摘要:

The occurrence of the quarantine pestThrips palmiin The Netherlands in 1992 and the difficulties encountered in enforcing legal eradication measures have stimulated discussion concerning a possible network of diagnostic services in Europe. Official services find it increasingly difficult to evaluate and adopt the latest diagnostic techniques produced by research institutes and universities. It is proposed to create international discipline‐oriented working groups to prepare draft identification methods for the official EPPO and EU quarantine pests. These draft methods could be harmonized by the EPPO Panel on Diagnostics and approved by EPPO Council. This is a two‐sided sword: internationally agreed identification methods are produced and a diagnostic network comes into being at the same t

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01432.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

As a spin‐off from fundamental molecular biological research, there has been a remarkable increase in new methods for diagnosis (i.e. detection and identification) in recent years. Because of their origin, these new methods all have in common that they use structural elements of the target organisms such as nucleic acids, lipids, fatty acids, proteins, polyamines and polysaccharides as a basis. These structural elements are either used as templates for development of so‐called probes for detection (and identification) or they are placed into man‐made patterns and used for identification/classification. The article presents the advantages and perspectives of the new methods compared with conventional ones. It may be noted that, in many studies, the specificity and reproducibility of the new methods has not been adequately treated or has even been only assumed. These features, which are closely linked with experimental and sampling error, lead to the principal pitfalls of the new methods, which are also rev

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01433.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Le principal problème posé par les méthodes d'immunodétection pour les champignons phytopathogènes est liéà la présence de très nombreuses protéines fongiques, dont certaines ne sont pas spécifiques de l'espèce. Aussi, chaque préparation fongique contient un nombre important d'antigènes parmi lesquels seuls quelques‐uns sont intéressants pour un diagnostic. Par quelques exemples, nous voulons illustrer les méthodes employées à l'INRA Versailles, où il a été décidé de concentrer les efforts sur une meilleure compréhension de la variabilité des protéines de champignons et d'utiliser des anticorps polyclonaux comme préliminaires à la production d'anticorps monoclonaux. Dans le cas dePeronospora viciaesur semences de pois, ou deVerticillium dahliaesur boutures de pélargonium, il n'y a qu'un pathogène du même genre présent dans la plante. Aussi, une grande spécificité n'est pas nécessaire, et il est facile de produire un bon antisérum détectant le mildiou du pois de semence ouV. dahliaedans les plantes, même si d'autres mildious ouVerticilliumréagissent avec l'antisérum. Si deux espèces proches d'un point de vue taxonomique sont présentes sur la même plante, par exempleSclerotinia sclerotiorumetBotrytis cinereasur pétales de colza, il est nécessaire d'éliminer les protéines non spécifiques par différents types d'absorption croisée. Si différentes espèces fongiques du même genre infectent la même plante, le problème est plus compliqué. Etudier et choisir des protéines spécifiques,

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01434.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Monoclonal antibody‐secreting cell lines were generated againstPyrenophora gramineaandPhylophthora fragariaeby various immunization techniques. The specificity of each antibody was investigated by DAS ELISA, membrane ELISA and IF. Antibodies generated againstP. fragariaewere found to be genus‐specific and could be used to detectP. fragariae‐infected strawberry roots. Antibodies developed againstP. gramineashowed marked isolate variability in twoPyrenophoraspp. The differences in isolate specificity were not consistent in all the assays investigated: antibodies reacted to some isolates by ELISA but did not detect them in other assays. An attempt was made to develop a membrane ELISA for detection of the fungi in seed material using the monoclonal antibodies that had been deve

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01435.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Polyclonal antisera for the immunological detection ofBotrytis alliiwere raised from cell wall, cytoplasmic and surface plate washings of two isolates of the pathogen. These antisera were used to develop an indirect ELISA for the detection of latentB. alliiinfection in stored onion bulbs. The antisera did not react with healthy onion tissue and showed only minimal recognition of fungi from other genera. Some cross reactivity was observed withB. cinereabut not with otherBotrytisspecies tested. In many instances following infection, fungal antigen could be detected earlier by ELISA than by direct plating. In tests on bulbs from a commercial store, the level ofB. alliiinfection was found to be 3.3% by both ELISA and direct plating, but this result was achieved 7 days sooner by ELISA.

