Progress 09/15/06 to 09/14/09
Outputs
OUTPUTS: A field study with winter wheat showed that treating seeds with the wild type M. anisopliae increased the average weight of each harvested seed (N = 2000 seeds) by 14.9%. UV micrographs of roots from field sites showed GFP-Metarhizium on ~40% of the roots. The low-virulence mutant (Mcl1) also colonized the wheat roots but increased seed weight by only 3.4%, while ΔMad2 that did not colonize root surfaces had no beneficial effect. The inability of the rhizosphere competent, but non-entomopathogenic M. anisopliae Mcl1 to significantly promote growth indicates that the efficacy of M. anisopliae results largely from suppression of insect pests. However, follow-up experiments in insect-free microcosms showed that M. anisopliae still managed to accelerate growth of seedlings, and we demonstrated that M. anisopliae acts as a biofertilizer, mobilizing inorganic phosphate. We initiated a field trail of M. anisopliae colonizing turf which confirmed that strain 2575 expressing gfp (GFP-2575) can survive long term in soils, but with fluctuating population levels. These population cycles coincided with temperature extremes causing precipitous drops in population levels. Effects such as reduced productivity of plants and insects at extreme temperatures detrimentally impact population structures of M. anisopliae. Population counts of the two knockout strains Mcl1 and Mad2 showed different trends. CFU counts of the avirulent M. anisopliae Mcl1 cycled with the seasons in a similar manner to the original GFP-2575, although the CFU counts of Mcl1 were 3-fold lower within 2 months of application. Evidently Metarhizium populations are not dependent on the presence of insect hosts for cycling. In spite of being infectious to insects, the ΔMad2 mutant showed a linear decrease in population i.e., without the intermittent increases in population size demonstrated by Mcl1, GFP-2575 and the native strains. This suggests that the ability to adhere to root surfaces plays an important part in maintaining population size, irrespective of the presence of insects. We used array based mutation accumulation assays to inventory M. anisopliae isolates recovered from the field site for altered patterns of gene regulation that can be used to predict any physiological changes for ecologically relevant traits. Three and a half years after release, 25 field recovered Metarhizium isolates differed from the input strain by an average of 5 out of 1,749 (0.026%) arrayed genes. In absolute terms, the number of genes subject to mutable expression was 1.31% of the arrayed genes showed altered expression among the 25 isolates. Summary. This study provided fundamental information on pathogen ecology including knowledge of microbial survival and activity within the environment and of the persistence of recombinant DNA and its transfer to the indigenous microflora. PARTICIPANTS: This research provided opportunities for a Post-Doc (Sibhao Wang) and three graduate students. it was also greatly facilitated by undergraduate help; in total 13 worked on the project. TARGET AUDIENCES: Scientists working on pest control and population structures of microbes in the field, regulators and advocacy groups PROJECT MODIFICATIONS: We had originally proposed using the glucorinidase gene as a marker for transgenic fungi. We have instead used the red fluorescent protein gene as it does not require a substrate to be incorporated into the medium
Impacts
We used 8 mobile elements from strain 2575 as probes in Southern blot analysis to assay for frequency of jumping events and to identify changes in copy number in rescued colonies. These data showed that M. anisopliae strain 2575 displayed no observable chromosomal changes over three and a half years in the field. Patterns of duplication, divergence and deletion of transposons were also analyzed using strains of known relatedness to strain 2575. Molecular clock data estimates indicate that strains 2575 and 23 diverged less than 40,000 years ago. The pattern of transposons in 23 was identical to 2575 suggesting chromosomal changes and recombination events had been minimal in this lineage. To further determine the extent to which relatedness is a predictor of recombination events we co-infected insects with strains labeled with with either gfp or rfp. Co-infecting insects with GFP-2575 and RFP-2575 resulted in ~23% of spores harvested from cadavers carrying both markers. However, no recombinants were observed coinfecting insects with 2575-gfp and either strain 23-rgf or the more distantly related strain 549-rfp. This suggests that strain 2575 has barriers to gene introgression from different strains.By comparing isolates we attempted to determine if all biological processes or molecular functions were equally mutable or conserved. As the isolates are from 5 separate plots some at least represent independent evolutionary experiments so commonalities in gene expression patterns would be evidence for selection rather than chance events. Our microarray experiments have the power to detect expression differentials of only 0.2 standard deviation units, which is of the order of 1.4-fold or more. However, at this level of differentiation, genes for secreted enzymes, virulence factors, and genes required for most metabolic pathways were highly conserved between all isolates. Comparison of Gene Ontology categories indicated that mutations disproportionately affected cell wall and stress responses. Sequence comparisons suggest that 8% of the arrayed unigenes encode cell wall proteins, consistent with the typical 5 to 10 % of a fungal genome being devoted to cell wall function. Whereas only 1.31% of the arrayed genes overall showed altered regulation in 25 field strains, 19% of cell wall proteins showed differential gene expression in at least one of these isolates. Aside cell wall proteins, out of 19 gene ontology categories only the stress response (12%) and cell polarity (13%) categories contained > 10% genes with mutable expression. We argue that cell wall and stress tolerance genes are likely to have a direct impact on fitness of individual conidia and germtubes. The cell wall is the interphase by which fungi interact with the environment, and plays a vital role in morphology, adhesion, resource acquisition, resistance to toxins and avoidance of phagocytosis. Genes encoding proteins that effect cell wall structure are likely therefore to be under strong selection. Likewise, the ability to tolerate stressful temperature, UV etc will be an essential component of survival in soil.
