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.     

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

A 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.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

The zinc complex catalyzed hydration of alkyl isothiocyanates

Based upon our preceding studies of the hydration of CO₂, COS and CS₂, accelerated by the carbonic anhydrase (CA) using simplified [ZnL₃OH]⁺ complexes as model catalysts, we calculated the hydration mechanisms of both the uncatalyzed and the [ZnL₃OH]⁺-catalyzed reactions (L = NH₃) of isothiocyanates RNCS on the B3LYP/6-311+G(d,p) level of theory. Interestingly, the transition state for the favored metal mediated reaction with the lowest Gibbs free energy is only slightly higher than in the case of CO₂ (depending on the attacking atom (N or S). Calculations under inclusion of solvent corrections show a reduction of the selectivity and a slight decrease of the Gibbs free energy in the rate-determining steps. The most plausible pathway prefers the mechanism via a Lindskog proton-shift transition state leading to the thermodynamically most stable product, the carbamatic-S-acid. Furthermore, powerful electron withdrawing substituents R of the cumulenic substrates influence the selectivity of the reaction to a significant extent. Especially the CF₃-group in trifluoromethylisothiocyanate reverses the selectivity. This investigation demonstrates that reaction principles developed by nature can be translated to develop efficient catalytic methods, in this case presumably for the transformation of a wide variety of heterocumulenes aside from CO₂, COS and CS₂. Figure Competing transition structures for the [ZnL₃OH]⁺-mediated activation of isothiocyanates Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transthiocarbamoylation of proteins by thiolated isothiocyanates

Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examined the effect of sulfhydryl molecules on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we observed significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addition, the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chemical insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl molecules by 6-HITC-ME and observed that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide containing Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins.     

Water flow induced transport of Pseudomonas fluorescens cells through soil columns as affected by inoculant treatment

Abstract Water flow induced transport of Pseudomonas fluorescens cells through soil columns was measured as affected by the inoculant treatment. Bacterial cells were introduced into the topsoil of columns, either encapsulated in alginate beads of different types or mixed with bentonite clay in concentrations ranging from 0.5 to 5.0% (w/v). Survival of bacterial cells was improved with the use of alginate or bentonite. Transport, as determined by destructive sampling of the columns, was reduced with the use of alginate encapsulation. Drying of the beads had no influence on transport. The presence of bentonite in the topsoil, either pre-mixed through the soil, or applied as a slurry together with the bacteria, also reduced transport, except when 0.5% was pre-mixed through the soil. P. fluorescens cells encapsulated in alginate beads prepared with water and supplemented with skim milk powder and bentonite showed the best survival during the time of the experiment and the most reduced transport compared to the control. Therefore, cells encapsulated in this way are suitable, due to their optimal survival and reduced spread, for use in a field experiment with genetically manipulated bacteria.     

Leaching of Pseudomonas aeruginosa, and transconjugants containing pR68.45 through unsaturated, intact soil columns

Abstract Leaching of genetically engineered microbes (GEMs) through soil is a significant concern related to groundwater quality. The objective of this study was to examine the leaching, survival and gene transfer of a genetically engineered microbe and indigenous recipients of pR68.45 in nonsterile, undisturbed soil columns. Pseudomonas aeruginosa PAO25, containing the plasmid R68.45, was added to the surface of undisturbed soil columns (10 cm diameter × 80 cm length). Unsaturated flow conditions were maintained by 100 ml daily additions of 2 mM CaCl₂ for a period of 70 days. The population of the GEM exhibited a significant ( P = 0.05) linear decline with time. The GEM leached only to a depth of 30–40 cm in 70 days. Transfer of pR68.45 was shown to occur from P. aeruginosa into the indigenous bacterial population although relatively low numbers of transconjugants were observed (log 2 cfu g⁻¹ dry soil). The number of transconjugants also decreased with depth and time. Leaching of transconjugants, however, occured more readily than that of the GEM, probably as a result of plasmid transfer into smaller, more mobile bacteria. At 70 days incubation, no GEMs were detected in the columns, while transconjugants were observed at several depths. These results demonstrate the importance of examining both the survival and movement of GEMs and transconjugants in soil.     

