Influence of calcareous soil on Cry3Bb1 expression and efficacy in the field

Greater than expected injury by western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) to Cry3Bb1 expressing maize hybrids (Zea mays L.) has been reported in southwestern Nebraska. Affected areas of some fields are often associated with high pH calcareous soils where maize growth is poor and iron chlorosis is common. As part of a comprehensive study to understand potential causes of unexpected injury, experiments were conducted during 2013 and 2014 to ascertain whether the calcareous soil conditions and associated poor maize growth negatively affect the expression of Cry3Bb1. Quantitative determination of Cry3Bb1 protein expression levels in root tissues was carried out on plants at V5–V6 growth stage using the enzyme-linked immunosorbent assay. Cry3Bb1 and non-Bt near isoline maize hybrids were artificially infested with Cry3Bb1-susceptible WCR eggs to measure survival and efficacy of Cry3Bb1 maize in calcareous and noncalcareous soils. Results showed that there was not a significant difference in expression of Cry3Bb1 protein between plants from calcareous and non-calcareous soils (18.9–21.2 μg/g fresh weight). Western corn rootworm survival was about sevenfold greater from the non-Bt isoline than Cry3Bb1 maize indicating that Cry3Bb1 performed as expected when infested with a Cry3Bb1 susceptible rootworm population. When survival from calcareous and non-calcareous soils was compared, no significant differences were observed in each soil. A significant positive correlation between soil pH and expression of Cry3Bb1 protein in roots was detected from samples collected in 2014 but not in 2013. No such correlation was found between soil pH and survival of WCR. Results suggest that Cry3Bb1 expression levels were sufficient to provide adequate root protection against WCR regardless of soil environment, indicating that lowered Cry3Bb1 expression is not a contributing factor to the greater than expected WCR injury observed in some southwestern Nebraska maize fields. Includes supplementary materials

Larval survival and plant injury of Cry1Ab-susceptible, -resistant, and -heterozygous genotypes of the sugarcane borer on transgenic corn containing single or pyramided Bt genes

Transgenic corn (Zea mays L.) products expressing multiple Bt proteins targeting a same group of insect pests have become commercially available in the U.S. The first commercialized pyramided Bt corn technologies for managing lepidopteran pests include Genuity® VT Triple Pro™ and Genuity® SmartStax™. In this study, larval survival and plant injury of Cry1Ab-susceptible (Cry1Ab-SS), -resistant (Cry1Ab-RR), and -heterozygous (Cry1Ab-RS) genotypes of the sugarcane borer, Diatraea saccharalis F., on five commercial corn hybrids were evaluated in a leaf tissue bioassay and two greenhouse trials during 2010–2011. The five hybrids included two non-Bt corn and three Bt corn hybrids representing three transgenic technologies: YieldGard® Corn Borer, Genuity® VT Triple Pro™, and Genuity® SmartStax™. YieldGard® Corn Borer corn contains a single Bt protein (Cry1Ab), while Genuity® VT Triple Pro™ and SmartStax™ contain two and three pyramided Cry genes targeting above-ground lepidopteran pests, respectively. An average of 75.5% of larvae survived after 6 d on non-Bt corn leaf tissue. The 6-d survivorship on Cry1Ab corn leaf tissue was 3% for Cry1Ab-SS, 19% for Cry1Ab-RS, and 35% for Cry1Ab-RR larvae, while none of the three insect genotypes survived for 6-d on the leaf tissue of the two pyramided Bt corn hybrids. After 21 d on whole plants in the greenhouse, 42.6–62.5% of larvae survived on non-Bt corn plants. Larval survivorship rates on YieldGard® Corn Borer plants after 21 d in the greenhouse were 4.7–5.6% for Cry1Ab-SS, 29.4–32.5 % for Cry1Ab-RS, and 36.6–45.6% for Cry1Ab-RR. Both pyramided Bt corn hybrids were very effective against D. saccharalis regardless of the insect genotype with 21-d survivorship of \u3c2% for Cry1Ab-SS and Cry1Ab- RS, and \u3c5% for Cry1Ab-RR. Cry1Ab-RS and -RR caused significant entry/exit holes and tunneling inside the plant stalks of non-Bt and YieldGard® corn plants, while they caused little injury on the two pyramided Bt corn hybrids. The data generated from both the leaf tissue and whole plant tests showed that the Cry1Ab-resistant D. saccharalis was susceptible to the pyramided Bt corn hybrids. The results suggest that the pyramided Bt corn containing Genuity ® VT Triple Pro™ or SmartStax™ traits should offer a means for Cry1Ab resistance management in D. saccharalis

