Exploring viral infections in honey bee colonies: insights from a metagenomic study in southern Brazil.

The decline in honey bee colonies in different parts of the world in recent years is due to different reasons, such as agricultural practices, climate changes, the use of chemical insecticides, and pests and diseases. Viral infections are one of the main causes leading to honey bee population declines, which have a major economic impact due to honey production and pollination. To investigate the presence of viruses in bees in southern Brazil, we used a metagenomic approach to sequence adults' samples of concentrated extracts from Apis mellifera collected in fifteen apiaries of six municipalities in the Rio Grande do Sul state, Brazil, between 2016 and 2017. High-throughput sequencing (HTS) of these samples resulted in the identification of eight previously known viruses (Apis rhabdovirus 1 (ARV-1), Acute bee paralysis virus (ABPV), Aphid lethal paralysis virus (ALPV), Black queen cell virus (BQCV), Bee Macula-like virus (BeeMLV), Deformed wing virus (DWV), Lake Sinai Virus NE (LSV), and Varroa destructor virus 3 (VDV-3)) and a thogotovirus isolate. This thogotovirus shares high amino acid identities in five of the six segments with Varroa orthomyxovirus 1, VOV-1 (98.36 to 99.34% identity). In contrast, segment 4, which codes for the main glycoprotein (GP), has no identity with VOV-1, as observed for the other segments, but shares an amino acid identity of 34-38% with other glycoproteins of viruses from the Orthomyxoviridae family. In addition, the putative thogotovirus GP also shows amino acid identities ranging from 33 to 41% with the major glycoprotein (GP64) of insect viruses of the Baculoviridae family. To our knowledge, this is the second report of a thogotovirus found in bees and given this information, this thogotovirus isolate was tentatively named Apis thogotovirus 1 (ATHOV-1). The detection of multiple viruses in bees is important to better understand the complex interactions between viruses and their hosts. By understanding these interactions, better strategies for managing viral infections in bees and protecting their populations can be developed.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Benefits Associated with the Interaction of Endophytic Bacteria and Plants

ABSTRACT The endophytic bacteria belong to a larger group of microorganisms that have their life-cycle partly or entirely inside the plant and are located in intra and inter-cellular spaces or in the vascular tissue. These bacteria can be found colonizing aerial parts or roots. This review aims to analyze the colonization strategies of endophytic bacteria through interaction with plants, as well as to highlight the metabolic influence of these organisms in plant tissues, which result in physiological and biochemical changes. Depending on the different mechanisms used internally to colonize a plant, these microorganisms are called obligate, facultative, or passive endophytes. Phytostimulation, biofertilization and biological control are mechanisms that result in the development of the plant through the production of plant hormones, bioavailability of nutrients and antagonistic action to phytopathogens, respectively. The association between endophytic bacteria and plants features important benefits such as significant increases in growth, plant biomass, length of roots, dry matter production, and grain yield. Studies show that there is a great diversity of endophytic bacteria colonizing plant structures that result in several benefits to the host plant.

Functional Genes of Microorganisms, Comprehending the Dynamics of Agricultural Ecosystems

ABSTRACT The microbial composition of different types,in ecosystems (including agro-ecosystems), has been investigated in a rapidly growing number of studies in the past few years. The importance of microorganisms, regarding the maintenance and stability of nutrients in agroecosystems, is a key to maintain the sustainability of a crop. Molecular tools to study microbial communities are possible through many methods such as RISA, DGGE, TGGE, clone libraries, T-RFLP, RAPD, SSCP and more recently NGS (Next-Generation Sequencing). DGGE is widely employed to characterize the diversity and the community dynamics of microorganisms in the environment, making possible to find out specific groups through functional genes, allowing access to data that cannot be obtained by cultural methods. The aim of this paper is to review the functional groups related to agroecosystems and to indicate the critical choice of DNA primers pairs and targeted DNA regions that may be used in PCR-based methods such as the DGGE technique in order to evaluate the microbial communities in a variety of environments.

Diversity of culturable Gram-negative bacteria isolated from irrigation water of two rice crop regions in Southern Brazil.

