Post-establishment movement of western corn rootworm larvae (Coleoptera: Chrysomelidae) in Central Missouri corn

If registered, transgenic corn, Zea mays L., with corn rootworm resistance will offer a viable alternative to insecticides for managing Diabrotica spp. corn rootworms. Resistance management to maintain susceptibility is in the interest of growers, the Environmental Protection Agency, and industry, but little is known about many aspects of corn rootworm biology required for an effective resistance management program. The extent of larval movement by the western corn rootworm, Diabrotica virgifera virgifera LeConte, that occurs from plant-to-plant or row-to-row after initial establishment was evaluated in 1998 and 1999 in a Central Missouri cornfield. Post-establishment movement by western corn rootworm larvae was clearly documented in two of four treatment combinations in 1999 where larvae moved up to three plants down the row and across a 0.46-m row. Larvae did not significantly cross a 0.91-m row after initial host establishment in 1998 or 1999, whether or not the soil had been compacted by a tractor and planter. In the current experiment, western corn rootworm larvae moved from highly damaged, infested plants to nearby plants with little to no previous root damage. Our data do not provide significant insight into how larvae might disperse after initial establishment when all plants in an area are heavily damaged or when only moderate damage occurs on an infested plant. A similar situation might also occur if a seed mixture of transgenic and isoline plants were used and if transgenic plants with rootworm resistance are not repellent to corn rootworm larvae.     

Polymorphic microsatellite loci from northern and Mexican corn rootworms (Insecta: Coleoptera: Chrysomelidae) and cross-amplification with other Diabrotica spp

The northern corn rootworm (Diabrotica barberi) and Mexican corn rootworm (Diabrotica virgifera zeae) are significant agricultural pests. For the northern corn rootworm, and to a lesser extent, the Mexican corn rootworm, high resolution molecular markers are needed. Here we present 14 polymorphic microsatellite loci isolated from libraries constructed using pooled northern and Mexican corn rootworm genomic DNA. Polymorphism in other Diabrotica, including the banded cucumber beetle, southern corn rootworm and western corn rootworm, is described.     

Western corn rootworm larval movement in SmartStax seed blend scenarios

Insect resistance management (IRM) can extend the lifetime of management options, but depends on extensive knowledge of the biology of the pest species involved for an optimal plan. Recently, the Environmental Protection Agency (EPA) registered seed blends refuge for two of the transgenic Bacillus thuringiensis (Bt) corn products targeting the western corn rootworm, Diabrotica virgifera virgifera LeConte. Larval movement between Bt and isoline plants can be detrimental to resistance management for high dose Bt products because the larger larvae can be more tolerant of the Bt toxins. We assessed movement of western corn rootworm larvae among four spatial arrangements of SmartStax corn (expressing both the Cry34/35Ab1 and Cry3Bb1 proteins) and isoline plants by infesting specific plants with wild type western corn rootworm eggs. Significantly fewer western corn rootworm larvae, on average, were recovered from infested SmartStax plants than infested isoline plants, and the SmartStax plants were significantly less damaged than corresponding isoline plants. However, when two infested isoline plants surrounded a SmartStax plant, a significant number of larvae moved onto the SmartStax plant late in the season. These larvae caused significant damage both years and produced significantly more beetles than any other plant configuration in the study (including isoline plants) in the first year of the study. This plant configuration would occur rarely in a 5% seed blend refuge and may produce beetles of a susceptible genotype because much of their initial larval development was on isoline plants. Results are discussed in terms of their potential effects on resistance management.     

Effect of Cry3Bb1-expressing transgenic corn on plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae)

Dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in specific combinations of transgenic corn expressing the Cry3Bb1 protein and nontransgenic, isoline corn was evaluated in a 2-yr field study. In total, 1,500 viable western corn rootworm eggs were infested in each subplot. Each year, plant damage and larval recovery were evaluated among four pedigree combinations (straight transgenic; straight nontransgenic corn; nontransgenic corn with a transgenic central, infested plant; and transgenic corn with a nontransgenic central, infested plant) on six sample dates between egg hatch and pupation. For each subplot, the infested plant, three successive plants down the row (P1, P2, and P3), the closest plant in the adjacent row of the plot, and a control plant were sampled. The number of western corn rootworm larvae recovered from transgenic rootworm-resistant plants adjacent to infested nontransgenic plants was low and not statistically significant in either 2001 or 2002. In 2001, significantly fewer larvae were recovered from transgenic rootworm-resistant plants than from nontransgenic plants when both were adjacent to infested, nontransgenic plants. In 2002, significantly more neonate western corn rootworm larvae were recovered from nontransgenic plants adjacent to infested, transgenic rootworm-resistant plants than nontransgenic plants adjacent to infested, nontransgenic plants on the second sample date. Together, these data imply that both neonate and later instar western corn rootworm larvae prefer nontransgenic roots to transgenic rootworm-resistant roots when a choice is possible. However, when damage to the infested, nontransgenic plant was high, western corn rootworm larvae apparently moved to neighboring transgenic rootworm-resistant plants and caused statistically significant, although only marginally economic, damage on the last sample date in 2001. Implications of these data toward resistance management plan are discussed.     

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.     

Planting transgenic insecticidal corn based on economic thresholds: consequences for integrated pest management and insect resistance management

A simulation model of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was used to investigate whether sampling and economic thresholds can improve integrated pest management (IPM) and insect resistance management (IRM) when transgenic insecticidal crops are used for insect pest management. When transgenic corn killed at least 80% of susceptible larvae, the calculated economic threshold increased linearly as the proportion of susceptible beetles surviving the toxin increased. The use of economic thresholds slightly slowed the evolution of resistance to transgenic insecticidal crops. In areas with or without rotation-resistant western corn rootworm phenotypes, the use of sampling and economic thresholds generated similar returns compared with strategies of planting transgenic corn, Zea mays L., every season. Because transgenic crops are extremely effective, farmers may be inclined to plant transgenic crops every season rather than implementing costly and time-consuming sampling protocols.     

Effects of row spacing and plant density on corn rootworm (Coleoptera: Chrysomelidae) emergence and damage potential to corn

Planting corn, Zea mays L., in row spacings less than the conventional width of 76 cm has been shown to increase grain yields. This study was conducted to determine if row spacing and plant density affected corn rootworm, Diabrotica virgifera virgifera LeConte and D. barberi Smith & Lawrence, adult emergence, larval injury to the roots, and plant tolerance to injury. Field experiments were conducted at Ames and Nashua, IA, in 1998, 1999, and 2000. Treatments were row spacings of 38 and 76 cm, and plant populations of 64,500 and 79,600 plants per hectare. Adult emergence was 31% greater in 38 cm compared with 76-cm rows. However, root injury was not significantly different between row spacings or plant populations. Row spacing alone did not significantly influence tolerance to injury, measured as root size and the amount of root regrowth. However, at one environment where precipitation was low, plants in 38-cm rows produced 25% more regrowth compared with plants in 76-cm rows. Root dry weight and regrowth were suppressed by 16 and 32%, respectively, at the high plant population. Although lodging was 51% lower in the 38-cm rows compared with the 76-cm rows, grain yields were not significantly different between row spacings. Reducing the row spacing of field corn from 76-38 cm should not increase the potential for injury from corn rootworm larvae.     

Influence of western corn rootworm (Coleoptera: Chrysomelidae) larval injury on yield of different types of maize

Two field experiments were conducted in 1995-1996 to determine if there are common yield responses among maize hybrids to larval western corn rootworm, Diabrotica virgifera virgifera LeConte injury. Three yellow dent hybrids, five white food grade dent hybrids, and a popcorn hybrid were included in the study. The minimum level of rootworm injury as measured by root damage ratings (3.2-4.2) that significantly reduced yield was similar across the hybrids included in the study. However, the pattern of yield response to different rootworm injury levels varied among hybrids. This suggests that maize hybrids may inherently differ in their ability to tolerate rootworm injury and partition biomass in response to injury and other stresses. The complex interaction among hybrid, level of injury, and other stresses suggests that a common western corn rootworm injury-yield relationship may not exist within maize.     

