B. napus physiology during drought stress and infection with V. longisporum

Physiological parameters were monitored in order to address the questions concerning B. napus-VL interaction under drought stress conditions. The first and foremost objective was to investigate whether VL-infection and the resulting accumulation of vascular occlusions have a negative impact on plant fitness under conditions of water deficit stress. In addition, the role of genotypic difference in

Chapter 4. B. napus - VL interaction under drought conditions Discussion

126

determining physiological responses under disease and drought stress conditions was studied. To answer the above questions, several physiological parameters in plants grown under optimal conditions were compared to those exposed to either drought, VL-infection or both stress factors. Results obtained from the different physiological measurements as well as gene expression analysis consistently showed that VL infection in B. napus did not cause a significant change to most of the physiological variables considered. That is, except for proline synthesis and water use efficiency, all other physiological parameters measured in this study were only significantly affected by drought stress. Neither VL alone nor its interaction with drought or the genotype had a significant effect on expression of drought inducible genes (Fig. 4.12), gas exchange and RWC parameters (Table 4.9).

Gas exchange and water use efficiency

In general, regardless of genotype and VL infection, stepwise decrease in gas exchange (transpiration rate, stomatal conductance, photosynthesis rate) and increase in WUE was observed as water supply dropped from full watering (100% FC) to 60% and 30% FC. Mixed model ANOVA showed that a significant reduction in gas exchange was only induced by drought. The remaining two factors and their interaction had no impact at all. Regarding WUE, in addition to the strong impact of drought, genotype x VL x drought interaction had a significant effect (Table 4.9). It was also noticed that the genotype SEM had a slightly higher WUE than Falcon. Besides, unlike VL-inoculated plants, 30% FC watering in mock-inoculated SEM plants significantly increased WUE (Fig. 4.7). Since increased WUE is a common evolutionary designed response of plants to drought stress (Blum, 2005), the above observations suggest two key points, namely the drought sensitivity of genotype SEM and the reduced impact of drought stress during infection with VL. In contrast to SEM, severe drought stress significantly increased WUE in VL-inoculated Falcon plants but not in mock-inoculated treatments, again suggesting a better reaction of this genotype to drought and the severe impact of VL-infection in this genotype as well. As it is shown for gas exchange parameters, VL infection has no significant impact on WUE of both genotypes (Table 4.9; Table 4.12).The impact of drought stress on B. napus gas exchange has been extensively investigated in various field and greenhouse studies. According to these studies, photosynthesis, transpiration and stomatal conductance are the major gas exchange parameters that are significantly reduced due to plant exposure to drought stress (Hashem et al., 1998; Naderikharaji et al., 2008;

Sangtarash et al., 2009).

Regarding the impact of VL, except for the slight reduction in transpiration rate and stomatal conductance in fully irrigated (100% FC) treatments, VL-infection alone and its interaction with drought

Chapter 4. B. napus - VL interaction under drought conditions Discussion

127

and genotype had no significant effect on gas exchange (Table 4.9; Table 4.12). Similar results have been reported by Floerl et al. (2008) who have shown no impact of VL-infection on photosynthesis and transpiration rates in B. napus. In another study with Arabidopsis, they also have shown that VL-infection has no negative impact on plant water and nutrient relation (Floerl et al., 2010). Similar observations have also been reported elsewhere in several crop species infected with other Verticillium species. Haverkort et al., 1990 found that V. dahliae alone and its interaction with drought had no impact on transpiration rate and stomatal conductance in early developmental stages of potato. In sunflower, stomatal conductance was unaffected by infection with V. dahliae (Sadras et al., 2000).

Verticillium albo-atrum infection in alfalfa caused no significant reduction in stomatal conductance and photosynthesis (Pennypacker et al., 1991). Besides drought stress, different concomitant factors such as fungal toxins are suggested as causes of stomatal closure that lead to depression in photosynthetic activity in Verticillium albo-atrum infected tomato plants (Lorenzini et al., 1997). In another pathosystem, Thorne et al. (2006) have shown that grapevine infection by the xylem-limited bacterium Xylella fastidiosa caused no effect on stomatal conductance and transpiration rate. In the B. napus-VL interaction, it is evident that vascular colonisation of VL and accumulation of occlusions is restricted to individual xylem vessels and adjacent vessels remained completely free and unaffected (Eynck et al, 2007). The presence of unaffected and fully functional vessels could provide adequate capacity for sufficient uptake of water and mineral nutrients. This could be one of the possible explanations why gas exchange is not affected by VL-infection and also why VL is not causing wilt symptoms in B. napus. Floerl et al. (2008) suggested that chlorotic and stunting symptoms of VL-infection in B. napus are not the result of limited water supply due to pathogen induced xylem obstruction. Induced accumulation of vessel occlusions due to infection with the bacterium Xylella fastidiosa was not causal for the water deficit and plant wilting symptoms in grapevine (Thorne et al., 2006). In contrast, a significant reduction of gas exchange due to drought stress induced by V. dahliae infection is reported in several crop species including pepper (Pascual et al., 2010), tomato (Bowden et al., 1990) and cotton (Hampton et al., 1990).

