortality. Significant difference between different treatments within each time point are marked witha different letter, while significant differences within each treatment across time points are indicated as * (comparing 2 h with 1 h),** (4 h with 2 h) and *** (24 h with 4 h) (using LSD, P < 0.05). Error bars indicate standard error.

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Fig. 6. Results of the immobility and mortality assay for Meloidogyne graminicola. Means, n = 3, are presented following ANOVAfor immobility, and following GLM for mortality. Significant difference between different treatments within each time point are markedwith a different letter, while significant differences within each treatment across time points are indicated as * (comparing 2 h with 1 h),** (4 h with 2 h) and *** (24 h with 4 h) (using LSD, P < 0.05). Error bars indicate standard error.

Discussion

Above-ground plant/herbivore/carnivore interactions,mediated by plant volatile organic compounds (VOCs),are well studied and these studies show that releasedVOCs attract specific carnivores to plants infested by her-bivores (Bruce et al., 2005; Bruce & Pickett, 2007). Bycontrast, below ground, although plants are capable of re-leasing VOCs in the rhizosphere, the biological signifi-cance of such compounds for plant-nematode interactionshas not been studied in depth. Plant-parasitic nematodesare responsive to many phytochemicals present in rootexudates, and these can act as hatching stimulants, re-pellents, attractants or inhibitors, and some can be toxicto nematodes (Zhao et al., 2000; Curtis et al., 2009). Itis plausible to hypothesise that J2 of Meloidogyne spp.are attracted or influenced by different volatile chemicalstimuli, as well as by other components of root exudates,including non-volatile compounds that emanate from thehost root during penetration and/or their migration insidethe root tissue using amphidial and phasmidial receptors(Curtis et al., 2007).

From our previous work we hypothesised that nema-tode attraction to roots might be a result of the recogni-

tion of a blend of attractants and repellents, which mightbe different in good, poor and non-hosts, and that thesemight affect nematode movement patterns (Dutta et al.,2011). The factors that determine whether the chemicalsignature of plant root exudates will be perceived as anattractive or a repellent cue still require elucidation.

This study showed, for the first time, that SLMs withphysical properties similar to plant VOCs (Ranganathan& Borges, 2009), present in tomato and rice root exudates,have an inhibitory impact on the motility of J2 of M. in-cognita and M. graminicola. They were seen to have anematostatic effect on both Meloidogyne species, they in-duced very little stylet thrusting, and only induced minutequantities of secretions after 16 h of exposure. Thus, wespeculate that SLMs present in both tomato and rice rootexudates might have allellopathic effects on these nema-todes, that they might act as repellents when perceivedfrom a certain distance, and that they have a nematostaticeffect when in contact with the J2. Roles for SLMs assemiochemicals (behaviour-modifying chemicals) that in-fluence the behaviour of invertebrate arthropods in otheraqueous systems have also been discovered in our labora-tory, e.g., mosquito oviposition cues (Carson et al., 2010),and host/non-hosts cues for Lepeophtheirus salmonis, an

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ectoparasite affecting salmonid fish (Mordue & Birkett,2009). Furthermore, strigolactone and other lipophilic lowmolecular weight compounds exuded from plant roots actas host signals for beneficial fungal symbionts and para-sitic weeds (Giovannetti et al., 1996; Nagahashi & Douds,1999; Akiyama & Hayashi, 2006).

Nematodes are influenced by volatile compounds: forinstance, Caenorhabditis elegans, a well-studied and ge-netically tractable experimental system, employschemotaxis-mediated movement in response to bacterialvolatile gradients, which are known to disperse up to afew metres through the soil, to locate pockets of bacteriain the soil (Troemel et al., 1997). In the presence of itsbacterial food, C. elegans moves more slowly and showsa significant decrease in its locomotory rate (Sanyal et al.,2004). It has been postulated that C. elegans responds toa blend of M. truncatula volatiles, including dimethyl sul-phide. The pine wood nematode, Bursaphelenchus xylo-philus, has also been shown to be attracted to volatiles offorest pine trees, such as α-pinene, β-pinene and longi-folene, which are found in the healthy xylem of Pinusmassoniana (Zhao et al., 2007). Rasmann et al. (2005)reported that maize roots release a volatile signal, (E)-caryophyllene, in response to feeding by larvae of thebeetle Diabrotica virgifera virgifera, and that this attractsan entomopathogenic nematode, Heterorhabditis megidis.Certain volatile fatty acids present in soil are toxic to ne-matodes, and formic, acetic, propionic and butyric acid,in their undissociated states, are strongly lipophilic, areable to cross membranes (both bacterial and cellular mem-branes) and can readily immobilise nematodes (Sayre &Starr, 1988).

Plant secondary metabolites play an important role inplant-nematode interactions, and phytochemicals are con-sidered as good biopesticide candidates or can serve asmodel compounds for the development of chemically syn-thesised derivatives with enhanced nematicidal activity.A number of plant compounds have been shown to affectplant nematodes negatively. For instance, in low concen-trations, salicylic acid strongly attracted J2 of M. inco-gnita and inhibited their motility, but at higher concentra-tions it displayed a nematicidal effect and was moderatelyinhibitive to hatching (Wuyts et al., 2006). In our labo-ratory, low concentrations (0.1%) of acetic acid acted asa repellent and at high concentration (1%) was nemato-toxic for J2 of Meloidogyne spp. (R. Curtis, unpubl.). Sim-ilarly, the nematotoxic/nematostatic effect of ToSLM andRiSLM described in this paper was also concentration-dependent. Therefore, once the active compound(s) are

identified it will be important to study the relationship be-tween concentration and nematode behaviour over a timecourse.

Multiple phenolic compounds have been tested fortheir effect on M. incognita by Mahajan et al. (1985,1992). Many were reported to kill nematodes and inhibithatching. The phenylpropanoid medicarpin (isoflavonoid-derivative) inhibited Pratylenchus penetrans motility inan in vitro assay (Balbridge et al., 1998). Tannic acidwas shown to have attractant effects while ferulic acidhad moderately repellent effects on M. incognita (Hewlettet al., 1997). Meloidogyne incognita was sensitive todegraded flavonols after 48 h of incubation (Mahajanet al., 1985). J2 of M. javanica and M. hapla havealso been shown to be positively stimulated by ascorbicacid, gibberellins or glutamic acid (Bird, 1959, 1962).Plant-parasitic nematodes are susceptible to essential oilsof plants (Chitwood, 2002) and, indeed, Meyer et al.(2006) found that some essential oil extracts were toxicto M. incognita eggs and J2. The pentacyclic alkaloid,serpentine, has been found to induce death and inhibithatching of M. incognita (Chandravadana et al., 1994).

Studies of the role and identity of host-derived semio-chemicals for nematodes can help to develop a fundamen-tal understanding of plant-nematode relationships. How-ever, semiochemicals derived from nematode-antagonisticplants have not been identified for exploitation (Chitwood,2002). Our study shows that SLMs present in the root exu-dates of tomato and rice can affect Meloidogyne spp. mo-bility negatively, and the identification and characterisa-tion of the active compound(s) present in these SLMs isunder way in our laboratory. The identification of nema-tode specific repellent/avoidance compounds from plantsis an important area of research that could lead to the de-velopment of plants that are technically ‘invisible’ to thenematodes (stealth plants). Such compounds might playa regulatory role by affecting nematode behaviour in therhizosphere, and could be incorporated into an integratedpest management programme, either by direct applicationas novel agents for nematode control or, alternatively, theproduction of these repellents could be induced, in planta,using natural plant activators, such as cis-jasmone (Birkettet al., 2000; Bruce et al., 2008).

Acknowledgements

This research was supported by a grant from the UK-India Education and Research Initiative (UKIERI) andRothamsted Research, which receives grant-aided support

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Effect of small lipophilic molecules on root-knot nematodes

from the Biotechnology and Biological Sciences ResearchCouncil. TKD receives a Ph.D. studentship from theBritish Council and the Indian Council of AgriculturalResearch. We thank Allison van-de-Meene, Bioimaging,Rothamsted Research, for helping to take some beautifulphotographs. We also thank Prof. Ken Evans for hiscritical evaluation and for editing this manuscript.

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