Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings.
Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea.