INTERUNIVERSITY PROGRAMME
IN
PHYSICAL LAND RESOURCES
Ghent University
Vrije Universiteit Brussel
Belgium
CONTRIBUTION OF MAIZE ROOT-DERIVED CARBON
TO STABLE SOIL ORGANIC MATTER
Promoter:
Prof. Dr. ir. Steven Sleutel
Master dissertation submitted in
partial
fulfillment
of
the
requirements for the degree of
Master of Science in Physical
Land Resources
by Marissa Permatasari Jayaputra
(Indonesia)
Academic Year 2014–2015
This is an unpublished M.Sc dissertation and is not prepared for further distribution.
The author and the promoter give the permission to use this Master dissertation
for consultation and to copy parts of it for personal use. Every other use is subject
to the copyright laws, more specifically the source must be extensively specified
when using results from this Master dissertation.
Gent, August 2015
The Promoter,
The Author,
Prof. Dr. ir. Steven Sleutel
Marissa Permatasari Jayaputra
ACKNOWLEDGEMENTS
I have finally arrived at the last phase of my journey here in Ghent University.
Certainly I could not have made it through these two years on my own. Thus I
would like to thank those who have taken part in bringing me to this finish line.
First of all, I would like to thank God for His unending love and kindness in my life,
without which I would not have come to this point and be the person I am now. It
is only in His grace alone can I find my comfort and strength.
Next I would like to thank my promoter, Prof. Steven Sleutel, and my tutor, An
Vanderhasselt, for all the encouragement, guidance, and input throughout this
thesis research. I am truly grateful for your support. I would also like to
acknowledge the financial support I received from Indonesia Endowment Fund for
Education, Indonesian Ministry of Finance, which enabled me to pursue my Master
degree here in Belgium. It was an honor for me to be one of the scholarship
recipient.
Furthermore, I also thank my lecturers back in Bogor Agricultural University. You
are the ones who gave me the idea to study for a Master degree and also showed
me the way to Ghent University. I am very glad now that I took your advices and
came here. Also thanks to Theresia for all the tears and laughter we have shared
these two years. Hope our friendship grows even more in the future. Last but not
least, thanks to my mom and brothers. I know I could not thank you enough for
keeping me at ‘home’ wherever I am. Thanks for reminding me that I am always
there in your heart.
Gratefully yours,
Marissa Permatasari Jayaputra
TABLE OF CONTENTS
TABLE OF CONTENTS ........................................................................................ i
LIST OF FIGURES ..............................................................................................iii
LIST OF TABLES ...............................................................................................iiv
LIST OF ABBREVIATIONS ................................................................................. v
ABSTRACT ........................................................................................................ vi
I. INTRODUCTION .............................................................................................. 1
1.1. Background ............................................................................................... 1
1.2. Research questions................................................................................... 2
II. LITERATURE REVIEW ................................................................................... 3
2.1. Maize (Zea mays L.).................................................................................. 3
2.2. Soil Organic Matter.................................................................................... 4
2.2.1. Nature of soil organic matter ............................................................... 4
2.2.2. Role of organic matter to soil fertility in croplands ............................... 4
2.3. Microbial mediated SOM decomposition ................................................... 5
2.4. Contribution of root vs. shoot-derived OM to native SOM .......................... 5
2.4.1. Roots vs. shoots as OM inputs in agricultural soils.............................. 5
2.4.2. Differences in C input from roots vs. shoots ........................................ 6
2.4.3. Stability of SOM from root-derived C vs shoot-derived C .................... 8
2.5. Experimental assessment of the importance of root- vs. shoot-derived C 10
2.5.1. Quantifying root biomass .................................................................. 10
2.5.2. Microrhizotrons ................................................................................. 11
2.5.3. 13C natural abundance ...................................................................... 11
2.5.4. 14C pulse labelling ............................................................................. 12
2.6. Physical fractionation .............................................................................. 13
III. MATERIALS & METHODS ........................................................................... 15
3.1. Comparison of maize root biomass as a function of cultivar and soil
texture .................................................................................................... 15
3.1.1. Maize cultivar experiment ................................................................. 15
3.1.2. Maize root extraction......................................................................... 16
3.2. Comparison of soil C distribution of silage vs. grain maize or non-maize
cropped soil to determine the relative stability of maize root-derived
SOM....................................................................................................... 17
3.2.1. Long-term field experiments.............................................................. 17
3.2.2. Soil sampling .................................................................................... 21
i
3.3. Physical fractionation to isolate specific soil fractions (intra microaggregate particulate organic matter) and quantification of maizederived C contribution ............................................................................ 22
3.3.1. Dispersion and size fractionation ...................................................... 22
3.3.2. Sequential sedimentation .................................................................. 23
3.3.3. Density separation ............................................................................ 25
3.4. C-content and C-isotope analysis ............................................................ 26
3.5. Statistical analysis ................................................................................... 27
IV. RESULTS AND DISCUSSION ..................................................................... 28
4.1. Maize root biomass ................................................................................ 28
4.2. Bulk soil OC content and content of maize derived C of silage maize
vs. grain maize or non-maize cropped soil ............................................. 29
4.2.1. Soil organic carbon ........................................................................... 29
13
4.2.2.
Measurement of
C ................................................................... 31
4.2.3.
Content of maize-derived C .......................................................... 33
4.3. Distribution of soil C and maize-derived C over physical fractions .......... 35
4.3.1.
Dry matter weight ......................................................................... 35
4.3.2.
Soil organic carbon ....................................................................... 36
4.3.3.
Maize-derived C ........................................................................... 40
4.4. Relative stability of shoot vs. root derived C ............................................ 46
V. CONCLUSION .............................................................................................. 48
REFERENCES .................................................................................................. 50
ii
LIST OF FIGURES
Figure 1. C inputs from different treatments through amendments, roots including
rhizodeposition, and above-ground crop residues in Ultuna long-term
field experiment with crop rotation of oat (1956), spring cereals (19571999), and silage maize plant (last 10 years) ..................................... 8
Figure 2. Equipment for 14C labelling in the field .............................................. 13
Figure 3. Locations of experimental plots for comparison of maize root
biomass ............................................................................................ 15
Figure 4. Root sampling from harvested rows in the Merelbeke maize cultivar
trial ................................................................................................... 16
Figure 5. Procedure of root sample preparation .............................................. 16
Figure 6. Locations of long term experimental trials in Belgium (top), Italy
(middle), and Germany (below) ........................................................ 18
Figure 7. Dispersion and size fractionation of soil aggregates < 4 mm ............ 23
Figure 8a. Steps in sequential sedimentation to separate the free soil clay
fraction. Sedimentation thermal bath (i) and at the beginning of
sedimentation cycle (ii) ..................................................................... 24
Figure 8b. Steps in sequential sedimentation to separate the free soil clay
fraction. at the end of sedimentation cycle (iii) and drying the <2μm
suspension (iv) ................................................................................. 25
Figure 9. Set-up of ultrasonic dispersion by means of an ultrasonic
microprobe ....................................................................................... 26
Figure 10. Root biomass of different maize cultivars measured from undisturbed
soil cores collected at 30 cm depth in three ILVO maize cultivar field
trials in Autumn 2014 ........................................................................ 28
Figure 11. Bulk soil OC-content of the sampled plough layer of selected
rotational treatments in three European long-term fields experiments
Bottelare (a), Tetto Frati (b), and Puch (c).. ...................................... 30
Figure 12. Estimated maize-derived C in the bulk soil for rotational treatments in
the three European field experiments. Bottelare (a), Tetto Frati (b),
and Puch (c). .................................................................................... 34
Figure 13. Distribution of OC from different soil fraction within crop rotation at
three long-term fields experiments. Bottelare (a) and Tetto Frati (b). 38
Figure 14. Comparison of the relative distribution of OC (% of SOC) over isolated
soil fractions for different crop rotations at three long-term field
experiments. Bottelare (a), Tetto Frati (b), and Puch (c). .................. 39
Figure 15 Distribution of maize-derived C from different soil fraction in crop
rotation at three long-term field experiments. Bottelare (a) and Tetto
Frati (b)............................................................................................. 43
Figure 16. Maize-derived C of sand fraction at Tetto Frati (a) and Puch (b). ...... 44
Figure 17. Maize-derived C of clay fraction in Tetto Frati. .................................. 45
Figure 18. Maize-derived C of silt & clay microaggregate fraction in Bottelare... 46
iii
LIST OF TABLES
Table 1.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
Table 7.
Table 8.
Table 9.
Table 10.
Table 11.
Table 12.
Table 13.
Table 14.
Measured maize dry biomass (2003) and estimated C input from
maize plant materials (1993-2003) ............................................................ 7
Treatments, type of mineral N fertilizer and organic amendment
applied to plots in Ultuna long-term experiment ...................................... 8
Relative contribution factor of OC from roots vs incorporated shoots to
total SOC........................................................................................................ 9
Advantages and disadvantages of 13C natural abundance .................. 12
Advantages and disadvantages of 14C pulse labelling .......................... 13
Crop rotation in Bottelare’s long-term experimental field ..................... 19
Cropping systems established in the Tetto Frati long-term
experimental field (since 1992)................................................................. 20
Crop rotation in Puch long-term experimental field ............................... 21
δ13C of the bulk soil samples in long-term field experiments. .............. 32
Dry matter (DM) weight of isolated soil fractions from three European
long-term field experiments with silage maize in rotation or
monoculture as compared to grain maize or non-maize rotations ...... 35
Distribution of SOC over different soil fractions (expressed on a bulk
soil basis) and in brackets the measured OC content (%OC) of each
individual fraction from three long-term field experiments.................... 37
Measured δ13C of the different isolated soil fractions in long-term field
experiments ................................................................................................. 40
Estimated fraction of maize-derived C (% of C in each fraction) of the
different isolated soil fractions in long-term field experiments ............. 41
Estimates of Maize root and shoot C inputs (over the full duration of
the field experiments) relative to the quantified contents of root and
shoot derived C in the plough layer ......................................................... 47
iv
LIST OF ABBREVIATIONS
δ13C
μm
ρ
‘E
‘N
C
cm
CO2
g
ha
kg
m.a.s.l
ml
mm
N
o
C
OC
OM
P
pH
POM
rpm
S
SOC
SOM
t
USDA
vs.
WRB
y
ratio of 13C/12C
micrometer
density
degree East
degree North
carbon
centimeter
carbon dioxide
gram
hectare
kilogram
meters above sea level
milliliter
millimeter
nitrogen
degree Celcius
organic carbon
organic matter
phosphorus
potential hydrogen
particulate organic matter
rotation per minute
sulphur
soil organic carbon
soil organic matter
ton
United States Department of Agriculture
versus
world reference base
years
v
ABSTRACT
The main objectives of this research are to investigate if root-derived organic
carbon (OC) is preferentially accumulated in soil OC relative to above-ground
biomass and if it also ends up in physically protected OC. The effect of maize
cultivar and difference in soil texture on root maize biomass production as well as
the stability of maize root vs .shoot-derived C were also studied. Intact soil cores
were taken from ILVO maize field trials in Belgium (Ravels, Merelbeke, and
Basselvelde) to test the effect of maize cultivar and site on root biomass. Maize
cultivar had a lesser control while soil texture was proven to have a more apparent
effect on root DM biomass at three ILVO maize field trials.
The study of stability of maize roots vs. shoots-derived C was conducted by
sampling long-term field experiments with silage vs. grain maize or non-maize
cropped soil in Belgium (Bottelare), Italy (Tetto Frati), and Germany (Puch).
Physical fractionation of collected soil samples was applied for further investigation
in order to quantify the maize-derived OC contribution. Even in the finer textured
soil at Puch, there seemed to be no preferential accumulation of maize root derived
C in the physically protected particulate organic matter (OM) pool. Instead this root
derived OC ended up partly in the sand fraction (coarse POM which is probably
not protected to a substantial degree) and in the silt and clay bound OM (sum of
free clay and silt&clay in microaggregates fractions). Root-derived C was proven
to be more stable than shoot-derived in Bottelaere and Tetto Fratti. Based on the
relative contribution factor of roots vs. shoot to SOC, the root-derived C is nearly 3
times more stable in the soil compared to shoot biomass and contributed most of
the OC stored in the soil at these locations.
The result of this study finally led to general conclusion that maize root-derived C
is more stable than shoot-derived C and it is not always accumulated in soil as
physically protected OM. It would be highly relevant to measure the C inputs from
root exudation to know its perhaps underestimated contribution to buildup or
maintanence of soil OC.
vi
I. INTRODUCTION
1.1. Background
The decline of soil organic carbon (SOC) has recently re-emerged as a major
environmental issue under research and there are considerable efforts to
understand stabilization of SOC against microbial decomposition. One approach
to maintain the organic carbon (OC) content in soil is to increase inputs of OC
derived either from roots or aboveground biomass. The input of aboveground
biomass in many agronomic systems has, however, decreased due to land
conversion and increased harvest indices. Besides there has been replacement of
permanent grassland by fodder crops, which often involve harvesting of the near
entire aboveground biomass, to be utilized as energy source or animal feed.
Furthermore, in Belgium and other EU countries nutrient legislation restricts
farmers to replace all OC exported as aboveground biomass by addition of
compost, animal manure or other organic fertilizers in the field. These issues have
led to decreasing fertility of arable soils, including in Flemish croplands (Sleutel et
al., 2003), and the need for more knowledge about the stability of organic matter
(OM) derived from above versus below ground carbon inputs is pertinent.
There are three major factors that determine the speed of decomposition of soil
organic matter (SOM): soil organisms, the physical environment, and the quality of
the organic matter (Bot and Benites, 2005). In other words, the speed of microbial
mediated decomposition of SOM is related to both its quality, its binding to the
mineral phase and its location in the soil matrix. Generally, when the rate of
addition of OC is less than the rate of decomposition, SOC content declines. On
the other hand, when the rate of addition is higher than the rate of decomposition,
SOC will accumulate.
