Beetle, contact and potato true seed transmission of Andean potato latent virus

A species of the flea beetle (Epitrix sp.) transmitted Andean potato latent virus (APLV) from Datura stramonium to D. stramonium, Nicandra physalodes and potato, and from N. physalodes to N. physalodes and D. stramonium. Although the acquisition and inoculation feeding times used were varied, this transmission always occurred at low efficiency. By contrast, APLV was very readily transmitted in tests in which infected plants of D. stramonium, N. physalodes, Nicotiana bigelovii and potato were brushed against healthy plants of one or more of these species. A low level of seed transmission of APLV occurred in potato.     

Eggplant mosaic virus, and its relationship to Andean potato latent virus

Eggplant mosaic virus (EMV), obtained from Solanum melongena L. from Trinidad, is readily transmitted by inoculation of sap to several solanaceous and a few non-solanaceous plant species. Purified preparations of EMV contain isometric particles 30 nm in diameter, and with sedimentation co efficients of either 111 or 53 S. The particles have thirty-two major morphological subunits. EMV is closely serologically related to Andean potato latent virus and has a similar host range, but is more virulent. Also, whereas EMV accumulates fastest in Nicotiana clevelandii leaves at 20–24 °C, Andean potato latent virus accumulates fastest at 15 °C, and fails to attain a serologically detectable concentration at 24 °C. A few symptomatologically or serologically distinguishable strains of EMV were obtained. EMV has properties typical of viruses of the Andean potato latent subgroup of the turnip yellow mosaic group of viruses, and its present cryptogram is */*:*/*:S/S:S/Cl.     

The relationship between the Andean strain of potato virus S and pepino latent virus

The titres obtained in microprecipitin tests with purified preparations of pepino latent virus (PepLV) and the Andean strain of potato virus S (PVS^A) using PepLV antiserum and two antisera to the ordinary strain of PVS (PVS°) indicated a close serological relationship between PepLV and PVS^A. Using antiserum to PVS°, both viruses were detected by ELISA when infective Chenopodium quinoa sap was diluted to 10^-5but not to 10^-6. Particles of both viruses were decorated equally well by antibodies to PVS^o, PVS^Aand PepLV in all virus-antiserum combinations. When PepLV was inoculated to C. quinoa, C. amaranticolor and potato plants, the symptoms induced closely resembled those of PVS^Ain these hosts. It is concluded that PepLV is an isolate of PVS^Afrom pepino.     

Host range, purification and properties of potato virus T

Potato virus T (PVT) infected nine species of tuber-bearing Solanum, most of them symptomlessly, and as a rule was transmitted through the tubers to progeny plants: two genotypes of S. tuberosum ssp. andigena were not infected. The virus was also transmitted by inoculation with sap to 37 other species in eight plant families. Chenopodium amaranticolor is useful as an indicator host, C quinoa as a source of virus for purification, and Phaseolus vulgaris as a local-lesion assay host; the systemic symptoms in Datura stramonium, Nicotiana debneyi and in these three species are useful for diagnosis. Attempts to transmit PVT by aphids failed, but the virus was transmitted through seed to progeny seedlings of four solanaceous species, and from pollen to seed of S. demissum. PVT was purified by clarifying sap with n-butanol or bentonite, followed by precipitation with polyethylene glycol, differential centrifugation and sedimentation in a sucrose density gradient. Purified preparations had an E260/E280 ratio of 1.18 and contained a single infective component with a sedimentation coefficient of 99 S. This component consisted of flexuous filamentous particles of about 640 times 12 nm that showed a characteristic substructure when stained with uranyl acetate. The virus particles contained a single species of infective single-stranded RNA, of molecular weight 2–2 times 106 daltons, and a single species of polypeptide of molecular weight about 27 000 daltons. PVT is serologically related to apple stem grooving virus but not to four other common potato viruses with flexuous filamentous particles. Apple stem grooving virus and PVT cause similar symptoms in several hosts, but also differ somewhat in host range and symptomatology. Apple stem grooving virus did not infect potato, caused additional symptoms in C. quinoa also infected with PVT, and its particles did not show the structural features specific to PVT. The two viruses are considered to be distinct. The cryptogram of PVT is R/1:2–2/(5): E/E: S/C.     

