| dc.description.abstracteng | Oilseed rape (Brassica napus) is one of the most important sources of vegetable oil in the world. Due to the growing demands in the biodiesel industry in particular, the global OSR production is gradually increasing since the last decade. Among other factors, the intensive production of OSR may have largely contributed to the emergence and increased economic importance of pest and diseases. One of the most important pathogens, Verticillium longisporum (VL), is a recently evolved vascular pathogen of crucifers. In recent periods, it has become a potential threat to OSR production in major OSR growing countries of the temperate region. This soil borne host-specific fungus causes foliar chlorosis, reduced growth, and premature senescence and ripening which ultimately leads to substantial yield losses. Unavailability of VL-effective fungicides and production of abundant and highly durable microsclerotia contributing to the soil inoculum are among the major factors that greatly hampers the management of VL. The only possible alternative management option available at present is the use of genotypes with enhanced resistance. The present study therefore focused on the identification of B. napus lines resistant to VL. Besides, the applicability of qPCR as an alternative method for the assessment of VL disease severity in the field was validated. Furthermore, mechanisms of cultivar related disease resistance and the significance of plant resistance mechanisms to VL under drought stress conditions were investigated.
Chapter two describes the resistance screening experiments conducted under greenhouse, outdoor and field conditions. Initially, a large number (>230) of B. napus DH lines and accessions were screened for VL resistance in multiple greenhouse experiments. Results of disease severity (AUDPC) and stunting effect assessments not only demonstrated the availability of VL-resistance in B. napus but also showed the presence of a wide range of variation in the level of resistance. Accordingly, B. napus lines that showed high degree of resistance in the greenhouse conditions were identified and recommended for further evaluation in the field. It was also found that the greenhouse resistance screening method used in this study, that involved the use of non-vernalized few-weeks old plants, provided consistent and reliable information. The method further enabled the screening of quite a large number of plants within a relatively small space and short period of time.
The outdoor screening experiment was conducted with the specific objective of identifying the sources/causes of variations in VL symptoms (mainly stunting and excessive branching) between greenhouse and field conditions. Results showed that stunting is significantly correlated with disease severity (genotype’s susceptibility). This is in agreement with greenhouse observations and suggests the possible effect of type/source of inoculum and method of inoculation on the development of this symptom. In contrast, increased branching as a result of VL infection was genotype dependent and is apparently not related to susceptibility to VL.
In the field experiments, more than 80 best performing lines selected based on previous greenhouse screening results were evaluated in three different locations that vary in climatic factors, soil conditions and level of natural disease infestation. From the field trials conducted for three consecutive seasons (from 2010/11 to 2012/13), it was understood that plant genotype, seasonal variations and locational differences have a significant influence on the extent of disease development and the resulting response of plants to VL infection. Like in greenhouse conditions, significant differences among genotypes were observed in the field. Accordingly, best performing B. napus DH lines were identified and recommended for use as parent materials in future breeding programs that aim at integrating VL resistance in commercial OSR varieties. The other interesting observation from the field experiments was the substantial variation of disease severity across locations. The highest disease severity was observed in Svaöv followed by Fehmarn. In contrast, the least disease level was recorded in Göttingen, where unlike the other locations trials were conducted with additional artificial application of inoculum. Regarding seasonal variations, comparisons were not conclusive as no complete data sets were available. Nevertheless, compared to the later seasons, disease severity in 2010/10 was relatively higher. More importantly, correlation analysis showed that disease index assessment results are poorly and only occasionally correlated across locations, years as well as with results of greenhouse experiments. In contrast, qPCR was the only field disease evaluation parameter that showed very strong correlations across locations. Furthermore, unlike disease index, this parameter was more significantly correlated to field disease index recordings in different locations and years, and with AUDPC and stunting effect results of greenhouse and outdoor experiments. With this method, detection of OSR infection in the field was possible as early as BBCH65 (50% flowering stage). However, distinct separation of resistance and susceptible varieties and differentiation of infestation levels across locations was achieved at BBCH80. Hence, for reliable quantification of VL disease severity and identification of differential plant resistance by qPCR in the field, sampling between growth stages BBCH70 and BBCH70 are recommended. In conclusion, the use of qPCR method for detection and quantification of VL infection in OSR field samples has several merits over the use of post-harvest stubble disease index screening. The consistency and rapidity of this method and possibly the cost effectiveness in comparing plant resistance or assessing disease epidemics in the field is much more reliable than post- harvest stubble disease index assessment. Nevertheless, further improvement of this method to achieve early (in late autumn or early spring) detection of infection or disease severity will provide a more applicable and timely information.
