Quantification of Cry1Ac and Cry1F Bacillus thuringiensis insecticidal proteins in selected transgenic cotton plant tissue types.

Quantitative enzyme-linked immunosorbent assays were used to characterize the geographical (locations) and temporal (through 6 wk) expression of CrylAc, from Bacillus thuringiensis variety kurstaki, and Cry1F, from B. thuringiensis variety aizawai, in transgenic cotton, Gossypium hirsutum L., plant structures. Terminal leaves, squares (flower buds), flowers, bolls (fruit), and mature leaves located five and eight nodes below the terminal apex were sampled during weeks 2, 4, and 6 after the initiation of anthesis. The effect of location (environment) significantly influenced protein expression levels, although similar trends were observed across locations. Cry1F was expressed at levels greater (1.1-29.0-fold) than that for CrylAc in all structures with exception to flowers. In contrast, the level of CrylAc in flowers was generally greater than Cry1F. Within each sampling period, concentrations of Cry1F in mature leaves (five and eight node) were greater than that for other structures. Expression was also greater for older, eight-node mature leaves than younger, five-node mature leaves. CrylAc expression in bolls was lowest compared with terminal leaves, squares, flowers, and mature leaves, which expressed at similar concentrations. Cry1F expression increased through time for mature leaves and terminal leaves; whereas, a decline in Cry1Ac protein concentration was observed for terminal leaves and bolls. The data presented here provides a means to understand observed levels of efficacy (patterns of insect damage) by comparing the spatial and temporal dynamics of expression for Cry1Ac and Cry1F in PhytoGen 440W transgenic cotton.

Efficacy of transgenic cotton expressing Cry1Ac and Cry1F insecticidal protein against heliothines (Lepidoptera: Noctuidae).

Cotton, Cossypium hirsutum L, plants expressing Cry1Ac and Cry1F (Phytogen 440W) insecticidal crystal proteins of Bacillus thuringiensis (Bt) Berliner, were evaluated against natural populations of tobacco budworm, Heliothis virescens (F.), and bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), across 13 southern U.S. locations that sustained low, moderate, and high infestations. The intrinsic activity of Phytogen 440W was compared with nontreated non-Bt cotton (PSC355) and with management strategies in which supplemental insecticides targeting heliothines were applied to Phytogen 440W and to PSC355 cotton. Infestations were composed primarily of bollworm, which is the least sensitive of the heliothine complex to Cry toxins. Therefore, damage recorded in these studies was primarily due to bollworm. Greater than 75% of all test sites sustained heliothine infestations categorized as moderate to high (10.6-64.0% peak damaged bolls in nontreated PSC355). Phytogen 440W, alone or managed with supplemental insecticide applications, reduced heliothine-damaged plant terminals, squares (flower buds), flowers, and bolls equal to or better (1.0-79.0-fold) than managing a non-Bt cotton variety with foliar insecticides across all infestation environments. Rarely (frequency of < or = 11% averaged across structures), sprayed Phytogen 440W reduced damaged structures compared with nontreated Phytogen 440W. Protection against heliothine-induced plant damage was similar across the three levels of infestation for each viable management strategy, with exception to damaged squares for nontreated Phytogen 440W. In situations of moderate to high heliothine infestations, cotton plants expressing Cry1Ac and Cry1F may sustain higher levels of damage compared with that same variety in low infestations. No significant difference in yield was observed among heliothine management strategies within each infestation level, indicating cotton plants may compensate for those levels of plant damage. These findings indicate Phytogen 440W containing Cry1Ac and Cry1F provided consistent control of heliothines across a range of environments and infestation levels.

Cross-resistance responses of CrylAc-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein vip3A.

