Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Entomology
Non Technical Summary
Transgenic Bt crops (e.g. corn, cotton) have been widely planted for pest management in the United States. Crop growers have gained substantial benefits by planting Bt crops. However, the rapid adoption of Bt crops also places a great threat to the sustainability of the technology because high selection pressure can accelerate resistance development in pest populations. Studies have shown that, due to the intense use of Bt crops, resistance alleles to single-gene Bt crops in several target pests has significantly increased. To date, field resistance that has resulted in control problems has been documented in at least 22 cases.The corn earworm (CEW) is considered to be the most costly crop pest in North America. In the southern region, where cotton is also planted, CEW, after corn senescence, also moves to other hosts, notably cotton, grain sorghum and soybean, for 2-3 additional generations. There is the potential for multiple exposures across generations to Bt proteins in both Bt corn and Bt cotton. During the past two decades, the rapid and large scale adoption of Bt crops has led to the development of resistance in CEW to Cry1A/Cry2A sweet corn in Maryland and to Cry1/Cry2 cotton and Cry1A/Cry2A corn in the U.S. southern region.To delay resistance development, the U.S. has adopted an insect resistance management (IRM) plan, known as the 'high dose/refuge' strategy, for planting Bt crops. Before 2010, only a 'structured refuge' strategy was approved for Bt corn IRM. However, growers' compliance with the structured refuge requirement has been an issue. Because of the compliance issues with the use of the "structured refuge" strategy, plus the availability of the pyramided Bt corn, the U.S. EPA approved a seed blend refuge strategy for planting pyramided Bt corn in the U.S. Corn Belt. The currently used seed blend strategy in the U.S. Corn Belt is at a rate of 95:5% Bt and non-Bt corn seeds. However, seed blend refuge has not been approved in the southern region of the U.S. where cotton is also planted. Several mathematical models have shown that the pyramiding/seed blend could be effective if the assumptions are met. One of these assumptions is that larval movement in the field is limited, which is usually not true for most pests. Comprehensive, scientific data to support the pyramiding/seed blend strategy are still lacking. Although there have been numerous studies related to Bt crop IRM during the past 20+ years, predominance of these studies have dealt with only single-gene resistance. Recently, there have been a few studies related to dual- or multiple-gene Bt resistance.In 2016 and 2017, notable ear damage and larval survival of CEW were observed on pyramided Cry1A.105/Cry2Ab2 corn plants in some of our experimental fields in northeast Louisiana. Results of initial bioassays showed that the LC50s of Cry1A.105 and Cry2Ab2 for the populations collected from the areas with control problems were as much as >909- and >25-fold higher than that of a known susceptible strain, respectively. In the proposed project, many more field CEW populations will be collected across the major corn planting areas in Louisiana. Laboratory bioassays and field trials will be conducted to document the current status of CEW resistance to Cry1A/Cry2A Bt corn in the state (Objective 1). A relatively new protein, Vip3A, has been incorporated into both Bt corn and Bt cotton. Vip3A has a different mode of action from the Cry1 and Cry2A proteins in the plants. Because of the occurrence of the CEW resistance to Cry1 and Cry2 proteins in the U.S., Vip3A becomes the only Bt protein in both Bt corn and Bt cotton that is still fully active against the Cry1A/Cry2A-resistant CEW in the U.S. Thus, preservation of the Vip3A susceptibility is essential for the sustainable use of Bt crop technology. There is no published data available about the baseline susceptibility of CEW populations to the Vip3A protein. In the proposed project, CEW larvae will be collected from corn fields across the U.S. southern region, and laboratory bioassays will be conducted to determine the Vip3A susceptibility to establish the baseline data for resistance monitoring in the region (Objective 2).A few recent studies have investigated the field resistance of CEW to Bt corn, but resistance allele frequency of CEW populations to the Bt proteins in the plants are still unknown, largely due to the difficulty in pairing, mating, and reproduction of CEW in the laboratory conditions. In recent years, we have tested several methods to improve the success of adult mating and reproduction of CEW. Based on the biology and behavior of CEW, we recently developed a modified F2 screening method that can be used to detect the Bt resistance alleles in the insect. In the proposed study, we will use our modified F2 screening method to detect resistance alleles and thus to estimate the resistance allele frequency in field CEW populations in the southern region to four common Bt proteins expressed in Bt corn: Cry1Ab, Cry1A.105, Cry2Ab2, and Vip3A protein (Objective 3).As mentioned above, the seed blend strategy of non-Bt and Bt corn has been used to provide susceptible refuge insect populations for IRM in the northern U.S. Corn Belt. Another major concern for the use of seed blends is larval movement among Bt and non-Bt plants in the seed blend may reduce the survival of the refuge insect population. Empirical data addressing this great concern related to CEW, the most important ear feeding target pest of the 2nd generation