Particle properties of parsnip yellow fleck virus

Particle preparations of parsnip yellow fleck virus (PYFV) isolates A-421 and P-121, representing the two major serotypes, were made by clarifying leal extracts with ether or butan-1-ol and concentrating the virus particles by precipitation with polyethylene glycol and differential centrifugation. The preparations contained c. 31 nm-diameter particles comprising two sedimenting components. Top component (T) consisted of stain-penetrable protein shells with A₂₆₀/A₂₈₀= 0.8–0.9, sedimentation coefficient (S₂₀) = 56 S (A-421) or 60 S (P-121), and buoyant density = 1.297 g/cm³. Bottom component (B) consisted of nucleoprotein particles, not penetrable by negative stain, with A₂₆₀/A₂₈₀= 1.9, sedimentation coefficient (S⁰_20.w) = 148 S (A-421) or 153 S (P-121), and buoyant density = 1.520 g/cm³ (A-421) or 1.490 g/cm³ (P-121). Yields of B component particles were up to c. 1 mg/100 g leaf tissue (both isolates); yields of T component particles were up to c. 0.6 mg (A-421) or 5.5 mg (P-121) per 100 g leaf tissue. PYFV particles were found to contain a single RNA species (mol. wt c. 3.4 × 10⁶, c. 9800 nucleotides), constituting 40% of the particle weight, and three polypeptide species, of mol. wt (× 10 ³) 30 , 26 and 24 (A-421) or 31 , 26 and 23 (P-121).     

Purification, properties and transmission of parsnip yellow fleck, a semi-persistent, aphid-borne virus

Parsnip yellow fleck virus (PYFV) is the commonest cause of virus-like symptoms in parsnip plants in Britain: it is sap-transmissible but systemically infects few species outside the Umbelliferae. It has isometric particles 29–31 mμ in diameter, a sedimentation coefficient of 167s, and loses infectivity in sap after dilution to 10^-3-10^-4, heating for 10 min at 57·5–65°C, or storage at room temperature for 4–7 days. Two isolates, from parsnip and Anthriscus sylvestris respectively, are only distantly serologically related. The aphid Cavariella aegopodii transmits PYFV in a semi-persistent manner from A. sylvestris but not from parsnip. Transmission by aphids apparently depends on the presence in A. sylvestris or other source plants of a second virus, anthriscus yellows (AYV), which is persistent in the vector and not manually transmissible. PYFV was therefore not transmitted by aphids from manually inoculated plants or from parsnip or other plants immune to AYV. In controlled experiments, C. aegopodii transmitted PYFV (both A. sylvestris and parsnip isolates) from chervil plants inoculated separately with PYFV and AYV, but not from plants inoculated only with PYFV.     

Transmission and differentiation of six viruses infecting hogweed (Heracleum sphondylium) in Scotland

Six viruses, code-named HV1-HV6, were transmitted manually and/or by aphids (Cavariella spp. from symptomless wild plants of hogweed (Heracleum sphondylium) in Scotland. HV1 was identified as parsnip yellow fleck virus (PYFV); anthriscus yellows virus, on which it depends for transmission by aphids, was presumably also present in the hogweed plants. HV2 was transmitted manually and by aphids and had very flexuous filamentous particles c. 700–750 nm long; it has affinities with the closteroviruses, and the name heracleum latent virus is proposed. HV3, HV4 and HV5 were transmitted manually, HV3 and HV5 also by aphids, but their particle morphology is unknown. HV6 was transmitted only by aphids and has very flexuous particles up to 1400 nm long; it is presumably a closterovirus distinct from HV2. All the viruses infected cultivated umbelliferous species experimentally but only PYFV is known to infect umbelliferous crops.     

