Location: Crops Pathology and Genetics Research
Project Number: 0500-00044-030-13
Start Date: Sep 28, 2012
End Date: Sep 27, 2014
1. Soil & root samples will be collected from field/greenhouse plots to represent communities associated with plant disease incidence or suppression. Trees affected by Prunus replant disease (PRD) may be sampled to represent an orchard-based replant problem apparently mediated by poorly understood soil microbial communities. Soil & fine root samples will be collected from healthy and PRD-affected trees in fumigated & non-fumigated plots in commercial orchards. Samples will be collected from healthy & PRD-affected plants in pasteurized (or fumigated) and non-treated pots of PRD soil, in greenhouse trials. Another disease that may be included in our sampling of soil is crown gall disease (CG) incited by A. tumefaciens. Samples will be collected from soil plots that are conducive or suppressive to populations of the pathogen; the suppressiveness may be experimentally manipulated using combinations of soil fumigation and soil amendment with selected composts. For each disease chosen for metagenomic examination, at least six replicate samples (roots & soil for PRD, soil for CG) will be collected per soil treatment per experiment. It is anticipated that multiple experiments will be conducted. The samples will be preserved at -80 °C immediately after collection. 2. Total DNA will be extracted and purified from the samples described above. Next generation sequencing will be used to “shotgun” sequence random fragments of the DNA, or, alternatively, the sequencing may occur after a series of semi-selective amplifications targeting rDNA or other genes of interest from diverse soil microbiota. Whether random or sequential runs of targeted sequencing are completed, the goal will be to represent all soil microbial community members (i.e., among archaea, bacteria, & eukaryotes) to the extent possible. Computer algorithms will be used to quality check the sequenced DNA fragments and assign legitimate sequences to operational taxonomic units (OTUs), genes of interest, and other groupings. Sequencing products will be classified as to the phylogenetic origin and the gene families that they contain using various metagenomic analysis tools including those developed in the Eisen lab. 3. Ordination methods will be used to identify collective and individual shifts in OTU or gene incidence that are associated with incidence or suppression of the soilborne diseases of interest. Shifts in OTU or gene incidence that are putatively implicated in disease incidence or suppression will be examined further using quantitative(q)PCR and other quantitative techniques to confirm the shifts in populations of OTUs and/or genes. For this purpose, qPCR primers will be designed, tested, and optimized using sequences from the OTUs or genes of interest. 4. Quantitative PCR primers and/or other appropriate technologies will be optimized and validated using the samples described above as well as additional field samples. Predictive uses of the assay methods may include quantification of target microbial community members in soils likely to be conducive or suppressive to PRD, CG, or other diseases of interest. Diagnostic uses may include detection of such pathogens in plant roots.