Inhibition of soil nitrifying bacteria communities and their activities by glucosinolate hydrolysis products

Transcription

1 Soil Biology & Biochemistry 32 (2000) 1261± Inhibition of soil nitrifying bacteria communities and their activities by glucosinolate hydrolysis products Gary D. Bending*, Suzanne D. Lincoln Department of Soil and Environment Sciences, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 13 July 1999; received in revised form 30 November 1999; accepted 23 February 2000 Abstract During microbial degradation of crucifer tissues in soil, a range of low molecular weight volatile S-containing compounds is produced. While a number of these compounds are known to have potent nitri cation inhibiting properties, the e ects of isothiocyanates (ITCs), which are derived from glucosinolates, are not known. We investigated the e ects of glucosinolate hydrolysis products on communities and activities of nitrifying bacteria in bioassays using contrasting sandy- and clay-loam soils. In both soils, ITCs reduced populations of NH 4 + -oxidizing bacteria and inhibited their growth. ITCs had no apparent inhibitory e ect on populations of NO 2 -oxidizing bacteria in sandy-loam, but did reduce growth of these bacteria in clay-loam. Individual application of an aliphatic and an aromatic ITC inhibited nitri cation of applied NH 4 + in the two soils, with the e ect being longer lived in sandy-loam relative to clay-loam. After 42 days, mineralization of N in sandy-loam amended with 2- phenethyl-itc was greater than in unamended soil, suggesting that this compound had a general fumigant e ect on the soil microbiota. ITCs were more e ective inhibitors of nitri cation than intact glucosinolates or nitriles. Phenyl-ITC was found to be the most toxic of the ITCs tested, but generally there were no di erences between the nitrifying inhibitory properties of aliphatic and aromatic ITCs. The capacity of 2-propenyl-ITC to inhibit nitri cation was shown to be less than that of dimethyldisulphide. However, when concentrations of 2-propenyl-ITC and dimethyl-sulphide, which had no e ect on nitri cation when applied to soil individually, were mixed, nitri cation was strongly inhibited. No such synergistic interaction was found for either of these compounds with dimethyl-disulphide. The signi cance of these ndings is discussed Elsevier Science Ltd. All rights reserved. Keywords: Nitrifying bacteria; Nitri cation; Isothiocyanate; Biofumigation; Glucosinolate 1. Introduction Plant tissues contain a great variety of secondary metabolites, with the speci c amounts and composition varying according to family and species, and the physical and chemical environment of the location in which individuals are growing (Bennett and Wallsgrove, 1994). The functions of many of these compounds within the living plant are generally understood, with most compounds considered * Corresponding author. Tel.: ; fax: address: gary.bending@hri.ac.uk (G.D. Bending). to act as defences against herbivores, pests or pathogens (Bennett and Wallsgrove, 1994). Secondary compounds are generally turned over rapidly within the plant, and with the notable exception of certain phenolic compounds, are mobilized and withdrawn from tissues undergoing senescence, so that in natural circumstances, concentrations of the secondary compounds in plant materials returned to soil are low (Harbourne, 1997). However, in the case of green manures and crop residues, plant tissues incorporated into soil contain their full complement of secondary compounds. In such situations, these compounds could in uence the activities of the soil microbiota, and thus the rates of mineralization processes. While the e ects of phenolic and /00/$ - see front matter Elsevier Science Ltd. All rights reserved. PII: S (00)

