巨细胞病毒中性红斑定量试验与高滴度病毒的制备

在巨细胞病毒(CMV)的研究中常需对病毒定量。CMV需低滴度传代,否则会产生没有感染性的缺损病毒颗粒;CMV的抗原性受其感染量的影响;检测CMV中和抗体或纯化病毒都需具备病毒空斑定量基础。另外,制备高感染滴度的无细胞病毒(游离病毒)是对CMV进行分子生物学研究的前提。本文建立了CMV微量板法中性红斑定量技术并比较了几种制备无细胞CMV的方法。     

特异扩增BBTV基因组Ⅲ、Ⅳ、Ⅰ编码区部分序列及其在检测中的应用

香蕉束顶病(BBTD)是香蕉植株的一种毁灭性病害,正在世界(包括中国)的许多香蕉种植区蔓延[1～7].其病原物为香蕉束顶病毒(Banana Bunchy Top Virus,BBTV),被列为我国第三类检疫对象.目前生产上主要采用培育脱毒组培苗来防治BBTD的发生,因此建立一种能快速、灵敏、特异地检测BBTV的方法就显得很重要.国内现在大多采用ELISA方法,但其灵敏度不够高,且需要制备特异性强的抗血清,否则较易出现假阳性.     

卫星RNA研究新进展：结构与功能表达

植物病毒卫星通常是指那些如果没有特定辅助病毒的帮助就不能复制,而它本身对于辅助病毒的复制是不必需的,且与辅助病毒的基因组无序列同源性的一类RNA分子。到目前为止,已报道有六组共26种植物病毒带有卫星。这些植物病毒卫星有的具有编码自身外壳蛋白的遗传信息,称卫星病毒;有的无此编码功能称为病毒卫星RNA。病毒卫星RNA在其发现的早期,其生物学功能尚不为人重视。然而,七十年代早期,在法国阿尔萨斯爆发的由黄瓜花叶病毒(Cucumber Mosaic Virus,CMV)侵染引起的番茄作物坏死病被确定与CMV卫星RNA有关,至此,关于卫星RNA的生物     

二羟丙氧甲基鸟苷(DHPG)体外抑制单纯疱疹病毒增殖的研究

二羟丙氧甲基鸟苷[9-(1,3-dihydroxy-2-Propoxymethyl)guanine,简称DHPG]是近几年合成的一种结构与无环鸟苷(ACV)类似的抗病毒化合物(图1)。已证明,它对单纯疱疹病毒1、2型(HSV-1、2),巨细胞病毒(CMV)、水痘带状疱疹病毒(VZV)具有较强的选择性抑制作用,尤以对CMV的作用,被认为是最有希望的抗CMV药物之     

黄瓜花叶病毒和烟草花叶病毒在双抗转基因线辣椒内的增殖

本试验以转化CMV-CP和TMV-CP基因的转基因线辣椒纯合系植株作为研究试材,比较了单独 或混合接种CMV和TMV后,转化线辣椒的抗病性表达特点,并测定了两种病毒在植株体内的病 毒含量.结果表明转化线辣椒不仅能抵抗CMV和TMV的单独侵染,而且还能抵抗CMV和TMV的 复合侵染.转化线辣椒表现为系统症状延迟出现7-15d,显症株率和病害严重度级别大幅度降低, CMV和TMV在接种叶、新生叶中的病毒含量明显减低.转基因线辣椒原生质体作为研究试材接 种CMV,测定病毒含量结果表明CMV病毒的增殖在转基因线辣椒原生质体内受到明显抑制. 在CMV接种浓度为40μg/mL,感染原生质体48h后,CP(-)植株原生质体内CMV是CP(+)的4.2倍 .这一结果揭示了转基因线辣椒具有抑制病毒增殖的抗病性.     

侵染天南星科植物病毒的分子鉴定及其生态学研究

通过病毒粒子部分提纯和形态学观察,发现侵染我国南方天南星科植物的病毒主要有线状和球状两种形态．经病毒基因组序列分析确定线状病毒为芋花叶病毒(DsMV);经血清学反应和序列分析确定球状病毒为黄瓜花叶病毒(CMV)．CMV CP基因序列同源性分析的结果表明,侵染天南星科的CMV是相对独立的种内变异类型,归属于亚组L同时,CMV存在对天南星科植物的适应性变异．对采自我国海南、湖南、浙江、上海等地的126个天南星科植物样品进行RNA核酸斑点杂交检测,获得病毒检测结果。海南省样品DsMV的检出率为73．3％,CMV的检出率为46．7％;湖南省样品DsMV的检出率为100％,CMV的检出率为38.5％;浙江省样品DsMV的检出率为93．0％,CMV的检出率为7．0％;上海市样品DsMV的检出率为100％,尚没有检测到CMV,首次证实了自然条件下CMV作为天南星科植物主要病毒的存在,在我国南方地区,该病毒对天南星科植物的自然侵染受到气候、季节和寄主等生态因子的影响。DsMV则在天南星科植物上普遍存在。     

侵染香蕉的黄瓜花叶病毒分离物衣壳蛋白的肽链分析

香蕉花叶病为华南香蕉生产的重要限制因素之一［‘1。特别是随着组培苗应用面积的扩大,发生越来越严重,一般地块发病率为20—40％,个别重病地块高达90％,损失严重。此病病原虽在1930年【’1就被鉴定为黄瓜花叶病毒（Cucumbermosaicvirus,CMV）,但至今对其研究不多。从有限的文献看,大多数作者【’·’嘟认为其病原为CMV的一个株系,即香蕉株系。但也有认为为二个株系［‘］、或三个株系的［’］。因此有关病原的调查鉴定就成为当前迫切需要解决的一个重要问题。1991～1993年我｛I’J在广东省广州市天河和黄埔区,以及顺德、番禹…     

两株黄瓜花叶病毒卫星RNA的竞争与共存研究

通过体外转录方法 ,将大小分别为 36 9nt和 385nt的 2个黄瓜花叶病毒 (Cucumbermosaicvirus,CMV)的卫星RNAYi和Yns共同与不含卫星的辅助病毒株CMV_CNa进行假重组 ,接种CMV系统寄主心叶烟。结果表明 :在接种5d的接种叶上同时检测到卫星RNA_Yi和卫星RNA_Yns;在系统叶上 ,接种 5d和 10d亦可同时检测到 2株卫星 ;但接种 15d ,在系统叶组织中只检测到卫星RNA_Yi。再将接种 5d的接种叶扩大接种到几种不同的指示植物后 ,经dsRNA抽提 ,也只获得 1条与卫星RNA_Yi大小相符的条带。通过假重组病毒株中分别获得卫星RNA并测序 ,确定2个卫星RNA的序列没有变化。卫星RNA_Yns和Yi在辅助病毒CMV_CNa作用下 ,表现出明显的竞争性 ,它们在辅助病毒中不能形成稳定的共存关系。     

利用表面等离子体共振仪检测黄瓜花叶病毒

目的：研究一种便捷、高效地检测黄瓜花叶病毒（CMV）的方法。方法：利用表面等离子体共振（SPR）技术检测CMV。首先用11-MUA修饰SPR金片,再用EDC／NHS活化,之后通过NHS酯基与CMV抗体结合,用BSA封闭未结合的NHS酯基。将SPR金片装入SPR仪,通入待检样品,通过折射率变化实时监测实验过程。结果：该方法检测CMV的灵敏度能够达到10ng／mL,具有良好的特异性,与同属的花生矮化病毒、番茄不孕病毒无交叉反应。结论：建立的SPR方法操作简单、灵敏度高、特异性好,是一种新的高效检测CMV的方法。     

巨细胞病毒的生物学的特性

巨细胞病毒(Cytomegalovirus,CMV)属疱疹病毒科,分布十分广泛,各种动物均有种特异的CMV。早在发现此病毒之前,已有人在肾病理组织切片中发现巨细胞,这种巨细胞核大,周围有亮区,胞核及浆内均可见包涵体。这类病变细胞常与婴儿肝、脾肿大、肺炎等有关。直到1949年Enders建立细胞培养技术后,CMV才培养成功。1954年Smith和weller分别从死亡婴儿的唾液腺及巨细胞包涵体病婴儿尿中分离到了CMV。现公认AD169为CMV的代表株。     

Potential implications of biopriming in banana (Musa spp) plantlets against banana bunchy top virus (BBTV)

Abstract

Transplant media as a means for the introduction of biological agents is currently being investigated in a variety of crops. This study aimed to investigate the impact of microbial inoculation in micropropagated banana plantlets to enhance their resistance against Banana bunchy top virus (BBTV). Virus indexed micropropagated plantlets of banana were subjected to root colonization followed by foliar spraying with bacterial strains Pseudomonas fluorescens Pf1, CHA0 and Bacillus subtilis EPB22 during primary and secondary hardening stage in the nursery, at the time of repotting and 3 months after planting in the pot. Microbe inoculated plantlets showed enhanced PR proteins and defense enzymes besides reducing banana bunchy top disease incidence under glasshouse condition. The results indicated the effective use of beneficial microbes in reducing the disease incidence of BBTV in tissue culture banana plantlets. In addition, the molecular characterization of endophytes isolated from banana plantlets, using SDS-PAGE and RAPD-PCR revealed that endophytes were categorized into two distinct groups. These results emphasize the significance of microorganisms in protection of young plantlets from transplanting stresses in field. Further, the use of beneficial microorganisms instead of chemicals sustains an ecological niche in the agricultural ecosystem.     

Identification and evaluation of two diagnostic markers linked to Fusarium wilt resistance (race 4) in banana (Musa spp.)

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (FOC4) results in vascular tissue damage and ultimately death of banana (Musa spp.) plants. Somaclonal variants of in vitro micropropagated banana can hamper success in propagation of genotypes resistant to FOC4. Early identification of FOC4 resistance in micropropagated banana plantlets is difficult, however. In this study, we identified sequence-characterized amplified region (SCAR) markers of banana associated with resistance to FOC4. Using pooled DNA from resistant or susceptible genotypes and 500 arbitrary 10-mer oligonucleotide primers, 24 random amplified polymorphic DNA (RAPD) products were identified. Two of these RAPD markers were successfully converted to SCAR markers, called ScaU1001 (GenBank accession number HQ613949) and ScaS0901 (GenBank accession number HQ613950). ScaS0901 and ScaU1001 could be amplified in FOC4-resistant banana genotypes (“Williams 8818-1” and Goldfinger), but not in five tested banana cultivars susceptible to FOC4. The two SCAR markers were then used to identify a somaclonal variant of the genotype “Williams 8818-1”, which lost resistance to FOC4. Hence, the identified SCAR markers can be applied for a rapid quality control of FOC4-resistant banana plantlets immediately after the in vitro micropropagation stage. Furthermore, ScaU1001 and ScaS0901 will facilitate marker-assisted selection of new banana cultivars resistant to FOC4.     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

Improved detection methods for fruit tree phytoplasmas

Phytoplasmas infecting fruit trees are considered quarantine organisms in Europe and North America. Detection often is hampered by their extremely irregular distribution in host plants. A sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. PCR methods require tedious preparation of DNA; also, the available primers are highly specific and exhibit some homology to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time required. We also developed new primers using rDNA sequence information from an Austrian isolate of European Stone Fruit Yellows (ESFY). These primers operate at high annealing temperatures and, thus, increase the specificity and decrease the risk of false positives. The primers could reliably detect the European phytoplasmas (AP, ESFY and PD) within a collection of isolates maintained in micropropagated periwinkle. Thus, they are suitable as general primers for phytoplasma detection. The primers also can be used for strain identification by direct PCR followed by RFLP analysis as demonstrated with micropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-scale testing of apple materialin vivo andin vitro.     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

Soft rot inciting <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> (syn. <Emphasis Type="Italic">Erwinia carotovora</Emphasis>) is unlikely to be transmitted as a latent pathogen in micropropagated banana

This study was undertaken to ascertain if the soft rot inciting Pectobacterium carotovorum/Erwinia carotovora would pass through the micropropagated bananas as a latent pathogen and cause disease during or post acclimatization. In vitro cultures of ‘Grand Naine’ were exposed to the pathogen by providing 100 μl of inoculum (0.001–1.0 at OD₆₀₀ nm) at the lower leaf axil. These cultures showed a gradual development of soft rot symptoms coupled with obvious bacterial colony growth on banana proliferation medium and consequent plant mortality within a month irrespective of the inoculum level employed. Plants carried forward to acclimatization following inoculation in vitro failed to establish ex vitro. Monitoring the normal field-grown suckers at culture initiation through PCR screening employing soft rot Erwinia primers did not show the amplification of the 119-bp fragment as seen with the pure cultures of pathogen. Further testing of micropropagated banana plants through soil inoculation, in vitro culturing and PCR screening ruled out the possibility of the pathogen surviving in micropropagated stocks in latent form as the organism outgrew and killed the cultures. It emerged that the infection possibly takes place in the nursery. This information will be of particular value for the plant tissue culture industry, plant pathologists and quarantine agencies.     

Endogenous Levels of Cytokinins, Indoleacetic Acid, Abscisic Acid, and Pigments in Variegated Somaclones of Micropropagated Banana Leaves

The banana (Musa spp. AAA) micropropagation shows a high incidence of off-types, among whose variegated plants are very common. Endogenous levels of growth regulators and pigment content were measured in normal and variegated leaves of the micropropagated banana plants growing in a greenhouse. Growth regulators were separated by high pressure liquid chromatography and submitted to enzyme-linked immunosorbent assay for quantification. Pigment content was measured using the colorimetric method. Green leaves contained 1.9 and 10 times more cytokinins compared with green and yellow sectors of variegated leaves, respectively. The levels of indoleacetic acid in normal leaves were significantly higher than those found in green and yellow sectors of variegated leaves; however, the levels of abscisic acid were lower in normal leaves. The lower content of chlorophylls in variegated leaves coincided with decreased endogenous levels of cytokinins, which indicated that variegation in banana leaves may be associated with alterations in the metabolism of this growth regulator. Received December 3, 1997; accepted February 2, 1998     

Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.     

A complete protocol using conductance for rapid detection of salmonellas in confectionery materials

Increased confidence in conductimetric detection of salmonellas was achieved by combining a bacteriophage-based test with use of a selenite cystine trimethylamine oxide dulcitol medium and a modified lysine decarboxylase broth. All 81 Salmonella isolates tested were detected and few of the 39 non-salmonellas gave false positives. Results from the screening of 43 inoculated product samples further support the use of this simple, rapid method for routine salmonella testing in the food industry.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.