Proposal to the Independent Projects/Research Opportunities Program
For a Project on
Detection of Phytoplasma in Trillium grandiflorum using 16s rDNA PCR
Submitted By
Nathan D. Wheeler
J. Peter Coppinger, Ph.D.
Executive Summary
A small population of Trillium grandiflorum in southeast Michigan exhibit symptoms
typical of infection by a plant pathogen belonging to the genus Phytoplasma; the typical white
flowers were either entirely or partially variegated. Phytoplasmic infections are usually confined
to phloem, and often result in the transformation of floral parts to leafy green structures,
potentially leading to sterility of the plant. T. grandiflorum, often referred to as White Trillium,
is a perennial monocot indigenous to the North Eastern portion of the United States. While it has
long been speculated that variegated Trillium is infected by a species of phytoplasma, it has
never been confirmed. Since no published information could be obtained concerning the
identification of the infectious agent in White Trillium, the primary focus of this on-going
research is to determine if a phytoplasmic species is associated with the symptoms. Using DNA
sequencing, our preliminary results suggest that this may be a previously un-described species or
subspecies. Continuing research will focus on verification that the phytoplasma are causing
symptoms in Trillium flowers.
This project will occur during Spring Quarter 2012
We are requesting $1000 for travel to field site and DNA sequencing reagents.
The student will be doing this project for academic credit
The work does require lab space, and we have arranged for space in O109
Project Description
We seek to understand a disease that potentially threatens a large population of Trillium
grandiflorum, a spring wildflower endemic to North America. No significant research has been
performed on this infection. This research could potentially identify a previously
undiscovered Trillium-infecting bacterium. Trillium exhibiting disease symptoms were
collected in April 2010 in Shiawassee County Michigan. DNA was isolated from flowers using
a plant genomic DNA extraction kit; flowers showing no symptoms were used as negative
controls. PCR was performed using pathogen-specific 16SrDNA primers. Sequencing of PCR
products revealed a 96-97% similarity to a group of pathogens known as phytoplasma; however,
none of the similar species were known to infect Trillium. This raises the possibility of a
previously undescribed phytoplasmic pathogen. Ongoing research will focus on identifying
whether phytoplasma in Trillium is the causal agent of disease symptoms in Trillium
grandiflorum. Also it is vital to obtain a complete 16s rDNA sequence (as complete as possible);
making to acquisition of new samples vital to future research.
Reasons for Grant Submission
As the quality of the samples decline with prolonged storage, it is critical to the success
of this project that we obtain fresh samples from the original field site in Southern Michigan.
This will require two full days in the field. Once fresh samples are collected, we must re-analyze
the DNA sequence of the infecting pathogen. This step will require a DNA extraction kit
(purchased from Promega™) and sequencing reagents. We are requesting $100 for travel to
assist with gas. PCR and DNA sequencing is performed at a cost of ~$20 per sample, allowing
for us to analyze 40 fresh samples. We feel that this is the minimum number of samples required
for publication-quality results.
In summary,

New samples must be collected from a field site in Southern Michigan. New samples are
needed to ensure consistent and quality DNA sequences.

New reagents are needed to analyze these new samples, including a DNA extraction kit
and DNA sequencing reagents.
Budget Request
Supplies/Consumables
Cost
DNA cloning and sequencing reagents
$800
Travel
$100
Plant Genomic DNA Extraction Kit
$100
Total Requested Budget
$1000