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Title: Identification and Quantification of Pathogenic Rhizoctonia solani and R. oryzae Using Real-time PCR

Author
item Okubara, Patricia
item SCHROEDER, KURT - WASHINGTON STATE UNIV.
item Paulitz, Timothy

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2008
Publication Date: 7/1/2008
Citation: Okubara, P.A., Schroeder, K., Paulitz, T.C. 2008. Identification and Quantification of Pathogenic Rhizoctonia solani and R. oryzae Using Real-time PCR. Phytopathology. Vol. 98, No. 7, 2008. p. 837-847.

Interpretive Summary: This manuscript reports the development and application of rapid, sensitive and specific molecular assays for diagnosing species of Rhizoctonia that cause root rot and bare patch of wheat and barley. Root diseases are estimated to cause yield losses of 10-15% yearly, or over $100 million to the industry in the Pacific Northwest. Losses can exceed 30% during transition from convention tillage to direct seeding. Tracking and managing Rhizoctonia root diseases will be greatly aided by our diagnostic assays. The assays helped to determine the major species and groups of Rhizoctonia associated with cereal production regions of the Pacific Northwest. Protocols for extracting pathogen DNA from root tissue and soil were developed, cutting diagnostic time to hours instead of days. The assays quantified 1 propagule pathogen per gram of soil, or 10- to 50-fold less than levels that cause disease in the field.

Technical Abstract: Rhizoctonia solani and R. oryzae are the principal causal agents of Rhizoctonia root rot in dryland cereal production systems of the Pacific Northwest. To facilitate the identification and quantification of these pathogens in agricultural samples, we developed SYBR Green I-based real-time quantitative polymerase chain reaction (Q-PCR) assays for R. solani AG-2-1, AG-8 and AG-10, three groups of R. oryzae, and a binucleate Rhizoctonia species (teleomorph Ceratobasidium) AG-I. The assays targeted the nuclear ribosomal DNA regions containing the internal transcribed spacers ITS1 and ITS2 and 5.8S DNA genes. Specificity at the subspecies level was obtained using ITS1 and ITS2 as target sites for the forward and reverse primers, respectively. Quantification was most reliable at or above 10 to 30 fg of mycelial DNA from cultured fungi, 1 to 5 pg of pathogen DNA from soil extracts, and 0.2 to 10 pg from root extracts. However, 100-fold lower levels of pathogen DNA could be specifically detected in all types of extracts. A survey of randomly-selected isolates demonstrated that R. solani AG-2-1, R. solani AG-10 and R. oryzae group II/III were the most prevalent. Blastn, primer-target duplex stability and phylogenetic analyses indicated that the Q-PCR assays are suitable for diagnosis of isolates from Australia, Israel, Japan and other countries.