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.     

Absence of genetic divergence between western corn rootworms (Coleoptera: Chrysomelidae) resistant and susceptible to control by crop rotation

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn, Zea mays L., in North America that has recently invaded Europe. A loss of ovipositional fidelity to cornfields has allowed the species to circumvent crop rotation as a means of control in part of its range in the United States. Analyses of variation at eight microsatellite loci provided no evidence for general genetic differentiation between samples of western corn rootworm collected in soybean, Glycine max (L.) Merr., fields and those collected in cornfields both inside and outside the rotation-resistance problem area. This result suggests that few or no barriers to gene flow exist between rotation-resistant and -susceptible rootworm populations. The implications of this result for the management of western corn rootworm in North America and Europe are discussed.     

Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.     

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.     

Larval sampling and instar determination in field populations of northern and western corn rootworm (Coleoptera: Chrysomelidae)

Abundance and head capsule width were measured for northern (Diabrotica barberi Smith & Lawrence) and western corn rootworm (D. virgifera virgifera LeConte) larvae recovered primarily from maize root systems but also from large soil cores each centered around a root system. Larvae for measurement derived from field populations under infestation and rotation regimes that allowed most specimens to be assigned to species. A frequency distribution of head capsule widths indicated three separate peaks for western corn rootworm, presumably representing frequency of the three larval instars, with no larvae measuring 280 or 420 microm in the valleys between peaks. Multiple normal curves fit to similar but partially overlapping peaks generated by northern corn rootworm suggested that division of first to second and second to third instar can best be made for this species at 267 and 406 microm, respectively (270 and 410 when measurements are made to the nearest 20 microm). These results implied that instar of individuals from mixed northern and western corn rootworm populations can be accurately judged from head capsule width without having to determine species. The relative abundance of western corn rootworm instars was similar in root systems removed from the center of 19-cm diameter x 19-cm deep soil cores and in soil cores from which the root systems were removed. Furthermore, the number of larvae from root systems correlated significantly with that from the surrounding soil. These results indicated that the former and much more convenient sampling unit can be used to estimate population developmental stage and possibly density, at least early in the season when these tests were done and young larvae predominated.     

Interaction between western corn rootworm (Coleoptera: Chrysomelidae) larvae and root-infecting Fusarium verticillioides

A greenhouse experiment was conducted to evaluate the effect of soil-dwelling larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, on infection of maize roots by the mycotoxin-producing plant-pathogenic fungus, Fusarium verticillioides (Saccardo) Nirenberg (synonym=Fusarium moniliforme Sheldon). The time and order of application of F. verticillioides and western corn rootworm were varied in three different treatments to investigate the influence of timing on root colonization of F. verticillioides and western corn rootworm larval development. Root feeding by western corn rootworm larvae increased root colonization by F. verticillioides (as determined by real-time polymerase chain reaction) up to 50-fold when a high inoculum (10(7) spores/plant) of F. verticillioides was applied before western corn rootworm eggs were added. This effect was stronger the earlier F. verticillioides was applied relative to the time of western corn rootworm egg application but was only significant for the high F. verticillioides inoculum density treatment; F. verticillioides colonization was not increased when a low F. verticillioides inoculum density (10(6) spores/plant) was applied. F. verticillioides slightly suppressed larval development in that the ratio of second- to third-instar larvae was higher in treatments with F. verticillioides than without F. verticillioides. F. verticillioides reduced western corn rootworm head capsule width when applied before or simultaneously with western corn rootworm. The results of this study are discussed focusing on conditions that favor root colonization by F. verticillioides and its influence on western corn rootworm larval development.     

Field-evolved resistance to Bt maize by western corn rootworm: predictions from the laboratory and effects in the field

Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) provide an effective management tool for many key insect pests. However, pest species have repeatedly demonstrated their ability to adapt to management practices. Results from laboratory selection experiments illustrate the capacity of pest species to evolve Bt resistance. Furthermore, resistance has been documented to Bt sprays in the field and greenhouse, and more recently, by some pests to Bt crops in the field. In 2009, fields were discovered in Iowa (USA) with populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, that had evolved resistance to maize that produces the Bt toxin Cry3Bb1. Fields with resistant insects in 2009 had been planted to Cry3Bb1 maize for at least three consecutive years and as many as 6years. Computer simulation models predicted that the western corn rootworm might evolve resistance to Bt maize in as few as 3years. Laboratory and field data for interactions between western corn rootworm and Bt maize indicate that currently commercialized products are not high-dose events, which increases the risk of resistance evolution because non-recessive resistance traits may enhance survival on Bt maize. Furthermore, genetic analysis of laboratory strains of western corn rootworm has found non-recessive inheritance of resistance. Field studies conducted in two fields identified as harboring Cry3Bb1-resistant western corn rootworm found that survival of western corn rootworm did not differ between Cry3Bb1 maize and non-Bt maize and that root injury to Cry3Bb1 maize was higher than injury to other types of Bt maize or to maize roots protected with a soil insecticide. These first cases of field-evolved resistance to Bt maize by western corn rootworm provide an early warning and point to the need to apply better integrated pest management practices when using Bt maize to manage western corn rootworm.     

