应用微量玻片凝胶双扩散技术鉴定马铃薯卷叶病毒

应用常规免疫双扩散不能鉴定马铃薯植株中的卷叶病毒(PLRV)。我们用微量玻片凝胶双扩散技术对马铃薯植株汁液中的PLRV进行了鉴定。试验说明,用这种方法可测出马铃薯茎的汁液最高稀释度为1:4,微量玻片凝胶双扩散与普通免疫双扩散灵敏度的比较试验表明,两者可测出提纯PLRV的最低浓度分别为1μg/ml和6μg/ml,但后者不能测出植物汁液中的PLRV。微量玻片凝胶双扩散是目前能够用于检测感病植物汁液中PLRV的最简便易行和可靠的血清学方法。     

马铃薯卷叶病毒中国株ORF1及5′端非编码区序列特点分析

马铃薯卷叶病毒（potato leafroll virus,PLRV）是一种严格由蚜虫以持久性方式传播的病毒,是黄化病毒属（Luteovirus）成员,可侵染马铃薯（Solanum tuberosum L）引起卷叶、黄化、矮缩、僵化及块茎网状坏死等症状,严重影响马铃薯的产量和品质,是一种世界范围的马铃薯主要病毒病害。因此,有效防治此病害具有重要经济意义。     

携病毒马铃薯茎尖分化成苗与脱毒率检测

以携带病毒的‘夏波蒂’马铃薯无菌苗为材料,38℃/4h热处理4周,剥离带1个叶原基的茎尖,接种至不同浓度激素组合的24种MS固体培养基上,22℃/16h培养30d后统计茎尖的愈伤组织诱导率和分化成苗率;RT-PCR检测茎尖再生苗3种病毒(PVX、PVY、PLRV)和纺锤块茎类病毒(PSTVd)的脱毒率。结果表明:茎尖分化成苗最适培养基为MS+1.0mg/L ZT+0.2mg/L NAA+2.0mg/L GA3,愈伤组织诱导率为76.25%,分化成苗率为26.25%;再生苗3种病毒PVX、PVY和PLRV的脱毒率分别为69.4%、91.7%和100%,纺锤块茎类病毒PSTVd脱毒率仅为8.3%,二次茎尖剥离后脱毒率增加到20.8%。     

马铃薯卷叶病毒的提纯

本文提出了一个应用液氮冷冻,一步提取,蔗糖垫层差速离心,Sephadex G-200柱层析以及蔗糖密度梯度离心法纯化马铃薯卷叶病毒的程序,改进后的马铃薯卷叶病毒提纯方法,使病毒产量达到1.18mg/kg酸浆组织,病毒提取物纯度比差速离心者更高,20％蔗糖垫层差速离心能够更加有效地去除宿主细胞成份,纯化病毒的OD260/280,260/240比值分别达到1.77和1.43。     

马铃薯卷叶病毒的分离、提纯及抗血清制备

同马铃署品种“小叶子×多子白”和“米拉”分离了马铃薯卷叶病毒。用0．2M磷酸缓冲液榨汁,滤渣经液氮冷冻,捣碎,匀浆以代替加入酶制剂,在差速离心后,用Scpbadex G—200凝胶过尊和蔗塘密度梯宝离心,从蚜虫接种的马铃薯和洋酸浆的茎叶中提纯了PLRV。其紫外最大吸收在260nm,最低吸收在240 nm,0．D 260／280nm 比值为1．70。 提纯能病毒粒体在电镜下观察为等轴20面体,表面形态亚单位清晰可见。粒体平均直径为23．9 8nm。用提纯的 PLRV免疫啄鼠阳家兔,在国内首次制备出高效价特异性的PLRV抗血清。用对流免疫电泳测得抗血清最高效价为1／4096。应用适当稀释的抗血清,以对流免疫电泳的方法,可直接诊断感染卷叶病的马铃薯茎叶中的PLRV。     

马铃薯卷叶病毒单克隆抗体的制备

采用杂交瘤技术,以马铃薯卷叶病毒(Potato Leafroll Virns,PLRV)为抗原,用直接将病毒注入脾脏和随后尾静脉注射的方法,免疫BALB/C小鼠。将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0融合。用Dot-ELISA和间接血凝试验筛选分泌抗马铃薯卷叶病毒抗体的阳性克隆,建立了分泌抗PLRV单克隆抗体的杂交瘤细胞株。用微量玻片双扩散法测定单克隆抗体亚类为IgG_1和IgG2a,轻链为λ。注射杂交瘤细胞株A_1、A_3、C_3和D_3于小鼠腹腔,制备出含高效价单克隆抗体的腹水。用获得的四种单克隆抗体对马铃薯卷叶病毒15个分离物进行了鉴定。     

马铃薯卷叶病毒基因间隔区转化的马铃薯抗病性研究

将本室合成、克隆的马铃薯卷叶病毒(Potato Leafroll Virus, PLRV)中国分离株的基因间隔区(intergenic sequence, IS)双链cDNA以正、反向两种方式分别构建于转化载体pROK2中,通过致瘤农杆菌介导,以马铃薯叶圆片为转化材料,转化马铃薯栽培品种Desiree,获得了转基因植株.卡那霉素抗性分析和PCR检测目的基因,证明PLRV IS双链cDNA已经整合到转基因马铃薯的染色体基因组中.将转基因植株移栽网棚用蚜虫接种PLRV,观察症状并用酶联免疫吸附测定(ELISA)检测转基因植株中PLRV含量.结果表明,表达PLRV IS正意和反意RNA的转基因植株,接种病毒后表现无症状或症状轻微,PLRV平均滴度均较未转基因对照植株低.表达正意RNA的转基因植株PLRV滴度降低43%～72%,表达反意RNA的转基因植株PLRV滴度降低72%～86%,由此可见,表达PLRV IS反意RNA的转基因马铃薯对PLRV抗性较强.     

应用聚合酶链式反应扩增和光敏生物素标记的cDNA探针检测马铃薯纺锤块茎类病毒

以含马铃薯纺锤块茎类病毒(potato spindle tuber viroid,PSTV)RNA的总核酸为模板,加入人工合成的互补DNA引物,用反转录酶合成PSTV cDNA;在聚合酶链式反应系统中,用两个PSTV特异性引物进行cDNA扩增,用以制备光敏生物素标记的PSTV cDNA探针。用此探针进行斑点杂交检测含PSTV的马铃薯核酸提取液和汁液均出现阳性杂交信号,而健康马铃薯的核酸提取液和汁液的结果均为阴性。光敏生物素标记探针检测纯化PSTV的灵敏度可达5pg;检测感染PSTV的马铃薯块茎汁液的可测出最高稀释度为1:400。     

用^32P标记cDNA探针进行核酸斑点杂交检测马铃薯卷叶病毒（PLRV）

利用克隆的马铃薯卷叶病毒（ＰＬＲＶ）外壳蛋白（ＣＰ）基因ｃＤＮＡ,用切口平移法制成＾３２Ｐ标记探针,通过核酸斑点杂交,对马铃薯卷叶病毒ＲＮＡ,提纯的马铃薯卷法病毒和感染ＰＬＲＶ的马铃薯茎、叶、声     

马铃薯卷叶病毒单克隆抗体的制备

马铃薯卷叶病毒(Potato leafroll virus,PLRV)在植物体内数量很少,难以提纯足够量病毒进行免疫,至今未见国内有制备PLRV单克隆抗体(McAb)的报道.本文以本室以前报道的方法提纯马铃薯卷叶病毒作为抗原,直接注入Balb/c小鼠脾脏,随后从尾静脉加强注射,采用杂交瘤技术建立了4个分泌抗PILRV McAb的杂交瘤细胞株。经微量免疫双扩散法鉴定,杂交瘤细胞株A1、A3、D3分泌的McAb均属gG1亚类,C3株者属IgG2a,它们的轻链都是λ型。将0.5ml含10~8个A1,A3,C3,D3杂交瘤细胞注入     

Application of enzyme-linked immunosorbent assay to the detection of potato leafroll virus in potato tubers

Factors affecting the detection of potato leafroll virus (PLRV) by enzyme-linked immunosorbent assay (ELISA) in tubers of field-grown potato plants with primary or secondary infection were studied. The reactions of extracts of virus-free potato tubers were minimised by pre-incubating the extracts at room temperature and by careful choice of the dilution of enzyme-conjugated globulin. PLRV was reliably detected in tubers produced by secondarily infected plants of all six cultivars tested. PLRV concentration was greater in heel-end than in rose-end vascular tissue of recently harvested tubers but increased in rose-end tissue when tubers stored at 4°C for at least 5 months were placed at 15–24°C for 2 wk. PLRV occurred at greater concentration in tubers from plants of cv. Maris Piper with natural or experimentally induced primary infection than in tubers from secondarily infected plants; again PLRV concentration was greater in heel-end than in rose-end vascular tissue. Plants whose shoots were infected earliest in the growing season were invaded systemically and produced the greatest proportion of infected tubers; plants infected late in the season also produced infected tubers but PLRV was not detected in their shoot tops. PLRV concentration in tubers from the earliest-infected plants was less than in tubers from later-infected plants. PLRV was detected reliably by ELISA in tubers from progenies that were totally infected but was not detected in all infected tubers from partially infected progenies. ELISA is suitable as a routine method of indexing tubers for PLRV, although the virus will not be detected in all infected tubers produced by plants to which it is transmitted late in the growing season.     

Detection of Potato Leaf Roll Virus in Intact Sprout Disks by Enzyme-linked Immunosorbent Assay

Potato leaf roll virus (PLRV) was detected by enzyme-linked immunosorbent assay (ELISA) when intact sprout, stem or leaf tissue disks were substituted for leaf or tuber extracts as test samples. Absorbance (A₄₀₅) values increased with increasing number of disks per plate well. Readings with sprout disks were significantly higher than those with disks cut from other plant tissues or with tuber sap. A₄₀₅ values obtained by using 7 or 5 sprout disks per well were near the maximum oneobtained with leaf sap. PLRV was slightly more efficiently detected by ELISA in light sprout disks than in etiolated sprout ones. When ten out of 34 healthy tubers were replaced by PLRV-infected ones in the tuber indexing test, the diseased samples werereliably detected with 5 etiolated sprout disks per well. The sprout disk sampling technique should be useful for qualitative evaluation of PLRV infection in sprouted potato tubers without necessity to wound them and using sprouts not long enough for maceration.     

Factors affecting the detection of potato leafroll virus in potato foliage by enzyme-linked immunosorbent assay

Using antiserum globulins that reacted only weakly with plant materials, potato leafroll virus (PLRV) at 10 ng/ml was detected consistently by enzyme-linked immunosorbent assay (ELISA). The reaction with PLRV particles was slightly impaired in potato leaf extracts that were diluted less than 10^-1 but not at greater dilutions. Antiserum globulins that reacted more strongly with plant materials could be used satisfactorily for coating microtitre plates but were unsuitable for conjugating with enzyme. The detection end-point of PLRV, in leaf sap of potato cv. Cara plants grown from infected tubers in the glasshouse, was about 10^-2 and the virus was reliably detected in extracts of composite samples of one infected and 15 virus-free leaves. PLRV concentration was much less in extracts of roots or stolons than in leaf extracts. The virus was detected in infected leaves of all 27 cultivars tested. PLRV was readily detectable 2 wk before symptoms of secondary infection developed in field-grown plants of cv. Cara and Maris Piper and remained so for at least 5 wk. Its concentration was slightly greater in old than in young leaves and was similar to that in glasshouse-grown plants. In field-grown plants of cv. Maris Piper with primary infection, PLRV was detected in tip leaves 21–42 days after lower leaves were inoculated by aphids; in some shoots it later reached a concentration, in tip leaves, similar to that in leaves with secondary infection. Symptoms of primary infection developed in the young leaves of some infected shoots but were inconspicuous and were not observed until at least a week after PLRV was detected by ELISA.     

Restricted multiplication of potato leafroll virus in resistant potato genotypes

Plants of a range of potato genotypes differing in rating for field resistance to potato leafroll virus (PLRV) were inoculated with the virus by grafting or by aphids (Myzus persicae). Plants of all genotypes tested became infected by each inoculation method and PLRV was detected by ELISA in the upper leaves of all genotypes within 26 days after grafting. Most genotypes with high resistance ratings developed only mild primary and secondary symptoms whereas those with low resistance ratings developed more pronounced symptoms. However, one genotype (G7461(4)) with a high resistance rating was very severely affected. The concentrations attained by PLRV in genotypes with high resistance ratings were only 1–10% of those in genotypes with low resistance ratings. These differences in virus concentration were found in young leaves of plants with primary or secondary infection, whether inoculated by grafting or by aphids and whether grown in the glasshouse or the field. In older leaves, differences in virus concentration between genotypes were at least as pronounced as those in younger leaves. In contrast, PLRV concentration in vascular tissue at the heel end of tubers of plants with primary infection was similar for all the genotypes tested. Although low PLRV concentration was consistently associated with high resistance rating it is not the only form of resistance to PLRV occurring in potato.     

Increased sensitivity of detection of plant viruses obtained by using a fluorogenic substrate in enzyme-linked immunosorbent assay

The fluorogenic substrate 4-methylumbelliferyl phosphate (MUP) of alkaline phosphatase was compared with the chromogenic substrate p-nitrophenyl phosphate (NPP) in tests for plant viruses by enzyme-linked immunosorbent assay (ELISA). In tests on leaf extracts of squash infected with prune dwarf virus, Chenopodium quinoa and apple infected with apple mosaic virus (ApMV), and potato infected with potato leafroll virus (PLRV), MUP increased sensitivity 2–16 times, the smallest and greatest increases being obtained with ApMV (in apple) and PLRV respectively. In similar tests on 21 dormant PLRV-infected potato tubers, sensitivity was increased 2–4 times with 13 tubers, but the two substrates gave the same detection end-points with eight tubers. When individual seeds of potato plants infected with the Andean potato calico strain of tobacco ringspot virus were tested, the virus was detected in virtually all seeds by MUP-ELISA, but detection by NPP-ELISA was inefficient unless absorbance values were measured after overnight incubation at 4 °C, instead of after 2 h at room temperature. In tests on Myzus persicae carrying PLRV and Sitobion avenae carrying barley yellow dwarf virus (BYDV), both viruses were consistently detected in a greater proportion of individual aphids by MUP-ELISA than NPP-ELISA irrespective of whether incubation was for 2 h at room temperature or overnight at 4 °C. The effeciency of detection of virus in single viruliferous aphids by MUP-ELISA was not decreased by grouping with one or four non-viruliferous aphids but was decreased (PLRV) or greatly decreased (BYDV) by grouping with nine. MUP-ELISA and transmission tests to Physalis floridana seedlings (2–3 day inoculation access periods) both detected PLRV in most individual M. persicae, but the results obtained with the two methods did not correlate completely. In similar tests for BYDV in individual S. avenae, virtually all aphids transmitted BYDV to oat seedlings during a 3-day inoculation access period but it was subsequently detected by MUP-ELISA in less than half of them. By contrast, MUP-ELISA detected PLRV in most viruliferous M. persicae even after they had fed for 3 days on Chinese cabbage, a non-host for this virus.     

Spread of potato leafroll virus is decreased from plants of potato clones in which virus accumulation is restricted

Tubers of eight potato clones infected with potato leafroll luteovirus (PLRV) were planted as ‘infectors’ in a field crop grown, at Invergowrie, of virus-free potato cv. Maris Piper in 1989. The mean PLRV contents of the infector clones, determined by enzyme-linked immunosorbent assay (ELISA) of leaf tissue, ranged from c. 65 to 2400 ng/g leaf. Myzus persicae colonised the crop shortly after shoot emergence in late May and established large populations on all plants, exceeding 2000/plant by 27 June. Aphid infestations were controlled on 30 June by insecticide sprays. Aphid-borne spread of PLRV from plants of the infector clones was assessed in August by ELISA of foliage samples from the neighbouring Maris Piper ‘receptors’. Up to 89% infection occurred in receptor plots containing infector clones with high concentrations of PLRV. Spread was least (as little as 6%) in plots containing infectors in which PLRV concentrations were low. Primary PLRV infection in guard areas of the crop away from infectors was 4%. Some receptor plants became infected where no leaf contact was established with the infectors, suggesting that some virus spread may have been initiated by aphids walking across the soil.     

