title: A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels.
abstract: OBJECTIVE
  To develop a real-time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels.
  
  
  RESULTS
  Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245-bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 μg of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R(2) = 0·9912; = 0·004). In field-inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0·82, P < 0·0001) and two visual techniques to assess grain quality (R = 0·88, P < 0·0001 and R = 0·81, P < 0·0001).
  
  
  CONCLUSIONS
  The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real-time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass.
  
  
  CONCLUSIONS
  Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.
authors.affiliation: Department of Plant Pathology, University of Arkansas, Division of Agriculture, Fayetteville, AR 72701, USA.
authors.forename: P
authors.initials: P
authors.lastname: Horevaj
authors.forename: E A
authors.initials: EA
authors.lastname: Milus
authors.forename: B H
authors.initials: BH
authors.lastname: Bluhm
mesh_terms: D018533:Biomass; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005670:Fusarium / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014255:Trichothecenes / Q000032:analysis; D014908:Triticum / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D014255:Trichothecenes; C007262:deoxynivalenol