A Real-Time PCR Differentiating subsp. From subsp. in Corn Seed.

Stewart's wilt of corn caused by the bacterium subsp. is a seed-borne disease of major phytosanitary importance. Many countries have imposed restrictions on corn seed imports from regions where the disease occurs to prevent the potential introduction of the pathogen. Current laboratory testing methods (enzyme-linked immunosorbent assay [ELISA] and polymerase chain reaction [PCR]) cannot readily distinguish subsp. from the closely related subspecies subsp. . However, subsp. , a nonpathogen on corn, is occasionally found on corn seed as part of the resident bacterial population and can yield false positive test results. A real-time PCR targeting the intergenic sequence was developed to specifically detect subsp. from corn seeds and distinguish it from subsp. . The assay successfully detected subsp. from corn seed, and subsp. -contaminated seed lots, which previously yielded false positives by ELISA and published PCR methods, were negative. The absence of subsp. and the presence of subsp. in this seed were confirmed by size differentiation of the amplicons in a conventional PCR. By distinguishing the two subspecies, the assays described would avoid false positive results and help prevent unnecessary restrictions on international movement of corn seed.