Leaching of isothiocyanates through intact soil following simulated biofumigation

Biofumigation can be used as an alternative to conventional soil fumigation to control soil-borne pests. With biofumigation, plant tissue with a natural content of glucosinolates (cruciferous plants) is damaged and incorporated into the topsoil. When the plant tissue is damaged, the glucosinolates come into contact with the endogenous enzyme myrosinase, which catalyse the hydrolysis of glucosinolates into various products depending on the reaction conditions. Isothiocyanates are among the potential products formed from these reactions. We investigated if the isothiocyanates from rape plant material were leached through the soil to drain depth when a heavy rainstorm followed the biofumigation. We applied isothiocyanates from rape plant material (1,480 μmol m⁻²) to four large (0.6 m diameter, 1.0 m long) intact soil monoliths from a loamy and a sandy soil and conducted a leaching experiment under semi-field conditions. The soil monoliths were irrigated with 70–90 mm (10 mm h⁻¹) and the concentrations of three isothiocyanates (3-butenyl, 4-pentenyl and 2-phenethyl) were monitored in the leachate. Between 0 and 14.8 mmol isothiocyanates were leached for each mol of isothiocyanates applied during application of 70–90 mm irrigation. The distribution coefficient estimated from leached concentrations was 0.04–1.19 for 3-butenyl, 0.04–1.15 for 4-pentenyl isothiocyanate and 0.037–0.97 for 2-phenethyl isothiocyanate. The concentration of total isothiocyanates in the leachate was in the same order of magnitude as the LD₅₀ of isothiocyanates for sensitive aquatic organisms.     

Glucosinolates in oilseed rape: secondary metabolites that influence interactions with herbivores and their natural enemies

Glucosinolates are sulphur‐containing secondary metabolites characteristic of Brassicaceous plants. Glucosinolate breakdown products, which include isothiocyanates, are released following tissue damage when hydrolytic enzymes act on them. The isothiocyanates have toxic effects on generalist herbivores when they attempt to feed on oilseed rape, Brassica napus, and also function as repellents. However, specialist herbivores such as Brevicoryne brassicae aphids, flea beetles, Psylliodes chrysocephala and the Lepidopteran pest, Pieris rapae, are adapted to the presence of glucosinolates and thrive on plants containing them. They may do this by avoiding tissue damage to prevent the formation of isothiocyanates or by metabolising or tolerating glucosinolates. For many specialist herbivores, the isothiocyanates function as attractants and glucosinolates can even be sequestered for defence against predatory insects. Thus, these herbivores have evolved resistance to host‐plant secondary metabolites and this type of evolutionary history may have given some insects an enhanced ability to adapt to xenobiotics. In an agricultural context, this may make pests better able to evolve resistance to artificially applied pesticides. The effect of increased glucosinolate content in making oilseed rape cultivars more susceptible to specialist pests was highlighted in a seminal article in the Annals of Applied Biology in 1995. This review of the literature considers developments in this area since then.     

The phloem mobility of glucosinolates

The transport properties of glucosinolates within Brassica napus are of interest as identification of the mechanism of transport could lead to lower levels being obtained in specific tissues such as the seeds. The phloem mobility of ³⁵S-gluconapin (but-3-enylglucosinolate) and ³⁵S-desulphogluconapin in oilseed rape plants has been inferred from tissue distribution patterns, as well as from observed coincident phloem mobility of ³H-gluconapin and ¹⁴C-sucrose. The measured relative phloem mobilities for sinigrin (2-propenylglucosinolate), ³H-gluconapin, ³⁵S-desulphogluconapin, ³⁵S-desulphosinigrin, ¹⁴C-tryptophan, ³H-AIB (-aminoisobutyric acid), and literature values for a reduced ³H-oligogalacutonide elicitor (degree of polymerization 6) and ¹⁴C-IAA (indolylacetic acid), have been compared with the predicted values obtained using the Kleier model for phloem mobility of xenobiotics. All the above compounds show phloem systemicity, demonstrated using the Ricinus assay, as predicted by the model. Log Kow (octanol-water partition coefficient) values for glucosinolates and desulphoglucosinolates measured at pH 4 and pH 7, and the effect of pH on uptake by oilseed rape embryos are provided as evidence against a weak acid trap mechanism acting in either the phloem mobility or the accumulation of glucosinolates in oilseed rape embryos. The phloem mobility of glucosinolates is explained by the intermediate permeability hypothesis. In conclusion, it would appear that glucosinolates like other groups of endogenous compounds have physicochemical properties allowing phloem mobility as predicted by the Kleier model.Keywords: Brassica napus, Ricinus communis, phloem mobility, glucosinolates, Kleier model.      

Feeding preferences of woodpigeons and flea-beetles for oilseed rape and turnip rape

Winter turnip rape Brassica rapa ssp. oleifera was shown to have reduced palatability to woodpigeons, enhanced susceptibility to adult flea beetle feeding and reduced susceptibility to larval flea-beetle infection when compared with winter oilseed rape (B. napus) cultivars in field trials. Levels of leaf waxes were negatively correlated with feeding preferences of adult flea beetles. Analysis of volatiles from damaged leaves showed that while all cultivars produce a similar range of nitriles, cyanoepithioalkanes and isothiocyanates, derived from 3-butenyl, 4-pentenyl and phenylethyl glucosinolates, B. rapa leaves produced relatively high levels of 1-methylpropyl isothiocyanate, although there was significant plant-to-plant variation. The possible involvement of this mustard oil glycoside and variation in epicuticular waxes in plant-herbivore interactions are discussed.     

Exploitation of host plant preferences in pest management strategies for oilseed rape (Brassica napus)

New control strategies for insect pests of arable agriculture are needed to reduce current dependence on synthetic insecticides, the use of which is unsustainable. We investigated the potential of a simple control strategy to protect spring‐sown oilseed rape, Brassica napus L. (Brassicaceae), from two major inflorescence pests: the pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), and the seed weevil, Ceutorhynchus assimilis (Paykull) (Coleoptera: Curculionidae), through exploitation of their host plant preferences. The strategy comprised, for the main crop, Starlight [an oilseed rape cultivar with relatively low proportions of alkenyl glucosinolates in the leaves (thereby releasing lower levels of attractive isothiocyanates than conventional cultivars)] and turnip rape, Brassica rapa (L.) (Brassicaceae), as a trap crop. We tested the system in laboratory, polytunnel semifield arena, and field experiments. The odours of Starlight were less attractive in olfactometer tests to both pests than those from a conventional cultivar, Canyon, and the plants were less heavily colonized in both polytunnel and field experiments. Turnip rape showed good potential as a trap crop for oilseed rape pests, particularly the pollen beetle as its odour was more attractive to both pests than that of oilseed rape. Polytunnel and field experiments showed the importance of relative growth stage in the system. As turnip rape flowers earlier than oilseed rape, beetles would be maintained on turnip rape past the damage‐susceptible growth stage of oilseed rape. The development of a pest control regime based on this strategy is discussed.     

The responses of the cabbage seed weevil Ceutorhynchus assimilis to volatile compounds from oilseed rape in a linear track olfactometer

The cabbage seed weevil, Ceutorhynchus assimilis Payk. [syn. Ceutorhynchus obstrictus (Marsham)] (Coleoptera: Curculionidae), a crucifer-feeding insect, is a pest of oilseed rape (Brassica napus L.). It is known to be attracted by isothiocyanates, crucifer-specific volatiles that are metabolites of the glucosinolates. The responses of this insect to other electrophysiologically-active volatiles from rape were tested in a linear track olfactometer. Attraction was demonstrated to nitriles (phenylacetonitrile, 4-pentenenitrile and 5-hexenenitrile), which are also glucosinolate metabolites, and to volatiles emitted by a wider spectrum of plant families ((Z)-3-hexen-1-ol and methyl salicylate). Combination of an isothiocyanate mixture with phenylacetonitrile increased attraction, but there was no such increase when the isothiocyanate mixture was combined with methyl salicylate. A mixture of 23 volatiles, emulating an attractive air-entrainment extract of oilseed rape, was not significantly attractive, although a high proportion of weevils (60%) turned towards it. The potential of these volatiles for inclusion into an isothiocyanate-based monitoring system is discussed.     

Insect herbivore counteradaptations to the plant glucosinolate-myrosinase system

The glucosinolate-myrosinase system found in plants of the Brassicales order is one of the best studied plant chemical defenses. Glucosinolates and their hydrolytic enzymes, myrosinases, are stored in separate compartments in the intact plant tissue. Upon tissue disruption, bioactivation of glucosinolates is initiated, i.e. myrosinases get access to their glucosinolate substrates, and glucosinolate hydrolysis results in the formation of toxic isothiocyanates and other biologically active products. The defensive function of the glucosinolate-myrosinase system has been demonstrated in a variety of studies with different insect herbivores. However, a number of generalist as well as specialist herbivores uses glucosinolate-containing plants as hosts causing large agronomical losses in oil seed rape and other crops of the Brassicaceae. While our knowledge of counteradaptations in generalist insect herbivores is still very limited, considerable progress has been made in understanding how specialist insect herbivores overcome the glucosinolate-myrosinase system and even exploit it for their own defense. All mechanisms of counteradaptation identified to date in insect herbivores specialized on glucosinolate-containing plants ensure that glucosinolate breakdown to toxic isothiocyanates is avoided. This is accomplished in many different ways including avoidance of cell disruption, rapid absorption of intact glucosinolates, rapid metabolic conversion of glucosinolates to harmless compounds that are not substrates for myrosinases, and diversion of plant myrosinase-catalyzed glucosinolate hydrolysis. One of these counteradaptations, the nitrile-specifier protein identified in Pierid species, has been used to demonstrate mechanisms of coevolution of plants and their insect herbivores.     

Glucosinolates and amines in Reseda media

The content of glucosinolates and amines in green parts of Reseda media has been investigated. Benzylglucosinolate, 2-phenethylglucosinolate, and m-hydroxybenzylglucosinolate occur in appreciable amounts accompanied by minor amounts of other glucosinolates, benzylamine and m-hydroxybenzylamine. Isolation and identification of these compounds was made using ion-exchange chromatography, high voltage electrophoresis, GC, MS, and ¹³C-NMR spectroscopy. The glucosinolates were transformed into corresponding nitriles and isothiocyanates by thioglucoside glucohydrolase-catalysed hydrolysis and to the corresponding carboxylic acids by acid-catalysed hydrolysis. The content of glucosinolates and amines in leaves and inflorescences of R. media has been determined by UV-spectroscopy and GC.     

Variation in the glucosinolate content of vegetative tissues of Chinese lines of Brassica napus L

The glucosinolate content of leaves, stems and roots of a range of Chinese oilseed rape (Brassica napus L.) breeding lines was analysed. Total content and spectrum of individual glucosinolates varied widely, and there was no correlation between seed and vegetative tissue glucosinolate content. Lines with low seed glucosinolates (00) did not necessarily have low glucosinolate content in vegetative tissues; nor did high seed glucosinolate lines always have high vegetative tissue content. There was no correlation between the glucosinolate content of leaf, stem, and root in any given line. It appears that glucosinolate synthesis and accumulation is under tissue-specific control, and the mutation which blocks accumulation of glucosinolates in seeds does not influence other tissues. The responses of these lines to elicitors was also examined. Methyl jasmonate and salicylic acid treatments produced increases in leaf indolyl and aromatic glucosinolates respectively. However, the extent of such increases differed widely between the lines, and there were other, less consistent, effects on other classes of glucosinolate. There seems to be greater variation in glucosinolate accumulation in rape than has previously been reported, and the lines described here have considerable potential for evaluating the effects of manipulating glucosinolate profiles on pest and disease interactions.     

Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols.

Organic isothiocyanates are widely distributed in plants and are responsible for a variety of beneficial and toxic biological effects. No direct and generic method for quantitating isothiocyanates has been described. Under mild conditions nearly all organic isothiocyanates (R-NCS) react quantitatively with an excess of vicinal dithiols to give rise to five-membered cyclic condensation products with release of the corresponding free amines (R-NH2). The products of the condensation of propyl-NCS with 1,2-ethanedithiol, 2,3-dimercaptopropanol, and 1,2-benzenedithiol have been isolated and identified as 1,3-dithiolane-2-thione, 4-hydroxymethyl-1,3-dithiolane-2-thione, and 1,3-benzodithiole-2-thione, respectively. Since 1,3-benzodithiole-2-thione (lambda max 365 nm and alpha m 23,000 M-1 cm-1) can be sensitively measured spectroscopically, the reaction of organic isothiocyanates with 1,2-benzenedithiol has been developed for analytical purposes. All aliphatic and aromatic isothiocyanates tested (except tert-butyl and other tertiary isothiocyanates) reacted quantitatively with an excess of 1,2-benzenedithiol. Thiocyanates, cyanates, isocyanates, cyanides, or related compounds did not interfere with this reaction under assay conditions. The method can be used to measure 1 nmol or less of pure isothiocyanates or isothiocyanates in crude mixtures. It can also be used to measure isothiocyanates in chromatographic fractions obtained from plant extracts and for the assay of the rate of cleavage of glucosinolates by myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1).     

Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS

Dietary glucosinolates are under intensive investigation as precursors of cancer-preventive isothiocyanates. Quantitation of the dose and bioavailability of glucosinolates and isothiocyanates requires a comprehensive analysis of the major dietary glucosinolates, isothiocyanates, and related metabolites. We report a liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the comprehensive analysis of the seven major dietary glucosinolates, related isothiocyanates, and putative amine degradation products. The parent glucosinolates were sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin. The LC-MS/MS analysis method for these compounds was developed and validated; a standard addition analysis protocol was used generally to avoid the requirement for stable isotopic standards. Where stable isotopic standards were available, internal standardization with these gave estimates in agreement with those obtained by the standard addition analysis protocol. For glucosinolates, negative ion electrospray LC-MS/MS analysis was performed. Isothiocyanates and amines were prederivatized to the corresponding thiourea and N-acetamides, respectively, and were quantified by positive ion electrospray LC-MS/MS. The limits of detection were 0.5-2 pmol; the recoveries for glucosinolates, isothiocyanates, and amines were 85-90%, 50-85%, and 60-70%, respectively; and the intra- and interbatch coefficients of variation were 1-4% and 3-10%, respectively. These methods provide facile access to comprehensive analytical data on the major dietary glucosinolates and related metabolites to quantify inputs and metabolic formation of these compounds in cancer prevention and related studies.     

Effect of zinc on the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell)

The effect of Zn²⁺ ions (in the form of ZnCl₂) in the ceoncentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell). In the “long-term” experiment zine ions influenced the seedlings during eight days of germination, whereas in the “short-term” experiment zinc ions acted only 72 h on seven days old intact seedlings. The biosynthesis of indole glucosinolates has been followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into both glucosinolates in experiments with, hypocotyl segments of the rape seedlings. Zinc ions at chronic “long-term” application increased the glucobrassicin and neoglucobrassicin level in the seedlings. The neoglucobrassicin content especially was increased. A “short-term” application of zinc ions increased the level of both glucosinolates at higher and lower concentrations, whereas medium concentrations (10^?4 and 10^?5 M) lowered their level. Zn²⁺ ions lowered absorption of³⁵SO₄ ^?2 ions by hypocotyl segments and simultaneously lowered the incorporation of³⁵S into glucobrassicin. On the contrary, the incorporation of³⁵S into neoglucobrassicin and proteins was stimulated. Zinc ions do exhibit a specific effect on neoglucobrassicin biosynthesis, on membrane permeability as against sulphate ions and on the incorporation of sulphur into proteins.     

In vitro activity of glucosinolate-derived isothiocyanates against postharvest fruit pathogens*

The authors assayed the ability of some reaction products, essentially isothiocyanates, derived from the myrosinase-catalysed hydrolysis (neutral pH) of six natural glucosinolates to inhibit germination and mycelial growth of Botrytis cinerea, Rhizopus stolonifer, Monilinia laxa, Mucor piriformis and Penicillium expansum, the leading postharvest fungal pathogens of fruit and vegetable crops. All of the tested products showed antifungal activity, although they proved less effective against mycelial growth than in germination control. The isothiocyanates produced by enzymatic hydrolysis of glucoraphenin, sinalbin and sinigrin were particularly effective because they completely inhibited conidic germination of all five pathogens considered. The sinigrin-derived isothiocyanate exhibited a wide pathogen-control spectrum, either inhibiting conidia germination altogether or delaying by 3–6 days the onset of mycelial growth compared to the control.     

Some glucosinolates of Farsetia aegyptia and Farsetia ramosissima

Air-dried leaves of Farsetia aegyptia and F. ramosissima have been analysed for their glucosinolates; the former was shown to contain at least six but chiefly allylglucosinolate, whilst the latter contains at least five but mainly but-3-enylglucosinolate with some 4-(methylthio)butylglucosinolate. Without the addition of extraneous thioglucosidase enzyme, both species gave predominantly nitrile degradation products of glucosinolates; but if extra enzyme were added, corresponding isothiocyanates became the major products instead. Varying the pH from the natural level for the plant also considerably affected the ratios of glucosinolate products.     