One susceptible and three Cry1Ac-resistant strains of tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), were used in laboratory studies to determine the level of cross-resistance between the Bacillus thuringiensis (Berliner) toxins Cry1Ac and Vip3A by using concentration-mortality and leaf tissue experiments. Concentration-mortality data demonstrated that the three Cry1Ac-resistant H. virescens strains, YHD2, KCBhyb, and CxC, were at least 215- to 316-fold resistant to Cry1Ac compared with the susceptible strain, YDK. Results from Vip3A concentration-mortality tests indicated that mortality was similar among all four H. virescens strains. Relative larval growth on Cry1Ac reflected concentration-mortality test results, because YHD2 larval growth was mostly unaffected by the Cry1Ac concentrations tested. Growth ratios for KCBhyb and CXC indicated that they had a more moderate level of resistance to Cry1Ac than did YHD2. Relative larval growth on Vip3A was highly variable at lower concentrations, but it was more consistent on concentrations of Vip3A above 25 microg/ml. Differences in larval growth among strains on Vip3A were not as pronounced as seen in Cry1Ac experiments. Mortality and larval growth also was assessed in leaf tissue bioassays in which YDK, CxC, and KCBhyb neonates were placed onto leaf disks from non-Bt and Bt cotton, Gossypium hirsutum L., for 5 d. Three Bt lines were used in an initial bioassay and consisted of two Vip3A-containing lines, COT203 and COT102, and a Cry1Ac-producing line. Mortality of KCBhyb and CXC was lower than that of YDK larvae in the presence of leaf tissue from the Cry1Ac-producing line. Additionally, increased larval growth and leaf tissue consumption on Cry1Ac-containing leaf disks was observed for KCBhyb and CXC. Mortality and larval weights were similar among strains when larvae were fed leaf tissue of either non-Bt, COT203, or COT102. A subsequent leaf tissue bioassay was conducted that evaluated four cotton lines: non-Bt, Cry1Ab-expressing, Vip3A-expressing, and pyramided-toxin plants that produced both Cry1Ab and Vip3A. Mortality levels were similar among strains when fed non-Bt, Vip3A-expressing, or pyramided-toxin leaf tissues. Mortality was higher for YDK than for KCBhyb or CXC on Cry1Ab-expressing leaf tissues. No differences in larval weights were observed among strains for any genotype tested. Results of these experiments demonstrate that cross-resistance is nonexistent between CrylAc and Vip3A in H. virescens. Thus, the introduction of Vip3A-producing lines could delay Cry1Ac-resistance evolution in H. virescens, if these lines gain a significant share of the market.

Genetic variation for resistance to Bacillus thuringiensis toxins in Helicoverpa zea (Lepidoptera: Noctuidae) in eastern North Carolina.

To evaluate resistance to Bacillus thuringiensis Berliner (Bt) toxins, adult female bollworms, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), were collected from four light trap locations in two eastern North Carolina counties from August to October during 2001 and 2002. Females were allowed to oviposit, and upon hatching, 24 neonates from each female (F1 lines) were screened for survival and growth rate on each of three diets: non-Bt diet, diet containing 5.0 microg/ml Cry1Ac toxin, or diet containing 5.0 microg/ml Cry2Ab toxin. These screens were designed to identify nonrecessive Bt resistance alleles present in field populations of bollworm. Of 561 and 691 families screened with both Cry1Ac- and Cry2Ab-containing diets in 2001 and 2002, respectively, no F1 lines were identified that seemed to carry a gene conferring substantial resistance to either Cry1Ac or Cry2Ab. Adults from F1 lines with growth scores in the highest (R) and lowest (S) quartiles were mated in four combinations, RxR, SxR, RxS, and SxS. Differences in growth rates of larvae from these crosses demonstrated that there is substantial quantitative genetic variation in eastern North Carolina populations for resistance to both Cry1Ac and Cry2Ab toxins. These findings, in addition to results suggesting partially dominant inheritance of resistance to Cry1Ac and Cry2Ab, are critically important for determining appropriate resistance management strategies that impact the sustainability of transgenic cotton, Gossypium hirsutum (L.).

Trichogramma releases in North Carolina cotton: why releases fail to suppress heliothine pests.

Field studies were conducted in 1996 and 1997 to determine the fate of naturally oviposited F3 heliothine eggs in cotton plots treated with augmentative releases of Trichogramma exiguum Pinto & Platner and nontreated plots. Four cohorts of newly oviposited eggs (< 24 h old) were followed in 1996 and two cohorts in 1997. In 1996, mean +/- SD percent parasitism, estimated by in-field studies following the fate of naturally oviposited eggs, ranged from 7 +/- 7 to 61 +/- 8% in T. exiguum release plots and 0 +/- 0 to 35 +/- 13% in control plots. The mean +/- SD percent of eggs hatched in T. exiguum release plots ranged from 1 +/- 2 to 11 +/- 4% and 7 +/- 4 to 28 +/- 10% in control plots. In 1997, mean +/- SD percent egg parasitism ranged from 27 +/- 4 to 40 +/- 3% in T. exiguum release plots and 15 +/- 18 to 25 +/- 8% in control plots. The mean +/- SD percent of eggs hatched in T. exiguum release plots ranged from 7 +/- 3 to 12 +/- 2% and 18 +/- 6 to 28 +/- 8% in control plots. Despite increased parasitism and reduced egg hatch in T. exiguum release plots, overall, there was no significant difference in larval density (all instars combined) between T. exiguum release and control plots. Combined analysis of the heliothine larval populations and egg fate data revealed that the additional egg mortality produced by released T. exiguum was offset by lower larval mortality in release plots. Because of the occurrence of compensatory mortality, the egg stage of heliothines is not an appropriate target for biological control using Trichogramma wasp releases.

