Soybean Cyst Nematode Effects on Soybean Aphid ... - Gratton Lab

PLANT-INSECT INTERACTIONS

Soybean Cyst Nematode Effects on Soybean Aphid Preference and Performance in the Laboratory S. C. HONG, J. DONALDSON,

AND

C. GRATTON1

Department of Entomology, University of WisconsinÑMadison, 237 Russell Labs, 1630 Linden Drive, Madison, WI 53706

Environ. Entomol. 39(5): 1561Ð1569 (2010); DOI: 10.1603/EN10091

ABSTRACT Herbivores on plants frequently interact via shared resources. Studies that have examined performance of herbivores in the presence of other herbivores, however, have often focused on above-ground feeding guilds and relatively less research has examined interactions between belowand above-ground consumers. We examine how soybean aphid, Aphis glycines (Matsumura) an above-ground phloem-feeding herbivore, interacts with a below-ground plant parasite, soybean cyst nematode, Heterodera glycines (Ichinohe) through their shared host plant, soybean (Glycine max L). Laboratory experiments evaluated the preference of alate (ßight-capable) soybean aphids toward plants either infected with soybean cyst nematode or uninfected controls in a simple choice arena. Alate soybean aphids preferred uninfected soybean over soybean cyst nematode-infected plants: 48 h after the releases of alate aphids in the center of the arena, 67% more aphids were found on control soybean compared with nematode infected plants. No-choice feeding assays were also conducted using clip cages and apterous (ßight-incapable) aphids to investigate effect of soybean cyst nematode infection of soybean on aphid performance. These studies had mixed results: in one set of experiments overall aphid population growth at 7 d was not statistically different between control and soybean cyst nematode-infected plants. A different experiment using a life-table analysis found that apterous aphids feeding on soybean cyst nematode-infected plants had signiÞcantly greater Þnite rate of increase (␭), intrinsic rate of increase (rm), and net reproductive rate (Ro) compared with aphids reared on uninfected (control) soybean plants. We conclude that the below-ground herbivore, soybean cyst nematode, primarily inßuences soybean aphid behavior rather than performance. KEY WORDS above- and below-ground interactions, indirect effects, induced plant responses, root herbivory, phytoparasite

Like many other agricultural Þeld crops, soybean plants (Glycine max [L.] Merr) are exposed to attacks by a variety of pests. For example, soybean aphids (Aphis glycines Matsumura) feed on plant phloem, bean leaf beetles (Cerotoma trifurcata Forster) attack leaves and roots, soybean cyst nematodes (Heterodera glycines Ichinohe) feed on roots, and viruses are intracellular parasites. Soybean aphid and soybean cyst nematode are considered the most serious pest and pathogen in soybean production and proÞtability in the United States (Noel 1992, Wrather 1998, Ragsdale et al. 2004). Since the Þrst report of soybean aphid outbreaks in the upper Midwest of the United States in 2000 (Alleman et al. 2002), soybean aphid, a native to East Asian countries, has become one of the serious economic pests of soybean (Macedo et al. 2003) in North America. In addition, soybean cyst nematode lives in the soil and is an endoparasitic obligate pathogen infecting and reproducing in the roots of soybean as well as pea and common bean and can cause serious yield losses (Wrather 1998). 1

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

Although soybean aphid and soybean cyst nematode share the same host, they are separated spatially in the plant parts they attack and temporally over the course of the season. Soybean aphid occurs aboveground (i.e., on leaves and stems) and feeds on the phloem while soybean cyst nematode occupies belowground parts (i.e., roots) of soybean and feeds intracellularly after degrading cell walls with enzymes (Niblack et al. 2006). In addition, there is temporal asynchrony in the colonization of soybean by soybean cyst nematode and soybean aphid during the growing season. Soybean cyst nematode occurs in the soil and infects soybean roots shortly after germination (Niblack et al. 2006), while soybean aphid colonizes plants in the mid-late summer after plants have established a canopy (Ragsdale et al. 2004, Onstad et al. 2005). This scenario may create the opportunity for the early colonizing soybean cyst nematode to inßuence the performance and/or preference of laterarriving soybean aphid indirectly via herbivore-induced responses in the shared host plant. Herbivore-induced plant responses often occur systemically within a plant (Karban and Baldwin 1997,

