Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests.

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 CrylAc. 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.

Liquid chromatographic determination of glyphosate and aminomethylphosphonic acid (AMPA) in environmental water: collaborative study.

A new method for determination of glyphosate and aminomethylphosphonic acid (AMPA) residues in environmental water was collaboratively studied by 6 laboratories. The method is simpler and shorter than previous methods. A filtered volume of water is evaporated to dryness and the residue is dissolved in a buffered EDTA solution. Glyphosate and AMPA are determined by liquid chromatography with postcolumn reaction detection. The method was validated over the range 0.50-5000 ppb, although one of the collaborating laboratories could not reliably quantitate below 1.0 ppb. Statistical analysis of the results showed that typical reproducibility relative standard deviations (RSDR) ranged from 11 to 20% for both glyphosate and AMPA, which compares very well with predicted values for this concentration range. Total variability (as measured by sR) increased with increasing fortification level. The method has been adopted official first action by AOAC.

Stereoselectivity of chloroperoxidase-dependent halogenation.

The stereoselectivity of chloroperoxidase halogenation of four substrates has been examined. Chloroperoxidase catalyzes the bromination, but not chlorination, of racemic 2-exo-methylbicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid (to the delta-lactone) and racemic bicyclo-[3.2.0]hept-2-en-6-one (to the 2-exo-bromo-3-endo-hydroxy-bromohydrin). These products are obtained in near quantitative yield and are racemic. The circumstances of the bromination strongly suggest that halogenation does not occur at the active site but rather by chloroperoxidase-catalyzed formation of Br2 and its release into solution. The inability of chloroperoxidase to halogenate these two alkenes at its active site most probably derives from a steric exclusion from the active site. The stereoselectivity of two additional substrates that undergo active site chlorination was determined. Methionine is quantitatively converted to a 50:50 ratio of the two methionine sulfoxide diastereomers. 2-Methyl-4-propylcyclopentane-1,3-dione is quantitatively chlorinated to 2-chloro-2-methyl-4-propylcyclopentane-1,3-dione. On the basis of optical rotation and proton nuclear magnetic resonance, this product is present as a 40:60 ratio of the racemic diastereomers. It is concluded that active site chlorination by chloroperoxidase proceeds without appreciable stereoselectivity.