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OPEN Soil properties and productivity

in two long-term crop rotations
differing with respect to organic
Received: 29 January 2018
Accepted: 29 November 2018 matter management on an Albic
Luvisol
Published: xx xx xxxx

S. Martyniuk1, D. Pikuła2 & M. Kozieł1

This study was based on a three-factor experiment carried out since 1980 on a loamy sand (Albic Luvisol)
in which arable crops were grown in two 4-years rotations: RotA (grain maize, winter wheat, spring
barley and silage maize) and RotB [grain maize, winter wheat plus mustard green manure (GM), spring
barley and grass–clover ley (GCL)]. The soil in RotB with an increased input of OM (GM and 1-year GCL)
accumulated significantly larger amounts of soil organic carbon and soil microbial biomass C, had higher
activities of dehydrogenase and acid phosphatase enzymes and gave significantly higher winter wheat
grain yields compared to the soil in RotA. However, in the absence of liming, the soil in RotB, contrary
to that in RotA, became more acidic, had reduced activity of alkaline phosphatase and lower contents
of Ca and Mg, and contained a diminished proportion of the >0.5 mm macroaggregates fraction. These
soil deteriorative effects of crop rotations delivering larger amounts of OM have not been reported so
far. In both rotations FYM applied once per 4-year rotation at 40 Mg ha−1 improved all the tested soil
properties and had mitigating effects on the negative changes found in the soil of RotB.

Biological, chemical and physical properties of mineral soils depend to a great extent on the content of soil
organic carbon (SOC) in their matrix and for this reason SOC, the main component of soil organic matter (SOM),
is regarded as an integrative and most important indicator of soil quality1–3. The quantity and quality of SOM in
soils depend on many edaphic and environmental factors, and change in response to various agriculture related
practices such as: soil tillage, organic amendments, crop rotation and mineral fertilization. Crop management
practices that help to maintain or enrich cultivated soils in organic matter (OM) include those which restrict min-
eralization of SOC, e.g. reduced tillage intensity4,5, and those which increase inputs and gains of OM in soils, such
as: applications of organic fertilizers and wastes (animal manures, green manures, composts, sewage sludge)2,3,6–9,
diversified crop rotations2–4, particularly those including grass-legume leys, and balanced mineral fertilization
increasing crop residues4,7. Interactive effects of organic amendments, crop rotations and mineral fertilizers on
SOM accumulation and other soil properties have been extensively studied, also in numerous long-term field
experiments2,4,10–13, however green manures were seldom included in these experiments. Green manuring
(GM) is most often practiced by ploughing down biomass of cover/catch crops in order to enrich soils in OM,
to improve soil structure and productivity4,8,14, to stimulate activity of soil dwelling organisms9,15, and to reduce
soil erosion, including nutrients leaching8,14. Thus, all good crop and soil management practices leading to the
accumulation OM in soils can be regarded as beneficial to their quality2,7–12. However, it has also been shown that
large inputs of OM, e.g. through long-term applications of high rates of cattle manure or manure + NPK, resulted
in the deterioration of the formation and stability of large soil aggregates13,16.
With respect to other soil properties, it was shown that externally added plant materials, like green manures
or composts, elevated soil pH2,17. However, we have found in our previous studies18 that after 16 years of growing

1
Department of Agricultural Microbiology, Institute of Soil Science and Plant Cultivation, Czartoryskich 8, 24-100,
Puławy, Poland. 2Department of Plant Nutrition and Fertilization, Institute of Soil Science and Plant Cultivation,
Czartoryskich 8, 24-100, Puławy, Poland. Correspondence and requests for materials should be addressed to S.M.
(email: [email protected])

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Inorganic fertilizer rate (kg ha−1 y−1)

Crop
rotation Plant N0 N1b N2 N3 P2O5 K2O
Grain maize 0 50 100 150 54 160
Winter wheat 0 50 100 150 54 100
RotA
Spring barley 0 30 60 90 54 85
Maize for silage 0 50 100 150 54 120
Grain maizea 0 50 100 150 54 160
Winter wheat 0 50 100 150 54 100
RotB
Spring barley 0 30 60 90 54 85
Grass-clover ley 0 50 100 150 54 120

Table 1. Inorganic fertilizer rates applied in RotA and RotB of the long-term experiment at Grabow, Poland.

