The N and S status in cropping systems with white cabbage as influenced by organic fertilizers

This article was downloaded by: [SLU Library]On: 07 November 2012, At: 00:42Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Acta Agriculturae Scandinavica, Section B - Soil &Plant SciencePublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/sagb20

The N and S status in cropping systems with whitecabbage as influenced by organic fertilizersSara Elfstrand a , Birgitta Båth a & Bengt Lundegårdh ba Department of Crop Production Ecology, SLU, Uppsala, Swedenb The Biodynamic Research Institute, Järna, SwedenAccepted author version posted online: 07 Jun 2012.Version of record first published: 08Aug 2012.

To cite this article: Sara Elfstrand, Birgitta Båth & Bengt Lundegårdh (2012): The N and S status in cropping systems withwhite cabbage as influenced by organic fertilizers, Acta Agriculturae Scandinavica, Section B - Soil & Plant Science, 62:8,711-719

To link to this article: http://dx.doi.org/10.1080/09064710.2012.698640

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss, actions,claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

ORIGINAL ARTICLE

The N and S status in cropping systems with white cabbage asinfluenced by organic fertilizers

SARA ELFSTRAND1, BIRGITTA BATH1 & BENGT LUNDEGARDH2

1Department of Crop Production Ecology, SLU, Uppsala, Sweden, 2The Biodynamic Research Institute, Jarna, Sweden

AbstractFresh and anaerobically digested red clover were compared as N and S sources in a incubation experiment without plantsand a pot experiment with white cabbage, both conducted in climate chambers. The hypothesis was that anaerobic digestionwould increase S availability in relation to N and that arylsulphatase activity would be higher in treatments with S deficiency.Besides the two red clover-based treatments, two treatments, one unamended and the other Biofer, an organic fertilizer with7% S containing by-products from the slaughter industry, were included in the experiments. The availability of S in relationto N was higher in biogas slurry than in fresh red clover. In the incubation, an equal percentage (approx. 50%) of N wasmineralized from all three fertilizers, while in the pot experiment, N mineralization was highest in the red clover treatment(approximately 70%). The highest S mineralization in both experiments occurred in the Biofer treatment. Growth of whitecabbage was higher in the biogas slurry than in the red clover treatment despite high N availability in the latter treatment.Immobilization of S due to more readily available C in the clover treatment could have reinforced the difference and given aless well adjusted relationship between N and S for white cabbage demand than in the biogas slurry treatment.Arylsulphatase activity in the bulk soil was higher in the red clover-based treatments than with Biofer, while the activity inthe rhizosphere soil did not differ between treatments. Arylsulphatase activity in the bulk soil was negatively correlated withwhite cabbage S concentration and positively correlated with N:S ratio in white cabbage shoots, while that in the rhizospheresoil was positively correlated with white cabbage S concentration.

Keywords: Anaerobic digestion, Brassica oleraceae L., nitrogen, red clover, sulphur, Trifolium pratense L., white cabbage.

Introduction

Sulphur (S) deficiency is recognized as one of the

major nutritional problems in northern European

crop production (Scherer, 2001; Nziguheba et al.,

2006). Brassica crops, such as white cabbage, are

particularly S-demanding. Cereals have been re-

ported to require 15�20 kg S ha�1, whereas rape

has been reported to require 30�40 kg S ha�1 (Zhao

et al., 1996) and cabbage 45�70 kg S ha�1

(Tabatabai, 1984). This high S demand may con-

stitute a problem in cropping systems with Brassica

crops where directly incorporated green manure

crops are the main nutrient source. The reason is

the low S content in green manures in relation to

nitrogen (N) and the high S requirement of Brassica

crops. Since N and S are closely associated in protein

synthesis, the S demand varies with the supply of N

to crops (Tabatabai, 1984) and S deficiency can limit

the uptake of available N (Eriksen et al., 2001).

In addition to direct incorporation, green manures

may also be harvested and used to produce biogas

via anaerobic digestion. The by-product from this

process, slurry, may be used as a fertilizer. In

contrast to fresh plant materials, in which N is

mainly organically bound, about 40�60% of the N in

biogas slurry occurs as mineral (mainly ammonium)

N (Nordberg & Edstrom, 1997; Bath & Ramert,

2000). The S in fresh plant material is mainly

organically bound, but may also occur in relatively

large amounts as sulphate (Bergmann, 1992), a

direct S source for plants. The S forms in biogas

slurry have not been investigated to the same extent

as those in fresh plant material, but a large propor-

tion appears to be in reduced form (Elfstrand et al.,

2007), that is in the form of sulphides, the stage in

Correspondence: Birgitta Bath, Department of Crop Production Ecology, SLU, Box 7043, SE-750 07 Uppsala, Sweden. Tel: �46 18 67 23 10.

