Progress 10/01/04 to 09/30/07
Outputs
The overall goal of this project is to establish genetic tools for studying the grape powdery mildew fungus, Erysiphe necator (synonym Uncinula necator), and to analyze its population structure. This fungus is an obligate parasite of grapevines and cannot be cultured artificial medium, i.e., it can only grow on living grape leaves, making it very difficult to work with experimentally. During 2007, we continued to develop molecular genetic markers based on polymerase chain reaction (PCR) and to apply them to samples of E. necator from Europe and North America. Reports from Europe suggest that E. necator comprises at least two independent evolutionary lineages, possibly multiple species that are genetically and reproductively isolated. Relevant to agriculture, these genetic subgroups may have some profound biological differences that affect disease management. This research is directed at comparing these genetic subgroups from Europe to populations of E. necator in the
US, where E. necator is thought to be native (it was introduced into Europe from the US in the middle of the 1800s). We acquired DNA of E. necator from European wine grapes (Vitis vinifera) from colleagues in France and Italy, where two genetic subgroups were previously described with other genetic markers. We also obtained DNA from E. necator from eastern North America, including New York, New Jersey, Ohio and Virginia; these were obtained from both cultivated and wild species of grapes. We recently acquired DNA of E. necator from Washington state, California and Australia, but these have not been analyzed yet. From many of the other samples, we have obtained nucleotide sequences from multiple genes to look for single nucleotide polymorphisms (SNPs) and to analyze their relatedness and population genetic structure. Isolates of E. necator were also obtained from two species of Parthenocissus, which are in the same family as grapes; these isolates were sequenced to study their
relatedness to populations of E. necator on cultivated grapes. In addition to analyzing E. necator populations using DNA sequences, we have begun to make crosses between New York isolates in the laboratory. Isolates are being grown on leaves in the lab so that they can mate and produce offspring for genetic analyses. Spores must mature for several months in a controlled environment that simulates winter conditions before further analysis can be done. We are also actively trying to clone the mating-type locus (MAT) in E. necator. The tangible outputs from this research are in the form of developing methods and PCR primers for genes specific to E. necator that can now be used for amplifying a set of genes for population studies of grape powdery mildew.
Impacts
Although we are still developing methods and acquiring DNA from various locations, we have some preliminary results that have changed our knowledge in this system. We found that the two genetic subgroups in Europe are distinct using the multiple gene sequences we obtained. More significantly, however, neither of these two subgroups is found in our sample from the eastern US. Furthermore, although we analyzed a relatively small sample of isolates, we found very little polymorphism in US populations, which is surprising because the eastern US is thought to be the center of origin for E. necator and therefore should be more diverse. More sampling is needed in the eastern US to test whether this region is truly the center of diversity. Additional sampling is needed also from the west coast of the US. In particular, based on some preliminary data, we hypothesize that the same genetic groups found in Europe will be found on the west coast of the US. We found that the
isolates of E. necator we obtained from Parthenocissus species are genetically very different from those sampled from grapes. The difference is large enough to separate these populations into a new species, distinct from E. necator. Further taxonomic work will be needed to make this a formal distinction. Efforts to clone the mating-type locus to date have been made by searching for DNA sequences in E. necator similar to the MAT locus in other fungi. This approach, which is useful for other fungi, has not yet succeeded. However, we have cloned and sequenced two genes that typically flank the MAT locus in other fungi. We are in the process of using these genes as a starting point for walking towards the MAT locus in E. necator. Knowledge of mating type, which we can easily assay by PCR once we clone the gene, should help us in genetic studies for making crosses. This marker would also aid in studying the role of sexual reproduction and recombination in field populations, which relates
to the ability of the fungus to adapt to fungicides and host plant resistance.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The overall goal of this project is to establish genetic tools for studying the grape powdery mildew fungus, Erysiphe necator (synonym Uncinula necator). This fungus is an obligate parasite of grapevines and cannot be cultured in vitro, making it very difficult to work with. During 2006 we continued to develop molecular genetic markers based on polymerase chain reaction (PCR). We have now designed PCR primers and succeeded in PCR-amplifying DNA from seven different nuclear genes: the ITS and IGS regions of nuclear ribosomal DNA, elongation factor-1-alpha (EF1-α), chitin synthase I (CHS-1), actin (ACT), 14-alpha-demethylase (CYP51), and beta-tubulin (TUB2). Additional genes are being tried based on conserved sequences among related fungi. We have also experimented with amplifying DNA from the fruiting bodies of E. necator collected from the field and have succeeded in amplifying the ITS region. This is significant because ITS may be a diagnostic marker showing
differences between genetic subgroups of this fungus that have marked biological differences.
Impacts
To date, the most significant impact of this work is establishing a set of genes that can be amplified for studying the population genetics and evolution of the grape powdery mildew fungus, Erysiphe necator (synonym Uncinula necator). It is extremely difficult to work with this fungus because it cannot be cultured, making progress relatively slow. With these tools, we can now address questions concerning the evolution of this fungus in native populations in the US. Reports from Europe suggest that E. necator comprises independent evolutionary lineages, possibly multiple species that are genetically and reproductively isolated. Relevant to agriculture, these genetic subgroups may have some profound biological differences that affect disease management. Future studies will be directed at discerning these differences, both genetic and biological. Development of genetic tools is the first step towards understanding the diversity of this economically important disease.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The overall goal of this project is to establish genetic tools for studying the grape powdery mildew fungus, Uncinula necator. This fungus is an obligate parasite of grapevines and cannot be cultured in vitro, making it very difficult to work with. During 2005 we worked with a collection of fungal isolates that we made the previous year, and extracted DNA from them for developing genetic markers. Because it is difficult to grow this fungus in any appreciable quantity (it must be grown on living leaves and the spores vacuumed off), we developed a method for amplifying the entire genome using polymerase chain reaction. We succeeded in performing this whole genome amplification on several isolates, and from these DNAs we have determined the sequences of the intergenic spacer regions of the ribosomal RNA genes. We are working on this region and several other candidate regions to look for variation that we can exploit as genetic markers. Progress on this front has been
slow to date because of the difficulties in working with minute quantities of DNA from this fungus.
Impacts
To date, our greatest impact is in getting whole genome amplification to work on small quantities of DNA. If we can perfect this method, we will be able to sample mildew colonies and extract DNA from minute quantities of fungal tissue collected in the field. This type of method may eventually lead also to detection methods for this fungus, although applications for sensitive detection techniques are not a high priority. Future development of genetic markers from small quantities of DNA will allow us to address basic questions on the genetics of the pathogen and the epidemiology of grape powdery mildew.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
This project began in October 2004, therefore, this report covers only three months of activity. The overall goal of this project is to establish genetic tools for studying the grape powdery mildew fungus, Uncinula necator. This fungus is an obligate parasite of grapevines and cannot be cultured in vitro, making it relatively difficult to work with. Nonetheless, during 2004 we have concentrated on developing techniques needed for working with this fungus in the laboratory. In particular, we have made a small collection of isolates from the field and acquired isolates of each of the two mating types from another lab. These isolates are being maintained in the laboratory on detached leaves from vines grown in the greenhouse. We have succeeded in extracting DNA from several isolates and will use these shortly for developing genetic markers.
Impacts
So far we have not accomplished anything of any impact in the three months duration of this project. However, we expect that the development of genetic markers in future years will allow us to address basic questions on the genetics of the pathogen and the epidemiology of grape powdery mildew.
Publications
- No publications reported this period
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