Article pubs.acs.org/JAFC
Isolation, Identification, and Autotoxicity Effect of Allelochemicals from Rhizosphere Soils of Flue-Cured Tobacco Xia Ren,†,‡ Xiaofeng He,‡ Zhongfeng Zhang,§ Zhiqiang Yan,‡ Hui Jin,‡,§ Xiuzhuang Li,‡ and Bo Qin*,‡ †
University of Chinese Academy of Sciences and ‡Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’ Republic of China § Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling and Integrated Management, Institute of Tobacco Research, Chinese Academy of Agricultural Sciences, Qingdao 266101, People’s Republic of China ABSTRACT: Autotoxicity, defined as a deleterious allelopathic effect among individuals of the same plant species, is considered as one of the factors that contributes to replant failure. Tobacco, as an important cultured and economic crop over the world, has been often hampered by replant failure. In view of the seriousness of this problem, the allelochemicals of flue-cured tobacco and their allelopathic effects were investigated. The extracts of rhizosphere soil exhibited phytotoxic activities against Lactuca sativa and autotoxic activities against tobacco itself. Bioassay-guided fractionation of the extract led to the isolation of six compounds, the structures of which were elucidated by spectroscopic analysis. Among them, β-cembrenediol (1), di-n-hexyl phthalate (2), and bis(2-propylheptyl) phthalate (3) showed observably phytotoxic activities against L. sativa seedlings and autotoxic activities on tobacco. The allelochemicals were then verified in the root zone soils of flue-cured tobacco by high-performance liquid chromatography (HPLC). These results provide new insights into the allelopathic mechanisms involved in the replant failure of flue-cured tobacco. KEYWORDS: allelochemicals, flue-cured tobacco, autotoxicity
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INTRODUCTION Flue-cured tobacco (Virginia tobacco), a member of the Solanaceae family, is an important economic crop and has a long cultivated history worldwide. As the main raw material for the cigarette industry, the quality of flue-cured tobacco is of the utmost importance in tobacco production. However, because of land-use efficiency and cultivation habits,1 flue-cured tobacco plants are often hampered by replant failure, resulting in serious problems, such as yield reductions, poor seedling growth, and poor leaf quality.2,3 The problem may be caused by many factors in the soil including the deterioration of soil physicochemical characteristics, nutrient imbalance, soilborne diseases, and autotoxicity.4−6 Autotoxicity, a type of intraspecific allelopathy through the release of compounds (allelochemicals) into the environment by volatilization, root exudation, decomposition, and leaching, influences the growth and development of the plant itself.6−8 Autotoxicity is often thought to be a major reason causing replant problems and has been documented in various plants.9−13 Root exudation includes the secretion of a diverse array of carbon-containing primary metabolites and more complex secondary compounds.14 The root exudates represent the largest direct inputs of plant chemicals into the rhizosphere environment; therefore, they are considered as the largest source of allelochemicals.15 In continuous cropping systems, plant roots repeatedly release the same types of exudates for years, resulting in autotoxicity in many crops.15−17 Previous studies have showed that water and ethanol extracts of tobacco rhizospheric soil significantly inhibited the seed germination of Lactuca sativa and tobacco.18 The composition of root exudates from tobacco was analyzed by GC-MS.19,20 However, low © XXXX American Chemical Society
