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Title: Development of an improved real-time PCR system for broad spectrum detection of diverse didymella bryoniae genotypes

Author
item Ling, Kai-Shu
item Wechter, William - Pat
item SOMAI, B - UNIV OF SOUTH AFRICA
item WALCOTT, R - UNIVERSITY OF GEORGIA
item KEINATH, A - CLEMSON UNIVERSITY

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 4/30/2008
Publication Date: 7/26/2008
Citation: Ling, K., Wechter, W.P., Somai, B.M., Walcott, R.R., Keinath, A.P. 2008. Development of an improved real-time PCR system for broad spectrum detection of diverse didymella bryoniae genotypes. Phytopathology. 98(6 supplement):S91.

Interpretive Summary:

Technical Abstract: Gummy stem blight (GSB) is a major disease of cucurbit crops (e.g., cantaloupe, cucumber and watermelon) in the southern United States. Didymella bryoniae (anamorph Phoma cucurbitacearum), the causal agent of GSB, is often isolated from infected tissues together with other Phoma spp. RAPD profiles and PCR primers have been used to differentiate two genotypes (RG I and RG II) of D. bryoniae from each other and from non-pathogenic Phoma spp. RG I isolates are more virulent on cucurbits and widespread in major cucurbit growing areas in the world. RG II isolates are less virulent with limited geographical distribution. Existence of different genotypes of D. bryoniae with various levels of virulence in field populations makes the use of sequence-based detection methods (e.g., real-time PCR) more challenging. The objectives of this study were to identify a conserved genome sequence derived from RAPD fragments from RG I and RG II isolates and to develop a real-time PCR system with a broad-spectrum reaction to D. bryoniae isolates. Three previously unpublished D. bryoniae specific primer sets (namely, 17-mer, 21-mer and 25-mer) derived from RAPD products were used in PCR. A conserved sequence region common to both genotypes of D. bryoniae was identified in PCR products generated from the 17-mer primer set. New primers and probe were designed and used in real-time PCR. This newly developed real-time PCR system detected a worldwide collection of D. bryoniae isolates (including 108 RG I and 20 RG II) with no cross reaction to Phoma spp. or to Colletotrichum, Alternaria, and Fusarium.