Host range, purification and serological properties of heracleum latent virus

Heracleum latent virus (HLV occurs commonly in wild plants of Heracleum sphondylium (hogweed) in Scotland without causing symptoms. It was transmitted manually or by aphids (Cavariella aegopodii, C. pastinacae or C. theobaldi) to 37 of 105 species in 11 of 18 families (especially to members of the Amaranthaceae, Chenopodiaceae, Solanaceae and Umbelliferae), but was not transmitted through seed of four species tested. It has very flexuous filamentous particles c. 730 × 12 nm in phosphotungstate, with obvious cross-banding of pitch 3–8 nm. Leaf extracts lost infectivity after 1–2 days at 22°C, 10 min at 40–50°C and after dilution 10^-4-10^-5. Infectivity in leaf extracts was not stabilised by addition of Mg²⁺, Ca²⁺ or Ni²⁺, but was abolished by EDTA. HLV was purified by bentonite clarification, precipitation with polyethylene glycol (mol. wt 6000), and differential centrifugation. Its properties resemble those of the tentative closterovirus, apple chlorotic leaf spot (ACLSV), but no serological relationship was detected to this or to any of 18 other filamentous viruses, including six definitive closteroviruses. No cross-protection was observed between HLV, ACLSV and apple stem grooving virus.     

Host range and some properties of potato mop-top virus

Potato mop-top virus (PMTV) was transmitted by inoculation of sap to twenty-six species in the Solanaceae or Chenopodiaceae and to Tetragonia expansa; species in eleven other plant families were not infected. The virus was cultured in inoculated leaves of Nicotiana tabacum cv. Xanthi-nc or in N. debneyi. Diagnostic local lesions were produced in Chenopodium amaranticolor. In winter, ten solanaceous species were slowly invaded systemically but the first leaves infected were those immediately above inoculated leaves. When transmitted to Arran Pilot potato by the vector Spongospora subterranea, PMTV induced all the main types of shoot and tuber symptoms found in naturally infected plants. Isolates of PMTV from different sources differed considerably in virulence. PMTV-containing tobacco sap lost infectivity when heated for 10 min at 80 °C, diluted to 10^-4, or stored at 20 °C for 14 weeks. Infectivity was partially stabilized by 0·02% sodium azide. When sap was centrifuged for 10 min at 8000 g, infectivity was mainly in the sediment. Infective sap contained straight rod-shaped particles about 20 nm wide, with lengths up to 900 nm and crossbands at intervals of 2·5 nm. Many of the particles were aggregated side-to-side, and the ends of most seemed damaged. The slight infectivity of phenol-treated leaf extracts was abolished by pancreatic ribonuclease. The present cryptogram of PMTV is R/*:*/*:E/E:S/Fu.     

Host range and properties of potato black ringspot virus

Potato black ringspot virus (PBRV), obtained from cultivated potato in Peru, was found to have a very wide host range resembling that of tobacco ringspot virus (TRSV-B), to which PBRV is distantly related serologically. However, PBRV caused the more severe symptoms in many species and, unlike TRSV B, infected Lycopersicon esculentum and Cyamopsis tetragonoloba. In Solanum tuberosum, PBRV caused necrotic spots and ringspots in systemically infected leaves in the year of infection and was readily transmitted through tubers to progeny plants, most of which developed no obvious symptoms although systemically infected. TRSV-B infected non-inoculated S. tuberosum leaves only sporadically, did not induce symptoms in them and was not transmitted through tubers to progeny plants. PBRV was cultured in Nicotiana clevelandii and infectivity was assayed in Cheno-podium amaranticolor or C. quinoa. Virus particles were purified from leaf extracts, after clarification using chloroform, by precipitation with 6% polyethylene glycol and differential centrifugation. Purified preparations contained 25 nm diameter isometric particles with somewhat angular outlines, sedimenting as three components (T, M and B) at 49, 84 and 117 S, and containing a single protein species of mol. wt 59 000. Preparations of PBRV nucleic acid contained two species, estimated by polyacrylamide gel electrophoresis in non-denaturing conditions to have mol. wt of about 25 10⁶ (RNA-1) and 15 10⁶ (RNA-2). Infectivity was associated with B particles, preparations of which contained RNA-1 and RNA-2, presumably in different particles. M particles contained RNA-2, were not infective and enhanced infectivity only slightly when added to B particles. Similar relative amounts of RNA-1 and RNA-2 were extracted from unfractionated virus using phenol or Pronase, but preparations obtained using phenol were much the more infective. PBRV has properties typical of nepoviruses; its present cryptogram is (R/1):2–5/41 + 15/28 or 2 1 5/46:S/S:S/*, nepovirus group.     