Bt corn, are still lacking. In the proposed study, we will conduct field cage trials to investigate larval movement and survival of CEW in seed blends (Objective 4). Recently, a single-gene Cry1A.105-resistant strain and a single Cry2Ab2-resistant strain of CEW have been isolated using the F2 screening method. By crossing these two single-gene resistant strains, a Cry1A.105/Cry2Ab2 dual gene resistant strain of CEW is being developed for the proposed project. The availability of the single-gene and dual-gene Bt resistant strains of CEW will provide an opportunity to test the phenotypic performance of different insect genotypes containing 0-4 resistance alleles. Objective 5 of the proposed project will employ laboratory assays to evaluate the phenotypic performance of all nine possible genotypes of a dual-gene Bt resistance in CEW on pyramided Bt plants. With the availability of the Cry1A.105/Cry2Ab2-dual gene Bt resistant strain of CEW mentioned in objective 5, we will conduct laboratory bioassays to evaluate the performance of heterozygous and homozygous genotypes of CEW possessing Cry1A.105/Cry2Ab2 dual-resistant genes on common single and pyramided Bt corn traits to identify Bt traits that can be used for managing the Cry1A/Cry2A resistance in CEW (Objective 6).The overall goal of this research project is to ensure the long-term success of transgenic Bt corn technology as a corn insect management tool for corn growers in Louisiana and other states of the U.S. southern region. The proposed project will include a series of studies and trials to provide supporting data to improve the current Louisiana Cooperative Extension Service recommendations. Results generated from this project also will provide essential information to researchers, industry, and regulatory agencies for developing effective IRM strategies for the sustainable use of transgenic Bt-crop technology. In addition, studying the role of Bt crop technology on an area-wide (landscape) basis will enhance feasibility of this project. Data from this study will result in popular articles and peer-reviewed publications. Results generated also will be presented at local, regional, national, and international meetings.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
The overall goal of this research project is to ensure the long-term success of transgenic Bt corn technology as a corn insect management tool for corn growers in Louisiana and other states of the U.S. southern region. The proposed project will include a series of studies and trials to provide supporting data to improve the current Louisiana Cooperative Extension Service recommendations. Results generated from this project also will provide essential information to researchers, industry, and regulatory agencies for developing effective IRM strategies for the sustainable use of transgenic Bt-crop technology. In addition, studying the role of Bt crop technology on an area-wide (landscape) basis will enhance feasibility of this project. Data from this study will result in popular articles and peer-reviewed publications. Results generated also will be presented at local, regional, national, and international meetings. The objectives are:Documenting corn earworm resistance to Cry1A/Cry2A Bt corn in Louisiana;Establishing baseline susceptibility of corn earworm populations to Vip3A in the southern region of the U. S.;Estimating resistance allele frequency in field populations of corn earworm to four common Bt proteins expressed in Btcorn: Cry1Ab, Cry1A.105, Cry2Ab2, and Vip3A;Investigating larval survival and movement of corn earworm in seed blends of non-Bt and Bt corn containing Viptera® trait;Evaluating phenotypical performance of nine genotypes of corn earworm possessing 0-4 Cry1A.105/Cry2Ab resistancealleles on MON 89034 corn;Examining larval survival and ear damage of Cry1A.105/Cry2Ab2-susceptible and -resistant populations of corn earworm onnon-Bt and Bt corn containing single- or pyramided Bt genes.
Project Methods
Obj. 1. CEW will be collected across the major corn areas in Louisiana. Field-collected larvae will be reared in 30-ml cups containing a meridic diet. Purified Cry1A.105 and Cry2Ab2 proteins will be provided by Bayer Crop Sciences (St. Louis, MO). Susceptibility of the F1 or F2 CEW to Bt proteins will be determined using a diet over-lay bioassay method. Larval mortality will be recorded on the 7th day after neonate release. In each bioassay, there will be four replications with 16-32 larvae in each replicate. Larval morality data will be subjected to probit analysis to calculate the median lethal concentrations. The number of larvae, larval development index, and area of kernel injury recorded from the field trails will be analyzed using a one-way analysis of variance (ANOVA) with the maize hybrid as the main factor.Obj. 2. CEW will be collected across the major corn areas across the U.S. southern region. Field-collected larvae will be reared in the meridic diet as described above. Vip3A protein will be provided by Syngenta Biotechnology (Research Triangle Park, NC). Susceptibility of F1 or F2 generation of the field-collected CEW to Vip3A will be determined using the diet over-lay bioassays as mentioned above. LC50s generated from this study will be used as baseline susceptibility data for resistance monitoring in the future.Obj. 3. The overall procedures of the F2 screen for detecting the resistance alleles to the four Bt proteins will be similar as used in our previous studies for detecting the Cry1Ab resistance alleles in the sugarcane borer, and Cry1F and Cry1A.105 resistance alleles in the