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

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 -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 biotest 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

Number of involved genes and heritability of clover rot (Sclerotinia trifoliorum) resistance in red clover (Trifolium pratense)

European red clover (Trifolium pratense) crops are challenged by clover rot, a devastating disease caused by Sclerotinia trifoliorum or, in some cases by S. sclerotiorum. No completely resistant cultivars are available and resistance breeding is hampered by the lack of knowledge on the number of involved resistance genes and the heritability of clover rot resistance. In this study, we estimated the number of major genes contributing to clover rot resistance by analysing 15 F1 progeny populations from pair crosses between ramets of resistant and susceptible genotypes. Parent plants were chosen from diverse, diploid populations, including wild material, landraces and cultivars. Young progeny plants were inoculated with ascospores, evaluated phenotypically and the segregation of disease scores was studied. Our results indicated that clover rot resistance may be conferred by three major effect genes, although segregation patterns suggested that there may be numerous minor effect genes involved as well. No proof was found for a maternal inheritance of clover rot resistance. To get insight in the heritability of clover rot resistance, we applied divergent selection by our high-throughput bio-test on an experimental diploid population: the original population (70.5 %), the first generation after selection for susceptibility (79.2 %) and the first generation after selection for resistance (62.3 %) differed significantly in susceptibility (p\0.001). The second generation after selection for resistance (60.0 %) was not more resistant than the first generation after selection for resistance. In the first generation of selection the heritability (h2) was on average 0.34. In the second generation of selection h2 was 0.07. These findings have important implications for resistance breeding

Influence of flower and flowering characteristics on seed yield in diploid and tetraploid red clover

Tetraploid red clover (Trifolium pratense L.) often produces insufficient seed to be economically interesting. Numerous studies have identified poor pollination due to long corolla tubes as its cause, but other studies disagree. Therefore, we investigated seed yield, corolla tube length and flowering traits in 244 plants from five diploid and five tetraploid cultivars during 2years. Tetraploids produced fewer seeds, fewer flower heads and fewer seeds per head, but an equal number of flowers per head when compared to diploids. Although corolla tubes were longer in tetraploid than in diploid red clover (P<0.001), no correlation between corolla tube length and seed yield was detected. Therefore, the corolla tube length is likely neither the main nor only cause of low seed yield in tetraploid red clover. Instead, three putative causes for low seed yield were identified: tetraploids produced less inflorescences per plant, possessed a lower degree of determinacy and had markedly less seeds per head when compared to diploids. Possible causes for such differences include lower pollen viability and higher rates of embryo abortion

Resistance of red clover to broad spectrum of Sclerotinia trifoliorum

In this research a diversity study on different European isolates of the pathogenic fungus S. trifoliorum will be performed using mycelial compatibility grouping and AFLP. The next step is the development of a bio-test to screen red clover plants for their resistance level against clover rot. The third step in this research is evaluating different European strains for their virulence and evaluating a broad spectrum of red clover varieties for their resistance against S. trifoliorum. Over 100 varieties will be evaluated, including cultivars, landraces and wild varieties. Finally the inheritage of clover rot resistance in red clover will be evaluated by a QTL study. Sclerotinia isolates have been collected from clover fields among different European countries. Mycelial compatibility has shown a large variability within fields. The DNA extraction has been optimized. Sequencing of the ITS-region will be used to determine the exact species of every isolate. Primer combinations are currently being tested for the AFLP study. Different culture media were tested for their capacity to induce the production of multiple big sclerotia. The most optimal medium is being used to produce sclerotia from every isolate. Sclerotia are induced to apothecia formation and formed ascospores will be used to construct the bio-test

Morphological and pathogenic characterization of genetically diverse Sclerotinia isolates from European red clover crops (Trifolium pratense L.)