In this study, we assessed the diversity of Gram-negative bacteria found in water used for irrigation of rice crops in two growing areas of southern Brazil. Samples were collected from the main irrigation channel and field drain area. Twenty-two bacterial species were found in Cachoeirinha and 28 in Camaquã. In both areas, the most frequent bacterial families were Enterobacteriaceae and Aeromonadaceae. Differences in microbial diversity were observed in both study areas. Thirty-five Gram-negative species were identified; however, only 15 were common in both locations. In addition, there were found pathogenic and drug-resistant species, such as Acinetobacter sp., Brucella spp., and Chryseobacterium meningosepticum. This study demonstrates the existence of a number of pathogenic species in aquatic ecosystems analyzed in three consecutive crop years, especially water used for rice production.

Microbial Diversity: Relevance and Relationship Between Environmental Conservation And Human Health

This work presents bibliographic data on the role and function of microbial diversity. The increasing use of probiotics and prebiotics foods has led to the studies on their actual functions in the human body. It is known that in the environment, microorganisms are extremely important in recycling of nutrients, balance of trophic chains, vital physiological activities in the plants and animals, as well as the conservation of natural habitats. In human food, these microscopic organisms contribute from flavoring products to the synthesis of antimicrobial substances and vitamins essential to living beings.

Bactérias endofíticas como agentes de controle biológico na orizicultura / Endophytic bacteria as biological control agents in rice fields

Esta publicação apresenta alguns dados sobre as interações entre as bactérias e as plantas hospedeiras, considerando os simbiontes e os patógenos das plantas de importância agrícola, com ênfase nos agroecossistemas de arroz irrigado. Neste trabalho foram abordados aspectos relacionados a essas bactérias endofíticas, tais como: (i) endofíticos e suas interações com as plantas cultivadas; (ii) rizobactérias promotoras do crescimento vegetal [plant growth-promoting rhizobacteria (PGPR)] e sua aplicabilidade no controle biológico; (iii) bactérias endofíticas versus PGPR no controle biológico de fitopatógenos; (iv) aplicação de recombinantes endofíticos na agrobiotecnologia. No final do artigo estão apresentadas as opiniões e perspectivas dos autores sobre as bactérias que vivem associadas às plantas cultivadas nos agroecossistemas.(AU)

This paper presents some data on the interactions of the bacteria and the host plants, considering the symbionts and pathogens of plants of agricultural importance, focusing on rice fields. This article addresses issues related to these endophytic bacteria, such as: (i) endophytes and their interactions with the cultivated plants; (ii) promoting rhizobacteria plant growth [plant growth-promoting rhizobacteria (PGPR)] and its application in biological control; (iii) endophytic bacteria PGPR against the biological control of plant pathogens; (iv) application of endophytic recombinant in agricultural biotechnology. This paper ends show the point of view and perspectives of the authors about bacteria that live associated with the plants grown in agroecosystems.(AU)

Mode of Action and Specificity of Bacillus thuringiensis Toxins in the Control of Caterpillars and Stink Bugs in Soybean Culture.

The bacterium Bacillus thuringiensis (Bt) produces delta-endotoxins that possess toxic properties and can be used as biopesticides, as well as a source of genes for the construction of transgenic plants resistant to insects. In Brazil, the introduction of Bt soybean with insecticidal properties to the velvetbean caterpillar, the main insect pest of soybean, has been seen a promising tool in the management of these agroecosystems. However, the increase in stink bug populations in this culture, in various regions of the country, which are not susceptible to the existing genetically modified plants, requires application of chemicals that damage the environment. Little is known about the actual toxicity of Bt to Hemiptera, since these insects present sucking mouthparts, which hamper toxicity assays with artificial diets containing toxins of this bacterium. In recent studies of cytotoxicity with the gut of different hemipterans, susceptibility in the mechanism of action of delta-endotoxins has been demonstrated, which can generate promising subsidies for the control of these insect pests in soybean. This paper aims to review the studies related to the selection, application and mode of action of Bt in the biological control of the major pest of soybean, Anticarsia gemmatalis, and an analysis of advances in research on the use of Bt for control hemipterans.

Receptors and Lethal Effect of Bacillus thuringiensis Insecticidal Crystal Proteins to the Anticarsia gemmatalis (Lepidoptera, Noctuidae).

Bioassays with insecticidal crystal proteins (ICPs) from Bacillus thuringiensis have demonstrated that Cry1Aa, Cry1Ac, and Cry1Ba are the most active toxins on larvae of the Anticarsia gemmatalis. The toxins Cry1Da and Cry1Ea are less toxic, and toxins Cry2Aa are not active. Binding of these ICPs to midgut sections of the A. gemmatalis larvae was studied using streptavidin-mediated detection. The observed staining patterns showed that Cry1Aa and Cry1Ac bound to the brush border throughout the whole length of the midgut. However, the binding sites of Cry1Ba were not evenly distributed in the midgut microvilli. The in vivo assays against larvae of 2nd instar A. gemmatalis confirmed the results from the in vitro binding studies. These binding data correspond well with the bioassay results, demonstrating a correlation between receptors binding and toxicity of the tested ICPs in this insect.

Indica rice cultivar IRGA 424, transformed with cry genes of B. thuringiensis, provided high resistance against Spodoptera frugiperda (Lepidoptera: Noctuidae).

Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice "IRGA 424", via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B delta-endotoxin in IRGA 424 plants transformed with the ubiquitin promoter. Data showed the production and dissemination of a high number of embryogenic calli in addition to obtaining plants transformed with the cry1Aa and cry1B genes until the reproductive phase. The feed bioassays with the transformed plants and Spodoptera frugiperda (JE Smith) larvae indicated high rates of mortality to the insect target. The highest corrected mortality rate achieved under laboratory conditions with Bt-rice plants transformed with the cry1B and cry1Aa genes was 94 and 84%, respectively. Thus, our results demonstrated the great potential of transformed Bt-rice plants in controlling the damage caused by these insect pests in rice paddy fields.

Microbiological and physical-chemical water quality of the rice fields in Sinos River's basin, Southern Brazil.

In this study, we evaluate spatial and temporal variations of the water's quality used in rice fields. Every 15 days during the different phases of cultivation of the rice-vegetative, reproductive, and maturity-samples were collected from the main irrigation channel, from the surface water in the field, and from the excess-water drainage channel. The differences in the values of 13 variables were analyzed by the analysis of variance system and by the Principal Component Analysis (PCA) technique. The results demonstrate that the values observed for the presence of total coliforms, heat-tolerant coliforms, and pH were higher in the irrigation water and that the calcium and magnesium components were greater in the drainage water. The PCA results indicate that three groups of variance exist and that these three account for 77 % of the observed variances. The first principal component, (39 % of the variances), includes the variables pH, phosphorus, potassium, carbon, and turbidity; the second (28.1 %), calcium, magnesium, and conductivity; while the third accounts for only 9.9 % of the variation and incorporates the variable biological thermotolerant coliforms. The spatial pattern resulting from the distribution of the sampling locations as regards the first two principal components indicates a difference between the irrigation and drainage waters. The variables of the first and third items (except for the turbidity in the second component) reach higher values in the irrigation water, while the variables associated with the second component have higher values in the drainage water. The information provided is important for the analysis of the influence exercised by plantation management decisions on the microbiological, physical, and chemical quality of the water. The results confirm the ability of paddy rice field to filter out some of the chemicals and coliforms from the irrigation water as it passes through the agro ecosystem.

Mortality of Oryzophagus oryzae (Costa Lima, 1936) (Coleoptera: Curculionidae) and Spodoptera frugiperda (J E Smith, 1797) (Lepidoptera: Noctuidae) Larvae Exposed to Bacillus thuringiensis and Extracts of Melia azedarach