2 1262 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269 terpenoid compounds contained in leaf litter on the soil biota have been studied (Zucker, 1983; Bremner and McCarty, 1993), little is known of the e ects of other secondary compounds contained in plant material returned to soil on soil organisms, or of possibilities for manipulating pro les of secondary compounds to control mineralization processes. It is well known that crucifer tissues have toxic e ects following incorporation into soil, resulting in inhibition of various fungal pathogens, plant parasitic nematodes, and seed germination (Brown and Morra, 1997). Further, there is evidence that the rate of mineralization of N from crucifer crop residues is slower than would be expected from their C-to-N ratios (Bending et al., 1998). The toxic properties of crucifer tissues have been attributed to the combined action of isothiocyanates (ITCs) derived from glucosinolates, and low molecular weight (MW) non-glucosinolate derived sulphur compounds, which are generated following incorporation of crucifer tissues into soil (Bending and Lincoln, 1999). Since these compounds are highly volatile, toxic e ects can occur at spatial locations that are removed from the point of origin, with the result that the process has been termed `biofumigation'(angus et al., 1994). Glucosinolates are a family of S-containing secondary compounds found in the Crucifereae. On tissue damage, glucosinolates, which are stored in the cell vacuole, come into contact with thioglucosidases, which are located in the cell wall, cytoplasm or in separate cells, (Poulton and Moller, 1993), and are hydrolysed to a number of toxic hydrolysis products, including ITCs and nitriles (Cole, 1976). The nature of the products formed depends on the types of glucosinolate present and the physical and chemical environment under which hydrolysis takes place (Fenwick et al., 1983). Bending and Lincoln (1999) demonstrated that quantities of ITC are produced during the early stages of decomposition of crucifer tissues in soil. Other volatile low MW sulphur compounds are formed following incorporation of crucifer tissues in soil, including dimethyl-sulphide, dimethyl-disulphide, carbon-disulphide and methanethiol (Lewis and Papavizas, 1970; Bending and Lincoln, 1999). These compounds are formed during microbial degradation of S- containing amino acids and sulphoxides, which are abundant in crucifer tissues (Banwart and Bremner, 1975). A number of the non-glucosinolate derived low MW S compounds produced during breakdown of crucifer tissues in soil, including carbon-disulphide and dimethyl-disulphide, are known to be highly e ective inhibitors of nitri cation, even at low concentrations (Powlson and Jenkinson, 1971; Bremner and Bundy, 1974). Although ITCs are considered to be more toxic than non-glucosinolate derived volatile S compounds (Lewis and Papavizas, 1971), their e ects on nitri cation processes are not known. Our aim was to determine the extent to which glucosinolate hydrolysis products act as nitri cation inhibitors, and also to determine the most e ective hydrolysis products, the concentrations at which the compounds are toxic, and the longevity of inhibitive e ects. Additionally, we investigated whether the inhibitive properties of ITCs are a ected by interaction with other low MW volatile sulphur compounds which are also produced during crucifer decomposition in soil. 2. Materials and methods 2.1. Soils Two contrasting soils were used in the study. Sandyloam soil was collected from the top 20 cm of a fallow eld at Wellesbourne, Warwickshire, UK. The soil is of the Wick series, with 14% clay, ph of 5.9 and an organic-c content of 0.8%. Clay-loam was collected from the top 20 cm of a fallow eld at Kirton, Lincolnshire, UK. This soil is of the Romney series, with 23% clay, ph of 7.5, and an organic-c content of 1%. Each soil was sieved (2 mm), air dried, and stored at 48C for up to two months. In each experiment, soil was moistened to 480 kpa and incubated at 158C for 5 days before use E ect of isothiocyanates on communities of nitrifying bacteria and nitri cation Sandy-loam and clay-loam soils were amended with a solution of (NH 4 ) 2 SO 4 to give a concentration of 80 mg NH 4 + ±N g 1 fw soil. Stock solutions of 2-propenyland phenethyl-itc, (Aldrich Chemical Company, Dorset, UK) were prepared by adding 10 mg of the pure compound to 5 ml distilled H 2 O, and sonicating for 30 min (Williams et al., 1993). Twenty g fw samples of soil were dispensed into 60 ml polystyrene containers and treated with 100 ml aliquots of the stock solution, to give a moisture content of 126 kpa and an allelochemical concentration of 10 mg g 1 dw soil. A control treatment in which distilled H 2 O replaced ITC was also included. Estimates of potential amounts of ITC that could be generated from crucifer green manures, based on incorporation in soil to 15 cm depth, and using equivalents of 2-propenyl-ITC derived from 2- propenyl-glucosinolate, range between 20 and 56 mg ITC g 1 soil (Williams et al., 1993; Kirkegaard and Sarwar, 1998). After mixing the soil thoroughly, the containers were incubated in the dark at 158C within 15 l plastic