0046-225X/10/1561Ð1569$04.00/0 䉷 2010 Entomological Society of America

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ENVIRONMENTAL ENTOMOLOGY

Bezemer and van Dam 2005) creating the possibility that herbivores specializing on different plant parts can inßuence each other. One example of this indirect interaction is between below- and above-ground herbivores (Karban and Baldwin 1997, van der Putten et al. 2001, van Dam et al. 2003, 2005, Bezemer and van Dam 2005). Several studies suggest that soybean respond to root infection by soybean cyst nematode through various changes in secondary metabolites as a result of changes in gene expression (Kim et al. 1987; Mazarei et al. 2002, 2007; Ithal et al. 2007a,b; Jones et al. 2007). These changes may affect performance and/or behavior of other herbivores, especially those secondarily attacking the plant. However, because of its recent arrival to North America, little is currently known of how soybean aphid preference and performance are affected by early infections of soybean by soybean cyst nematode. In this study, we conducted a series of greenhouse experiments to examine soybean aphid preference and performance on soybean plants infected (2 or 3 wk earlier) with soybean cyst nematode. We hypothesized that because of the expected induced changes in plant quality caused by belowground feeding by soybean cyst nematode, soybean aphid preference for and performance on infected soybean plants would be lower compared with uninfected plants. Materials and Methods Aphid and Nematode Colonies Soybean Aphid Colony. Soybean aphid collected from soybean Þelds at the University of WisconsinÕs Arlington Agricultural Research Station (Columbia Co, WI) were maintained on soybean plants (cultivar ÔWilliamsÕ) in a greenhouse. Soybean cultivar ÔWilliamsÕ was used for this study because it is also a susceptible host to soybean cyst nematode (McGinnity et al. 1980). Soybean aphid colonies were maintained in either a cage or a bench top in the greenhouse. New young soybean plants (vegetative stages V1Ð2: one or two fully expanded trifoliates) were replenished to maintain a healthy soybean aphid colony every 2 or 3 wk, depending on plant quality and age of plants (reproductive stages ⱖR1 were removed from the colony). Plants were maintained in a greenhouse at 18 Ð 25⬚C under a 16:8 h L:D photoperiod and fertilized with 20:20:20 (N:P:K) weekly. H. glycines Inoculum. Mass production of soybean cyst nematode was carried out to obtain inoculum for preference and performance assays. Soybean cyst nematode lives in the soil and is an endoparasitic obligate pathogen infecting and reproducing in the roots of soybean as well as pea and common bean. Eggs hatch in response to plant-derived exudates and the second-stage juvenile (J2) is infectious, attacks roots, and migrates intracellularly to vascular tissues where the feeding is initiated. Gravid females develop 50 to several hundred eggs within their bodies that remain partially exposed to the external soil matrix. After the

