Comparison of responses of ascospores and mycelium by ELISA with anti‐mycelium and anti‐ascospore antisera for the development of a method to detect Sclerotinia sclerotiorum on petals of oilseed rape

Abstract

The goal of this project was the development of a serological test for the detection of Sclerotinia sclerotiorum on oilseed rape petals. Since the fungus exists in two forms on petals, as ascospores and mycelium, the responses of anti-mycelium and anti-ascospore antisera against these two kinds of antigens were compared. Two anti-mycelium sera, Smy and Smy', were produced against mycelial soluble extracts at different concentrations (0.3 mg and 0.1 mg of protein ml-I). Smy gave the greatest response level with eight S. sclerotiorum mycelial extracts tested. It had a very superior level of recognition to that of anti-ascospore serum, Ssp, when mycelium was tested as antigen. In contrast, Ssp and Smy were equally reactive when exposed to ascospores but the sensitivity of the assay was low. For each antiserum, the solution from which ascospores had been removed reacted similarly to the original suspension containing ascospores. A collection of fungi likely to be found on oilseed rape petals was examined. Cross-reactions were produced with both antisera, especially with Botrytis cinerea for which greater cross-reactivities were produced with Smy. The cross-absorption of antiserum Smy with a mycelial extract of B. cinerea considerably reduced this cross-reaction. The choice of antiserum for the development of a reliable detection system is discussed.

Detection of Sclerotinia sclerotiorum Using a Monomeric and Dimeric Single-Chain Fragment Variable (scFv) Antibody

“…This result is perhaps not unexpected and may be explained by the fact that Botrytis is genetically similar to Sclerotinia, and therefore the antigen that is being recognized may be common to the two phytopathogenic fungi (30). A similar cross-reactivity between the two taxonomically related fungi was previously observed for polyclonal antibodies that were used in ELISA-based experiments (16). B. cinerea has been observed to infect flower petals as part of its disease cycle on other plants (31); however, gray mold caused by B. cinerea is rarely a problem in canola production in western Canada (5), so it is typically not observed on petals or on other plant tissues of canola.…”

“…Previous attempts at using antibodies to detect S. sclerotiorum were based on polyclonal antibodies , . Antibody production requires the immunization of animals followed by collection of serum containing the antibodies generated by the immune systems of the animals.…”

Immuno-impedimetric Biosensor for Onsite Monitoring of Ascospores and Forecasting of Sclerotinia Stem Rot of Canola

“…The specificity and the possible cross reactivity of anti- S. sclerotiorum antibody with fungi commonly found in crop fields have been investigated by a number of groups 41 – 44 . Jamaux and Spire 41 , 42 reported that anti- S. sclerotiorum polyclonal antibodies cross react with closely related species such as S. trifoliorum and S. minor , but do not cross-react with 30 other fungi which could possibly be found in different crop fields such as canola, wheat, sunflower, lettuce, soybean, etc. However, according to Bom and Boland 43 , the cross-reactivity among Sclerotinia spp.…”

“…is not anticipated to be a significant source of error in the field because S. homoeocarpa , S. trifoliorum and S. minor are not typically found in the phyllosphere of canola due to their lack of spores, their host range, or both. The cross-reaction to Botrytis cinerea 41 , 42 may cause problem in the polyclonal anti- S. scleroriorum antibody application. To overcome the cross-reactivity problem two approaches could be adopted to improve the antibody specificity for field application viz to perform absorption of S. sclerotiorum antigen with polyclonal anti- B. cinerea antibody as described by Jamaux and Spire 41 or to develop monoclonal antibodies.…”