Soil disinfection to prevent transmission of tobacco rattle virus to potatoes and flower bulbs

In July 2007, potato tubers cv. Russet Burbank (RB) with necrotic arcs and spots were detected in three fields in Buffalo County, Wisconsin and one field in Benson County, Minnesota. Umatilla Russet (UR) potatoes harvested from the west half of a field in Swift County, MN had similar, but visually distinct necrotic lesions. Portions of one field in Minnesota were abandoned, and the stored potato crop from two fields in Wisconsin was rejected by processors, representing a total crop loss due to tuber necrosis. Tuber symptoms displayed in both cultivars resembled those described for corky ringspot caused by Tobacco rattle virus (TRV) (4). Total RNA was isolated from necrotic tuber tissue crushed in liquid nitrogen and extracted using the Total RNA Isolation Kit (Promega Corp., Madison, WI). These extracts were tested for the presence of TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 and identical to nucleotides 6113 to 6132 within the 3' terminal open reading frame of TRV RNA-1 (3). The expected 463-bp fragments were amplified from RB tubers. Nucleotide sequences from a Wisconsin and Minnesota isolate (GenBank Accession Nos. EU569290 and EU569291, respectively) were 99 to 100% identical to the corresponding region in a published TRV sequence (GenBank Accession No. AF055912). A 396-bp fragment was amplified from UR tubers and sequence data (GenBank Accession No. EU569292) indicated a unique 63 nucleotide sequence was substituted for a 129 nucleotide sequence spanning residues 227 to 357 of the 463-bp amplicon from the RB TRV isolates. Seven fragments were sequenced from different UR tubers and the 396-bp fragment was identical among them. The sequence outside the substituted region had 92% identity to the published TRV sequence. Amplification of the full-length TRV RNA2 using primers 179/180 located in the 5' and 3' untranslated regions (2) was successful for 28 and 0% of the RB and UR samples, respectively, suggesting that the RNA2 is not present in these strains or has undergone significant mutation. TRV-infected sap from both potato cultivars was mechanically transmitted to tobacco cv. Samsun NN and these plants subsequently tested positive for TRV by ELISA using ATCC antiserum PVAS 820. Ninety tubers exhibiting mild to severe symptoms of TRV were planted in the greenhouse. Each tuber was bisected laterally; necrotic tissue was removed from one half of the tuber and tested for the presence of TRV using RT-PCR protocols described above for RNA1. The remaining half was bisected horizontally and both sections were planted. Foliage from each emerged plant was subsequently also tested by RT-PCR for TRV RNA1. All RB tubers from Wisconsin tested positive for TRV, but only 7 of 24 emerged plants tested positive. Only 72% of the UR tubers and 4 of 25 emerged plants tested positive. TRV has been confirmed in California, Colorado, Florida, Idaho, Michigan (1), Oregon, and Washington. To our knowledge, this is the first report of corky ringspot in potato caused by TRV in Minnesota and Wisconsin.

The studies focus on an ultrastructural analysis of the phenomenon of intercellular and systemic (vascular) transport of tobacco rattle virus (TRV) in tissues of the infected plants. TRV is a dangerous pathogen of cultivated and ornamental plants due to its wide range of plant hosts and continuous transmission by vectors—ectoparasitic nematodes. Two weeks after infection with the PSG strain of TRV, tobacco plants of the Samsun variety and potato plants of the Glada variety responded with spot surface necroses on inoculated leaf blades. Four weeks after the infection a typical systemic response was observed on tobacco and potato leaves, necroses on stems and lesions referred to as corky ringspot. Ultrastructural analysis revealed the presence of two types of TRV virions: capsidated and non-capsidated forms in tobacco and potato tissues. In the protoplast area, viral particles either occurred in a dispersed form or they formed organised inclusions of virions. We demonstrated for the first time the presence of non-capsidated-type TRV in the vicinity of and inside plasmodesmata. Capsidated particles of TRV were observed in intercellular spaces of the tissues of aboveground and underground organs. Expanded apoplast area was noted at the cell wall, with numerous dispersed non-capsidated-type TRV particles. These phenomena suggest active intercellular transport. Our ultrastructure studies showed for the first time that xylem can be a possible route of TRV systemic transport. We demonstrated that both capsidated and non-capsidated virions, of varied length, participate in long-distance transport. TRV virions were more often documented in xylem (tracheary elements and parenchyma) than in phloem. Non-capsidated TRV particles were observed inside tracheary elements in a dispersed form and in regular arrangements in potato and tobacco xylem. The presence of TRV virions inside the bordered pits was demonstrated in aboveground organs and in the root of the tested plants. We documented that both forms of TRV virions can be transported systemically via tracheary elements of xylem.

