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.

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.

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.