Artichoke Italian latent virus causes artichoke patchy chlorotic stunting disease

Artichoke patchy chlorotic stunting (APCS) is the most serious disease affecting artichoke in Greece. It is widespread in Argolis, the main artichoke centre in Greece, where the local thornless artichoke, cv. “Argos”, is cultivated. The disease was first noticed in 1982, but to growers it was known since earlier times. Data collected during 1980–1990 revealed its wide distribution in the area, its patchy dispersal in fields, and its annual radial increase of the patches during the years, as well as a centripetal symptomatological severity gradient within each patch. These observations indicated a soil-borne nature for the disease. Field surveys for several viruses indicated a correlation of APCS with artichoke Italian latent nepovirus (AILV), a soil-borne virus known to occur in such patches and transmitted by the vector nematode Longidorus fasciatus Roca et Lamberti. In experiments fulfilling Koch's postulates, it was possible to reproduce the symptoms of APCS, demonstrating that AILV is a cause of the disease, if not the only one. Field surveys also revealed the ubiquitous occurrence of artichoke mottled crinkle virus (AMCV), independently of any particular symptoms. In the same survey, broad bean wilt virus (BBWV), also, was recovered from artichoke in Greece for the first time.     

Artichoke latent virus: characterisation, ultrastructure and geographical distribution

An isolate of artichoke latent virus (ALV-I) obtained from a symptomless artichoke plant in Southern Italy was characterised and compared with ALV isolates from other countries. ALV occurs in California and throughout the western part of the Mediterranean basin but of Mediterranean countries east of Italy, it was found only in Israel and Turkey. ALV-I was readily transmissible by inoculation of sap to a moderate range of hosts, was transmitted in a non-persistent manner by Aphis fabae, Brachicaudus cardui and Myzus persicae, but was not seed transmitted. The virus has flexuous rod-shaped particles measuring c. 12 nm × 746 nm with a sedimentation coefficient of 145 S and a buoyant density of 1·31 g/cm³. The particles contain single stranded RNA with a mol. wt of 3 × 10⁶ and protein composed of a single polypeptide species with a mol. wt of 33 000. Cylindrical cytoplasmic inclusions consisting of pinwheels and laminated aggregates were present in cells of naturally and artificially infected plants. ALV isolates from different geographical origin were indistinguishable from ALV-I biologically, morphologically, serologically and ultrastructurally. These properties place ALV in the Potyvirus group, but it was serologically unrelated to 12 other potyviruses 10 of which occur commonly in Italy.     

Effects of Artichoke Latent Virus Infection on the Production of Artichoke Heads

Virus-free and artichoke latent virus (ALV) infected plants of ‘Brindisino’ artichoke obtained by in vitro propagation, were studied over a period of three years to evaluate the effect of ALV infection on artichoke field performance and to determine the infection rate of healthy plants. ALV infection caused qualitative and quantitative changes ‘Brindisino’ artichoke such as leaf and bract discoloration, opening of head apex, delay of first harvest, shortening of head stalk, reduction of head width and a dramatic decrease of yield. Due to the high infection rate of healthy plants during the trial period, the differences in the production of heads between the two groups of plants decreased in the last year of experiment.     

Detection of Artichoke Latent Virus by an Indirect Dot-ELISA

An indirect Dot-ELISA was compared with DAS-ELISA for detecting Artichoke Latent Virus (ALV) both in purified preparations and in crude sap from “Spinoso sardo” artichoke leaves. Antigen diluted samples (1 μl) were first spotted on nitrocellulose (NC) and polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated in rabbit anti-ALV IgG, then in goat anti-rabbit IgG—alkaline phosphatase conjugate, and finally exposed to substrate and examined for a coloured precipitate. The minimum detection levels for ALV by Dot-ELISA were 125 pg of purified virus and 1/2,000 dilution of virus-infected sap on NC, and 83.3 pg of purified virus and 1/4,000 dilution of virus-infected sap on PVDF, as compared with 50 ng of purified virus and 1/1,000 dilution of virus-infected sap detectable by DAS-ELISA. This indirect Dot-ELISA proved to be more sensitive and more economical than DAS-ELISA, and can be completed in as little as 5—6 hours.     

