Project : USDA ARS

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Research Project: Managing Insects in the Corn Agro-Ecosystem

Location: Corn Insects and Crop Genetics Research

2018 Annual Report

Objectives
Objective 1: Improve knowledge of the ecology, genetics, and behavior of key corn pests, especially corn rootworm and lepidopteran species, such as European corn borer, corn earworm and western bean cutworm, in relation to pest abundance and insect resistance to transgenic corn. Sub-objective 1.A. Correlate genetic markers with phenotypic traits of interest in European corn borer, western corn rootworm and western bean cutworm. Sub-objective 1.B. Determine how larval movement and adult dispersal influence insect resistance to transgenic corn. Objective 2: For corn agro-ecosystems, determine potential impacts of changing farming practices on the demographics and ecology of pest and non-pest arthropods, such as the monarch butterfly. Sub-objective 2.A. Assess the potential value of neonicotinoid insecticide seed treatments to growers of major row crops under different agronomic conditions. Sub-objective 2.B. Develop strategies for improving monarch butterfly habitat in modern farm landscapes. Objective 3: Characterize genetic and biochemical responses associated with corn defenses to rootworm and lepidopteran caterpillar pest injury to enhance conventional and transgenic crop protection strategies. Sub-objective 3.A. Develop genetic markers and genomic tools for western corn rootworm, European corn borer, western bean cutworm, and other pests of corn. Sub-objective 3.B. Characterize genetic regulation of surface lipids on corn silks and assess protective capacity of these lipids on corn earworm feeding.

Approach
Field-resistance in western corn rootworm (WCR) to Cry3Bb1 Bt toxins and in European corn borer (ECB) to Cry1Fa and Cry1Ab toxins will be mapped by using genotyping-by-sequencing (GBS) protocols and single nucleotide polymorphism (SNP) markers. Pedigrees will be constructed from Bt resistant and susceptible individuals of both species. GBS will be performed by constructing genomic DNA libraries from non-size-selected fragments, to which adapters with a unique barcode are ligated. A population mapping approach using a high density of SNP markers will be taken to identify loci that differ significantly between E- and Z-race ECB. The premise of population mapping is similar to quantitative trait loci (QTL) mapping, but SNPs with skewed frequencies between races are assumed to result from either selection for race-specific adaptations or genetic drift facilitated by restricted gene flow. GBS-derived SNP markers will be used to estimate gene flow among WCR populations. The strategy will be to estimate Wright's neighborhood area for WCR, the radius of which constitutes a measure of the typical distance genes move per generation. Dispersal and survival rates of fall armyworm (FAW) will be measured in field plot trials. FAW egg masses will be placed in the whorl of vegetative-stage corn plants surrounded by uninfested plants. Living and dead larvae on plants will be counted periodically using destructive sampling. Planting arrangements will include various combinations of Bt and non-Bt isoline plants. Flight behavior and capacity of WCR will be characterized and compared across three Cry3Bb1-resistant and two susceptible strains using flight mills to determine the degree to which resistance affects dispersal. In collaboration with other ARS laboratories and as a NP304 effort, a comprehensive review of the literature will be conducted to provide information on the usefulness of seed treatment with neonicotinoid insecticide in controlling target pests and protecting crops from yield loss. A series of studies will be conducted related to milkweed species selection (larval performance and oviposition preference), and milkweed plant establishment (determining patch sizes and maximizing sustainability of milkweeds). Initially, studies will focus on four Asclepias species found in Iowa: common milkweed, A. syriaca, swamp milkweed, A. incarnata, butterfly weed, A. tuberosa, and whorled milkweed, A. verticillata. Also, studies will be conducted to determine how to maintain these plants in the landscape while avoiding loss from plant competition. This will require identifying the best companion plants for the targeted milkweed species. Two sources of corn germplasm will be used to screen for silk activity against corn earworm (CEW). These corn lines and CEW resistant checks will be grown in the field. Emerged silks will be harvested, immediately frozen in liquid nitrogen, lyophilized, and ground to a powder using a knife mill. One cohort of powdered silks will be used for surface lipid metabolic analysis, while another will be used for CEW bioassays, allowing the metabolomic analysis and CEW feeding studies to be conducted in parallel.

Progress Report
Resistance of western corn rootworm (WCR) to transgenic Bt-corn has been increasing in the Corn Belt, creating an urgent need for better understanding of this pest's dispersal behavior, which strongly influences the rate of resistance development and the rate at which resistance spreads following development. Genotyping-by-sequencing has been completed for adults collected in fields along two 120-mile transects in the species' native home range of eastern Colorado and western Kansas for both 2014 and 2016. Analyses of both the 2014 and 2016 samples indicate that 88% of the rootworm population will disperse approximately 0.2 miles over an average lifetime. However, 12% of the population migrating out of the natal field will disperse farther than 170 miles. A laboratory flight mill study was completed, showing that western corn rootworm larvae reared under high density, as would be expected in a non-Bt field or Bt field infested with a Bt-resistant population, resulted in greater flight activity of adults. A major collaborative effort led by ARS researchers in Ames, Iowa, has completed reviews of the vast literature on the prevalence and risk posed by these pests, which have revealed the factors (sometimes region-dependent) that put individual fields at increased or decreased risk of infestation. Along with an overview paper, the four papers profiling the sporadic early-season pests of corn, soybean, cotton, and wheat targeted by neonicotinoid seed treatments were published as a group. Data are being collected from other scientists and the literature to perform a meta-analysis on the value of neonicotinoid seed treatments to the farmer based on pest pressure and region of the country, which will help address this knowledge gap. Research was conducted to evaluate insect crop pest resistance to insecticidal agents. The level and range of Western bean cutworm (WBC) resistance to transgenic Cry1F toxin was estimated using laboratory toxin overlay and plant tissue bioassays from 13 sample sites with a geographic range from Nebraska to New York. The expression of Cry1Fa and Vip3A toxins within transgenic corn tissues at the time when typically fed upon by WBC larvae were quantified using enzyme linked immunosorbent assays. Transcriptome-based data identified genes differentially-expressed in the midgut between Cry1F-exposed and -unexposed WBC larvae. The candidate Bt resistance genes were down-regulated in Cry1Ab and Cry1Ac resistant corn borer. Additionally, the differential expression of microRNAs was associated with a Drosophila insecticide resistance model and a Bt resistance trait in corn borer. In the Drosophila model, resistance traits are associated with structural and functional variants in enzymes within xenobiotic detoxification pathways. Whole genome sequencing approaches were applied to assemble a WBC draft genome using linked reads, but remained disjoined. Long-read sequence data are being generated to improve the assembly. A draft WCR genome was assembled and is currently being annotated using two transcriptome datasets: 1) an adult, egg and larval stages transcriptome used to annotate the resistance gene family and functional information obtained by targeted gene knockdown by RNA interference, and 2) a reference transcriptome that was applied to identify differentially-regulated transcripts involved in resistance to cell death in Cry3Bb1- and Cry34/35Ab1-exposed larvae. In addition, a genome assembly of soybean aphid biotype 1 was completed and annotated, and whole genome re-sequencing data were collected from biotypes 1, 2, 3, and 4 to identify biotype-specific mutations that may determine aphid abilities to feed on cultivars with resistance to aphis glycines (Rag) traits. These activities were conducted under an ARS-wide arthropod genomics research (AGR) working group coordinated by the project, which generates intra-agency technology transfer and coordinates research efforts. This umbrella also included participation in the Scientific Advisory Council (SAC) coordinated by the Acting Chief Scientific Information Officer (CSIO), and in the Virtual Research Support Core (VRSC) that developed computational pipelines for use on ARS high-performance computer resources. Research is continuing towards developing strategies for increasing habitat for monarch butterflies to counter loss of milkweeds in herbicide-tolerant corn and soybeans. The second season is underway for 45 monarch/pollinator habitats that were planted November 2016, mostly on growers’ non-crop lands. Preliminary data suggest that weed seed banks will be problematic for some