Publications
- No publications reported this period
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: our field study with winter wheat showed that treating seeds with the wild type M. anisopliae increased the average weight of each harvested seed (N = 2000 seeds) by 14.9%. UV micrographs of roots from field sites placed on agar showed fluorescent growth by GFP-Metarhizium on ~40% of the roots. The low-virulence mutant (Mcl1) also colonized the wheat roots but increased seed weight by only 3.4%, while ΔMad2 that did not colonize root surfaces had no beneficial effect. The inability of the rhizosphere competent, but non-entomopathogenic M. anisopliae Mcl1 to significantly promote growth indicates that the efficacy of M. anisopliae results largely from suppression of insect pests. However, follow-up experiments in insect-free microcosms showed that M. anisopliae still managed to accelerated growth of seedlings. We initiated a field trail of M. anisopliae colonizing turf which confirmed that strain 2575 expressing gfp (GFP-2575) can survive long term in soils, but with fluctuating population levels. While there has been an overall decline in CFU counts intermittent increases in the CFU counts occur each year. These population cycles coincided with temperature extremes causing precipitous drops in population levels. effects such as reduced productivity of plants and insects at extreme temperatures detrimentally impact population structures of M. anisopliae. Population counts of the two knockout strains Mcl1 and Mad2 showed different trends. CFU counts of M. anisopliae Mcl1 cycled with the seasons in a similar manner to the original GFP-2575, although the CFU counts of Mcl1 were 3-fold lower within 2 months of application. Evidently Metarhizium populations are not dependent on the presence of insect hosts for cycling. In spite of being infectious to insects, theΔMad2 mutant showed a linear decrease in population i.e., without the intermittent increases in population size demonstrated by Mcl1, GFP-2575 and the native strains. This suggests that the ability to adhere to root surfaces plays an important part in maintaining population size, irrespective of the presence of insects. The Mad2 mutant does not show different sensitivity to temperature extremes than the wild type, at least in laboratory conditions, suggesting that the fluctuations in population sizes shown by rhizosphere competent strains of M. anisopliae depends on a temperature dependent interaction with plants rather than on differential survival of dormant spores. we used array based mutation accumulation assays were used to inventory M. anisopliae isolates recovered from the field site for altered patterns of gene regulation that can be used to predict any physiological changes for ecologically relevant traits. Three and a half years after release, 25 field recovered Metarhizium isolates differed from the input strain by an average of 5 out of 1,749 (0.026%) arrayed genes. In absolute terms, the number of genes subject to mutable expression was 1.31% of the arrayed genes showed altered expression among the 25 isolates. PARTICIPANTS: This research provided opportunities for a Post-Doc (Sibhao Wang) and three graduate students. it was also greatly facilitated by undergraduate help; in total 13 worked on the project. TARGET AUDIENCES: Scientists working on pest control and population structures of microbes in the field, regulators and advocacy groups PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We used 8 mobile elements from strain 2575 as probes in Southern blot analysis to assay for frequency of jumping events and to identify changes in copy number in rescued colonies. These data showed that M. anisopliae strain 2575 displayed no observable chromosomal changes over three and a half years in the field. Patterns of duplication, divergence and deletion of transposons were also analyzed using strains of known relatedness to strain 2575. Molecular clock data estimates indicate that strains 2575 and 23 diverged less than 200,000 years ago. The pattern of transposons in 23 was identical to 2575 suggesting chromosomal changes and recombination events had been minimal in this lineage. To further determine the extent to which relatedness is a predictor of recombination events we co-infected insects with strains labeled with with either gfp or rfp. Co-infecting insects with GFP-2575 and RFP-2575 resulted in ~23% of spores harvested from cadavers carrying both markers (Fig. . However, no recombinants were observed coinfecting insects with 2575-gfp and either strain 23-rgf or the more distantly