Glucosinolates and biofumigation: fate of glucosinolates and their hydrolysis products in soil

The bioactive hydrolysis products of glucosinolates, particularly the isothiocyanates, can be used to control soil pests and weeds by incorporating glucosinolate-containing plant material in soil—a practice known as biofumigation. The fate of glucosinolates and their hydrolysis products in soil determines both the efficacy and environmental impact of biofumigation. Knowledge of the processes by which these compounds are sorbed, degraded or otherwise lost from the soil is fundamental to developing effective, but environmentally benign biofumigation strategies. Effective biofumigation relies on maximum hydrolysis of the glucosinolate in the plant tissue to generate high isothiocyanate concentrations in the soil after incorporation. This is favoured by maximum cell disruption, by addition of water, and a high soil temperature. Residual glucosinolates are very weakly sorbed, readily leached and are microbially degraded and mineralised in soil. In contrast, isothiocyanates are strongly sorbed by the organic matter in soil, react strongly with nucleophilic groups present in soil, and are prone to volatilization losses in addition to microbial degradation and mineralisation. These loss processes are influenced by soil type, water content and temperature. Using appropriate incorporation strategies, sufficiently high isothiocyanate concentrations (>100 nmol g⁻¹) can be achieved in soil using biofumigation for effective suppression of susceptible pests. The relatively rapid sorption and degradation of the isothiocyanates in the period of days after incorporation minimizes the risks of persistence in the environment or leaching. Biofumigation is therefore a promising technique which can be further developed to form part of IPM (Integrated Pest Management) strategies to reduce reliance on synthetic pesticides with minimal unintended impacts on the environment.     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Regulation and function of specifier proteins in plants

Specifier proteins are responsible for the diversification of biologically active products formed upon myrosinase-catalyzed glucosinolate hydrolysis and are therefore assumed to have an impact on the defensive function of the glucosinolate–myrosinase system. Among glucosinolate hydrolysis products, the generation of epithionitriles and organic thiocyanates requires the presence of epithiospecifier protein (ESP) and thiocyanate-forming protein (TFP), respectively, while myrosinase alone is sufficient for the production of isothiocyanates. Both ESP and TFP also promote the formation of simple nitriles upon myrosinase-catalyzed glucosinolate hydrolysis. Only little is known about the biological effects of epithionitriles and thiocyanates. Moreover, simple nitriles have repeatedly been reported to be less toxic to plant pathogens and herbivorous insects than the correponding isothiocyanates. Thus, it has remained an open question how plants benefit from the presence of specifier proteins. In this review, we survey the biological effects of different types of glucosinolate hydrolysis products on insects and pathogens as well as the current knowlegde on the developmental, organ specific and stimuli-mediated regulation of specifier proteins. Integrating these findings can help us to better understand the ecological functions of plant specifier proteins as well as the co-evolution of glucosinolate-containing plants and their insect herbivores.     

Leaching of Cryptosporidium parvum oocysts, Escherichia coli, and a Salmonella enterica serovar Typhimurium bacteriophage through intact soil cores following surface application and injection of slurry

Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.     

Leaching losses of inorganic N and DOC following repeated drying and wetting of a spruce forest soil