Effect of four cropping systems on variant western corn rootworm (Coleoptera: Chrysomelidae) adult and egg densities and subsequent larval injury in rotated maize

The cultural practice of rotating corn, Zea mays L., with soybean, Glycine max (L.) Merrill, to manage larval injury by the western corn rootworm, Diabrotica virgifera virgifera LeConte, was used extensively throughout east central Illinois and northern Indiana until the mid-1990s. The effectiveness of this management tactic diminished due to a shift in the ovipositional behavior of the western corn rootworm. The variant western corn rootworm has since spread as far as northwestern Illinois, southern Wisconsin, southern Michigan, and eastern Ohio. The objective of this study was to evaluate the influence of four cropping systems on adult and egg densities of the western corn rootworm and to quantify the level of root injury in rotated corn after each system. The four cropping systems used included: 1) corn; 2) soybean; 3) double-cropped winter wheat, Triticum aestivum L., followed by soybean; and 4) winter wheat. Research trials were conducted near Monmouth (northwestern), DeKalb (northern), and Urbana (east central), IL, during 2003 and 2004. Results indicated variant western corn rootworm adults can be found in all four treatments at each location and consequently no crop was immune to oviposition or root injury by corn rootworm larvae in rotated corn the following season. Adults were found primarily in corn and soybean, whereas egg densities were greatest in corn plots in all three locations in both years of the study. Root injury by larvae was most abundant in corn following corn at all three sites. Of the four systems evaluated, the use of wheat demonstrated the most potential for preventing yield reducing levels of root injury in rotated corn.     

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.     

Comparative susceptibility of western corn rootworm (Coleoptera: Chrysomelidae) adults to selected insecticides in Kansas

Susceptibility of adult populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, to several insecticides was evaluated in seven Kansas counties, including Dickinson, Ford, Finney, Pottawatomie, Republic, Riley, and Stevens, between 1996 and 2002. All populations surveyed were highly susceptible to methyl parathion with the largest difference in susceptibility of only three-fold based on 16 complete bioassays for the populations from six counties over a 5-yr period. Noticeable decreases in carbaryl susceptibility were found in populations collected from Republic County between 1997 and 2001 when the cucurbitacin-carbaryl-based bait SLAM was widely used as an areawide management approach for adult corn rootworm control. However, the lowered carbaryl susceptibility returned to previous levels 1 yr after the use of SLAM was halted in the managed (treated) cornfields. This change implies possible dispersal of insects into the relatively small managed area from surrounding untreated cornfields and / or some fitness cost associated with carbaryl resistance within the population. Relative susceptibility of western corn rootworm adults also was evaluated for seven commonly used insecticides, including bifenthrin, carbaryl, chlorpyrifos, cypermethrin, fipronil, malathion, and methyl parathion. They were tested with corn rootworm adults collected from a single cornfield. Methyl parathion and bifenthrin were highly toxic to corn rootworm adults, and cypermethrin, chlorpyrifos, carbaryl, and malathion were only slightly less toxic. Although fipronil was highly toxic to adult rootworms, its activity was much slower than that of other insecticides. Thus, bifenthrin and methyl parathion were among the most effective in killing corn rootworm adults.     

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.     

Ovarian development and ovipositional preference of the western corn rootworm (Coleoptera: Chrysomelidae) variant in east central Illinois

The rotation of maize, Zea mays L., and soybean, Glycine max (L.) Merr., has been the traditional cultural tactic to manage the western corn rootworm, Diabrotica virgifera virgifera LeConte, in the Corn Belt. The reduced effectiveness of this rotation as a pest management tool in east central Illinois, northern Indiana, and southern Michigan can be explained by the shift in the ovipositional behavior of the new variant of western corn rootworm. The objective of this study was to evaluate the influence of maize, soybean, oat, Avena sativa L., stubble, and alfalfa, Medicago sativa L., on the ovarian development and ovipositional preferences of the variant western corn rootworm. Field research was conducted near Urbana, IL, during 1998-2000. Gravid females were present throughout the season in all crops, and due to the prolonged period in which western corn rootworm females can lay eggs, none of the crops were immune from oviposition. Results indicated that the western corn rootworm variant oviposits in maize, soybean, oat stubble, and alfalfa In 1998 and 1999, maize was the preferred oviposition site among crops; however, in 2000, maize, soybean, and oat stubble treatments had similar densities of western corn rootworm eggs. Lack of oviposition preference of the western corn rootworm variant demonstrated in this experiment represents a reasonable explanation of why the effectiveness of the rotation strategy to control western corn rootworm has diminished.     

