Effects of glucosinolates and their enzymatic hydrolysis products via myrosinase on the root-knot nematode Meloidogyne incognita (Kofoid et White) Chitw

The root-knot nematode Meloidogyne incognita (Kofoid et White) Chitw. is responsible for large yield losses in several horticultural crops. Fumigation with chemicals has been efficient in fighting this soil pest, but it clearly shows a negative environmental impact. Thus, it is necessary to find an environmentally friendly alternative to control this nematode and meet the requirements imposed by world regulation to ban some chemical fumigants in the world after 2005. The glucosinolate-myrosinase system, typical of the Brassicaceae family, appears to be an important natural alternative for the control of several soilborne pests and pathogens. The aim of this study was to evaluate, in vitro, the biocidal activity of 11 glucosinolates and their degradation products on second-stage juveniles of the root-knot nematode M. incognita expressed by the nematicidal (LD(50)) and immobilization effects, after 24 and 48 h. None of the intact glucosinolates had any biological effect. After myrosinase addition, their hydrolysis products (essentially isothiocyanates) resulted in highly different biocidal activities. Among the hydrolysis products of the tested glucosinolates, 2-phenylethyl, benzyl, 4-methylthiobutyl, and prop-2-enyl isothiocyanate showed the stronger activity, with an LD(50) at concentrations of 11, 15, 21, and 34 microM, respectively. On the basis of the in vitro test results, new genotypes of Brassicaceae had been selected for high content in the roots of the glucosinolates generating the more active isothiocyanates and their agronomic performances verified in view of a full-field application as catch crop plants. With this aim, the qualitative and quantitative glucosinolate contents in the roots of these potentially nematicidal plants are also reported and discussed.     

Changes in glucosinolate concentrations, myrosinase activity, and production of metabolites of glucosinolates in cabbage (Brassica oleracea Var. capitata) cooked for different durations

In cabbage, glucosinolates such as sinigrin are hydrolyzed by plant myrosinase to allyl isothiocyanate (AITC), allyl cyanide, and, in the presence of an epithiospecifier protein, 1-cyano-2,3-epithiopropane (CEP). Isothiocyanates have been implicated in the cancer-protective effects of Brassica vegetables. The effect of processing on the hydrolysis of glucosinolates was investigated in cabbage. Cabbage was steamed or microwaved for six time durations over 7 min. Glucosinolate concentrations were slightly reduced after microwave cooking (P < 0.001) but were not influenced after steaming (P < 0.05). Myrosinase activity was effectively lost after 2 min of microwave cooking and after 7 min of steaming. Hydrolysis of residual glucosinolates following cooking yielded predominantly CEP at short cooking durations and AITC at longer durations until myrosinase activity was lost. Lightly cooked cabbage produced the highest yield of AITC on hydrolysis in vitro, suggesting that cooking Brassica vegetables for a relatively short duration may be desirable from a health perspective.     

Correlation of glucosinolate content to myrosinase activity in horseradish (Armoracia rusticana)

Fully developed horseradish (Armoracia rusticana Gaertn., Mey., & Scherb.) roots from 27 accessions and leaves from a subset of 9 accessions were evaluated for glucosinolates and myrosinase enzyme activity. Eight different glucosinolates were detected (based on HPLC retention times as desulfoglucosinolates) in both root and leaf tissues. The sum of these glucosinolates, referred to as total, ranged from 2 to 296 micromol g(-1) of dry weight (DW) in both tissues. Four glucosinolates (sinigrin, glucobrassicin, neoglucobrassicin, and gluconasturtiin) were detected in major quantities. In fully developed roots, sinigrin concentration represented approximately 83%, gluconasturtiin approximately 11%, and glucobrassicin approximately 1% of the total glucosinolates. Approximately the same proportions of individual glucosinolates appeared in fully developed leaves, except that glucobrassicin was substituted by neoglucobrassicin and gluconasturtiin concentration was significantly lower (<1%). At least four other glucosinolates were detected in very small quantities (<1%) in both roots and leaves. Myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) is the enzyme responsible for the hydrolysis of the parent glucosinolates into biologically active products. Very little is known about myrosinase activity and the correlation of its activity to total and individual glucosinolates in plant tissues. Significant differences in myrosinase activity were detected between the roots and leaves, ranging from 1.2 to 57.1 units g(-1) of DW. Data showed no correlation between myrosinase activity and total and/or individual glucosinolates in the roots. However, in the leaves, significant correlations were found between myrosinase activity and total glucosinolates (0.78 at P = 0.01) and between myrosinase activity and sinigrin (0.80 at P = 0.01). Glucosinolates content and myrosinase activity were also correlated in young and fully developed roots and leaves and during tissue crushing. Glucobrassicin concentration in the roots and neoglucobrassicin concentration in the leaves were significantly higher in young than in fully developed tissue. Crushing of the tissue resulted in rapid hydrolysis of sinigrin and glucobrassicin, as expected, from the presence of myrosinase. Likewise, myrosinase activity declined rapidly after crushing, perhaps due to inactivation by the reaction products and/or the depletion of its substrates.     

Behavior of glucosinolates in pickling cruciferous vegetables

Crucifer species, which include widely consumed vegetables, contain glucosinolates as secondary metabolites. Cruciferous vegetables are consumed in Japan in salt-preserved or pickled form as well as cooked and raw fresh vegetables. In this study, changes in contents of glucosinolates during the pickling process were investigated. 4-Methylthio-3-butenyl glucosinolate, a major glucosinolate in the root of Japanese radish, daikon (Raphanus sativus L.), was detected in pickled products with a short maturation period but not in those with a long maturation period. As a model pickling experiment, fresh watercress (Nasturtium officinale) and blanched watercress were soaked in 3% NaCl solution for 7 days. The results showed that the ratio of indole glucosinolates to total glucosinolates increased during the pickling process, whereas total glucosinolates decreased. Myrosinase digestion of glucosinolates in nozawana (Brassica rapa L.) indicated that indole glucosinolates, especially 4-methoxyglucobrassicin, were relatively resistant to the enzyme. The effect of pickling on glucosinolate content and the possible mechanism are discussed in view of degradation by myrosinase and synthetic reaction in response to salt stress or compression during the pickling process.     

Biotransformation of glucosinolates epiprogoitrin and progoitrin to (R)- and (S)-Goitrin in Radix isatidis

Radix isatidis is an important traditional Chinese medicine with antiviral efficacy. (R)- and (S)-Goitrin are its main bioactive constituents in the 2010 edition of the Chinese Pharmacopoeia. (R)- and (S)-Goitrin are the breakdown products of epiprogoitrin and progoitrin from R. isatidis. The biotransformation of glucosinolates epiprogoitrin and progoitrin to (R)- and (S)-goitrin, however, is still unclear. In the current paper, the biotransformation of glucosinolates was studied. First, the high-performance liquid chromatography methods to analyze glucosinolates and their breakdown products were developed. Then, the biotransformation of individual glucosinolates such as epiprogoitrin and progoitrin was investigated under different pH conditions. Lastly, their biotransformation under five extraction environments was studied. The results showed that (R)- and (S)-goitrin were the most noteworthy breakdown products. Their relative transformation rates were about 70-80%, and the influence of different extraction environments on the transformation rate was not significant. These results would serve as a theoretical basis for industrial production and quality control and would be helpful for further studies on the biotransformation of glucosinolates.     

Extraction and determination of glucosinolates from soil

The use of glucosinolate-containing plants as soil-incorporated biofumigants for pest and disease control has raised questions regarding the fate of glucosinolates in soil; however, no method for routine analysis of glucosinolates in soil has been reported. A simple method to extract glucosinolates from soil with quantification as desulfoglucosinolates by HPLC is presented. The method involves two extractions with 70% methanol at room temperature, centrifugation, and filtration prior to the desulfation step. The desulfoglucosinolates are then quantified by HPLC using established protocols for plant tissue analysis. There were no significant interfering peaks from the soil extracts, and the method provided high extraction efficiencies (around 100%) for both aromatic (benzyl) and aliphatic (2-propenyl) glucosinolates when amended at a wide range of realistic field soil concentrations (1.6-120 nmol/g of soil). The method was equally effective in three diverse Australian soils that varied in organic matter, clay content, and pH. The method was effective in air-dried or field-moist soil, although evidence for rapid glucosinolate degradation in field-moist soil indicates that extraction of moist soils should be performed as soon as possible after sampling. The method is compatible with field soil sampling at remote sites and utilizes the same equipment and protocols already established for plant tissue analysis. Extraction of glucosinolates in the field following incorporation of Indian mustard (Brassica juncea) and rape (Brassica napus) green manure crops was also tested. Eight different glucosinolates contained in the plant tissues were identified and quantified in soil extracts at concentrations ranging from 0.11 to 21.7 nmol/g of soil.     

