Responses of micropropagated peach cultivars to thermotherapy for the elimination of prunus necrotic ringspot virus

Peach (Prunus persica (L.) Batsch) shoot-cultures infected with prunus necrotic ringspot virus (PNRSV) were selected for evaluating the responses of in vitro grown shoots of cvs Hermosa and Summerset to thermotherapy. The survival of shoot-cultures during thermotherapy was improved by selection of the optimum concentration of 6-benzylamino purine in the medium and optimum age of shoots for treatment. Alternating high and low temperature thermotherapy regimes were more effective in decreasing virus titre than constant high temperatures. Of the regimes tested, the most effective inhibition of PNRSV combined with a high survival of shoots was obtained by applying 38°C for 16 h in light alternating with 28 ^CC for 8 h in darkness for 18 days for Hermosa and 22 days for Summerset. Following this treatment 90% of Hermosa and 40% of Summerset shoot-cultures were virus-free as determined by enzyme-linked immunosorbent assay. Relatively large (about 10 mm) apices excised from these shots regenerated into virus-free plants. The advantage of the in vitro system for thermotherapy is discussed.     

The occurrence, hosts and properties of lilac chlorotic leafspot virus, a newly recognised virus from Syringa vulgaris

Lilac chlorotic leafspot virus (LCLV), a hitherto undescribed virus, was isolated from three of 65 lilacs (Syringa vulgaris) with chlorotic leafspotting symptoms growing in S.E. England. The virus was transmitted readily by sap-inoculation to 21 of 52 species from eight of 20 families, but it was not seed-borne in four hosts or transmitted in the semi-persistent manner by any of four aphid species. The virus was moderately stable in vitro; sap from Chenopodium quinoa was infective after 10 min at 60 but not 65 ^oC, after 8–16 days at 20 ^oC or 25–30 wk at 2 ^oC, and after dilution to 10^-3 but not 10^-4. Up to 180 mg of purified virus per kg leaf tissue were obtained from C. quinoa by clarification of buffered leaf extracts with 8% (v/v) n-butanol, followed by one cycle of differential centrifugation and molecular permeation chromatography on controlled pore glass beads (700 Å, 120–200 mesh). LCLV has fragile flexuous filamentous particles which, when intact, mostly measured c. 12-5 times 1500–1600 nm; the helical substructure (pitch c. 3–7 nm) was clearly visible on some particles mounted in uranyl acetate. The particles sedimented as a single component (sedimentation coefficient 96 S; buoyant density 1–302 g cm^-3) and contained c. 5% nucleic acid and a single polypeptide of mol. wt 27 times 10³. Although these properties place LCLV in the closterovirus group, the virus showed no serological relationship to any of six closteroviruses (beet yellows, beet yellow stunt, carnation necrotic fleck, apple chlorotic leafspot, apple stem grooving and potato virus T) and differed from other recognised or possible members of this group in host range and/or symptoms induced in indicator species. The infrequent occurrence of LCLV in lilac in S.E. England indicates that the virus could probably be eradicated by selecting only virus-free plant material for propagation.     

Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.     

Elimination of virus and rapid propagation of disease-free sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. cultivar NCo376) using apical meristem culture

The use of apical meristem culture for simultaneous virus elimination and shoot proliferation in sugarcane was assessed. Virus-free plants were propagated from Sugarcane mosaic virus and Sugarcane yellow leaf virus-infected material of the South African commercial cultivar, NCo376. A combination of thermotherapy by hot water treatment of stem sections (nodes) and subsequent germination of vegetative buds at 40°C and optimal meristem size were key factors for the production of virus-free plants. Only meristems of 2 mm in length or of a smaller size (but >0.5 mm) resulted in virus-free sugarcane. Shoot induction and proliferation via direct organogenesis were achieved on Murashige and Skoog nutrient medium supplemented with 0.1 mg l⁻¹ 6-benzyladenine and 0.015 mg l⁻¹ 6-furfurylaminopurine (KIN). The established protocol provides for the rapid proliferation of virus-free shoots from infected sugarcane plants and approximately 1,300 shoots were propagated from a single 2 mm meristem in 11 weeks. Plants remained virus-free when tested 12 months later.     

Some properties of grapevine Bulgarian latent virus

Grapevine Bulgarian latent virus (GBLV), obtained from symptomless vines grown in Bulgaria, was readily transmitted by inoculation of sap to a restricted range of hosts. The virus was re-inoculated into virus-free rooted cuttings and seedlings of several grapevine cultivars without inducing symptoms. Purified preparations of GBLV contained isometric particles c. 30 nm in diameter which sedimented as three components at 52 , c. 120 and 1275, respectively. The slow-sedimenting component (T) contained non-infective protein shells, whereas the two heavier classes of particles (J5_X, B₂) each contained one molecule of single-stranded ribonucleic acid (RNA) with mol. wts of 2.1 and 2.2 times 10⁶ daltons. The RNA content of B₁ and B₂ components were 39 and 41 % by particle weight respectively. The capsid was composed of one type of protein with a mol. wt of 54000. GBLV did not react with antisera to any of twenty-eight viruses with isometric particles. Its present cryptogram is R/1: 2-2/41 +2-1/39:S/S:S/*.     