Oryzophagus oryzae (Costa Lima 1936) (Coleoptera: Curculionidae) and Spodoptera frugiperda (J E Smith, 1797) (Lepidoptera: Noctuidae) cause important crop losses in southern Brazil. Control is possible by the use of the bacteria Bacillus thuringiensis and extracts of Melia azedarach. This study aimed to evaluate the mortality, in vivo, of O. oryzae and S. frugiperda submitted to two isolates of B. thuringiensis and the aqueous extract of M. azedarach. The LC50 for O. oryzae due to bacteria was 5.40μg/mL (Bt 2014-2) and due to plant extract 0.90μg/mL. For S. frugiperda, the Bt 1958-2 bacterial suspension (1.10(10)UFC/mL) caused a 100% of corrected mortality, showing that the purified Cry proteins caused a CL10 of 268μg/mL five days after the treatments, and M. azedarach toxins caused a CL50 173μg/mL four days after the treatment. Corrected mortality for O. oryzae and S. frugiperda in the interaction between the bacterial and plant toxins were 11 and 6%, respectively. In the PCR analysis of B. thuringiensis isolates, DNA fragments were enlarged and corresponded to the cry1 and cry2 genes for Bt 1958-2. Thus, it could be concluded that the usage of Bt 2014-2 active against O. oryzae larvae; Bt 1958-2 for S. frugiperda and, for both the insect species, M. azedarach aqueous extract could be used.

Toxic effects of essential plant oils in adult Sitophilus oryzae (Linnaeus) (Coleoptera, Curculionidae)

Toxic effects of essential plant oils in adult Sitophilus oryzae (Linnaeus) (Coleoptera, Curculionidae). Stored grains are subject to losses in quality nutritional value and in sanitation from the time they are stored to the time they are consumed. Botanical insecticides may offer an alternative solution for pest control. The objective was to test the insecticidal properties of the essential oils of Cymbopogon citratus (leaf), Zingiber officinale (root) and Mentha sp. (leaf). The efficacy of these oils was tested to control the rice weevil, S. oryzae, using hydrodistillation. Chemical analysis of the essential oils was carried out by gas chromatography. Major components of C. citratus were geranial (48 percent) and neral (31 percent), of Z. officinale were α-zingibereno (13 percent), geranial (16 percent), neral (10 percent) and α-farneseno (5 percent) and of Mentha sp. was menthol (92 percent). Bioassays were carried out by fumigation and topical application. In topical application assays, the essential oil of C. citratus had greater toxicity (LC50 0.027 µL mL-1) and shorter exposure time than the oils of the other two plants. After 24 h and 48 h, 70 percent and 100 percent mortality of S. oryzae occurred, respectively. In fumigation assays, essential oil of Z. officinale had a lower LC50 (1.18 µL cm-2) and 70 percent mortality after 24 h exposure. Therefore, we recommend the use of essential oils of C. citratus and Z. officinale to control the rice weevil S. oryzae.

Efeito tóxico de óleos essenciais de plantas medicinais em adultos de Sitophilus oryzae (Linnaeus) (Coleoptera, Curculionidae). Grãos armazenados estão sujeitos a perdas na qualidade física, sanitária e nutricional, desde sua maturação fisiológica até o momento do consumo. Uma alternativa para o manejo de pragas são os inseticidas botânicos. O presente trabalho teve como objetivo avaliar o efeito inseticida de óleos essenciais de Cymbopogon citratus (folha), Zingiber officinale (raiz) e Mentha sp. (folha), obtidos através do método de hidrodestilação,no controle de adultos do gorgulho-do-arroz, S. oryzae. A caracterização química dos compostos encontrados nos óleos essenciais foi realizada por cromatografia gasosa, sendo observados como principais componentes para C. citratus: geranial (48 por cento) e neral (31 por cento), para Z. officinale: 16 por cento de geranial, 13 por cento de α-zingibereno, 10 por cento de neral e 5 por cento de α-farneseno e para Mentha sp.: 92 por cento de mentol. Os bioensaios foram realizados por aplicação tópica e fumigação. Os resultados da aplicação tópica mostraram que o óleo essencial de C. citratus apresenta maior toxicidade quando comparado à Mentha sp.e ao Z. officinale, pois apresentou uma CL50 de 0.027 µL mL-2 e um menor período de exposição, causando 70 por cento e 100 por cento de mortalidade de adultos de S. oryzae, respectivamente, 24 e 48 horas após dos tratamentos. Nos ensaios com fumigação, em adultos de S. oryzae, o óleo essencial de Z. officinale, apresentou a menor CL50 (1,18 µL cm-2) e o menor período de exposição, causando 70 por cento de mortalidade em 24 horas após a aplicação dos tratamentos. Desse modo conclui-se que os óleos de C. citratus e Z. officinale podem ser recomendados no controle de adultos do gorgulho-do-arroz S. oryzae.

