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Effect of d -pinitol isolated and identified from Robinia pseudoacacia against cucumber powdery mildew
Scientia Horticulturae 176 (2014) 38–44
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Effect of d-pinitol isolated and identified from Robinia pseudoacacia against cucumber powdery mildew Jia Chen, Guang-Hui Dai ∗ Laboratory of Plant Health and Natural Product & Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 22 June 2014 Accepted 25 June 2014 Keywords: d-Pinitol Sphaerotheca fuliginea Erysiphe cichoracearum Robinia pseudoacacia Control effect
a b s t r a c t The Robinia pseudoacacia plant extract has been reported to have protective effect against cucumber powdery mildew (Sphaerotheca fuliginea). In this study, the active compound of R. pseudoacacia was isolated from the plant extract, and the structure was identified to be d-Pinitol by nuclear magnetic resonance. d-Pinitol, as a known compound, has important physiological activity on plant osmotic tolerance and medicinal activities on human health. This study is the first to report the antifungal activity of d-pinitol on an obligate parasite S. fuliginea and tried to develop a phytochemical fungicide. The evaluation in the growth chamber showed that the control effect of d-pinitol and its formulation against cucumber powdery mildew disease was 80.7% and 94.4%, at a concentration of 0.5 mg/ml. This formulation was further tested in a greenhouse to evaluate its control effect against cucumber (S. fuliginea) and tobacco (Erysiphe cichoracearum) powdery mildew under natural condition. The efficacy of d-pinitol in both trials was better than that of positive control at the recommended concentration. The content of d-pinitol in this plant was 25 mg/g (w/w, dry leaves weight) as determined by gas chromatography. The abundant biomass of d-pinitol in R. pseudoacacia plant extract makes it more economical in practical use. Our study provides a base for the future exploitation of d-pinitol as a natural phytochemical fungicide. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sphaerotheca fuliginea (known as Podosphaera xanthii and P. fuliginea), an obligate parasitic fungus that causes cucurbit powdery mildew, is one of the most destructive diseases in greenhouses and fields throughout the world (Glawe, 2008). The application of fungicides is presently the main practice in most cucurbit crops to control powdery mildew (McGrath, 1996). These fungicides generally carry a high risk of developing resistance in their targets, because such fungicides have specific modes of action and powdery mildew fungus has a high potential for resistance development (McGrath, 2001). Concerning about health and the environment, the demand to replace chemical fungicides with biological control is stronger than ever. Numerous studies on alternative strategies to control powdery mildew have been carried out to reduce the input of fungicides. Cucumber was shown to be affected by light quality in resisting powdery mildew (Wang et al., 2010). Chitosan and salicylic acid, elicitors of plant defenses, induced systemic acquired
∗ Corresponding author. Tel.: +86 021 34206923; fax: +86 021 34206923. E-mail address: [email protected] (G.-H. Dai). http://dx.doi.org/10.1016/j.scienta.2014.06.035 0304-4238/© 2014 Elsevier B.V. All rights reserved.
resistance to powdery mildew on cucumber (Conti et al., 1996; Moret et al., 2009). Fawe et al. (1998) studied the silicon-mediated resistance to powdery mildew and found the accumulation of flavonoid phytoalexins in cucumber. A foliar spray of micronutrient solutions on cucumber also induces local and systemic protection to control the same disease (Reuveni et al., 1997). Cow’s milk has also been found to be effective in controlling powdery mildew, and the action modulator was further studied (Bettiol, 1999; Medeiros et al., 2012). The commercial mycopesticide Vertalec® based on Lecanicillium longisporum was evaluated for simultaneous control of aphids and powdery mildew on vegetable crops in protected facilities (Kim et al., 2008). In recent research, natural plant extracts have been studied to control powdery mildew. The extracts of Achyranthes japonica (Miq) Nakai (Amaranthaceae; whole plant), Rumex crispus L. (Polygonaceae; roots), and Inula viscosa were screened for their efficiency (Kim et al., 2004; Wang et al., 2004). In Greece, the efficacy of Milsana® (a registered name given to a formulated plant extract of Reynoutria sachalinensis) against powdery mildew in greenhouse-grown cucumbers was also investigated over a 3-year period under high disease pressure (Konstantinidou-Doltsinis and Schmit, 1998). However, the active components of these plant extracts were not known.
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Our group has previously found that extracts from Robinia pseudoacacia Linn. have high protective effect against the cucumber powdery mildew fungus S. fuliginea (Jin et al., 2006; Zhang et al., 2008). In this study, we isolated and purified the active compound of R. pseudoacacia by a bioassay-guided fractionation technique (Chaubal et al., 2005), and its structure was identified as d-pinitol by nuclear magnetic resonance (NMR). The amount of this active compound in R. pseudoacacia was determined by gas chromatography. The efficacy of d-pinitol against cucumber powdery mildew was evaluated under growth chamber; its formulation was further tested in controlling the cucumber and tobacco powdery mildew under greenhouse conditions. 2. Materials and methods 2.1. Plant and fungal material R. pseudoacacia leaves were collected at Henan Province, China, in the autumn of 2008 and were grinded into powders after being dried at room temperature. The fungal material of S. fuliginea was detached and identified from the heavily infected cucumber leaves in the greenhouse. The cucumber cultivar ‘Baoyang’ 5th (Shanghai Baoshan Vegetables Science Institute, China), which is highly susceptible to S. fuliginea, was used in all the experiments. The fungal was maintained on the young true leaves of cucumber seedlings in the Laboratory of Plant Health and Natural Product of Shanghai Jiao Tong University, China. The culture condition was as follows: temperature of 22 ± 1 ◦ C, photoperiod of 16 h and humidity of 50%–75%. The pathogen was transferred to a new seedling every 2 weeks. 2.2. Biological assay of potted seedlings The control effect of extracts against the cucumber powdery mildew fungus S. fuliginea was tested according to the Chinese National Agriculture Industry Standard: Guideline for laboratory bioassay of pesticides – Potted plant test for fungicide control of powdery mildew on cucurbits: NY/T 1156.11 – 2008. Cucumber seedlings at the first true-leaf stage were sprayed with the different concentrations of extracts from each stage of isolation until the leaves were fully spread. After 24 h of treatment, they were inoculated with S. fuliginea conidial suspensions (concentration of 105 conidia ml−1 ). The treated seedlings were cultured for 7–10 days at 22 ± 1 ◦ C and at a 16 h light/8 h dark photoperiod. Each pot with five cucumber plants was used with six replicates per treatment. Experiments were repeated three times. The evaluation was started when the percentage of diseased leaves in the water control group was more than 80%. The incidence of powdery mildew was visually evaluated on individual leaves and ranked as a percentage infected area using an index of 0, 1, 3, 5, 7, 9, and 11, where 0 = no symptoms, 1 = 0–5%, 3 = 5–15%, 5 = 15–25%, 7 = 25–50%, 9 = 50–75%, and 11 = 75–100%, according to the guideline mentioned above. The disease index and control efficacy were calculated according to the following equations: Disease index (%) =
the number of diseased plant leaves in this range × the disease range total plant leaves investigated × 11
× 100
Control efficacy (%) =
D − D T C DC
× 100
where DT = the disease index of treatment and DC = the disease index of water control.
