ABSTRACT

A technological milestone in genetic engineering resulted in the production and deployment of Bollgard® cotton varieties expressing the Cry1Ac protein, for the control of certain lepidopterous pests of cotton. Commercially available in several varieties since 1996, this product has provided an effective and specific alternative to the use of synthetic insecticides for the control of tobacco budworm, Heliothis virescens; cotton bollworm, Helicoverpa zea; and pink bollworm, Pectinophora gossypiella. In an attempt to increase efficacy, expand spectrum of activity, and mitigate or postpone the development of resistance, a stacked product, Bollgard II^TM, has been developed, expressing both Cry1Ac and Cry2Ab2 proteins. The combined activity of the two proteins in Bollgard II^TM provides increased efficacy against the budworm/bollworm complex, enhanced spectrum of activity against beet armyworm, Spodoptera exigua; and fall armyworm, Spodoptera frugi[perda (Table 1). The Environmental Protection Agency recently granted registration for this product, and Bollgard II^TM will be launched in the 2003 cotton-growing season.

Bollgard® cotton has consistently provided outstanding control of the tobacco budworm, Heliothis virescens; and pink bollworm, Pectinophora gossypiella across the cotton belt, in a diverse number of cotton varieties (Perlak et al., 2001). Although the Cry1Ac protein has very good activity against all budworm/bollworm complex of pests, it has been well demonstrated that especially under high infestations, bollworm larvae have been found feeding in fresh flowers and on small bolls under the bloom tags of Bollgard® cotton (Brickle et al., 2001) and more recently in Bollgard IITM cotton (Gore et al., 2001).

The study was designed to evaluate the expression profile and bioactivity of different parts of the cotton flower relative to vegetative parts. Large leaves, terminal leaves, squares, flowers and small bolls (under bloom tags) were sampled from three isolines, DP50, DP50B (Bollgard®) and DP50B II (Bollgard II^TM), and the flowers were teased out into the bracts, calyx, petals, anthers, and ovules. These tissue types were freeze-dried, finely powdered and utilized in all assays. The ELISA and the tobacco budworm quantitative bioassay (Greenplate, 1999) were conducted to study the expression profile. Activity against bollworm larvae was ascertained using a diet-based assay, where the tissue in agar was overlaid on diet (2% tissue in 0.2% agar), and infested with first instar larvae. Readings were taken seven days after infestation.

All data (weight data, quantitative ELISA and the tobacco budworm quantitative bioassay data) were subjected to a two-way analysis of variance using PROC GLM (SAS, Version 8), and means for each treatment were separated (P<0.05) using Fisher's Protected Least Significance. Bollworm bioassay results were analyzed using the mean weights of surviving larvae (Table 2). All Bollgard® and Bollgard II^TM tissues gave significantly lower weight of survivors compared to DP50. Mean weight of surviving larvae fed on Bollgard IITM were significantly lower in weight relative to those fed on Bollgard®, for all tissues. None of the Bollgard II^TM survivors developed past the second larval stage, seven days post infestation, demonstrating that it would provide a high degree of bollworm control.

Results obtained using ELISA (Table 3) and quantitative bioassays (Table 4) show that all tissues under investigation expressed the Cry1Ac alone or in combination with the Cry2Ab2 proteins, and in many instances, at levels comparable with expression in terminal leaf or square tissues, which are customarily sampled. The results from the quantitative bioassay showed that both Bollgard® and Bollgard II^TM did not show significant differences in expression profile among the nine tissue types that were studied. Significantly higher lepidopteran activity was demonstrated by Bollgard IITM relative to Bollgard®, across all tissue types, clearly demonstrating the added value provided by the Cry2Ab2 gene in Bollgard II^TM.

These studies have clearly demonstrated that most of the floral tissue types examined express the relevant proteins at levels comparable to those found in leaf tissues. It is well understood that Bollgard® provides good but not complete control of the bollworm. Field observations on surviving bollworm larvae in blooms and bloom tags could at least in part be the result of the preferential feeding of bollworm larvae on these plant parts. These and all other studies in our laboratories have shown that mortality measurements alone do not provide the whole picture when considering in planta control provided by Bacillus thuringiensis toxins. Damage to plant parts and developmental data on surviving larvae should be taken into account before arriving at conclusions with regard to spray thresholds or economic injury. Clearly, Bollgard II^TM would provide significantly superior control of bollworms and also has added value from the standpoint of resistance management.