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The paper reports about the development and evaluation of two methods, a PCR-based assay and an enzyme-linked immunosorbent assay (ELISA), for the detection of the common bunt fungus Tilletia caries (syn. T. tritici) in young wheat plants. Using the published primer pair Tcar2A/Tcar2B for polymerase chain reaction (PCR) with DNA from axenic cultures of T. caries or from T. caries-infected plants, we obtained a single band after electrophoresis of the amplification products. By PCR the bunt pathogen could be detected in shoots (EC 12) as well as in leaves (EC 13–14) of infected plants. Immunological detection was performed using a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with biotinylated detection antibodies. The antibodies were obtained after injection of mycelial homogenates of axenic cultures of T. caries into rabbits. The detection limit was 16 pg DNA per 100 mg plant fresh weight for the PCR and 7 ng/ml fungal protein for the ELISA, respectively. Except for the closely related T. controversa, no cross-reactions with other fungi were observed with both methods. While it was possible to detect teliospores of T. caries by PCR, the ELISA did not react with spore extracts. Analysis by ELISA of shoots of individual plants grown from inoculated seeds revealed that at EC 10 all plants were infected. There was, however, a large variability in the amount of T. caries present in the plants. This observation and reports in the literature indicate quantitative differences in the degree of colonization of the tissue between individual plants even in a given variety. Regarding the use of modern diagnostics to assist in the development of resistant varieties we therefore suggest that for the wheat– T. caries pathosystem the non-quantitative PCR-assay employed here is less suited than the ELISA that allows precise quantification of the amount of fungal antigen present in the plant. However, to routinely employ the ELISA in resistance breeding further development work is needed. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Coat protein genes of six members of the genus Allexivirus, Garlic viruses -A, -B, -C, -D, -E and -X, were over-expressed in bacteria and used to prepare polyclonal antibodies. In western blot analysis, Garlic virus D (GarV-D) and GarV-E were closely related serologically and there were weaker relationships between some other pairs, particularly GarV-E with GarV-A, GarV-X with GarV-A, GarV-X with GarV-B and GarV-X with GarV-C. Sequence analysis suggested that there was no simple relationship between amino acid identity and serological properties. [ABSTRACT FROM AUTHOR]

Protein extracts from Tobacco mosaic virus (TMV) infected young tobacco leaves exhibiting strong symptoms contain higher amounts of ubiquitin conjugates in comparison with non-infected control plants presumably due to stress reactions of host cells. Western blots with antibodies against TMV indicate as well an increase in coat protein or in ubiquitinated coat protein subunits. Immunogold labelling of infected leaf material revealed the accumulation of large amounts of coat protein in opaque inclusions which also reacted strongly with ubiquitin antibodies. These inclusions appear in close vicinity to chloroplasts in chlorotic areas of the leaf. It is concluded that most of the ubiquitin conjugates found in Western blots are due to ubiquitinated coat protein. As a consequence, a considerable amount of coat protein cannot be used for correct capsid construction as the stabilizing lysine residues are blocked by ubiquitination. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Polyclonal antibodies were raised in rabbits against MLO associated with faba bean (Viciafaba L.) phyllody which exists in the Sudan. Two indirect ELISA methods were able to detect the MLO antigens. In the former, the whole antigen was directly coated onto plates, while in the second, only the F(ab'), fragments of the IgG were used to coat the ELISA plates. Higher detectable efficiency was obtained when the F(ab'), method was used. Moreover the obtainable antiserum was found to exhibit a high degree of specificity through which the MLO associated with faba bean phyllody in the Sudan, are serologically differentiated from other isolates of MLO existing in the Sudan as well as European MLO isolates maintained at Versailles, and Spiroplasma atri, causal agent of Citrus Stubborn Disease. The positive reactions obtained with this antiserum against MLO phyllody naturally existing in the Sudan on Crotalana saltiana and some Catharanthus roseus demonstrate that these plants are potential reservoirs of the disease in the Sudan. The same antiserum was used in order to distinguish healthy and diseased plant preparations using the membrane ELISA method (dot-blot). [ABSTRACT FROM AUTHOR]