concentrations and the difficult collection process of root exudates led to these studies being focused on allelopathic potential of tobacco, and the specific allelochemicals isolated from flue-cured tobacco and their autotoxic effect on replant failure have not been reported so far. In light of the serious threat of autotoxic effect on flue-cured tobacco plant, we decided to investigate the allelochemicals of flue-cured tobacco and their allelopathic effects. In this study, the root exudates of flue-cured tobacco were collected from the rhizosphere soil, and the chemicals were isolated and purified to characterize the allelochemicals and evaluate their phytotoxic activity, with the aim of elucidating the continuous cropping obstacle of this plant with autotoxic allelopathy. The results of our research indicated that three of the purified compounds from rhizosphere soil observably showed phytotoxic activities against young seedlings of L. sativa and tobacco. The allelochemicals were then verified in the root zone soil of flue-cured tobacco by high-performance liquid chromatography (HPLC). Our results suggest that flue-cured tobacco exploits allelopathy as one main factor in replant failure and provide new insights into the allelopathic mechanisms involved in fluecured tobacco replant failure. To the best of our knowledge, this is the first report of these allelochemicals and their phytotoxic activities. Received: June 22, 2015 Revised: September 25, 2015 Accepted: September 28, 2015
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DOI: 10.1021/acs.jafc.5b03086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Bioassays. Inhibitory activities on the growth of tobacco and L. sativa seedlings were evaluated using the plate-culture method. The seeds of tobacco and L. sativa were soaked in 10% hypochlorite for 7 min, washed five times by distilled sterile water, and then germinated on filter paper with a photoperiod condition of 16/8 h day/night at 22 °C for 7 and 3 days, respectively. Then, the tested seedlings were transferred to 6-well plates (VWR Scientific, Inc., Shanghai, China), and the extracts of tobacco rhizospheric soil chemicals isolated were diluted with dimethyl sulfoxide (DMSO) with aliquots (10 μL) added to each of the treated wells (10 μL of DMSO was added as control). Then, the plants were incubated in a constant-temperature humidity chamber at 22 °C, under a 16 h light/8 h dark photoperiod (L. sativa seeds for 3 days, tobacco seeds for 7 days). The extracts of tobacco rhizospheric soil were prepared at final concentrations of 400, 200, and 100 μg/mL, and compounds 1−6 were prepared for 200, 100, 50, and 25 μg/mL. Three replicates were performed per treatment. The root length and stem length were measured at the end of the experiment. Inhibitory ratios (%) were determined by the formula [(control length − plant length treated)/control length] × 100. Statistical Analysis. All data were subject to an analysis of variance using SPSS Statistics 18. Each value was expressed as the mean ± standard error (SE). The significant differences in seedling growth between the treatments and control were calculated using a one-way analysis of variance (ANOVA) followed by a Fisher’s least significant difference (LSD) test. Identification and Quantitation of Allelochemicals in the Soil. Identifications of the compounds in the soil of flue-cured tobacco were completed by comparing the retention times to the standards of compounds 1−3. The quantitation of compound 1 in the soil was also done. The standard curves were performed using the linear regression method, and peak areas at 210 nm were plotted versus concentrations. The regression equation of compound 1 was y = 14780525x + 58587 (r2 = 0.9989).
MATERIALS AND METHODS 1
General Experimental Instruments and Procedures. H and13C NMR spectra were performed on a Bruker AM-400BB instrument (Bruker, Karlsruhe, Germany) with TMS as internal standard, operating at 400 and 100 MHz, respectively. EI-MS was measured with a VG ZAB-HS instrument (VG, East Sussex, UK), and ESI-ION trap was measured with an Esquire 6000 instrument (Bruker Daltonics, Billerica, MA, USA). Column chromatography (CC) used Sephadex LH-20 (25−100 mm) (Pharmacia Fine Chemical Co., Ltd., Berlin, Germany), silica gel 60 RP-18 (230−400 mesh) (Merck, Rahway, NJ, USA), and silica gel (200−300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). TLC was performed on precoated silica gel 60 F254 plates (Qingdao Haiyang Chemical Co., Ltd.), and spots were detected by ultraviolet (UV) illumination and sprayed with 5% H2SO4 in C2H5OH (v/v). HPLC Analysis. The HPLC analysis was carried out on a HP1100 system (Agilent, Santa Clara, CA, USA) with a model G1315D diode array, and the column used was a 150 mm × 4.6 mm, 5 μm, GraceSmart Rp C18 (Grace Corp., Deerfield, IL, USA). The solvent of methanol for HPLC analysis was of HPLC gradient grade (Anhui Fulltime Co., Ltd., Hefei, China). Compounds were monitored at 211 and 254 nm, and UV spectra were recorded between 200 and 400 nm. The mobile phase, which was composed of methanol (A) and water (B), was programmed as follows: 0−30 min, A 80%. The flow rate with an injection volume of 20 μL was 0.8 mL/min at a column temperature of 35 °C. Plant Material. The plants were collected from Mengtong of Shandong province, China, in 2014. The tobacco seeds are of Zhongyan 100 and were kindly provided by Tobacco Research Institute of Chinese Academy of Agricultural Sciences (CAAS). The seeds of L. sativa were purchased from a seed company in Shandong. Soil Sample. Soil samples surrounding the root of flue-cured tobacco were collected from Mengtong (the same location where fluecured tobacco was collected) of Shandong province, China, in 2014. Five different soil samples were collected about 15 cm deep around the root of flue-cured tobacco. The collected samples were dried in the dark at room temperature and then stored at 4 °C until they were extracted and analyzed. Soil samples were crushed and then passed through a 1 mm screen sieve to remove root contaminants, with visible root fibers picked out by hand. A sieved sample (100 g) was extracted ultrasonically with methanol three times (30 min each). The filtrate was concentrated in a vacuum to near dryness on a rotary evaporator. Then, the dry residue from the evaporation was redissolved in 100% MeOH and passed through a 0.45 μm nylon membrane filter prior to HPLC analysis. Extraction and Isolation of Allelochemicals from Rhizosphere Soils. The tobacco plants were carefully uprooted, and clump soil was shaken off. The roots with rhizosphere soil were quickly washed in a container with distilled water, and 10 L of filtrate was collected. After clarification filtration, the resultant filtrate was adsorbed by macroporous adsorptive resin (XAD-4), eluting with ethanol. Then the desorbed solution was concentrated in a vacuum to near dryness on a rotary evaporator, yielding the exudates (1.8 g). The exudates was submitted to silica gel CC (25 × 125 cm) with chloroform/EtOAc [1:0 (2 L), 10:1 (3 L), 5:1 (2.5 L), 2:1 (3 L), 1:1 (1 L), 0:1 (2 L)]−methanol to give fractions A−F by means of TLC analysis. Fractions A, D, and E showed significant inhibitory activities in the bioassays against lettuce seedlings. Fraction A was separated by Sephadex LH-20 column (2.5 × 160 cm, 20 mg) (1:1 CHCl3/ CH3OH), then passed through silica gel CC (1.2 × 60 cm, 10 mg), and eluted with petroleum/acetone (60:1, v/v) to produce compound 3. Fraction D was separated by Sephadex LH-20 column (2.5 × 160 cm, 20 mg) (1:1, CHCl3/CH3OH) to produce compound 4, then passed through silica gel CC (1.2 × 60 cm,10 mg), and eluted with petroleum/EtOAc (3:1, v/v) to produce compounds 2 and 5. Fraction E was separated by Sephadex LH-20 column (2.5 × 160 cm, 10 mg) (1:1 CHCl3/CH3OH), then passed through silica gel CC (1.2 × 60 cm, 10 mg), and eluted with petroleum/EtOAc (3:1, v/v) to produce compound 1.
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RESULTS Isolation of Phytotoxic Chemicals from Rhizosphere Soil of Flue-Cured Tobacco. The isolation of compounds from rhizosphere soil of flue-cured tobacco was conducted by a bioassay-directed fractionation approach. The results showed that the crude root exudates from the rhizosphere soil of fluecured tobacco had significant inhibitory effects on seedling growth of tobacco and L. sativa (Figure 1), indicating that there are potential inhibitors in the extracts of rhizosphere soil that affect the growth of tobacco plants themselves and other plant seedlings. The allelopathic effects of rhizosphere soil extracts
Figure 1. Phytotoxic effects of flue-cured tobacco root exudates against lettuce and tobacco seedlings at concentrations of 100, 200, and 400 μg/mL, respectively. Values are presented as a percentage of the mean compared to the control. Means significantly lower than the DMSO controls are indicated with one asterisk (∗) (Dunnett’s one-sided t test; p < 0.05) or two asterisks (∗∗) (p < 0.01). Error bars are one standard error of the mean. N = 3. B
DOI: 10.1021/acs.jafc.5b03086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Structures of the compounds isolated from rhizosphere soil of flue-cured tobacco.