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2.3. Isolation and identification of the active compound Ethanol, methanol, n-butyl alcohol, and ethyl acetate (analytical grade) were purchased from Shanghai Lingfeng Reagent Co. (Shanghai, China). Zhang et al. (2008) already found that the active component was in the aqueous fraction when the crude extracts of R. pseudoacacia were partitioned with four solvents (petroleum ether, hexyl acetate, n-butyl alcohol, and water). The isolation and the purification were thus performed according to Zhang et al. (2008). The concentrated ethanol extracts (20 g) were dissolved in water and then extracted with n-butyl alcohol. The water phase was condensed at a volume of 200 ml and then purified using a macroporous resin (HZ841, East China University of Science and Technology, China). The water phase and ethanol phase partitions were collected and concentrated. The water-soluble protein was separated from the water phase extracts using the removal of protein-Sevag method. The remaining water phase was fractioned by silica gel (100–200 mesh) column chromatography (CC) (3 cm × 60 cm; 1:20 g) with ethyl acetate (300 ml), different concentrations of ethyl acetate/methanol/water (9:1:0.5 [600 ml], 3:1:0.2 [300 ml], 1:1:0.1 [600 ml], and 1:3:0.2 [300 ml]), and finally, methanol (200 ml). The fractions of 10 ml volume were collected. The progress of CC separation was monitored by thin layer chromatography (TLC), using an isopropyl-alcohol/ammonia/water concentration of 8:1:1 as the developing system and 5% phosphomolybdic acid in ethanol as the chromogenic reagent. Fractions showing a similar chemical property in the TLC test were combined, and finally nine major fractions (1–9) were obtained. The fraction showing the best control effect in the bioassay was purified by repeated crystallization from MeOH to yield white crystals (compound A). The melting point of compound A was measured on a WRS1A Digital Melting Point Apparatus (Shanghai YICE Apparatus & Equipments Co., China). Optical rotation was determined using a Perkin–Elmer 341 polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DRX-500 spectrometer. Chemical shifts were expressed in ı (ppm).
2.4. Quantitative analysis of d-pinitol The content of d-pinitol was determined by a gas chromatograph (GC; SHIMADZU Japan GC-2010) according to the method of Sun et al. (1999), using a 30 m fused silica capillary column with polydimethylsiloxane as stationary phase (Rtx-5, 0.25 mm ID, 0.25 m film thickness) and a flame ionization detector (FID). The column was heated to 150 ◦ C and maintained for 3 min, increased to 200 ◦ C at 5 ◦ C/min and to 300 ◦ C at 10 ◦ C/min, and finally kept at 300 ◦ C for 17 min. Purified helium as the carrier gas was allowed to flow at a rate of 30 cm/s. Powdered plant material of R. pseudoacacia leaves (4 g) were extracted with 100 ml of 25% ethanol at 60 ◦ C for 1 h. The extract was filtered through Whatman no.1 filter paper, and the filtrate was adjusted to 100 ml with distilled water. The filtrate was kept on to pass through a 0.2 mm nylon membrane filter. A 0.1 ml sample was dried under vacuum and then derivatized with 1.0 ml of trimethylsilylimidazole (TMSI) dissolved in pyridine (1:4, v/v) at 90 ◦ C for 1 h. After cooling to room temperature for 1 h, a 1 l solution was injected into the GC using a Shimadzu autosampler. The objective peak areas corresponding to the d-pinitol concentrations were recorded. The standard solution of d-pinitol (Sigma, China) was prepared in MilliQ water at concentrations of 0.2, 0.6, 1, 1.5, and 2 mg/ml. The standard solutions of each concentration (0.1 ml) were dried and derivatized before testing by gas chromatography. The standard curve was obtained by using standard solutions as the external
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Fig. 1. Control efficacy of R. pseudoacacia extracts derived from different isolation stages against cucumber powdery mildew. The control efficacy of the two partitions separated by macroporous resin (A), the control efficacy of water partition after Sevag protein removal (B), and the control efficacy of nine fractions isolated from column chromatography (C) are shown. Control efficacy was calculated by biological assay at potted seedlings. Fractions isolated by each treatment were condensed to the same concentration of 5 mg/ml (A), 3 mg/ml (B), and 2 mg/ml (C). The columns and error bars represent their mean control efficiencies and standard errors of three replicates, respectively. Means followed by the same letter are not significantly different at a level of 0.05 according to Duncan’s test.
