Establishing a Corky Ringspot Disease Plot for Research Purposes

A method to establish two experimental corky ringspot disease (CRS) plots that had no prior CRS history is described. CRS is a serious disease of potato in the Pacific Northwest caused by tobacco rattle virus (TRV) and transmitted primarily by Paratrichodorus allius. ‘Samsun NN’ tobacco seedlings were inoculated with viruliferous P. allius in the greenhouse before they were transplanted into the field soil at the rate of 3,000 plus seedlings/ha. Care was taken to keep soil around plants in the greenhouse and transplants in the field moist to avoid vector mortality. The vector population in the soil of one of the fields was monitored by extraction, examination under microscope and bioassay on tobacco seedlings to ascertain that they were virus carriers. Presence of virus in tobacco bioassay plants was determined by visual symptoms on tobacco leaves and by testing leaves and roots using ELISA. Although TRV transmission was rapid, there was loss of infectivity in the first winter which necessitated a re-inoculation. After two years of planting infected tobacco seedlings, 100% of soil samples collected from this field contained viruliferous P. allius. In the second field, all five commercial potato cultivars, known to be susceptible, expressed symptoms of CRS disease indicating that the procedure was successful.     

Control of Paratrichodorus allius and Corky Ringspot Disease in Potato with Shank-injected Metam Sodium

Corky ringspot disease (CRS) of potato produces necrotic areas in tubers that are considered quality defects that can lead to crop rejection. CRS is caused by tobacco rattle virus that is vectored by stubby-root nematodes (Paratrichodorus spp., Trichodorus spp.) at very low population densities, making disease management difficult and expensive. Fumigation with metam sodium (MS) is a common practice to control soil-borne fungi and increase potato yield. MS is generally applied in water via chemigation (water-run, WR) but is ineffective at controlling CRS when WR-applied, even at high rates. Therefore, WR MS is often used in combination with 1,3-dichloropropene (1,3-D), aldicarb or oxamyl to attain adequate CRS control. Between 1996 and 2000, fields with a history of CRS were treated with WR MS, shank-injected MS, and/or 1,3-D, and tubers were evaluated for symptoms of CRS. Shank injection of MS (SH MS) at depths of 41 cm, 15 and 30 cm, or 15, 30 and 45 cm controlled CRS over 3 years of testing. All rates of 280 liters/ha or greater were effective. Shank injection of metam potassium (MP) at rates of 448 liters/ha was also effective. 1,3-D controlled CRS alone or in combination with WR or SH MS. Proper shank application of MS or MP may adequately control CRS without the additional cost of other nematicides at low (<10 P. allius/250 g soil) to moderate (10 to 30 P. allius/250 g soil) populations of the nematode vector. Although SH MS was superior to WR MS, additional research is necessary to determine if this practice would be sufficient at higher CRS disease pressure or if addition of other nematicides would be necessary.     

THE USE OF TISSUE CULTURES TO PRODUCE VIRUS-FREE CLONES FROM INFECTED POTATO VARIETIES

By growing the excised apical meristems of sprouts from the potato varieties King Edward and Arran Victory, infected respectively with potato paracrinkle virus and potato virus S , virus-free plants were obtained. Although the method failed to produce virus-free plants from varieties infected with potato virus X , this virus also seems not to be present in apical meristems, for no virus could be demonstrated in callus tissue that developed from excised meristems less than 200 μ across. The concentration of tobacco mosaic virus in tomato roots and tobacco stems is also much less near the growing point than in older cells, but there is no evidence that the meristematic region is virus-free.     

