Release Of Isothiocyanate Research Articles

Comparative Studies of Extracts Obtained from Brassica oleracea L. Plants at Different Stages of Growth by Isolation and Determination of Isothiocyanates: An Assessment of Chemopreventive Properties of Broccoli.

The main goal of this work was to develop analytical procedures for the isolation and determination of selected isothiocyanates. As an example, particularly sulforaphane from plants of the Brassicaceae Burnett or Cruciferae Juss family. The applied methodology was mainly based on classical extraction methods and high-performance liquid chromatography coupled with tandem mass spectrometry. Moreover, the effect of temperature on the release of isothiocyanates from plant cells was considered. The cytotoxic activity of the obtained plant extracts against a selected cancer cell line has also been included. The results allow evaluating the usefulness of obtained plant extracts and raw sprouts regarding their content of isothiocyanates-bioactive compounds with chemopreventive properties.

N-Methylation of Self-Immolative Thiocarbamates Provides Insights into the Mechanism of Carbonyl Sulfide Release.

Hydrogen sulfide (H2S) is an important biomolecule, and self-immolative thiocarbamates have shown great promise as triggerable H2S donors with suitable analogous control compounds; however, thiocarbamates with electron-deficient payloads are less efficient H2S donors. We report here the synthesis and study of a series of N-methylated esterase-triggered thiocarbamates that block the postulated unproductive deprotonation-based pathway for these compounds. The relative reaction profiles for H2S release across a series of electron-rich and electron-poor N-Me aniline payloads are examined experimentally and computationally. We show that thiocarbamate N-methylation does block some side reactivity and increases the H2S release profiles for electron-poor donors. Additionally, we show that isothiocyanate release is not a competitive pathway, and rather that the reduced efficiency of electron-poor donors is likely due to other side reactions.

Sisymbrium Officinale (the Singers’ Plant) as an Ingredient: Analysis of Somatosensory Active Volatile Isothiocyanates in Model Food and Drinks

Sisymbrium officinale (L.) Scop. (hedge mustard) is a wild common plant of the Brassicaceae family. It is known as “the singers’ plant” for its traditional use in treating aphonia and vocal disability. The plant is rich in glucosinolates and isothiocyanates; the latter has been demonstrated to be a strong agonist in vitro of the Transient Receptor Potential Ankirine 1 (TRPA1) channel, which is involved in the somatosensory perception of pungency as well as in the nociception pathway of inflammatory pain. Volatile ITCs are released by the enzymatic or chemical hydrolysis of GLSs (glucosinolates) during sample crushing and/or by the mastication of fresh plant tissues when the plant is used as an ingredient. Some functional food and drink model preparations have been realised: honey enriched with seeds and flowers, infusions, cold drink (voice drink), artisanal beer, and a fermented tea (kombucha). Using SPME-GCMS chromatography, we analysed samples of the plant and of the food preparations adopting conditions that simulate the release of isothiocyanates (ITCs) during oral assumption. Two active compounds, iso-propylisothiocyanate and 2-butylisothiocyanate, have been assayed. The concentration of ITCs varies according to temperature, pH, grinding conditions, and different plant organs used. Kombucha-type fermentation seems to eliminate the ITCs, whereas they are retained in beer. The ITCs’ concentration is higher when entire seeds and flowers are used.

Inactivation of Thioglucosidase from Sinapis alba (White Mustard) Seed by Metal Salts.

The glucosinolates which are specialized plant metabolites of Brassica vegetables are prone to hydrolysis catalyzed by an endogenous enzyme myrosinase (thioglycoside hydrolase, thioglucosidase) that exists in Brassica plant tissue causing volatile isothiocyanates release. Currently existing literature data on the inactivation of myrosinase is insufficient in particular for use in the analysis of volatile and odor compounds in vegetables rich in glucosinolates. In this study, the impact of different metal salts in effective inactivation of enzyme activity was investigated by solid-phase microextraction (SPME) and GC/MS system in aqueous samples and kohlrabi matrix. A saturated solution of calcium chloride which is commonly used to stop enzyme activity in plant tissue inactivates the myrosinase–glucosinolate system. However, even without the participation of myrosinase, it changes the reaction pathway towards nitrile formation. The model experiment shows that optimum efficiency in inhibition of the enzyme system shows iron(III) ions, silver ions, and anhydride sodium sulfate resulting in no volatile products derived from glucosinolates. However, in the kohlrabi matrix, the strongest enzyme inhibition effect was observed for silver salt resulting in no volatile products, also both anhydrous Na2SO4 and saturated CaCl2 solution seem to be useful inhibitors in flavor studies.