standards. The d-pinitol content of R. pseudoacacia leaf was calculated using standard curve. 2.5. d-Pinitol formulation Commercial auxiliary surfactants were added to increase the control efficacy of the d-pinitol solution. The formulation (soluble concentrate SL) included: d-pinitol: 20%; Terwet 245: 6%; ethylene glycol: 5%; and Terwet 3001: 4%. The Terwet 245 (polyalkylene oxide derivative of a synthetic alcohol) and Terwet 3001 (alkyl polyglucoside) used in this study were gifts from Hunsman Co. (America). Purified d-pinitol (98%) was dissolved in distilled water to produce different concentration solutions: 0.025, 0.05, 0.1, 0.2, 0.5, and 1 mg/ml. The d-pinitol formulation and the chemical control (commercial fungicide: tebuconazole 250 g/L, EW, Bayer) were diluted in distilled water to produce a series of concentrations (0.025, 0.05, 0.1, 0.2, 0.5, and 1 mg a.i./ml). The formulation
component (without d-pinitol) was diluted at the same rate as the d-pinitol formulation. Distilled water was used as control. The control effect of these diluted solutions was then tested. The inoculation, incubation, and evaluation in the experiment were conducted as described in Section 2.2. 2.6. Greenhouse trials of the d-pinitol formulation The control effect of the d-pinitol formulation against cucumber powdery mildew, S. fuliginea, was evaluated at a farm in Shanghai, China. The trial was carried out in a greenhouse (100 m2 ) from April 7th (sowing date) until May 29th (last assessment), 2010. The same susceptible cucumber cultivar ‘Baoyang’ 5th was used. Plants were arranged in a complete randomized block design with four replicates. Treatments were randomly assigned with 30–33 plants in each block. The following treatments were applied: 20% d-pinitol formulation (SL) at the rate of 0.13, 0.2, 0.25, 0.33, and 0.5 mg/ml;
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the commercial biofungicide Azoxystrobin (SC 250 g/L) purchased from Syngenta Co. at a rate of 0.17 mg/ml; and water as control. Application started when the first symptoms of powdery mildew occurred naturally; treatment was applied twice at 7-day intervals. The control effect of the d-pinitol formulation was evaluated against another powdery mildew fungus, Erysiphe cichoracearum, on tobacco at a farm in Kunming City, Chinese Yunnan Province, which is the most famous tobacco producing area. The positive control Triadimephon is the most common commercial fungicide for tobacco powdery mildew control locally. The trial was carried out from October 2012 (transplanting date) until January 2013 (last assessment). The tobacco cultivar K326 susceptible to E. cichoracearum was used in the greenhouse assay. Plants were arranged in a complete randomized block design with three replicates. The following treatments were applied: the 20% d-pinitol formulation (SL) at a rate of 0.5 mg/ml; commercial fungicide triadimefon (EC 20%) purchased from Jiannong Co. (China) at a rate of 0.1% v/v; and water was used as control. Application started when the percentage of diseased leaves of powdery mildew was above 50%; treatment was applied three times at 7-day intervals. Disease severity on individual leaves was assessed visually. The disease severity index was evaluated as described by Zhang et al. (2008), and the control efficacy for each treatment was calculated as follows:
Control efficacy (%) = 1 −
DC0 · DT1 DC1 · DT0
× 100
where DT0 = disease index of the treatment before application, DT1 = disease index of the treatment after application, DC0 = disease index of the water control before application, and DC1 = disease index of the water control after application. 2.7. Statistical analysis Data on the control effect against cucumber powdery mildew disease was analyzed by analysis of variance (ANOVA) using an SPSS procedure. Means followed by the same letter were not significantly different at a level of 0.05 according to Duncan’s test. 3. Results 3.1. Isolation and identification of d-pinitol from R. pseudoacacia extracts A previous study (Zhang et al., 2008) demonstrated that the water phase extracts of R. pseudoacacia have a protective effect of 74.7% at a concentration of 10 mg/ml, but the active compound remains unknown. In this study, we improved the isolation of the active compound using a modified method. After separation of the water phase extracts of R. pseudoacacia using a macroporous resin, the water phase had an 82.3% control efficacy at a concentration of 5 mg/ml, whereas the ethanol phase had a 33.1% control efficacy at the same concentration (Fig. 1A). The control efficacy of the water phase increased from 70.9% to 83% at a concentration of 3 mg/ml after the water-soluble proteins were separated by removal of protein-Sevag method (Fig. 1B). To purify the active compounds, the water phase extracts without soluble proteins were isolated by silica gel CC. These fractions were divided into nine groups through TLC analysis. The bioassay of the nine fractions showed that fraction 5 had the highest control efficacy of 85.9% at a concentration of 2 mg/ml (Fig. 1C). Fraction 5 became a white needle crystal after crystallization from MeOH and was named compound A. The properties of compound A were as follows: m.p. 185–186 ◦ C (literature value), [˛]D 20 : +67 (c 2.5, H2 O) (literature data); data for compound A (C7 H14 O6 ): 1 H NMR (500 MHz, D O): ı 4.10 (m, H-1, H-6), 3.91 (dd, H-2), 3.44 (t, 2
41
Fig. 2. Structure of d-pinitol. The active compound isolated from the R. pseudoacacia leaf extract was identified to be d-pinitol.
H-3), 3.74 (t, H-4), 3.85 (t, H-5), 3.69 (s, MeO); 13 C NMR (125 MHz, D2 O): ı 72.50 (d, C-1), 70.65 (d, C-2), 83.60 (d, C-3), 72.95 (d, C4), 71.36 (d, C-5), 72.29 (d, C-6), 60.56 (q, MeO). These spectral data and physicochemical properties indicated that compound A is d-pinitol (Fig. 2), as previously isolated from Limeum pterocarpum (Abdoulaye et al., 2004). To investigate the content of d-pinitol in R. pseudoacacia, leaf samples were extracted with low concentration ethanol, separated with GC, and monitored with FID after derivation. In the preliminary experiments, the GC column temperature condition was estimated by the standard of d-pinitol. The curve of the peak area (Y) versus the d-pinitol standard concentration (X) was plotted. The linear regression equation was Y = 715708X + 51390, R2 = 1. Compared with the size of the peaks of the d-pinitol standard that appeared at the same time, the content of d-pinitol in R. pseudoacacia was 25 mg/g of the dry weight of the leaf powder. 3.2. Control effect of d-pinitol and its formulation against powdery mildew disease of cucumber and tobacco In the growth chamber trial, d-pinitol purified from R. pseudoacacia was dissolved in distilled water and tested for cucumber powdery mildew disease control. The control efficacy increased from 7.8% to 88.9% as the dose increased from 0.025 mg/ml to 1 mg/ml, suggesting that d-pinitol has a dose-dependent control activity (Fig. 3). The apparent increase in control effect can be observed in the formulation study. The control efficacy of d-pinitol formulation increased by 10–20% compared with pure d-pinitol solution at the same concentration, suggesting that the d-pinitol formulation is more effective than pure d-pinitol. The d-pinitol formulation solvent had no effect on the disease, suggesting that the added auxiliary solvent can only help increase the control effect of d-pinitol. The commercial chemical fungicide tebuconazole was significantly better than the d-pinitol formulation at a concentration of less than 0.5 mg/ml, but equally effective as the d-pinitol formulation at concentrations above 0.5 mg/ml. A negative impact on the cucumber plant was not observed in the group treated with the d-pinitol formulation at all doses, whereas the leaves of the tebuconazole-treated group turned yellow and wilted at high concentrations of more than 0.5 mg/ml. Although the control effect was not as effective compared with that of the chemical control, the cucumber grew better when treated with d-pinitol. In the greenhouse trial, the control effect evaluation of d-pinitol formulation at different rates on cucumber powdery mildew, S. fuliginea, compared with a commercial fungicide Azoxystrobin (SC 250 g/L) was realized in suburban Shanghai. The evaluations were recorded and the disease severity index was calculated. The results showed that the severity of cucumber powdery mildew in all
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Fig. 3. Dose–effect curve of d-pinitol against cucumber powdery mildew compared with its formulation (SL), solvent, and tebuconazole. Values are means and standard deviations of three replicates. Although the d-pinitol formulation at a concentration of 0.5 mg/ml or lower is significantly less efficient than the commercial chemical fungicide tebuconazole, its control efficacy elevated to nearly the same as that of tebuconazle with the increase in concentration to more than 0.5 mg/ml. Formulated d-pinitol showed improved performance over d-pinitol, whereas the solvent exhibited no effect on cucumber powdery mildew.
