Helicoverpa zea (Boddie), a pest of cotton that also occurs in field corn, is commonly controlled through the use of foliar-applied insecticides or transgenic crops expressing Bt genes. To prevent the selection of resistant populations, refuge systems have been implemented into the agroecosystem. Historically, structured refuge compliance among growers has been low, leading to the commercialization of seed blended refugia. To test the viability of seed blended refugia in the southern U.S., field studies were conducted in Mississippi and Georgia during the 2016, 2017, and 2018 growing seasons. To quantify adult H. zea emergence from structured and seed blended refuge options, emergence traps were utilized. Kernel damage and moth emergence timings were recorded. Various percentages of stand loss ranging from 0% to 50% were also simulated to determine yield effects in unprotected seed blended refugia. Lastly, H. zea feeding and emergence in a two-gene field corn variety expressing Cry1A.105 and Cry2Ab2 were compared to non-Bt field corn. When compared to a structured refuge, H. zea adult moth emergence from seed blended refugia did not significantly differ. Kernel damage was not different between seed blended treatments and structured refuge treatments. Moth emergence timings were not significantly delayed between the structured refuge and seed blended refuge treatments. Significant yield losses were observed when stand loss was simulated at various levels in field corn, suggesting that there is an opportunity to see yield losses in an unprotected seed blended refuge field corn landscape. Kernel damage did not significantly differ between field corn expressing Cry1A.105 and Cry2Ab2 and non-Bt field corn, possibly due to H. zea resistance to the Cry genes. However, there was a significant difference in emergence from two-gene expressing field corn and non-Bt field corn. This suggests that there may be high pupal mortality in two-gene corn plots. Based on these data, seed blended refuge could be a viable option to replace structured refuge strategies in the southern U.S., however, if left unprotected, yield loss could be observed in a case of high boring insect pressure. The significant loss of refuge plants can also compromise refuge effectiveness.

Transgenic corn and cotton expressing crystalline (Cry) insecticidal proteins from Bacillus thuringiensis (Bt) were commercially introduced in 1996. This technology has greatly improved the control of several key lepidopteran insect pests tobacco budworm, Heliothis virescens (Farbricius), pink bollworm, Pectinophora gossypiella (Saunders), southwestern corn borer, Diatraea grandiosella (Dyar), and European corn borer, Ostrinia nubilalis (Hübner). The corn earworm, Helicoverpa zea, has been more difficult to control using Bt crops and supplemental insecticide applications are often needed to prevent economic losses. A major threat to the longevity of transgenic technology is the evolution of resistance, especially when an insect pest infests both Bt corn and cotton in the same growing season. Similar Cry proteins are currently expressed in both corn and cotton commercial production systems. At least one generation per year develops on Bt corn before infesting cotton. Given that H. zea infests both crop hosts at some point every year, the objectives of this study were to evaluate the contribution and influence of Genuity VT3 PRO corn (expressing Cry1A.105 and Cry2Ab) on H. zea density, fitness, and selection on BollGard II cotton (expressing Cry1Ac and Cry2Ab). Non-Bt and VT3 PRO corn fields were sampled for larval density and any observed larvae were collected. Non-Bt field corn supported 61% more larvae compared to VT3 PRO fields. Larvae infesting non-Bt corn developed faster than those infesting VT3 PRO corn. Larvae collected from VT3 PRO corn had significantly higher pupal weight two out of the three years of this study. Pupae from VT3 PRO corn also had longer pupal duration two out of three years compared to pupae of larvae collected from non-Bt corn. Offspring from larvae reared on VT3 PRO corn had a higher LC50 compared to offspring from larvae reared on non-Bt corn. H. zea susceptibility to Bt is highly variable but the results presented here indicate that dual-gene corn hybrids such as VT3 PRO can select for H. zea populations with a higher propensity for causing damage in Bt cotton.

