GENERAL CONCLUSIONS AND PERSPECTIVES FOR FUTURE RESEARCH
7.2. Future objectives and perspectives for future research
This thesis opens perspectives for further research. The AFLP primers that were developed for S. trifoliorum can be used to screen more isolates. For example, the genetic diversity among S. trifoliorum strains from different host crops such as white clover, alfalfa, pea and common bean could be studied using our AFLP primers.
Although we found no population structure among S. trifoliorum isolates from red clover, subpopulations might be present between S. trifoliorum isolates from different host crops.
Sclerotinia isolates differed in aggressiveness and an analysis of the infection process suggested that aggressive isolates invest more energy in penetrating the host tissue rather than spending energy growing on the surface of the leaf. More research would be needed to investigate how aggressive isolates can penetrate more cells with an equal number of appressoria. In other words, why do the appressoria of aggressive isolates have a higher success rate? Possibly, the higher success rate of penetration in aggressive isolates is due to a different mechanism of host cell recognition or host cell infection. In that case, resistance to these isolates may be based on other resistance genes or QLRs, and resistance breeding should include aggressive isolates as well as less aggressive isolates to provide protection against a broad spectrum of Sclerotinia isolates.
We have shown that collected ascospores can be stored for more than one year while viability remains high. In the future, viability should be measured during longer periods to determine the shelf life of ascospores. Ascospore production should also be optimised for S. sclerotiorum isolates from red clover crops, as this species can also cause clover rot in red clover. Various protocols have been described for ascospore production in S. sclerotiorum (Dillard et al., 2001), but our attempts to produce S.
sclerotiorum ascospores were not successful until now.
Our high-throughput bio-test is a valuable tool for breeding programmes. We will continue to use it in the ILVO breeding programme and in future research. We suggest to apply our high-throughput bio-test at the beginning of each selection cycle.
However, if infrastructure is limiting, the bio-test would be most useful in the first cycle of selection when the genetic diversity is largest. Successive applications in the second and third cycle will increase homozygocity for major QRLs. Future research should design an optimal breeding scheme to improve clover rot resistance by means of the high-throughput bio-test, along with other important traits. Selection for clover rot resistance must leave sufficient genetic variation to allow the necessary progress in other important traits such as resistance to rust disease, plant vigour and seed yield.
Although we found no pathotypes among Sclerotinia isolates from red clover, the effect of the isolate in resistance breeding remains largely unknown. Some interaction between Sclerotinia isolates and genotypes or cultivars was present, suggesting that there is an influence of the isolate in resistance breeding. Does screening with one S.
trifoliorum isolate result in resistance against all other S. trifoliorum isolates? The
answer to this question might be similar as observed in previous studies, such as the EUCARPIA multi-site crown rust evaluation on ryegrasses (Schubiger et al., 2007;
Schubiger et al., 2010). In these trials, 33 perennial, 15 Italian and 3 hybrid ryegrass cultivars were evaluated at 24 sites in 11 European countries for resistance to crown rust (Puccinia coronata) in field conditions. Crown rust susceptibility of Italian and hybrid ryegrass cultivars was significantly different among sites, just as we found for clover rot susceptibility in red clover cultivars. Yet despite the interaction between cultivars and sites, the correlation between the susceptibility at particular sites and the average susceptibility at all sites was significant for all but a few cases in the three ryegrass species. Moreover, the ranking of cultivars according to average crown rust susceptibility was highly similar in four trial years between 2001 and 2010 (Schubiger et al., 2007; Schubiger et al., 2010). In other words, the resistance level of ryegrass cultivars to crown rust remained more or less the same when evaluated against different isolates in different environmental conditions. Godoy et al. (2005) found similar results in resistance to white mould (S. sclerotiorum) among 47 sunflower hybrids. Hybrids showed different levels of resistance in different environments.
