樱桃病毒A北京分离物外壳蛋白的原核表达及抗血清制备

根据前期研究获得的樱桃病毒A北京分离物(Cherry virus Aisolate Beijing,CVA-BJ)外壳蛋白基因序列设计引物,将此基因克隆到原核表达载体pET-28a后转化大肠杆菌BL21(DE3),以终浓度为1 mmol/L的IPTG进行诱导表达。Ni珠吸附法纯化表达产物后免疫家兔制备抗血清。间接ELISA测得抗血清效价为1∶2048。以纯化后的蛋白和樱桃叶片总蛋白为检测材料,Western blot分析表明抗血清具有高度特异性。间接ELISA方法检测樱桃病叶,结果呈阳性,与RT-PCR检测结果一致。表明制备的抗血清可用于感病樱桃样品中CVA的检测。     

我国部分地区樱桃病毒病害初步调查和病原检测

对山东泰安、辽宁大连和北京的樱桃病毒病发生情况进行调查,发现8个果园/栽培区均有病毒病发生,主要症状为叶片皱缩、畸形、卷叶、花叶、植株矮缩等。采集20份样品,利用12种病毒的引物进行RT-PCR检测。结果表明,在样品中扩增出与樱桃病毒A(Cherry virus A,CVA)、李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),李矮缩病毒(Prune dwarf virus,PDV)、李树皮坏死与茎痘伴随病毒(Plum bark necrosis stem pitting-associated virus,PBNSPaV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃小果病毒-1(Little cherry virus-1,LChV-1)预期大小一致的目的片段;序列分析表明,与GenBank中注册所测的病毒核苷酸序列均具有较高的一致性。其中,大连、泰安和北京样品均检测到CVA;大连和北京样品中检测到PNRSV和PDV;北京样品中检测到PBNSPaV;大连苗木样品枝条中检测到CGRMV和LChV-1。这是在我国樱桃上首次检测到LChV-1。     

Monoclonal antibodies to specific components of the Dutch elm disease pathogen Ophiostoma ulmi

The potential of polyclonal antisera and monoclonal antibodies to differentiate the EAN and NAN aggressive subgroups of Ophiostoma ulmi was explored. Polyclonal antisera, when tested by ELISA, cross-reacted widely with unrelated species and failed to distinguish between the two aggressive subgroups but small quantitative differences were found, particularly between antigens secreted overnight, by EAN and NAN germlings. Monoclonal antibodies were raised in mice against mycelial homogenates. From two fusions, 33 cell lines were raised that secreted antibodies positive for O. ulmi. Approximately one third were non-specific; 11 were specific either to species or subspecies. Two cell lines differentiated mycelial antigens of the aggressive isolates of O. ulmi from those of the non-aggressive subgroup, but not antigens from surface washings. Only quantitative differences were detected between the EAN and NAN aggressive subgroups. Almost all the monoclonal antibodies and antiserum recognized antigens present in surface washings of cultures on solid medium, in cell-free extracts of mycelial homogenates, in cell-free culture fluids, and in substances secreted overnight by germinating spores. Specific detection of such molecules promises to provide a highly sensitive mechanism for studying early pathogen/host plant interactions. Most of the monoclonal antibodies appeared to have potential diagnostic value; they gave readings twofold to tenfold higher with extracts from diseased than from healthy tissue. However, one cell line that secreted antibodies specific to O. ulmi cross-reacted strongly with extracts of healthy tissue.     

Development of monoclonal antibody-based immunological assays for the detection of live propagules of Rhizoctonia solani in soil

