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J. Agric. Food Chem. 2005, 53, 554−561
Degradation Studies on Benzoxazinoids. Soil Degradation Dynamics of (2R)-2-O-β-D-Glucopyranosyl-4-hydroxy-(2H)1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and Its Degradation Products, Phytotoxic Allelochemicals from Gramineae FRANCISCO A. MAC´ıAS,* ALBERTO OLIVEROS-BASTIDAS,† DAVID MAR´ıN, DIEGO CASTELLANO, ANA M. SIMONET, AND JOSEÄ M. G. MOLINILLO Grupo de Alelopatı´a, Departamento de Quı´mica Orga´nica, Facultad de Ciencias, Universidad de Ca´diz, C/Repu´blica Saharaui s/n, Apartado 40, 11510 Puerto Real, Ca´diz, Spain
Wheat (Triticum aestivum L.) has been found to possess allelopathic potential and studies have been conduced to apply wheat allelopathy for biological weed control. 2,4-Dihydroxy-(2H)-1,4-benzoxazin3(4H)-one (DIBOA) is a common product found in wheat, corn, and rye exudates and it can be released to the environment by that way. In this report, the stability of DIBOA is studied in two soils from crop lands of wheat cv. Astron and cv. Ritmo. These varieties were selected by their concentrations of DIBOA and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) from aerial parts and by the bioactivities of their aqueous extracts in the growth of wheat coleoptile sections. The degradation rate of DIBOA in these soils was measured in laboratory tests during 90 h by high-pressure liquid chromatography methods. These analyses demonstrate that DIBOA was transformed primarily into 2-benzoxazolinone (BOA). This transformation was similar in both soil types with an average half-life of 43 h. The degradation studies for BOA show its biotransformation to 2-aminophenoxazin-3-one (APO) with a half-life of 2.5 days. Therefore, BOA is an intermediate product in the biotransformation from DIBOA to APO in these wheat crop soils and is consistent with previous findings. APO was not degraded after three months in soil, suggesting that its degradation rate in soil is very slow. KEYWORDS: bioactivity
Benzoxazinoids; DIBOA-Glc; DIBOA, BOA; biodegradations; soil; Triticum aestivum;
INTRODUCTION
Some cereal plants produce a series of benzoxazinoid compounds (cyclic hydroxamic acids). The number of this group of natural products is small (1), but they possess diverse biological activities. These compounds are involved in the defense of plants against fungi (2) and insects (3) as well as in allelopathic interactions (4,5). The most important benzoxazinoids reported (Figure 1) are 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-(2H)1,4-benzoxazin-3(4H)-one (DIMBOA), which are present in wheat, maize, and rye and have been found in members of families Acanthaceae, Rannunculaceae, and Scrophulariaceae (6). These compounds are present as glycosides (Figure 1) in plants, being released as aglycones by the activity of the enzyme β-glucosidase (7, 8). Moreover, these aglycones are unstable in solution and soil, being transformed to 2-benzoxazolinone (BOA), 7-methoxy-2* To whom correspondence should be addressed. Tel.: +34(956)016370; fax: +34(956)016193; e-mail:
[email protected]. † Laboratorio de Quı´mica Ecolo ´ gica, Departamento de Quı´mica, Facultad de Ciencias, Universidad de Los Andes, Nu´cleo Universitario Pedro Rinco´n Gutie´rrez, La Hechicera, Me´rida 5101-A, Venezuela.
benzoxazolinone (MBOA), and other degradation products (1) (Figure 1). These transformations depend on the chemical and biological conditions. Some of these transformation products are more biologically active than the original ones (9). Transport of allelochemicals to the soil can occur mainly by leaching of the foliar parts (10, 11), exudation from root (12), decomposition of plant residues by microbial action (13), or by direct transformation by microbes associated to the roots (14). Previous publications have dealt with the isolation, characterization, and biological activity of the degradation products (15-17). However, the dynamic aspects of the degradative processes of these compounds such as half-life, concentration dependence of the half-life, or the influence of other microbial substrate on degradation have not been researched. Here, we report the stability and degradation studies of DIBOA-Glc and its derivatives in different conditions and soils used to cultivate two wheat varieties (Triticum aestiVum cv. Astron and cv. Ritmo). Our objectives were to study the influence of soil microbes in the degradation dynamics of these compounds. Understanding this process will allow us to elucidate the influence of these microbes on chemical defense strategies in
10.1021/jf048702l CCC: $30.25 © 2005 American Chemical Society Published on Web 01/04/2005
Biodegradation of Benzoxazinoids in Soil
Figure 1. Structures of benzoxazinoids and their degradation products mentioned in the text.