Anticancer properties of erucin, an H2 S-releasing isothiocyanate, on human pancreatic adenocarcinoma cells (AsPC-1).

Plants of the Brassicaceae family are well-known for containing the glucosinolate myrosinase system, which is able to release isothiocyanates after plant biotic and abiotic lesions. Erucin (ERU; 1-isothiocyanato-4-(methylthio)-butane), an isothiocyanate particularly abundant in arugula (Eruca sativa Mill., Eruca vesicaria L., etc.), derives from the hydrolysis of the glucosinolate glucoerucin by the enzyme myrosinase. Many other natural isothiocyanates influence cancer cells and, in particular, induce antiproliferative effects at relatively high concentrations. Similar antiproliferative effects have also been shown by the newly emerging gasotransmitter hydrogen sulfide (H2 S) and by H2 S-releasing compounds. In a previous study, our group demonstrated that isothiocyanates release H2 S in biological environments. In this work, we demonstrated the H2 S-donor properties of ERU in pancreatic adenocarcinoma cells (AsPC-1) and delineated its profile as a chemopreventive or anticancer agent. Indeed, ERU showed significant antiproliferative effects: ERU inhibited AsPC-1 cell viability at relatively high concentrations (30-100μM). Moreover, ERU inhibited cell migration, altered the AsPC-1 cell cycle, and exhibited proapoptotic effects. Finally, ERU inhibited ERK1/2 phosphorylation. This mechanism is particularly important in AsPC-1 cells because they are characterized by a mutation in KRAS that determines KRAS hyperactivation followed by MAP-kinase hyperphosphorylation, which plays a pivotal role in pancreatic cancer proliferation, growth, and survival.

Constant Isothiocyanate-Release Potentials across Biofumigant Seeding Rates.

Biofumigation is an integrated pest-management method involving the mulching of a glucosinolate-containing cover crop into a field in order to generate toxic isothiocyanates (ITCs), which are effective soil-borne-pest-control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest-control outcomes and develop best practices for choosing biofumigants, growth conditions, and mulching methods that allow the greatest potential isothiocyanate release. We measured the glucosinolate concentrations of six different commercial varieties of three biofumigant plant species: Brassica juncea (ISCI99, Vitasso, and Scala) Raphanus sativus (Diablo and Bento), and Sinapis alba (Ida Gold). The plants were grown in the range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, the highest ITC-release potentials were achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. The highest ITC-release potential occurred at the 50% flowering growth stage across the species. The seeding rate had a minor impact on the ITC-release potential of R. sativus but had no significant effects on the ITC-release potentials of the B. juncea or S. alba cultivars.

Abstract A197: Synthesis and in vitro anti-prostate cancer activities of androgen receptor ligand-isothiocyanate hybrid drug