Partial Characterization of Artichoke Virus M

A virus with filamentous particles 697 nm in length was isolated from artichoke plants in Southern Italy and identified as a new possible member of Carlavirus group, for which the name artichoke virus M (AVM) is suggested. AVM could not be transmitted by sap inoculation to herbaceous hosts and was always present in artichoke in mixed infections with other viruses. Virus particles had a buoyant density in CsCl of 1.31 g × cm^?3 and contained a single species of nucleic acid with an apparent size of 7.5 Kb and a single coat protein species with a mol. wt of 31,000. The virus was distantly related serologically to carnation latent and poplar mosaic carlaviruses but not to other members of the group including the recently described artichoke latent S carlavirus. Cytological alterations consisted of complex cytoplasmic inclusions composed of deranged organelles, lipid droplets and accumulations of membranes.     

Production and fingerprinting of virus-free clones in a reflowering globe artichoke

Most of the 24 viruses which infect globe artichoke are detrimental to the crop’s performance and hamper the development of a nursery activity in the respect of current EU legislation. We describe a procedure to sanitize globe artichoke “Brindisino” from Artichoke Italian latent virus (AILV) and Artichoke latent virus (ArLV), while preserving its valuable early flowering trait. ArLV was successfully eliminated by meristem-tip culture, while AILV was removed when two rounds of meristem-tip culture were spaced out with in vitro thermotherapy. In vivo thermotherapy, followed by meristem-tip culture, was also successful in producing virus-free material but was less efficient in terms of the number of plants recovered post treatment. Due to the multi-clonal composition of the populations at present in cultivation, the selected and sanitised clones were fingerprinted by applying microsatellite and AFLP (amplified fragment length polymorphism) markers. One AFLP primer combination produced 28 informative fragments used to evaluate genetic relatedness among the clones in study. Our results demonstrates that AFLP-based molecular fingerprinting enables to verify the true to clone correspondence in nurseries, ensure the effective correspondence between the real and the declared identity of a clone, so that to avoid commercial frauds, and might represents a valuable tool for assessing somaclonal variation occuring during ‘in vitro’ propagation.     

Purification and properties of the arginase from Jerusalem artichoke tubers

Arginase [l-arginine amidinohydrolase] in Jerusalem artichoke tubers occurs in a particulate fraction from which it was released in active form by detergent treatment. The particulate enzyme was purified 450-fold with ca 3% yield. The enzyme has a MW of ca 140 000 and pI of 5.3. The enzyme required Mn²⁺ for activity and was unstable when Mn²⁺ was removed. In tissue extracts the K_m for arginine was ca 1OmM, but when purified the K_m (arginine) was 145 mM. The artichoke arginase was shown to be more substrate specific than other plant and animal arginases which have been described, and to be very sensitive to competitive inhibition by indospicine, ornithine and citrulline.     

Preliminary Studies on Black Spot of Globe Artichoke

Studies were conducted to determine the cause of an apparently new disorder of globe artichoke ( Cynara scotymus L.) characterised by a localised black necrosis in the receptacle of the flower buds. The effects of single and mixed infections by artichoke latent potyvirus (ALV) and broad bean wilt virus – French artichoke (BBWV-FA) in buds with necrosis of three globe artichoke cultivars were studied. Virus-free plants developed diseased buds irrespective of whether the plants were 1 or 2 years old, but the presence of ALV and/or BBWV-FA significantly increased black spot occurrence on 2-year-old plants. Virus effects were dependent on cultivar, cv. Capitan being less susceptible to virus infection than cvs Camus de Bretagne and Castel. The head size of the globe artichoke was found to be related to the rate of necrosis, with a higher incidence for terminal buds.
ALV and BBWV-FA did not cause the disorder but favour its development probably by weakening plants through increased leaf transpiration. The results are discussed in relation to a possible physiological disorder exacerbated by the presence of ALV and/or BBWV-FA.     

Structure-based design and experimental engineering of a plant virus nanoparticle for the presentation of immunogenic epitopes and as a drug carrier

Biomaterials research for the discovery of new generation nanoparticles is one of the most active areas of nanotechnoloy. In the search of nature-made nanometer-sized objects, plant virus particles appear as symmetrically defined entities that can be formed by protein self-assembly. In particular, in the field of plant virology, there is plenty of literature available describing the exploitation of plant viral cages to produce safe vaccine vehicles and nanoparticles for drug delivery. In this context, we have investigated on the use of the artichoke mottled crinkle virus (AMCV) capsid both as a carrier of immunogenic epitopes and for the delivery of anticancer molecules. A dual approach that combines both in silico tools and experimental virology was applied for the rational design of immunologically active chimeric virus-like particles (VLPs) carrying immunogenic peptides. The atomic structures of wild type (wt) and chimeric VLPs were obtained by homology modeling. The effects of insertion of the HIV-1 2F5 neutralizing epitope on the structural stability of chimeric VLPs were predicted and assessed by detailed inspection of the nanoparticle intersubunit interactions at atomic level. Wt and chimeric VLPs, exposing on their surface the 2F5 epitope, were successfully produced in plants. In addition, we demonstrated that AMCV capsids could also function as drug delivery vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first systematic predictive and empirical research addressing the question of how this icosahedral virus can be used for the production of both VLPs and viral nanoparticles for biomedical applications.     

Cytoplasmic streaming and ionic regulation inNitella flexilis having artificial cell saps of low ionic strength

The cell sap of the internode ofNitella flexilis was replaced with the isotonic artificial pond water of high Ca²⁺-concentration (0.1 mM KCl, 0.1 mM NaCl, 10 mM CaCl₂ and 275 mM mannitol) and changes in osmotic value and concentrations of K⁺, Na⁺ and Cl⁻ of the cells were followed. When the operated cells were incubated in the artificial pond water containing 0.1 mM each of KCl, NaCl, CaCl₂, they survived for only a short period of time (<10 hr). The cells did not absorb ions from the artificial pond water and showed a conspicuous decrease in the rate of cytoplasmic streaming. In such cell the concentration of K⁺ in the protoplasm decreased significantly. In order to reverse normal concentration gradients of K⁺ and Na⁺ across the protoplasmic layer, the cells of low vacuolar ionic concentrations were incubated in the artificial cell sap (90 mM KCl, 40 mM NaCl, 15 mM CaCl₂, 10 mM MgCl₂). It was found that the cells rapidly absorbed much K⁺, Na⁺ and Cl⁻ and survived for a longer period (1–2 days). During this period the rate of cytoplasmic streaming was nearly normal. Furthermore, the cell lost much mannitol, indicating an enormous increase in permeability to it. Since both absorption of ions and leakage of mannitol at 1 C occurred at nearly the same rates as at 22 C, the processes are assumed to be passive.     

cDNA cloning of artichoke mottled crinkle virus RNA and localization and sequencing of the coat protein gene

We report the cDNA cloning of the genomic RNA of artichoke mottled crinkle virus (AMCV), which is a member of Tombusvirus group. AMCV has a monopartite positive sense RNA genome, which is not polyadenylated at the 3 end. The genome size is 4.8 kb.We have localized and sequenced the open reading frame (ORF) encoding the coat protein. Unlike most monopartite positive-strand RNA plant viruses, the ORF is not located near the 3 end, but like other members of the Tombusvirus group, CyRSV (cymbidium ringspot virus), TBSV-cherry (tomato bushy stunt virus cherry strain) and CNV (cucumber necrosis virus) it starts ca. 2.7 kb downstream of the 5 end and stops ca. 1 kb upstream of the 3 end. This ORF predicts a polypeptide chain of 387 amino acids.Comparison of the coat proteins of AMCV, TBSV-BS3, TBSV-cherry and CNV confirms that, within the Tombusvirus group, there exists a high degree of similarity among coat proteins but that this similarity is not uniformly distributed among domains. In particular, the N-terminal region, thought to make contact with the phosphate groups of the viral RNA, and the C-terminal region, considered the most immunogenic portion of the capsid, are found to be the least homologous.     

Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

Properties of a carlavirus causing a latent infection of pepino (Solanum muricatum)

Pepino (Solanum muricatum) cuttings imported from Chile contained a latent virus which was transmitted by inoculation of sap to Chenopodium quinoa but not to 21 other species. The virus was transmitted by the aphid, Myzus persicae. In C. quinoa sap, the virus lost infectivity when diluted between 10^-3 and 10^-4, heated for 10 min between 65 and 70 °C, or stored at room temperature for 4 to 6 days. The virus particles were straight or slightly flexuous filaments 660 to 680 nm long. Up to 15 mg virus per 100 g C. quinoa leaves was obtained by clarification with a mixture of chloroform and carbon tetrachloride. Purified preparations had A_max/A_min= 1.11, A₂₆₀/A₂₈₀= 1–30, A^0.₂₆₀^1%= 2.8, and contained a single sedimenting component with a sedimentation coeficient of 149s and a buoyant density in CsCl of 1–318. The virus particles contained 5.5% of single-stranded RNA of mol. wt 2.4×10⁶ (estimated by gel electrophoresis of undenatured RNA) and sedimentation coefficient 38.5S, and a single polypeptide of mol. wt 33 000. The virus is distantly serologically related to potato S and carnation latent viruses and is considered a new member of the carlavirus group. The name pepino latent virus is proposed. The cryptogram for this virus is R/1: 2.4/5–5: E/E: S/Ve/Ap.     

Impact of seaweed sap foliar application on growth,yield, and tuber quality of potato (Solanum tuberosum L.)

Field experiments were carried out during winter 2015–2016 and 2016–2017 at research farm of Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India to study the growth, productivity, quality, and profitability of potato (cv. Kufri Jyoti) cultivation as influenced by foliar application of different seaweed extracts, i.e., Kappaphycus alvarezii sap (K sap) and Gracilaria edulis sap (G sap). The experiment was laid out in a randomized complete block design comprised of two different concentrations (5% and 10%) of each K sap, G sap, and GA₃-free K sap, and a control plot (water spray), with three replications. Foliar feeding with 10% K sap along with recommended dose of fertilizer (200 kg N, 150 kg P, and 150 kg K ha^?1) brought about significant enhancement in plant height, being statistically similar with 10% G sap. Similar treatment resulted in maximum tuber bulking rate (17.62 and 18.93 g m^?2 day^?1 at 50–70 DAP in year 1 and year 2, respectively) and tuber yield (26.37 and 29.77 t ha^?1 in year 1 and year 2, respectively), accounting 32.11% and 24.87% yield enhancement over control. Maximum nutrient (N, P, and K) uptake as well as best values of quality traits in terms of ascorbic acid, reducing sugar content, and specific weight of potato tuber was recorded with economically viable treatment having 10% K sap spray. Results suggested that the application of seaweed sap at higher concentration along with the recommended dose of fertilizer was the potential alternative way for better potato production without harming the ecological sustainability.

     

A critical assessment of the value of petiole sap analysis in optimizing the nitrogen nutrition of the potato crop

The importance of standardizing on sampling procedures was shown by examining diurnal variation of petiole sap nitrate-N concentration, [NO₃ ^--N], and its variation within the plant. There was important variability in petiole sap [NO₃ ^--N] about the general decline in values through the season, in time, between cultivars, between nitrogen fertilizer treatments, and between replicates. We have shown the lack of a consistent relation between petiole sap [NO₃ ^--N] and rates of uptake of nitrogen either in the period around the time of sampling, or integrated over the whole growing season. Finally, we have examined the literature and have been unable to find reports of critical, independent tests of the use of petiole sap [NO₃ ^-] to guide the application of fertilizer supplements. We conclude that a new, critical attitude should be taken to testing petiole sap [NO₃ ^-] if the technique is to be used properly and to its seeming potential. Comparisons were made between measurements using a hand-held system and an auto-analyser.     

Isotopic composition and concentration of total nitrogen and nitrate in xylem sap under near steady-state hydroponics

After root uptake, nitrate is effluxed back to the medium, assimilated locally, or translocated to shoots. Rooted black cottonwood (Populus trichocarpa) scions were supplied with a NO₃⁻-based (0.5 mM) nutrient medium of known isotopic composition (δ¹⁵N), and xylem sap was collected by pressure bombing. To establish a sampling protocol, sap was collected from lower and upper stem sections at 0.1–0.2 MPa above the balancing pressure, and after increasing the pressure by a further 0.5 MPa. Xylem sap from upper stem sections was partially diluted at higher pressure. Further analysis was restricted to sap obtained from intact shoots at low pressure. Total-, NO₃⁻-N and, by difference, organic-N concentrations ranged from 6.1–11.0, 1.2–2.4, and 4.6–9.4 mM, while discrimination relative to the nutrient medium was −6.3 to 0.5‰, −23.3 to −11.5‰ and − 1.3 to 4.9‰, respectively. There was diurnal variation in δ¹⁵N of total- and organic-N, but not NO₃⁻. The difference in δ¹⁵N between xylem NO₃⁻ and organic-N suggests that discrimination by nitrate reductase is near 25.1 ± 1.6‰. When this value was used in an isotope mass balance model, the predicted xylem sap NO₃⁻-N to total-N ratio closely matched direct measurement.     

Transport of magnesium ions in the phloem of Ricinus communis L. seedlings

During growth of Ricinus communis seedlings, magnesium ions are mobilized in the endosperm, taken up by and accumulated to very high levels (150 μmol·g FW^?1) in the cotyledons, and translocated to hypocotyl and roots. The magnesium gain from days 6 to 7 in the cotyledons and the seedling axis necessitates a total up-take rate of 600 nmol·h^?1-seedling^?1 and the phloem translocation rate must amount to 200 nmol·h^?1. seedling^?1. The phloem loading of magnesium and the regulatory properties of this process were investigated, making specific use of the ability to collect pure phloem sap from the cut hypocotyl of 6-d-old Ricinus seedlings. The concentration of magnesium in sieve-tube sap (5 mM) was fairly constant under many incubation conditions, e.g. incubation in magnesium-free buffer, incubation with different cations (K⁺, Na⁺, NH ₄ ⁺ ) or anions (Cl^?, NO ₄ ^- , SO ₄ ^2- ), or incubation with sucrose and amino acids. Even addition of magnesium chloride to the cotyledons did not enhance phloem loading of magnesium ions. Therefore the high magnesium content of the cotyledons was sufficient for continuous phloem loading of magnesium, irrespective of external ionic conditions. Also, the flow rate of sieve-tube sap did not influence the magnesium concentration in the sap. Only the incubation with sulfate and phosphate ions increased the magnesium-ion concentration in the phloem. Magnesium sulfate offered to the cotyledons caused a threefold increase of magnesium ions in the sieve-tube sap, which was inhibited by Na⁺, NH ₄ ⁺ and Ca²⁺ in rising order, but not by K⁺. Incubation with phosphate for a prolonged period (8 h) led to an increased mobilization of intra-cotyle-donary magnesium and an enhanced phloem loading of mobilized magnesium. It is concluded that phosphate availability is a decisive factor for mobilization and translocation of magnesium ions within the plant.     

How aphids decide what is good for them: experiments to test aphid feeding behaviour on Tanacetum vulgare (L.) using different nitrogen regimes

Leaf-chewing herbivores select food with a protein/carbohydrate ratio of 0.8–1.5, whereas phloem sap, which aphids feed on, has a ratio of ~0.1. Enhanced N fertilization increases the amino acid concentration in phloem sap and elevates the N/C ratio. The study examines: (1) whether aphids select between plants of different N nutrition, (2) whether feeding time correlates with the amino acid composition of phloem sap, and (3) at which stage of probing aphids identify the quality of the plant. Uroleucon tanaceti (Mordvilko) and Macrosiphoniella tanacetaria (Kaltenbach), specialist aphids feeding on tansy (Tanacetum vulgare L.), were reared on this host plant grown essentially hydroponically (in Vermiculite) in the greenhouse on 1, 3, 6, or 12 mM NH₄NO₃. One and 3 mM NH₄NO₃ corresponds to the situation found in natural tansy stands. Aphid stylet penetration was monitored by electrical penetration graphs whilst phloem sap was sampled by stylectomy. Both aphid species settled 2–3 times more frequently on plants fertilized with 6 or 12 mM NH₄NO₃. The phloem sap of these plants contained up to threefold higher amino acid concentrations, without a change in the proportion of essential amino acids. No time differences were observed before stylet penetration of plant tissue. After the first symplast contact, most aphids penetrated further, except M. tanacetaria on low-N plants, where 50% withdrew the stylet after the first probing. The duration of phloem feeding was 2–3 times longer in N-rich plants and the time spent in individual sieve tubes was up to tenfold longer. Aphids identified the nutritional quality of the host plant mainly by the amino acid concentration of phloem sap, not by leaf surface cues nor the proportion of essential amino acids. However, U. tanaceti infestation increased the percentage of methionine plus tryptophan in phloem tenfold, thus manipulating the plants nutritional quality, and causing premature leaf senescence.