Microsclerotia from Metarhizium robertsii: Production, ultrastructural analysis, robustness, and insecticidal activity.

Microsclerotia (MS) are considered one of the most promising propagules for use as active ingredients in biopesticides due to their tolerance to abiotic factors and ability to produce infective conidia for the control of pests. Therefore, the objective of this research was to establish the conditions required to induce the formation of microsclerotia in Metarhizium robertsii Mt004 and to study its development process, tolerance to abiotic factors and insecticidal activity of MS-derived conidia. M. robertsii started to form hyphal aggregates after 2 days and looked more compact after 8 days. MS were mature and pigmented after 20 days. The final yield was 2.0 × 103 MS/mL and MS size varied between 356.9 and 1348.4 µm. Ultrastructure analysis revealed that mature MS contained only a few live cells embedded in an extracellular matrix. Mature MS were more tolerance to UV-B radiation, heat and storage trials than conidia from Solid State Fermentation. MS-derived conidia were as virulent as conidia against Diatraea saccharalis larvae. These results showed that MS are promising propagules for the development of more persistent and efficient biopesticides for harsh environmental conditions. Our findings provide a baseline for production and a better understanding of microsclerotia development in M. robertsii strains.

Compatibilidad de un bioinsecticida a base de Beauveria bassiana con agroquímicos: efecto en la germinación y la actividad insecticida sobre Diatraea saccharalis

Los barrenadores del tallo del género de Diatraea spp. (Lepidoptera:Crambidae) han sido reconocidos por ser la plaga de mayor impacto en el cultivo de caña de azúcar, en el que causan importantes pérdidas económicas. Para su control, una cepa colombiana de Beauveria bassiana (Bals.) Vuill (aislamiento Bv062) que demostró alta virulencia frente a larvas de D. saccharalis (Fabricius), fue utilizada para el desarrollo de un bioinsecticida formulado como un concentrado emulsionable (CE). Con el fin de integrar este bioinsecticida en un esquema de manejo integrado de plagas, es necesario evaluar su compatibilidad con los agroquímicos utilizados comúnmente en el cultivo de caña de azúcar. Para tal fin se evaluó el efecto in vitro de 11 agroquímicos (fungicidas, insecticidas y herbicidas) sobre la germinación y actividad insecticida de conidios formulados. Inicialmente el CE y los agroquímicos fueron ajustados al doble de la concentración recomendada y mezclados en proporción 1:1, se incubaron durante dos horas, y se determinó el porcentaje de germinación. Los conidios expuestos a los agroquímicos fueron también purificados y utilizados para un bioen-sayo frente a larvas de segundo instar de D. saccharalis. El CE resultó ser compatible con los herbicidas e insecticidas evaluados, manteniendo una germinación y eficacia superior al 80%. Por el contrario, el CE fue incompatible con todos los fungicidas evaua-dos. Estos resultados permiten sugerir recomendaciones de aplicación del bioplaguicida a base de B. bassiana Bv062, junto con insecticidas y herbicidas empleados en el cultivo de caña, pero se recomienda no aplicarlo de manera simultánea con fungicidas como el benomil y del grupo de triazoles. Se recomienda validar estos resultados en condiciones de campo.

Sugarcane stem borers of the genus Diatraea spp. (Lepidoptera:Crambidae) have been recognized as the pest of the greatest impact on sugarcane crops causing significant economic losses. For its control, a colombian strain of Beauveria bassiana (Bals.) Vuill (code Bv062) that showed a high virulence against D. saccharalis (Fabricius) larvae was used for a biopesticide development, formulated as emulsifiable concentrate (EC). In order to include the EC into an integrated pest management (IPM) scheme, it is necessary to evaluate its compatibility with the agrochemicals commonly used in sugarcane crops. For this purpose, the in vitro effect of 11 agrochemicals (fungicides, insecticides and herbicides) on the germination and insecticidal activity of Bv062 formulated conidia was evaluated. Initially, the EC and agrochemicals were adjusted to double the recommended concentration and mixed in a 1:1 ratio. The mixes were incubated for two hours, and the germination percentage was calculated. The conidia exposed to the agrochemicals were also purified and used for a bioassay against second instar larvae of D. saccharalis. The EC was compatible with the herbicides and insecticides, maintaining germination and efficacy higher than 80%. In contrast, EC was incompatible with all the fungicides evaluated. These results allow us to suggest recommendations for the application of the biopesticide based on B. bassiana Bv062 with insecticides and herbicides used in sugarcane crops, but it is not recommended to apply it simultaneously with fungicides such as benomyl and the triazole group. It is recommended to validate these results under field conditions.

Natural Coinfection between Novel Species of Baculoviruses in Spodoptera ornithogalli Larvae.

