European corn borer oviposition response to soil fertilization practices and arbuscular mycorrhizal colonization of corn

Authors

  • Ebony G. Murrell,

    Corresponding author
    1. Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin 53706 USA
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    • Present address: Pennsylvania State University, Department of Ecosystem Science and Management, 411 Agricultural Sciences and Industries Building, University Park, Pennsylvania 16802 USA.

  • Crystal R. Hanson,

    1. Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin 53706 USA
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  • Eileen M. Cullen

    1. Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin 53706 USA
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    • Present address: Department of Plant Science, California State Polytechnic University, Pomona, California 91768 USA.


  • Corresponding Editor: D. P. C. Peters.

Abstract

Soil fertility and resulting crop plant nutrition contribute to optimal crop yields in both conventional and organic farming systems. Additionally, soil management practices can affect the colonization and efficacy of arbuscular mycorrhizae (AM), which in turn may improve crop resilience to drought and soil nutrient deficiencies. Soil mineral fertilization and AM colonization have been shown to affect herbivorous insect oviposition response and performance. However, the below-ground interaction of fertilization practices and AM colonization on plant nutrition and insect oviposition response has been largely unexplored. To test this, we obtained soils from agricultural fields managed under 3 different soil fertilization practices for 5 continuous years: Synthetic fertilizers only with a 2-year corn-soybean rotation (conventional farming, or CONV), dairy manure with a 4-year alfalfa/oat-alfalfa-corn-soybean rotation (standard organic farming, or STDO), and dairy manure + 4-year alfalfa/oat-alfalfa-corn-soybean rotation with biannual gypsum applications (organic basic cation saturation ratio farming, or BCSR). Soils from these treatments have been previously shown to vary significantly in their Ca:Mg:K ratios and also in S content. We reared field corn plants in these soils in a greenhouse, then used them to conduct oviposition choice assays with the corn insect pest Ostrinia nubilalis (European corn borer, or ECB). Colonization of AM on plant roots did not significantly differ among treatments. Plant tissue minerals (primarily S, Fe, and Cu) varied significantly among treatments but were not affected by AM colonization. However, the number of ECB eggs laid per plant per trial varied significantly by soil fertilization treatment, plant height, and AM colonization, with significant interaction effects. Female oviposition response was positively correlated with AM colonization and height in low mineral soil fertilization treatments (STDO and CONV), but moths showed a negative response to AM colonization in BCSR plants as plant height increased. Our results indicate that both fertilization practices and mycorrhizal associations can interact to modify oviposition in pest insects, which may have significant implications for the utilization of fertilization practices for pest insect suppression.

Introduction

Optimal fertilization of crops has become increasingly important, in light of projected human population growth and agricultural intensification on existing crop lands globally (Bongaarts 2009, FAO 2011). Novel crop production paradigms and management practices will play a role in meeting the growing demand for food, fiber, and fuel while preserving land and water resources (Foley et al. 2005, Pretty 2008). In order to enhance production efficiency while preserving ecosystem services in agricultural production systems, nutrient applications should be applied not only for their direct effect on crop yields but also in consideration of their effects on both below-ground and above-ground communities. Fertilization practices can affect below-ground processes such as microbial activity (Tiquia et al. 2002, Kautz et al. 2004) and colonization of plant roots by mycorrhizae (Mäder et al. 2000, Treseder 2004), symbiotic fungi that not only facilitate nutrient availability and uptake naturally, but may also improve crop resilience to drought and other climatic conditions (Kaewchai et al. 2009).

Both fertilization practices and below-ground processes can affect insect responses to crops. Not surprisingly many of the nutrients that are important to plant growth also improve the performance of the herbivorous insects that feed upon them (reviewed by Awmack and Leather 2002), but there are exceptions (reviewed by Awmack and Leather 2002). Within agricultural systems, the type of fertilizer used can greatly affect insect performance. Some herbivorous insect species have been shown to lay fewer eggs upon, or show reduced performance when feeding upon, crops grown with organic fertilizers as opposed to synthetic fertilizers (Phelan et al. 1995, Morales et al. 2001, Hsu et al. 2009, Cardoza 2011). Also, plants that have been reared in organically managed soils can achieve a ratio of microminerals (Mg, Ca, Fe, Al, Mn, Cu, Zn) that may make them less susceptible to herbivorous insects (Phelan 1997).