Kinetics of the stability of broccoli (Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during pressure/temperature treatments

The Brassicaceae plant family contains high concentrations of glucosinolates, which can be hydrolyzed by myrosinase yielding products having an anticarcinogenic activity. The pressure and temperature stabilities of endogenous broccoli myrosinase, as well as of the synthetic isothiocyanates sulforaphane and phenylethyl isothiocyanate, were studied in broccoli juice on a kinetic basis. At atmospheric pressure, kinetics of thermal (45-60 degrees C) myrosinase inactivation could be described by a consecutive step model. In contrast, only one phase of myrosinase inactivation was observed at elevated pressure (100-600 MPa) combined with temperatures from 10 up to 60 degrees C, indicating inactivation according to first-order kinetics. An antagonistic effect of pressure (up to 200 MPa) on thermal inactivation (50 degrees C and above) of myrosinase was observed indicating that pressure retarded the thermal inactivation. The kinetic parameters of myrosinase inactivation were described as inactivation rate constants (k values), activation energy (Ea values), and activation volume (Va values). On the basis of the kinetic data, a mathematical model describing the pressure and temperature dependence of myrosinase inactivation rate constants was constructed. The stability of isothiocyanates was studied at atmospheric pressure in the temperature range from 60 to 90 degrees C and at elevated pressures in the combined pressure-temperature range from 600 to 800 MPa and from 30 to 60 degrees C. It was found that isothiocyanates were relatively thermolabile and pressure stable. The kinetics of HP/T isothiocyanate degradation could be adequately described by a first-order kinetic model. The obtained kinetic information can be used for process evaluation and optimization to increase the health effect of Brassicaceae.     

Development and validation of a spectrophotometric method for quantification of total glucosinolates in cruciferous vegetables

Given their putative role in chemoprevention, validated methods are needed for quantification of total glucosinolates. Based on the colorimetric reaction of ferricyanide with 1-thioglucose, released by alkaline hydrolysis of glucosinolates, we developed a simple and sensitive method for spectrophotometric quantification of total glucosinolates in cruciferous vegetables. Lyophilized and ground vegetables are extracted with 80% boiling methanol. Extracted glucosinolates are isolated using a strong anion exchange column and then hydrolyzed with 2 N NaOH to release 1-thioglucose. Ferricyanide is added, and the decrease in absorbance is measured at 420 nm, with final values adjusted for background. Recovery of internal standard (sinigrin) was 107%. Intra- and interassay coefficients of variation were 5.4% and 15.8%, respectively. Dose response was linear with sinigrin and amount of plant material extracted (R(2) ≥ 0.99). Using sinigrin, the lower limit of quantification was 0.6 mg. This straightforward method may be an alternative to time-consuming and costly chromatographic methods.     

Use of anion-exchange membrane extraction for the high-performance liquid chromatographic analysis of mustard seed glucosinolates

A new one-step extraction using anion-exchange membranes for the HPLC determination of glucosinolates in mustard seeds is reported. The exchange of glucosinolates on the membranes was studied using sinigrin in solutions and sinigrin added as an internal standard to seeds of yellow mustard. By varying time of extraction, membrane size, and sample size, the optimal conditions for maximum glucosinolate recovery were determined and the following procedure was adopted: 0.2 g of ground mustard seeds are heated in 20 mL of boiling water for 5 min. After cooling, samples are transferred to plastic centrifuge tubes, 9-cm(2) membranes are added, and suspensions are shaken on a mechanical shaker for 2.5 h. Glucosinolates are then eluted from the membranes with 25 mL of 1 N KCl by shaking again for 2.5 h. Using this procedure, the sinigrin extraction from solutions and from mustard seeds was linear with 80% recovery. Seeds of yellow, brown, oriental, and Indian mustard were analyzed by this procedure; excellent reproducibility, with coefficients of variation in the range 1.0-4.3% were obtained. This method offers a simple and inexpensive alternative to complicated and tedious procedures for glucosinolate isolation/purification required for chromatographic determinations.     