In vitro effect of Bacillus thuringiensis strains and Cry proteins in phytopathogenic fungi of paddy rice-field

Cry1Ab and Cry1Ac strains and proteins synthesized by Bacillus thuringiensis thuringiensis and B. thuringiensis kurstaki were assessed in the following phytopathogens: Rhizoctonia solani,Pyricularia grisea,Fusarium oxysporum and F. solani, which had their micelial growth decreased after incubation in the presence of the bacterial strains. As to Cry proteins, there were no inhibition halo development in the assessed concentrations.

As cepas e proteínas Cry1Ab e Cry1Ac sintetizadas por Bacillus thuringiensis thuringiensis e B. thuringiensis kurstaki, foram avaliadas nos fitopatógenos: Rhizoctonia solani,Pyricularia grisea,Fusarium oxysporum e F. solani, os quais tiveram seu crescimento micelial reduzido após a incubação na presença das cepas bacterianas. Em relação às proteínas Cry, não houve formação de halo de inibição nas concentrações avaliadas.

Histopatologia de lagartas de Spodoptera frugiperda (Lep., Noctuidae) infectadas por Bacillus thuringiensis aizawai e com ovos de Campoletis flavicincta (Hym., Ichneumonidae) / Histopathology of larvae of Spodoptera frugiperda (Lep., Noctuidae) infected by Bacillus thuringiensis aizawai and with eggs of Campoletis flavicincta (Hym., Ichneumonidae)

Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera, Noctuidae) está entre os insetos-praga que mais danificam culturas, sendo possível seu controle com produtos à base de Bacillus thuringiensis Berliner, 1911 (Bt). Um dos principais parasitóides de S. frugiperda é Campoletis flavicincta (Ashmead, 1890) (Hymenoptera, Ichneumonidae). A interação de Bt aizawai e de C. flavicincta através da análise histopatológica do ovo do parasitóide em lagartas de S. frugiperda infectadas foi avaliada. Lotes de lagartas de segundo ínstar foram analisados: 1) parasitadas e infectadas por Bt aizawai (Xentari/Abbott Laboratórios do Brasil Ltda) a 0,5mg ml-1 e 2) apenas parasitadas por C. flavicincta (controle). Todas as lagartas foram fixadas em "Bouin Hollande", em intervalos de 6, 12 e 24 horas posteriores à infecção. Após o preparo das lagartas em blocos de parafina, foram efetuados cortes histológicos de 6µm de espessura. A análise histológica de lagartas parasitadas e infectadas indicou não ter havido alteração no ovo do parasitóide, resultante da ação do bacilo. Esse resultado indica que ovos de C. flavicincta não são afetados por Bt aizawai no interior do corpo de lagartas de S. frugiperda.

Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera, Noctuidae) is one of the most important insect pests that affect several crops, and it can be controled with Bacillus thuringiensis Berliner, 1911 (Bt) products. One of the main parasitoids of S. frugiperda is Campoletis flavicincta (Ashmead, 1890) (Hymenoptera, Ichneumonidae). The interaction between Bt aizawai and C. flavicincta by histopathological analysis of the egg of parasitoid in S. frugiperda infected larvae was evaluated. Groups of second instar larvae were analyzed: 1) parasitized and infected by Bt aizawai (Xentari /Abbott Laboratórios do Brasil Ltda) at 0.5mg ml-1 and 2) only parasitized by C. flavicincta (control). All the larvae were fixed in "Bouin Hollande", at intervals of 6, 12 and 24 hours after the infection. After the preparation of the larvae in paraffin blocks, histological cuts of 6µm thickness were made. The histological analysis of parasitized and infected larvae indicated no alteration in the egg of the parasitoid, as a result of the action of the bacillus. This result suggests that eggs of C. flavicincta are not affected by Bt aizawai inside of the body of larvae of S. frugiperda.