Anin vitro procedure to eradicate potato viruses X,Y, and S from Russet Norkotah and two of its strains

Summary A tissue culture method was developed for the eradication of three important potato viruses, PVX, PVY, and PVS, from the Russet Norkotah variety and two of its strains (TXNS 112 and TXNS 278). The method combined the use of liquid medium, thermotherapy, and chemotherapy. Initially, virus-free plants were inoculated with PVX, PVY, and PVS and, after 10 d, tested quantitatively for virus titer by ELISA to determine the initial virus concentration. The apical tip and the roots were removed from the inoculated plants, and only stem sections from inoculated plants were cultured in liquid medium containing MS inorganic salts, vitamins, and ribavirin (40 μM or 80 μM). After 5 d, half of the plants were subjected to thermotherapy and half were kept at room temperature. At 6 wk, the uppermost node (5–7 mm) was removed and recultured, and plants were tested then and again after 6 wk using ELISA to identify the virus-free plants. Ribavirin alone eradicated the viruses from some plants; however, more virus-free plants were obtained when the treatments included heat. Additionally, thein vitro culture technique using liquid medium promoted rapid lateral shoot elongation and resulted in significantly faster plant production. Also this approach, which required less skilled labor, produced more plants than the meristem culture method for virus eradication. A detailed procedure for elimination of multiple viruses is described. This procedure resulted in production of more than 10% virus-free plants.     

Virus Content and Virulence of Polymyxa betae Keskin Isolates Obtained from Different Regions in Europe

Polymyxa betae isolates were obtained by means of bait plants from a large number of soil samples collected in eastern Germany. Additional P. betae isolates were received from several institutions in western Germany and abroad. Isolates were grown on sugarbeet seedlings and tested for the presence of beet necrotic yellow vein virus (BNYVV) and beet soilborne virus (BSBV). BNYVV was only present in isolates from western Germany and abroad but absent in all isolates from eastern Germany., In contrast, BSBV was detected in more uniform geographic distribution in 14 out of 33 P. betae isolates tested. The virulence of P. betae isolates was estimated on the basis of the extent of resting spore formation in the root system of sugarbeet seedlings. Differences in virulence were found among virus-free as well as virus-carrying P. betae isolates. The mean value of virulence ratings was distinctly lower with BNYVV-carrying isolates and slightly lower with BSBV-carrying isolates as compared to virus-free isolates.     

Sex‐ and age‐related differences in c‐fos expression in dog olfactory bulbs

Sex‐ and age‐related differences in cognitive abilities are frequently reported. However, the sex‐ and age‐related differences in dog olfaction due to biological system are still poorly understood. We examined c‐fos expression in dog olfactory bulbs by immunohistochemistry approaches. The c‐fos is mainly expressed in the olfactory glomerular layer (GL), mitral cell layer (ML) and granule cell layer (GRL). We found that a higher density of c‐fos‐positive cells could be detected in the ML of olfactory bulbs of adult female dogs compared with that in males and the c‐fos‐positive cells in females' olfactory bulbs are more distinct. Sex‐related differences in c‐fos expression also appeared in the GL of olfactory bulbs in juvenile dogs. We also discovered that the density of c‐fos‐positive cells in the GRL of adult dogs was much higher than that in the GRL of juvenile dogs. Our results indicate that cells in the olfactory bulbs of female dogs are more active than those in males and female dogs may have much stronger ability for long‐time memory of odours than male dogs. Furthermore, our results also suggest that adult dogs may have much stronger ability for long‐time memory of odours and can deal with more complicated odour information than juvenile dogs.     

The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh.