Determination of glucosinolates in canola seeds using anion exchange membrane extraction combined with the high-pressure liquid chromatography detection

A rapid, simple, and reliable method for the determination of individual glucosinolates in canola seeds was developed using a semiquantitative extraction of glucosinolates with anion exchange membranes and HPLC detection. In this one-step extraction procedure, a membrane (7 cm(2)) is placed in the seed suspension prepared by grinding and boiling 0.8 g of seeds in 20 mL of water. After 10 min of shaking on the mechanical shaker, the membrane is removed from the suspension, washed, and transferred to a vial containing 5 mL of 1 N tetramethylammonium chloride. The glucosinolates are eluted from the membrane by shaking the membrane for 10 min with the eluting solvent. The glucosinolate content in membrane eluates is determined by HPLC using sinigrin standards. A coefficient of variation ranging from 1.9 to 7.6% for aliphatic glucosinolates indicated very good reproducibility of the method. Because of the instability of 4-hydroxyglucobrassicin, the coefficient of variation for the determination of this indolyl glucosinolate was 13.9%. To verify the results of the membrane extraction/HPLC detection, this new method was compared with the existing colorimetric and GC procedures. Very good correlation (R(2) = 0.98) was obtained between the total glucosinolates determined by the membrane extraction/HPLC method and the palladate colorimetric procedure for 17 canola varieties. Concentrations of individual glucosinolates in five canola varieties were compared with the GC data. Very good agreement between these two methods was obtained for aliphatic glucosinolates. However, the membrane extraction/HPLC method yielded slightly higher values for 4-hydroxyglucobrassicin than the GC method, possibly indicating that the decomposition of this glucosinolate was reduced during the sample extraction with the membranes. The simplicity and low cost of the membrane extraction/HPLC method make it an attractive alternative to the existing procedures for glucosinolate analysis in canola seeds.     

High-performance liquid phase separation of glycosides. 5. Determination of individual glucosinolates in cabbage and rapeseed by laser-induced fluorescene capillary electrophoresis via the enzymatically released isothiocyanate aglycon.

A capillary electrophoresis (CE) method was developed for the profiling and determination of individual glucosinolates (GSs) via their isothiocyanate degradation products upon myrosinase digestion. The resulting isothiocyanates, the structures of which are reflective of the parent GS's, were then converted to their corresponding amines via base hydrolysis or reaction with 1, 2-benzenedithiol. Subsequently, the amines were fluorescently labeled to allow their sensitive detection by laser-induced fluorescence (LIF). The CE method involved the use of in situ charged micelles for the separation of isothiocyanates and their corresponding fluorescently labeled amines by micellar electrokinetic capillary chromatography (MECC). The term "in situ charged micelles" refers to micelles formed by complexing the polar hydroxyl groups of glycosidic surfactants with borate. The MECC method with on-column LIF detection was applied to the determination of GSs in white cabbage, rapeseed leaves, and rapeseed roots.     

Soluble and total myrosinase activity in defatted Crambe abyssinica meal

Crambe defatted meal contains 4-6% w/w of glucosinolates, with epiprogoitrin accounting for >90% of the total. This feature limits the use of the meal as feed due to the antinutritional properties of myrosinase-glucosinolate breakdown products. In this context, myrosinase activity assumes particular importance. In this study the total and soluble myrosinase activities have been evaluated directly on defatted meals of eight Crambe abyssinica varieties. The pH-stat method, which is the most suitable for assays in heterogeneous solid-water systems, was used. The total myrosinase activity in C. abyssinica varieties, determined using epiprogoitrin as substrate, ranged from 288 to 653 units g(-1). These activity values were up to 26 times higher than those obtained using other substrates, namely, sinigrin, glucosinalbin, glucotropaeolin, progoitrin, and glucoraphenin. Crambe myrosinase is unusual in that, unlike other Brassicaceae containing a typical main glucosinolate, it does not show the same specificity toward its natural substrates.     

Variation of glucosinolates in wild radish (Raphanus raphanistrum) accessions

Glucosinolate composition was determined in wild radish accessions from eight states in the northeastern and southern United States to determine the variability of production among accessions. Glucosinolates were evaluated from roots, leaves, flowers, primary, and secondary branches. Seventeen glucosinolates were identified, with glucoerucin, glucoraphenin, glucobrassicin, and gluconasturtiin contributing 90% to 100% of the total glucosinolates. Flowers contained the highest glucosinolate concentrations, 12.07 to 55.36 μmol/g, but flowers contributed only 5.3 to 21.3% to the total glucosinolates. Of the eight accessions, the Mississippi accession produced significantly higher levels of total glucosinolates and glucosinolates which can be degraded to isothiocyanates per plant, totals of 618.97 and 563.53 μmol/plant, respectively. Total plant biomass did not differ between accessions indicating a difference in the ability of the Mississippi accession to produce glucosinolates. Further studies are needed to determine if this accession would consistently produce higher glucosinolate levels under different environmental conditions.     

Preparation and biological activity of four epiprogoitrin myrosinase-derived products

(5R)-5-Vinyl-1,3-oxazolidine-2-thione, (2S)-1-cyano-2-hydroxy-3-butene, and two diastereoisomeric erythro-(2S)- and threo-(2S)-1-cyano-2-hydroxy-3,4-epithiobutanes were prepared in pure form starting from (2S)-2-hydroxybut-3-enyl glucosinolate (epiprogoitrin). This glucosinolate was isolated in almost pure form using ripe seeds of Crambe abyssinica and then hydrolyzed under different conditions. The hydrolysis was carried out using either myrosinase immobilized on nylon, to produce (5R)-5-vinyl-1,3-oxazolidine-2-thione, or the endogenous myrosinase contained in defatted crambe meals, to produce the other epiprogoitrin-derived products. After purification and physicochemical characterization, all four myrosinase degradation products were tested for their biological activity. A bioassay on Lactuca sativa was chosen as a simple test to determine their apparent action on living tissues. (5R)-5-Vinyl-1,3-oxazolidine-2-thione negatively affected mainly root growth, whereas (2S)-1-cyano-2-hydroxy-3-butene affected the early phase of germination, and both (2S)-1-cyano-2-hydroxy-3,4-epithiobutane diastereoisomers appeared to negatively affect both germination and root growth at doses 5-10 times lower than those of (2S)-1-cyano-2-hydroxy-3-butene or (5R)-5-vinyl-1,3-oxazolidine-2-thione.     

Aspergillus flavus transformation of glucosinolates to nitriles by an arylsulfatase and a β-thio-glucosidase

Biofumigation is a biocontrol method that uses volatile compounds to combat soil pathogens. We investigated a biofumigation process based on the green manure of Brassicaceae. These plants contain the glucosinolate-myrosinase system which releases inhibitory compounds such as isothiocyanate into the soil. However, the biocontrol effectiveness is often lower than expected, possibly due to microbial transformation of the isothiocyanates. In order to identify the possible function of microorganisms, their interaction with glucosinolates and glucosinolate-derived products was investigated. We report the ability of a soil isolate of Aspergillus flavus to grow in liquid culture and convert 2-propenyl and 2-phenylethyl glucosinolate and their desulfo-derivatives, to nitriles. This finding would suggest the existence of an arylsulfatase and a β-thio-glucosidase, different from myrosinase, which could direct glucosinolate conversion in soil towards nitriles rather than isothiocyanates. If confirmed in soil, our observations could at least partially explain the low concentrations of isothiocyanates after biofumigation.     

马铃薯田线虫病害发生及防治

近些年马铃薯土传病害发生严重,除了真菌、细菌等病原物引起的病害外,线虫病成为威胁马铃薯生产的又一大病害.危害马铃薯的植物寄生线虫主要有马铃薯腐烂茎线虫(Ditylenchus destructor)、鳞球茎线虫(Ditylenchus dipsaci)、马铃薯孢囊线虫(马铃薯白线虫(Globodera rostochi...     