Abstract Prostate cancer (PCa) is the most common male malignancy in U.S.. In contrast to the localized early-stage disease, prognosis of castration-resistant prostate cancer (CRPC), i.e., the relapsed/metastatic advanced disease phenotype, is extremely poor. Because the sustained androgen receptor (AR) signaling remains one of the central driving forces of CRPC, new-generation nonsteroid AR antagonist has been developed in recent years, exemplified by the approval of enzalutamide (Enz) in 2012 for post-chemo and in 2014 for chemotherapy-naïve CRPC. However, drug resistance inevitably develops and survival benefits of Enz in CRPC patients are short-lived. Isothiocyanates (ITCs), e.g., sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), are active metabolites of dietary glucosinolate from cruciferous vegetables with broad anti-PCa activities, such as suppressing AR expression, downregulating antiapoptotic proteins, inducing cell cycle arrest and apoptosis, and inhibiting PCa stem-like cells, etc. SFN-rich extract was recently tested in recurrent PCa patients and notably prolonged PSA doubling time without severe adverse events. By modifying Enz structure, we designed and synthesized AR ligand-ITC hybrid drug, aimed at antagonizing androgen-stimulated AR transcriptional activity and meanwhile downregulating AR expression and inducing other anticancer effects in PCa cells. To increase water solubility, ITCs were further reacted with N-acetyl cysteine (NAC) to obtain NAC conjugates. A representative hybrid drug NAC conjugate 2-63 was selected for in vitro study. In PBS buffer (pH 7.4), 2-63 gradually decomposes and releases free ITC with a half-life of about 3.4h. Measured using MTT assay, 2-63 was more tolerable in noncancerous RWPE-1 cell, significant differences were observed between RWPE-1 and PCa LNCaP cells, suggesting NAC conjugate could be a water-soluble carrier of AR ligand-ITC hybrid drug. The slower release of ITC constrains the concentration of electrophilic ITC within limit that is tolerated in healthy tissue. The newly designed hybrid drug chemical scaffold retains AR antagonist activity, evidenced by the inhibition of ARE-regulated luciferase activity in kb2 cell and the suppression of DHT-stimulated proliferation of LNCaP cell. 2-63 also dose- and time-dependently inhibited growth of CRPC VCaP cell and disrupted mitochondrial membrane potential, more effectively than SFN and Enz. Analyzed using Western blot, 2-63 downregulated the expression of full-length AR (fAR) and AR splice variants 7 (AR-V7). The AR downregulating properties were also observed in LNCaP and in 22Rv1 cells carrying different AR mutants. Since AR-V expression is one of the mechanisms causing clinical resistance to Enz treatment, targeting both fAR and AR-Vs using the newly discovered AR ligand-ITC hybrids may provide a new modality to improve the prognosis in CRPC patients. Citation Format: Siyu Ou, Liping Xu, Zhihui Qin. Synthesis and in vitro anti-prostate cancer activities of androgen receptor ligand-isothiocyanate hybrid drug [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A197.

Investigation of isothiocyanate release from electrospun modified poly(L-lactic acid)/mustard powder composite fibers

Mustard seed containing glucosinolates can be used as a natural source of antimicrobial volatile isothiocyanate (ITC) through enzymatic hydrolysis. ITC can be used in active food packaging to inhibit the proliferation of spoilage microorganisms. In this study, antibacterial composite fibers were prepared by encapsulating mustard powder (MP) in carrier materials of electrospun poly(L-lactic acid) (PLLA)/poly[(lactic acid)-co-lysine] (PLAL) fibers. Uniaxial composite fibers (PLLA/MP, PLLA/PLAL/MP) and coaxial composite fibers (PLLA/PLAL/MP-PLLA, PLLA/MP-PLLA/PLAL) were fabricated by electrospinning, and these fibers could be used to produce a series of volatile antibacterial substances, ITCs, which were extracted by hexane under proper temperature and moisture. The morphologies, chemical structures, thermal properties and hydrophilicities of the electrospun composite fibers were investigated. Ultraviolet spectrophotometry (UV) was used to evaluate the controlled ITC release behaviors from these fibers. The results indicated that the mean particle diameter was 975±126 nm and the main interaction between the MP and the carrier polymers was physical. The addition of MP and PLAL could improve the hydrophilic property of PLLA and change the thermal properties. Different fibers exhibited distinct ITC release behaviors, and the ITC release period of the coaxial fibers (approximately 14 days) was longer than that of the uniaxial fibers (approximately 8 days). These fibers have bright prospects in antibacterial ITC controlled release. UV can be used to investigate ITC release from electrospun modified PLLA/MP fibers by hexane extracting under the proper temperature and moisture.

Myrosinase Compatible Simultaneous Determination of Glucosinolates and Allyl Isothiocyanate by Capillary Electrophoresis Micellar Electrokinetic Chromatography (CE-MEKC).

