CONFIRMATION OF CLAVIBACTER MICHIGANENSIS SUBSP. SEPEDONICUS INFECTIONS USING A MULTIPLEX REAL-TIME PCR ASSAY FOR RING ROT DIAGNOSIS (NDSU)
Vegetable and Forage Crops Production Research
2012 Annual Report
1a.Objectives (from AD-416):
Confirmation of Clavibacter michiganensis subsp. sepedonicus infections using a multiplex real-time PCR assay for ring rot diagnosis and potato seed lot screening.
1b.Approach (from AD-416):
Real-time PCR primers and fluorescently labeled Taqman probes were previously developed for detection of three unique areas of the bacterial genome of C. michiganensis subsp. sepedonicus (Cms), the causal agent of bacterial ring rot (BRR) of potato. These genomic regions code for cellulose genes (CelA and CelB) that are primary virulence determinants for Cms. These reagents will be used for additional screening of bacterial potato endophytes by a multiplex real-time PCR to verify specificity and reliability of the procedure for Cms detection and identification.
This is the final report for this project. A molecular test was used in conjunction with an antibody-based test for detection of strains of Potato virus Y (PVY) and other viruses. Numerous samples from the central United States were analyzed in order to determine the incidence of the different strains of PVY prevalent in different parts of the country. Results showed the recombinant “N:O” strain has become the predominant virus. This indicates a change from the previously prevalent “O” strain of the virus. This is significant because the “N:O” strain, and not the “O” strain, is capable of producing severe symptoms in potato tubers thereby reducing quality of the crop. Other necrotic strains of PVY were also detected. An improved detection technique, called IC-RT-PCR was developed for rapid detection and identification of diverse strains of PVY. This test is coupled with the traditional ELISA test and is an important improvement to existing methods of detection of this important virus. Potato mop top virus (PMTV) and Tobacco rattle virus (TRV) are both important soil-borne viruses and were also detected in several locations. PMTV in particular was found to be more widespread than previously reported. Partial genetic sequence of the TRV and PMTV isolates were determined and compared to those from the US and Europe. Detection of these viruses alerts growers, processors, field-men, extension agents, and diagnostic laboratories to the presence of these important, and hard to control, viruses in several potato-producing regions of the United States.
This project investigates the role of various viruses, especially potato virus Y, tobacco rattle virus, and potato mop top virus, in production of necrosis in potato tubers which contributes to objective 2 of the related in-house project, "Determine host resistance options, epidemiological parameters and develop diagnostic tests for emerging pests and pathogens of potato".