Isolation and biochemical characterization of a basic myrosinase from ripe Crambe abyssinica seeds,highly specific for epi-progoitrin

On the basis of previous studies on the mechanism-based inhibition, activation, and active site structure of myrosinase(s) isolated from Sinapis alba and other cruciferous seeds, crambe myrosinase shows uncommon properties and behavior. For this reason homogeneous crambe myrosinase was isolated and investigated to establish the most important physicochemical features, including kinetic properties determined with the epimers progoitrin (R) and epi-progoitrin (S) as substrates, with and without ascorbate as an activator. The results of this study demonstrate that crambe myrosinase is highly specific for epi-progoitrin due to a better stabilization of the enzyme-substrate complex. This stabilization is caused by additional hydrogen bonding that only epi-progoitrin can set up between its hydroxyl group and a suitable residue in the hydrophobic pocket where the "docking" of the glucosinolates side chain takes place.     

Variation of glucosinolates in vegetable crops of Brassica oleracea

Glucosinolates were evaluated in 5 groups and 65 accessions of Brassica oleracea (50 broccoli, 4 Brussels sprouts, 6 cabbage, 3 cauliflower, and 2 kale) grown under uniform cultural conditions. Glucosinolates and their concentrations varied among the different groups and within each group. The predominant glucosinolates in broccoli were 4-methylsulfinylbutyl glucosinolate (glucoraphanin), 3-butenyl glucosinolate (gluconapin), and 3-indolylmethyl glucosinoate (glucobrassicin). Glucoraphanin concentration in broccoli ranged from 0.8 micromol g(-1) DW in EV6-1 to 21.7 micromol g(-1) DW in Brigadier. Concentrations of the other glucosinolates in broccoli varied similarly over a wide range. In Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolates were sinigrin (8.9, 7.8, 9.3, and 10.4 micromol g(-1) DW, respectively) and glucobrassicin (3.2, 0.9, 1.3, and 1.2 micromol g(-1) DW, respectively). Brussels sprouts also had significant amounts of gluconapin (6.9 micromol g(-1) DW). Wide variations in glucosinolate content among genotypes suggest differences in their health-promoting properties and the opportunity for enhancement of their levels through genetic manipulation.     

Genetic variation in leaf and stem glucosinolates in resynthesized lines of winter rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Glucosinolates are secondary components characteristic for the Brassicaceae with complex biological functions. Glucosinolates in the seeds are anti-nutritive when feeding animals and their inheritance have been extensively investigated. Much less is known about the genetics of glucosinolates in leaves and stems, which may attract some insects, while repelling others. They may also inhibit bacterial processes of importance when using green biomass for the production of biogas. The objective of this study was to analyse the genetic variation of total and individual glucosinolates in the green material of rapeseed. For this 28 resynthesized winter rapeseed lines were tested at two locations. There was a large variation in leaf glucosinolate content between 0.10 and 4.75 μmol/g dry matter. The predominant leaf glucosinolates are the alkenyle glucosinolates progoitrin, gluconapin and glucobrassicanapin. The line R53 is exceptional, while combining a relative high content of the indole glucosinolate glucobrassicin with low alkenyle glucosinolates in the leaves. The total glucosinolate concentration in the stems and leaves is not correlated with the seed glucosinolate concentrations. Heritabilities are above h2 = 0.60 for progoitrin, h2 = 0.65 for gluconapin, h2 = 0.30 for glucobrassicanapin and h2 = 0.52 for total glucosinolate content in the leaves. In conclusion, resynthesized rapeseed is an important genetic resource to modify the leaf glucosinolate content and composition of rapeseed.     

氮、硫供应水平对芜菁硫代葡萄糖苷含量的影响

硫代葡萄糖苷（GS）含量的多少是芜菁品质好坏的重要指标。本文采用沙培试验、 高效液相色谱法（HPLC）研究不同氮（80 kg/hm2、 160 kg/hm2和240 kg/hm2）和硫（10 kg/hm2和60 kg/hm2）供应水平对白玉芜菁叶和根中硫代葡萄糖苷组成和含量的影响。结果表明, 白玉芜菁叶片中有7种硫代葡萄糖苷,根中有9种硫代葡萄糖苷。叶片中总硫代葡萄糖苷含量是根部总硫代葡萄糖苷含量的1.16倍。增加硫供应水平,显著增加了根和叶中各种硫苷含量;随着氮供应水平的增加,吲哚族硫苷含量占总硫苷含量的比例显著增加。白玉芜菁中硫代葡萄糖苷的含量决定于氮和硫的平衡供应,在氮N 160 kg/hm2、 硫S 60 kg/hm2供应水平下硫代葡萄糖苷含量最高。