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female dies, she becomes a leathery and spherical cyst within which the eggs remain and are later released into the soil (Niblack et al. 2006). Initial inoculum of soybean cyst nematode eggs was obtained from cysts collected from soil in soybean Þelds with history of nematode infection in central Wisconsin (Portage and Adams Co.). Soybean cyst nematode cysts were extracted from soils using a slightly modiÞed procedure of Jenkins (1964) with a sucrose density ßoatation method and sieving through screens (32Ð74 ␮m) to collect nematode cysts. Soybean nematode cysts then were gently crushed (Faghihi and Ferris 2000) to release eggs. A subsample of eggs was counted under a dissecting microscope to determine egg density (number of eggs per milliliter). Soybean plants used for soybean cyst nematode inoculum were grown in a soil mixture consisting of Þeld soil, sand, and vermiculite (1:1:2 ratio). The soil mixture was pasteurized in the autoclave at 80⬚C for 30 min. Soybean seeds were germinated on a moistened paper towel until the radicle of soybean seeds was ⬇2Ð3 cm long. Germinated seeds were placed in holes of the soil mixture in a 7.6 cm2 pot. Approximately 3,300 soybean cyst nematode eggs suspended in water were carefully dispensed into the hole with a germinated seed by a syringe and the hole was covered with soil. Plants were maintained in an aphid-free greenhouse under growing conditions described previously. A similar procedure as described above was followed to prepare inoculum for laboratory experiments with soybean aphid. At ⬇50 d after inoculation, soybean cyst nematode cysts were harvested from the soybean plant roots using slightly modiÞed procedure of Jenkins (1964). Cysts were crushed to recover eggs using the method used by Faghihi and Ferris (2000) and extracted eggs were used to inoculate soybean seedlings for laboratory assays within 24 h after extraction. Soybean Aphid Preference Experiments Laboratory experiments were conducted to determine if the effect of prior infection by soybean cyst nematode of soybean affected soybean aphid preference and performance. Soybean plants used for all aphid preference and performance experiments were between vegetative stage two (V2) and three (V3), that is, three fully expanded trifoliates, ⬇21 d postnematode infection. The method of soybean cyst nematode inoculation and harvest was identical as described in H. glycine inoculum section unless otherwise noted. Soybean cyst nematode egg densities for inoculations varied between 10,000 and 30,000 eggs/plant, depending on inoculum availability and the number of replicates. At the end of experiments, the actual soybean cyst nematode egg density in each replicate plot was determined using the same nematode extraction procedure described above. Soybean plants without nematodes were used as controls. Three preference tests were conducted (Exp. I: April 2007, Exp. II: May, 2008, and Exp. III: June 2008)

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HONG ET AL.: SOYBEAN APHID AND SOYBEAN CYST NEMATODE INTERACTIONS

in the laboratory with soybean plants with initial soybean cyst nematode egg densities of ⬇20,000, 20,000, and 30,000 eggs/plant, respectively. In other studies, average soybean cyst nematode densities that they surveyed varied from 1,700 to 33,600 eggs/100 cm3 depending on soybean maturity group and location (Riggs et al. 2000, Wang et al. 2000, Mitchum et al. 2007). At the end of the experiment, soybean cyst nematode infected plants had 294 cysts ⫾ 34.5/pot (mean ⫾ SE), while control plants never had any nematode cysts. Two plants (i.e., soybean cyst nematode infected and control plants), were placed in an experimental arena with their positions in the cage randomly assigned. Experimental arenas consisted of large Plexiglas cages (34 ⫻ 55 ⫻ 55 cm, L ⫻ W ⫻ H). Alate (ßight-capable) soybean aphids were collected from a colony cage. In Exp. I, a petri dish containing 25 alate aphids was placed at the height of base of the plants (⬃10 cm) equidistant from the two test plants. After 24 h, the number of alate aphids on each plant was counted. This study was conducted on two consecutive days with Þve replications per day. In Exps. II and III, alatae collected from the colony were starved for ⬇8 h before releasing them into the arena as in Exp. I. Twenty and 30 aphids were released into the Plexiglas cage for Exp. II and Exp. III, respectively, with 10 replications. Number of alate aphids attached on each plant was counted at 16, 24, 40, and 48 h after release. All assays were conducted in the greenhouse under 95% shade cloth to minimize aphid orientation to artiÞcial lights in the greenhouse. The analysis of data collected in 2007 (i.e., Exp. I) was conducted separately from those obtained from Exp. II and III. Counts of soybean aphids in preference experiments were analyzed by Þtting aphid counts using a Poisson generalized linear mixed model (GLMM) with log-link function (PROC GLIMMIX, SAS Institute 2002). GLMM allows for a Poisson regression (aphid counts) with the inclusion of random effects in the predictor (McCulloch and Searle 2001). For Exp. II and III (2008), a Poisson generalized linear mixed model with repeated measures was Þtted to the data because observations were made four times over the 48 h duration of the experiment. Data analysis for Exp. II and III was conducted on pooled data. The Þxed effects were treatment and time. The random effects were experimental trials, block (cage), sampling error, and plot error. Compound symmetry (CS) was used for the within-subject covariance structure. Pairwise mean comparisons among treatment and time effects were made using least-squares means (LSMEANS) method. Soybean Aphid Performance on Soybean Cyst Nematode-Infected Plants Clip-Cage Experiments. Performance studies were conducted in 2008 (May, June, July; Exp. IV, V, VI, respectively). Soybean plants (soybean cyst nematode-infected and controls) at the start of these experiments had three fully expanded trifoliates (i.e., V3

