Effects of Root and Foliar Applications of 24-Epibrassinolide on Fusarium Wilt and Antioxidant Metabolism in Cucumber Roots

in HortScience

Root and foliar applications of 24-epibrassinolide (EBL), an immobile phytohormone with antistress activity, were evaluated for their effects on reducing fusarium wilt and their influence on antioxidant and phenolic metabolism in roots of cucumber plants (Cucumis sativus L. cv. Jinyan No. 4). EBL pretreatment significantly reduced disease severity together with improved plant growth and reduced losses in biomass regardless of application methods. EBL treatments significantly reduced pathogen-induced accumulation of reactive oxygen species (ROS), flavonoids, and phenolic compounds, activities of defense-related and ROS-scavenging enzymes. The enzymes included superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, catalase as well as phenylalanine ammonia-lyase and polyphenoloxidase. There was no apparent difference between two application methods used. EBL applications triggered a slight increase in H2O2 concentration followed by increases in the transcript levels of WRKY transcription factor and defense-related genes. This study demonstrated that EBL enhanced resistance to fusarium wilt by a novel mechanism that was not related to its active transport or increase in antioxidant system.

Abstract

Root and foliar applications of 24-epibrassinolide (EBL), an immobile phytohormone with antistress activity, were evaluated for their effects on reducing fusarium wilt and their influence on antioxidant and phenolic metabolism in roots of cucumber plants (Cucumis sativus L. cv. Jinyan No. 4). EBL pretreatment significantly reduced disease severity together with improved plant growth and reduced losses in biomass regardless of application methods. EBL treatments significantly reduced pathogen-induced accumulation of reactive oxygen species (ROS), flavonoids, and phenolic compounds, activities of defense-related and ROS-scavenging enzymes. The enzymes included superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, catalase as well as phenylalanine ammonia-lyase and polyphenoloxidase. There was no apparent difference between two application methods used. EBL applications triggered a slight increase in H2O2 concentration followed by increases in the transcript levels of WRKY transcription factor and defense-related genes. This study demonstrated that EBL enhanced resistance to fusarium wilt by a novel mechanism that was not related to its active transport or increase in antioxidant system.

Cucumber (Cucumis sativus L.) is one of the major greenhouse vegetables in the world and is very vulnerable to fusarium wilt caused by Fusarium oxysporum (FO) (Ahn et al., 1997; Ye et al., 2004). Fusarium pathogen infects the roots and then moves into the stems resulting in rapid wilting and death of shoots. Disease control is normally dependent on the use of resistant varieties. Chemical control with fungicides is not very effective.

Brassinosteroids (BRs) are a new group of phytohormones that are structurally similar to animal and insect steroid hormones. They control a broad range of processes, including seed germination, stem elongation, cell division and expansion, xylem differentiation, plant growth, and apical dominance (Azpiroz et al., 1998; Clouse and Sasse, 1998; Li et al., 1996; Sasse, 2003; Szekeres et al., 1996). In addition to its roles in plant growth and development, there is considerable evidence that BRs exert antistress effects on plants such as those caused by heat, cold, drought, and salt (Anuradha and Rao, 2001; Dhaubhadel et al., 1999, 2002; Kagale et al., 2007; Ogweno et al., 2008). The potential role of BRs in pathogen defense has also been the topic of recent studies. Potato plants sprayed with BRs had a lower incidence of infection by Phytophthora infestans (Khripach et al., 1996). BR-induced disease resistance was also noted in barley, potato tubers, and cucumber plants (Khripach et al., 2000). Recently, BRs have been shown to induce disease resistance in tobacco and rice against a broad range of pathogens (Nakashita et al., 2003). However, most of the early evidence about the protective activities of BRs against plant diseases is based on field observations but not field experiments (Khripach et al., 2000).

Plants have a natural array of defense mechanisms to protect themselves from pathogenic organisms. Recently, the generation and scavenging system of reactive oxygen species (ROS) has attracted increasing attention with regard to their roles in the defense of plants against biotic stresses. It is known that low-dose ROS could function as a signal for inducing stress tolerance, whereas high-dose ROS has been implicated in the oxidative damage and the hypersensitive response (HR) (Apel and Hirt, 2004; Laloi et al., 2004). Evidence for the role of ROS in HR has mainly been obtained from observations on localized infections of foliar tissues by obligate biotrophic or necrotrophic pathogens and relatively little is known about oxidative metabolism in plant resistance to pathogens that invade plant vascular system (Garcia-Limones et al., 2002; Mehdy et al., 1996). Although BRs are known as a kind of immobile hormones, it has been shown to enhance transport of auxin (Symons and Reid, 2004; Symons et al., 2008). It is interesting to investigate whether BRs are able to induce resistance in plant parts that have not received BRs directly. Furthermore, little is known about the mechanism by which BRs induce resistance. The increased resistance to sprouting and diseases in BR-treated potato tubers were found to be associated with enhancement of the synthesis of abscisic acid (ABA) as well as phenolic and terpenoid substances (Khripach et al., 2000). In cucumber plants, increased activities of peroxidase (POD) and polyphenoloxidase (PPO) enzymes, which are involved in the metabolism of polyphenols, have been suggested as a factor contributing to BR-induced disease resistance (Khripach et al., 2000). BR-induced resistance in tobacco was not associated with an increase in salicylic acid levels or induction in pathogenesis-related gene (PR) expression, suggesting that the mechanism of BR-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible resistance (Nakashita et al., 2003). Most recently, we have found that BR-induced tolerance to a broad range of stresses was dependent on H2O2 accumulation generated by NADPH oxidase (Xia et al., 2009).

