Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue8-9.Aug2000:
Soil Biology & Biochemistry 32 (2000) 1261±1269
www.elsevier.com/locate/soilbio
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 eects of
isothiocyanates (ITCs), which are derived from glucosinolates, are not known. We investigated the eects 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 eect on populations of NOÿ
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
eect being longer lived in sandy-loam relative to clay-loam. After 42 days, mineralization of N in sandy-loam amended with 2phenethyl-ITC was greater than in unamended soil, suggesting that this compound had a general fumigant eect on the soil
microbiota. ITCs were more eective 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 dierences 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 eect 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. 7 2000 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.: +44-1789-470382; fax: +44-1789470552.
E-mail 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 eects of phenolic and
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 4 3 - 2
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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 eects
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
eects 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
eects 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 Scontaining 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 eective
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 eects 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 eective
hydrolysis products, the concentrations at which the
compounds are toxic, and the longevity of inhibitive
eects. Additionally, we investigated whether the inhibitive properties of ITCs are aected 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 ÿ480 kPa and incubated at 158C for
5 days before use.
2.2. Eect of isothiocyanates on communities of
nitrifying bacteria and nitri®cation
Sandy-loam and clay-loam soils were amended with
a solution of (NH4)2SO4 to give a concentration of 80
ÿ1
fw soil. Stock solutions of 2-propenylmg NH+
4 ±N g
and phenethyl-ITC, (Aldrich Chemical Company, Dorset, UK) were prepared by adding 10 mg of the pure
compound to 5 ml distilled H2O, 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 ÿ126 kPa and an allelochemical concentration of 10 mg gÿ1 dw soil. A control
treatment in which distilled H2O 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 2propenyl-glucosinolate, range between 20 and 56 mg
ITC gÿ1 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
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
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
ÿ
were taken for determination of NH+
4 ±N, NO2 ±N
ÿ
and NO3 ±N. Ten g fw soil was shaken with 50 ml 0.5
M K2SO4 for 30 min, and the suspension ®ltered
ÿ
through a Whatman No. 1 ®lter. NH+
4 ±N, NO2 ±N
ÿ
and NO3 ±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
ÿ
Method, using media selective for NH+
4 - and NO2 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 CaCl2 and 5 ml
dichloromethane (DCM) containing 5 mg mlÿ1 benzylITC (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.
2.3. 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
ÿ1
fw soil, and 2-propenyl-ITC and pheNH+
4 ±N g
nethyl-ITC stock solutions and/or distilled H2O were
added to give ITC concentrations ranging from 0 to 20
mg gÿ1 dw soil, and a moisture content of ÿ126 kPa.
For each soil a control treatment receiving distilled
H2O 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 NOÿ
3 ±N analysis, as
described above.
2.4. Eects of glucosinolate-derived allelochemicals on
nitri®cation
The eect of a variety of glucosinolate hydrolysis
1263
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
gÿ1 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 ÿ126 kPa, and
an allelochemical concentration of 10 mg gÿ1 dw soil.
A control treatment receiving distilled H2O was also
included. To investigate the eect 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 3butene-nitrile treatments. Soils were incubated for 21
days under the conditions described above before determination of NOÿ
3 ±N.
2.5. Interaction of isothiocyanates with nonglucosinolate derived volatile S compounds
The eect 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
ÿ1
fw soil and a moisture
to give 100 mg NH+
4 ±N g
content of ÿ126 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 gÿ1 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 NOÿ
3 ±N.