The bollworm, Helicoverpa zea (Boddie), is an economically important pest of cotton and is becoming more difficult to control in the United States. This project was designed to develop a better understanding of the effects of resistance development and plant structures on the efficacy of Bt cotton and foliar insecticides. Experiments examined larval behavior and damage in current cotton varieties expressing proteins from Bacillus thuringiensis. Other experiments examined the effects of floral structures on the control of bollworm using foliar insecticides. Results suggest that larval feeding and avoidance behavior is dependent on point of eclosion. Fruiting form damage increased in two-gene cotton compared to earlier research but was low in cotton expressing the Vip3A protein. Increases in damage can lead to more frequent applications of foliar insecticides. Floral structures can hinder insecticide efficacy by reducing exposure. Results from these experiments will be important for refining management recommendations for bollworm in Bt cotton.

Helicoverpa zea, commonly referred to as corn earworm, has been a major pest of corn and cotton, along with many major crops grown in United States. Migration of this pest plays a major role in its distribution and successful survival. Part of the current resistance management strategy for transgenic crops, using non-Bt crops as refuges, is totally based on the movement of the adult populations between the Bt and non-Bt fields, and successful exchange of genetic material between the rare resistant and susceptible populations. To study the movement and migration patterns, and their implications in resistance management, suitable molecular genetic markers were comparatively selected, followed by a study of temporal variations in populations from north Mississippi. The dominant marker system Inter Simple Sequence Repeats (ISSR) was selected for the study based on higher polymorphism (5.0) and PIC (0.34) values compared to Simple Sequence Repeats (SSR) and Sequence Related Amplification Polymorphism (SRAP) marker systems. 53 ISSR loci were used in understanding the temporal variations in H. zea. Percent polymorphism and heterozygosity levels showed variation among the twelve collections tested. Early spring putative migrants were distinct from the rest of the generations. Population differentiation was higher in the beginning of the season and then declined by end of the season (pairwise FST = 0.341, early in March and 0.025, late in August). Average heterozygosity levels recorded were 0.11, coinciding with 1993 and 2002 data. The result of this study supports the existence of spring migration of adults and their contribution to the local gene pool. The extent of genetic exchange between the putative migrants and the local populations varied among three years. There is variation in population densities from migrants and local emergence in one of the three years. The results of this study indicate a need for continuous monitoring for genetic changes and their possible implications for resistance management in transgenic crops in Mississippi.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objectives of this study were to evaluate the effects of Bt toxin and seeds treatment with insecticides (imidacloprid + thiodicarb) on initial growth of corn and the control of Spodoptera frugiperda (J. E. Smith) in soil; the effect of Bt corn and insecticides (seed treatment with imidacloprid + thiodicarb and spray application of novaluron and/or methomyl) for the control of S. frugiperda in crop shoots during early and late planting seasons; and the effects of Bt corn and insecticides control of S. frugiperda on the occurrences and damages from Diatraea sp. and Helicoverpa zea (Boddie) pests. Three experiments