I. Species-specific primer design

A.Obtain ribosomal gene region sequences of turfgrass species and their pathogens

1)Sequences obtained from GenBank

a) Rhizoctonia solani (Brown Patch)

b) Sclerotinia homeocarpa (Dollar Spot)

c) Gauemannomyces graminis (Take-all Patch)

d) Dreschlera poae (Melting Out)

e) Colletotrichum graminicola (Anthracnose)

f) Magnaporthe poae (Summer Patch)

g) Pythium ultimum (Pythium Blight)

h) Annual bluegrass

i) Creeping bentgrass

j) Zea mays

2)Sequencing done in the lab

a) R. solani isolates

b) Isolates of S. homoeocarpa

c) Procedure used:

i. Dellaporta extraction of DNA from a pure fungal culture

ii. Polymerase Chain Reaction of extracted DNA using ITS-1 and ITS-4 primers in a Perkin-Elmer PCR

   machine

iii. Gel electrophoresis of PCR product to determine if DNA amplification was successful

iv. Cloning of PCR fragment using a modified version of TOPO TA Cloning by Invitrogen®

v. Mini-prep of clones (Medhat Nakhla)

vi. DNA was prepared for sequencing using the Wizard® Plus SV Minipreps DNA Purification process by

   Promega

vii. DNA was sent for sequencing to the Biotechnology Center on the University of Wisconsin-Madison

   campus

B. Compared sequences of R. solani and S. homoeocarpa to other sequences obtained

1) Used the Genetics Computer Group program to align DNA sequences

2) Regions conserved at the species level were used to design specific primers for both R. solani and S. homoeocarpa

a) Criteria used to design primer

i. Primer length = 20-27 nucleotides

ii. High G+C content

iii. Avoid complementarity at primer ends

iv. Frame a sequence 200-500 base pairs long

b) Compared primer with other sequences to test specificity

c)Blasted primer on GenBank

3) Order primer
II. Test designed primer in the lab

A. Used the general primers, ITS-1 and ITS-4, as positive controls for the primer tests (bands should be visible for PCR

   products of all samples)

B. Ran PCR tests with ITS-1 and designed primer on the following samples

1) R. solani

2) S. homoeocarpa

3) Fusarium

4) Bipolaris

5) Annual Bluegrass

6) Kentucky Bluegrass

7) Creeping Bentgrass
III. Reduced time of diagnosis

A. DNA extraction

1) Dellaporta extraction (1½ hrs.). In order to reach the goal of 4 hours, a faster extraction method was sought out.

2) Rapid DNA extraction (½ hr.). This method was designed for use on whiteflies, but was found to be successful on

   plant and fungal DNA.

B. Polymerase chain reaction

1) Initially the Perkin-Elmer PCR machine (running time = 4 hrs.) was used.

2) The Rapid Cycler PCR machine (running time = 1 hr.) was employed to do the same work.