Corky ringspot (CRS) believed to be caused by tobacco rattle virus (TRV) is an important disease of potatoes in northeastern Florida, U.S.A. Although potato cultivars such as Pungo possessing resistance to CRS are available, other varietal characteristics such as susceptibility to bacterial wilt (caused by Pseudomonas solanacerarum E.F.Sm.), tendency to produce dark potato chips, and low yield potential in Florida’s sub-tropical climate, have limited their use. Since 1970 most potato fields in northeast Florida have been fumigated with chemicals such as DD for control of Belonolaimus longicaudatus Rau, Meloidogyne incognita (Kofoid & White) Chitwood, and Tylenchorhynchus claytoni Steiner, as well as nematodes in five other plant parasitic genera. There were reports from other sections of Florida that following soil fumigation, numbers of Trichodorus christiei Allen (the vector of TRV) on other crops sometimes increased to levels exceeding those in non-treated controls. Experiments were therefore initiated during 1970 to determine whether numbers of T. christiei would increase on potatoes following soil fumigation and whether incidence and severity of CRS would be affected by any observed increases; and, whether non-volatile carbamate and organophosphate nematicides would reduce incidence and severity of CRS.

Fifty-nine potato cultivars or breeding clones were planted near Umatilla, OR and/or Pasco, WA, in fields known to be infested with tobacco rattle virus (TRV) and vector nematodes,Paratrichodorus allius Jen. (Sid.). Tubers from these field plots were cut and examined for corky ringspot (CRS) symptoms. Reverse transcription-polymerase chain reaction (RT-PCR) for TRV was conducted on tissue samples from symptomatic and asymptomatic tubers. Sixty-five percent of the symptomatic and 42% of the asymptomatic tissue samples from CRS symptomatic tubers contained detectable TRV. Approximately 2% of plants grown from either symptomatic or asymptomatic tubers contained TRV when tested by ELISA, whereas 20% and 12% of plants grown from symptomatic and asymptomatic tubers, respectively, were positive for TRV by RT-PCR. These results suggest that RT-PCR is a more sensitive assay for detection of TRV. Systemic infections by TRV were detected more often in foliage of CRS-susceptible genotypes. Daughter tubers exhibiting symptoms of CRS, and which contained RT-PCR-detectable TRV, were produced on plants of three genotypes, including one from an asymptomatic parent tuber.

Two Julesburg sandy-loam fields in Weld County, Colorado, with a history of Corky Ringspot (CRS) caused by tobacco rattle virus (TRV) were injected with Telone-C as fall or spring applications and planted to potatoes. Deep soil sample cores taken from the experimental fields were planted toNicotiana tabacum L. var. Samsun serving as TRV bait and indicator plants. Systemic infection of bait plants and assay of the bait plant roots for TRV indicate that potential TRV inoculum was present which escaped the effects of fumigation treatments. TRV was found to be widely but erratically distributed throughout the experimental fields. The low incidence of CRS in tubers harvested from the treated fields suggests that fumigation may have short term benefits but potential inoculum exists which could lead to infection after the effects of fumigation have dissipated. The total yield of tubers was increased in the two cultivars planted (Norgold and Norland) in the fumigated fields receiving spring or fall applications. The yield of U.S. No 1 Grade potatoes in the Norland cultivar was increased significantly by both fall and spring fumigation. These yield increases were directly related to the reduced incidence ofVerticillium wilt observed in the fumigated soils and reported as a personal communication from unpublished data.

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.

