Detection of Acidovorax avenae ssp. avenae in Rice Seeds Using BIO-PCR

The bacterium, Acidovorax avenae ssp. avenae causes several important plant diseases including bacterial stripe of rice, bacterial stalk rot of corn, bacterial leaf blight of oats, and red stripe of sugarcane and millet. Although the organism is seed-borne in rice, no reliable seed assay is available. A semiselective liquid medium based on d-sorbitol and l-pyroglutamic acid (SP medium), and two sets of polymerase chain reaction (PCR) primers were designed for use in a BIOPCR assay for detection of A. avenae ssp. avenae in rice seeds. External primer set, Aaaf3 and Aaar2, and internal primer set, Aaaf5 and Aaar2, designed from a 619 bp fragment of the internal transcribed spacer region of the 16S–23S rDNA of A. avenae ssp. avenae strain CAa4 were specific at the subspecies level. A nested-PCR assay produced the expected DNA product from 58 rice strains tested but not with DNA from 27 strains of A. avenae ssp. avenae from corn and other hosts. Furthermore, the primers failed to amplify a PCR product from two strains of A. avenae ssp. cattleyae, 10 strains of A. avenae ssp. citrulli, or 64 other bacteria. When A. avenae ssp. avenae was enriched prior to PCR by incubating washings of naturally contaminated rice seeds for 12 h in SP liquid medium, populations of the pathogen increased over 1000-fold, whereas populations of saprophytic bacteria remained stable. Populations of the target bacterium routinely reached PCR-detectable levels after seed samples containing as few as 1–2 target cells and up to 104 cfu of other bacteria were soaked overnight in phosphate buffer and then enriched for 12 h in SP liquid medium. Assays of 64 naturally infected rice seed samples resulted in 53 being positive by BIO-PCR and 34 by classical PCR. The BIO-PCR assay provides a sensitive, reliable tool for the specific detection of A. avenae ssp. avenae in rice seeds. The use of trade, firm or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable