Article pubs.acs.org/JAFC
Antifungal Activity of Compounds Extracted from Cortex Pseudolaricis against Colletotrichum gloeosporioides Jing Zhang,†,‡,# Li-Ting Yan,§,# En-Lin Yuan,†,‡,∥ Hai-Xin Ding,⊥ Huo-Chun Ye,†,‡ Zheng-Ke Zhang,†,‡ Chao Yan,†,‡ Ying-Qian Liu,*,§ and Gang Feng*,†,‡ †
Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Science, Haikou 571010, People’s Republic of China ‡ Key Laboratory of Monitoring and Control of Tropical Agricultural and Forest Invasive Alien Pests, Ministry of Agriculture, Haikou 571010, People’s Republic of China § School of Pharmacy, Lanzhou University, Lanzhou 730000, People’s Republic of China ∥ College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China ⊥ Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science & Technology Normal University, Nanchang 330013, People’s Republic of China ABSTRACT: Cortex Pseudolaricis is the root bark of Pseudolarix amabilis Rehder, found only in China, and has been widely used in folk antifungal remedies in traditional Chinese medicine. In order to find the natural antifungal agents against mango anthracnose, eight compounds, namely pseudolaric acid A (1), ethyl pseudolaric acid B (2), pseudolaric acid B (3), pseudolaric acid BO-β-D-glucoside (4), piperonylic acid (5), propionic acid (6), 3-hydroxy-4-methoxybenzoic acid (7), and 4-(3-formyl-5methoxyphenyl) butanoic acid (8) were isolated from the ethanol extracts of Cortex Pseudolaricis by bioassay-guided fractionation and evaluated for in vitro antifungal activity against Colletotrichum gloeosporioides Penz. Results demonstrated that all of the eight compounds inhibited the mycelial growth of C. gloeosporioides at 5 μg/mL. Among them, pseudolaric acid B and pseudolaric acid A showed the strongest inhibition with the EC50 values of 1.07 and 1.62 μg/mL, respectively. Accordingly, both Pseudolaric acid B and Pseudolaric acid A highly inhibited spore germination and germ tube elongation of C. gloeosporioides. Dipping 100 μg/mL pseudolaric acid B treatment exhibited more effective suppression on postharvest anthracnose in mango fruit when compared to the same concentration of carbendazim. Scanning electron microscopy observations revealed that pseudolaric acid B caused alterations in the hyphal morphology of C. gloeosporioides, including distortion, swelling, and collapse. Pseudolaric acid B caused the mycelial apexes to show an abnormal growth in dimensions with multiple ramifications in subapical expanded areas with irregular shape. These findings warrant further investigation into optimization of pseudolaric acid B to explore a potential antifungal agent for crop protection. KEYWORDS: Pseudolarix amabilis, antifungal activity, Colletotrichum gloeosporioides, pseudolaric acid B, mango
