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The hornworm (Erinnyis ello) is an important pest of cassava and rubber tree with high capacity of migration and can be controlled with biological control agents such as baculoviruses. Baculoviruses are rod-shaped enveloped viruses with circular double-stranded DNA genomes infecting insects of the order Lepidoptera. Prior to large-scale use of certain baculovirus, it is essential to assess its genetic and morphological information. This work aimed to characterize the baculovirus isolate occurring in Erinnyis ello populations at small farms in Cruzeiro do Sul, Acre State, Brazil and to develop parameters for field management using this isolate. Viral particles were purified from infected larvae through ultracentrifugation in sucrose gradient. After treatment and negative staining with uranyl acetate 2%, viral particles were visualized using transmission electron microscope. The virus with ovicylindrical occlusion body presenting only one virion per envelope and size less than 0.5 μm was identified as a member of the genus Betabaculovirus. Viral DNA was extracted through phenol/chloroform cycles, digested with different restriction enzymes and separated with agarose gel electrophoresis. The restriction enzyme patterns obtained with the enzymes Bam HI and Hind III revealed seven and three DNA fragments, respectively; cleavage with Pst I, generated eight molar fragments and three submolar fragments and Eco RI digestion resulted in 21 fragments, with some submolar bands. Comparison of the DNA restriction pattern from ErelGV isolated in Acre with that described for ErelGV isolated from SC State showed high similarity. This isolate named as ErelGV-Acre presented potential for control of E. ello populations.

Spodoptera frugiperda (fall armyworm) is one of the most important maize pests in the world and the baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a natural pathogen of this pest, has been used as a biopesticide for its control. At present, in vivo strategies at the commercial scale are employed by multiplying the virus in the host insect in biofactory facilities; however, in vitro large-scale production is an interesting alternative to overcome the limitations of baculoviruses massal production. This study aimed to develop the process of the SfMNPV in vitro production by evaluating the effects of different multiplicities of infection (MOI) and nutritional supplements, morphological and molecular analysis of the infection on the growth of Sf9 cells and virus production. The Bioreactor Stirred Tank Reactor (STR) approach with glutamine-supplemented Sf-900 III serum free culture medium, combined with the MOI of 1.0, showed the best viral production performance, with a specific productivity above 300 occlusion bodies (OBs)/cell and volumetric productivity of 9.0 × 1011 OBs/L.

Abstract: The objective of this work was to evaluate the response of an in vitro host range to Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a pathogenic virus to the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), for the further development of a biopesticide based on cell culture systems. The cell lines from Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis (UFL-AG-286), and S. frugiperda (IPLB-SF-21AE and Sf9) were tested for their susceptibility to a highly-virulent Brazilian isolate of SfMNPV. The cytopathic effects induced by the virus, the production of viral particles, and the synthesis of viral polypeptides were examined and compared. Both S. frugiperda cell lines showed hypertrophy of cell nuclei and production of many polyhedra. The SDS-Page of radiolabed proteins showed that the cell protein synthesis was shutoff, while an intense band of about 30 kDa, recognized as polyhedrin, was synthesized. The other cell lines did not show polyhedra production, although some of them underwent morphological changes and protein synthesis shutdown in response to virus infection. The SF-21 and Sf9 cell lines are recommended for further in vitro production of SfMNPV.

Resumo: O objetivo deste trabalho foi avaliar as informações qualitativas e quantitativas sobre as interceptações de pragas ausentes não regulamentadas e quarentenárias em material vegetal importado. As informações sobre interceptações de pragas pelo serviço de quarentena vegetal da Embrapa, no período de 1977 a 2013, foram obtidas em um banco de dados, atas laboratoriais e listas de interceptação publicadas. As interceptações foram analisadas de acordo com regulamentação das pragas, espécie vegetal, parte vegetal importada e origem. O material foi categorizado em sementes botânicas e material de propagação vegetativa. No período do levantamento, foram interceptadas 75 espécies de pragas em 114 eventos de interceptação. Fungos, vírus e ácaros constituem a maior parte das interceptações, e a maioria delas ocorreu em lírio, oliveira, trigo, uva, arroz, batata e maçã. A taxa média anual de infestação/infecção do material vegetal analisado é de 2% dos processos de importação. O material para propagação vegetativa apresentou maior taxa de infestação/infecção por pragas do que as sementes botânicas. Das espécies-pragas interceptadas, 63% não são regulamentadas como quarentenárias para o Brasil. Esses resultados indicam a necessidade premente de revisão da atual lista de pragas quarentenárias do País.

