Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

Molecular and Biochemical Examination of Spraing Disease in Potato Tuber in Response to Tobacco rattle virus Infection

Field-grown tubers of potato were examined for infection by Tobacco rattle virus (TRV) and consequent production of corky ringspot or spraing symptoms. A microarray study identified genes that are differentially expressed in tuber tissue in response to TRV infection and to spraing production, suggesting that hypersensitive response (HR) pathways are activated in spraing-symptomatic tubers. This was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of a selected group of HR-related genes and by histochemical staining of excised tuber tissue with spraing symptoms. qRT-PCR of TRV in different regions of the same tuber slice showed that nonsymptomatic areas contained higher levels of virus relative to spraing-symptomatic areas. This suggests that spraing formation is associated with an active plant defense that reduces the level of virus in the infected tuber. Expression of two of the same plant defense genes was similarly upregulated in tubers that were infected with Potato mop-top virus, a virus that also induces spraing formation.

Detection of Tobacco rattle virus (TRV) in Phryma leptostachya L.: A New Native Plant Host in Minnesota

To our knowledge, this is the first report of TRV infection in P. leptostachya. The repeated isolation of TRV from plants in isolated, uncultivated habitats suggests that TRV may be endemic to North America. Further studies are needed to determine if infected native perennial plants could serve as a potential TRV reservoir that could cause corky ringspot infection in potato. Accepted for publication 9 October 2014. Published 29 December 2014.

The Potato Corky Ringspot Pathogen, Tobacco rattle virus, Occurs in Native Habitats in Minnesota

When corky ringspot of potato, caused by Tobacco rattle virus, was first reported in Minnesota in 2008, it was thought that TRV did not occur naturally in Minnesota. Results reported here indicate that TRV occurs in Minnesota in areas with no known history of cultivation to potatoes or other crops, that these TRV isolates are variable, and that some are similar to virus isolated from potato with corky ringspot symptoms. These data suggest that corky ringspot incidence in Minnesota is due to the presence of endemic rather than introduced sources of TRV. Accepted for publication 10 September 2011. Published 28 October 2011.

First Report of Tobacco rattle virus Associated with Corky Ringspot in Potatoes Grown in North Dakota

Tobacco rattle virus (TRV) belongs to the genus Tobravirus and causes a stem mottle of potato (Solanum tuberosum) foliage and necrotic arcs and rings in tubers referred to as corky ringspot. This virus is generally transmitted by a number of species of stubby-root nematode. The virus is widespread and has been reported in California, Colorado, Florida, Idaho, Michigan, Oregon, Washington, Minnesota, and Wisconsin (2). In the spring of 2009, we received potato tubers of cv. Russet Burbank with internal necrotic arcs very similar to those caused by TRV from potato storages located in Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions of two tubers from each location using the Total RNA Isolation kit (Promega Corp., Madison WI). These extracts were tested for TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 (Primer A) and identical to nucleotides 6113 to 6132 (Primer B) within the 3′ terminus of TRV-SYM RNA-1 (GenBank Accession No. X06172) (3). The expected 463-bp amplicons from two separate tuber samples from each county were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. The sequences obtained from the four clones at both locations were found to be identical to each other and were 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession Nos. EU315226.1 and AF055912.1, respectively). Since sequences from all four clones were identical, only one of the sequences was submitted to Genbank (Accession No. GQ223114) and thus represents a consensus sequence. The extracts also tested positive in RT-PCR with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8-kbp amplicon (1). No evidence was found by RT-PCR for several other viruses that cause tuber necrosis in potato (Potato mop top virus, Tomato spotted wilt virus, Alfalfa mosaic virus, and tuber necrosis strains of Potato virus Y). The virus was mechanically transmitted by inoculating sap from symptomatic tubers from both counties to tobacco cv. Samsun NN, which showed typical bright yellow patches and spots on leaves 2 weeks postinoculation. TRV was confirmed in tobacco by RT-PCR from total RNA extracted from tobacco leaves with both sets of the aforementioned primers. To our knowledge, this is the first report of TRV in North Dakota and the first report of corky ringspot disease of potato in this state. References: (1) J. M. Crosslin et al. Virus Res. 96:99, 2003. (2) N. C. Gudmestad et al. Plant Dis. 92:1254, 2008. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992.

Detection of Tobacco Rattle Virus RNA in Processed Potato Chips Displaying Symptoms of Corky Ringspot Disease

In Apr. 2008, commercially purchased processed potato chips were observed with dark brown arcs and rings typical of corky ringspot disease. This disease is caused by infection with tobacco rattle virus (TRV). A portion of RNA 1 of TRV was amplified by reverse transcription polymerase chain reaction (RT-PCR) from each of eight discolored chips from three different bags purchased at three locations. Sequence analysis of the 463-bp amplicons confirmed that the products were indeed TRV in origin and were 97% identical to TRV sequences of isolates originating in Washington, Florida, and Wisconsin. Extracts from the symptomatic RT-PCR-positive chips were not infectious when mechanically inoculated onto tobacco leaves. To the author's knowledge, this is the first report of the detection of plant virus RNA in a food product after high-temperature frying.