Mycorrhizal colonization of plants can also affect herbivorous insects' response to their host plants by improving plant nutrition in ways that also benefit the insects (reviewed by Hartley and Grange 2009, Koricheva et al. 2009). Mycorrhizal colonization can also interact with specific mineral abundances to affect plant-insect associations. Gall fly performance has been shown to be enhanced by N fertilization of the thistle Cirsium arvense L. only when mycorrhizae were also present (Gange and Nice 1997). Also, a recent study by Barber et al. (2013) demonstrated that (1) different farming practices (organic vs. conventional fertilization) affected colonization rates of arbuscular mycorrhizae (AM) in the roots of cucumber plants, (2) soil obtained from farms that regularly employed these farming practices also differed significantly in their mineral content, and (3) together, these nutrient and AM characteristics may have altered plant characteristics (plant tissue nutrients and flower production) in ways that could have then altered the response of insect species dependent upon those plants (pollinators and herbivorous insects).

On the other hand, mycorrhizae have the potential to negatively affect insect response and herbivorous insect performance by altering plant chemical defenses (Hartley and Grange 2009, Koricheva et al. 2009). The presence of mycorrhizae in plant roots have been shown to increase production of jasmonic acid in plant tissues (Jung et al. 2012), and in corn plants AM colonization can increase concentrations of DIMBOA (Song et al. 2011), both of which are defense compounds that are detrimental to herbivorous insects (Song et al. 2011, Jung et al. 2012). Gravid female insects will avoid ovipositing on plants with greater jasmonic acid-production capability (Sánchez-Hernández et al. 2005). While significant oviposition deterrence of herbivorous insects to DIMBOA concentrations has not been exclusively demonstrated (Traynier et al. 1994, Konstantopoulou et al. 2004), herbivorous moths do tend to avoid ovipositing on early-growth corn plants (Hussein and Kameldeer 1988, Derridj et al. 1989), the development stage at which DIMBOA concentrations are highest (Klun and Robinson 1969). This creates no clear-cut prediction for how oviposition response of herbivorous insects may actually change in response to both increased fertilization and AM colonization.

To date no one has investigated whether interactions between farming fertilization practices and mycorrhizal colonization actually alter pest insect response to crops. In this study, we tested the following hypotheses: (1) corn plants (Zea mays L.) grown in different organic and conventional soil fertilization treatments will differ in AM associations; (2) corn plants exhibit different mineral profiles due to soil fertilization treatments; and (3) oviposition response by the corn pest Ostrinia nubilalis (Hübner) will be shaped by the interactive effects of soil fertilization treatments and AM colonization of roots.

Methods

Study system

Ostrinia nubilalis, commonly known as the European corn borer (ECB), was introduced to North America in the early 1900s (Brindley and Dicke 1963) and is now located throughout north central and eastern North America (Capinera 2000). Its populations are currently reduced in the United States due to widespread planting of genetically modified Bt corn which produces a toxin lethal to the phytophagus ECB larvae (Hutchison et al. 2010), but it continues to pose a threat to non-Bt corn, which includes certified organic field corn, as the USDA National Organic Program regulations prohibit the use of transgenic crops (USDA-AMS 2014). Ostrinia nubilalis larvae, which are chewing insects, feed upon many parts of the plant including the stalk, tassel, and corn ears, although they do not feed on the roots (Capinera 2000).

Oviposition behavior in adult female ECBs is highly responsive to chemical stimuli (Udayagiri and Mason 1995, Phelan et al. 1996, Udayagiri and Mason 1997; reviewed by Renwick and Chew 1994). Though ECBs preferentially oviposit on corn, they also respond to volatiles of, and will readily oviposit on, other plant species (Udayagiri and Mason 1995), which suggests that ECBs are responding to chemical conditions within their host plants that are not plant species-specific. Studies with corn plants have shown that not only does O. nubilalis oviposition rate change significantly in response to different fertilization practices (Phelan et al. 1995), but also to mineral profiles within individual plants (Phelan et al. 1996, Phelan 1997). This broad response to chemical composition of host plants makes O. nubilalis a good model organism, as well as an economically important one, in which to assess fertilization-mycorrhiza mediated oviposition response.

The soils used in this study were obtained from long-term experimental plots located in a 12.14-ha field at the University of Wisconsin-Madison Arlington Agricultural Research Station in Arlington, WI. The specific fertilization regimen of the field plots, as well as abundances of Ca, K, Mg, and S in the soil of each plot, are described in detail in Murrell and Cullen (2014). To briefly summarize, there are thirty-two 0.31-ha randomized plots that have been grown in an organic 4-year alfalfa/oat-alfalfa-corn-soybean rotation since 2007–2008. Half of these organic plots (standard organic, or STDO) have received only annual applications of liquid dairy manure as fertilizer based on soil test and crop nutrient needs (Laboski et al. 2006). The remaining 16 plots have received the same calibrated annual manure applications, plus amendment with Hurlbut Hi-Cal Lime (CaCO3) in 2007 and gypsum (CaSO4.2H2O) in 2008 and 2009. The purpose of these additions was to achieve soil Ca:Mg:K ratios that are purported to maximize calcium and other plant nutrient cation uptake by crops, in keeping with the Basic Cation Saturation Ratio Hypothesis, or BCSR (Albrecht 1975, Schonbeck 2001). We therefore refer to the organic plots with high calcium lime fertilization as BCSR plots. Directly adjacent to the certified organic plots are eight 0.30-ha plots (conventional, or CONV) that have been planted in a 2-year corn-soybean rotation and fertilized with only conventional NPK fertilizers urea (CO(NH2)2), anhydrous ammonia (NH3), and granular 5-14-42 (N-P2O5-K20) since 2008 in the year corn is planted. Soil cores obtained from the plots in 2012 showed that the soil mineral composition differs significantly among the three different soil fertility treatments, with the Ca:Mg:K ratio significantly higher in BCSR plots than in either STDO or CONV plots, and S abundance highest in BCSR plots and lowest in CONV plots (Murrell and Cullen 2014).

Soil collection and plant rearing

On September 3, 2012 we collected soil from the four plots each of BCSR, STDO, or CONV fertilization in which corn was currently planted. Approximately 15 L of soil was collected from each plot. Soil of each treatment was hand sifted a 1.3 cm mesh sieve, mixed to create a composite soil for that soil type, and air-dried.

Plants were reared in a single greenhouse, with a 16:8 L:D cycle. Day and night temperatures were 29.8° ± 0.6°C and 19.7° ± 1.4°C (mean ± SE), respectively. We double-lined 3.79-L plastic pots with plastic bags in order to prevent nutrient loss via water and filled with soil to a total dry weight of 4.2 kg. A total of 15 STD, 15 BAL, and 14 CONV pots were created. Remaining soil was saved for nutrient analysis. In each pot we planted three Viking 6710 variety corn seeds and added 200 mL of deionized (DI) water containing 0.2243 g/L urea, 0.4243 g/L diammonium phosphate, and 0.187 g/L potassium chloride. Pots were then watered up to 5 kg total pot weight. Plants were watered as needed to approximately 5 kg for the following two weeks, after which each pot was thinned to a single plant. After 1 month of growth, corn plants were fertilized again with 100 mL of DI water containing 0.3 g/L urea and 0.37 g/L potassium chloride. Plants were then grown until at least 6 weeks of age and minimum V5 development stage (minimum/maximum leaf height 73.66/125.10 cm) prior to use in the experiment. This height corresponds with the height of corn plants in southern WI in mid-June (Andraski and Lowery 1992), the same time in which the first annual flight of O. nubilalis occurs in this region (Cullen et al. 2014).

ECB rearing

Ostrinia nubilalis used for this experiment were Z-strain eggs obtained from a laboratory colony established by the USDA-ARS CICGRU laboratory in Ames, Iowa. Larvae hatched from these eggs were reared on artificial diet at 26.7°C day temperature, 21.1°C night temperature and 16:8 L:D cycle. Adults of mixed sexes were housed in small cages in a separate environmental chamber under the same conditions and provided with 20% sucrose solution. Trays of DI water were placed in the bottom of the chamber to provide adequate humidity, and the cages were also misted with DI water daily.