Bioprospecting in potato fields in the Central Andean Highlands: Screening of rhizobacteria for plant growth-promoting properties

The Central Andean Highlands are the center of origin of the potato plant (Solanum tuberosum). Ages of mutualism between potato plants and soil bacteria in this region support the hypothesis that Andean soils harbor interesting plant growth-promoting (PGP) bacteria. Therefore, the aim of this study was to isolate rhizobacteria from Andean ecosystems, and to identify those with PGP properties. A total of 585 bacterial isolates were obtained from eight potato fields in the Andes and they were screened for suppression of Phytophthora infestans and Rhizoctonia solani. Antagonistic mechanisms were determined and antagonistic isolates were further tested for phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and production of NH₃- and indole-3-acetic acid (IAA). PGP was studied in healthy and R. solani diseased plantlets under growth room conditions. Performance was compared to the commercial strain B. subtilis FZB24^® WG. Isolates were dereplicated with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), and identified with 16S rRNA gene sequencing and multi locus sequence analysis (MLSA). A total of 10% of the isolates were effective antagonists, of which many were able to solubilize phosphate, and produce IAA, ACC deaminase, NH₃ and hydrogen cyanide (HCN). During growth room experiments, 23 antagonistic isolates were associated with plant growth-promotion and/or disease suppression. Ten isolates had a statistically significant impact on test parameters compared to the uninoculated control. Three isolates significantly promoted plant growth in healthy plantlets compared to the commercial strain, and seven isolates outperformed the commercial strain in in vitro R. solani diseased plantlets.     

Host range, purification and some properties of two carlaviruses from hop (Humulus lupulus): hop latent and American hop latent

Hop latent virus (HLV) occurs in virtually all commercial hop plants in England, without causing apparent symptoms. It was transmitted between hop plants in a non-persistent manner by the aphid Phorodon humuli, but was not seed-borne in hop. The virus infected six species in four families out of 40 in 13 families which were inoculated, but infection was systemic only in Dianthus deltoides and hop. Only Phaseolus vulgaris and Chenopodium murale developed symptoms. Purification of HLV from hop extracts was hampered by aggregation of virus particles but this was minimised either by resuspending pellets in phosphate-buffered saline containing Tween 20 or by avoiding ultra-centrifugation. Virus was purified from extracts treated with Triton X-100 by precipitation with polyethylene glycol (PEG) followed either by centrifugation through sucrose density gradients or by exclusion chromatography through columns of Sephadex G-25 and Sepharose 4B. Purified preparations contained filamentous particles c. 675 × 14 nm composed of c. 6% single stranded RNA of mol. wt c. 2.9 × 10⁶ and a single protein species of mol. wt c 33 000. Immunosorbent electron microscopy (IEM) decoration tests suggested that HLV was serologically related to carnation latent, Helenium virus S, lily symptomless and Nerine latent viruses. American hop latent virus (AHLV) was found in two introductions to England from Corvallis, USA in 1975 and 1976. It was transmitted between hop plants in the non-persistent manner by P. humuli. The virus infected 17 species in seven families out of 41 species in 13 families which were mechanically inoculated and was systemic in nine species. It did not cause symptoms in any of five English hop cultivars. C. quinoa was a convenient propagation host and countable local necrotic lesions and ringspots occurred in leaves of Datura stramonium. AHLV was purified by PEG precipitation and centrifugation in sucrose density gradients. Preparations contained filamentous particles c. 680 × 15 nm composed of c. 6% single-stranded RNA of mol. wt c. 3.0 × 10⁶ and a single protein species of mol. wt c. 33 000. In IEM decoration tests AHLV was serologically related to Nerine latent virus but did not react with antisera to 14 other carlaviruses.     