were conducted under field conditions and natural infestation of pests, in Itaara and Santiago, in Rio Grande do Sul state, Brazil, during the 2008/2009 planting seasons. The initial growth of corn was not affected by the production of Bt toxin (Cry1Ab) and insecticide seed treatment in areas without pest infestation. The toxin (Cry1Ab) present in the Bt corn as well as seeds treatment in non-transformed corn effectively protected the plants to cut-off S. frugiperda. Seeds treatment enables the reduction of damages caused by S. frugiperda in Bt corn (Cry1Ab) to the shoots of crops in early infestations. The Bt corn (Cry1Ab) is effective in controlling S. frugiperda, especially when infestations are low to moderate. When high infestations of S. frugiperda occurred in late planting, insecticide spray on Bt corn (Cry1Ab) resulted to less damages and fewer lavae development comparatively with unsprayed plots. Insecticide treatment of nontransformed corn seeds during early infestations of Diatraea sp. and Bt corn reduced the percentage of stems attacked and injuries. However, insecticides sprayed for the control of S. frugiperda had no effect on the percentage of stems attacked and the injuries caused by Diatraea sp.. Application of insecticides to seeds or spraying for the control of S. frugiperda did not affect the percentage of corn ears attacked and the injuries caused by H. zea, while the Bt corn (Cry1Ab) reduced the percentage of ears attacked by H. zea despite not having much effect in reducing the ear-feeding injuries. / O objetivo do estudo foi avaliar: o efeito da toxina Bt e do tratamento de semente com inseticidas (imidacloprido + tiodicarbe), no crescimento inicial das plantas de milho e no controle de Spodoptera frugiperda (J. E. Smith) no solo; o efeito do milho Bt e dos inseticidas (imidacloprido + tiodicarbe em tratamento de semente e novalurom e/ou metomil em pulverizações), no controle de S. frugiperda na parte aérea da cultura, na semeadura do cedo e do tarde; e os efeitos do milho Bt e dos inseticidas para o controle de S. frugiperda, na ocorrência e na injúria da Diatraea sp. e da Helicoverpa zea (Boddie). Foram realizados três experimentos em condições de campo e com infestação natural dos insetos-praga, em Itaara e Santiago, RS, durante a safra 2008/09. O crescimento inicial das plantas de milho não é afetado pela produção da toxina Bt (Cry1Ab) e pelos inseticidas em tratamento de semente, em áreas sem infestação de insetos-praga. A toxina Cry1Ab, presente no milho Bt, assim como o tratamento de semente no milho convencional, protegem de forma eficiente as plantas do corte de S. frugiperda. O tratamento de semente, possibilita a redução da injúria na parte aérea da cultura causada por S. frugiperda em milho Bt (Cry1Ab), em infestações precoces. O milho Bt (Cry1Ab) é eficiente no controle de S. frugiperda, especialmente quando as infestações são baixas a moderadas. Em época de semeadura, na qual ocorrem altas infestações de S. frugiperda, o milho Bt (Cry1Ab) quando pulverizado com inseticida, apresenta em relação ao sem pulverização, menos injúria e menor número de lagartas grandes. O tratamento de semente com inseticidas em milho convencional, quando em infestações precoces de Diatraea sp. e o milho Bt, reduzem as injúrias e o percentual de colmos atacados, porém os inseticidas pulverizados para o controle de S. frugiperda, não têm efeito sofre as injúrias e no percentual de colmos atacados por Diatraea sp.. Os inseticidas aplicados na semente ou em pulverizações para o controle de S. frugiperda, não afetam as injúrias e o percentual de espigas atacadas por H. zea, porém o milho Bt (Cry1Ab) reduz o percentual de espigas atacadas por H. zea, apesar de não ter efeito significativo na redução das injúrias nas espigas.