FYM N rates
(Mg ha−1) (kg ha−1) 1989 1993 1997 2001 2005 2009 2013
0 0 7.65 (1.45) 6.31 (0.88) 9.20 (1.79) 7.45 (1.69) 5.10 (1.22) 3.50 (1.18) 0.50 (0.10)
0 100 14.71 (2.48) 17.58 (2.36) 13.00 (2.28) 13.55 (2.81) 12.00 (2.45) 3.20 (0.88) 0.60 (0.20)
0 150 15.77 (2.42) 18.85 (2.40) 13.60 (2.36) 14.85 (2.50) 13.20 (2.30) 4.80 (1.09) 0.50 (0.10)
40 0 8.18 (1.53) 6.30 (0.85) 9.60 (1.70) 8.65 (2.05) 7.40 (1.83) 5.30 (1.65) 0.50 (0.20)
40 100 15.73 (2.64) 18.08 (2.38) 12.60 (2.34) 16.50 (3.21) 12.90 (2.63) 5.30 (1.55) 0.50 (0.20)
40 150 17.50 (2.66) 19.30 (2.52) 12.80 (2.35) 20.40 (3.10) 14.80 (2.87) 7.20 (2.02) 0.50 (0.20)
Mean 13.26 (2.20) 14.40 (1.90) 11.80 (2.14) 13.57 (2.56) 10.90 (2.23) 4.88 (1.44) 0.52 (0.17)

Table 2. Yields (Mg ha−1) of mustard green manure fresh and dry matter (in brackets) grown after winter wheat
harvest in Rot B of the long-term experiment at Grabow, Poland.

arable plants in a cropping system with an increased input of OM, due to growing grass-clover ley (GCL) and
mustard green manure (GM), soil pH decreased in comparison to that in similar cropping system, but without
GCL and GM. This long-term field experiment is still running and the main objectives of the present study were
(i) to characterize changes in biological, chemical and physical soil properties after 33 years of growing arable
crops in two 4-years rotations differing primarily with respect to organic matter management systems and (ii) to
elucidate possible drivers of these changes.

Results and Discussion

Organic matter input. The long-term field experiment (initiated in 1980) which provided the results pre-
sented in this work includes two 4-years crop rotations (RotA and RotB) with the following order of crops: grain
maize, winter wheat, spring barley and silage maize – for RotA and: grain maize, winter wheat plus mustard GM,
spring barley (with undersown grass–clover) and grass–clover ley (GCL) – for RotB. These rotation fields have
been divided into four replicated blocks and within each block different application rates of FYM and inorganic N
fertilized were varied in a split-plot design. All soil amendments applied in this experiment, particularly mineral
N fertilizer and FYM rates, were the same in both rotations (Table 1). However, the compared crop rotations dif-
fered substantially in organic matter management systems, particularly with respect to fresh organic matter input,
which was markedly larger in RotB than in RotA as a result of growing mustard GM (after winter wheat) and GCL
in the former one. With the exception of the year 2013, when mustard yields were low due to the dry autumn in
this year, in previous years mustard above-ground biomass ranged from about 3 Mg ha−1 fresh weight (FW) in
the unfertilized treatments to more than 20 Mg of FW ha−1 (3.1 DM ha−1) in the treatments with the highest rates
of FYM and mineral N fertilizer (Table 2). We did not measure biomass yields of mustard roots, but results of
other studies19 indicate that at approximately 20 Mg FW ha−1 of mustard above-ground parts, this plant provides
also about 2.5 Mg FW ha−1 of roots. Thus, depending on the fertilization treatments, the soil in RotB receives, per
rotation, from about 6 Mg ha−1 to 23 Mg ha−1 of fresh organic matter (mustard) more than that in RotA. As the
forth crop in each rotation cycle, silage maize or GCL are grown in RotA and RotB, respectively. It was estimated
that incorporation of grass leys or GCLs can provide soils with up to 10 Mg ha−1 of dry organic matter20,21. On
the other hand, comparisons made by Jarchow & Liebman21 indicate that corn can produce approximately four
to six times less root biomass than C3 grasses or grass-legume mixtures. Taking these results into considerations,
it seems reasonable to assume that GCLs enrich the soil of RotB in considerably larger amounts of fresh organic
matter (residues of roots and lower parts of stems) than silage maize grown in RotA. This increased and long-last-
ing input of organic matter (GM and GCL) in RotB has caused both beneficial and also some negative changes in
selected soil properties and winter wheat yields obtained in this rotation as compared to those in RotA.