Analysis of the occurrence and distribution of tobacco rattle virus in field soil and disease in a subsequent tulip crop

The occurrence and distribution of tobacco rattle virus (TRV) in field plots was determined by soil bait-testing and disease incidence in tulips subsequently grown on these plots was studied. The virus occurred in patches, calculated as 1.5 m × 3.6 m. The presence of virus was not correlated with numbers of potential vector trichodorid nematodes. Of three trichodorid nematode species present, only Paratrichodorus teres transmitted TRV which, as with virus isolates obtained in bait-tests and from infected tulips, reacted in serological tests with an antiserum prepared against a Dutch isolate of pea-early browning virus (PEBV). Virus prevalence in a subsequent tulip crop was 0.8% and in a sample of tulip plants, virus was recovered only from plants showing virus symptoms. Plots from which TRV was recovered in bait-tests yielded significantly more virus diseased tulips than plots which tested negative for virus. Growing bait-plants in field-plots, as compared with greenhouse tests using soil collected as a series of sub-samples, resulted in an underestimate of the occurrence of TRV.     

Characterization of Pratylenchus penetrans from Ten Geographically Isolated Populations Based on Their Reaction on Potato

Single female cuhures of Pratylenchus penetrans were established from soil and root samples collected from 10 geographically isolated locations in North America. The resultant isolates were used to evaluate nematode egression from and multiplication on roots of potato clones to distinguish intraspecific differences among isolates. The 10 nematode isolates were statistically separated into four groups based on percentage of nematodes that egressed from the P. penetrans-resistant potato done L 118-2. The Cornell (CR), Wisconsin (WI), Long Island (LI), and Adirondack (AD) isolates, selected as representative isolates of each of the four groups, exhibited 53%, 39%, 25%, and 10% egression from L118-2, respectively. Reproduction of these four isolates was measured on three potato cultivars (Russet Burbank, Butte, and Hudson) and two breeding lines (NY85 and L118-2). The LI and AD isolates reproduced well on all five potato clones. The CR isolate reproduced well on Russet Burbank and NY85 but significantly less on Butte, Hudson, and L118-2. Reproduction of the WI isolate was less than the LI and AD isolates but more than the CR isolate on all potato clones tested except Russet Burbank. Reproduction of the WI isolate on Russet Burbank was less than the other three isolates. Based on these results, four distinct intraspecific variants of P. penetrans are proposed: Cornell, Wisconsin, Long Island, and Adirondack.     

Ultrastructural studies of plasmodesmatal and vascular translocation of tobacco rattle virus (TRV) in tobacco and potato

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.     

Effects of In-furrow and Water-run Oxamyl on Paratrichodorus allius and Corky Ringspot Disease of Potato in the Klamath Basin

Corky ringspot disease (CRS) of potato (Solanum tuberosum) is caused by the tobacco rattle virus (TRV), which is vectored by stubby-root nematodes, Paratrichodorus spp. and Trichodorus spp., and is a significant threat to potato quality and production in many areas of the western United States. Between 2002 and 2005, fields with a history of CRS were planted to potato and treated with various combinations of in-furrow (IF) and chemigated (water run, WR) oxamyl [Methyl N'N'-dimethyl-N-[(methyl carbamoyl)oxy]-1-thiooxamimidate] applications. Soil samples were collected to determine how Paratrichodorus allius populations responded to the various treatment regimes (2002-2004); potato tubers were evaluated for symptoms of CRS in 2004-2005. Applications of oxamyl to potato (1.1 kg a.i./ha) did not cause significant mortality of P. allius but did prevent the populations from increasing. Oxamyl applications that began at 55 days after planting (DAP) or later did not control CRS and were not different from the untreated control. However, application schedules that began early-season, either IF at planting, early WR (33 - 41 DAP), or both, significantly reduced CRS expression in cv. Yukon Gold. Therefore, oxamyl applications must be made early in the growing season to be effective in controlling CRS. Effects of oxamyl on CRS may be due to nematostatic action that suppresses feeding activity during early field season when most virus transmission probably occurs.     

