Breeding for resistance to clover rot (Sclerotinia spp.) in red clover (Trifolium pratense)

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    Red clover (Trifolium pratense L.) is a perennial forage crop grown in mixture with grasses or as a pure stand. It is valued for its nitrogen fixation capacity, benefits in organic farming, high quality forage, and beneficial effects on the soil structure. However, the lack of persistence is a major problem in red clover and the lack of winter hardiness and diseases are the most important reasons. Clover rot (clover cancer or Sclerotinia crown and root rot) is an important disease in European red clover crops, caused by the ascomycete fungi Sclerotinia trifoliorum Erikks. or Sclerotinia sclerotiorum Lib. de Bary, Clover rot is difficult to control because its development is highly dependent on weather conditions. Moreover, resistance breeding is hampered by the lack of useable bio-tests and by the lack of knowledge on the genetic and phenotypic variation among Sclerotinia isolates. In this context, the objectives of this thesis were to analyse the phenotypic and genetic diversity among Sclerotinia populations from European red clover crops, to construct bio-tests for Sclerotinia, to identify possible sources of resistance and factors related with resistance, and to acquire insight in the heritability of clover rot resistance.
    In the first part of this dissertation the focus was on the genetic diversity among a collection of 192 Sclerotinia isolates from 25 locations in 12 European countries. Mycelial compatibility grouping provided a first idea of genetic diversity, while an AFLP study disclosed the genetic diversity in more detail. Two Sclerotinia species were present on red clover: S. trifoliorum was found in most locations while S. sclerotiorum was found in locations Fr.A and Fr.B. Genetic differences were found among isolates between and within locations, but there were no subpopulations and genetic distance was not correlated with geographic distance. Within-location variance accounted for 79.2% and among location variance for 20.8% of the genetic variation within S. trifoliorum isolates, indicating that the degree of population differentiation is low. A species specific PCR based on the B-tubulin gene was constructed to quickly discriminate between S. trifoliorum and S. sclerotiorum.
    Bio-tests were developed for Sclerotinia on red clover. First, ascospore production for S. trifoliorum was optimised. A high-throughput bio-test was optimised: young plants are inoculated a with suspension of ascospores or mycelium fragments and incubated on an ebb and flow table covered with an opaque plastic cap. The disease is scored on a scale from 1 to 5 after incubation at 15°C during 10 days for mycelium fragment inoculation and 14 days for ascospore inoculation. Additionally, we constructed an in vitro bio-test on detached leaves to analyse plant - pathogen interactions more precisely. Leaves are incubated on 0.5% water agar, inoculated with a mycelium plug or by spraying an ascospore suspension and the percentage of leaf damage is calculated after incubation. Both bio-tests were fairly correlated.
    The phenotypic variation was studied among a sub collection of 30 Sclerotinia isolates. Mycelial growth rate, production of sclerotia and secretion of oxalate were measured and aggressiveness was assessed by both bio-tests. Growth rate, sclerotia production, oxalate production and pathogenicity according to both bio-tests differed between isolates. While fast growing isolates and isolates that were aggressive on detached leaves were more aggressive on plants, isolates that produced numerous sclerotia were less aggressive. A microscopic analysis of the infection process disclosed that the speed of ascospore germination was similar among isolates, yet aggressive isolates grew shorter secondary mycelia and attained more successful penetrations after 48h.
    To identify possible sources of resistance in red clover germplasm, a diverse collection of over 121 red clover accessions was screened. Plants were evaluated in the field and susceptibility to clover rot was determined. Accessions differed significantly in susceptibility to clover rot but no completely resistant accessions were found. Isoflavone levels, branching, growth habit and resistance to mildew and virus disease did not affect clover rot susceptibility. High yielding plants and plants susceptible to rust were more resistant to clover rot. Tetraploid cultivars were less susceptible by 11.7% than diploid cultivars. Cultivars were generally less susceptible than landraces and wild accessions. Two tetraploid cultivars and one diploid landrace were significantly more resistant. Cultivars and some landraces from both ploidy levels may be the most suitable sources of resistance.
    In a final part, the inheritance of clover rot resistance was investigated. Analysis of segregation ratios in progenies from 15 pair crosses between ramets of resistant and susceptible genotypes suggested that clover rot resistance was conferred by three major quantitative resistance genes, while at least a part of the resistance was conferred by minor effect genes. Clover rot resistance was not maternally inherited. To estimate the heritability of clover rot resistance, we applied divergent selection by the high-throughput bio-test on an experimental population. Our high-throughput bio-test altered the resistance level in both directions in the first generation of selection, but only marginal progress was made in the second generation. The heritability was on average 0.34 in the first cycle of selection and 0.07 in the second cycle of selection.
    The insights obtained during this research offer a basis for resistance breeding. The influence of isolates in resistance breeding is expected to be small. Allocating sources of resistance is difficult, yet recent cultivars and landraces from different genetic backgrounds may be the most suitable. Mass selection is ineffective, yet successive cycles of family selection against a strong selection pressure may increase clover rot resistance. Tetraploidisation of diploid populations may provide an additional level of protection. Finally, attention should be paid to other ways of controlling clover rot, such as culture techniques and biocontrol agents, as they may prevent the disease even more efficiently than cultivars with low susceptibility.

    Translated title of the contributionVeredeling naar resistentie tegen klaver rot (Sclerotinia spp.) in rode klaver (Trifolium pratense)
    Original languageEnglish
    Place of PublicationGent
    Print ISBNs978-90-5989-629-1
    Publication statusPublished - 5-Jul-2013


    • B390-phytopathology
    • B390-breeding

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