Glucosinolate changes in developing pods of single and double low varieties of autumn-sown oilseed rape (B. napus)

Single and double low varieties of oilseed rape were grown in the 1987/88 and 1988/89 seasons to study changes in the concentrations of total and individual glucosinolates within pods during development. Total glucosinolate concentration in seeds of all varieties increased during development when expressed on a fresh weight basis. The levels of the major alkenyl glucosinolates present in the seed; 2–hydroxy-3–butenyl, 3–butenyl and 4–pentenyl had been reduced in the transition from single to double low varieties. The major indole glucosinolates in the seed, 4–hydroxy-3–indolylmethyl and 3–indolylmethyl were present in the same amounts in single and double low varieties but in the latter represented a greater proportion of the total seed glucosinolate content. A decline in the total glucosinolate concentration in the pod walls with time together with the analogous profile of individual glucosinolates in the seeds and pod walls suggests that the pod wall is a major site of seed glucosinolate synthesis. Other plant parts may also have an important role to play in provision of intact glucosinolates or precursors to the pod walls for glucosinolate biosynthesis.     

Phosphatases from pollen of Brassica campestris and Lilium regale

Soluble and wall-bound acid phosphatases isolated from rape seed pollen showed similar properties except for the pH optimum curve which was elevated for the cell wall enzyme. About 50 % of the phosphatase activity of washed pollen wall preparations could be solubilized with Triton X-100, compared with only ca 20% for the corresponding preparation from lily pollen. A comparison of the wall-bound acid phosphatase of rape seed and lily pollen showed a marked difference in specificity towards fructose-6-phosphate and glucose-6-phosphate. A Mg²⁺-dependent alkaline pyrophosphatase was obtained from rape seed pollen but this activity could not be detected in cell wall preparations.     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Oilseed rape seeds with ablated defence cells of the glucosinolate-myrosinase system. Production and characteristics of double haploid MINELESS plants of Brassica napus L

Oilseed rape and other crop plants of the family Brassicaceae contain a unique defence system known as the glucosinolate-myrosinase system or the 'mustard oil bomb'. The 'mustard oil bomb' which includes myrosinase and glucosinolates is triggered by abiotic and biotic stress, resulting in the formation of toxic products such as nitriles and isothiocyanates. Myrosinase is present in specialist cells known as 'myrosin cells' and can also be known as toxic mines. The myrosin cell idioblasts of Brassica napus were genetically reprogrammed to undergo controlled cell death (ablation) during seed development. These myrosin cell-free plants have been named MINELESS as they lack toxic mines. This has led to the production of oilseed rape with a significant reduction both in myrosinase levels and in the hydrolysis of glucosinolates. Even though the myrosinase activity in MINELESS was very low compared with the wild type, variation was observed. This variability was overcome by producing homozygous seeds. A microspore culture technique involving non-fertile haploid MINELESS plants was developed and these plants were treated with colchicine to produce double haploid MINELESS plants with full fertility. Double haploid MINELESS plants had significantly reduced myrosinase levels and glucosinolate hydrolysis products. Wild-type and MINELESS plants exhibited significant differences in growth parameters such as plant height, leaf traits, matter accumulation, and yield parameters. The growth and developmental pattern of MINELESS plants was relatively slow compared with the wild type. The characteristics of the pure double haploid MINELESS plant are described and its importance for future biochemical, agricultural, dietary, functional genomics, and plant defence studies is discussed.     

In vitro Propagation and Isozyme Polymorphism of the Medicinal Plant Hypericum brasiliense

Responses of 20 d-old plants of two Brassica napus L. cultivars Dunkeld and Cyclon to NaCl salinized soil [electrical conductivity 2.4 (control), 4.0, 8.0 or 12.0 dS m⁻¹] were examined. The salt tolerant line Dunkeld had significantly higher fresh and dry masses of shoots, and seed yield than salt sensitive line Cyclon in all salinities. The effect of salt stress on reduction in total leaf soluble sugars was markedly greater in Dunkeld as compared to that in Cyclon. No effect of salt stress was observed on leaf soluble proteins but there was a slight increase in total free amino acids of both cultivars. Leaf proline content increased markedly in both cultivars and Dunkeld had greater proline content than Cyclon at all salinities. Salt stress had no significant effect on seed oil content and erucic acid content of seed oil, however, content of glucosinolates in the seed meal increased and Cyclon had greater content of glucosinolates than Dunkeld. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue