The association between species of Trichodorus and Paratrichodorus vector nematodes and serotypes of tobacco rattle tobravirus

Virus transmission bait tests with single trichodorid nematodes from England, the Netherlands, Scotland or Sweden showed that a substantial degree of specificity occurs between trichodorid vector species and tobravirus serotypes. This specificity was more apparent with associations between Paratrichodorus vector species and tobravirus serotypes than with those between Trichodorus species and tobravirus serotypes. P. pachydermus transmitted PRN-serotype tobacco rattle virus (TRV) isolates, P. teres ORE-serotype isolates and P. anemones TRV isolates which did not react with any of the antisera used, but which could be distinguished from all other isolates by their symptomatology in Chenopodium test plants. T. viruliferus, T. primitivus and T. cylindricus transmitted RQ-serotype isolates and the latter species also transmitted TRV isolates reacting with TCB2 and pea early-browning SP5-antisera. Several TRV isolates transmitted by T. cylindricus failed to react with any of the antisera used.     

Tobraviruses—plant pathogens and tools for biotechnology

The tobraviruses, Tobacco rattle virus (TRV), Pea early‐browning virus (PEBV) and Pepper ringspot virus (PepRSV), are positive‐strand RNA viruses with rod‐shaped virus particles that are transmitted between plants by trichodorid nematodes. As a group, these viruses infect many plant species, with TRV having the widest host range. Recent studies have begun to dissect the interaction of TRV with potato, currently the most commercially important crop disease caused by any of the tobraviruses. As well as being successful plant pathogens, these viruses have become widely used as vectors for expression in plants of nonviral proteins or, more frequently, as initiators of virus‐induced gene silencing (VIGS). Precisely why tobraviruses should be so effective as VIGS vectors is not known; however, molecular studies of the mode of action of the tobravirus silencing suppressor protein are shedding some light on this process.     

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.     

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 potential of reflective mulching in combination with insecticide sprays for control of aphid-borne viruses of iris and tulip in Tasmania

Silver coloured plastic mulches and weekly insecticide sprays were examined individually and in combination for efficacy in reducing spread of tulip breaking potyvirus (TBV) in tulip, and these plus mineral oil treatments, alone and in combination with insecticide and mulch, were evaluated for their effect on iris mild mosaic potyvirus (IMMV) spread in bulbous iris. In the iris trial, significant reductions in virus spread were noted for all treatments, with the combined treatment of mulch and insecticide giving the best virus control. However, with little virus spread there were no significant treatment effects on TBV spread in tulips. Reduced numbers of aphid vectors were trapped over unsprayed mulched than non-mulched plots. Mulch treatments had no effect on stem length or harvested bulb weight while the insecticide treatment (tulips only) and treatments incorporating mineral oil significantly reduced both stem length and bulb weight. This work suggests that enhanced virus management in flowerbulb production may be achieved by incorporating reflective mulches in current virus control strategies.     

Augusta disease in tulip - a reassessment

In an experiment in which the roots of field-grown tulip were commonly infected with tobacco necrosis virus (TNV), Augusta disease did not develop in the year of infection or when progeny bulbs were grown in the field or glass-house. When tulip bulbs of other stocks, including grades of 11 and 12 cm circumference, were forced, the disease developed sporadically, in some instances as the result of infection with TNV from the soil in which they were planted and in others as a result of infection by bulb-borne virus. The incidence of disease produced by current year infection was increased by warming the plunge bed. Different strains of TNV were obtained from field-grown plants with Augusta disease and different strains of the virus produced the disease when inoculated to tulip. Some, but not all, naturally diseased plants contained satellite virus, which therefore does not cause or prevent disease development. The disease was produced in some plants by TNV transmitted by Olpidium brassicae, but neither a vector nor a non-vector isolate of O. brassicae completed its life cycle in tulip. However, Olpidium-like zoospores were observed in some washings of tulip roots from TNV-infested soils. TNV was not obtained from all tulip plants with necrotic leaf symptoms resembling Augusta disease. Some were infected with tomato bushy stunt virus or cucumber mosaic virus, or with another agent that was transmitted by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and carried by bulbs of up to 11 cm circumference.     

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.     