Relations of the semi-persistent viruses, parsnip yellow fleck and anthriscus yellows, with their vector, Cav arietta aegopodii

Studies were made of the relations of parsnip yellow fleck virus (PYFV) and its helper virus, anthriscus yellows (AYV), with their aphid vector, Cavariella aegopodii. Apterous insects were more efficient vectors than alates; apterous nymphs were as efficient as apterous adults. C. aegopodii never transmitted PYFV in the absence of AYV, but aphids carrying both viruses infected some test plants with one or other virus alone. C. aegopodii that fed first on a source of AYV and then on a source of PYFV transmitted both viruses to test plants, but aphids that fed on the sources in the reverse order transmitted only AYV. Test plants receiving some aphids from a source of AYV, and others from a source of PYFV, became infected only with AYV. C. aegopodii acquired AYV or the AYV/PYFV complex from plants in a minimum acquisition access time (AAT) of 10–15 mm and inoculated the viruses to test plants in a minimum inoculation access time (IAT) of 2 min. Increasing either AAT or IAT, or both, to 1 h or longer increased the frequency of transmission of each virus. Starving the insects before the acquisition feed on AYV or AYV/PFYV sources did not affect transmission. Aphids already carrying AYV acquired PYFV from plants in a minimum AAT of only 2 min; they acquired and inoculated PYFV in a minimum total time of 12 min. The data suggest that AYV is confined to deeply lying tissues whereas PYFV is distributed throughout the leaf. C. aegopodii transmitted both PYFV and AYV in a semi-persistent manner: the aphids retained both viruses for up to 4 days but lost them on moulting. Neither virus was passed to progeny of viruliferous adults. Earlier results suggesting that AYV is a persistent virus may have been caused by contamination of the AYV culture with carrot red leaf virus.     

The role of the helper virus, anthriscus yellows, in the transmission of parsnip yellow fleck virus by the aphid Cavariella aegopodii

Transmission of parsnip yellow fleck virus (PYFV) by the aphid Cavariella aegopodii occurs only when the aphids are also carrying the helper virus, anthriscus yellows (AYV). None of five other viruses tested was able to act as helper. In experiments in which aphids were allowed to feed through membranes on crude or treated extracts from infected plants, aphids already carrying AYV acquired PYFV, but virus-free aphids failed to acquire either AYV or PYFV. PYFV was not transmitted by insects injected with haemolymph from aphids carrying both viruses, or with purified preparations of PYFV. PYFV was transmitted when AYV-carrying aphids, except those whose stylets had been removed, were contaminated externally with PYFV preparations. Ultraviolet irradiation of infected leaves did not prevent aphids from acquiring AYV, presumably because it is confined to deeply-lying tissues. AYV-carrying aphids could acquire PYFV from u.v.-irradiated leaves after acquisition access times of 2 h but not after feeds of only 2 or 15 min (which are adequate on unirradiated leaves), suggesting that PYFV is present in all parts of the leaf. No ‘helper agent’ distinct from AYV itself was detected in these experiments or in experiments on minimum acquisition feeding time or maximum period of persistence in the aphid. U.v.-inactivated PYFV competed with infective PYFV for retention sites in AYV-carrying aphids, whereas AYV apparently did not. It is suggested that there is no helper agent for PYFV, other than AYV particles. The possibility that there is one for AYV is not excluded.     

The Mechanical transmission and some properties of a virus disease of cow-parsnip,Heracleum sphondylium L

The described virus of cow-parsnip,Heracleum sphondylium L., was found in three ruderal localities of Greater Praha. The symptoms are manifested by decolorations which consist of bright yellow areas spreading from the centre of the leaf blade along the main veins. These symptoms appear severely in May. Under higher temperatures and in a chronic stage of infection the symptoms are more or less masked. The disease is mechanically transmissible to parsley, coriander, parsnip, dill, sowbane,Chenopodium quinoa and C.giganteum. The author failed to transmit the disease to celery, carrot, caraway and to 27 species of differential host plants, he failed in the transmission of the virus by the dodder,Cuscuta campestris YUNCK., too. Thermal inactivation point of the virus lies between 51° and 55° C. Infectivity of extracted sap was lost after 2 days at room temperature.     

Specificity of the effect of sap-transmissible viruses in increasing the accumulation of luteoviruses in co-infected plants

The concentration of potato leafroll luteovirus (PLRV) (c. 1300 ng/g leaf) in singly infected Nicotiana clevelandii plants was increased up to 10-fold in plants co-infected with each of several potyviruses, or with narcissus mosaic potexvirus, carrot mottle virus or each of three tobravirus isolates. With the tobraviruses, PLRV concentration was increased equally by co-infection with either NM-type isolates (coat protein-free cultures containing RNA-1) or M-type isolates (particle-producing cultures containing RNA-1 and RNA-2). In contrast, the accumulation of PLRV was not substantially affected by co-infection with either of two nepoviruses, cucumber mosaic cucumovirus, broad bean mottle bromovirus, alfalfa mosaic virus, pea enation mosaic virus or parsnip yellow fleck virus. The specificity of these interactions between PLRV and sap-transmissible viruses was retained in tests made in Nicotiana benthamiana and when beet western yellows luteovirus was used instead of PLRV.     

Host plant, host plant chemistry and the polyembryonic parasitoid Copidosoma sosares: indirect effects in a tritrophic interaction

Host plant identity and host plant chemistry have often been shown to influence host finding and acceptance by natural enemies but comparatively less attention has been paid to the tritrophic effects of host plant and host plant chemistry on other natural enemy fitness correlates, such as survivorship, clutch size, body size, and sex ratio. Such studies are central to understanding both the selective impact of plants on natural enemies as well as the potential for reciprocal selective impact of natural enemies on plant traits. We examined the effects of host plant and host plant chemistry in a tritrophic system consisting of three apiaceous plants (Pastinaca sativa, Heracleum sphondylium and H. mantegazzianum), the parsnip webworm (Depressaria pastinacella) and the polyembryonic parasitic wasp Copidosoma sosares. All of these plants produce furanocoumarins, known resistance factors for parsnip webworms. Furanocoumarin concentrations were correlated neither with the presence nor the number of webworms on a given plant. Concentrations of two furanocoumarins were negatively associated with C. sosares fitness correlates: isopimpinellin with the likelihood that a given webworm would be parasitized and xanthotoxin with both within‐brood survivorship (of all‐male and mixed‐sex broods) and clutch size. Brood sex ratio and body sizes of individual wasps were not correlated with furanocoumarin chemistry. Because additive genetic variation exists in P. sativa for furanocoumarin chemical traits, these are subject to selection by webworms through herbivory. Third trophic level selective impacts on furanocoumarin traits may include selection for reduced production of those chemicals that affect parasitoid survivorship yet do not influence host plant choice by the herbivore. That such might be the case is suggested by patterns of furanocoumarin production in populations of P. sativa with different histories of infestation; in the Netherlands, where parasitism rates of webworms by C. sosares are high, plants produce lower levels of all linear furanocoumarins and proportionately less isopimpinellin than do midwestern U.S. populations of P. sativa, where natural enemies of the webworm are effectively absent.     

Multiple copies of virG enhance the transient transformation of celery,carrot and rice tissues by Agrobacterium tumefaciens

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical -glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.Current address: Department of Agronomy, Purdue University     

Application of Beneficial Microorganisms to Seeds during Drum Priming

Five microbial plant growth promoters or biocontrol agents (Pseudomonas fluorescens CHA0, Pseudomonas sp. AB842, Bacillus subtilis MBI600, Trichoderma harzianum T22 and T. virens G20) were assessed for ability to proliferate on seeds of carrot, parsnip and leek. In small-scale priming systems, both pseudomonads and MBI600 (when applied as cells) at levels between 10⁵ and 10⁶ cfu g^?1 seed were able to colonise all seeds at the end of priming (240 h) despite initial poor recovery after addition of the cells in some cases. Pf CHA0 was a particularly aggressive seed coloniser often comprising the total pseudomonad population at the end of priming. Drying the seed after priming resulted in <1 log₁₀ cfu g^?1 seed loss for the pseudomonads but greater losses for MBI600 on carrot and leek seed. Application of spores of MBI600 resulted in little loss in cfu g^?1 seed following addition of the cells and these levels were maintained throughout the priming period and after drying back. Both T22 and G20 were recovered from carrot and parsnip at the end of priming and in general reflected survival of the inoculum rather than proliferation. T22 and G20 could not be recovered from leek seed following priming. Comparisons were made between proliferation and survival in large-scale drum priming with post-priming application of microorganisms. Pf CHA0 proliferated on carrot and parsnip seed during drum priming and survived the drying back process whereas there was no recovery when applied as a post-priming treatment. In contrast, MBI600 could not be recovered when applied during priming as cells, whereas recovery was good when applied post-priming as spores. T22 spores could be applied in either manner. Post-priming application of metalaxyl and thiabendazole had variable effects on microorganism recovery. The significance of the application of microorganisms to seeds during priming is discussed.     

Cryptococcus neoformans and Cryptococcus gattii Isolated from the Excreta of Psittaciformes in a Southern Brazilian Zoological Garden

Cryptococcus neoformans, a major pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from soils, avian excreta and plant material. To further study potential saprophytic sources of this yeast in the Southern Brazilian State Rio Grande do Sul, we analyzed fecal samples from 59 species of captive birds kept in cages at a local Zoological Garden, belonging to 12 different orders. Thirty-eight environmental isolates of C. neoformans were obtained only from Psittaciformes (Psittacidae, Cacatuidae and Psittacula). Their variety and serotype were determined, and the genetic structure of the isolates was analyzed by use of the simple repetitive microsatellite specific primer M13 and the minisatellite specific primer (GACA)₄ as single primers in the PCR. The varieties were confirmed by pulsed-field gel electrophoresis (PFGE). Thirty-three isolates (87%) were from the var. grubii, serotype A, molecular type VNI and five (13%) were Cryptococcus gattii, serotype B, molecular type VGI. All the isolates were mating type α. Isolates were screened for some potential virulence factors. Quantitative urease production by the environmental isolates belonging to the C. gattii was similar to the values usually obtained for clinical ones.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

Fatty acids of plant vacuolar membrane lipids

Fatty acid (FA) composition of vacuolar membrane lipids from storage tissues of umbelliferous plants, viz., parsnip (Pastinaca sativa L.), parsley (Petroselinium crispum L.), and carrot (Daucus carota L.) is studied by gas-liquid chromatography and the FA biosynthetic pathways are considered. Vacuolar membrane lipids are characterized by high (78% of the total FA pool) content of unsaturated FA among which linoleic acid is predominant. Its content in vacuolar lipids of parsnip, parsley and carrot is 53.5, 55.1, and 54.9%, respectively. Parsnip and parsley vacuolar lipids contain large amounts of hexadecadienoic C16:2ω6 acid (8.0 and 4.6%, respectively). The content of α-linolenic acid in vacuolar lipids of tested plants varies from 4.8 to 7.3%. Palmitic acid (18.0–20.7%) predominates among saturated FA. High content of linoleic and hexadecadienoic acid in parsnip and parsley vacuolar lipids is suggestive of a crucial role of the microsomal ω6 fatty-acid desaturase fad2 gene in resistance and acclimation of plants to low temperatures.     

Serological Examination of Some Strains That Are in the Mycobacterium avium-intracellulare-scrofulaceum Complex But Do Not Belong to Schaefer's Serotypes

One hundred strains belonging to the Mycobacterium avium-intracellulare-scrofulaceum (MAIS) complex but not agglutinating with antisera type-specific for Schaefer''s 23 MAIS serotypes were examined using antisera against seven other such strains. Four of the 100 strains were found to be of the same serotype as one of the 7 against which antisera were prepared; 4 other strains were of the same serotype as another of those against which antisera were prepared. Although the strains against which antisera were prepared were serologically distinct from each other, no strains serologically identical to 5 of them were found. This suggests that numerous serotypes might have to be defined if strains such as those examined are to be assigned to their respective serotypes.     

Effect of nitrogen level on acid phosphatase activity of eight isolates of ectomycorrhizal fungus Paxillus involutus cultured in vitro

The higher levels of nitrogen in ammonium form stimulated the growth of mycelia and increased the accessible as well as the total acid phosphatase activity of Paxillus involutus (Batsch) Fr. isolates grown in pure culture. Rates of mycelia growth and acid phosphatase activities varied widely from one isolate to another. Scots pine (Pinus sylvestris L.) seedlings were inoculated with different P. involutus isolates in axenic conditions. Shoots of pine seedlings with mycorrhizae contained more phosphorus than shoots of non-mycorrhizal seedlings. The relations between growth and phosphatase activity of P. involutus isolates and their efficiency in supplying the host plant with phosphate are discussed.     

Fatty acid composition of lipids from the vacuolar membranes of the roots of root vegetables

The fatty acid (FA) composition of vacuolar membrane lipids from the storage tissues of parsnip (Pastinaca sativa L.), parsley (Petroselinum crispum L.), and carrot (Daucus carota L.) was studied by gasliquid chromatography, and possible pathways of the biosynthesis of these acids are considered. A high level of unsaturated FAs (up to 78% of the total FA amount) was characteristic of these membrane lipids with the predominance of linoleic acid, which content in vacuolar lipids of parsnip, parsley, and carrot was 53.5, 55.1, and 54.4%, respectively. A high content of hexadienoic acid (C16:2ω6) was characteristic of the vacuolar lipids of parsnip and parsley (8.0 and 4.6%. respectively); the content of α-linolenic acid in the vacuolar lipids of these plants was 4.4–7.3%. Palmitic acid predominated among the saturated FAs (18.0–20.4%).     

Interactions between seed priming treatments and nine seed lots of carrot, celery and onion. II. Seedling emergence and plant growth

Samples of three seed lots of each of three cultivars of carrot, celery and onion were primed in polyethylene glycol solution for two weeks at 15 °C. Seedling emergence was recorded in the field for carrot and onion and in the glasshouse for celery. Compared to the untreated control, priming increased the percentage seedling emergence in certain poorly-emerging seed lots of carrot and celery, but had no effect on onion. Mean emergence times were reduced by priming in all seed lots, by 3–5, 5–8 and 3–9 days in carrot, celery and onion respectively. The largest effects occurred in the slowest-emerging seed lots. There were significant interactions between priming and seed lots within cultivars in carrot and celery and between priming and cultivars in celery and onion. Priming generally reduced the spread of emergence times, but the effects were not statistically significant in carrot. Drying back the primed seeds had no effect on percentage emergence in onion, but reduced it (compared to primed seed which had not been dried-back) in certain carrot and celery seed lots. Primed and dried-back seeds emerged later than primed seeds, by up to 1·5, 2·6 and 2·6 days in carrot, celery and onion respectively. The spread of emergence times was generally larger for primed and dried-back seeds than for primed seeds, but the differences were not always statistically significant. Plant fresh weights were recorded 9, 7 and 12 wk after sowing for carrot, celery and onion, respectively. In each species, mean plant weight was inversely related to seedling emergence time; thus plants grown from primed seed were always heavier than the controls, by up to 33%, 182% and 47% in carrot, celery and onion respectively.     

The isolation and identification of parsley viruses occurring in Britain*

Severe stunting of parsley plants, with leaf chlorosis and reddening was reported from four localities in Britain in 1968-70. Affected plants were collected from thirteen sites in Bedfordshire, Buckinghamshire, Cheshire and Bristol, and five viruses (designated PV1-PV5) were isolated from them. The viruses were distinguished by electron microscopy, host range and type of aphid transmission. From diagnostic reactions in a range of host species and its transmission by Cavariella aegopodii Scop., the most frequently isolated virus (PV4) and the principal cause of the parsley disease was identified as carrot mottle virus (CMotV). The other four viruses were infrequently isolated. PV1, PV2 and PV3 were transmitted in the non-persistent manner by Myzus persicae Sulz. Each was purified and identified serologically as western celery mosaic virus, cucumber mosaic virus and broad bean wilt respectively. PV5 was not fully identified, but was transmitted by C. aegopodii in the presence of CMotV and had particles ca. 500 nm in length. Each of these viruses was re-transmitted to parsley, but induced slight symptoms or none.     

Incidence of pea seedborne mosaic virus pathotypes in the US National Pisum germplasm collection

One hundred and eighty-nine isolates of pea seedborne mosaic virus (PSbMV) were obtained from seedlings of 435 pea (Pisum sativum) germplasm introductions originally acquired from India, Turkey, Latin America and Europe. Fifty-eight per cent of the isolates were identified as belonging to pathotype P-1, 22% to pathotype P-4, and 7% to the mild form of the lentil pathotype L-1. Some isolates could not be classified into any known pathotype and need further study. No isolate appeared to present a threat to peas beyond that already known to exist with previously described forms of the virus.     

DNA methylation and Dc8-GUS transgene expression in carrot (Daucus carota L.)

Summary DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.Abbreviations ABA Abscisic Acid - 5-azaC 5-azacytidine - GUS -glucuronidase - Dc8 carrot promoter