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A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz
Physiological and Molecular Plant Pathology xxx (2017) 1e6
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A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz Xinyi Zhang a, Pinyan Qin b, Jiahang Song a, Fei Qi a, Wei Tian a, * a b
School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin'an 311300, Zhejiang, China School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Lin'an 311300, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2016 Received in revised form 19 February 2017 Accepted 21 February 2017 Available online xxx
The enzymatic activities (POD, PPO, CAT, PAL), as protective enzymes in the leaves of Atractylodis maceocephalae koidz, were tested by treating different concentrations of polysaccharides isolated from Sclerotium rolfsii sacc (P. S. rolfsii) at 0 mg/l, 20 mg/l, 200 mg/l and 400 mg/l against S. rolfsii, and atractylenolides as a phytoalexin in the rhizome of A. maceocephalae were evaluated in compared with control. It was evident that the plant under stress by pathogen has instigated the significant synthesis and accumulation of atractylenolides and the higher enzymes activities were described on the eight day after fungal elicitor inoculation than the control group. Furthermore, the treatments of A. maceocephalae seedlings with P. S.rolfsii increased disease index development caused by S. rolfsii. The disease index is lowest when inoculated at a concentration as low as 20 mg/ml. In general, these results indicated that P. S. rolfsii may be useful as a fungal elicitor, which can enhance resistance and triggered innate immunity in A. maceocephalae, and had the potential to suppress the disease on A. maceocephalae when P. S. rolfsii at 20 mg/l, were used to inoculate the root of A. maceocephalae. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Fungal elicitor Resistance response Enzyme activity Atractylenolides Disease index
1. Introduction The Atractylodis maceocephalae koidz (A. maceocephalae) is a traditional Chinese medicine and a perennial herbal plant [6], which is mainly planted in Zhejiang and Jiangsu. Past researches have confirmed that A. maceocephalae can strengthen the spleen and replenish Qi, dry dampness, and promote diuresis, hidroschesis, and tocolysis [9,30]. There are many studies on A. maceocephalae, including identified and isolated several compounds, a volatile oil [2], lactones [2,24], and polysaccharides [34]. The rhizome of A. maceocephalae has been used for thousands of years with its particular pharmacologic activities [12,32], in which research has focused on immunomodulation, diuresis, anti-tumor activity, anti-sepsis, anti-inflammation, hypoglycemic effects, and anti-senescence, with emphases on the nervous system, digestive system, and uterine smooth muscle [35]. Continuous cropping of A. maceocephalae has been a major issue, and has resulted in plant
* Corresponding author. Nurturing Station for the State Key Laboratory of Subtropical Silviculture, School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang, China. E-mail addresses: [email protected] (X. Zhang), [email protected] (W. Tian).
infections. How to enhance disease-resistance of A. maceocephalae is an inevitable problem [17,36,39]. Production of the Chinese herb has been lost partly because of plants infected with Sclerotium rolfsii sacc (S. rolfsii), which is Mycelia Sterilia of Deuteromycotina [16,21] which is a soilinhabiting pathogen. S. rolfsii is one of the important diseases affecting grain, horticultural plants, forests, and herbs in the tropics and subtropics. Southern blight, also referred to as white mildew, mainly hurts plants close to the surface of the stems and roots. As a stem-based disease, a dark brown, moist, amorphous, concave disease spot appears; when wet, the disease part appears to be radial spun silk with a white mycelium. The scab will expand horizontally around the base of the stem, resulting in yellow leaves, and even death [28]. Fungal elicitors are specific chemical signals derived from fungal cells. Fungal elicitors, when combined with plants, can induce genes to be expressed rapidly with high specificity and selectivity, which will lead to activation of secondary metabolic pathways and accumulate secondary metabolites [22,26,31]. Fungal elicitors are the surface structure or secretions of cells, which are rich in fungal mycelia, the degradation products of fungal mycelia, fermentation broth, and fungal secretions. The fungal elicitors consist of polysaccharides, peptides, glycolipid proteins, oligosaccharides, fatty
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Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
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acids, glycoproteins, and other substances [23]. Fungal elicitors enhance the biomass and secondary metabolites of plants, such as polysaccharides, and improve enzymatic activity inside plants [7]. Hu et al. have demonstrated that oligosaccharides from Dothiorella gregaria as an elicitor can induce expression of the resistance gene, increase resistant metabolism, and accumulate resistant products rapidly [28]. Not all of fungal elicitors can promote an effect on the secondary metabolites, and some fungal elicitors can break the production of secondary metabolites and inhabit the growth of the plant [5,11,18]. Research involving S. rolfsii has focused on attacking the growth of plants, which perpetuates as sclerotia on plant debris and in the soil [27]. Nevertheless, very little research has addressed polysaccharides from S. rolfsii (P. S. rolfsii) as fungal elicitors to induce the production of secondary metabolites that effect the growth of plants. Our previously research has showed that polysaccharides isolated from Chrysanthemum indicum can trigger systemic acquired resistance (SAR) with a certain concentration in A. maceocephalae [1]. While it is a heroic guess that is polysaccharides isolated from S. rolfsii may be induce SAR with a certain concentration and utilize its elicitor activities against S. rolfsii in A. maceocephalae. Our aim was to research potential fungal elicitor activities of the polysaccharides originated from S. rolfsii, whether the activities of P. S. rolfsii can tangle together with other biomolecules in A. maceocephalae and their level in S. rolfsii- inoculated rhizome of A. maceocephalae in response to P. S. rolfsii treatment in comparison with the control. Our experiment was (i) to analyze the activities of peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL), the protective enzymes in A. maceocephalae. (ii) to assess the level of atractylenolides -Ⅱ, a phytoalexins of A. maceocephalae. (iii) to evaluate the disease index (DI), resistance response in A. maceocephalae. 