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1.
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   

2.
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   

3.
Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.  相似文献   

4.
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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7.
Polyclonal and monoclonal antibodies were raised against secreted proteins from an anastomosis group 8 isolate of Rhizoctonia solani and tested for reactivity to field isolates from anastomosis groups 2-1, 3,4 and 8. Polyclonal antibodies raised against total secreted proteins cross-reacted in immunoblotting experiments with all R. solani isolates. However, immunoreactive proteins specific to Ag-8 isolates were evident. Monoclonal antibodies to secreted proteins were raised which reacted with fewer proteins and showed a greater degree of specificity for AG-8 isolates. Two monoclonals were selected for further study. One, an IgM monoclonal antibody, reacted with all R. solani isolates, recognizing a 40-kDa protein specific to AG-8 isolates and proteins of lower molecular weight in isolates from other anastomosis groups. The other, an IgG monoclonal antibody, was more specific, reacting with 38, 40 and 55-kDa proteins from AG-8 isolates and cross-reacting with few isolates from other anastomosis groups. Preliminary results on the induction of antigens recognized by the monoclonal antibodies are presented. The monoclonal antibodies characterized here are useful for the identification of isolates of R. solani and may also be used as probes to clarify the relationships between anastomosis groups.  相似文献   

8.
A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.  相似文献   

9.
The isolate BOR-3, collected in Slovakia in 1996, was recently identified as a natural recombinant between an M and D type of Plum pox virus (PPV). Biological assays demonstrated its capacity to be aphid- and graft-transmitted to various Prunus spp. hosts. A study was carried out to determine the further presence of PPV recombinants in two epidemiologically distinct areas – Slovakia and France. Tools based on PPV-M and D subgroup typing, targeting P3–6K1, CI and CP regions of the PPV genome were used for recombinant identification. Closely related recombinant variants were detected in different Prunus spp. during a survey conducted in Slovakia in 2001, but not within a set of selected PPV isolates from France collected between 1985 and 2001. Sequence analysis of the (Cter)NIb–(Nter)CP region of 10 recombinant isolates from Slovakia showed their high homology, reaching more than 98%. All the recombinant isolates shared the same recombination breakpoint situated in the C terminus of the NIb gene. Our study demonstrates that the PPV recombinants are viable and competitive with conventional PPV-M and D isolates. The present work indicates that the occurrence of recombinants within PPV isolates might be more common than previously assumed.  相似文献   

10.
For many years, Plum pox virus (PPV) was considered to be transmissible by seed, increasing the fear of long-distance spread of the disease. In the late 1970s, it was claimed on the basis of biological transmission of the virus to herbaceous indicator plants and the development of serological diagnosis based on polyclonal antibodies, that PPV was seed-transmitted, with a different infection rate according to the plant species and part of the seed which was tested. In the 1990s, PPV was characterized into four different types, and specific monoclonal antibodies were produced for them. These new and more sensitive diagnostic techniques, together with RT-PCR with different sets of specific primers, were used to approach once again the problem of PPV transmission through seeds. The virus was detected in seed coats and cotyledons, but embryonic tissue and seedlings obtained from germinated seeds never showed symptoms, and gave negative results for PPV with both ELISA and PCR assays. No PPV isolate is currently recognized to be seed transmitted, so vertical transmission of PPV from infected mother plants to their progeny does not occur. Hypothetically, the only possibility of seed transmission would arise from a mutation in the helper component of the virus, associated with high susceptibility of the infected Prunus cultivar.  相似文献   

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12.
Two monoclonal antibodies were obtained against the apple proliferation phytoplasma that provide easy, rapid, specific and sensitive serological detection. They reacted specifically by using ELISA and immunofluorescence techniques with apple proliferation-infected periwinkles and apple trees from different regions in northern Italy and Slovenia, but not with several other phytoplasma isolates. We did not observe any monoclonal antibody reaction even using phytoplasmas belonging to the same phylogenetic group such as European stone fruit yellows and pear decline. Two serological techniques, immunofluorescence and ELISA, were compared with DAPI staining and PCR. From July until leaf fall ELISA was as sensitive as PCR but was more rapid and convenient than PCR; immunofluorescence was useful for specific detection of apple proliferation phytoplasma on roots throughout the year. Serological techniques could be conveniently applied in the roots, stems and leaves of apple trees depending on specific phenological stages of the plants.  相似文献   

13.
Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.  相似文献   

14.
Plum pox potyvirus (PPV) isolates show quantitative and qualitative differences in host plants, symptomatology, aphid transmissibility and molecular biology. In order to gain an overview of the different PPV strains that exist in Germany, several PPV isolates, mainly deriving from southern Germany, were analysed by polymerase chain reaction (PCR), the reaction yielding a 243 bp product. All PPV isolates tested were amplified and the amplified fragments were analysed by restriction endonuclease digestion. An Rsa I restriction site polymorphism in the amplified fragments allowed the discrimination of two groups of isolates.  相似文献   

15.
A large-scale serological characterisation of Plum pox virus (PPV) isolates was carried out with 19 monoclonal antibodies (MAbs), including the universal MAb5B and the following strain-specific MAbs: AL (specific to PPV-M), 4DG5 (specific to PPV-D), TUV and AC (specific to PPV-C), and EA24 (specific to PPV-EA). The study involved 108 PPV isolates of different geographical origin (Albania, Bulgaria, Cyprus, Czech Republic, Egypt, France, Germany, Greece, Italy, Hungary, Moldova, Romania, Slovakia, Spain, Turkey and Yugoslavia) and hosts (almond, apricot, peach, plum and cherry). The inter- and intra-strain serological relationships of PPV isolates were evaluated by DASI-ELISA. High serological variability was detected, not only between strains, but also among isolates of the same strain. Computer-assisted analysis of serological data support the hypothesis of the existence of two distinct subclusters, denoted PPV-M1 and PPV-M2, which seem to prevail in Mediterranean and Eastern–Central European countries, respectively.  相似文献   

16.
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17.
Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.  相似文献   

18.
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19.
Very limited information is available on the origin, diversity and evolution of Plum pox virus (PPV) ‘Turkey’ (T) strain. Phylogenetic analyses based on partial sequences of 421 isolates and complete genome sequences of 57 isolates, representing the geographical distribution of PPV-T in Turkey, revealed the existence of several monophyletic and, in some cases, geographically limited groups within the PPV-T strain (Ankara-Konya1-Kayseri, Ankara-Balkan, Istanbul, Konya2 and Balkan). PPV-T diversity (0.018%) was found to be greater than that of PPV strains D and Rec but lower than that of the M strain when including the newly described and divergent M-Istanbul isolates, suggesting a long evolutionary history for PPV-T. The European part of Turkey in the Balkans, close to Bulgaria where PPV was identified for the first time, appears as a likely centre of origin for PPV-T isolates. The colonization of various parts of Turkey by diverse isolates from that region, followed by secondary local spread, is the most likely scenario for the diffusion of PPV-T in Turkey.  相似文献   

20.
Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVYNTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVYNTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVYNTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVYE strain. To distinguish PVY‐AGA and PVY‐MON from other PVYNTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVYN‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVYN‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.  相似文献   

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