RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious pest of maize (Zea mays L.) in the U.S. Corn Belt and parts of Europe. Transgenic maize hybrids expressing at least one of the four currently available insecticidal toxins from Bacillus thuringiensis (Bt) Berliner, currently the most widely adopted control method in continuous maize, have faltered due to the emergence of resistance. The resistance mechanisms of WCR to Bt toxins are not fully understood. We identified metabolic profiles of susceptible and resistant WCR larvae fed on maize hybrids expressing each of three available Cry3 proteins (eCry3Ab1, mCry3A, and Cry3Bb1) targeting corn rootworms and a control non-Bt maize via an untargeted metabolomics approach. Over 580 unique metabolites found in WCR larvae were classified into different pathways (amino acids, carbohydrates, cofactors and vitamins, energy, lipid, nucleotide, peptide, and xenobiotics). By exploring shifts in WCR larval metabolome exclusively by Bt toxins, several candidate metabolites and metabolic pathways were identified in susceptible and resistant larvae that may be involved in defense against or recovery from Bt ingestion by these larvae. These findings would provide mechanistic insights into altered metabolic pathways associated with the resistance mechanisms of WCR to Bt toxins.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), poses a serious threat to maize (Zea mays L.) growers in the U.S. Corn Belt. Transgenic corn expressing Bacillus thuringiensis (Bt) Berliner is the major management tactic along with crop rotation. Bt crops targeting WCR populations have been widely planted throughout the Corn Belt. Rootworms have developed resistance to nearly all management strategies including Bt corn. Therefore, there is a need for new products that are not cross-resistant with the current Bt proteins. In this study, we evaluated the susceptibility of WCR strains resistant and susceptible to Cry3Bb1 to the biological insecticide Spear-T (GS-omega/kappa-Hexatoxin-Hv1a) alone and combined with Cry3Bb1 protein. The activity of Hv1a alone was similar between Cry3Bb1-resistant and susceptible strains (LC50s = 0.95 mg/cm2 and 1.50 mg/cm2, respectively), suggesting that there is no cross-resistance with Cry3Bb1 protein. Effective concentration (EC50), molt inhibition concentration (MIC50), and inhibition concentration (IC50) values of Hv1a alone were also similar between both strains, based on non-overlapping confidence intervals. Increased mortality (64%) was observed on resistant larvae exposed to Hv1a (0.6 mg/cm2) + Cry3Bb1 protein (170.8 µg/cm2) compared to 0% mortality when exposed to Cry3Bb1 alone and 34% mortality to Hv1a alone (0.3 mg/cm2). The time of larval death was not significantly different between Hv1a alone (3.79 mg/cm2) and Hv1a (0.6 mg/cm2) + Cry3Bb1 (170.8 µg/cm2). New control strategies that are not cross-resistant with current insecticides and Bt proteins are needed to better manage the WCR, and Hv1a together with Cry3Bb1 may fit this role.

As one of the consequences of global warming, the gradual dispersion of western corn rootworm (WCR) is becoming a serious maize pest in Europe and all over the world. The WCR was introduced to Europe from the USA. First it was detected in Europe near Beograd in 1992. The WCR has spread from its initial infestation point to a range of several hundred kilometres, affecting many countries in the region including Hungary first in 1995. The structure of a healthy, WCR free maize field shows straight rows in a clear order and upstanding maize stalks. WCR infection can cause wilted broken corn stalks randomly lying on the ground. That means the damage itself causes physical and visible disorder in the maize field. Our goal is to assess and identify the disorders and structural changes caused by WCR using polarimetic radar images (ALOS PALSAR). The initial results of our assessment will be presented in this paper. This project has been implemented in the framework of the tender called by the Ministry of Environment and Water and with the support of the Hungarian Space Office. To develop and extend the method is worthwhile as the WCR monitoring system is expeditious, objective and economical. The development of the system will create the possibility to monitor the spread of WCR on larger areas and to forecast the following year's damage. The amount of information can be used efficiently in plant protection; moreover can be useful for farmers, pesticide producers, state authorities and research institutes.