3 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261± tubs, through which moist air was continually passed to ensure an aerobic environment. Every 7th day over 42 days, ve replicate containers from each treatment were taken for determination of NH + 4 ±N, NO 2 ±N and NO 3 ±N. Ten g fw soil was shaken with 50 ml 0.5 M K 2 SO 4 for 30 min, and the suspension ltered through a Whatman No. 1 lter. NH + 4 ±N, NO 2 ±N and NO 3 ±N were quanti ed using an EnviroFlow 5012 ow injection system (Tecator AB, Sweden). After 1, 3 and 7 days, ve separate replicates of each soil were harvested, and populations of nitrifying bacteria determined using the Most Probable Number Method, using media selective for NH and NO 2 - oxidizing bacteria (Alexander, 1982; Schmidt and Belser, 1982). No measurement was made of the heterotrophic nitrifying population. After 7 days, concentrations of ITCs remaining were determined using the method of Brown et al. (1991). Five g fw of soil was placed into a glass screw-top jar, to which was added 2.5 ml 100 mm CaCl 2 and 5 ml dichloromethane (DCM) containing 5 mg ml 1 benzyl- ITC (Aldrich Chemical Company, Dorset, UK) as an internal standard. After shaking the suspension for 30 min, the samples were centrifuged at 1500 rpm for 5 min. The DCM layer was removed using a Pasteur pipette, and ITCs were analysed by g.c. using a Hewlett±Packard Sigma 3 gas chromatograph tted with a BP-10 capillary column (12 m 530 mm 680 mm, SGE, UK). Injector and detector temperatures were set at 2508C, and ITCs were eluted using a programme to increase column temperature from 30 to 2008C in 18 min. Compounds were detected using a ame ionization detector In uence of isothiocyanate concentration on nitri cation Using the procedures described above, sandy-loam and clay-loam soils were each amended with 80 mg NH + 4 ±N g 1 fw soil, and 2-propenyl-ITC and phenethyl-itc stock solutions and/or distilled H 2 O were added to give ITC concentrations ranging from 0 to 20 mg g 1 dw soil, and a moisture content of 126 kpa. For each soil a control treatment receiving distilled H 2 O was also included. There were ve replicates for each treatment. Soils were incubated under the conditions described above. After 21 days, at which time approximately 20% of the added NH + 4 remained in the control treatment, ve replicate containers from each treatment were taken for NO 3 ±N analysis, as described above E ects of glucosinolate-derived allelochemicals on nitri cation The e ect of a variety of glucosinolate hydrolysis products on nitri cation was investigated. Six ITCs including aliphatic and aromatic types, an aliphatic and an aromatic nitrile, together with intact 2-propenyl-glucosinolate were used (Table 1). Twenty g fw samples of sandy-loam, amended with 80 mg NH 4 + ±N g 1 fw soil, were dispensed into 60 ml polystyrene containers and treated with 100 ml aliquots of ITC stock solution, to give a moisture content of 126 kpa, and an allelochemical concentration of 10 mg g 1 dw soil. A control treatment receiving distilled H 2 O was also included. To investigate the e ect of time of exposure on the inhibitory properties of the compounds, additional treatments, in which lids were screwed tightly onto containers to stop volatilization, were included for control, 2-propenyl-ITC, phenethyl-itc and 3- butene-nitrile treatments. Soils were incubated for 21 days under the conditions described above before determination of NO 3 ±N Interaction of isothiocyanates with nonglucosinolate derived volatile S compounds The e ect on nitri cation of the interaction between ITC and other non-glucosinolate derived low MW volatile S compounds, which are also produced during decomposition of crucifer