related strain 549-rfp. This suggests that strain 2575 has barriers to gene introgression from different strains.By comparing isolates we attempted to determine if all biological processes or molecular functions were equally mutable or conserved. As the isolates are from 5 separate plots some at least represent independent evolutionary experiments so commonalities in gene expression patterns would be evidence for selection rather than chance events. Our microarray experiments have the power to detect expression differentials of only 0.2 standard deviation units, which is of the order of 1.4-fold or more. However, at this level of differentiation, genes for secreted enzymes, virulence factors, and genes required for most metabolic pathways were highly conserved between all isolates. Comparison of Gene Ontology categories indicated that mutations disproportionately affected cell wall and stress responses. Sequence comparisons suggest that 8% of the arrayed unigenes encode cell wall proteins, consistent with the typical 5 to 10 % of a fungal genome being devoted to cell wall function. Whereas only 1.31% of the arrayed genes overall showed altered regulation in 25 field strains, 19% of cell wall proteins showed differential gene expression in at least one of these isolates. Aside cell wall proteins, out of 19 gene ontology categories only the stress response (12%) and cell polarity (13%) categories contained > 10% genes with mutable expression. We argue that cell wall and stress tolerance genes are likely to have a direct impact on fitness of individual conidia and germtubes. The cell wall is the interphase by which fungi interact with the environment, and plays a vital role in morphology, adhesion, resource acquisition, resistance to toxins and avoidance of phagocytosis. Genes encoding proteins that effect cell wall structure are likely therefore to be under strong selection. Likewise, the ability to tolerate stressful temperature, UV etc will be an essential component of survival in soil.
Publications
- St. Leger, R.J. 2008 Studies on adaptations of Metarhizium anisopliae to life in the soil. Journal of Invertebrate Pathology 98: 271-276
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: We are investigating the movement, persistance and potential genetic changes in transgenic Metarhizium in the rhizosphere, using marker genes and functional genomic tools for identifying genetic changes. The marker genes are the green and the red fluorescent protein genes. These are being used to mark wild type strains, an attenuated strain in which an immune evasion gene has been deleted and a strain that cannot attach to plant surfaces because of deletion of the Metarhizium adhesion 2 gene. These strains were realized on a University of Maryland field site this April. The experimental set up is a randomized block design with four treatments and five replications per treatment. The treatments were applications of 0 (control) and 1) RFP, GFP-2575 (pathogenic and root exudate competent); 2) GFP-2575ΔMcl (to test the hypothesis that populations are maintained by cycling through insects) and 3) RFP_2575ΔMAD2 (to test the hypothesis that populations are maintained on
root exudates). Conidia were applied with a hydraulic sprayer (ca. 4 liters/90m2) followed by irrigation to carry spores into the ground.
PROJECT MODIFICATIONS: We had originally proposed using the glucorinidase gene as a marker for transgenic fungi. We have instead used the red fluorescent protein gene as it does not require a substrate to be incorporated into the medium
Impacts
we are currently: 1) investigating maintenance, stability, proliferation, die off and pathogenicity of the genetically marked fungus; 2) using microarrays to investigate mutational capacity of M. anisopliae, and 3) determining whether survivorship in soil depends on recycling of fungi through insects. We have been particularly interested in monitoring strain stability and possible dissemination of its genetic material by determining whether fungi retain the marker elements (gfp and rfp)in their original form. We have found that Metarhizium can form stable diploids which quickly fragment to produce aneuploids. The possibility that intense systematic deployment of transgenic strains in field conditions could result in gene transfer to other strains is also being scrutinized. Other results include obseving that spiders are rapid disseminators of transgenic fungi and confirmation that GFP-2575ΔMcl does not infcet insects in the field
Publications
- No publications reported this period
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