Effect of insecticides on Trichogramma exiguum (Trichogrammatidae: Hymenoptera) preimaginal development and adult survival.

The effect of insecticides on Trichogramma exiguum Pinto & Platner emergence, adult survival, and fitness parameters was investigated. Insecticides tested were lambda cyhalothrin, cypermethrin, thiodicarb, profenophos, spinosad, methoxyfenozide, and tebufenozide. All insecticides, with the exception of methoxyfenozide and tebufenozide, adversely affected Trichogramma emergence from Helicoverpa zea (Boddie) host eggs when exposed at different preimaginal stages of development (larval, prepupal, or pupal). Regardless of the developmental stage treated, none of the insecticides tested had a significant effect on the sex ratio or frequency of brachyptery of emerged females. However, the mean life span of emerged T. exiguum females significantly varied among insecticide treatments, and was significantly affected by the developmental stage of parasitoid when treated. Based on LC50 values, spinosad and prophenofos were the most toxic compounds to female T. exiguum adults, followed by lambda cyhalothrin, cypermethrin, and thiodicarb. Insecticides field-weathered for four to 6 d on cotton leaves showed no activity against female T. exiguum adults.

Life history traits of Helicoverpa zea (Lepidoptera: Noctuidae) on non-Bt and Bt transgenic corn hybrids in eastern North Carolina.

Transgenic varieties of field corn that express the CrylAb B. thuringiensis (Bt) toxin in ear tissue present the potential of reducing ear feeding by the corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae), and for reducing the size of populations of the insect infesting other host crops. Life history parameters of H. zea feeding on ears of conventional and Bt field corn varieties were measured in field plots in eastern North Carolina in 1997 and 1998. Transformation events investigated were Mon-810 and Bt-11. Bt corn was found to cause a steady mortality of larvae during development, but permitted approximately 15-40% survival to the prepupal stage compared with non-Bt corn. Mortality of prepupae and pupae from Bt corn was also higher than from non-Bt corn, reducing overall adult production by 65-95%. The larvae that did survive grew more slowly on Bt than on non-Bt corn, and produced pupae that weighed 33% less. Pupation and adult eclosion were delayed by 6-10 d by feeding on Bt corn ears. Corn varieties expressing Bt in ear tissue have the potential to reduce H. zea ear feeding by up to 80%, and the potential to reduce populations emerging from ear-stage corn fields to infest cotton, soybean and other crops by around 75%. To have a measurable effect on area-wide populations, Bt corn varieties would need to be planted in large proportions of corn fields. Extensive planting of varieties such as those tested here, having only moderate effects on H. zea, would raise concerns about rapid evolution of resistance.

Trichogramma exiguum (Hymenoptera: Trichogrammatidae) releases in North Carolina cotton: evaluation of heliothine pest suppression.

