ABSTRACT This study investigated the interaction between two pest biological control agents, the parasitoid wasp Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) and the entomopathogen Bacillus thuringiensis (Bacillales: Bacillacea) (Bt). The aim of this study was to evaluate if the presence of Bt (formulated products Agree®, Dipel® and HD1 and HD11 strains) interferes in the oviposition preference of T. pretiosum to eggs of Helicoverpa zea (Lepidoptera: Noctuidae). Using an olfactometry test, the eggs of H. zea were bathed with the commercial formulations, with the Bt suspensions or distilled water, and offered to the parasitoid wasps in order to evaluate parasitism. The results showed that H. zea eggs sprayed with commercial formulations and Bt strains did not interfere in the choice made by the parasitoid. The parasitoid wasp is not able to distinguish between eggs with or without B. thuringiensis treatment, independently of strains suspension or commercial formulations. Therefore, these two control agents may be used together without negative interaction.

A quantidade de carbono e nitrogênio usados para produzir biopesticidas à base de Bacillus thuringiensis (Berliner) pode influenciar a qualidade final do produto. O objetivo deste trabalho foi testar meios com diferentes níveis de carbono e nitrogênio: meio 1 - glicose de milho a 1.5% + farinha de soja a 0,5%, meio 2 - glicose de milho a 3,0% + farinha de soja a 1,0%, meio 3 - glicose de milho a 1,0% + farinha de soja a 3,0% e meio 4 - Luria Bertani (LB) + sais (FeSO4, ZnSO4, MnSO4 e MgSO4). O inóculo semente foi produzido usando 150 ml de meio LB mais sais incubados por 18h a 30ºC, sob agitação de 200 rpm. A cepa utilizada foi 344 (B. thuringiensis var tolworthi - pertencente ao Banco de Microorganismos da Embrapa). O pH foi medido a intervalos regulares. Após 96h de cultivo, o pH de todos os meios testados tenderam ao básico (entre 6,91 e 8,15), o maior número de esporos foi de 4,39 x 10(9) sporos/ml no meio 3, onde o teor de proteína usado foi o mais alto. A produção de massa celular foi maior no meio 3, com total acumulado de 39,3 g/l. A mortalidade de larvas de Spodoptera frugiperda (J.E. Smith) de dois dias de idade nos meios 3 e 4 foi de 100%. A CL50 para o meio 3 foi de 8,4 x 10(6) esporos/ml. Os meios alternativos usados promoveram crescimento satisfatório de Bt, sendo o meio 3 o mais promissor para ser usado na produção de biopesticida à base de Bt.

The amount and sources of carbon and nitrogen used to produce Bacillus thuringiensis (Berliner)-based biopesticide may influence the quality of the final product. The objective of this research was to test different levels of carbon and nitrogen: medium 1 - 1.5% maize glucose + 0.5% soy flour, medium 2 - 3.0% maize glucose + 1.0% soy flour, medium 3 - 1.0% maize glucose + 3.0% soy flour and medium 4 - Luria Bertani (LB) + salts (FeSO4, ZnSO4, MnSO4, MgSO4). The seed culture was produced in LB medium plus salt, under agitation (200 rpm) for 18h at 30ºC. The strain 344 of Bt was used (B. thuringiensis var tolworthi - belonging to the Embrapa's Bt Bank). The pH was measured at regular intervals. and After culturing for 96h, the pH of the four tested media was basified (6.91 and 8.15), the number of spores yielded 4.39 x 10(9) spores/ml in medium 3, where the amount of protein is high. The dry biomass weight accumulated in media 3 was 39.3 g/l. Mortality of 2-day-old larvae Spodoptera frugiperda (J.E. Smith) was 100% when using Bt produced in media 3 and 4. CL50 for medium 3 was 8.4 x 10(6) spores/ml. All tested media were satisfactory to Bt growth, and medium 3 wass the most promising to be used on a large scale Bt-based biopesticide production.

Foram utilizados 22 isolados de vírus amostrados em diferentes regiões produtoras de milho do Brasil. Os vírus foram purificados e suas suspensões fornecidas a lagartas sadias do 3º e 4º ínstar de Spodoptera frugiperda (J.E. Smith). A mortalidade foi avaliada diariamente, e as lagartas infectadas foram congeladas logo após sua morte, o que em geral ocorreu do 5º ao 7º dia após ingestão do vírus. Os isolados foram usados em seis concentrações (10³ a 10(8) poliedros/ml) e uma testemunha (água). Os percentuais de mortalidade, duração do período larval e período pupal, peso de pupa e a concentração letal (CL50) foram determinados para todos os isolados. Foram observadas diferenças significativas entre todos os isolados e concentrações testadas para todos os parâmetros avaliados, e também foi constatada a presença da interação isolado x concentração, exceto para período pupal. Os padrões de amplificação de 54 marcadores RAPD, sendo 41 polimórficos, foram utilizados para avaliar a distância genética e a sua correlação com os índices de mortalidade das lagartas. A divergência genética calculada pelo coeficiente Jaccard utilizando os dados moleculares permitiu dividir os isolados em dois grupos, com um elevada confiabilidade. O agrupamento não apresentou associação com a taxa de mortalidade causada pelos isolados ou com sua distribuição geográfica. No entanto, um fragmento de RAPD OPW04.2280 apresentou-se altamente associado com a mortalidade das lagartas e com a CL50, explicando 23% e 65% da variação fenotípica para essas características entre os isolados virais, respectivamente.

