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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » Grain Quality and Structure Research » Research » Publications at this Location » Publication #253271
Title: Development of a Real-Time PCR Assay for Fusarium thapsinum in Grain Sorghum

Author
item Tilley, Michael - Mike
item NOLL, LANCE - Kansas State University
item Bean, Scott
item LITTLE, CHRISTOPHER - Kansas State University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/15/2010
Publication Date: 10/24/2010
Citation: Tilley, M., Noll, L.W., Bean, S., Little, C.R. 2010. Development of a Real-Time PCR Assay for Fusarium thapsinum in Grain Sorghum. Meeting Abstract. Cereal Foods World. 55:A72.

Interpretive Summary:

Technical Abstract: Grain mold is a yield-limiting disease that impacts caryopsis viability and quality of sorghum. The objective of the study was to develop an assay that will allow investigators to identify the presence and amount of disease observed in field trials to F. thapsinum levels without laborious culture work and DNA Grain mold is a yield-limiting disease that impacts caryopsis viability and quality of sorghum. The objective of the study was to develop an assay that will allow investigators to identify the presence and amount of disease observed in field trials to F. thapsinum levels without laborious culture work and DNA sequencing. Field grown samples from four sorghum lines, Sureno, Tx2911, SC170, and Tx430, that vary in grain mold resistance were scored visually and given a grain mold rating. Samples were milled and Fusarium colony forming units were determined by plating flour dilutions onto potato dextrose agar. After subculturing, colonies representing different species were evaluated by PCR amplification of a region of the fungal translation elongation factor 1-alpha (TEF-1 alpha) gene and DNA sequence comparisons were made. Fusarium spp. present in the field included F. thapsinum, F. verticillioides, F. proliferatum and F. equiseti. Purified genomic DNA from the major pathogen, F. thapsinum, was used to develop specific real time PCR methods based upon variable regions within the TEF-1 alpha gene. Results confirmed specific detection of F. thapsinum and lack of cross reactivity between other Fusarium species. Based on genomic DNA, the limit of detection was 0.1 pg fungal DNA. Standard curves were developed using dilutions of genomic DNA and DNA extracted from experimentally inoculated and field samples were analyzed. Analysis of sorghum flour from field grown samples demonstrated that the assay provides a highly specific method for identification and quantification of F. thapsinum.