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INTRODUCTION Mango (Mangifera indica L. Anacardiaceae) fruit is an important tropical and subtropical crop with high commercial value due to its bright color, favorable flavor and taste, and rich nutrition.1,2 However, mango is highly susceptible to various fungal pathogens, resulting in huge economic losses. Anthracnose disease, caused by Colletotrichum gloeosporioides Penz., is one of the most common and serious diseases affecting mango.3 The pathogen causes severe symptoms of black lesions on leaves, twigs, and blossoms and may attack immature fruit as a latent infection. The disease appears progressively until fruit ripening during storage.4 In general, control of mango anthracnose disease is achieved by applications of fungicides, such as benomyl and prochloraz.5 However, repeated and exclusive application of chemical-based fungicides often results in increased chemical resistance in pathogens,6 undesirable effects on nontarget organisms, and the potential risks to human health and environmental pollution.7,8 Therefore, identification and exploitation of natural products as an alternative strategy to control anthracnose diseases has been proposed instead of synthetic-based fungicides. Previous studies © 2014 American Chemical Society
have reported a number of natural compounds isolated from medicinal plants, herbs, and antagonistic microorganisms which have exerted antifungal activity against C. gloeosporioides in vitro and controlled mango anthracnose disease.9−13 Pseudolarix amabilis Rehder (Pseudolarix kaempferi Goeden) is a rare endemic species of Pinaceae found only in south China. The root and trunk bark of P. amabilis, named Cortex Pseudolaricis (Tu-Jin-Pi) in traditional Chinese medicine, have been used traditionally to treat skin diseases caused by fungal infections.14 Increasing evidence demonstrates that some constituents of Cortex Pseudolaricis possess biological activity such as antifungal activity,15,16 anticancer activity,17,18 and peroxisome proliferator-activated receptor activity.19 Ethanol extracts of Cortex Pseudolaricis showed strong in vitro antifungal activity against Rhizoctonia cerealis, the main pathogen causing wheat sharp eyespot.20 However, the Received: Revised: Accepted: Published: 4905
February 25, 2014 May 12, 2014 May 13, 2014 May 13, 2014 dx.doi.org/10.1021/jf500968b | J. Agric. Food Chem. 2014, 62, 4905−4910
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Pseudolaric Acid A (1). White amorphous powder; mp 212−214 °C; 1H NMR (400 MHz, CDCl3) δ ppm: 7.28 (1H, brd, H-15), 6.57 (1H, dd, J = 10.8, 14.8 Hz, H-14), 5.94 (1H, d, J = 14.4 Hz, H-13), 5.58 (1H, m, H-8), 3.49 (1H, d, J = 3.6 Hz, H-3), 2.97(1H, dd, J = 8.4, 14.0 Hz, H-5), 2,46 (2H, d, J = 14.4 Hz, H-9), 2.24 (1H, m, H-5), 2.17 (3H, s, COOCH3), 1.96 (3H, s, H-17), 1.88 (2H, d, J = 6.8 Hz, H6), 1.82 (2H, d, J = 10.4 Hz, H-1), 1.80 (2H, m, H-2), 1.76 (3H, s, H19), 1.60 (3H, s, H-12). 13C NMR (400 MHz, CDCl3) δ(ppm): 173.4 (C-2), 173.3 (C-18), 169.6 (COCH3), 144.9 (C-13), 139.2 (C-8), 138.8 (C-15), 127.6 (C-16), 123.1 (C-7), 121.5 (C-14), 90.6 (C-4), 83.2 (C-11), 55.3 (C-10), 49.0 (C-3), 33.2 (C-1), 30.6 (C-5), 28.3 (C12), 27.2 (C-9), 26.7 (C-6), 26.5 (C-19), 24.3 (C-2), 21.7 ( COCH3), 12.5 (C-17) ; ESI-MS m/z: 389 [M + H]+. Ethyl Pseudolaric Acid B (2). Yellow oily matter; 1H NMR (400 MHz, CDCl3) δ: 7.18 (1H, m, H-8), 7.13 (1H, d, J = 10.8 Hz, H-15), 6.51 (1H, dd, J = 7.2, 11.6 Hz, H-14), 5.85 (1H, d, J = 12.4 Hz, H-13), 4.22 (2H, q, J = 7.2 Hz, 18-OCHH2CH3), 3.72 (3H, s, OCHH3), 3.11 (1H, d, J = 8.4 Hz, H-3), 3.07 (1H, dd, J = 4.4, 11.2 Hz, H-5), 2.91 (1H, dd, J = 6.0, 13.6 Hz, H-6), 2.76 (1H, d, J = 8.8 Hz, H-9), 2.63 (1H, d, J = 15.2 Hz, H-9), 2.14 (3H, s, COOCH3), 2.05 (1H, m, H-6), 1.96 (3H, s, H-17), 1.84−1.71 (5H, m, H-1,2,5), 1.60 (3H, s, H-12), 1.30 (3H, t, J = 8.4 Hz, 18-OCHH2CH3). 13C NMR (400 MHz, CDCl3) δ: 172.8 (C-18), 172.8 (C-20), 169.4(COCH3), 168.0 (C-19), 144.2 (C-13), 141.6 (C-8), 138.4 (C-15), 134.4 (C-7), 127.8 (C-16), 121.6 (C-14), 90.0 (C-4), 83.6 (C-11), 60.6 (18OCH2CH3), 55.1 (C-10), 51.9 (-OCH3), 49.2 (C-3), 33.2 (C-1), 30.6 (C-5), 28.3 (C-12), 27.6 (C-9), 24.2 (C-2), 21.6 (−COCH3), 20.0 (C6), 14.2 (18-OCHH2CH3), 12.5 (C-17) ; ESI-MS m/z: 483 [M + Na]+. Pseudolaric Acid B (3). Yellowish amorphous powder; mp 148−151 °C; 1H NMR (400 MHz, CDCl3) δ: 7.28 (1H, t, J = 15.6 Hz, H-15), 7.22 (1H, d, J = 7.6 Hz, H-8), 6.59 (1H, dd, J = 3.6, 11.6 Hz, H-14), 5.94 (1H, d, J = 15.2 Hz, H-13), 3.72 (3H, s, OCHH3), 3.11 (1H, d, J = 3.6 Hz, H-3), 3.07 (1H, dd, J = 5.6, 8.0 Hz, H-5), 2.87 (1H, dd, J = 6.0, 9.2 Hz, H-6), 2.76 (1H, dd, J = 8.8, 6.0 Hz, H-9), 2.63 (1H, m, H9), 2.14 (3H, s, COOCH3), 2.05 (1H, m, H-6), 1.96 (3H, s, H-17), 1.84−1.71 (5H, m, H-1,2,5), 1.60 (3H, s, H-12). 13C NMR (400 MHz, CDCl3) δ: 172.8 (C-18), 172.8 (C-20), 169.4 (COCH3), 168.0 (C19), 144.2 (C-13), 141.6 (C-8), 138.4 (C-15), 134.4 (C-7), 127.8 (C16), 12.16 (C-14), 90.0 (C-4), 83.6 (C-11), 55.1 (C-10), 51.9 ( OCH3), 49.2 (C-3), 33.2 (C-1), 30.6 (C-5), 28.3 (C-12), 27.6 (C-9), 24.2 (C-2), 21.6 (COCH3), 20.0 (C-6), 12.5 (C-17) ; ESI-MS m/z: 455 [M + Na]+. Pseudolaric Acid BO-β-D-Glucoside (4). White powder; mp 109−113 °C; 1H NMR (400 MHz, CDCl3) δ: 7.31 (1H, t, J = 12.0 Hz, H-15), 7.26 (1H, d, J = 15.2 Hz, H-8), 6.39 (1H, dd, J = 4.0, 11.2 Hz, H-14), 6.06 (1H, d, J = 11.2 Hz, H-13), 4.92 (1H, d, J = 9.6 Hz, anomeric-H), 3.72 (3H, s, OCHH3), 3.25 (1H, d, J = 7.2 Hz, H-3), 3.04 (1H, dd, J = 5.6, 8.0 Hz, H-5), 2.87 (1H, dd, J = 6.0, 9.6 Hz, H-6), 2.74 (1H, dd, J = 9.2, 6.0 Hz, H-9), 2.59 (1H, m, H-9), 2.17 (3H, s, COOCH3), 1.97 (1H, m, H-6), 1.89 (3H, s, H-17), 1.77−1.71 (5H, m, H-1,2,5), 1.57 (3H, s, H-12), 3.47−3.87 (sugar). 13C NMR (400 MHz, CDCl3) δ: 172.8 (C-18), 172.8 (C-20), 169.4 (COCH3), 168.0 (C19), 144.2 (C-13), 141.6 (C-8), 138.4 (C-15), 134.4 (C-7), 127.8 (C16), 121.6 (C-14), 90.0 (C-4), 83.6 (C-11), 55.1 (C-10), 51.9 ( OCH3), 49.2 (C-3), 33.2 (C-1), 30.6 (C-5), 28.3 (C-12), 27.6 (C-9), 24.2 (C-2), 21.6 (COCH3), 20.0 (C-6), 12.5 (C-17); ESI-MS m/z: 595 [M + H]+, 617 [M + Na]+. Piperonylic Acid (5). White powder; mp 196−199 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.7 (1H, s, COOH), 7.63(1H, dd, J = 1.2, 6.8 Hz, H-6), 7.34 (1H, d, J = 1.2 Hz, H-2), 6.97 (1H, d, J = 8.0 Hz, H5), 6.02 (2H, s, OCH2O). 13C NMR (400 MHz, DMSO-d6) δ: 166.7 (COOH), 151.2 (C-4), 147.5 (C-3), 125.0 (C-6), 124.8 (C-1), 108.9 (C-2), 108.1 (C-5), 102.0 (OCH2O); ESI-MS m/z: 167 [M + H]+, 205 [M+K]+. C8H6O4 ; Propionic Acid (6). White crystals ; mp 130−132 °C; 1H NMR (400 MHz, DMSO-d6) δ: 11.9 (1H, s, COOH), 2.16 (2H, t, J = 7.2 Hz, H-2), 1.46 (2H, t, J = 6.8 Hz, H-3), 1.23 (3H, s, H-4). 13C NMR (400 MHz, DMSO-d6) δ: 174.5 (COOH), 33.6 (C-2), 28.5 (C-3), 24.5 (C-4); ESI-MS m/z: 86 [M + H]+.
