Influence of arachidonic acid and phytoviruses on the hormonal system of potato plants grown in vitro

Changes in the phytohormonal system during the development of antivirus resistance was investigated. This resistance was induced by arachidonic acid (AA) in potato plants (Solanum tuberosum L.) grown in vitro. The plants in tubes were treated with AA 7 days before infection with tobacco mosaic virus or potato X virus. These plants were used for ABA, IAA, and cytokinin quantification. As a rule, phytoviruses increased ABA and cytokinin levels and decreased that of IAA in both immunized and unimmunized plants. AA significantly decreased the phytovirus content in potato plants at least during 3 weeks. In general, AA immunization acted as a limiting factor for increasing the ABA and cytokinin concentrations during virus pathogenesis in experimental plants. However, no regularity in the changes of the IAA concentration was noted. The data obtained showed that AA-induced changes in the hormonal system could be an important factor in the mechanism of plant resistance against phytopatogenic viruses.     

Induction of Phytohemagglutinin Activity by Arachidonic Acid in in vitro Potato Plants

Tube-grown potato (Solanum tuberosum L., cv. Nevskii) plants treated with arachidonic acid (AA) were used as a model to study the activity of phytohemagglutinins (PHA) during induction of the plant antiviral defense system. Plant treatment with 10^–8 M AA and also their inoculation with potato viruses X, Y, and M resulted in the increased activity of PHAs in potato shoots. The inducer of antiviral resistance behaved as a modulator of the PHA activity providing for its various levels during the development of viral infection. During the development of AA-induced systemic resistance, the level of phytohemagglutinin activity did not essentially depend on the nature of the viral pathogen. We suggested that the mechanism of AA-induced plant antiviral defense was connected with changes in the PHA activity.     

The Effect of Arachidonic Acid and Viral Infection on the Phytohemagglutinin Activity during the Development of Tobacco Acquired Resistance

The effects of arachidonic acid (AA) on the development of viral infection and the activity of phytohemagglutinins in Nicotiana tabacum L. plants were studied. Cv. Samsun NN was used, which displayed a genotypically determined hypersensitive response to tobacco mosaic virus (TMV) infection. When tobacco leaf disks were treated with 10^–9 to –10^–7 M AA, viral reproduction was suppressed by 90–100%. The AA concentration of 10^–8 M was optimal for the improvement of plant virus resistance. Tobacco leaves maintained virus resistance for at least two weeks. Both AA treatment and TMV inoculation were accompanied by an enhanced lectin activity, which may indicate the involvement of lectins in the development of plant defense responses. Lectin accumulation was observed in the intact plants developing systemic resistance and in the detached leaves characterized by local resistance.     

Effect of Chitosan on Systemic Viral Infection and Some Defense Responses in Potato Plants

The development and the possible mechanism of the chitosan-induced resistance to viral infection were investigated in potato plants. The plants were sprayed with a solution of chitosans (1 mg/ml) with the mol wt of 3, 36, and 120 kD. After 1, 2, 3, or 4 days, the treated leaves were cut off and mechanically infected with the potato virus X (PVX). The disks cut out from the inoculated leaves were used for determining virus accumulation, callose content, and ribonuclease and -1,3-glucanase activities. In another set of experiments, the plants were infected with PVX within 1, 4, or 8 days after chitosan treatment, and the number of systemically infected plants was determined. It was found that, a day after treatment, the plants acquired a resistance to viral infection. The disks from the chitosan-treated leaves, as compared to the control, accumulated less amount of virus. The chitosan treatment also significantly decreased the number of systemically infected plants as compared to the control. After 2–3 days, the resistance disappeared or even gave way to an increased susceptibility to the infection; subsequently, the resistance increased again. The extent of the resistance correlated with the callose content and the level of ribonuclease activity observed on the infection day. The resistance towards the infection with PVX is probably mediated by the callose and ribonuclease induction. The cultivation of test-tube potato plants from the cuttings previously infected with PVX on the chitosan-containing nutrient medium did not eradicate the viral infection from the plants.     

Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus

Summary Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant court-noué disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a court-noué disease.Abbreviations GFLV-F13 grapevine fanleaf virus F13 isolate - ArMV arabis mosaic virus - CP coat protein - MS Murashige and Skoog - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - VPg genome linked viral protein - TMV tobacco mosaic virus - PVX potato virus X - PVY potato virus Y - TRV tobacco rattle virus - +CP CP expressing - -CP control plant, not expressing CP - CPMP coat protein-mediated protection - CPMCP coat crotein-mediated cross protection     

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.     

Accumulation of potato virus Y is enhanced in Solatium brevidens also infected with tobacco mosaic virus or potato spindle tuber viroid

The accumulation of potato virus Y?(PVY?) and potato leaf roll virus (PLRV) was studied in plants of Solanum brevidens co-infected with each of six viruses or a viroid. Virus could not be detected by ELISA in plants of S. brevidens infected solely with PVY. However, accumulation of PVY was increased c. 1000-fold in plants doubly infected with tobacco mosaic virus or potato spindle tuber viroid (PSTVd). PVY titres in doubly infected plants of S. brevidens were between 1% and 0.1% of those found in the PVY-susceptible interspecific Solanum hybrid DTO-33. Double infections of 5. brevidens by PVY and alfalfa mosaic virus or potato viruses M, S, T or X did not significantly enhance PVY accumulation. Accumulation of PLRV was not enhanced in plants co-infected with any of the six viruses or PSTVd.     

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.     

Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana

Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.     

The effects of temperature on the availability and acquisition of potato leafroll luteovirus by Myzus persicae

The concentration of potato leafroll luteovirus (PLRV) did not differ in potato plants with secondary infections grown at 15°C or 27°C. Detached leaves of plants grown at 15°C or 27°C were used as sources of PLRV for peach-potato aphids (Myzus persicae Sulz.) both at 15°C and 27°C. At comparable temperature during virus acquisition, aphids which fed on leaves of plants kept previously at 15°C contained more viral antigen detected by ELISA than aphids which fed on leaves of plants grown at 27°C. The aphids which acquired PLRV at 27°C contained evidently more viral antigen than those which acquired PLRV at 15°C. The greatest amount of PLRV was found in the aphids which acquired the virus at 27°C from the leaves of plants kept at 15°C. The ability of M. persicae to transmit PLRV to Physalis ftoridana Rydb. generally decreased with decrease in the amount of PLRV in vectors.     

Purification and Identification of a Rhabdovirus from Thunbergia alata

A subgroup 2 rhabdovirus was isolated in south-east Queensland from black-eyed Susan (Thunbergia alata) with symptoms of vein yellowing, vein clearing and leaf distortion. Bacilliform particles accumulated in the perinuclear space of infected plants and measured 69 ± 7 × 161 ± 8 nm in unfixed preparations. The virus was not transmitted mechanically. Purified preparations of the Thunbergia alata rhabdovirus (TaRV) contained four major proteins with molecular weights of 80 kD, 48 kD, 40 kD and 35 kD, similar to those of datura yellow vein virus (DYW), a newly described rhabdovirus from Australia. The 80 kD protein was identified as the viral glycoprotein. In immunoblots, the two largest proteins of TaRV reacted strongly with antiserum to DYW, but were serologically distinct from sonchus yellow net, cereal chlorotic mottle, potato yellow dwarf and lettuce necrotic yellows viruses. TaRV is considered to be a strain of DYW.     

Effect of Plant Oligoadenylates on Viral Infection and Antistress and Growth-Regulating Properties in Potato Plants Grown in vitro

Biological properties of plant oligoadenylates (POA) were studied in in vitro grown potato (Solanum tuberosum L.) plants. POA were synthesized by ATP polymerization in the presence of an enzyme preparation isolated from leaves of wild potato Solanum chacoense Bitt. treated with resistance inducers. The concentration of viral antigen was determined by the method of immunoenzyme analysis. In the course of virus elimination by the apical meristem method, pretreatment of potato tuber sprouts with POA increased the survival rate of meristem explants and, in several cultivars, enhanced morphogenesis and increased the yield of virus-free regenerants. Prolonged application of POA in the nutrient medium for microcutting of potato tube plants infected in vitro with X-, S-, and M-viruses increased their height due to internode lengthening. The effect of this treatment on virus reproduction was ambiguous: various combinations of virus species, the duration of growth on POA-containing media, and POA concentration resulted in either statistically significant inhibition or a significant enhancement of virus reproduction. Possible mechanisms of the antistress effect of POA, in connection with their effect on virus reproduction, are discussed.     

Factors underpinning the responsiveness and higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes

Responses to Potato virus A (PVA, genus Potyvirus) segregate to three phenotypic groups in a diploid cross between Solanum tuberosum subsp. andigena and a highly interspecific potato hybrid. The aim of this study was to compare gene expression between the progeny genotypes which react with hypersensitive response (HR) to PVA, allow PVA accumulation in inoculated leaves but restrict PVA infection to the inoculated leaf by blocking systemic movement [non-necrotic resistance (nnr)], or are susceptible (S) and systemically infected with PVA. Expression levels of ca 10 000 genes were compared using probes arranged in a microarray format, and real-time RT-PCR was applied for quantitative comparison of the expression of selected defense-related genes (DRGs). Results showed that a few DRGs were autoactivated in HR genotypes at an early stage of plant growth in the absence of PVA infection, which was not observed in the two other phenotypic groups (nnr and S). More detailed studies on the DRGs encoding a beta-1,3-glucanase, a chitinase and a basic PR-1b protein showed that autoactivation of the genes was not evident in vitro and up to 2 weeks of growth in soil in a controlled growth cabinet but was apparent 2 weeks later. Hence, autoinduction of these DRGs in the HR genotypes could be associated with growth stage, environmental factors or both. Furthermore, a number of other DRGs were induced in the inoculated leaves of HR genotypes as a response to infection with PVA, which was not observed in nnr and S genotypes. These results provide some novel information about factors underpinning the higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes and suggest that part of the resistance is attributable to additional ‘minor’ genes functioning simultaneously, hence adding to the overall responsiveness and level of resistance against infection. These results also imply that some genotypes might be more responsive to chemical induction of pathogen and pest resistance, which could be considered in screening of progenies in plant-breeding programs.     

Boosting the Immune System of Solanaceous Plants by Lysophosphatidylcholine

An in vitro treatment of solanaceous plants, viz. potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.), with lysophosphatidylcholine promoted their resistance to phytopathogens, such as Phytophthora infestans (Mont.) de Bary and potato virus Y. The systemic and long-term lysophospholipid-induced response was independent of the phytopathogen type. It was concluded that lysophosphatidylcholine, a product of phosphatidylcholine hydrolysis with phospholipase A₂, plays an important role in the regulation of plant immune system.     

Restricted distribution of potato leafroll virus antigen in resistant potato genotypes and its effect on transmission of the virus by aphids

Enzyme-linked immunosorbent assay was used to measure the concentration of potato leafroll virus (PLRV) antigen in different parts of field-grown secondarily infected plants of three potato genotypes known to differ in resistance to infection. The antigen concentration in leaves of cv. Maris Piper (susceptible) was 10–30 times greater than that in cv. Pentland Crown or G 7445(1), a breeder's line (both resistant). Differences between genotypes in antigen concentration were smaller in petioles and tubers (5–10-fold) and in above-ground stems (about 4-fold), and were least in below-ground stems, stolons and roots (about 2-fold). PLRV antigen, detected by fluorescent antibody staining of tissue sections, was confined to phloem companion cells. In Pentland Crown, the decrease in PLRV antigen concentration in leaf mid-veins and petioles, relative to that in Maris Piper, was proportional to the decrease in number of PLRV-containing companion cells; this decrease was greater in the external phloem than in the internal phloem. The spread of PLRV infection within the phloem system seems to be impaired in the resistant genotypes. Green peach aphids (Myzuspersicae) acquired < 2800 pg PLRV/aphid when fed for 4 days on infected field-grown Maris Piper plants and < 58% of such aphids transmitted the virus to Physalis floridana test plants. In contrast, aphids fed on infected Pentland Crown plants acquired <120 pg PLRV/aphid and <3% transmitted the virus to P. floridana. The ease with which M. persicae acquired and transmitted PLRV from field-grown Maris Piper plants decreased greatly after the end of June without a proportionate drop in PLRV concentration. Spread of PLRV in potato crops should be substantially decreased by growing cultivars in which the virus multiplies to only a limited extent.     

Rates of growth and increase ofMyzus persicae on virus-infected potatoes according to type of virus-vector relationship

Growth, reproduction and survival (=performance) of the aphidMyzus persicae Sulzer was measured on virus-free and virus-infected potato plants The principle objective was to evaluate if various viral infections affected aphid performance differently, and if so, whether any order in the performance response of the aphid was discernible according to the type of virus-vector relationship. Three viruses varying in their dependency onM. persicae as a vector were used. Plants infected with potato leafroll virus (PLRV), a circulative virus highly dependent uponM. persicae for dispersal and transmission, were superior hosts as determined by the significantly greater mean relative growth rate (MRGR) and intrinsic rate of increase (r_m) ofM. persicae compared with those of aphids reared on other plants. Plants infected with potato virus Y, a noncirculative virus less dependent uponM. persicae for dispersal than PLRV, were intermediate in their quality based upon intermediate MRGR and r_m values. Plants infected with potato virus X, a nonvectored virus independent ofM. persicae, were least suitable hosts along with the group of virus-free plants according to the lower MRGR and r_m values.     

Microbial populations,fungal species diversity and plant pathogen levels in field plots of potato plants expressing theBacillus thuringiensis var.tenebrionis endotoxin

The environmental release of genetically engineered (transgenic) plants may be accompanied by ecological effects including changes in the plant-associated microflora. A field release of transgnic potato plants that produce the insecticidal endotoxin ofBacillus thuringiensis var.tenebrionis (Btt) was monitored for changes in total bacterial and fungal populations, fungal species diversity and abundance, and plant pathogen levels. The microflora on three phenological stages of leaves (green, yellow and brown) were compared over the growing season (sample days 0, 21, 42, 63 and 98) for transgenic potato plants, commercial Russet Burbank potato plants treated with systemic insecticide (Di-Syston) and commercial Russet Burbank potato plants treated with microbialBtt (M-Trak). In addition, plant and soil assays were performed to assess disease incidence ofFusarium spp.,Pythium spp.,Verticillium dahliae, potato leaf roll virus (PLRV) and potato virus Y (PVY). Few significant differences in phylloplane microflora among the plant types were observed and none of the differences were persisent. Total bacterial populations on brown leaves on sample day 21 and on green leaves on sample day 42 were significantly higher on the transgenic potato plants. Total fungal populations on gree leaves on sample day 63 were significantly different among the three plant types; lowest levels were on the commerical potato plants treated with systemic insecticide and highest levels were on the commercial potato plants treated with microbialBtt. Differences in fungal species assemblages and diversity were correlated with sampling dates, but relatively consistent among treatments.Alternaria alternata, a common saprophyte on leaves and in soil and leaf litter, was the most commonly isolated fungus species for all the plant treatments. Rhizosphere populations of the soilborne pathogensPythium spp.,Fusarium spp. andV. dahliae did not differ between the transgenic potato plants and the commercial potato plants treated with systemic insecticide. The incidence of tuber infection at the end of the growing season by the plant pathogenV. dahliae was highest for the transgenic potato plants but this difference was related to longer viability of the transgenic potato plants. This difference in longevity between the transgenic potato plants and the commercial + systemic insecticide potato plants also made comparison of the incidence of PVY and PLRV problematic. Our results indicate that under field conditions the microflora of transgenicBtt-producing potato plants differed minimally from that of chemically and microbially treated commerical potato plants.