Field studies were conducted in 1996 and 1997 to reevaluate the use of augmentative releases of Trichogramma wasps for heliothine management in cotton. In 1996, nine releases of Trichogramma exiguum Pinto & Platner, spaced 3-4 d apart, were made into three 0.4-ha cotton plots. Six weekly releases were made in 1997, each containing two T. exiguum cohorts developmentally staggered by 45 degrees C degree-days. Field release rates, estimated from laboratory and field quality control data, averaged 108,357 T. exiguum female female per hectare per cohort per release in 1996 and 193,366 female female per hectare per cohort per release in 1997. In 1996, mean +/- SD adult emergence under laboratory conditions for released cohorts was 92 +/- 7%; 62 +/- 5% of emerged adults were females, 3 +/- 2% of females displayed brachyptery (nonfunctional wings), mean female longevity under laboratory conditions was 15 +/- 4 d, and mean +/- SD field emergence was 97 +/- 2%. Quality control measurements were similar in 1997. In 1996, mean +/- SD percent parasitism of heliothine eggs in field plots on the sampled dates ranged from 67 +/- 4 to 83 +/- 5% in T. exiguum release plots and 25 +/- 9 to 55 +/- 8% in control plots. In 1997, parasitism levels ranged from 74 +/- 4 to 89 +/- 5% in T. exiguum release plots and 18 +/- 18 to 69 +/- 11% in control plots. Despite increased parasitism levels in T. exiguum release plots, there were no significant differences in density of fifth instars, boll damage, or yield between T. exiguum release and control plots. Therefore, it is concluded that Trichogramma augmentation is not an effective heliothine management tool in North Carolina cotton.

Comparative production of Helicoverpa zea (Lepidoptera: Noctuidae) from transgenic cotton expressing either one or two Bacillus thuringiensis proteins with and without insecticide oversprays.

Transgenic cotton, Gossypium hirsutum (L.), expressing either one or two Bacillus thuringiensis ssp. kurstaki Berliner (Bt) proteins was compared with the conventional sister line in field experiments with regard to production of bollworm, Helicoverpa zea (Boddie), and bolls damaged by bollworm. The relative numbers of bollworms that developed on Bollgard (Monsanto Co., St. Louis, MO), Bollgard II (Monsanto Co.), and conventional cotton were estimated under nontreated conditions in 2000 and both insecticide-treated and nontreated conditions in 2001-2002 in North Carolina tests. Averaged across seven field studies under nontreated conditions, Bollgard cotton generated statistically similar numbers of large (L4-L5) bollworm larvae compared with the conventional variety; however, Bollgard cotton produced significantly fewer damaged bolls and bollworm adults than the conventional variety. Production of large larvae, damaged bolls, and adults was decreased dramatically by Bollgard II cotton as compared with Bollgard and conventional varieties. When comparing insecticide-treated and nontreated cotton genotypes, both Bt cotton sustained less boll damage than the conventional variety averaged across insecticide regimes; furthermore, Bollgard II cotton had fewer damaged bolls than the Bollgard variety. When averaged across cotton genotypes, pyrethroid oversprays reduced the numbers of damaged bolls compared with the nontreated cotton. Insecticide-treated Bollgard cotton, along with insecticide-treated and nontreated Bollgard II cotton reduced production of bollworm larvae, pupae, and adults. However, the addition of pyrethroid oversprays to Bollgard II cotton seemed to be the best resistance management strategy available for bollworm because no bollworms were capable of completing development under these conditions.

Relationship between cereal leaf beetle (Coleoptera: Chrysomelidae) egg and fourth-instar populations and impact of fourth-instar defoliation of winter wheat yields in North Carolina and Virginia.

Cereal leaf beetle, Oulema melanopus (L.), has become a serious pest of small grains in the mid-Atlantic region of the United States. Existing thresholds for implementing control measures allowed too much leaf damage and consequent yield loss to occur before recommending treatment. Information on beetle biology and crop response to injury, both prerequisites for developing new management strategies, was lacking for this region. A 3-yr project was initiated to generate an area wide cereal leaf beetle biological and yield impact database for winter wheat, and to evaluate the injury and yield loss potential of different population densities. Over the study period, beetle populations were evaluated at 26 winter wheat field locations in Virginia and North Carolina. Eggs and larvae, classified to instar, were counted twice each week from February to June. Replicated insecticide versus noninsecticide treatments were conducted at each location where leaf defoliation and yield were documented. Results showed that the relationship between 50th percentile egg and fourth-instar population estimates were in strong agreement (y = 0.36x - 0.01; r2 = 0.79). Potentially detrimental larval infestations were forecast before appearance of foliage injury from egg populations present during the stem elongation to flag leaf emergence developmental stages. A significant positive linear relationship between total fourth instar per stem population estimates and percent flag leaf defoliation was detected (y = 20.29x + 1.34; r2 = 0.60). A weaker but still significant relationship between the total fourth-instar population estimates and percent yield loss was found (y = 11.74x + 6.51; r2 = 0.26), indicating that factors in addition to flag leaf injury, primarily by fourth instars, also contributed to reduced yields.