E-mail: [email protected]

Acta Agriculturae Scandinavica Section B � Soil and Plant Science, 2012; 62: 711�719

(Received 31 January 2012; revised 11 May 2012; accepted 23 May 2012)

ISSN 0906-4710 print/ISSN 1651-1913 online # 2012 Taylor & Francis

http://dx.doi.org/10.1080/09064710.2012.698640

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

decomposition of organic S prior to oxidation into

sulphate.

The mineralization of sulphate esters, which plays

an important role in the release of sulphate (Scherer,

2001), is mediated by sulphatase enzymes originat-

ing from microorganisms and plants (Knauff et al.,

2003). Plants respond to an insufficient S supply by

increased production and excretion of sulphatases to

a higher extent than at sufficient S supply. The

ability to exude sulphatases also differs between

plant species and has been shown to be higher with

Brassica species than with grasses at S deficiency

levels (Knauff et al., 2003). Arylsulphatase is a

generally occurring sulphatase enzyme and may

hence be used as an indicator of biochemical

mineralization intensity of organic sulphate esters

in the soil (Kotkova et al., 2008).

Due to differences in availability and the fact that

both total N and S content and N:S ratio can vary

considerably between different forms of organic

fertilizers (Eriksen, 2005), it is difficult to assess

their agronomic significance in terms of N and S

supply (Scherer, 2001). In the present study, two

forms of red clover (fresh and anaerobically digested

red clover shoots) were compared as N and S sources

in white cabbage production. The hypothesis was

that anaerobic digestion would increase the avail-

ability of S in relation to N compared with fresh red

clover shoots. A further hypothesis was that arylsul-

phatase activity would be higher in treatments with

S deficiency and thus functions as an indicator of the

S status of the cropping systems.

Materials and methods

To quantify the amount of N and S released from the

different organic amendments, an initial incubation

experiment without a crop (Experiment I) was

conducted prior to a pot experiment with white

cabbage (Experiment II). Besides the red clover and

biogas slurry treatments, two control treatments, one

unamended and one with Biofer 6-3-12, were

included in each of the two experiments. Biofer, an

organic fertilizer with 7% S, contains by-products

from the slaughter industry such as meat, bone and

blood meal (Gyllebo Godning AB, Lantmannen AB,

Sweden).

Preparation of red clover

Red clover (Trifolium pratense L. var. Vivi) was grown

in a greenhouse and fertilized with a complete liquid

fertilizer (Wallco 51-10-43�micro; Cederoth Inter-

national AB), starting two months after sowing.

After three months, the red clover was harvested

by cutting with scissors close to the stem base and

used in the incubation experiment (Experiment I).

The re-growth of clover was cut after one month to

prevent it from flowering. The second re-growth was

harvested after an additional month and used in the

pot experiment with white cabbage (Experiment II).

The biogas slurry was produced from ensiled,

fresh red clover shoots in an earlier experiment (Bath

& Elfstrand, 2008). The clover was rowed and left

lying in the field to dry for two days. Bulk masses of

3150 kg clover, DM content 43%, were ensiled in

large bales. Biodigestion was carried out in a biogas

digester with a total volume of 30 m3, to which was

added 7000 l water and 3000 l inoculants in the form

of cattle slurry. The silage was fed continuously into

the digester, starting with small amounts that were

gradually increased. The anaerobic digestion, a one

step mesophilic (378C) process, ran for approxi-

mately 100 days.

For analysis the red clover was dried at a tem-

perature of 308C for four days and thereafter milled

while the biogas slurry and Biofer were stored frozen

until analysis.

Characteristics of soil and fertilizers

The soil used in the two experiments consisted of 9%

clay, 20% silt and 71% sand and had an organic

matter content of 2.5%. Soil texture was character-

ized by mechanical fractionation (Ljung, 1987) and

organic matter concentration by loss on ignition,

corrected for H2O in the clay fraction. Before

chemical characterization, the soil was dried at

358C and sieved (mesh 2 mm).

The total amount of C, N and S in the incubated

soil and the amounts added with fertilizers are shown

in Table I, as is the N:S ratio for the two experi-

ments. In both experiments the fertilizer doses were

based on the same amount of N.