2. Materials and methods 2.1. Fungal cultures and fungal elicitor polysaccharide preparation The fungal elicitor was prepared from the solid culture of the isolate of S. rolfsii, which were derived from China General Microbiological Culture Collection Center called BJJ-528. Solid cultures were initiated from potato dextrosc agar (PDA) with 9 cm petri dishes. The cultures were incubated in the dark and 25 C for 6 day. The mycelia were washed by sterile water, and the elicitor was transferred into sterile 250 ml conical flasks, sterilized by autoclaving, filtered with acetone and dried. The finally solid was used as crude extract of fungal elicitor. Crude polysaccharides were collected from crude extract of fungal elicitor which were grind into homogenate and diluted with to 50 ml in volumetric flask. The total sugar content was measured by phenol-sulfuric acid method referring to glucose standard curve [13], which was calculated regression equation: A ¼ 0.0145c þ 0.0039, r ¼ 0.9992 [14]. Then we figured out the conversion factor (f) of fungal elicitor by f ¼ W÷CD (W: the weight of polysaccharose (mg); C: the concentration of glucose (mg/mL); D: the dilution ratio of polysaccharose) [14]. So the conversion factor (f) was worked out 7.655. Finally, the polysaccharide content of fungal elicitor was figured out, which was 100% in the crude extract by the conversion coefficient P%¼(C D F÷W) 100%. 2.2. The pretreatment of A. maceocephalae A. maceocephalae seeds were purchased from cultivation base, Dafeng, Yancheng City, AnHui province, China. Seeds (50 seeds per treatment, 15 pots in total) were soaked for 24 h in 25 C water
before sowing in soil and were cultivated in a greenhouse at 25e27 C with the maximum illumination intensity of 80 000 lx, photoperiod of 16/8 h (day/night), their nutrition substrate was peat soil and perlite (3: 1, v/v). When A. maceocephalae seedlings grew to a certain period of five to seven leaves, 15 pots were divided into five groups with three duplicates randomly, including 0 mg/L (CK), 20 mg/L, 200 mg/L, 400 mg/L fungal elicitor with S. rolfsii incubated respectively, and a blank control check (BCK) (Table 1). Fifth days after inoculation, the leaves of A. maceocephalae seedlings in the treatment groups and control groups were sampled separately, then the enzyme activity assay were measured and recorded the day before spray and inoculation and on 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h after spray and inoculation. 2.3. Enzyme extraction and assays The true leaves from different concentrations (0 mg/L, 20 mg/L, 200 mg/L, 400 mg/L) and BCK were harvested at different times (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h) after P. S. rolfsii treatment and were stored at 80 C. The frozen leaf sections (1 g) for each treatment were homogenized in sodium borate buffer (0.1 mol/L, pH 8.8, 1 mmol/L EDTANa2, 5 mmol/L mercaptoethanol) in a mortar and pestle. Then the mixture was centrifuged at 10,000 g for 18 min at 4 C. The supernatant was collected under the same condition for use as crude enzyme extracts. The activities of PAL, POD, PPO, CAT were assayed according to the Moerschbacher's method [15], the Guaiacol methol [19], the colorimetry methol [42], and UV spectrophotometry [37] respectively. 2.4. Atractylenolides determined by HPLC Sun Fire C18 (250 mm 4.6 mm, 5 mm) chromatographic column was used for HPLC, with the column temperature maintained at 30 C. Gradient elution was formed on a mobile phase which consisted of two solutions, methyl alcohol (A) and distilled water (B). Initially, the mobile phase consisted of a ratio of 85:15 (A: B, v/ v); after 14 min, this was changed to a ratio of 95:5, which was maintained just 1min; from 15 to 22min, solvent A was reach 95%, and from 22 to 26min, A was keeping in 100%. The flow rate was 1 mL/min. Stock solution of atractylenolides -Ⅰ, -Ⅱ and -Ⅲ of standard substance were prepared by dissolving an accurately weighed sample in an appropriate volume of methanol to get a final concentration of 0.120 mg/ml, 0.137 mg/ml, 0.114 mg/ml, respectively. A. maceocephalae samples roots were accurately weighed 0.05 g in 1 ml volumetric flask, then dilute with methanol to 1 mL. 2.5. Bioassay for fungal elicitor- induced disease resistance in A. maceocephalae A. maceocephalae seedlings were treated with fungal elicitors respectively with the three concentrations (20 mg/L, 200 mg/L, 400 mg/L), CK (0 mg/L) and BCK. Afterwards, the seedlings were
Table 1 The handing methods of different leaves. Experiment
20 mg/L
200 mg/l
400 mg/L
Hypha suspension
water
1 2 3 4 5
þ e e e e
e þ e e e
e e þ e e
þ þ þ þ e
e e e e þ
þSpraying different concentrations of fungal elicitor extract; -no treatment.
Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
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The results were analyzed by SPSS, using the Statistics 19.0 package, and excel 2010. And standard error was figured out, and the significant difference was compared using SPSS.
also significant differences between the CK and treated groups, especially in the 24 and 168 h after elicitation (Fig. 1B). The different treatment groups were much higher than the CK groups, and low (20 mg/l) and high concentrations (400 mg/ml) were significantly different in PPO activity of A. maceocephalae leaves. Significantly higher CAT activity was expressed in the plants treated with 20, 200, and 400 mg/l of P. S. rolfsii fungal elicitors (Fig. 1C). Infected plant CAT activity exceeded the CK plants, and the treatment groups (400 mg/l) were much higher than other treatment groups. Although CAT activity presented larger fluctuations in all doses of fungal elicitors 192 h after spraying, 20 mg/ml of P. S. rolfsii had a significantly higher CAT activity than CK. The PAL activity of A. maceocephalae seeding treated with the fungal elicitor, P. S. rolfsii, was not significantly different in previous periods (0e144 h); however, in the case of later growth (144e192 h) there were significant differences in the 4 treatments (the concentrations of fungal elicitors were 0, 20, 200, and 400; Fig. 1D). Significantly lower PAL activity was shown in the host plant induced with 0 mg/l than doses of 20, 200, and 400 mg/l of P. S. rolfsii. An apparent high peak appeared in 400 mg/l treatment group which was significantly different in the CK.
3. Results
3.2. Effect of P. S. rolfsii-induced resistance on atractylenolides
The P. S. rolfsii as elicitors used in the study showed some differences in enzyme activities, atractylenolide, and disease index at different concentration.