fall armyworm. For the F2 screen, 3rd to 5th instars of CEW will be collected from corn fields across the southern region of the U. S. Field-collected larvae will be reared on the meridic diet until the pupal stage. Newly emerged males and newly emerged females will be placed in each mesh cage. Two days after adult emergence, females will be removed from the adult cages and placed individually into 3.8-10 L paper containers for oviposition. Ninety F1 larvae of each family-line will be reared on a meridic diet. All F1 pupae of each family-line will be placed in mesh cages. Eggs of F2 families will be used for the F2 screen for detecting resistance alleles to the four Bt proteins. The F2 screen for detecting resistance alleles to the four Bt proteins will be conducted in 128-cell trays containing a surface overlaying diagnostic dose of 5 µg/cm2 for Vip3A and 10 µg/cm2 for the other three proteins. For each F2 family line, a total of 128 F2 neonates will be screened. The number of live larvae and larval stage will be recorded at the 7th day after inoculation. To confirm if a potential resistant family identified in the F2 screen actually possesses resistance alleles, laboratory strains of the potential resistant family will be established from the survivors in the F2 screen. These strains will be re-evaluated in laboratory bioassaysand for larval survival on whole Bt corn plants in the greenhouse or on ears removed from field-grown plants.The Bayesian mode will be used to calculate the resistance allele frequencies to the Bt proteins.Obj. 4.F1 progeny of field populations from non-Bt sweet corn plants will be used as the insect source for the study. A corn hybrid containing the Agrisure Viptera®3111 trait and a closely related non-Bt corn hybrid will be obtained from Syngenta for the study. A total of four trials will be conducted at the LSU AgCenter's Stations. For each of the trials, eight planting patterns will be evaluated with 5 rows and 21 plants in a row in each planting pattern. The eight planting patterns will be arranged in a randomized complete block design with four replications. At the R1 plant stage, 35 F1 neonates of a field-collected CEW population will be manually placed on ear silks of the central plant in each plot. Number of live larvae and larval stages on each ear, as well as area of ear kernel injury in cm2 will be recorded when the majority of larvae on non-Bt plants reach the 4th -5th instars. The number of live larvae per ear will be analyzed using two-way ANOVA with planting pattern and DC/row as the two main factors.Obj. 5.MON 89034 traits expressing Cry1A.105/Cry2Ab2 and genetically closely-related non-Bt corn hybrids will be provided by Bayer Crop Sciences. Bt corn and the related non-Bt corn hybrids will be planted in open-fields at LSU AgCenter's Research farms. Four CEW strains that are currently maintained in the PI's laboratory will be used as the original insect sources in the study. In addition, reciprocal crosses among the four original strains will be conductedto generate five F1 heterozygous genotypes containing 1-3 resistance alleles.In each trial, corn ears of non-Bt and Bt plants, along with shanks and husks, will be collected from open-field plots at R1-R2 stages. Five neonates of a CEW genotype will be placed on the silks of the ear and the ear containing neonates will be placed into 5.7L plastic containers with paper towel underneath. The insect assay containers will be placed in the insect rearing room described above. Neonate-to-larval survival, pupation time, pupal body weight, number of pupae produced from each ear, and ear kernel damage area will be recorded. For each combination of corn hybrid and insect genotype, there are four replications with 50 ears in each replication. We expect to use at least 15 males and 15 females for each pupal group (replication) for measuring the reproductive effects. These pupae will be placed in a 20-L mesh cage. The cages will be maintained in insect rearing room. Eggs laid on the gauze cloth that is placed on the top of the cage will be harvested daily and counted by visual observation. Data on the number of pupae produced, pupal body weight, neonate-to-pupa developmental time, and number of progeny produced will be analyzed using two-way ANOVA. In addition, the phenotypic performance of a genotype containing 1-3 resistance alleles in the dual-gene resistance system will be also assessed using a similar method to that used in estimating the effective (functional) dominance levels (DML) of resistance in a single-gene system.Obj. 6. A Bt-susceptible (S1S1S2S2), a Cry1A.105/Cry2Ab2 dual-gene resistant (R1R1R2R2), and an F1 heterozygous (R1S1R2S2) lines will be tested in this study. Common commercial Bt corn traits will be included in the tests. Corn hybrids will be planted in the open fields at LSU AgCenter's farms. Ears including shanks and husks will be collected from fields at R1-R2 stages and five neonates of a genotype will be infested on the silks of each ear. Ears containing neonates will be placed in the insect rearing rooms as described above. For each treatment combination of corn hybrid and insect genotype, there will be four replications with 10 ears in each replication. Biological parameters measured will include neonate-to-pupal developmental time, neonate-to-pupation rate, pupal body weight, and ear kernel damage area. Data on neonate-to-pupation rate, pupal body weight, neonate-to-pupa developmental time, and area of kernel injury will be then analyzed using two-way ANOVA with insect genotypes and corn hybrids as the two main factors.