Two Brassica napus genotypes with different resistances to Sclerotinia sclerotiorum were used to clarify whether or not the epicuticular wax is involved in the defence mechanism of B. napus to S. sclerotiorum. The total wax and wax constituents were significantly higher in the susceptible cultivar than in the resistant cultivar, except for esters. Infection by S. sclerotiorum increased the content of aldehydes in both cultivars, while increasing the content of alkanes and unknowns in the resistant cultivar. More crystalloid rods were observed on adaxial surfaces of leaves of the susceptible cultivar than on those of the resistant cultivar. The resistance to S. sclerotiorum was correlated to the responses of wax content but not the amount of total wax. The up-regulation of transcription of a wax-related gene and a pathogenesis-related gene (PR1) in the pathogen-challenged resistant cultivar also supported this observation. After inoculation, the increase in phenylalanine ammonia lyase (PAL) activity in the resistant cultivar and the decrease in the susceptible cultivar were correlated with their resistance to disease. The content of alkanes, alcohols, ketones and total wax were negatively correlated with the activities of PAL and polyphenoloxidase. After wax removal, the resistant cultivar developed more necrotic spots compared to seedlings with intact wax, whilst no significant change was observed for susceptible seedlings. These results show that epicuticular wax contributed more to the defence of resistant cultivars than susceptible cultivars. The leaf epicuticular wax, defence enzymes and salicylic acid-dependent signalling pathway all contribute to defence against S. sclerotiorum in B. napus.
Sclerotinia sclerotiorum is a necrotrophic ascomycete that attacks more than 400 plant species, including economically important crops such as rapeseed (Brassica spp.; Purdy, 1979; Li et al., 2006). In China, S. sclerotiorum affects both the yield and quality of rapeseed. In the principal rapeseed production area along the Yangtze River, where almost 4·7 million ha are infected by S. sclerotiorum annually, losses of 5–25% in yield and infection of up to 50% have been recorded (Shuang et al., 2006).
In practice, foliar fungicide application is recommended for the disease management. However, awareness of environmental pollution from fungicides has led to the development of alternative measures, such as the use of oilseed rape cultivars resistant to S. sclerotiorum. Zhongshuang 9, an oilseed rape cultivar with high resistance to S. sclerotiorum and high yield, bred by multiple crossing and the microspore culture technique, was registered and released in China in 2002 (Wang et al., 2004). The activities of phenylalanine ammonia lyase (PAL), exochitinase, β-1,3-glucanase, peroxidase (POD) and polyphenoloxidase (PPO) in Zhongshuang 9 were higher than those in other susceptible cultivars after inoculation with S. sclerotiorum (Wang et al., 2004). The defence responses mounted against S. sclerotiorum appear to be multifactorial and other defence mechanisms might exist in Zhongshuang 9. Zhao & Meng (2003) reported that single-locus QTLs and epistatic interactions played important roles in Sclerotinia resistance of rapeseed. Yang et al. (2007) reported that more than 300 transcripts, including those putatively associated with jasmonic acid biosynthesis and signalling, reactive oxygen species metabolism, and cell wall structure and function, increased or decreased in abundance in S. sclerotiorum-challenged rapeseed.
Protection of plants against pathogens depends on constitutive and induced defence mechanisms. Some of these mechanisms are common to all types of assaults, such as tissue reinforcement and the production of antimicrobial metabolites; others are tailored to combat specific types of pathogen interactions (McDowell & Dangl, 2000). Luo & Zhou (1994) reported that the resistant rapeseed cultivars have some common features such as glaucous appearance caused by greater wax cover, upward and more compact branching, and strong stems. Epicuticular waxes on the surfaces of higher vascular plants are believed to play important roles in plant defence against bacterial and fungal pathogens (Jenks et al., 1994; Alcerito et al., 2002).
Chemically, epicuticular waxes consist of a very heterogeneous mixture of hydrocarbons, long-chain fatty acids, fatty aldehydes, primary and secondary alcohols, ketones and esters (Jenks et al., 1994; Alcerito et al., 2002). The morphology, arrangement and density of crystals and hydrophobicity of the wax components determine vital functions for plants such as the relative adhesion of water, pesticides and fungal spores. Wax components and chemical characteristics of Digitaria sanguinalis and Festuca arundinacea showed important roles in determining how effectively these plants were able to combat invasion of Curvularia eragrostidis (Wang et al., 2008). Needles from a resistant selection of eastern white pine had a significantly higher percentage of stomata that were occluded with wax, and fewer germ tubes penetrating stomata than needles from a susceptible selection (Smith et al., 2006). Alkane and ester proportions in rose leaf cuticular wax were most closely associated with blackspot disease susceptibility ratings (Goodwin et al., 2007).