In the current study, atractylenolides -Ⅰ, -Ⅱ, and -Ⅲ, which was recorded in the roots of A. maceocephalae with four treatment groups (doses of 0, 20, 200, and 400 mg/l.P. S. rolfsii) and a BCK group by HPLC showed a declining trend (Fig. 2). Obviously, the BCK was higher than the other treatment groups. Fig. 2a indicated that atractylenolides -Ⅰpresented higher in all doses of P. S. rolfsii than CK, especially there were significant differences between 20 mg/l P. S. rolfsii treatment and CK. Fig. 2b lactone Ⅱ gradually declined during the first days and increased in the last days (Fig. 2b), but the CK (0 mg/l) was always at the bottom among all doses (0, 20, 200, and 400 mg/l). Atractylenolide-Ⅱappeared to be simulated on day 4, and there were significant differences between the treated and CK and apparently higher than the CK. In contrast, atractylenolide -Ⅲ showed a stable trend, while the high concentration peaked on day 2 and there were no significant differences among the 4 treatment groups 2 days later (Fig. 2c).
continuously cultured for 30 day before disease development, then to calculate out morbidity degree. According Level 5 division method [40], we can figure up DI. Every potting would be repeated three times. The control efficiency was calculated using the P following formulas: DI¼( (disease score * number of infected leaves for each score)/(total inoculated leaves *highest score) 100%, which can represent both disease incidence and symptom severity. The disease score were divided into 0 to 5 grades: the percentage of the lesion size is 0% called 0 grade; the percentage of the lesion size is under 20% called 1 grade; the percentage of the lesion size is 21%e40% called 2 grade; the percentage of the lesion size is 41%e60% called 3 grade; the percentage of the lesion size is 61%e 80% called 4 grade; the percentage of the lesion size is 81%e100% called 5 grade [40]. 2.6. Statistical analysis
3.1. Effect of P. S. rolfsii-induced resistance on protective enzymes of A. maceocephalae The inoculation process guaranteed high synchronization of the control and treatment groups at different concentrations of the fungal elicitor extract. Furgure1 showed that the enzymes activities of BCK was still in steady state and belowed each treatment groups. To compare the effect of the different enzymes, we developed a comparable procedure that included different concentrations of fungal elicitor extract with the passage of time. Although the results obtained in four independent experiments had varied concentrations of fungal elicitor extract, the overall behavior of the curves for the measured enzyme activity, as well as the relationship between the different concentrations of fungal elicitor extract (including CK), were always similar. A few days earlier the enzyme activity was significantly higher than the CK. Fig. 1A, B, and D presented a downward trend, and the treatment curve was less than or close to the control curve from days 2e6. As shown in Fig. 1 the treatment curve was more like the CK curve. A few days thereafter, the enzyme activity had a rising trend and was higher than the CK group. The POD activity of A. maceocephalae seeding treated with the fungal elicitor, P. S. rolfsii, was not significantly different in previous periods (0e96 h); however, in the case of later growth (96e192 h) there were significant differences in the for 4 treatments (the concentrations of fungal elicitors were 0, 20, 200, and 400 mg/l; Fig. 1A). The POD activity showed a rising trend during the 192-h investigation, including infected and uninfected plants. The seedlings sprayed with 20 mg/l of fungal elicitors had significantly greater POD activity (P < 0.05) compared with the CK group (0 mg/ l) 96 h after elicitation. The first high peak at the time of the 200 mg/l concentration of fungal elicitors occurred at 144 h, and the 20 and 400 mg/l concentrations occurred at 8 days. POD activity of the 20 mg/l concentration was initially >200 and 400 mg/l during the treatment times. The PPO activity of A. maceocephalae seeding treated with the fungal elicitor, P. S. rolfsii, was in constant unrest, but there were
3.3. Effect of P. S. rolfsii-induced resistance on the disease index of A. maceocephalae The DI of A. maceocephalae investigate was calculated (Fig. 3). The host plants inoculated with S. rolfsii, but plants not treated with the P. S. rolfsii fungal elicitor (BCK) exhibited a 20.58% disease index after 29 days; however, the disease index after 29 days was clearly increased to 37.35% and 21.08% when plants were treated with 200 and 400 mg/mL of the P. S. rolfsii fungal elicitor, respectively. There was an apparent increase trend in the disease index in all doses of P. S. rolfsii, At concentrations as high as 200 or 400 mg/L, the disease index was superior to the CK after treatment 15 days. While the DI of 20 mg/l concentration was inferior to another groups (including BCK and CK). 4. Discussion In the current study, the crude polysaccharide content of S. rolfsii was approximately 100%, which induced resistance in A. maceocephalae. The hyphal walls of S. rolfsii contain a remarkably high amount of polysaccharides [8], however, there was a deficiency in our study regarding the further separation and purification of crude polysaccharides of S. rolfsii, and analyzing the structure of P. S. rolfsii. In contrast, because S. rolfsii has only recently been shown
Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
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Fig. 1. Time course induction of POD, PPO, CAT, PAL activity (Ug1min1) of A. maceocephalae leaves in response to treatment by different doses of 0, 20, 200 and 400 mg/l purified P. S. rolfsii, and BCK group respectively. Each value is the mean of five repetitions ± standard error and vertical bars shows standard errors (±SE). Different letters indicate statistical significance according to SPSS19.0 (P 0.05).
