Identification and Phytotoxicity of a New Glucosinolate Breakdown

Jul 6, 2014 - Its identity was confirmed by LC-UV and high resolution LC-MS/MS ... of Meadowfoam (Limnanthes alba) Seed Meal for Weed Management...
0 downloads 0 Views 729KB Size
Download PDF
Article pubs.acs.org/JAFC

Identification and Phytotoxicity of a New Glucosinolate Breakdown Product from Meadowfoam (Limnanthes alba) Seed Meal Suphannika Intanon,*,† Ralph L. Reed,‡ Jan F. Stevens,‡ Andrew G. Hulting,† and Carol A. Mallory-Smith† †

Department of Crop and Soil Science, Oregon State University, 109 Crop Science Building, Corvallis, Oregon 97331, United States Department of Pharmaceutical Sciences, Oregon State University, 203 Pharmacy Building, Corvallis, Oregon 97331, United States

‡

S Supporting Information *

ABSTRACT: Meadowfoam (Limnanthes alba Hartw. ex Benth.) is an oilseed crop grown in the Willamette Valley of Oregon. Meadowfoam seed meal (MSM), a byproduct after oil extraction, contains 2−4% glucosinolate (glucolimnanthin). Activated MSM, produced by adding freshly ground myrosinase-active meadowfoam seeds to MSM, facilitates myrosinase-mediated formation of glucosinolate-derived degradation products with herbicidal activity. In the activated MSM, glucolimnanthin was converted into 3-methoxybenzyl isothiocyanate (“isothiocyanate”) within 24 h and was degraded by day three. 3Methoxyphenylacetonitrile (“nitrile”) persisted for at least 6 days. Methoxyphenylacetic acid (MPAA), a previously unknown metabolite of glucolimnanthin, appeared at day three. Its identity was confirmed by LC-UV and high resolution LC-MS/MS comparisons with a standard of MPAA. Isothiocyanate inhibited lettuce germination 8.5- and 14.4-fold more effectively than MPAA and nitrile, respectively. Activated MSM inhibited lettuce germination by 58% and growth by 72% compared with the control. Results of the study suggest that MSM has potential uses as a pre-emergence bioherbicide. KEYWORDS: glucolimnanthin, breakdown products, soil amendment, methoxyphenylacetic acid

■

INTRODUCTION

Myrosinase is present in leaf, stem, root, and seed of glucosinolate-containing plants and is separated from vacuoles containing glucosinolates.12−14 Enzymes with myrosinase-like activity are also present in soil microorganisms.15−17 Even though myrosinase is active in other Brassicaceae seed meals after the cold crushing procedure,18,19 it is rarely detected in MSM because of heating to denaturing temperature during the oil extraction.20 Activation by adding myrosinase from freshly ground meadowfoam seeds to MSM results in the quantitative conversion of glucolimnanthin 1 to GBPs.11 MSM has potential utility in agriculture as a soil amendment to enhance plant growth,20 suppress weeds,11,21−23 and inhibit soil pests such as nematodes24 and insects.25 It has been reported to suppress downy brome (Bromus tectorum L.)11,21 and velvetleaf (Abutilon theophrasti Medik.).22 The phytotoxic activity of GBPs in MSM applied followed by soil incorporation depends on various factors including soil pH, soil temperature, glucosinolate concentration in seed meal, and the presence of myrosinase enzyme. Because GBPs have greater herbicidal activity than the parent glucosinolate,11 we tested the hypothesis that activated MSM exerts greater herbicidal activity than myrosinase-inactivated MSM after soil incorporation. To gain a further understanding of MSM building on an earlier study,11 we investigated temporal changes in biodegradation of glucolimnanthin 1 and GBPs under soil conditions. In the process, we identified

Meadowfoam (Limnanthes alba Hartw. ex Benth.) is a species in the Limnanthaceae family, order Brassicales, which is native to southern Oregon and northern California.1 Meadowfoam is grown as an oilseed crop in Oregon. Meadowfoam oil is 98% unsaturated and is rich in long-chain 20:1, 22:1, and 22:2 fatty acids.2 The oil extracted from meadowfoam seed possesses a unique oxidative stability that makes it useful for a wide range of products including cosmetics, lubricants, rubber additives, plastics, and biodiesel.3−5 About 70% of the biomass of harvested seed remains following meadowfoam oil extraction. This byproduct, known as meadowfoam seed meal (MSM), has limited commercial uses. Finding additional uses for the seed meal would make the crop more economical to produce. The principle plant secondary metabolite in meadowfoam is a glucosinolate, glucolimnanthin 1 (Figure 1). MSM contains 2−4% glucolimnanthin 1,6 which is one of more than 130 identified glucosinolates.7,8 Other glucosinolates are predominately found in Brassicaceous plants, and more than one type of glucosinolate is commonly found in Brassicaceae tissues.9 Glucosinolate structures consist of β-thioglucoside N-hydroxysulfates with a side chain and a sulfur-linked β-D-glucopyranose moiety.7 Glucosinolate variations are based on differentiation of the side chains. The side chain in glucolimnanthin 1 is an aromatic ring of 3-methoxybenzyl. When plant cells are ruptured, the glucosinolates in the vacuoles are converted by myrosinase into glucosinolate breakdown products (GBPs) via hydrolysis.10 3-Methoxyphenylacetonitrile (nitrile 2) and 3methoxybenzyl isothiocyanate (isothiocyanate 3) were detected in relatively greater amounts than other GBPs.11 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7423

