Searching for VL resistance factors in OSR xylem sap

Understanding mechanisms of VL-resistance was one of the major objectives of the present study. To this end, the investigations on xylem sap residing cultivar-related V. longisporum resistance factors in oilseed rape were undertaken in different experiments involving greenhouse studies, in vitro bioassays and biochemical analyses. Three B. napus genotypes with differential resistance towards VL were used.

Greenhouse studies were conducted for the purpose of verifying resistance responses of the genotypes and collection of xylem sap. In vitro bioassays and biochemical analyses were performed to study the effects of OSR xylem sap constituents on VL growth.

Analysis of the disease evaluation data obtained from assessment of disease index and quantification of VL DNA (Fig. 3.2) showed the development of very slight symptoms or disease level in genotype Aviso and SEM, confirming their resistance to VL. On the other hand, the very fast and high level of disease

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development in cultivar Falcon demonstrated its high susceptibility to VL. Further evidences for the consistent response of the three genotypes to VL-infection was shown in agronomic traits where unlike SEM, significantly higher reduction of plant height, stem thickness and shoot dry mater accumulation due to infection occurred in cultivar Falcon (Figure 3.3). Correlation analysis also showed very strong, significant, positive correlation within disease or agronomic parameters and negative correlations between disease and agronomic parameters (Table 3.6). VL-resistance of genotype SEM (SEM 05-500256) and susceptibility of Falcon under different experimental conditions (greenhouse, outdoor and field) was also shown in another independent disease screening experiments (Chapter 2). Furthermore, similar results from previously field and greenhouse disease screening studies have been reported (Knüfer, 2013; Eynck et al., 2009b; Rygulla et al, 2007b). Greenhouse and field studied by Keunecke (2009) also demonstrated the resistance of cultivar AVISO to VL.

From the phenotypic and molecular greenhouse disease evaluation results described in Chapter 2 and Chapter 4 and from previous studies on mechanisms of VL resistance in OSR (Eynck et al., 2009b;

Obermeier et al., 2013), it is well known that 21-28 DPI is the critical time point for resistance and susceptible genotypes to show marked differences in disease symptoms. That means, the level of accumulation of resistant factors in resistant genotypes at this time point is sufficient to reduce or restrict further growth and development of the pathogen. Hence, in order to compare the nature of VL growth in xylem sap of resistant (SEM) and susceptible (Falcon) plants, xylem sap derived from different aged plants was used in in vitro bioassay. Assessment of xylem sap fungal growth by spectrophotometry revealed that regardless of plant genotype, xylem sap served as a suitable medium for the growth of VL.

Initiation of spore germination and further fungal growth was detectable 2-3 days after incubation. Later 5 days after incubation, robust fungal growth that covered the whole area of the microtitre plate wells was observed. Compared to its typical slow growth on artificial medium (such as PDA) which usually takes several weeks to cover a full radius of a 90mm Petri dish (Fig. 1.2), VL growth on xylem sap seems a bit faster. Further inspection of fungal growth even one week after incubation shows a similar story, no significant growth difference in xylem sap derived from plants with contrasting response to VL infection. Similarly, comparisons of fungal growth in xylem sap of mock and VL-inoculated plants also revealed the absence of infection induced VL-resistance factor contributing to significant reduction of in vitro VL growth. Nevertheless, whether xylem sap constitutes of resistant and susceptible plants differentially affect fungal sporulation, was not investigated here.