To understand the dependency of SOC level on the amount of organic matter
added to the soil from various crops, it is crucial to know the contribution of root
and aboveground biomass or litter-derived OC inputs to SOM. Most often, primarily
the aboveground litter inputs have been considered as the most important
contributor to SOC in simple organic matter balance approaches. Campbell et al.
(1991), however, already found no differences in treatments with or without litter
inputs and suggested that root inputs may be more important in maintaining SOC
levels. Later on Balesdent and Balabane (1996) reported that root-derived OC has
a slower decomposition than shoot material. The role of root-derived C has since
then therefore more and more been recognized in contributing to SOM, maintaining
microbial populations and stabilizing aggregates. Since Balesdent and Balabane
(1996)’s pioneering study several researchers reported that root-derived C
contributes relatively more to SOC compared to aboveground biomass. Puget and
Drinkwater (2011) reported that since after harvesting, roots still remain in the soil,
they should make a bigger contribution to increase the soil organic matter pool via
physical entrapment of this root material, opposite to above ground biomass.
1
Multiple mechanisms have been forwarded to preferentially protect root C from
being decomposed (Puget and Drinkwater, 2001; Rasse et al., 2005):
underestimation of C from rhizodeposition, biochemical recalcitrance of roots, and
enhanced physical and chemical protection of root-derived C. These mechanisms
would explain why proportionally more SOC is root-derived.
The aim of this research is to investigate firstly if root-derived C is preferentially
accumulated in SOC relative to aboveground biomass. Campbell et al. (1991)
suggested that the OC input of roots contributed more to the stable SOC pool
because root fragments are physically protected into soil aggregates. This
hypothesis has to date not been unequivocally tested. Aside from some work on
sand sized macroaggregates, the role of particulate organic matter (POM) that is
physically protected in silt sized aggregates has not been considered before.
Nonetheless, such fine intra-aggregate OM may represent a considerable fraction
of intermediately protected SOM (Vitro et al., 2010).
To investigate if root-derived SOC preferentially ends up in physically protected
OC, i.e. microaggregate occluded. Physical stabilization of SOM is operative on a
time scale of years to decades. Therefore the relative stability of below- vs. above
ground biomass derived SOC and specifically physically protected OC needs to be
studied in field experiments of sufficient long-term span. Ideally observations are
made of comparable field rotations with either only below or above-ground biomass
application. Maize, being cropped as grain or silage maize and with a distinct
13
C/12C isotopic ratio than C3 crops, practically offers the opportunity to do so in
existing European long-term field experiments.
In the present thesis research soil was collected from applicable long-term field
experiment with maize in various rotations. Physical fractionation of collected soil
samples was used to investigate several research questions.
1.2. Research questions
Research questions for this research are:
1) What is the effect of maize cultivar and difference in soil texture on root maize
biomass production?
2) Is root maize biomass-derived soil C relatively more stable than shoot-derived
C?
3) Does the maize root derived C preferentially stabilize in physical entrapped
SOM?
4) Does soil texture through physical protection affect the relative stabilization of
root maize derived C?
2
II. LITERATURE REVIEW
2.1. Maize (Zea mays L.)
Zea mays L. (maize) is known as a cultivated crop originated from Mesoamerican
region (Mexican highlands). Maize is an annual plant for which natural vegetative
reproduction does not occur, thus it is reproduced only by seed. It can be grown
as a dry land or irrigated crop in various environment ranging from sea level up to
3,800 metres with growing seasons from 42 to 400 days. Each cultivation region
has produced a range of maize cultivars with various morphological and
physiological traits. Based on the soil condition, maize grows best in well-drained
and nutrient-rich soils with pH (CaCl2) between 5.5 and 7.0. It should be noted that
maize is sensitive to saline soils. Nutrient status is important to be evaluated before
planting maize as it is very important for its productivity. Nitrogen is particularly
yield limiting in maize production. Phosphorus, potassium, zinc, and molybdenum
may also be necessary in some cases (Australian Government, 2008).
As a plant which evolved under tropical condition, maize is a C4 plant which means
that its efficiency in carbon utilization is higher than a C3 plant. Typical maize plant
has a height of 1 – 4 m. Its leaves are broad. A mature maize plant can have up to
30 leaves with various size, leaf number, and orientation. Tropical maize usually
develop more leaves than temperate cultivars. Judging from its usage, maize is a
very important crop worldwide. It can be consumed as food starting from baby corn
to mature grain or to be processed further into corn syrup or maize meal. However
the major output of maize is still as feed stock (40% in tropical areas and ≤85% in
developed countries) in the form of green chop, dry forage, silage or grain. The
major non-food usage of maize is corn starch which is used to produce fuel ethanol
or used in paper industry (Australian Government, 2008).
The type of maize used in this research are grain maize and silage maize as
explained below (Australian Government, 2008):
1. Grain maize is usually harvested after 130 to 150 days from planting. Moisture
content of the kernels is around 28-34% when physiological maturity is reached
whereas optimum storage requires grain moisture to be approximately 12%
hence drying is an important step. Harvest is usually carried out at moisture
content of 18%.
2. Silage maize is grown similarly to grain maize but with higher planting density.
Anaerobic fermentation which improves digestibility and nutrient preservation
of ensiled material occurs in silage, which is basically a moist preserved fodder.
Thus silage maize are usually harvested when the dry matter content is around
30-35%. Its production involves chopping, compaction, and careful packing to
exclude air. Yield for silage maize is around 6-10 times that of grain maize.
3
2.2. Soil Organic Matter
2.2.1. Nature of soil organic matter
Soil organic matter (SOM) is a product of the microbial mediated decomposition
process of once living organisms (plants or animal) returned to the soil. At any
certain point in time, it consists of a range of materials ranging from the intact
original tissues of plants and animals to the substantially transformed mixture of
materials, often termed as humus (Bot and Benites, 2005). There are four main
sources of organic matter input into the soil: plant roots, plant shoots, root
exudates, and soil microorganisms. The relative importance of these sources
depends on climatic parameters, management, soil inherent processes as well as
land-use. For example, a high input of dissolved organic matter in deeper soil can
be expected under humid climate conditions and when podzolisation is the main
factor of soil forming processes (Rumpel and Kögel-Knabner, 2011).
Thus, SOM can be also simply divided into two fractions depending on SOM’s
association with the soil mineral phase. Firstly a light fraction, which consists of
mineral-free OM composed of partly decomposed plant animal residues. This
fraction has a specific density lower than soil minerals and a rapid turn over time.
A second, heavy fraction, which composed of more processed decomposition
products, is associated with soil minerals and demonstrates a slower turn over
(Alvarez and Alvarez, 2000).
There is a mutual benefit between SOM and soil organisms (including
microorganisms). Soil organisms use SOM as food and when they break down the
organic matter, they will release any excess nutrients (N, P and S) into the soil in
the available forms that plants can use for their growth. This release process is
called mineralization. Besides nutrients, microorganisms also produce waste
products that are less decomposable than the original plant and animal material,
but many other organisms can use these metabolites (Bot and Benites, 2005).
2.2.2. Role of organic matter to soil fertility in croplands
The OM content is relatively low in agricultural soils. Not with standing, OM is an
important component of the soil, determining soil fertility. There are several
functions of SOM, and it also depends where the SOM is placed in the soil. On the
soil surface, OM as plant residues, helps protect the soil from the effect of rainfall,
wind and sun, preventing soil erosion. From an agricultural viewpoint, OM within
the soil serves two main important functions: it acts as a nutrient fund and it
improves soil structure. As a nutrient fund, organic matter contributes to fertility of
the soil, since SOM derived from plant residues contains all of the essential
nutrients for plants. Furthermore, decomposition of OM also affects the stable
organic fraction that adsorbs and holds nutrients in available form for plants. SOM
is also essential in obtaining a good physical structure, and it contributes in many
4
processes in soil favourably enhancing chemical and physical properties.
Properties influenced by SOM are: soil structure, water holding capacity, diversity
and activity of microorganisms, and nutrient availability for crop and soil. SOM also
plays a part in the cation exchange capacity of the soil and contributes in essential
nutrients, trace elements for plants, and during decaying process, it will
continuously be a source of nutrients for plant growth (Bot and Benites, 2005).
2.3. Microbial mediated SOM decomposition
The carbon cycling process in terrestrial ecosystems is the continuous
transformation of organic and inorganic carbon compounds by plants and microand macro-organisms between the soil, plants and the atmosphere (Bot and
Benites, 2005). Decomposition of OM is a biological process that includes the
physical breakdown of OM and further biochemical transformation of complex
organic molecules of dead material into simpler organic and inorganic molecules
(Juma, 1998). When plant residues are returned to the soil, decomposer organisms
directly utilize a variety of organic compounds. There are three major factors that
determine the speed of decomposition: soil organisms, the physical environment,
and the quality of the OM. Continuous addition of decaying plant residues to the
soil surface contributes to the biological activity and the carbon cycling process in
the soil. Decomposition of SOM, root growth, and decaying plant residues also
contribute to the biological activity and the carbon cycling process in the soil.
Fresh residues consist of former microbial remnants, micro- and macro fauna, old
plant roots, plant residues, and manure. Plant residues contain mainly complex
carbon compounds that originate from cell walls, such as lignin, cellulose,
hemicellulose, etc. Microorganisms change the organic structures of fresh residues
into carbon products in the soil during the decomposition process. There are many
different types of molecular organic products of this process in the soil. Many
simple molecules are directly synthesized from plants or other living organisms.
These simple molecules, such as sugars, amino acids, and cellulose are in
available form and ready to be consumed by other organisms (Bot and Benites,
2005). In summary, biochemical composition of the source OM applied to the
soil logically affects its degradability in the soil environment.
2.4. Contribution of root vs. shoot-derived OM to native SOM
2.4.1. Roots vs. shoots as OM inputs in agricultural soils
From the source of origin, OM could be differentiated as aboveground and
belowground-derived. Roots and shoots are important sources of SOM and after
decay these plant fragments at first become a major constituent of the particulate
organic matter (POM) fraction. Recently, research has focused on the
contribution of roots to bulk SOM rather than the shoots parts. But little is known
about the specific contribution of these sources of OM to various soil fractions,
5
including POM As mentioned before, roots have become the focus of main interest
for researchers because after harvesting, roots still remain in the soil, and because
roots make a bigger contribution to increase the SOM pool than shoots (Puget and
Drinkwater, 2001).
Estimations of the OC input from belowground biomass production to the soil are
needed to study the effectiveness of OM application and crop rotations in cultivated
soils. The photosynthates that are translocated beneath the ground are present
as living roots and rhizomes, exudates, sloughed off and dead root tissues,
microbial tissues and metabolites. Measurement of the quantity and distribution
of root-derived C material in the soils requires the use of a tracer, due to the
presence of indigenous OM (Balesdent and Balabane, 1992), as will be further
explained in part 2.4.
2.4.2. Differences in C input from roots vs. shoots
Several researches have suggested that the input of OC from roots could be
greater than the input of OC from shoots to the stable SOC pool, due to the
constant release of C from roots and the rhizosphere to the soil. There is a
distinction between interactions of aboveground and belowground biomass with
the soil microbial system, because OC inputs from roots include root
production and exudation and both are in closer contact with the soil than
above ground derived OC. OC inputs from shoots mainly come from litter and
has no direct contact with biological activity in the soil (Puget and Drinkwater,
2001). The influence of roots on SOC pools could also be relatively higher than the
influence of aboveground OC inputs because of the continuous release of OC from
roots and the complex nature of the rhizosphere to soil interface. The dominant
role of roots in contributing to the stabilization of SOC has been noticed, but there
is a lack of studies that attempt to differentiate between aboveground and
belowground OC inputs because of the difficulties in quantifying belowground
biomass (Puget and Drinkwater, 2001). In addition only a few studies reported the
belowground OC inputs in situ compared to the aboveground OC inputs.
Campbell et al. (1991) and Fisher et al. (1994) suspected that roots are an
important source of SOM. This statement was mainly hypothesized from carbon
balance studies, where aboveground biomass inputs could only explain one part
of the SOC stocks and their variations. They reported that the input of roots to SOC
is greater than of shoots and that roots contribute more to the stable SOC pool
because the root-derived OC is physically protected into soil aggregates. Since
then, the importance of roots for SOC sequestration was underlined by the fact that
they have a high potential to be stabilized in soil, as reviewed by Rasse et al.
(2006). Despite their importance as a soil OC source, root C fluxes to the soil
are to date still poorly understood mainly due to uncertainties associated
6
with the measurement of total root C input, in particular from root exudation and
root cell sloughing.
Kätterer et al. (2011) mentioned that the amount of belowground OC inputs
attributed to extra-root OC becomes an important issue related to this topic. The
extra-root OC can be defined as turnover and cell sloughing of epidermal root
tissues during the growing season, and its soluble compounds released by
exudation from the roots. Bolinder et al. (1999) assumed that this extra-root OC
will contribute to about 65–100% of the measurable root biomass and predicted
that this contribution will increase the resistance of SOC. However, this assumption
is not yet proved and still an open issue. The root-derived OC input has been
claimed to delay decomposition of crop residues and native SOM, but it could also
even accelerate the decomposition rate of both crop residues and other organic C
present in the rhizosphere (Kätterer et al., 2011).
Measured dry biomass and estimated cumulative OC input from maize plant by
Rasse et al. (2006) summed to a total OC input of 5,020 g C/m2, where 60% of this
total was contributed from leaves and stems incorporated to soil and the remaining
40% was the roots contribution (Table 1). Clearly, roots had an important role of C
input into the soil especially for the top of 15 cm soil profile. Kätterer et al. (2011)
studied the contribution of OM from roots and the effect of organic amendments
and mineral N fertilizers on the crop and soil to this process in the Ultuna long-term
field experiment. All treatments with combination of six organic amendments and
mineral N fertilizers are shown in Table 2. His result showed that the total C inputs
varied between treatments, the contribution of C input from roots to the SOC is
higher compared to above-ground residues (Figure 1).
