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Title: An Improved Real-Time PCR System for Broad-Spectrum Detection of Didymella bryoniae, the Causal Agent of Gummy Stem Blight of Cucurbits

Author
item Ling, Kai-Shu
item Wechter, William - Pat
item SOMAI, BENESH - University Of South Africa
item WALCOTT, RONALD - University Of Georgia
item KEINATH, ANTHONY - Clemson University

Submitted to: Seed Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/22/2010
Publication Date: 10/26/2010
Citation: Ling, K., Wechter, W.P., Somai, B.M., Walcott, R.R., Keinath, A.P. 2010. An Improved Real-Time PCR System for Broad-Spectrum Detection of Didymella bryoniae, the Causal Agent of Gummy Stem Blight of Cucurbits. Seed Science and Technology. 38:692-703

Interpretive Summary: One of the most serious diseases of cucurbit crops (e.g., cantaloupe, cucumber and watermelon) is caused by a seed-borne fungus (Didymella bryonia) resulting in gummy stem blight (GSB) or black rot of cucurbits. A low level of contaminated seed could potentially result in a major disease epidemic under favorable environmental conditions. A reliable and rapid method in detection of D. bryoniae is not yet available. In the present study, an improved real-time PCR assay was developed which demonstrated a sensitive and broad-spectrum reactivity to a worldwide collection of D. bryoniae with no cross reactivity to several other fungal pathogens. Implementation of such molecular tool could provide an accurate and timely detection of the causal agent thus to provide clean and healthy cucurbit seed for the U.S. industry. Early and accurate detection of the causal agent (D. bryoniae) in plants also could lead to less fungicide uses, thus to benefit environment.

Technical Abstract: Gummy stem blight (GSB) is a major disease of cucurbit crops (e.g., cantaloupe, cucumber and watermelon). Didymella bryoniae (anamorph Phoma cucurbitacearum), the causal agent of GSB, is often co-isolated from infected tissues with other Phoma spp. RAPD profiles and PCR primers have been used to delineate two genotypes, RAPD Group (RG) I and RG II, of D. bryoniae and distinguish them from non-pathogenic Phoma spp. The current real-time PCR diagnostic assay (designated as DB2) is limited in scope because it detects only RG I but not RG II isolates of D. bryoniae. The objectives of this study were to identify a genomic sequence conserved across RG I and RG II isolates and to develop a real-time PCR system that could detect diverse D. bryoniae isolates. A conserved sequence region common to both genotypes of D. bryoniae was identified in PCR products generated from the DB17 primer set. New primers (designated as DB3) and a new probe were designed and used in real-time PCR. This real-time PCR system accurately detected a worldwide collection of 111 RG I and 17 RG II D. bryoniae isolates but did not amplify DNA from 15 isolates of Phoma sp., Colletotrichum sp., Alternaria sp., or Fusarium sp.. The DB3 real-time PCR assay detected the presence of the pathogen in DNA samples prepared from each individual seed that was naturally infested with D. bryoniae. The success of such sensitivity assay suggests that the newly developed DB3 system could be useful in seed and plant health diagnostics.