Source: OHIO STATE UNIVERSITY submitted to NRP
REMEMBERING A FORGOTTEN PEST: BT RESISTANCE AND MONITORING IN EUROPEAN CORN BORER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1028991
Grant No.
2022-33522-37838
Cumulative Award Amt.
$499,928.00
Proposal No.
2022-03045
Multistate No.
(N/A)
Project Start Date
Sep 1, 2022
Project End Date
Aug 31, 2025
Grant Year
2022
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
(N/A)
Non Technical Summary
Transgenic crops have controlled insects for over 20 years. Recently, Bt crops have failed due to emerging resistance in many insects. Resistance threatens the durability and sustainability of a critical and valuable biotechnology control tactic for insects. We will investigate resistance in the European corn borer, Ostrinia nubilalis. European corn borer (ECB) was a classic example of successful control by Bt-corn. In 2018, Bt-corn expressing the toxin Cry1F failed to control ECB in 4 fields in Nova Scotia, Canada. This is the first ever report of a field failure by ECB. We suspect that the emergence of Cry1F resistance in Nova Scotia poses a severe risk for further spread across North America, as well as accelerates resistance evolution in the remaining Bt traits. This project will expand our understanding of Bt resistance in European Corn Borer to develop strategies that prevent resistance evolution and protect Bt durability. Our proposal focuses on 1) Estimating the risck and spread of Cry1F resistance; 2) Determine the potential and risk for additional Cry resistance in ECB; 3) Investigate the efficacy of other Cry proteins and cross resistance with Cry1F. Our team will implement an international and interdisciplinary approach to understand the risk that ECB resistance poses for the continued durability and sustainability of transgenic biotechnology.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110113070%
2113110107015%
2113110108015%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology; 1070 - Ecology; 1080 - Genetics;
Goals / Objectives
European Corn Borer (Ostrinia nubialis), was once the most important insect pest of corn in North America. Newer, transgenic corn with insecticidal proteins (known as Bt or Cry proteins) provided excellent control for almost 25 years, until resistance occured in Nova Scotian corn fields.We suspect that the emergence ofCry resistance in Nova Scotia poses a severe risk for further spread across North America, as well as accelerates resistance evolution in the remaining Bt proteins. Ouroverall project goal isexpand our understanding of Bt resistance in European Corn Borer, develop strategies that prevent resistance evolution and protect Bt durability. Our objectives are:1) Estimate risk and spread of Cry1F resistance2) Determine the potential and risk for additional Cry resistance in ECB3)Investigate efficacy of other Cry proteins and cross resistance with Cry1F
Project Methods
Objective 1. We will collect ECB from natural populations during the spring and autumn of 2023, 2024 and 2025. These will mostly include adults collected from pheromone traps or from sweep samples along roadsides near corn fields. Samples will be sent to the Michel Laboratory for DNA extraction and single nucleotide polymorphism (SNP) genotyping. Using previously established SNP markers that are linked with the Cry1F resistance phenotype, we will estimate the frequency of Cry1F resistance. When possible, we will cross any males and females and perform either F1 mating (with resistance individuals from University of Guelph colonies) or F2 screens.Objective 2. We will estimate the frequency of resistance to additional Cry proteins (Cry1Ab, Cry1A.105/Cry2Ab) using F2 screening of natural ECB populations. Live adults will be collected at several locations in the US and Canada and be sent to either the Michel or Smith laboratories. Adults will be placed in single pair mating cages, with the resulting F1 larvae being distributed in individual rearing plates or plastic cups. Pupae from this F1 generation will be sexed and set up in single pair mating cages. The resulting F2 generation will be screened using Cry protein assays against the 3 proteins. All larvae will be genotyped using our Cry1F molecular marker. In addition, we will perform laboratory selection on Cry1F resistant and susceptible colonies to determine if Cry1F resistance results in faster evolution for resistance to other proteins. These colonies will be exposed to increasing dosages of Cry protein using diet bioassays to select for resistance.Objective 3. We will determine the efficacy (e.g. LC50s) of the three other additional Cry proteins to control ECB. Using either field collected larvae or the F1 generation from collected adults, we will perform both diet bioassays with protein overlays and on-plant/leaf discs. Larvae will be monitored for mortality and data will be analyzed using standard probit analysis for mortality and efficacy.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Our target audience were researchers (academic, federal, provincial and industry), crop consultants and corn producers. Changes/Problems:ECBs were difficult to collect in 2024. However, we are still maintaining 1 colony collected from Ohio that we will assay in January 2025. Thees will be screened for Cry2Ab resistance, as well as for any SNPs linked to resistance. What opportunities for training and professional development has the project provided?This project is providing funding for Yamikani N'gona, a Ph.D. student in the Michel Laboratory. Yami is learning molecular genetic techiques, ECB sampling and collecting, and has made contacts with agricultural industry. How have the results been disseminated to communities of interest?We have provided several presentations in 2023-2024. Much ouf our work will be ready for publication in 2024-2035 What do you plan to do during the next reporting period to accomplish the goals?Objective 1:Analyzeexpression of ABCC2 genes in ECB (candidate genes for Cry1F resistance) Objective 1:Develop a CRISPR assay for ABCC2 gene knockdown Objective 2:F1 screens of ECB collected in 2024. Objcective 3: Screen Cry1F resistant larvae for suvival on other Cry proteins