Beneficial effects. The soil in RotB receiving higher amounts of OM accumulated significantly more SOC
than the soil in RotA, irrespective of FYM application and mineral N rates (Fig. 1). Averaged across all the treat-
ments, the SOC content in RotB was almost 12% higher than that in RotA. In RotA the unamended soil (N-0,
no FYM) contained the lowest amount (6.1 g kg−1) of SOC, while the highest content of SOC (7.43 g kg−1) was

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Figure 1. Organic C and microbial biomass C contents in soil as influenced by crop rotation (I), manure
application (II) and mineral N rate (III) in the long-term field experiment at Grabow, Poland. Error bars
denote standard deviations (n = 3). LSD(P ≤ 0.05): for C org.: I = 0.52, II = 0.65, III = 0.23, Interactions = not
significant; for microbial biomass C: I = 11.5, II = 10.1, III = 11.2, Interactions = not significant.

found in the soil fertilized with N-150 and FYM. In RotB the corresponding values of SOC were 6.67 g kg−1 and
8.5 g kg−1, respectively. In both rotations and at all N rates soil amendment with 40 Mg ha−1 of FYM resulted in
significantly higher SOC contents compared to the unmanured soil. The soil amended with 40 Mg ha−1 of FYM in
RotA was 13.5% richer in SOC, on average for N rates, than the unmanured soil. In RotB this increase was signifi-
cantly higher and amounted to 16.2%. In general, mineral N fertilization had also a beneficial effect on SOC accu-
mulation. This effect was insignificant when FYM was not applied in RotA, but in RotB in the absence of FYM
the soil treated with 100 and 150 kg N ha−1 contained significantly larger amounts of SOC than the unfertilized
soil (N-0). When manure was used, application of 150 kg N ha−1 resulted in the accumulation of the maximum
amounts of SOC in both rotation (Fig. 1).
Soil microbial biomass C (SMBC) contents and soil enzymes (dehydrogenase, acid phosphatase) activities
were also significantly affected by all the treatments studied in this experiment (Fig. 1, Table 3). In general, these
parameters had significantly higher values in RotB than in RotA and when soil was amended with FYM compared
to the unmanured soil in both rotations. The exception was the acid phosphatase activity, which was significantly
higher in the unmanured soil at N-0 than in the soil amended with FYM at N-0, but only in RotB (Table 3). SMBC
was unaffected by mineral N rates only in the case of the unmanured soil in RotB, but in all other treatments

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Amendments Enzymes
Manure N mineral Alkaline
Crop rotation (Mg ha−1) (kg ha−1) phosphatase Acid phosphatase Dehydrogenase
0 0 8.63 33.23 17.28
0 100 6.57 37.53 16.80
0 150 5.57 38.06 16.38
Mean 6.92 36.28 16.81
A
(grain maize – winter wheat – 40 0 14.63 47.87 35.67
spring barley – silage maize)
40 100 11.77 51.77 29.50
40 150 9.70 55.67 27.07
Mean 12.03 51.77 30.74
Overall mean 9.48 44.02 23.78
0 0 6.66 69.57 33.07
0 100 6.03 59.03 27.07
0 150 4.63 51.87 18.47
Mean 5.77 60.17 26.20
B
(grain maize – winter wheat† – 40 0 11.0 62.97 41.17
spring barley – GCL‡)
40 100 8.43 62.50 31.43
40 150 6.60 61.80 23.43
Mean 8.68 62.42 32.01
Overall mean 7.22 61.30 29.11
For: I = 1.22, For: I = 3.72, For: I = 3.09,
II = 0.58, II = 2.48, II = 1.47,
III = 0.61 III = n.s. III = 1.76.
LSD (P ≤ 0.05) Interactions: Interactions: Interactions:
II/I = 0.82 II/I = 3.29 II/I = 2.08
III/I = n.s. III/I = 3.11 III/I = 2.49
III/II = 0.86 III/II = 3.11 III/II = 2.40

Table 3. Activity of acid and alkaline phosphatase (µg pNP g−1 h−1) and dehydrogenase (µg formazan g−1
24 h−1) in soil as influenced by crop rotation (I), manure application (II) and N fertilizer rates (III) in the long-
term experiment at Grabow, Poland. †plus mustard green manure; ‡grass-clover ley.

microbial biomass C decreased as N rates increased (Fig. 1). Similar responses to N rates were also found for
dehydrogenase activities, but in the case of this enzyme its activity was not significantly affected by N rates only
in the unmanured soil of RotA (Table 3). Although the overall effect of N fertilization on the activity of acid
phosphatase was insignificant, interactions were significant (Table 3). Irrespective of FYM application in RotA,
both N rates were beneficial for the activity of this enzyme, particularly in comparison to the N-0 treatment, but
in the case of RotB the opposite was true, especially in the unmanured soil. The results shown above are in accord
with those reported earlier in numerous studies all over the world, which clearly indicate that various organic
amendments (FYM, slurry, composts, straw and other plan residues) and diversified crop rotations, in particular
those comprising grasses or grass-legume leys, beneficially affect different soil processes and properties, including
accumulation of SOC, SMBC and stimulation of soil biological activities2–8,11. There is also a general consensus
that proper soil fertility management practices on croplands, particularly with respect to N fertilization, enhance
SOC sequestration and are beneficial for soil microorganisms and their activity, mainly due to a greater return of
crop residues to the soil3,4,7,22,23. However, there are also reports showing that mineral N fertilizers, particularly
their high rates, can reduce SMBC and soil enzymes activities5,24, and our results with respect to SMBC and the
dehydrogenase activity (Fig. 1, Table 3) are in accordance with these reports.
In 2013 winter wheat was grown in both rotations and it gave significantly higher grain yields in all the treat-
ments of RotB than in the corresponding treatments of RotA (Fig. 2), even though mineral fertilization (NPK)
was the same in these rotations (Table 1). Ten Berge et al.25 reported that mean DM yields of all other crops grown
in this experiment were also higher in RotB than in RotA. The lowest grain yield (2.55 Mg ha−1) was obtained in
RotA on plots without FYM and N application, while in the same treatment in RotB winter wheat yielded over 1
Mg ha−1 more. Grain yields on the manured plots at N-0 were 4.4 Mg ha−1 and 4.87 Mg ha−1 for RotA and RotB,
respectively. Beneficial effects of FYM addition on winter wheat yields were also clearly visible at both N rates
(N-100 and N-150). In both rotations the maximum grain yields were obtained at the highest N rate (150 kg ha−1)
and FYM application, but in RotB the yield was almost 10% higher than that in RotA (Fig. 2). The higher SMBC
content (Fig. 1) and more active microorganisms, as indicated by the increased dehydrogenase and acid phos-
phatase activities (Table 3), transforming larger amounts of fresh OM and the stored SOC (Fig. 1) in RotB were
probably responsible for a better performance of winter wheat (Fig. 2) and other crops grown in this rotation
than those grown in RotA25, despite less favorable soil pH in the former one (Fig. 3). The lack of yield reduction
in response to soil acidification is uncommon, although not unknown in the scientific literature. For example,
Schroder et al.26 monitored soil acidification as a result of over 30 years use of increasing rates (up to 272 kg N
ha−1) of various N mineral fertilizers and found that although soil pH levels decreased to <5.0 already after 10
years of N fertilization, no significant reductions of winter wheat grain yield were observed during 25 consecutive

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Figure 2. Winter wheat grain yield as influenced by crop rotation (I), manure application (II) and mineral N
rate (III) in the long-term field experiment at Grabow, Poland. Error bars denote standard deviations (n = 4).
LSD(P ≤ 0.05): for I = 0.34, II = 0.30, III = 0.29, Interactions: I x II and I x III = not significant, II x III = 0.40.

Figure 3. Soil acidity (pH in H2O) as influenced by crop rotation (I), manure application (II) and mineral N
rate (III) in the long-term field experiment at Grabow, Poland. Error bars denote standard deviations (n = 3).
LSD(P ≤ 0.05): for I = 0.31, II = 0.20, III = 0.09, Interactions: I x II and I x III = not significant, II x III = 0.13.

growing seasons. It is important to add that in this experiment winter wheat was grown in monoculture and no
organic amendments were added to the soil. It seems reasonable to assume that in our experiment yield decreases
will occur in the future and that the delay in the appearance of negative effects of a stronger soil acidification in
RotB on crop yields results from the beneficial effects (as discussed above) of a higher input of organic matter
(fresh GM, GCL) in this rotation as compared to that in RotA.