Plant wax is believed to be responsive to environmental conditions. Abiotic stresses, arising from exposure to climatic extremes such as drought, heat, and UV irradiation, commonly induce changes in the amount and composition of waxes (Jenks et al., 1994; Alcerito et al., 2002). However, whether fungal pathogen infection influences the plant epicuticular wax is still not clear. In Brassica, it has been shown that the glaucous B. napus is less susceptible to Alternaria brassicae than the non-glaucous B. rapa (Tewari & Skoropad, 1976; Conn & Tewari, 1989). The objective of this study was to clarify whether or not epicuticular wax is involved in the defence mechanism of B. napus to S. sclerotiorum. Therefore, in the current study, the effects of S. sclerotiorum infection on the B. napus’ epicuticular wax morphology, composition, and the up-regulation of wax-related gene (KCS1) and pathogenesis-related gene (PR1) transcription were examined. The responses of wax composition and crystal structure to infection by S. sclerotiorum were also analysed.
Materials and methods
Seeds of two Brassica napus genotypes, the Sclerotinia-resistant cultivar Zhongshuang 9 (ZS9) and the susceptible cultivar Yuyou 19 (YY19), were obtained from the Chongqing Rapeseed Engineering Research Center, Beibei, Chongqing, China. Three seeds were sown in pots (10 cm diameter and 10 cm depth) filled with a mixture of 1·5 kg of oven-dried soil (Inceptisol) and sand (2:1, v:v). Seedlings were thinned to one plant per pot, 5 days after germination. Plants were grown in a greenhouse (22 ± 2°C, 16 h/8 h photoperiod regime) and irrigated every 3 days until the five-leaf stage. A total of 68 pots per genotype were used in the experiment and leaves from four independent biological replicates were prepared for analysis (three biological replicates for gene quantitative analysis).
Sclerotinia sclerotiorum spray inoculation
The strain of S. sclerotiorum used in this study was isolated from lesions of diseased stems of field-grown B. napus. Sclerotia were subcultured onto solid potato dextrose agar (PDA) media under light (24 h per day) for 3 days. Then agar plugs (0·5 cm) were removed with a sterilized cork borer from the leading edge of the mycelia and were added into the liquid culture medium at the rate of 10 plugs per 100 mL. The medium was cultured at 25°C and 200 rpm for 5 days, and homogenized using an homogenizer for 30 s to form a mycelial suspension. The suspension was then diluted to a final concentration with an optical density of 1·0–2·0 at 600 nm, and sprayed onto plant leaves. Leaves of uninoculated plants were treated similarly with a PDA suspension without the mycelia. Plants, uninoculated and inoculated, were placed in a humidity chamber for 24 h, and then transferred into a greenhouse (22 ± 2°C, 16 h/8 h photoperiod regime). Twenty-four, 48 and 72 h after inoculation, the second intact leaves from the top of the plant were harvested from uninoculated and inoculated plants for enzyme activity measurements and RNA extraction, and the leaves harvested 72 h after inoculation were also used for gas exchange measurements, wax extraction and scanning electron microscopy (SEM) observation.
Assessment of resistance to S. sclerotiorum
The resistance to S. sclerotiorum was rated according to a disease severity index based on symptoms observed and a disease severity formula as described previously (Purdy, 1979; Li et al., 2006) with minor modification. The disease was classified into five levels: 0 (no lesions), 1 (lesion area smaller than 1 cm2), 2 (lesions smaller than 2 cm2), 3 (lesion area smaller than 4 cm2), and 4 (lesion area larger than 4 cm2). The lesion diameters were measured to calculate the lesion areas. The disease severity index (DSI) ranged from 0 (no disease) to 100 (whole plant dead) and was calculated for each plant by using the following formula:
DSI = Σ(class × no. of leaves in class) × 100/(total no. of leaves × 4)
where 4 is the maximum disease rating value. Disease severity indexes for each cultivar were determined by rating eight replicate plants.
Wax layer removal
To examine whether wax removal influences gas exchange indexes and infection of leaves, the epicuticular wax layer was removed from the adaxial and abaxial sides of 16 plants per genotype using Blu-Tack (Bostik) as previously described (Mohammadian et al., 2007). This method proved to be more efficient and safe compared to chloroform removal in an in vivo pre-experiment. The removal efficiency of the wax was evaluated by SEM. Half of the plants with wax removed were sprayed with S. sclerotiorum inoculum as described above. Seventy-two hours after inoculation, the disease index was calculated and the gas exchange indexes and activity of defence enzymes from all wax-removed plants were measured.
Leaf gas exchange measurements
Gas exchange parameters were measured between 10:00 and 11:00 h in the second fully expanded leaf from shoot top using a LI–6400 Portable Photosynthesis System (LI–COR Inc.). The leaves were measured under their respective CO2 atmospheric concentrations during growth (400 μmol mol−1 CO2) with a red/blue LED light source (LI-6400-02B) mounted onto a 6-cm2 clamp-on leaf chamber. Gas exchange parameters were calculated as described by Voncaemmerer & Farquhar (1981). Four plants were measured in each treatment (uninoculated or inoculated).
Wax extraction and chemical analysis
The leaves used for gas exchange measurements were dipped in 10 mL chloroform containing 5 μg hexadecane (Sigma) for 30 s at room temperature. Hexadecane was added as an internal standard. The raw wax extractions were filtered through sterilized cotton to remove leaf dust, dried using a nitrogen stream, and then derivated with 100 μL BSTFA (N,O–bis(trimethylsilyl) trifluoroacetamide) for 20 min at 100°C, and the surplus BSTFA was evaporated under nitrogen. The sample was redissolved in 1 mL hexane for wax analysis using GC–2010 (Shimadzu Technologies Co.) equipped with a flame ionization detector (FID). The GC column was a 30 m × 0·25 mm × 0·2 μm DM–5 capillary column and the carrier gas was nitrogen. The injector and FID detector temperatures were set at 300°C and 320°C, respectively. The oven temperature for the GC was programmed with an initial temperature of 80°C and increased at 15°C min−1 to 260°C, where the temperature remained unchanged for 10 min. The temperature was then increased at 5°C min−1 to 320°C, where the temperature was held for 15 min. Quantification was based on FID peak areas and internal standard. Compound identification was based on co-injection with commercial standards and analysis of a subsample in a GC/MS–GP2010. The total amount of unknown constituents was calculated from the cumulative peak areas for all unidentified peaks. Total wax amount per unit leaf area was calculated as the total of all wax constituents including unknown peaks not identified by GC/MS, and expressed as micrograms per total leaf area (μg cm−2). After wax extraction, the leaf was scanned with an Epson Expression/STD 4800 scanner and the leaf area was measured using WinFOLIA leaf analysis software (Regent).
Scanning electron microscopy (SEM) analysis
For both inoculated and uninoculated treatments, the second fully expanded leaf from the top of each genotype was collected 72 h after inoculation and prepared for SEM analysis. A piece measuring 0·2 × 0·5 cm was cut from the middle of each leaf and fixed to an aluminum stub with double-sided adhesive tape. Stubs were coated with gold and placed in the low-vacuum, variable-pressure chamber of the Hitachi S3500 scanning electron microscope and photographed with a digital camera. Images were collected at three predetermined normal and spot locations.
Enzyme activity assays
About 0·5 g fresh leaf from inoculated or uninoculated plants was homogenized to a fine paste in a prechilled mortar with 50 mm borate buffer (pH 8·8) containing mercaptoethanol. The homogenate was centrifuged at 8000 g for 20 min at 4°C and the supernatant was directly used to measure phenylalanine ammonia lyase (PAL) activity according to Rosler et al. (1997). About 0·5 g fresh leaf was homogenized in 5 mL 50 mm phosphate buffer (pH 7·8), centrifuged at 8000 g for 20 min at 4°C, and the supernatant was used to measure activities of peroxidase (POD) and polyphenoloxidase (PPO). Activity of POD was measured as described by Olmos et al. (1997) and that of PPO was measured according to Sun & Song (2003). The enzyme activity was expressed as units per g fresh weight.
RNA isolation and synthesis of cDNA
The second intact leaves from inoculated plants were harvested 0, 24, 48 and 72 h after inoculation, flash frozen in liquid nitrogen and stored at −80°C until RNA extraction. Total RNA was isolated from leaf tissue using TransZol Plant Mini kit (TransGen) and the DNA was digested using DNase I (Takara) according to the manufacturer's instructions. RNA integrity was checked on an agarose gel and quantified by DNA Trace Measurement (NanoDrop Technologies Inc.). First-strand cDNA was synthesized from 2·5 μg total RNA using TransScript II (TransGen) and oligo-dT18 in a 20 μL reaction.
Quantitative RT-PCR (qRT-PCR)
Total RNA was isolated using TransZol kit (TransGen). Purified RNA was treated with DNase I (TaKaLa) and RNA integrity was checked on a 1% (w/v) agarose gel. Total RNA was reverse transcribed in a 20 μL reaction using an oligo-dT18 primer and TransScript First-Strand cDNA Synthesis SuperMix kit (TransGen), according to the manufacturer's instructions. The cDNA was then diluted 30 times and 1 μL was used as template for the real-time PCR experiment. Primers FKCS1 (5′-GGGACCGTGCTGGTTCAGCTAA-3′) and RKCS1 (5′-CGCTCGTCTTCCGGTTTGTAGCA-3′) were designed to amplify the β-ketoacyl-CoA synthase KCS1 and primers FPR1 (5′-GAGAAGGCTAACTATAACCACGATTCGA-3′) and RPR1 (5′-GGTTCCACCATTGTTACACCTCACTT-3′) were designed to amplify the pathogenesis-related gene PR1 from B. napus. Primers FACT7 (5′-GTGACAATGGAACTGGAATGGTGA-3′) and RACT7 (5′-GGACTGAGCTTCATCACCAACGTAA-3′) were used to amplify the internal control actin gene. The reference gene actin7 was used to normalize for differences of the total RNA amount.
Real-time PCR was performed on an Mx3000P (Stratagene). Samples were amplified in a 25 μL reaction containing 2 × TransStart Eco Green qPCR SuperMix (TransGen) and 200 nm each primer. The thermal profile consisted of one cycle at 95°C for 2 min followed by 40 cycles of 95°C for 10 s, 55°C for 20 s and 72°C for 20 s. For each run, data acquisition and analysis was carried out using MxPro v. 4.10 (Stratagene). The relative quantities of gene expression for sample comparison were calculated using the comparative Ct (2−ΔΔCt) method (Wong & Medrano, 2005).
The data obtained were subjected to analysis of variance (anova) using GenStat v 13.0. The contents of total waxes and wax constituents were tested by a two-way anova for significance between cultivars, inoculation and their interactions. The activities of enzymes and photosynthesis index were tested by multifactorial anova for significance between cultivars, inoculation, wax removal or inoculation time and their interactions. The disease index was tested by a one-way anova for significance between wax and wax removal for each cultivar. Mean values were separated by protected Fisher's least significant difference (LSD).
Effects of leaf wax on the infection of S. sclerotiorum
The necrotic lesions appeared on leaves of B. napus 24 h after inoculation and increased with culture times. Seventy-two hours after inoculation, the disease index of ZS9 was significantly lower than that of YY19 (Fig. 1) as expected. When the epicuticular waxes were removed from leaves, resistant seedlings developed many more necrotic spots, resulting in a higher disease index compared to seedlings with wax intact, while no significant change was observed for susceptible seedlings.
Effects of S. sclerotiorum inoculation on leaf waxes
The leaf epicuticular waxes of B. napus included alkanes, alcohols, long-chain fatty acids, ketones, aldehydes, esters and unknowns, among which the contents of alkanes accounted for 60% (ZS9) and 52% (YY19) of the total wax. The amounts of total wax and wax constituents were significantly higher in YY19 than in ZS9 except for esters (Table 1; Fig. 2).
Table 1. Analysis of variance of main effects (cultivar, inoculation and wax) and their interactions for total amount of waxes and wax constituents, activities of enzymes and photosynthesis index of Brassica napus inoculated with Sclerotinia sclerotiorum
The responses of the contents of wax constituents to S. sclerotiorum inoculation differed between the two B. napus cultivars. Five days after inoculation, the content of esters in leaves of ZS9 decreased significantly, the contents of alkanes, aldehydes and unknowns increased significantly, while the contents of alcohols, acids, ketones and total wax did not change significantly (Fig. 2). In YY19, the contents of aldehydes increased significantly after inoculation, the contents of unknowns decreased significantly and there was no significant change for the other wax constituents or total wax.