antifungal effects of Cortex Pseudolaricis on mango anthracnose and other horticultural diseases has not been previously investigated. Our preliminarily experiments showed that ethanol extract from Cortex Pseudolaricis had a significant inhibitory activity against C. gloeosporioides (data not shown). Therefore, we further conducted a phytochemical study on the extract using a bioactivity-guided purification process, isolated and identified the main metabolites, and then evaluated their antifungal activity.
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MATERIALS AND METHODS
General. The Cortex Pseudolaricis was collected in Yinzhou District, Ningbo City, Zhejiang Province of the People’s Republic of China (29°46′6″E, 121°19′5″N) and identified by Associate Professor Jianyin Li from the Pharmacy Institute of Lanzhou University. The commercial fungicide carbendazim (nonformulated analytical grade, 98% purity) (Jiangsu Bailing Agrochemical Co., Ltd., China) was used as a positive control. C. gloeosporioides was obtained from Postharvest Pathology and Preservation Laboratory, Environment and Plant Protection Institute of Chinese Academy of Tropical Agricultural Science. It is carbendazim-sensitive and isolated from mango fruit. Analytical thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were performed with silica gel plates using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd.). Melting points were taken on a Kofler melting point apparatus and are uncorrected. Mass spectra were recorded on a Bruker Daltonics APEXII49e spectrometer with ESI source as ionization. 1H and 13C NMR spectra were recorded at 400 and 100 MHz on a Bruker AM400 spectrometer using TMS as reference (Bruker Company, U.S.A.). Isolation of Pseudolaric Acids. The Cortex Pseudolaricis (20 kg) was ground and percolated with 95% ethanol. After filtration and removal of the solvent, the ethanol extract was dissolved in 5 L of 5% NaHCO3 solution to make a suspension and extracted with ethyl acetate to give a neutral ethyl acetate-soluble fraction. The aqueous solution was then treated with 5% HCl solution to about pH 6 and extracted with ethyl acetate again to afford an acidic ethyl acetatesoluble fraction. The acidic ethyl acetate-soluble fraction was subjected to silica gel column chromatography using a gradient solvent system of petrolumm-ethyl acetate (4:1), and then chloroform−methanol (10:1), and three major fractions, 1−3, were collected. Fraction 1 was then chromatographed sequentially over silica gel column using a solvent system of petroleum−ethyl acetate (4:1) to afford Pseudolaric acid A (1, PAA), a silica gel column petroleum−ethyl acetate (5:1), to afford Ethyl Pseudolaric acid B (2, EPAB). Fraction 2 was recrystallized from petroleumether−ethyl acetate (4:1) to afford pseudolaric acid B (3, PAB) (12.2 g). Fraction 3 was chromatographed sequentially over silica gel column using a solvent system of chloroform−methanol (20:1 to 1:1) to yield pseudolaric acid BOβ-D-glucoside (4, PABG) (2.5 g). Isolation of Other Compounds. The dried Cortex Pseudolaricis (20 kg) was ground and ultrasonically extracted with 95% ethanol. After filtration and removal of the solvent, the ethanol extract was dissolved in 5 L of 5% NaHCO3 solution to make a suspension and immediately extracted with ethyl acetate to give a neutral ethyl acetatesoluble fraction. The aqueous solute was then treated with 5% HCl solution to about pH 2−3 and extracted with ethyl acetate again to afford an acidic ethyl acetate-soluble fraction (177g). The acidic ethyl acetate-soluble fraction was subjected to silica gel column chromatography using a gradient solvent system of petroleum−acetone (5:1− 1:1), and three major fractions 1−3 were collected. The first of these fractions was then chromatographed sequentially over silica gel column using a solvent system of petroleum−acetone (3:1) to afford compound 5. Fraction 2 was chromatographed sequentially over silica gel column using a gradient solvent system of petroleum−chloroform−acetone (5:2:2), and then petroleum−chloroform−acetone (5:1:2), and then petroleum−chloroform (1:1), to afford compounds 6, 7, and 8, respectively. 4906
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3-Hydroxy-4-methoxybenzoic Acid (7). White amorphous powder; mp 155−157 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.6 (1H, s, COOH), 7.54 (1H, dd, J = 1.6, 6.8 Hz, H-6), 7.43 (1H, d, J = 0.8 Hz, H-2), 7.01 (1H, d, J = 8.4 Hz, H-5), 6.10 (1H, d, J = 8.0 Hz, C-3 OH), 3.80 (3H, s, −OCH3). 13C NMR (400 MHz, DMSO-d6) δ: 167.2 (COOH), 152.7 (C-4), 148.4 (C-3), 125.0 (C-6), 123.2 (C-1), 111.9 (C-2), 108.9 (C-5), 55.7 (OCH3); ESI-MS m/z: 169 [M + H]+, 191 [M + Na]+. 4-(3-Formyl-5-methoxyphenyl)butanoic Acid (8). White amorphous powder; mp: 99−102 °C; 1H NMR (400 MHz, DMSO-d6) δ: 12.3 (1H, s, COOH), 9.82 (1H, s, CHO), 7.41 (2H, t, J = 7.2 Hz, H-2, H-6), 6.83 (1H, d, J = 8.8 Hz, H-4), 3.79 (3H, s, OCH3), 3.34 (1H, s, H-9), 2.17 (1H, t, J = 7.6 Hz, H-7), 1.46 (1H, t, J = 6.8 Hz, H-7), 1.23 (2H, m, H-8), 1.15 (1H, dd, J = 6.4, 16.4 Hz, H-9). 13C NMR (400 MHz, DMSO-d6) δ: 174.5 (COOH), 167.3 (CHO), 151.1 (C-5), 147.3 (C-3), 123.5 (C-1), 121.7 (C-6), 115.1 (C-4), 112.7 (C-2), 55.6 (OCH3), 33.7 (C-7), 28.5 (C-9), 24.5 (C-8) ; ESI-MS m/z: 223 [M + H]+, 245[M + Na]+. In Vitro Effect on Mycelial Growth of C. gloeosporioides. The effects of the compounds from Cortex Pseudolaricis on the mycelial growth against C. gloeosporioides were assessed using Poison Food Technique in solid media.21 First, the compounds were dissolved in acetone and water containing Tween-80, then mixed with sterile molten potato dextrose agar to obtain final concentrations ranging from 0.3125 to 40.0 μg/mL. The PDA was poured into 9 cm Petri plates (15 mL) that were then inoculated with 4 mm plugs of C. gloeosporioides. Three replicate plates were used per treatment, and PDA containing a corresponding concentration of acetone in water as a normal growth control. After the fungal growth in the control had completely covered the Petri dishes, mycelial growth diameters were measured and inhibition percentages relative to the control with acetone were calculated using the formula from Agarwal:22
treatment consisted of 3 replicates with 10 fruits per replicate, and the experiment was repeated twice. Scanning Electron Microscopy (SEM). SEM observations on the hyphae of C. gloeosporioides was conducted according to the method described by Feng et al.23 A mycelial disk (4 mm diameter) was taken from the periphery of the colony grown on PDA medium containing 2.5 μg/mL (EC80) of test compounds and incubated for 24−72 h at 28 °C. The youngest C. gloeosporioides hyphae were chosen from the margin of the mycelia, and the samples were routinely fixed in 2.5% glutaraldehyde (prepared previously with 0.1 mol/L phosphate buffer, pH 7.2) at 4 °C overnight, briefly postfixed for 2 h at 4 °C in 1% OsO4 in the same buffer, and then dehydrated in a graded ethanol series, critical point dried, and gold coated. SEM observations were performed with an FEI Quanta 400 Thermal FE Environment Scanning Electron Microscope at an accelerating voltage of 20 kV. Statistical Analyses. Data were presented as means and standard deviations. ANOVA was used to determine whether there were significant (P < 0.05) treatment effects between groups, and the means were compared using the Duncan’s multiple range test. Differences among different treatments were analyzed using SAS version 9.13. Dose−response curves were analyzed using a four-parameter log− logistic model24 using R software (version 2.15.2, R Foundation for Statistical Computing, Vienna, Austria) with the drc module.25 Means and standard deviations were obtained using the raw data, and the EC50 values were obtained from the parameters in the regression curves. The graphs were generated with Sigma Plot (version 11, Systat Software Inc., San Jose, CA, U.S.A.).
