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United States Department of Agriculture

Agricultural Research Service

Research Project: CONFIRMATION OF CLAVIBACTER MICHIGANENSIS SUBSP. SEPEDONICUS INFECTIONS USING A MULTIPLEX REAL-TIME PCR ASSAY FOR RING ROT DIAGNOSIS (NDSU)
2010 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. Documets SCA with N. Dakota State University. Formerly 5354-21220-002-20S (5/08).


3.Progress Report

In spring of 2009, we received potato tubers (Solanum tuberosum) cv. Russet Burbank with internal tuber necrotic arcs very similar to that caused by tobacco rattle virus. These tubers originated from Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions in two tubers from each location. These extracts were tested for TRV by reverse transcription PCR (RT-PCR) using primers within the 3’ terminal open reading frame of TRV RNA-1. The extracts also tested positive with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8 kb amplicon. The amplicon sequences obtained from both locations were found to be 100% similar to each other. These sequences were found to be 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession numbers EU315226.1 and AF055912.1, respectively). In addition to the sequencing, the sap from symptomatic tubers was mechanically transmitted to tobacco cv. Samsun NN, which showed bright yellow patches and spots on leaf at two weeks post inoculation. The presence of TRV was confirmed by reverse transcription PCR.

In spring of 2010, we received another set of potato tubers (cv. Russet Burbank) from Grand Forks County in North Dakota. The tubers had internal multiple concentric necrotic arcs and circles. None of the symptomatic tubers tested positive for Tobacco rattle virus (TRV), Tomato spot wilted virus (TSWV), Alfalfa mosaic virus (AMV), Potato leaf roll virus (PLRV), or the necrotic strains of Potato virus Y (PVY) by RT-PCR. These tubers were positive for Potato mop-top virus (PMTV) when tested with enzyme-linked immunosorbent assay. Extracts were tested for PMTV by reverse transcription RT-PCR using two different sets of primers. The first primer set used was C189/H360 complimentary to the CP protein of PMTV and is expected to yield an amplicon of about 460 bp upon RT-PCR. The amplicon generated from the necrotic lesions were cloned and sequenced. A second set of primers, pmtF4/pmtR4 designed to bind to a region in RNA 2 of PMTV yielded about 417 bp amplicon which was cloned and sequenced similarly. The sequences from all the six tuber lesions were identical to each other for the respective primer sets. Thus a consensus sequence for each of the primer pair was submitted to GenBank. The sequences were found to be 99% and 100% identical to the corresponding regions of the PMTV isolates from Northern Europe (GenBank Number AM503629 and AY187010 respectively). The freeze dried necrotic tuber tissue was sent to a USDA laboratory in WA.

Progress on the project was monitored by email and phone conversations.

This project investigates the role of various viruses, especially tobacco rattle virus and potato mop top virus, in production of necrosis in potato tubers which contributes to Objective 2 of the in-house project.


Last Modified: 12/27/2014
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