Characterizing the Relationship Between Western Corn Rootworm (Coleoptera: Chrysomelidae) Larval Survival on Cry3Bb1-Expressing Corn and Larval Development Metrics

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a significant pest of field corn, Zea mays L. (Poales: Poaceae), across the United States Corn Belt. Widespread adoption and continuous use of corn hybrids expressing the Cry3Bb1 protein to manage the western corn rootworm has resulted in greater than expected injury to Cry3Bb1-expressing hybrids in multiple areas of Nebraska. Single-plant bioassays were conducted on larval western corn rootworm populations to determine the level of resistance present in various Nebraska counties. The results confirmed a mosaic of susceptibility to Cry3Bb1 across Nebraska. Larval development metrics, including head capsule width and fresh weight, were measured to quantify the relationship between the level of resistance to Cry3Bb1 and larval developmental rate. Regression and correlation analyses indicate a significant positive relationship between Cry3Bb1 corrected survival and both larval development metrics. Results indicate that as the level of resistance to Cry3Bb1 within field populations increases, mean head capsule width and larval fresh weight also increase. This increases our understanding of western corn rootworm population dynamics and age structure variability present in the transgenic landscape that is part of the complex interaction of factors that drives resistance evolution. This collective variability and complexity within the landscape reinforces the importance of making corn rootworm management decisions based on information collected at the local level

Susceptibility of Cry1Ab Maize-Resistant and -Susceptible Strains of Sugarcane Borer (Lepidoptera: Crambidae) to Four Individual Cry Proteins

Sugarcane borer, Diatraea saccharalis (F.), is a major target of Bt maize in South America and many areas of the US mid-south region. Six laboratory strains of D. saccharalis were established from six single-pair F2 families possessing major resistance alleles to Cry1Ab maize hybrids. Susceptibility of the six strains was evaluated on diet treated with each of four purified trypsin-activated Bt proteins, Cry1Ab, Cry1Aa, Cry1Ac, and Cry1F. Bt susceptibility of the six strains was compared with that of known Cry1Ab-susceptible and -resistant strains of D. saccharalis. At least two of the six strains demonstrated a similar level (\u3e526-fold) of resistance to Cry1Ab as shown in the known Cry1Ab-resistant strain, while resistance levels were relatively lower for other strains (116- to 129-fold). All the six strains were highly cross-resistant to Cry1Aa (71- to 292-fold) and Cry1Ac (30- to 248-fold), but only with a low level to Cry1F (\u3c7-fold). Larval growth of all six strains was also inhibited on Bt-treated diet, but, except for Cry1F, the growth inhibition of the six strains was considerably less than that of the Cry1Ab-susceptible larvae. The results provide clear evidence that the observed resistance to Cry1Ab maize in the six strains is a result of resistance to the Cry1Ab protein in the plants. The low level of cross-resistance between Cry1A and Cry1F suggests that pyramiding these two types of Bt proteins into a plant could be a good strategy for managing D. saccharalis

Occurrence and larval movement of \u3ci\u3eDiatraea saccharalis\u3c/i\u3e (Lepidoptera: Crambidae) in seed mixes of non-\u3ci\u3eBt\u3c/i\u3e and \u3ci\u3eBt\u3c/i\u3e pyramid corn

Background: Larval movement of target pest populations among Bt and non-Bt plants is a major concern in the use of a seed mixture refuge strategy for Bt resistance management. In this study, occurrence and larval movement of the sugarcane borer, Diatraea saccharalis (F.), were evaluated in four planting patterns of non-Bt and Bt plants containing Genuity® SmartStax™ traits in 2009– 2011. The four planting patterns were: (1) a pure stand of 27 Bt plants; (2) one non-Bt plant in the center, surrounded by 26 Bt plants; (3) a pure stand of 27 non-Bt plants; (4) one Bt plant in the center, surrounded by 26 non-Bt plants. Studies were conducted under four conditions: (1) open field with natural infestation; (2) greenhouse with artificial infestations; open field with artificial infestations (3) on the center plants only and (4) on every plant. The major objective of this study was to determine whether refuge plants in a seed mixture strategy could provide a comparable refuge population of D. saccharalis to a “structured refuge” planting. Results: Larvae of D. saccharalis showed the ability to move from infested plants to at least four plants away, as well as to adjacent rows, but the majority remained within the infested row. However, the number of larvae found on the non-Bt plants in the mixture plantings was not significantly reduced compared with the pure stand of non-Bt corn. Conclusion: The results of this study show that refuge plants in a seed mixture may be able to provide a comparable refuge population of D. saccharalis to a structured refuge planting