Fig. 5. Disease index of tobacco powdery mildew E. cichoracearum treated with dpinitol formulation (SL 20%) and triadimefon (EC 20%) in the greenhouse trial. The assessment of the disease index was carried out seven days after each application. Values are means and standard deviations of three replicates. Both d-pinitol formulation and the chemical fungicide triadimefon were effective on tobacco powdery mildew. The d-pinitol treatment at a rate of 0.5 mg/ml significantly reduced the disease severity compared with the fungicide triadimefon at the recommended rate of 0.1% v/v (p ≤ 0.01).
The disease index of tobacco powdery mildew E. cichoracearum is presented in Fig. 5. Both d-pinitol formulation and the chemical fungicide triadimefon reduced the disease severity. The d-pinitol treatment at a rate of 0.5 mg/ml was significantly better than the fungicide at the recommended rate of 0.1% v/v (p ≤ 0.01). The control effect of this formulation was 58.1% after the third application, which was more effective than that of triadimefon (37.6%). 4. Discussion
Fig. 4. Disease index of cucumber powdery mildew S. fuliginea treated with different rates of formulated d-pinitol and Azoxystrobin in the greenhouse trial. The disease severity index of powdery mildew was recorded before the first application and seven days after each application. Values are means and standard deviations of four replicates. The severity of cucumber powdery mildew gradually decreased when treated with increasing doses of d-pinitol formulation, and also improved after the second application in most of the treatment groups compared with the first one.
treatments decreased significantly after the first application (Fig. 4). The control efficacy of the formulation was calculated by the disease severity index as follows: 52.2% (d-pinitol 0.5 mg/ml), 39.4% (dpinitol 0.37 mg/ml), 37.0% (d-pinitol 0.25 mg/ml), 21.9% (d-pinitol 0.2 mg/ml), and 23.5% (Azoxystrobin 0.17 mg/ml). The severity of this plant disease also decreased after the second application in most of the treatment groups compared with the first one. The control efficacy increased to 68.6% (d-pinitol 0.5 mg/ml), 55.6% (dpinitol 0.37 mg/ml), 39.2% (d-pinitol 0.25 mg/ml), 21.5% (d-pinitol 0.2 mg/ml), and 56.7% (Azoxystrobin 0.17 mg/ml). The efficacy of Azoxystrobin reached 56.7% at 0.17 mg/ml, whereas a similar efficacy of d-pinitol formulation of 55.6% at 0.37 mg/ml was observed after second application.
R. pseudoacacia, a widely distributed species in most temperate regions, have interesting biological and medical activities (Pharriss and Russell, 1967). Tian et al. (2001) isolated robinlin from ethanolic extracts of this plant, which showed high lethality to brine shrimp. A low-molecular-weight cationic peptide isolated from R. pseudoacacia seeds has inhibitory effects against seven bacteria (Talas-Ogras et al., 2005). In this study, an active component against cucumber powdery mildew disease was isolated from the leaves of R. pseudoacacia, which was later demonstrated to be d-pinitol. dPinitol, as a known compound, was isolated for the first time from Pinus lambertiana and studied as an important tolerance molecular against osmotic press (Gottlieb and Brauns, 1951; Nelson et al., 1999; Sun et al., 1999). Its derivatives, and metabolites thereof, were shown to be useful compositions in nutritional and medicinal treatment, associated with insulin resistance, anti-inflammatory, and antihelmintic activity (Asuzu et al., 1999; Fonteles et al., 2000; Singh et al., 2001; Kim et al., 2007). d-Pinitol was also effective in inhibiting the growth of Helicoverpa armigera, had larvicidal activity against Aedes aegypti, and is an oviposition attractant against Battus philenor (Chaubal et al., 2005; Dreyer et al., 1979; Papaj et al., 1992). Our study is the first to report its antifungal activity on an obligate parasite S. fuliginea of powdery mildew. The natural products physcion, riboflavin, and osthol were already studied for the control of cucumber powdery mildew (Wang et al., 2009; Yang et al., 2008). Milsana, a formulated extract from R. sachalinensis, was commercialized as a fungicide to control cucumber powdery mildew, and the protective mechanism was also studied (Konstantinidou-Doltsinis et al., 2006). However, these
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kinds of plant material are not easy obtained, and the high costs of these natural products limit their application in practice. Considering the application of R. pseudoacacia leaf extracts in cucumber powdery mildew control, we evaluated the d-pinitol content in R. pseudoacacia dry leaves by GC. The occurrence of d-pinitol and its derivative had been reported in many plants, being especially abundant in pine and legumes (Baumgartner et al., 1986; Gottlieb and Brauns, 1951; Van Boven et al., 2001). d-Pinitol is the most dominant component of the low-molecular-weight carbohydrate fraction of soybeans, and the tropical content varies from 20 to 25 mg/g (Phillips et al., 1982; Binder and Haddon, 1984; Kuo et al., 1997). Our study confirmed that the d-pinitol content of R. pseudoacacia dry leaves content was 25 mg/g, and the abundant biomass of d-pinitol in this plant extract makes it more economical in the future practical use. The bioassay results showed that the control effect of d-pinitol formulation against cucumber powdery mildew is dose dependent, and higher rates are more effective than lower ones against this disease regardless of the application time. The low rate application of d-pinitol formulation was low-effective and failed to control powdery mildew. The severity of the powdery mildew at the very beginning was different between each treatment. The disease severity index values in low rate d-pinitol treatments were higher than those of high rate treatments, especially of the water treatment. This result may be attributed to the fact that the low rate application of d-pinitol formulation at high disease severity index had low efficacy on disease control. Artificial inoculation after d-pinitol application in the growth chamber trial suggested that dpinitol can serve as a preventive treatment, whereas application after the powdery mildew occurred naturally in the greenhouse trial indicated that d-pinitol can also have a cure effect on the plant disease. Another greenhouse trial that lasted for two years verified that d-pinitol has effect on both the causal agent of the cucumber powdery mildew S. fuliginea and E. cichoracearum (unpublished data). E. cichoracearum has also been reported to be the causal agent of tobacco powdery mildew. In this study, the d-pinitol effect was tested against E. cichoracearum on other host plant, namely, tobacco, a very important economical crop worldwide. The result showed that d-pinitol had a control effect on this disease. Our study demonstrated that d-pinitol isolated and identified from Robinia pseudoacacia, with abundant biomass, possesses an antifungal activity against the fungal phytopathogen S. fuliginea and E. cichoracearum of the powdery mildew disease. The formulation study also provided a possibility for a new natural phytochemical fungicide to control this disease. Further studies of d-pinitol on the modes of action will be conducted.
Acknowledgments This study was supported by the Agricultural Science and Technology Achievements Transformation Fund Programs (2008GB2C000095) and the Shanghai Leading Academic Discipline Project (B209). The authors thank Dr. Zamir Punja for his assistance in English writing, Zhang Zhiyuan for his prior study, and Kang Suzhen and Zhang Zhen for their technical assistance.