Table 1. Measured maize dry biomass (2003) and estimated C input from maize
plant materials (1993-2003) (source: Rasse et al. (2006))
Dry biomass (kg/ha)
C input (g/m2)
Grains
8980 ± 760
Cobs
1490 ± 130
Leaves (including husks)
3911 ± 422
1531
Stems (including stubble)
3644 ± 390
1510
Roots 0-15 cm
1198 ± 312
756
Roots 15-30 cm
556 ± 261
351
Roots 30-45 cm
455 ± 182
287
Roots 45-60 cm
326 ± 162
206
Roots 60-75 cm
303 ± 175
191
Roots 75-90 cm
298
188
7
Table 2. Treatments, type of mineral N fertilizer and organic amendment applied
to plots in Ultuna long-term experiment (source: Kätterer et al. (2011))
ID
Treatment
Fertilizera
Organic
(80 kg N/ha/year)
amendment
A
Bare fallow
B
Control
C
Calcium nitrate
Ca(NO3)2
D
Amonium sulfate
(NH4)2SO4
E
Calcium cyanamid
CaCN2
F
Straw
Straw
G
Straw + N
Ca(NO3)2
Straw
H
Green manure
Grass
I
Peat
Peat
J
Farmyard manure
Farmyard manure
Kb
Farmyard manure
CaCN2
Farmyard manure
L
Sawdust
Sawdust
M
Peat + N
Ca(NO3)2
Peat
N
Sawdust + N
Ca(NO3)2
Sawdust
O
Sewage sludge
Sewage sludge
a
All treatments icluding bare fallow applied with 20 kg P and 35-38 kg K /ha/year
b
Calcium cyanamid was applied in K treatment only in 1956, manure applied in
1956 and 1960. Total application of N was about 60 kg higher in K than J treatment
during that time period.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
Figure 1. C inputs from different treatments through amendments, roots including
rhizodeposition, and above-ground crop residues in Ultuna long-term field
experiment with crop rotation of oat (1956), spring cereals (1957-1999),
and silage maize plant (last 10 years). Source: Kätterer et al. (2011)
2.4.3. Stability of SOM from root-derived C vs shoot-derived C
Puget and Drinkwater (2001) examined that root-derived C was retained to a
greater extent than shoot-derived C in several labile C pools and the greater
retention of root-derived C suggested more than one mechanism. They
concluded at least three mechanisms are involved in retention of root-derived C:
8
biochemical recalcitrance of root-derived C, physical protection of root-derived
POM within the aggregates, and root C input (root exudates and turn over). The
greater proportion of root-derived C was found as occluded POM within the clay
and silt fraction. The measurable root biomass was equivalent with the annual input
of fresh root C incorporated into soil from 3 different processes: root exudation,
turn over, and cell sloughing (Barber, 1979). The differences in decomposition
rates for root- and shoot-derived C were partly due to the differences in the
decaying process. Puget and Drinkwater (2001) also concluded, that these results
had important implications for the long-term dynamics of SOM formation. During
the first growing season, the origin of litter coming from shoots played an important
role to determine the inputs of C, and this process was followed by hairy vetch
incorporation. At the end of the growing season, almost one-half of the root-derived
C was still present in the soil, while only 13% of the shoot derived C remained.
Table 3. Relative contribution factor of OC from roots vs incorporated shoots to
total SOC (source: Rasse et al. (2005))
Method & crop type
Duration
Relative
References
(months) contribution
I. Root system grown in situ
Maize
132
1.50
Barber (1979)
Maize
Balesdent and Balabane
48
1.75
(1996)
Maize
180
1.70
Bolinder et al. (1999)
Maize
152
3.30a
Clapp et al. (2000)
Hairy vetch (Vcia Villosa)
5
3.70
Puget and Drinkwater
(2001)
Alfalfa
24
2.70
Rasse (unpublished data)
average
90
2.40
II. Incubation: shoot and root material mixed into soil
Barley (Hordeum vulgare)
Broadbent and Nakashima
60
1.33
(1974)
Medicago sp.
1
1.22
Amato et al. (1984)
Medicago sp.
24
1.45
Amato et al. (1984)
Miscanthus giganteus
20
1.26
Beuch et al. (2000)
Clover (Trifolium repens)
3
1.30
de Neergaard et al. (2002)
Ryegrass
3
1.24
de Neergaard et al. (2002)
Average
18
1.30
III. Incubation: litter-bag experiments
Fagus sylvatica
Schenu and Schauerman
36
1.55
(1994)
Festuca vivipara
2
1.50
Robinson et al. (1997)
Festuca vivipara
13
2.10
Robinson et al. (1997)
Poa liguralis
21
0.94
Moretto et al. (2001)
Stipa clarazii
21
0.86
Moretto et al. (2001)
Stipa tenuissima
21
0.77
Moretto et al. (2001)
Lepidium lasiocarpum
3
1.33
Parker et al. (1984)
Average
17
1.29
In a simulation study, Molina et al. (2001) stated that maize roots contributed 1.8
times more C to SOM on the long term than the above-ground biomass. Other
9
findings from several researchers were compiled by Rasse et al. (2005) and
indicated that the relative contribution of maize roots to SOC had was on average
2.4 times the contribution from shoot C (Table 3). Rasse et al. (2005) used two
different methods to estimate the relative contribution of root OC to SOM: a
conservative method from estimation of root biomass and secondly considering
root exudation and root cell sloughing, as mentioned in Barber (1979). Rasse et al.
(2005) corrected data from Balesdent and Balabane (1996) by including estimates
of root exudation and turnover. The same correction was also made for estimates
of relative root contributions from Barber (1979). They concluded that the values
of the relative contribution of root C to SOM in Table 3 were in fact even
underestimated. This was also supported by the results of Puget and Drinkwater
(2001), which showed that the relative contribution roots vs shoots was even 3.7
based on the total of root contribution to SOC.
2.5. Experimental assessment of the importance of root- vs.
shoot-derived C
Kätterer et al. (2011) argued that in situ measurements in long term field
experiments are superior over other methods for quantifying humification values,
because insignificant annual changes only accumulate over time under natural
conditions and only eventually become detectable. There are, however, only a few
long-term field experiments with detailed soil and crop records available, in which
the overall quality of measurements enables to analyse changes in root-derived
SOC with sufficient precision. For instance, there has not always been enough
consideration of soil bulk density dynamics, changes in topsoil depth and other
confounding factors. Using SOC or SOM analyses and conversions between these
can also introduce some errors.
2.5.1. Quantifying root biomass
The auger sampling method is the most general method to quantitatively measure
root length and root biomass, where the soil is augured, then washed and sieved
to separate the roots from the soil. Measurement of root biomass by weighing the
oven-dried root sample (Livesley et al., 1999). Benomar et al. (2013) also
distinguished living from dead roots by colour and flexibility.
The study of soil management effects on plant root system requires a high cost in
terms of time and involves laborious work for collecting the sample and manually
separating the live roots from dead roots and other OM residues. Alternatively root
washers with opening size-strainer and filter have been constructed.
Disadvantages of this method is the cost and the fact that the opening size of the
strainer needs to be changing according to the size of roots and soil texture
(Benjamin and Nielsen, 2004).
10
2.5.2. Microrhizotrons
A fairly recent method developed to determine the rooting density based on the
root length through an observation window is called microrhizotrons (Itoh, S.,
1985). The microrhizotrons method are easy, low cost, and less damaging to the
soil in the field (Itoh, S., 1985). However, microrhizotrons are only able to estimate
the most dynamic roots (<1 mm) and not roots with larger diameter (>1 mm) for
which isotope techniques as 14C pulse labelling and 13C natural abundance may
be more suitable (Majdi and Andersson, 2005).
2.5.3. 13C natural abundance
The 13C natural abundance technique has been used in characterization of SOM
studies to distinguish the origin of OM between biomass produced by C3 and C4
plants. Crop type regulates the stable carbon isotopic compositions (δ13C) in SOC
due the different isotopic signatures in assimilated CO2 form as a result of
photosynthetic 13C discrimination. Thus, stable C isotopic techniques have been
widely used to quantify the relative contributions of C3 and C4 plants to SOC
(Wang et al., 2015). In line with Balesdent and Balabane (1992), δ13C value is
−27‰ in plants with C3 pathway and the δ13C value is −12‰ for C4 plants.
Balesdent and Balabane (1992) used this 13C natural abundance technique to
estimate SOC derived from field-grown maize at harvest time (at the end of
one growing season). They concluded that the 13C-abundance combined with
physical fractionation provided a good estimation of sand sized root material
compared to weighting techniques of root biomass estimation. The maize C could
be distinguished from SOC derived from previous crops. Bolinder et al. (1999)
as well used 13C natural abundance to compare different treatments in maize plants
with either aboveground residue removal or return to the soil after harvest and with
a bare soil treatment. The δ13C in continuous silage maize was higher compared
to silage maize in rotation with barley-wheat and barley-hay, which shows that
under continuous silage maize there was more maize derived-C in the SOM.
Werth and Kuzyakov (2008) discussed the advantages and disadvantages of this
technique in comparison to 14C tracer methods (see 2.4.4) to quantify the
contribution of root derived C to soil respiration and the soil microbial biomass
(Table 4). Werth and Kuzyakov (2009) stated that there are two assumptions for
13
C natural abundance technique: δ13C value of the roots is the same as the δ13C
isotope signature of root-derived CO2 and δ13C value of microbial biomass
corresponds to the microorganism respiration that produced δ13C isotope signature
of CO2. The first assumption was fully accepted and tested by other researchers,
while contradiction was rising for the second assumption. They finally suggested
that isotopic fractionation between SOM/exudates, microbial biomass, and
11
microbial CO2 under the controlled conditions should be considered when using
13
C natural abundance techniques.
Table 4. Advantages and disadvantages of 13C natural abundance (source: Werth
and Kuzyakov (2008))
No Advantages
Disadvantages
1
Continuous labelling of plants and
Very low sensitivity of the
soil pools
contribution of plant-derived C to
CO2 and to microbial biomass
2
No labelling equipment required
Only incorporation of plant-derived C
into pools with high turnover rates
during one vegetation period is
possible
3
No radioactivity precautious
Applicable ony on pure C3 or C4 soil
necessary
4
Easy usage under laboratory and
Contamination with air CO2 is
field conditions
possible
5
High variation of C in CO2 or
microbial biomass is possible
6
Results are strongly affected by 13C
fractionation
7
Results are strongly affected by
preferential isotope utilisation
8
Expensive purchase costs and
indvidual analyses
2.5.4. 14C pulse labelling
Another option to determine the quantity and fate of root-derived C in soil are 14C
labelling techniques in combination with incubation studies. Radioactivity is used
in the measurement of 14C pulse labelling. After a plant is pulse labelled by
exposure to an atmosphere with 14CO2, 14C activity can be measured with liquid
scintillation counter from the collected oxidized CO2 of plant or soil sample. The
14
C-pulse labelling technique also allows to measure the distribution of recently
assimilated C at specific plant development stages in the plant and soil. The
contribution of plant-derived C to below-ground pools (root and soil) can however,
only be determined for the whole growth period by repeated labelling pulses (Werth
and Kuzyakov, 2008).
Figure 2 shows a schematic diagram of 14C labelling equipment used in the field
(Werth and Kuzyakov, 2008; Kisselle et al., 1999). A summarize of the advantages
and disadvantages of the 14C pulse labelling technique by Werth and Kuzyakov
(2008) are shown in Table 5.
12
Figure 2. Equipment for 14C labelling in the field
Table 5. Advantages and disadvantages of 14C pulse labelling (source: Werth and
Kuzyakov (2008))
No. Advantages
Disadvantages
1.
High sensitivity of the contribution
Uncompleted distribution of labelled
of plant-derived C to CO2 and to
C between plant organs below
microbial biomass
ground pools if sampling is done too
early after labelling
2.
Information on distribution of
Recalculation of total rhizodeposition
assimilated C in individual stages
is suitable only for linear growth
of plant development
periods
3.
Allows estimating the incorporation Provides only distribution of recently
of plant C into pools with low and
assimilated C at specific
very low turnover rates
development stages of plants
4.
One or many pulses are possibly
Both non-recent and recent
easy to handle
assimilates can be traced if labelling
pulses are repeated
5.
Cheap purchase costs and
No recalculation of distribution to
individual analyses
whole growth period
6.
Radioactivity hazards
7.
Laborious labelling sessions with
chambers required
2.6. Physical fractionation
Fractionation of OM implies the separation of the total OM into different parts that
are thought to be functionally homogeneous with respect to physicochemical
properties and turnover rate. The separation can be carried out by chemical or
more often by physical means: e.g. sieving, flotation, dispersion. The difference in
C content between fractions of an agricultural field and the corresponding native
vegetation for example can yield information about the mechanisms of C
sequestration (Six et al., 2002a; Del Galdo et al., 2003). Likewise comparison of
13
distribution of C and 13C in physical fractions from fields with or without above
ground maize residue incorporated will enhance mechanistic insight.
The use of physical fractionation in SOM studies have become important over the
past three decades. Physical fractionation of soil involves various degrees of
dispersion, recognizing the significance of interactions between organic and
inorganic soil components in the turnover of SOM. There are several contradictory
views on the suspected relevance of fractions isolated by size and density
fractionation, especially related to SOC sequestration on the long term
(Christensen, B.T, 2001; Moni et al., 2012). Physical fractionation is nowadays
preferably used over chemical fractionation because it minimizes the disruption of
SOM due to chemical processes (Christensen, B.T, 2001). It also recognizes the
relevance of location of SOM within the soil matrix. Indeed, physical fractionation
isolates SOM based on its size and level of organo-mineral interaction (Torn et al.,
2009). According to Moni et al. (2012), general treatments of soil dispersion are
sonication, slaking, interference with water jet, mixing, and wet sieving. These
treatments were categorized as ‘moderate’ dispersion. Other dispersion treatments
involve chemical agents and high energy sonication and were categorized as
‘strong’ dispersion.
