Development Of Serological Assay For Fusarium Graminearum And Its Application On Resistance Evaluation Of Wheat Cultivars

Polyclonal antibody against Fusarium graminearum, the causal pathogen of wheat scab, was produced, and the enzyme-linked immumosorbent assay (ELISA) for qualitative and quantitative detection of the pathogen was developed. Main research results were followed:1. Preparation of antigensThe mycelial mass was collected from the culture according with the growth standard curve of F. graminearum. The mycelia was pulverised with quartz sand in liquid nitrogen and homogenised with a low volume of extraction buffer in a mortar. The antigen/protein concentration was determined by using Coomassie Brilliant Blue G-250 method. The final concentration of antigen was adjusted to 1mg/mL, and stored at-20℃until needed.2. Production of polyclonal antibodyThe polyclonal antibody against the mixed antigens prepared from the mycelia of three strains (BDX3-2, BDX4-5, HwH4-5) of F. graminearum was developed by immunizing rabbits (cultivar Da erbai). The best antiserum selected from three rabbits tested was precipitated by 3.9 molar saturated ammonium sulphate solution, and then the proteins were purified by Protein A column. The high titer of antibody was at 1: 64000. The sensitivity and specificity of the antibody were analyzed using ELISA. Results revealed that the antibodies have good sensitivities and specificities and no cross reaction with other genus of fungus.3. Detection of F. graminearum in different wheat varieties using ELISABiomass of F. graminearum in ten infected wheat cultivars were quantified by using the indirect ELISA technique. Among the ten wheat cultivars tested, Yangmai No. 10 was visually assessed with 17.3% of diseased ear rate, while detected as negative (no pathogen) by using the indirect ELISA. For other cultivars, the data obtained from ELISA test were positive corelation with the data obtained from visualassessment (r=0.74, P=0.01, n=9). It concluded that the polyclonal antibody was able to detect F. graminearum in wheat, and the ELISA technique developed in this study could provide an early and accurate diagnosis of F. graminearum in wheat.