Effects of S. sclerotiorum inoculation on crystal structure of leaf wax
The SEM analysis showed that the crystal structure of epicuticular waxes on leaves of B. napus included plates, short rods, branching rods and dendrites (Fig. 3). Compared to ZS9, more rods appeared on the adaxial surface of leaves of YY19. The abaxial and adaxial sides of leaves showed no significant difference in crystal structure. After inoculation, branching rods and dendrites fused and their amounts decreased, while the amounts of plates and short rods in both cultivars increased. The changes of crystalloid structure on the adaxial surface of the leaf of ZS9 were much more dramatic than in YY19.
Effects of S. sclerotiorum inoculation and wax on gas exchange indexes
The net photosynthesis rates (Pn), stomatal conductance (gs), intercellular CO2 (Ci), and transpiration rates (Tr) were significantly affected by cultivars, inoculation and wax, except for the insignificant influence of inoculation on Ci and of wax on Tr (Table 1). The Pn, gs and Tr of inoculated leaves were significantly higher than those of the control for both cultivars (Fig. 4). When the leaf wax was removed using Blu-Tack, the Pn of leaves with wax removed was significantly higher than that of leaves with intact wax for both cultivars. Wax removal decreased the gs and Ci for ZS9, while no significant change was observed in YY19.
After wax removal, inoculation increased Pn, gs and Tr for ZS9, whereas for YY19 only gs was significantly higher in inoculated leaves.
Effects of S. sclerotiorum inoculation on activities of defence enzymes
The activities of PAL, POD and PPO in leaves of B. napus were significantly affected by cultivar, inoculation and the times after inoculation (Table 1). The activities of PAL in leaves of inoculated plants were significantly higher than those of uninoculated plants for ZS9, but were significantly lower than those of uninoculated plants for YY19 except for an insignificant difference 48 h after inoculation (Fig. 5). The activities of POD in leaves of inoculated plants were significantly lower than those of uninoculated plants for both cultivars except for insignificant changes for ZS9 24 h and 48 h after inoculation. The activities of PPO of inoculated plants were significantly higher than those of uninoculated plants 24 h and 48 h after inoculation in cultivar ZS9. There was no significant effect of inoculation on PPO activity for YY19. For inoculated plants, the activities of PAL, POD and PPO in leaves of ZS9 were significantly higher than those in leaves of YY19 except for POD 72 h after inoculation.
After wax removal, the activities of PAL in leaves of both cultivars increased significantly, the activity of POD for both cultivars and of PPO for YY19 did not change significantly and the PPO activity of ZS9 decreased significantly (Table 2). In leaves with wax removed, inoculation increased the activities of PAL, POD and PPO in ZS9 but not YY19.
Table 2. Percentage changes of enzyme activities 72 h after inoculation as influenced by wax removal in leaves of Brassica napus
Correlation analysis between leaf wax content and gas exchange indexes and defence enzymes
Correlation analysis (Table 3) showed that no significant relationship was observed between the content of waxes and Pn and Tr except for aldehydes, which were positively correlated with Pn. Ci and gs were negatively correlated with the contents of most wax constituents and total wax. The activities of PAL and PPO were negatively correlated with the contents of alkanes, alcohols, ketones and total wax. No significant relationship was observed between the contents of wax constituents and the activity of POD except for the content of aldehydes.
Table 3. Correlation analysis between wax constituents and photosynthesis and enzyme activities 72 h after inoculation of leaves of Brassica napus with Sclerotinia sclerotiorum
Pn, net photosynthesis rate (μmol m−2s−1); gs, stomatal conductance (mol m−2s−1); Ci, concentration of intercellular CO2 (μmol CO2 mol−1); Tr, transpiration rate (mmol m−2s−1); PAL, phenylalanine ammonia lyase; POD, peroxidase; PPO, polyphenoloxidase.
Effects of S. sclerotiorum inoculation on expression of KCS1 and PR1
To test the influence of S. sclerotiorum infection on expression of a wax gene and a pathogenesis-related gene, the expression of the plant KCS1 and pathogenesis-related PR1 genes were monitored by qRT-PCR at different times after inoculation (Fig. 6). The transcripts of KCS1 in the resistant cultivar increased 24 h after inoculation and remained at a stable level until 72 h after inoculation, while no significant change was observed in the susceptible cultivar. Compared to KCS1, the transcripts of PR1 increased significantly for both cultivars except for a decrease for YY19 24 h after inoculation.
