A study on JA- and BTH-induced resistance of Rosa rugosa ‘Plena’ to powdery mildew (Sphaerotheca pannosa)
Different concentrations of jasmonic acid (JA) and benzothiadiazole (BTH) were sprayed on 2-year-old Rosa rugosa‘Plena’ seedlings. The induced resistance of JA and BTH to Sphaerotheca pannosa (Wallr.) and the changes of their related physiological indices were investigated. Results showed that JA and BTH treatments had inhibitory impacts on S. pannosa infection. The optimal concentration of JA and BTH was 0.5 mmol/L for the disease-resistance induction of the leaves, its inductive effect was up to 66.36% for BTH and 54.49% for JA. Our results confirmed that exogenous JA and BTH significantly improved R. rugose ‘Plena’ resistance to S. pannosa. When treated with JA and BTH, activities of the three defense enzymes (POD, PPO, and PAL) increased significantly. Contents of total phenolics, flavonoids, and lignin also increased significantly. It is inferred from these results that exogenous JA and BTH could improve the resistance of R. rugose ‘Plena’ to S. pannosa through enhancing activities of the defensive enzymes and accumulation of secondary metabolites in the leaves.
KeywordsBenzothiadiazole Defensive enzyme Jasmonic acid Powdery mildew Sphaerotheca pannosa (Wallr.) Rosa rugosa‘Plena’ Secondary metabolism
Roses, one of the most important commercial crops, have a long history in the service of humankind (Rout et al. 1999; Uggla and Carlson-Nilsson 2005; Pati et al. 2006). Rosa rugosa ‘Plena’, belonging to the genus Rosa, is a perennial shrub with multiple values such as for ornamentals, and sources of food and medicines (Zhang et al. 2014). In recent years, the exploitation and industrial production of Rosa-based products have been growing rapidly. However, with the extensive cultivation and application of R. rugose ‘Plena’, it has been reported to be affected by severe pests and diseases (Yan et al. 2017). Powdery mildew, Sphaerotheca pannosa (Wallr.), is one of the most common and important plant diseases and has a significant negative impact on plant development, even leading to death of the plant (Pasini et al. 1997; Mortensen and Gislerød 2005). Traditional control by pesticides on the pathogenic bacteria pollutes environment and damages its pharmacological efficacy and edible value (Zhao et al. 2017). Studies on the induction of plant disease resistance provide new ideas for exploring environmentally friendly methods of disease control (Zhao et al. 2003; Wang 1994; Chen et al. 2007; Terry and Joyce 2004; Walters et al. 2005).
Peroxidase (POD) is an important active oxygen scavenger in plant cells. It catalyzes the oxidation reaction of phenolic substances and synthesis of coniferyl alcohol, a precursor of lignin. It participates in a polymerization reaction at the final step of lignin synthesis, so it has a close correlation with plant disease resistance (He 2001; Shivakumar et al. 2003; Yanti 2015). Phenylalanine ammonia-lyase (PAL) is another defensive enzyme and involved in the generation and sediment of lignin in cells. Polyphenol oxidase (PPO) can catalyze the oxidation of phenolic substances to quinones which have a higher toxicity to pathogenic bacteria as compared with phenolic substances. The PAL and PPO both promote the lignification of cell walls to defend against various pathogenic bacteria (Wang et al. 2005; Babu et al. 2015). A previous study showed that increasing PAL, PPO and POD activities could reflect the dynamics of plant disease resistance and be considered as a biochemical index (Ran et al. 2004; Duzan et al. 2005).
Phenolic substances can accumulate in plants that have been infected by pathogenic bacteria or were treated by inductors. These phenolic substances could inhibit the growth and spore production of pathogenic bacteria and in addition, they would induce the generation of flavonoids such as pisatin and phaseolin to prevent secondary infection by some pathogenic bacteria (Chen et al. 2010a, b; Aires et al. 2011). When infected by pathogenic bacteria, treatment by a biological agent or induction by physical and chemical factors increases the lignin content significantly, leading to the thickening of cell walls which is beneficial to prevent the spread of infection of pathogenic bacteria (Ren et al. 2007). Plant secondary metabolites play important roles in improvement of disease resistance.
Jasmonic acid (JA) is a key hormone in plant resistance to pathogenic bacteria and pests. It is a signal molecule inducing the expression of resistance genes (Niu et al. 2011; Liu et al. 2006; Gaige et al. 2010). When plants are infected by fungi, jasmonic acid can induce physiological changes and form a defensive structure (Lorenzo et al. 2004). Benzothiadiazole (BTH) is an artificially synthesized inductor, the most commonly used chemical systemic-acquired resistance (SAR) inducer (Perazzolli et al. 2008). It does not display any noticeable direct activity against pathogens, but does increase crop resistance to diseases caused by viruses, bacteria and fungi by activating SAR-signaling pathways (Bovie et al. 2004) and plant priming (Kohler et al. 2002). Recent studies have shown that BTH has significant induced-resistance to some fungal diseases of crops such as: downy mildew in cucumber, powdery mildew in melon, Sclerotinia sclerotiorum in cauliflower, Alternaria solan in potato, Peronospora hyoscyami in tobacco (Bokshi et al. 2003; Perez et al. 2003; Cheng et al. 2006; Chen et al. 2011; Sun et al. 2012; Sillero et al. 2012). However, it is still not well known if induced-resistance to powdery mildew of R. rugosa‘Plena’ could be induced by applying JA or BTH, and how their resistance-physiological index might be changed.
In this study, we investigated the effects of JA and BTH on R. rugosa‘Plena’ resistance. The results obtained will provide a reference to the study of disease resistance inducing of R. rugosa‘Plena’ and might provide new ideas for exploring environmentally friendly methods of plant disease control.
Materials and methods
2-year-old healthy R. rugosa‘Plena’ seedlings with similar growth vigor were used.
Induction and inoculation methods
Based on preliminary experimental results, three concentrations of JA and three of BTH were used in this experiment. JA and BTH at concentrations of 0.1, 0.5 and 1.0 mmol/L were sprayed on the leaves of R. rugosa‘Plena’. The inductive agents were evenly sprayed on the leaves at a dosage of 20 ml per seedling. Each concentration treated 40 seedlings. S. pannosa inoculation was carried out by leaf spraying 2 days (48 h) after spraying with JA or BTH. Spores of S. pannosa were obtained from powdery mildew-infected leaves on outdoor cultivated R. rugosa‘Plena’ and were diluted with distilled water to a suspension liquid with a concentration of 3 × 104 spores/ml for inoculation. Distilled water spraying alone was used as the control 1 (CK1), and distilled water + spores inoculation was control 2 (CK2). Three replications were conducted for each treatment.
Leaf samples were collected on the 1st, 3rd, 5th, 7th, 9th, 11th and 14th day the second day after inductive agents were sprayed for 24 h, as well as on the 1st, 3rd, 5th, 7th, 9th and 12th day after S. pannosa was inoculated. Samples were immediately sealed into plastic bags, stored in a freezer at −80 °C for measurement.
Index measured and methods
Statistics on disease injury
Rank of S. pannosa infected leaves of Rosa rugosa‘Plena’
Lower than 1/4 of the ratio disease spots area to total leaf area
1/4–1/2 of the ratio disease spots area to total leaf area
1/2–3/4 of the ratio disease spots area to total leaf area
More than 3/4 of the ratio disease spots area to total leaf area
PAL was measured according to the method described by Hu et al. (2009) and Yan et al. (2013a, b). A 0.2 g leaf sample was weighed and ground in a mortar with liquid nitrogen, and 5-ml of an ice-cold borate buffer (5 mmol/L, pH 8.8) was added. The homogenates were centrifuged at 6000 r min−1 for 20 min at 4 °C. The supernatant was used as a crude extract for PAL activity assay. A 0.05 ml supernatant was mixed with 1.0 ml of 0.02 mol/L phenylalanine and 2.95 ml distilled water to produce a 4-ml reaction system. The mixtures were shaken well and transferred to a quartz cuvette and measured at 290 nm. The mixtures were placed in a thermostatic water bath at 30 °C for 30 min, and measured again at 290 nm. The reaction system without substrate had 1.0 ml distilled water added and was taken as the control. The results were expressed as U min−1 g−1 Fw.
PPO measurement is based on Yan et al. (2013a, b). A 0.5 g leaf sample was weighed and ground under liquid nitrogen, and 5-ml of 0.05 mol/L phosphate buffer (pH 7.0) added. The homogenate was centrifuged at 4000 r min−1 for 15 min at 4 °C. A 0.1 ml supernatant was mixed with 1.5 ml of 0.02 mol/L pyrocatechol and 1.5 ml phosphate buffer. The reaction system was left standing at 30 °C for 2 min and then measured at 398 nm. One unit of enzyme activity was defined as an increase in absorbance of 0.01/min.
Peroxidase (POD) was measured as described by Yan et al. (2013a, b) and Li et al. (2016). A 0.3 g leaf sample was weighed and placed into a pre-cooled mortar for grinding and 5 ml of 0.2 mol/L sodium phosphate buffer (pH 7.4) was added. The homogenate was centrifuged at 3000 r min−1 at 4 °C for 10 min. The supernatant was used as a crude enzyme solution and 0.1 ml of the supernatant and 2 µl guaiacol were transferred to a quartz cuvette. Optical density was record at 470 nm immediately within 2 min after adding 0.6% H2O2. The control mixture contained no hydrogen peroxide. Enzyme activity was measured at an absorbance of OD470, one unit of enzyme activity was defined as an increase in absorbance of 0.01/min. Enzyme activity was expressed as U min−1 g−1 Fw.
Total phenolics and flavonoids were measured according to the measurement of Lin et al. (1995) with a minor modification. A 0.2 g leaf sample was ground in a pre-cooled mortar with 5 ml of a 1% HCl-methanol solution. The liquid was centrifuged at 6000 r min−1 at 4 °C for 20 min, and then measured at 280 nm after standing at 4 °C for 24 h. Total phenolic content was calculated on the basis of a gallic acid standard curve (y = 0.0066x + 0.0077, R2 = 0.9963), expressed by mg g−1. Flavonoid content was measured at 325 nm, expressed by mg g−1.
Lignin content was measured according to Yu et al. (2013) and Wang et al. (2015) with a minor modification. A 0.2 g leaf sample was ground in 95% ethyl alcohol. The liquid was filtered and washed in 95% ethyl alcohol three times and subsequently three times in a mixture of ethanol and n-hexane (1:2, V/V). The sediment obtained was dissolved in 25% acetyl bromide glacial acetic acid, and then placed in a water bath of 70 °C for 30 min. A 0.9 mL of 2 mol/L NaOH, 3 mL glacial acetic acid and 0.1 mL 7.5 mol/L hydroxylamine hydrochloride were added in sequence to the reaction system and centrifuged at 4500 r min−1 for 10 min. A 20 μL supernatant was diluted by adding three mL of distilled water and measured at 280 nm.
The data were analyzed using SPSS 19. One-Way ANOVA and LSD testing was used for significance analysis of difference.
Results and analysis
Effects of JA and BTH on leaf morbidity ratio
Disease index of S. pannosa on R. rugosa‘Plena’ leaves treated with JA and BTH after inoculation
Days after inoculation (d)
Percentage of infected leaves (%)
Disease index (%)
Inductive effects (%)
Effects on defensive enzymes of Rosa rugosa ‘Plena’ induced by JA and BTH
Effects on total phenolic content of Rosa rugosa ‘Plena’ induced by JA and BTH
The total phenolic content of the 0.5 mmol/L JA treatment was higher than that of CK1 and CK2 at all time periods (p < 0.05). The maximum value was on the seventh (fifth) day which was 1.68-times higher than CK1 and 1.47-fold higher than CK2. Total phenolic content of the 0.5 mmol/L BTH treatment showed a similar trend to that of the 0.5 mmol/L JA treatment, total phenolic content was 1.90- and 1.66-fold higher than CK1 and CK2 (p < 0.05), respectively.
Effects on flavonoid content of Rosa rugosa ‘Plena’ induced by JA and BTH
Effects on lignin content of Rosa rugosa ‘Plena’ induced by JA and BTH
Discussion and conclusions
Co-actions of multi disease-tolerance factors are usually involved in induced tolerance of plants for protection from infection by pathogens but are not depend on a single disease-resistant factor. Plant disease resistance is commonly induced by enzymatic actions. The activity of protective enzymes is usually increased when plants are infected by pathogens or are treated by inductive agents, which is considered an important mechanism for plant disease resistance induction (Mao et al. 2005). POD, PPO, and PAL are three vital protective enzymes in plant disease resistance reactions. Increasing their activities is considered a biochemical index of plant disease resistance (Chen et al. 2010a, b). POD plays a role in plant disease resistance by catalyzing phenolics to form quinones (Li et al. 2016). PPO and POD participate in lignin synthesis, leading to thickness of cell walls to protect plants from infection. (Shivakumar et al. 2003; Chen et al. 2010a, b; Yanti 2015). PAL is a key and rate-limiting enzyme in catalyzing l-phenylalanine to trans-cinnamic acid, it can improve the disease resistance of plants by promoting the synthesis of phenolic substances and lignin (Xue et al. 1983; Zhang et al. 1987). Total phenolics and flavonoids were higher in disease resistant cultivars than in disease-sensitive ones (Chen et al. 2010a, b); for example, BTH treatment improved cucumber resistance to downy mildew by inducing synthesis of phenolics and lignin (Wang et al. 2005).
In this study, different concentrations of JA and BTH increased activities of PAL, PPO, and POD in R. rugosa‘Plena’ leaves. This is in agreement with that reported by Yu et al. (2013) and Chen et al. (2011). The optimal concentration for both exogenous BTH and JA was 0.5 mmol/L for inducing R. rugosa‘Plena’ resistance to S. pannosa. After treatment with BTH and JA, the total content of phenols, flavonoids and lignin were higher than in the controls. The results in this study are consistent with melon resistance to powdery mildew (Chen et al. 2010a, b) and cucumber resistance to downy mildew (Wang et al. 2005), but with a difference in induction time possibly due to differences in the stress response ratio of different plants to inductive agents. The results suggest that BTH- and JA-induced increase in activities of the protective enzymes and contents of secondary metabolites is a possible mechanism of enhanced resistance of R. rugosa‘Plena’ to S. pannosa.
Plant disease resistance levels depend on a response time and the ratio and quantity of accumulated resistant substances after the expression of resistance genes. Inductive agents have functions to induce the expression of disease resistance genes and subsequent synthesis and accumulation of disease resistant chemicals (Chen et al. 2010a, b). An improvement in disease resistance is the optimal selection for plants in defending against pathogen infections (Zeng et al. 2008). Our results demonstrated that different concentrations of JA and BTH induced increased resistance of R. rugosa‘Plena’ leaves to S. pannosa. Leaves treated with 0.5 mmol/L BTH had the maximum resistance to S. pannosa; its inductive effect reached 66.36%. Leaves treated with 0.5 mmol/L JA showed the optimal induction to plant disease resistance with its inductive effect reaching 54.49%. Both JA and BTH can improve the resistance to powdery mildew of R. rugosa‘Plena’. This result is similar to the research of Chen et al. (2010a, b) and Niu et al. (2011) on melon and wheat. The optimum concentration of induction was different for different species. To conclude, BTH and JA treatments could increase the resistance of R. rugosa‘Plena’ to S. pannosa by improving the activities of the protective enzymes and the accumulation of secondary metabolites.
Plants use different defense signaling pathways to carry out the most effective response to pathogens (Thomma et al. 1998). At present, it is generally accepted that there are two main signaling pathways to induce disease resistance in plants. They are salicylate-dependent defense-response pathways and jasmonate-dependent defence-response pathways (Balbi and Devoto 2008). BTH, as a salicylic acid analogue, can induce the production of the salicylic acid pathway (Sun et al. 2012). Therefore, we concluded that JA- and BTH- induced resistance of R. rugosa‘Plena’ to S. pannosa might be related to the activation of salicylic acid and jasmonic acid pathways.
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