In Experiment I, about the same amounts of N

and C was added with the two forms of green

manure-based treatments (Table I). Since C abducts

in the biogas process, this suggests losses of N. The

losses may have occurred at the harvesting and post-

harvest of red clover and the further treatment of the

slurry after the anaerobic digestion process. As a

consequence of this, the N:S ratio was lower in the

biogas slurry treatment than in the red clover

treatment. In Experiment II, differences in C input

were larger while the difference in N:S ratio between

the two forms of green manure-based treatments was

smaller.

Incubation experiment (Experiment I)

The incubation experiment was conducted in 1-L

vessels without lids, with three replicates per treatment

712 S. Elfstrand et al.

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

organized in a completely randomized design in a

dark climate chamber set at 158C. Before the start of

the experiment, the soil (750 g DW per vessel) was

pre-incubated at 20% of water-holding capacity

(WHC) for two weeks. After the pre-incubation

period Biofer, red clover and biogas slurry were

added in amounts corresponding to approximately

0.2 g N. Red clover (1.7 g DW stems and 1.3 g DW

leaves) was cut into pieces of approximately 0.01 m

size before being incorporated into the soil. The

water content in the unamended control, Biofer and

red clover treatments was adjusted to the same level

as in the biogas slurry treatment (37% of WHC).

The fertilizers and added water were mixed thor-

oughly into the soil. The water content was adjusted

daily based on weight loss by adding distilled water.

Destructive sampling for analysis of min �N (NO3,

NH4) and SO4 was carried out with three pots per

treatment after 0, 1, 4, 7, 14, 28 and 56 days. The

samples were frozen immediately after sampling.

Pot experiment with white cabbage (Experiment II)

The pot experiment conducted in a climate chamber

had a completely randomized design with four

replicates. The temperature was monitored continu-

ously during the experimental period and the mean

temperature was 16.291.48C (SD). The soil was

pre-incubated in pots (2 kg FW, 1.98 kg DW, soil per

pot) covered with plastic film at 20% of WHC for

two weeks. After the pre-incubation period, 0.5 g N

per pot was added as Biofer, red clover or biogas

slurry. The amount of N added was based on the

expected N requirement, which was calculated

assuming exponential growth (DW�0.011e0.14*day)

for the first 54 days of the experiment and linear

growth (DW�0.21*day�5.6) from day 55 onwards

(Ekbladh, 2007). The calculated growth curve was

based on a starting weight of the white cabbage

transplant of 0.005 g DW and on an N concentration

of 3% of DW.

The red clover was incorporated on the day of

harvest. An amount of 5.7 g DW stems and 3.7 g

DW leaves, cut into pieces of approximately 0.01 m

size, was added to each pot. The water content in the

unamended control, Biofer and red clover treat-

ments was adjusted to the same level as in the biogas

slurry treatment, that is, to 44% of WHC. The

fertilizers and added water were mixed thoroughly

into the soil and the pots were placed in the climate

chamber. After seven days, four-day-old seedlings of

white cabbage (Brassica oleraceae L. var. Nordpol F1)

were planted, one in each pot, and the growing

regime in the chamber was set to 16 h of daylight

(350 mmol m�2 s�1) with 75% humidity during the

day and 90% during the night. For the first 42 days,

the WHC level in each pot was adjusted weekly by

adding distilled water to its initial weight at planting.

After 42 days, sampled plants (see below) were used

to correct for current aboveground plant weight. The

water content at this time was adjusted twice a week,

while after 56 days it was adjusted three times a

week. The pots were rotated clockwise every other

week in the climate chamber.

Samples of bulk soil for analyses of mineral N and

sulphate were obtained by destructive sampling on

days 42, 49, 56 and 63 after planting. On the last

sampling occasion, soil for analysis of arylsulpha-

tase was sampled in all treatments. Bulk soil was

defined as soil loosely adhering to the roots,

removed by shaking the roots vigorously. The

remaining soil, which was defined as rhizosphere

soil, was removed from the root surface with a small

brush. The sampled soil was frozen immediately

after sampling.

White cabbage shoots were sampled destructively

from four plants per treatment on days 42, 49, 56

and 63 after planting. The shoots were cut 0.005 m

below the cotyledons and weighed for FW, then

dried at 808C for 48 hours and weighed again. The

samples were milled prior to analysis.

Table I. Total amounts (g) of C, N and S in soil and three fertilizers; green manure based (fresh red clover and red clover digested in a

biogas reactor) and Biofer (an organic fertilizer based on by-products from the slaughter industry) in an incubation experiment (750 g DW

soil) without plants (Experiment I) and in a pot experiment (1980 g DW soil) with white cabbage (Experiment II), and the N:S ratio in soil

and added fertilizers.

Total C (g) Total N (g) Total S (g) N:S

I II I II I II I II

Soil 10.5 27.9 0.83 2.18 0.15 0.40 6:1 5:1

Biofer 0.7 1.9 0.19 0.50 0.18 0.47 1:1 1:1

Green manure-based treatments

Red clover 1.2 3.8 0.17 0.48 0.007 0.02

Leaves 0.6 1.6 0.089 0.24 0.004 0.01 22:1 24:1

Stems 0.6 2.2 0.078 0.25 0.003 0.01 26:1 25:1

Biogas slurry 1.3 2.7 0.20 0.50 0.017 0.03 12:1 17:1

N and S in cropping systems 713

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

Analyses

The total content of C and N in soil, plants and

fertilizers was analysed on a LECO analyser (CN

2000 USA) according to the Dumas method (Brem-

ner & Hauk, 1982) except for N in the biogas slurry,

which was analysed according to the Kjeldahl

method (Bremner & Mulvaney, 1982). Analyses of

S content were performed using an inductively

coupled plasma emission spectrometer (Perkin El-

mer Optima 3000 DV) after wet digestion with nitric

acid (HNO3). Total C, N and S contents were

calculated based on dry weight (1058C).

Analyses of mineral N in soil samples (NO3, NH4)

were carried out after soil samples had been milled

frozen and extracted with 250 mL 2 M KCl per 100

g FW soil (Linden, 1981). The extract was analysed

by colorimetry on a TRAACS 800 (Bran Lubbe).

For analysis of sulphate, 5 g FW soil were shaken on

a rotary shaker with 25 mL of 500 mg L�l P-solution

(16 mM KH2PO4) for 30 min (Tabatabai, 1982).

Extracts were centrifuged for 10 min at 4000 rev

min�1 and filtered. Extracts were then Millipore-

filtered (0.22 mm) before determination of sulphate

concentration by ion chromatography (Dionex).

Arylsulphatase activity was determined according

to Tabatabai (1994) with a few modifications. Field-

moist soil (equivalent to 1 g DW) was incubated

together with 1 mL 0.1 M p-nitrophenylsulphate

solution and 4 mL acetate buffer (0.5 M, pH 5.8) for

1 h in a rotating water bath at 378C. After addition of

1 mL 0.5 M CaCl2 and 4 mL 0.5 M NaOH, the

samples were filtered and the concentration of

p-nitrophenol released was determined at 400 nm

(Perkin-Elmer Lambda 25 UV/vis). The arylsulpha-

tase activity was calculated based on soil dry weight

(1058C).

Statistical analyses and calculations

Due to the irregular sampling occasions, data on

N and S mineralization in the incubation experiment

(Experiment I) and arylsulphatase in the pot experi-

ment (Experiment II) were analysed with a general

linear model to compare treatment means at each

sampling occasion separately. All other data from soil

and plant samples were analysed with a general linear

model with treatment and day, and the interactions

between these, as model components. When signifi-

cant effects (pB0.05) of treatment or day were

found, Tukey’s test was used to compare means.

When the residuals indicated that the require-

ments of normal distribution and equal variances

were not fulfilled, the data were either log- or arcsin-

transformed (shoot N and S concentrations) before

being subjected to analysis of variance. However,

untransformed data are shown in tables and figures.

The analyses were performed in Minitab 16 (Mini-

tab Inc, State College, PA, USA).

Net N and S mineralization from the fertilizers

was calculated by the difference method, that is as

½ððN or S in treatment � N or S in controlÞ=N or S in controlÞ � 100�:

Results

Mineralization of N and S

The apparent net N mineralization from the fertili-

zers in the incubation experiment (Experiment I)

differed initially (Figure 1). At days 0 and 1, more N

had been mineralized from the biogas slurry and red

clover than from the Biofer, whereas after day 14 the

net N mineralization in the Biofer treatment had

exceeded that in the red clover treatment (pB0.001

0

10

20

30

40

50

60

days

% m

in N

0 1 4 7 14 28 56 0 1 4 7 14 28 56

Biofer

Red clover

Biogas slurry

-20

0

20

40

60

80

100

120

140

days

%S

O42

-

(a) (b)

Figure 1. Apparent mineralization of N (a) and SO42�-S (b) from three fertilizers; green manure based (fresh red clover and red clover

digested in a biogas reactor) and Biofer (an organic fertilizer based on by-products from the slaughter industry) expressed as percentage of

the unamended control (the difference method) in an incubation experiment (750 g DW soil) without plants. Symbols represent treatment

means and bars represent SEM (n�3).