Bacterial diversity in rice-field water in Rio Grande do Sul

As bactérias são as mais diversas formas de vida na terra e somente uma fracão dessas espécies têm sido identificadas. Esse trabalho objetivou a análise da diversidade de bactérias mesófilas, heterotróficas presentes em amostras de água de irrigacão de lavouras de arroz do Estado do Rio Grande do Sul, Brasil. As amostras foram coletadas em cinco regiões produtoras de arroz, sendo em seguida o isolamento, a multiplicacão e a caracterizacão bacteriana. A identificacão das espécies foi baseada em testes bioquímicos (Sistema API, bioMérieux). A diversidade bacteriana foi determinada pelo índice de Shannon & Weaner e Pielou. Os resultados obtidos quanto a diversidade de bactérias revelaram 26 espécies. Representantes dos gêneros Bacillus, Corynebacterium, Staphylococcus e Lactobacillus foram os mais abundantes com uma distribuicão homogênea em todas as regiões orizícolas estudadas, assim como nas áreas correspondentes ao canal de irrigacão e a parcela de cultivo do arroz. A água do canal de irrigacão apresentou maior diversidade (H'= 1,00) quando comparada à parcela (H' = 0,77). O canal também apresentou uma distribuicão mais uniforme das espécies (e = 0,73) enquanto a parcela (e = 0,59) teve a maior dominância que foi representada pelo gênero Bacillus. As regiões e as fases da cultura não influenciaram significativamente à diversidade bacteriana nas áreas de estudo (F4,12=1,04; P>0,05). Os resultados revelam que o manejo da cultura pode influenciar nas populacões bacterianas da água.

Histopathology of Anticarsia gemmatalis Hübner (Lepidoptera; Noctuidae) treated with Nucleopolyhedrovirus and Bacillus thuringiensis serovar kurstaki

A Anticarsia gemmatalis é responsável pelo uso de inseticidas químicos na cultura da soja, ocasionando um significativo aumento nos custos das lavouras e um grande desequilíbrio no ecossistema. O uso de agentes microbianos, como Bacillus thuringiensis sorovar kurstaki (Btk) e Vírus de Poliedrose Nuclear de Anticarsia gemmatalis (VPNAg), é uma alternativa para o controle químico de insetos-praga. Na análise da interacão de bactérias e vírus entomopatogênicos, considera-se importante o modo de acão in vitro desses agentes de controle microbiano. Assim, o presente trabalho objetiva a análise histopatológica do sistema digestivo das lagartas de A. gemmatalis, após a interacão dos entomopatógenos Btk e VPNAg, representados nas formulacões Dipel e Baculovirus anticarsia, respectivamente. As avaliacões foram realizadas com lagartas de 2º ínstar, onde a mortalidade foi avaliada diariamente, e a histopatologia foi realizada com lagartas coletadas nos períodos de 1, 3, 6, 12 e 24 horas após a aplicacão dos tratamentos. Os resultados dos ensaios in vivo, revelam que o tratamento referente à associacão VPNAg/Btk (98.68 per center de mortalidade) foi mais eficiente que VPNAg (81.28 per center de mortalidade) isoladamente, porém o Btk isoladamente causou 100 per center de mortalidade. Os tratamentos mostraram diferencas significativas (P menor 0,05) entre AgNPV e Btk,AgNPV e AgNPV/Btk. As análises de patogenicidade do VPNAg e Btk em lagartas de A. gemmatalis sugerem que os produtos Dipel e Baculovirus anticarsia foram mais eficientes, quando utilizados simultaneamente, pois a acão do VPNAg foi intensificada quando utilizada em associacão com Btk, provocando alteracões no intestino médio das lagartas a partir de 6 horas após os tratamentos. Quando os entomopatógenos foram utilizados isoladamente, as alteracões das células intestinais foram observadas apenas 12 horas após a aplicacão dos tratamentos.