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growth stage). Replication varied among experiments for control and soybean cyst nematode, respectively (n ⫽ 10, 10 for Exp. IV, n ⫽ 6, 9 for Exp. V, and n ⫽ 19, 16 for Exp. VI). Harvests of plants at the end of the experiment found that soybean cyst nematode infected plants had 734 ⫾ 133 cysts/pot below-ground, while control plants never had any nematodes. A single neonate aphid collected from the lab colony was placed inside a clip cage attached to the uppermost trifoliate on a test plant. Clip cages were made from petri dish of 50 mm diameter ⫻ 9 mm (Benton Dickinson and Co., Franklin Lakes, NJ) as described in Donaldson and Gratton (2007). Aphids were monitored daily until they molted into adults. Neonates that molted into alatae (with wings) were excluded from the analysis because these have different fecundity than apterae. New aphid offspring in each cage were recorded 7 d after the Þrst production of offspring. Mean difference in aphid fecundity over the 1 wk period between two treatments was examined via a mixed-model analysis of variance (ANOVA) (PROC MIXED, SAS Institute 2002). Life Table Experiment. A life table assay (Exp. VII) was conducted to evaluate the performance of soybean aphid on soybean cyst nematode-infected and control plants using the approach detailed in Myers and Gratton (2006). Brießy, neonate aphids (⬍24 h old) were placed individually into clip cages attached on leaves from the uppermost trifoliates of soybean plants from soybean cyst nematode-infected and control soybean plants (n ⫽ 16 each). Harvests of plants at the end of the experiment found that soybean cyst nematode-infected plants had 1,151 ⫾ 129 cysts/pot below-ground, while no control plants had any nematodes. Because of neonate mortality and occasional development of alate adults (that were excluded from the experiment) rather than apterous adults, Þnal replication was 7 and 13 for control and soybean cyst nematode infected-plants, respectively. Plants with aphids were maintained in the greenhouse under the same conditions as described in the clip-cage experiments above. After neonate aphids molted to adults, survivorship and daily fecundity of each female was examined, with new offspring removed from the clip cage daily. A life table analysis was conducted for the aphid cohorts reared on two treatments (i.e., soybean cyst nematode-infected versus control plants) using the lx (daily survivorship schedule) and mx (average daily fecundity) of aphids on control or soybean cyst nematode-infected plants. The intrinsic rate of increase (rm), net reproductive rate (Ro), and mean generation time (T) were calculated. Randomization tests (Monte Carlo analysis, 1,000 iterations) were conducted using PopTools 3.0 (Hood 2008) to determine if there were differences in life table parameters between two treatments by randomly shufßing replicates between treatments and recalculating life table parameters (Myers and Gratton 2006). Only adult apterous survivorship was used for this analysis. Survival curves of aphids on both treatments were compared using Kaplan-Meier survivorship analysis

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Fig. 1. Alate soybean aphid count on soybean cyst nematode infected and control plants in 2007 (a, 24 h postrelease) and 2008 (b). Pairwise comparisons, *, P ⬍ 0.05; ●, P ⬍ 0.10; NS, P ⬎ 0.1. Vertical bars represent SEM.