2.6. Statistical analysis
Experiments were repeated twice, with similar results
obtained each time. Results from only one experiment
are presented. Signi®cance of dierences between treatments were determined by analysis of variance. In the
case of analysis of the eects 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
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
Common occurrence of parent glucosinolatea
Allelochemical
Structural formula
MW
2-Propenyl-glucosinolate
CH21CHCH2CSNOSO3ÿ (glucose)
Methyl-isothiocyanate
2-Propenyl-isothiocyanate
Butyl-isothiocyanate
Phenyl-isothiocyanate
Benzyl-isothiocyanate
CH3NCS
CH21CHCH2NCS
CH3(CH2)3NCS
C6H5NCS
C6H5CH2NCS
Phenethyl-isothiocyanate
C6H5CH2CH2NCS
3-Butene-nitrile
3-Phenyl-propionitrile
CH21CHCH2CN
C6H5CH2CH2CN
415.5 Black mustard, brown mustard, Indian mustard, cabbage, brussels
sprout, cauli¯ower
73.1 Horseradish, Indian mustard, cauli¯ower
99.2 (as 2-propenyl-glucosinolate)
115.2 Horseradish, Indian mustard, cabbage, brussels sprout
135.2 Horseradish, Indian mustard
149.2 Indian cress, nasturtium, horseraddish, Indian mustard, raddish,
cabbage, brussels sprout
163.2 Watercress, Indian mustard, radish, cabbage, brussels sprout,
cauli¯ower
67.1 (as 2-propenyl-glucosinolate)
131.2 (as phenethyl-isothiocyanate)
a
(Fenwick et al., 1983).
dierences between treatments were compared by LSD
using the transformed data.
3. Results
3.1. Eect 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 eect
on populations of NOÿ
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
NOÿ
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
was rapid in the clay-loam, with
applied NH+
4
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.
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
1265
Fig. 2. Populations of NOÿ
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 NOÿ
3 was delayed in soil
treated with the ITCs during the ®rst 35 days following application (Fig. 4a). However, after 42 days, NOÿ
3
in soil treated with 2-phenethyl-ITC was signi®cantly
higher than in the control soil. There was no dierence
between the amounts of NOÿ
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
ÿ1
fw soil (data
NOÿ
2 ±N pools were less than 1 mg g
not shown).
The total mineral N pool for each treatment was
determined at each harvest, by combining the NH+
4 ±
ÿ
N, NOÿ
2 ±N and NO3 ±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 dierences
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.
3.2. 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 dierences between control and 2-propenyl-ITC, control and phenethyl-ITC and propenyl-ITC and phenethyl-ITC, respectively; : signi®cantly dierent (P < 0.05), ns: not signi®cantly dierent. Bars represent +/ÿ standard error of the mean. (a) Sandy-loam; (b)
Clay-loam.
1266
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
Fig. 4. Formation of NOÿ
3 in unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-ITC (Q). From top to bottom, signi®cance of dierences between control and 2-propenyl-ITC, control and phenethyl-ITC and propenyl-ITC and phenethyl-ITC, respectively; :
signi®cantly dierent (P < 0.05), ns: not signi®cantly dierent. Bars represent +/ÿ standard error of the mean. (a) Sandy-loam; (b) Clay-loam.
a concentration of 0.5 mg gÿ1 dw soil, while phenethylITC caused signi®cant inhibition of nitri®cation at
concentrations down to 2.5 mg gÿ1 dw soil (Fig. 5).
However, at concentrations above 5 mg gÿ1 dw soil,
ITC type made no signi®cant dierence 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 gÿ1. However, in the
case of 2-propenyl-ITC, increasing concentration
above 5 mg gÿ1soil had no further inhibitive eect.
The eect of the glucosinolate-derived chemicals on
nitri®cation is shown in Fig. 6. All the compounds sig-
ni®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 eect
on inhibitory properties. Additionally, there was little
dierence between the inhibitory eect 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 eect 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; BuI, butyl-ITC; Bz-I, benzyl-ITC; Ph-I, phenyl-ITC; Pe-I, phenethylITC; Bu-N, 3-butenenitrile; Pp-N, 3-phenyl-propionitrile, Sin, 2-propenyl-glucosinolate).
3.3. Eect of allelochemicals on nitri®cation
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
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).
3.4. Interaction of isothiocyanates with nonglucosinolate derived volatile S compounds
When applied individually, 2-propenyl-ITC and
dimethyl-sulphide had no signi®cant eect 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 eects 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,
1267
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 gÿ1 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 2propenyl-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 gÿ1 soil (Choesin and Boerner,
1991), while Isatis tinctoria has been shown to release
up to 4 mg of indole glucosinolates per gÿ1 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 sucient to produce
allelopathic suppression of plant species, our results indicate that this concentration could aect N mineraliz-
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 eect, 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 dierences in the potential
of dierent 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 gÿ1 dw soil, there was no dierence 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-propenylITC, it is able to exert its toxic eects for longer. This
was con®rmed by the relative eects of capping on
ITC toxicity, which had a great eect on inhibition of
nitri®cation caused by 2-propenyl-ITC, but had relatively less eect on inhibition caused by phenethylITC.