January 6, 2014 July 1, 2014 July 6, 2014 July 6, 2014 dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429

Journal of Agricultural and Food Chemistry

Article

Figure 1. Production of glucolimnanthin breakdown products in soil amendment with meadowfoam seed meal. in acetonitrile and separated on a 250 mm × 4.6 mm Phenomenex Luna 5 μm C18 column (Phenomenex, Torrance, CA) eluted with a linear gradient of 35−40% of 0.1% formic acid in acetonitrile and aqueous 0.1% formic acid in H2O at 1 mL/min. The eluted compound 4 was identified using LC-UV at λ 274 nm (SPD-10Avp UV−vis detector, Shimadzu Scientific Instruments, Columbia, MD) and high resolution LC-MS/MS (Triple TOF 5600, AB SCIEX, Framingham, MA). Mass spectrometric analysis utilized negative full scan and product ion scan modes. A DuoSpray source was operated at 550 °C and −4.5 kV using a declustering potential of −80 V and a collision energy of 20 eV. The commercial standard was analyzed using LC-UV and high resolution LC-MS/MS under the same conditions as the suspension extract. The retention times and mass spectra of the standard and compound 4 were compared. Glucolimnanthin and Its Breakdown Products in Soil. Soil incubations were conducted in 15 mL centrifuge tubes using a completely randomized design with three replications. Dry soil (1.94 g) was incubated with either 0.06 g of MSM or activated MSM in a tube. After addition of 750 μL of deionized H2O to thoroughly hydrate the seed meal amended soil, the tubes were laid horizontally. Nonamended soil was used as a control. The extraction method for glucolimnanthin 1 and its breakdown products from the soil was developed by modifying the method of Stevens et al.11 Each incubated soil tube received 6 mL of 70% MeOH. The tube was shaken, sonicated for 10 min, and allowed to stand for 60 min. The mixture was centrifuged for 5 min at 3000 rpm. The supernatant was centrifuged for 10 min at 13 000 rpm. The MeOH concentration in the supernatant was increased to 90% to prevent further enzymatic degradation of glucolimnanthin 1. The analyses of glucolimnanthin 1 and GBPs were performed using HPLC.11 The injection volume was 30 μL. A Waters 2996 photodiode array detector (Waters Corporation, Milford, MA) at λ 274 nm was used to calculate peak areas for all compounds. Analyte concentrations were determined from calibration curves constructed for each analyte using the external standard method. The glucolimnanthin 1 and GBPs were quantified on 0, 1, 2, 3, 6, 12, and 18 DAI. The experiment was repeated. Petri Plate Bioassay. In a comparative assay of relative toxicity, each GBP was used to test the response of lettuce germination and growth. A 10 cm diameter Petri plate (VWR International, Visalia, CA) served as a bioassay study chamber. Test compounds were dissolved in EtOH and diluted in various concentrations. Test solutions (480 μL) were added on top of an 8.3 cm diameter germination blotter paper. Based on preliminary testing, the following concentrations were chosen for dose−response study of the test compounds. Concentrations of test compounds were adjusted by considering the potential losses during the evaporative period. Nitrile 2 solutions were prepared to give 0, 0.38, 3.77, 7.54, 15.1, 22.6, 30.1, 37.7, and 75.4 μmol/plate. Isothiocyanate 3 was prepared to deliver 0, 0.033, 0.33, 0.67, 1.33, 2, 2.66, and 3.33 μmol/plate, and MPAA 4 was prepared at 0, 0.048, 0.48, 4.8, 9.6, 19.2, 28.8, 38.4, and 48 μmol/plate.

another glucolimnanthin 1-related compound and evaluated the phytotoxicity of soil amended with either nonactivated or activated MSM on seed emergence and growth.

■

MATERIALS AND METHODS

Chemicals. Glucolimnanthin 1 (1-S-(2-(3-methoxyphenyl)-N(sulfonatooxy)ethanimidoyl)-1-thio-β-D-glucopyranose) was extracted from MSM.11 3-Methoxyphenylacetonitrile (nitrile 2) (Sigma-Aldrich Chemicals, St. Louis, MO), 3-methoxybenzyl isothiocyanate (isothiocyanate 3) (Oakwood Products, West Columbia, SC), and 3methoxyphenylacetic acid (MPAA 4) (TCI America, Portland, OR) were purchased from the indicated commercial sources. Materials. MSM (Natural Plant Products, Salem, OR) was processed using a coffee grinder (Proctor Silex E160B, Southern Pines, NC) and sieved through 1 mm mesh before use. MSM was used at 3:100 MSM/dry soil. Activated MSM was produced by adding 1:100 freshly ground meadowfoam seed/MSM to provide active myrosinase.11 Nonactivated MSM contained only ground MSM. Soil was collected at 0−20 cm in 2010 from a site near Sweet Home, Oregon, USA (44°25′5″ N, 122°42′43″ W), where no herbicide application had been made for at least 4 years. The soil was a Newburg sandy loam (coarse-loamy, mesic Fluventic Haploxerolls) with an organic matter content of 3.2% and pH of 6.1. The soil was ground, passed through a 2 mm sieve, air-dried for 7 days, and kept in a closed container until use. Isolation and Identification of a Compound 4. Compound 4 was detected in soil amended with MSM and activated MSM and was not found in the untreated control during the analyses of glucolimnanthin 1 and its breakdown products. Isolation and identification of compound 4 were performed using the following procedures. Soil amended with 3% by weight of MSM was mixed thoroughly in an 11 cm × 11 cm × 3.5 cm plastic box, and H2O was added to reach 30% moisture content. The MSM amended soil was incubated in the sealed box at room temperature for 4 days before extraction. The moist soil was transferred into a glass centrifuge tube, and CH2Cl2 was added (1.5 mL/g of moist soil). The tube was shaken, sonicated for 3 min, and allowed to stand for 15 min. The supernatant was transferred to a glass tube, and the extraction was repeated for two more cycles. The supernatants were combined and then dried using a rotary evaporator. The resultant residue was redissolved in MeOH (2 mL/g dry soil). The methanolic resuspension was extracted with hexane three times to remove nonpolar compounds. The remaining MeOH suspension was evaporated using a rotary evaporator, and resuspended in MeOH before purification using a 2.5 cm × 50 cm column of Sephadex LH-20 (Sigma-Aldrich Chemicals, St. Louis, MO) eluted with 100% degassed MeOH at a flow rate of 1.6 mL/min. Elution was monitored using high performance liquid chromatography (HPLC) as previously described.11 The fractions containing compound 4 were collected and concentrated using a rotary evaporator. The fractions were redissolved 7424

dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429

Journal of Agricultural and Food Chemistry

Article

Control plates received EtOH only. Plates were placed in a fume hood for a 3-h evaporative period to remove EtOH. After 3 h, each plate received 6 mL of deionized H2O. Sixteen lettuce seeds (Lactuca sativa L. “Black Seeded Simpson”, Plantation Products, Norton, MA) were placed in a 4 × 4 grid on top of a germination blotter paper. The Petri plate was sealed with a layer of Parafilm (American National Can, Menasha, WI). Lettuce seeds were grown in the incubator at 20/15 °C day/night temperature with a 14 h photoperiod. Seeds were counted as germinated when the hypocotyl, radicle, or hypocotyl plus radicle measured 2 mm, and seedling growth was evaluated by measuring radicle and hypocotyl length on day 7. The experiment was performed in a completely randomized design with three replications of each test compound and was repeated. Phytotoxicity of Meadowfoam Seed Meal. Filter paper was placed at the bottom of a 132 mL pot to prevent soil loss. Pots were filled with 116.4 g of soil and amended with 3.6 g of either MSM or activated MSM. The untreated control was 120 g of soil without amendment. Lettuce seeds were sown nine seeds per pot at an approximate depth of 0.4 cm on 0, 6, and 12 days after MSM incorporation (DAI). Each pot was watered daily. Soil moisture in each pot was maintained at 37.5% by weighing the pots. The experiments were conducted in the greenhouse with 25/20 °C day/ night temperature and a 14 h photoperiod. Seedling emergence was counted, and shoot biomass was harvested 21 days after planting. The biomass was dried at 60 °C for 72 h and weighed. The experiment was structured in a randomized complete block design with three replications and was repeated. Statistical Analyses. Concentrations of glucolimnanthin 1 and GBPs and lettuce germination, emergence, hypocotyl and radicle growth, and biomass were analyzed using analysis of variance (ANOVA) with means separated using a least significant difference (LSD) test at a 0.05 level. For the phytotoxicity of GBPs in the lettuce germination study, dose−response curves were obtained by a nonlinear regression using a log−logistic equation (eq 1),26,27

y = C + (D − C)/[1 + (x /I50)b ]

Figure 2. Time-of-flight (TOF) mass spectrum of 3-methoxyphenylacetic acid (MPAA) produced from soil amended with 3% by weight of meadowfoam seed meal, showing the molecular ion (observed m/z 165.0559, calculated m/z 165.0552, 4.2 ppm error). The inset shows an MS−MS fragmentation of the parent peak at m/z 165.

(1)

where y represents germination (percent of untreated control) at GBP concentration x, C is the mean response at the greatest GBP concentration (lower limit), D is the mean response when the GBP concentration is zero (upper limit), b is the slope of the line at I50, and I50 is the GBP concentration required for 50% germination reduction. The regression parameters, 95% confidence interval for each GBP, and lack of fit test were obtained using the package drc in the statistical program R.28 The relative I50 level was calculated by the ratio of one GBP to another GBP. The statistical program R v. 3.0.2 was used for all the statistical analyses.29

■

RESULTS AND DISCUSSION Glucolimnanthin 1 and Its Breakdown Products. The analytical data of the composition of MSM and activated MSM are provided in Supporting Information, Figure S1. When MSM was incubated with soil, a previously unknown metabolite 4 of glucolimnanthin 1 was observed (Figure 1). Its identity was confirmed by LC-UV and by high resolution LC-MS/MS with a 4.2 ppm error compared with the exact mass of MPAA 4 (Figure 2). The observed spectrum of an MPAA 4 standard had 2.4 ppm error compared with the calculated mass of MPAA 4 (Supporting Information, Figure S2). Glucolimnanthin 1 was converted into isothiocyanate 3 by active myrosinase from freshly ground meadowfoam seed (Figure 3B). The production of isothiocyanate 3 occurred quickly within 30 min after soil incorporation with activated MSM. Maximal concentrations of isothiocyanate 3 were observed at 1 DAI, after which they decreased >90% of the maximum by 2 DAI. Within 6 days, glucolimnanthin 1 in MSM (Figure 3A) degraded slower to trace amounts compared with

Figure 3. Concentrations of glucolimnanthin and its breakdown products in soil amended with meadowfoam seed meal (A) and activated meadowfoam seed meal (B). On day 0, the extraction started 30 min after meal incorporation. Symbols and bars represent means and standard errors of two studies (n = 6).

activated MSM (Figure 3B), and isothiocyanate 3 was not detected. Isothiocyanate 3 was the major degradation product and was produced rapidly. Similar findings were observed in soil incorporation of Brassicaceous plants. Brown et al.30 7425

dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429

Journal of Agricultural and Food Chemistry

Article

reported the isothiocyanate degradation from soil amended with rapeseed (Brassica napus L.) meal, which was detected in 2 h but decreased to trace amounts at 2 DAI. The maximum concentrations of detected meadowfoam isothiocyanate 3 were similar to allyl isothiocyanate from sinigrin decomposition in soil.31 Allyl isothiocyanate reached maximum levels within 1 DAI and remained detectable until 7 DAI. Morra and Kirkegaard32 suggested that greater isothiocyanate production and its retention could occur by disruption of glucosinolatecontaining tissue to provide greater contact between glucosinolate-containing tissue and myrosinase. However, isothiocyanate disappeared rapidly, possibly because it easily binds with free amino acids and proteins.13 Nitrile 2 occurs in MSM because of the heat involved in the commercial process, but it is minimally produced by enzymatic degradation of glucolimnanthin 1.11 From 0 to 3 DAI, nitrile 2 concentrations with MSM or activated MSM were not different (p > 0.05) (Figure 3). Nitrile 2 concentrations decreased from 3 to 18 DAI in MSM and from 3 to 12 DAI in activated MSM. Vaughn et al.22 reported that the large amount of nitrile 2 in MSM was possibly a driving factor for its phytotoxicity. MPAA 4 appeared on 2 DAI of MSM and 3 DAI of activated MSM (Figure 3). While nitrile 2 decreased after 3 DAI, MPAA 4 increased. MPAA 4 occurred last and remained detectable at 12 DAI. Soil was amended with nitrile 2 alone to determine whether nitrile 2 was the parent compound of MPAA 4. The metabolite mass was consistent with the calculated mass of standard MPAA 4 (mass error 4.8 ppm; Supporting Information, Figure S3). A possible pathway for MPAA 4 formation from glucolimnanthin 1 via nitrile 2 involves hydrolytic conversion of nitrile 2 to its corresponding amide followed by hydrolysis of the amide into its corresponding carboxylic acid, MPAA 4 (Figure 1). However, MPAA 4 was detected when soil was incubated with either MSM or activated MSM within 3 DAI, when nitrile 2 concentration had not decreased. A potential explanation is that the MPAA 4 formation may not have been only from nitrile 2 degradation but also derived from glucolimnanthin 1 through nitrile 2. MPAA 4 was not detected in incubation of MSM in the absence of soil. It was also possible that MPAA 4 was not present as a simultaneous metabolite but occurred through enzymatic activity mediated by soil microbes. MPAA 4 production may have been facilitated by nitrilases from soil microorganisms, which are known to catalyze nitriles into their corresponding carboxylic acids.33 Petri Plate Bioassay. Herbicidal efficacy of each GBP was of interest. Commercially available GBPs were used to evaluate their phytotoxicity. The required concentrations of GBPs for suppression of seed germination and growth are important for developing MSM as a bioherbicide. Nitrile 2, isothiocyanate 3, and MPAA 4 showed potent inhibition of lettuce germination and growth (Figures 4 and 5). Isothiocyanate 3 had the most effective herbicidal activity followed by MPAA 4 and nitrile 2. The amounts required to reduce germination by 50% (I50) were 1.4 μmol/plate for isothiocyanate 3, 11.9 μmol/plate for MPAA 4, and 20.2 μmol/plate for nitrile 2. I50 values for the GBPs were different from each other (p < 0.05). The relative potency of isothiocyanate 3 was 8.5- and 14.4-fold greater than MPAA 4 and nitrile 2, respectively. The relative potency of MPAA 4 was 1.7-fold greater than nitrile 2. Both hypocotyl and radicle length decreased with increasing concentrations of GBPs (Figure 5). Isothiocyanate 3 provided the greatest inhibition of hypocotyl and radicle length followed by MPAA 4 and nitrile 2,

