Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests.

Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.

An Alcaligenes strain emulates Bacillus thuringiensis producing a binary protein that kills corn rootworm through a mechanism similar to Cry34Ab1/Cry35Ab1.

Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.

A selective insecticidal protein from Pseudomonas for controlling corn rootworms.

The coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.

Western corn rootworm (Coleoptera: Chrysomelidae) dispersal and adaptation to single-toxin transgenic corn deployed with block or blended refuge.

A simulation model of the temporal and spatial dynamics and population genetics of western corn rootworm, Diabrotica virgifera virgifera LeConte, was created to evaluate the use of block refuges and seed blends in the management of resistance to transgenic insecticidal corn (Zea mays L.). This Bt corn expresses one transgenic corn event, DAS-59122-7, that produces a binary insecticidal protein toxin (Cry34Ab1/Cry35Ab1) and provides host-plant resistance. The model incorporates the latest information about larval and adult behavior. Results of this modeling effort indicate that the seed-blend scenarios in many cases produced equal or greater durability than block refuges that were relocated each year. Resistance evolved in the most likely scenarios in 10-16 yr. Our standard analysis presumed complete adoption of 59122 corn by all farmers in our hypothetical region, no crop rotation, and 100% compliance with Insect Resistant Management (IRM) regulations. As compliance levels declined, resistance evolved faster when block refuges were deployed. Seed treatments that killed the pest when applied to all seeds in a seed blend or just to seeds in Bt corn blocks delayed evolution of resistance. Greater control of the pest population by the seed treatment facilitated longer durability of the transgenic trait. Therefore, data support the concept that pyramiding a transgenic insecticidal trait with a highly efficacious insecticidal seed treatment can delay evolution of resistance.

Effect of Bacillus thuringiensis cry3Bb1 protein on the feeding behavior and longevity of adult western corn rootworms (Coleoptera: Chrysomelidae).

The first transgenic corn hybrids expressing the Bacillus thuringiensis (Bt) Cry3Bb1 protein to control corn rootworm (Diabrotica spp.) larvae were registered for commercial use in 2003. This study was conducted to investigate the effect of Cry3Bb1 protein in combination with a cucurbitacin bait on adult feeding and longevity of both organophosphate-resistant and -susceptible western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). In choice and no-choice tests, possible repellency to the Bt protein was quantified by comparing beetle consumption of cellulose disks treated with three concentrations of Bt in combination with a feeding stimulant (Invite EC) to disks treated with stimulant alone. A lethal-time assay also was conducted to examine survival of beetles exposed to Bt protein in their diet. Results from these assays indicate that adult rootworms are not significantly deterred by the presence of Cry3Bb1 on the treated discs and that ingestion of toxin does not adversely affect adult longevity.

Node-injury scale to evaluate root injury by corn rootworms (Coleoptera: Chrysomelidae).

Corn rootworm larval feeding on corn roots can significantly reduce grain yield by interfering with photosynthetic rates, limiting the uptake of water and nutrients, and by increasing the plant's susceptibility to lodging. Of the techniques developed to measure the efficacy of corn rootworm larval control tactics, root damage ratings have generally been adopted as the standard because sampling roots is relatively efficient. Historically, the primary scales used for scoring root injury from corn rootworm larval feeding have been the 1-6 and 1-9 scales. A critical deficiency of those scales, however, is that each increase in a root-rating score does not reflect a linear increase in the actual amount of injury to the root system. This results in injury scores that are expressed qualitatively. We developed the node-injury scale to more accurately quantify corn rootworm larval injury based on the proportion of nodal roots that contain feeding injury. With the node-injury scale, the relationship between the numerical scale and the amount of root injury is linear and intuitive. In this article, we describe the node-injury scale, discuss sampling issues to consider when using the scale, and suggest the minimum node-injury score that causes economic damage under varying degrees of environmental stress.

In-field labeling of western corn rootworm adults (Coleoptera: Chrysomelidae) with rubidium.

Field and laboratory studies were conducted in 2000 and 2001 to determine the feasibility of mass marking western corn rootworm adults, Diabrotica virgifera virgifera LeConte, with RbCl in the field. Results showed that application of rubidium (Rb) in solution to both the soil (1 g Rb/plant) and whorl (1 g Rb/plant) of corn plants was optimal for labeling western corn rootworm adults during larval development. Development of larvae on Rb-enriched corn with this technique did not significantly influence adult dry weight or survival. Rb was also highly mobile in the plant. Application of Rb to both the soil and the whorl resulted in median Rb concentrations in the roots (5,860 ppm) that were 150-fold greater than concentrations in untreated roots (38 ppm) 5 wk after treatment. Additionally, at least 90% of the beetles that emerged during the first 3 wk were labeled above the baseline Rb concentration (5 ppm dry weight) determined from untreated beetles. Because emergence was 72% complete at this time, a significant proportion of the population had been labeled. Results from laboratory experiments showed that labeled beetles remained distinguishable from unlabeled beetles for up to 4 d postemergence. The ability to efficiently label large numbers of beetles under field conditions and for a defined period with virtually no disruption of the population provides an unparalleled opportunity to conduct mark-recapture experiments for quantifying the short-range, intrafield movement of adult corn rootworms.

Effects of row spacing and plant density on corn rootworm (Coleoptera: Chrysomelidae) emergence and damage potential to corn.

Planting corn, Zea mays L., in row spacings less than the conventional width of 76 cm has been shown to increase grain yields. This study was conducted to determine if row spacing and plant density affected corn rootworm, Diabrotica virgifera virgifera LeConte and D. barberi Smith & Lawrence, adult emergence, larval injury to the roots, and plant tolerance to injury. Field experiments were conducted at Ames and Nashua, IA, in 1998, 1999, and 2000. Treatments were row spacings of 38 and 76 cm, and plant populations of 64,500 and 79,600 plants per hectare. Adult emergence was 31% greater in 38 cm compared with 76-cm rows. However, root injury was not significantly different between row spacings or plant populations. Row spacing alone did not significantly influence tolerance to injury, measured as root size and the amount of root regrowth. However, at one environment where precipitation was low, plants in 38-cm rows produced 25% more regrowth compared with plants in 76-cm rows. Root dry weight and regrowth were suppressed by 16 and 32%, respectively, at the high plant population. Although lodging was 51% lower in the 38-cm rows compared with the 76-cm rows, grain yields were not significantly different between row spacings. Reducing the row spacing of field corn from 76-38 cm should not increase the potential for injury from corn rootworm larvae.