Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material.
Fusarium graminearum (teleomorph, Gibberella zeae) is the predominant causal agent of Fusarium head blight (FHB) of wheat resulting in yearly losses through reduction in grain yield and quality and accumulation of fungal generated toxins in grain. Numerous fungal genes potentially involved in virulence have been identified and studies with deletion mutants to ascertain their role are in progress. Although wheat field trials with wild-type and mutant strains are critical to understand the role these genes may play in the disease process, the interpretation of field trial data is complicated by FHB generated by indigenous species of F. graminearum. This report describes the development of a SYBR green-based real time PCR assay that quantifies the total F. graminearum genomic DNA in a plant sample as well as the total F. graminearum genomic DNA contributed from a strain containing a common fungal selectable marker used to create deletion mutants. We found our method more sensitive, reproducible and accurate than other similar recently described assays and comparable to the more expensive probe-based assays. This assay will allow investigators to correlate the amount of disease observed in wheat field trials to the F. graminearum mutant strains being examined.
Dyer
Rex B
RB
USDA Agricultural Research Service, National Center for Agricultural Utilization Research, United States. dyerr@ncaur.usda.gov <dyerr@ncaur.usda.gov>
Kendra
David F
DF
Brown
Daren W
DW
D052160:Benzothiazoles; D015169:Colony Count, Microbial / Q000379:methods*; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D004274:DNA, Recombinant / Q000032:analysis; D003959:Diamines; D005670:Fusarium / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D009154:Mutation; D009930:Organic Chemicals / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D015203:Reproducibility of Results; D012639:Seeds / Q000382:microbiology; D012680:Sensitivity and Specificity; D014908:Triticum / Q000382:microbiology*
D052160:Benzothiazoles; D004271:DNA, Fungal; D004274:DNA, Recombinant; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I