to have structural polysaccharides, few studies exist regarding activity. The present results showed that P. S. rolfsii can also induce the activities of resistance-related enzymes and phytoalexin increased. The result indicate that P. S. rolfsii is a fungal elicitor, which is a broad spectrum inducer of the A. maceocephalae immunity system and has potential applications for A. maceocephalae to control the development of southern blight. P. S. rolfsii triggers resistance-related gene expression, transcription, and translation, and effects the change of internal proteins always needed to be determined or documented. Thus, we are need of thorough research involving P. S. rolfsii as a fungal elicitor in A. maceocephalae. The POD enzyme plays an important role in the polymerization of monolignols dividing into lignin and the intensification of the cell wall, which results in a rapid tissue hardness to protect plants [25]. P. S. rolfsii expressed a key elicitor capacity of POD activity higher than CK and BCK (Fig. 1A), and this might be significantly responsive to changes in P. S. rolfsii-induced secondary metabolites [3]. The host plant treated by P. S. rolfsii had a high POD activity, even after 120 h of elicitation, which is similar to a previous report in which the expression of natural resistance induced by the host plant was found for a long time. CAT is a key enzyme of elimination of free oxygen, enhancing plant disease resistance [10]. PPO plays a key role in disease resistance because of an ability to oxidize phenol and transform to quinones, lignin, and phytoalexin. It is well-
known that quinones are harmful for pathogenic bacteria, while lignin and phytoalexin can protect plants. PAL is involved in the shikimate and phenylpropanoid pathways, which is necessary for the synthesis of defense-related compounds [20]. The variation of PAL activity is intimately linked with the synthesis of phenols. The POD, CAT, PPO, and PAL activities showed a significant increase after S. rolfsii treatment. The current study results indicate that the increase in enzyme activity is one of the important mechanisms of P. S. rolfsii fungal-induced resistance against the disease of A. maceocephalae, especially southern blight. A. maceocephalae produce atractylenolide -Ⅰ, atractylenolide -II and atractylenolide eIII, which was remarkable increased when the A. maceocephalae be subjected to higher-incidence of southern blight, so it can as a special kind of intrinsic terpenoid metabolites [33,38]. In our previous work, the atractylenolides have been verified to consist in the rhizome of the A. maceocephalae conduces to the expression of S. rolfsii infection and that atractylenolides, as phytoalexin synthesis, is a representative of a defence reaction fundamental in resistance of plants to S. rolfsii [41]. In particular, atractylenolide -II and -III have displayed fungistasis in A. maceocephalae, of which elevated levels in a typical pattern of phytoalexins, in answer to an eliciting treatment [41]. Therefore, in this study P. S. rolfsii, a fungal elicitor, can induce production of phytoalexin, as the disease-resistant signal conduction, in A. maceocephalae. In Fig. 2, the content of atractylenolides expressed higher
Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
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Fig. 2. Time course induction of atractylenolide-I, atractylenolide-II and atractylenolide-III (mg/g) of A. maceocephalae rhizomes in response to treatment by different doses of 0, 20, 200 and 400 mg/l purified P. S. rolfsii and BCK, respectively. Each value is the mean of five repetitions ± standard error and vertical bars shows standard errors (±SE). Different letters indicate statistical significance according to SPSS19.0 (P 0.05).
Fig. 3. Time course induction of DI in A. maceocephalae in response to treatment by different doses of 0, 20, 200, 400 mg/l and CK purified P. S. rolfsii and BCK, respectively. Each value is the mean of five repetitions ± standard error and vertical bars shows standard errors (±SE). Different letters indicate statistical significance according to SPSS19.0 (P 0.05).
Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
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level in P. S. rolfsii treatment groups than in blank control group, which means the fungal elicitor- P. S. rolfsii can induce the production of phytoalexin, stimulate the intrinsic metabolites of terpenoid compounds, and triggers the immune response in plants. Fungi are vitally important plant pathogens and even cause production drawdown [29], while plants have developed a system to perceive molecular signatures derived from fung wall fragments to trigger plant immunity, and thereby to attack by pathogens [4]. Our novel finding that P. S. rolfsii can elevate the level of resistance in A. maceocephalae, which is of extremely significance because no P. S. rolfsii as a fungal elicitor was reported to express any type of resistance in plants, thus provide strongly intuitionistic evidence that P. S. rolfsii have an elicitor function that can defend against the incursion of pathogens and induce defense responses in A. maceocephalae, and biosynthesis of antimicrobial compounds. Our discovery offers a new interesting research direction in the immune response of the plants response to fungal wall fragments. 5. Conclusions In conclusions, dose of the P. S. rolfsii affected the disease index for southern blight in A. maceocephalae (Fig. 3). And they acted as a fungal elicitor, induced the defence enzymes and trigged the conduction of phytoalexins in A. maceocephalae. The dose of 200 mg/l P. S. rolfsii produced higher enzymes activities, atractylenolide eII, and decreased disease index than control, but the disease index dropped to 5.71% spraying with 20.0 mg/l purified P. S. rolfsii 12 days after elicitation and even bellowed BCK. Moreover, it is necessary to do the depth research about whether the certain concentration of P. S. rolfsii induced the allergic reaction in A. maceocephalae. Acknowledgments This study was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No.LY13C140006. We gratefully thank Dr. Wei Tian, Nurturing Station for the State Key Laboratory of Subtropical Silviculture, School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang, China. References [1] M. Adhilakshmi, P. Latha, V. Paranidharan, D. Balachandar, K. Ganesamurthy, R. Velazhahan, Biological control of stem rot of groundnut (Arachis hypogaea L.) caused by Sclerotium rolfsii Sacc. with actinomycetes, Archives Phytopathology Plant Prot. 47 (3) (2014) 298e311. [2] H. Baoshan, S. jiansu, C. zhongliang, Isolation and identification of atractylenolide lV from atractylodes macrocephala koidz, Acta Bot. Sin. 34 (08) (1992) 614e617. [3] R.N. Bennett, R.M. Wallsgrove, Secondary metabolites in plant defence mechanisms, New Phytol. 127 (4) (1994) 617e633. [4] T. Boller, G. Felix, A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors, Plant Biol. 60 (60) (2009) 379e406. [5] B. Bu, D. Qiu, H. Zeng, L. Guo, J. Yuan, X. Yang, A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton, Plant cell Rep. 33 (3) (2014) 461e470. [6] C.F. Chau, S.H. Wu, The development of regulations of Chinese herbal medicines for both medicinal and food uses, Trends Food Sci. Technol. 17 (6) (2006) 313e323. [7] J. Chen, C. Wang, L. Shi, J. Yin, Effects of six kinds of fungal elicitors on growth of dendrobium hybrid. Cultivar ‘088’ tissue culture seedlings, Agric. Sci. Technol. 15 (03) (2015) 28e31. [8] I. Chet, Y. Henis, X-ray analysis of hyphal and sclerotial walls of Sclerotium rolfsii Sacc, Can. J. Microbiol. 14 (7) (1968) 815e816. [9] J.N.M. College, Dictionary Traditional Drugs, Shanghai Scientific and Technical Publishers, Shanghai, 1986. [10] R.F. Dalisay, J.A. Ku c, Persistence of induced resistance and enhanced peroxidase and chitinase activities in cucumber plants, Physiological Mol. Plant Pathol. 47 (4) (1995) 315e327.
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A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz Xinyi Zhang a, Pinyan Qin b, Jiahang Song a, Fei Qi a, Wei Tian a, * a b
School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin'an 311300, Zhejiang, China School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Lin'an 311300, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2016 Received in revised form 19 February 2017 Accepted 21 February 2017 Available online xxx
The enzymatic activities (POD, PPO, CAT, PAL), as protective enzymes in the leaves of Atractylodis maceocephalae koidz, were tested by treating different concentrations of polysaccharides isolated from Sclerotium rolfsii sacc (P. S. rolfsii) at 0 mg/l, 20 mg/l, 200 mg/l and 400 mg/l against S. rolfsii, and atractylenolides as a phytoalexin in the rhizome of A. maceocephalae were evaluated in compared with control. It was evident that the plant under stress by pathogen has instigated the significant synthesis and accumulation of atractylenolides and the higher enzymes activities were described on the eight day after fungal elicitor inoculation than the control group. Furthermore, the treatments of A. maceocephalae seedlings with P. S.rolfsii increased disease index development caused by S. rolfsii. The disease index is lowest when inoculated at a concentration as low as 20 mg/ml. In general, these results indicated that P. S. rolfsii may be useful as a fungal elicitor, which can enhance resistance and triggered innate immunity in A. maceocephalae, and had the potential to suppress the disease on A. maceocephalae when P. S. rolfsii at 20 mg/l, were used to inoculate the root of A. maceocephalae. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Fungal elicitor Resistance response Enzyme activity Atractylenolides Disease index
1. Introduction The Atractylodis maceocephalae koidz (A. maceocephalae) is a traditional Chinese medicine and a perennial herbal plant [6], which is mainly planted in Zhejiang and Jiangsu. Past researches have confirmed that A. maceocephalae can strengthen the spleen and replenish Qi, dry dampness, and promote diuresis, hidroschesis, and tocolysis [9,30]. There are many studies on A. maceocephalae, including identified and isolated several compounds, a volatile oil [2], lactones [2,24], and polysaccharides [34]. The rhizome of A. maceocephalae has been used for thousands of years with its particular pharmacologic activities [12,32], in which research has focused on immunomodulation, diuresis, anti-tumor activity, anti-sepsis, anti-inflammation, hypoglycemic effects, and anti-senescence, with emphases on the nervous system, digestive system, and uterine smooth muscle [35]. Continuous cropping of A. maceocephalae has been a major issue, and has resulted in plant
* Corresponding author. Nurturing Station for the State Key Laboratory of Subtropical Silviculture, School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang, China. E-mail addresses: [email protected] (X. Zhang), [email protected] (W. Tian).
infections. How to enhance disease-resistance of A. maceocephalae is an inevitable problem [17,36,39]. Production of the Chinese herb has been lost partly because of plants infected with Sclerotium rolfsii sacc (S. rolfsii), which is Mycelia Sterilia of Deuteromycotina [16,21] which is a soilinhabiting pathogen. S. rolfsii is one of the important diseases affecting grain, horticultural plants, forests, and herbs in the tropics and subtropics. Southern blight, also referred to as white mildew, mainly hurts plants close to the surface of the stems and roots. As a stem-based disease, a dark brown, moist, amorphous, concave disease spot appears; when wet, the disease part appears to be radial spun silk with a white mycelium. The scab will expand horizontally around the base of the stem, resulting in yellow leaves, and even death [28]. Fungal elicitors are specific chemical signals derived from fungal cells. Fungal elicitors, when combined with plants, can induce genes to be expressed rapidly with high specificity and selectivity, which will lead to activation of secondary metabolic pathways and accumulate secondary metabolites [22,26,31]. Fungal elicitors are the surface structure or secretions of cells, which are rich in fungal mycelia, the degradation products of fungal mycelia, fermentation broth, and fungal secretions. The fungal elicitors consist of polysaccharides, peptides, glycolipid proteins, oligosaccharides, fatty
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acids, glycoproteins, and other substances [23]. Fungal elicitors enhance the biomass and secondary metabolites of plants, such as polysaccharides, and improve enzymatic activity inside plants [7]. Hu et al. have demonstrated that oligosaccharides from Dothiorella gregaria as an elicitor can induce expression of the resistance gene, increase resistant metabolism, and accumulate resistant products rapidly [28]. Not all of fungal elicitors can promote an effect on the secondary metabolites, and some fungal elicitors can break the production of secondary metabolites and inhabit the growth of the plant [5,11,18]. Research involving S. rolfsii has focused on attacking the growth of plants, which perpetuates as sclerotia on plant debris and in the soil [27]. Nevertheless, very little research has addressed polysaccharides from S. rolfsii (P. S. rolfsii) as fungal elicitors to induce the production of secondary metabolites that effect the growth of plants. Our previously research has showed that polysaccharides isolated from Chrysanthemum indicum can trigger systemic acquired resistance (SAR) with a certain concentration in A. maceocephalae [1]. While it is a heroic guess that is polysaccharides isolated from S. rolfsii may be induce SAR with a certain concentration and utilize its elicitor activities against S. rolfsii in A. maceocephalae. Our aim was to research potential fungal elicitor activities of the polysaccharides originated from S. rolfsii, whether the activities of P. S. rolfsii can tangle together with other biomolecules in A. maceocephalae and their level in S. rolfsii- inoculated rhizome of A. maceocephalae in response to P. S. rolfsii treatment in comparison with the control. Our experiment was (i) to analyze the activities of peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL), the protective enzymes in A. maceocephalae. (ii) to assess the level of atractylenolides -Ⅱ, a phytoalexins of A. maceocephalae. (iii) to evaluate the disease index (DI), resistance response in A. maceocephalae. 