苜蓿花叶病毒外壳蛋白基因对红三叶的遗传转化及转基因植株的抗病性分析

以红三叶(Trifolium pratense L．)子叶为转化体,用农杆菌介导法将外源的苜蓿花叶病毒外壳蛋白基因AMV4转入红三叶,经过筛选、分化和再生,得到了具有卡拉霉素抗性的转基因植株。对这些植株进行PCR、Southern印迹杂交和Northern杂交分析,结果表明,外源目的基因已经整合到红三叶基因组中并且得到了表达。对Northern分析呈阳性的植株进行了抗病性检测,结果表明,表达苜蓿花叶病毒外壳蛋白基因的植株病症减轻,发病率、病情指数及病毒积累量都明显低于对照,有的甚至不表现症状,达到了免疫的程度。     

The elimination of cocksfoot streak virus, cocksfoot mild mosaic virus and cocksfoot mottle virus from Dactylis glomerata by shoot tip and tiller bud culture

Shoot tips from 0.2 to 4.8 mm long and tiller buds 0.2 to 5.1 mm long were cultured from cocksfoot ( Dactylis glomerata ) plants known to be infected with combinations of cocksfoot streak, cocksfoot mild mosaic and cocksfoot mottle viruses. Regenerated plants were tested for the presence of viruses by electron microscopy, serology and the expression of symptoms. All viruses were eliminated by culturing shoot tips and tiller buds less than 1.2 mm long but the maximum explant size capable of regenerating healthy plants depended upon the infecting virus. Viruses were not detected in shoot tip and tiller bud samples of similar size to the explants that gave healthy plants. The method is of value in eliminating viruses from desirable stocks of D. glomerata that must be vegetatively propagated.     

Temporal dynamics of spread of four viruses within mixed species perennial pastures

Six mixed species, perennial pastures at two locations, A (four pastures) and B (two pastures), were sampled at regular intervals over periods of 10 to 22 months. The predominant plant species present were white clover (Trifolium repens), perennial ryegrass (Lolium perenne) and kikuyu grass (Pennisetum clandestinum). To determine the extent to which incidences of viruses transmitted in different ways change in the same pastures over time, samples of each plant species were taken at random on every visit and tested for virus presence. To help identify factors that might explain changes in virus incidence, records were also made of aphid presence, pasture management practices, grazing regimes, sward height and the relative proportions of different plant species within the swards. Samples of white clover were tested for presence of Alfalfa mosaic virus (AMV) and White clover mosaic virus (WCMV), ryegrass for Barley yellow dwarf virus (BYDV) and Ryegrass mosaic virus (RyMV), and kikuyu grass for BYDV and potyvirus infection. AMV and WCMV were detected in white clover, and BYDV and RyMV in ryegrass at both locations but often with wide incidence fluctuations for the individual viruses. AMV incidences in white clover ranged from 0% to 19% at A, and from 27% to 100% at B. WCMV incidences in white clover fluctuated between 9% and 46% at B, but never exceeded 1% at A. RyMV incidences in ryegrass fluctuated between 3% and 34% at A, and 19% and 73% at B. BYDV incidences in ryegrass ranged from 0% to 6% at A and 4% to 17% at B. In kikuyu grass, an unknown potyvirus and BYDV were detected twice (1% incidence) and once (4% incidence) respectively at B, and the unknown potyvirus only once (2% infection) at A. During repeated trapping of aphids in four pastures (two each at A and B), numbers of aphids caught varied widely between trapping dates and between individual pastures on the same trapping date. The species caught were Acyrthosiphon kondoi, A. pisum, Aphis craccivora, Rhopalosiphum padi and Therioaphis trifolii. Except in summer, when only T. trifolii was caught, A. craccivora was the most abundant. The trends in incidence for each virus within each pasture were compared with those from the other pastures sampled over identical periods to determine whether there was any commonality. For RyMV in ryegrass, overall incidence trends within the different pastures at both locations resembled each other during the same sampling periods. Within pastures at the same location there was commonality in incidence trends for RyMV and BYDV in ryegrass, but with AMV in white clover periods of similarity were rare even when pastures were adjacent and managed identically. Unravelling the individual effects of alterations in season, vector numbers, mowing, intermittent heavy grazing and pasture species composition on virus incidence proved difficult due to complex interactions between these and other factors influencing new spread or declining virus occurrence.     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

The occurrence and effects of red clover necrotic mosaic virus in red clover (Trffbliumpratense)