Forest soils are frequently subjected to dry–wet cycles, but little is known about the effects of repeated drying and wetting and wetting intensity on fluxes of , and DOC. Here, undisturbed soil columns consisting of organic horizons (O columns) and organic horizons plus mineral soil (O + M columns) from a mature Norway spruce stand at the Fichtelgebirge; Germany, were repeatedly desiccated and subsequently wetted by applying different amounts of water (8, 20 and 50 mm day⁻¹) during the initial wetting phase. The constantly moist controls were not desiccated and received 4 mm day⁻¹ during the entire wetting periods. Cumulative inorganic N fluxes of the control were 12.4 g N m⁻² (O columns) and 11.4 g N m⁻² (O + M columns) over 225 days. Repeated drying and wetting reduced cumulative and fluxes of the O columns by 47–60 and 76–85%, respectively. Increasing (0.6–1.1 g N m⁻²) and decreasing fluxes (7.6–9.6 g N m⁻²) indicate a reduction in net nitrification in the O + M columns. The negative effect of dry–wet cycles was attributed to reduced net N mineralisation during both the desiccation and wetting periods. The soils subjected to dry–wet cycles were considerably drier at the final wetting period, suggesting that hydrophobicity of soil organic matter may persist for weeks or even months. Based on results from this study and from the literature we hypothesise that N mineralisation is mostly constrained by hydrophobicity in spruce forests during the growing season. Wetting intensity did mostly not alter N and DOC concentrations and fluxes. Mean DOC concentrations increased by the treatment from 45 mg l⁻¹ to 61–77 mg l⁻¹ in the O tlsbba columns and from 12 mg l⁻¹ to 21–25 mg l⁻¹ in the O + M columns. Spectroscopic properties of DOC from the O columns markedly differed within each wetting period, pointing to enhanced release of rather easily decomposable substrates in the initial wetting phases and the release of more hardly decomposable substrates in the final wetting phases. Our results suggest a small additional DOC input from organic horizons to the mineral soil owing to drying and wetting.     

High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet–visible (UV–Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV–Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.     

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis–Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts.     

The synthesis and enzymic hydrolysis of (E)-2-[2,3-2H2]propenyl glucosinolate: confirmation of the rearrangement of the thiohydroximate moiety

(E)-2-[2,3-2H2]propenyl glucosinolate was synthesised starting from (E)-[3,4-2H2]but-3-en-1-ol, which was produced by reduction of but-3-yn-1-ol with deuterium gas in the presence of Lindlar's catalyst. The synthesis of (E)-2-[2,3-2H2]propenyl glucosinolate was completed via the nitro intermediate to form the basic desulphoglucosinolate skeleton. The (E)-2-[2,3-2H2]propenyl glucosinolate was fully characterised and deuterium NMR spectroscopy used to examine the rearrangement of the thiohydroximate to the isothiocyanate and thiocyanate.     

Leaching of phage from Class B biosolids and potential transport through soil

The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium.     

Trichoderma spp. tolerance to Brassica carinata seed meal for a combined use in biofumigation

Biofumigation by Brassicaceae green manure or seed meal incorporation into soil is an ecological alternative to chemical fumigation against soil-borne pathogens, based on the release of glucosinolate-derived compounds. This study aimed at investigating the tolerance of the beneficial fungus Trichoderma to these compounds in view to combined utilization with Brassica carinata seed meal (BCSM). Forty isolates of Trichoderma spp. were tested in vitro for tolerance to toxic volatiles released by BCSM and in direct contact with the meal. They were found to be generally less sensitive than the assayed pathogens (Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum), even if a fungistatic effect was observed at the highest dose (10 μmole of sinigrin). Most of them also were able to grow on BCSM and over the pathogens tested. A preliminary experiment of integrating BCSM with Trichoderma in soil was carried out under controlled conditions with the patho-system P. ultimum—sugar beet. BCSM incorporation increased pathogen population, but reduced disease incidence, probably due to indirect mechanisms. The greatest effect was achieved when BCSM was applied in combination with Trichoderma, regardless of meal ability to release isothiocyanate. These findings suggest that disease control can be improved by this integrated approach. This study also highlighted that a reduction of allyl-isothiocyanate concentration in soil could occur due to the activity of some Trichoderma isolates. This effect could protect resident or introduced Trichoderma isolates from depressing effects due to the biocidal compounds, but, on the other hand, could reduce the efficacy of biofumigation against target pathogens.     

Leaching of boron through sewage sludge amended soil: the role of clinoptilolite

A laboratory experiment was conducted to determine the release of boron from soil-sewage sludge mixtures by leaching using a clinoptilolite type natural zeolite, before land application of the sewage sludge. Soil columns were filled up with the clinoptilolite soil after mixing with sewage sludge at a rate of 30 tons ha(-1) and with two different particle sizes (0.1-0.25 and 1.0-2.0 mm) of clinoptilolite each at the concentrations of 1% and 2%. The particle size and the application rate of clinoptilolite affected both boron leaching from soil compared to the control treatment (soil and sewage sludge mixture). The total soluble boron leached from a soil column varied from 66-92% depending on the applications of clinoptilolite and reached 96% for the control treatment, following application of 80 cm depth of water in all treatments. In the cases of the 1% application rate of 0.1-0.25 and 1-2 mm sized clinoptilolite 78% and 92% of the total boron leached, respectively. While at 2% application rate of 0.1-0.25 and 1-2 mm zeolite, 66% and 87% of total soluble boron leached, respectively. Boron concentrations in the soil layers increased as application rate increased and particle size of clinoptilolite decreased because of its high adsorption capacity. Adsorption isotherms indicated that clinoptilolite had a high adsorption capacity for boron compared to the sewage sludge and soil.     

The spatial variability of soil resources following long-term disturbance

The spatial distributions of selected soil properties in two adjacent sites in southwest Michigan were examined to evaluate the potential effects of chronic disturbance on resource heterogeneity. One site was a cultivated field that had been cleared, plowed, and cropped annually for decades prior to sampling while the other, uncultivated field was cleared of original forest in 1960 after which it was mown annually but never plowed or cropped. We took replicate samples from a 330-point unaligned grid across the sites for soil pH, gravimetric moisture, inorganic phosphorus, total carbon, and net nitrification and nitrogen mineralization potentials. Soils in the cultivated site contained less than half as much carbon as in the uncultivated site, but had higher levels of inorganic phosphorus and moisture, and higher soil pH. Potential net nitrogen mineralization and nitrification rates did not differ between sites. Geostatistical analysis showed that almost all properties examined were strongly autocorelated within each site; structural variance as a proportion of sample variance ranged from 30–95% for all properties, and for any given property differed little between sites. The distance over which this dependence was expressed, however, was for all properties but pH substantially less in the uncultivated site (7–26 m) as compared to the tilled site (48–108m), especially for total C and net nitrification and N mineralization. These results suggest that the spatial pattern and scale of soil variability can differ markedly among edaphically identical sites and that these differences can be related to disturbance history.     

Nematicidal effect of Lepidium sativum on activity and reproduction of potato cyst nematode Globodera rostochiensis in soil

Abstract

In Iran, potato cyst nematode (Globodera rostochiensis) jeopardizes the traditionally high yields of potatoes in Hamadan Province in the west of Iran. Biofumigation is an eco-friendly method for integrated management of plant parasitic nematodes. In the laboratory, water extracts of water cress, fenugreek and dill similarly reduced viability of second stage juveniles after 3?h of exposure, and decreased hatching of encysted eggs to less than 1%. Pre-treatment and combined tests similarly decreased hatch. The nematicidal efficiency of top green manure of Lepidium sativum on the survival of nematode was tested on a susceptible cv in microplots. The weights of biofumigated plants increased. Anti-hatching properties of water cress applied as a biofumigant reduced hatch by average of 56%. Reproduction rates were lowered to below one, and final populations of cysts and their egg contents were reduced by nearly 60% in treated soil. Biofumigation at a 1% amendment rate was sufficient to bring about these results, which were comparable with those achieved with 2 and 3% rates. Nematicidal isothiocyanates released after incorporating glucosinolate-containing brassica plants are fully biodegradable and less toxic than their synthetic equivalents, and their use is considered a safer alternative to soil fumigants such as methyl bromide.