Bioreactor Production Process of Spodoptera frugiperda multiple nucleopolyhedrovirus Biopesticide.

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.

Isolation of a Novel Alphabaculovirus (Baculoviridae) from Automeris liberia (Cramer, 1780) (Lepidoptera: Saturniidae) in African Oil Palms in Brazil.

A novel baculovirus observed to infect Automeris liberia (Cramer) (bullseye moth) is here described. Caterpillars of A. liberia with symptoms of viral infection were collected from African oil palm plantations in Tailândia, PA, Brazil. Macerated caterpillars were then offered to caterpillars of Automeris cinctistriga (Felder & Rogenhoper), leading to viral symptoms and death before pupation. A transmission electron microscope was used for virus ultrastructural identification. The presence of viral occlusion bodies (OBs) containing multiple nucleocapsids was observed and such features are compatible with Alphabaculovirus (Baculoviridae). Molecular detection by PCR with primers for polyhedrin gene (polh) and for late expression factor-8 gene (lef-8), confirmed that this isolate belonged to Alphabaculovirus genus. To our knowledge, this is the first record of a baculovirus isolated from or associated to Automeris. The name Automeris liberia nucleopolyhedrovirus (AuliNPV) is proposed for the new virus.

Evaluation of key parameters for developing a Metarhizium rileyi-based biopesticide against Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize: laboratory, greenhouse, and field trials.

BACKGROUND: The entomopathogenic fungus Metarhizium rileyi is known to cause natural infections in some caterpillars. In this multiyear study, we carried out laboratory, greenhouse and three field trials with the aim of developing a sustainable option for control of the fall armyworm (FAW) in maize. Association of M. rileyi with Spodoptera frugiperda baculovirus (SfMNPV) and delivery strategies were also investigated. RESULTS: The selected isolate (CG381) was effective in the laboratory at a low concentration (~ 4 × 103 conidia cm-2 ), killing >95% of FAW larvae within 8 days. In the greenhouse assay, applications of conidia suspended in water or as dry powder in maize whorls (~ 6.3 × 106 conidia per plant) produced similar larval mortalities (88%-96%). In the field trials, conventional spraying of unformulated conidia (0.6 and 1.2 × 1012 conidia ha-1 ) caused low larval mortalities (27-31%). Simultaneous application of either unformulated or oil-based formulations of M. rileyi conidia and S. frugiperda baculovirus (SfMNPV) to plant rows caused larval mortalities comparable with each of the pathogens applied alone. However, when a formulation containing both pathogens was sprayed directly into the whorls, the overall mortality of S. frugiperda larvae due to pathogens (mostly to M. rileyi infections) reached ~ 59%, twofold higher than the other treatments with conventional spraying, whereas in the control, the overall mortality was only 1%. CONCLUSION: Optimizing exposure of S. frugiperda larvae to an inoculum of virulent entomopathogens through directed applications to maize whorls is critical to produce satisfactory mortality levels and is promising for integrated pest management.

Optimization of Large-Scale Production of Chrysodeixis includens nucleopolyhedrovirus for Its Use as a Biopesticide.

The baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) is pathogenic to Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) larvae, known as soybean looper, which is an important pest of soybean and bean. In this study, some parameters were tested to overcome the difficulties in the in vivo production of ChinNPV aiming to increase its use as a biopesticide. First, different combinations of larval instars (3rd and 4th instars), larval incubation temperatures (23 °C and 26 °C), and rearing densities (individually and 10 larvae/cup) were compared for larval weight and the production of occlusion bodies (OBs). A positive correlation (p< 0.001) was observed for OB production and larval weight. Fourth instar larvae produced more OBs than third instar larvae (p<0.05); however, no significant differences in OBs/larva (p>0.05) were observed for larvae kept in groups or individually. Therefore, a second assay was performed using fourth instar larvae incubated at 26 °C and two larval densities (10 larvae/cup and 40 larvae/cup). The losses of insects and OB production were evaluated as well as the influence of storage temperatures post-mortem (-20 °C, 4 °C, and 15 °C) in the OB yield. As expected, insect losses due to cannibalism or microbial contamination were greater (p<0.05) with the increase in larval density, although no difference was observed in OBs/larva (p>0.05). In addition, the storage temperature post-mortem did not influence the OB yield (p>0.05). The average production of ChinNPV OBs was 3×1010 OBs/40 larvae cup. The results demonstrate the viability of rearing C. includens in groups to enhance the mass production and reduce virus production costs.