Between 1 and 3 days prior to the beginning of each block of the experiment, adults between 1 and 6 days old were removed from the colony and placed in small plastic tubs. Each tub contained five females and two males to ensure fertilization, in order to maximize the egg production of females (Fadamiro and Baker 1999). Each cage contained a bottle of sucrose solution and was misted with DI water daily until the moths were used for the oviposition study.

Oviposition study

Three 1.22 m × 1.22 m × 1.37 m pop-up greenhouses (“cages”) were placed in a row in the center of the greenhouse, with all cage doors facing west. Cage positions were labeled as “North”, “Middle”, and “South”. One corn plant from each soil type was randomly selected for each cage, for a total of 3 plants/cage. For the first four blocks of the experiment only previously unused plants were selected; for the final three blocks, the plants were selected from the pool of plants used in the first four blocks. The total number of individual plants used in the experiment was 36 (12 of each soil type), with 21 plants (7 of each soil type) being used twice. No single plant was reused within a 7-day period, and no two replicates shared the same 3-plant combination.

For each replicate, one plant was placed in the north, south, and east walls of each cage. To eliminate the effect of position within the cages, we switched the orientations of the three soil types so that no two cages within the same block had the same plant orientation, and that the orientations of all cages differed from one block to the next. Leaves on all plants were bent down approximately 30 cm from the stalk of the plant to prevent plants of different soil types from touching or overlapping.

During each block, 5 female and 3 adult male ECB moths (isolated as described previously) were released into the 3 cages, respectively, and left there for 2 days. A bottle of 20% sucrose solution was left in the center of the cage to provide nourishment to the adult moths. After 2 days all moths were collected, female mortality recorded, and the remaining moths were killed to prevent accidental reuse of any moths. Plants were then removed from cages and inspected for eggs. We recorded the number of egg masses found on each plant, the number of eggs per mass, and total number of eggs per plant. All eggs were then immediately removed from the plant and destroyed.

Upon completion of the experiment we removed the broadest, healthiest leaf from each plant for plant tissue nutrient analysis. These samples were air-dried, ground, and sent to the University of Wisconsin Soil and Plant Analysis Laboratory (SPAL 2005) for P, K, Ca, Mg, S, Zn, B, Mn, Fe, Cu, Al, and Na using inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES, SPAL 2005).

To collect roots for the mycorrhizal analyses, we removed each root mass from the soil and rinsed it thoroughly in DI water. Roots 5–8 cm beneath the base of the stem were then removed and stored in a solution of 50% ethanol. To assess AM colonization, we cleared 1 cm sections of preserved roots in 10% KOH solution, dyed them in a solution of 5% distilled vinegar and Shaeffer black ink (Vierheilig et al. 1998). We then slide-mounted 24 randomly selected stained root sections for each plant, scanned 5 random intersections per root at 20× magnification (McGonigle et al. 1990) for a total of 120 root intersections/plant, and recorded the number of intersections/plant in which AM hyphae plus arbuscules and/or vesicles were present. Arbuscules are finely branched hyphae located within plant root cells, while vesicles are lipid-filled hyphal pockets produced by the fungus (Peterson et al. 2004). Both are relatively permanent structures and unique to AM (Peterson et al. 2004), and are therefore more useful for identifying AM establishment in the root than counting presence of hyphae alone.

Statistical analyses

To determine effects of soil treatments on plant properties, we compiled plant tissue nutrient profiles using principle component analysis (PCA) with a varimax rotation (PROC FACTOR, SAS 9.3). We tested effects of soil fertilization and number of root intersections colonized by AM arbuscules and/or vesicles (AM colonization), and nutrient principle components (PCs) using MANOVA (PROC GLM, SAS 9.3). As a follow-up test to any significant effects within the MANOVA, we compared the magnitude of standardized canonical coefficients of the dependent variables to determine which variables contributed most strongly to the significant effect (Scheiner 2001). We also tested for differences in AM colonization among soil fertilization treatments using ANOVA. As height was recorded prior to each trial (therefore producing two heights for each plant that was reused), we ran a separate mixed model (PROC GLIMMIX, SAS 9.3) to determine effect of soil treatment on height, a block effect of plant reuse (initial vs. final height for plants used twice), and the reuse × soil type interaction, with individual plant included as a random effect.