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Effect of d-pinitol isolated and identified from Robinia pseudoacacia against cucumber powdery mildew Jia Chen, Guang-Hui Dai ∗ Laboratory of Plant Health and Natural Product & Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 22 June 2014 Accepted 25 June 2014 Keywords: d-Pinitol Sphaerotheca fuliginea Erysiphe cichoracearum Robinia pseudoacacia Control effect
a b s t r a c t The Robinia pseudoacacia plant extract has been reported to have protective effect against cucumber powdery mildew (Sphaerotheca fuliginea). In this study, the active compound of R. pseudoacacia was isolated from the plant extract, and the structure was identified to be d-Pinitol by nuclear magnetic resonance. d-Pinitol, as a known compound, has important physiological activity on plant osmotic tolerance and medicinal activities on human health. This study is the first to report the antifungal activity of d-pinitol on an obligate parasite S. fuliginea and tried to develop a phytochemical fungicide. The evaluation in the growth chamber showed that the control effect of d-pinitol and its formulation against cucumber powdery mildew disease was 80.7% and 94.4%, at a concentration of 0.5 mg/ml. This formulation was further tested in a greenhouse to evaluate its control effect against cucumber (S. fuliginea) and tobacco (Erysiphe cichoracearum) powdery mildew under natural condition. The efficacy of d-pinitol in both trials was better than that of positive control at the recommended concentration. The content of d-pinitol in this plant was 25 mg/g (w/w, dry leaves weight) as determined by gas chromatography. The abundant biomass of d-pinitol in R. pseudoacacia plant extract makes it more economical in practical use. Our study provides a base for the future exploitation of d-pinitol as a natural phytochemical fungicide. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sphaerotheca fuliginea (known as Podosphaera xanthii and P. fuliginea), an obligate parasitic fungus that causes cucurbit powdery mildew, is one of the most destructive diseases in greenhouses and fields throughout the world (Glawe, 2008). The application of fungicides is presently the main practice in most cucurbit crops to control powdery mildew (McGrath, 1996). These fungicides generally carry a high risk of developing resistance in their targets, because such fungicides have specific modes of action and powdery mildew fungus has a high potential for resistance development (McGrath, 2001). Concerning about health and the environment, the demand to replace chemical fungicides with biological control is stronger than ever. Numerous studies on alternative strategies to control powdery mildew have been carried out to reduce the input of fungicides. Cucumber was shown to be affected by light quality in resisting powdery mildew (Wang et al., 2010). Chitosan and salicylic acid, elicitors of plant defenses, induced systemic acquired
∗ Corresponding author. Tel.: +86 021 34206923; fax: +86 021 34206923. E-mail address: [email protected] (G.-H. Dai). http://dx.doi.org/10.1016/j.scienta.2014.06.035 0304-4238/© 2014 Elsevier B.V. All rights reserved.
resistance to powdery mildew on cucumber (Conti et al., 1996; Moret et al., 2009). Fawe et al. (1998) studied the silicon-mediated resistance to powdery mildew and found the accumulation of flavonoid phytoalexins in cucumber. A foliar spray of micronutrient solutions on cucumber also induces local and systemic protection to control the same disease (Reuveni et al., 1997). Cow’s milk has also been found to be effective in controlling powdery mildew, and the action modulator was further studied (Bettiol, 1999; Medeiros et al., 2012). The commercial mycopesticide Vertalec® based on Lecanicillium longisporum was evaluated for simultaneous control of aphids and powdery mildew on vegetable crops in protected facilities (Kim et al., 2008). In recent research, natural plant extracts have been studied to control powdery mildew. The extracts of Achyranthes japonica (Miq) Nakai (Amaranthaceae; whole plant), Rumex crispus L. (Polygonaceae; roots), and Inula viscosa were screened for their efficiency (Kim et al., 2004; Wang et al., 2004). In Greece, the efficacy of Milsana® (a registered name given to a formulated plant extract of Reynoutria sachalinensis) against powdery mildew in greenhouse-grown cucumbers was also investigated over a 3-year period under high disease pressure (Konstantinidou-Doltsinis and Schmit, 1998). However, the active components of these plant extracts were not known.
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Our group has previously found that extracts from Robinia pseudoacacia Linn. have high protective effect against the cucumber powdery mildew fungus S. fuliginea (Jin et al., 2006; Zhang et al., 2008). In this study, we isolated and purified the active compound of R. pseudoacacia by a bioassay-guided fractionation technique (Chaubal et al., 2005), and its structure was identified as d-pinitol by nuclear magnetic resonance (NMR). The amount of this active compound in R. pseudoacacia was determined by gas chromatography. The efficacy of d-pinitol against cucumber powdery mildew was evaluated under growth chamber; its formulation was further tested in controlling the cucumber and tobacco powdery mildew under greenhouse conditions. 2. Materials and methods 2.1. Plant and fungal material R. pseudoacacia leaves were collected at Henan Province, China, in the autumn of 2008 and were grinded into powders after being dried at room temperature. The fungal material of S. fuliginea was detached and identified from the heavily infected cucumber leaves in the greenhouse. The cucumber cultivar ‘Baoyang’ 5th (Shanghai Baoshan Vegetables Science Institute, China), which is highly susceptible to S. fuliginea, was used in all the experiments. The fungal was maintained on the young true leaves of cucumber seedlings in the Laboratory of Plant Health and Natural Product of Shanghai Jiao Tong University, China. The culture condition was as follows: temperature of 22 ± 1 ◦ C, photoperiod of 16 h and humidity of 50%–75%. The pathogen was transferred to a new seedling every 2 weeks. 2.2. Biological assay of potted seedlings The control effect of extracts against the cucumber powdery mildew fungus S. fuliginea was tested according to the Chinese National Agriculture Industry Standard: Guideline for laboratory bioassay of pesticides – Potted plant test for fungicide control of powdery mildew on cucurbits: NY/T 1156.11 – 2008. Cucumber seedlings at the first true-leaf stage were sprayed with the different concentrations of extracts from each stage of isolation until the leaves were fully spread. After 24 h of treatment, they were inoculated with S. fuliginea conidial suspensions (concentration of 105 conidia ml−1 ). The treated seedlings were cultured for 7–10 days at 22 ± 1 ◦ C and at a 16 h light/8 h dark photoperiod. Each pot with five cucumber plants was used with six replicates per treatment. Experiments were repeated three times. The evaluation was started when the percentage of diseased leaves in the water control group was more than 80%. The incidence of powdery mildew was visually evaluated on individual leaves and ranked as a percentage infected area using an index of 0, 1, 3, 5, 7, 9, and 11, where 0 = no symptoms, 1 = 0–5%, 3 = 5–15%, 5 = 15–25%, 7 = 25–50%, 9 = 50–75%, and 11 = 75–100%, according to the guideline mentioned above. The disease index and control efficacy were calculated according to the following equations: Disease index (%) =
the number of diseased plant leaves in this range × the disease range total plant leaves investigated × 11
× 100
Control efficacy (%) =
D − D T C DC
× 100
where DT = the disease index of treatment and DC = the disease index of water control.
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2.3. Isolation and identification of the active compound Ethanol, methanol, n-butyl alcohol, and ethyl acetate (analytical grade) were purchased from Shanghai Lingfeng Reagent Co. (Shanghai, China). Zhang et al. (2008) already found that the active component was in the aqueous fraction when the crude extracts of R. pseudoacacia were partitioned with four solvents (petroleum ether, hexyl acetate, n-butyl alcohol, and water). The isolation and the purification were thus performed according to Zhang et al. (2008). The concentrated ethanol extracts (20 g) were dissolved in water and then extracted with n-butyl alcohol. The water phase was condensed at a volume of 200 ml and then purified using a macroporous resin (HZ841, East China University of Science and Technology, China). The water phase and ethanol phase partitions were collected and concentrated. The water-soluble protein was separated from the water phase extracts using the removal of protein-Sevag method. The remaining water phase was fractioned by silica gel (100–200 mesh) column chromatography (CC) (3 cm × 60 cm; 1:20 g) with ethyl acetate (300 ml), different concentrations of ethyl acetate/methanol/water (9:1:0.5 [600 ml], 3:1:0.2 [300 ml], 1:1:0.1 [600 ml], and 1:3:0.2 [300 ml]), and finally, methanol (200 ml). The fractions of 10 ml volume were collected. The progress of CC separation was monitored by thin layer chromatography (TLC), using an isopropyl-alcohol/ammonia/water concentration of 8:1:1 as the developing system and 5% phosphomolybdic acid in ethanol as the chromogenic reagent. Fractions showing a similar chemical property in the TLC test were combined, and finally nine major fractions (1–9) were obtained. The fraction showing the best control effect in the bioassay was purified by repeated crystallization from MeOH to yield white crystals (compound A). The melting point of compound A was measured on a WRS1A Digital Melting Point Apparatus (Shanghai YICE Apparatus & Equipments Co., China). Optical rotation was determined using a Perkin–Elmer 341 polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DRX-500 spectrometer. Chemical shifts were expressed in ı (ppm).
2.4. Quantitative analysis of d-pinitol The content of d-pinitol was determined by a gas chromatograph (GC; SHIMADZU Japan GC-2010) according to the method of Sun et al. (1999), using a 30 m fused silica capillary column with polydimethylsiloxane as stationary phase (Rtx-5, 0.25 mm ID, 0.25 m film thickness) and a flame ionization detector (FID). The column was heated to 150 ◦ C and maintained for 3 min, increased to 200 ◦ C at 5 ◦ C/min and to 300 ◦ C at 10 ◦ C/min, and finally kept at 300 ◦ C for 17 min. Purified helium as the carrier gas was allowed to flow at a rate of 30 cm/s. Powdered plant material of R. pseudoacacia leaves (4 g) were extracted with 100 ml of 25% ethanol at 60 ◦ C for 1 h. The extract was filtered through Whatman no.1 filter paper, and the filtrate was adjusted to 100 ml with distilled water. The filtrate was kept on to pass through a 0.2 mm nylon membrane filter. A 0.1 ml sample was dried under vacuum and then derivatized with 1.0 ml of trimethylsilylimidazole (TMSI) dissolved in pyridine (1:4, v/v) at 90 ◦ C for 1 h. After cooling to room temperature for 1 h, a 1 l solution was injected into the GC using a Shimadzu autosampler. The objective peak areas corresponding to the d-pinitol concentrations were recorded. The standard solution of d-pinitol (Sigma, China) was prepared in MilliQ water at concentrations of 0.2, 0.6, 1, 1.5, and 2 mg/ml. The standard solutions of each concentration (0.1 ml) were dried and derivatized before testing by gas chromatography. The standard curve was obtained by using standard solutions as the external
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Fig. 1. Control efficacy of R. pseudoacacia extracts derived from different isolation stages against cucumber powdery mildew. The control efficacy of the two partitions separated by macroporous resin (A), the control efficacy of water partition after Sevag protein removal (B), and the control efficacy of nine fractions isolated from column chromatography (C) are shown. Control efficacy was calculated by biological assay at potted seedlings. Fractions isolated by each treatment were condensed to the same concentration of 5 mg/ml (A), 3 mg/ml (B), and 2 mg/ml (C). The columns and error bars represent their mean control efficiencies and standard errors of three replicates, respectively. Means followed by the same letter are not significantly different at a level of 0.05 according to Duncan’s test.