Guinea grass (Megathyrsus maximus)is widely used as a forage crop in Brazil, but its production faces disease threats. Recently, new symptoms, such as oblong to irregular brown lesions on leaves, have emerged in M. maximus across the country. Particularly severe symptoms were observed in the BRS Tamani cultivar, and some other genotypes showed lesion coalescence and intense leaf necrosis. This study aimed to identify the cause of these symptoms. A fungus was isolated from the lesions and, using morphological measurements and molecular analysis of the ITS, GPDH and TEF-1α genes, it was identified as Bipolaris yamadae. Pathogenicity tests confirmed its ability to cause disease in M. maximus cv. Tamani. A field survey of 205 M. maximus genotypes showed severe symptoms in 8.35 % of the materials.

Abstract Chrysodeixis includens has become the major Lepidopteran pest of soybean crops, especially in the Brazilian Cerrado (savanna) region. A native isolate of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from this region, Buritis, MG, was assessed for its biological and molecular features. In addition, in vitro co-infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), another virus of an important soybean pest, was tested. The ChinNPV-Buritis isolate presented an average LC50 of 7,750 occlusion bodies (OBs)/ml of diet in C. includens larvae. Analysis of restriction endonuclease profiles of viral DNA revealed similarities with previously described ChinNPV isolates IE, IF, and IG from Brazil, although the presence of submolar bands indicates genetic heterogeneity. Optical microscopy analysis in conjunction with quantitative PCR (qPCR) demonstrated in vitro infection of this isolate in IPLB-SF-21AE, Sf9, and BTI-Tn-5B1-4 cell lines, but the amount of ChinNPV tends to decrease through serial passages. The qPCR method developed in this study successfully detected both AgMNPV and ChinNPV from cell culture and from infected larvae. The cell line Tn-5B1-4 is indicated for future development of in vitro production and co-infection studies.

Abstract: The objective of this work was to evaluate the phytosanitary aspect of two accessions of maize (Zea mays) seeds from the United States introduced to Brazil, regarding the presence of Wheat mosaic virus (WMoV). Two to three weeks after sowing, symptomatic leaves were tested by Elisa using specific antiserum to WMoV. The reaction was positive, and leaf samples were analyzed by real-time PCR and amplified PCR products were sequenced. The WMoV isolates had 99 to 100% nucleotide identity with isolates from Australia and the United States. Until now, there is no report of the presence of this virus in Brazil. According to the federal law on plant protection, the plants were burned to avoid the introduction of this exotic pest in the country. The obtained results show WMoV interception in Brazil.

ABSTRACT: Apple plants from France introduced in Brazil for research purposes were subjected to a phytosanitary analysis at the Plant Quarantine Laboratory of Embrapa Genetic Resources and Biotechnology (Cenargen). After grafting onto healthy apple rootstock, some plants showed phytoplasma-infection symptoms. Polymerase Chain Reaction (PCR) tests yielded DNA fragments of the expected size for phytoplasmas. DNA sequencing revealed an identity of the 16S rDNA nucleotide sequence of 98-99% with ‘Candidatus Phytoplasma solani’. This phytoplasma species is responsible for losses in European apple orchards and has not been reported in Brazil. According to the Federal Legislation on Plant Protection, the plants were incinerated to avoid the introduction of this exotic pest in Brazil.