RESUMO

Granuloviruses (GVs) Betabaculovirus associated with the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), especially those of the type I, have scarcely been studied. These GVs might be an effective alternative for the biocontrol of this insect. In this study, the native GVs SfGV-CH13 and SfGV-CH28 were isolated from FAW larvae and characterized for morphology, molecular traits, and insecticidal activity. The elapsed time between symptomatic infection of larvae and stop feeding as well as the weight of larvae before death or prior to pupation were also evaluated. Both GVs had ovoid shape and a length of 0.4 µm. They had the same DNA restriction profiles and their genome sizes were about 126 kb. The symptomatic infection with the tested GVs mainly caused flaccidity of larva body and discoloration of integument. The integument lysis was only observed in 8% of infected larvae. Infected larvae gradually stopped feeding. Overall, these symptoms are characteristic of infections caused by type I GVs, which are known as monoorganotropic or slow-killing GVs. The median lethal dose (LD50) values for SfGV-CH13 and SfGV-CH28 isolates were 5.4 × 102 and 1.1 × 103 OBs/larva, respectively. The median lethal time (LT50) ranged from 17 to 24 days. LT50 values decreased as the viral dose was increased. The elapsed time from symptomatic infection until pupation and body weight of larvae (third instar) were higher with SfGV-CH28 than SfGV-CH13. Both granulovirus isolates were able to kill the FAW larvae from the 12th day.

Assuntos

RESUMO

The ability of the isolate VG008 of S. frugiperda granulovirus (SpfrGV) to enhance the infectivity of the isolate SfCOL of S. frugiperda multiple nucleopolyhedrovirus (SpfrMNPV) was evaluated on S. frugiperda larvae. Bioassays were performed with mixtures by using different proportions 90%:10% (M1), 95%:5% (M2) and 97.5%:2.5% (M3) of SfCOL:VG008, respectively. All mixtures showed higher insecticidal activity that SfCOL. The mixture M3 showed the highest enhancement of SfCOL reducing 11.40 times the Mean Lethal Concentration and 96 h in the Mean Time to Death. The enhancer activity of proteins derived from VG008 (GVPs) were also evaluated in mixture with SfCOL. The GVPs increased 27% larval mortality caused by SfCOL and damaged the peritrophic membrane of S. litura larvae, suggesting that the key point in this enhancing activity is the initial step of the larva colonization, the midgut infection. M3 was formulated and evaluated under greenhouse conditions in maize plants using different doses. The highest efficacy was obtained with the highest dose of M3 (8 × 1011 OBs/ha), which was similar to that found when formulated SfCOL was applied using an approximately twofold higher dose. The viral mixture M3 was selected as the active ingredient for developing a new biopesticide for a more efficient management of the pest in the field.

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BACKGROUND: The hornworn Erinnyis ello is the major pest of natural rubber crops in Colombia, mainly controlled using toxic chemical insecticides. The use of E. ello Betabaculovirus is an environmentally sustainable alternative for its control. The aim of the present work was to characterize a prototype biopesticide formulation and evaluate its efficacy under different conditions. RESULTS: Quality control evaluations of formulated biopesticide revealed that all the parameters evaluated were under the permissible level. The lethal concentrations LC50 and LC90 of the biopesticide were 4.3 × 103 and 5.5 × 104 occlusion bodies (OBs) mL-1 , respectively. Biopesticide efficacies against second and fourth instar larvae under greenhouse conditions were higher than 80%. Evaluation of two application rates in a clonal garden resulted in 84% and 88% efficacy, comparable to that obtained with the chemical. The biopesticide in a commercial plantation showed efficacies between 74% and 82%. Biopesticide post-application persistence was estimated at least in 1 week under field natural conditions. Results allowed selection of the lowest evaluated dose (1 × 1011 OBs ha-1 ) as the basis for further field evaluations. CONCLUSION: Formulated ErelGV showed high efficacy to control the hornworm in rubber crops and high potential to be included in integrated pest management programs, thus it could be an interesting alternative to replace agrochemicals. © 2018 Society of Chemical Industry.

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RESUMO

In this report, we described the genome of a novel baculovirus isolated from the monocot insect pest Mocis latipes, the striped grass looper. The genome has 134,272 bp in length with a G + C content of 38.3%. Based on the concatenated sequence of the 38 baculovirus core genes, we found that the virus is a betabaculovirus closely related to the noctuid-infecting betabaculoviruses including Pseudaletia unipuncta granulovirus (PsunGV), Trichoplusia ni granulovirus (TnGV), Helicoverpa armigera granulovirus (HearGV), and Xestia c-nigrum granulovirus (XecnGV). The virus may constitute a new Betabaculovirus species tentatively named Mocis latipes granulovirus (MolaGV). After gene content analysis, five open reading frames (ORFs) were found to be unique to MolaGV and several auxiliary genes were found including iap-3, iap-5, bro-a, bro-b, and three enhancins. The virus genome lacked both chitinase and cathepsin. We then looked at the evolutionary history of the enhancin gene and found that betabaculovirus acquired this gene from an alphabaculovirus followed by several duplication events. Gene duplication also happened to an endonuclease-like gene. Genomic and gene content analyses revealed both a strict collinearity and gene expansion into the genome of the MolaGV-related species. We also characterized the granulin gene using a recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and found that occlusion bodies were produced into the nucleus of infected cells and presented a polyhedral shape and no occluded virions within. Overall, betabaculovirus genome sequencing is of importance to the field as few genomes are publicly accessible. Mocislatipes is a secondary pest of maize, rice, and wheat crops in Brazil. Certainly, both the discovery and description of novel baculoviruses may lead to development of greener and safer pesticides in order to counteract and effectively control crop damage-causing insect populations.

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Six complete genome sequences of Cydia pomonella granulovirus (CpGV) isolates from Mexico (CpGV-M and CpGV-M1), England (CpGV-E2), Iran (CpGV-I07 and CpGV-I12), and Canada (CpGV-S) were aligned and analyzed for genetic diversity and evolutionary processes. The selected CpGV isolates represented recently identified phylogenetic lineages of CpGV, namely, the genome groups A to E. The genomes ranged from 120,816 bp to 124,269 bp. Several common differences between CpGV-M, -E2, -I07, -I12 and -S to CpGV-M1, the first sequenced and published CpGV isolate, were highlighted. Phylogenetic analysis based on the aligned genome sequences grouped CpGV-M and CpGV-I12 as the most derived lineages, followed by CpGV-E2, CpGV-S and CpGV-I07, which represent the most basal lineages. All of the genomes shared a high degree of co-linearity, with a common setup of 137 (CpGV-I07) to 142 (CpGV-M and -I12) open reading frames with no translocations. An overall trend of increasing genome size and a decrease in GC content was observed, from the most basal lineage (CpGV-I07) to the most derived (CpGV-I12). A total number of 788 positions of single nucleotide polymorphisms (SNPs) were determined and used to create a genome-wide SNP map of CpGV. Of the total amount of SNPs, 534 positions were specific for exactly one of either isolate CpGV-M, -E2, -I07, -I12 or -S, which allowed the SNP-based detection and identification of all known CpGV isolates.

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RESUMO

El uso de baculovirus como agentes de control biológico de insectos plaga, se ha convertido en una estrategia efectiva que se ha implementado gradualmente en diferentes sistemas productivos a nivel mundial. Para el desarrollo de un bioplaguicida a base de baculovirus, es necesario contar con una metodología para determinar el título viral en el producto en proceso y terminado. Para tal fin, en este trabajo se diseñó y optimizó una técnica de cuantificación viral (Betabaculovirus) mediante PCR cuantitativo (q-PCR). Se utilizó una sonda TaqMan diseñada sobre el gen de granulina, altamente conservado. Para la técnica de q-PCR se determinó la especificidad, sensibilidad y reproducibilidad, encontrando que puede detectar y cuantificar aislamientos del género Betabaculovirus provenientes de cinco especies diferentes de insectos (granulovirus de Tecia solanivora, Phthorimaea operculella,Erinnyis ello, Tuta absoluta y Spodoptera frugiperda) incluso de diferente origen geográfico, pero no detecta aislamientos del género Alphabaculovirus (nucleopoliedrovirus de Spodoptera ornithogalli, Diatraea saccharalis o S. frugiperda). El límite mínimo de detección de la técnica fue de 6,4 x 10-4 ng de ADN, lo que equivale a 1,25 x 10(5) copias del gen. Así mismo, la variación intra e inter ensayos fue mínima, demostrando la reproducibilidad de la misma. La aplicabilidad de la técnica fue evaluada para la detección de granulovirus en muestras de larva, suelo, y para determinar la concentración viral en un bioplaguicida formulado como concentrado emulsionable. En conclusión, la técnica de q-PCR desarrollada fue reproducible, sensible y específica, con aplicabilidad en estudios de persistencia viral en campo, control de infecciones en crías de insectos y control de calidad de bioplaguicidas a base de betabaculovirus.

The use of baculovirus as a biocontrol agent is an effective strategy, which has been gradually implemented in different production systems worldwide. For the development of a biopesticide based on baculovirus, it is necessary to have a methodology to determine the viral concentration in the process and in the finished product. In this study, a technique for viral quantification by quantitative PCR (q-PCR) was designed and optimized; therefore we used a TaqMan probe designed on granulin gene which is highly conserved. The specificity, sensitivity and reproducibility of the technique were determined. The q-PCR was able to detect and quantify isolates from the genus Betabaculovirus from five different insects species (granulovirus from Tecia solanivora, Phthorimaea operculella, Erinnys ello, Tuta absoluta and Spodoptera frugiperda) even from different geographic origins, while other isolates of baculovirus as from the genus Alphabaculovirus (nucleopolyhedrovirus from Spodoptera ornithogalli, Diatraea saccharallis or S. frugiperda) were not detected. The minimum detection limit of the technique was 6.4 x 10-4 ng /µl of DNA, equivalent to 1.25 x 10³ gene copies. Additionally, intra- and inter-assays variation was minimal, demonstrating the reproducibility of technique. The applicability of the technique was evaluated for detecting granulovirus from samples of larva and soil, and to determine the virus concentration in the biopesticide formulated as emulsifiable concentrate. In conclusion, quantitative PCR was a technique reproducible, sensitive and specific to allow viral persistence studies in field, viral infection control in rearing and quality control of a biopesticide based on betabaculovirus.