2. Materials and methods 2.1. Fungal cultures and fungal elicitor polysaccharide preparation The fungal elicitor was prepared from the solid culture of the isolate of S. rolfsii, which were derived from China General Microbiological Culture Collection Center called BJJ-528. Solid cultures were initiated from potato dextrosc agar (PDA) with 9 cm petri dishes. The cultures were incubated in the dark and 25 C for 6 day. The mycelia were washed by sterile water, and the elicitor was transferred into sterile 250 ml conical flasks, sterilized by autoclaving, filtered with acetone and dried. The finally solid was used as crude extract of fungal elicitor. Crude polysaccharides were collected from crude extract of fungal elicitor which were grind into homogenate and diluted with to 50 ml in volumetric flask. The total sugar content was measured by phenol-sulfuric acid method referring to glucose standard curve [13], which was calculated regression equation: A ¼ 0.0145c þ 0.0039, r ¼ 0.9992 [14]. Then we figured out the conversion factor (f) of fungal elicitor by f ¼ W÷CD (W: the weight of polysaccharose (mg); C: the concentration of glucose (mg/mL); D: the dilution ratio of polysaccharose) [14]. So the conversion factor (f) was worked out 7.655. Finally, the polysaccharide content of fungal elicitor was figured out, which was 100% in the crude extract by the conversion coefficient P%¼(C D F÷W) 100%. 2.2. The pretreatment of A. maceocephalae A. maceocephalae seeds were purchased from cultivation base, Dafeng, Yancheng City, AnHui province, China. Seeds (50 seeds per treatment, 15 pots in total) were soaked for 24 h in 25 C water
before sowing in soil and were cultivated in a greenhouse at 25e27 C with the maximum illumination intensity of 80 000 lx, photoperiod of 16/8 h (day/night), their nutrition substrate was peat soil and perlite (3: 1, v/v). When A. maceocephalae seedlings grew to a certain period of five to seven leaves, 15 pots were divided into five groups with three duplicates randomly, including 0 mg/L (CK), 20 mg/L, 200 mg/L, 400 mg/L fungal elicitor with S. rolfsii incubated respectively, and a blank control check (BCK) (Table 1). Fifth days after inoculation, the leaves of A. maceocephalae seedlings in the treatment groups and control groups were sampled separately, then the enzyme activity assay were measured and recorded the day before spray and inoculation and on 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h after spray and inoculation. 2.3. Enzyme extraction and assays The true leaves from different concentrations (0 mg/L, 20 mg/L, 200 mg/L, 400 mg/L) and BCK were harvested at different times (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h) after P. S. rolfsii treatment and were stored at 80 C. The frozen leaf sections (1 g) for each treatment were homogenized in sodium borate buffer (0.1 mol/L, pH 8.8, 1 mmol/L EDTANa2, 5 mmol/L mercaptoethanol) in a mortar and pestle. Then the mixture was centrifuged at 10,000 g for 18 min at 4 C. The supernatant was collected under the same condition for use as crude enzyme extracts. The activities of PAL, POD, PPO, CAT were assayed according to the Moerschbacher's method [15], the Guaiacol methol [19], the colorimetry methol [42], and UV spectrophotometry [37] respectively. 2.4. Atractylenolides determined by HPLC Sun Fire C18 (250 mm 4.6 mm, 5 mm) chromatographic column was used for HPLC, with the column temperature maintained at 30 C. Gradient elution was formed on a mobile phase which consisted of two solutions, methyl alcohol (A) and distilled water (B). Initially, the mobile phase consisted of a ratio of 85:15 (A: B, v/ v); after 14 min, this was changed to a ratio of 95:5, which was maintained just 1min; from 15 to 22min, solvent A was reach 95%, and from 22 to 26min, A was keeping in 100%. The flow rate was 1 mL/min. Stock solution of atractylenolides -Ⅰ, -Ⅱ and -Ⅲ of standard substance were prepared by dissolving an accurately weighed sample in an appropriate volume of methanol to get a final concentration of 0.120 mg/ml, 0.137 mg/ml, 0.114 mg/ml, respectively. A. maceocephalae samples roots were accurately weighed 0.05 g in 1 ml volumetric flask, then dilute with methanol to 1 mL. 2.5. Bioassay for fungal elicitor- induced disease resistance in A. maceocephalae A. maceocephalae seedlings were treated with fungal elicitors respectively with the three concentrations (20 mg/L, 200 mg/L, 400 mg/L), CK (0 mg/L) and BCK. Afterwards, the seedlings were
Table 1 The handing methods of different leaves. Experiment
20 mg/L
200 mg/l
400 mg/L
Hypha suspension
water
1 2 3 4 5
þ e e e e
e þ e e e
e e þ e e
þ þ þ þ e
e e e e þ
þSpraying different concentrations of fungal elicitor extract; -no treatment.
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The results were analyzed by SPSS, using the Statistics 19.0 package, and excel 2010. And standard error was figured out, and the significant difference was compared using SPSS.
also significant differences between the CK and treated groups, especially in the 24 and 168 h after elicitation (Fig. 1B). The different treatment groups were much higher than the CK groups, and low (20 mg/l) and high concentrations (400 mg/ml) were significantly different in PPO activity of A. maceocephalae leaves. Significantly higher CAT activity was expressed in the plants treated with 20, 200, and 400 mg/l of P. S. rolfsii fungal elicitors (Fig. 1C). Infected plant CAT activity exceeded the CK plants, and the treatment groups (400 mg/l) were much higher than other treatment groups. Although CAT activity presented larger fluctuations in all doses of fungal elicitors 192 h after spraying, 20 mg/ml of P. S. rolfsii had a significantly higher CAT activity than CK. The PAL activity of A. maceocephalae seeding treated with the fungal elicitor, P. S. rolfsii, was not significantly different in previous periods (0e144 h); however, in the case of later growth (144e192 h) there were significant differences in the 4 treatments (the concentrations of fungal elicitors were 0, 20, 200, and 400; Fig. 1D). Significantly lower PAL activity was shown in the host plant induced with 0 mg/l than doses of 20, 200, and 400 mg/l of P. S. rolfsii. An apparent high peak appeared in 400 mg/l treatment group which was significantly different in the CK.
3. Results
3.2. Effect of P. S. rolfsii-induced resistance on atractylenolides
The P. S. rolfsii as elicitors used in the study showed some differences in enzyme activities, atractylenolide, and disease index at different concentration.
