Construction of a bio-test for infecting red clover plants with Sclerotinia trifoliorum

Sclerotinia trifoliorum causes clover cancer in red clover crops. Clover cancer is difficult to control and completely resistant red clover varieties are not available. Breeding for resistant red clover varieties is being slowed down because little is known about the diversity of European S. trifoliorum populations and because of the lack of bio-tests that are useable in breeding programs. The first objective of this research was to develop a reliable high-throughput bio-test, useable in breeding programs. The second objective was to optimise another bio-test, based on isolated leaves, for more precise studies. First, we optimised a method for ascospore production of S. trifoliorum. Once produced, the ascospores were used to evaluate the effects of climate conditions, ascospore concentration and plant age on the high-throughput bio-test. For the bio-test on isolated leaves, the effects of infection method, incubation conditions, incubation period, ascospore concentration, leaf growth stage and mechanical damage were evaluated. In the high-throughput bio-test, disease levels rose with increasing ascospore concentration up to 20,000 spores/ml. The plant age had a small, yet significant effect on the disease level. For the isolated leaf bio-test, the most effective and most repeatable infection method was spraying of an ascospore suspension. Disease levels continued to increase with rising concentrations and incubation time did not interact with plant susceptibility levels. The youngest completely opened leaf yielded the most repeatable results. Both bio-tests were shown to be correlated and could be valuable instruments for breeding programs and for studying plant-pathogen interactions.