Negative effects. In this experiment no soil liming was performed to show soil buffering properties of FYM,
and Fig. 3 shows that this effect was evident in both rotations, although less pronounced in RotB. For the purpose
of this study only treatments with 40 Mg ha−1 of FYM were selected, as similar rates (30–40 Mg ha−1) are most

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Figure 4. Ca and Mg contents in soil as influenced by crop rotation (I), manure application (II) and mineral N
rate (III) in the long-term field experiment at Grabow, Poland. Error bars denote standard deviations (n = 3).
LSD(P ≤ 0.05): for Ca: I = 0.32, II = 0.43, III = 0.36, Interactions = not significant; for Mg: I = 2.11, II = 2.02,
III = 1.25, Interactions = not significant.

often used in practice by farmers and also because higher rates (60 Mg ha−1 and 80 Mg ha−1) of FYM applied in
this experiment were only slightly more effective in maintaining soil pH than FYM at 40 Mg ha−1. The soil pH val-
ues in the case of unmanured plots of RotA ranged from 5.3 in the N-0 treatment to 4.9 and 4.67 when 100 kg ha−1
and 150 kg ha−1 of N were applied, respectively. Soil pH in the unmanured plots of RotB had significantly lower
values, which were 4.7, 4.6 and 4.3 for N-0, N-100 and N-150 treatments, respectively (Fig. 3). In the absence of
liming increasing fertilizer N rates caused stronger soil acidification, particularly in the unmanured plots of both
rotations, thus confirming results of previous studies5,27-29.
Since soil amendments, particularly mineral N fertilizer and FYM rates, were the same in both rotations we
attribute the lower values of soil pH in RotB to growing mustard GM and GCL in this rotation, which had ben-
eficial effects on crop yields, as discussed above. Higher crop yields25 (Fig. 2) indicate higher plant offtake and
export of nutrients (including basic cations (K+, Ca2+, Mg2+), from the soil in RotB than that in RotA, and this
was probably the main cause for the more intensive soil acidification in RotB compared to RotA (Fig. 3). Indeed,
Fig. 4 shows that the exchangeable Ca and Mg contents in the soil of all the treatments in RotB were significantly
lower than those in RotA. Although the chemical mechanisms for soil pH change by plant residues are not fully

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Amendments Aggregate content (g kg soil−1)

Manure N rates
Crop rotation (Mg ha−1) (kg ha−1) >0.5 mm 0.5-0.25 mm <0.25 mm
0 0 615.7 181.7 202.6
0 100 617.7 172.0 210.3
0 150 615.7 188.0 196.3
Mean 616.3 180.6 203.1
A
(grain maize – winter wheat – 40 0 718.3 126.3 155.4
spring barley – silage maize)
40 100 700.3 146.0 153.7
40 150 650.0 167.0 183.0
Mean 689.6 146.4 164.0
Overall mean 652.9 163.5 183.6
0 0 517.0 281.0 202.0
0 100 510.0 271.0 219.0
0 150 526.3 249.0 224.7
Mean 517.8 267.0 215.2
B
(grain maize – winter wheat† – 40 0 539.3 272.0 188.7
spring barley – GCL‡)
40 100 548.3 252.0 199.7
40 150 508.3 277.7 214.0
Mean 532.0 267.2 200.8
Overall mean 524.9 267.1 208.0
For: I = 12.5, For: I = 16.0,
For: I = 30.4, II = 44.0,
II = n.s., II = 19.6,
LSD (P ≤ 0.05) III = n.s.
III = n.s. III = n.s.
Interactions = n.s.
Interactions = n.s Interactions = n.s.

Table 4. Contents of different aggregate size classes in air dry soil as influenced by crop rotation (I), manure
application (II) and mineral N fertilizer (III) in the long-term field experiment at Grabow, Poland. †Plus
mustard green manure; ‡grass-clover ley.

understood, it is generally accepted that the most significant proton (H+) and hydroxyl ion (OH−) generating
processes occur during C and N transformations in soils27,29. CO2 production during microbial decomposition
of fresh organic matter (GM and GCL) in Rot B might have been another soil acidifying factor in this rotation.
Contrary to the acid phosphatase, the activity of alkaline phosphatase was significantly lower in all the treat-
ments of RotB compared to RotA (Table 3), probably due to more acidic soil reaction in RotB than that in RotA30.
In both rotations soil manuring had beneficial effects on the alkaline phosphatase, but increasing rates of fer-
tilizer N significantly reduced the activity of this enzyme and these results agree with those obtained in other
studies5,31,32.
To get insight into the soil structure (aggregation) as influenced by the experimental factors we have sieved dry
soil samples through a set of sieves to separate two macroaggregate fractions (>0.5 mm and 0.5–0.25 mm) and
one microaggregate fraction (<0.25 mm) and results of these analyses are shown in Table 4. The soil in RotA con-
tained significantly more of the >0.5 mm macroaggregate fraction than the soil in RotB, but the opposite was true
for the smaller (0.5–0.25 mm) macroaggregates and the microaggregate fraction, indicating that the formation of
large aggregates in RotB was reduced. Although it has been reported that in soils treated yearly and for a long time
with high rates of organic amendments the dispersion of large aggregates may be increased13,16, in the case of our
study the decreased share of the >0.5 mm macroaggregate fraction in the soil of RotB might result mainly from
the lower contents of bivalent cations, Ca2+ and Mg2+ (Fig. 4) in this soil compared to the soil in RotA, as these
ions, particularly Ca2+, are known to play an important role in soil aggregation through the formation of bridges
between clays and SOM particles10,33. Similarly to the results of previous studies13,34, FYM applied to the soil at the
rate 40 Mg ha−1 increased the content of the >0.5 mm macroaggregate fraction and reduced the microaggregate
fraction in comparison to the unmanured soil, but this effect was significant only in Rot A (Table 4).
In summary, this work has shown that the soil in RotB with an increased input organic matter (GM, GCL)
accumulated significantly larger amounts of SOC and SMBC, had higher activities of dehydrogenase and acid
phosphatase enzymes and gave significantly higher winter wheat grain yields compared to the soil in RotA with-
out GM and GCL. However, the soil in RotB, contrary to that in RotA, has become more acidic, had lower con-
tents of Ca and Mg and contained a diminished proportion of the >0.5 mm aggregates fraction. Thus, these
results are in accordance with those reported earlier that arable crop rotations providing soil with larger amounts
of organic matter, e.g. through growing GM or GCL, exert many beneficial effects on soil properties and produc-
tivity. Nevertheless, under some circumstances, e.g. in the absence of soil liming, such rotations may have also
deteriorative effects on some properties (e.g. soil pH and aggregation) of loamy sandy soils (Luvisols). Regardless
of the crop rotation, FYM applied once per 4-year rotation at the rate of 40 Mg ha−1 exerted beneficial effects on
all the tested soil properties, including mitigating effects on the negative changes found in the soil of RotB.