The total of 22 Baculovirus isolates surveyed in different corn producing regions in Brazil were used against fall armyworm, Spodoptera frugiperda (J.E. Smith). The viruses were purified and their suspensions were used to feed fall armyworm larvae from 4th and 5th instar. The mortality rate was checked daily and the infected larvae were frost after death, what generally occurred between the 5th and 7th day after virus ingestion. The 22 Baculovirus isolates were used in six concentrations (from 10³ to 10(8) polyhedra/ml) and one check treatment with water. Mortality rate, larval period, pupal period, pupa weight and lethal concentration (LC50) were determined for all isolates. Significant differences were found among all isolates and different concentrations, also interaction between isolate x virus concentration for all characteristics evaluated, except for pupal period. Amplification patterns of 54 RAPD markers, being 41 polymorphic among the isolates, were used to evaluate the genetic distance and its correlation with the fall armyworm larvae mortality rate. The genetic diversity calculated by the Jaccard's coefficient using the molecular data allowed a division of the isolates into two groups, with a high level of confidence. These groups did not present any association with the mortality rate caused by the isolates or with their geographical distribution. However, a RAPD fragment OPW04.2280 was highly associated with the larvae mortality rate and with LC50, explaining 23 and 65% of the phenotypic variation for these traits among the isolates, respectively.

O total de 3408 cepas de Bacillus thuringiensis foi isolado de 1448 amostras de solo provenientes de 10 estados, em quatro regiões brasileiras, abrangendo 96 municípios. As cepas foram avaliadas contra lagartas de Spodoptera frugiperda (J. E. Smith) sendo que apenas 62% mataram entre 81% e 100%, e 1758 não causaram mortalidade. O Sul destacou-se com 16,6% de cepas eficientes (mortalidade>75%) do total obtido por região, seguido das regiões Centro Oeste (3,1%), Sudeste (1,1%), e Nordeste (0,4%).

A total of 3408 strains of Bacillus thuringiensis were collected from 1448 soil samples in 10 Brazilian states, four different geographical regions, covering 96 counties. These strains were evaluated against Spodoptera frugiperda (J.E. Smith) larvae. Only 62% killed between 81% and 100% and 1758 caused no mortality. Highest proportion of efficient strains (larval mortality above 75%) was found from the total isolated per region in the South Region (16.6%), followed by Western Central (3.1%), Southeast (1.1%) and Northeast Region (0.4%).

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is the most important maize insect pest in Brazil and its damage can reduce yield up to 34%. The objective of this work was to utilize the PCR technique to Identify Bacillus thuringiensis (B.t) strains that control S. frugiperda in maize crops. Bioassays showed that 16 strains were very effective against S. frugiperda, 15 of them collected from soil samples and one (T09) obtained at the Institute Pasteur. The DNA of these strains were probed with cryI general primers and their proteins were analised by SDS-PAGE eletrophoresis. All strains present positive results for cryI genes. To further characterize these strains, specific cryI primers were employed. PCR technique showed that some strains harbour the same cryI genes. The only difference was the amplification of an unexpected fragment of approximately 160bp when a mixture of cryIB and cryID specific primers was used. Analysis by phase contrast microscope showed that crystal proteins produced by these strains were all bipyramidal crystals. Also, eltrophoretic analysis of proteins by SDS-PAGE showed the same protein banding pattern for most of the strains.

Recentemente, proteínas inseticidas vegetativas (Vips), identificadas no sobrenadante da cultura de certas cepas de Bacillus thuringiensis (B.t.), foram capazes de controlar alguns insetos-praga economicamente importantes. Como avaliação do potencial de aplicabilidade destas proteínas contra a lagarta-do-cartucho (Spodoptera frugiperda Smith), o inseto-praga mais importante da cultura do milho, as características e o efeito de mortalidade do sobrenadante da cultura de cinco cepas de B.t. foram investigadas. Notáveis diferenças entre as cepas foram detectadas, não somente em termos de eficiência na mortalidade dos insetos, mas também nas diferenças de mortalidade entre os sobrenadantes aquecido e não-aquecido, tratamentos estes que foram usados para distinguir a fração protéica inseticida termo-sensível de outra fração termo-estável, de natureza não-protéica (b-exotoxinas). Os padrões qualitativo, quantitativo e temporal de secreção da proteína total no meio (sobrenadante) foram avaliados por espectrofotometria e eletroforese em gel de poliacrilamida. As cepas apresentaram distintas taxas de crescimento e tempos de secreção de proteínas em relação a densidade de células na cultura. Além disso, o padrão de bandas eletroforéticas também variou de maneira cepa-específica, em ambas condições desnaturantes e não-desnaturantes. Polipeptídeos que apresentaram peso molecular semelhantes aos esperados para proteínas Vip3A identificadas anteriormente foram encontrados em cepas com alta taxa de mortalidade do sobrenadante. Os dados sugerem a possibilidade e a utilidade de pesquisar frações inseticidas de natureza protéica (do tipo Vip) em sobrenadantes de B.t., como forma para desenvolver alternativas específicas e eficientes de controle biológico que possam ser empregadas em programas de manejo integrado de S. frugiperda em milho tropical.