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RESULTS Structure Elucidation of the Bioactive Constitutes. Bioassay-guided separation of the components in the ethanol extract of the root bark of Cortex Pseudolaricis afforded eight major bioactive metabolites (Figure 1). All these compounds were purified by column chromatography, and their structures were characterized by 1H NMR, 13C NMR, and ESI-MS data,
I(%) = [(C − d) − (T − d)]/(C − d) × 100 where d is diameter of the cut fungus (4 mm), I is the inhibition (%), and C and T are the average colony diameters of the mycelium of the control and treatment, respectively. Spore Germination Assay. The inhibitory activity of compounds (1−8) on spore germination and germ-tube elongation of C. gloeosporioides were assessed by microscopic observation.20 Spore suspensions (1 × 105 spores/mL) were prepared by seeding conidia in a 0.05% Tween 80 solution. The acetone solution of the test compounds was diluted with conidial suspension to obtain six different final concentrations of 0.5, 1, 5, 10, 20, and 40 μg/mL. Then, 30 μL of these cultures were removed to concave slide and incubated in a humidity chamber at 28 °C and 90% relative humidity. Three replicates were performed and conidial suspension containing a corresponding concentration of acetone in water was regarded as control. After incubation at 28 °C for 12 h, the number of germinated spores and the length of germ tubes were examined by counting and measuring approximately 100 conidia in each field of three randomly selected fields under a biological microscope photographic system at 400× magnification. The experiments were repeated three times. In Vivo Trials on Mango Fruits. Mango fruit (Mangifera indica), cv. “Tai-Nong 1#”at the green mature stage were harvested from “Yangguang Farm” located in Sanya City, Hainan Province of China). The surface of the mango fruit was sterilized by immersion in 70% ethanol for 1 min and thereafter inflicting one wound (1 mm deep) in the middle of each fruit with a sterile needle. Then, 5 μL of spore suspension (104 spores/mL) of C. gloeosporioides were inoculated into wounded site. The inoculated fruit were incubated in a sterile box overnight at 28 °C prior to being dipped in the diluted compounds that were prepared by dissolution in 5 mL of 10% DMSO and then diluted with sterile distilled water to final concentrations of 50 and 100 μg/mL. The compound treatments were compared with carbendazim and 0.5% DMSO treatment that served as a negative control. The treated fruits were stored in a moisture chamber at 28 °C and 80% RH for 9 d. Disease developments were assessed by measuring the diameter of the anthracnose lesions on the mango fruit.9 Each
Figure 1. Chemical structures of compounds 1−8. 4907
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and their structures also were confirmed by direct comparison with previously reported spectroscopic data.26,27 On the basis of the following data, the constituents were identified as pseudolaric acid A (1, PAA), ethyl pseudolaric acid B (2, EPAB), pseudolaric acid B (3, PAB), pseudolaric acid BO-βD-glucoside(4, PABG), piperonylic acid (5), propionic acid (6), 3-hydroxy-4-methoxybenzoic acid (7), and 4-(3-formyl-5methoxyphenyl)butanoic acid (8). Effects of Compounds on Mycelial Growth in Vitro. To evaluate the antifungal activity, the effects of the eight compounds on C. gloeosporioides mycelial growth on solid media were determined (Figure 2). The results showed that the
Table 1. EC50 Values (μg/mL) of Active Components from Cortex Pseudolaricis against Spore Germination and Germ Tube Elongation of C. gloeosporioidesa EC50 (95% FL) treatments Pseudolaric acid A ethyl Pseudolaric acid B Pseudolaric acid B Pseudolaric acid BO-β-D-glucoside piperonylic acid propionic acid 3-hydroxy-4-methoxybenzoic acid 4-(3-formyl-5-methoxyphenyl)butanoic acid carbendazim (98%) a
spore germination
germ tube elongation
5.69 11.42 3.80 23.57 >40 >40 >40 >40
1.14 2.78 0.75 6.35 >40 >40 >40 >40
21.34
2.04
EC50 was determined by probit-log analysis. FL, fiducial limits.
Figure 2. Dose−response curves of the test compounds on inhibition of C. gloeosporioides mycelial growth. ● = pseudolaric acid A; ○ = ethyl pseudolaric acid B; ▼ = pseudolaric acid B; △ = pseudolaric acid-O-βD-glucoside; and ■ = commercial standard carbendazim. Figure 3. Efficacy of active components from Cortex Pseudolaricis in controlling anthracnose disease on mango fruits (Disease incidence was determined 9 days after inoculation). Each bar represents the mean of three independent experiments ± standard deviation. Different letters indicate that the means are significantly different at P < 0.05.
PAB, PAA, EPAB, and PABG had high dose-dependent activities against the C. gloeosporioides mycelial growth with the EC50 value of 1.62, 3.7, 1.07, and 11.30 μg/mL, respectively. In particular, PAB and PAA showed a greater mycelial growth inhibition than that of carbendazim (2.37 μg/mL) (Figure 2). By contrast, compounds piperonylic acid, propionic acid, 3hydroxy-4-methoxybenzoic acid, and 4-(3-formyl-5-methoxyphenyl) butanoic acid showed weaker inhibitory activity, whose mycelial growth inhibitions were all less than 30% at 40 μg/mL. Effects of Compounds on Spore Germination and Germtube Elongation. PAB and PAA exhibited greater inhibition of spore germination and germ tube elongation than did the carbendazim standard (Table 1). PAB had an effective concentration of 50% (EC50) value of of 3.80 μg/mL on spore germination and 0.75 μg/mL on germ tube elongation. Effects of PAB on Control of C. gloeosporioides in Mango Fruit. The effects of four active compounds, including PAB, PAA, EPAB, and PABG, on the postharvest anthracnose of mango fruit were demonstrated in Figure 3. Results indicated that all the compounds tested at 50 and 100 μg/mL significantly suppressed lesion diameter of anthracnose in mango fruit inoculated with spore suspension of C. gloeosporioides. Smaller lesion development was achieved at higher concentrations of test compounds (P < 0.05). PAB treatment showed the greatest efficiency in controlling mango anthracnose, where the lesion diameters were 87.61% and 46.67% lower than those in control and carbendazim-treated fruit.