(Parmer and Machin 1995) in the R package ÔsurvivalÕ (R Development Core Team 2008). Effect of soybean cyst nematode infection on soybean aphid fecundity was examined by Þtting aphid counts using a Poisson generalized linear model with log-link function (PROC GLIMMIX, SAS Institute 2002). The Þxed effects were treatment and day. The random effects were soybean plant nested within each treatments and plot error. A Þrst-order autoregressive (AR[1]) was used for covariance structure. LSMEANS was used to compare between the two treatments.

24 and 40 h postrelease this effect was marginally signiÞcant. By the end of the experiment (48 h) there were ⬇67% more alate aphids on control plants compared with soybean cyst nematode-infected plants (Fig. 1b; Treatment: F 1, 133 ⫽ 3.89; P ⫽ 0.05; Time: F 3, 133 ⫽ 13.22; P ⬍ 0.0001; Treatment ⫻ Time: F 3,133 ⫽ 0.35; P ⫽ 0.79).

Results

Clip-Cage Experiments. The results of three aphid performance studies varied (Fig. 2) with differences in aphid performance among treatments in only one of the three clip cage experiments. In Exp. IV and V (Fig. 2), there were no signiÞcant differences in mean percapita aphid increase between control and soybean cyst nematode-infected plants after 7 d (Exp. IV: F1, 38 ⫽ 0.33; P ⫽ 0.57; Exp. V: F1, 13 ⫽ 0.27; P ⫽ 0.61). There was a signiÞcant difference between the two treatments in Exp. VI when per capita increases in

Soybean Aphid Preference Experiments In the Þrst preference trial (Exp. I), alate aphids were twice as common on control soybean plants compared with soybean cyst nematode-infected plants (Fig. 1a; F1, 17 ⫽ 12.81, P ⫽ 0.002). The same pattern was observed in Exp. II and III. Alate aphids were increasingly more common on control plants relative to soybean cyst nematode-infected plants: by

Soybean Aphid Performance on Soybean Cyst Nematode-Infected Plants

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Fig. 2. Apterous soybean aphid performance on soybean cyst nematode infected and control plants in laboratory studies. *, P ⬍ 0.05; ●, P ⬍ 0.10; NS, P ⬎ 0.1. Vertical bars represent SEM.

aphids was ⬇35% higher on soybean cyst nematodeinfected soybean (F1, 33 ⫽ 4.48; P ⫽ 0.04). Life Table Experiment. In a clip-cage experiment where aphids were followed as a cohort over their lives (Exp. VII), apterous aphids reared on soybean cyst nematode-infected soybean performed better than those on control plants. Soybean aphid reared on soybean cyst nematode-infected soybean had ⬇74% higher net reproductive rate (Table 1; Ro), 28% higher intrinsic rate of increase (rm) and 8% higher Þnite rate of increase (␭) compared with aphids developing on control plants (Randomization test; P ⬍ 0.05 for all Table 1. plants

Estimation of life table parameters of soybean aphids reared in clip cages on soybean cyst nematode infected and control

n Uninfected Soybean cyst nematode-infected P valuea a

parameters). Fecundity during the early mature stage (i.e., 7Ð12 d in Fig. 3a) was higher in aphids reared on soybean cyst nematode-infected plants (3.3 per aphid) than on control plants (2.8 per aphid, Treatment: F1, 19 ⫽ 5.04; P ⫽ 0.04; Day: F24, 456 ⫽ 6.97; P ⬍ 0.0001; Interaction: F24, 456 ⫽ 0.8; P ⫽ 0.74). However, there was no difference in adult survivorship between soybean cyst nematode-infected and control plants: soybean aphid took on average 5Ð 6 d to reach adulthood from neonate (Fig. 3a) and survivorship of aphids on control and soybean cyst nematode-infected plants did not differ over the course of the

8 13

Based on n ⫽ 1,000 randomizations.

Finite rate of increase (␭)

Intrinsic rate of increase (rm,)

Net reproductive rate (Ro)

Generation time (T)

1.339 1.452 0.037

0.292 0.373 0.043

18.125 31.538 0.016

8.531 7.861 0.866

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Fig. 3.