Novel vegetative insecticidal proteins (Vips) identified in the supernatant of Bacillus thuringiensis (B.t.) cultures have shown to provide adequate control over a wide spectrum of economically important crop pests. To evaluate the potential applicability of these proteins against fall armyworm (Spodoptera frugiperda Smith) larvae, the most important insect pest for tropical maize, the characteristics and mortality effects of culture supernatants from five B.t. strains were investigated. Striking differences among strains were detected, not only in terms of efficiency in killing the insect, but also regarding to mortality effects of heated and non-heated supernatants, which were used to distinguish the heat-sensitive protein-derived insecticidal fraction from a thermostable one, with a non-protein nature (b-exotoxinas). The qualitative, quantitative and temporal patterns of total protein secretion in the medium (supernatant) were assessed through spectrophotometry and polyacrylamide gel electrophoresis. The strains showed remarkably distinct rates of growth and timing for protein secretion relative to cell density in culture. Moreover, the electrophoretic-banding patterns also varied in a strain-specific manner, both in denaturing and non denaturing conditions. Polypeptides displaying a molecular weight that is very close to the expected for previously identified Vip3A proteins were found for the strains with high supernatant-mortality ratios. The data suggest the feasibility and usefulness of searching for protein-derived (Vip-like) insecticidal fractions in B.t. supernatants as a mean of developing especific and efficient alternatives of biological control to be employed in integrated pest management programs of S. frugiperda in tropical maize.

A survey of the natural enemies of S. frugiperda larvae was done during the years of 1992/93 and 94/95, in Cascavel region, Parana State. The most frequent parasitoids were Campoletis sp. (Hymenoptera: Ichneumonidae), which reached 47.0% of parasitism and Archytas marmoratus (Diptera: Tachinidae), which reached 15.4%. Of all Baculovirus found, 10.8% were NPV (nuclear polihedrosis virus) and only 1% was GV (granulosis virus).

Diferentes concentrações de vírus da poliedrose nuclear (VPN) foram aplicadas em suspensão aquosa, usando-se pulverizador (acoplado a um trator ou costal-manual), para o controle de larvas de Spodoptera frugiperda (Smith), em milho (Zea mays L.). A mortalidade das larvas variou com o equipamento de aplicação. Na aplicação via trator, foi necessária uma dose de pelo menos 2,5 x 10(12) corpos de inclusões poliédricas (CIP)/ha para se ter eficiência comparável a que se obteve com o pulverizador costal-manual (70,2%). Para este tipo de pulverizador pode-se usar 2,5 x 10(11) CIP/ha, porém o efeito residual foi muito curto. Maior persistência foi obtida com doses acima de 1,25 x 10(12) CIP/ ha (93,4% de mortalidade). A ocorrência de parasitóides na área experimental propiciou um aumento médio na taxa de mortalidade larval (16,4%), independente da metodologia de aplicação. Entre os parasitóides, a maior ocorrência foi de Campoletis flavicincta (Ashmead) (53,0%), seguido por Chelonus insularis (Cresson) (31,3%), e Eiphosoma spp. (15,6%).

Different concentrations of nuclear polyhedrosis virus were applied as aqueous suspensions using a tractor mounted or a backpack-manual sprayer to control Spodoptera frugiperda (Smith) larvae on maize (Zea mays L.). Larval mortality varied with the application equipment. For the tractor-mounted sprayer application of at least 2.5 x 10(12) P.I.B./ha was necessary to attain a control comparable to that obtained with the backpack-manual sprayer application (70.2%). For this application method it can be used the dose of 2.5 x 10(11) PI.B./ ha, butNPV persistence was very short. A greaterNPV persistence was obtained with doses over 1.25 x 10(12) P.I.B./ha (93.4% mortality). The occurrence of parasitoids in the experimental area increased larval mortality rate (16.4%), independent of the application method. Among the parasitoids, the most prevalent was Campoletis flavicincta (Ashmead) (53.0%), followed by Chelonus insularis (Cresson) (31.3%), and Eiphosoma spp. (15.6%).