Gerzabek et al. (2001) used a combination of physical fractionation and the 13C
natural abundance of soil fractions to assess the effect of organic and mineral
fertilizer treatments on SOC-organic changes associated with different soil size
fractions. His results showed that most of the silt fraction was dominated with SOC
due to the increased amount of sewage. Guggenberger et al. (1994) supported this
argument by saying that the increase in C-organic content is equivalent to the
decrease in particle size. Gerzabek et al. (2001), amongst many others, found that
according to mass balance calculations, the sand fraction was most sensitive to
treatment changes. The conforming measurement between 13C content of the plant
material/organic fertilizer and 13C content of C-organic from which it originates from
allowed the variations in δ13C between different fraction sizes to be used as
fingerprint (Gerzabek et al., 1997).
14
III. MATERIALS & METHODS
This research is composed of two main parts. A first smaller part involves a
comparison of maize root biomass in function of cultivar and soil texture (3.1). The
second bulk part of this thesis research is the use of physical fractionation to isolate
specific soil fractions from selected crop rotation objects sampled from long-term
field experiments (3.2). Specifically, soil cropped with silage maize was compared
with grain maize or with non-maize rotations to estimate maize-(root)-derived C
accumulation in the isolated soil fractions. To determine the maize-derived C the
distinct 13C/12C-natural abundance of maize will be used.
3.1. Comparison of maize root biomass as a function of cultivar and soil
texture
3.1.1. Maize cultivar experiment
Maize root biomass was determined at harvest time of the 2014 growing season.
Maize cultivar trials have been annually conducted in five locations situated in
Belgium. Out of these trials, three locations were selected based on their difference
in soil texture (Figure 3). The three maize cropped fields selected for this thesis
were located in Ravels, Merelbeke, and Bassevelde, having a sandy, sandy
loam, and clay soil texture, respectively.
Each location was planted with eight cultivars of maize (Kalientes, MAS 17E,
LG30.224, LG30.222, Ronaldinio, NK Falkone, LG3020, and Banguy) with three
replication plots for each cultivar.
Figure 3. Locations of experimental plots for comparison of maize root biomass
15
3.1.2. Maize root extraction
Root biomass including surrounding soil was collected from the plots in each
location using a special stainless steel auger (10 cm x 30 cm) until 30 cm depth.
Samples were taken in duplicate from rows in the middle of each plot, after the
above-ground biomass was harvested (at a dry matter content of 30%) (Figure 4).
The soil-root samples were immediately stored in a refrigerator at 40C.
Figure 4. Root sampling from harvested rows in the Merelbeke maize cultivar trial
Root biomass was isolated from the soil through soil dispersion and sieving. Before
being washed, the soil cores were submerged in a peptisation fluid (Na-hexa-metaphosphate) in 10l buckets and these were shaken on a reciprocal shaker for 24
hours. The objective of adding this dispersive fluid is to facilitate the removal of the
soil surrounding the roots. The resulting suspensions were poured onto a 1 mm
sieve and were consecutively washed thoroughly with running water. Visible soil
particulate organic matter, dead roots, and small stones were removed manually
from the sieve. After being sieved the root samples were then oven-dried for two
days at 700C and weighed (Figure 5). Finally, the root samples were ground into
powder using a plant’s grinder.
Figure 5. Procedure of root sample preparation
16
3.2. Comparison of soil C distribution of silage vs. grain maize or non-maize
cropped soil to determine the relative stability of maize root-derived
SOM
3.2.1. Long-term field experiments
Long-term field experiments are useful instruments to compare the stability of
applied OM. One option is to do so by comparing silage and grain-maize
treatments. Maize’s distinct 13C/12C ratio may allow quantification of the specific
contribution of maize derived C to the bulk SOM. Any maize-derived C in silage
maize plots would be primarily root-borne. In absence of a comparative grain maize
treatment, with shoot material incorporation, the soil 13C/12C of the silage maize
treatment could be compared to other crop rotations, to determine the relative
stability of maize root-derived C compared to other plant material.
The second part of this research included three long term experimental trials,
located in Belgium, Italy, and Germany (Figure 6). These locations were chosen
because of the length of the trials, their monocultures or rotations of both silage
maize and grain maize, and inclusion of treatments devoid of animal manure
application, which is mostly enriched in 13C as well. Soil texture differed between
these locations (Bottelare: sandy loam, Tetto Frati: loam, and Puch: silt loam) and
this difference could give an additional insight to the importance of aggregate
formation in preferential stabilization of maize root-derived C in soil fractions.
The first experiment is a 12-years old field experiment located in Bottelare,
Belgium (HoGent experimental farm). In 2006, the crop rotation experiment was
established on a sandy loam (USDA) soil at the experimental farm of Ghent
University in Bottelare (50°57’ N, 03°49’ E). The clay (< 2 μm), silt (2-20 μm) and
sand (20-2000 μm) content is 68, 411 and 521 g/kg, respectively. Climate is fully
humid temperate with warm summers (Kottek et al. 2006) with approximately 836
mm of annual precipitation and a mean annual temperature of 9°C (KMI
meteorological station, Melle situated at 50°59’N, 03°49’E).
There are 11 different crop rotations laid out in combination to two levels of nitrogen
input (0 kg N and 150 kg N). Seven out of the eleven crop rotations were chosen
to be sampled for this thesis research. The plots that were sampled for the 0 kg N
treatment consisted of two crop rotations (monoculture maize for grain and silage
maize in rotation with Italian ryegrass that is harvested). For the 150 kg N level,
five crop rotations were included: two of them the same ones as with the 0 kg N
treatment, two crop rotations where grass clover is alternated with maize for silage,
and lastly a rotation of 5 different agricultural crops including maize. All crop
rotations were laid out in 3 replication plots (Table 6).
17
Figure 6. Locations of long term experimental trials in Belgium (top), Italy (middle),
and Germany (below)
18
Table 6. Crop rotation in Bottelare’s long-term experimental field
Crop rotationa (N = 0 kg/ha/year)
2013
2014
2015
2016
2017
2018
2019
M
M
M
M
M
M
P
M+IR↓
M+IR↓
M+IR↓
M+IR↓
M+IR↓
M+IR↓
P
M+IR↑
M+IR↑
M+IR↑
M+IR↑
M+IR↑
M+IR↑
P
GK
GK
GK
GK
GK
GK
P
M↑
M↑
GK
GK
M↑
M↑
P
M↑
GK
GK
M↑
M↑
GK
P
GK
GK
M+IR↑
M+IR↑
GK
GK
P
SW+YM
FB
P
M↓
WW+YM
FB
P
FB
M↓
P
M↓
FB
M
P
EW+YM
SW
P
M↓
EW+YM
WW
P
M↓
EW
P
M↓
M↓
EW
P
Crop rotationa (N = 150 kg/ha/year)
2013
2014
2015
2016
2017
2018
2019
M
M
M
M
M
M
P
M+IR↓
M+IR↓
M+IR↓
M+IR↓
M+IR↓
M+IR↓
P
M+IR↑
M+IR↑
M+IR↑
M+IR↑
M+IR↑
M+IR↑
P
GK
GK
GK
GK
GK
GK
P
M↑
M↑
GK
GK
M↑
M↑
P
GK
M↑
GK
M↑
M↑
GK
P
GK
GK
M+IR↑
M+IR↑
GK
GK
P
FB
SW+YM
P
M↓
WW+YM
FB
P
FB
M↓
P
M↓
FB
M
P
EW+YM
SW
P
M↓
EW+YM
WW
P
M↓
EW
P
M↓
M↓
EW
P
- aM: Maize; IR: Italian Ryegrass; GK: Grass Clover; SW: Summer Wheat; YM: Yellow
Mustard; FB: Fodder Beet; EW: protein crop like peas; P: Potato; WW: Winter Wheat;
↓: incorporated (not harvested); ↑: stubble incorporated (harvested).
- Crop rotations in bold indicate selected crop for this thesis research.
The second field experiment is located in Tetto Frati, Carmagnola, Italy, and has
been running since 1992 (23 years) and still ongoing. The site is located at a
latitude of 450N, in the western area of the Po plain (229 m.a.s.l.), and is
characterized by a scarcely weathered alluvial soil that, according to the Soil
Taxonomy of USDA Soil Conservation Service (USDA), is classified as a Typic
Udifluvent (Grignani et al., 2007). The climate is temperate sub-continental,
characterized by two main rainy periods, in spring (April and May) and autumn
(September-November). During the experimentation period, from 1993 to 2003,
the mean annual precipitation was 792 mm and the mean annual temperature was
11.8 0C.
The experimental design was a randomized block with three replicates. The plot
surface was 75 m2, to allow common farm machines to be used. Before the
experiment started, maize for grain fertilized with urea was cultivated on the whole
surface for a period of more than 10 years. There are 6 cropping systems with 9
different managements of fertilizer inputs. Only three of the laid out nitrogen levels
(0 kg N, 170 kg N, and 250 kg N) were sampled from two different cropping systems
19
for this thesis research: grain maize monoculture and silage maize monoculture,
again from 3 replicate plots (Table 7).
Table 7. Cropping systems established in the Tetto Frati long-term experimental
field (since 1992)
Fertilization
Cropping Systema
ManageMs
Mg
Mr
Ml
Mu
SA/PM
mentsb
N0
Ms0
Mg0
Mr0
Ml0
Mu0
SA0
SLow
MsSLow MGSLow MrSLow MlSLow MsSLow
SHigh
MsSHigh MgSHigh MrSHigh MlSHigh
FLow
MsFLow MgFLow MrFLow MlFLow
PMFLow
FHigh
MsFHigh MgFHigh MrFHigh MlFHigh
N100
Ms100
Mg100
Mr100
Ml100
N170
Ms170
Mg170
Mr170
Ml170
N250
Ms250
Mg250
Mr250
Ml250
N350
Ms350
Mg350
Mr350
Ml350
- aMs: silage maize; Mg: grain maize; Mr: Italian Ryegrass in autumn and winter, silage
maize in spring and summer; Ml: grass ley in rotation with silage maize; Mu: lucerne ley
in rotation with silage maize; SA: set-aside in the period 1992-2000; PM: permanent
meadow since 2001.
- bN0: urea 0 kg N/ha/year; SLow: bovine slurry 170 kg N/ha/year; SHigh: bovine slurry
250 kg N/ha/year; FLow: farmyard manure 170 kg N/ha/year; FHigh: farmyard manure
250 kg N/ha/year; N100: urea 100 kg N/ha/year; N170: urea 170 kg N/ha/year; N250:
urea 250 kg N/ha/year, N350: urea 350 kg N/ha/year.
- Crop rotations in bold indicate selected crop for this thesis research.
The third field experiment is located in Puch, Southern Germany, and has been
an experimental trial since 1983 (32 years). A luvisol (WRB) derived from loess
sediments (clay: 18%, silt: 73%, sand: 9%) with pH values below 6.5 in 1983 and
around 6.2 in 1994 (Heitkamp et al., 2012). Bulk density was estimated to 1.5 g
cm3 in the plough layer (25 cm).
There are two different crop rotations: silage maize monoculture followed by winter
wheat and winter barley with various additions of organic fertilizers and at different
level of nitrogen input were planted in this field. In the alternative crop rotation
silage maize was replaced by sugar beets, all other conditions kept equal. The
silage maize-winter wheat-winter barley rotation was sampled for 3 different levels
of nitrogen inputs (0 kg N, 100 kg N, and 200 kg N) and the sugar beets based
rotation for only the 200 kg N object. For each treatment 3 replicant plots were
sampled from the experimental field (Table 8).
20
Table 8. Crop rotation in Puch long-term experimental field
Crop rotation with silage maizea
MS
WW
WB
Fertilizer
1*
2**
1*
2**
1*
2**
Organic
0 kg/ha/year
0 kg/ha/year
0 kg/ha/year
Mineral
0 kg N/ha/year
0 kg N/ha/year
0 kg N/ha/year
Organic
0 kg/ha/year
0 kg/ha/year
0 kg/ha/year
Mineral
100 kg N/ha/year
100 kg N/ha/year
100 kg N/ha/year
Organic
0 kg/ha/year
0 kg/ha/year
0 kg/ha/year
Mineral
200 kg N/ha/year
200 kg N/ha/year
200 kg N/ha/year
Organic
Strawb
Strawb
Strawb
Mineral
200 kg N/ha/year
200 kg N/ha/year
200 kg N/ha/year
Organic
Slurry
Slurry
Slurry
Slurry
Slurry
Slurry
Mineral
60 kg
50 kg
0 kg
25 kg
0 kg
25 kg
N/ha/year N/ha/year N/ha/year N/ha/year N/ha/year N/ha/year
Crop rotation with sugar beeta
Fertilizer
SB
WW
WB
1*
2**
1*
2**
1*
2**
Organic
0 kg/ha/year
0 kg/ha/year
0 kg/ha/year
Mineral
0 kg N/ha/year
0 kg N/ha/year
0 kg N/ha/year
Organic
0 kg/ha/year
0 kg/ha/year
0 kg/ha/year
Mineral
100 kg N/ha/year
100 kg N/ha/year
100 kg N/ha/year
Organic
0 kg/ha/year
0 kg/ha/year
0 kg/ha/year
Mineral
200 kg N/ha/year
200 kg N/ha/year
200 kg N/ha/year
Organic
Strawb
Strawb
Strawb
Mineral
200 kg N/ha/year
200 kg N/ha/year
200 kg N/ha/year
-
aMS:
Silage Maize; WW: Winter Wheat; WB: Winter Barley; SB: Sugar Beet
accumulating
*during period 1984-1998; **during period 1999-2004
Crop rotations in bold indicate selected crop for this thesis research.
bafter
3.2.2. Soil sampling
Soil samples were collected using a 30 mm diameter auger as a composite from
10 different sampling points within each plot. The samples were collected to a
depth of the plough layer, which was 23 cm in Bottelare, 30cm in Tetto Frati, and
25 cm in Puch. Soil samples were first sieved on a 4 mm sieve, while still in a moist
condition to avoid disruption of microaggregates. The sieved soil was then air-dried
for 7-10 days. After drying, a small subsample was ground into powder by a
stainless steel ball mill for further bulk soil C-content and 13C/12C-isotope analysis
(see part 3.4). The remaining non-ground sieved sample was further used for
physical fractionation (see part 3.3).
21
3.3. Physical fractionation to isolate specific soil fractions (intra microaggregate particulate organic matter) and quantification of maizederived C contribution
Soil samples <4 mm were further physically fractionated to investigate relative
occurrence of maize root derived C in different soil fractions. A combination of size
and density separation procedures was used in this research, partly based on a
fractionation scheme developed by Vitro et al. (2008). As mentioned in Vitro et al.
(2008), the physical fractionation method was specifically developed to solve
uncontrolled soil dispersion and specifically maintain silt sized aggregates. In most
adopted physical fractionation procedures used, silt-size aggregates are not
separated from other soil fractions due to the unspecific action of dispersing
agents. The isolation of silt-sized particles and stable aggregates and OM held
within from larger aggregates in which they are partly stored requires a very
specific sequence of physical fractionation steps. The adopted fractionation
scheme consisted several steps: dispersion and size fractionation of soil
aggregates (see part 3.3.1), sedimentation (see part 3.3.2), and lastly a sequential
density fractionation in combination with ultrasonic dispersion to isolate silt-size
fractions (see part 3.3.3).
3.3.1. Dispersion and size fractionation
Based on Virto et al (2008), a specific sieving apparatus causing disruption of
macroaggregates without concomitant disruption of silt sized water stable
microaggregates was designed for sieving of the bulk soils. In this first step of the
fractionation procedure 15 g of soil sample was weighed in a 30 ml plastic bottle
and 10 glass beads were added into this bottle (diameter +/- 5mm) as well. A 2 cm
diameter opening in the plastic bottle’s cap was covered with a 53 µm mesh size
nylon cloth. This 30 ml bottle was attached onto the inner side of the larger 250 ml
PE bottle’s cap, then inserted into a 250 ml bottle with 200 ml of deionized water
added. The 250 ml bottle were laterally shaken on a reciprocal shaker for 4 hours
at a rate of 150 rpm.
The purpose of adding the glass beads was to disrupt gently soil macroaggregates
(i.e. > 53 µm) and to release microaggregates held within. After being shaken, the
larger bottle contained the major part of the < 53 µm soil material (primary particles
and microaggregates) while some < 53 µm sized sediment remained inside the 30
ml bottle and most material was in fact of size > 53 µm. Then, the suspension in
the 30 ml bottle was poured onto a 53 µm sieve and rinsed with deionized water.
The fraction passing through the sieve was combined with the previously collected
< 53 µm material from the 250 ml bottle (Figure 7). This fraction was kept in
suspension until further separation by repeated sedimentation. The sand-size (>
53 µm) fraction remaining on the 53 µm sieve was oven-dried (1050C) for 24 hours
inside pre-weighed aluminium cups. After being weighed on an analytical balance
the soil was ground into powder by a stainless steel ball mill for subsequent Ccontent and 13C/12C-isotope ratio analysis (see part 3.4).
22
Figure 7. Dispersion and size fractionation of soil aggregates < 4 mm
3.3.2. Sequential sedimentation
In a second step, repeated sedimentation was used to further subdivide the <53
µm soil fraction into free clay (<2 µm) and silt-sized (2-53 µm) material.
Sedimentation method was preferred over sieving to preserve silt-size
microaggregates with less disruption during sedimentation compared to wet
sieving. The sedimentation time of the clay-size (<2 µm) fraction was calculated
using Stokes’s Law, which accounts for height of the water column and its
temperature. The suspension containing the <53 µm soil fraction was poured into
1000 ml sedimentation tubes and deionized water was added approximately until
28 cm height of the water column and the temperature was maintained at 290C by
placing the sedimentation tubes in a thermostatic water bath (Figure 8a).
The time needed for one sedimentation cycle when height of water and
temperature were maintained at 28 cm and 290C, respectively for each sample
was 18 hours, 48 minutes, and 33 seconds. The equation proposed by Stoke’s law
was used to count the velocity of falling material to settle down at the bottom of the
sedimentation tube and calculated the time of sedimentation by velocity and height
of water:
23
2
× [𝑟 2 (𝑑 − 𝑑𝑤) × 𝑔]
𝑣=9
𝑛
ℎ
𝑣
where, v is the velocity of the falling material, 2/9 is the emperical factor for
intermediate calculation, r2 is the equivalent radius, d is the particle density of the
clay mineral, it is assumed by 2.5 g/cm3, dw is the density of water, n is the viscosity
of water, h is the height of water, and t is the time needed for sedimentation. Dw
and n were calculated based on the function of temperature.
𝑡=
Figure 8a. Steps in sequential sedimentation to separate the free soil clay fraction.
Sedimentation thermal bath (i) and at the beginning of sedimentation
cycle (ii)
The number of consecutive sedimentation cycle needed for near complete removal
of clay was different for each field experiment (Bottelare: 3-4 cycles, Tetto Frati: 45 cycles, and Puch: 8-9 cycles). Differences in texture controlled the required
number of sedimentation cycles, the more clay, the larger the number needed for
sedimentation cycles. At the end of each sedimentation cycle (18 hours 48 minutes
33 seconds), the 2-53 µm sized sediment remained in the bottom of the tube and
the suspension with < 2 µm material, i.e. the free clay fraction, was aspirated and
collected in a 10l sealed plastic bucket. Later, this < 2 µm suspension was dried
on a hot plate and the remaining clay material was collected into pre-weighed
aluminium cups and weighed (Figure 8b).
In the end of the second step of the soil fractionation procedure, there were 3
different soil fractions: the sand-sized fraction (from the first step) containing
mainly sand sized particulate organic matter, the free clay-sized fraction bound
OM, and the silt-sized fraction contained clay bound OM, which was further
separated by a combination of density separation and ultrasonic dispersion (see
part 3.3.3).
24
Figure 8b. Steps in sequential sedimentation to separate the free soil clay fraction.
At the end of sedimentation cycle (iii) and drying the < 2 µm suspension
(iv)
3.3.3. Density separation
The final step of the full fractionation procedure involved density separation of the
bulk silt-size fraction (2-53µm) into free and occluded POM and mineral bound OM.
The silt-size fraction was still composite of free POM < 53 µm, silt-sized
microaggregates, and free silt particles. Fractionated at density 1.6 g/cm3
differentiated firstly 2 fractions: a light fraction (ρ<1.6 g/cm3) consisting of nonoccluded uncomplexed light OM, from a composite heavy fraction (ρ>1.6 g/cm3).
The objective of density separation is to isolate these fractions regardless of their
size, only based on the density.
The 1.6 g/cm3 heavy liquid for density fractionation was prepared using sodium
polytungstate (SPT) (Sometu - Europe, Germany). About 10 ml of the soil
suspension containing the silt-sized fraction collected from the preceding
sequential sedimentation (3.3.2) was placed in a 65 ml Nalgene centrifuge tube
(Oak ridge company). Then, 30 ml of 1.8 g/cm3 SPT was added into the centrifuge
tube so that the density of resulting suspension became 1.6 g/cm3. The tubes were
gently shaking and rotated by hand. The tubes were then placed in a swinging
bucket rotor centrifuge (Eppendorf 5810 R) at 4000 rpm for 30 minutes with the
adapters installed. In order to calculate the centrifugation time to release fraction
with density 1.6 g/cm3 from 2.65 g/cm3 (density of organo-mineral), there are many
factor to take into account such as temperature, particle diameter of heavy fraction,
height of suspension, and height of decantation. Combination between these
factors and speed of rotation will determine the time needed to settle the heavy
fraction in the bottom of tube. After being centrifuged a vacuum set up was used
to aspirate the supernatant including all floating material onto a 47 mm diameter
Supelco nylon 66 membrane filter with opening of 0.45 µm on a milipore unit. The
filters were rinsed with about 50 ml of deionized water to remove remaining SPT.
In this way non-occluded light OM was isolated on the filters, which were then dried
in the oven at 1050C and weighed. For further 13C/12C and C-content analysis a
subsample of the filtrate was scraped from the filter and transferred directly into Nicups used by the elemental analyser. The remaining >1.6 g/cm3 sample was left
25
in the centrifuge tube. Then 40 ml of 1.6 g/cm3 SPT was added and ultrasonic
dispersion was used to disrupt the silt-sized microaggregates and release the
particulate OM held within.
The 50 ml 1.6 g/cm3 suspension was dispersed by means of an ultrasonic probe
Model CV33 (Figure 9) for 5 minutes. An ice bath has been used in order to avoid
excessive increasing of the temperature in the 50 ml and to avoid reduction in
cavitation during sonication. The energy output of the ultrasonic probe was
calibrated by following the rise in temperature of 150 ml water inside a Dewar
vessel upon contiuous ultrasonication. The 5 minutes ultrasonication was equal to
an average energy output of 440 J/ml, which would result in near complete
disruption of all silt sized aggregates.
The occluded silt sized intra-microaggregates light POM fraction was then
recovered after centrifugation of the ultrasonically dispersed suspension at 4000
rpm for 1 hour. This suspension then undergone the same procedure as in case of
the previously collected non-occluded uncomplexed light OM fraction. The soil
material remaining in the tube was called a heavy fraction, consisting of nonoccluded and occluded silt-sized minerals and occluded clay-size particles
(microaggregates silt&clay).
Figure 9. Set-up of ultrasonic dispersion by means of an ultrasonic microprobe
3.4. C-content and C-isotope analysis
Measurements of C-content and the 13C/12C-ratio and from all different isolated soil
fractions: the sand-sized fraction, the free clay-sized fraction, the free OM light
fraction (non-occluded light OM fraction), the occluded silt-sized light POM fraction,
and the silt-sized fraction (microaggregates silt&clay) were performed using a PDZ
Europa ANCA-GSL elemental analyser interfaced with a Sercon 20-20 IRMS from
SysCon electronics (SerCon, Cheshire, UK) at the UGent Isotope Bioscience
Laboratory.
26
The equation proposed by Balesdent and Mariotti (1996) was used for the
conversion of the measured δ13C values into a proportion of maize-derived C of
each soil fraction:
δ13 𝐶 𝑠𝑎𝑚𝑝𝑙𝑒 − δ13 𝐶 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
𝑚𝑎𝑖𝑧𝑒 𝑑𝑒𝑟𝑖𝑣𝑒𝑑 𝐶 =
∆ δ13 𝐶
13
13
where δ C sample is the δ C of the soil sample or soil fraction, δ13C control is the
δ13C of a control treatment (e.g. from a plot with only C3 plants cropped or with a
starting situation of maize derived C present), and Δ δ13C is the difference in the
13
C/12C between maize plant and C3-plants. According to trial results of Dignac et
al. (2005) average values for 13C/12C of maize and wheat of −13‰ and −26‰ were
used respectively.
3.5. Statistical analysis
Means with standard errors were calculated for DM, C%, C content and % maizederived C of bulk soils and soil fractions. Statistical analysis was performed with
Spotfire Splus 8.2. Root biomass dry matter for each location was analyzed with
two-way ANOVA with soil texture and cultivar as a factor, while C-content and
maize derived C for each fraction were analyzed with one-way ANOVA with crop
rotation as a factor. The Kolmogorov-Smirnov and the Modified Levene tests were
used to test the normal distribution of data and equality of variance
(homoscedasticity), respectively. Multiple mean comparison using Tukey’s test
was used to identify differences between treatments when the ANOVA indicated a
level of significance p < 0.05.
27
IV. RESULTS AND DISCUSSION
4.1. Maize root biomass
The dry matter (DM) weight of maize root biomass from eight cultivars at three
ILVO maize field trials are presented in Fig. 10. There were no significant
differences in root DM of the different maize cultivars. In spite of relatively large
variation between cultivars in Merelbeke, there was no interactive effect of field site
and cultivar either. However, there was a significant difference in mean maize root
DM between the field trials. At first sight this difference could most probably be
ascribed to their different soil texture. Merelbeke, where the soil texture is a sandy
loam had the highest root biomass from all cultivars compared to the two other
fields. Although there was no significant interactive effect between location and
cultivars, it appeared that at Merelbeke the response of different cultivars to site
existed only for some of the cultivars such as NK Falcone, Kalientes and LG30.224.
The response of a crop’s rooting system to soil texture would evidently depend on
the cultivar. Each cultivar has specific growth requirement for their development.
When the medium growth’s (soil) condition not meet with the requirements, maize
would not grow optimally leading to a decrease of maize’s DM both in maize aboveground biomass and root biomass.
a
b
b
Figure 10. Root biomass of different maize cultivars measured from undisturbed
soil cores collected at 30 cm depth in three ILVO maize cultivar field
trials in Autumn 2014. Each bar represents the average of 3 field
replicates, vertical error bars represent standard deviation. The same
letters denote no significant difference between means DM location
(P < 0.05) by Tukey’s multiple mean comparison test
Texture is an important characteristic of soil and can affects water holding capacity,
drainage properties and therefore root growth. Maize has low resistance to drought
stress and requires continued water supply throughout their growth. Root
28
development is also directly affected by structure, which depends on soil texture.