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Methods
Study site and experimental design. The study was based on a three-factor long-term field experiment,
which has been established in 1980 at the Grabow Experimental Station (Lat: 51°21′N; Long: 21°40′E), belonging
to the Institute of Soil Science & Plant Cultivation in Pulawy, Poland. The soil was classified as an Albic Luvisol
(FAO, 1998) and is of loamy sand texture (70% sand, 25% silt, 5% clay). The climate at the site is temperate with
a mean annual rainfall of about 560 mm and a mean annual temperature of 7.8 °C. The experiment includes two
4-years crop rotations (RotA and RotB) with the following order of crops: grain maize, winter wheat, spring bar-
ley and silage maize – for RotA and grain maize, winter wheat plus mustard GM, spring barley (with undersown
grass–clover) and grass–clover ley (GCL) – for RotB. Till 2008 potatoes were grown in both rotations, which were
then replaced by grain maize in view of the rapid expansion of grain maize in Poland. Barley straw and wheat
straw are harvested in both rotations. Mustard (Sinapis alba L.) for green manuring is sown in the third decade of
August, shortly after disk harrowing of winter wheat stubble. At the beginning of November green mustard bio-
mass is disked and about two weeks later incorporated into the soil by ploughing. The grass–clover sward is har-
vested in three to four cuts per year and removed. Grass-clover sod is disked and ploughed down in the autumn.
Within each rotation field, application rates of FYM and inorganic N fertilizer were varied in a split-plot design
replicated in four blocks per field. Five FYM rates were assigned to main plots, starting in autumn 1979. FYM
was applied in both rotations once per 4-year cycle, in the autumn preceding potatoes (grain maize), at rates of 0,
20, 40, 60 and 80 Mg ha−1. FYM was incorporated by disc harrowing and ploughing, one or 2 days after applica-
tion. On average, FYM had a pH of about 8.4 and contained 5.4 (N), 3.5 (P2O5) and 5.8 (K2O) kg Mg−1 (fresh wt.
basis). In 1988 N fertilizer rates were introduced as a third orthogonal factor. Four N rates were assigned to plots
within each main plot, that is per FYM rate. Each plot measures 8 × 5 m gross, or 6.25 × 4 m net harvested area.
In this experiment no soil liming has been done to show a “liming effect” of different rates of manure. Mineral
fertilizes rates for particular plants are given in Table 1. N was applied as ammonium nitrate (34% N), P as triple
superphosphate (45% P2O5) and K as potassium chloride (60% K2O). In this experiment conventional soil tillage
system is used, with skimming in order to cover stubble, followed by mouldboard ploughing to 25 cm.

Soil sampling and analyses. In 2013, when winter wheat was grown in RotA and RotB, replicated wheat
plots from selected treatments (Tables 3 and 4) were sampled for the purpose of this study. At the beginning of
June soil samples (ten per plot) were taken between plant rows from a 0–25 cm depth using a soil corer (30 mm
internal diameter). Field moist soil samples were passed through a sieve with 2 mm openings and stored at 4 °C.
Microbial biomass C was determined by the chloroform-fumigation-extraction method and calculated accord-
ing to the following formula: Cmic = EC/kEC, where EC = soluble C in fumigated samples – soluble C in control
(un-fumigated) samples and kEC = 0.4535. An Automated N/C Analyzer (Multi N/C 2100, Analytik Jena, Jena,
Germany) was used to measure C contents in soil extracts. Dehydrogenase activity was estimated using TTC
(2,3,5-triphenyltetrazolium chloride) as the substrate36 and, in the case of phosphatases (acid and alkaline),
p-nitrophenyl phosphate (PNP) was used as the substrate30.
Subsamples of field moist soil were also sieved through 5 mm screens and air-dried at room temperature.
These samples were used to assess basic parameters of soil structure (aggregation) using a dry soil sieving method
similar to that described by Nimmo & Perkins37. To separate macroaggregates and microaggregates, portions
(200 g) of air dried soil samples were hand sieved through a set of two nested sieves with 0.5 mm and 0.25 mm
openings to obtain two macroaggregate size fractions (>0.5 mm and 0.5–0.25 mm) and one microaggregate frac-
tion (<0.25 mm) collected in the bottom container. The aggregates retained in each compartment were weighed
and expressed in g per kg of soil dry matter (DM).
Determinations of: C org. (PN-ISO 14235 – Soil Quality – Determination of organic carbon by sulfochromic
oxidation), exchangeable Ca2+ and Mg2+ (extracted with 1.0 M ammonium acetate and measured by AAS) and
soil pH (potentiometrically in 1:2.5 suspension of soil in H2O [ISO 10390 – Soil Quality – Determination of
pH]) were performed by the certified chemical laboratory of the Institute of Soil Science and Plant Cultivation in
Pulawy, Poland.
The data were subjected to the 3 way analysis of variance (ANOVA) with significance of differences assessed at
P ≤ 0.05, using the FR-ANALWAR software based on Microsoft Excel.

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Acknowledgements
This work was supported by the Tasks 1.4 and 2.2 of the State Project IUNG-PIB founded by Polish Ministry of
Agriculture and Rural Development.

Author Contributions
S.M. designed the study, analyzed the data and wrote the paper; D.P. supervises the experiment, collected soil
samples and contributed to the physicochemical soil analysis; M.K. performed the microbial and enzymatic soil
analyses; all authors discussed the results and reviewed the manuscript.

Additional Information
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