Potato mop-top virus (PMTV) was best detected in field soils by air-drying them for more than a week before remoistening and growing seedlings of Nicotiana tabacum or N. debneyi for a 6–10 week period. Infection of N. tabacum was assessed by inoculating sap from roots and shoots to Chenopodium amaranticolor. Similar inoculations from N. debneyi were far less convenient for detecting PMTV than recording leaf symptoms, but slightly more efficient. Air-dry soil retained PMTV infectivity for 9 months, when passed through a 50 μ sieve or when diluted with 10³ but not 10⁴ parts of steamed soil. Tobacco seedlings were not infected when their roots were steeped in PMTV-containing tobacco sap. Infective soils contained Spongospora subterranea, spore balls of which resisted air-drying for more than a year and passed a 50 μ sieve. Roots of susceptible seedlings were infected with PMTV when exposed to spore balls of S. subterranea taken from powdery scabs on PMTV-infected potato tubers, or to suspensions obtained by steeping, in nutrient solution, roots infected with virus-carrying cultures of S. subterranea. Plants in several families were hosts of S. subterranea, but probabilities of infection when exposed to spore balls differed greatly between families and only species of Solanaceae were good hosts. The ten species infected with PMTV when grown in infective soil or when exposed to spore balls of S. subterranea taken from PMTV-infected potato tubers are all members of this family. PMTV seems to be carried internally in S. subterranea spore balls and survived in them for at least a year. PMTV was transmitted by S. subterranea to Arran Pilot potato, causing yellow blotches in some leaves and spraing in many tubers. However, when newly infected with PMTV in the field, not all Arran Pilot tubers developed spraing. Also, although many spraing-affected or symptomless but PMTV-infected tubers carried PMTV-containing spore balls of S. subterranea, powdery scabs were rarely found near the centres of the rings of primary spraing. PMTV became established in virus-free soil when PMTV-infected tubers carrying S. subterranea were planted as ‘seed’ but not when virus-free tubers bearing powdery scabs were used. 5. subterranea seems the main, and possibly the only, vector of PMTV in the soils examined. S. subterranea did not transmit potato aucuba mosaic virus from potato to N. debneyi or Capsicum annuum.     

Augusta disease in tulip - a reassessment

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

Determination of Onion yellow dwarf virus concentration levels on onion bulb and leaf by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)

With the aim of understanding virus movement and fluctuations in the virus concentration in bulb and leaves of onion (Allium cepa L.) plants after infection, Onion yellow dwarf virus (OYDV) was analysed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). OYDV concentrations were higher in onion leaves of plants grown from tested bulbs compared with bulbs, although the virus was successfully detected in bulb of onion.     

Investigations of carnation viruses

Carnation vein mottle virus (CarVMV) is rare in glasshouse carnations in Britain, although locally common in Dianthus barbatus in private gardens. In Sim carnations free from other viruses, CarVMV caused slight diffuse chlorotic mottling in the younger leaves, decreased flower yield by c. 22%, and caused flower breaking in cvs William Sim and Dusty. In non-Sim cultivars Pink Shibiuya, Orchid Beauty and Vesta, leaf symptoms and flower breaking were more pronounced. In mixed infections with carnation mottle virus, symptoms were much more severe. CarVMV was not eliminated from carnation or D. barbatus plants grown for 4 wk at 37^oC, and only rarely from cuttings then taken from them, but it was readily eliminated by meristem-tip culture. Myzus persicae adults or nymphs acquired and transmitted the virus within a total time of 4 min, and remained infective for 30–60 min if feeding, or for 75 min if starved. The carnation aphid, M. persicae f. dianthi, transmitted the virus much less efficiently. The virus was not transmitted by dodder (Cuscuta campestris), or through seed of D. barbatus or Chenopodium quinoa. The maximum infective dilution in sap of D. barbatus, carnation and C. quinoa ranged from 10^-2 to 10^-5. The virus withstood 10 min at 60 but not 65^oC, up to 9 days at c. 18^oC or 3–4 wk at c. 2^oC. CarVMV infected twenty-two of 107 plant species in six of thirty-seven families; suscepts were confined to the Chenopodiaceae, Caryophyllaceae and closely allied families. C. quinoa was the best local lesion assay host. Seedling clones of D. barbatus, selected as resistant to carnation mottle virus, proved the best indicator and propagation species. Up to 50 mg virus/kg tissue were obtained by butanol clarification followed by differential and density gradient centrifugation. The preparations contained a single sedimenting component, s_20w= 144S, and had flexuous filamentous particles, c. 790 times 12 run; the particles contained a single polypeptide, mol. wt 34800, and 5% of a single-stranded ribonucleic acid (RNA) with nucleotide base ratios of G21: A25: C25: U29. Serologically CarVMV was related distantly to turnip mosaic (cabbage black ring strain), pea mosaic, watermelon mosaic (Strain 2) and bean yellow mosaic viruses, more closely to pepper veinal mottle virus, but unrelated to twelve other potyviruses. CarVMV is not at present a danger to carnation crops in Britain, but the recent trend of sending carnation plants to overwinter outdoors in warmer countries involves potential risks of more rapid spread by effective vector races of M. persicae.     