The aim of the present study was to evaluate the effect of 24-epibrassinolide (EBL; Fig. 1) application to roots or shoots on the development of fusarium wilt and changes in metabolism of antioxidant and phenolic compounds in roots of cucumber plants. Subsequently, we determined to what extent these changes could be associated with EBL-induced resistance.

Fig. 1.
Fig. 1.

The chemical structure of 24-epibrassinolide.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

Materials and Methods

Greenhouse experiments.

Cucumber (Cucumis sativus L.) cv. Jinyan No. 4 was used because of its known susceptibility to fusarium wilt [Fusarium oxysporum (Schlechtend:Fr) f. sp. cucumerinum (Owen) Snyder & Hansen] (FO) (Ye et al., 2004). Seeds were sterilized in 1% (w/v) NaClO and germinated in perlite. After emergence, batches of eight seedlings were grown hydroponically in a plastic tank (13 L) filled with 10 L half-strength Enshi nutrient solution (Yu and Matsui, 1994). Plants were incubated in a greenhouse maintained at 32/22 °C (day/night) with a relative humidity of 85% and a photoperiod of 12 h with an average photosynthetic flux of 800 μmol·m−2·s−1.

When cucumber seedlings were at the two-leaf stage, EBL (Sigma) was applied to either roots (rEBL) or shoots (sEBL) 2 d before FO inoculation. EBL is one of the brassinosteroids with high physiological activity (Sasse, 2003). There were four treatments: untreated non-FO-infected (control), untreated FO-infected (FO), rEBL + FO, and sEBL + FO. EBL was dissolved in ethanol and was applied either to roots by adding it to the nutrient solutions to give a final concentration of 0.1 μM or to shoots by spraying with a concentration of 0.2 μM at 10 mL/plant. Our preliminary experiment showed that EBL at 0.2 μM had the highest physiological activity for spraying, whereas EBL concentration higher than 0.1 μM in a nutrient solution induced phytotoxicity (Yu et al., 2004). The final concentration of ethanol in nutrient and spray solutions was 0.1% (v/v), at which ethanol has a negligible effect on cucumber plants (Yu and Matsui, 1997). The same concentration of ethanol was applied to roots or shoots of plants that had not received EBL treatments. Two days after EBL applications, all treatments except the untreated and uninfected control were inoculated with FO by adding a conidial suspension to the nutrient solutions. FO inoculation was carried out 2 d after EBL application because EBL had the highest physiological activity at Day 2 in our preliminary experiment. The FO suspension was prepared by culturing the pathogen in a potato sucrose liquid medium at 28 °C for 6 d (Yu and Komada, 1999) and added to the nutrient solutions to give a final concentration of 104 conidia/mL. Each treatment had 16 plants and was replicated three times. The day for inoculation was designated 0 d. The experiment was ended at Day 12 after inoculation (dpi) when control plants had ≈10 leaves and FO plants showed wilting symptoms or yellowing leaves. The percentage of leaf yellowing or wilting plants per plot was rated at Days 0, 2, 4, 6, 8, 10, and 12 after FO inoculation (dpi). At Day 12, plants were harvested for assessment of root rot and vascular bundle browning on a scale of 0 to 4 as follows: 0, healthy without any browning; 1, white root with scarce browning; 2, light root rot and browning; 3, mild root rot and browning; 4, severe root rot and browning (Ye et al., 2004). Dry weights of roots, shoots, and total plants per plot were then determined after drying the plants at 80 °C for 3 d.

Antioxidant enzyme activity determination.

The antioxidant enzyme extraction and the activity determination were carried out as described in our early studies (Ding et al., 2007; Zhou et al., 2004). Samples of 0.5 g of root material, which was ≈10 cm from the root tip, were homogenized in 3 mL 25 mm HEPES buffer (pH 7.8) containing 0.2 mm EDTA and 2% (w/v) polyvinylpyrrolidone. The homogenate was centrifuged for 20 min at 12,000 g and the supernatant obtained was used for antioxidant enzyme analysis, including superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT). All operations were carried out at 0 to 4 °C. An aliquot of the extract was used to determine its protein content by the method of Bradford (1976) using bovine serum albumin as the standard.

Superoxide dismutase [electrical conductivity (EC) 1.15.1.1] was measured by the photochemical method as described by Giannopolitis and Ries (1977). Ascorbate peroxidase (APX; EC 1.11.1.11) was measured according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm (E = 2.8 mm·cm−1). The activity of GPX (EC 1.11.1.7) was assayed according to the method of Cakmak and Marschner (1992). Catalase EC 1.11.1.6) activity was assayed in a reaction mixture containing 25 mm phosphate buffer (pH 7.0), 10 mm H2O2, and the enzyme. The decomposition of H2O2 was followed at 240 nm (E = 39.4 mm·cm−1) (Cakmak and Marschner, 1992).

Determination of activities of phenylalanine ammonia-lyase and polyphenoloxidase and contents of flavonoids and phenolics.

Phenylalanine ammonia-lyase (PAL) and PPO were extracted according to Camacho-Cristóbal et al. (2002) using another root sample than the one used for antioxidant enzyme analysis described previously. The activity of PAL was assayed with L-phenylalanine as the substrate (Zucker, 1965). PPO activity was assayed by measuring the increase in absorbance at 370 nm with caffeic acid as the substrate (Ruiz et al., 1999). Total soluble phenolics were extracted according to Arnaldos et al. (2001) and assayed by absorbance at 765 nm using caffeic acid as a standard after the addition of Folin-Ciocalteu reagent (Ruiz et al., 1999). Flavonoids were extracted and determined according to Tekel'ova et al. (2000).

Determination of hydrogen peroxide and malonaldehyde content.

The content of hydrogen peroxide (H2O2) was determined by the absorbance at 410 nm monitoring titanium peroxide complex according to the methods described by Patterson et al. (1984). The thiobarbituric acid (TBA) test, which determines malonaldehyde (MDA) as an end product of lipid peroxidation in the roots, was used to measure MDA. Root samples (0.5 g) were homogenized with inert sand in 80:20 (v/v) ethanol/water followed by centrifugation at 3000 g for 10 min. A 1-mL aliquot of appropriately diluted sample was added to a test tube with an equal volume of either 1) –TBA solution containing 20% (w/v) trichloroacetic acid and 0.01% (w/v) butylated hydroxytoluene; or 2) +TBA solution containing that plus 0.65% (w/v) TBA. The mixtures were then heated in a water bath at 95 °C for 25 min. The reaction was stopped by placing the reaction tubes in an ice bath, the samples were then centrifuged at 3000 g for 10 min, and the absorption of the supernatant was read at 440, 532, and 600 nm. MDA equivalents were calculated according to the method of Hodges et al. (1999).

Total RNA extraction and gene expression analysis.

To determine the changes in the transcript of defense-related genes, roots of cucumber plants were sampled at Day 2 after EBL spray. Total RNA was extracted from roots using Trizol according to the supplier's recommendation. Residual DNA was removed with purifying column. One microgram total RNA was reverse-transcribed using 0.5 μg of Oligo (dT) 12-18 (Invitrogen) and 200 units of Superscript II (Invitrogen) following the supplier's recommendation. On the basis of EST sequences, the gene-specific primers were designed (Table 1) and used for amplification.

Table 1.

Primers used for real-time polymerase chain reaction assays.

Table 1.

Quantitative real-time polymerase chain reaction (PCR) was performed using the iCycler iQ™ Real-time PCR Detection System (Bio-Rad, Hercules, CA). PCRs were performed using the SYBR Green PCR Master Mix (Applied Biosystems). The PCR conditions consisted of denaturation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using software provided with the iCycler iQ™ Real-time PCR Detection System. The identity of the PCR products was verified by single-strand sequencing using MegaBACE 1000 DNA analysis system (Amersham Biosciences). To minimize sample variations, mRNA expression of the target gene was normalized relative to the expression of the housekeeping gene actin. All experiments were repeated three times for cDNA prepared for two samples of cucumber roots. The quantification of mRNA levels is based on the method of Livak and Schmittgen (2001).

Statistical analysis.

There were 16 plants per treatment and three replicates laid out in fully randomized complete blocks. Mean values of three replicates for all data were compared by using Duncan's multiple range test (P = 0.05).

Results

Untreated plants inoculated with pathogenic FO started to wilt 2 d after inoculation and by 12 d had reached a wilting percentage of 75.4 ± 7.5% (Figs. 2 and 3A). Plants treated with EBL applied either to roots or shoots showed a delay in the onset of wilting and after 12 d wilting percentage decreased to 39.4% ± 4.2% and 30.9% ± 11.7%, respectively. The roots of FO plants showed a vascular bundle browning index of 2.9 ± 0.2 at Day 12 (Fig. 3B). The corresponding values for EBL-treated plants were 1.6 ± 0.1 by root application and 1.4 ± 0.1 by shoot application. FO inoculation caused a reduction in dry weight of 67.1% in roots and 51.7% in shoots (Table 2). This reduction was significantly alleviated by EBL applications, in which corresponding reductions in dry weight were 34.3% and 17.9% when it was applied to the roots and 28.6% and 15.4% when it was applied to the shoots, respectively.

Table 2.

Effects of 24-epibrassinolide (EBL) application on the biomass accumulation in cucumber plants with or without inoculation of Fusarium oxysporum (FO).z

Table 2.
Fig. 2.
Fig. 2.

Effect of 24-epibrassinolide (EBL) application on the severity of fusarium wilt caused by Fusarium oxysporum (FO) on cucumber plants. Control, untreated plants without FO inoculation; FO, untreated plants inoculated with FO; rEBL + FO, plants were supplied with 0.1 μM EBL through the roots before FO inoculation; sEBL + FO, plants sprayed with EBL at 0.2 μM EBL before FO inoculation, EBL was applied 2 d before FO inoculation. Photographs were taken at 10 d after FO inoculation.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

Fig. 3.
Fig. 3.

Effect of 24-epibrassinolide (EBL) application on the incidence of fusarium wilt (A) and browning index of vascular bundle (B) caused by Fusarium oxysporum (FO) in cucumber plants. Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

FO inoculation resulted in an overall increase in the activities of antioxidant enzymes (Fig. 4). GPX and APX activities peaked at Day 4, whereas SOD and CAT activities peaked at Day 8 after FO inoculation. EBL application to roots or shoots brought about a decrease in the activities of these antioxidant enzymes. The activities of these antioxidant enzymes in EBL-treated plants were slightly higher than those of control plants in most cases. There were no apparent differences in the activities of SOD, GPX, APX, and CAT whether EBL was applied to roots or shoots.

Fig. 4.
Fig. 4.

Effects of 24-epibrassinolide (EBL) application on the activities of superoxide dismutases (SOD, A), ascorbate peroxidase (APX, B), glutathione peroxidase (GPX, C), and catalase (CAT, D) in roots of cucumber plants with or without inoculation of Fusarium oxysporum (FO). Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

There was a gradual increase of PPO and PAL activities in roots after exposure to FO (Fig. 5A–B). PPO activity increased by 16.6%, 46.3%, and 45.1%, whereas PAL activity increased by 17.6%, 24.3%, and 25.1%, respectively, at 4, 8, and 12 d after inoculation. A pretreatment of roots or shoots with EBL significantly attenuated this increase with EBL application to shoots having more significant effects than application to roots.

Fig. 5.
Fig. 5.

Effects of 24-epibrassinolide (EBL) application on the activities of polyphenoloxidase (A), phenylalanine ammonia-lyase (B), flavonoids content (C), and total phenolics content (D) in roots of cucumber plants with or without inoculation of Fusarium oxysporum (FO). Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

Accompanied with the change in PPO and PAL activities, FO inoculation also resulted in a gradual increase in flavonoids and total phenolics in roots (Fig. 5C–D). At Day 12, flavonoids and total phenolics increased by 343.0% and 63.4%, respectively. EBL applications to roots and shoots both significantly attenuated this increase. In comparison with the FO treatment without EBL, EBL application to shoots decreased flavonoids by 64.3%, 50.6%, and 57.3%, respectively, at Days 4, 8, and 12 after FO inoculation. A similar trend was also observed in total phenolics. It was interesting to note that flavonoid content for EBL spray treatment was not significantly different from that of control plants.

Changes in antioxidant enzymes were accompanied by changes in H2O2 and MDA contents. EBL applications resulted in a slight increase in H2O2 content in roots regardless of whether it was supplied to roots or shoots (Fig. 6A). H2O2 content in roots significantly increased after FO inoculation and reached a value 1.5 times higher than that of the control at 12 d. This increase, however, was significantly attenuated by both EBL treatments. Similarly, MDA content also significantly increased with time (Fig. 6B). FO-induced increase in MDA, however, was significantly reduced by both EBL treatments, especially by the foliar spray. At Day 12, MDA content in roots of FO, rEBL + FO, and sEBL + FO increased by 173.8%, 152.3%, and 87.4%, respectively, compared with control plants.

Fig. 6.
Fig. 6.

Effects of 24-epibrassinolide (EBL) application on the contents of hydrogen peroxide (H2O2, A) and malonylaldehyde (MDA, B) in roots of cucumber plants with or without inoculation of Fusarium oxysporum (FO). Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

To further study the role of H2O2 in BR-induced stress tolerance, we examined expression of genes encoding proteins involved in H2O2 signaling and antioxidative/defense responses in roots of BR-treated plants. The transcript levels of WRKY30 (transcription factor), PR-1 (pathogenesis-related proteins), PAL (phenylalanine ammonia-lyase), and cAPX (cytosolic ascorbate peroxidase) were all upregulated in roots for plants at 3 d after EBL application to shoots (Fig. 7). However, no differences were found at 5 d after EBL treatment (data not shown).

Fig. 7.
Fig. 7.

Effects of 24-epibrassinolide (EBL) foliar application on transcripts abundance of defense-related genes in roots of cucumber plants. Roots were sampled at 2 d after EBL foliar spray. Genes were analyzed by quantitative real-time polymerase chain reaction using the gene-specific primer pairs shown in Table 1. Data were normalized to an internal actin control, and the data delta CT method was used to obtain the relative expression levels for each gene. Values for control samples were arbitrarily set to 1.0. Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

Citation: HortScience horts 44, 5; 10.21273/HORTSCI.44.5.1340

Discussion

Although BRs have been implicated in a broad range of stress responses in plants, relatively little is known about their role in pathogen defense. In our study, EBL application to either roots or shoots significantly increased the resistance to fusarium pathogens as evidenced by a decreased severity of fusarium wilt. This result confirmed the role of BRs in plant response to pathogen attack. BRs have been found to induce resistance in tobacco plants against tobacco mosaic virus, the bacterial pathogen Pseudomonas syringaem and the fungal pathogen Oidium sp. In rice, BRs induced resistance to Magnaporthe grisea and Xanthomonas oryzae, which cause rice blast and bacterial blight, respectively (Nakashita et al., 2003). Early evidence for BR-induced resistance to diseases was, however, obtained by local application to the foliage for protection against aerial diseases or to the roots for protection against root diseases (Khripach et al., 2000). In this regard, our finding that EBL application to shoots suppressed the severity of a disease originating from root infection is interesting because BRs are immobile in the plant with little transport from shoots to roots (Symons and Reid, 2004; Symons et al., 2008). It is likely that EBL application to shoots might generate a secondary signal, which was transmitted from shoots to roots. This opens new possibilities for its application in agricultural production because foliar spraying is more practical than drenching soils.

Protective activities of BRs against plant diseases have been indicated based on evaluations from field trials and greenhouse experiments, but its mechanism at the molecular level remains to be clarified. Increases in the activities of POD and PPO enzymes were suggested as a factor contributing to EBL-induced disease resistance (Khripach et al., 2000). It has been shown that BR-induced resistance in tobacco is not correlated with increases in salicylic acid levels or induction of pathogenesis-related gene (PR) expression, suggesting that the mechanism of BR-induced resistance is distinct from SAR and wound-inducible resistance (Nakashita et al., 2003). To date, there is little evidence about the interaction of BRs with other hormones in the pathogenesis. Recently, evidence is emerging that BRs may affect the long-distance transport of auxin (IAA) (Jager et al., 2007). IAA has been found to reduce diseases caused by Pythium ultimum on tomato plants and Phytophthora infestans on potato plants (Gravel et al., 2007; Noel et al., 2001; Terrile et al., 2006). It is, therefore, possible that EBL application to shoots attenuated the severity of fusarium wilt partly by the promotion of IAA transport from shoots to roots.

An increased rate in ROS-scavenging metabolism was frequently observed in localized infection of foliar tissues by obligate biotrophic or necrotrophic pathogens associated with a rapid HR (Mehdy et al., 1996; Ye et al., 2006). In this study, we found that the significant increases in activities of ROS-scavenging enzymes SOD, APX, GPX, and CAT, together with increased levels of H2O2 and MDA after FO infection were greatly attenuated by EBL pretreatment, suggesting that less peroxidative stress occurred in the EBL-treated plants. It needs to be noted that high levels of ROS cause cell death; low levels of ROS, however, have regulatory roles in plant stress responses. Application of ABA and SA as well as exposure to low temperature all resulted in a transient elevation of H2O2, leading to an increased tolerance to salt, high light, heat, and oxidative stress (Dat et al., 1998; Prasad et al., 1994; Zhang et al., 2001). It has been proposed that ROS plays a critical role in induced tolerance by activating or inducing stress response-related factors such as MAP kinases, transcription factors, antioxidant enzymes, dehydrins, and low-temperature-induced, heat-shock, and pathogenesis-related proteins (Gechev et al., 2006). In agreement with these studies, we found that EBL application induced an elevation of H2O2 accompanied with transiently increased transcript level of defense related genes such as PAL and cAPX. It is possible that secondary signals such as H2O2 were responsible for the expression of defense-related genes such as PAL, leading to an increased resistance to pathogens and a lower level of diseases. However, it remains unclear how H2O2 elevation in root tissues was induced in plants by foliar spray with EBL.

Flavonoids and phenolics can act as scavengers of free radicals such as ROS (Heim et al., 2002). Increased flavonoid synthesis and increased activities of PPO and PAL were often observed after infection by pathogens (Gallet et al., 2004; Modafar and Boustani, 2001) and were also found in our study. In agreement with the changes in disease severity and the ROS metabolism, FO-induced increases in activities of PPO and PAL, and associated accumulation of flavonoids and phenolics were significantly attenuated by the EBL pretreatment either to roots or shoots. All these results indicated that BR-induced resistance to FO is not attributed to changes in phenolic metabolism. However, phenolic metabolism may play an important role in the defense against pathogen attack.

In summary, we have found that the application of EBL either to shoots or to roots alleviated the symptoms of fusarium infection and reduced pathogen-induced oxidative stress, flavonoids, and phenolic compounds. Foliar EBL application triggered a slight increase in H2O2 concentration followed by increases in the transcript levels of defense-related genes in roots. This study provided evidence that BRs could induce resistance to the pathogen by a novel long-distance signal relay mechanism. This finding is of importance not only for basic understanding of the role of hormone, but also for potential use of such compounds in agriculture and horticulture.

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    • Search Google Scholar
    • Export Citation
  • KagaleS.DiviU.K.KrochkoJ.E.KellerW.A.KrishnaP.2007Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stressesPlanta225353364

    • Search Google Scholar
    • Export Citation
  • KhripachV.ZhabinskiiV.GrootA.D.2000Twenty years of brassinosteroids: Steroidal plant hormones warrant better crops for XXI centuryAnn. Bot. (Lond.)86441447

    • Search Google Scholar
    • Export Citation
  • Khripach V.A. V.N. Zhabinskii R.P. Litvinovskaya M.I. Zavadskaya E.A. Savel'eva I.I. Karas A.V. Kilcchevskii and S.N. Titova. 1996. A method for protection of potato from phytophthorosis. Pat. Appl. BY 960346.

  • LaloiC.ApelK.DanonA.2004Reactive oxygen signalling: The latest newsCurr. Opin. Plant Biol.7323328

  • LiJ.NagpalP.WitartV.McMorrisT.C.ChoryJ.1996A role for brassinosteroids in light-dependent development of Arabidopsis Science272398401

    • Search Google Scholar
    • Export Citation
  • LivakK.J.SchmittgenT.D.2001Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT methodMethods25402408

    • Search Google Scholar
    • Export Citation
  • MehdyM.G.SharmaY.K.SathasivanK.BaysN.W.1996The role of activated oxygen species in plant disease resistancePlant Physiol.98365374

  • ModafarC.E.L.BoustaniE.E.L.2001Cell wal bound phenolic acid and ligin contents in date palm as related to its resistance to Fusarium oxysporum Biol. Plant.44125130

    • Search Google Scholar
    • Export Citation
  • NakanoY.AsadaK.1981Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplastsPlant Cell Physiol.22679690

  • NakashitaH.YasudaM.NittaT.AsamiT.FujiokaS.AraiY.SekimataK.TakatsutoS.YamaguchiI.YoshidaS.2003Brassinosteroid functions in a broad range of disease resistance in tobacco and ricePlant J.33887898

    • Search Google Scholar
    • Export Citation
  • NoelG.M.A.M.MadridE.A.BottiniR.LamattinaL.2001Indole acetic acid attenuates disease severity in potato–Phytophthora infestans interaction and inhibits the pathogen growth in vitroPlant Physiol. Biochem.39815823

    • Search Google Scholar
    • Export Citation
  • OgwenoJ.O.SongX.S.ShiK.HuW.H.MaoW.H.ZhouY.H.YuJ.Q.NoguésS.2008Brassinosteroids alleviate heat-induced inhibition of photosynthesis by increasing carboxylation efficiency and enhancing antioxidant systems in Lycopersicon esculentum J. Plant Growth Regul.274957

    • Search Google Scholar
    • Export Citation
  • PattersonB.D.MackaeE.A.MackaeI.1984Estimation of hydrogen peroxide in plants extracts using titanium (iv)Anal. Biochem.139487492

  • PrasadT.K.AndersonM.D.MartinB.A.StewartC.R.1994Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxidePlant Cell66574

    • Search Google Scholar
    • Export Citation
  • RuizJ.M.GarciaP.C.RiveroR.M.RomeroL.1999Response of phenolic metabolism to the application of carbendazin plus boron in tobaccoPlant Physiol. Biochem.106151157

    • Search Google Scholar
    • Export Citation
  • SasseJ.M.2003Physiological actions of brassinosteroids: An updateJ. Plant Growth Regul.22276288

  • SymonsG.M.ReidJ.B.2004Brassinosteroids do not undergo long-distance transport in pea. Implications for the regulation of endogenous brassinosteroid levelsPlant Physiol.13521962206

    • Search Google Scholar
    • Export Citation
  • SymonsG.M.RossJ.J.JagerC.E.ReidJ.B.2008Brassinosteroid transportJ. Expt. Bot.591724

  • SzekeresM.NemethK.Koncz-KalmanZ.MathurJ.KauschmannA.AltmannT.RedeiG.P.NagyF.SchellJ.KonczC.1996Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and deetiolation in Arabidopsis Cell85171182

    • Search Google Scholar
    • Export Citation
  • Tekel'ovaD.RepcakM.ZemkovaE.TothJ.2000Quantitative changes of dianthrones, hyperforin and flavonoids content in the flower ontogenesis of Hypericum perforatum Planta Med.66778780

    • Search Google Scholar
    • Export Citation
  • TerrileM.C.OlivieriF.P.BottiniR.CasalongueC.A.2006Indole-3-acetic acid attenuates the fungal lesions in infected potato tubersPhysiol. Plant.127205211

    • Search Google Scholar
    • Export Citation
  • XiaX.J.WangY.J.ZhouY.H.TaoY.MaoW.H.ShiK.AsamiT.ChenZ.X.YuJ.Q.2009Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumberPlant Physiol.150801814

    • Search Google Scholar
    • Export Citation
  • YeS.F.YuJ.Q.PengY.H.ZhengJ.H.ZouL.Y.2004Incidence of fusarium wilt in Cucumis sativus L. is promoted by cinnamic acid, an autotoxin in root exudatesPlant Soil263143150

    • Search Google Scholar
    • Export Citation
  • YeS.F.ZhouY.H.SunY.ZouL.Y.YuJ.Q.2006Cinnamic acid causes oxidative stress in cucumber roots, and promotes incidence of fusarium wiltEnviron. Exp. Bot.56255262

    • Search Google Scholar
    • Export Citation
  • YuJ.Q.HuangL.F.HuW.H.ZhouY.H.MaoW.H.YeS.F.NoguésS.2004A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus J. Expt. Bot.5511351143

    • Search Google Scholar
    • Export Citation
  • YuJ.Q.KomadaH.1999Hinoki (Chamaecyparis obtusa) bark, a substrate with anti-pathogen properties that suppress some root diseases of tomatoSci. Hort.811324

    • Search Google Scholar
    • Export Citation
  • YuJ.Q.MatsuiY.1994Phytotoxic substances in the root exudates of Cucumis sativus LJ. Chem. Ecol.202131

  • YuJ.Q.MatsuiY.1997Effects of root exudates of cucumber (Cucumis sativus) and allelochemicals on uptake by cucumber seedlingsJ. Chem. Ecol.23817827

    • Search Google Scholar
    • Export Citation
  • ZhangX.ZhangL.DongF.GaoJ.GalbraithD.W.SongC.2001Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba Plant Physiol.12614381448

    • Search Google Scholar
    • Export Citation
  • ZhouY.H.YuJ.Q.HuangL.F.NoguesS.2004The relationship between CO2 assimilation, photosynthetic electron transport and water–water cycle in chill-exposed cucumber leaves under low light and subsequent recoveryPlant Cell Environ.2715031514

    • Search Google Scholar
    • Export Citation
  • ZuckerM.1965Induction of phenylalanine deaminase by light and its relation to chlorogenic acid synthesis in potato tuber tissuePlant Physiol.40779784

    • Search Google Scholar
    • Export Citation

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Contributor Notes

This work was supported by the National Key Project of Scientific and Technical Supporting Programs Funded by Ministry of Science & Technology of China (2006BAD07B02, 2008BADA6B02), National Basic Research Program of China (2009CB119000), and the National Natural Science Foundation of China (30500344, 30671428).

To whom reprint requests should be addressed; e-mail jqyu@zju.edu.cn.

  • View in gallery

    The chemical structure of 24-epibrassinolide.

  • View in gallery

    Effect of 24-epibrassinolide (EBL) application on the severity of fusarium wilt caused by Fusarium oxysporum (FO) on cucumber plants. Control, untreated plants without FO inoculation; FO, untreated plants inoculated with FO; rEBL + FO, plants were supplied with 0.1 μM EBL through the roots before FO inoculation; sEBL + FO, plants sprayed with EBL at 0.2 μM EBL before FO inoculation, EBL was applied 2 d before FO inoculation. Photographs were taken at 10 d after FO inoculation.

  • View in gallery

    Effect of 24-epibrassinolide (EBL) application on the incidence of fusarium wilt (A) and browning index of vascular bundle (B) caused by Fusarium oxysporum (FO) in cucumber plants. Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

  • View in gallery

    Effects of 24-epibrassinolide (EBL) application on the activities of superoxide dismutases (SOD, A), ascorbate peroxidase (APX, B), glutathione peroxidase (GPX, C), and catalase (CAT, D) in roots of cucumber plants with or without inoculation of Fusarium oxysporum (FO). Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

  • View in gallery

    Effects of 24-epibrassinolide (EBL) application on the activities of polyphenoloxidase (A), phenylalanine ammonia-lyase (B), flavonoids content (C), and total phenolics content (D) in roots of cucumber plants with or without inoculation of Fusarium oxysporum (FO). Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

  • View in gallery

    Effects of 24-epibrassinolide (EBL) application on the contents of hydrogen peroxide (H2O2, A) and malonylaldehyde (MDA, B) in roots of cucumber plants with or without inoculation of Fusarium oxysporum (FO). Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

  • View in gallery

    Effects of 24-epibrassinolide (EBL) foliar application on transcripts abundance of defense-related genes in roots of cucumber plants. Roots were sampled at 2 d after EBL foliar spray. Genes were analyzed by quantitative real-time polymerase chain reaction using the gene-specific primer pairs shown in Table 1. Data were normalized to an internal actin control, and the data delta CT method was used to obtain the relative expression levels for each gene. Values for control samples were arbitrarily set to 1.0. Data are the means of three replications within an experiment with ses. Different letters are significantly different between the treatments at 5% level according the Tukey tests.

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  • JagerC.E.SymonsG.M.GlancyN.E.ReidJ.B.RossJ.J.2007Evidence that the mature leaves contribute auxin to the immature tissues of pea (Pisum sativum L.)Planta226361368

    • Search Google Scholar
    • Export Citation
  • KagaleS.DiviU.K.KrochkoJ.E.KellerW.A.KrishnaP.2007Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stressesPlanta225353364

    • Search Google Scholar
    • Export Citation
  • KhripachV.ZhabinskiiV.GrootA.D.2000Twenty years of brassinosteroids: Steroidal plant hormones warrant better crops for XXI centuryAnn. Bot. (Lond.)86441447

    • Search Google Scholar
    • Export Citation
  • Khripach V.A. V.N. Zhabinskii R.P. Litvinovskaya M.I. Zavadskaya E.A. Savel'eva I.I. Karas A.V. Kilcchevskii and S.N. Titova. 1996. A method for protection of potato from phytophthorosis. Pat. Appl. BY 960346.

  • LaloiC.ApelK.DanonA.2004Reactive oxygen signalling: The latest newsCurr. Opin. Plant Biol.7323328

  • LiJ.NagpalP.WitartV.McMorrisT.C.ChoryJ.1996A role for brassinosteroids in light-dependent development of Arabidopsis Science272398401

    • Search Google Scholar
    • Export Citation
  • LivakK.J.SchmittgenT.D.2001Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT methodMethods25402408

    • Search Google Scholar
    • Export Citation
  • MehdyM.G.SharmaY.K.SathasivanK.BaysN.W.1996The role of activated oxygen species in plant disease resistancePlant Physiol.98365374

  • ModafarC.E.L.BoustaniE.E.L.2001Cell wal bound phenolic acid and ligin contents in date palm as related to its resistance to Fusarium oxysporum Biol. Plant.44125130

    • Search Google Scholar
    • Export Citation
  • NakanoY.AsadaK.1981Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplastsPlant Cell Physiol.22679690

  • NakashitaH.YasudaM.NittaT.AsamiT.FujiokaS.AraiY.SekimataK.TakatsutoS.YamaguchiI.YoshidaS.2003Brassinosteroid functions in a broad range of disease resistance in tobacco and ricePlant J.33887898

    • Search Google Scholar
    • Export Citation
  • NoelG.M.A.M.MadridE.A.BottiniR.LamattinaL.2001Indole acetic acid attenuates disease severity in potato–Phytophthora infestans interaction and inhibits the pathogen growth in vitroPlant Physiol. Biochem.39815823

    • Search Google Scholar
    • Export Citation
  • OgwenoJ.O.SongX.S.ShiK.HuW.H.MaoW.H.ZhouY.H.YuJ.Q.NoguésS.2008Brassinosteroids alleviate heat-induced inhibition of photosynthesis by increasing carboxylation efficiency and enhancing antioxidant systems in Lycopersicon esculentum J. Plant Growth Regul.274957

    • Search Google Scholar
    • Export Citation
  • PattersonB.D.MackaeE.A.MackaeI.1984Estimation of hydrogen peroxide in plants extracts using titanium (iv)Anal. Biochem.139487492

  • PrasadT.K.AndersonM.D.MartinB.A.StewartC.R.1994Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxidePlant Cell66574

    • Search Google Scholar
    • Export Citation
  • RuizJ.M.GarciaP.C.RiveroR.M.RomeroL.1999Response of phenolic metabolism to the application of carbendazin plus boron in tobaccoPlant Physiol. Biochem.106151157

    • Search Google Scholar
    • Export Citation
  • SasseJ.M.2003Physiological actions of brassinosteroids: An updateJ. Plant Growth Regul.22276288

  • SymonsG.M.ReidJ.B.2004Brassinosteroids do not undergo long-distance transport in pea. Implications for the regulation of endogenous brassinosteroid levelsPlant Physiol.13521962206

    • Search Google Scholar
    • Export Citation
  • SymonsG.M.RossJ.J.JagerC.E.ReidJ.B.2008Brassinosteroid transportJ. Expt. Bot.591724

  • SzekeresM.NemethK.Koncz-KalmanZ.MathurJ.KauschmannA.AltmannT.RedeiG.P.NagyF.SchellJ.KonczC.1996Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and deetiolation in Arabidopsis Cell85171182

    • Search Google Scholar
    • Export Citation
  • Tekel'ovaD.RepcakM.ZemkovaE.TothJ.2000Quantitative changes of dianthrones, hyperforin and flavonoids content in the flower ontogenesis of Hypericum perforatum Planta Med.66778780

    • Search Google Scholar
    • Export Citation
  • TerrileM.C.OlivieriF.P.BottiniR.CasalongueC.A.2006Indole-3-acetic acid attenuates the fungal lesions in infected potato tubersPhysiol. Plant.127205211

    • Search Google Scholar
    • Export Citation
  • XiaX.J.WangY.J.ZhouY.H.TaoY.MaoW.H.ShiK.AsamiT.ChenZ.X.YuJ.Q.2009Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumberPlant Physiol.150801814

    • Search Google Scholar
    • Export Citation
  • YeS.F.YuJ.Q.PengY.H.ZhengJ.H.ZouL.Y.2004Incidence of fusarium wilt in Cucumis sativus L. is promoted by cinnamic acid, an autotoxin in root exudatesPlant Soil263143150

    • Search Google Scholar
    • Export Citation
  • YeS.F.ZhouY.H.SunY.ZouL.Y.YuJ.Q.2006Cinnamic acid causes oxidative stress in cucumber roots, and promotes incidence of fusarium wiltEnviron. Exp. Bot.56255262

    • Search Google Scholar
    • Export Citation
  • YuJ.Q.HuangL.F.HuW.H.ZhouY.H.MaoW.H.YeS.F.NoguésS.2004A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus J. Expt. Bot.5511351143

    • Search Google Scholar
    • Export Citation
  • YuJ.Q.KomadaH.1999Hinoki (Chamaecyparis obtusa) bark, a substrate with anti-pathogen properties that suppress some root diseases of tomatoSci. Hort.811324

    • Search Google Scholar
    • Export Citation
  • YuJ.Q.MatsuiY.1994Phytotoxic substances in the root exudates of Cucumis sativus LJ. Chem. Ecol.202131

  • YuJ.Q.MatsuiY.1997Effects of root exudates of cucumber (Cucumis sativus) and allelochemicals on uptake by cucumber seedlingsJ. Chem. Ecol.23817827

    • Search Google Scholar
    • Export Citation
  • ZhangX.ZhangL.DongF.GaoJ.GalbraithD.W.SongC.2001Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba Plant Physiol.12614381448

    • Search Google Scholar
    • Export Citation
  • ZhouY.H.YuJ.Q.HuangL.F.NoguesS.2004The relationship between CO2 assimilation, photosynthetic electron transport and water–water cycle in chill-exposed cucumber leaves under low light and subsequent recoveryPlant Cell Environ.2715031514

    • Search Google Scholar
    • Export Citation
  • ZuckerM.1965Induction of phenylalanine deaminase by light and its relation to chlorogenic acid synthesis in potato tuber tissuePlant Physiol.40779784

    • Search Google Scholar
    • Export Citation
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