showed dose-dependent alterations. This was in accordance with previous studies.18 It was also found that the extracts showed a stronger inhibitory effect on tobacco than L. sativa and implied remarkable autotoxicity. Six compounds (Figure 2), including β-cembrenediol (1),21 di-n-hexyl phthalate (2),22 bis(2-propylheptyl) phthalate (3),23 ergosta-5-en-3-ol (4),24 1-tetradecene (5),25 and octacosane (6),26 were isolated from rhizosphere soil and characterized by spectroscopic methods. Phytotoxic Activities of the Purified Compounds on L. sativa Seedlings. The bioassay technique is commonly used to identify whether isolated compounds have allelopathic behavior. Compounds 1−6, isolated and purified from the rhizosphere soil of flue-cured tobacco, were evaluated for phytotoxic activities against the seedling growth of L. sativa. Results indicated that compounds 1, 2, and 3 decreased the growth of the tested seedlings at different degrees, and the inhibitory effects of the three compounds showed dosedependent alterations, whereas compounds 4, 5, and 6 had only a slight inhibitory activity (Figure 3). Compound 1 showed a significantly stronger inhibitory activity than the other two compounds at higher concentrations (≤100 μg/mL). Compounds 2 and 3 showed weaker activity than that of compound 1 at low concentrations (≤50 μg/mL), whereas all three compounds were much more effective at high concentrations (≥100 μg/mL). At higher concentrations, the inhibitory effects on the root growth of L. sativa are more significant than those on stem length. The root length of L. sativa seedlings treated with compound 1 at 200 μg/mL was reduced by 88.09% compared to the control, the seedlings showed wilting symptoms, and the roots turned yellow. Compound 1 arrested the growth of the treated plants at higher concentrations, but did not kill them. Identification of Allelochemicals in Soil Samples. To achieve verification of the allelochemicals released from the flue-cured tobacco plant into soil during its growth, HPLC was used to confirm the existence of the allelochemicals in the zone soil by comparing their retention times with those of the standards of compounds 1−3 (Figure 4). With comparison to the chromatogram of the rhizosphere soil, compounds 1, 2, and 3 appeared in the chromatogram of soil samples. It was then verified that compounds 1, 2, and 3 should be released into the soil environment and act as allelochemicals by the plant in the course of its growth and development. The standard curves were obtained by use of the linear regression method, and peak areas at 210 nm were plotted to
Figure 3. Phytotoxic effects of compounds 1−6 on lettuce seedling stem length (A) and root length (B) at concentrations of 25, 50, 100, and 200 μg/mL, respectively. Values are presented as a percentage of the mean compared to the control. Means significantly lower than the DMSO controls are indicated with one asterisk (∗) (Dunnett’s onesided t test; p < 0.05) or two asterisks (∗∗) (p < 0.01). Error bars are one standard error of the mean. N = 3.
calculate concentrations; thus, the concentration of compound 1 in the soil was determined as 2.23 μg/g. Autotoxicity of the Allelochemicals on Flue-Cured Tobacco Seedlings. Results showed that compounds 1, 2, and 3 isolated as the allelochemicals of flue-cured tobacco had remarkable phytotoxicity against tobacco seedlings (Table 1). When tobacco seedlings were treated with compounds 1 and 2 at 200 μg/mL, their root and stem lengths were significantly reduced by 60.26 and 42.31% and by 45.45 and 39.80%, respectively. This indicated that tobacco can release some organic compounds into the surrounding environment with autotoxic allelopathy to the plant itself. Allelochemicals are low-molecular-weight compounds released from living or decomposed plant tissues during growth. C
DOI: 10.1021/acs.jafc.5b03086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. HPLC analysis of the rhizosphere soil and zone soil of flue-cured tobacco. (A) Compounds 1, 2, and 3 were confirmed in the HPLC chromatogram of the rhizosphere soil and had LC retention times of 14.40, 9.49, and 14.26 min, respectively. (B) Compounds 1, and 2, and 3 are shown in the HPLC chromatogram of the zone soil and had LC retention times of 14.10, 9.36, and 14.10 min, respectively.
tobacco. It had been found that some of the terpenoids and esters secreted from plants exhibit strong allelopathic potential at low concentrations.33 Our results are in good accordance with the reports in the literature, but the isolation and determination of these two types of allelochemicals of fluecured tobacco are reported for the first time. Autotoxicity, which is defined as a deleterious allelopathic effect among individuals of the same species, has been documented in various crops.34,35 Autotoxicities in eggplant and peanut had been reported as one possible factor that contributes to replant failure.36,37 Tobacco synthesizes various secondary metabolites during its growth and development; it is known that the natural products are one of the most important factors to evaluate the qualities of tobacco leaf and cigarette. The culture of flue-cured tobacco has often been hampered by replant failure exhibiting autotoxic effects on the seedlings of
Many plants could produce various allelochemicals such as phenols, terpenoids, and alkaloids and inhibit the growth of others. Compound 1 belongs to the terpenoids, which are the second type of allelochemicals, mostly released through volatilization and root exudation into soil to influence the growth of neighboring plant or themselves. Compound 1 is an important surface exudate of tobacco leaf, which had been reported to exhibit various activities, such as antifungal activity27,28 and insecticide activity,29 and might be leached into the soil, so that it is detected in soil samples. Compounds 2 and 3 are phthalate derivatives, which had been detected in root exudates of many crops and inhibit germination and seedling growth.30,31 A prior study had found that esters and terpenoids have strong allelopathic potential in low concentration.32 These two types of allelochemicals might be released into the soil environment through root and/or leaf exudates by flue-cured D
DOI: 10.1021/acs.jafc.5b03086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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100 μg/mL
0.22 ± 0.06e 0.31 ± 0.04d 0.32 ± 0.03c
50 μg/mL
0.42 ± 0.03c 0.45 ± 0.08b 0.48 ± 0.03b
25 μg/mL
root length (cm, mean ± SE)
0.48 ± 0.05b 0.52 ± 0.02a 0.53 ± 0.02a
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AUTHOR INFORMATION
control
0.56 ± 0.01a 0.56 ± 0.01a 0.56 ± 0.01a
200 μg/mL
0.77 ± 0.04d 0.84 ± 0.07d 0.85 ± 0.01d
Corresponding Author
*(B.Q.) Phone: +86-931-4968372. Fax: +86-931-8277088. Email:
[email protected]. Funding
This work was supported by the National Natural Science Foundation of China (No. 31070386, 21302195, and 31300290), 135 key cultivation program of the Chinese Academy of Sciences, and the open project of Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling and Integrated Management, Institute of Tobacco Research, Chinese Academy of Agricultural Sciences (IPM 201404). Notes
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0.99 ± 0.04c 1.04 ± 0.02c 1.03 ± 0.01c
REFERENCES
Different letters in the same row refer to the Duncan test, p < 0.05.
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a
1 2 3
50 μg/mL 25 μg/mL
1.24 ± 0.03b 1.32 ± 0.02b 1.35 ± 0.05a
control
1.41 ± 0.04a 1.41 ± 0.04a 1.41 ± 0.04a
compd
1.12 ± 0.03c 1.29 ± 0.04b 1.21 ± 0.09b
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The authors declare no competing financial interest.
stem length (cm, mean ± SE)
Table 1. Autotoxic Effect of Compounds from Root Exudates of Flue-Cured Tobacco on Tobacco Seedlinga
this plant itself. It is considered that the homogeneity of both allelochemical and chemical compositions of tobacco accumulated in the soil environment is the major reason for allelopathic autotoxicity in the continuous cropping systems, but the specific allelochemicals released into the soil environment responsible for the autotoxic allelopathy have not been clarified until now. In this study, compounds 1−3 isolated from the root exudates of flue-cured tobacco showed observably phytotoxic activities against L. sativa and tobacco plant itself. With the results of bioassay and verification in the root zone soil by HPLC, the three compounds were determined as allelochemicals released into the soil environment by flue-cured tobacco and inhibiting its own growth. The results suggest that fluecured tobacco exploits allelopathy as one main factor in replant failure. It is interesting to note that compound 1 has a strong inhibitory effect on L. sativa as well, and the treated Lactuca seedlings showed wilting symptoms at a concentration of 200 μg/mL. Even though the potential mechanism of action needs further study to be elucidated, our findings suggest that the high levels of compound 1 in tobacco may be utilized in many applications.
0.37 ± 0.07d 0.41 ± 0.06c 0.42 ± 0.05b
200 μg/mL
Journal of Agricultural and Food Chemistry
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