Acquisition and transmission of potato mop-to furovirus by a culture of Spongospora subterranea f.sp. subterranea derived from a single cystosorus

Potato mop-top virus (PMTV) was detected by ELISA in primary zoospores from four out of six isolates of Spongospora subterranea f.sp. subterranea. One virus-free isolate (N) of S. subterranea was used to acquire PMTV from potato roots and to transmit the virus to healthy plants. A mono-fungal culture of S. subterranea (isolate N) was derived by infecting tomato plant roots with a single cystosorus. The culture was used successfully to acquire PMTV from the roots of infected Nicotiana debneyi plants that had been manually inoculated with virus isolates, and subsequently to transmit the virus to healthy bait plants. These experiments confirm that S. subterranea is a vector of PMTV. Two PMTV isolates that had been maintained by manual inoculation for 19 and 21 passages were also acquired and transmitted by the fungus culture.     

Differentiation of Two New York Isolates of Pratylenchus penetrans Based on Their Reaction on Potato

The behavior of two isolates of Pratylenchus penetrans on six potato clones was assessed to test the hypothesis that these nematode isolates from New York were different. Four potato cultivars (Superior, Russet Burbank, Butte, and Hudson) and two breeding lines (NY85 and L118-2) were inoculated with nematode isolates designated Cornell (CR) and Long Island (LI). Population increase and egression of nematodes from roots were used to distinguish resistance and susceptibility of the potato clones. Based on numbers of eggs, juveniles, and adults in their roots 30 days after inoculation, potato clones Butte, Hudson, and L118-2 were designated resistant to the CR isolate and susceptible to the LI isolate. More eggs were found in the roots of all plants inoculated with the LI isolate than with the CR isolate. The clones NY85 and L118-2 were inoculated with the CR and LI isolates in a 2 x 2 factorial experiment to assess differences in nematode egression. Egression was measured, beginning 3 days after inoculation, for 12 days. The rates of egression were similar for the four treatments and fit linear regression models, but differences were detected in numbers of egressed nematodes. More nematodes of the CR isolate than the LI isolate egressed from L118-2. Differences in egression of females was particularly significant and can be used as an alternative or supplement to reproduction tests to assess resistance in potato to P. penetrans and to distinguish variation in virulence.     

The role of weed hosts and the distribution and activity of vector nematodes in the ecology of tobacco rattle virus

At 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.     

Effects of Salicylate on Systemic Invasion of Tobacco Plants by Various Viruses

Salicylate watered onto soil in which White Burley tobacco plants were grown represents a reversible stress characterized by stomatal closure, slight slackening of plant growth and low chlorophyll loss. Salicylate affected viral pathogenesis in opposite ways. It had no effect against local and systemic infections by potato virus X (PVX), potato virus Y⁰ (PVY⁰) or tobacco mosaic virus (TMV), whereas it completely prevented systemic infection by alfalfa mosaic virus (AIMV) or tobacco, rattle virus (TRV) in a high proportion of treated plants. When infection moved from leaves inoculated with AIMV or TRV, the tendency to limit systemic spread was shown by the restriction of systemic infection to very limited areas erratically distributed in some uninoculated leaves. The salicylate-induced restriction of AIMV or TRV infectivity to inoculated leaves did not appear due to inhibition of virus multiplication because the inoculation of potentially resistant leaves of salicylate-reated plants resulted in virus antigen accumulation comparable to that of untreated controls. Salicylate may therefore inhibit some long distance virus transport function. Salicylate appears able to evoke true hypersensitivity only against systemic viruses able to induce local necrotic lesions, probably by activating some genetic information for resistance that is normally not expressed.     