Partial purification and some properties of wineberry latent, a virus obtained from Rubus phoenicolasius

Wineberry latent virus (WLV) was obtained from a single symptomless plant of American wineberry (Rubus phoenicolasius) originally imported from the United States of America. On graft inoculation, WLV infected but induced no distinctive symptoms in several Rubus species including those used as indicators for known Rubus viruses. It was not seed-borne in wineberry. WLV was mechanically transmitted to several herbaceous species but induced local lesions in only a few; it was weakly systemic in some Chenopodium species. Infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 70°C, and storage either for 8 days at 18°C or for 32 days at 4°C. Sap from infected plants contained flexuous filamentous particles c. 510°12 nm. WLV was partially purified by extracting infected C. quinoa leaves in 0·05 M tris-HCl buffer (pH ₇) containing 0·2% thio-glycerol and 10% (v/v) chloroform and concentrating virus by precipitation with 7% (w/v) polyethylene glycol (PEG, mol. wt 6000) and 0·1 NaCl. The virus was then pelleted through a 30% (w/v) sucrose pad containing 7% PEG+0·1 M NaCl and finally sedimented through a sucrose density-gradient. These preparations had A_260/280 ratios of 1·26, contained end to end aggregates of WLV particles and formed a partly polydispersed peak in the analytical ultracentrifuge. WLV did not react with antisera to four potex-viruses, or to apple chlorotic leaf spot or apple stem grooving viruses.     

Host species-dependent population structure of a pollen-borne plant virus, Cherry leaf roll virus

Cherry leaf roll virus (CLRV) belongs to the Nepovirus genus within the family Comoviridae. It has a host range which includes a number of wild tree and shrub species. The serological and molecular diversity of CLRV was assessed using a collection of isolates and samples recovered from woody and herbaceous host plants from different geographical origins. Molecular diversity was assessed by sequencing a short (375-bp) region of the 3' noncoding region (NCR) of the genomic RNAs while serological diversity was assessed using a panel of seven monoclonal antibodies raised initially against a walnut isolate of CLRV. The genomic region analyzed was shown to exhibit a significant degree of molecular variability with an average pairwise divergence of 8.5% (nucleotide identity). Similarly, serological variability proved to be high, with no single monoclonal antibody being able to recognize all isolates analyzed. Serological and molecular phylogenetic reconstructions showed a strong correlation. Remarkably, the diversity of CLRV populations is to a large extent defined by the host plant from which the viral samples are originally obtained. There are relatively few reports of plant viruses for which the genetic diversity is structured by the host plant. In the case of CLRV, we hypothesize that this situation may reflect the exclusive mode of transmission in natural plant populations by pollen and by seeds. These modes of transmission are likely to impose barriers to host change by the virus, leading to rapid biological and genetic separation of CLRV variants coevolving with different plant host species.     

Host range, properties and purification of raspberry bushy dwarf virus

Raspberry bushy dwarf virus (RBDV) was found in all plants of Lloyd George raspberry with bushy dwarf disease and occurred occasionally in plants of some other cultivars. It was transmitted by inoculation of sap to fifty-five other species in twelve families of flowering plants and infected most of them symptomlessly. It caused systemic symptoms in some species of Amaranthaceae, Chenopodiaceae and Cucurbitaceae, and necrotic local lesions in some Leguminosae. It did not induce bushy dwarf disease when returned to Lloyd George raspberry. Chenopodium quinoa was used for propagating the virus and Vigna cylindrica for local lesion assay. In C. quinoa sap, RBDV lost infectivity when diluted 10^-4, heated for 10 min at 65 °C or stored for 4 days at 22 °C. Preparations made by twice precipitating the virus at pH 4·8 and resuspending it at pH 7·0, followed by ultracentrifugation and exclusion chromatography in columns of 2 % agarose beads, contained isometric particles about 33 nm in diameter, which sedimented as two components, with sedimentation coefficients of 111 and 116S. Only a few particles, all of them disrupted, were seen in preparations mounted in phosphotungstate, but the particles were well preserved in uranyl formate provided that they were first dispersed in a saxlt such as MgCl₂ instead of distilled water. Many particles were oval in outline as though distorted during drying. No serological relationship was detected between RBDV and twenty-four other isometric viruses nor between RBDV and the filamentous virus apple chlorotic leafspot, to which it was previously thought to be related. An isolate of loganberry degeneration virus was serologically indistinguishable from RBDV.     