Mouse monoclonal antibodies (mAbs) and rabbit (polyclonal) antiserum were used to develop DIAGNOSTIC-ELISA, double-antibody-sandwich-ELISA (DAS-ELISA), DIP-STICK and immuno-fluorescence colony staining immunoassays for the specific detection of Rhizoctonia solani in soil. mAbs were raised against an anastomosis group 4 isolate of R. solani. Mice were immunized using either phosphate-buffered saline (PBS) suspensions of lyophilized mycelium plus Quil A adjuvant, or with a solubilized acetone precipitate prepared from cell-free surface washings from solid slant cultures. Polyclonal antisera were raised in rabbits using PBS suspensions of lyophilized mycelium and Quil A adjuvant. Hybridoma supernatants and rabbit antisera were screened by ELISA. Four of the cell lines raised produced mAbs that were species-specific. They recognized antigens from R. solani by ELISA and immunofluorescence, but not other related or unrelated species of soil-borne fungi. The remaining cell line produced mAbs that cross-reacted slightly, by ELISA, with antigens from R. cerealis. These mAbs did not recognize R. cerealis by immunofluorescence, or other related or unrelated soil-borne fungi, by ELISA.     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

Preliminary account of the phytosanitary status of stone-fruit trees in Lebanon

Investigations were carried out in the main stone-fruit growing areas of Lebanon to assess the phytosanitary condition of commercial orchards. The presence of virus and virus-like diseases and their identification was ascertained through: (i) field surveys, (ii) sap transmission to herbaceous hosts, (iii) graft transmission to woody indicators; and (iv) ELISA and IEM tests. The mean infection level was 25%. ranging from 5% in apricot to 45% in cherry. The following viruses were identified: apple chlorotic leaf spot trichovirus (ACLSV), prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered.     

Serological reaction of beet western yellows and potato leafroll viruses in ELISA

The detection of potato leafroll virus (PLRV) and beet western yellows virus (BWYV) and their serological relatedness were investigated by the double sandwich ELISA (DS). In both pure preparations and crude plant extracts, an unequivocal detection of each of these viruses was obtained by DS, provided the homologous antisera were used. No detection was achieved when the heterologous antisera were used, although purified virus could be detected when using the heterologous antisera for coating, providing the homologous antisera were used as conjugates. Therefore, for routine screening of BWYV or PLRV infection in plants, the DS method with homologous antisera can be used. However, it does not seem that BWYV infection in potato could be detected by the routine DS aimed for PLRV screening. In immunosorbent electron microscopy, PLRV and BWYV could be easily detected by coating grids with either homologous or heterologous antiserum. This and the possibility of trapping each virus in ELISA plates with the heterologous antiserum indicate serological cross-reactivity between PLRV and BWYV.     

Sanitary status of stone-fruit trees in East Anatolia (Turkey) with particular reference to apricot

Field surveys were carried out in the main stone-fruit-growing areas of East Anatolia (Turkey) to assess the sanitary status of varietal collections, mother blocks and commercial orchards. The presence of virus and virus-like diseases was ascertained by enzyme-linked immunosorbent assay (ELISA), sap transmission to herbaceous hosts, graft transmission to peach cv. GF305 and molecular hybridization tests. A total of 1019 samples was tested by ELISA (859 apricot, 120 cherry, 21 almond and 19 peach). The sanitary status of apricot was extremely satisfactory, as the infection level was less than 0.3%. Cherry and almond, however, showed 21% and 33% infection respectively. The viruses identified were apple chlorotic leaf spot trichovirus (ACLSV), prune dwarf ilarvirus (PDV) and prunus necrotic ringspot ilarvirus (PNRSV). The commonest virus was PDV. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV) and the nepoviruses tomato black ring (TBRV), raspberry ringspot (RpRSV), strawberry latent ringspot (SLRV), cherry leaf roll (CLRV), arabis mosaic (ArMV) and tomato ringspot (ToRSV) were not encountered. Peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) were not detected either.     

Development of ELISA for Ophiostoma ulmi using antigen-coated wells

A simple sensitive ELISA test has been developed that detects either antibodies to, or antigens of, the Dutch elm pathogen, Opiostoma ( Ceratocystis ) ulmi. The dilution end point for antisera raised in mice againt mycelial antigens was 10^-4. The minimum amount of antigen detected in wells passively coated with antigens solubilized in phosphate-buffered saline was 500 pg/ml. The assay proved an effective method for rapidly screening large numbers of hybridoma supernatants for monoclonal antibodies that recognize O. ulmi antigens and it has also been used to detect fungal antigens in saline extracts of diseased tissue. Non-specific binding of preimmune antiserum to plant extracts was markedly higher in extracts from diseased than non-diseased tissue. Production by the diseased host or by the pathogen in vivo of a protein A or lectin-like molecule that binds non-specifically to immunoglobulins is postulated.     

Prunus necrotic ringspot virus Early Invasion and Its Effects on Apricot Pollen Grain Performance

ABSTRACT The route of infection and the pattern of distribution of Prunus necrotic ringspot virus (PNRSV) in apricot pollen were studied. PNRSV was detected both within and on the surface of infected pollen grains. The virus invaded pollen during its early developmental stages, being detected in pollen mother cells. It was distributed uniformly within the cytoplasm of uni- and bicellular pollen grains and infected the generative cell. In mature pollen grains, characterized by their triangular shape, the virus was located mainly at the apertures, suggesting that PNRSV distribution follows the same pattern as the cellular components required for pollen tube germination and cell wall tube synthesis. PNRSV also was localized inside pollen tubes, especially in the growth zone. In vitro experiments demonstrated that infection with PNRSV decreases the germination percentage of pollen grains by more than half and delays the growth of pollen tubes by approximately 24 h. However, although PNRSV infection affected apricot pollen grain performance during germination, the presence of the virus did not completely prevent fertilization, because the infected apricot pollen tubes, once germinated, were able to reach the apricot embryo sacs, which, in the climatic conditions of southeastern Spain, mature later than in other climates. Thus, infected pollen still could play an important role in the vertical transmission of PNRSV in apricot.     

樱桃小果病毒1号胶体金免疫层析试纸的研发与应用

近年来，病毒病在甜樱桃主产区的发生呈上升趋势，制约了甜樱桃产业升级和发展。能够侵染甜樱桃的病毒种类中，樱桃小果病毒1号(little cherry virus 1,LChV-1)导致樱桃果实缩小，彻底丧失经济价值，对甜樱桃产量影响较大。LChV-1通常具有潜伏侵染的特性，在樱桃苗期难以通过症状进行判断。同时，多数品种对LChV-1敏感，因此该病毒的早期检测对甜樱桃的生产尤为重要。传统的病毒检测手段需要专业仪器，不能满足田间快速检测的需求。本研究获取了LChV-1外壳蛋白的多克隆抗体，并研发了一种能够准确检测LChV-1病毒的胶体金免疫层析试纸，确定了胶体金的最佳抗体标记浓度为0.24 mg/mL,检测线最佳重组蛋白浓度为0.25 mg/mL,质检线最佳羊抗兔IgG抗体浓度为0.04 mg/mL。运用该试纸检测只需10～15 min,检测时间短、成本低、结果容易判断，可用于田间快速检测。本研究为樱桃小果病的综合防控提供了有效的监测和检测手段。     

Detection of spore balls of Spongospora subterranea on potato tubers by enzyme-linked immunosorbent assay

A rabbit was immunized with a homogenate of spore balls (cystosori) of Spongospora subterranea which also contained some potato tuber debris. The resultant polyclonal antiserum contained antibodies which reacted against extracts of spore balls and healthy tubers. Antibodies to the tuber debris were removed by incubating the blood serum with an extract from a S. subterranea -free tuber. In enzyme-linked immunosorbent assay (ELISA), the γ-globulin fraction of the absorbed antiserum reacted with dilute tuber extracts containing the equivalent of as little as 0·08 spore balls per ml. The reaction with spore balls was inhibited in high concentrations of tuber sap. Extracts of peel from symptomless tubers which had been stored in contact with scabbed tubers also reacted with the γ-globulin, presumably because the symptomless tubers became contaminated with spore balls. However, spore balls in soil were only weakly detected when high numbers were present. The γ-globulin did not react with the plasmodial stage of the pathogen, or with 15 other micro-organisms tested, including resting spores of Plasmodiophora brassicae.     