The trial at Ravels was laid out on a sand textured soil, displayed the lowest root
biomass and largest proneness to drought period when stress can occur and affect
the root development. The maize root biomass was lowest at Ravels. Instead, a
loamy texture would have provided more available water to the plant and root
system may have better developed under these conditions. A further analysis of
the above ground biomass production will reveal if maize growth was hindered
overall in the lighter textured soil of Ravels. If so, the hypothesized reduced growth
of the root system at Ravels due to water shortage seems plausible.
It is not possible at this stage to identify soil texture as the sole responsible factor
controlling the root development. Alongside with soil texture, soil phosphorus (P)
availability is important for the early growth and development of maize and its root
system. P-availability affects root morphological and physiological characteristics
that are important for P uptake since P is relatively unavailable and immobile in
many soils (Hajabbasi and Schumacher, 1994). Since sufficient P-was applied in
all three trials at the exact same dose, nutrient effects would at first seem to be
irrelevant. However, due to the often general low availabilty of P, differences in Pstatus of the soils may still have a controlling effect on root development. Sandy
soils in Flanders often display a substantial availability of P because they have
become P-saturated. If so at Ravels, there is less need for roots to expand, which
means there is no increasing in surface area of roots and root length and leads to
low root biomass DM. Comparison of the P-status of the sites will need to be
conducted in particular to support this argument.
4.2. Bulk soil OC content and content of maize derived C of
silage maize vs. grain maize or non-maize cropped soil
4.2.1. Soil organic carbon
There was no significant difference in the bulk soil OC-content between crop
rotations at the Bottelare and Puch field experiments (Fig. 11). Soil at Bottelare
and Tetto Frati had a similar OC-content of 10-12 g C/kg soil. There appeared to
be a higher soil OC-content when grain maize was cultivated compared to the other
rotations. So at first, there seems to be no significant additional build-up of soil OC
if above-ground biomass was incorporated in Bottelare, even after 12 years of
continuous grain maize cropping. This gives a first indication that indeed aboveground OM matters less to maintenance of soil OC when compared to other
sources of OM.
Yet there existed not significant difference in bulk soil OC-content with silage maize
for any individual treatment. However, a contrast test between the MG0, MG150
on the one hand and MS+IR0, MS+IR150 and MS150 on the other revealed a
highly significantly (p<0.01) elevated C content in the group of grain maize plots.
Higher OC-content in maize grain was expected because of the additional
contribution of C-input from maize above-ground biomass (Fig. 11a).
29
At Tetto Frati, the MG0 treated plot had a significantly higher (p<0.001) OC content
than each of the MS plots (MS0, MS170 and MS250). Hence only at 0 kg N
application did grain maize cultivation result in accumulation of SOC relative to
silage maize (Fig. 11b). At Puch the soil OC-content was low, amounting just 8-9
g C/kg soil. No grain maize objects were included and this site could only be used
to quantify the relative stability of maize root biomass derived C (Fig. 11c).
a
a
a
a
a
a
a
(a)
b
a
a
ab
a
(b)
a
a
a
a
(c)
Figure 11. Bulk soil OC-content of the sampled plough layer of selected rotational
treatments in three European long-term fields experiments Bottelare
(a), Tetto Frati (b), and Puch (c). Each bar represents the average of
3 field replicates, error bars represent standard deviation. The same
letters denote no significant difference between means (P < 0.05) by
Tukey’s multiple mean comparison test.
30
4.2.2. Measurement of 13C
Stable C isotopic techniques have been widely used to quantify the relative
contributions of C3 and C4 plants to SOC. According to Dignac et al. (2005), 13C
value is -26‰ in plants with C3 pathway (i.e. wheat) and the 13C value is -13‰ for
C4 plants (i.e. maize). The value of 13C of the ploughed sample of selected crop
rotations in the long-term fields experiments are presented in Table 9.
Bulk soil at Tetto Frati had 13C values from -15.98‰ to -17.22‰, which is relatively
close to the pure maize’s 13C of -12‰ to -13‰. So, these plots are suspected to
contain SOM which is largely C4 plant derived. In contrast at Bottelare and Puch
similar values of 13C around -24‰ to -25‰ were found, which suggests the SOM
to have a low contribution of C4 plant-derived OM, in spite of maize cropping for 12
and 32 years at these sites, respectively.
SOM in the Tetto Frati soils had the closest value of 13C to pure maize-biomass,
because maize had been cultivated for 22 years without any other crop in rotation.
Although in Puch maize was even cropped for almost 32 years, the contribution of
C from maize was by far less than at Tetto Frati. Probably because maize was
cropped in a 3-year rotation with winter wheat and winter barley, so the
accumulation of OC from maize would indeed be lower than in Tetto Frati.
To be able to calculate the amount of maize-derived C in the soil, a field object
needs to act as a relative control or otherwise the 13C values need to be compared
to reference C3 plant value of -26‰. Thus, the selected reference rotational
treatments for Bottelare and Puch were the ‘fodder beet’ and ‘sugar beet’ crop
rotations, where maize was replaced by these crops. For Tetto Frati, we assumed
a 13C value of -26‰ to calculate the SOM’s maize-derived C content, since there
is no non-maize cropped reference object in this field experiment.
31
Table 9. δ13C of the bulk soil samples in long-term field experiments. The
treatments that were considered as reference to be further used for
calculation of the % of maize derived C are marked in bold (Average of 3
field replicates ± standard deviation)
Level
of N
13C vs PDB
Crop rotation
Codeb
(kg
(‰)
N/ha)
Bottelare
monoculture grain maize
0
MG0
-23.64±0.15
silage maize + Italian ryegrass
0
MS+IR0
-24.16±0.17
harvested
monoculture grain maize
150
MG150
-23.70±0.29
silage maize + Italian ryegrass
150
MS+IR150
-24.34±0.34
harvested
silage maize + grass clover
150
MS150
-25.17±0.52
fodder beet + 4 other crops
150
FB150
-25.57±0.15
grass clover + silage maize
150
GK150
-25.23±0.05
Tetto Fratia
monoculture grain maize
monoculture silage maize
monoculture silage maize
monoculture grain maize
monoculture silage maize
Puch
silage maize + winter wheat + winter
barley
silage maize + winter wheat + winter
barley
silage maize + winter wheat + winter
barley
sugar beet + winter wheat +
winter barley
0
0
170
250
250
MG0
MS0
MS170
MG250
MS250
-15.98±0.30
-16.91±0.45
-17.22±0.68
-16.56±0.21
-18.27±0.27
0
MS0
-24.77±0.42
100
MS100
-24.81±0.01
200
MS200
-24.99±0.16
200
SB200
-25.97±0.05
- a No non-maize rotation was sampled at Tetto Frati and comparison of accumulation of
maize-derived C of MG and MS treatments assumed a C3-crop 13C of -26‰
- b MG: grain maize; MS: silage maize; FB: fodder beet; GK: grass clover; SB: sugar beet;
IR: Italian ryegrass; numbers denote N-fertilizer appication rates in kg N/ha/year
32
4.2.3. Content of maize-derived C
Fig. 12 shows the estimated content of maize-derived C in the bulk soil per longterm field experiment. The amount of maize-derived C was varied from 0.3 to 10 g
C/kg soil, where the soils at Tetto Frati had the highest maize-derived C, followed
by Bottelare, and a very low amount of maize-derived C was measured at Puch.
There were no significant differences between crop rotation/fertilizer aplication
objects in the case of Puch. This is not very surprising since only three silage maize
objects were compared at Puch. However, crop rotation did affect the content of
maize-derived C at the two other field experiments.
At Bottelare, there seemed to be a low (approx. 0.3 g C/kg in GK150) background
concentration of maize-derived C, probably due to past cultivation of maize or
application of 13C-enriched manure, preceding the field experiment. Analogous to
bulk soil OC-content, the highest content of maize-derived C was obtained in the
grain maize objects with 1.8 g C/kg soil (MG150) and 10 g C/kg (MG0) soil at
Bottelare and Tetto Frati, respectively. However, the grain maize object’s maizederived C content was only significantly higher than that of the corresponding
silage maize treatment for the 150 kg N fertilized plots (MG150 vs. MS150) in
Bottelare.
We have no measure of the initial amount of maize derived C of the Tetto Frati
soils, but the very high 13C would suggest a long-term cultivation of maize or
application of manure from maize fed animals. Indeed it is known that prior to
instalment of the field trial in 1992, there was 10 years of monoculture grain maize,
however with only urea used as N-source. Still, regardless of this large 13C background there were significantly higher contents of maize derived C in the MG
compared to the MS treatments (P<0.05). This was the case both for the 0 kg N
(MG0 vs.MS0) and for the 250 kg N applications (MG250 vs. MS250).
33
b
b
ab
ab
a
a
(a)
a
b
b
bc
c
(b)
a
a
a
(c)
Figure 12. Estimated maize-derived C in the bulk soil for rotational treatments in
the three European field experiments. Bottelare (a), Tetto Frati (b), and
Puch (c). Error bars represent standard deviations around means (3
field replicates). Different letters denote significant differences between
means (P< 0.05) by Tukey’s multiple mean comparison test
34
4.3. Distribution of soil C and maize-derived C over physical
fractions
4.3.1. Dry matter weight
Physical fractionation was used to isolate different soil fractions which would
contain SOC at various degrees of decomposition as well as protection against
microbial decomposition. The quantified masses of the different soil fractions are
presented in Table 10. There were large differences between the three sites. The
sand-sized fraction represented maximally 61.15±11.46% (MS+IR0), 34.07±2.36%
(MG0), 12.97±0.77% (MS0) of the total soil from Bottelare, Tetto Frati, and Puch,
respectively. The silt-sized fraction as a whole represented 35-41% of the total soil
mass in Bottelare, 55-65% at Tetto Frati, and in Puch 72-76%. So the content of
silt&clay microaggregate seemed to be primarily determined by soil texture and to
a much smaller extent by crop rotation. The DM weight of the two isolated silt-sized
light fractions (non-occluded light OM and occluded POM) were minor, as were to
be expected since these comprised non-mineral soil material only.
Table 10. Dry matter (DM) weight of isolated soil fractions from three European
long-term field experiments with silage maize in rotation or monoculture
as compared to grain maize or non-maize rotations (Average of 3 field
replicates ± standard deviation)
Crop
rotation
> 53µm
(sand)
Weight of fraction (g/100g soil)
2-53 µm (silt)
< 2 µm (free
nonsilt&clay
clay)
occluded
microaggregate
light OM
occluded
light silt
POM
Bottelare
MG0
MS+IR0
MG150
MS+IR150
MS150
FB150
GK150
56.08±3.40
61.15±11.46
54.94±2.16
58.26±3.04
56.27±2.04
54.79±2.43
56.93±4.99
2.24±0.50
2.04±0.39
2.22±0.80
2.15±0.59
1.77±0.19
2.36±0.19
1.94±0.24
40.46±3.13
35.49±10.03
41.59±2.15
37.64±4.24
41.80±2.00
40.46±3.31
40.71±6.12
0.04±0.00
0.16±0.22
0.23±0.19
0.08±0.08
0.02±0.02
0.08±0.07
0.06±0.05
0.18±0.01
0.11±0.05
0.21±0.17
0.15±0.06
0.18±0.03
0.20±0.04
0.09±0.07
Tetto Frati
MG0
MS0
MS170
MG250
MS250
34.07±2.36
34.03±1.70
31.51±2.72
29.65±0.81
30.87±1.69
5.18±0.42
5.15±0.13
5.43±0.10
5.17±0.28
5.20±0.20
61.32±1.53
58.87±1.41
65.08±4.50
65.40±0.50
55.36±15.81
0.07±0.07
0.05±0.00
0.04±0.01
0.16±0.07
0.14±0.09
0.42±0.37
0.62±0.32
0.44±0.38
0.78±1.12
0.13±0.09
Puch
MS0
MS100
MS200
SB200
12.97±0.77
12.67±0.77
12.49±1.62
12.29±0.83
11.57±1.65
11.20±0.97
11.91±0.67
11.33±1.00
76.61±1.43
75.76±1.19
76.60±1.81
72.34±2.08
0.14±0.00
0.23±0.09
0.12±0.01
0.10±0.01
0.57±0.91
0.11±0.04
0.12±0.05
0.10±0.12
a
MG: grain maize; MS: silage maize; FB: fodder beet; GK: grass clover; SB: sugar beet;
IR: Italian ryegrass; numbers denote N-fertilizer application rates in kg N/ha/year
The DM distribution differs amongst the field trials, evidently due to their differences
in soil texture with most sand in case of Bottelare, followed by Tetto Frati, and
Puch. The content of silt-sized fraction (combination of microaggregate silt&clay,
35
non-occluded light OM, and occluded light silt POM) seemed to differ a bit with
rotational treatment. There was however, no consistent pattern between silage and
grain maize objects. Silage maize (MS) at Puch had similar DM distribution over
the isolated soil fractions. So there was no significant effect of N fertilizer in the DM
at Puch. The sugar beet crop rotation (SB) had less DM in the silt-sized fraction.
The DM distributions showed differences with reports of soil texture analysis from
literature on the three experiments. The content of sand DM at Tetto Frati in
particular (about 30%) is lower than the reported sand content of 47%. It is not
clear what causes this difference but the consistent result for all field replicates and
treatments at all sites indicated a correct analysis of DM partitioning in the physical
fractionations.
4.3.2. Soil organic carbon
Table 11 shows the amount of OC-content present in the five different isolated soil
fractions for the three long-term field experiments. In total, 43-49% of the total SOC
was associated with the microaggregate silt-sized fraction (i.e. silt and clay in
microaggregate and silt-sized POM). Within this size fraction, OC in occluded silt
POM was greater than OC in the non-occluded light OM. This means that about 012% of SOC was physically protected in the microaggregate in the Bottelare soils.