To test oviposition response of the ECBs, we analyzed effects of plant height, soil treatment, number of intersections containing AM arbuscules/vesicles, and all interactions on the total number of eggs per plant per replicate using mixed model analyses (PROC GLIMMIX, SAS 9.3), with a Poisson distribution corrected for overdispersion (SAS/STAT(R) 2009). A block effect of plant reuse was also included in the model to test for a priori effects of plant reuse on oviposition response. Individual test replicate (block × cage) and individual plant (for resused plants) were included as a random effects.

Results

Colonization of AM arbuscules/vesicles did not significantly differ among soil fertilization treatments (ANOVA df = 2, 33, F = 1.51, p = 0.2346). Principle component analysis yielded three PCs (Table 1) of eigenvalues 3.0235, 2.9132, and 2.8268, respectively. Collectively the 3 PCs account for 73.15% of the variation in the nutrient data. Plant tissue nutrients were not significantly altered by AM arbuscule/vesicle colonization (MANOVA Pillai's Trace df = 3, 30, F = 0.22, p = 0.8815), but did differ significantly by soil fertilization treatment (MANOVA Pillai's Trace df = 6, 62, F = 8.70, p < 0.0001). Standardized canonical coefficients (PC1 = 0.0629, PC2 = 2.8611, PC3 = −1.4189) indicate that PC2 nutrients, of which the strongest contributors to PC2 were S, Fe, and Cu, accounted for the greatest difference among soil fertilization treatments. Each of these minerals were of highest concentration in BCSR plants and lowest in CONV plants (Fig. 1). In keeping with the pattern of the individual minerals, there is a distinct separation of soil fertilization treatments along the PC2 axis (Fig. 2A, B). Nutrients contributing to the PC1 axis (Ca, Mg, Mn) exhibited no variation among soil fertilization treatments (Fig. 2A, C) despite the significant difference in Ca:Mg in the soil nutrient profiles (Murrell and Cullen 2014).

Figure 1.

Plant tissue nutrient concentrations, shown on a log scale (mean + SE).

Figure 2.

Ordination of individual plants along (A) PC1 and PC2, (B) PC2 and PC3, (C) PC1 and PC3. Data points are colored according to soil fertilization treatment.

Table 1. Principle components axes for plant tissue nutrients. Nutrients that contribute greatly to each PC (>40) are marked with an asterisk.Thumbnail image of

Plant height was 94.74 ± 2.29 cm for CONV (mean ± SE), 98.53 ± 2.90 cm for STDO, and 100.13 ± 3.37 cm for BCSR. Plant height significantly differed by soil treatment (mixed model F = 5.26, df = 2, 51, p = 0.0084) with CONV plants significantly shorter than STDO or BCSR plants, but no difference in plant height between organic treatments. As expected, plants were significantly taller the second time they were used (F = 74.73, df = 1, 51, p < 0.0001), but there was no significant reuse × soil treatment interaction (F = 1.80, df = 2, 51, p = 0.1750).

The mixed model for number of ECB eggs laid showed no significant a priori effects of plant reuse on oviposition (Table 2), but there was a significant 3-way interaction among plant height, AM arbuscule/vesicle colonization, and soil treatment. For BCSR plants moth oviposition responded positively to height in plants with low AM colonization, but this response to height became significantly more negative as AM colonization increased (Table 2, Fig. 3A). In STDO treatments the opposite effect was observed, with oviposition response increasing significantly as both height and AM colonization increased (Fig. 3B). The number of eggs oviposited per plant was much lower on CONV plants (36.68 ± 7.04) than on either STDO plants (70.84 ± 16.10) or BCSR plants (95 ± 23.41), and oviposition responses to height and/or AM colonization were not significant in the CONV soil treatment (Fig. 3C). The number of eggs laid per replicate was 203 ± 32, and oviposition rates did not significantly differ among replicates (ANOVA F = 1.14, df = 18, 38, p = 0.3516) or by block (ANOVA F = 0.41, df = 6, 50, p = 0.8688).

Figure 3.

Effects of AM colonization and plant height on number of ECB eggs per plant per trial, for (A) BCSR plants, (B) STDO plants, (C) CONV plants. Though AM colonization was analyzed as a continuous variable in the mixed model, results are displayed here categorically as low (0–12.5%), medium (12.5–25%), and high (>25%) percentages of root intersections colonized. Points represent actual data, while lines represent predicted relationships estimated by the mixed model.