standards. The d-pinitol content of R. pseudoacacia leaf was calculated using standard curve. 2.5. d-Pinitol formulation Commercial auxiliary surfactants were added to increase the control efficacy of the d-pinitol solution. The formulation (soluble concentrate SL) included: d-pinitol: 20%; Terwet 245: 6%; ethylene glycol: 5%; and Terwet 3001: 4%. The Terwet 245 (polyalkylene oxide derivative of a synthetic alcohol) and Terwet 3001 (alkyl polyglucoside) used in this study were gifts from Hunsman Co. (America). Purified d-pinitol (98%) was dissolved in distilled water to produce different concentration solutions: 0.025, 0.05, 0.1, 0.2, 0.5, and 1 mg/ml. The d-pinitol formulation and the chemical control (commercial fungicide: tebuconazole 250 g/L, EW, Bayer) were diluted in distilled water to produce a series of concentrations (0.025, 0.05, 0.1, 0.2, 0.5, and 1 mg a.i./ml). The formulation
component (without d-pinitol) was diluted at the same rate as the d-pinitol formulation. Distilled water was used as control. The control effect of these diluted solutions was then tested. The inoculation, incubation, and evaluation in the experiment were conducted as described in Section 2.2. 2.6. Greenhouse trials of the d-pinitol formulation The control effect of the d-pinitol formulation against cucumber powdery mildew, S. fuliginea, was evaluated at a farm in Shanghai, China. The trial was carried out in a greenhouse (100 m2 ) from April 7th (sowing date) until May 29th (last assessment), 2010. The same susceptible cucumber cultivar ‘Baoyang’ 5th was used. Plants were arranged in a complete randomized block design with four replicates. Treatments were randomly assigned with 30–33 plants in each block. The following treatments were applied: 20% d-pinitol formulation (SL) at the rate of 0.13, 0.2, 0.25, 0.33, and 0.5 mg/ml;
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the commercial biofungicide Azoxystrobin (SC 250 g/L) purchased from Syngenta Co. at a rate of 0.17 mg/ml; and water as control. Application started when the first symptoms of powdery mildew occurred naturally; treatment was applied twice at 7-day intervals. The control effect of the d-pinitol formulation was evaluated against another powdery mildew fungus, Erysiphe cichoracearum, on tobacco at a farm in Kunming City, Chinese Yunnan Province, which is the most famous tobacco producing area. The positive control Triadimephon is the most common commercial fungicide for tobacco powdery mildew control locally. The trial was carried out from October 2012 (transplanting date) until January 2013 (last assessment). The tobacco cultivar K326 susceptible to E. cichoracearum was used in the greenhouse assay. Plants were arranged in a complete randomized block design with three replicates. The following treatments were applied: the 20% d-pinitol formulation (SL) at a rate of 0.5 mg/ml; commercial fungicide triadimefon (EC 20%) purchased from Jiannong Co. (China) at a rate of 0.1% v/v; and water was used as control. Application started when the percentage of diseased leaves of powdery mildew was above 50%; treatment was applied three times at 7-day intervals. Disease severity on individual leaves was assessed visually. The disease severity index was evaluated as described by Zhang et al. (2008), and the control efficacy for each treatment was calculated as follows:
Control efficacy (%) = 1 −
DC0 · DT1 DC1 · DT0
× 100
where DT0 = disease index of the treatment before application, DT1 = disease index of the treatment after application, DC0 = disease index of the water control before application, and DC1 = disease index of the water control after application. 2.7. Statistical analysis Data on the control effect against cucumber powdery mildew disease was analyzed by analysis of variance (ANOVA) using an SPSS procedure. Means followed by the same letter were not significantly different at a level of 0.05 according to Duncan’s test. 3. Results 3.1. Isolation and identification of d-pinitol from R. pseudoacacia extracts A previous study (Zhang et al., 2008) demonstrated that the water phase extracts of R. pseudoacacia have a protective effect of 74.7% at a concentration of 10 mg/ml, but the active compound remains unknown. In this study, we improved the isolation of the active compound using a modified method. After separation of the water phase extracts of R. pseudoacacia using a macroporous resin, the water phase had an 82.3% control efficacy at a concentration of 5 mg/ml, whereas the ethanol phase had a 33.1% control efficacy at the same concentration (Fig. 1A). The control efficacy of the water phase increased from 70.9% to 83% at a concentration of 3 mg/ml after the water-soluble proteins were separated by removal of protein-Sevag method (Fig. 1B). To purify the active compounds, the water phase extracts without soluble proteins were isolated by silica gel CC. These fractions were divided into nine groups through TLC analysis. The bioassay of the nine fractions showed that fraction 5 had the highest control efficacy of 85.9% at a concentration of 2 mg/ml (Fig. 1C). Fraction 5 became a white needle crystal after crystallization from MeOH and was named compound A. The properties of compound A were as follows: m.p. 185–186 ◦ C (literature value), [˛]D 20 : +67 (c 2.5, H2 O) (literature data); data for compound A (C7 H14 O6 ): 1 H NMR (500 MHz, D O): ı 4.10 (m, H-1, H-6), 3.91 (dd, H-2), 3.44 (t, 2
41
Fig. 2. Structure of d-pinitol. The active compound isolated from the R. pseudoacacia leaf extract was identified to be d-pinitol.
H-3), 3.74 (t, H-4), 3.85 (t, H-5), 3.69 (s, MeO); 13 C NMR (125 MHz, D2 O): ı 72.50 (d, C-1), 70.65 (d, C-2), 83.60 (d, C-3), 72.95 (d, C4), 71.36 (d, C-5), 72.29 (d, C-6), 60.56 (q, MeO). These spectral data and physicochemical properties indicated that compound A is d-pinitol (Fig. 2), as previously isolated from Limeum pterocarpum (Abdoulaye et al., 2004). To investigate the content of d-pinitol in R. pseudoacacia, leaf samples were extracted with low concentration ethanol, separated with GC, and monitored with FID after derivation. In the preliminary experiments, the GC column temperature condition was estimated by the standard of d-pinitol. The curve of the peak area (Y) versus the d-pinitol standard concentration (X) was plotted. The linear regression equation was Y = 715708X + 51390, R2 = 1. Compared with the size of the peaks of the d-pinitol standard that appeared at the same time, the content of d-pinitol in R. pseudoacacia was 25 mg/g of the dry weight of the leaf powder. 3.2. Control effect of d-pinitol and its formulation against powdery mildew disease of cucumber and tobacco In the growth chamber trial, d-pinitol purified from R. pseudoacacia was dissolved in distilled water and tested for cucumber powdery mildew disease control. The control efficacy increased from 7.8% to 88.9% as the dose increased from 0.025 mg/ml to 1 mg/ml, suggesting that d-pinitol has a dose-dependent control activity (Fig. 3). The apparent increase in control effect can be observed in the formulation study. The control efficacy of d-pinitol formulation increased by 10–20% compared with pure d-pinitol solution at the same concentration, suggesting that the d-pinitol formulation is more effective than pure d-pinitol. The d-pinitol formulation solvent had no effect on the disease, suggesting that the added auxiliary solvent can only help increase the control effect of d-pinitol. The commercial chemical fungicide tebuconazole was significantly better than the d-pinitol formulation at a concentration of less than 0.5 mg/ml, but equally effective as the d-pinitol formulation at concentrations above 0.5 mg/ml. A negative impact on the cucumber plant was not observed in the group treated with the d-pinitol formulation at all doses, whereas the leaves of the tebuconazole-treated group turned yellow and wilted at high concentrations of more than 0.5 mg/ml. Although the control effect was not as effective compared with that of the chemical control, the cucumber grew better when treated with d-pinitol. In the greenhouse trial, the control effect evaluation of d-pinitol formulation at different rates on cucumber powdery mildew, S. fuliginea, compared with a commercial fungicide Azoxystrobin (SC 250 g/L) was realized in suburban Shanghai. The evaluations were recorded and the disease severity index was calculated. The results showed that the severity of cucumber powdery mildew in all
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Fig. 3. Dose–effect curve of d-pinitol against cucumber powdery mildew compared with its formulation (SL), solvent, and tebuconazole. Values are means and standard deviations of three replicates. Although the d-pinitol formulation at a concentration of 0.5 mg/ml or lower is significantly less efficient than the commercial chemical fungicide tebuconazole, its control efficacy elevated to nearly the same as that of tebuconazle with the increase in concentration to more than 0.5 mg/ml. Formulated d-pinitol showed improved performance over d-pinitol, whereas the solvent exhibited no effect on cucumber powdery mildew.