Multiple components of the resistance of potatoes to potato leafroll virus

In glasshouse experiments the ranking of potato genotypes for resistance to infection with potato leafroll virus (PLRV) using three concentrations of aphid-borne inoculum was the same as their field resistance ratings. In field-grown plants this resistance to infection increased in all genotypes as the plants aged but its rate of increase differed between genotypes. In tests on field-grown plants infected by aphid- or graft-inoculation, the proportion of virus-free progeny tubers increased the later the date of inoculation but was greater in resistant than in susceptible genotypes. This trend was most pronounced in the resistant clone G7445(1), in which the virus failed to move from the foliage to the tubers of some plants infected in glasshouse tests. The spread of PLRV will thus be minimised in crops of resistant compared with susceptible genotypes for three reasons: plants have greater resistance to infection, systemic spread of virus from their foliage to tubers is less likely and, as shown previously, the low concentration of virus particles in leaf tissue makes infected plants less potent sources of inoculum for aphids.     

Detection of potato leafroll and potato mop-top viruses by immunosorbent electron microscopy

Attachment of virus particles to antiserum-coated electron microscope grids (immunosorbent electron microscopy) provided a test that was at least a thousand times more sensitive than conventional electron microscopy for detecting potato leafroll (PLRV) and potato mop-top (PMTV) viruses. The identity of the attached virus particles was confirmed by exposing them to additional virus antibody, which coated the particles.
PLRV particles (up to 50/μm² of grid area) were detected in extracts of infected potato leaves and tubers, infected Physalis floridana leaves, and single virus-carrying aphids. On average, Myzus persicae yielded 10–30 times more PLRV particles than did Macrosiphum euphorbiae .
PMTV particles (up to 10/μm² of grid area) were detected in extracts of inoculated tobacco leaves, and of infected Arran Pilot potato tubers with symptoms of primary infection. Particles from tobacco leaves were of two predominant lengths, about 125 nm or about 290 nm, and fewer particles of other lengths were found than in previous work, in which partially purified or purified preparations of virus particles were examined, using grids not coated with antiserum.     

Restricted distribution of potato leafroll virus antigen in resistant potato genotypes and its effect on transmission of the virus by aphids

Enzyme-linked immunosorbent assay was used to measure the concentration of potato leafroll virus (PLRV) antigen in different parts of field-grown secondarily infected plants of three potato genotypes known to differ in resistance to infection. The antigen concentration in leaves of cv. Maris Piper (susceptible) was 10–30 times greater than that in cv. Pentland Crown or G 7445(1), a breeder's line (both resistant). Differences between genotypes in antigen concentration were smaller in petioles and tubers (5–10-fold) and in above-ground stems (about 4-fold), and were least in below-ground stems, stolons and roots (about 2-fold). PLRV antigen, detected by fluorescent antibody staining of tissue sections, was confined to phloem companion cells. In Pentland Crown, the decrease in PLRV antigen concentration in leaf mid-veins and petioles, relative to that in Maris Piper, was proportional to the decrease in number of PLRV-containing companion cells; this decrease was greater in the external phloem than in the internal phloem. The spread of PLRV infection within the phloem system seems to be impaired in the resistant genotypes. Green peach aphids (Myzuspersicae) acquired < 2800 pg PLRV/aphid when fed for 4 days on infected field-grown Maris Piper plants and < 58% of such aphids transmitted the virus to Physalis floridana test plants. In contrast, aphids fed on infected Pentland Crown plants acquired <120 pg PLRV/aphid and <3% transmitted the virus to P. floridana. The ease with which M. persicae acquired and transmitted PLRV from field-grown Maris Piper plants decreased greatly after the end of June without a proportionate drop in PLRV concentration. Spread of PLRV in potato crops should be substantially decreased by growing cultivars in which the virus multiplies to only a limited extent.