Eliminating Tobacco Rattle Virus from Viruliferous Paratrichodorus allius and Establishing a New Virus-Vector Combination

A reliable method to eliminate tobacco rattle virus (TRV) from viruliferous Paratrichodorus allius populations was developed. This virus is vectored by P. allius in the Pacific Northwest and causes corky ringspot disease (CRS) of potato. The viruliferous nematodes that were reared on ''Vernema'' alfalfa or ''770'' scotch spearmint for at least 3 months did not transmit TRV to ''Samsun NN'' tobacco, a suitable indicator plant, and did not cause CRS symptoms on ''Russet Norkotah'' tubers. A new isolate of TRV was introduced into a nonviruliferous population of P. allius. First, tobacco plants were inoculated with a field population of P. allius that transmitted an isolate of TRV that caused severe symptoms on potato. The tobacco roots were then washed free from soil and dipped in 0.525% sodium hypochlorite to remove the initial nematode inoculum. After the disinfected tobacco plants recovered and began to grow, the virus-free population of P. allius was introduced around the root system to acquire the new virus isolate from tobacco roots. The newly established virus-vector combination caused CRS symptoms on ''Russet Norkotah'' that were characteristic of the more virulent virus isolate, indicating that the virus-free P. allius population had reacquired virus.     

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.     

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.     

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.     

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.     

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.     

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.     

Hatch and Reproduction of Globodera tabacum tabacum in Response to Tobacco,Tomato, or Black Nightshade

The effects of broadleaf tobacco, tomato, and black nightshade on juvenile hatch and reproduction of Globodera tabacum tabacum were determined in laboratory and greenhouse experiments. Root exudates from nightshade stimulated greater egg hatch than those from either ''Rutgers'' tomato or ''86-4'' tobacco. Hatch was greater at higher proportions of root exudates for all three plant species. Root exudates from plants greater than 3 weeks old stimulated more hatch than younger plants. No regression relationships existed between plant age and nematode batch. In other experiments, hatch from eggs in cysts was higher for tomato and nightshade after 10 weeks in greenhouse pots compared to tobacco and bare soil. Numbers of second-stage juveniles in eggs in cysts produced from a previous generation on the same host were highest on nightshade and less on tomato and tobacco. Cysts of variable age recovered from field soil had increased hatch in both root exudates or water compared to recently produced cysts from plants in growth chambers. Globodera t. tabacum may be subject to both host and environmentally mediated diapause.     

寡雄腐霉发酵液对番茄生长的影响及对灰霉病的防治作用

为了研发对番茄灰霉病高效、稳定、安全的生物农药,试验利用自主分离获得的寡雄腐霉菌株制备发酵液,采用盆栽试验研究寡雄腐霉发酵液对番茄生长的影响和对灰霉病的防治效果及机制,并在大田生产中验证其生防效果。结果表明,盆栽试验中,寡雄腐霉发酵液促进健康番茄植株生长,植株总生物量和根系生物量分别增加9.5%和15.4%,提高了植株叶绿素含量、根系活力及氮、磷、钾吸收量,并使带病番茄植株的发病率和病情指数分别降低57.2%和60.3%,相对防治效果达60.3%,施用寡雄腐霉发酵液对番茄叶片细胞膜具有保护性,降低丙二醛含量,提高病原性相关酶""超氧化物歧化酶、多酚氧化酶和苯丙氨酸解氨酶活性。后续田间试验中寡雄腐霉发酵液对番茄灰霉病的防治效果达71.2%。说明寡雄腐霉发酵液能有效防治番茄灰霉病,还具有促进番茄生长的作用,并且可诱导番茄植株对病原菌的防御作用,应用前景广泛。     

Effects of Initial Nematode Density on Population Dynamics of Globodera rostochiensis on Resistant and Susceptible Potatoes

The influence of resistant and susceptible potato cultivars on Globodera rostochiensis population density changes was studied at different nematode inoculum levels (Pi) in the greenhouse and field. Soil in which one susceptible and two resistant cultivars were grown and fallow soil in pots was infested with cysts to result in densities of 0.04-75 eggs/cm³ soil. A resistant cultivar was grown in an infested field with Pi of 0.7-16.7 eggs/cm³ soil. Pi was positively correlated with decline of soil population densities due to hatch where resistant potatoes were grown in the greenhouse and in the field but not in fallow soil. However, Pi was not correlated with in vitro hatch of G. rostochiensis cysts in water or potato root diffusate. Under continuous culture o f a resistant cultivar, viable eggs per cyst declined 60-90% per plant growth cycle (4 weeks) and the number of cysts containing viable eggs had decreased by 77% after five cycles. The rate of G. rostochiensis reproduction on both resistant and susceptible cultivars was negatively correlated with Pi. These data were used to predict the effect of resistant and susceptible potato cultivars on G. rostochiensis soil population dynamics.     

Pathogenicity of Criconemoides xenoplax to Prune and Plum Rootstocks

Elimination of Criconemoides xenoplax from a prune orchard soil by fumigation with ethylene dibromide at the rate of 42 μliter/liter of soil (equivalent to about 13 gal/acre) improved the growth of Myrobalan plum, Addition of this nematode to Myrobalan seedlings or young ''Marianna 2624'' plants propagated from cuttings resulted in destruction of cortical root tissue, darkening of roots, alteration of water stress, lowering of nutrient levels in leaves, and reduction in plant weight. C. xenoplax increased on all nine Prunus cerasifera varieties and hybrids tested, including those used commonly as rootstocks for prunes and plums. Rhizoctonia solani isolated from Myrobalan seedlings infected with C. xenoplax caused lesions on the hypocotyls of young Myrobalan seedlings in the laboratory, but had no effect on older seedlings in the greenhouse, and did not alter the effect of C. xenoplax.     

Efficacy of a Novel Nematicidal Seed Treatment against Meloidogyne incognita on Cotton

The efficacy of abamectin as a seed treatment for control of Meloidogyne incognita on cotton was evaluated in greenhouse, microplot, and field trials in 2002 and 2003. Treatments ranging from 0 to 100 g abamectin/100 kg seed were evaluated. In greenhouse tests 35 d after planting (DAP), plants from seed treated with abamectin were taller than plants from nontreated seed, and root galling severity and nematode reproduction were lower where treated seed were used. The number of second stage juveniles that had entered the roots of plants from seed treated with 100 g abamectin/kg seed was lower during the first 14 DAP than with nontreated seed. In microplots tests, seed treatment with abamectin and soil application of aldicarb at 840 g/kg of soil reduced the number of juveniles penetrating seedling roots during the first 14 DAP compared to the nontreated seedlings. In field plots, population densities of M. incognita were lower 14 DAP in plots that received seed treated with abamectin at 100 g/kg seed than where aldicarb (5.6 kg/ha) was applied at planting. Population densities were comparable for all treatments, including the nontreated controls, at both 21 DAP and harvest. Root galling severity did not differ among treatments at harvest.     

Vector Capability of Xiphinema americanum sensu lato in California

Seven field populations of Xiphinema americanum sensu lato from California''s major agronomic areas were tested for their ability to transmit two nepoviruses, including the prune brownline, peach yellow bud, and grapevine yellow vein strains of tomato ringspot virus and the bud blight strain of tobacco ringspot virus. Two field populations transmitted all isolates, one population transmitted all tomato ringspot virus isolates but failed to transmit bud blight strain of tobacco ringspot virus, and the remaining four populations failed to transmit any virus. Only one population, which transmitted all isolates, bad been associated with field spread of a nepovirus. As two California populations of Xiphinema americanum sensu lato were shown to have the ability to vector two different nepoviruses, a nematode taxonomy based on a parsimony of virus-vector relationship is not practical for these populations. Because two California populations of X. americanum were able to vector tobacco ringspot virus, commonly vectored by X. americanum in the eastern United States, these western populations cannot be differentiated from eastern populations by vector capability tests using tobacco ringspot virus.     

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.     