Global large-scale adoption of Bt transgenic crops has provided effective management of key insect pests and have greatly reduced insecticide use. However, some field populations of several insect species have evolved resistance to Bt crops in the field, which threatens the continuing success of Bt crops. The cotton bollworm (Helicoverpa zea) is among the first pest reported to have field-evolved resistance to Cry1Ac and Cry2Ab, but the underlying mechanisms remain largely unknown. To determine the current resistance status of the field populations of H. zea and to elucidate the mechanisms of Bt resistance in this pest, I conducted a series of experiments including bioassays of field populations as well as biochemical and molecular comparisons of midgut proteases and putative Cry1Ac receptors between Cry1Ac-susceptible and -resistant strains. Diet incorporation bioassays of six field populations of H. zea collected from Tifton, Georgia USA in 2008 and 2009 indicated that, comparing to LAB-S, a susceptible laboratory strain, all six field populations were significantly resistant to Cry1Ac toxin and one of three field strains was significantly resistant to Cry2Ab toxin. Across the five populations, survival on leaf-discs producing Cry1Ac was positively correlated to the lethal concentration that kills 50% of the population (LC₅₀) for Cry1Ac from diet bioassays. These results support previous findings of field-evolved resistance to Bt crops in H. zea and suggest an overall increase in resistance to Cry1Ac from 2002 to 2009.One of the six field population, which was designed as GA and had 55-fold resistance to Cry1Ac, was further selected with Cry1Ac in the laboratory to generate a more resistant strain, which was designated as GA-R and had 560-fold resistance to Cry1Ac. Total protease activity of the midgut extracts from GA-R and GA strains is significantly lower than that from the susceptible laboratory strain LAB-S. Among the proteases contributing to the total activity, trypsin-like and chymotrypsin-like activities of GA-R and GA midgut extracts are significantly lower than that from the susceptible strain, while no difference in elastase-like activity is evident. Decreased proteolytic activity was correlated to the decreased Cry1Ac activation rate of midgut extracts of the GA-R and GA strains. Cytotoxicity assays with H. zea midgut cells show that the product of Cry1Ac protoxin digested with GA-R and GA midgut extracts has significantly lower cytotoxicity when compared with that digested with the susceptible strain midgut extracts. Transcriptional analysis of a limited number of protease genes did not identify specific proteases involved in the decline in Cry1Ac activation in GA-R and GA. These results indicate that the decreased Cry1Ac activation rate by midgut proteases is involved in the field originated Cry1Ac resistance in the H. zea GA-R and GA strains. I also compared the cDNA sequences and expression levels of the putative Cry1Ac receptors cadherin, aminopeptidase 1 (APN1), alkaline phosphatase 2 (ALP2) and ATP-binding cassette subfamily C member 2 (ABCC2) in LAB-S and GA-R. No indels (insertions and deletions) were found in the cDNA sequences of the resistant alleles of the four receptors, relative to those of the susceptible alleles. While there were no amino acid point mutations in the resistant alleles of ALP2 and ABCC2, we found 2 and 14 consistent amino acid point mutations in the resistant alleles of cadherin and APN1, respectively. However, neither cadherin nor APN1 point mutations were genetically linked to Cry1Ac resistance in GA-R. Quantitative RT-PCR analysis revealed no differences in the transcripts of the four receptors between the two strains. Taken together, these results indicate that the four receptors are not involved in Cry1Ac resistance in the GA-R strain of H. zea.

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-10-10T17:24:48Z No. of bitstreams: 1 TeseACMR.pdf: 2424700 bytes, checksum: 8f6c5b8a916ee974192846236c7e1dc9 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T18:33:03Z (GMT) No. of bitstreams: 1 TeseACMR.pdf: 2424700 bytes, checksum: 8f6c5b8a916ee974192846236c7e1dc9 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T18:33:09Z (GMT) No. of bitstreams: 1 TeseACMR.pdf: 2424700 bytes, checksum: 8f6c5b8a916ee974192846236c7e1dc9 (MD5) / Made available in DSpace on 2016-10-20T18:33:16Z (GMT). No. of bitstreams: 1 TeseACMR.pdf: 2424700 bytes, checksum: 8f6c5b8a916ee974192846236c7e1dc9 (MD5) Previous issue date: 2016-06-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / This study objective to identify the diversity of predators through the use of serology producing specific antiserums for the main pests found in maize, Spodoptera frugiperda, Helicoverpa armigera and H. zea, Rhopalosiphum maidis and uses it to determine their predators. Pest samples were macerated in 0.85% saline solution. The macerated were centrifuged and the supernatants used as immunizing antigens for obtaining antiserum. For this purpose, a rabbit was immunized with 3.0 ml of the immunizing antigen on the lymph node region. Homologous serological tests were performed in double diffusion in agar. Homologous serological reactions were positive after seven days of antigen inoculation. The technique has sensitivity to detect predation of the four pests studied in this paper. The tests were positive for a prey in the digestive tract of the predator to 96 hours of ingestion. Regarding the number of preys, there was no difference in the lines, they only grew stronger and sharper. In laboratory, D. luteipes, O. vnigrum and C. externa presented preference for R. maidis and E. annulipes for S. frugiperda. In tests where predators were collected in the field, serological tests showed that there was a certain food preference of D. luteipes and O. v-nigrum by S. frugiperda. E. annulipes, H. axyridis, E. connexa and C. externa, by the aphid. After a single injection of antigen in rabbit’s lymph node region, it was possible to get an antiserum specific for pests of corn. / O presente trabalho teve como objetivo identificar a diversidade alimentar dos predadores através do uso da serologia produzindo os antissoros específicos para as principais pragas encontradas na cultura do milho Spodoptera frugiperda, Helicoverpa armigera, Helicoverpa zea, Rhopalosiphum maidis. Amostras das pragas foram maceradas em solução salina NaCl (0,85%). Os macerados foram centrifugados, e os sobrenadantes, utilizados como antígenos imunizantes para obtenção do antissoro. Para esse propósito um coelho foi imunizado com 3,0 ml do antígeno imunizante na região do linfonódulo. Testes serológicos homólogos foram realizados em dupla difusão em ágar. Reações serológicas homólogas foram positivas após sete dias após da imunização do antígeno. A técnica tem sensibilidade para detectar predação de das quatro pragas estudadas nesse trabalho. Os testes foram positivos para uma presa no trato digestivo do predador até 96hs de sua ingestão. Com relação ao número de presas ingeridas não houve diferença nas linhas de precipitação, apenas ficaram mais fortes e nítidas. Em laboratório a preferência alimentar de Doru luteipes, Olla v-nigrum e Chrysoperla externa apresentaram preferência alimentar por R. maidis e Euborellia annulipes por S. frugiperda. Nos testes onde os predadores foram coletados no campo os testes serológicos mostraram que houve uma preferência alimentar de D. luteipes e O. v-nigrum por S. frugiperda. E. annulipes, H. axyridis, E. connexa, e C. externa por pulgão. Concluiu-se que após uma única injeção do antígeno na região do linfonódulo do coelho, foi possível obter-se antissoro específico para as pragas do milho.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-28T12:40:36Z No. of bitstreams: 1 Luziani Rezende Bestete.pdf: 403712 bytes, checksum: 4a5246d287630547b7bb138f6fc4cd2f (MD5) / Made available in DSpace on 2016-11-28T12:40:36Z (GMT). No. of bitstreams: 1 Luziani Rezende Bestete.pdf: 403712 bytes, checksum: 4a5246d287630547b7bb138f6fc4cd2f (MD5) Previous issue date: 2011-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The tomato fruitworm (TFW), Helicoverpa zea (Boddie) (Lep.: Noctuidae) is considered an important pest in several crops. The purpose of the study was to evaluate the use of castor bean oil, garlic and tobacco extracts to control H. zea larvae, and the influence of these products on the egg parasitoid Trichogramma pretiosum Riley (Hym: Trichogrammatidae). Larval mortality of TFW was investigated when treated with the alternative products at different concentrations through ingestion and contact aplication. Tests of selectivity and susceptibility of T. pretiosum were performed by spraying TFW eggs prior and post parasitism. Castor bean oil at 3% (v/v) through ingestion and contact treatments of TFW larvae caused mortality of 44 and 20%, respectively. Garlic extract at all concentrations and type of application resulted in TFW mortality equal or greater than the tobacco extract. The number of eggs parasitized by T. pretiosum