Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: In order to model natural as well as anthropogenic dispersal scenarios, we investigated evolutionary processes in a green fluorescent protein tagged strain of Metarhizium robertsii following transfer from a semitropical to a temperate soil community. Adaptive changes occurred over four years despite recurrent genetic bottlenecks and lack of recombination with locally well adapted strains. By coupling microarray-based functional analysis with DNA hybridizations we determined that expression of cell wall and stress response genes evolved at an accelerated rate in multiple replicates, whereas virulence determinants, transposons, and chromosome structure were unaltered. The mutable genes were enriched for TATA boxes possibly because they are larger mutational targets. In further field trials, we showed that the new mutations increased the fitness of M. robertsii in the new range by enhancing saprophytic associations, and these benefits were maintained in subsequent years. Consistent with selection being habitat rather than host specific, populations of an avirulent mutant cycled with seasons similarly to the wild type, whereas a mutant unable to adhere to plant roots showed a linear decrease in population. PARTICIPANTS: A post doc (Sibao Wang) and graduate student (Tammatha O Brien) have worked on this project, along with 4 undergraduates TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our results provide a mechanistic basis for understanding postrelease adaptations, show that agents can be selected that lack gene flow and virulence evolution, and describe a means of genetically containing transgenic strains by disrupting the Mad2 gene. Predicting the consequences of different types of human intervention has become an increasingly important challenge in light of habitat fragmentation, climate change, invasive species, and genetically modified introductions. Genetically modified Metarhizium strains represent a major new arsenal for combating agricultural pests. However, the current predictive data base for risk assessment of genetically engineered microbes is very small. Our study provided information on survival of individual genotypes (clones) of microbes in nature and experimentally derived information on gene transfer risks between strains. Such knowledge about the fate of genotypes at the population and ecosystem levels reduces uncertainty about the efficacy, survivability, and environmental risk posed by any biocontrol agent.
Publications
- Wang, S. O Brien, T., Pava-Ripoll, M and St. Leger, R.J. 2011 Local adaptation of an introduced transgenic insect fungal pathogen due to new beneficial mutations. Proc. Natl. Acad. Sci. USA 108: 20449-20454.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: OUTPUTS: We are continuing our multi-year analysis with M. anisopliae GFP-2575 to provide fundamental information on pathogen ecology including knowledge of microbial survival and activity within the environment, the impact of abiotic and biotic factors on a cycling population structure, and the persistence of recombinant DNA and its (so far) lack of transfer to the indigenous microflora. Seasonal fluctuations in temperature/rainfall also directly impact fungal activity and variable titers of fungi in winters confirm that multi-year studies are required to discriminate between these effects. Air and soil temperature (2.5 cm down), depths freezing occurs, cloudiness, wind direction and speed and soil moisture content (usually ranging between 15-26%) are therefore being monitored. Quantitative multi-year data on the impact of these parameters increases the likelihood of producing predictive tools that will provide consistent risk assessment results in applied microbial control projects. We are also doing new trials by field testing "evolved" strains of M. anisopliae that derive from our survey of field isolates and have diverged from the introduced strain. We are comparing the performance in field trails of the parent strain with that of three independently evolved isolates i.e., from different field plots, rescued 3.5 years after application. E1 was chosen as it has a large number of changes; E2 and E3 were chosen as representative isolates with an average number of changes. The E isolates are normal for growth rate, colony morphology and level of conidia production. They inherit from the parent strain the gfp gene and can therefore be identified by fluorescence. Co-infecting M. sexta larvae with E-strains and RFP-2575 showed that E3 and E1 had slightly increased and reduced virulence, respectively (determined by the proportion of GFP-spores on cadavers). PARTICIPANTS: PARTICIPANTS: A post doc (Sibao Wang) two graduate students (Liang Cai and Tammatha O Brien) have worked on this project, along with 6 undergraduates TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In order to model natural as well as anthropogenic dispersal scenarios, we investigated evolutionary processes in a green fluorescent protein tagged strain of Metarhizium robertsii following transfer from a semitropical to a temperate soil community. Adaptive changes occurred over four years despite recurrent genetic bottlenecks and lack of recombination with locally well adapted strains. By coupling microarray-based functional analysis with DNA hybridizations we determined that expression of cell wall and stress response genes evolved at an accelerated rate in multiple replicates, whereas virulence determinants, transposons, and chromosome structure were unaltered. The mutable genes were enriched for TATA boxes possibly because they are larger mutational targets. In further field trials, we showed that the newmutations increased the fitness of M. robertsii in the newrange by enhancing saprophytic associations, and these benefits were maintained in subsequent years. Consistent with selection being habitat rather than host specific, populations of an avirulent mutant cycled with seasons similarly to the wild type, whereas a mutant unable to adhere to plant roots showed a linear decrease in population. Our results provide a mechanistic basis for understanding postrelease adaptations, showthat agents can be selected that lack gene flow and virulence evolution, and describe a means of genetically containing transgenic strains by disrupting the Mad2 gene.
