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The long-term aims of this research are to survey, identify, characterize and improve understanding of forest decline in relation to the pathogens that cause tree diseases, particularly those pathogens that are of invasive, hybrid, and superrace populations. We endeavor to unravel the primary genetic mechanisms generating phenotypic and genotypic changes in pathogenic oomycetes and fungi in the presence of hybridization or in the absence of sexual recombination. We further endeavor to compare population genetics of suspect invasive species to foreign populations and determine whether discoveries in America represent native or invasive pathogens. These goals may exceed the 5-year term of the umbrella project but significant progress should be demonstrable in 5-years.
<P>Detailed objectives include:<OL> <LI> Measuring, recording, and reporting on characteristics of specific forest decline conditions.<LI> Developing and employing sensitive systems for detection of specific tree pathogens.<LI>Determining incidence, distribution, and host range of specific tree pathogens in specific forests and geographic regions.<LI>Identifying species of pathogens and formally describing new species.<LI> Delimiting species concepts in the process of distinguishing new species.<LI> Detecting potentially invasive pathogens in forest ecosystems.<LI>Determining whether newly discovered pathogens are native or introduced.<LI>Characterizing the nuclear condition of emergent, hybrid species and superrace strains of Phytophthora.<LI>Characterizing changes in nuclear condition and ploidy of asexual and sexual progeny in Phytophthora in comparison to parental isolates.<LI>Mapping DNA markers and SNPs of specific strains in relation to published genomes of Phytophthora.<LI>Characterizing changes in phenotype (aggressiveness, race, fungicide sensitivities) in asexual and sexual progeny.<LI>Analyzing the genetic heterogeneity in clonal progeny using codominant and dominant DNA markers.<LI>Correlating markers to genetic changes in fungicide sensitivity phenotypes.<LI>Correlating markers to genetic changes in race phenotypes.<LI>Correlating markers to genetic changes in virulence phenotypes.<LI>Mapping DNA markers for avirulence genes in Phytophthoa strains and progeny.<LI>Correlating markers with chromosomal rearrangements near avirulence genes.<LI>Providing experimental support for hypotheses of mechanisms of phenotypic change in asexual superrace straons and in populations of hybrid species of Phytophthora.
NON-TECHNICAL SUMMARY: This project is one of discovery, understanding, and characterization of invasive plant pathogens, including description of new species, as well as new races, and pathotypes of established pathogens. The importance to the mission of the MAES is in the science-based effort of protecting the natural resources (forests), crops (nursery and potato), and plant industry (landscape and arboriculture) of Michigan and the United States from destructive pathogens. This effort of protection includes clarifying systematic and biological information to improve the decision making of government agencies on regulatory and quarantine issues of disease control. Additionally, the nature of this project is to approach better understanding of fundamental genetic mechanisms of variation in virulence, and sudden shifts in race, fungicide resistance, and host preference of pathogens. The project emphasizes invasive pathogens of forest trees, particular pathogens that are model systems of invasive forest pathogens, recent hybridizations leading to shifts in tree host preference, pathogens expected to expand their geographical range with climate change, newly recognized pathogens that need description, and unexplored genetic systems that lead to sudden shifts in race and fungicide sensitivity. The project also includes biogeographical biodiversity surveys of forest pathogens of the Rocky Mountain and Alaska community bar-coding effort led by the PI, several state surveys, collaborative state and regional surveys, to supplement knowledge of native pathogens, as part of forest disease risk assessment, forest health evaluation, and monitoring for invasive pathogens. The project includes determining the native or introduced status of emergent pathogens, quantifying damage levels by pathogens that are poorly understood, improving technology for the diagnosis of tree diseases, investigating tree diseases of unknown etiology such as physiological leaf scorch (Swartz et al 2009), white pine branch mortality,and designing and recommending relevant integrated pest management programs and disease control methods.
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APPROACH: Michigan DNR foresters or regional USFS foresters will provide approximate locations of forest decline situations based in IR aviation FHP surveys and public reporting, and relay information to PI in cooperative agreements. The PI, cooperators, students, and volunteers will locate trees showing leaf scorch, dieback, root and butt rots, cankers and declines. Approximate tree location will be recorded by GPS coordinates or street addresses and recorded by mapping. At each forest site, twenty trees will be classified as healthy or declining based on visual symptoms, including dieback class, twig mortality, main branch mortality, loss of apical dominance, and presence of active cankers. For each sample the frequency and distribution of cankered twigs and branches will be estimated. The year of occurrence of branch dieback will be estimated and the presence of trunk cankers will be recorded. Additional trees will be sampled until a minimum of ten trees from the declining class are selected in each stand to compare incidence and severity of branch dieback in trees on each site. Species-specific oligonucleotide primers will be designed and tested for real-time quantitative PCR detection, and optimized for identification of the select pathogen in diseased plant tissues. Presence of particular pathogens will be determined with qPCR technology. Identification of pathogens will be by DNA sequencing, microscopic morphology, or combinations of these techniques. Climatic, physiographic, and edaphic data will be collected and overlaid on the maps. Pathogens will be isolated and cultures and morphological features will be examined microscopically for comparison to relevant taxonomic monographs. Relevant herbarium specimens will be compared in morphological study. Latin diagnosis will be published and Type specimens submitted to national herbaria. Phylogenetic analyses using multigene sequence databases, will be employed to delimit known species and putative new species. Collections of isolates of pathogenic species of suspect origin will be compared to collections of the species from other regions or continents in cooperative studies using population genetic analyses based on microsatellite and AFLP marker systems. Flow cytometric analyses of hyphae, zoospores and gametangia with determinations of DNA content per nucleus compared to haploid standards. Illumina sequencing and bioinformatics analysis. Replicated inoculations of excised leave, harvested tubers, or entire plants of host differentials, and evaluations of plant susceptibility responses, in incubators. Comparisons of growth rates and responses of isolates in "Poison food" assays in petri dishes. Microsatellite and AFLP genetic analysis of single-zoospore derived progeny of different phenotype. Six avirulence genes including a cluster of three have been located on the mapped linkage groups of P. infestans by high-density AFLP analysis using 30,000 markers. The cloned genes will be available for use as labeled-probes in Southern hybridizations of the separated chromosome fragments in situ. Synthesis of combined results will conclude the work.