was reduced by castor bean oil, while other biological parameters were not affected. Garlic extract positively interacted with T. pretiosum resulting in greater number of eggs parasitized, although it was observed a lower number of individuals emerging per parasitized egg in selectivity test. Castor bean oil was toxic to TFW larvae in both types of application. Garlic extract produced better results when ingested by TFW larvae and it was superior when compared to tobacco extract to control TFW larvae. Based on the results, castor bean oil and garlic extract are compatible with T. pretiosum, but the former one should be applied after the parasitoid release. Therefore, castor bean oil and garlic extract are promising as phytosanitary tools to control H. zea larvae. / A broca-grande (BRG), Helicoverpa zea (Boddie) (Lep.: Noctuidae) é considerada uma praga importante em diversas culturas. Assim, neste trabalho foi avaliado o uso do óleo de mamona e os extratos de alho e fumo para o controle de H. zea, e a influência desses sobre o parasitóide de ovos Trichogramma pretiosum Riley (Hym.: Trichogrammatidae). Foram estudadas as vias de aplicação por ingestão e contato, em diferentes concentrações, sobre a mortalidade larval da BRG. Testes de seletividade e susceptibilidade de T. pretiosum foram realizados através da pulverização dos ovos de BRG antes e após o parasitismo. O óleo de mamona a 3% (v/v) via ingestão e contato ocasionou 44 e 20% de mortalidade de larvas, respectivamente. O extrato de alho em todas as concentrações e em ambas as vias de aplicação foi sempre igual ou superior ao extrato de fumo, ocasionando as maiores mortalidades de larvas. O número de ovos da BRG parasitados por T. pretiosum foi afetado pelo óleo de mamona, porém os demais parâmetros não foram influenciados. O extrato de alho interagiu positivamente com T. pretiosum obtendo maior número de ovos parasitados, porém no teste de seletividade reduziu o número de indivíduos emergidos por ovo. O óleo de mamona mostrou toxicidade em ambas às vias de aplicação para a BRG. O extrato de alho foi mais eficiente para o controle da BRG quando comparado ao extrato de fumo, atuando principalmente por ingestão. Com base nos resultados, o óleo de mamona e o extrato de alho são compatíveis com a utilização de T. pretisoum, porém o óleo de mamona deve ser utilizado após a liberação do parasitóide. A utilização do óleo de mamona e extratos de alho é viável para o emprego em programas de manejo fitossanitário de H. zea.

Field corn, Zea mays L., has been introduced to the market with pyramided Bacillus thuringiensis (Bt) corn technologies. These technologies reduce kernel damage from ear feeding caterpillar pests, including corn earworm, Helicoverpa zea (Boddie). The first generation Bt traits in field corn demonstrated limited activity on corn earworm feeding on grain in ears. The pyramided corn technologies have greater cumulative protein concentrations and improved expression throughout the plant, so these corn traits should provide an effective management tool against these pests. In addition, reduced kernel injury may also have a direct effect on physical grain quality. The results for this trial showed no relationship between number of damaged kernels and yield was observed for corn hybrid expressing the Herculex®, YieldGard®, or Genuity VT Triple Pro® technologies. A significant relationship between both damaged kernels and test weight and damaged kernels and aflatoxin concentrations was observed in two technologies.

Transgenic corn hybrids expressing Bt toxins are widely deployed to control pests such as Helicoverpa zea (Boddie). However, Bt resistance can reduce the efficacy of Bt corn hybrids. Volunteer corn expressing Bt toxins may impact Bt resistance management of pests. Surveys in Mississippi revealed the occurrence of fall season volunteer corn in high densities. Helicoverpa zea larvae were found feeding during both vegetative and reproductive stages. However, Bt parentage and low to moderate water and nitrogen stresses did not significantly influence H. zea larval growth and development on Bt positive volunteer plants. Additionally, pollen mediated Bt gene flow to non-Bt refuge ears from Bt hybrids significantly reduced H. zea larval growth on cross-pollinated refuge ears. The implications of these findings are that volunteer corn in most Mississippi corn production regions will have little impact on H. zea Bt resistance management, but resistance management could be compromised in more southern regions.