Table 2. Mixed model analysis of effects of plant reuse, plant height, number of root intersections colonized by AM arbuscules/vesicles (AM colonization), and interactions on the total number of eggs oviposited on each plant per trial. Significant effects are in boldface.Thumbnail image of

Discussion

While previous studies have demonstrated that organic and conventional fertilization practices can affect mycorrhizal and mineral profiles of plants (Barber et al. 2013), and that fertilization practices can affect adult insect oviposition response (Phelan et al. 1995), this is the first study to characterize the interactive effects of AM colonization and plant tissue minerals on insect oviposition response. We show that AM colonization of roots was not altered by the three soil fertilization treatments, but that plant mineral profiles were affected by soil management history, particularly for the PC2 micronutrients (S, Fe, and Cu). The conditional response of O. nubilalis to AM colonization is especially interesting given that this insect does not feed upon corn roots as larvae, and adult ECBs are likely only nectar feeders (Leahy and Andow 1994), thus unlikely to have any direct association with plant roots.

Plant growth and mineral profiles in this study were consistent with the corn plants reared in the same soil treatments for the Murrell and Cullen (2014) study. In that study, the minerals S, Fe, and Cu significantly differed in plant tissues among the three treatments, having the highest concentration in BCSR plants and lowest in CONV. Additionally, development of CONV plants was significantly slower than plants in either of the organic fertilization treatments (Murrell and Cullen 2014). While the plants in this study were reared for a much shorter time period, the differences in plant mineral profiles by soil treatment were the same and delayed development (e.g., shorter plant height) was evident in the CONV plants.

At first glance our oviposition results appear to contradict those of Phelan et al. (1995). In that study ECB moths showed consistently higher oviposition response to plants reared in conventionally farmed soil, while in our study we found the opposite response. However, in Phelan et al. (1995) the conventional corn plants were significantly taller than their organic counterparts, while our CONV plants were noticeably shorter than either the BCSR or the STDO plants. Since AM colonization did not differ among our soil treatments, the low oviposition response of ECBs to our CONV plants is most likely due to the overall nutrient deficiency of these plants relative to their organic (STDO and BCSR) counterparts. Copper plays a vital role photosynthesis (Maksymiec 1997) in plants, iron is a component of hemoproteins and chlorophyll structure (Imsande 1998, Yosefi et al. 2011) and sulfur contributes to numerous aspects of plant physiology, including chloroplast membranes, amino acids, and vitamins (Imsande 1998). All of these nutrients were least abundant in the CONV plants. The exact mechanism by which lower mineral abundance altered the chemical properties of the plants (such as plant volatiles), or physically altered the plants (such as changes in plant coloration, height, etc.) to affect oviposition response is unknown.

Within the organic treatments the oviposition responses were much more complex. In STDO plants moth oviposition response increased as both height and AM colonization increased. While AM colonization was not significantly associated with any of the nutrients we tested, AM have been shown to facilitate uptake of plant phosphorus (reviewed by Treseder 2013), nitrogen, (Hawkins et al. 2000) and sulfur (Allen and Shachar-Hill 2009). It is possible that AM are facilitating nutrient uptake in our STDO plants, rendering them more attractive to ovipositing ECBs, but we are statistically unable to detect this relationship within the small number of plants per soil fertilization treatment (n = 12).

In contrast to the response to STDO plants, ECB moths showed an increasingly negative oviposition response to plant height in BCSR plants as AM colonization increased. Since the BCSR soil contained the highest mineral supplementation, it is possible that increased AM colonization produced no tangible effect on plant mineral composition. We hypothesize that under these conditions in which plants are healthy and nutrients are abundant, ECBs may respond secondarily to defense compounds produced by corn-mycorrhiza associations (jasmonic acid, DIMBOA, etc.).