Clover rot, an important disease in European red clover crops, is caused by Sclerotinia trifoliorum or Sclerotinia sclerotiorum. Until today, little is known about the variation in aggressiveness among Sclerotinia isolates from red clover. Aggressiveness has never been correlated with morphological characteristics. Rapidly growing isolates may be more aggressive, but this was never investigated in S. trifoliorum before. Also nothing is known about the link between sclerotia production and aggressiveness. Oxalic acid is an important pathogenicity factor in Sclerotinia species, but its effect on aggressiveness is unknown in S. trifoliorum isolates. For this study, we selected 30 Sclerotinia isolates from 25 locations Europe: 26 S. trifoliorum isolates and 4 S. sclerotiorum isolates from two locations in France (Fr.A and Fr.B). For each isolate, the in vitro growth speed, sclerotia production, oxalate production and aggressiveness were analysed and correlations were estimated between aggressiveness and the other characteristics. Aggressiveness was assessed in vitro on detached leaves and in a greenhouse on young plants. Our isolates differed significantly in growth speed, sclerotia production, oxalate production and aggressiveness. The infections on detached leaves and young plants revealed interaction between isolates and plant genotypes and between isolates and cultivars, but there was no indication that pathotypes exist. In vitro growth speed and in vitro aggressiveness on detached leaves were positively correlated with aggressiveness on young plants, while sclerotia production was negatively correlated with aggressiveness on young plants. These factors can be used as predictors of aggressiveness of Sclerotinia isolates from red clover crops

Improving seed yield in red clover through marker assisted parentage analysis

Red clover (Trifolium pratense L.) is an important perennial forage crop in Europe. Unfortunately, seed production of modern cultivars is often unsatisfactory and breeding progress for higher seed yield is slow. We evaluated whether marker assisted parentage analysis could identify progeny plants with two high seed-yielding parents in a diploid selection trial. Furthermore, we studied if breeding for high seed yield could be speeded up by selecting progeny plants with two high seed-yielding parents, compared to traditional selection on the mother plant only. In this study, 111 genotypes with excellent vegetative traits from a first cycle selection trial were allowed to pollinate each other in isolation. After seed harvest, ten high seed-yielding plants were identified and their seeds were used to grow ten half-sib progeny populations, with 1,121 plants in total. Two multiplex primer sets targeting 18 SSR loci were designed and used to fingerprint parental and progeny plants. Parentage was reliably determined for 1,083 progeny plants: 135 plants were the result of a cross between two high seed-yielding parents. In a 2-year seed yield trial, the seed yield of these 135 progeny plants was compared to that of a control group of 216 progeny plants from the same ten high seed-yielding mother plants but with no selection on the male contribution, as is current practice. Compared to the control group, progeny plants with two high seed-yielding parents had significantly higher seed yields, 23.0 and 75.9 % higher in 2012 and 2013 respectively, and a significantly higher thousand seed weight, 6.6 and 5.7 % higher in 2012 and 2013 respectively. Seed yield was strongly correlated with flower abundance (r = 0.643) and plant volume (r = 0.593 in 2012 and r = 0.509 in 2013) and negatively correlated with susceptibility to mildew (r = -0.145). We conclude that breeding programs aiming at increasing seed yield in diploid red clover cultivars would strongly benefit from the integration of marker assisted parentage analysis to identify those progeny plants that are the result of a cross between two high seed-yielding plants. This goal cannot be achieved with traditional family selection where only the seed yield of the mother plant is known

Aggressiveness study on Sclerotinia isolates from red clover crops

Sclerotinia trifoliorum Erikks. causes clover rot (clover cancer, Sclerotinia crown and root rot) in red clover crops (Trifolium pratense L.), an important disease in Europe. Little is known about the aggressiveness of Sclerotinia isolates and aggressiveness studies were never conducted on a European scale. In this study we compared the aggressiveness of 30 Sclerotinia isolates isolated from red clover crops in 25 locations in 12 European countries using a plant-based bio-test. Plants from 6 red clover cultivars with different resistance levels were spray inoculated at the age of 12 weeks with 1 to 1.5 ml mycelium fragment suspension per plant. After 10 days incubation, plants were scored on a scale from 1 (healthy plant) to 5 (dead plant) and the disease index was calculated. The experiment was repeated 3 times and all repetitions were highly correlated. Average disease indices ranged from 52.6% to 82.7%. Significant differences were detected between isolates and between cultivars, but there was no isolate – cultivar interaction. Based on these results, the most aggressive isolates can be selected for resistance breeding. Future work should investigate whether the differences in aggressiveness are due to a higher growth speed or due to a higher secretion of cell-wall degrading components and pathogenicity factors