Comparative phylogenies of yellow fever isolates from Peru and Brazil

We recently reported phylogenetic evidence to support the presence of enzootic transmission foci of yellow fever virus (YFV) in Peru [Bryant et al., Emerg. Infect. Dis. (2003)]. Because the prevailing paradigm of YFV transmission in Brazil is that of 'wandering epizootics' rather than discrete enzootic foci, we have now compared the molecular phylogenies of YFV isolates from Peru and Brazil, and re-examined the question of virus mobility by mapping the spatio-temporal distribution of genetic variants from these areas. Sequences were obtained for two genomic regions from 50 strains of YFV collected between 1954 and 2000 comprising 223 codons of the structural proteins (premembrane and envelope genes, 'prM/E'), and a distal region spanning the carboxy terminus of NS5 and part of the 3' non-coding region ('EMF'). Peruvian and Brazilian isolates formed two monophyletic clades with no evidence to support recombination between lineages. Variation within both coding and non-coding regions revealed similar substitution rates and overall levels of diversity within each clade. The branching structure of the prM/E and EMF trees of Brazilian sequences showed strong agreement of intra-lineage relationships; in contrast, the EMF sequences of Peruvian isolates failed to fully support the subclade structure of the prM/E phylogeny. These phylogenies suggest that transmission cycles of YFV in Peru and Brazil may sometimes be locally maintained within specific locales, but have also on occasion become very widely dispersed.     

Partial purification and some properties of a latent CO2 reductase from green potato tuber chloroplasts

We have partially purified the CO2 reductase, present in green potato tuber chloroplasts, as a latent form. Illumination of the chloroplasts in the absence of substrate, bicarbonate, activated the enzyme, which could then be obtained in soluble forms. Purification of the enzyme was achieved by (NH4)2SO4 fractionation (0-30%) and adsorption and elution from a DEAE-Sephadex A-50 column. The final preparation showed 15-fold purification and 50% recovery of the activity. The pH optimum for CO2 reductase was 8.0. Hepes and Tricine buffers showed maximum activity whereas Tris/phosphate or borate failed to show any activity. The enzyme reaction was sensitive to the presence of metal ions like Fe3+, Hg2+, Cu2+, Mo6+ and Zn2+, however, a threefold activation was observed with Fe2+. The metal requirement for CO2 reductase was evident from the observed inhibition by metal chelators like o-phenanthroline, alpha, alpha'-dipyridyl, bathocuproine, 8-hydroxyquinoline etc. Out of these o-phenanthroline was the strongest inhibitor and its concentration for 50% inhibition was 40 microM. The presence of Fe2+ ions in the reaction mixture protected the enzyme from heat denaturation upto 50 degrees C. Maximum enzyme activity was observed at 15 degrees C. The enzyme activity showed a 30-s lag period and the maximum was reached in 90 s. Supplementation of sodium dithionite in the reaction activated enzyme activity threefold, suggesting involvement of dithiol groups in the catalytic activity. There was strong inhibition by -SH inhibitors like 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide and -SH reagents like dithiothreitol, 2-mercaptoethanol and cysteine. Various nucleotide coenzyme tried inhibited the enzyme strongly.     

Host range, purification and some properties of rubus Chinese seed-borne virus

A previously undescribed virus, for which the name rubus Chinese seed-borne virus (RCSV) is proposed, was isolated from a single, symptomless plant of an unidentified Rubus species grown from seed collected in the wild in the People's Republic of China, Experimentally RCSV infected 23 out of 39 spp. in six out of eight families. The virus was seed-transmitted in Chenopodium quinoa (100%) and Nicotiana bigelowii (27%). RCSV was not transmitted by the nematodes Xiphinema diversicaudatum or X. index. The particles of RCSV were isometric, c. 30 nm in diameter with some penetrated by negative stains. In thin sections particles were found in double walled tubular structures with an outer membrane enclosing one or more tubules. In crude extracts some particles were found within single-walled tubules. Two virus-associated bands were seen in sucrose density gradients of purified preparations. The upper band was not infective and consisted of penetrated particles apparently devoid of nucleic acid. The lower, infective band was resolved into two components, of density 1.452 and 1.461 g/ml, in caesium chloride isopycnic gradients. There were two polypeptides (mol. wts c. 47 000 and 25 200 daltons) and two nucleic acid species (one of mol. wt c. 1.4 × 10⁶ daltons; the second was poorly defined by the methods used but was of higher molecular weight). RCSV was distantly related serologically (6–7 SDI) to the type isolate of strawberry latent ringspot virus (SLRV) and also reacted with antisera to serologicaly distinct grape and olive isolates of SLRV. It did not react with antisera to 10 other isometric viruses.