Changes in the Hyphae Morphology of C. gloeosporioides. To reveal the antifungal mechanism of PAB, the hyphae morphology of C. gloeosporioides treated with 2.4 μg/mL (EC80) PAB was observed by scanning electron microscopy. C. gloeosporioides mycelium grown in PDA with 1% acetone (control) displayed characteristic morphology with lengthened, regular, homogeneous hyphae of constant diameter with smooth external surfaces and rounded apexes (Figure 4A,B). In the PAB treatment at 24 h, the hyphae morphology showed undulations along the hyphal border, a little swelling localized along the hyphae and at their extremities, and the formation of anomalous apex bifurcations (Figure 4C,D). Macroscopic morphologic alterations of C. gloeosporioides were more profound when the PAB treatment time was extended to 48 h. The apexes of C. gloeosporioides treated with PAB showed an irregular growth in dimension, with multiple ramifications in subapical expanded areas with an irregular shape (Figure 4E); several swellings of ovoidal or spherical shape were evident and were localized in the subterminal position near the apexes, or in the middle position along the hyphae (Figure 4F). Seventy-two hours after PAB treatment, hypha were distorted and collapsed (Figure 4G). 4908
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report on the potential agricultural use of these compounds isolated from Cortex Pseudolaricis against C. gloeosporioides in vitro and in vivo. The present study evaluated the in vitro antifungal activity of eight compounds from the ethanol extracts of Cortex Pseudolaricis and their capability in controlling postharvest anthracnose. It was previously reported that pseudolaric acid analogues from Cortex Pseudolaricis were the active components.27−29 Such observation was also supported by our study, in which pseudolaric acid analogues PAA, PAB, EPAB, and PABG, effectively suppressed C. gloeosporioides in mango fruit. PAB showed the strongest efficacy and had greater inhibitory activity than fungicide carbendazim. These results indicate that PAB has great potential to control postharvest mango anthracnose as a natural product based alternative to synthetic fungicides. Currently, natural PAA and PAB can only be obtained from the root bark of P. kaempferi. Recently, PAB was successfully achieved by manual synthesis in laboratory,18 which could provide an ample supply of this compound and introduce a variety of novel structural modifications to the PAB molecule based on synthesis for structure activity relationships. Further studies need to be expanded to other important plant pathogenic fungi, and the indicated SAR need to be conducted on these compounds. Research on the fungicidal mechanism of action for PAB is being carried out. Zhang et al. proposed that the mechanism of PAB could be unique and different from antibiotics with the known mechanisms of amphotericin B, nystatin, ketoconazole, and clotrimazole. It was been reported that PAB resulted in DNA damage and inhibited cell division.30,31 Recently, Wong et al. showed that the antitumor activity of PAB could be associated with its ability to directly interact with tubulin and identified the microtubules as the molecular target of PAB.18 In agricultural fungi, tubulin is one kind of important fungicide target. Both benzimidazole and zoxamide fungicides inhibit phytopathogenic fungus by binding to β-tubulin.32,33 In our present study, PAB showed stronger inhibition of mycelial growth and germ tube elongation than spore germination. SEM images revealed that PAB treatments caused hyphal tip swelling, malformed branches, and swollen, distorted, concave-collapsed hyphae in C. gloeosporioides. Morphological changes induced by PAB are similar to those caused by tubulin inhibitors, such as benzimidazole fungicides.34 Therefore, these findings suggest that PAB may have a similar function in inhibiting mycelial growth and germ-tube elongation in C. gloeosporioides.
Figure 4. Scanning electron micrographs of the hyphae of C. gloeosporioides grown on PDA plates with or without PAB at 28 °C. (A, B) control (1% acetone); (C, D) PAB (24 h); (E, F) PAB (48 h), and (G) PAB (72 h).
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DISCUSSION In recent years, consumer demand for fresh fruits and vegetables without chemical residues has increased.26 Susceptibility of anthracnose disease caused by C. gloeosporioides is one of the most serious problems affecting the mango industry worldwide. As alternatives to synthetic fungicides, the use of natural antifungal compounds isolated from plants to control mango anthracnose disease has been investigated, but few if any satisfactory compounds have been found.9−13 The root bark of P. kaempferi has been successfully used in traditional Chinese medicine as an antifungal remedy and more recently, its major constituents have been isolated and characterized.10,23,27 Although the antifungal activity and possible mechanisms of PAB against medical fungi have been well demonstrated, the effects of PAB and the pseudolaric acid analogues on plant phytopathogenic fungi were not documented. This is the first
AUTHOR INFORMATION
Corresponding Authors
*Phone: +86-898-66969260; fax: +86-898-66969211; e-mail:
[email protected] (Y.Q.L). *Phone: +86-898-66969260; fax: +86-898-66969211; e-mail:
[email protected] (G.F.). Author Contributions #
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (31201538), Natural Science Foundation of Hainan Province of China (310086), Funda4909
dx.doi.org/10.1021/jf500968b | J. Agric. Food Chem. 2014, 62, 4905−4910
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mental Research Fund for Central-level Nonprofit Research Institutes (1630042012002, Young Talents in Tropical Agriculture 2013hzs1J002), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-69).
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