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Interestingly, fungal growth on filtered xylem sap was slightly reduced, but did not significantly affect fungal growth. The minor protein binding property of the syringe filter used to prepare filtered xylem sap treatments might adsorb some substances that are required by the fungus and this might cause the slight reduction of fungal growth. Results of protein assay also showed slightly reduced amounts of total soluble proteins in filtered samples. Again, the difference in concentration of total soluble proteins in xylem sap of resistant and susceptible genotypes was not significant (Table 3.3). Even the infection-induced slight increase in protein concentration occurred similarly in both susceptible and resistant genotypes. Similar results have been reported in tomato where infection with V. albo-atrum caused a general increase of xylem sap proteins irrespective of plant resistance to the disease (Dixon and Pegg, 1972). The above mentioned facts are in strong agreement with the present bioassay results where no significant effect of plant genotype, VL-infection and filtration of xylem sap on in vitro growth of VL is shown. Unfortunately, compared to other plant fluids or tissue extracts, very little is known about the composition and role of xylem sap constituents in plants in general and in B. napus in particular. In agreement with the findings of the present study, the work by Floerl el al. (2008) showed no effect of VL-infection on B. napus total xylem sap protein concentration. This study further demonstrated B.

napus xylem sap as a suitable medium for the growth of VL. However, in contrast to this work, the study used a single susceptible genotype and found significant reduction of in vitro growth of VL on xylem sap of infected plants. As it is shown in another pathosystem, several factors such as symbiotic or pathogenic interactions can determine the level and composition of xylem sap proteins and other constitutes (Subramanian et al, 2009).

The other xylem sap constitutes related to plant defence are plant hormones. The role of the well-known defence hormone SA in B. napus-VL interaction was investigated in previous studies. Results provided a strong evidence for the lack of correlation between the enhanced accumulation of SA in xylem sap or tissue extracts and cultivar-related resistance of OSR against VL. These studies rather demonstrated VL-infection induced increased accumulation of SA in susceptible cultivars than in resistant plants (Ratzinger et al., 2009; Siebold, 2012; Kamble et al., 2013). Similarly, in V. dahliae-Arabidopsis interaction, impairment of SA does not cause either high sensitivity to disease or any change in symptom development (Veronese et al., 2003). Other plant hormones such as JA and ABA seems to have no or insignificant role in Arabidopsis- and B. napus- VL interaction (Ratzinger et al., 2009;

Veronese et al., 2003).

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The other interesting observation of the present study that exactly correlates with natural (field) lifecycle of the pathogen is its enhanced in vitro growth on xylem sap of older aged plants (Fig. 3.8). As thoroughly discussed in previous chapters (Chapter 1 and 2) and section 5.1 of this chapter as well, VL shows a long biothrophic latency period during early stages of plant development. Late in the growing season, the pathogen undergoes fast development and rapidly disseminate to the above-hypocotyl shoot part, leading to the development of the typical phenotypic symptoms (Knüfer, 2013). It is possible that VL can sense signals of plant developmental stages present in OSR xylem sap. This may possibly explain why the fungus displayed fast in vitro growth in xylem sap of older plants. The increased accumulation xylem sap sugars in older plants which is also known for other crop species such as tomato (Wang and Bergeson, 1974)and Arabidopsis (Yu et al., 2013) could also be a possible explanation for enhanced growth of VL in xylem sap of older aged plants.

This is a first study on functional analysis of cultivar related VL-resistance factors in winter OSR xylem sap. It provided concrete evidence that OSR xylem sap, irrespective of plant genotype, provide a suitable nutritional and chemical environment for the growth of VL. The slightly increased total soluble protein and sugar content in xylem sap of infected plants also demonstrates possible VL-induced changes in the composition or level of OSR xylem sap constitutes. However, since these quantitative changes were not significantly different between resistant and susceptible genotypes, it can be concluded that OSR soluble xylem sap constituents are not playing a role as major resistance factors for cultivar-related winter oilseed rape resistance against VL. This is in strong agreement with the findings of previous studies on mechanisms of VL-resistance in OSR that demonstrated the significant role of cell wall bound metabolites and physical barriers in resistance of OSR to V. longisporum (Eynck et al., 2009b; Obermeier et al., 2013). Nevertheless, further studies that encompass a large number of genotypes and assessment of other parameters such as fungal sporulation are suggested.