tissues in soil, was investigated. Twenty g fw samples of sandy-loam were placed into 60 ml polystyrene containers and amended to give 100 mg NH 4 + ±N g 1 fw soil and a moisture content of 126 kpa. Aluminium lids were screwed rmly onto the containers. A syringe needle was inserted through the lid of the container and aliquots of 2-propenyl-ITC, dimethyl-sulphide and dimethyldisulphide injected onto the wall of the container, approximately 1 cm above the soil surface, to give a headspace concentration equivalent to 10 mg g 1 dw soil. The holes in the lids were sealed with blu-tak, and the containers left for 30 min at room temperature, by which time the compounds had volatilized. Containers were set up with each compound singly, and in all possible combinations. Unamended control soil was also included. The soils were shaken and then incubated at 158C for 21 days before analysis of NO 3 ±N Statistical analysis Experiments were repeated twice, with similar results obtained each time. Results from only one experiment are presented. Signi cance of di erences between treatments were determined by analysis of variance. In the case of analysis of the e ects of 2-propenyl- and phenethyl-itc on nitri cation over a 42-day period, data was not normally distributed and was subject to log transformation to stabilise the variance. None of the other datasets required transformation. Statistical

4 1264 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269 Table 1 Properties and occurrence of the allelochemicals used in the study Allelochemical Structural formula MW Common occurrence of parent glucosinolate a 2-Propenyl-glucosinolate CH 2 1CHCH 2 CSNO S O 3 (glucose) Black mustard, brown mustard, Indian mustard, cabbage, brussels sprout, cauli ower Methyl-isothiocyanate CH 3 NCS 73.1 Horseradish, Indian mustard, cauli ower 2-Propenyl-isothiocyanate CH 2 1CHCH 2 NCS 99.2 (as 2-propenyl-glucosinolate) Butyl-isothiocyanate CH 3 (CH 2 ) 3 NCS Horseradish, Indian mustard, cabbage, brussels sprout Phenyl-isothiocyanate C 6 H 5 NCS Horseradish, Indian mustard Benzyl-isothiocyanate C 6 H 5 CH 2 NCS Indian cress, nasturtium, horseraddish, Indian mustard, raddish, cabbage, brussels sprout Phenethyl-isothiocyanate C 6 H 5 CH 2 CH 2 NCS Watercress, Indian mustard, radish, cabbage, brussels sprout, cauli ower 3-Butene-nitrile CH 2 1CHCH 2 CN 67.1 (as 2-propenyl-glucosinolate) 3-Phenyl-propionitrile C 6 H 5 CH 2 CH 2 CN (as phenethyl-isothiocyanate) a (Fenwick et al., 1983). di erences between treatments were compared by LSD using the transformed data. 3. Results 3.1. E ect of isothiocyanates on communities of nitrifying bacteria and nitri cation Application of phenethyl-itc for 3 days reduced the population of NH 4 + -oxidizing bacteria in sandy-loam (Fig. 1a). Growth subsequently recovered, but after 7 days the size of the population remained lower than in the unamended control soil. 2-Propenyl-ITC did not signi cantly reduce the size of the NH 4 + -oxidizing bacteria population, but it did inhibit growth of the population between 3 and 7 days. Application of both ITCs reduced populations of NH 4 + -oxidizing bacteria in clay-loam (Fig. 1b). 2-Propenyl-ITC inhibited recovery of the population for longer than phenethyl-itc. Although 2-propenyl-ITC had no signi cant e ect on populations of NO 2 -oxidizing bacteria in sandyloam, phenethyl-itc appeared to stimulate the population in the rst 3 days (Fig. 2a). Application of ITCs to the clay-loam did not diminish the population of NO 2 -oxidizing bacteria (Fig. 2b). However, both ITCs reduced the growth rate of this population between 1 and 3 days following application. In sandy-loam, application of ITCs reduced the metabolism of applied NH 4 + after 14 days, causing a delay in the assimilation of NH 4 + for at least 42 days following application to soil (Fig. 3a). Metabolism of applied NH 4 + was rapid in the clay-loam, with amounts of the compound becoming very low in all treatments after 14 days (Fig. 3b). However, the rate of NH 4 + assimilation was signi cantly slower in soils treated with ITCs. Fig. 1. Populations of NH 4 + -oxidizing bacteria in control unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-itc (Q). (a) Sandy-loam; (b) Clay-loam.

