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A Sensitive PCR-Based Assay to Detect Neotyphodium Fungi in Seed and Plant Tissue of Tall Fescue and Ryegrass Species

USDA/ARS, Oregon State University, 3450 S.W. Campus Way, Corvallis, OR 97331

View larger version (87K): [in a new window]   Fig. 1. Alignment of Neotyphodium spp. tub2 sequences isolated from infected seed from tall fescue and ryegrass species. Sequence designation: TUB2-Fa, Festuca arundinacea (Genbank accession no. AY865627); TUB2-Lm, Lolium multiflorum (Genbank accession no. AY865629); TUB2-Lp, Lolium perenne (Genbank accession no. AY865628); and TUB2-Cp-Claviceps purpurea (Genbank accession no. AF062646). CLUSTAL X sequence alignment is in 5'–3' orientation. Primer names and their sequences are located above the tub2 sequence alignment at their annealing sites (see boxes). Arrows denote direction of amplification. Asterisks denote conservation among all tub2 sequences shown.

View larger version (31K): [in a new window]   Fig. 2. Determination of optimal temperature range for touchdown PCR reaction for detection of Neotyphodium sp. Numbers above lanes denote the temperature range (°C) used in the touchdown step. MW, Invitrogen 1 kb plus DNA ladder. (A) PCR reaction using 1 ng DNA from N. coenophialum fungus isolated from tall fescue. (B) PCR reaction using 1 ng DNA isolated from 20 N. coenophialum E+ seeds (tall fescue cv. Kentucky 31, 90% infected).

View larger version (37K): [in a new window]   Fig. 3. Determination of DNA concentration necessary for PCR detection of Neotyphodium sp. Numbers above the lanes represent the amount of DNA used in the PCR reaction. MW- Invitrogen 1 kb plus DNA ladder. (A) DNA isolated from N. coenophialum fungus isolated from tall fescue. (B) DNA extracted from 20 E+ seeds (tall fescue cv. Kentucky 31, 90% infected).

View larger version (30K): [in a new window]   Fig. 4. Amplification of products using DNA extracted from seed samples from tall fescue and ryegrasses. DNA extraction was performed on 20-seed samples from the following grasses: tall fescue cv. Kentucky 31, up to 90% infected; Italian ryegrass cv. Jumbo, 34% infected; and perennial ryegrass cv. Express, 64% infected. Lanes: MW, Invitrogen 1 kb plus DNA ladder; (1) N. coenophialum, tall fescue; (2) E+ seed, tall fescue; (3) E– seed, tall fescue; (4) N. lolii, perennial ryegrass; (5) E+ seed, perennial ryegrass; (6) E– seed, perennial ryegrass; (7) E+ seed, Italian ryegrass; (8) E– seed, Italian ryegrass; (9) 0.5 ng of Claviceps pupurea DNA; (10) water control, MW, Invitrogen 1 kb plus DNA ladder. Reactions of seed samples used approximately 1 ng of isolated DNA, and pure fungal DNA controls utilized 0.1 ng (lanes 1 and 4).

View larger version (21K): [in a new window]   Fig. 5. Limits of N. coenophialum detection in tall fescue seed. DNA was isolated from tall fescue cv. Kentucky 31, E+ seed (90%) and E– seed (0%). The percentages above the lanes represent the final proportions of DNA isolated from E+ (90%) seed combined with the DNA isolated from E– seed (0%). Approximately 1 ng of isolated DNA/reaction. MW, Invitrogen 1 kb plus DNA ladder.

View larger version (18K): [in a new window]   Fig. 6. Detection of N. coenophialum in different tissues of E+ tall fescue cv. Kentucky 31 plants. Each reaction used 1 ng of template DNA. Lanes: MW, Invitrogen 1 kb plus DNA ladder; (1) seed; (2) developing seed head, at time of flowering; (3) stem from reproductive tiller; (4) vegetative tiller 1; (5) vegetative tiller 2; (6) crown; (7) roots; (8) water control, MW, Invitrogen 1 kb plus DNA ladder.