A real-time PCR differentiating Pantoea stewartii subsp. stewartii from P. stewartii subsp. indologenes in corn seed

Authors: Pal, Narinder; Gardner, Candice; BLOCK, CHARLES - Iowa State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/13/2018
Publication Date: 4/29/2019
Citation: Pal, N., Gardner, C.A., Block, C.C. 2019. A real-time PCR differentiating Pantoea stewartii subsp. stewartii from P. stewartii subsp. indologenes in corn seed. Plant Disease. https://doi.org/10.1094/PDIS-06-18-0936-RE.
DOI: https://doi.org/10.1094/PDIS-06-18-0936-RE

Interpretive Summary: Stewart's wilt of corn disease is caused by the bacterium, Pantoea stewartii subsp. stewartii. The pathogen is indigenous to North America. It is present on corn seeds, and within seeds at a low frequency, and can be transmitted to the plants grown from infected seeds. Because seeds can serve as a means of moving the pathogen across geographical boundaries, several countries have imposed restrictions on importation of corn seed from the United States to prevent introduction of the pathogen. They require seed to be certified as free of Pantoea stewartii subsp. stewartii based on field inspection of seed parent plants or laboratory testing of seeds, or both. Currently available laboratory methods for seed testing cannot readily differentiate subsp. stewartii from another closely-related subspecies, P. stewartii subsp. indologenes, which is not pathogenic on corn but occasionally present on corn seed of tropical or sub-tropical origin. The inability of current laboratory methods to distinguish between the two closely-related subspecies can result in false-positive test results and thus impact trade. We have developed a seed testing method based on genetic sequence which distinguishes between the subspecies and specifically identifies subsp. stewartii, the pathogen. Use of the new PCR assay method eliminates false positives from subsp. indologenes obtained when using ELISA or previously-published PCR methods, and helps prevent unnecessary restrictions on the international movement of corn seed.

Technical Abstract: Stewart's wilt of corn caused by the bacterium Pantoea stewartii subsp. stewartii, is a seed transmitted disease of major phytosanitary importance. Many countries have imposed restrictions on corn seed imported from areas where the disease is present to prevent introduction of the pathogen. Current lab methods (ELISA and PCR) cannot readily distinguish P. stewartii subsp. stewartii from the closely-related, non-pathogenic (on corn) subspecies P. stewartii subsp. indologenes, occasionally present on corn seeds of tropical or sub-tropical origin as part of the resident bacterial population, and can thus yield false-positive test results. A real-time PCR targeting cpsAB intergenic sequence was developed to specifically detect P. stewartii subsp. stewartii in seed wash from corn seeds. The assay successfully detected P. stewartii subsp. stewartii from naturally-infected corn seed lots while tests of P. stewartii subsp. indologenes-infested corn seed lots of tropical or subtropical origin were negative. Absence of subsp. stewartii and presence of subsp. indologenes in tropical/subtropical lots was further confirmed by size differentiation of cpsAB amplicons in a conventional PCR. By distinguishing the two subspecies, the assays described eliminate false-positive test results obtained using ELISA and previously published PCR methods, and help prevent unnecessary restrictions on international movement of corn seed.