In the current study, atractylenolides -Ⅰ, -Ⅱ, and -Ⅲ, which was recorded in the roots of A. maceocephalae with four treatment groups (doses of 0, 20, 200, and 400 mg/l.P. S. rolfsii) and a BCK group by HPLC showed a declining trend (Fig. 2). Obviously, the BCK was higher than the other treatment groups. Fig. 2a indicated that atractylenolides -Ⅰpresented higher in all doses of P. S. rolfsii than CK, especially there were significant differences between 20 mg/l P. S. rolfsii treatment and CK. Fig. 2b lactone Ⅱ gradually declined during the first days and increased in the last days (Fig. 2b), but the CK (0 mg/l) was always at the bottom among all doses (0, 20, 200, and 400 mg/l). Atractylenolide-Ⅱappeared to be simulated on day 4, and there were significant differences between the treated and CK and apparently higher than the CK. In contrast, atractylenolide -Ⅲ showed a stable trend, while the high concentration peaked on day 2 and there were no significant differences among the 4 treatment groups 2 days later (Fig. 2c).
continuously cultured for 30 day before disease development, then to calculate out morbidity degree. According Level 5 division method [40], we can figure up DI. Every potting would be repeated three times. The control efficiency was calculated using the P following formulas: DI¼( (disease score * number of infected leaves for each score)/(total inoculated leaves *highest score) 100%, which can represent both disease incidence and symptom severity. The disease score were divided into 0 to 5 grades: the percentage of the lesion size is 0% called 0 grade; the percentage of the lesion size is under 20% called 1 grade; the percentage of the lesion size is 21%e40% called 2 grade; the percentage of the lesion size is 41%e60% called 3 grade; the percentage of the lesion size is 61%e 80% called 4 grade; the percentage of the lesion size is 81%e100% called 5 grade [40]. 2.6. Statistical analysis
3.1. Effect of P. S. rolfsii-induced resistance on protective enzymes of A. maceocephalae The inoculation process guaranteed high synchronization of the control and treatment groups at different concentrations of the fungal elicitor extract. Furgure1 showed that the enzymes activities of BCK was still in steady state and belowed each treatment groups. To compare the effect of the different enzymes, we developed a comparable procedure that included different concentrations of fungal elicitor extract with the passage of time. Although the results obtained in four independent experiments had varied concentrations of fungal elicitor extract, the overall behavior of the curves for the measured enzyme activity, as well as the relationship between the different concentrations of fungal elicitor extract (including CK), were always similar. A few days earlier the enzyme activity was significantly higher than the CK. Fig. 1A, B, and D presented a downward trend, and the treatment curve was less than or close to the control curve from days 2e6. As shown in Fig. 1 the treatment curve was more like the CK curve. A few days thereafter, the enzyme activity had a rising trend and was higher than the CK group. The POD activity of A. maceocephalae seeding treated with the fungal elicitor, P. S. rolfsii, was not significantly different in previous periods (0e96 h); however, in the case of later growth (96e192 h) there were significant differences in the for 4 treatments (the concentrations of fungal elicitors were 0, 20, 200, and 400 mg/l; Fig. 1A). The POD activity showed a rising trend during the 192-h investigation, including infected and uninfected plants. The seedlings sprayed with 20 mg/l of fungal elicitors had significantly greater POD activity (P < 0.05) compared with the CK group (0 mg/ l) 96 h after elicitation. The first high peak at the time of the 200 mg/l concentration of fungal elicitors occurred at 144 h, and the 20 and 400 mg/l concentrations occurred at 8 days. POD activity of the 20 mg/l concentration was initially >200 and 400 mg/l during the treatment times. The PPO activity of A. maceocephalae seeding treated with the fungal elicitor, P. S. rolfsii, was in constant unrest, but there were
3.3. Effect of P. S. rolfsii-induced resistance on the disease index of A. maceocephalae The DI of A. maceocephalae investigate was calculated (Fig. 3). The host plants inoculated with S. rolfsii, but plants not treated with the P. S. rolfsii fungal elicitor (BCK) exhibited a 20.58% disease index after 29 days; however, the disease index after 29 days was clearly increased to 37.35% and 21.08% when plants were treated with 200 and 400 mg/mL of the P. S. rolfsii fungal elicitor, respectively. There was an apparent increase trend in the disease index in all doses of P. S. rolfsii, At concentrations as high as 200 or 400 mg/L, the disease index was superior to the CK after treatment 15 days. While the DI of 20 mg/l concentration was inferior to another groups (including BCK and CK). 4. Discussion In the current study, the crude polysaccharide content of S. rolfsii was approximately 100%, which induced resistance in A. maceocephalae. The hyphal walls of S. rolfsii contain a remarkably high amount of polysaccharides [8], however, there was a deficiency in our study regarding the further separation and purification of crude polysaccharides of S. rolfsii, and analyzing the structure of P. S. rolfsii. In contrast, because S. rolfsii has only recently been shown
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Fig. 1. Time course induction of POD, PPO, CAT, PAL activity (Ug1min1) of A. maceocephalae leaves in response to treatment by different doses of 0, 20, 200 and 400 mg/l purified P. S. rolfsii, and BCK group respectively. Each value is the mean of five repetitions ± standard error and vertical bars shows standard errors (±SE). Different letters indicate statistical significance according to SPSS19.0 (P 0.05).
