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1.
An Italian isolate of plum pox potyvirus (PPV) from apricot, Ispave 17, was used as antigen for production of monoclonal antibodies. Six clones secreting specific antibodies to PPV were obtained. All these monoclonal antibodies were used to test a collection of different Italian PPV isolates, collected from plum, apricot and peach orchards, and other European isolates (including PPV-D and PPV-M serotypes), using DAS-ELISA, SDS-PAGE, western blot and GIEM. In western blot analysis, the PPV-M and PPV-D coat protein, detected directly from crude peach GF305 extracts, showed different electrophoretic mobility, the coat protein of PPV-M being slightly larger than that of PPV-D. ELISA tests, performed with fixed dilutions of antibodies and limiting dilutions of clarified samples, showed with some monoclonal antibodies a marked difference between PPV-M and PPV-D strains, at ratios greater than 1:40 (w/v). Also in GIEM some monoclonal antibodies gave a good labelling reaction only with PPV-D serotype. With the help of this differentiation, it was found that all Italian isolates tested were of the D serotype and none of the severe M strain of PPV, which has not been reported in Italy. 相似文献
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M. CAMBRA M. ASENSIO M. T. GORRIS E. PREZ E. CAMARASA J. A. GARCI J. J. MOYA D. L
PEZ-ABELLA C. VELA A. SANZ 《EPPO Bulletin》1994,24(3):569-577
Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity. 相似文献
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Plum pox potyvirus on sour cherry in Moldova 总被引：1，自引：0，他引：1
Plum pox potyvirus (PPV) was identified in six cultivars of sour cherry in the collection orchard of the Moldavian Horticultural Research Institute. Study of biological properties of the sour cherry isolate in herbaceous indicators showed its similarity or identity with the PPV isolate of Van Oosten and a significant difference from isolates widespread in Moldova. A purified viral preparation was used to develop antiserum with a working titre of 1:1024. Comparative serological examination of the sour cherry and conventional plum PPV isolates using ELISA, ISEM and SDS-PAGE of the protein capsid could not differentiate these isolates. The sour cherry isolate was transferred to plum resulting in weak but distinctive PPV symptoms in susceptible cv. Sopernitsa. 相似文献
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Six isolates of plum pox potyvirus from different Mediterranean countries were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), peptide mapping and Western blotting after improved purification of virions using a protease inhibitor cocktail that reduced coat protein degradation. One isolate (Spanish isolate 3.3 from plum) differed from the others in possessing a smaller coat protein (approximately 34 instead 36 kDa) with a possible deletion in the surface-exposed amino-terminal region. Infectivity of the viruses after proteolysis, assessed using a local lesion host, was significantly reduced. Protease digestion conditions were established to generate a 28 kDa resistant core of the viral coat protein. Such conditions (longer incubation times or an increase in the enzyme concentration) differed from the milder ones reported for other potyviruses. Implications of the results in relation to the production and screening of virus-specific monoclonal antibodies are discussed. 相似文献
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ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants. 相似文献
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Kensaku Maejima Hideo Hoshi Masayoshi Hashimoto Misako Himeno Takeshi Kawanishi Ken Komatsu Yasuyuki Yamaji Hiroshi Hamamoto Shigetou Namba 《Journal of General Plant Pathology》2010,76(3):229-231