The cultivation of sterile giant miscanthus ( Miscanthus × giganteus , M × g ) for bioenergy and bioproducts has expanded into grain‐cropped land in the United States (US) as local markets developed for this high‐yielding perennial grass (10–30 Mg DM ha −1 ). However, the magnitude of spatial and temporal variability in yield within US Corn Belt fields, along with impacts on economic return and sustainable land management, is poorly understood. This study established a diagnostic model relating remote sensing‐derived vegetation indices to ground truth data from 105 hand‐harvested stem biomass samples, which were strategically selected to represent the full range of vegetation index observations. The high‐resolution satellite‐sensed vegetation indices captured > 90% of the yield variation measured within fields. This model was then used to predict yield variability and assess economic performance across four of the first commercial M × g fields in the Corn Belt state of Iowa, US. Significant spatial variability in biomass dry matter (DM) yields (9.3–18.1 Mg DM ha −1 ) and net profits ($83 to $1211.5 ha −1 ) was observed. All fields were profitable in all site‐years. When low profit occurred, it was explained by limited management experience of the crop in Iowa. The breakeven yield at a selling price of $130 Mg −1 varied from 9.0–12.1 Mg ha −1 at 15% moisture content (7.6–10.3 Mg DM ha −1 ). Breakeven prices ranged from $73 to $122.4 Mg −1 , matching ranges used in the Department of Energy Billion Ton Report (US Department of Energy, 2023). Notably, M × g yield and profits were commensurate with grain crops particularly with favorable precipitation. This study provides insight on the M × g management “learning curve”, performance on marginal land and in drought conditions, and demonstrates that addressing yield gaps, reducing costs, and implementing precision agriculture strategies can enhance profitability. These findings emphasize the value of remote sensing technologies in guiding sustainable and competitive commercial‐scale M × g production.

Knockdown of RNA interference pathway genes in western corn rootworm, Diabrotica virgifera virgifera, identifies no fitness costs associated with Argonaute 2 or Dicer-2

The development of new biopesticides to control the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is urgent due to resistance evolution to various control methods. We tested an air-dried non-live preparation of Chromobacterium species Panama (Csp_P), against multiple corn rootworm species, including Bt-resistant and -susceptible WCR strains, northern (NCR, D. barberi Smith & Lawrence), and southern corn rootworm (SCR, D. undecimpunctata howardi Barber), in diet toxicity assays. Our results documented that Csp_P was toxic to all three corn rootworms species based on lethal (LC50), effective (EC50), and molt inhibition concentration (MIC50). In general, toxicity of Csp_P was similar among all WCR strains and ~ 3-fold less toxic to NCR and SCR strains. Effective concentration (EC50) was also similar among WCR and SCR strains, and 5-7-fold higher in NCR strains. Molt inhibition (MIC50) was similar among all corn rootworm strains except NCR diapause strain that was 2.5–6-fold higher when compared to all other strains. There was no apparent cross-resistance between Csp_P and any of the currently available Bt proteins. Our results indicate that Csp_P formulation was effective at killing multiple corn rootworm strains including Bt-resistant WCR and could be developed as a potential new management tool for WCR control.

Remote sensing observations that vary in response to plant growth and senescence can be used to monitor crop development within and across growing seasons. Identifying when crops reach specific growth stages can improve harvest yield prediction and quantify climate change. Using the Level 2 vegetation optical depth (VOD) product from the European Space Agency’s Soil Moisture and Ocean Salinity (SMOS) satellite, we retrospectively estimate the timing of a key crop development stage in the United States Corn Belt. We employ nonlinear curves nested within a hierarchical modeling framework to extract the timing of the third reproductive development stage of corn (R3) as well as other new agronomic signals from SMOS VOD. We compare our estimates of the timing of R3 to United States Department of Agriculture (USDA) survey data for the years 2011, 2012, and 2013. We find that 87%, 70%, and 37%, respectively, of our model estimates of R3 timing agree with USDA district-level observations. We postulate that since the satellite estimates can be directly linked to a physiological state (the maximum amount of plant water, or water contained within plant tissue per ground area) it is more accurate than the USDA data which is based upon visual observations from roadways. Consequently, SMOS VOD could be used to replace, at a finer resolution than the district-level USDA reports, the R3 data that has not been reported by the USDA since 2013. We hypothesize the other model parameters contain new information about soil and crop management and crop productivity that are not routinely collected by any federal or state agency in the Corn Belt.