Figure 4. Phytotoxicity of glucolimnanthin breakdown products on lettuce germination. Symbols represent means of samples of two studies (n = 6).

Figure 5. Phytotoxicity of glucolimnanthin breakdown products on lettuce hypocotyl length (A) and radicle length (B). Symbols and bars represent means and standard errors of samples of two studies (n = 6).

respectively (p < 0.05), which was the same as for germination inhibition. Several published studies of Brassicaceae glucosinolates9,32 and meadowfoam glucolimnanthin 1 23−25 have shown 7426

dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429

Journal of Agricultural and Food Chemistry

Article

seed meal incorporation so waiting before planting into MSM treatments was necessary to prevent crop injury. A study conducted by Rice et al.18 suggested that lettuce sown into 3% white mustard seed meal-amended soil required approximately 35 DAI for crop safety. The length of phytotoxicity varied depending on glucosinolate-containing species, application methods, and environmental conditions such as soil moisture and soil temperature.37 On the 12 DAI planting date, there was no difference in lettuce emergence but there was a difference in lettuce biomass. The lettuce biomass was greater in MSM compared with untreated control and activated MSM. These results were possibly due to the protein and fiber content in MSM,6 which may be utilized as N and C sources by soil microorganisms. Similar fertilizer effects were found after other Brassicaceae seed meal applications.18 However, the reduced lettuce biomass in activated MSM compared with MSM was potentially due to greater phytotoxicity and nonselective inhibition of soil microorganisms. When the concentration of the remaining phytotoxic compounds was no longer toxic to lettuce seeds, it still may have inhibited soil microbial activity. Snyder et al.38 suggested the mixed effects of different glucosinolate-containing Brassicaceae seed meals on plants and soil microbes involved with the N cycle. The soil microbes may not have completely recovered and utilized the C and N sources provided by the activated MSM application. Intanon37 reported that between 0 and 7 days after activated MSM incorporation, microbial biomass C and N increased. Microorganisms immobilized C and N sources, and then C and N became available to plants during microbial turnover.39 The harvest time at 21 days after seeding may not be long enough to observe the fertilizer effect from the activated MSM application.37 The low number of emerged lettuce seedlings in all treatments on the 0 DAI planting date possibly was due to seeds being buried during the initial irrigation. When seeds were planted 6 and 12 days later, the soil was more settled so seeds were not buried as deep. Other possible reasons were a CO2 flush or microbial respiration. Andalo et al.40 suggested that the response on seed germination to elevated CO2 as a result of wetting dry soil is species specific. Lettuce seed germination was inhibited with increasing the concentration of CO2 to 6.4% and 12.8%.41 The increase of CO2 concentration during germination had no effect on seed germination in mouse-ear cress (Arabidopsis thaliana (L.) Heynh.).40 After wetting of the soil, the moisture can induce metabolic processes such as respiration by seeds and microbes. During the respiration process, heat was released, which possibly reduced germination.42 Although isothiocyanate 3 was shown to have the greatest toxicity, its presence in the soil amended with activated MSM was very short-lived (Figure 3B). The rapid degradation of this bioactive compound in the soil should allow the use of activated MSM for weed control preplant. After the loss of MSM phytotoxicity, subsequent fertilizer effects were observed. Thus, planting crops in the field at least 6 days after MSM application may protect the crop from allelochemicals and increase competitiveness against emerging weeds. The results of this study improve the current knowledge that activated MSM has greater herbicidal activity compared with nonactivated MSM. The temporal change in biodegradation of glucolimnanthin 1 and GBPs under soil conditions were evaluated. In addition, the newly identified metabolite, MPAA 4, was discovered as a breakdown product of glucolimnanthin