Publications
- Wang, S. O Brien, T., Pava-Ripoll, M and St. Leger, R.J. 2011 Local adaptation of an introduced transgenic insect fungal pathogen due to new beneficial mutations. Proc. Natl. Acad. Sci. USA 108: 20449-20454.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: We are continuing our multi-year analysis with M. anisopliae GFP-2575 to provide fundamental information on pathogen ecology including knowledge of microbial survival and activity within the environment, the impact of abiotic and biotic factors on a cycling population structure, and the persistence of recombinant DNA and its (so far) lack of transfer to the indigenous microflora. Seasonal fluctuations in temperature/rainfall also directly impact fungal activity and variable titers of fungi in winters confirm that multi-year studies are required to discriminate between these effects. Air and soil temperature (2.5 cm down), depths freezing occurs, cloudiness, wind direction and speed and soil moisture content (usually ranging between 15-26%) are therefore being monitored. Quantitative multi-year data on the impact of these parameters increases the likelihood of producing predictive tools that will provide consistent risk assessment results in applied microbial control projects. We are also doing new trials by field testing "evolved" strains of M. anisopliae that derive from our survey of field isolates and have diverged from the introduced strain. We are comparing the performance in field trails of the parent strain with that of three independently evolved isolates i.e., from different field plots, rescued 3.5 years after application. E1 was chosen as it has a large number of changes; E2 and E3 were chosen as representative isolates with an average number of changes. The E isolates are normal for growth rate, colony morphology and level of conidia production. They inherit from the parent strain the gfp gene and can therefore be identified by fluorescence. Co-infecting M. sexta larvae with E-strains and RFP-2575 showed that E3 and E1 had slightly increased and reduced virulence, respectively (determined by the proportion of GFP-spores on cadavers), while the virulence of E2 was unaltered (see "note" on page 14). The chosen E isolates were re-screened againsts large arrays (3,563 genes) representing ~ one third of the genome PARTICIPANTS: A post doc (Sibao Wang) and graduate student (Liang Cai) have worked on this project, along with 4 undergraduates TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The trial has so far shown that the evolved strain survive better in the field. Levels of evolved strains E1, E2 and E3 (expressing gfp) in the outer and inner rhizospheres are being compared with the parent strain (expressing rfp), as an indicator of adaptation to field conditions. Recombinants between an E isolate and the parent strain (e.g., expressing GFP and RFP) were assayed for levels of gene expression with QT-PCR to quantify the % of recombinants with E1, E2 or E3 patterns of gene expression. Most recombinants have a preponderance of the E strain gene expression patterns, this further indicates that the changes in gene expression have survival value compared to the parental patterns. The data so far therefore suggests that mutated isolates recovered from the field are better adapted to the field environment than the parental phenotype. Improved survival of the field isolates suggests that differences in gene expression identified by microarray analysis can be used to narrow down candidate genes involved in adaptation.
Publications
- No publications reported this period
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