Fig. 5. Disease index of tobacco powdery mildew E. cichoracearum treated with dpinitol formulation (SL 20%) and triadimefon (EC 20%) in the greenhouse trial. The assessment of the disease index was carried out seven days after each application. Values are means and standard deviations of three replicates. Both d-pinitol formulation and the chemical fungicide triadimefon were effective on tobacco powdery mildew. The d-pinitol treatment at a rate of 0.5 mg/ml significantly reduced the disease severity compared with the fungicide triadimefon at the recommended rate of 0.1% v/v (p ≤ 0.01).
The disease index of tobacco powdery mildew E. cichoracearum is presented in Fig. 5. Both d-pinitol formulation and the chemical fungicide triadimefon reduced the disease severity. The d-pinitol treatment at a rate of 0.5 mg/ml was significantly better than the fungicide at the recommended rate of 0.1% v/v (p ≤ 0.01). The control effect of this formulation was 58.1% after the third application, which was more effective than that of triadimefon (37.6%). 4. Discussion
Fig. 4. Disease index of cucumber powdery mildew S. fuliginea treated with different rates of formulated d-pinitol and Azoxystrobin in the greenhouse trial. The disease severity index of powdery mildew was recorded before the first application and seven days after each application. Values are means and standard deviations of four replicates. The severity of cucumber powdery mildew gradually decreased when treated with increasing doses of d-pinitol formulation, and also improved after the second application in most of the treatment groups compared with the first one.
treatments decreased significantly after the first application (Fig. 4). The control efficacy of the formulation was calculated by the disease severity index as follows: 52.2% (d-pinitol 0.5 mg/ml), 39.4% (dpinitol 0.37 mg/ml), 37.0% (d-pinitol 0.25 mg/ml), 21.9% (d-pinitol 0.2 mg/ml), and 23.5% (Azoxystrobin 0.17 mg/ml). The severity of this plant disease also decreased after the second application in most of the treatment groups compared with the first one. The control efficacy increased to 68.6% (d-pinitol 0.5 mg/ml), 55.6% (dpinitol 0.37 mg/ml), 39.2% (d-pinitol 0.25 mg/ml), 21.5% (d-pinitol 0.2 mg/ml), and 56.7% (Azoxystrobin 0.17 mg/ml). The efficacy of Azoxystrobin reached 56.7% at 0.17 mg/ml, whereas a similar efficacy of d-pinitol formulation of 55.6% at 0.37 mg/ml was observed after second application.
R. pseudoacacia, a widely distributed species in most temperate regions, have interesting biological and medical activities (Pharriss and Russell, 1967). Tian et al. (2001) isolated robinlin from ethanolic extracts of this plant, which showed high lethality to brine shrimp. A low-molecular-weight cationic peptide isolated from R. pseudoacacia seeds has inhibitory effects against seven bacteria (Talas-Ogras et al., 2005). In this study, an active component against cucumber powdery mildew disease was isolated from the leaves of R. pseudoacacia, which was later demonstrated to be d-pinitol. dPinitol, as a known compound, was isolated for the first time from Pinus lambertiana and studied as an important tolerance molecular against osmotic press (Gottlieb and Brauns, 1951; Nelson et al., 1999; Sun et al., 1999). Its derivatives, and metabolites thereof, were shown to be useful compositions in nutritional and medicinal treatment, associated with insulin resistance, anti-inflammatory, and antihelmintic activity (Asuzu et al., 1999; Fonteles et al., 2000; Singh et al., 2001; Kim et al., 2007). d-Pinitol was also effective in inhibiting the growth of Helicoverpa armigera, had larvicidal activity against Aedes aegypti, and is an oviposition attractant against Battus philenor (Chaubal et al., 2005; Dreyer et al., 1979; Papaj et al., 1992). Our study is the first to report its antifungal activity on an obligate parasite S. fuliginea of powdery mildew. The natural products physcion, riboflavin, and osthol were already studied for the control of cucumber powdery mildew (Wang et al., 2009; Yang et al., 2008). Milsana, a formulated extract from R. sachalinensis, was commercialized as a fungicide to control cucumber powdery mildew, and the protective mechanism was also studied (Konstantinidou-Doltsinis et al., 2006). However, these
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kinds of plant material are not easy obtained, and the high costs of these natural products limit their application in practice. Considering the application of R. pseudoacacia leaf extracts in cucumber powdery mildew control, we evaluated the d-pinitol content in R. pseudoacacia dry leaves by GC. The occurrence of d-pinitol and its derivative had been reported in many plants, being especially abundant in pine and legumes (Baumgartner et al., 1986; Gottlieb and Brauns, 1951; Van Boven et al., 2001). d-Pinitol is the most dominant component of the low-molecular-weight carbohydrate fraction of soybeans, and the tropical content varies from 20 to 25 mg/g (Phillips et al., 1982; Binder and Haddon, 1984; Kuo et al., 1997). Our study confirmed that the d-pinitol content of R. pseudoacacia dry leaves content was 25 mg/g, and the abundant biomass of d-pinitol in this plant extract makes it more economical in the future practical use. The bioassay results showed that the control effect of d-pinitol formulation against cucumber powdery mildew is dose dependent, and higher rates are more effective than lower ones against this disease regardless of the application time. The low rate application of d-pinitol formulation was low-effective and failed to control powdery mildew. The severity of the powdery mildew at the very beginning was different between each treatment. The disease severity index values in low rate d-pinitol treatments were higher than those of high rate treatments, especially of the water treatment. This result may be attributed to the fact that the low rate application of d-pinitol formulation at high disease severity index had low efficacy on disease control. Artificial inoculation after d-pinitol application in the growth chamber trial suggested that dpinitol can serve as a preventive treatment, whereas application after the powdery mildew occurred naturally in the greenhouse trial indicated that d-pinitol can also have a cure effect on the plant disease. Another greenhouse trial that lasted for two years verified that d-pinitol has effect on both the causal agent of the cucumber powdery mildew S. fuliginea and E. cichoracearum (unpublished data). E. cichoracearum has also been reported to be the causal agent of tobacco powdery mildew. In this study, the d-pinitol effect was tested against E. cichoracearum on other host plant, namely, tobacco, a very important economical crop worldwide. The result showed that d-pinitol had a control effect on this disease. Our study demonstrated that d-pinitol isolated and identified from Robinia pseudoacacia, with abundant biomass, possesses an antifungal activity against the fungal phytopathogen S. fuliginea and E. cichoracearum of the powdery mildew disease. The formulation study also provided a possibility for a new natural phytochemical fungicide to control this disease. Further studies of d-pinitol on the modes of action will be conducted.
Acknowledgments This study was supported by the Agricultural Science and Technology Achievements Transformation Fund Programs (2008GB2C000095) and the Shanghai Leading Academic Discipline Project (B209). The authors thank Dr. Zamir Punja for his assistance in English writing, Zhang Zhiyuan for his prior study, and Kang Suzhen and Zhang Zhen for their technical assistance.
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