John T. Greenplate

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Examination of midgut luminal proteinase activities in six economically important insects

A biochemical survey of insect midgut fluid proteinase activities was conducted to determine which proteinase inhibitors could play roles in insect control strategies. In this study, midgut juices from six insects of economic importance have been isolated and their proteolytic activities classified. Three lepidopterans (black cutworm, corn earworm, and tobacco budworm) and the boll weevil have maximal midgut fluid proteolytic activity in vitro at pH 10–11. Soybean trypsin inhibitor inhibits lepidopteran and boll weevil activities by 63–72%, indicating that their major proteinases are “trypsin-like” serine proteinases. Varying levels of inhibition were seen against the midgut fluid proteinase activities whereas bovine trypsin was inhibited 95–98% by each of four different trypsin inhibitors. Southern corn rootworm and Colorado potato beetle have maximal midgut fluid proteolytic activity in vitro at pH 6–7. E-64 inhibits southern corn rootworm (69%) and Colorado potato beetle (100%) midgut fluid proteinase activity, demonstrating that their major activities are provided by cysteine proteinases. Feeding studies found no mortality nor stunting associated with feeding high levels of trypsin inhibitors to boll weevil or tobacco budworm larvae despite the fact that these proteins were very good inhibitors 60–78% inhibition) of midgut proteolytic activity in vitro.

Toxicity and Characterization of Cotton Expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 Proteins for Control of Lepidopteran Pests

Abstract Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with Cry1Ac. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S. frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30–80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S. frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20–69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.

Resistance Monitoring of Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis Insecticidal Protein in China

Abstract Sensitivities of Helicoverpa armigera (Hübner) field populations to Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac were monitored during 1998–2000 in China. A total of 41 strains was sampled, and most of them were collected from Bt cotton planting regions. The range of IC50 values (concentration producing 50% inhibition of larval development to third instar) among different populations in 1998, 1999, and 2000 were 0.020–0.105 μg/ml, 0.016–0.099 μg/ml, and 0.016–0.080 μg/ml, respectively. Diagnostic concentration studies (IC99) showed that the percentage of individuals reaching third instar ranged from 0 to 4.35%, with only eight of the 41 tested populations showing values above 0%. Also interesting was a trend over successive years in which fewer populations contained individuals that survived the diagnostic concentration by reaching third instar. Considering these data, it was determined that the field populations sampled during the 3-yr study were susceptible to Cry1Ac protein, and that movement toward resistance among H. armigera populations was not apparent.

The design and implementation of insect resistance management programs for Bt crops

Cotton and corn plants with insect resistance traits introduced through biotechnological methods and derived from the bacterium Bacillus thuringiensis (Bt) have been widely adopted since they were first introduced in 1996. Because of concerns about resistance evolving to these Bt crops, they have been released with associated IRM programs that employ multiple components and reflect the input of academic, industrial and regulatory experts. This paper summarizes the current status of Bt crop technologies in cotton and corn, the principles of IRM for Bt crops and what they mean for the design of IRM programs. It describes how these IRM programs have been implemented and some of the key factors affecting successful implementation. Finally, it suggests how they may evolve to properly steward these traits in different geographies around the world. The limited number of reported cases of resistance after more than 15 years of intensive global use of Bt crops suggest that this exercise has been broadly successful. Where resistance issues have been observed, they have been associated with first generation technologies and incomplete or compromised IRM programs (i.e., inadequate structured refuge). Next generation technologies with multiple pyramided modes of action, together with the implementation of IRM strategies that are more dependent upon manufacturing and less dependent upon grower behavior, such as seed mixes, should further enhance IRM programs for Bt crops.

Cholesterol oxidase: an oöstatic and larvicidal agent active against the cotton boll weevil, Anthonomus grandis

The enzyme cholesterol oxidase (E.C. 1.1.3.6), purified from Streptomyces culture filtrate was previously found to have oral insecticidal activity on neonate larvae of the boll weevil (Anthonomus grandis grandis Boheman) from a laboratory population. In the present study, second instar larvae were also controlled by the enzyme at diet concentrations similar to those which control neonates (12 day LC50 = 2.4 μg.ml−1 in diet). Larvae from field‐collected adults were similarly susceptible to cholesterol oxidase in the diet. When ingested by adult females during the mating/pre‐oviposition period, cholesterol oxidase greatly reduced subsequent oviposition (83% reduction in eggs laid as compared to controls) and larval survival (97% reduction from controls). Dissection of treated adult females revealed poorly developed ovaries and few developing oöcytes. These studies were conducted to further evaluate the utility of cholesterol oxidase in a program to establish boll weevil‐resistant transgenic cotton.

Field-Evolved Resistance: Assessing the Problem and Ways to Move Forward

ABSTRACT “Field-evolved resistance” is defined as a “genetically based decrease in susceptibility of a population to a toxin caused by exposure to the toxin in the field.” The key component of “field-evolved” resistance is that it does confer decreased susceptibility to an insecticide in the field. Another key component is that the decrease in susceptibility to the insecticide is because of previous exposure of the target insect to the toxin in the field. Several studies have reported field-evolved resistance to crops engineered to express proteins from the bacterium, Bacillus thuringiensis (Bt). However, there has not been a consistent standard in the application of the definition of field-evolved resistance for Bt crops. The inconsistency in applying the definition arises from differences in the methods used to detect resistance, the ecology of the interaction between the pest and the Bt crop, and the effective dose the pest encounters while feeding on the Bt crop. Using case studies of reported resistance to Bt crops, it is demonstrated resistance does not come in a single form, and that in most cases, resistance can still be managed.

Studies on the mode of action of cholesterol oxidase on insect midgut membranes

Cholesterol oxidase (EC 1.1.3.6.) is an insecticidal protein known to have potent activity against the boll weevil, milder activity against a number of lepidopteran species, and virtually no activity against other insects. Several factors that could explain its species-dependent differential activity were examined. We compared cholesterol concentrations and rates of cholesterol oxidation in the midgut membranes from larvae of boll weevil (Anthonomus grandis grandis Boheman), southern corn rootworm (Diabrotica undecimpunctata howardi Barber), tobacco budworm (Heliothis virescens Fabricius), and yellow mealworm (Tenebrio molitor Linnaeus). Results showed that cholesterol concentration alone could not account for the differences in insecticidal activity and that midgut brush-border membranes of all species tested were generally susceptible to oxidation by cholesterol oxidase in vitro. We also demonstrated that cholesterol oxidase stability in the midgut environment was similar for the species tested and thus could not account for the differential activity. However, comparison of the pH of the insect midgut fluids with the pH optimum of cholesterol oxidase indicated that the lower sensitivity of lepidopteran larvae to the enzyme may be partially due to the alkaline nature of their midgut environments. In some species, oxidation caused significant changes in the activities of brush-border membrane alkaline phosphatase, and these changes did correlate with the susceptibility of the insect to cholesterol oxidase. Arch. Insect Biochem. Physiol. 34:429–442, 1997.

The economic impacts of second generation Bt cotton in West Africa: empirical evidence from Burkina Faso

West Africa has been slow in adopting agricultural biotechnology. The most progressive stance has been taken by Burkina Faso, which began field testing Bt cotton in 2003. This paper reports the first three years of Bt cotton field trials, which found that Bt cotton increased cotton yields by an average of 20% and reduced insecticide applications by two-thirds. While the technical success of Bt cotton is encouraging, the tests were confined to small scale plots and unit returns found on the experiment station. This paper extrapolates the field test results to the national scale using an economic model, which predicts the economic impacts of introducing Bt cotton in the Burkina Faso cotton sector. The model results found that Bt cotton would generate benefits of

Characterization of the Activity Spectrum of MON 88702 and the Plant-Incorporated Protectant Cry51Aa2.834_16

106 per year under typical pest density conditions.