Vorkommen von Tobacco Rattle-Virus in Roggen (Secale cereale) in der Bundesrepublik Deutschland

Natural occurrence of tobacco rattle virus in rye (Secale cereale) in the Federal Republic of Germany Near Bergen/Dumme in northern Germany, in an area known for the occurrence of tobacco rattle virus (TRV) on potatoes, 5–10 % of rye plants of one experimental plot were severely stunted and showed distinct chlorotic mottling of leaves. In these plants TRV was identified on the basis of particle morphology and serology (Immunoelectron microscopy and ELISA). TRV was also detected in Capsella bursa-pastoris and Stellaria media from an area adjacent to the rye plot but not in nearby commercial rye fields.     

Properties of spinach yellow mottle, a distinctive strain of tobacco rattle virus

A virus (isolate SYM) obtained from spinach plants in England with a severe yellow mottle disease induced symptoms resembling those of tobacco rattle virus (TRV) in several indicator species but caused systemic necrosis in Chenopodium amaranticolor and C. quinoa. It was transmitted to bait plants grown in soil containing the nematode Trichodorus primitivus. Purified virus preparations contained rod-shaped particles that were predominantly of four modal lengths: 188 nm (L particles), 101 nm (S particles), 57 nm and 48 nm (together called VS particles), containing RNA with mol. wts of 2.4, 1.5, 0.7 and 0.6 million, respectively. L particles (s°₂₀= 300 S) and S particles (230 S) greatly outnumbered VS particles (c. 150 S). All particles contained a single polypeptide species with estimated mol wt of 24 700, slightly larger than those previously reported for tobraviruses. Purified L particles were infective but both L and S particles were needed to induce the production of virus nucleoprotein particles. VS particles were not infective and apparently had no qualitative or quantitative effect on infection by L or by L plus S particles. S particles carried determinants for serological specificity and ability to invade C. amaranticolor systemically. Isolate SYM produced pseudo-recombinants with isolate PRN of TRV. Also, isolates CAM, OR and PRN of TRV, and isolate SYM, were found to be distantly related by three kinds of serological test. No relationship was detected between these isolates and pea early-browning virus in gel-diffusion precipitin tests or electron microscope serological tests, but a distant relationship between isolate SYM and pea early-browning virus was found by micro-precipitin tests. Isolate SYM therefore has closer affinities with TRV than with pea early-browning virus and is considered to be a distinctive strain of TRV.     

Tests for transmission of cherry leaf roll virus using Longidorus, Paralongidorus and Xiphinema nematodes

The ability of 10 nematode species to transmit three strains of cherry leaf roll virus (CLRV) was tested by three methods: (1) virus-infected source plants and virus-free bait plants were grown concurrently in nematode-infested soil, (2) as for (1) but virus source plants were removed before bait plants were planted, and (3) nematodes were extracted from soil after access to virus source plants, and were added to pots containing bait plants. The occurrence of galls on roots showed that nematodes fed both on source and on bait plants in all experiments and, in some experiments, CLRV was detected by direct assays (slash tests) of Longidorus elongatus, L. leptocephalus and Paralongidorus maximus. Although the nematodes readily transmitted control viruses, for which they are known to be vectors, CLRV was detected by root assays in only a few bait plants exposed to L. elongatus, L. macrosoma, Xiphinema diversicaudatum or L. leptocephalus + X. vuittenezi in tests by method 1. The recovery of CLRV in these tests is interpreted as being due to contamination. These results add to the increasing circumstantial evidence against the involvement of nematodes in the transmission of CLRV. Other possible mechanisms of spread are discussed.     

Development of potato blight Phytophthora infestans after planting infected seed tubers

About 1000 blight-infected seed potato tubers, usually of the cultivar King Edward, were planted for 9 yr and the subsequent plants examined until the disease had developed in the plots. Haulm infection originated each year from the seed tubers and occurred first on basal leaves. When tubers were inoculated with a complex race of P. infestans this race was recovered from the leaves and from the soil near the seed tuber. Transmission of infection from soil to leaves was demonstrated by splash of artificially contaminated soil to leaves suspended above the soil. In 4 yr, plants were grown on flat rows as well as on ridges. In 2 yr, when emergence was almost complete, infected stems were observed on otherwise normal plants. In the first year 0.6% grew on ridges and 3.0% on the flat and in the second all grew on the flat (5.3%). Only seven of the 43 plants had more than one infected stem. Flat plots had a significantly higher number of stemdplant than ridge plots, but this bore no relation to numbers of infected stems. When flat plots which had developed affected plants had soil replaced as ridge plots, no further infected stems were observed. Such stems continued to develop on flat plots. No prematurely dead stems were observed below soil level when all plants were dug. Underground portions of most infected stems showed little evidence of P. infestans which was found only at about soil level. Infection appeared to occur first in this area.     