Purification and serology of virions of impatiens necrotic spot tospovirus

Impatiens necrotic spot tospovirus (INSV) virions were purified using a procedure devised for tomato spotted wilt tospovirus (TSWV) from systemically infectedNicotiana benthamiana plants grown at 33 °C day/26 °C night and a photoperiod of 14 hours. With plants grown at 24/18 ° C purification was unsuccessful. In SDS-PAGE the protein pattern of INSV was similar to that reported for TSWV, except the appearance of a single G2 protein band. A polyclonal antiserum, prepared against virions, reacted in Western blots with INSV nucleoprotein and glycoproteins but only with TSWV glycoproteins. In DAS ELISA the antiserum reacted with both INSV and TSWV infected plant sap and, after absorption with TSWV, only with INSV. In TAS ELISA the antiserum trapped both INSV and TSWV nucleoproteins and glycoproteins as detected by specific monoclonal antibodies, and, after absorption with TSWV, only the homologous proteins. This appears to be the first report of the purification of INSV virions and the production of an antiserum reacting with both nucleoprotein and glycoprotein antigens.     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato viruses A and Y in potato leaves and sprouts

With the enzyme-linked immunosorbent assay (ELISA) potato virus A (PVA) could be detected reliably in potato sprouts, especially when these were young and sappy. The detection of this virus in leaves of glasshouse-grown potato plants was less reliable. The tobacco veinal necrosis strain of potato virus Y (PVY^N) was readily demonstrated in foliage of glass-house-grown potato plants using an antiserum to this strain. Plants infected with the common strain (PVY^O) did not react in ELISA with this antiserum. In young sappy sprouts, using the PVY^N antiserum, PVY^N could be detected reliably when samples with PVY^O were excluded, as the reaction of samples infected with the latter virus was intermediate between PVY^N-diseased and PVY-free samples. PVY was also detected in plants inadvertently infected during the experiments.     

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.     

Assessment of the inheritance of resistance and tolerance in cherry (Prunus sp.) to Blumeriella jaapii,the causal agent of cherry leaf spot

Cherry leaf spot (CLS), caused by Blumeriella jaapii, is a serious fungal disease of sour cherry (Prunus cerasus). Cultivar Montmorency, the major cultivar grown in the United States, is highly susceptible to CLS. As many as 10 fungicide sprays can be required each growing season to combat this disease; therefore, developing CLS‐resistant cultivars is a top breeding priority. Germplasm previously reported to be resistant or tolerant to CLS was acquired and incorporated into the sour cherry breeding programme at Michigan State University (MSU) and included three cherry species: sour cherry, sweet cherry (P. avium), and the wild species P. canescens. This study aimed to: (i) compare the CLS disease progression profile of the susceptible cultivar Montmorency with those of the resistant and tolerant germplasm; and (ii) gain an understanding of the inheritance of these resistance and tolerance traits by evaluating the host response of progeny individuals belonging to families derived from this germplasm. Significant differences were observed between the susceptible Montmorency and the tolerant and resistant accessions in their response to CLS and its progression during the growing season. Evaluation of the CLS host responses of progeny individuals derived from this germplasm supported a dominant two‐gene model for P. canescens‐derived resistance and a recessive gene model for sweet cherry‐derived tolerance. These insights into disease progression and trait inheritance improve the efficiency and potential success of breeding sour cherry cultivars with durable resistance to CLS.     

Infection of alfalfa pollen bySclerotinia sclerotiorum

Blossom blight, caused bySclerotinia sclerotiorum, has become an important disease of alfalfa (Medicago sativa L.) in seed production areas of western Canada. Studies using light microscopy and scanning and transmission electron microscopy revealed that pollen grains of alfalfa are susceptible to infection byS. sclerotiorum. Ascospores ofS. sclerotiorum germinated readily in water with or without pollen grains. Examinations of ascospore—pollen mixtures incubated at room temperature (20–22°C) for 5 days revealed that numerous pollen grains were infected byS. sclerotiorum by direct hyphal penetration through the equatorial germinative pores or through the exine and intine layers of the pollen wall without the formation of infection cushions or appressoria. After penetration, hyphae ramified within the pollen grains, causing plasmolysis of the cytoplasmic membrane and eventual disintegration of the pollen cytoplasm. The study suggests that alfalfa pollen may play a role in the epidemiology of blossom blight in alfalfa.