5 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261± Fig. 2. Populations of NO 2 -oxidizing bacteria in control unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-itc (Q). (a) Sandy-loam; (b) Clay-loam. ITCs inhibited nitri cation in both soils. In the case of sandy-loam, formation of NO 3 was delayed in soil treated with the ITCs during the rst 35 days following application (Fig. 4a). However, after 42 days, NO 3 in soil treated with 2-phenethyl-ITC was signi cantly higher than in the control soil. There was no di erence between the amounts of NO 3 formed in the control and 2-propenyl-ITC treated soils at this time. Application of 2-propenyl-ITC inhibited nitri cation in clayloam for at least 14 days following application, while addition of phenethyl-itc signi cantly inhibited nitri- cation for at least 35 days (Fig. 4b). In all cases, soil NO 2 ±N pools were less than 1 mg g 1 fw soil (data not shown). The total mineral N pool for each treatment was determined at each harvest, by combining the NH 4 + ± N, NO 2 ±N and NO 3 ±N pools (data not shown). It was found that the mineral±n pool in clay-loam treated with phenethyl-itc was signi cantly lower (P < 0.05) than that in unamended soil between 21 and 35 days. Also, the mineral-n pool of sandy-loam was signi cantly higher (P < 0.05) in soil treated with 2-propenyl-ITC, relative to control soil, after 42 days. At all other times, there were no signi cant di erences between the sizes of the total mineral-n pools in ITCtreated and unamended soil. After 7 days, none of the ITCs were detected in any of these soils In uence of isothiocyanate concentration on nitri cation 2-Propenyl-ITC signi cantly reduced nitri cation at Fig. 3. Metabolism of applied NH 4 + in unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-itc (Q). From top to bottom, signi cance of di erences between control and 2-propenyl-ITC, control and phenethyl-itc and propenyl-itc and phenethyl-itc, respectively; : signi cantly di erent (P < 0.05), ns: not signi cantly di erent. Bars represent +/ standard error of the mean. (a) Sandy-loam; (b) Clay-loam.

6 1266 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269 Fig. 4. Formation of NO 3 in unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-itc (Q). From top to bottom, signi cance of di erences between control and 2-propenyl-ITC, control and phenethyl-itc and propenyl-itc and phenethyl-itc, respectively; : signi cantly di erent (P < 0.05), ns: not signi cantly di erent. Bars represent +/ standard error of the mean. (a) Sandy-loam; (b) Clay-loam. a concentration of 0.5 mg g 1 dw soil, while phenethyl- ITC caused signi cant inhibition of nitri cation at concentrations down to 2.5 mg g 1 dw soil (Fig. 5). However, at concentrations above 5 mg g 1 dw soil, ITC type made no signi cant di erence to the degree of inhibition of nitri cation. Increasing the concentration of phenethyl-itc enhanced the degree of nitri- cation inhibition up to 10 mg g 1. However, in the case of 2-propenyl-ITC, increasing concentration above 5 mg g 1 soil had no further inhibitive e ect E ect of allelochemicals on nitri cation The e ect of the glucosinolate-derived chemicals on nitri cation is shown in Fig. 6. All the compounds signi cantly inhibited nitri cation. While the ITCs inhibited nitri cation by 35±65%, the nitriles caused 10± 15% inhibition. The intact glucosinolate induced over 20% inhibition. Increasing length of the side chain of the aromatic or aliphatic ITC had no apparent e ect on inhibitory properties. Additionally, there was little di erence between the inhibitory e ect of aromatic and aliphatic isothiocyanates, although phenyl-itc caused markedly more inhibition than the other ITCs. Preventing escape of the glucosinolate-derived compounds by capping bottles caused 27% and 7% more inhibition of nitri cation by 2-propenyl- and phenethyl-itc, respectively (signi cant P < 0.05). However, capping had no e ect on the inhibitive properties of 3-butene-nitrile. Fig. 5. In uence of 2-propenyl-ITC (T) and phenethyl-itc (Q) concentration on nitri cation of added NH 4 + after 21 days in sandyloam. Fig. 6. Inhibition of nitri cation of added NH 4 + by aliphatic and aromatic isothiocyanates (ITC), nitriles and intact 2-propenyl-glucosinolate after 21 days (Me-I, methyl-itc; Pr-I, 2-propenyl-ITC; Bu- I, butyl-itc; Bz-I, benzyl-itc; Ph-I, phenyl-itc; Pe-I, phenethyl- ITC; Bu-N, 3-butenenitrile; Pp-N, 3-phenyl-propionitrile, Sin, 2-propenyl-glucosinolate).