We describe a simple method of purifying Pseudomonas syringae pv. syringae (P. s. pv. s.) specific polyclonal antibodies which are directed against a cell surface protein sized 31 kD. The actual purification step of the polyclonal antibodies occurs with denatured proteins after an SDS-PAGE gel electrophoresis and western blotting. Polyclonal antibodies were obtained which recognized a 31 kD protein. On a western blot no cross reaction of the purified polyclonal "monospecific 31 kD antibodies" with other proteins from the same strain was observed. However, a positive "mono- specific 31 kD antibody" reaction was only visible when either protein extracts from several selected pathogenic P. s. pv. 5. strains from different hosts, or two isolates of Pseudomonas syringae pv. pisi (P. s. pv. p.) were used. An indication that this 31 kD protein is specific to P. s. pv. s. is that no cross reaction was found with protein extracts from the tested pathogenic and non-pathogenic pseudomonads. However, a differentiation of the tested P. s. pv. s. and P. s. pv. p. isolates was possible when cells were grown above 30 °C. Then protein extracts from P. s. pv. s. revealed the lack of the 31 kD protein but not P. s. pv.p. protein extracts. Polyclonal antibodies raised in rabbits against the 31 kD protein band from the western blot proved to be specific enough to agglutinate whole P. s. pv. s. strain R32 cells or for detecting a single 31 kD protein band on a western blot when whole cell protein extracts of P. s. pv. s. strain R32 were used. [ABSTRACT FROM AUTHOR]

Inorganic pyrophosphatase (PPase) from E. coli was used as label in enzyme immunoassay for the detection of different plant viruses. The use of PPase-conjugated antibodies and tetrasodium pyrophosphate as a substrate in ELISA provides some advantages in detection of plant viruses in purified preparations and extracts from plant specimens: high sensitivity, negligible level of background reactions in control samples, bright blue-greenish colour which allows to detect the reaction visually and high stability of PPase-conjugated antibodies. [ABSTRACT FROM AUTHOR]

A panel of monoclonal antibodies prepared against cowpea severe mosaic virus was divided into two groups based upon presence or absence of competition with a biotinylated antibody in competition ELISA. Antibodies that did compete could be further characterized by comparison of competition curves obtained from assays performed in simultaneous and consecutive manners. These two types of competition assays provide an easy method to define initially relationships among epitopes on antigens and to characterize specific immunoreactivity of different antibodies to the antigen. [ABSTRACT FROM AUTHOR]

Different polyclonal antisera and enzyme-linked immunosorbent assay (ELISA) procedures have been tested for their potential to detect tomato spotted wilt virus (TSWV). The virus could efficiently be detected in high dilutions of sap from infected plants, and at low concentrations of purified virus and nucleocapsid protein preparations in the cocktail ELISA and the double antibody sandwich ELISA (DAS-ELISA). Amounts of 1 to 3 ng of virus protein still gave positive readings using purified preparations, while sap could be diluted approximately 100,000 times. Differences in the detection level were observed using nucleocapsid protein antiserum (anti-N-serum) and the antiserum against intact virus particles (anti-TSWV-serum), but both antisera showed to be powerful sera for the detection of TSWV. Using anti-N-serum, TSWV could be detected in highly diluted extracts of different hosts, and also in leaf extracts or intact tissues stored for 30 days under different conditions. These results indicate that the TSWV nucleocapsid protein remains antigenic for long periods. [ABSTRACT FROM AUTHOR]

In thin sections of wheat rachis and stem, embedded in Spurr's resin, prefixed with glutaral-dehyde and postfixed with osmium, large amounts of barley thionins were localized in cell walls with anti-thionin-protein A/gold. Reduced binding to thionin was detected in cell walls penetrated by Fusarium culmorum, suggesting that cell wall thionins were degraded by the fungus. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

When purified banana bunchy top virus (BBTV) was used to immunize Balb/c mice, all 480 wells from one fusion contained hybridomas, and culture fluid from 92% of them showed a positive reaction to BBTV using plate trapped antigen (PTA) ELISA (also known as indirect ELISA). After a third screening, 84 hybridoma cell lines retained their ability to produce BBTV-specific antibodies. Fifty-eight monoclonal cell lines with the ability to produce BBTV-specific antibodies were obtained when these hybridomas were cloned by limiting dilution to single cells. The antibodies produced by these monoclonal hybridomas were mostly isotype IgG2a. The titres of antibodv in culture supernatant and ascitic fluid of one clone were 10³ and 104, respectively, while those of another clone were as high as 104 and 106. Hybridomas produced 40 to 62 times more anti-BBTV specific antibodies in mouse ascites than in culture. The minimum active concentration of purified immunoglobulin produced by three hybridoma ranged from 0.03 to 0.06 μg/ml, while the minimum detectable concentration of BBTV antigen was 0.26 μg/ml with PTA ELISA. Antibody trapped antigen (ATA) ELISA (also known as direct ELISA) was 16 times more sensitive than PTA ELISA in detecting BBTV. Although PTA ELISA could not detect BBTV in crude extract of diseased tissues, ATA ELISA was able to detect the virus even when the extract was diluted 1/ 512. [ABSTRACT FROM AUTHOR]