plants that produce hydroxamic acids and to propose which chemical structures are responsible for the allelopathic behavior observed. To ensure the maximum accuracy in the study, soils from different wheat varieties were used and compared, and the influence of soil chemical reactivity and spontaneous degradation processes was taken into account by using the suitable standards for the different analysis. MATERIALS AND METHODS Contents of DIBOA and DIMBOA in Six Triticum aestiWum Varieties. The contents of DIBOA and DIMBOA were determined in six wheat varieties (Triticum aestiVum cv. Hill, Portal, Ritmo, Astron, Stakado, and Solist). The varieties were cultivated in a growth chamber at controlled temperature and humidity (25 °C and 68%) with a photoperiod of 16 h light/8 h dark provided by white light fluorescent lamps. After 7 days of growth, seedlings were extracted with acidified MeOH (1% AcOH) in an ultrasonic bath for 5 min. The resulting mixture was filtered, and the residue was extracted again with acidified MeOH. The two solutions were combined and the solvents were eliminated by distillation in vacuo at 30 °C. The residue was dissolved in water and loaded in a Sep-Pak C18. Then, it was washed with acidified water and with an acidified solution of aqueous methanol (MeOH:H2O:AcOH, 60:40:1). The resulting solutions were combined, the solvents were eliminated by distillation in vacuo, and the residue was dissolved in 1 mL of MeOH:H2O:HOAc (60:40:1) for their HPLC analysis. The six wheat varieties were cultivated in experimental crop lands (see below). They were harvested in their stage 21 (BBCH scale) (18) and were immediately stored at -20 °C until their study. One kilogram of the complete plant was extracted with 3 L of distilled water by rain simulation (24 h) by means of a Pluviotron Rain Simulation Equipment (19). The resulting aqueous extract was filtered (98%. 1H and 13C NMR spectra were recorded using MeOH-d4 as solvent in a Varian INOVA spectrometer at 399.99 and 100.577 MHz, respectively. The resonance of residual methanol for 1H and 13C was set to δH 3.30 and δC 49.00, respectively, and used as internal reference. HPLC data were acquired by using a Varian ProStar system, equipped with two solvent delivery modules (Model 210), an autosampler (Model 410), a PDA detector (diode array UV-vis system, Model 330), a Quadrupole MS/MS detector (Model 1200L), and a Phenomenex SYNERGI 4 micron Fusion RP80 column (250-460 mn). Triticum aestiWum Cultivars (cv. Astron and cv. Ritmo). The soil samples were collected in November 2002 from fields where wheat crops of cv. Astron and Ritmo were grown at the Institute of Agriculture and Development Center, Rancho de la Merced (Jerez de la Frontera, Spain). Conventional fertilization procedures were used without using herbicides and fungicides. The crop terrains were mechanically treated to eliminate weeds prior to planting. After approximately one month
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Table 1. Physicochemical Analysis of Wheat Crop Soil exchange capacity (mequiv/100 g) exchangeable cations (mequiv/100 g) Ca2+ Mg2+ Na+ K+ carbonates (% w/w) active limestone (% w/w) assimilable phosphorus (Olsen, ppm) total organic content (ppm) organic nitrogen (% w/w) pH (H2O:1/2.5) pH (KCl) assimilable potassium (ppm) clay (% w/w) sand (% w/w) slime (% W/W) textural classification
33.91 28.96 3.08 0.29 1.58 18.25 8.81 8.7 1.35 0.08 8.33 7.31 635 52.6 15.4 32.0 clay
of growth (growing stage 21-BBCH scale), samples of each variety were sown in individual parcels (300 m2, 12 plots of 25 m2). Wheat samples were collected and stored at -20 °C prior to their analysis. Soil Collection. The selected parcels for the cultivation contained similar physicochemical characteristics at the moment of their cultivation (Table 1) and did not contain any other plant species in growth. After harvest, samples of soil from the vicinities of the plants were taken to get the maximum density of the microbial population associated with the plant underground parts. The samples were taken at the 1520-cm depth, in a radius of 3 cm around the plant lines. One hundred random soil samples were collected for each crop (0.014 m3). The soil was sieved at 2 mm, placed in plastic bags, and preserved at -20 °C until its study. Before the degradation studies, dry plant material was removed from the samples as well as calcareous stones by a sieve that allows a distribution of soil particles smaller than 1 mm. Soil pH was measured in a 1:2.5 (v/v) aqueous extract and in KCl solution. Total organic nitrogen (TON) was determined according to the Kjeldhal method, and total organic carbon (TOC) was determined by oxidation with potassium dichromate. Available phosphorus was extracted with sodium bicarbonate (23) and determined by colorimetry, according to the method of Murphy and Riley (24). Ca, Mg, Na, and K were determinated by flame photometry (25). Experiment Design. Soil samples (10 g) were placed in 50-mL vials and test solutions (5 mL of sterilized Milli-Q water with 10 mg of DIBOA-Glc or DIBOA, and 2.5-50 mg of BOA) were added to them. Samples of these solutions were collected using a glass syringe. Sample amounts, sampling periods, and treatments prior to the analysis are described below. The resulting suspension is homogenized by using a vortex mixer (VWR International) at room temperature. For control samples, soil was sterilized by washing with methanol (30 mL/5 g soil, 12 h) and drying at 100 °C over a period of three consecutive days (Gallenkamp Hotbox Oven). An aqueous solution of the tested substance without soil was added as water control to evaluate spontaneous degradation of the substance. These samples were analyzed by the same procedure and at the same times as those containing nonsterilized soil samples. Recovery extractions in water and sterile soil control samples were used to correct the determinations in the degradation experiments. Sampling and Processing of Soil. Samples (0.5 mL) of incubation solution were taken from the soil at different time periods after homogenization of the soil-water suspension. Samples were preserved at -20 °C after the addition of methanol (1 mL) to avoid degradation between the end of the experiment and the moment of the analysis. Samples were preserved at -20 °C after the addition of methanol (1 mL) to avoid degradation between the end of the experiment and the moment of the analysis. The sampling intervals were determined by means of preliminary studies, in which degradation of each compound was achieved (12 h for DIBOA-Glc, 12 h DIBOA and 24 h for BOA).
For HPLC analysis, solutions were centrifuged in a Selecta Microfiger BL 71379 apparatus at 13.000 rpm for 10 min and then filtered (