However, the ranking of genotypes did not significantly change from one location to another because the differential responses were only a matter of scale. This type of genotype - environment interaction is known as non-crossed or quantitative interaction. Our results indicate that the same may be true for clover rot resistance in red clover.
As two Sclerotinia species cause clover rot in red clover, the same question can be asked for the two species: does selection for resistance against S. trifoliorum also confer resistance to S. sclerotiorum? Results of Pratt and Rowe (1995) indicate that responses to both pathogens are similar in alfalfa and that selection to S. trifoliorum may also confer resistance to S. sclerotiorum. Moreover, the host of origin would not be an important determinant for the virulence of Sclerotinia isolates on alfalfa (Pratt and Rowe, 1995). Our results showed similar responses of red clover to both pathogens, possibly indicating that selection against one Sclerotinia pathogen may confer resistance to the other, although more research would be needed to prove this hypothesis. It would be interesting to set up a European scale multi-site evaluation for clover rot on various red clover cultivars. Such study would allow to investigate the interactions between clover rot resistance, local isolates from both Sclerotinia species and environmental conditions on a European scale.
Our study indicated that environmental conditions may play a key role in clover rot resistance. For example, a cold treatment prior to inoculation decreased susceptibility to clover rot in our study. Probably, a cold treatment induces the production of proteins with a dual function in plant defence and cold tolerance. More research should be done in this direction. When selecting for winter hardiness in the field, one partly selects for clover rot resistance, because clover rot is one of the factors weakening plants in wintertime. In other words, plants with a good resistance to clover rot are favoured in winter hardiness but the question remains whether plants with higher winter hardiness are also more resistant to clover rot.
Analysis of segregation ratios in progenies of pair crosses between resistant and susceptible genotypes suggest that clover rot resistance is determined by three major effect QLRs and numerous minor effect QLRs. Future research should confirm this hypothesis by making crosses between resistant F1 plants. The segregation of the F2
progeny should confirm whether there are indeed three major QLRs involved. We have estimated h2 in an experimental diploid population containing European cultivar germplasm. Future research should estimate h2 in other populations, as it may be different. Until now, no estimates for h2 are available for tetraploid populations. It is unclear if the heritability would be similar in tetraploid cultivar germplasm. As the genetic diversity might be lower in tetraploids, the heritability may be lower as well.
To localise possible QRLs in the genome, the progeny populations from our pair crosses between resistant and susceptible plants can be used as mapping populations for a quantitative trait loci (QTL) study. A linkage map with SSR (simple sequence repeat) markers is already available for red clover (Sato et al., 2005), and a QTL study may allow the detection of QRLs. Until now, the genome of red clover has not been sequenced. Sequencing the genome of red clover may allow the identification of even more QRLs through sequence homology with related Fabaceae species. At least a part of the resistance to clover rot may be situated at the level of entry in host tissue.
Future research should investigate this possibility in more detail.
We found that tetraploids were more resistant than diploids, yet it largely remains unclear why. It would be interesting to investigate exactly why tetraploids are more resistant than diploids.
Finally, in this thesis we focussed on disease resistance as a means to limit damage by clover rot. Yet the decreasing heritability of clover rot resistance after successive generations of selection and the major influence of environmental conditions on the disease development may indicate that complete resistance to clover rot can never be achieved. Instead, other methods to control clover rot may be more effective than the use of resistant cultivars. Future research should investigate integrated control strategies for clover rot, rather than focussing on resistance breeding only. The feasibility to use methods such as biocontrol agents (Contans® WG) and biofumigation to control clover rot should be investigated. Previous studies reported suppressed growth of S. sclerotiorum in the presence of organic residues of cabbage (Brassica oleracea) (Li et al., 2006) and Indian mustard (Brassica juncea) (Larkin and Griffin, 2007). Crop rotation with cabbage and mustard crops and mixing their organic residues in the soil may help to prevent clover rot in subsequent red clover crops, but more research would be needed to prove this hypothesis.
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