areas. Three manuscripts were published and another submitted on studies related to monarch caterpillar growth, development and oviposition on nine milkweed species native to Iowa. All the milkweed species were useful to monarch butterflies, but common milkweed, Asclepias syriaca, and swamp milkweed, A. incarnata, stood out as the most valuable. A three year field study was completed to investigate effects of different densities (one, five and ten plants per 25 m2) of common and swamp milkweed on monarch oviposition. Female monarchs oviposited in all three patch densities when given a choice of three patches. Optimal patch size, however, will depend on the goals of the land manager and their interest in a higher number of eggs per stem or a higher number of eggs per patch. In another study, milkweed plants along four mile length gravel roadsides were mapped to within 4-5 cm with GPS units. There was an average of 1,423 stems per hectare, which is higher than the estimates from other reports. If all gravel roadsides in Iowa counties could establish milkweed, and those stems naturally established in roadside habitats, Iowa could provide 209 million stems to be utilized by females with minimal maintenance. Another study was started fall 2017 to determine the best way to establish common milkweed into brome grass areas. From 2015-2016, maize breeders working on the NIFA-Organic Agriculture Research and Extension Initiative, “Breeding non-commodity corn for organic production systems”, observed ten experimental lines that seemed resistant to heavy natural infestations of fall armyworm while the lines were grown at winter nursery in Puerto Rico. In 2017, seeds were obtained to conduct a single row screen of these lines in Ames, Iowa. Twenty plants from each line were grown in addition to a resistant check, Mp708, and susceptible check, B73. At the V7 stage, each plant was infested with 60 neonatal fall armyworms and leaf damage ratings were given at 14d. Using a standard, 0-9, visual rating scale where 0 is no damage and 9 is almost complete destruction of the maize whorl, three of the experimental lines exhibited resistance to fall armyworm, similar to the resistant check, Mp708. Seed increases for all of the lines were made and a replicated study is being conducted in 2018. Two of the three apparently resistant experimental lines have similar pedigrees suggesting common resistance mechanisms. Both lines include parents that are maize germplasm originally collected from St. Croix, an island in the Caribbean Sea and constituent district of the United States Virgin Islands. A request was made to the USDA-ARS North Central Regional Plant Introduction Station to receive all available maize germplasm collected from the St. Croix area. This germplasm is being grown in a replicated trial to test for fall armyworm leaf feeding resistance in 2018. A doubled haploid set of maize lines were produced from a composite population. The composite population was developed by interbreeding four maize populations with parentage of Piura 208 (PI 503849) and a susceptible maize inbred. Piura 208 provides a new (non-maysin) source of silk feeding resistant to the corn earworm. In 2018, 1100 doubled haploid lines were successfully produced and grown in Ames, Iowa for seed increase. An additional round of seed increase will be necessary in winter nursery. These lines will permit genetic and biochemical dissection of the corn earworm resistance observed in Piura208.

Accomplishments
1. Risk factors for early-season sporadic insect pests. Neonicotinoid seed treatments are extensively used to protect against sporadic early-season insect pests, but there are growing concerns about consequences to the environment including pollinators. ARS scientists in Ames, Iowa led a multi-location effort to profile each pest listed on neonicotinoid seed treatment labels for corn, cotton, soybean, and wheat, focusing on frequency of infestations without preventative measures, and the factors that affect risk of an economic infestation. Risk factors can include region, soil type, topography, cropping landscape, planting date, rotation scheme, tillage, weeds, and many other variables. The goal was to help farmers make more-informed decisions about the need for seed treatments given the conditions at hand. The results are being used by the Environmental Protection Agency to help inform their cost-benefit analyses of this class of insecticide and are being used by university scientists to develop a decision support tool for farmers.

U.S. DEPARTMENT OF AGRICULTURE

Corn Insects and Crop Genetics Research: Ames, IA