At Tetto Frati, the % of physically protected C was larger with 3-20% of SOC and
at Puch intermediate with 4-14% of SOC. In case of Puch, with a finer texture, only
9% sand and 70% silt, microaggregation and containment of OM held within, could
be expected to be larger. This was also the case when compared to the coarser
soil at Bottelare. However the texture of the Tetto Frati and Bottelare soils was in
fact omparable and still much more microaggregate occluded POM was present in
the Tetto Frati soil. Soil aggregation, however, depends not only on texture but also
on content of CaCO3 (4.7% at Tetto Frati) and clay type (presumably vermiculiterich near the Po river at Tetto Frati). for the free clay-sized fraction (i.e. not part of
microaggregate) amounted 10.31±1.85%, 14.27±1.04%, and 33.17±3.68% on
average of the total soil OC in case of the Bottelare, Tetto Frati, and Puch soils,
respectively. So clearly a comparatively much larger part of soil OC was claybound in the Puch soil. The reason for this is not clear, other than perhaps the
relatively low contribution of C in this experiment.
The sand and silt fraction are particularly enriched in C compared to the clay
fraction in Bottelare and Tetto Frati. Even though the sand fraction had smaller Ccontent than the silt&clay microaggregate fraction, its contribution to soil OC was
bigger. While, in Tetto Frati and Puch, this fraction seems to be contributed to SOC.
There were only significance differences between crop rotation treatments for
Bottelare’s sand fraction and Puch’s non-occluded light fraction.
36
Table 11. Distribution of SOC over different soil fractions (expressed on a bulk soil
basis) and in brackets the measured OC content (%OC) of each
individual fraction from three long-term field experiments (Average of 3
field replicates ± standard deviation). Different letters denote significant
differences between means (P < 0.05) by Tukey’s multiple mean
comparison test
Crop
rotation
Bottelare
MG0
MS+IR0
MG150
MS+IR150
MS150
FB150
GK150
Tetto Frati
MG0
MS0
MS170
MG250
MS250
Puch
MS0
MS100
MS200
SB200
> 53µm
(sand)
C-content (g C/kg soil)
2-53 µm (silt)
< 2 µm
nonsilt&clay
(free clay)
occluded
microaggregate
light OM
occluded
light silt
POM
5.73±5.45
(1.00±0.91)
3.58±2.53
(0.55±0.29)
2.82±1.97
(0.51±0.33)
3.01±1.18
(0.52±0.20)
3.22±0.91
(0.57±0.15)
2.01±1.15
(0.37±0.22)
3.42±0.95
(0.59±0.12)
1.06±0.35
(4.68±0.95)
1.14±0.20
(5.82±2.07)
1.21±0.41
(5.56±0.83)
1.15±0.19
(5.55±1.13)
0.85±0.03
(4.85±0.39)
1.25±0.09
(5.30±0.08)
0.95±0.16
(4.90±0.27)
3.75±0.53
(0.93±0.12)
3.37±1.39
(0.92±0.21)
5.05±0.54
(1.21±0.08)
4.90±1.34
(1.29±0.20)
4.42±1.16
(1.07±0.33)
3.63±1.43
(0.91±0.37)
3.85±1.05
(0.95±0.22)
0.10±0.03
(28.17±10.09)
0.36±0.45
(24.46±3.20)
0.75±0.53
(34.04±4.58)
0.10±0.11
(14.67±1.38)
0.02±0.00
(7.92±0.33)
0.27±0.28
(33.17±4.28)
0.25±0.21
(39.67±1.69)
0.78±0.04
(43.81±5.46)
0.53±0.31
(44.75±7.12)
0.62±0.27
(37.08±13.32)
0.68±0.33
(45.27±7.12)
0.62±0.03
(35.26±9.11)
0.90±0.12
(44.07±3.98)
0.44±0.33
(44.72±7.85)
3.17±0.15c
(0.93±0.08)
2.43±0.22ab
(0.71±0.03)
1.99±0.28a
(0.64±0.13)
2.13±0.15a
(0.72±0.07)
2.84±0.28bc
(0.92±0.14)
1.74±0.17
(3.36±0.09)
1.60±0.14
(3.11±0.20)
1.60±0.08
(2.95±0.09)
1.57±0.11
(3.03±0.05)
1.71±0.04
(3.29±0.13)
3.99±0.76
(0.65±0.11)
4.05±1.06
(0.69±0.19)
4.12±0.91
(0.63±0.14)
4.63±0.28
(0.71±0.04)
3.41±1.14
(0.62±0.10)
0.41±0.12
(34.47±4.14)
0.15±0.01
(28.77±2.20)
0.13±0.04
(30.10±3.21)
0.49±0.29
(28.75±5.01)
0.48±0.33
(32.05±2.75)
0.55±0.43
(14.68±2.82)
1.27±0.71
(20.55±3.26)
1.83±1.59
(40.47±1.26)
2.38±3.40
(30.17±0.22)
0.34±0.13
(29.63±7.51)
1.12±0.19
(0.87±0.16)
1.03±0.24
(0.81±0.14)
1.17±0.21
(0.94±0.06)
1.20±0.17
(0.98±0.10)
2.61±0.28
(2.26±0.17)
2.41±0.20
(2.17±0.34)
3.04±0.28
(2.56±0.33)
2.90±0.35
(2.55±0.11)
2.86±1.05
(0.38±0.14)
2.28±1.01
(0.30±0.14)
2.86±0.17
(0.37±0.03)
2.25±0.30
(0.31±0.05)
0.44±0.05ab
(31.05±3.50)
0.71±0.28a
(31.68±0.31)
0.38±0.04ab
(32.65±1.92)
0.25±0.01b
(25.85±0.30)
1.19±1.84
(28.47±7.06)
0.39±0.20
(35.53±4.93)
0.41±0.13
(34.53±1.75)
0.28±0.32
(29.09±4.62)
The distribution of OC from different isolated soil fractions are presented in Fig. 13.
It is clear that most of the crop treatments had most C in their sand and
microaggregate silt&clay fractions. If all the isolated fractions were summed up for
each crop rotation as represented by each bar, the highest amount of total OC was
found in the grain maize cropped soil at Bottelare and Tetto Frati. Fig. 14 shows
the relative distribution of the bulk soil OC over the isolated soil fractions. In case
at Puch site, OC more found in the clay-sized fraction than in the sand-sized
37
fraction, which logical because of the clay soil texture. Generally, the occluded light
silt POM at Tetto Frati soils contained more OC compared to other sites. This is
because Tetto Frati had been more than 20 years of cultivation and almost of the
SOC has been transformed to occluded light silt POM.
(a)
(b)
(c)
Figure 13. Distribution of OC from different soil fraction within crop rotation at three
long-term fields experiments. Bottelare (a) and Tetto Frati (b). Each bar
represents the average of 3 field replicates
38
(a)
(b)
(c)
Figure 14. Comparison of the relative distribution of OC (% of SOC) over isolated
soil fractions for different crop rotations at three long-term field
experiments. Bottelare (a), Tetto Frati (b), and Puch (c). Each bar
represents the average of 3 field replicates
39
4.3.3. Maize-derived C
4.3.3.1. 13C and % of maize-derived C
Generally, the 13C value decreased from coarse to fine fractions (Table 12). This
result is opposite with similar studies (Gerzabek et al., 2001; Balesdent and
Mariotti, 1996), where increasing values of 13C were found with decreasing of
fraction size. The non-occluded light OM fraction had very similar 13C values as
the occluded light silt POM, indicating a close relation between both fractions
(Gerzabek et al., 2001). The low incorporation of relatively young maize derived C
in these fractions could be an indication of their high stability (Gerzabek et al.,
2001).
Crop rotation affected the size fraction’s 13C (Table 12). The sand-sized fraction
in all three long-term field experiments had values of 13C ranging from -15.47‰,
i.e. relatively close to pure maize’s 13C, all the way to -24.65‰, closer to C3vegetation 13C values. These 13C values were higher when compared to other
soil fractions. So, the sand fraction appear to be preferentially contain of maizederived C, over other soil fractions and bulk soil. All three field experiments
revealed these similar patterns of 13C values over the different isolated fractions.
The non-occluded light and occluded light POM fractions had 13C values relatively
close to C3 vegetation, which indicated little maize-derived C in silt sized POM,
compared to other soil fractions.
Table 12. Measured δ13C of the different isolated soil fractions in long-term field
experiments (Average of 3 field replicates ± standard deviation)
13C vs PDB (‰)
Crop
rotation
Bottelare
MG0
MS+IR0
MG150
MS+IR150
MS150
FB150
GK150
Tetto Frati
MG0
MS0
MS170
MG250
MS250
Puch
MS0
MS100
MS200
SB200
2-53 µm (silt)
nonsilt & clay
occluded
microaggregate
light OM
> 53µm
(sand)
< 2 µm
(clay)
-23.15±1.87
-24.40±0.55
-23.92±0.67
-24.58±0.91
-25.13±0.34
-26.09±0.45
-25.25±0.23
-26.04±0.75
-26.03±0.51
-25.50±0.31
-26.18±0.69
-26.56±0.46
-26.28±0.19
-26.42±0.65
-25.98±0.019
-26.05±0.03
-25.29±0.07
-26.92±0.13
-26.75±0.07
-26.38±0.07
-26.14±0.13
-26.22±0.21
-26.21±0.12
-26.23±0.15
-26.04±0.31
-25.93±0.28
-26.58±0.15
-26.20±0.26
-26.61±0.04
-26.83±0.19
-26.34±0.57
-26.88±0.34
-26.76±0.22
-27.54±0.14
-26.93±0.28
-14.77±0.81
-15.28±0.68
-15.44±0.56
-16.77±0.76
-15.12±0.59
-21.04±0.43
-19.45±1.17
-19.26±0.40
-19.19±0.35
-17.86±0.26
-19.58±1.05
-19.59±0.45
-20.02±0.70
-21.26±0.58
-19.57±0.32
-24.19±1.37
-23.72±0.48
-23.53±0.17
-22.88±0.59
-24.00±0.63
-23.01±1.36
-23.80±0.61
-26.79±0.81
-23.54±0.14
-24.43±0.55
-22.23±0.84
-24.85±0.30
-24.94±0.50
-26.26±0.87
-24.94±0.38
-25.21±0.10
-25.53±0.23
-25.13±0.23
-26.41±0.06
-26.35±0.12
-26.63±0.18
-27.15±0.20
-27.90±0.04
-27.97±0.04
-27.83±0.05
-27.70±0.12
-27.97±0.20
-28.16±0.06
-27.83±0.50
-28.19±0.48
40
occluded
light silt
POM
The % of C which was maize-derived accumulated in the soil fractions is presented
in Table 13. Based on the calculation of 13C values, the biggest contribution of
maize-derived C was present in the sand-sized OM and microaggregate contained
silt & clay bound OM. In the Bottelare experiment, relatively most maize-derived C
was present in the sand fraction. This is because the newly accumulated maize C
is probably still very much present as POM (i.e. only partly transformed to the plant
material) and has not been converted yet to finer POM (i.e. in the occluded siltsized light POM fractions) nor to silt and clay bound OM to a large extent. An
exception appeared to be the MG150 treatment, where the microaggregate
silt&clay of maize-derived C was considerable.
In the Tetto Frati soils, there was clearly the biggest contribution of maize-derived
C amongst the three studied sites with about 80% of maize-derived C accumulated
in the sand-sized fraction. This is probably so because continuous grain maize was
planted over 10 years at Tetto Frati before onset of the trial and during the
experiment for 22 years there was no other crop rotation beside monoculture maize
in the field. Strangely, at Puch the non-occluded light fraction contained no maizederived C at all. This was even the case for the free clay fraction as well. Instead,
most maize-derived C accumulated in the sand fraction. Sugar beet and fodder
beet contained no maize-derived C at all, as expected, because these crop
rotations were used as a reference objects to calculate the maize-derived C for the
other crop rotation.
Table 13. Estimated fraction of maize-derived C (% of C in each fraction) of the
different isolated soil fractions in long-term field experiments (Average
of 3 field replicates ± standard deviation)
Crop
rotation
maize-derived C (%)
2-53 µm (silt)
nonsilt&clay
occluded
microaggregate
light OM
>53µm
(sand)
<2 µm
(clay)
Bottelare
MG0
MS+IR0
MG150
MS+IR150
MS150
FB150
GK150
22.73±11.93
12.95±1.22
16.61±4.10
11.62±3.91
7.33±0.75
0.00
6.35±3.18
3.28±5.17
2.61±4.51
5.85±3.64
2.65±3.41
0.09±0.30
0.00
1.24±1.49
5.96±1.13
2.45±0.29
8.12±0.83
3.40±1.48
0.00
0.00
1.77±1.35
2.79±2.42
2.69±0.91
2.27±2.39
3.66±3.57
4.47±0.76
0.00
2.51±0.65
6.39±1.07
4.89±2.02
8.29±3.05
4.54±3.26
5.41±1.41
0.00
4.23±1.72
Tetto
Frati
MG0
MS0
MS170
MG250
MS250
86.39±6.24
82.45±5.26
81.26±4.34
71.01±5.82
83.72±4.55
38.17±3.27
50.39±9.00
51.86±3.04
52.41±2.73
62.58±2.04
49.38±8.07
49.33±3.47
45.99±5.35
36.45±4.48
49.47±2.46
13.89±10.54
17.51±3.68
18.97±1.30
24.00±4.57
15.41±4.87
23.00±10.49
16.95±4.68
0.00
18.92±1.09
12.10±4.23
Puch
MS0
MS100
MS200
SB200
30.03±10.10
10.22±8.29
9.83±2.27
0.00
3.76±4.18
0.00
0.00
0.00
5.24±0.93
5.65±2.07
3.67±0.23
0.00
0.00
0.00
0.00
0.00
1.48±1.72
1.08±2.22
2.41±2.17
0.00
41
occluded
light silt
POM
4.3.3.2. Distribution of maize-derived C over soil fractions
Analoguous to bulk soil OC, the contribution of shoot-derived C can be calculated
by subtracting the maize-derived C in the grain maize objects by the silage maize.