isothiocyanates to be the major phytotoxic metabolites. Vaughn et al.22 reported that meadowfoam isothiocyanate 3 had phytotoxic effects on velvetleaf and wheat (Triticum aestivum L.) radicle growth, but they were unable to detect isothiocyanate 3 in nonactivated MSM. However, Stevens et al.11 reported that meadowfoam nitrile 2 had greater activity than the isothiocyanate 3 in preventing coleoptile emergence of downy brome. The difference between the studies may have been due to experimental conditions and target species. In our study, isothiocyanate 3 had the greatest phytotoxic effects on lettuce germination and growth. It is possible that phytotoxicity does not depend on a single chemical compound. A combination of all GBPs may result in phytotoxicity. A synergistic or antagonistic response of GBP combinations for herbicidal activity may exist. Phytotoxicity of Meadowfoam Seed Meal in Soil. Greenhouse studies were conducted to evaluate the direct use of MSM as a soil amendment material for inhibition of seed emergence and growth. Lettuce emergence and growth were different among amendment materials and planting times (Table 1). Activated MSM had greater phytotoxicity on lettuce Table 1. Greenhouse Studies on Lettuce Emergence and Growth in Soil Amended with 3% by Weight of Meadowfoam Seed Meal (MSM) or Activated MSM treatments

lettuce emergencea (% of sown seeds)

unamended MSM activated MSMc

57.4 ± 50.0 ± 25.9 ±

unamended MSM activated MSMc

79.6 ± 70.4 ± 77.8 ±

unamended MSM activated MSMc

81.5 ± 77.8 ± 75.9 ±

lettuce biomass (mg/plant)

Planted on 0 DAIb 5.30 a 9.7 10.24 a 4.1 4.68 b 2.7 Planted on 6 DAI 5.30 nsd 9.4 9.37 13.9 2.87 8.5 Planted on 12 DAI 8.45 nsd 10.0 7.03 19.3 3.41 10.9

± 1.30 a ± 0.87 b ± 1.00 b ± 0.89 nsd ± 3.29 ± 2.61 ± 0.65 b ± 2.44 a ± 1.83 b

a

Data are from two experiments, with each value representing the mean of six replicates ± the standard error of the mean. Different letters within a column indicate significant differences at the 0.05 level within a planting date. bPlanting dates after meal incorporation (DAI). c 1:100 freshly ground meadowfoam seed/total meadowfoam seed meal. dNot significant.

emergence than MSM when lettuce seeds were sown on 0 DAI (p < 0.05). Lettuce emergence was reduced by 55% in activated MSM compared with the untreated control. On the 0 DAI planting date, the lettuce seedling biomass was reduced by 58% in MSM and 72% in activated MSM treatments compared with the untreated control. Adding active myrosinase from freshly ground meadowfoam seed to MSM increased phytotoxicity and suppression of lettuce growth. Similar results were reported in another study where activated MSM with 18-h incubation or addition of iron provided greater inhibition on coleoptile emergence of downy brome compared with MSM.11 Other glucosinolate-containing seed meals such as brown mustard (Brassica juncea (L.) Czern.)18,34,35 and white mustard (Sinapis alba L.)35,36 have been reported to have potential uses as bioherbicides without requiring activation. No differences in lettuce emergence and biomass were found among treatments when lettuce seeds were planted 6 days after 7427

dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429

Journal of Agricultural and Food Chemistry

Article

(17) Sakorn, P.; Rakariyatham, N.; Niamsup, H.; Nongkunsarn, P. Rapid detection of myrosinase-producing fungi: A plate method based on opaque barium sulphate formation. World J. Microbiol. Biotechnol. 2002, 18, 73−74. (18) Rice, A. R.; Johnson-Maynard, J. L.; Thill, D. C.; Morra, M. J. Vegetable crop emergence and weed control following amendment with different Brassicaceae seed meals. Renew. Agric. Food Syst. 2007, 22, 204−212. (19) Borek, V.; Morra, M. J. Ionic thiocyanate (SCN−) production from 4-hydroxybenzyl glucosinolate contained in Sinapis alba seed meal. J. Agric. Food Chem. 2005, 53, 8650−8654. (20) Linderman, R. G.; Davis, E. A.; Masters, C. J. Response of conifer seedlings to potting medium amendment with meadowfoam seed meal. In Issues in New Crops and New Uses; Janick, J., Whipkey, A., Eds.; ASHS Press: Alexandria, VA, 2007; pp 138−142. (21) Machado, S. Allelopathic potential of various plant species on downy brome: Implications for weed control in wheat production. Agron. J. 2007, 99, 127−132. (22) Vaughn, S. F.; Boydston, R. A.; Mallory-Smith, C. A. Isolation and identification of (3-methoxyphenyl)acetonitrile as a phytotoxin from meadowfoam (Limnanthes alba) seedmeal. J. Chem. Ecol. 1996, 22, 1939−1949. (23) Vaughn, S. F.; Palmquist, D. E.; Duval, S. M.; Berhow, M. A. Herbicidal activity of glucosinolate-containing seedmeals. Weed Sci. 2006, 54, 743−748. (24) Zasada, I. A.; Weiland, J. E.; Reed, R. L.; Stevens, J. F. Activity of meadowfoam (Limnanthes alba) seed meal glucolimnanthin degradation products against soilborne pathogens. J. Agric. Food Chem. 2012, 60, 339−345. (25) Bartelt, R. J.; Mikolajczak, K. L. Toxicity of compounds derived from Limnanthes alba seed to fall armyworm (Lepidoptera: Noctuidae) and European corn borer (Lepidoptera: Pyralidae) larvae. J. Econ. Entomol. 1989, 82, 1054−1060. (26) Seefeldt, S. S.; Jensen, J. E.; Fuerst, E. P. Log-logistic analysis of herbicide dose-response relationships. Weed Technol. 1995, 9, 218− 227. (27) Streibig, J. C.; Rudemo, M.; Jensen, J. E. Dose-response curves and statistical models. In Herbicide Bioassay; Streibig, J. C., Kudsk, P., Eds.; CRC Press: Boca Raton, FL, 1993; pp 29−53. (28) Knezevic, S. Z.; Streibig, J. C.; Ritz, C. Utilizing R software package for dose-response studies: the concept and data analysis. Weed Technol. 2007, 21, 840−848. (29) R Core Team R: A language and environment for statistical computing; R Foundation for Statistical Computing: Vienna, Austria, 2013. (30) Brown, P. D.; Morra, M. J.; McCaffrey, J. P.; Auld, D. L.; Williams, L. Allelochemicals produced during glucosinolate degradation in soil. J. Chem. Ecol. 1991, 17, 2021−2034. (31) Borek, V.; Morra, M. J.; Brown, P. D.; McCaffrey, J. P. Allelochemicals produced during sinigrin decomposition in soil. J. Agric. Food Chem. 1994, 42, 1030−1034. (32) Morra, M. J.; Kirkegaard, J. A. Isothiocyanate release from soilincorporated Brassica tissues. Soil Biol. Biochem. 2002, 34, 1683−1690. (33) O’Reilly, C.; Turner, P. D. The nitrilase family of CN hydrolysing enzymes − a comparative study. J. Appl. Microbiol. 2003, 95, 1161−1174. (34) Earlywine, D. T.; Smeda, R. J.; Teuton, T. C.; Sams, C. E.; Xiong, X. Evaluation of oriental mustard (Brassica juncea) seed meal for weed suppression in turf. Weed Technol. 2010, 24, 440−445. (35) Handiseni, M.; Brown, J.; Zemetra, R.; Mazzola, M. Herbicidal activity of Brassicaceae seed meal on wild oat (Avena fatua), Italian ryegrass (Lolium multif lorum), redroot pigweed (Amaranthus retrof lexus), and prickly lettuce (Lactuca serriola). Weed Technol. 2011, 25, 127−134. (36) Hansson, D.; Morra, M. J.; Borek, V.; Snyder, A. J.; JohnsonMaynard, J. L.; Thill, D. C. Ionic thiocyanate (SCN) production, fate, and phytotoxicity in soil amended with Brassicaceae seed meals. J. Agric. Food Chem. 2008, 56, 3912−3917.

1, and it has phytotoxic activity. The study benefits meadowfoam growers as well as provides an alternative bioherbicide for use on organic farms and other high value crops for which reduced synthetic herbicide applications are desired.