BackgroundLarvae of the Western Corn Rootworm (WCR) feeding on maize roots cause heavy economical losses in the US and in Europe. New or adapted pest management strategies urgently require a better understanding of the multitrophic interaction in the rhizosphere. This study aimed to investigate the effect of WCR root feeding on the microbial communities colonizing the maize rhizosphere.Methodology/Principal FindingsIn a greenhouse experiment, maize lines KWS13, KWS14, KWS15 and MON88017 were grown in three different soil types in presence and in absence of WCR larvae. Bacterial and fungal community structures were analyzed by denaturing gradient gel electrophoresis (DGGE) of the16S rRNA gene and ITS fragments, PCR amplified from the total rhizosphere community DNA. DGGE bands with increased intensity were excised from the gel, cloned and sequenced in order to identify specific bacteria responding to WCR larval feeding. DGGE fingerprints showed that the soil type and the maize line influenced the fungal and bacterial communities inhabiting the maize rhizosphere. WCR larval feeding affected the rhiyosphere microbial populations in a soil type and maize line dependent manner. DGGE band sequencing revealed an increased abundance of Acinetobacter calcoaceticus in the rhizosphere of several maize lines in all soil types upon WCR larval feeding.Conclusion/SignificanceThe effects of both rhizosphere and WCR larval feeding seemed to be stronger on bacterial communities than on fungi. Bacterial and fungal community shifts in response to larval feeding were most likely due to changes of root exudation patterns. The increased abundance of A. calcoaceticus suggested that phenolic compounds were released upon WCR wounding.

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

Ammonia (NH3) emissions from fertilizer application is a highly uncertain input to chemical transport models (CTMs). Reducing such uncertainty is important for improving predictions of ambient NH3 and PM2.5 concentrations, for regulatory and policy purposes and for exploring linkages of air pollution to human health and ecosystem services. Here, we implement a spatially and temporally resolved inventory of NH3 emissions from fertilizers, based on high-resolution crop maps, crop nitrogen demand and a process model, as input to the Comprehensive Air Quality Model with Extensions (CAMx). We also examine sensitivity to grid resolution, by developing inputs at 12 km × 12 km and 4 km × 4 km, for the Corn Belt region in the Midwest United States, where NH3 emissions from chemical fertilizer application contributes to approximately 50% of anthropogenic emissions. Resulting predictions of ambient NH3 and PM2.5 concentrations were compared to predictions developed using the baseline 2011 National Emissions Inventory, and evaluated for closure with ground observations for May 2011. While CAMx consistently underpredicted NH3 concentrations for all scenarios, the new emissions inventory reduced bias in ambient NH3 concentration by 33% at 4 km × 4 km, and modestly improved predictions of PM2.5, at 12 km × 12 km (correlation coefficients r = 0.57 for PM2.5, 0.88 for PM-NH4, 0.71 for PM-SO4, 0.52 for PM-NO3). Our findings indicate that in spite of controlling for total magnitude of emissions and for meteorology, representation of NH3 emissions and choice of grid resolution within CAMx impacts the total magnitude and spatial patterns of predicted ambient NH3 and PM2.5 concentrations. This further underlines the need for improvements in NH3 emission inventories. For future research, our results also point to the need for better understanding of the effect of model spatial resolution with regard to both meteorology and chemistry in CTMs, as grid size becomes finer.

Diabrotica virgifera virgifera Le Conte or western corn rootworm (WCR) is a major pest of cultivated maize, Zea mays L., introduced into Europe in 1992. Since then, the beetle spread through Central Europe, leading to a continuous pest population in 11 European countries. This article presents an economic assessment of different damage abatement strategies against this invasive species in the Hungarian maize sector. A bio‐economic model, using Monte Carlo sampling, estimates the potential damage from WCR under no control and the value created by Bt maize, seed treatment, soil insecticides and cultural control. At the same time, potential market shares for the different damage abatement options under perfect information are deducted. The potential damages under a no control scenario are estimated at €176/ha for grain maize farmers on average, which points out the need for well‐designed damage abatement strategies. For land‐constrained farmers cultural control is a valuable damage abatement strategy, being the optimal choice in 69% of the cases. In monoculture Bt maize is the best option as it creates the highest value in 78% of the cases. However, as Bt maize active against WCR is not deregulated in the European Union, soil insecticides in 54% of the cases and seed treatment in 46% of the cases are the rational choices. As the value created by Bt maize is positive, not deregulating Bt maize in Hungary leads to benefits foregone ranging from €16/ha for land‐constrained grain farmers to €49/ha in the case of silage maize under monoculture. Finally, the results of the sensitivity analysis can be used to develop a multi‐criteria tool to aid farmers in applying the appropriate damage abatement strategy. This could decrease the dependency of farmers on scouting techniques and economic thresholds of WCR presence.