The functional food Cruciferous vegetables contain glucosinolates which are decomposed by the myrosinase enzyme upon tissue damage. The isothiocyanates are the most frequent decomposition products. Because of their various bioactivities, these compounds and the myrosinase is of high interest to many scientific fields. Development of a capillary electrophoresis method capable of myrosinase-compatible, simultaneous quantification of glucosinolates and isothiocyanates. Capillary electrochromatography parameters were optimised, followed by optimisation of a myrosinase-compatible derivatisation procedure for isothiocyanates. Vegetable extracts (Brussels sprouts, horseradish, radish and watercress) were tested for myrosinase activity, glucosinolate content and isothiocyanate conversion rate. Allyl isothiocyanate was quantified in some food products. The method allows quantification of sinigrin, gluonasturtiin and allyl isothiocyanate after myrosinase compatible derivatisation in-vial by mercaptoacetic acid. The chromatograhpic separation takes 2.5 min (short-end injection) or 15 min (long-end injection). For the tested vegetables, measured myrosinase activity was between 0.960-27.694 and 0.461-26.322 µmol/min/mg protein, glucosinolate content was between 0-2291.8 and 0-248.5 µg/g fresh weight for sinigrin and gluconastrutiin, respectively. The possible specificity of plants to different glucosinolates was also shown. Allyl isothiocyanate release rate was different in different vegetables (73.13 - 102.13%). The method could also be used for quantification of allyl isothiocyanate from food products. The presented capillary electrophoresis method requires a minimal amount of sample and contains only a few sample preparation steps, and can be used in several applications (glucosinolate determination, myrosinase activity measurement, isothiocyanate release estimation). Copyright © 2016 John Wiley & Sons, Ltd.

Aphid-deprivation from Brassica plants results in increased isothiocyanate release and parasitoid attraction

In response to insect herbivory, plants release volatile blends that differ quantitatively, and in many cases also qualitatively, from those of undamaged plants. These altered blends can attract the herbivore’s natural antagonists, and such herbivore-induced volatile blends have often been interpreted as co-evolved plant–insect antagonist signals. When comparing volatile profiles of infested Brassica oleracea var. gemmifera L. plants (Brassicales: Brassicaceae), on which Brevicoryne brassicae L. (Hemiptera: Aphididae) aphids are feeding, with previously infested plants (i.e., deprived of aphids four hours prior to volatile collection), we found that the emission of a particular volatile compound, the glucosinolate breakdown product allyl isothiocyanate, was significantly higher in the latter. We used dual choice olfactometry to evaluate attractiveness of plants and aphids to Diaeretiella rapae (M’Intosh 1855) (Hymenoptera: Braconidae), a parasitoid of B. brassicae. Previously infested plants deprived of the herbivore attracted significantly more parasitoids than infested plants, although aphid odours per se proved to be attractive. Mechanical damage approximating aphid stylet insertion remained without effect on emission of allyl isothiocyanate and on parasitoid response. The unexpected higher parasitoid attraction to previously infested than infested plants is discussed here from the perspective of the plant and the parasitoid, as well as from the herbivore. Results suggest that while the host-finding mechanism of D. rapae has not evolved to allow this parasitoid a discrimination between herbivore-infested and previously infested plants, the herbivore seems able to suppress emission of a key volatile, thereby reducing its own olfactory detectability to its specialised natural antagonist.

Synergistic inhibition of the seed germination by crude glycerin and defatted oilseed meals

The main co-products of biodiesel and biolubricant chains are defatted oilseed meals and crude glycerin, generally considered as by-products and mainly used in animal feeding, bioenergy production and other minor uses. However, these co-products could have other interesting features, and high value added outlets could be developed in order to significantly improve the overall economic and environmental impact of the entire biodiesel chain. When defatted seed meals derive from some specific oilseed crops belonging to Brassicaceae they are characterized by high levels of glucosidic molecules potentially able to release biologically active compounds by means of an enzymatic hydrolysis reaction. The second biodiesel chain by-product, crude glycerin, contains glycerol and some impurities (including water) up to 20%. This manuscript reports in vitro and in vivo bioassay results on the application of crude glycerin and defatted seed meals of some Brassicaceae species (Brassica carinata, Brassica nigra, Barbarea verna, Crambe abyssinica) in seed germination inhibition. These features open new perspectives for obtaining bio-based products for weed control, starting from both main co-products of the biodiesel chain. Indeed, the results confirmed the inhibitory effect of crude glycerin and seed meals used alone and, moreover, showed a synergistic activity when combined. As expected, the inhibitory activity was correlated to glycerol concentrations and to the quali–quantitative release of isothiocyanates from defatted seed meals.