■

ASSOCIATED CONTENT

S Supporting Information *

HPLC analysis of MSM and activated MSM, mass spectrum of an MPAA 4 standard, and an extraction of soil incubated with nitrile 2. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Telephone (541) 737-5754; fax (541) 737-1589; e-mail [email protected]. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Kleiman, R. Chemistry of new industrial oilseed crops. In Advances in New Crops; Janick, J., Simon, J. E., Eds.; Timber Press: Portland, OR, 1990; pp 198−199. (2) Knapp, S. J.; Crane, J. M. Fatty-acid diversity of section inflexae Limnanthes (meadowfoam). Ind. Crops Prod. 1995, 4, 219−227. (3) Burg, D. A.; Kleiman, R. Preparation of meadowfoam dimer acids and dimer esters, and their use as lubricants. J. Am. Oil Chem. Soc. 1991, 68, 600−603. (4) Hirsinger, F. New annual oil crops. In Oil Crops of the World; Röbbelen, G., Downey, R. K., Ashri, A., Eds.; McGraw-Hill: New York, 1989; pp 518−532. (5) Steiner, J. J.; Griffith, S. M.; Mueller-Warrant, G. W.; Whittaker, G. W.; Banowetz, G. M.; Elliott, L. F. Conservation practices in western Oregon perennial grass seed systems: II. Meadowfoam rotation crop manament. Agron. J. 2006, 98, 177−186. (6) Purdy, R. H.; Craig, C. D. Meadowfoam: New source of longchain fatty acids. J. Am. Oil Chem. Soc. 1987, 64, 1161−1174. (7) Fahey, J. W.; Zalcmann, A. T.; Talalay, P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 2001, 56, 5−51. (8) Agerbirk, N.; Olsen, C. E. Glucosinolate structures in evolution. Phytochemistry 2012, 77, 16−45. (9) Brown, P. D.; Morra, M. J. Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination. Plant Soil 1996, 181, 307−316. (10) VanEtten, C. H.; Tookey, H. L. Glucosinolates in cruciferous plants. In Effects of Poisonous Plants on Livestock; Keeler, R. F., Van Kampen, K. R., James, L. F., Eds.; Academic Press: New York, 1978; pp 507−520. (11) Stevens, J. F.; Reed, R. L.; Alber, S.; Pritchett, L.; Machado, S. Herbicidal activity of glucosinolate degradation products in fermented meadowfoam (Limnanthes alba) seed meal. J. Agric. Food Chem. 2009, 57, 1821−1826. (12) Borek, V.; Morra, M. J.; McCaffrey, J. P. Myrosinase activity in soil extracts. Soil Sci. Soc. Am. J. 1996, 60, 1792−1797. (13) Fenwick, G. R.; Heaney, R. K.; Mullin, W. J.; VanEtten, C. H. Glucosinolates and their breakdown products in food and food plants. Crit. Rev. Food Sci. Nutr. 1982, 18, 123−201. (14) Thangstad, O. P.; Evjen, K.; Bones, A. Immunogold-EM localization of myrosinase in Brassicaceae. Protoplasma 1991, 161, 85− 93. (15) Gimsing, A.; Kirkegaard, J. Glucosinolates and biofumigation: Fate of glucosinolates and their hydrolysis products in soil. Phytochem. Rev. 2009, 8, 299−310. (16) Rakariyatham, N.; Butrindr, B.; Niamsup, H.; Shank, L. Screening of filamentous fungi for production of myrosinase. Braz. J. Microbiol. 2005, 36, 242−245. 7428

dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429

Journal of Agricultural and Food Chemistry

Article

(37) Intanon, S. Bioherbicide use of plant defensive compounds in meadowfoam seed meal. Ph.D. Dissertation, Oregon State University, Corvallis, OR, 2013. (38) Snyder, A.; Morra, M. J.; Johnson-Maynard, J.; Thill, D. C. Seed meals from Brassicaceae oilseed crops as soil amendments: influence on carrot growth, microbial biomass nitrogen, and nitrogen mineralization. HortScience 2009, 44, 354−361. (39) Brookes, P. The soil microbial biomass: Concept, measurement and applications in soil ecosystem research. Microbes Environ. 2001, 16, 131−140. (40) Andalo, C.; Godelle, B.; Lefranc, M.; Mousseau, M.; TillBottraud, I. Elevated CO2 decreases seed germination in Arabidopsis thaliana. Global Change Biol. 1996, 2, 129−135. (41) Negm, F. B.; Smith, O. E. Effects of ethylene and carbon dioxide on the germination of osmotically inhibited lettuce seed. Plant Physiol. 1978, 62, 473−476. (42) Justice, O. L.; Bass, L. N. Principles and Practices of Seed Storage; USDA: Washington, D. C., 1978; p 289.

7429

dx.doi.org/10.1021/jf5018687 | J. Agric. Food Chem. 2014, 62, 7423−7429