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Soybean aphid fecundity (a) and survivorship (b) on soybean cyst nematode infected and control plants.

experiment. Survival or mortality of 50% aphids in each group occurred at 12 and 20 d for control and nematodeinfected plants, respectively (Fig. 3b; ␹2(1) ⫽ 0.1; P ⫽ 0.80). In addition, mean generation time (T), was not signiÞcantly different (P ⫽ 0.87; Table 1). Discussion Soybean herbivores with two different feeding modes and feeding locations on the shared host can interact indirectly via the host plant. The most consistent Þnding of these experiments was that aphid behavior (i.e., preference) was more sensitive to the effects of below-ground feeding by soybean cyst nematode than was aphid performance. Alate soybean aphids signiÞcantly preferred control soybean plants compared with soybean cyst nematode-infected plants while soybean cyst nematode-infected plants had either no effect or a positive effect on aphid population growth. Despite variability in the outcomes of repeated experiments, in no case was aphid performance worse when feeding on soybean cyst nematode infected plants. Although the mechanisms of interaction between soybean cyst nematode and soybean aphid in soybean are not currently understood, the results from our

preference experiments clearly indicate that alate soybean aphids signiÞcantly prefer uninfected soybean plants over plants infected by soybean cyst nematode. Short and long-range cues are likely responsible for aphid plant selection (Dixon 1997). Volatiles chemical signals are known to play a role in the host selection (especially landing) process of aphids (Chapman et al. 1981, Nottingham and Hardie 1993, Powell et al. 2006). However, there is no evidence of which we are aware that indicates that soybean cyst nematode infection or other endoparasitic nematodes (below-ground) induces plant volatiles emissions from leaves (i.e., above-ground) which could be sensed by alighting aphids. Olson et al. (2008) for example found that cotton plants damaged by root-knot nematode (Meloidogyne incognita [Kofoid and White] Chitwood) did not emit systemically induced volatiles attractive to parasitoids of corn earworm. However, the potential role of plant volatile compounds in inßuencing soybean aphid preference can not be ruled out because other plant stressors have been shown to inßuence aphid preference via changes in volatile emissions (Fereres et al. 1989, 1999, Medina-Ortega et al. 2009, Werner et al. 2009). For example, the aphid virus vectors, Sitobion avenae (Fabricius) (Fereres et al. 1989), Rhopalosiphum padi (L.) (Medina-Ortega et

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al. 2009), and Myzus persicae (Sulzer) (Werner et al. 2009) exhibited greater preference for plants infected with viruses than for control plants. Further studies are necessary to investigate if infection of soybean by below-ground herbivore (i.e., nematode) systemically mediates release of volatile compounds from leaves in response to nematode infection and whether aphids are responding to these long-medium range signals for plant selection. An additional mechanism that aphids use to evaluate host suitability is by inserting their stylets into host plant tissues after landing on plants (Nault and Styer 1972, Powell et al. 2006). Attacks by herbivore and/or pathogens can induce profound changes in plant allelochemistry (Bezemer et al. 2004, Kaplan et al. 2008a) and distribution of metabolites within plants (Kaplan et al. 2008b). These changes can also result in systemic resistance and affect performance and preference of secondary plant attackers (Stout et al. 2006). Soybean responds to various stresses by biotic and abiotic factors such as wounding (Wegulo et al. 2005), fungi (McDonald and Cahill 1999, Cheong et al. 2000, Wegulo et al. 2005, Zhang et al. 2008), bacteria (Cheong et al. 2000), viruses (Kelley et al. 2006), nematodes (Ithal et al. 2007a,b, Mazarei et al. 2007), and aphids (Diaz-Montano 2006, Li et al. 2008). At a cellular level, soybean responds to root infection by soybean cyst nematode with various changes in production of secondary metabolites, such as ßavonoids (Ithal et al. 2007b, Jones et al. 2007), and in structure of roots (Kim et al. 1987). These changes may therefore affect both aphid preference through probingrelated evaluation of the plant, and later aphid feeding performance, once settling on the plant has occurred. Given that in these experiments, the difference between aphids on control versus soybean cyst nematode-infected plants increases over time, we speculate that one possible scenario by which aphids evaluate plant suitability is by randomly alighting on plants and then either using short-range volatile cues emanating from plants or by using contact or gustatory cues once landing on a plant has occurred to evaluate plants, although the mechanism by which this occurs is not known for these aphids. If plants are deemed suitable, aphids remain on plants and begin to feed whereas if plants are deemed unsuitable, the aphids realight and randomly redistribute themselves on plants. As the process repeats itself over a several-day period, more and more aphid accumulated on preferred (control) plants (Powell et al. 2006). We found that performance and population growth of aphids feeding on soybean cyst nematode-infected plants compared with control plants was either small (and positive) or neutral. This is in contrast to other research which has found negative effects of belowground herbivory on aphids. For example, green peach aphid (Myzus persicae Sulzer) produced 43.8% fewer offspring on English plantain (Plantago lanceolata L.) damaged by nematodes (Pratylenchus penetrans [Cobb] Filipjev and Schuurmans Stekhoven) infected plants compared with uninfected (Wurst and van der Putten 2007). In Þeld cage studies, Kaplan et