Within-host interactions of Metarhizium rileyi strains and nucleopolyhedroviruses in Spodoptera frugiperda and Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Members of the family Baculoviridae have been quite successfully used as biocontrol agents against some lepidopterans. Likewise, a number of fungi are important natural enemies of these pests. An interesting approach to increase control efficacy could be the combination of a given nucleopolyhedrovirus (NPV) and a fungus, since they possess distinct modes of action. As a first step towards this goal, we assessed the interaction between NPV (either AgMNPV-79 or SfMNPV-6nd) and the entomopathogenic fungus Metarhizium rileyi (either CG1153 or CG381), using Anticarsia gemmatalis and Spodoptera frugiperda as hosts. In sequential applications of these pathogens, per os inoculation of an NPV (leaf discs with 2.5 × 104 occlusion bodies) either two days before or two days post-spraying of its counterpart fungal strain (5 × 103 conidia.cm-2 sprays) usually resulted in an antagonistic effect. When both pathogens were simultaneously applied at different combined dosages, usually an additive effect was seen. Interestingly, a number of dead larvae showing signs of co-infections (partially with soft integument and partially mummified) were recorded. However, mixes with lower dosages of both pathogens did not cause significantly higher insect mortalities compared to low dosages of the fungus applied alone. The advantages and disadvantages of the simultaneous applications of NPV and M. rileyi aiming at the management of either A. gemmatalis or S. frugiperda were discussed.

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.

Characterization of a Chrysodeixis includens nucleopolyhedrovirus Isolate from Brazilian Cerrado and Assessment of its Co-Infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus

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.

Interception of 'Candidatus Phytoplasma solani' in Brazil in apple tree propagative material imported from France / Interceptação de 'Candidatus Phytoplasma solani' no Brasil em material propagativo de macieira importado da França

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.(AU)

Plantas de macieira originárias da França introduzidas no Brasil para fins de pesquisa foram submetidas à análise no Laboratório de Quarentena Vegetal da Embrapa Recursos Genéticos e Biotecnologia (Cenargen). Após realizar a enxertia em porta-enxertos sadios, algumas plantas apresentaram sintomas típicos de infecção por fitoplasmas. Os testes por meio da técnica de reação em cadeia da polimerase (PCR) obtiveram fragmentos de DNA do tamanho esperado para fitoplasmas. O sequenciamento de DNA revelou uma identidade de 98-99% da região 16 rDNA dos fitoplasmas encontrados com o fitoplasma 'Candidatus Phytoplasma solani'. Essa espécie de fitoplasma é responsável por perdas em pomares de maçã europeus e não foi relatada no Brasil. De acordo com a Legislação Federal de Proteção de Plantas, as plantas de macieira foram incineradas para evitar a entrada dessa praga exótica no Brasil.(AU)

Genome Assembly of Schlumbergera Virus X Infecting Prickly Pear (Opuntia cochenillifera) in Brazil.

Potexviruses frequently infect plants from the family Cactaceae. We report the complete genome sequence of a new Schlumbergera virus X (SchVX) isolate. The genome has 6,615 nucleotides and shares 94% nucleotide identity with SchVX-K11 from Schlumbergera. This is the first sequence of an isolate of SchVX from the genus Opuntia.

Biological and molecular characterisation of Bidens mosaic virus supports its assignment as a member of a distinct species in the genus Potyvirus.

The complete nucleotide (nt) sequence of Bidens mosaic virus (BiMV) isolate SP01 was determined and shown to consist of 9,557 nt. Since it shared highest identities in the nt sequence of the whole genome (66-73 %) and in the aa sequence of the polyprotein (60-76 %) with viruses of the potato virus Y subgroup, it was compared with them genetically and biologically. Phylogenetic analysis showed that the closest relative of BiMV is sunflower chlorotic mottle virus, from which it, however, differed significantly in various respects. These results indicate that BiMV should represent a distinct species in the genus Potyvirus.