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.     

Identification of jasmonic acid and its methyl ester as gum-inducing factors in tulips

The purpose of this study was to identify endogenous factors that induce gummosis and to show their role in gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) stems. Using procedures to detect endogenous factors that induce gum in the stem of tulips, jasmonic acid (JA) and methyl jasmonate (JA-Me) were successfully identified using gas–liquid chromatography–mass spectrometry. Total amounts of JA and JA-Me designated as jasmonates in tulip stems were also estimated at about 70–80 ng/g fresh weight, using deuterium-labeled jasmonates as internal standards. The application of JA and JA-Me as lanolin pastes substantially induced gums in tulip stems with ethylene production. The application of ethephon, an ethylene-generating compound, however, induced no gummosis although it slightly affected jasmonate content in tulip stems. These results strongly suggest that JA and JA-Me are endogenous factors that induce gummosis in tulip stems.     

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.     

Occurrence of Melon Necrotic Spot Virus in Crete (Greece)

Since 1982 melon plants cv. Galia grown in plastic houses showed severe leaf and stem necrosis. A virus isolated from affected plants infected only Cucurbitaceae. Systemic infections were only developed in melon and Luffa acutangula, while a severe hypocotyl necrosis was observed in watermelon. Purified virus sedimented in sucrose gradients as a single component and reacted only with antisera to melon necrotic spot virus (MNSV). Seed transmission (22.5%) of the virus was observed m melon plants grown from seed of naturally infected melons, but the virus could not be detected in triturated seeds. The virus could be isolated from leachate of contaminated soil and melon plants became infected when grown in contaminated soil or when watered with suspensions of virus. These properties suggest that the virus is an isolate of MNSV.     

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.     

Accumulation and persistence of a nuclear polyhedrosis virus of the cabbage looper in the field

A nuclear polyhedrosis virus of the cabbage looper, Trichoplusia ni, was applied to foliage of Fall-planted broccoli and cabbage plants. Soil bioassays demonstrated that appreciable quantities of active virus accumulated in the upper 1 cm of the soil of plots sprayed with 10, 20, and 100 larval equivalents (LE)/acre as well as in unsprayed plots. After completion of the spray program, soil from plots treated with 8 foliar applications of 10 LE/acre contained only slightly more virus than the soil from unsprayed plots, but considerably more virus accumulated in plots treated with 20 and 100 LE/acre. The accumulation of virus continued after the completion of spraying and considerable quantities of active virus persisted through the winter.     

Tulip chlorotic blotch virus, a second potyvirus causing tulip flower break

Tulip chlorotic blotch virus (TCBV), an apparently undescribed potyvirus found in field grown tulips in Australia, causes symptoms in tulip leaves and flowers identical to those induced by tulip breaking virus (TBV). TCBV was transmitted mechanically to 14 of 34 species in four of 13 families. Nicotiana clevelandii is a suitable propagation host and Chenopodium amaranticolor a local-lesion assay host. TCBV was transmitted from tulip to tulip and TV. clevelandii by the aphid Myzus persicae. Unlike TBV it was not transmitted to Lilium formosanum either by M. persicae or by manual inoculation. Leaf extracts from TCBV-containing TV. clevelandii were infective after dilution to l0^-3 but not 10-⁴ and after heating for 10 min at 50°C but not 60°C; infectivity and particle recovery were adversely affected by freezing at -20°C. TCBV particles were purified (c. 1 mg/100g g N. clevelandii leaf) from tissue extracts in 0·3 M citrate buffer containing 10 mM EDTA and 0·2% (v/v) 2-mercaptoethanol at pH 7·4 by clarification with 8·5% (v/v) n-butanol followed by differential centrifugation and sucrose density gradient centrifugation. Purified particles measured c. 720 × 12 nm. Virus particle antigen was readily detected in leaf and tepal extracts of tulip by enzyme-linked immunosorbent assay. A distant serological relationship was found between particles of TCBV and those of bean yellow mosaic virus but no serological relationship was found to TBV or four other potyviruses.     

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.