Over the past 150 years, land-use changes from native ecosystems to row crop corn and soybeans have negatively impacted a variety of ecosystem services in the United States (US) Corn Belt, including nutrient attenuation, water storage, landscape heterogeneity and habitat. In landscapes dominated by agricultural land use, restoring floodplains through United States Department of Agriculture (USDA) Natural Resource Conservation Service (NRCS) programs (e.g., Emergency Watershed Protection program) could offer disproportionate opportunities to positively enhance ecosystem service benefits - yet, identifying and prioritizing opportunities for conservation practices, including floodplain easements, to maximize environmental outcomes and financial resources remains challenging. This research sought to identify, prioritize, and evaluate costs associated with potential floodplain easements across the state of Iowa. Leveraging key geospatial data layers and criteria, including proportion of field within the 2-, 5-, and 10-year floodplain, field boundaries, and land use characteristics, we identified 2,767 high priority easement locations based on the frequency of inundation of the fields, representing a total area of 57,500 ha (0.4% of the state area). Approximately 138,500 additional suitable floodplain easements were also identified for potential consideration. To estimate acquisition, direct, and total costs associated with potential easements, we used a variety of data, including soils data and a crop productivity index, land value data, and partial enterprise budgets. Estimated costs to restore only high priority easement opportunities total approximately $995,000,000. This analysis aims to assist natural resource practitioners and conservation planners in identifying, prioritizing, and estimating costs for potential floodplain easement locations and can be applied across the state of Iowa and in other regions where data exist.

Crops producing toxins derived from the bacterium Bacillus thuringiensis (Bt) are widely planted to manage insect pests including western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), which is a significant pest of maize in the United States and Europe. However, the widespread use of Bt maize places intense selection pressure on pest populations to evolve resistance, and field‐evolved resistance to Bt maize by western corn rootworm has been documented in the United States. In conjunction with non‐Bt refuges, fitness costs of Bt resistance can delay resistance evolution. Fitness costs arise in the absence of Bt toxin when individuals with resistance alleles have lower fitness than Bt‐susceptible genotypes. We quantified the level of resistance and fitness costs of resistance for a strain of western corn rootworm with laboratory‐selected resistance to transgenic maize (Zea mays L.) producing Bt toxin Cry3Bb1. Survival to adulthood on Cry3Bb1 maize was more than twofold higher for resistant insects vs. susceptible insects, which is similar to the magnitude of resistance first observed in the field. Fitness costs were measured in two experiments; the first used maize hybrids and the second used inbred lines. The experiment with maize hybrids compared resistant and susceptible strains while the experiment with maize inbreds compared resistant, susceptible and heterozygous genotypes. The only non‐recessive fitness cost detected (i.e. cost affecting heterozygotes) was for adult size. Recessive fitness costs (i.e. costs affecting the resistant strain) were observed for developmental rate, female survival and egg viability. However, when reared on non‐Bt maize, the resistant strain also displayed higher fecundity, higher survival for males and greater adult longevity compared to the susceptible strain. These results suggest that resistance to Bt maize by western corn rootworm may not impose substantial fitness costs, and consequently, may evolve quickly and persist once present.

Our research assessed the condition of maize (Zea mays) crops in the Ternopil region, specifically examining infestations by insect pests, particularly the western corn rootworm (Diabrotica virgifera virgifera Le Conte). We also analyzed the biological characteristics and distribution of the western corn rootworm across Ukraine and within the Ternopil region. The study of insect pests was conducted on farmland in Birky and Sosnivka villages (Kremenets district, Ternopil region) and on a household plot in Polivtsi village (Chortkiv district, Ternopil region). We identified three key insect pests on the maize crops: D. virgifera virgifera, Swedish fly (Oscinella frit), and European corn borer (Ostrinia nubilalis). The western corn rootworm is a significant invasive species that poses a major threat to maize cultivation. It impacts crop yield, product quality, and increases pest control costs. D. virgifera virgifera has spread extensively throughout Ukraine, with its peak presence recorded in 2022. By 2023, its presence in 18 Ukrainian regions highlighted the urgent need for comprehensive phytosanitary monitoring and control measures. The most affected areas were the Chernivtsi, Zakarpattia, Mykolaiv, and Kirovohrad regions. Conversely, some areas, like the Ivano-Frankivsk region, experienced significant decreases in infestation levels. In 2023, the western corn rootworm was detected for the first time in the Poltava and Chernihiv regions, demonstrating its dynamic spread. We observed the Swedish fly on maize crops during the 3-4 leaf stage. Damage from this pest, characterized by deformation and yellowing along the central leaf vein, appeared as elongated spots. The European corn borer is another common maize pest; its flight was recorded in early July. However, a drop in temperature and precipitation during this period interrupted egg-laying and the pest's development. Our analysis also explored the relationship between western corn rootworm spread and average daily temperatures. During the study period, temperature conditions were favorable for the pest’s development. Notably, short-term temperature drops below -10°C had no negative impact on egg overwintering.

Molecular cloning and functional characterization of an endogenous endoglucanase belonging to GHF45 from the western corn rootworm, Diabrotica virgifera virgifera