Anin vitro procedure to eradicate potato viruses X,Y, and S from Russet Norkotah and two of its strains

Summary A tissue culture method was developed for the eradication of three important potato viruses, PVX, PVY, and PVS, from the Russet Norkotah variety and two of its strains (TXNS 112 and TXNS 278). The method combined the use of liquid medium, thermotherapy, and chemotherapy. Initially, virus-free plants were inoculated with PVX, PVY, and PVS and, after 10 d, tested quantitatively for virus titer by ELISA to determine the initial virus concentration. The apical tip and the roots were removed from the inoculated plants, and only stem sections from inoculated plants were cultured in liquid medium containing MS inorganic salts, vitamins, and ribavirin (40 μM or 80 μM). After 5 d, half of the plants were subjected to thermotherapy and half were kept at room temperature. At 6 wk, the uppermost node (5–7 mm) was removed and recultured, and plants were tested then and again after 6 wk using ELISA to identify the virus-free plants. Ribavirin alone eradicated the viruses from some plants; however, more virus-free plants were obtained when the treatments included heat. Additionally, thein vitro culture technique using liquid medium promoted rapid lateral shoot elongation and resulted in significantly faster plant production. Also this approach, which required less skilled labor, produced more plants than the meristem culture method for virus eradication. A detailed procedure for elimination of multiple viruses is described. This procedure resulted in production of more than 10% virus-free plants.     

Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum)

Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.     

Some properties of an isolate of tobacco rattle virus from Northern Ireland

A virus obtained from soil in which potato plants had shown severe spraing symptoms induced symptoms on indicator plants typical of tobacco rattle virus (TRY). Purified virus preparations of a local-lesion isolate contained particles of two modal lengths, 192 nm and 94 nm containing RNA molecules of mol. wt 2.4 × 10⁶ and 1.23 × 10⁶. Virus coat protein had a mol. wt of c. 21 500. The virus was serologically distantly related to TRY (SYM) and pea early browning virus (PEBV) SP5, but did not react with TRY (CAM) or TRY (PRN) antisera. However, cDNA hybridisation indicated that the virus was more closely related to TRY (PRN) than either TRY (SYM) or PEBV (SP5). The virus isolate has been designated TRY (NI).     

Failure of British isolates of beet western yellows virus to infect potato

Potato cultivars were tested for susceptibility to two British isolates of beet western yellows virus originally obtained from sugar beet and oil seed rape. Neither isolate was transmitted by Myzus persicae to virus-free potato plants, either by itself or in association with potato leafroll virus.     

Delivery of macromolecules to plant parasitic nematodes using a tobacco rattle virus vector

Plant parasitic nematodes cause significant damage to crops on a worldwide scale. These nematodes are often soil dwelling but rely on plants for food and to sustain them during reproduction. Complex interactions occur between plants and nematodes during the nematode life cycle with plant roots developing specialized feeding structures through which nematodes withdraw nutrients. Here we describe a novel method for delivering macromolecules to feeding nematodes using a virus-based vector [tobacco rattle virus (TRV)]. We show that the parasitic nematode Heterodera schachtii will ingest fluorescent proteins transiently expressed in plant roots infected with a TRV construct carrying the appropriate protein sequence. A prerequisite for this delivery is the presence of replicating virus in root tips prior to the formation of nematode-induced syncytia. We show also that TRV vectors expressing nematode gene sequences can be used to induce RNAi in the feeding nematodes.     

Immunogold Localization of Tobacco Rattle Virus Particles within Paratrichodorus anemones

Unequivocal evidence of the viral nature of virus-like particles observed at the specific site of retention of tobacco rattle virus (TRV) in Paratrichodorus and Trichodorus nematodes has not previously been available. A new staining technique using safranin-O, which does not affect viral antigenicity, was used with an antiserum raised against the coat protein of TRV and prepared for use with immunogold labelling. Application of this method enabled the occurrence and localization of particles of TRV to be confirmed in the pharynx of the natural vector of the virus, Paratrichodorus anemones, and provided unequivocal evidence that the particles observed were TRV particles. The TRV particles were observed attached only to the cuticle lining the posterior tract of the pharyngeal lumen of the vector. Therefore, the specific site of retention of TRV particles in P. anemones is apparently more localized than reported to occur in other vector trichodorid species.