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al. (2009) found that several genera of nematodes reduced the abundance of tobacco aphids (Myzus persicae Blackman) at the beginning of the colonization period (i.e., July) while no effect of nematodes were detected at the peak of aphid population later in August. The heterogeneity in aphid performance responses across replicate experiments may suggest that even if a response does exist, it may be relatively small compared with variability introduced by other factors. For example, experiments were carried out at two different times of the year which could have predisposed plants to be more or less sensitive to soybean cyst nematode infection. In addition, although the laboratory experiments were conducted in well-controlled greenhouse setting, seasonal changes are known to inßuence aphid and soybean physiology grown in the greenhouse (Gols et al. 2007). Finally, subtle differences in the types of performance assays used (clip cage versus life-table assays) could have differentially inßuenced aphid performance. For example, the life table assay indicated that aphid population growth was higher for aphids reared on soybean cyst nematode-infected plants, presumably because of increases in aphid fecundity. It is possible that the disturbance of daily removals of offspring (in life table experiments) and/or difference in densities within cages may have interacted with plant quality differences caused by soybean cyst nematode infections. In life table assays, aphid densities were consistently low while in clip cage assays populations were allowed to grow. Thus, we suggest that soybean cyst nematodemediated effects on aphid performance are sufÞciently small that, despite efforts to maintain consistency between experiments, other variability inherent in the experimental conditions may have overwhelmed this response. The results from our laboratory study provide insights on the relationship between below- and above-ground herbivores on soybean. Despite the variable outcome of aphid performance on soybean cyst nematode-infected soybean, the evidence of a nematode effect on aphid behavior (i.e., preference) indicates a potential role of soybean cyst nematode in inßuencing aphid populations. Interestingly, in the choice study winged-aphids exhibited a signiÞcant preference to undamaged plant over soybean cyst nematode-infected plant while in the performance assay, wingless-aphids showed no differences in performance on nematode-infected plants compared with the control plants. A challenge for future studies will be to investigate the relationship between nematodes and aphids in soybean Þelds and whether changes in aphid behavior in response to nematodes can inßuence population responses in the Þeld and whether aphid performance will be equally insensitive to below-ground soybean cyst nematode infection under more natural conditions. Acknowledgments We thank Ann E. MacGuidwin for help with nematode protocols and Carly Myers for help in the lab. We appreciate the comments of three anonymous reviewers who signiÞ-

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cantly contributed to improving this manuscript. This work was funded by USDA Cooperative State Research, Education and Extension Service (CSREES) Grant 2004-35302-14726 and UW Hatch Grant (WIS01285).

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