An obvious follow-up to this experiment would be a more controlled laboratory experiment, in which mycorrhizal inoculation and key nutrients (S, Fe, Cu, N, etc.) were manipulated independently within soils, plants were reared within these soils, and oviposition response of ECBs were tested based upon these nutrient and AM manipulations. It would also be necessary to test both mineral profiles of the plants, and possibly upregulation of genes responsible for defensing production, in order to elucidate the specific mechanisms by which soil nutrients and mycorrhizal presence/abundance may interact to alter plant chemistry in ways that affect ECB oviposition behavior. While our study does not isolate these specific mechanisms, it nevertheless demonstrates that both farming practices and AM colonization can alter corn plants in ways that affect ECB oviposition behavior. Further research will be necessary to determine exactly how these effects occur.

Unlike Barber et al. (2013), plants reared in our conventional vs. agricultural soils did not significantly differ in the amount of mycorrhizal colonization. Verbruggen et al. (2010) found a similar lack of difference in the amount of colonization, but AM community composition did differ significantly between conventionally and organically farmed soils. There are at least two possible explanations for our results. The first is that, unlike similar studies comparing AM in conventional and organic fields, our study utilizes experimental plots that are within the same 12.14 ha field (STDO and BCSR) and an adjacent 0.3 ha field (CONV) of uniform soil type (Plano Silt Loam) and have been subjected to nearly identical farming practices apart from crop rotation and fertilization treatment. It is possible that AM colonization in other studies is affected by additional factors (differences in farming practices, soil types, etc.) other than simply the type of fertilizer applied to the fields. The second possibility is that our organic plots were not old enough to manifest significant changes to the AM community. Verbruggen et al. (2010) showed a significant positive correlation between AM species richness and the amount of time since conversion to organic management, with the strongest effects present in plots 8–16 years post-conversion. Our organic plots were 4–5 years post-conversion, which may have been an insufficient amount of time for differences in AM abundance or species richness to manifest. However, as we did not assess actual AM community diversity in our study, we can only surmise whether either or both of these mechanisms accounted for lack of observed difference in AM colonization.

Implications for application

Although arbuscular mycorrhizae have likely long been important to agricultural crops, they have only relatively recently begun to receive attention in a production agriculture context (Hamel 1996). Mycorrhizal inoculation has been promoted as a biofertilizer to improve crop yield through increased availability, uptake, or absorption of nutrients by crop plants (reviewed by Kaewchai et al. 2009). However, it is known that the benefits of mycorrhizae can vary depending on the amount of nutrients in the soil (Gill et al. 2013) and the composition of plant and mycorrhizal communities (van der Heijden and Horton 2009). Our results, combined with previous studies that have shown interactions between mycorrhizae and soil fertilization, show that pest response, and subsequently top-down herbivory, can be altered by the bottom-up effects of mycorrhizae by plant nutrition interaction. This may provide an additional explanation for why the “more-is-better” approach to mycorrhizal inoculation does not consistently improve yields (Smith and Smith 2011), and highlights the importance of examining multitrophic effects of mycorrhizal inoculation.

Insect response to mycorrhizal inoculations is not generally considered in an applied agricultural context. However, as organic farmers rely, in part, on soil and crop nutrient management practices to suppress insect pest populations (USDA-AMS 2014), manipulation of below-ground processes to limit above-ground pest populations has become increasingly relevant. Moreover, improved understanding of these mechanisms are applicable beyond the organic-conventional binary to help address challenges of intensification of food production while maintaining long-term resilience and sustainability of agroecosystems (FAO 2011).

Our study demonstrates that mycorrhizal amendments might help to suppress pest populations in crops, but only when applied in tandem with a specific fertilization regimen. In our study, the lowest predicted ECB oviposition response corresponded with high AM colonization + gypsum (BCSR), and low AM colonization + conventional fertilizers (CONV). However, our study only assessed corn plant and insect response to these treatments at the vegetative stage. Further studies on mycorrhiza-fertilization manipulations are needed to determine which nutrient/AM combinations can minimize pest infestation while optimizing crop yield.

Acknowledgments

We would like to thank K. G. Bidne for supplying ECB eggs and assisting us with ECB rearing protocols, K. Shelley for his information on soil fertilizer applications applied to experimental field plots, X. Feng, C. M. Herren and A. R. Ives for their assistance with statistical analyses, V. A. Borowicz and T. D. Johnson for their assistance with mycorrhiza staining and identification procedures, and S. A. Steffan and two anonymous reviewers for comments on the manuscript. This study was funded by USDA NIFA Organic Agriculture Research and Extension Initiative Grant 2010-51300-21282.

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