The potential use of five isothiocyanates (ITCs) (allyl, butenyl, benzyl, 2-phenylethyl and 4-methylthiobutyl-ITC) to control Monilinia laxa was tested by in vitro and in vivo trials. In in vitro trials, ITC activity on spore germination and mycelial growth was evaluated. The 4-methylthiobutyl-ITC was more efficient in controlling M. laxa than the other ITCs, showing the lowest values of ED50 and ED95, respectively, 0.04 and 0.10mgL−1 for conidia germination and 0.30 and 0.52mgL−1 for mycelial growth. In addition, a significant reduction in conidia germination was observed after only 90min of ITC vapour exposure. In in vivo trials, artificially infected nectarines and peaches were exposed for 3–6h in an ITC-enriched atmosphere, resulting in a substantial difference in the pathogen control from the in vitro tests. Among the 5 ITCs tested, only allyl and butenyl-ITC reduced brown rot by more than 85% after 3–4 days of fruit incubation at 20°C but in the case of butenyl-ITC, some phytotoxic effects can occur. Similar results were obtained in further experiments with allyl and butenyl-ITC vapours produced in situ from defatted meal of Brassica carinata and Brassica rapa, respectively. Although further studies are necessary to exclude any detrimental effects on fruit quality, this study provides experimental evidence that supports the use of biofumigation based on ITCs, allyl-ITC in particular, as a technique to control postharvest brown rot in nectarine and peach fruit.

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.

The presence in all Brassicaceae plant organs of high amounts of glucosinolates, and of the enzyme myrosinase that catalyses their hydrolysis, linked to the high biocidal activity of some glucosinolate enzymatic hydrolysis derivative products (mainly isothiocyanates and nitriles) have suggested the practical possibility of amending soil with these natural biocidal compounds by the cultivation and green manure of selected species of this family. The application of this technique even at full field level has given, in recent years, interesting applicative results, with clear advantages for the following crop yield (e.g. strawberry), if compared with an untreated soil or a conventional green manure, evidencing that the glucosinolate-myrosinase system may provide a natural alternative for methyl bromide soil fumigation.In this paper, some preliminary results of a study on the possibility of drying some of these selections to produce biocidal pellets to be used as organic treatments in addition or in alternative to biocidal green manure are reported and discussed. The first target was to limit, during drying, glucosinolate leakage and myrosinase activity loss; in this way, in fact, dry plants should be able to produce in soil the biocidal compounds when watered by irrigation. Using a simple drying up lab technique, it was possible, for some selections, to lose <40% of starting glucosinolate and to preserve myrosinase activity at a sufficient level. These results could be further improved using industrial dehydration plant. The dried plants, after water addition, showed, in vitro, a good fungitoxic activity on Pythium ssp. and Rhizoctonia solani, confirming the results obtained in similar tests with pure glucosinolate and pure myrosinase.These results open interesting applicative perspectives for this new raw material probably even as a natural alternative to methyl bromide. (C) 2004 Elsevier B.V. All rights reserved.

The use of oil with a high level of erucic acid in some industrial sectors appears to offer excellent prospects from a technological and environmental point of view. This study was carried out to determine the potential of a new Eruca sativa spp. oleiformis selection named Nemat as a source of high erucic oil in the Po Valley environment. This species, classified as catch crop of the nematode Heterodera schachtii, showed a wide adaptability both in spring and autumn sowing and permitted a generally satisfactory productive yield over 3-year trials, with a grain yield ranging between 1.9 and 2.2tha−1 (autumn sowing). Seeds presented an oil concentration around 290gkg−1 with significant higher amount in spring sowing, while fatty acid composition was characterised by a long chain content higher than 53%. The residual meal presented a high level of thiofunctionalised glucosinolates (GLs), showing some interesting perspectives in integrated pest management.

Crambe abyssinica is becoming a reliable source of industrial oil with good technical characteristics because of its high erucic acid content. The possibility of using the industrial defatted meal of crambe as a feed could further promote the interest in this crop, but is prevented by the high glucosinolate content (70-150 μmol/g). Therefore the possibility of reducing this parameter by selection was evaluated and a search for a new quick analytical procedure begun. When hydrolysed by myrosinase (thioglucoside glucohydrolase EC 3.2.3.1.), at pH 6.5 or higher, epi-progoitrin, the main glucosinolate in crambe seed ( > 90% of total glucosinolates), gives (5R)-5-vinyl-1,3-oxazolidine-2-thione quantitatively. The latter can be easily estimated by high-performance liquid chromatography. This is the background of the proposed analytical procedure that permits rapid analysis of about 100 samples per day using about 25 mg of crambe meal (five seeds) for assay. The method can also be used to select for glucosinolate content in rapeseed and, probably with some adjustment, all crops that have a 2-hydroxy-glucosinolate as the main glucosinolate.

The fungitoxicity of allyl‐isothiocyanate (AITC) vapour against Penicillium expansum, the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L−1 AITC‐enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 103 conidia mL−1 to almost 80% at 1 × 106 conidia mL−1. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole‐resistant strain of P. expansum, reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum.

(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.

Starting from homochiral 1,3-oxazolidine-2-thiones 1, a two-step sequence led to homotopic 2,3-dihydro-oxazolo[2,3-b]quinazolin-5-one derivatives 3. The sequence was developed and studied regarding its scope and limitations.

Original chemo-enzymatic processing of epi-progoitrin (1), a glucosinolate extracted from crambe presscake, produces (5R)-5-vinyl-1,3-oxazolidine-2-thione (2) in enantiomerically pure form. The reactivity range of this chiron is investigated.

A comparison of the effect of isothiocyanates and nitriles derived from some glucosinolates, namely, epi-progoitrin, sinalbin, glucotropaeolin, glucocheirolin, and glucoraphenin, on human erythroleukemic in vitro cultured cells was studied. Many studies have in fact evidenced that a consumption of vegetable containing glucosinolates could reduce the development of colorectal cancer. In the experimental conditions used, the production of isothiocyanates and nitriles from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The obtained results demonstrated that in general nitriles are considerably less potent than the corresponding isothiocyanates in inhibiting cancer cell growth. Particularly, the isothiocyanates inhibitory activity on K562 cells growth is higher in the case of products derived from epi-progoitrin, glucotropaeolin, glucoraphenin, and glucocheirolin; while for nitriles the higher activity in inhibiting K562 cells growth is showed by sinalbin-derived product. Considering the antiproliferative activity found for isothiocyanates and nitriles, further studies will be aimed to the possible application of glucosinolate-derived products as chemopreventive cancer agents for the reduction of colorectal cancer.

Glucosinolate degradation products (GLDPs), mainly isothiocyanates, obtained by myrosinase-catalysed hydrolysis of glucosinolates (GLs), are an important group of natural bioactive substances. The fungitoxic activity of four GLDPs, chosen on the basis of their side-chain structures, was assayed against Pythium irregulare and Rhizoctonia solani. The effectiveness of the degradation products (DPs) of sinigrin (alkenyl GL), epi-progoitrin (hydroxy-alkenyl GL), glucoiberin and glucoerucin (thiofunctionalised GLs) in inhibiting P irregulare oospore germination and R solani soil colonisation were tested in a closed system, using an artificially infected soil. The fungitoxic activity of these GLDPs varied according to their side-chain structure. As in previous in vitro studies, the thiofunctionalised GLDPs were found the most effective, producing complete inhibition of P irregulare oospore germination (0.01 mu mole g(-1) soil) and R solani soil colonisation (0.5 mu mol eg(-1) soil), but even sinigrin DP showed a fungitoxic activity higher than that of epi-progoitrin DP. (C) 2000 Society of Chemical Industry.

Bioactive compounds were produced from natural glucosinolates, secondary plant metabolites, using myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) isolated from ripe seeds of Sinapis alba. The enzyme was immobilized on granular nylon 6.6 with the crosslinking technique. Immobilized myrosinase displayed extraordinary operational and storage stability. Using a small thermostatted continuous packed-bed bioreactor, the enzyme activity was unchanged after 15 days of continuous use at 37 degrees C and after >1 year of storage at room temperature. The bioreactor was particularly efficient in producing pure isothiocyanates, but it was less efficient for pure nitrile production.

Chiral 1,3-oxazolidine-2-thiones were prepared in enantiopure form from renewable resources through an enzymatic process involving immobilized myrosinase (thioglucoside glucohydrolase E.C. 3.2.3.1).

A commercial crude sulfatase from snail (Helix pomatia) was purified by ion exchange, affinity and FPLC gel-filtration chromatography. The enzyme isolated, which was ca. 37-fold more active than the commercial one, was covalently immobilized on nylon 6.6 by the cross linking method. Immobilized sulfatase was used to produce some desulfo-glucosinolates (DSGLs) on the gram-scale, starting from glucotropaeolin, glucoraphenin and epi-progoitrin, and for HPLC analyses of glucosinolates (GLs) contained in cruciferous material and rapeseed in particular. The immobilized enzyme, as well as allowing the standardization of the HPLC method for GLs analysis, seems to be an efficient system for producing DSGLs which are interesting starting materials to prepare bio-active structures through further chemical modifications.

Epi-progoitrin, the major glucosinolate of Crambe abyssinica, was readily extracted from defatted crambe meal. A simple biotechnological process using immobilized myrosinase allowed a high-yielding chemo- and stereocontrolled transformation of epi-progoitrin into epi-goitrin, which was obtained in pure stereochemical form. Many diverse chemical modifications can be envisaged on the derived epi-goitrin which should be considered as a novel sophisticated starting material for the production of enantiomerically pure fine chemicals.

Recent studies report the suppressive activity of Brassica plant green manure against soilborne phytopathogenic fungi as an ecological alternative to methyl-bromide in horticulture. This fungitoxic activity is due to the presence in all plant tissues of the glucosinolate (GL)-myrosinase system which, in presence of cellular damage, produces isothiocyanates and/or nitriles. These compounds present a strong bio-activity which depends on their chemical structure. The above ground biomass of Cleome hassleriana, of the Cleomaceae family, contains a large quantity of glucocapparin (methyl-glucosinolate) and glucocleomin (2-hydroxy-2-methylbutyl-glucosinolate). This composition is interesting because one of the hydrolysis products of glucocapparin is methyl-isothiocyanate, which is a biocidal ingredient of some commercial fumigants. Two years of full field Cleome cultivation in Northern Italy demonstrated that at the flowering stage, a selection named ISCI2 can produce more than 3.5 t/ha of dry matter with more than 25 kg/ha of glucosinolates. To demonstrate the fungitoxic activity of C. hassleriana green manure some experiments were carried out in pots under controlled conditions. Fresh C. hassleriana tissues collected at flowering stage were ground and mixed with natural Pythium spp. infested soil. Total fungi strongly increased in all fresh tissue treatments. On the contrary, a treatment with a common green manure crop increased Pythium propagules four-fold while after three weeks C. hassleriana treatment reduced Pythium by 88% as compared to untreated soil. These results suggest further studies to verify the potential use of C. hassleriana as an efficient tool to control some soil-borne pathogens in horticulture.

Epidemiological and pharmacological studies have shown that colorectal cancer development could be reduced by consuming vegetables that contain glucosinolates. In view of this the effect of some glucosinolates and their isothiocyanate (ITC)-derived products on in vitro cell growth was studied. We report the isolation and characterization of ITCs derived from glucosinolates by using HPLC, GC-MS, and NMR techniques. The in vitro activity of ITCs on human erythroleukemic K562 cells has been investigated by using two alternative approaches: the in situ and pre-mix methods. No differences in antiproliferative activity were found comparing the effect of ITCs produced either of these methods. In the experimental conditions used, the production of ITCs from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The ITCs' inhibitory activity on K562 cells growth is particularly evident in the cases of ITCs derived from sinigrin, progoitrin, epi-progoitrin, glucotropaeolin and glucocheirolin. Finally, the antiproliferative activity of the ITCs obtained from glucoraphenin, taken as an example, was determined on other tumor cell lines with a different origin and hystotype. Considering the antiproliferative activity found for ITCs these compounds could be considered potentially responsible for the reduction of colorectal cancer associated with diets rich in cruciferous vegetables. Further studies will be aimed at the possible application of glucosinolate-derived products as chemopreventive cancer agents.

The present study assayed the effect of six isothiocyanates (ITCs), produced by the enzymatic hydrolysis of glucosinolates, on fungal pathogens of pear. Sample pear fruits were artificially inoculated through induced wounds with conidial suspensions of Botrytis cinereaRhizopus stoloniferMonilinia laxaMucor piriformis or Penicillium expansum and were then treated with ITCs. Of the six ITCs tested, the ITC from glucoraphenin showed the highest effectiveness after 6 days at 20°C, against M. laxaB. cinerea and M. piriformis. The effectiveness of the ITC from glucoraphenin against M. laxa was assayed in two further trials to test the effect of ITC concentration on different concentrations of inoculum and to determine the duration of the curative effect of this ITC. ITC concentration directly affected fungus control capacity. The highest ITC concentration (3.6 mg mL−1) afforded pathogen control at the highest level of pathogen concentration (106 conidia mL−1) after 6 days at 20°C. Its curative effect was evident up to 40 h after inoculation.