Lewis and Papavizas (1971) found that 2-propenylITC was considerably more eective 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 dierent organisms vary in their susceptibility to individual
volatile S compounds, and that eective 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 tis-
sues 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 2propenyl-ITC and dimethyl-sulphide act on microbes
or proteins in the same metabolic process, while
dimethyl-disulphide acts on dierent 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 aected
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., 1982. 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.,
1994. 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., 1998. Fate of nitrogen
from crop residues as aected by biochemical quality and the microbial biomass. Soil Biology & Biochemistry 30, 2055±2065.
Bending, G.D., Lincoln, S.D., 1999. Characterisation of volatile sulphur compounds produced during decomposition of Brassica juncea in soil. Soil Biology & Biochemistry 31, 695±703.
Banwart, W.L., Bremner, J.M., 1975. Evolution of volatile sulfur
compounds from soils treated with sulfur-containing organic materials. Soil Biology & Biochemistry 8, 439±443.
Bennett, R.N., Wallsgrove, R.M., 1994. Secondary metabolites in
plant defence mechanisms. New Phytologist 127, 617±633.
Borek, V., Elberson, L.R., McCarey, J.P., Morra, M.J., 1995.
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
Toxicity of aliphatic and aromatic isothiocyanates to eggs of the
Black Vine Weevil (Coleoptera: Curculionidae). Journal of
Economic Entomology 88, 1192±1196.
Bremner, J.M., Bundy, L.G., 1974. Inhibition of nitri®cation in soils
by volatile sulfur compounds. Soil Biology & Biochemistry 6,
161±165.
Bremner, J.M., McCarty, G.W., 1993. Inhibition of nitri®cation in
soil by allelochemicals derived from plants and plant residues. In:
Bollag, J.M., Stotzky, G. (Eds.), Soil Biochemistry, vol. 8. Marcel
Dekker, New York, pp. 181±218.
Brown, P.D., Morra, M.J., McCarey, J.P., Auld, D.L., Williams,
L., 1991. Allelochemicals produced during glucosinolate degradation in soil. Journal of Chemical Ecology 17, 2021±2034.
Brown, P.D., Morra, M.J., 1997. Control of soil-borne plant pests
using glucosinolate-containing plants. Advances in Agronomy 61,
167±231.
Canessa, E.F., Morell, J.J., 1995. Eect of mixtures of carbon disul®de and methyl isothiocyanate on survival of wood-colonizing
fungi. Wood and Fiber Science 27, 207±224.
Choesin, D.N., Boerner, R.E.J., 1991. Allyl isothiocyanate release
and the allelopathic potential of Brassica napus (Brassicaceae).
American Journal of Botany 78, 1083±1090.
Cole, R.A., 1976. Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae. Phytochemistry
15, 759±762.
Drobnica, L., Zemanova, M., Antos, K., Kristian, P., Stullerova, A.,
Knoppova, V., Nemec, P., 1967. Antifungal activity of isothiocyanates and related compounds. Applied Microbiology 15, 701±
709.
Elliot, M.C., Stowe, B.B., 1971. Distribution and variation of indole
glucosinolates in Woad (Isatis tinctoria L.). Plant Physiology 48,
498±503.
Fenwick, G.R., Heaney, R.K., Mullin, W.J., 1983. Glucosinolates
and their breakdown products in food and food plants. In: Furia,
T.E. (Ed.), Critical Reviews in Food Science and Nutrition. CRC
Press, Florida, pp. 123±201.
Halkier, B.A., Du, L., 1997. The biosynthesis of glucosinolates.
Trends in Plant Science 2, 425±430.
Harbourne, J.B., 1997. Role of phenolic secondary metabolites in
1269
plants and their degradation in nature. In: Cadisch, G., Giller,
K.E. (Eds.), Driven By Nature: Plant Litter Quality and
Decomposition. CAB International, Wallingford, UK, pp. 67±74.
Jenkinson, D.S., 1988. The determination of microbial biomass carbon and nitrogen in soil. In: Wilson, J.R. (Ed.), Advances in
Nitrogen Cycling in Agricultural Ecosystems. CAB International,
Wallingford.