Corky ringspot disease (CRS) of potato is caused by tobacco rattle virus (TRV). The virus is transmitted by the stubby root nematode (Paratrichodorus alUus) in the Pacific Northwest potato-producing regions. Alfalfa (Medicago sativa L.) and Scotch spearmint (Mentha cardiaca Baker) rarely serve as hosts for TRV. Therefore, P. allius reared on these plants for 1 to 3 months are cleansed of TRV in greenhouse trials. However, weeds in alfalfa and Scotch spearmint rotation crops may serve as hosts for the virus. In greenhouse trials, hairy nightshade (Solanum sarrachoides), prickly lettuce ( Lactuca serriola ), henbit ( Lamium amplexicaule ) and, green foxtail (Setaria viridis) grown alone were found to be suitable hosts of P. alUus, whereas Powell amaranth (Amaranthus powellii) was not. ~rlrnliferous P. aUius added to hairy nightshade, prickly lettuce, henbit, green foxtall, or Powell amaranth in mixtures with alfalfa and/or Scotch spearmint occasionally remained virulfferous over a 3- to 4-month period, whereas P. allius maintained on weed-free alfalfa or Scotch spearmint became virus-free after 1 to 2 months. Potato grown in soil containing P. allius that had been maintained on weed-alfalfa or weed-Scotch spearmint mixtures for 3 to 4 months exhibited slight to severe CRS symptoms on new tubers, whereas potato following weed-free Scotch spearmint or alfalfa were free from CRS symptoms. Severe CRS symptoms on potato tubers were only observed when potatoes were grown in soil containing P. allius that were maintained on hairy nightshade or hairy nightshade mixtures with alfalfa or Scotch spearmint. These preliminary data suggest that the presence of weeds that serve as hosts of both TRV and P. alUus may nnllify the positive effects of growing alfalfa or Scotch spearmint for CRS control. Targeted control efforts of known weed hosts may be required to successfully eliminate CRS from fields using alfalfa and Scotch spearmint rotational crops.

Potato is the fourth most important agricultural commodity in Michigan and is grown for table stock, chip processing, and seed. Tubers are either processed or fresh packed immediately following harvest or sent to storage. Tubers of potato cv. FL1879 representing two separate fields in Huron County were removed from separate storage facilities in February and March of 2007, and substantial internal necrosis was observed in 1 to 2% of the tubers. Symptoms included arcs similar to those caused by Tobacco rattle virus (TRV). This virus is a member of the genus Tobravirus and is transmitted by a number of species of stubby-root nematodes (Paratrichodorus or Trichodorus spp.). Stubby-root nematodes have been reported previously from Michigan (1). To confirm the presence of TRV, nucleic acid extractions were made from these symptomatic tubers (3). Samples were initially tested for TRV by reverse transcription (RT)-PCR using primers specific to the 16 kDa open reading frame on genomic RNA-1 (2). Samples from both storage facilities were positive. The 463-bp RT-PCR product, amplified with these primers, was sequenced (GenBank Accession No. EU315227) and was 99.6% identical to the corresponding region of two TRV isolates from Florida and Washington (GenBank Accession Nos. AF055912 and EU315226, respectively). In addition, sap from cv. FL1879 tuber samples was used to transmit the virus mechanically to tobacco cv. Samsun NN, which produced typical TRV symptoms following inoculation, and sap extracts from the tobacco leaves also tested positive with antiserum specific to TRV upon subsequent ELISA testing. Corky ringspot can result in substantial losses, with entire potato fields being rejected because of internal tuber damage. Once found, fields must be considered permanently at risk to this disease due to the large host range of both the virus and the nematode vector. This disease has been previously found in the United States in California, Colorado, Florida, Idaho, Washington, Oregon, and it is likely present in Indiana. To our knowledge, this is the first report of corky ringspot and TRV on potato in Michigan.

SUMMARY 16 and N5 are naturally occurring tobravirus isolates that produce symptoms in herbaceous plants similar to those induced by strains of tobacco rattle virus (TRV). In immunosorbent electron microscopy tests, however, they reacted with antisera to particles of pea early-browning virus (PEBV), not TRV. Furthermore, these tests indicated that 16 was related to the British serotype of PEBV and N5 to the Dutch. Pseudo-recombinant isolates were produced by reassortment of the genome parts of 16 or N5 with those of TRV, in any combination, but not in most combinations with those of PEBV. However, 16 RNA-2 was replicated in plants inoculated also with RNA-1 from an isolate of the British serotype of PEBV, but the PEBV RNA-1 was imperfectly packaged by 16 coat protein, and the virus particles seemed to have only limited stability. Nucleic acid hybridization experiments showed that the RNA-1 sequences of both 16 and N5 were similar to those of TRV strains. 16 RNA-2 contained sequences resembling those of the British serotype of PEBV, but with some TRV-like sequences at the 3′ and 5′ ends, whereas N5 RNA-2 contained more extensive TRV-like 3′ and 5′ ends flanking sequences that were related, but perhaps not closely, to those of the Dutch serotype of PEBV. Thus, the RNA-2 species of 16 and N5 were recombinant molecules that contained sequences typical of both TRV and PEBV, and which probably had separate but similar evolutionary origins. As a result of their hybrid nature, 16 and N5 were part of the gene pool and had the pathogenicity of TRV, while possessing the serological properties of PEBV.