The epicuticular wax of plants is generally considered as the first barrier to invasion of pathogenic fungi (Wang et al., 2008). Previous studies have shown that epicuticular waxes are associated with resistance of corn to Aspergillus flavus (Russin et al., 1997). However, in the current study, the total amount of wax of the resistant cultivar was significantly lower than that of the susceptible cultivar. In a study of Furtado et al. (2009), the total amount of wax in leaves of soyabean also showed no consistency with resistance to Phakopsora pachyrhizi. The study of some Arabidopsis mutants impaired in cutin monomer biosynthesis revealed that an increased permeability of the cuticle facilitated the release of antifungal compounds to the plant surface, thus conferring resistance to Botrytis and Sclerotinia (Bessire et al., 2007). These results suggested that less wax in a resistant cultivar might produce a more permeable cuticle, altering the plant's perception of its environment leading to change in its physiology.
Although the resistant and susceptible cultivars showed no difference in wax constituents, the significantly higher contents of aldehydes and alkanes in the susceptible cultivar than those in the resistant cultivar might be the reason for the higher disease index in the susceptible cultivar. Garbay et al. (2007) reported that the amount of PR1 expression was correlated to the presence or absence of some particular lipid constituents in the epicuticular wax layer. Hansjakob et al. (2011) reported that conidial germination, differentiation and prepenetration processes of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) were triggered by very-long-chain aldehydes, minor constituents of barley leaf wax. Bourdenx et al. (2011) reported that CER1 over-expression dramatically increased the production of the odd-carbon-numbered alkanes, together with a substantial accumulation of iso-branched alkanes, and increased susceptibility to bacterial and fungal pathogens.
In the current study, although disease resistance was not consistent with the total amount of wax, the wax layer did have a positive effect on protecting the plant from infection by S. sclerotiorum, particularly for the disease resistant cultivar. The removal of the epicuticular wax layer resulted in much more severe infection by S. sclerotiorum in the resistant cultivar, but had no significant effect on the susceptible cultivar. In experiments where epicuticular waxes were removed from pine needles before seedlings were infected with white pine blister rust, resistant seedlings without wax developed approximately the same number of infection spots as susceptible seedlings with wax intact (Smith et al., 2006). The removal of the epicuticular wax layer from leaves of Hordeum chilense allowed appressoria of Puccinia hordei to develop more easily over stomata (Patto & Niks, 2001). These results imply that epicuticular wax may be more important for disease-resistant cultivars than disease-susceptible cultivars in protecting plants from fungal invasion. Cytological and chemical analyses also revealed that the inhibition of rust preinfection structures in irg1 mutants of Medicago truncatula was due to complete loss of the abaxial epicuticular wax crystals and reduced surface hydrophobicity (Uppalapati et al., 2012). It is suggested that the leaf wax constituents on adaxial and abaxial sides of the two tested cultivars in the current study might also be different and could be correlated to their disease resistance. This needs further analysis in the future.
In the current study, although no significant change was observed for the total amount of waxes in both cultivars 5 days after inoculation, the responses of wax constituents to S. sclerotiorum infection differed between the two cultivars. For ZS9, the contents of alkanes and unknowns increased and that of esters decreased, while for YY19, the contents of unknowns decreased. This indicated that fungal infection influenced wax biosynthesis but the defensive mechanism related to wax differed between cultivars. This was supported by the difference between expression patterns of the wax-related gene (KCS1) and the pathogenesis-related gene (PR1).
The β-ketoacyl-CoA synthase (KCS) in the elongase system is the rate-limiting enzyme for very-long-chain fatty acid (VLCFA) biosynthesis and therefore controls VLCFA synthesis (Millar & Kunst, 1997; Todd et al., 1999). The transcripts of KCS1 in the resistant cultivar increased 24 h after inoculation and afterwards remained at stable levels, while no significant change was observed for the susceptible cultivar. The pathogenesis-related protein 1 (PR1) gene is a marker in the SA-dependent signalling pathway. PR1 was induced after challenging ZS9 with S. sclerotiorum at three different time points. Although PR1 was also up-regulated 48 h and 72 h after YY19 was inoculated, the transcript amount was much lower than that of ZS9. In Arabidopsis thaliana mutants npr1 and ein2, which are hypersusceptible to S. sclerotiorum, induction of the plant defence genes PDF1.2 and PR1 was reduced (Guo & Stotz, 2007). In contrast to the susceptible cultivar, KCS1 and PR1 were strongly induced in the pathogen-challenged resistant cultivar, illustrating that the PR1 and KCS1-dependent pathway contributed to defence against S. sclerotiorum. The expression of the PR1 is reduced 100-fold in the cer6 mutant (CER6 is an epidermis-specific Arabidopsis KCS) compared to wildtype Arabidopsis (Garbay et al., 2007). Coordination between defence responses and cuticle biosynthesis could be subject to complex regulation at several different levels, including gene silencing, transcription, RNA stability, translation, protein degradation, post-translational protein modification, protein localization and protein–protein interactions (Reina-Pinto & Yephremov, 2009). Although progress has now been made, molecular mechanisms of this regulation are still poorly studied.