The effect of cherry leaf roll virus infection on the performance of birch pollen and studies on virus replication in germinating pollen

The rates of germ tube elongation in vitro of pollen from cherry leaf roll virus (CLRV)-infected birch (Betula pendula) did not differ significantly from those of pollen from virus-free trees. Differences in percentage germination of pollen collected from trees at different sites were significant but percentages of germination of pollen from virus-infected and virus-free trees did not differ greatly from one another. In vivo, pollen from infected trees germinated on healthy and CLRV-infected stigmas and callose plugs formed in both types of tissues. However, the extent of callose plug formation was greater in the pollen tubes of virus-free grains germinating in infected stigmas than in reciprocal crosses. CLRV coat antigen was detected by ELISA in stigmatic tissue, from healthy trees, on which virus-carrying pollen grains had germinated. Using fluorescein isothiocyanate conjugated to CLRV-specific γ-globulin, viral coat antigen was detected throughout germ tubes from virus-carrying but not virus-free pollen germinating in vitro. Protoplasts released following Driselase digestion of pollen germ tubes from virus-infected trees contained CLRV antigen detectable by ELISA. During germination of virus-infected pollen there was little synthesis of viral coat protein or nucleic acid but, following inoculation with purified virus particles, protoplasts made from healthy germinating pollen produced increasing amounts of CLRV-specific antigen implying that CLRV replicated in this system.     

Failure of British isolates of beet western yellows virus to infect potato

Potato cultivars were tested for susceptibility to two British isolates of beet western yellows virus originally obtained from sugar beet and oil seed rape. Neither isolate was transmitted by Myzus persicae to virus-free potato plants, either by itself or in association with potato leafroll virus.     

钾对兰州百合鳞茎中多酚类物质积累代谢及其抗氧化活性的影响

为探究施钾对兰州百合鳞茎中多酚类物质的积累、抗氧化能力及差异代谢物的影响,该研究以兰州百合鳞茎为试材,通过固定氮素(N)和磷素(P)用量,设置不同钾(K)浓度处理,即K_(0)(不施肥)、K_(1)(447.6 mg·L^(-1))、K_(2)(671.4 mg·L^(-1))、K_(3)(895.2 mg·L^(-1)),采用福林-肖卡法、溴甲酚绿比色法、香草醛比色法、DPPH法、铜离子还原能力(CUPRAC)法测定不同K浓度处理下兰州百合鳞茎中多酚类物质含量及其抗氧化活性,并采用LC-MS法分析多酚类物质的差异代谢物,并进行差异代谢物筛选,功能注释及富集分析,为兰州百合的优质栽培提供理论依据。结果表明:(1)不同K浓度处理下兰州百合鳞茎中多酚类物质的含量及其抗氧化活性存在显著差异(P<0.05),与K_(0)相比,K_(1)、K_(2)、K_(3)均能促进鳞茎多酚类物质的积累及其抗氧化能力的提高,其中以K_(2)(671.4 mg·L^(-1))效果最佳。(2)相关性分析表明,兰州百合鳞茎多酚类物质含量与抗氧化活性指标呈极显著(P<0.01)相关关系,相关系数为0.451~0.959。(3)K_(0)、K_(2)浓度处理下兰州百合鳞茎中存在89种多酚类及相关化合物,其中52种相对含量显著上调,37种相对含量显著下调,且显著富集到黄酮类及苯丙类化合物生物合成的通路上。研究认为,兰州百合的最佳施钾量(671.4 mg·L^(-1))能有效促进鳞茎中多酚类物质的积累并提高其抗氧化能力。     

The effect of asparagus virus infection on asparagus tissue culture

The impact of asparagus virus I (AV-I), a potyvirus, and asparagus virus II (AV-II), an ilarvirus, on micropropagation of field-grown asparagus was studied. Apical shoot tips excised from singly or doubly-infected plants were slow to develop roots and had a 15 to 75% reduction in survival in culture, respectively, compared to those excised from virus-free plants. The four virus infection groups were ranked: virus-free >AV-II>AV-I>AV-I & II for capacity of explants to both root and survivein vitro. Micropropagated plants infected with AV-II exhibited slight reductions in fresh and dry weights, with greater reductions associated with infection with AV-I and double infection, compared to the virus-free controls. Eighty-one virus-infected apical shoot tips yielded 7 (8.6%) virus-free clones, as determined by rub inoculation on indicator plants.     

Detection of lymphoid leukemia colony-forming cells in abelson virus infected mice: Differences in inbred strains

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A-MuLV), pseudotype Molony murine leukemia virus (M-MuLV). Infection of these mice with 10⁴ focus-forming units of A-MuLV (M-MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A-MuLV-infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 10³ cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane-primed BALB/c recipient. Colony-forming leukemia cells could be detected in the marrow of A-MuLV-infected mice as early as 8 days after virus incoluation. The number of colony-forming leukemia cells increased as a function of time after virus inoculation. Colony-forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin-C, provide this “helper” function. Only in the presence of untreated or mitomycin-C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated. Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 10³ microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin-C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.     

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.