Zilda Maria A. Ribeiro

Evaluation of seven viral isolates as potential biocontrol agents against Pseudoplusia includens (Lepidoptera: Noctuidae) caterpillars.

The caterpillar Pseudoplusia includens (Walker, 1857) (Lepidoptera, Noctuidae), known as soybean looper, is a pest that has recently assumed greater importance in soybean in Brazil. Isolates of nucleopolyhedroviruses (NPVs) of this pest have been identified from cotton in Guatemala and soybean farms in Brazil, providing an interesting perspective of potential use of viral insecticide against the insect in lieu to chemical insecticides. With the objective to contribute to the characterization studies of this virus, morphological and molecular analyses and biological activity were carried out with seven P. includens viral isolates (I-A to I-G). Electron microscopy of viral samples, purified from macerated infected larvae, showed particles with typical morphology of the Baculoviridae family, genus Alphabaculovirus (Nucleopolyhedrovirus - NPV) presenting virions with only a single nucleocapsid per envelope (SNPV) occluded in a protein matrix, forming occlusion bodies (OB). This virus was then classified as P. includens single nucleopolyhedrovirus (PsinSNPV). OB particles analyzed in SDS-polyacrylamide gel showed an intense band corresponding in size to NPV polyhedrin protein. DNA restriction profiles of the PsinSNPV isolates showed differences in the fragment size and number suggesting the existence of genotypic variants, except between I-E and I-F profiles that were similar. Among the isolates tested for infectivity against P. includens, I-A, I-E and I-F were the most virulent. Survival times (ST(50)) varied according to viral concentration, with significant differences among isolates for the three higher concentrations.

Produção de inóculo de Alternaria cassiae

In order to adjust the methodology of inoculum production, an isolate of the fungus Alternaria cassiae, selected as a biological control agent for sicklepod (Senna obtusifolia) was studied under different culture media and mycelial growth conditions. Homemade V-8 juice medium without CaCO3 addition, incubation on 200-250 mL of medium (around 50% of the flask volume) with agitation at 250 rpm during 48 hours, followed by mycelial mass growth in continuous dark were appropriate conditions for a large scale production of inoculum. The sporulation was aided by incubation of the mycelial mass at 25oC. The use of 300 mL of mycelial mass per pan (29.9 by 44.5 by 5.0 cm ) to induce the sporulation provided rapid dryness and ensured the conidium integrity, indicated by higher infectivity of the inoculum.

Identification of a new nucleopolyhedrovirus from naturally-infected Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae) larvae on poplar plantations in South Brazil

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81+/-2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [(35)S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.

Padrões isoenzimáticos e morfologia de isolados de Alternaria spp. patogênicos a Senna obtusifolia

ABSTRACTIsoenzyme patterns and morphology of isolates of Alternaria species pathogenic to Senna obtusifolia morfologicos. Portanto, esta tecnica tem potencial para serusada na separacao destas especies de Alternaria. A ocorrenciade mais de uma especie deste genero fungico em S. obtusifoliafoi constatada, ampliando o numero de patogenos a seremavaliados no programa de desenvolvimento de umbioherbicida para esta especie de planta daninha.Palavras-chave: biocontrole, patogeno, plantadaninha, caracterizacao.alternata. Isoenzyme patterns confirmed the results ofmorphological criteria. The occurrence of more than onespecies of this fungus infecting S. obtusifolia was found,widening the range of suitable pathogens to be evaluated inbioherbicide programs for sicklepod control.Um grande numero de especies de Alternaria temsido relatado causando manchas foliares em diversas plantascultivadas e nao cultivadas, dentre estas, algumas especiesdaninhas que infestam culturas economicamenteimportantes. Um bioherbicida foi desenvolvido a partir deAlternaria cassiae Jurair & Khan (Simons, 1982) eregistrado nos Estados Unidos para controle do fedegoso(Senna obtusifoliaI L.) (Charudattan, 1996). No Brasil,onde esta planta daninha vem-se tornando um serioproblema, particularmente em areas cultivadas com soja[Glycine max (L.) Merril], um isolado de A. cassiae , coletadono Distrito Federal (DF), demons trou grande potencial paraser utilizado contra biotipos brasileiros desta planta(Figueiredo et al ., 1992). Outros isolados de Alternaria spp.foram obtidos de folhas e hastes de S. obtusifolia , coletadasem areas do DF, em diferentes ocasioes, e incorporados aColecao de Fungos Agentes de Controle Biologico daEmbrapa-Recursos Geneticos e Biotecnologia, Brasilia, DF.Este trabalho teve como objetivos caracterizar esses isoladospor meio de caracteristicas morfologicas e analise deisoenzimas em gel de poliacrilamida e do estudo dascaracteristicas morfologicas.Foram utilizadas culturas monosporicas de 13isolados de Alternaria spp. (Tabela 1), dentre os quais, oisolado CG594, anteriormente identificado como A. cassiae(Figueiredo et al., 1992). Estes isolados foram previamenteavaliados quanto a patogenicidade, nas hospedeiras originaise mantidos em tubos de ensaio contendo o meio de batata-dextrose-agar (BDA) a 4

Complete Genome Sequences of Six Chrysodeixis includens Nucleopolyhedrovirus Isolates from Brazil and Guatemala

ABSTRACT The baculovirus, Chrysodeixis (formerly Pseudoplusia) includens nucleopolyhedrovirus (ChinNPV), is a new Alphabaculovirus pathogenic to Chrysodeixis includens. Here, we report the complete genome sequences of six ChinNPV isolates. The availability of these genome sequences will provide information on ChinNPV molecular genetics, promoting understanding of its pathogenicity, diversity, and evolution.

The genome sequence of Condylorrhiza vestigialis NPV, a novel baculovirus for the control of the Alamo moth on Populus spp. in Brazil

Condylorrhiza vestigialis (Lepidoptera: Cambridae), commonly known as the Brazilian poplar moth or Alamo moth, is a serious defoliating pest of poplar, a crop of great economic importance for the production of wood, fiber, biofuel and other biomaterials as well as its significant ecological and environmental value. The complete genome sequence of a new alphabaculovirus isolated from C. vestigialis was determined and analyzed. Condylorrhiza vestigialis nucleopolyhedrovirus (CoveNPV) has a circular double-stranded DNA genome of 125,767bp with a GC content of 42.9%. One hundred and thirty-eight putative open reading frames were identified and annotated in the CoveNPV genome, including 38 core genes and 9 bros. Four homologous regions (hrs), a feature common to most baculoviruses, and 19 perfect and imperfect direct repeats (drs) were found. Phylogenetic analysis confirmed that CoveNPV is a Group I Alphabaculovirus and is most closely related to Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Choristoneura fumiferana DEF multiple nucleopolyhedrovirus CfDEFMNPV. The gp37 gene was not detected in the CoveNPV genome, although this gene is found in many NPVs. Two other common NPV genes, chitinase (v-chiA) and cathepsin (v-cath), that are responsible for host insect liquefaction and melanization, were also absent, where phylogenetic analysis suggests that the loss these genes occurred in the common ancestor of AgMNPV, CfDEFMNPV and CoveNPV, with subsequent reacquisition of these genes by CfDEFMNPV. The molecular biology and genetics of CoveNPV was formerly very little known and our expectation is that the findings presented here should accelerate research on this baculovirus, which will facilitate the use of CoveNPV in integrated pest management programs in Poplar crops.