714 S. Elfstrand et al.

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

on all occasions). However, at the end of the

incubation, equal percentages (approximately 50%)

of N had been mineralized from all three fertilizers

(p�0.05 at day 56). The highest net SO42�-S

mineralization throughout the incubation was mea-

sured in the Biofer treatment. The explanation for

the high variance for sulphate on days 0�4 and the

high percentages may be an uneven soil distribution

of the Biofer pellets.

The mineralization pattern of N and S between

day 42 and day 63 as measured in the pot experiment

with white cabbage (Experiment II) is shown in

Figure 2. The percentage figures at day 56 after

planting could be compared with corresponding

figures after 56 days of incubation (Figure 1). Note

also that the content of N and S in the roots is not

included and that the scale on the y-axis representing

the dose of N and S differs between treatments for S.

The amount of N mineralized was highest in the red

clover treatment throughout the experiment

(pB0.001). Soil mineral N in the red clover treat-

ment was higher than in the other treatments from

day 42 to day 56 (pB0.001), with the exception of

the slurry treatment at day 42. The amount of S

mineralized was highest in the Biofer treatment

(pB0.001). The soil SO42�-S content in the Biofer

treatment was higher compared with in the red

clover-based treatments, throughout the experiment

(pB0.0001 on all occasions), but there were no

differences in soil SO42�-S content between the two

green manure-based treatments. The low level of

SO42-S in the soil in the fresh red clover treatment is

hard to distinguish at this dissolution.

White cabbage growth and concentration of N and S

The white cabbage growth in the pot experiment

(Experiment II) differed between treatments already

at the first destructive harvest, on day 42 after

planting (Figure 3a). The highest shoot growth,

with the exception of the biogas slurry treatment

on day 42 and day 49, with no signs of a declining

growth rate even at harvest, was found in the Biofer

treatment (pB0.001). The shoot dry weight was

higher in the biogas slurry treatment than in the red

clover treatment on all occasions (pB0.001) except

day 42. The white cabbage in the red clover

treatment only increased its dry weight between

days 56 and 63 (pB0.001). The fertilized treatments

had higher aboveground yields of cabbage than the

unamended control (pB0.001 on all sampling

occasions).

Figure 2. Mineralization (g) of N and S from three fertilizers; green manure based (fresh red clover and red clover digested in a biogas

reactor) and Biofer (an organic fertilizer based on by-products from the slaughter industry) in a pot experiment (1 980 g DW soil) with

white cabbage. Mineralization is based on the sum of bulk soil min-N and SO42-S contents by the difference method and aboveground

crop N and S uptake after subtracting the corresponding values in the unamended control. The percentage figures at day 56 after planting

could be compared with corresponding figures in the incubation experiment without plants, Figure 1. The scale on the y-axis is based on

the dose of N or S per pot and therefore differs between treatments for S.

N and S in cropping systems 715

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

Starting from day 49, white cabbage in the red

clover treatment had higher N concentration than all

other treatments (pB0.001), and peaked at above

5% on day 56 (Figure 3b). In contrast, the N

concentration in the Biofer and biogas slurry treat-

ments decreased during the time of the experiment

but differed among themselves from day 49 through

to harvest (pB0.001). The crop S concentration

(Figure 3c) was, on average over sampling dates, six

times higher in the Biofer treatment than in the other

treatments, although the concentration declined

towards day 63 (pB0.001 compared with previous

sampling occasions).

The N:S ratio (Figure 3d) in the white cabbage

shoots was higher in the two green manure-based

treatments than in the Biofer treatment on all

sampling occasions (pB0.001). The ratio also dif-

fered between the red clover and biogas slurry

treatment on all sampling occasions (pB0.001)

except at final harvest. The biogas slurry treatment

was the only treatment that showed a change in crop

N:S ratio over time, with a significant increase at

final harvest (pB0.001).

Arylsulphatase activity in bulk and rhizosphere soil

At harvest (day 63 after planting), arylsulphatase

activity in the bulk soil was higher in both red clover-

based treatments than in the Biofer and unamended

control treatments (pB0.001), while arylsulphatase

activity in the rhizosphere soil did not differ between

treatments (Figure 4).

Arylsulphatase activity in the bulk soil (day 63,

Figure 4) was negatively correlated with white

cabbage shoot S concentration (r��0.52,

pB0.05 with all treatments included in the correla-

tion analysis, r��0.81, pB0.01 with only

amended treatments included) and positively with

shoot N:S ratio (r�0.82, pB0.001 with all treat-

ments were included in the correlation analysis,

Figure 3. White cabbage (a) shoot DW development, (b) N concentration, (c) S concentration and (d) shoot N:S ratio on days 42, 49, 56

and 63 after planting in a pot experiment with 1980 g DW soil per pot. The treatments included pots with unnamed soil and pots amended

with one of three fertilizers; green manure based (fresh red clover and red clover digested in a biogas reactor) and Biofer (an organic

fertilizer based on by-products from the slaughter industry). Symbols represent treatment means9SEM (n�4).

716 S. Elfstrand et al.

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

r��0.79, pB0.01 with only amended treatments

included). The arylsulphatase activity in the rhizo-

sphere soil was only correlated with shoot S con-

centration (r�0.55, pB0.05), and only when all

four treatments were included in the analysis.

Discussion

Availability of S in relation to N

Even when the same amount of N was applied and

an equal percentage of N was mineralized from the

fertilizers (Figure 1), white cabbage DW yield

differed between treatments, with the highest yield

achieved in the Biofer treatment (Figure 3a). It is not

likely that the initial difference in mineralization

pattern could explain this difference in DW growth,

as the white cabbage N concentration (Figure 3b) in

all treatments was above or in the range of the

deficiency limit for vegetable crops, which is around

1.5% N (Bergmann, 1992). A possible explanation

for the low growth and DW yield in the green

manure-based treatments compared with the Biofer

treatment is lack of S. Critical S values derived from

oilseed rape leaf tissue have shown that a concentra-

tion below 0.35% S in young, fully developed leaves

is in the range of severe S deficiency, growth is

retarded but no macroscopic symptoms are visible

between 0.35% and 0.55% S and at 0.65% S

maximum yield may be obtained (Haneklaus &

Schnug, 1994). The S concentration in white

cabbage in this experiment was well above 0.65%

in the Biofer treatment until final harvest, but the red

clover and biogas slurry treatments and the un-

amended control had an S concentration well below

the 0.35% deficiency limit (Figure 3c).

The N:S ratio, which is sometimes suggested to be

more stable at different plant growth stages than

total S (Scherer, 2001), was higher (20�30) in the

green manure-based treatments than the recom-

mended ratio (10�15) (Tabatabai, 1984; Zhao

et al., 1996) and higher than in the Biofer treatment

(Figure 3d). Between days 42 and 56 the ratio also

differed between the green manure-based treat-

ments, being higher in the red clover treatment

than in the biogas slurry treatment. However, both

green manure-based fertilizers were too low in S in

relation to N. Hence, when using green manure-

based fertilizers it is important to use a complemen-

tary fertilizer high in S such as Biofer, particularly in

crops with a high S demand.

On day 56, mineralisation of SO42�-S in both

green manure-based treatments was higher in the pot

experiment with white cabbage (Experiment II) than

in the incubation experiment (Experiment I), 35 and

44% compared to �6 and 0% for red clover and

slurry respectively (Figures 1 and 2). Higher miner-

alization of SO42�-S in the presence of plants

is in line with Castellano and Dick (1991) and can

be attributed to mineralization of organic S from root

exudates and senescing roots and stimulation of S

mineralization by plant uptake and release of exu-

dates. The N mineralization was higher in the pot

experiment with white cabbage (Experiment II) than

in the incubation experiment (Experiment I) for the

red clover but not the biogas slurry treatment, where

organically bound N is stabilized in recalcitrant

compounds (Nordberg & Edstrom, 1997; Bath &

Ramert, 2000). This resulted in a higher N avail-

ability in soil and a higher N concentration in plants

in the red clover treatment.

However, the high soil min-N content and high

plant N concentration did not promote cabbage

growth in the red clover treatment before day 56

(Figure 3). The difference in cabbage growth be-

tween the green manure-based treatments was prob-

ably an effect of lower S input in the red clover

treatment. In line with this, the symptoms of S

deficiency in the cabbage crop appeared earlier and

were more severe in the red clover than in the biogas

slurry treatment. Immobilization of S due to more

readily available C in the clover treatment could have

reinforced the difference in S availability between the

green manure-based treatments. Nziguheba et al.

(2006) showed that the mineralization of S is largely

completed within 16 h of residue addition to soil.

The rapid mineralization can be explained by a high

content of compounds such as protein and amino

acids that are very labile to microbial decomposition

0

10

20

30

40

50

Biofer Red clover Biogas slurry Control

Ary

lsu

lph

ata

se a

ctiv

ity

(µ

g P

NP

g D

W s

oil -

1 h -1

)

Rhizosphere soil

Bulk soil

Figure 4. Arylsulphatase activity, measured as the amount of

p-nitrophenol (PNP) produced per g DW soil and hour, in bulk and

rhizosphere soil. The activity was measured in a pot experiment

(1980 g DW soil) with white cabbage at harvest (day 63 after

planting). The treatments included pots with unnamed soil and

pots amended with one of three fertilizers; green manure based

(fresh red clover and red clover digested in a biogas reactor) and

Biofer (an organic fertilizer based on by-products from the

slaughter industry). Bars represent treatment means9SEM

(n�4).

N and S in cropping systems 717

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

(Tabatabai & Chae, 1991). In our experiment, how-

ever, immobilization of S would not have been regis-

tered, since the first sampling took place after 24 h.

Arylsulphatase activity and its correlation to N and S

status of the cropping systems

Sinsabaugh et al. (1993) suggested that the sub-

strate-enzyme feedback mechanisms regulating soil

enzyme activity could be used to indicate nutrient

limitations, since high enzyme activities could in-

dicate substrate deficiency, while low activities in-

dicate synthesis suppression when substrate is

available in high concentrations (Dilly & Nannipieri,

1998). In line with this, we expected the arylsulpha-

tase activity to be highest in treatments with poor S

status. The S concentration and N:S ratios of the

white cabbage crop were used as indicators of S

availability in the treatments. The strong correlation

between arylsulphatase activity and both white

cabbage shoot S concentration and shoot N:S ratio

suggests that arylsulphatase activity in the bulk soil

may serve an indicator of the S status in controlled

pot experiments (Figure 4). However, arylsulphatase

activity in the rhizosphere soil did not follow the

same pattern as in the bulk soil. On the contrary, the

Biofer treatment that had the highest crop S

concentration at harvest also tended to have higher

arylsulphatase activity. The rhizosphere soil can be

expected to better reflect the current S status of the

crop than the bulk soil, since the root can alter the

availability of nutrients in the rhizosphere. Thus, it is

possible that the contradictory pattern of the activity

levels in the rhizosphere and bulk soil in the Biofer

treatment can be explained by a recent change in

crop S status. Indeed, the shoot S concentration in

the Biofer treatment declined significantly in the

week before harvest of the white cabbage (Figure 3).

As arylsulphatase activity has been shown to increase

in response to S deficiency in plants (Knauff et al.,

2003), it is possible that the observed decline in white

cabbage S status stimulated arylsulphatase activity in

the rhizosphere. It could also be a microbial response

to S deficiency in the rhizosphere, as plant uptake of S

increased with plant growth. However, since S con-

centration was only measured in the bulk soil and not

in the rhizosphere, this scenario cannot be confirmed

by this experiment.

The unamended control treatment also deviated

from the hypothesized pattern, that is, the relation-

ship between low S status and high arylsulphatase

activity. However, this can be explained by lower

availability of C in the control treatment compared

with the treatments amended with organic fertilizers,

and also by the poor growth of the white cabbage

crop, which was likely to result in less exudation and

rhizodeposition by the crop. These factors probably

resulted in an overall lower microbial activity and

consequently a lower enzymatic activity level in this

treatment. This illustrates the difficulty in correlating

the arylsulphatase activity to S status of a complex

cropping system.

References

Bath, B. & Elfstrand, S. (2008) Use of red clover-based green

manure in leek cultivation. Biological Agriculture and

Horticulture, 25, 269�286.Bath, B. & Ramert, B. (2000). Organic household wastes as a

nitrogen source in leek production. Acta Agriculturae Scandi-

navica, Section B, Soil and Plant Science, 49, 201�208.Bergmann, W. (1992) Nutritional Disorder of Plants: Development,

Visual and Analytical Diagnosis (New York: G. Fisher).

Bremner, J. M. & Hauk, R. D. (1982) Advances in methodology

for research on nitrogen transformation in soil. In F. J.

Stevenson (ed.) Nitrogen in Agricultural Soils (Madison, WI:

American Society of Agronomy), pp. 467�502.

Bremner, J. M. & Mulvaney C. S. (1982) Nitrogen-total. In A. L.

Page, R. H. Miller & D. R. Keeney (eds.), Methods of Soil

Analysis, American Society of Agronomy (Madison, WI: Soil

Science Society of America), pp. 595�624.

Castellano, S. D. & Dick, R. P. (1991) Cropping and sulphur

fertilization influence on sulphur transformations. Soil

Science Society of America Journal, 55, 114�121.Dilly, O. & Nannipieri, P. (1998) Intracellular and extracellular

enzyme activity in soil with reference to elemental cycling.