In Bottelare’s occluded light silt POM fraction from 0 kg N plot shows the rootderived C is 0.02 g C/kg soil and shoot-derived C is 0.03 g C/kg soil. In the silage
maize object, the root-derived contributes up to three times as shoot-derived C
relatively though given the lower bulk soil OC-content. Similar observation shows
higher contribution of root-derived C in Bottelare’s 150 kg N plots for other soill
fractions. At Tetto Frati, the shoot-derived C was large with 0.55 g C/kg compared
to 0.02 g/kg root C at the 250 kg N/ha level. At the 0 kg N level, the contribution of
root and shoot C to occluded POM was similar (0.09 g C/kg). Consequently, there
seems to be no preferential protection of root C in the occluded light silt POM
fraction. Microaggregation does not explain the long-term relative higher stability
of root vs shoot-derived.
Fig. 15 shows the distribution of maize-derived C over the isolated soil fractions in
g C/kg soil. The sand fraction had the highest amount of maize-derived C in the
Bottelare soils. In case of the Tetto Frati and Puch soils, the silt&clay
microaggregate fraction contained almost the same amount of maize-derived C as
the corresponding sand fraction. An exception is the MS0 object at Puch, which
had a very large content of sand maize C.
In the case of the Bottelare experiment (Fig. 15a), clearly most maize-derived C
was present in the sand fraction. After 12 years, indeed still a lot of the newly
entered C (from roots and shoots) would reside as relatively coarse particulate OM,
i.e. in the sand fraction. In the Tetto Frati (Fig. 15b) soils, a much larger share of
maize-derived C was already present in the silt&clay microaggregate and free clay
fractions. The Tetto Frati experiment had lasted longer than Bottelare, 22 y vs. 12
y, and possibly more POM has already been degraded by microbial decomposition
and has ended up and stabilized in the silt & clay microaggregate fraction. Soil
texture also is an important factor that affects more clay in Tetto Frati.
42
(a)
(b)
(c)
Figure 15 Distribution of maize-derived C from different soil fraction in crop
rotation at three long-term field experiments. Bottelare (a) and Tetto
Frati (b). Each bar represents the average of 3 field replicates
Due to large variation amongst field replicates as opposed to differences between
treatment differences in content of maize C were only significant in a few cases.
Fig. 16, 17, and 18 shows the maize derived-C between soil fractions to compare
silage vs. grain maize monocultures with corresponding level of N fertilizer applied
and the contribution to OC. Only figures with significant difference between means
of the treatments shown.
43
c
bc
ab
a
a
(a)
b
a
a
(b)
Figure 16. Maize-derived C of sand fraction at Tetto Frati (a) and Puch (b). Each
bar represents the average of 3 field replicates, error bars represent
standard deviation. Different letters denote significant differences
between means (P < 0.05) by Tukey’s multiple mean comparison test
If it is assumed that all maize-derived C in the silage maize objects is root-derived,
then all additional stored maize C in grain maize fields must be derived from
maize’s above-ground biomass. Maize-derived C was 2.74 g C/kg and 2.01 g C/kg
in the sand fraction at Tetto Frati in the plot without N fertilizer of grain maize
(MG0) and silage maize (MS0), respectively (Fig. 16a). So the maize shoot-derived
C is 0.74 g C/kg, while the maize root derived C is 2.01 g C/kg. Thus, the
contribution of root biomass-derived C to SOC was more than twice as large.
Compared to the 250 kg N fertilizer application, maize-derived C from grain maize
(MG) was significantly lower than the silage maize’s object (MS). Since maizederived C in MS250 higher than MG250, it is difficult to estimate the shoot-derived
C. However, it is clear that the maize-derived C in MS250 was derived from the
root-derived C and the relatively high of these value probably because there is a
contribution from root exudates. The maize root-derived C was observed to be
highest at Tetto Frati and lowest at Puch (Fig. 16a and b respectively) while maize
root-derived C in Bottelare was in between those two site (figure not shown).
44
In the clay fraction at Tetto Frati, the amount of maize-derived C was slightly lower
than in the sand fraction but silage maize have higher maize-derived C than the
grain maize both for treatment without N and 250 kg N/ha fertilizer (Fig. 17).
b
a
a
a
a
Figure 17. Maize-derived C of clay fraction in Tetto Frati. Each bar represents the
average of 3 field replicates, error bars represent standard deviation.
The same letters denote no significant difference between means
(P < 0.05) by Tukey’s multiple mean comparison test
For the Bottelare field experiment, the maize-derived C in the silt&clay
microaggregate fraction (Fig. 18) showed a similar trend as the sand fraction in
Tetto Frati. Maize-derived C was 0.23 g C/kg and 0.08 g C/kg of grain maize and
silage maize, respectively in the plots without N fertilizer application.. The same
applied to treatment with 150 kg N/ha fertilizer, the maize shoot-derived C was
even 0.25 g C/kg. In contrast with Tetto Frati (figure not shown), the maize rootderived C was larger than maize shoot-derived C and amounted 1.99 g C/kg for
wihout N fertilizer application while for 250 kg N fertilizer was 1.71 g C/kg.
There was significantly more occluded maize C in the light silt POM fraction in the
grain maize (MG) compared to silage maize (MS) in both different N fertilizer
application at Bottelare and Tetto Frati except the plot without N fertilizer
application at Tetto Frati (figure not shown). The contribution of maize root-derived
were found to be very low, thus it implies that there was more maize shoot-derived
C accumulation in the occluded light silt POM fraction.
45
d
c
bc
abc
ab
a
Figure 18. Maize-derived C of silt & clay microaggregate fraction in Bottelare
Each bar represents the average of 3 field replicates, error bars
represent standard deviation. Different letters denote no significant
difference between means (P < 0.05) by Tukey’s multiple mean
comparison test
4.4. Relative stability of shoot vs. root derived C
Estimates of C inputs from maize root vs shoot derived C are presented in Table
14. Grignani et al (2006) used a root and above-ground biomass ratio of 0.22 to
estimate the C input from maize roots. They estimated root C inputs of 1.7 t C/ha
for 0 kg N applied and 2.0 t C/ha for N-fertilized plots at Tetto Frati. DM stalks and
leaves production was 9 t DM for 0 kg N grain maize and 13 t DM for the N-fertilized
plots. With a C content of 0.45 kg/kg, this equals to 4.1 and 5.9 t C/ha inputs from
maize stalks and leaves. The total root and stalks+leaves C inputs can be simply
calculated by multiplying with the number of years maize grown (Table 14). Prior
to the onset of the Tetto Frati trial, the soil was cultivated with monoculture grain
maize, these C inputs for these 10 years are included in the root and shoot C inputs
estimates. As a remark it should be noted that root exudation was not included in
most estimates of root C inputs. Root exudation is extremely difficult to quantify
and is mostly estimated from DM production. Barber and Martin (1976) and Barber
(1979) suggested that root exudation, turnover and cell sloughing represent an
annual input of fresh root C into cropped soils equivalent to that of the measurable
root biomass. Therefore, we used 200% of the measured root biomass C as an
estimate of the total input of fresh root C into soils.
Overall, 2% to 25% of maize root-derived C was eventually incorporated into SOC.
These values varied within the fields and treatments. Often humification
coefficients of 0.2-0.35 are suggested for plant materials. Our calculations indicate
a lower stability of maize derived C, however these data are not entirely
comparable. A humification coefficient applies to stability of organic matter over 1
year, while the present study considered 12, 22 and 32 years. Tetto Frati had the
hig of maize root-derived C, followed by Bottelare, and then Puch. The crop
rotations with 0 kg N fertilizer treatment seems to be have higher maize root
46
derived-C compared to the other N fertilizer treatments. There is no effect of N
fertilizer application, in fact it could be a limitation. The maize root-derived C is
mostly accumulated in the sand and microaggregate silt&clay fraction.
Table 14. Estimates of Maize root and shoot C inputs (over the full duration of the
field experiments) relative to the quantified contents of root and shoot
derived C in the plough layer
Maize
root C
input
(t C / ha)
Maize
shoot C
input
(t C /
ha)a
Maize
root
derived
SOC
(t C /
ha)
Maize
shoot
derived
SOC
(t C /
ha)
Maize
root C
input/rootderived
SOC
Maize
shoot C
input/shootderived SOC
Bottelare
(12 y)
MG0
MS+IR0
MG150
MG+IR150
MS150
40.00
40.00
48.00
48.00
48.00
49.00
0.00
70.00
0.00
0.00
3.80
3.80
1.10
3.40
1.10
1.70
0.00
5.20
0.00
0.00
0.09
0.19
0.09
0.02
0.02
0.04
0.07
-
Tetto Frati
(22 y + 10 y GM)b
MG0
MS0
MS170
MG250
MS250
114.00
114.00
128.00
128.00
128.00
148.00
59.00
187.00
18.00
59.00
28.50
28.50
22.20
22.20
22.20
14.30
4.80
10.10
14.40
4.80
0.25
0.25
0.21
0.17
0.17
0.10
0.08
-
Puch
(32 y)
MS0
MS100
MS200
36.00
42.00
42.00
0.00
0.00
0.00
2.90
2.90
2.40
0.00
0.00
0.00
0.08
0.07
0.06
-
Crop rotation
-
a
root and shoot C inputs estimated based on DM production and root C input
data by Grignani et al. (2007)
- b accounting for the number of years with maize cultivated. Root exudation C
inputs was assumed to amount 100% of biomass, analogous to Rasse et al.
(2005)
Rasse et al. (2005) calculated a relative contribution factor of roots vs. shoots to
SOC, expressed as the ratio: (root-derived soil C/total root C input)/(shoot-derived
soil C/total shoot-C input). This ratio was 0.3 and 2.7 for the 0 kg N and 150 kg N
grain maize objects at Bottelare and 2.6 and 2.3 for the 0 kg N and 250 kg N objects
at Tetto Frati. A similar value of this relative contribution factor was obtained by
Rasse et al. (2005) of 2.4. This confirm that in the Tetto Frati and Bottelare sites
root biomass derived C is 2-3 times more stable in the soil compared to shoot
biomass and contributed most of the OC stored in the soil. This would support the
hypothesis that in fact soil C is mostly composed of root C. However the
contribution of root exudation compared to root biomass cannot be inferred from
the present study’s experimental data.
47
V. CONCLUSION
The main objectives of this research were to investigate if root-derived C is
preferentially accumulated in SOC relative to above-ground biomass derived C and
if it also ends up in physically protected OC. The effect of maize cultivar and
difference in soil texture on root maize biomass production as well as the stability
of maize root vs. shoot-derived C were also studied.
Maize cultivar had a lesser control while field site was proven to have a more
apparent effect on root DM biomass at three ILVO maize field trials. Based on the
result from other researches, drier sandy soil would be expected to provoke a more
expanded rooting system. However the opposite result was observed at Ravels
although it has a more sandy soil texture compared to the other sites. Possibly
limited available plant-water led to drought stress and if so perhaps general crop
growth was limited, including the root DM production. But this requires further
confirmation. Other factors like P-status of the sites or a direct textural effect as
well influence the root biomass and should be looked at as well.
Root-derived C was proven to be more stable than shoot-derived in two long-term
field experiments, viz. at Bottelare (Bel) and at Tetto Fratti (It). The contribution of
maize root-derived C to SOC was more than twice as large compared to shootderived C at both locations. Based on the relative contribution factor of roots vs.
shoots to SOC, the root-derived C is also 2-3 times more stable in the soil
compared to shoot biomass and contributed most of the OC stored in the soil at
these locations. This result is in line with recent literature on the effect of crop
residue incorporation on soil OC. More and more the consensus is growing that
root C matters much more in maintenance of SOC. The most current simulation
models to date, however, do not recognize the relatively much larger stability of
root-vs shoot derived C. This error is mainly due to the fact that it is difficult to
measure root C inputs, especially from root exudation. Elaborate field study of longterm effects of either root or shoot biomass incorporation are required to update
these models and such work cannot be replaced by any lab or pot experiment on
short or medium term.
Most of the maize root-derived C was found to be part of the silt&clay
microaggregates and sand fraction. Thus it implies that root-derived C did not
specifically accumulate in soil as physically protected particulate organic matter. In
fact at Puch, none of the maize root-derived C ended up in the occluded light siltsized POM fraction.
The location with the finest texture and also contained the most material of the
silt&clay microaggregates fraction was in fact Puch. This implies that supposedly
the content of microaggregates silt&clay including the non-occluded light OM and
occluded light silt-sized POM fractions was high and most of the maize-derived C
was accumulated in these fractions, mainly in the occluded light silt-sized POM
fraction. However, at Puch, the maize-derived C was least found in the occluded
light silt-sized POM fraction. So while a finer texture promotes the content of the
silt&clay microaggregates fraction, it did not promote incorporation of maize-root
derived C in particulate organic matter occluded within microaggregates. So even
in finer textured soils, there seems to be no preferential accumulation of maize root
derived C in the physically protected POM pool. Instead this root derived C ends
48
up partly in the sand fraction (coarse POM which is probably not protected to a
substantial degree) and in the silt and clay bound OM (sum of free clay and
silt&clay microaggregates fraction).
The result of this study finally led to general conclusion that maize root-derived C
is more stable than shoot-derived C but it is not always accumulated in soil as
physically protected C. This same research could also be repeated on the long
term in order to have an idea whether the physically protected status of this maize
derived C may change in time. It would also be very valuable to have sound
measures of the C-input by root exudation as a comparison to what was studied in
this research. Given the fact that not particulate OM contributed to accumulation of
root C, it seems realistic to hypothesize that instead C-input from root exudates
play a to date underestimated role in maintenance or build up of SOC.
49
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