Suppression of Verticillium dahliae by glucosinolate-containing seed meal amendments

The biofumigation potential of glucosinolate (GSL)-containing seed meals to Verticillium dahliae was systematically evaluated. Cultivars of four different species of Brassicaceae were assessed using a standardized bioassay with microsclerotia-infested sterile quartz sand amended with defatted seed meals at a rate of 0.4 % (vol/vol). Additionally, the GSL profile and isothiocyanate (ITC) release of the meals were determined. Brassica napus and Sinapis alba seed meals failed to reduce the number of viable microsclerotia significantly due to low concentrations and/or toxicity of the ITCs released. Amendments with B. juncea and B. carinata seed meals eliminated all microsclerotia in the sterile sand. The meals had 2-propenyl GSL concentrations between 82.8 and 108.1 μmol g−1. ITC release efficiencies ranged between 62.5 and 100 %. The expected amounts of 2-propenyl ITC released by the amendments suggest that the elimination of microsclerotia by the sinigrin-containing seed meals was predominantly an ITC-related effect. But in natural infested soils the efficiencies of B. juncea seed meal amendments were reduced. Depending on soil characteristics practical meal amendment rates of 4.0 t ha−1 or higher may be necessary to achieve sufficient Verticillium control. Additionally, the temporal pattern of ITC release of the amended B. juncea seed meal in two natural soils compared to sterile sand was determined. Maximum ITC concentrations were measured between 3 and 6 h after application, before concentrations decreased continuously and reached a minimum after 48 h. In summary, the findings suggest that sinigrin-containing seed meal amendments can be a useful tool in the management of Verticillium, especially on sandy soils with low organic carbon contents.

Concentration- and time-dependent effects of isothiocyanates produced from Brassicaceae shoot tissues on the pea root rot pathogen Aphanomyces euteiches.

Isothiocyanates (ITCs) hydrolyzed from glucosinolates (GSLs) in Brassicaceae tissue are toxic to soil organisms. In this study, the effect of aliphatic and aromatic ITCs from hydrated dry Brassicaceae shoot tissues on the mycelium and oospores of the pea root rot pathogen Aphanomyces euteiches was investigated. The profile and concentrations of GSLs in two test Brassicaceae species, Sinapis alba and Brassica juncea, and the ITCs from the dominant hydrolyzed parent GSLs were monitored. The concentrations of dominant ITCs and pathogen exposure time were evaluated in in vitro experiments. The greatest effect on the pathogen was observed from aliphatic ITCs hydrolyzed from B. juncea tissue, and the effect depended on the ITC concentration and exposure time. ITCs were more effectively hydrolyzed from B. juncea GSLs than from S. alba GSLs; i.e., the ITC/GSL ratio was higher in B. juncea than in S. alba tissue, giving a different release pattern. The release of phenylethyl isothiocyanate, which was common to both species, followed a pattern similar to that of the dominant ITC in each crop species. This suggests that traits other than GSL content, e.g., plant cell structure, may affect the release of ITCs and should therefore influence the choice of species used for biofumigation purposes.

First evidence for a major cover crop effect on arbuscular mycorrhizal fungi and organic maize growth

Arbuscular mycorrhizal fungi are increasingly used in organic cropping systems to increase yields. Although cover crops are largely used in organic farming, there is little knowledge on the impact of cover crops on native mycorrhizal fungi. Here, we studied the effect of cover crop diversity on mycorrhizal colonization in subsequent organic maize cultivars differing in the level of genetic diversity. Experiments were conducted from 2010 to 2012 in a Mediterranean environment. First Indian mustard (Brassica juncea L. Czern.), hairy vetch (Vicia villosa Roth), a mix of seven cover crop species (Mix), and natural vegetation (Control) were cultivated as winter cover crops. Then, an organically and a conventionally bred maize hybrid and three organically bred composite cross populations were cultivated. Mycorrhizal propagule dynamics were measured. Results at juvenile stage show a higher mycorrhizal colonization in maize plants grown after hairy vetch, of 35.0 %, and Mix cover crops, of 29.4 %, compared to Indian mustard, of 20.9 %, and Control, of 21.3 %. The potential of soil mycorrhization decreased of 56.5 % following Indian mustard, higher than that of other cover crops, of 34.1–47.3 %. This finding could be explained by the release of isothiocyanates in soils. Moreover, maize shoot biomass, nitrogen, and phosphorus content across all maize genotypes at juvenile stage increased with mycorrhizal colonization. These findings provide the first evidence of the greater role played by cover crop identity in the enhancement of early mycorrhizal colonization of the subsequent crop and of soil mycorrhizal activity.

Release of isothiocyanates does not explain the effects of biofumigation with Indian mustard cultivars on nematode assemblages

While soil biota play an essential role in ecosystem services, the plant-pathogenic fraction may have a large, negative economic impact on food and feed production. For decades, the use of so-called fumigants (=general biocides) has been a common practice for controlling soil pathogens, including plant-parasitic nematodes. Due to their adverse environmental impact, many fumigants have been banned. Biofumigation – a possible alternative – encompasses the incorporation of mulched Brassicaceous debris into topsoil, and its mode of action is based on the conversion of glucosinolates (GSLs) into nematicidal isothiocyanates (ITCs). Contrary to the relatively well-characterized impact of biofumigation on plant-parasitic nematodes, the effects on the non-parasitic part of the community is largely unknown. We investigated the field effects of biofumigation with four Indian mustard (Brassica juncea) cultivars on both plant-parasitic and free-living nematodes. Prior to biofumigation, GSL contents of B. juncea were determined, and from this, the expected ITC concentrations in the topsoil were calculated. As positive controls, two concentrations of 2-propenyl ITC – corresponding to the average expected ITC concentration, and two times the concentration predicted for the highest producer – were directly applied to wheat (=non Brassica control) plots. Although biofumigation resulted in significant changes for most nematode taxa, none of these shifts could be attributed to the release of ITCs. Moreover, none of the two directly applied ITC concentrations resulted in effects on the nematode community distinct from the water control. We therefore conclude that the observed changes in nematode assemblages are related to intense mechanical disturbance, green manure and the absence of host plants for obligatory plant-parasitic nematodes, rather than to the release of ITCs.

Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts

The consumption of brassica sprouts as raw vegetables provides a fair amount of glucosinolates (GLs) and active plant myrosinase, which enables the breakdown of GLs into health-promoting isothiocyanates (ITCs). This study reports the determination of the main constituents related to human health found in edible sprouts of two Brassica oleracea varieties, broccoli and Tuscan black kale, and two Raphanus sativus varieties, Daikon and Sango. Radish sprouts exhibited the highest ability to produce ITCs, with Daikon showing the greatest level of conversion of GLs into bioactive ITCs (96.5%), followed by Sango (90.0%). Tuscan black kale gave a value of 68.5%, whereas broccoli displayed the lowest with 18.7%. ITCs were not the exclusive GL breakdown products in the two B. oleracea varieties, since nitriles were also produced, thus accounting for the lower conversion observed. Measuring the release of plant ITCs is a valuable tool in predicting the potential level of exposure to these bioactive compounds after the consumption of raw brassica sprouts.

The Impact of Biofumigation and Chemical Fumigation Methods on the Structure and Function of the Soil Microbial Community

Biofumigation (BIOF) is carried out mainly by the incorporation of brassica plant parts into the soil, and this fumigation activity has been linked to their high glucosinolate (GSL) content. GSLs are hydrolyzed by the endogenous enzyme myrosinase to release isothiocyanates (ITCs). A microcosm study was conducted to investigate the effects induced on the soil microbial community by the incorporation of broccoli residues into soil either with (BM) or without (B) added myrosinase and of chemical fumigation, either as soil application of 2-phenylethyl ITC (PITC) or metham sodium (MS). Soil microbial activity was evaluated by measuring fluorescein diacetate hydrolysis and soil respiration. Effects on the structure of the total microbial community were assessed by phospholipid fatty acid analysis, while the impact on important fungal (ascomycetes (ASC)) and bacterial (ammonia-oxidizing bacteria (AOB)) guilds was evaluated by denaturating gradient gel electrophoresis (DGGE). Overall, B, and to a lesser extent BM, stimulated microbial activity and biomass. The diminished effect of BM compared to B was particularly evident in fungi and Gram-negative bacteria and was attributed to rapid ITC release following the myrosinase treatment. PITC did not have a significant effect, whereas an inhibitory effect was observed in the MS-treated soil. DGGE analysis showed that the ASC community was temporarily altered by BIOF treatments and more persistently by the MS treatment, while the structure of the AOB community was not affected by the treatments. Cloning of the ASC community showed that MS application had a deleterious effect on potential plant pathogens like Fusarium, Nectria, and Cladosporium compared to BIOF treatments which did not appear to inhibit them. Our findings indicate that BIOF induces changes on the structure and function of the soil microbial community that are mostly related to microbial substrate availability changes derived from the soil amendment with fresh organic materials.

Duration of control of two soilborne pathogens following incorporation of above‐ and below‐ground residues of Brassica juncea into soil

The persistence of control of primary infections caused by two soilborne fungal plant pathogens, Rhizoctonia solani and Gaeumannomyces graminis var. tritici, following the incorporation of above‐ground parts (AP), below‐ground parts (BP) or both (AP+BP) of Brassica juncea into soil was examined through an experiment in controlled conditions. Control was quantified by measuring disease incidence in bioassays where inoculum was introduced at different dates after the incorporation of plant residues. All types of residue showed an unexpected long‐term persistence that lasted at least 13 days, while the predominant glucosinolates contained in AP (20·9 µmol sinigrin g−1 dry matter) and BP (2·3 µmol gluconasturtiin g−1 dry matter) were hydrolysed in less than 3 days. Temporal trends in the efficacy of the residues behaved mostly in a quadratic manner, suggesting that the noxious effect of residues may be attributable to the release of isothiocyanates during the first days following incorporation, but that other mechanisms are most likely to contribute to lasting persistence. Across all treatments, AP and AP+BP suppressed R. solani by 54 and 63%, respectively, and G. graminis var. tritici by 40 and 40%, respectively, compared with controls. While BP did not cause any additional detectable effect when combined with AP, they had a significant effect when incorporated alone (approximately 20% suppression of both species), suggesting the existence of a complex interaction between these two types of residue.

Factors that impact on the ability of biofumigants to suppress fungal pathogens and weeds of strawberry

This research aimed at identifying factors that improve the efficacy of biofumigants for soil disinfestation against strawberry ( Fragaria× ananassa) pathogens and weeds. In laboratory bioassays, volatiles released from the macerated roots of a Brassica rapa/ Brassica napus biofumigant crop were six times more effective at suppressing the growth of the strawberry pathogen Rhizoctonia fragariae than shoots. Furthermore, the toxicity of the volatiles from the biofumigant crop to R. fragariae increased as plant developmental stage progressed. These results related to the release of higher quantities and greater diversity of isothiocyanates (ITCs) from the roots of mature biofumigant plants than from their shoots. Separate bioassays showed that volatiles from the biofumigant crop suppressed the growth of six different soil-borne pathogens ( Alternaria alternata, Colletotrichum dematium, Cylindrocarpon destructans, Fusarium oxysporum, Pythium ultimum, Phytophthora cactorum, and R. fragariae) of strawberry and seven Trifolium spp. (clover) weeds. The suppression of Trifolium spp. by biofumigant volatiles did not relate to their seed size, but might relate to their hard-seededness or genotype. In the field, rotary incorporation of the biofumigant crop did not produce detectable levels of ITCs in soil; reduce the survival of the strawberry pathogens, P. cactorum and C. destructans; or affect populations of culturable soil microflora. Yet, it suppressed the growth of emerging weeds by 40% and the growth of P. cactorum by 20%. We hypothesise that allelochemicals other than ITCs, such as nitriles, or other biological mechanisms might also play a role in biofumigation. A key to improving the efficacy of biofumigation in the field seems to lie in the development of application technologies that can macerate and incorporate biofumigants evenly in soils, in addition to incorporating biofumigants under optimal edaphic conditions for release of ITCs.

A new method for collecting isothiocyanates released from plant residues incorporated in soil

AbstractA method was developed for collecting isothiocyanates (ITCs) from compost and their detection by gas chromatography–mass spectrometry. Identification of the compounds was based on retention time, molecular weight and previously published information (Wiley Database, Hewlett Packard ChemStation Library). The release of ITCs from freeze‐dried plant material of Brassica carinata, Cleome spinosa and Tropaeolum majus cultivars was investigated by incorporation in compost contained in pots. Allyl isothiocyanate, methyl isothiocyanate and benzyl isothiocyanate, respectively, were released after hydrolysis of plant tissues. Abundance of ITCs was monitored for 12 days and was shown to decrease rapidly following incorporation until the fourth day for all types of plant tissue.