SUMMARYAt a site in eastern Scotland, nine common species of arable weeds were infected with tobacco rattle virus (TRV), and some of these, notably Viola arvensis and Stellaria media, comprised an overwintering reservoir of the virus. TRV was seed‐borne both in naturally and in experimentally infected V. arvensis (2–10%), and occasionally in other weed species. In the glasshouse at 20 oC a naturally infective population of vector nematodes (Tricho‐dorus spp.) kept in soil free of plants retained its infectivity for 20 wk, although few Trichodorus survived for this period. In the field, the incidence of TRV infection in potato (spraing disease) in plots kept free of weeds for 1–5 years was 3–4 times that in weed‐infested plots but Trichodorus numbers did not differ appreciably between the two treatments. Presumably the virus is retained for long periods in its vectors and these feed on potato more frequently when other hosts are not available. Weeds are probably important in the long term as hosts of both TRV and its vectors, but in the short term weed control seems unlikely to prevent potato spraing because of the long persistence of TRV in vector populations.In the field, Trichodorus accumulated near the interface between topsoil and subsoil, and the incidence of spraing was greatest where the topsoil was shallowest. When cucumber seedlings were exposed to virus‐carrying Trichodorus, TRV reached a greater concentration in roots at 20 oC than at 24 oC, and the virus was not detected in roots at 29 oC. In a sandy soil, TRV was transmitted only when the water content exceeded 15%, and at least 30 % water was needed for maximum transmission. Annual records of rainfall and spraing disease suggest that spraing is most prevalent when the summer is wettest.TRV is not confined to cultivated land. Stabilized sand dunes supporting a pure stand of Ammophila armaria were colonized by Trichodorus pachyder‐mus, but TRV was detected only where the plant community had enlarged to include V. arvensis and other dicotyledons. In such situations, TRV may be introduced in the seed of V. arvensis, and the movement of soil by wind probably contributes to the dispersal of Trichodorus.

Tobacco rattle virus (TRV), genus Tobravirus, is able to infect a broad range of plant species, possesses worldwide distribution and naturally infects a very large number of cultivated as well as ornamental plants. Members of the plant ectoparasitic nematode genera Trichodorus and Paratrichodorus transmit TRV in a semi-persistent way in a non-replicative process, where virus particles are transferred to the host plant through vector feeding on root epidermal and root hair cells. Our investigations concentrated on ultrastructural and anatomical effects of TRV strain PSG infection in situ following introduction into potato and tobacco tissues by Trichodorus primitivus. Our anatomical observations indicated necrotic changes of rhizodermis and cell wall deformations in primary cortex parenchyma and external phloem layers as a consequence of the interactions between Trichodorus–TRV–host plants. Ultrastructural analyses revealed TRV particles in rhizodermis, cortex and vascular roots of potato and tobacco tissues. These results indicate that TRV strain PSG was transferred into roots and also transported from cell to cell in all root tissues. Complete TRV PSG particles of two lengths were documented in companion cells, phloem fibre and parenchyma, as well as for the first time in immature and mature xylem tracheary elements and xylem parenchyma. Our findings suggested that TRV was systemically transported from the place of direct transfer by the vector to above-ground plant organs (especially leaves). The presence of TRV PSG particles in mesophyll and vascular leaf tissues confirmed our thesis. Moreover, we concluded that TRV systemic movement occurred not only in the phloem, but especially in the xylem, because virus particles were more frequently observed in xylem parenchyma and xylem tracheary elements.

Growing resistant potato cultivars is of little help in avoiding damage by spraing, because of regionally differing strains of tobacco rattle virus and of special requirements of the processing industry. On soil, fumigated in 1971 during autumn with a solution of dichloropropene-dichloropropane (660 g/l) at 250 l/ha, the vectorsTrichodorus spp., were effectively controlled and the tuber disorder did not appear in the next year's potato crop. But potatoes from the treated plots had an unacceptable off-flavour. In the second crop after treatment, control was still good and there was no difference in flavour between potatoes from treated and untreated plots. Disease incidence was significantly lower after spring barley than after sugarbeet, maize, or potatoes. Kringerigheid bij aardappelen wordt voornamelijk veroorzaakt door tabaksratelvirus (TRV) dat wordt overgebracht doorTrichodorus-aaltjes, die voorkomen in lichte en organische gronden. Het telen van resistente aardappelrassen biedt slechts beperkte mogelijkheden om schade door kringerigheid te voorkomen, doordat regionaal verschillende stammen van het virus voorkomen (Tabel 1) en de industrie zeer bepaalde rassen wenst. Grondontsmetting met 250 l DD/ha in de herfst gaf een effectieve doding vanTrichodorus in de bovenste 60 cm van de grond en kringerigheid trad slechts op in een zeer laag percentage van de knollen. Dit effect werd ook een jaar later nog waargenomen (Tabel 2). Grondontsmetting met 100 l DD/ha was onvoldoende en de toepassing van tweemaal 250 l nauwelijks beter. De aardappelen die geteeld waren in het jaar direkt na de grondontsmetting hadden echter een ontoelaatbaar afwijkende smaak. In het daaropvolgende jaar verschilden ze niet in smaak van die van de onbehandelde veldjes. Op beide proefvelden was het percentage knollen met kringerigheid lager na de voorvrucht zomergerst dan na biet, mais en aardappel (Tabel 3). Uit deze proefresultaten blijkt, dat de ziekte bij ‘Bintje’ kan worden bestreden door de teelt van zomergerst, en bij ‘Eersteling’ en ‘Bintje’ door grondontsmetting. Aangezien door grondontsmetting geen volledige doding vanTrichodorus in de grond wordt bereikt, mag worden aangenomen dat de ziekte zal terugkeren wanneer gewassen worden geteeld waarop deze aaltjes zich vermeerderen en waaruit ze het virus kunnen opnemen. De voorkeur moet daarom worden gegeven aan een geintegreerde bestrijding van de ziekte door toepassing van grondontsmetting en vruchtwisseling met zomergerst, waarbij zorg zal moeten worden besteed aan de bestrijding van onkruidwaardplanten van TRV. De kans op smaakafwijking bij aardappelen kan worden beperkt door eerst zomergerst of mais te telen na de grondontsmetting met DD, waarbij rekening zal moeten worden gehouden met een stikstofeffect van de grondontsmetting.

Soil samples from corky ringspot (CRS) problem fields of potato in the states of Washington and Oregon were collected and planted withNicotiana tabacum ‘Samsun NN’ tobacco to bait tobacco rattle virus (TRV) and to increaseParatrichodorus allius populations, the vector of TRV. Pathogenicity of three isolates of TRV was assessed on Russet Burbank and Russet Norkotah plants usingP. allius as the vector. The most severe CRS tuber symptoms were with TRV from Pasco, WA, followed by Umatilla, OR, and Mattawa, WA, indicating a distinct variation in virulence among virus isolates. The lowest number ofP. allius that transmitted TRV resulting in CRS symptoms on Russet Norkotah was three nematodes per 250 cm3 soil. When potato plants were exposed toP. allius at different times in their growth, tubers on older plants were more resistant than younger tubers to CRS. Severity of CRS tuber symptoms was correlated with age of potato plants in pot culture. Reproduction ofP. allius on potato roots did not influence the incidence and severity of tubers symptoms. Under similar conditions, Bintje, a variety known to be resistant to CRS, served as a host forP. allius, yet remained asymptomatic. The protocol employed in these studies is appropriate for testing potato germplasm for resistance to CRS.

Molecular characterization of variants of a new ‘rule-breaking’ tobacco rattle virus RNA2 in potatoes

The molecular weights of proteins from several tobacco rattle viruses (or tobraviruses) have been determined by electrophoresis with 'marker' proteins in acrylamide gels of different strengths. Plotting KR, a measure of the effect of varying the gel strength, against molecular weight gave a value of 23,000 +/- 2,800 for tobacco rattle virus (TRV) strain CAM and similar values for other strains of TRV and pea early-browning virus. A value of 21,700 +/- 700 was obtained for TRV strain PRN by sedimentation to equilibrium in 6 M guanidine hydrochloride. These estimates are similar to those made by a statistical reappraisal of published data on amino acid composition, and a value of about 22,000 is proposed for TRV coat proteins.