Belowground resistance to western corn rootworm in lepidopteran-resistant maize genotypes

Several maize, Zea mays L., inbred lines developed from an Antiguan maize population have been shown to exhibit resistance to numerous aboveground lepidopteran pests. This study shows that these genotypes are able to significantly reduce the survival of two root feeding pests, western corn rootworm, Diabrotica virgifera virgifera LeConte, and southern corn rootworm, Diabrotica undecimpunctata howardi Barber. The results also demonstrated that feeding by the aboveground herbivore fall armyworm, Spodoptera frugiperda (J. E. Smith), before infestation by western corn rootworm reduced survivorship of western corn rootworm in the root tissues of some, but not all, genotypes. Likewise, the presence of western corn rootworm in the soil seemed to increase resistance to fall armyworm in the whorl in several genotypes. However, genotypes derived from the Antiguan germplasm with genetic resistance to lepidopterans were still more resistant to the fall armyworm and both rootworm species than the susceptible genotypes even after defense induction. These results suggest that there may be intraplant communication that alters plant responses to aboveground and belowground herbivores.     

Delaying corn rootworm resistance to Bt corn

Transgenic crops producing Bacillus thuringiensis (Bt) toxins for insect control have been successful, but their efficacy is reduced when pests evolve resistance. To delay pest resistance to Bt crops, the U.S. Environmental Protection Agency (EPA) has required refuges of host plants that do not produce Bt toxins to promote survival of susceptible pests. Such refuges are expected to be most effective if the Bt plants deliver a dose of toxin high enough to kill nearly all hybrid progeny produced by matings between resistant and susceptible pests. In 2003, the EPA first registered corn, Zea mays L., producing a Bt toxin (Cry3Bb1) that kills western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. The EPA requires minimum refuges of 20% for Cry3Bb1 corn and 5% for corn producing two Bt toxins active against corn rootworms. We conclude that the current refuge requirements are not adequate, because Bt corn hybrids active against corn rootworms do not meet the high-dose standard, and western corn rootworm has rapidly evolved resistance to Cry3Bb1 corn in the laboratory, greenhouse, and field. Accordingly, we recommend increasing the minimum refuge for Bt corn targeting corn rootworms to 50% for plants producing one toxin active against these pests and to 20% for plants producing two toxins active against these pests. Increasing the minimum refuge percentage can help to delay pest resistance, encourage integrated pest management, and promote more sustainable crop protection.     

Multiplex polymerase chain reaction method for differentiating western and northern corn rootworm larvae (Coleoptera: Chrysomelidae)

Western corn rootworm, Diabrotica virgifera virgifera LeConte, and northern corn rootworm, D. barberi Smith and Lawrence, are sympatric species and serious pests of corn cultivation in North America. Comparison of nucleotide sequence of mitochondrial cytochrome oxidase I and II was used to design polymerase chain reaction (PCR) primers that discriminate immature stages of the two species based on differences in amplicon size. Multiplex PCR can be used to give a positive test for each species in a single amplification reaction. This provides a method to identify field caught larvae and facilitates investigations of larval interaction and competition between the species.     

Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae)

The major proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatin-zymogram analysis of the midgut proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25kDa and several minor proteinases. Similar proteinase activity pH profiles were exhibited by root-fed and diet-fed rootworms with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on the artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine proteinases. Furthermore, rootworm proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine proteinase inhibitor-10', PCPI-10', obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm proteinase activity. PCPI-10' and E-64 were of comparable potency as inhibitors of southern corn rootworm proteinase activity (IC(50) =31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC(50) =121nM). Incorporation of PCPI-10' into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI-10' by transgenic corn plants in the field is a potentially attractive method of host plant resistance to these Diabrotica species.     

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP‐47Aa, from an isolate of Pseudomonas mosselii. PIP‐47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP‐47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP‐47Aa show significant protection from root damage by WCR. PIP‐47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP‐1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP‐47Aa is a novel insecticidal protein for controlling the corn rootworm pests.     

Population patterns of Mexican corn rootworm (Coleoptera: Chrysomelidae) adults indicated by different sampling methods

The Mexican corn rootworm, Diabrotica virgifera zeae Krysan & Smith, is a serious pest of corn, Zea mays L., in several areas of Texas. Recent demonstrations of areawide adult control suggest this tactic has promise for rootworm management, but additional information regarding treatment thresholds and sampling methodology is needed. In 2000 and 2001 we examined the influence of distance into the field on rootworm captures by CRW and Pherocon AM traps, the fidelity of trap captures to population estimates from visual counts of beetles on plants (whole plant samples), and the seasonal population patterns indicated by each sampling method. Only the CRW trap consistently indicated reduced trap captures at the field margin compared with other distances. However, trends for the AM trap and whole plant samples suggested sampling on the field margin should be avoided. Population estimates at other distances into the field (2-30 m) were usually statistically similar. Thus, monitoring does not require trap placement far into the field. Both trap types indicated population peaks after flowering in corn, whereas plant samples indicated peak populations during tasseling and flowering. Both the CRW trap and plant samples showed the proportion of female beetles increased as the season progressed, but the CRW trap underestimated the proportion of females until after flowering. Regressions relating captures by traps to counts from plant samples indicated efficiency of both traps increased with increasing plant development. Our findings should increase acceptance of the CRW trap by producers and consultants and provide a rationale for development of improved, plant growth stage-specific treatment thresholds.     

