Molecular diversity of the Metarhizium anisopliae lineage in an agricultural field

Entomopathogenic fungal isolates identified by morphology as Metarhizium anisopliae may belong to different species when identified by molecular characters. We isolated Metarhizium spp. from an experimental agricultural field under both conventional and organic farming regimes using Tenebrio molitor as bait insect to assess the molecular diversity within the soil. Isolates were analyzed using DNA sequencing and applying SSR markers. Within the former M. anisopliae lineage, we found M. brunneum (86.3%), M. robertsii (11.3%) and M. majus (3.4%) in the soil samples. Several genotypes of each species were identified based on SSR markers. Differences in abundance of the species and their genotypes suggest different adaptations to the soil environment of the agricultural field. There were no effects of conventinal or organic farming regimes on diversity of the fungi

Assessing potential hybridization between a hypothetical gene drive-modified Drosophila suzukii and nontarget Drosophila species.

Genetically engineered gene drives (geGD) are potentially powerful tools for suppressing or even eradicating populations of pest insects. Before living geGD insects can be released into the environment, they must pass an environmental risk assessment to ensure that their release will not cause unacceptable harm to non-targeted entities of the environment. A key research question concerns the likelihood that nontarget species will acquire the functional GD elements; such acquisition could lead to reduced abundance or loss of those species and to a disruption of the ecosystem services they provide. The main route for gene flow is through hybridization between the geGD insect strain and closely related species that co-occur in the area of release and its expected dispersal. Using the invasive spotted-wing drosophila, Drosophila suzukii, as a case study, we provide a generally applicable strategy on how a combination of interspecific hybridization experiments, behavioral observations, and molecular genetic analyses can be used to assess the potential for hybridization

Preventive application of an entomopathogenic fungus in cover crops for wireworm control

Efficacy of the Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) strain ART2825 for control of wireworms (Agriotes obscurus (L.), Coleoptera: Elateridae) was examined in a semi-field pot experiment. Pots were treated in late summer during sowing of spring oat as a cover crop. Survival of wireworms was assessed four weeks after their release in October 2013, and 30 weeks after release in April 2014. Viability and persistence of the fungus was determined by counting colony forming units from substrate samples and microsatellite analyses of recovered Metarhizium isolates. The number of colonies detected in the substrate in October 2013 increased with increasing concentrations of applied conidia, and no significant reduction was observed at the second evaluation date in April 2014. Increasing conidia application rates significantly increased mycosis and reduced wireworm survival, to a level comparable to that of treatment using insecticide-coated oat seeds. The preventive application of M. brunneum conidia to reduce wireworm populations in cover crops, preceding a damage-sensitive crop like potatoes, may be a promising biocontrol strategy

Virulence of in vivo and in vitro produced conidia of Metarhizium brunneum strains for control of wireworms

Wireworms are the soil inhabiting larvae of click beetles and can cause severe damage to arable crops such as potatoes (Solanum tuberosum, L.). Several strains of the entomopathogenic fungus Metarhizium brunneum (Petch) are pathogenic to wireworms. In this study, three European strains of M. brunneum were tested in the laboratory against the most damaging wireworm species in Europe, Agriotes lineatus (L.), Agriotes obscurus (L.) and Agriotes sputator (L.). A Swiss strain, isolated from an A. obscurus cadaver, proved to be most effective, killing up to 73% of A. lineatus and 83% A. obscurus individuals, respectively. The median lethal time (LT50) was 21 days post inoculation (dpi) for A. lineatus and 14 dpi for A. obscurus. The strain did not lose virulence through subsequent cultivation on artificial medium and thus seems to be suitable for mass production as a biocontrol agent for wireworm control

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers▿

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed

Assessing potential hybridization between a hypothetical gene drive-modified Drosophila suzukii and non-target Drosophila species

Genetically engineered gene drives (geGD) are potentially powerful tools for suppressing or even eradicating populations of pest insects. Before living geGD insects can be released into the environment, they must pass an environmental risk assessment (ERA) to ensure that their release will not harm valued and protected entities of the environment. A key research question concerns the likelihood that non-target species will acquire the functional GD elements; such acquisition could lead to the loss of those species and to a disruption of the ecosystem services they provide. The main route for gene flow is through hybridization between the GD insect strain and closely related species that co-occur in the area of release. Using the invasive spotted-wing drosophila, Drosophila suzukii, as a case study, we demonstrate how a combination of interspecific hybridization experiments, behavioral observations, and molecular genetic analyses can be used to assess the potential for hybridization.Funding provided by: AgroscopeCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100022575Award Number

Development of a SNP-based tool for the identification and discrimination of Melolontha melolontha and Melolontha hippocastani

The European (Melolontha melolontha L.) and Forest (M. hippocastani F.) cockchafer are widespread pests throughout Central Europe. Both species exhibit a 3-5-year life cycle and occur in temporally shifted populations, which have been monitored and documented for more than 100 years. Visual identification of adults and larvae belonging to these morphologically similar species requires expertise and, particularly in the case of larvae, is challenging and equivocal. The goal of the study was the development of an efficient and fast molecular genetic tool for the identification and discrimination of M. melolontha and M. hippocastani. We established a collection of both species from Switzerland, Austria and Northern Italy in 2016, 2017 and 2018. An approximately 1550 bp long fragment of the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene was amplified and sequenced in 13 M. melolontha and 13 M. hippocastani beetles. Alignment of the new sequences with reference sequences (NCBI GenBank and BOLDSYSTEMS databases) and subsequent phylogenetic analysis revealed consistent clustering of the two species. After the identification of M. melolontha and M. hippocastani species-specific single nucleotide polymorphisms (SNPs) in the CO1 alignment, we developed an effective SNP tool based on the ABI PRISM (R) SNaPshot (TM) Multiplex Kit for the rapid and accurate species discrimination of adults and larvae.ISSN:0007-4853ISSN:1475-267