Ocorrência de parasitóides de Spodoptera frugiperda (J. E. Smith)(Lep., Noctuidae) em lavouras de milho em Cachoeirinha, RS / Occurrence of Spodoptera frugiperda (J. E. Smith) (lep. Noctuidae) parasitoids in maize crops in Cachoeirinha, RS, Brazil

O Rio Grande do Sul é um dos maiores produtores de milho do país, porém pouco se conhece em termos de inimigos naturais dos principais insetos-praga da cultura. O objetivo do presente trabalho foi realizar um levantamento dos parasitóides da lagarta-do-cartucho do milho, Spodoptera frugiperda. Para tanto, foram efetuadas amostragens em áreas de lavouras de milho do Instituto Riograndense do Arroz (IRGA), em Cachoeirinha, RS, nos anos agrícolas 1999/2000 e 2000/2001. A coleta dos insetos foi realizada semanalmente, durante o ciclo da cultura. As lagartas foram individualizadas e mantidas em laboratório até a emergência dos adultos ou dos parasitóides. Foram coletadas 1425 lagartas no primeiro período de amostragens e 518 no segundo. Destas, 18,11 por cento e 22,01 por cento encontravam-se parasitadas, com a presença dos seguintes parasitóides: Chelonus sp., Cotesia sp. e Exaticolus sp. (Hym., Braconidae), Campoletis flavicincta e Ophion sp. (Hym., Ichneumonidae) e Archytas incertus e Lespesia archippivora (Dip., Tachinidae). Houve predomínio de C. flavicincta, correspondendo a 63,57 por cento e 76,32 por cento dos parasitóides, respectivamente para o primeiro e segundo período de amostragens. Em C. flavicincta, ocorreu hiperparasitismo por Conura sp. (Chalcididae) e Aphanogmus sp. (Ceraphronidae).

PCR and bioassays screening of Bacillus thuringiensis isolates from rice-fields of Rio Grande do Sul, specific to lepidopterans and coleopterans

Visando a selecão de seis grupos de genes cry de Bacillus thuringiensis (Bt), que codificam proteínas ativas para coleópteros e lepidópteros pragas do arroz, 46 isolados de Bt provenientes de amostras de solos das regiões orizícolas do Rio Grande do Sul (RS), foram testados por PCR. Os isolados de Bt foram crescidos em Agar Nutriente durante 12 h e submetidos a extracão de DNA total. Os fragmentos amplificados foram analisados em géis de agarose (1-1,5 per center). Os resultados referentes ao total de isolados selecionados mostraram que 56,51 per center foram potencialmente específicos a lepidópteros (cry1, cry2 e cry9) e 21,73 per center a coleópteros (cry3 e cry7/8), tendo sua distribuicão homogênea entre as regiões orizícolas do RS. Apenas os genes cry2 foram localizados somente na região Litoral. Nos bioensaios com lagartas de Spodoptera frugiperda o isolado Bt 2027-1 obteve a maior mortalidade corrigida (25 per center), o qual havia sido pré-selecionado pela presenca de genes cry9. Para a mesma espécie, os testes de toxicidade através de proteínas purificadas de Bt aizawai HD68 revelaram uma DL50 de 0,95 mg/larva. Dois isolados de Bt causaram 100 per center de mortalidade às larvas de Oryzophagus oryzae, tendo esses sido pré-selecionados pela presenca de genes cry3. Os resultados dos bioensaios confirmam a predicão da atividade de Bt por PCR, a qual deve estar diretamente relacionada aos genes cry que codificam as proteínas inseticidas específicas.

Genes de Bacillus thuringiensis: uma estratégia para conferir resistência a insetos em plantas / Bacillus thuringiensis genes: an approach to confer insect resistance to plants

Os insetos constituem uma das principais causas de danos à produçäo agrícola no mundo. O controle de insetos tem sido realizado por meio de agroquímicos e, em muito menor escala, pelo emprego de inseticidas biológicos. As plantas transgênicas resistentes a insetos representam uma nova alternativa no combate aos insetos-praga das lavouras. A bactéria entomopatogênica Bacillus thuringiensis Berlinier (Bt) é a fonte dos genes de resistência nas chamadas plantas-Bt, produzidas comercialmente. No presente trabalho de revisäo, säo abordados os aspectos relacionados à bactéria Bt como fonte de genes de resistência a insetos-pragas, plantas geneticamente modificadas, vantagens do uso de plantas-Bt, bem como perspectivas dessa ferramenta biotecnológica