7 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261± Fig. 7. Inhibition of nitri cation of added NH 4 + by volatile sulphur compounds singly and in combination (M: dimethyl sulphide, D: dimethyl disulphide, I: 2-propenyl-isothiocyanate) Interaction of isothiocyanates with nonglucosinolate derived volatile S compounds When applied individually, 2-propenyl-ITC and dimethyl-sulphide had no signi cant e ect on nitri cation, while the dimethyl-disulphide application inhibited the process by 30% (Fig. 7). However, when the sub-inhibitory concentrations of 2-propenyl-ITC and dimethyl-sulphide were mixed, nitri cation was inhibited by over 35%. Application of either of the sub-inhibitory concentrations of 2-propenyl-ITC or dimethyl-sulphide with dimethyl-disulphide caused no greater inhibition than when dimethyl-disulphide was applied alone. Similarly, when all three compounds were applied together, the inhibition of nitri cation was the sum of the inhibition caused by the combined application of 2-propenyl-ITC and dimethyl-sulphide, and the application of dimethyl-disulphide. 4. Discussion Our results demonstrate that ITCs inhibit nitri cation processes by direct e ects on the size of communities of nitrifying bacteria, and by reducing their nitrifying activities. Other allelochemicals, such as terpenes, have been shown to inhibit nitri cation by causing immobilization of mineral-n as the soil microbiota utilizes the compound as a C source (Bremner and McCarty, 1993). In our study, the mineral-n pool in soil amended with phenethyl-itc was shown to be smaller than in unamended control soil between 21 and 35 days, which could indicate immobilization of soil mineral-n as the compound was utilized by soil microbes as a C source. However, the ITC had disappeared from the soil 14 days before this point in time, either by means of volatilization or microbial degradation. Additionally, phenethyl-itc has a C-to-N ratio of 7.7, which is similar to that of the soil biomass (Jenkinson, 1988). Metabolism of this compound would therefore result in extra mineralization of N rather than immobilization. Reduction of the total mineral-n pool by this compound therefore probably arose from inhibition of mineralization of native soil organic matter in treated relative to unamended soil. Nitri cation was shown to be inhibited at concentrations of 0.5 mg 2-propenyl-ITC g 1 dw soil, which is about 1% of the amounts which could potentially be formed following incorporation of Brassica crop residues or green manures into soil (Williams et al., 1993). Further, the fact that sub-lethal concentrations of 2- propenyl-itc interact synergistically with dimethyl-sulphide to inhibit nitri cation suggests that the actual capacity of ITCs to inhibit nitri cation processes will not only depend on the amount of ITCs generated during decomposition, but also on the amounts of compounds with which it interacts. Since ITCs are known to inhibit the growth and activity of soil saprophytic fungi (Drobnica et al., 1967), decomposition processes could also be subject to inhibition by ITCs. Inhibition of decomposition and mineralization processes by ITCs acting together with other low MW S compounds which are characteristically generated during decomposition of crucifer tissues could help to explain why mineralization of N from cruciferous crop residues can be slower than expected from their C-to-N ratios (Bending et al., 1998). Selection of crucifer varieties within rotations on the basis of glucosinolate pro les could be used as a tool to manage the mineralization of N from crop residues, and thus improve synchrony with needs of following crops. Further, recent progress in understanding the metabolic pathways of glucosinolate biosynthesis has led to the possibility of making quantitative and qualitative manipulation of the glucosinolate pro les of plant tissues (Halkier and Du, 1997). This could be of considerable potential for modifying the rate at which N is mineralized from crop residues incorporated in soil. Additionally, ITCs are released into the soil by growing crucifer roots. Soil from pots in which Brassica nigra was grown, possessed concentrations of 0.3 mg 2-propenyl-ITC g 1 soil (Choesin and Boerner, 1991), while Isatis tinctoria has been shown to release up to 4 mg of indole glucosinolates per g 1 fw root over 6 weeks (Elliot and Stowe, 1971). Although the amount of ITC detected by Choesin and Boerner (1991) was not considered to be su cient to produce allelopathic suppression of plant species, our results indicate that this concentration could a ect N mineraliz-

8 1268 G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269 ation processes, particularly at points close to the root surface where concentrations would be much higher. There was evidence in our study that over the long term, ITCs may promote mineralization of soil N. This could have arisen from a fumigant e ect, in which ITCs killed a portion of the biomass, the tissues of which were subsequently degraded by surviving organisms, resulting in mineralization of N from the dead tissues. Such processes occur following fumigation of soil with chloroform (Jenkinson and Powlson, 1976). There were considerable di erences in the potential of di erent glucosinolate hydrolysis products to inhibit nitri cation, with nitriles showing little toxicity, and ITCs showing varying toxicity. In vitro investigations of the toxicity of ITCs to soil fungi and black vine weevil eggs showed that aromatic ITCs are more toxic than those of the aliphatic type, and that increasing the size of the aliphatic side chain attached to aromatic ITCs increases toxicity (Drobnica et al., 1967; Borek et al., 1995). However, in our study there was no evidence that toxicity to nitrifying bacteria was related to either property. Both toxicity and volatility will control the extent to which ITCs inhibit soil organisms. Our results demonstrated that while 2-propenyl-ITC was more toxic than phenethyl-itc when applied to soil at concentrations less than 2.5 mg g 1 dw soil, there was no di erence at higher concentrations. Sarwar et al. (1998) showed that while a drop of 2-propenyl-ITC will volatilise within 5 min at room temperature, phenethyl-itc takes more than 72 h. Presumably, the change in relative toxicity of the two ITCs was related to the slower volatilization and loss of phenethyl-itc from soil, so that while this compound is less toxic than 2-propenyl- ITC, it is able to exert its toxic e ects for longer. This was con rmed by the relative e ects of capping on ITC toxicity, which had a great e ect on inhibition of nitri cation caused by 2-propenyl-ITC, but had relatively less e ect on inhibition caused by phenethyl- ITC. Lewis and Papavizas (1971) found that 2-propenyl- ITC was considerably more e ective than other low MW S compounds as an inhibitor of the fungal plant pathogen Aphanomyces euteiches. This is in contrast with our ndings, which has shown dimethyl-disulphide to be a more potent inhibitor of nitri cation than 2-propenyl-ITC. It is therefore evident that di erent organisms vary in their susceptibility to individual volatile S compounds, and that e ective utilization of biofumigation could depend on targeting crucifer tissues with speci c compositions of volatile S compound precursors for the organism or process for which management is needed. It was demonstrated that low MW volatile S compounds produced during decomposition of crucifer tissues in soil interact to inhibit nitri cation. Similarly, Canessa and Morrell (1995) demonstrated that concentrations of the industrial fumigants methyl-itc and carbon disulphide, which were sub-lethal when applied singly, inhibited the growth of pathogenic fungi colonizing pine wood. The toxicity of ITCs and volatile S compounds is known to arise from their capacity to bind to proteins, resulting in alteration of the tertiary structure of enzymes and the inhibition of metabolic processes (Brown and Morra, 1997). 2-Propenyl-ITC and dimethyl-sulphide interacted synergistically with each other, but not with dimethyl-disulphide, to inhibit the activities of nitrifying organisms. This suggests that 2- propenyl-itc and dimethyl-sulphide act on microbes or proteins in the same metabolic process, while dimethyl-disulphide acts on di erent microbes or proteins. The composition of volatile S compounds produced during crucifer decomposition in soil is determined by the nature of precursor compounds in the plant material and the physical and chemical environment of the soil (Banwart and Bremner, 1975; Bending and Lincoln, 1999). Improved understanding of the ways in which the toxicity of volatile S compounds is a ected by synergistic interactions will be of fundamental importance for utilizing biofumigation as a practical tool. Acknowledgements We thank the Biotechnology and Biological Sciences Research Council for nancial support; Simon Elliot for conducting the mineral-n analyses; and Julie Jones for statistical advice. References Alexander, M., Most probable number method for microbial populations. In: Miller, R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2, 2nd ed. Soil Science Society of America, Madison, pp. 815±821. Angus, J.F., Gardner, P.A., Kirkegaard, J.A., Desmarchelier, J.M., Biofumigation: isothiocyanates released from Brassica roots inhibit growth of the take-all fungus. Plant and Soil 162, 107± 112. Bending, G.D., Turner, M.K., Burns, I.G., Fate of nitrogen from crop residues as a ected by biochemical quality and the microbial biomass. Soil Biology & Biochemistry 30, 2055±2065. 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