Purified potato virus A (PVA) was used for immunization to produce monoclonal antibodies (MAb). The type of ELISA with purified PVA or non-purified PVA, played an essential role in selecting MAb with different specificity. Two MAb's (MAb-1 and MAb-2) were selected, using indirect ELISA (I-ELISA) with purified PVA. Competition experiments suggested that MAb-1 and MAb-2 reacted with the same epitope on purified PVA (epitope 1). ELISA, IEM and SDS-PAGE-immunoblotting experiments showed that epitope-1 was only present on purified PVA but not on non-purified PVA, suggesting that this epitope was introduced during the purification. Assays at different steps during purification indicated that epitope-1 was only exposed after plant components and reducing agents were removed from the PVA extract. Three MAb's (MAb-3, MAb-4 and MAb-5) were selected by indirect double antibody sandwich ELISA (IDAS-ELISA) with non-purified PVA. These MAb's reacted in I-ELISA or IDAS-ELISA with purified PDA as well as with non-purified PVA and might be useful for routine diagnosis. MAb-3, 4 and 5 cross-reacted with some other potyviruses in I-ELISA and in IDAS-ELISA. MAb-1 cross-reacted with 5 out of 7 other potyviruses in I-ELISA, but not in IDAS-ELISA. [ABSTRACT FROM AUTHOR]

Detection of plant viruses by ELISA using horseradish peroxidase for antibody labelling (ELISA-peroxidase) has been standardized by evaluating variants of the procedure, regarding composition and concentration of buffers and additives. Immunoglobulins (IgG) are isolated from antisera by precipitation with ammonium-sulphate and by purification with DEAE-52 (Whatman) cellulose. IgG are conjugated with horseradish peroxidase by a modified oxidation-periodate method. In ELISA-peroxidase 0.05 M carbonate-bicarbonate coating buffer pH 9.6 has been substituted by 0.01 M carbonate buffer pH 9.2. Extraction buffer is used with 0.5 % bovine serum albumin (BSA), without polyvinylpyrrolidone (PVP). Samples are diluted in phosphate buffered saline (PBS) pH 7.2 with 0.05% Tween 20 and 0.5% BSA. IgG are conjugated with horseradish peroxidase, diluted in 0.1 M Tris-HCl, pH 7.4 with 0.05 % Tween 20 and 1 % BSA. The substrate is incubated in the darkness for 20 min at room temperature. ELISA-peroxidase proved to be equivalent in sensitivity and specificity with ELISA using alkaline phosphatase for antibody labelling. Its advantage is a lower cost of chemicals used in the test. [ABSTRACT FROM AUTHOR]

Antigen-antibody reactions in agar gel, as demonstrated by the double diffusion technique, between cotton seed globulins and the antisera specific to each of the tested Fusarium oxysporum f. sp. vasinfectum isolates as well as the antiserum of F. moniliforme revealed that all the tested antisera of F. oxysporum f. sp. vasinfectum reacted with seed globulins except the Menoufi cultivar globulins. No precipitin lines were detected in the reaction between the antigen of the cotton cultivar Acala SJ2 versus the antiserum of P10 isolate. The 5 cultivars behaved differently with each fungal antiserum to the extent that they could be distinguished accordingly. When the seed globulins of the susceptible cultivars (Giza 74 and Bahtim 110) reacted with antiserum of the tested F. oxysporum f. sp. vasinfectum isolates, more precipitin lines were formed than the resistant cultivars. On the other hand, no obvious reaction was detected in case of F. moniliforme antiserum. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (MCAs) specific for Grapevine Fanleaf Virus (GFV) were obtained by fusion of a non-secreting myeloma cell line with the spleen cells from Balb/c mice immunized with GFV isolate F 13. The MCAs were tested with a panel of GFV isolates, using both crude extracts and purified virus particles. Some of the MCAs recognize all 41 isolates tested and are suitable as a diagnostic reagent for GFV detection. Certain MCAs do not bind to some of the isolates, while others show a lower reactivity for certain isolates than for the immunogen F 13. MCAs make it possible to distinguish GFV isolates which until now could not be distinguished by using polyclonal antiserum. [ABSTRACT FROM AUTHOR]

Upright dieback, caused byPhomopsis vacciniiShear (teleomorphDiaporthe vacciniiShear), is an economically important disease that could threaten blueberry and cranberry production. Diagnosis of the disease by traditional phytopathological methods is difficult. We report the successful detection ofP. vacciniiby direct tissue blot immunoassay (DTBIA) and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). The infected samples obtained from Lithuania are the findings reported on the fungus in Europe. [ABSTRACT FROM AUTHOR]