Chapter three mainly focused on the investigation of cultivar related VL-resistance factors in winter OSR xylem sap, an environment where the pathogen spends most part of its life cycle. Three B. napus genotypes with known differential resistance towards VL were used. Greenhouse studies were conducted to obtain xylem sap and verify resistance responses of the genotypes. In vitro bioassays and biochemical analyses were performed to study the effects of OSR xylem sap constituents on the growth of VL. From in vivo phenotypic (AUDPC and stunting effect) and molecular (qPCR) disease severity evaluations, the resistance of genotype SEM and susceptibility of Falcon was confirmed. Further evidences for the consistent response of these genotypes to VL-infection were obtained from the assessment of stem thickness and biomass yield. In vitro fungal growth analysis showed uniform growth of VL on xylem sap of 28 DPI old susceptible and resistant genotypes. Similarly, comparisons of fungal growth in xylem sap of mock and VL-inoculated plants revealed the absence of infection induced VL-resistance, which would contribute to significant reduction in VL growth. Quantification of total soluble protein content also showed no significant difference between xylem sap of resistant and susceptible genotypes. Even the infection-induced slight increase in protein concentration was similar in both susceptible and resistant genotypes. Interestingly, a time course independent study using the resistant genotype AVISO and susceptible cultivar Falcon indicated that regardless of plant resistance to VL, xylem sap collected from older plants provide enhanced fungal growth. This phenomenon correlates with natural lifecycle of the pathogen on field grown OSR plants. The increased accumulation of xylem sap sugars in older plants could also be one of the possible explanations for enhanced growth of VL in xylem sap of older plants. This is a first study on functional analysis of cultivar related VL-resistance factors in winter OSR. It provided concrete evidence that OSR xylem sap, regardless of disease resistance, provides a favourable 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 constituents. However, since these quantitative changes were not significantly different between resistant and susceptible genotypes, it can be concluded that soluble xylem sap constituents do not play a role as major resistance factors for cultivar-related resistance of winter OSR against VL. This is in strong agreement with previous studies on the mechanisms of VL-resistance in OSR that demonstrated the significant role of cell wall bound metabolites and physical barriers. Further studies with large number of genotypes and assessment of other parameters such as effects of xylem sap constituents on fungal sporulation are suggested.
In the last chapter, the study on the response of B. napus to the combined effects of VL infection and drought stress was conducted. Previously, substantial amounts of vascular occlusions that obstruct xylem vessels have been detected in resistant VL-infected OSR genotypes. This mechanism of resistance to the pathogen may however alter the rate of water and nutrient transport and consequently plant response to drought stress. To investigate whether genotypic VL resistance is associated with a reduced drought tolerance, drought resistance of VL-resistant and susceptible winter OSR genotypes were studied in combination with infection with VL. Furthermore, the influence of drought stress on the response of plants to VL infection was investigated. This study was conducted in a controlled pot experiment where seedlings of the VL susceptible cultivar Falcon and the tolerant line SEM were inoculated with VL and exposed to three watering levels (optimum, moderate deficiency and severe deficiency i.e. watering at 100, 60 and 30% field capacity). Analysis of disease parameters (AUDPC, stunting and qPCR) showed a significantly lower rate and level of disease development in the resistant genotype across all watering regimes. Likewise, regardless of the water supply at different field capacity levels, high disease severity and stunting effects were observed in the susceptible cultivar. Furthermore, the amount of fungal DNA was up to 31fold in Falcon as compared to SEM. qPCR results showed that levels of fungal DNA were positively correlated with the intensity of drought stress. At 49 DPI, the respective average fungal DNA in dry hypocotyl tissue at 100, 60, and 30% field capacity was 27.1, 29.0 and 36.0 ng/g in SEM and 839.1, 1,032.4 and 1,096.4 ng/g in Falcon; indicating more pronounced effect of VL during drought stress, particularly on susceptible B. napus varieties. Significant changes in physiological parameters (gas exchange, relative water content, proline accumulation and water use efficiency) and up-regulation of drought stress marker genes confirmed the reaction of both genotypes to drought stress. On the other hand, neither VL alone nor its interaction with drought or the genotype had any significant effect on physiological parameters. Further comparisons of the drought induced physiological changes under mock- and VL-inoculation conditions showed a cultivar-independent trend of a slightly reduced impact of drought stress during VL infection. The main and interactive effects of VL and drought on biomass yield and other agronomic traits (stem diameter, branching and phenological growth stage) were significant but the magnitude of their impact was dependent on differential disease and physiological responses of the genotypes. In general, the consistent and interrelated results from ANOVA, correlation, regression and principal component analyses of the present comprehensive study not only proved that VL-resistance mechanisms have no additive negative consequence on plant performance under drought stress but also demonstrate effective functioning of the quantitative VL-resistance mechanisms even under conditions of severe drought stress. Nevertheless, despite the stable VL-resistance under water deficit conditions and the slightly smaller effects of drought on infected plants, simultaneous exposure of OSR to both stresses can cause considerable yield loss. | de |