Progress 09/01/23 to 08/31/24
Outputs
Target Audience:The PI (St. Leger) and graduate students Mike Nan and Huiyu Sheng gave talks at the Entomological Society of America meeting Changes/Problems:Basically, we are a year behind where we should be becausewe had to repeat our 2022 releasein October 2023 What opportunities for training and professional development has the project provided?Graduate students Mike Nan and Huiyu Sheng areworking on this project. The project has also allowed us to employ three undergraduates who acquire a range of wet lab and bioinformatic skills. This project is excellent for training as it incorporates genomics, bioinformatics, biotechnology, molecular biology, microbiology, microscopy and fundamental "big picture" scientific principles revolving around mutualism, host- pathogen biology and evolution. The conceptual framework serves as a tool for exposing students to non-canonical ideas regarding mutualism and pathogenicity within a tritrophic framework of microbes, plants and insects. How have the results been disseminated to communities of interest?Mostly in conferences, and in an article submitted to Fungal Biology What do you plan to do during the next reporting period to accomplish the goals?We will continue to compare the potential of arthropod-mediated dispersal of different strains (different host ranges and ability to colonize roots) to non-targeted deployment areas. By collecting hundred of isolates of each strain of M. robertsii we will survey divergence of life history traits within each introduced M. robertsii strain to provide a predictive basis for the evolutionary potential and invasion ecology of different genetic backgrounds of engineered Metarhizium if containment fails. In order to understand life history traits of each strain, we will assay field isolates (40 from each field plot) for changes in virulence against insects and changes in the ability to colonize plant roots. Huiyu Sheng will graduate this October but will continue working on the project as a post-doc. She has conducted lab analysis of the 8 strains (genomes plus wet lab studies) that will result in two papers crediting this grant (in preperation, hopefully submitted by the end of the year)
Impacts
What was accomplished under these goals?
The field trial (main aim of the project) had originally commenced October 2022 with the application of eight strains of Metarhizium (representing a range of interactions with insects and plants) to a plot of turf; we intended to assess their survival and any change in properties in spring and at regular intervals thereafter. However, unbeknownst to us and the field site manager, there was a water pipe under the field site. This leaked in April and was replaced, involving some of the replicate application sites being excavated. Because of the loss of replicates, we repeated the release at a neighboring site inOctober 2023 (the trial requires the fungi to be applied in the fall). We sampled background levels of Metarhizium spp, scarabaeid larvae (particularly June bugs), tipulid larvae, lepidopteran larvae and collembolans at the new site. Pitfall traps embedded in the soil at set distances in and around the plots are being used to collect arthropods to determine the extent to which transgenic fungal strains can be recovered from arthropods within an intense deployment area in comparison to the extent found in the surrounding and remote sampling areas.To date we have found very littledispersal.Over the last year we havemonitoredthe longevity and habitat of the M. robertsii populations.Samples are being obtained by collecting soil adhering loosely to the roots (outer rhizosphere), soil adhering after shaking but subject to removal by washing (inner rhizosphere) and non-rhizospheric (bulk) soil from evenly spaced locations within grass free paths to monitor dispersal of the 8 strains of transgenic fungi. Population levels per weight of soil are being determined following serial plate dilution studies on a cyclohexamide-based selective medium. We are scanning with UV to distinguish isolates expressing GFP or Cherry or both GFP and Cherry from each other and indigenous strains of Metarhizium spp. The results show that all 8 of the fungi have persisted in the field sites but at very different levels. Th strain we call 2457 which grows at higher temps in the laboratory is surviving well; surprising so also is 1120 which is an early diverged Metarhizium with less ability than most of the other strains to interact with plant roots in the lab (plant roots help strains persist in harsh environments). We are investigating this further in soil samples from the diversity of plants at the site, as failure to detect rhizosphere competence in the lab against a couple of plant species may not translate to incompetence in the field against numerous plants.
Publications
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The PI (St. Leger) and graduate student Mike Nangave several invited talks at conferences including at the Entomological Society of America meeting and the Society of Invertebrate Pathology meeting. Changes/Problems:The field trial (main aim of the project) commenced October 2022 with the application of eight strains of Metarhizium; we intended to assess their survival and any change in properties in spring and at regular intervals thereafter. However, unbeknownst to usthere was a water pipe under the field site. This leaked in April and was replaced, involving some of the replicate application sites being excavated. Because of the loss of replicates, we repeated the release in a neighboring site in October 2023 (the trial requires the fungi to be applied in the fall). What opportunities for training and professional development has the project provided?Graduate student Mike Nan is working full time on this project. The project has also allowed us to employ three undergraduates who acquire a range of wet lab and bioinformatic skills. This project is excellent for training as it incorporates genomics, bioinformatics, biotechnology, molecular biology, microbiology, microscopy and fundamental "big picture" scientific principles revolving around mutualism, host- pathogen biology and evolution. The conceptual framework serves as a tool for exposing students to non-canonical ideas regarding mutualism and pathogenicity within a tritrophic framework of microbes, plants and insects. How have the results been disseminated to communities of interest?Mostly in conferences, and in an article submitted to Fungal Biology What do you plan to do during the next reporting period to accomplish the goals?We will compare the potential of arthropod-mediated dispersal of different strains (different host ranges and ability to colonize roots) to non-targeted deployment areas. Pitfall traps embedded in the soil at set distances in and around the plots are beingused to collect arthropods to determine the extent to which transgenic fungal strains can be recovered from arthropods within an intense deployment area in comparison to the extent found in the surrounding and remote sampling areas. By collecting hundred of isolates of each strain of M. robertsii we will survey divergence of life history traits within each introduced M. robertsii strain to provide a predictive basis for the evolutionary potential and invasion ecology of different genetic backgrounds of engineered Metarhizium if containment fails. In order to understand life history traits of each strain, we will assay field isolates (40 from each field plot) for changes in virulence against insects and changes in the ability to colonize plant roots.
Impacts
What was accomplished under these goals?
The field trial (main aim of the project) commencedOctober 2022 with the application of eight strains of Metarhizium (representying a range of interactions with insects and plants) to a plot of turf; we intended to assess their survival and any change in properties in spring and at regular intervals thereafter. However, unbeknownst to us and the field site manager, there was a water pipe under the field site. This leaked in April and was replaced, involving some of the replicate application sites being excavated. Because of the loss of replicates, werepeated the release ata neighboring site this October (the trial requires the fungi to be applied in the fall). Because of this delay there has been very little financial draw on the project. We didhoweversamplebackground levels ofMetarhiziumspp, scarabaeid larvae (particularly June bugs), tipulid larvae, lepidopteran larvae and collembolans. The trial having commenced again we are monitoring the migration and longevity of the M. robertsii populations. Samples are being obtained by collecting soil adhering loosely to the roots (outer rhizosphere), soil adhering after shaking but subject to removal by washing (inner rhizosphere) and non-rhizospheric (bulk) soil from evenly spaced locations within grass free paths to monitor dispersal of the 8 strains of transgenic fungi. Population levels per weight of soil are being determined following serial plate dilution studies on a cyclohexamide-based selective medium. We are scanning with UV to distinguish isolates expressing GFP or Cherry or both GFP and Cherry from each other and indigenous strains of Metarhizium spp.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
St. Leger 2023 Complex outcomes of Metarhizium-mediated infections. Fungal biology
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