Diabrotica Research Articles

Evaluation of Liquid and Granular Insecticide Formulations at Planting for Larval Corn Rootworm Control, 2017A

Evaluation of Liquid and Granular Insecticide Formulations at Planting for Larval Corn Rootworm Control, 2017A

BT Corn Rootworm Hybrid and Registered Insecticidal Materials for Management of Rootworm Larvae in Corn and Its Impact on Yield in Eastern South Dakota, 2016

BT Corn Rootworm Hybrid and Registered Insecticidal Materials for Management of Rootworm Larvae in Corn and Its Impact on Yield in Eastern South Dakota, 2016

Populacioni nivo imaga kukuruzne zlatice u periodu od 2005-2009 godine

Western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is one of the most important and potentially most dangerous pests of maize. Since its occurrence in Serbia in 1992, it has been present in almost all areas under maize cultivation. Alongside with all preventive measures, first of all, the crop rotation, which is considered the most economic one, a great attention has been paid to the forecast of the pest occurrence based on the flight dynamics and the population level. The flight dynamics, sex dominance, variations in the population level depending on the trap types and climate factors were observed on two locations: Crepaja in three-year continuous cropping (2005-2007) and in the experimental field of the Maize Research Institute, Zemun Polje in the duration of five years (2005-2009). According to the flight dynamics of WCR adults, the highest population level of this pest was observed in 2005, while the comparison of the number of adults over locations showed greater abundance of the pest in Crepaja than in Zemun Polje, which was attributed to extreme favourability of agro-ecological conditions on this location for the WCR development. The 2008-2009 period was characterised by the reduced number of WCR, which coincided with the extremely arid conditions that do not favour the development of this pest.

Distinct fitness costs associated with the knockdown of RNAi pathway genes in western corn rootworm adults.

RNA interference (RNAi) based approaches can potentially be used to control insect pests. These approaches may depend on the usage of microRNA (miRNA) or double stranded RNA (dsRNA) mediated gene knockdown, which likely involves proteins that regulate these pathways, such as Argonaute 1 (Ago1), Argonaute 2 (Ago2), Dicer 1 (Dcr1), Dicer 2 (Dcr2), and Drosha in insects. We previously performed functional characterization of Ago2 and Dcr2 of western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and observed that knockdown of Ago2 and Dcr2 ameliorated the lethal effect induced by the dsRNA-mediated knockdown of an essential gene in WCR, thereby confirming the involvement of Ago2 and Dcr2 in the dsRNA pathway. In the current study, we identified and characterized additional members of the Argonaute and Dicer gene families, namely Ago1, Ago3, Aubergine, and Dcr1, in a previously developed WCR transcriptome. We also identified a Drosha homolog in the same transcriptome. We evaluated the impacts on WCR adult fitness associated with the dsRNA-mediated knockdown of Ago1, Ago2, Dcr1, Dcr2, and Drosha genes. Among these putative RNAi pathway genes, only the knockdown of Ago1 incurred significant fitness costs such as reduced survival and oviposition rate, as well as decreased egg viability. The present study, to our knowledge, represents the first report showing that Ago1 is critical to the survival of insect adults. Our findings suggest that Ago1 plays an essential role in broader life stages of an insect than previously thought. Importantly, since fitness costs were not observed, downregulation or loss of function of RNAi pathway genes such as Ago2 or Dcr2 may confer resistance to pest control measures that rely on the normal functions of these genes. However, the precise roles of these genes under field conditions (i.e., in the presence of possible viral pathogens) requires further investigation.

Quantitative Trait Loci Mapping of Western Corn Rootworm (Coleoptera: Chrysomelidae) Host Plant Resistance in Two Populations of Doubled Haploid Lines in Maize (Zea mays L.).

Over the last 70 yr, more than 12,000 maize accessions have been screened for their level of resistance to western corn rootworm, Diabrotica virgifera virgifera (LeConte; Coleoptera: Chrysomelidae), larval feeding. Less than 1% of this germplasm was selected for initiating recurrent selection or other breeding programs. Selected genotypes were mostly characterized by large root systems and superior root regrowth after root damage caused by western corn rootworm larvae. However, no hybrids claiming native (i.e., host plant) resistance to western corn rootworm larval feeding are currently commercially available. We investigated the genetic basis of western corn rootworm resistance in maize materials with improved levels of resistance using linkage disequilibrium mapping approaches. Two populations of topcrossed doubled haploid maize lines (DHLs) derived from crosses between resistant and susceptible maize lines were evaluated for their level of resistance in three to four different environments. For each DHL topcross an average root damage score was estimated and used for quantitative trait loci (QTL) analysis. We found genomic regions contributing to western corn rootworm resistance on all maize chromosomes, except for chromosome 4. Models fitting all QTL simultaneously explained about 30 to 50% of the genotypic variance for root damage scores in both mapping populations. Our findings confirm the complex genetic structure of host plant resistance against western corn rootworm larval feeding in maize. Interestingly, three of these QTL regions also carry genes involved in ascorbate biosynthesis, a key compound we hypothesize is involved in the expression of western corn rootworm resistance.

Response of Maize Hybrids With and Without Rootworm- and Drought-Tolerance to Rootworm Infestation Under Well-Watered and Drought Conditions.

Anecdotal data in the past have suggested that the effect of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), on maize yield is greater under drought and the effect of drought is greater under rootworm infestations, but no field experiments have controlled both moisture and rootworm levels. Field studies were conducted in 2012, 2013, and 2014 with treatments in a factorial arrangement of western corn rootworm infestation levels, and maize hybrids (with and without tolerance to drought and rootworm feeding). The experiment was repeated under well-watered and drought conditions in adjacent plots. Leaf water potential and stomatal conductance data suggested significant plant stress was achieved in the drought plots toward the end of the season each year and maize hybrids only played a minor role. In particular, in 2012 and 2013 yield was dramatically lower for the drought experiment than for the well-watered experiment. However, the impacts of rootworm infestation level and maize hybrids on water potential, stomatal conductance, and yield were variable across years and between experiments. In fact, the only year that the main effect of rootworm infestation levels significantly impacted yield was in 2014, when an extremely high infestation level was added and this was only for the well-watered portion of the experiment. Overall, rootworm infestation level played a relatively minor role in maize productivity and it did not appear that soil moisture level influenced that to a large degree.

Sequestration and activation of plant toxins protect the western corn rootworm from enemies at multiple trophic levels.

Highly adapted herbivores can phenocopy two-component systems by stabilizing, sequestering and reactivating plant toxins. However, whether these traits protect herbivores against their enemies is poorly understood. We demonstrate that the western corn rootworm Diabrotica virgifera virgifera, the most damaging maize pest on the planet, specifically accumulates the root-derived benzoxazinoid glucosides HDMBOA-Glc and MBOA-Glc. MBOA-Glc is produced by D. virgifera through stabilization of the benzoxazinoid breakdown product MBOA by N-glycosylation. The larvae can hydrolyze HDMBOA-Glc, but not MBOA-Glc, to produce toxic MBOA upon predator attack. Accumulation of benzoxazinoids renders D. virgifera highly resistant to nematodes which inject and feed on entomopathogenic symbiotic bacteria. While HDMBOA-Glc and MBOA reduce the growth and infectivity of both the nematodes and the bacteria, MBOA-Glc repels infective juvenile nematodes. Our results illustrate how herbivores combine stabilized and reactivated plant toxins to defend themselves against a deadly symbiosis between the third and the fourth trophic level enemies.

Transgenic Bt Corn, Soil Insecticide, and Insecticidal Seed Treatment Effects on Corn Rootworm (Coleoptera: Chrysomelidae) Beetle Emergence, Larval Feeding Injury, and Corn Yield in North Dakota.

Northern, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), and western, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), corn rootworms are economic pests of corn, Zea mays L. in North America. We measured the impacts of corn hybrids incorporated with Cry3Bb1, Cry34/35Ab1, and pyramided (Cry3Bb1 + Cry34/35Ab1) Bacillus thuringiensis Berliner (Bt) proteins, tefluthrin soil insecticide, and clothianidin insecticidal seed treatment on beetle emergence, larval feeding injury, and corn yield at five locations from 2013 to 2015 in eastern North Dakota. In most cases, emergence was significantly lower in Bt-protected corn than in non-Bt corn hybrids. Exceptions included Wyndmere, ND (2013), where D. barberi emergence from Cry34/35Ab1 plots was not different from that in the non-Bt hybrid, and Arthur, ND (2013), where D. v. virgifera emergence from Cry3Bb1 plots did not differ from that in the non-Bt hybrid. Bt hybrids generally produced increased grain yield compared with non-Bt corn where rootworm densities were high, and larval root-feeding injury was consistently lower in Bt-protected plots than in non-Bt corn. The lowest overall feeding injury and emergence levels occurred in plots planted with the Cry3Bb1 + Cry34/35Ab1 hybrid. Time to 50% cumulative emergence of both species was 5-7 d later in Bt-protected than in non-Bt hybrids. Tefluthrin and clothianidin were mostly inconsequential in relation to beetle emergence and larval root injury. Our findings could suggest that some North Dakota populations could be in early stages of increased tolerance to some Bt toxins; however, Bt corn hybrids currently provide effective protection against rootworm injury in eastern North Dakota.

A Maize Inbred Exhibits Resistance Against Western Corn Rootwoorm, Diabrotica virgifera virgifera.

Insect resistance against root herbivores like the western corn rootworm (WCR, Diabrotica virgifera virgifera) is not well understood in non-transgenic maize. We studied the responses of two American maize inbreds, Mp708 and Tx601, to WCR infestation using biomechanical, molecular, biochemical analyses, and laser ablation tomography. Previous studies performed on several inbreds indicated that these two maize genotypes differed in resistance to pests including fall armyworm (Spodoptera frugiperda) and WCR. Our data confirmed that Mp708 shows resistance against WCR, and demonstrates that the resistance mechanism is based in a multi-trait phenotype that includes increased resistance to cutting in nodal roots, stable root growth during insect infestation, constitutive and induced expression of known herbivore-defense genes, including ribosomal inhibitor protein 2 (rip2), terpene synthase 23 (tps23) and maize insect resistance cysteine protease-1 (mir1), as well high constitutive levels of jasmonic acid and production of (E)-β-caryophyllene. In contrast, Tx601 is susceptible to WCR. These findings will facilitate the use of Mp708 as a model to explore the wide variety of mechanisms and traits involved in plant defense responses and resistance to herbivory by insects with several different feeding habits.

Diet improvement for western corn rootworm (Coleoptera: Chrysomelidae) larvae.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious insect pest of corn (Zea mays L.) in the United States and parts of Europe, and arguably one of the world’s most expensive pests to control. Several diet formulations are currently used by industry and public researchers to evaluate WCR larvae in diet-toxicity bioassays. However, a publicly available diet that produces normative insects that are physiologically similar to WCR larvae reared on corn roots will accelerate development of management technologies. We report a new diet formulation that supports improved weight gain, larval development and survival compared with the only public diet for WCR that is currently available in the refereed literature. The formulation was created by using response surface methods combined with n-dimensional mixture designs to identify and improve the formulation of key ingredients. Weight gain increased two-fold, and survival and molting rates increased from 93% and 90%, respectively when reared on the public diet, to approximately 99% for both survival and molting at 11 days when reared on our new formulation. This new formulation provides a standardized growth medium for WCR larvae that will facilitate comparison of research results from various working groups and compliance with regulatory requirements.

Previous crop and rotation history effects on maize seedling health and associated rhizosphere microbiome

To evaluate crop rotation effects on maize seedling performance and its associated microbiome, maize plants were grown in the greenhouse in soils preceded by either maize, pea, soybean or sunflower. Soils originated from a replicated field experiment evaluating different four-year rotation combinations. In the greenhouse, a stressor was introduced by soil infestation with western corn rootworm (WCR) or Fusarium graminearum. Under non-infested conditions, maize seedlings grown in soils preceded by sunflower or pea had greater vigor. Stress with WCR or F. graminearum resulted in significant root damage. WCR root damage was equivalent for seedlings regardless of soil provenance; whereas F. graminearum root damage was significantly lower in maize grown in soils preceded by sunflower. Infestation with WCR affected specific microbial taxa (Acinetobacter, Smaragdicoccus, Aeromicrobium, Actinomucor). Similarly, F. graminearum affected fungal endophytes including Trichoderma and Endogone. In contrast to the biological stressors, rotation sequence had a greater effect on rhizosphere microbiome composition, with larger effects observed for fungi compared to bacteria. In particular, relative abundance of Glomeromycota was significantly higher in soils preceded by sunflower or maize. Defining the microbial players involved in crop rotational effects in maize will promote selection and adoption of favorable crop rotation sequences.