Leaf relative water content

As previously described, reduction in leaf relative water content (RWC) was observed due to water deficit and the effect was particularly significant at severe drought stress and at the later time point (Table 4.4). A week after initiation of drought treatments (28 DPI), RWC of stressed plants was slightly decreased but the reduction was not statistically significant except in infected Falcon plants. Since drought treatments began at 21 DPI and plants at this time point were still small (BBCH 50; lower bud development stage), this might have led to low water demand and consequently to a low rate of

Chapter 4. B. napus - VL interaction under drought conditions Discussion

128

transpiration. As a result, the soil moisture difference between normally watered and drought stressed treatments at 28 DPI might have not been large enough to show significant differences. Kumar and Elson (1992) have shown a significant effect of plant growth stage on leaf RWC of drought-stressed B.

napus plants. At 49 DPI, however, due to large biomass production, applied water might be used quickly and reduction in leaf RWC was observed shortly after re-watering. Accordingly, 13% and 14% reduction in RWC was observed in mock- and VL-inoculated SEM plants, respectively. Interestingly, even at this time point, the reduction in cultivar Falcon was lower and insignificant, resulting in 10.3 and 10.1% loss at moderate and severe drought stress treatments, respectively. Depending on the plant cultivar or intensity of drought, up to 40% reduction of leaf RWC in B. napus have been reported in several studies (Ullah et al., 2012; Khalili et al., 2012; Sepehri and Golparvar, 2011; Good and Zaplachinski, 1994; Khalili et al., 2012). Regarding the disease factor, irrespective of plant age and genotype, no impact of VL-infection on RWC was observed (Table 4.9). Similarly, Reusche et al. (2014) recently reported no effect of the wilt inducing vascular pathogen V. dahliae on leaf water content of Arabidopsis. In pepper as well, leaf RWC remained unchanged until four weeks after inoculation with Verticillium dahliae (Goicoechea, et al, 2000). In contrast, Reusche et al. (2012) showed a rather positive impact of V. longisporum in Arabidopsis where infection provided improved leaf water content under drought stress condition.

Proline content

Accumulation of substantial amounts of free proline as a response to a wide range of biotic and abiotic stress is a common phenomenon in different groups of organisms (Delauney and Verma, 1993). In plants, stress-induced accumulation of proline has multiple positive roles in stress adaptation, recovery and signaling. It is involved in intracellular osmotic adjustment between cytoplasm and vacuole, protects photosynthetic organelles, stabilizes redox balance and influences programmed cell death which triggers HR during infection with avirulent pathogens, and regulates plant growth and development during stress conditions (Szabados and Savouré, 2010). In the present study, exposure of B. napus plants to drought stress caused up to 18-fold increase of proline accumulation. Drought-induced synthesis of free proline was positively correlated with the intensity of drought in plants in this study, with the highest amount quantified in plants supplied with water at 30% FC. Similarly, a gradual pattern of increase in proline concentration with increase in intensity and duration of drought (Ghaffari et al., 2011; Omidi, 2010) and salinity stress (Saadia, et al., 2012) has previously been reported in B. napus.

Other factors responsible for variations in drought-induced proline synthesis were genotype and plant part. The initial leaf proline content was slightly higher in cultivar Falcon than in SEM. Drought stress

Chapter 4. B. napus - VL interaction under drought conditions Discussion

129

however induced significantly higher proline synthesis in SEM, regardless of VL-infection and plant part (Fig. 4.10). It is possible that due to its sensitivity to drought, this genotype is responding with higher accumulation of proline. High proline accumulation does not necessarily reflect the level of drought tolerance in plants since it is rather a stress sensor and an indicator of the plant water status (Sundaresan and Sudhakaran, 1995; Hanson et al., 1977). This is in strong agreement with physiological, agronomic and disease evaluation results that clearly showed drought sensitivity and VL-resistance of this genotype. Expression analysis of the specific proline metabolism gene Δ1-pyrroline-5-carboxylate synthase1 (P5CS1) also revealed relatively increased expression of this gene in Falcon compared to SEM (Fig. 4.12). Saadia, et al. (2012) noted a maximum expression of the P5CS1 gene in a drought sensitive B.

napus line. On the other hand, several studies showed strong association of high proline concentration and improved drought tolerance in several crop species including B. napus (Ghaffari et al., 2011; Saadia, et al., 2012), soybean (Silvente et al., 2012), alfalfa (Kang et al., 2011) and rice (Bunnag and Pongthai, 2013). Considering the impact of VL-infection in proline synthesis, it was noticed that drought induced proline accumulation in leaf and hypocotyl tissue was significantly higher in the VL-resistant genotype SEM (Fig. 4.10). In pepper, Goicoechea, et al. (2000) suggested an increased proline accumulation as a sensor of wilt damage caused by V. dahliae infection. In other pathosystems, high proline concentration is associated with resistance of Arabidopsis (Fabro et al., 2004) and tobacco (Senthil-Kumar and Mysore, 2012) against avirulent strains of Pseudomonas syringae via triggering of HR. In our B. napus-VL interaction however, since VL resistance does not involve induction of HR, the involvement of proline (as a scavenger of ROS) in plant resistance is not likely. The second interesting factor that showed significant differences in the amount of drought-induced proline accumulation was the plant part. Irrespective of any other factor considered in this experiment, it was found that the drought-induced total amount of proline was significantly higher in leaf than in hypocotyl tissue (Table 4.5; Fig. 4.10). As a key regulator of drought stress adaptation and signaling, it is not surprising that proline is present in high concentration in leaf tissues. Higher production of drought induced proline in the leaf than in stem and root tissue has been previously reported in potato (Ghorbanli et al., 2012) and in the ornamental plant Matthiola incana (El-Quesni et al., 2012).

Comparison of drought induced changes in rate of physiological processes in the presence and absence of VL infection

The most interesting and important observation regarding physiological parameters was the difference in physiological changes between mock- and VL-inoculated plants. Comparison of changes in gas exchange, hypocotyl proline and WUE due to drought stress under mock- and VL-inoculation conditions

Chapter 4. B. napus - VL interaction under drought conditions Discussion

130

indicated that the impact of drought stress seems to be reduced during infection with VL. For instance, in mock-inculcated treatments, the rate of transpiration, stomatal conductance and photosynthesis at 30% FC was reduced 4-, 6- and 2-folds in SEM and 4-, 5- and 3-fold in Falcon, respectively. During infection with VL, the respective reduction of the three gas exchange parameters was 2-, 3-, and 1-fold in SEM and 2-, 2-, and 1-fold in Falcon. In case of hypocotyl proline content, 10- and 18-fold increase due to severe drought stress was observed in mock-inoculated SEM and Falcon plants, respectively.

Whenever the disease factor was added, proline accumulation was increased only by 7-fold in SEM and 4-fold in Falcon. Analysis of WUE also showed a similar trend for genotype SEM (Table 4.5). The reduced impact of drought stress during VL-infection in both VL-resistant and VL-susceptible B. napus genotypes may be associated with infection induced anatomical changes in xylem tissue. According to Eynck et al.

(2007) and Eynck et al. (2009b), colonization of xylem vessels with VL and accumulation of VL-induced vascular occlusions in B. napus are restricted to individual vessels and other adjacent vessels remain completely uninfected and free of obstructions. The presence of a sufficient number fully functional vessel might provide efficient uptake and transport of water and this may explain why plants are not suffering from drought stress during VL-infection. Yadeta and Thomma et al. (2013) also suggested that if less numbers of vessels are closed by occlusions, the host plant will not suffer from drought stress.

Furthermore, a recent study in Arabidopsis has shown enhanced drought tolerance of infected plants due to VL-induced de novo xylem formation (Reusche et al., 2014). Xu et al. (2008) showed viral infection induced an increase in osmoprotectant and antioxidant substances providing better drought tolerance to several crop species. Enhanced drought tolerance associated with mycorrhizal fungi has been reported in many crops such as wheat (Ellis et al., 1985; Abdel-Fattah and Abdul-Wasea, 2012), lettuce (Ruiz-Lozano et al., 1995), onion (Nelsen and Safir, 1982), common bean (Aroca et al., 2007), rosemary (Sánchez-Blanco et al., 2003), and pigeon pea (Qiao et al., 2011). Stimulation of increased accumulation of osmolites and sugars, improved nutrient uptake and root growth, reduced plant surface area of water loss etc. are among the mechanisms of mycorrhizal induced drought tolerance reported previously. It is also well known that an increase in xylem vessel density and diameter provides improved water absorption during drought which is closely associated with drought tolerance in tree (Qian and Ning, 2012) and annual crop species (Kulkarni et al., 2008).

4.4.3 Expression of drought responsive genes during drought stress and infection with V. longisporum