Impacts
What was accomplished under these goals? Objctive 1: We genotyped European corn borers (ECB) throughout many locations in the US in August of 2023. These were either collected by the Michel laboratory or sent to our lab by collaborators. Combined with 2022 collections, we genotyped over 300 individuals at 6 single nucleotide polymorphism markers to estimate the frequency of resistant alleles. In addition, a subset of these individuals were sent to the University of Tennessee for genomic sequencing. The frequency of alleles linked to Cry1F resistance in Canada varied from <1% to greater than 90%. In 2023, we identified the allele most likely causing Cry1F resistance in individuals collected from New Hampshire, Ohio and Iowa. All individuals were heterozygous and the overall frequency across the US remained low. Our manuscript is in preparation with a submission goal of January 2025. Objective 2 and 3: -In October of 2023, our group assisted collecting ECB from Bt ears expressing Cry1A.105 and Cry2Ab2 in Connecticut (with the assistance of Dr. Kelsey Fisher). We were unable to establish a colony from our small number of collections but were able to genotype these individuals. These collections suggest that resistance to these other Cry proteins may be emerging. -In August of 2024, we collected 75 ECB larvae from Milan, Ohio. These individuals are stored under cold temperature and will form the basis of F2 screening in January of 2025. We will screen families for survival on Cry2Ab2, and for the presence of putative resistant SNP alleles.

Publications


    Progress 09/01/22 to 08/31/23

    Outputs
    Target Audience:Our target audience included researchers (academic, federal, provincial and industry), crop consultants and corn producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have assigned a PhD student, Yamikani Ng'Ona, to this project. Yamikani was also awarded a fellowship from the Foundation of Food and Agricultural Research, with matching funds from Bayer CropSciences. For Year 1 of the project, we also assigned a post-doctoral researcher (Dr. Yasmine Farhan) to work on Canadian collections. How have the results been disseminated to communities of interest?We have published our methodology for genotyping ECB samples. We have also presented our research at the Entomological Society of America-North Central Branch Annual Meeting. Our preliminary work was discussed at the annual meeting of Corn Entomologists (NC246:Ecology and Management of Arthropods in Corn). We have also had continuing conversations with Bayer CropSciences personnel. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: 1) Finish genotyping of 2022 and 2023 samples. Submit publication 2) Develop method for quick genotyping of Cry1F (LAMP-PCR based) Objective 2: 1) Attempt to establish colonies of 2023 collected samples and run bioassays for Bt resistance. 2) Expand collection effort in 2024 3)Measure ABCC2 gene expression in susceptible and resistant ECB Objective 3: 1) Run bioassays on Cry resistant ECB (Guelph) 2) Run bioassays on any ECB families with different arrangements of SNP genotypes

    Impacts
    What was accomplished under these goals? Objective 1: -We identified 6 molecular markers (single nucleotide polymorphisms and a deletion) that were associated with resistance to Cry1F in European corn borers collected in Canada. These polymorphisms caused amino acid changes in the ABCC2 gene, including a deletion that caused a premature stop codon. These were published in Farhan et al. 2023. -In 2022, we collected European corn borer from several areas in the northeastern US (3 populations from New York and 1 population from New Hampshire). These samples were sent to the Michel Lab for genotyping with the Cry1F molecular markers. We found alleles associated with resistance, but none of the over 250 samples carried the mutation that led to a premature stop. As resistance to Cry1F has not been detected in the US, our data supports the hypothesis that the deletion is the causal mutation. -In August of 2023, we collected European corn borer from similar sites in 2022 (New York and New Hampshire), with additional sites in Ohio and Connecticut. In this case, we not only collected adults, but we also managed to collect live larvae. We hope to establish populations in Year 2 of this project. We also collected additional populations in Eastern Canada, including from fields that may have unexpected injury on corn expressing Cry1A.105/Cry2Ab. Live larvae are being reared at the University of Guelph. -We have optimized DNA extractions from historical collections to test for the presence of resistance alleles. Objective 2: -We ran bioassays on samples collected in 2022 from Eastern Canada and the Maritime provinces. Objective 3: -We have made crosses with our resistant colonies and are beginning selection experiments.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2023 Citation: Farhan Y, Smith JL, Sovic MG, Michel AP. Genetic mutations linked to field-evolved Cry1Fa-resistance in the European corn borer, Ostrinia nubilalis. Sci Rep. 2023 May 18;13(1):8081. doi: 10.1038/s41598-023-35252-y. PMID: 37202428; PMCID: PMC10195855.