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species⁵, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by^9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by^{2,6,7,9,10,11,17}). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)^3,13, ecology^2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals¹⁴ or compounds¹⁵, new cultural approaches^1,4,19, or the selection and development of resistant cultivars^7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging⁷. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.     

Predisposition of Broadleaf Tobacco to Fusarium Wilt by Early Infection with Globodera tabacum tabacum or Meloidogyne hapla

In greenhouse experiments, broadleaf tobacco plants were inoculated with tobacco cyst (Globodera tabacum tabacum) or root-knot (Meloidogyne hapla) nematodes 3, 2, or 1 week before or at the same time as Fusarium oxysporum. Plants infected with nematodes prior to fungal inoculation had greater Fusarium wilt incidence and severity than those simultaneously inoculated. G. t. tabacum increased wilt incidence and severity more than did M. hapla. Mechanical root wounding within 1 week of F. oxysporum inoculation increased wilt severity. In field experiments, early-season G. t. tabacum control by preplant soil application of oxamyl indirectly limited the incidence and severity of wilt. Wilt incidence was 48%, 23%, and 8% in 1989 and 64%, 60%, and 19% in 1990 for 0.0, 2.2, and 6.7 kg oxamyl/ha, respectively. Early infection of tobacco by G. t. tabacum predisposed broadleaf tobacco to wilt by F. oxysporum.     

Root bacteria from nematicidal plants and their biocontrol potential against trichodorid nematodes in potato

Trichodorid nematodes (Nematoda: Trichodoridae) are vectors of tobacco rattle virus (TRV), one of the causal agents of spraing disease in potato. Root bacteria from nematicidal plants and their control potential against Trichodoridae were the focus of this study. Bacteria isolated from the roots of 12 nematicidal plants and potato were characterized for their production of hydrolytic enzymes, hydrogen cyanide, phenol oxidation ability and antifungal activity towards the potato pathogen Rhizoctonia solani. Based on these functional traits, bacteria isolates were selected and tested in greenhouse conditions on potato (cv. Saturna) for their effect on plant growth, and screened for nematicidal activity against Paratrichodorus pachydermus and Trichodorus primitivus in naturally infested soil. Sixteen bacteria isolates out of 44 reduced nematode densities by 50–100%. Nine selected isolated were further tested by bacterizing potato tubers (cv. King Edward) which were planted in a trichodorid and TRV-infested soil. Four bacterial isolates consistently reduced nematode densities (by 56.7–74.4%) with no visual negative effect on plant growth. These isolates were tentatively identified, partly by fatty acid methyl ester (FAME) analysis as: Stenotrophomonas maltophilia, Bacillus mycoides, Pseudomonas sp., and one unidentified bacterium. The isolates originated from potato, Plantago major, Thymus vulgaris and Asparagus officinalis, respectively. Two Pseudomonas isolates obtained from Zinnia elegans and selected for their strong nematicidal activity in soil screening tests, did not reduce the nematode population when tested on potato. It is concluded that plants releasing nematicidal compounds may harbour nematode-antagonistic bacteria as well.     

Relationship Between Time of Infection with Heterodera glycines and Soybean Yield

Experiments were conducted to determine the relationship between time of infection by Heterodera glycines and soybean growth in the greenhouse and yield of plants grown in the field. Soybean cultivar Essex seedlings growing in the greenhouse were inoculated with H. glycines at 2, 4, or 6 weeks after planting. Seedling growth was inhibited by H. glycines infection at 2 or 4 weeks after planting but not at 6 weeks. Infection of Essex by H. glycines in the field was delayed 2-6 weeks by nematicides. Yields were significantly increased when H. glycines infection was delayed 2 weeks by nematicide treatment. Essex yields were highest when infection was delayed 6 weeks, equalling the yield of the H. glycines-resistant cultivar Asgrow 5474. The effect of H. glycines on soybean growth in the greenhouse and yields in the field declined when infection was delayed 6 weeks. Thus, soybean sensitivity to H. glycines seemed to diminish with age of the soybean plants.