Spodoptera ornithogalli (Guenée) (Lepidoptera: Noctuidae) is an important pest in different crops of economic relevance in America. For its control, strategies that include chemicals are usually used; so, the description of entomopathogens would be very useful for the formulation of biopesticides. In this regard, two different baculoviruses affecting S. ornithogalli were isolated in Colombia, with one of them being an NPV and the other a GV. Ultrastructural, molecular, and biological characterization showed that both isolates possess the 38 core genes and are novel species in Baculoviridae, named as Spodoptera ornithogalli nucleopolyhedrovirus (SporNPV) and Spodoptera ornithogalli granulovirus (SporGV). The bioassays carried out in larvae of S. ornithogalli and S. frugiperda showed infectivity in both hosts but being higher in the first. In addition, it was observed that SporGV potentiates the insecticidal action of SporNPV (maximum value in ratio 2.5:97.5). Both viruses are individually infective but coexist in nature, producing mixed infections with a synergistic effect that improves the performance of the NPV and enables the transmission of the GV, which presents a slowly killing phenotype.

Metarhizium rileyi biopesticide to control Spodoptera frugiperda: Stability and insecticidal activity under glasshouse conditions.

The insect-pathogenic fungus Metarhizium rileyi is highly sensitive to nutritional and environmental conditions which makes it difficult to produce as a stable biopesticide. In this study, a Colombian isolate of this fungus was produced in bulk, and conidia were formulated as an emulsifiable concentrate (EC). The stability of formulated conidia was studied. Conidial viability was maintained at >85 % viability for 12 m under refrigeration and for >three months at 18 °C. The pH values were stable, while contaminant content was significantly reduced. The efficacy of the EC to control Spodoptera frugiperda (Smith) was correlated with the storage time using different mathematical models, and conservative values of six and 12 months at 8 °C and 18 °C respectively, were established. Finally, the EC was evaluated in maize plants under glasshouse conditions. The LC50 and LC90 were estimated to be 1.17 × 104 and 4.03 × 106 conidia/mL respectively and a 57 % reduction in recent damage of plants was achieved. This study demonstrated the potential of M. rileyi formulated as EC to control S. frugiperda in maize. Therefore, it is necessary to continue developing this biopesticide, in order to deliver a new tool to be integrated in pest management programs.

Identification of Diatraea spp. (Lepidoptera: Crambidae) based on cytochrome oxidase II.

Diatraea spp. (Lepidoptera: Crambidae) are a group of insects that are agriculture pests in many economically relevant crops such as sugarcane, sorghum, corn and rice. Recognized species for this genus respond differentially to natural enemies used in their biological control, emphasizing the importance of species in a regional approach. Currently, identification is based on the male genitalia. However, the availability of specimens collected from field and subjectivity based on the character recognition can seriously hamper species identification, and therefore result in inadequate pest management. To overcome this, individuals of Diatraea spp. preliminarily classified male genitalia and obtained from reared conditions and the field (both derived from natural populations occurring in Colombia) were analyzed using genitalic morphometry and molecular biology specifically using a fragment of the cytochrome oxidase subunit II (CO II) mitochondrial gene. Although morphometric analysis did not show any overriding results regarding genitalia morphology, the bioinformatics analyses of CO II sequences resulted in an adequate classification of the individuals within the recognized species. It also, revealed that the occurrence of clades associated with geographical distribution may be associated with cryptic species. The latter was also confirmed by a Single-Strand Conformation Polymorphism (SSCP) methodology evaluating the same fragment of CO II. This experimental approach allows properly recognizing each species and in consequence is proposed as an effective tool in Diatraea species identification.

Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda.

BACKGROUND: Baculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use. RESULTS: Genome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus' life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport. CONCLUSIONS: The SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring in nature. This feature could be the cause of its high insecticidal power and therefore SfColA becomes a great candidate for bioinsecticide formulations.

Selección de un sistema de atomización para la formación de micropartículas de Eudragit S100 en lecho fluido / Selecting a spray system for the formation of Eudragit S100 microparticles in a fluid bed

La elaboración de micropartículas en lecho fluido es de gran interés en la industria farmacéutica, alimentaria y agrícola, ya que este tipo de formulación permite controlar la liberación de ingredientes activos y su estabilidad y funcionalidad mediante la formación de pequeñas partículas sólidas. El lecho fluido es el equipo comúnmente usado en la industria para realizar el proceso y éste puede tener dos sistemas de atomización: superior (Top spray) e inferior (Bottom spray). En este trabajo se evaluaron los dos sistemas de atomización con miras a la formación de micropartículas de Eudragit® S100. Se realizaron experimentos para ajustar los factores críticos del proceso y los niveles de éstos utilizando un diseño factorial multinivel y trabajando en un lecho fluido marca Glatt GmbH D – 01277. Los factores evaluados fueron la temperatura de entrada, la presión interna de la cámara y la velocidad de flujo. El material de recubrimiento consistió en un polímero del ácido metacrílico denominado Eudragit® S100 y como núcleo se empleó talco malla 325. El sistema seleccionado para la microencapsulación fue el de atomización superior (Top spray). Las condiciones de atomización establecidas fueron velocidad de flujo de 4,12 mL/min (flujo 8 rpm), temperatura de entrada de80ºC y presión interna de la cámara de 1 y 3 bares. Las micropartículas presentaron formas y tamaños homogéneos, menores de 100 μm.Las condiciones fijadas para el sistema de atomizaciónsuperior se pueden aplicar al desarrollo de procesos de microencapsulación de diferentes principios activos utilizando como polímero Eudragit® S100.