In some Brassica species, the epicuticular wax is organized into three layers, i.e. a continuous sheet, flat crystals and upright crystals (Conn & Tewari, 1989). In the current study, plates, short rods, branching rods and dendritic crystals were observed on leaves of B. napus. No significant difference was observed between the two cultivars. Inoculation increased the amounts of plates and short rods, particularly in the resistant cultivar, reduced branching rods and dendritic crystals. This indicated that both cultivars adapted to fungal invasion by changing their structure of wax crystalloid. This increase of plates might increase the coverage of wax on the leaf, reducing the direct contact between spore/hyphae and leaf surface, which might decrease the invasion of S. sclerotiorum. Such a change of the crystalloid structure implied that the plant might also take physical measures to prevent fungal invasion.
Physiologically, the roles of PAL and POD in defence against pathogens have been demonstrated in many plants. In A. thaliana, PAL was involved in the synthesis of lignin and salicylic acid and finally resulted in the resistance of the plant to pathogens (Mauchmani & Slusarenko, 1996). In the current study, the increase of PAL activity in the resistant cultivar and the decrease in the susceptible cultivar after inoculation were well correlated with their resistances to disease. The two cultivars also showed different responses of POD and PPO to infection with S. sclerotiorum, indicating that different disease-resistant cultivars had different physiological responses to fungal invasion and the increase of PPO activity might be more important in resistant cultivars. After inoculating with Sclerotinia mycelia, Huang et al. (1999) also found that the increase of PPO activity in the resistant B. napus cultivar was higher than that in the susceptible cultivar. The negative correlation between the activities of PAL and PPO and the contents of alkanes, alcohols, ketones and total wax also suggested that the initiation of antioxidative enzyme activity was more important for plants with lower wax cover when they were infected by S. sclerotiorum.
Generally, the net photosynthesis rate (Pn) decreased in infected leaves as the disease progressed (Bessire et al., 2007). However, in the current study, the Pn increased in infected leaves for both B. napus cultivars. The difference might be related to the region of leaf that was used for measurements. Scholes & Farrar (1986) reported that the Pn in the regions between pustules reduced after barley was infected with brown rust (Puccinia hordei), but the rate of gross photosynthesis was increased in rusted leaves in comparison to control tissue. In the current study, the gas exchange indexes on regions where no significant disease spot could be observed were measured. The negative correlation between the contents of total wax and Ci and gs also implied that higher wax cover over the leaf surface might inhibit gas diffusion across stomata and thus influence photosynthesis. The wax coverage at the entrance of stomata in Leucadendron lanigerum increased resistance to gas diffusion and as a consequence decreased stomatal conductance, transpiration and photosynthesis (Mohammadian et al., 2007). The increase of Pn after wax removal for both cultivars also supported this point. For the plants with wax removed, inoculation increased the Pn and Tr for the resistant cultivar but decreased for the susceptible cultivar. This indicates that wax was more important for the resistant cultivar than the susceptible cultivar and the non-stomatal factors influenced photosynthesis of B. napus.
In conclusion, these results suggest that the leaf epicuticular wax was not directly correlated to defence against S. sclerotiorum for the disease susceptible cultivar, but was rather correlated to the defence of the resistant cultivar. In response to infection by S. sclerotiorum, the leaf epicuticular wax, defence enzymes and PR1-dependent SAR pathway all contributed to defence against the pathogen.
This work was funded by the National Natural Science Foundation of China (31000122, 31270450), the Fundamental Research Funds for the Central Universities (XDJK2011C007) and the Natural Science Foundation Project of CQ CSTC (cstc2012jjA80022). The authors are grateful to Lv Jun and Zheng Jun for their assistance in measuring photosynthesis and epicuticular waxes.