Fernando H. Valicente

Bacillus thuringiensis survey in Brazil: geographical distribution and insecticidal activity against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

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%).

Levantamento dos inimigos naturais da lagarta do cartucho do milho, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), na região de Cascavel, PR

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).

Application rate trials with a nuclear polyhedrosis virus to control Spodoptera frugiperda (Smith) on maize

Diferentes concentracoes de virus da poliedrose nuclear (VPN) foram aplicadas em suspensao 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 aplicacao. Na aplicacao via trator, foi necessaria uma dose de pelo menos 2,5 x 1012 corpos de inclusoes poliedricas (CIP)/ha para se ter eficiencia comparavel a que se obteve com o pulverizador costal-manual (70,2%). Para este tipo de pulverizador pode-se usar 2,5 x 1011 CIP/ha, porem o efeito residual foi muito curto. Maior persistencia foi obtida com doses acima de 1,25 x 1012 CIP/ ha (93,4% de mortalidade). A ocorrencia de parasitoides na area experimental propiciou um aumento medio na taxa de mortalidade larval (16,4%), independente da metodologia de aplicacao. Entre os parasitoides, a maior ocorrencia foi de Campoletis flavicincta (Ashmead) (53,0%), seguido por Chelonus insularis (Cresson) (31,3%), e Eiphosoma spp. (15,6%).

Effect of Baculovirus spodoptera isolates in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae and their characterization by RAPD

Foram utilizados 22 isolados de virus amostrados em diferentes regioes produtoras de milho do Brasil. Os virus foram purificados e suas suspensoes fornecidas a lagartas sadias do 3o e 4o instar de Spodoptera frugiperda (J.E. Smith). A mortalidade foi avaliada diariamente, e as lagartas infectadas foram congeladas logo apos sua morte, o que em geral ocorreu do 5o ao 7o dia apos ingestao do virus. Os isolados foram usados em seis concentracoes (103 a 108 poliedros/ml) e uma testemunha (agua). Os percentuais de mortalidade, duracao do periodo larval e periodo pupal, peso de pupa e a concentracao letal (CL50) foram determinados para todos os isolados. Foram observadas diferencas significativas entre todos os isolados e concentracoes testadas para todos os parâmetros avaliados, e tambem foi constatada a presenca da interacao isolado x concentracao, exceto para periodo pupal. Os padroes de amplificacao de 54 marcadores RAPD, sendo 41 polimorficos, foram utilizados para avaliar a distância genetica e a sua correlacao com os indices de mortalidade das lagartas. A divergencia genetica calculada pelo coeficiente Jaccard utilizando os dados moleculares permitiu dividir os isolados em dois grupos, com um elevada confiabilidade. O agrupamento nao apresentou associacao com a taxa de mortalidade causada pelos isolados ou com sua distribuicao geografica. 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 variacao fenotipica para essas caracteristicas entre os isolados virais, respectivamente.

Solid-state fermentation of Bacillus thuringiensis tolworthi to control fall armyworm in maize

The well-known entomopathogenic bacterium Bacillus thuringiensis (Bt) produces a spore-crystal complex which is responsible for its biocide characteristic, and the bacterium can be obtained by fermentation, either in liquid or semi-solid substrates. This paper presents a successful way to achieve solid-state fermentation of active Bt var. tolworthi (Btt) against Spodoptera frugiperda (fall armyworm) in corn. More than 10 9

Detection of airborne genetically modified maize pollen by real‐time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi‐urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real‐time PCR analysis. Our results suggest that this ‘GM pollen monitoring by bioaerosol sampling and PCR screening’ approach might represent an useful aid in the surveillance of GM‐free areas, centres of origin and natural reserves.

Identificação através de PCR dos genes CryI de cepas de Bacillus thuringiensis Berliner eficientes contra a lagarta do cartucho, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

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.

Insecticidal activity of culture supernatants from Bacillus thuringiensis Berliner strains against Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) larvae

Recentemente, proteinas 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 avaliacao do potencial de aplicabilidade destas proteinas contra a lagarta-do-cartucho (Spodoptera frugiperda Smith), o inseto-praga mais importante da cultura do milho, as caracteristicas e o efeito de mortalidade do sobrenadante da cultura de cinco cepas de B.t. foram investigadas. Notaveis diferencas entre as cepas foram detectadas, nao somente em termos de eficiencia na mortalidade dos insetos, mas tambem nas diferencas de mortalidade entre os sobrenadantes aquecido e nao-aquecido, tratamentos estes que foram usados para distinguir a fracao proteica inseticida termo-sensivel de outra fracao termo-estavel, de natureza nao-proteica (b-exotoxinas). Os padroes qualitativo, quantitativo e temporal de secrecao da proteina total no meio (sobrenadante) foram avaliados por espectrofotometria e eletroforese em gel de poliacrilamida. As cepas apresentaram distintas taxas de crescimento e tempos de secrecao de proteinas em relacao a densidade de celulas na cultura. Alem disso, o padrao de bandas eletroforeticas tambem variou de maneira cepa-especifica, em ambas condicoes desnaturantes e nao-desnaturantes. Polipeptideos que apresentaram peso molecular semelhantes aos esperados para proteinas Vip3A identificadas anteriormente foram encontrados em cepas com alta taxa de mortalidade do sobrenadante. Os dados sugerem a possibilidade e a utilidade de pesquisar fracoes inseticidas de natureza proteica (do tipo Vip) em sobrenadantes de B.t., como forma para desenvolver alternativas especificas e eficientes de controle biologico que possam ser empregadas em programas de manejo integrado de S. frugiperda em milho tropical.

A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL50 of 19.49 ng/ml for 2nd instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

Characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate that does not liquefy the integument of infected larvae

Here we report the characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate, named SfMNPV-6nd, that does not cause the liquefaction of the host integument. The sequencing of the chitinase A (v-chiA) gene from SfMNPV-6nd revealed that it had a frameshift mutation that greatly reduced size of the putative enzyme. In order to evaluate the suitability of SfMPNV-6nd as a biopesticide, this isolate was compared with the highly virulent SfMNPV-19. Our results showed that the LC(50) of the two isolates were not significantly different, but that SfMNPV-6nd took a longer period of time to kill second instar S. frugiperda than SfMNPV-19.