Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.Downy mildew caused by Plasmopara halstedii (Farlow) Berlese et De Tony is one of the economically most important diseases of sunflowers, Helianthus annuus L. The fungus, which is an obligate parasite of sunflowers, occurs in all areas where sunflowers are cultivated extensively except Australia, South Africa, and possibly parts of North Africa (21, 23). Systemic downy mildew infection alters the development of vegetative and generative parts of the plant, as well as its metabolism (26,32). Inoculation of sunflower plants with P. halstedii at the two-leaf stage through apical buds greatly inhibits stem elongation. The yield of infected plants is usually less than 25% that of uninfected plants. There is no fungicide to control this disease after infection has occurred.Little information is available on the epidemiology and biochemistry of P. halstedii or its relationship with its sunflower host. Seven physiological races of P. halstedii have been identified (10,11,24), and they are capable of attacking a wide range of sunflower genotypes. In France, in addition to the three races-1, A (equivalent to American race 4), and B (equivalent to American race 3)-classically encountered (19), two new races, designated C and D, have been detected recently (13). In the Red River Valley of North Dakota, Minnesota, and Manitoba, all but race 1 have been identified (24). Some sunflower varieties carry resistance genes (Pl) against P. halstedii races present in France and in the United States (15,17,18,20,28). Genetic variation in the pathogen appears limited, since no random amplification of polymorphic DNA variation was found among isolates from races 1, A, and B or between isolates of the same race, and very few (89% similarity) polymorphisms were identified among all races of P. halstedii present in France (22). Downy mildew of sunflowers may result from oospores in the soil (6). Contamination of seeds by P. halstedii has also been implicated in the establishment of the disease (6). The only effective control tec...
The asexual phase of the life cycle of Plasmopara halstedii, the causal agent of downy mildew of sunflower, plays a key role in the propagation of the disease. We investigated the morphological and ultrastructural changes that occur during the asexual development of the pathogen. Direct examination of infected cotyledons confirmed the presence of sporangiophores. In contact with water, important ultrastructural changes occurred, affecting the surface of zoosporangia, which became smoother, and their cytoplasm, which differentiated into flagellate zoospores. The subsequent encystment of zoospores was characterized by the synthesis of a cell wall and the loss of the flagella. In addition, two monoclonal antibodies (MAbs) specific for P. halstedii were used to analyze the immunochemical changes associated with these modifications. MAb 16A6, which bound to a 48-kDa glycoprotein, mainly labeled the surface of mobile or encysted zoospores and of mother cells of germ tubes. Conversely, MAb 2F9, which recognized highly glycosylated antigens, labeled the surface of zoosporangia and of flagellate zoospores, but not the encysted zoospores. These results provide new insights into the morphological and ultrastructural changes associated with the release and the encystment of zoospores which may be interesting targets for the development of new antimicrobial products.
Un sérum polyclonal a été produit en utilisant les conidiophores broyés de Peronospora viciae comme 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 de P. viciae par 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 graines) employée actuellement pour détecter le mildiou dans les lots de graines.
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