Jenkinson, D.S., Powlson, D.S., 1976. The eects of biocidal treatments on metabolism in soil. Part V: A method for measuring
soil biomass. Soil Biology & Biochemistry 8, 209±213.
Kirkegaard, J.A., Sarwar, M., 1998. Biofumigation potential of brassicas. Part I: variation in glucosinolate pro®les of diverse ®eld
grown brassicas. Plant and Soil 201, 71±79.
Lewis, J.A., Papavizas, G.C., 1970. Evolution of volatile sulphurcontaining compounds from decomposition of crucifers in soil.
Soil Biology & Biochemistry 2, 239±246.
Lewis, J.A., Papavizas, G.C., 1971. Eect of sulfur-containing volatile compounds and vapours from cabbage decomposition on
Aphanomyces euteiches. Phytopathology 61, 208±214.
Poulton, J.E., Moller, B.L., 1993. Glucosinolates. In: Lea, P.J. (Ed.),
Methods in Plant Biochemistry, vol. 9. Academic Press, London,
pp. 209±237.
Powlson, D.S., Jenkinson, D.S., 1971. Inhibition of nitri®cation in
soil by carbon disul®de from rubber bungs. Soil Biology &
Biochemistry 3, 267±269.
Sarwar, M., Kirkegaard, J.A., Wong, P.T.W., Desmarchelier, J.M.,
1998. Biofumigation potential of brassicas. Part III: in vitro toxicity of isothiocyanates to soil-borne fungal pathogens. Plant and
Soil 201, 103±112.
Schmidt, E.L., Belser, L.W., 1982. Nitrifying bacteria. In: Miller,
R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2, 2nd
Ed. Soil Science Society of America, Madison, pp. 1027±1042.
Williams, L., Morra, M.J., Brown, P.D., McCarey, J.P., 1993.
Toxicity of allyl isothiocyanate-amended soil to Limonius californicus (Mann) (Coleoptera: Elateridae). Journal of Chemical
Ecology 19, 1033±1046.
Zucker, W.V., 1983. Tannins: does structure determine function? An
ecological perspective. The American Naturalist 121, 335±365.
www.elsevier.com/locate/soilbio
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 eects of
isothiocyanates (ITCs), which are derived from glucosinolates, are not known. We investigated the eects 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 eect on populations of NOÿ
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
eect being longer lived in sandy-loam relative to clay-loam. After 42 days, mineralization of N in sandy-loam amended with 2phenethyl-ITC was greater than in unamended soil, suggesting that this compound had a general fumigant eect on the soil
microbiota. ITCs were more eective 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 dierences 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 eect 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. 7 2000 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.: +44-1789-470382; fax: +44-1789470552.
E-mail 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 eects of phenolic and
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 4 3 - 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 eects
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
eects 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
eects 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 Scontaining 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 eective
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 eects 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 eective
hydrolysis products, the concentrations at which the
compounds are toxic, and the longevity of inhibitive
eects. Additionally, we investigated whether the inhibitive properties of ITCs are aected 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 ÿ480 kPa and incubated at 158C for
5 days before use.
2.2. Eect of isothiocyanates on communities of
nitrifying bacteria and nitri®cation
Sandy-loam and clay-loam soils were amended with
a solution of (NH4)2SO4 to give a concentration of 80
ÿ1
fw soil. Stock solutions of 2-propenylmg NH+
4 ±N g
and phenethyl-ITC, (Aldrich Chemical Company, Dorset, UK) were prepared by adding 10 mg of the pure
compound to 5 ml distilled H2O, 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 ÿ126 kPa and an allelochemical concentration of 10 mg gÿ1 dw soil. A control
treatment in which distilled H2O 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 2propenyl-glucosinolate, range between 20 and 56 mg
ITC gÿ1 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
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
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
ÿ
were taken for determination of NH+
4 ±N, NO2 ±N
ÿ
and NO3 ±N. Ten g fw soil was shaken with 50 ml 0.5
M K2SO4 for 30 min, and the suspension ®ltered
ÿ
through a Whatman No. 1 ®lter. NH+
4 ±N, NO2 ±N
ÿ
and NO3 ±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
ÿ
Method, using media selective for NH+
4 - and NO2 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 CaCl2 and 5 ml
dichloromethane (DCM) containing 5 mg mlÿ1 benzylITC (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.
2.3. 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
ÿ1
fw soil, and 2-propenyl-ITC and pheNH+
4 ±N g
nethyl-ITC stock solutions and/or distilled H2O were
added to give ITC concentrations ranging from 0 to 20
mg gÿ1 dw soil, and a moisture content of ÿ126 kPa.
For each soil a control treatment receiving distilled
H2O 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 NOÿ
3 ±N analysis, as
described above.
2.4. Eects of glucosinolate-derived allelochemicals on
nitri®cation
The eect of a variety of glucosinolate hydrolysis
1263
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
gÿ1 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 ÿ126 kPa, and
an allelochemical concentration of 10 mg gÿ1 dw soil.
A control treatment receiving distilled H2O was also
included. To investigate the eect 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 3butene-nitrile treatments. Soils were incubated for 21
days under the conditions described above before determination of NOÿ
3 ±N.
2.5. Interaction of isothiocyanates with nonglucosinolate derived volatile S compounds
The eect 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
ÿ1
fw soil and a moisture
to give 100 mg NH+
4 ±N g
content of ÿ126 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 gÿ1 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 NOÿ
3 ±N.
2.6. Statistical analysis
Experiments were repeated twice, with similar results
obtained each time. Results from only one experiment
are presented. Signi®cance of dierences between treatments were determined by analysis of variance. In the
case of analysis of the eects 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
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
Common occurrence of parent glucosinolatea
Allelochemical
Structural formula
MW
2-Propenyl-glucosinolate
CH21CHCH2CSNOSO3ÿ (glucose)
Methyl-isothiocyanate
2-Propenyl-isothiocyanate
Butyl-isothiocyanate
Phenyl-isothiocyanate
Benzyl-isothiocyanate
CH3NCS
CH21CHCH2NCS
CH3(CH2)3NCS
C6H5NCS
C6H5CH2NCS
Phenethyl-isothiocyanate
C6H5CH2CH2NCS
3-Butene-nitrile
3-Phenyl-propionitrile
CH21CHCH2CN
C6H5CH2CH2CN
415.5 Black mustard, brown mustard, Indian mustard, cabbage, brussels
sprout, cauli¯ower
73.1 Horseradish, Indian mustard, cauli¯ower
99.2 (as 2-propenyl-glucosinolate)
115.2 Horseradish, Indian mustard, cabbage, brussels sprout
135.2 Horseradish, Indian mustard
149.2 Indian cress, nasturtium, horseraddish, Indian mustard, raddish,
cabbage, brussels sprout
163.2 Watercress, Indian mustard, radish, cabbage, brussels sprout,
cauli¯ower
67.1 (as 2-propenyl-glucosinolate)
131.2 (as phenethyl-isothiocyanate)
a
(Fenwick et al., 1983).
dierences between treatments were compared by LSD
using the transformed data.
3. Results
3.1. Eect 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 eect
on populations of NOÿ
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
NOÿ
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
was rapid in the clay-loam, with
applied NH+
4
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.
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
1265
Fig. 2. Populations of NOÿ
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 NOÿ
3 was delayed in soil
treated with the ITCs during the ®rst 35 days following application (Fig. 4a). However, after 42 days, NOÿ
3
in soil treated with 2-phenethyl-ITC was signi®cantly
higher than in the control soil. There was no dierence
between the amounts of NOÿ
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
ÿ1
fw soil (data
NOÿ
2 ±N pools were less than 1 mg g
not shown).
The total mineral N pool for each treatment was
determined at each harvest, by combining the NH+
4 ±
ÿ
N, NOÿ
2 ±N and NO3 ±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 dierences
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.
3.2. 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 dierences between control and 2-propenyl-ITC, control and phenethyl-ITC and propenyl-ITC and phenethyl-ITC, respectively; : signi®cantly dierent (P < 0.05), ns: not signi®cantly dierent. Bars represent +/ÿ standard error of the mean. (a) Sandy-loam; (b)
Clay-loam.
1266
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
Fig. 4. Formation of NOÿ
3 in unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-ITC (Q). From top to bottom, signi®cance of dierences between control and 2-propenyl-ITC, control and phenethyl-ITC and propenyl-ITC and phenethyl-ITC, respectively; :
signi®cantly dierent (P < 0.05), ns: not signi®cantly dierent. Bars represent +/ÿ standard error of the mean. (a) Sandy-loam; (b) Clay-loam.
a concentration of 0.5 mg gÿ1 dw soil, while phenethylITC caused signi®cant inhibition of nitri®cation at
concentrations down to 2.5 mg gÿ1 dw soil (Fig. 5).
However, at concentrations above 5 mg gÿ1 dw soil,
ITC type made no signi®cant dierence 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 gÿ1. However, in the
case of 2-propenyl-ITC, increasing concentration
above 5 mg gÿ1soil had no further inhibitive eect.
The eect of the glucosinolate-derived chemicals on
nitri®cation is shown in Fig. 6. All the compounds sig-
ni®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 eect
on inhibitory properties. Additionally, there was little
dierence between the inhibitory eect 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 eect 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; BuI, butyl-ITC; Bz-I, benzyl-ITC; Ph-I, phenyl-ITC; Pe-I, phenethylITC; Bu-N, 3-butenenitrile; Pp-N, 3-phenyl-propionitrile, Sin, 2-propenyl-glucosinolate).
3.3. Eect of allelochemicals on nitri®cation
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
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).
3.4. Interaction of isothiocyanates with nonglucosinolate derived volatile S compounds
When applied individually, 2-propenyl-ITC and
dimethyl-sulphide had no signi®cant eect 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 eects 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,
1267
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 gÿ1 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 2propenyl-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 gÿ1 soil (Choesin and Boerner,
1991), while Isatis tinctoria has been shown to release
up to 4 mg of indole glucosinolates per gÿ1 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 sucient to produce
allelopathic suppression of plant species, our results indicate that this concentration could aect N mineraliz-
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 eect, 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 dierences in the potential
of dierent 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 gÿ1 dw soil, there was no dierence 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-propenylITC, it is able to exert its toxic eects for longer. This
was con®rmed by the relative eects of capping on
ITC toxicity, which had a great eect on inhibition of
nitri®cation caused by 2-propenyl-ITC, but had relatively less eect on inhibition caused by phenethylITC.
Lewis and Papavizas (1971) found that 2-propenylITC was considerably more eective 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 dierent organisms vary in their susceptibility to individual
volatile S compounds, and that eective 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 tis-
sues 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 2propenyl-ITC and dimethyl-sulphide act on microbes
or proteins in the same metabolic process, while
dimethyl-disulphide acts on dierent 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 aected
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., 1982. 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.,
1994. 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., 1998. Fate of nitrogen
from crop residues as aected by biochemical quality and the microbial biomass. Soil Biology & Biochemistry 30, 2055±2065.
Bending, G.D., Lincoln, S.D., 1999. Characterisation of volatile sulphur compounds produced during decomposition of Brassica juncea in soil. Soil Biology & Biochemistry 31, 695±703.
Banwart, W.L., Bremner, J.M., 1975. Evolution of volatile sulfur
compounds from soils treated with sulfur-containing organic materials. Soil Biology & Biochemistry 8, 439±443.
Bennett, R.N., Wallsgrove, R.M., 1994. Secondary metabolites in
plant defence mechanisms. New Phytologist 127, 617±633.
Borek, V., Elberson, L.R., McCarey, J.P., Morra, M.J., 1995.
G.D. Bending, S.D. Lincoln / Soil Biology & Biochemistry 32 (2000) 1261±1269
Toxicity of aliphatic and aromatic isothiocyanates to eggs of the
Black Vine Weevil (Coleoptera: Curculionidae). Journal of
Economic Entomology 88, 1192±1196.
Bremner, J.M., Bundy, L.G., 1974. Inhibition of nitri®cation in soils
by volatile sulfur compounds. Soil Biology & Biochemistry 6,
161±165.
Bremner, J.M., McCarty, G.W., 1993. Inhibition of nitri®cation in
soil by allelochemicals derived from plants and plant residues. In:
Bollag, J.M., Stotzky, G. (Eds.), Soil Biochemistry, vol. 8. Marcel
Dekker, New York, pp. 181±218.
Brown, P.D., Morra, M.J., McCarey, J.P., Auld, D.L., Williams,
L., 1991. Allelochemicals produced during glucosinolate degradation in soil. Journal of Chemical Ecology 17, 2021±2034.
Brown, P.D., Morra, M.J., 1997. Control of soil-borne plant pests
using glucosinolate-containing plants. Advances in Agronomy 61,
167±231.
Canessa, E.F., Morell, J.J., 1995. Eect of mixtures of carbon disul®de and methyl isothiocyanate on survival of wood-colonizing
fungi. Wood and Fiber Science 27, 207±224.
Choesin, D.N., Boerner, R.E.J., 1991. Allyl isothiocyanate release
and the allelopathic potential of Brassica napus (Brassicaceae).
American Journal of Botany 78, 1083±1090.
Cole, R.A., 1976. Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae. Phytochemistry
15, 759±762.
Drobnica, L., Zemanova, M., Antos, K., Kristian, P., Stullerova, A.,
Knoppova, V., Nemec, P., 1967. Antifungal activity of isothiocyanates and related compounds. Applied Microbiology 15, 701±
709.
Elliot, M.C., Stowe, B.B., 1971. Distribution and variation of indole
glucosinolates in Woad (Isatis tinctoria L.). Plant Physiology 48,
498±503.
Fenwick, G.R., Heaney, R.K., Mullin, W.J., 1983. Glucosinolates
and their breakdown products in food and food plants. In: Furia,
T.E. (Ed.), Critical Reviews in Food Science and Nutrition. CRC
Press, Florida, pp. 123±201.
Halkier, B.A., Du, L., 1997. The biosynthesis of glucosinolates.
Trends in Plant Science 2, 425±430.
Harbourne, J.B., 1997. Role of phenolic secondary metabolites in
1269
plants and their degradation in nature. In: Cadisch, G., Giller,
K.E. (Eds.), Driven By Nature: Plant Litter Quality and
Decomposition. CAB International, Wallingford, UK, pp. 67±74.
Jenkinson, D.S., 1988. The determination of microbial biomass carbon and nitrogen in soil. In: Wilson, J.R. (Ed.), Advances in
Nitrogen Cycling in Agricultural Ecosystems. CAB International,
Wallingford.
Jenkinson, D.S., Powlson, D.S., 1976. The eects of biocidal treatments on metabolism in soil. Part V: A method for measuring
soil biomass. Soil Biology & Biochemistry 8, 209±213.
Kirkegaard, J.A., Sarwar, M., 1998. Biofumigation potential of brassicas. Part I: variation in glucosinolate pro®les of diverse ®eld
grown brassicas. Plant and Soil 201, 71±79.
Lewis, J.A., Papavizas, G.C., 1970. Evolution of volatile sulphurcontaining compounds from decomposition of crucifers in soil.
Soil Biology & Biochemistry 2, 239±246.
Lewis, J.A., Papavizas, G.C., 1971. Eect of sulfur-containing volatile compounds and vapours from cabbage decomposition on
Aphanomyces euteiches. Phytopathology 61, 208±214.
Poulton, J.E., Moller, B.L., 1993. Glucosinolates. In: Lea, P.J. (Ed.),
Methods in Plant Biochemistry, vol. 9. Academic Press, London,
pp. 209±237.
Powlson, D.S., Jenkinson, D.S., 1971. Inhibition of nitri®cation in
soil by carbon disul®de from rubber bungs. Soil Biology &
Biochemistry 3, 267±269.
Sarwar, M., Kirkegaard, J.A., Wong, P.T.W., Desmarchelier, J.M.,
1998. Biofumigation potential of brassicas. Part III: in vitro toxicity of isothiocyanates to soil-borne fungal pathogens. Plant and
Soil 201, 103±112.
Schmidt, E.L., Belser, L.W., 1982. Nitrifying bacteria. In: Miller,
R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2, 2nd
Ed. Soil Science Society of America, Madison, pp. 1027±1042.
Williams, L., Morra, M.J., Brown, P.D., McCarey, J.P., 1993.
Toxicity of allyl isothiocyanate-amended soil to Limonius californicus (Mann) (Coleoptera: Elateridae). Journal of Chemical
Ecology 19, 1033±1046.
Zucker, W.V., 1983. Tannins: does structure determine function? An
ecological perspective. The American Naturalist 121, 335±365.