For the first time, plum pox virus (PPV) has been detected in commercial Japanese apricot (Prunus mume) trees in Tokyo, Japan. These trees had ringspot or mottle on leaves, color breaking of petals and, occasionally, mild ringspots
and malformation on fruits. The virus was identified based on the morphology of virus particles, serology, and RT-PCR. The
amplified nucleotide fragment shared 100% identity with a partial coat protein gene of PPV-D isolates. 相似文献
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M. RAVELONANDRO R. SCORZA A. CALLAHAN J. CORDTS M. MONSION M. FUCHS R. DELBOS J. BACHELIER D. GONSALVES J. DUNEZ 《EPPO Bulletin》1994,24(3):713-719
Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R2 progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums. 相似文献
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H. LECOQ M. RAVELONANDRO C. WIPF-SCHEIBEL M. MONSION B. RACCAH J. DUNEZ 《EPPO Bulletin》1994,24(3):555-559
Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment. 相似文献
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Pál Salamon László Palkovics 《European journal of plant pathology / European Foundation for Plant Pathology》2002,108(9):903-907
Plum pox virus (PPV) was found naturally infecting blackthorn (Prunus spinosa L.) plants in different regions in Hungary. The virus was identified on the basis of reactions with indicator plants, by DAS–ELISA tests and by RT–PCR. PPV isolated from blackthorn (PPV-BT-H) caused yellow lesions with a necrotic centre on Chenopodium foetidum L. indicating that it belongs to the intermediate pathotype. The coat protein gene of the blackthorn isolate was cloned, sequenced and compared with other PPV sequences. The BT-H isolate and the Hungarian plum isolate SK68 belong to different subclusters of the M group in contrast to the Hungarian almond isolate (PA) which belongs to the D group. Detecting PPV on blackthorn demonstrated that this plant may be an important source and reservoir for PPV in Hungary. 相似文献
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Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K1 with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K1 typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period. 相似文献
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葡萄A病毒（Grapevine virus A,GVA）为线性病毒科（Flexiviridae）葡萄病毒属（Vitivirus）的代表种,是葡萄皱木复合病（rugose wood complex disease）的重要病原之一,可引起葡萄嫁接成活率下降、春季萌芽延迟、生长减弱甚至衰退死亡等危害\[1,2\]。GVA为线状单链RNA病毒,基因组共编码5个开放阅读框（ORF1\|5）,其中ORF4 编码外壳蛋白（coat protein, CP）,是病毒粒子包裹和系统移动所必需的功能蛋白\[3,4\]。GVA自然寄主为葡萄,机械摩擦可侵染本氏烟等草本寄主\[2\],由于嫁接和无性繁殖材料调运等因素造成该病毒远距离传播,目前在世界多个国家和地区均有发生。 相似文献
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应用改进的多克隆抗体Western blot技术研究柑桔速衰病毒蛋白 总被引：1，自引：0，他引：1
本文利用多克隆抗体发展了一种无背景的Western blot技术并用于研究柑桔速衰病毒(CTV)的蛋白。结果表明,利用CTV兔多克隆抗体1212和1052发展的Western blot技术可以检测到感染CTV的墨西哥酸橙或Citrus excelsa植株内CTV的4种蛋白P1、P2、P3和P4。在健株或病株内杂交反应均无非特异性背景。从不同寄主上分离到的CTV不同分离物的蛋白条带是不同的。利用1212和1052抗体均可以检测到感染6个CTV分离物的墨西哥酸橙幼苗内的P1、P2和P3。利用1052抗体能检测到感染严重型分离物T36、T3和Mm2的墨西哥酸橙幼苗内微弱的P4,但感染轻型分离物T30、T26和T4的幼苗内则检测不到。利用1212抗体检测不到P4。在C. excelsa内,1212和1052抗体均能检测到感染所有分离物的病株内的P1。在感染T3、T26、T4或Mm2的病株内能检测到P2,但在感染T30和36分离物的病株内则检测不到。在感染T36、T3、T26、T4和Mm2的病株内可检测到P3,但在感染T30的病株内则检测不到。在大多数植物内,P1、P2、P3和P4的分子量分别约为25kDa,24kDa,21kDa和18kDa。在感染T36分离物的C. excelsa植株体内,P1和P3的分子量分别约为27kDa和22kDa,比感染其它分离物的C. excelsa和墨西哥酸橙内的P1和P3分子量略大。P1、P2和P3的分子量与利用单克隆抗体检测的CP,CP1和CP2的分子量相等。因此,P1、P2和P3可能是CTV的外壳蛋白CP,CP1和CP2。P4的特性不清楚。研究结果也表明,利用特异性的多克隆抗体进行的Western blot是研究CTV的一种有用的技术。应用该技术,病株内不同的CTV分离物或株系就可以通过特异性蛋白条带区分开来。 相似文献
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Pascal Gentit René-Pierre Delbos Thierry Candresse Jean Dunez 《European journal of plant pathology / European Foundation for Plant Pathology》2001,107(5):485-494
A pathogen was transmitted from apricot trees showing symptoms of viral infection to GF305 peach seedlings which reacted by stunting, shortened internodes and chlorotic mottling. The agent was transmitted to cherry, apricot, peach and plum by grafting and to several herbaceous hosts by mechanical inoculation. Isometric nepovirus-like particles of 30–31 nm diameter extracted from infected Chenopodium quinoa sedimented as two peaks in sucrose gradients. These particles contained two single stranded RNAs of approximately 5.9 and 7.9 kb, and a single coat protein subunit of 53.7 kDa. No cross-reactions were observed with a number of nepoviruses infecting fruit trees. Inoculation of purified particles to herbaceous or woody hosts reproduced the same symptoms caused by the original isolate. Sequencing of a 2.2 kbp cDNA clone covering the 3 end of the small genomic RNA identified an open reading frame encoding a 317 aa N-truncated protein exhibiting significant similarities with the coat protein of nepoviruses. The 1257 nt long 3 non-coding region showed up to about 65% homology to the equivalent region of members of the subgroup C of nepoviruses. The properties of this pathogen do not match those of any previously described nepovirus. It should therefore be considered as a new member of the subgroup C of nepoviruses, for which the name of Apricot latent ringspot virus (ALRSV) is proposed.The nucleotide sequence reported in this work has been deposited in the EMBL databank under the accession number AJ278875. 相似文献
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Surveys were carried out in the main stone-fruit growing areas of Albania to assess the phytosanitary status of Prunus in conimercial orchards and varietal collections. The presence of virus and virus-like diseases and their identification was ascertained through field observations, sap transmission to herbaceous hosts, graft transmission to woody indicators, ELISA and IEM tests. The mean infection level was 42%. In particular, infections in apricot and almond were 12 and 16%, respectively, i.e. lower than in plum and cherry (47 and 56%, respectively). The following viruses were identified: plum pox potyvirus (PPV). apple chlorotic leaf spot trichovirus (ACLSV). prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. PPV infection was very severe in plum, and limited in apricot and peach. Apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered in Primus. 相似文献
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Kensaku Maejima Misako Himeno Osamu Netsu Kazuya Ishikawa Tetsuya Yoshida Naoko Fujita Masayoshi Hashimoto Ken Komatsu Yasuyuki Yamaji Shigetou Namba 《Journal of General Plant Pathology》2014,80(2):176-183
Sharka disease, caused by plum pox virus (PPV), is the most serious viral disease of stone fruit trees. Among the eight known strains of the virus, PPV-D is the most important due to its recent global spread. Although enzyme-linked immunosorbent assay (ELISA) is the most common approach for diagnosing sharka, it involves time-consuming steps and requires expensive equipment and trained technicians. In this study, an on-site PPV detection kit based on immunochromatography was developed using polyclonal antibodies against the coat protein (CP) of a PPV-D isolate. The immunochromatographic (IC) assay kit was as sensitive as a commercial ELISA system for detecting Japanese PPV-D isolates. Moreover, it was easy to use (a one-step procedure), and results could be obtained on-site within 15 min without special laboratory equipment. The IC assay kit detected the virus from every aerial part of symptomatic Japanese apricot trees. In a detailed study of viral localization in leaves, the most suitable plant parts for use in the IC assay were symptomatic mesophyll tissues and the region from the petiole to the main vein. A positive reaction was also observed using the CP of other major (PPV-M and PPV-Rec) and minor (PPV-EA, PPV-W, and PPV-T) strains. 相似文献