Seed treatment of maize with Bacillus pumilus strain INR-7 affects host location and feeding by Western corn rootworm, Diabrotica virgifera virgifera

Western corn rootworm (WCR), Diabrotica virgifera virgifera is an important pest of maize and the costs of control and yield loss are estimated at $1 billion per year in the USA. As a specialist herbivore, WCR has evolved to use host odors and secondary plant metabolites as cues for host location and phagostimulants. This study reports that rhizobacteria applied as seed treatments to maize affect host-seeking behavior of WCR larvae. The results of a dual-choice test showed that a significantly higher percentage (76%) of WCR larvae chose untreated control plants than plants treated with the single Bacillus pumilus strain INR-7 (24%). In no-choice feeding tests, WCR larvae-fed INR-7-treated plants weighed significantly less than larvae-fed untreated plants or plants treated with bacilli blends. Overall, the results demonstrate that B. pumilus INR-7 can enhance resistance of maize against damage by WCR larvae. The implication of these findings is discussed in the context of using beneficial rhizobacteria in integrated pest management of corn rootworm.

Sublethal Effects of vATPase-A and Snf7 dsRNAs on Biology of Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber.

RNA interference is a powerful tool against corn rootworm. Adults and neonates of southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), were exposed to the LC50 of vATPase-A and Snf7 double-stranded RNAs (dsRNAs), and the effects on female fecundity, egg viability, male fitness as measured by sperm viability and mating capacity, larval recovery along with dry weight, and instar determination 10 d after exposure to dsRNA, were determined. Significant reductions were observed for a number of parameters in dsRNA-exposed rootworms relative to control treatments. Female fecundity and larval recovery were significantly reduced after exposure to both dsRNAs. In addition, larval dry weight and recovery of 2nd and 3rd instars along with dry weight for 3rd instars were significantly reduced after neonate exposure to vATPase-A dsRNA. Neither dsRNA affected male capacity to mate or sperm viability after exposure to the respective LC50s. After 10 d of feeding on untreated corn roots, neonates that survived exposure for 2 d to the vATPase-A dsRNA LC50 exhibited lower dry weight than the control. There was significant gene knockdown in adult males and females after exposure for 5 d to LC50 of vATPase-A and Snf7 dsRNAs. The parameters are discussed in terms of fitness and possible outcomes after deployment of corn hybrids expressing dsRNAs.

Control of Western Corn Rootworm (Diabrotica virgifera virgifera) Reproduction through Plant-Mediated RNA Interference

RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control.

SummaryThe 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.

Colonization history of the western corn rootworm (Diabrotica virgifera virgifera) in North America: insights from random forest ABC using microsatellite data

First described from western Kansas, USA, the western corn rootworm, Diabrotica virgifera virgifera, is one of the worst pests of maize. The species is generally thought to be of Mexican origin and to have incidentally followed the expansion of maize cultivation into North America thousands of years ago. However, this hypothesis has never been investigated formally. In this study, the genetic variability of samples collected throughout North America was analysed at 13 microsatellite marker loci to explore precisely the population genetic structure and colonization history of D. v. virgifera. In particular, we used up-to-date approximate Bayesian computation methods based on random forest algorithms to test a Mexican versus a central-USA origin of the species, and to compare various possible timings of colonization. This analysis provided strong evidence that the origin of D. v. virgifera was southern (Mexico, or even further south). Surprisingly, we also found that the expansion of the species north of its origin was recent—probably not before 1100 years ago—thus indicating it was not directly associated with the early history of maize expansion out of Mexico, a far more ancient event.

Monolith Root Sampling Elucidates Western Corn Rootworm Larval Feeding Injury in Maize

New experimental techniques are needed to investigate spatial relationships of subterranean herbivorous insects. This study was conducted to determine if monolith soil–root sampling techniques could quantify maize (Zea mays L.) root system spatial characteristics after western corn rootworm (Diabrotica virgifera virgifera LeConte) larval feeding. A 2‐yr field study evaluated larval injury to maize root systems under controlled western corn rootworm larval infestation. Root damage and dry weight were measured in a 20‐cm‐diam. root ball that was excavated using standard “garden‐fork” techniques. Monolith sampling was used to remove a soil block (40 cm long by 5 cm wide by 30 cm deep) in which root length was measured after grid sampling. Standard excavation revealed that larval feeding removed about one full whorl of adventitious roots and reduced root dry weight by 21%. Total root length per monolith (sum of root lengths across all grid samples) was reduced ∼26% by larval feeding. Root length density (cm of root length cm−3 of soil) was determined for each grid sample, and the distribution of root length density in the monoliths was visualized using contour plots. These plots revealed dramatic feeding injury reductions in root length density in the portion of the root system closest to the plant stem. Monolith techniques, which provided quantitative estimates of root length density changes to the adventitious root system in response to larval feeding injury, may thus be applicable for future ecological field studies investigating spatial relationships of subterranean herbivorous insects and their plant hosts and predators.

Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing

Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 μg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.

Integrated modelling of efficient crop management strategies in response to economic damage potentials of the Western Corn Rootworm in Austria

The spread of the Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) challenges farmers in intensive maize production regions. We model efficient crop management strategies in response to economic damage potentials of the invasive WCR in Austria. A spatially explicit integrated modelling framework has been developed to calculate economic damage potentials from maize yield losses for a past (1975–2005) and a future (2010–2040) period with climate change. The economic damage potentials determine the choice of efficient crop management strategies considering insecticide applications, crop rotations with gradual maize limitations, fertilization intensities and irrigation. The integrated modelling framework includes the crop rotation model CropRota, the bio-physical process model EPIC, and the non-linear land use optimization model BiomAT. Typical crop rotations are simulated by CropRota at the municipality level. They are input to EPIC to simulate crop yields at the 1km pixel resolution, which are part of the gross margin calculations entering BiomAT. Results of economic damage potentials with a 10% maize yield loss range between 3€/ha and 180€/ha, depending on the location, and increase to between 14€/ha and 903€/ha at 50% maize yield loss. The analysis of economic damage potentials shows a high regional variability. Moreover, the model results show that a decrease in maize shares combined with moderate fertilization levels is more efficient for WCR control than insecticide use. However, further crop management strategies have to be developed in order to reduce maize yield and economic losses.

Transgenic Corn Response to Nitrogen Rates under Corn Rootworm Pressure

Core Ideas Bacillus thuringiensis rootworm traits and corn rootworm feeding affected N uptake and root mass. N uptake by DK44‐61 SSX at V6 lagged DK46‐60 VT3 and DK 44‐92 RR2. DK44‐92 RR2 had a larger root system at V6 than either DK46‐60 VT3 or DK44‐61 SSX. Rootworm injury limited N uptake, root mass of DK44‐92 at V12 compared to Bt hybrids. Root mass, trait efficacy, and root injury interplay with N rate have insect resistance management implications. Recommended N rates for corn (Zea mays L.) typically consider yield, economic, and environmental dimensions, but not optimal protection by Bacillus thuringiensis (Bt) Berliner‐based traits against corn rootworm (CRW) (Diabrotica spp.). This research explored N rate effects on CRW trait performance under CRW pressure. The experiment featured a split‐plot randomized complete block design with factorial treatment combinations of N rates and hybrids differing in CRW traits. Root mass, N uptake, CRW efficacy, and grain yield data were collected from two locations in 2011. Root mass at growth stage V6 (before CRW injury) increased with N rate and was greater in the Roundup Ready (RR2) hybrid (Monsanto, St. Louis, MO) by 30.6%. Nitrogen uptake was similar between VT Triple (VT3) (Monsanto, St. Louis, MO) and the RR2 hybrids, which exceeded the SmartStax (SSX) (Monsanto, St. Louis, MO: Dow AgroSciences Indianapolis, IN) hybrid by 12.5%. By growth stage V12, CRW injury in the RR2 vs. Bt hybrids reduced N uptake by 42.0%, Beetle emergence was reduced 66 to 91% by Bt hybrids. Yield response to N differed between hybrids at only one site: the CRW‐injured RR2 hybrid was unable to utilize increasing N rates while the SSX hybrid did not plateau. Recommended N rates (134–185 kg N per ha−1) were sufficient to optimize root mass, N uptake, root protection, and yield under a low stress, modest CRW‐pressure environment: however, low N resulted in poor performance of these hybrids, suggesting further exploration of N rate effects when N loss or denitrification is present.