to have structural polysaccharides, few studies exist regarding activity. The present results showed that P. S. rolfsii can also induce the activities of resistance-related enzymes and phytoalexin increased. The result indicate that P. S. rolfsii is a fungal elicitor, which is a broad spectrum inducer of the A. maceocephalae immunity system and has potential applications for A. maceocephalae to control the development of southern blight. P. S. rolfsii triggers resistance-related gene expression, transcription, and translation, and effects the change of internal proteins always needed to be determined or documented. Thus, we are need of thorough research involving P. S. rolfsii as a fungal elicitor in A. maceocephalae. The POD enzyme plays an important role in the polymerization of monolignols dividing into lignin and the intensification of the cell wall, which results in a rapid tissue hardness to protect plants [25]. P. S. rolfsii expressed a key elicitor capacity of POD activity higher than CK and BCK (Fig. 1A), and this might be significantly responsive to changes in P. S. rolfsii-induced secondary metabolites [3]. The host plant treated by P. S. rolfsii had a high POD activity, even after 120 h of elicitation, which is similar to a previous report in which the expression of natural resistance induced by the host plant was found for a long time. CAT is a key enzyme of elimination of free oxygen, enhancing plant disease resistance [10]. PPO plays a key role in disease resistance because of an ability to oxidize phenol and transform to quinones, lignin, and phytoalexin. It is well-
known that quinones are harmful for pathogenic bacteria, while lignin and phytoalexin can protect plants. PAL is involved in the shikimate and phenylpropanoid pathways, which is necessary for the synthesis of defense-related compounds [20]. The variation of PAL activity is intimately linked with the synthesis of phenols. The POD, CAT, PPO, and PAL activities showed a significant increase after S. rolfsii treatment. The current study results indicate that the increase in enzyme activity is one of the important mechanisms of P. S. rolfsii fungal-induced resistance against the disease of A. maceocephalae, especially southern blight. A. maceocephalae produce atractylenolide -Ⅰ, atractylenolide -II and atractylenolide eIII, which was remarkable increased when the A. maceocephalae be subjected to higher-incidence of southern blight, so it can as a special kind of intrinsic terpenoid metabolites [33,38]. In our previous work, the atractylenolides have been verified to consist in the rhizome of the A. maceocephalae conduces to the expression of S. rolfsii infection and that atractylenolides, as phytoalexin synthesis, is a representative of a defence reaction fundamental in resistance of plants to S. rolfsii [41]. In particular, atractylenolide -II and -III have displayed fungistasis in A. maceocephalae, of which elevated levels in a typical pattern of phytoalexins, in answer to an eliciting treatment [41]. Therefore, in this study P. S. rolfsii, a fungal elicitor, can induce production of phytoalexin, as the disease-resistant signal conduction, in A. maceocephalae. In Fig. 2, the content of atractylenolides expressed higher
Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
X. Zhang et al. / Physiological and Molecular Plant Pathology xxx (2017) 1e6
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Fig. 2. Time course induction of atractylenolide-I, atractylenolide-II and atractylenolide-III (mg/g) of A. maceocephalae rhizomes in response to treatment by different doses of 0, 20, 200 and 400 mg/l purified P. S. rolfsii and BCK, respectively. Each value is the mean of five repetitions ± standard error and vertical bars shows standard errors (±SE). Different letters indicate statistical significance according to SPSS19.0 (P 0.05).
Fig. 3. Time course induction of DI in A. maceocephalae in response to treatment by different doses of 0, 20, 200, 400 mg/l and CK purified P. S. rolfsii and BCK, respectively. Each value is the mean of five repetitions ± standard error and vertical bars shows standard errors (±SE). Different letters indicate statistical significance according to SPSS19.0 (P 0.05).
Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002
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level in P. S. rolfsii treatment groups than in blank control group, which means the fungal elicitor- P. S. rolfsii can induce the production of phytoalexin, stimulate the intrinsic metabolites of terpenoid compounds, and triggers the immune response in plants. Fungi are vitally important plant pathogens and even cause production drawdown [29], while plants have developed a system to perceive molecular signatures derived from fung wall fragments to trigger plant immunity, and thereby to attack by pathogens [4]. Our novel finding that P. S. rolfsii can elevate the level of resistance in A. maceocephalae, which is of extremely significance because no P. S. rolfsii as a fungal elicitor was reported to express any type of resistance in plants, thus provide strongly intuitionistic evidence that P. S. rolfsii have an elicitor function that can defend against the incursion of pathogens and induce defense responses in A. maceocephalae, and biosynthesis of antimicrobial compounds. Our discovery offers a new interesting research direction in the immune response of the plants response to fungal wall fragments. 5. Conclusions In conclusions, dose of the P. S. rolfsii affected the disease index for southern blight in A. maceocephalae (Fig. 3). And they acted as a fungal elicitor, induced the defence enzymes and trigged the conduction of phytoalexins in A. maceocephalae. The dose of 200 mg/l P. S. rolfsii produced higher enzymes activities, atractylenolide eII, and decreased disease index than control, but the disease index dropped to 5.71% spraying with 20.0 mg/l purified P. S. rolfsii 12 days after elicitation and even bellowed BCK. Moreover, it is necessary to do the depth research about whether the certain concentration of P. S. rolfsii induced the allergic reaction in A. maceocephalae. Acknowledgments This study was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No.LY13C140006. We gratefully thank Dr. Wei Tian, Nurturing Station for the State Key Laboratory of Subtropical Silviculture, School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang, China. References [1] M. Adhilakshmi, P. Latha, V. Paranidharan, D. Balachandar, K. Ganesamurthy, R. Velazhahan, Biological control of stem rot of groundnut (Arachis hypogaea L.) caused by Sclerotium rolfsii Sacc. with actinomycetes, Archives Phytopathology Plant Prot. 47 (3) (2014) 298e311. [2] H. Baoshan, S. jiansu, C. zhongliang, Isolation and identification of atractylenolide lV from atractylodes macrocephala koidz, Acta Bot. Sin. 34 (08) (1992) 614e617. [3] R.N. Bennett, R.M. Wallsgrove, Secondary metabolites in plant defence mechanisms, New Phytol. 127 (4) (1994) 617e633. [4] T. Boller, G. Felix, A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors, Plant Biol. 60 (60) (2009) 379e406. [5] B. Bu, D. Qiu, H. Zeng, L. Guo, J. Yuan, X. Yang, A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton, Plant cell Rep. 33 (3) (2014) 461e470. [6] C.F. Chau, S.H. Wu, The development of regulations of Chinese herbal medicines for both medicinal and food uses, Trends Food Sci. Technol. 17 (6) (2006) 313e323. [7] J. Chen, C. Wang, L. Shi, J. Yin, Effects of six kinds of fungal elicitors on growth of dendrobium hybrid. Cultivar ‘088’ tissue culture seedlings, Agric. Sci. Technol. 15 (03) (2015) 28e31. [8] I. Chet, Y. Henis, X-ray analysis of hyphal and sclerotial walls of Sclerotium rolfsii Sacc, Can. J. Microbiol. 14 (7) (1968) 815e816. [9] J.N.M. College, Dictionary Traditional Drugs, Shanghai Scientific and Technical Publishers, Shanghai, 1986. [10] R.F. Dalisay, J.A. Ku c, Persistence of induced resistance and enhanced peroxidase and chitinase activities in cucumber plants, Physiological Mol. Plant Pathol. 47 (4) (1995) 315e327.
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Please cite this article in press as: X. Zhang, et al., A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz, Physiological and Molecular Plant Pathology (2017), http://dx.doi.org/10.1016/j.pmpp.2017.02.002