Field measures of western corn rootworm (Coleoptera: Chrysomelidae) mortality caused by Cry34/35Ab1 proteins expressed in maize event 59122 and implications for trait durability

Maize, Zea mays L., has been transformed to express the Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis strain PS149B1. These two proteins act together as a binary insecticidal protein that is effective against corn rootworm (Coleoptera: Chrysomelidae) species. The design of the resistance management plan to preserve the long-term durability of this trait largely depends on the level of rootworm mortality induced by Cry34/35Ab1 corn rootworm-protected maize (frequently referred to as "dose" in this context). Here, we report on studies that showed Cry34/35Ab1-expressing maize event 59122 caused 99.1 to 99.98% mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae, after adjusting adult emergence numbers for density-dependent mortality. In two of three studies, there was a short delay in time to 50% adult emergence from 59122 maize plots compared with control plots, although emergence was completed at approximately the same time from both types of maize. These data support an expectation that alleles conferring resistance to the Cry34/35Ab1 proteins in western corn rootworm will be functionally nearly completely to completely recessive on 59122 maize and that there is unlikely to be assortative mating of Cry34/35Ab1-resistant and susceptible rootworms. When incorporated into simulation models of rootworm adaptation to transgenic maize, these findings suggest that a 20% refuge is likely to be highly effective at prolonging the durability of 59122 maize.     

Evaluation of soil‐applied insecticides with Bt maize for managing corn rootworm larval injury

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is considered one of the most significant insect pests of maize in North America. Larvae of other secondary subterranean pests such as grape colaspis, Colaspis brunnea (F.), and Japanese beetle, Popillia japonica Newman, can also injure maize seedlings and cause yield loss. In the past decade, maize hybrids containing Bt proteins have been used to manage the western corn rootworm; additionally, seeds are commonly treated with a neonicotinoid and fungicide combination to control secondary pests. Recently, soil‐applied insecticides have been used in conjunction with rootworm Bt hybrids (and seed‐applied insecticides) in areas with perceived risk for increased rootworm larval or secondary pest damage. We conducted a series of trials from 2009 to 2011 that examined multiple rootworm Bt hybrids and their near‐isolines, along with two soil‐applied insecticides, to determine whether the Bt plus insecticide combination resulted in an increased level of efficacy or yield. We also sampled for Japanese beetle and grape colaspis larvae to determine their potential for reducing yield. Densities of secondary pests in our trials were low and likely had no effect on maize yield. The addition of a soil‐applied insecticide to rootworm Bt hybrids improved efficacy only once across 17 location‐years, when overall corn rootworm injury was highest; an improvement in yield was never observed. Our results suggest that the use of a soil‐applied insecticide with a rootworm Bt hybrid should only be considered in scenarios with potentially significant rootworm larval populations. However, potential negative consequences related to trait durability when soil insecticides are used with rootworm Bt maize should be considered.     

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.     

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.     

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.     

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.     

Supercooling and cold-hardiness in eggs of western and northern corn rootworms

Oviposition by northern corn rootworms, Diabrotica barberi Smith and Lawrence, and western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), key pests of corn in the Great Plains of the USA, occurs in the soil during late summer. Overwintering eggs are exposed to variable soil moisture and temperatures below ?5 °C. The winter mortality of eggs in the soil is a primary factor that determines the potential for larval injury to corn the following spring. Our studies aimed to determine the comparative supercooling capacities of northern and western corn rootworm eggs and to assess egg mortality following brief exposure to extreme low temperature, ranging from ?12.0 to ?21.5 °C, under three moisture regimes. Eggs of northern corn rootworm were supercooled to a temperature as low as ?27 °C, and survived supercooling to a greater extent than did western corn rootworm eggs. Moisture treatment prior to supercooling had little effect on northern corn rootworm eggs. Western corn rootworm eggs were more resistant than northern corn rootworm eggs to the effects of desiccation followed by supercooling. The survival of northern corn rootworm eggs was better than western corn rootworms under dry conditions, followed by exposure to temperatures of ?12.0 and ?17.5 °C, but was very low at ?21.5 °C, regardless of the moisture regime. The results suggest that moisture and temperature may interact in the soil environment to determine the overwintering survival of corn rootworms. It is evident from these studies that both rootworm species experience mortality at temperatures well above the supercooling points of the eggs, but that differences exist in the effects of substrate moisture treatments on the cold‐hardiness of eggs from the two species.