Zeitschrift fur Pflanzenernahrung und Bodenkunde, 161, 243�248.Ekbladh, G. (2007) Plant Analysis as a Tool to Determine Crop

Nitrogen Status. Doctoral theses no. 2007:109, Appendix IV.

SLU, Uppsala.

Elfstrand, S., Bath, B. & Martensson, A. (2007). Influence of

various forms of green manure amendment on soil microbial

community composition, enzyme activity and nutrient levels

in leek. Applied Soil Ecology, 36, 70�82.Eriksen, J. (2005). Gross sulphur mineralisation-immobilisation

turnover in soil amended with plant residues. Soil Biology &

Biochemistry, 37, 2216�2224.Eriksen, J., Nielsen, M., Mortensen, J. V. & Schjorring, J. K.

(2001) Redistribution of sulphur during generative growth of

barley plants with different sulphur and nitrogen status. Plant

and Soil, 230, 239�246.Haneklaus, S. & Schnug, E. (1994) Diagnosis of crop sulphur

status and application of X-ray fluorescence spectroscopy for

the sulphur determination in plant and soil materials.

Sulphur in Agriculture, 18, 31�40.Knauff, U., Schulz, M. & Scherer, H. W. (2003) Arylsulfatse

activity in the rhizosphere and roots of different crop species.

European Journal of Agronomy, 19, 215�223.Kotkova, B., Balık, J., Cerny, J., Kulhanek, M. & Bazalova, M.

(2008) Crop influence on mobile sulphur content and

arylsulphatase activity in the plant rhizosphere. Plant, Soil

and Environment, 54, 100�107.Linden, B. (1981) Movements and distribution of ammonium-

and nitrate-N in the soil. II. Methods of sampling and

analysing mineral nitrogen. Avdelningen for Vaxtnaringslara,

Rapport, 137. SLU; Uppsala, Sweden. (In Swedish; English

summary).

Ljung, G. (1987) Mekanisk analys � Beskrivning av en rationell

metod for jordartbestamning. Avdelningen for Lantbrukets

Hydroteknik, Avdelnings meddelande, 87. SLU; Uppsala,

Sweden. (In Swedish)

718 S. Elfstrand et al.

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012

Nordberg, A. & Edstrom, M. (1997) Co-digestion of a ley crop

silage, source-sorted municipal solid waste and municipal

sewage sludge. In W. Verstraete (ed.) Anaerobic Conversion for

Environmental Protection, Sanitation and Re-use of Residues

REUR technological series 52, FAO reginal office for Europe

(Belgium: University Gent), pp. 162�172.

Nziguheba, G., Smolders, E. & Merckx, R. (2006) Mineralization

of sulphur from organic residues assessed by inverse isotope

dilution. Soil Biology & Biochemistry, 38, 2278�2284.Scherer, H. W. (2001) Sulphur in crop production � Invited

paper. European Journal of Agronomy, 14, 81�111.Sinsabaugh, R. L., Antibus, R. K., Linkins, A. E., McClaugherty,

C. A., Rayburn, L., Repert, D. & Weiland, T. (1993). Wood

decomposition over a first-order watershed: Nitrogen and

phosphorus dynamics in relation to extracellular enzyme

activities. Ecology, 74, 1586�1593.

Tabatabai, M. A. (1982) Sulfur. In A. L. Page, R. H. Miller & D. R.

Keeney (eds.) Methods of Soil Analysis, Part 2 (Madison, WI:

SSSA), pp. 501�538.

Tabatabai, M. A. (1984) Importance of sulphur in crop produc-

tion. Biogeochemistry, 1, 45�62.Tabatabai, M. A. (1994) Soil enzymes. In R. W. Weaver & S. H.

Mickelson (eds.) Methods of Soil Analysis, Part 2 Microbio-

logical and Biochemical Properties � SSSA Book Series 5

(Madison, WI: SSSA), pp. 814�818.

Tabatabai, M. A. & Chae, Y. M. (1991) Mineralization of sulphur

in soils amended with organic wastes. Journal of Environ-

mental Quality, 20, 684�690.Zhao, F. J., Hawkesford, M. J., Warrilow, A. G. S., McGrath, S. P.

& Clarkson, D. T. (1996). Responses of two wheat varieties to

sulphur addition and diagnosis of sulphur deficiency. Plant and

Soil, 181, 317�327.

N and S in cropping systems 719

Dow

nloa

ded

by [

SLU

Lib

rary

] at

00:

42 0

7 N

ovem

ber

2012