Assessing the Direct Effect of Disease-Suppressive Soil Amendments on Rhizoctonia solani
A field experiment conducted from 2007-2009 addressed the impact of various soil amendments on potato disease suppression and soil microbial community characteristics. However, data for specific pathogen levels in soil were not collected. My faculty advisor and I met to determine the best method to isolate, quantify, and combine total DNA (by treatment) from soil samples collected from the field experiments in the past three years. The DNA extraction, quantification and combination methods were decided upon. All of the soil DNA samples (360 samples) have been extracted, quantified and combined by treatment. Experiments of quantitative real time polymerase chain reaction (qRT-PCR) detection of Rhizoctonia solani DNA and analysis of this data are expected to be completed according to the time table described in the grant proposal.
1. Monitor Rhizoctonia solani DNA levels in soils over the course of the growing season in response to the amendment of soils with a conifer-based compost, a rapeseed rotation crop, the biological control fungi hypovirulent Rhizoctonia solani (Rhs1A1)and Trichoderma virens, and the biological control bacterium Bacillus subtilis.
2. Correlate Rhizoctonia solani levels in the soil with observed tuber diseases and yield data.
Both objectives remain in progress as stated. In order to monitor R. solani levels, qRT-PCR of extracted total soil genomic DNA will be performed. While all DNA has been extracted and prepared for qRT-PCR, the actual qRT-PCR is in progress. Similarly, for the second objective, qRT-PCR data is required to correlate the R. solani levels with the tuber disease and yield data collected from the field experiments.
The project has three basic phases. The first phase is extraction of total DNA from soil samples. Beginning in August 2010, DNA extractions of soil samples collected in the field commenced. DNA was extracted from soil samples collected before planting, two weeks post-emergence, and two weeks post-harvest for the previous three years. DNA was extracted using a soil DNA extraction kit (SurePrep Soil DNA Isolation Kit, Fisher Scientific, Pittsburgh, PA). This resulted in 360 DNA samples from three years of field trials. DNA samples were then quantitated using a benchtop fluorometer (Qubit Fluorometer, Invitrogen, Carlsbad, CA). Since all samples were essentially equal in yield, they were combined by treatment. The combined DNA was then purified and concentrated using a DNA purification kit (QiaQuick PCR Purification Kit, Qiagen, Valencia, CA). Purified samples were then stored at -20 degrees Celsius until qRT-PCR could be performed.
The second phase of the project is to qRT-PCR of the combined soil DNA samples. This requires a nucleic acid primer for the selective amplification of DNA from R. solani. This nucleic acid primer was selected from a detailed literature search. The primer and all the reagents for qRT-PCR have been procured and the qRT-PCR experiments are currently in progress, with a projected completion date of February 2011. The third and final phase is data analysis of the qRT-PCR data. The data analysis will be performed using systems analysis software (SAS) version 9.1. The projected completion of data analysis is May of 2011. Once all data has been collected and analyzed, outreach meetings will be attended over the course of the summer and fall of 2011, as well as publications in local outreach newsletters and scientific journals to disseminate findings.
The project is currently progressing slightly behind schedule. DNA extractions have taken a little longer than anticipated and the projected completion date for the qRT-PCR experiments has been pushed back by a month. However, the project is still on track for completion by the end of May in 2011. Soil DNA extractions, quantification of samples, and combination by treatment have all gone according to plan and no difficulties have been encountered thus far. As such, no changes have been made to the qRT-PCR experiments or goals of the experiment.
Impacts and Contributions/Outcomes
While the project is only approximately 1/3 complete, no outcomes have yet been determined. However, a large potential for significant contributions to sustainable agriculture is possible. Our field experiments have revealed how the overall microbial communities respond to particular soil amendments, but how these treatments impact the growth and survival of R. solani (a major soil-borne potato pathogen) has not been determined. Assessing the impact of particular soil amendments on the pathogen, rather than relying on pathology data, is necessary to understand how to prevent disease without the use of economically and environmentally costly chemical pesticides. By providing evidence on the effect and ability of composts, rotation crops, and biological control organisms to deter the growth of the pathogen itself, it will be possible to tailor management practices to suppress these plant diseases. Combining data on the survival of the pathogen in the soil with potato yield and quality assessments will also aid in determining more effective management strategies that can provide effective and sustainable control options to growers.
University of Maine
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