In 1976, red clover necrotic mosaic virus (RCNMV) was identified in red clover variety trials at the Scottish Colleges of Agriculture and at the trial centres of the National Institute of Agricultural Botany (NIAB) in Northumberland, Dyfed, Devon and Cambridge. In 1977, RCNMV was also found in two commercial crops of red clover in South Wales. The only previous finding of this virus in Britain was in 1971.
In red clover leaves RCNMV causes veinal chlorosis, often followed by severe necrosis and deformation; the plants become stunted. All cultivars tested were infected either in field or glasshouse experiments and three of the four most susceptible cultivars were tetraploids. Yield losses in cv. Hungaropoly averaged 57% over three cuts. RCNMV was transmitted manually but not through seed or by aphids {Acyrthosiphon pisum and Myzus persicae) or weevils (Apion spp. and Sitona lineatus). Seedlings became infected when grown in pots containing RCNMV-infected plants or soil from infected sites, and the roots of infected test seedlings contained an Olpidium sp. which may be the vector.
White clover mosaic virus (WCMV), also common in red clover at some sites, was less damaging than RCNMV and in a glasshouse experiment decreased yield by only 22%. An unidentified seed-borne virus with spherical particles c. 33 nm in diameter was the only virus detected in clover seedlings screened for RCNMV.     

不同三叶草对二斑叶螨生长发育和繁殖的影响

【目的】明确红三叶、白三叶和杂三叶3种寄主对二斑叶螨Tetranychus urticae Koch生长发育和繁殖的影响,以及3种寄主间二斑叶螨的生物学差异。【方法】在室内(25±1)℃条件下,采用离体叶片法研究了3种三叶草对二斑叶螨种群参数的影响。【结果】3种三叶草对二斑叶螨生长发育、雌成螨寿命以及繁殖力有显著影响(P<0.05)。在杂三叶上,二斑叶螨发育历期、产卵前期明显比红三叶和白三叶上要长,而且雌成螨寿命和产卵期也明显缩短。每雌产卵量在红三叶上最高(150.87粒),白三叶次之(139.43粒),而杂三叶上最低(86.95粒)。二斑叶螨在3种三叶草上的存活曲线均为Ⅰ型,存活率高低依次为红三叶、白三叶和杂三叶。净增殖率(R0)、内禀增长率(rm)和周限增长率(λ)在红三叶上最高、杂三叶上最低,而平均世代周期(T)和种群加倍时间(Dt)在杂三叶上最长、红三叶上最短。【结论】3种三叶草对二斑叶螨的适合度具有一定差异,红三叶和白三叶对二斑叶螨具有更高的适合度。     

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

Transgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full-length CP, the core region of the CP, and full-length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full-length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene-encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.     

Interaction between Sitona lepidus and red clover lines selected for formononetin content

Adult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

Tests for resistance to white clover mosaic virus in red and white clover

A range of red and white clover cultivars was tested for immunity to white clover mosaic virus. All plants became infected although some showed no symptoms. Enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) revealed significant differences in virus concentration between red clover cultivars and between clones of white clover artificially infected with the virus. These differences could not, however, be related to relative yield losses.     

Incidence and severity of pest and disease damage to white clover foliage at 16 sites in England and Wales

From 1985 to 1987, leaves of white clover ( Trifolium repens ) cv. Grasslands Huia were examined for damage by pests and fungal diseases on seven occasions at up to 16 sites in England and Wales. Pest damage was recorded at all sites on all sampling dates. Over all sampling dates, the mean number of leaves damaged by slugs ranged from 23% to 67%, and that by weevils ( Sitona spp.) ranged from 3% to 62%. Also, up to 30% of leaves were damaged by other, unidentified pests. At individual sites, total pest damage frequently exceeded 90% of leaves. The area of leaf damaged by pests ranged from 2 - 12%. Fungal diseases were recorded in July and September but not in May, and were more prevalent in September 1985 than in September 1986 or 1987. Black blotch ( Cymadothea trifolii ) was the most frequently recorded disease, and the mean number of leaves damaged ranged from 4% to 21%. The mean area of leaves covered by lesions was 1–2%. Infections by viruses were assessed on two occasions, using indicator plants and electron microscopy, and only a very low incidence of arabis mosaic virus and red clover necrotic mosaic virus was recorded.     

Elimination in vitro of two Grapevine Nepoviruses by an Alternating Temperature Regime*

A 40 day in vitro treatment with 6 h at 39°C followed by 18 h at 22°C was effective in eliminating both grapevine fanleaf virus (GFLV) and arabis mosaic virus (AMV) from the developing shoot tips (2 mm) of grapevine shoot tip cultures. Longer treatment durations with consecutive 12 h periods at 35°C and 22°C eliminated GFLV in some cases, but did not eliminate AMV.     

The effect of shoot and root genotype on phosphorus concentrations of shoots and roots

Genotypes of two morphologically different populations of white clover (Trifolium repens L.) were reciprocally and self-grafted. Ungrafted stolon tips were also grown as controls. Grafting per se had no significant effect on shoot size, root size, leaflet width or shoot and root % P. The scion genotype had a significant effect on shoot and root size, and leaflet width. Neither scion nor rootstock genotype had a significant effect on either shoot or root % P. However, there was a significant scion × rootstock × P level interaction for shoot % P. This along with other evidence suggests that conflicting results regarding effects of scion and rootstock on % P content of plants within species is probaby due to the interaction of scion and rootstock with environment.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.     

小滴玻璃化法脱除蝴蝶兰种苗建兰花叶病毒的研究

以感染建兰花叶病毒(Cymbidium mosaic virus,CymMV)的蝴蝶兰(Phalaenopsis aphrodite)品种‘满天红’为试材,通过筛选蔗糖预培养浓度、预培养时间、PVS2(Plant vitrification solution 2,PVS2)处理时间三个关键因素,建立蝴蝶兰茎尖小滴玻璃化超低温脱毒体系,将再生的茎尖诱导类原球茎,再分化成苗,经RT-PCR检测CymMV的脱除情况,阴性结果的再生植株进行增殖和诱导生根。结果显示:最佳预培养为:BM+0.6 mol·L-1蔗糖处理1~2 d,超低温茎尖的成活率为70%~76.7%,再生率为53.3%~56.7%;PVS2最佳处理时间为60~90 min,超低温茎尖的成活率为73.3%~76.7%,再生率为50.0%~56.7%。再生植株经RT-PCR检测,CymMV的脱除率为50%。该研究为兰科植物脱除CymMV提供了理论和技术基础。     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Molecular Variability and Distribution of Cassava Mosaic Begomoviruses in Nigeria

Several begomovirus species and strains causing Cassava mosaic disease (CMD) have been reported from cassava in Africa. In Nigeria, African cassava mosaic virus (ACMV) was the predominant virus in this important crop, and East African cassava mosaic virus (EACMV), first reported from eastern Nigeria in 1999, was also found occasionally. A survey was conducted in 2002 to resolve the diversity of the virus types present in cassava in Nigeria and to further understand the increasing complexity of the viruses contributing to CMD. A total of 234 leaf samples from cassava with conspicuous CMD symptoms were collected in farmers’ fields across different agroecological zones of Nigeria and subjected to polymerase chain reaction (PCR) with type‐specific primers. In addition and, to provide a full characterization of the viruses present, DNA‐A genome components of several viruses and informative genome fragments were sequenced. In Nigeria, ACMV proved to be the dominant virus with 80% of all samples being positive for ACMV. The East African cassava mosaic Cameroon virus (EACMCV) prevalent in Cameroon and Ivory Coast was detected in single infections (2%) and in mixed infections (18%) with ACMV. There was no indication for other virus strains of EACMV present in the country. The EACMCV samples collected showed a high nucleotide sequence identity >98% and resembled the described sequence of a Cameroon isolate (EACMCV‐CM) more than an Ivory Coast isolate, EACMCV‐CM[CI]. Evidence is provided that the EACMCV has reached epidemiological significance in Nigeria.     

Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid     

Cytopathology of Shoot Apical Meristem of Hop Plants Infected with Hop Stunt Viroid11)

Shoot apical meristems of hop plants infected with hop stunt viroid (HSV) were examined for cytopathic changes. No measurable changes in ultrastructure were found in shoot tip 0.2 mm long bearing apical dome and two pairs of the primordia, whereas in the 3rd leaf primordium cell walls were undulate and/or frequently were irregular in thickness. These cell wall abnormalities were not observed in comparable shoot tips of the uninoculated hop plants or HSV-free hop plants obtained by meristem tip culture.     

病毒唑对大花蕙兰CyMV及ORSV脱毒效果初探

在大花蕙兰茎尖培养中结合病毒唑处理,探讨对建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)的脱除效果。结果表明,病毒唑添加到培养基中,处理茎尖2周,脱毒效果低于经病毒唑处理3个月后的幼苗,经病毒唑处理幼苗再结合茎尖培养脱毒效果最好。病毒唑处理对大花蕙兰CyMV的脱除效果优于对ORSV的脱除效果。