Predicting western corn rootworm (Coleoptera: Chrysomelidae) larval injury to rotated corn with Pherocon AM traps in soybeans

Crop rotation for portions of east central Illinois and northern Indiana no longer adequately protects corn (Zea mays L.) roots from western corn rootworm, Diabrotica virgifera virgifera LeConte. Seventeen growers in east central Illinois monitored western corn rootworm adults in soybean (Glycine max L.) fields with unbaited Pherocon AM traps during 1996 and 1997. In the following years (1997 and 1998), growers left untreated strips (no insecticide applied) when these fields were planted with corn. Damage to rotated corn by rootworms was more severe in untreated than in treated strips of rotated corn, ranging from minor root scarring to a full node of roots pruned. Densities of western corn rootworms in soybean fields from 1996 were significantly correlated with root injury to rotated corn the following season. Adult densities from 1997 were not significantly correlated with root injury in 1998, due to heavy precipitation throughout the spring of 1998 and extensive larval mortality. Twenty-eight additional growers volunteered in 1998 to monitor rootworm adults in soybean fields with Pherocon AM traps based on recommendations that resulted from our research efforts in 1996 and 1997. In 1999, these 28 fields were rotated to corn, and rootworm larval injury was measured in untreated strips. Based on 1996-1997 and 1998-1999 data, a regression analysis revealed that 27% of the variation in root injury to rotated corn could be explained by adult density in soybeans the previous season. We propose a sampling plan for soybean fields and a threshold for predicting western corn rootworm larval injury to rotated corn.     

Examining Cuphea as a potential host for western corn rootworm (Coleoptera: Chrysomelidae): larval development

In previous crop rotation research, adult emergence traps placed in plots planted to Cuphea PSR-23 (a selected cross of Cuphea viscosissma Jacq. and Cuphea lanceolata Ait.) caught high numbers of adult western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), suggesting that larvae may have completed development on this broadleaf plant. Because of this observation, a series of greenhouse and field experiments were conducted to test the hypothesis that Cuphea could serve as a host for larval development. Greenhouse-grown plants infested with neonates of a colonized nondiapausing strain of the beetle showed no survival of larvae on Cuphea, although larvae did survive on the positive control (corn, Zea mays L.) and negative control [sorghum, Sorghum bicolor (L.) Moench] plants. Soil samples collected 20 June, 7 July, and 29 July 2005 from field plots planted to Cuphea did not contain rootworm larvae compared with means of 1.28, 0.22, and 0.00 rootworms kg(-1) soil, respectively, for samples collected from plots planted to corn. Emergence traps captured a peak of eight beetles trap(-1) day(-1) from corn plots on 8 July compared with a peak of 0.5 beetle trap(-1) day(-1) on 4 August from Cuphea plots. Even though a few adult beetles were again captured in the emergence traps placed in the Cuphea plots, it is not thought to be the result of successful larval development on Cuphea roots. All the direct evidence reported here supports the conventional belief that rootworm larvae do not survive on broadleaf plants, including Cuphea.     

Binary insecticidal crystal protein from Bacillus thuringiensis,strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays

A family of novel binary insecticidal crystal proteins, with activity against western corn rootworm, Diabrotica virgifera virgifera LeConte, was identified from Bacillus thuringiensis Berliner. A binary insecticidal crystal protein (bICP) from B. thuringiensis strain PS149B1 is composed of a 14-kDa protein (Cry34Abl) and a 44-kDaprotein (Cry35Ab1). These proteins have been co-expressed in transgenic maize plants, Zea mays L., and effectively control western corn rootworm larvae under field conditions. Laboratory experiments were conducted to better understand the contribution of each component protein to the in vivo activity of the bICP. The 14-kDa protein is active alone against southern corn rootworm, Diabrotica undecimpunctata howardi Barber, and was synergized by the 44-kDa protein. In mixtures, the concentration of the 14-kDa protein had a greater impact on efficacy than the 44-kDa component. Although both proteins are clearly required for maximal insecticidal activity, laboratory results did not support the formation of a stable, fixed-ratio complex of the two component proteins.     

Effect of soybean varieties on survival and fecundity of western corn rootworm

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn (Zea mays L.) in North America and has evolved resistance to crop rotation by ovipositing in alternate crops such as soybeans [Glycine max (L.) Merr.]. Through experiments with plants grown in the greenhouse and the field, we tested whether soybeans with resistance to the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), affected survival, fecundity, and consumption of soybean for D. v. virgifera. Soybean varieties tested included those types resistant to A. glycines (Rag1 and rag1/rag3) and a susceptible near isoline of the Rag1 variety. Females were provided with a diet of corn tissue for 4 d after which they were fed a diet of tissue from one of three soybean varieties for 4 d, starved for 4 d, or fed corn tissue. When fed greenhouse grown plants, strains differed significantly in survival and consumption, but consumption did not differ by variety of soybean. Diet treatment only affected fecundity; individuals fed corn continuously had greater fecundity than those individuals fed soybeans. In the experiment with plants grown in the field, leaf consumption differed among strains and individuals fed corn continuously had greater fecundity than the other treatments. Soybean varieties with Rag1 and rag1/rag3 resistance to A. glycines did not appear to affect the fitness of D. v. virgifera. Thus, planting of these A. glycines-resistant soybean varieties should not directly affect the spread of rotation-resistant D. v. virgifera.     

Mortality of western corn rootworm larvae on MIR604 transgenic maize roots: field survivorship has no significant impact on survivorship of F1 progeny on MIR604

Mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to MIR604 transgenic corn, Zea mays L., expressing the modified Cry3A (mCry3A) protein relative to survivorship on corn with the same genetic background without the gene (isoline corn) was evaluated at three Missouri sites in both 2005 and 2006. We made these comparisons by using wild-type western corn rootworm at three different egg densities (6,000, 3,000, and 1,500 eggs per m) so that the role of density-dependent mortality would be known. The mortality due to the mCry3A protein was 94.88% when averaged across all environments and both years. Fifty percent emergence of beetles was delayed approximately 5.5 d. Beetles were kept alive and their progeny evaluated on MIR604 and isoline corn in the greenhouse to determine whether survivorship on MIR604 in the field for one generation increased survivorship on MIR604 in the greenhouse in the subsequent generation. There was no significant difference in survivorship on MIR604 in greenhouse assays between larvae whose parents survived isoline and larvae whose parents survived MIR604 in the field the previous generation, indicating that many susceptible beetles survived MIR604 in the field the previous season along with any potentially resistant beetles. The data are discussed in terms of rootworm insect resistance management.     

In-field labeling of western corn rootworm adults (Coleoptera: Chrysomelidae) with rubidium

Field and laboratory studies were conducted in 2000 and 2001 to determine the feasibility of mass marking western corn rootworm adults, Diabrotica virgifera virgifera LeConte, with RbCl in the field. Results showed that application of rubidium (Rb) in solution to both the soil (1 g Rb/plant) and whorl (1 g Rb/plant) of corn plants was optimal for labeling western corn rootworm adults during larval development. Development of larvae on Rb-enriched corn with this technique did not significantly influence adult dry weight or survival. Rb was also highly mobile in the plant. Application of Rb to both the soil and the whorl resulted in median Rb concentrations in the roots (5,860 ppm) that were 150-fold greater than concentrations in untreated roots (38 ppm) 5 wk after treatment. Additionally, at least 90% of the beetles that emerged during the first 3 wk were labeled above the baseline Rb concentration (5 ppm dry weight) determined from untreated beetles. Because emergence was 72% complete at this time, a significant proportion of the population had been labeled. Results from laboratory experiments showed that labeled beetles remained distinguishable from unlabeled beetles for up to 4 d postemergence. The ability to efficiently label large numbers of beetles under field conditions and for a defined period with virtually no disruption of the population provides an unparalleled opportunity to conduct mark-recapture experiments for quantifying the short-range, intrafield movement of adult corn rootworms.