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01436.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Un sérum polyclonal a été produit en utilisant les conidiophores broyés dePeronospora viciaecomme immunogène. Deux méthodes ELISA ont été comparées: une méthode indirecte (ACP‐ELISA) et une méthode sandwich révélée avec un système d'amplification à la biotinestreptavidine (DAS‐ELISA‐biotine). En DAS‐ELISA‐biotine, le champignon mélangéà des graines saines était mieux révélé qu'en ACP‐ELISA; 2,5ng de protéines deP. viciaepar ml pouvaient alors être détectés. Les principaux agents pathogènes et saprophytes des graines de pois (13 genres testés) n'étaient pas reconnus en DAS‐ELISA‐biotine. Cette méthode a été appliquée à l'analyse de lots de graines. Pour chaque lot, 32 groupes de 30 graines ont été prélevés et le tampon de lavage des graines a été analysé après une nuit de macération des graines, sous agitation. Des graines individuelles ont également été testées. La majorité des lots analysés présentaient un faible degré d'infection (de 2,5 à 10 ng de protéines fongiques par ml). De plus, le degré d'infection des graines individuelles était très hétérogène au sein d'un lot. Cette méthode ELISA s'est avérée plus fiable que la méthode traditionnelle (observation microscopique des oospores dans l'eau de lavage des g

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01437.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Baiting with apple fruit and stimulation of sporulation with paraquat are sensitive tests currently used on strawberry planting/propagating material for the detection ofColletotrichum acutatum.A monoclonal antibody/ELISA technique could make testing shorter and easier. Several monoclonal antibodies distinguishedC. acutatum in vitro; one recognized 45 out of 48 isolates believed to beC. acutatum, but not 18 out of 19 typical of closely related C.gloeosporioides.A less specific monoclonal antibody reacted positively with 66 out of 67Colletotrichumisolates, indicating it was genus‐specific. The antigen was mainly associated with conidia. A dot‐blot test using PALL Biodyne membrane gave qualitative results more quickly and simply than ELISA, reducing time taken from 24 to 3 h. Specific monoclonal antibodies distinguishedC. acutatumfromC. gloeosporioideson strawberry petioles, but were less sensitive than either plating or paraquat tests, detecting only eight out of 50 potential positives compared with at least 30 out of 50 with the other tests. An immunofluorescence technique confirmed that conidia were the main source of antigen. A specific monoclonal antibody also reacting to mycelium was produ

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01438.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Relatively rapid, highly specific monoclonal antibody‐based enzyme‐linked immunosorbent and immunofluorescent assays have been developed to detect and visualizeNectria galligenain woody tissue. Hybridoma cell lines were raised by co‐immunization of mice with extracts from freeze‐dried cultures ofN. galligenaand murine polyclonal antiserum to a related pathogen,N. haematococca.This method appeared to reduce the number of hybridoma cell lines that secreted cross‐reactive antibodies. From two fusions, ten cell lines that recognizedN. galligenaand notN. cinnabarinawere raised. Hybridoma supernatants were screened by ELISA using antigen‐coated wells. Monoclonal antibodies from one cell line, NG‐IE4, proved useful in the detection of the pathogen in woody tissue by ELISA and antibodies from another cell line, NG‐DF11, have been used to immunolocalize the fungus in woody tissue by immunofluorescence. Further applicati

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01439.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Polyclonal antisera were raised to whole and sonicated fractions of resting spores ofPlasmodiophora brassicae, the causal agent of clubroot disease. Cross reactivity of antisera was assessed against a wide range of soil‐borne pathogens. Antiserum raised to whole resting spores was used to probe artificially infested soils by indirect immunofluorescence. Resting spores could be detected at a concentration as low as to 1 × 102spores per g in artificially infested soil, and were readily identified in naturally infested soils, with negligible fluorescence in soil thought to be clubroot‐free. Antiserum raised to sonicated fractions of the resting spore was used in an indirect ELISA assay of soil. When bound antiserum was labelled with anti‐rabbit IgG conjugated to alkaline phosphatase, there was a negative correlation between absorbance levels and resting spore numbers in artificially infested soil. In contrast a close to linear response, with a correlation coefficient of 0.971, was recorded when infested soils were assayed with bound antiserum labelled with protein‐A pe

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01440.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Extracellular pectinase isozymes were determined by polyacrylamide gel electrophoresis (PAGE) for isolates ofGanodermacollected from a range of hosts and environments in south‐east Asia. Findings indicate these techniques to be of use in the differentiation of strains isolated from different hosts and environments. Average linkage cluster analysis of pectinase isozyme banding patterns separated isolates according to host of origi

ISSN:0250-8052    

DOI:10.1111/j.1365-2338.1995.tb01441.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY