Bachelor of Science

Agricultural Sciences

Jens Treffner

B.Sc. Thesis

Assessment of water and nitrogen

limitations to paddy performance

using stable isotopes

Supervisor:

Prof. Dr. Georg Cadisch

August 2008

University of Hohenheim

Institute of Plant Production and Agroecology in the Tropics

and Subtropics

Garbenstraße 13, 70599

Stuttgart, Germany

Abstract

i

Abstract Assessment of water and nitrogen limitations to paddy rice performance using

stable isotopes

The lowlands in north-west Vietnams Yen Chau district are mainly used for the cultivation of

paddy rice (Oryza sativa L.) for subsistence purposes. During the last decades the country‟s

agricultural sector has gone through major changes driven mainly by population growth. The

increase in agricultural productivity to match the people‟s needs was mainly achieved by the

clearance of upland forests for the cultivation of cash crops like maize (Zea Mays L.) and

cassava (Manihot esculenta Crantz). These procedures have a large impact through erosion

and nutrient fluxes downstream. Rice, the main food staple, is grown in flooded fields. De-

pending on the availability of rain and irrigation water, the rice paddies are cultivated either

once in the rainy season or twice, by supplemental irrigation water provided by an artificial

lake. The overall crop productivity of the paddies is hampered by water shortages, mainly

during the first season. In order to assess the impacts of seasonal conditions, fertilizers and

uplands-sediments on the lowlands crop, rice grains were analyzed. The method of Isotope

Ratio Mass Spectrometry (IRMS) was used to examine the composition of nitrogen and car-

bon stable isotopes 15N and 13C. Prior ecological research using this method permits conclu-

sions on the environmental conditions during plant growth. Analysis performed during this

study proved the hypothesis that the isotopic composition of rice grains shows clear relations

to growing conditions, particularly for 13C. The relative extents and proportions of the limita-

tions on crop performance caused by lack of nitrogen and water shortages could be de-

picted. In order to make the results given in this study and the used methodology universally

valid, further research and exacting sample selection and preparation is suggested.

List of Acronyms

ii

List of Acronyms and Abbreviations

‰ “per mille”, parts per thousand

12C stable carbon isotope, normal mass and 12 neutrons

13C stable carbon isotope, heavier with 13 neutrons

C3 Plants using the enzyme Ribulose-1,5-bisphosphat-Carboxylase/-

Oxygenase for carbon fixation; pathway

C4 Plants using the enzyme Phosphoenolpyruvat-Carboxylase for

carbon fixation; pathway

IRMS Isotope Ratio Mass Spectrometry

LAI Leaf Area Index

m meter

NUE Nitrogen Use Efficiency

RUBISCO Ribulose-1,5-Bisphosphate, carbon fixing enzyme

SPAD Soil Plant Analysis Development

SSA Specific Surface Area

WUE Water Use Efficiency

Author‟s declaration

iii

Author’s declaration

I, Jens Treffner (Matr. No.: 397603, University of Hohenheim) hereby affirm that I

have written this Bachelor of Science Thesis with the title

“Assessment of water and nitrogen limitations to paddy rice performance using sta-

ble isotopes”

independently and entirely by myself.

All authors mentioned or cited in this work and all sources have been cited. No work

has been included in this thesis without the authors being listed.

I further affirm that this Bachelor of Science Thesis was not submitted in the frame-

work of any other examination process.

Stuttgart, _____________________ Signature ____________________________

Acknowledgements

iv

Acknowledgements

I would like to thank my supervisor Prof. Dr. Georg Cadisch for giving me the opportunity to

perform this thesis work in the Institute of Plant Production in the Tropics and Subtropics.

Furthermore I would like to thank him for helpful advice during the time of my study.

I would also like to thank Dr. Thomas Hilger and Ph.D. student Petra Schmitter who both

patiently explained details about the study area, answered questions and provided data and

material. They also introduced me to the methods of scientific working and guided my way

through the whole study, for which I am very grateful.

More thanks is directed towards Stefan Becker-Fazekas. He patiently explained technical

details and technical devices and was an important help for my lab work.

Lastly, I would like to thank everybody in the Institute of Plant Production in the Tropics and

Subtropics for additional help, advice and their friendliness.

Index

v

Index

Abstract i

List of Acronyms and Abbreviations ii

Author’s declaration iii

Acknowledgements iv

Index v

LIST OF FIGURES VII

LIST OF TABLES VIII

A. Introduction 1

I. AN INSIGHT INTO NORTH-WEST VIETNAM’S AGRICULTURE 1

II. AIM OF THE STUDY 2

B. Literature Review 3

I. DEFICIENCIES OF NUTRIENTS IN PLANT PRODUCTION 3

II. LOWLAND RICE PADDIES, SOILS, WATER AND NUTRIENTS 4

III. STABLE ISOTOPES IN PLANT RESEARCH 6

1. Carbon isotopes 6

a) Theoretical considerations and denotations on 13C 6

b) Physiological and physical processes leading to carbon isotope fractionation 7

(1) Fractionation of C isotopes by carbon-fixing enzymes 8

(2) Fractionation by diffusion 8

(3) Fractionation as a result of environmental conditions 10

c) The use of 13C as an indicator for water use efficiency and competition in

agriculture 10

2. Nitrogen isotopes 12

a) Processes leading to nitrogen isotope fractionation 12

b) Environmental impacts on 15N 12

IV. REVIEW OF RESULTS AND CONCLUSIONS FROM FORMER RESEARCH 13

Index

vi

C. Material and Methods 15

I. DETAILS ABOUT THE STUDY AREA 15

II. AGRICULTURAL PRACTICES AND IRRIGATION 16

III. DEVELOPMENT OF CRITERIA FOR SAMPLE SELECTION 18

IV. SAMPLE PREPARATION FOR IRMS 20

V. FUNCTION OF MASS SPECTROMETERS 20

D. Results and Discussion 21

I. DISTRIBUTION OF GRAIN YIELD 21

1. Yield along the cascades BM1 and BP5 in the first season 21

2. Yield along the cascades BM0, BM1 and BP5 in the second season 22

II. RESULTS AND DISCUSSION OF THE IRMS – CARBON 23

1. Discrimination for 13C along the cascades in the first season 23

a) Graphical display by selection 23

b) Graphical display by fertigation 25

2. Discrimination for 13C along the cascades in the second season 27

a) Graphical display by selection 27

b) Graphical display by fertigation 30

III. RESULTS AND DISCUSSION OF THE IRMS – NITROGEN 31

1. Grain-Nitrogen and 15N, first season 31

2. Grain-Nitrogen and 15N, second season 33

IV. SUMMARY OF RESULTS AND DISCUSSION 35

V. Conclusion 39

Bibliography ix

Annex 1 xiv

Annex 2 xvi

List of figures

vii

LIST OF FIGURES

Figure 1: a) geographic map of Vietnam showing the Son La province; b) map of Son La

province with main roads and water bodies; c) map of the Yen Chau district, depicting the

sub-catchment of Chieng Khoi commune and main roads and water bodies (Source: (Hertel,

2007))………………………………………………………………………………. ....................... 15

Figure 2: a) Average precipitation and temperature of Yen Chau city recorded from 2000

until 2006; b) Precipitation and temperature during the research period in 2007; ............... 16

Figure 3: a) Erosion sediments in a paddy field after heavy storm event in the beginning of

the second season; b) Example of uplands cultivation with steep slopes and a settlement

with homegarden (Source: Schmitter) ................................................................................. 17

Figure 4: Top-down view of lower BM0 (bare soil) and BM1 cascade during first season 2007

(Source: Schmitter) ............................................................................................................. 18

Figure 5: Bottom-top view of BP5 cascade during the first season in 2007 (Source:

Schmitter) ........................................................................................................................... 18

Figure 6: Schematic draft of the cascades BM0, BM1 and BP5. ......................................... 19

Figure 7: Yield in Ban Me 1 cascade, first season ............................................................... 21

Figure 8: Yield in Ban Put cascade, first season ................................................................. 21

Figure 9: Yield in Ban Me 0 cascade, second season ......................................................... 22

Figure 10: Yield in Ban Me 1 cascade, second season ....................................................... 22

Figure 11: Yield in Ban Put 5 cascade, second season ....................................................... 22

Figure 12: Selection BM1, unfertilized section marked by empty symbols, fertilized by filled

symbols............................................................................................................................... 23

Figure 13: Selection BP5, unfertilized section marked by empty symbols, fertilized by filled

symbols............................................................................................................................... 23

Figure 14: Unfertilized sections, circles for BM1 and triangles for BP5 ................................ 25

Figure 15: Fertilized sections, circles for BM1 and triangles for BP5 ................................... 25

Figure 16: Selection BM0, unfertilized section marked by empty symbols, fertilized by filled

symbols............................................................................................................................... 27

Figure 17: Selection BM1, unfertilized section marked by empty symbols, fertilized by filled

symbols............................................................................................................................... 28

Figure 18: Selection BP5, unfertilized section marked by empty symbols, fertilized by filled

symbols............................................................................................................................... 29

Figure 19: Unfertilized sections; diamonds for BM0, circles for BM1 and triangles for BP5 . 30

Figure 20: Fertilized sections; diamonds for BM0, circles for BM1 and triangles for BP5 ..... 30

Figure 21: Discrimination of 15N and distribution of grain-nitrogen along the cascade Ban Me

1;......................................................................................................................................... 31

Figure 22: Discrimination of 15N and distribution of grain-nitrogen along the cascade Ban Put

5;......................................................................................................................................... 32

Figure 23: Discrimination of 15N and distribution of grain-nitrogen along the cascade Ban Me

0;......................................................................................................................................... 33

Figure 24: Discrimination of 15N and distribution of grain-nitrogen along the cascade Ban Me

1;......................................................................................................................................... 33

Figure 25: Discrimination of 15N and distribution of grain-nitrogen along the cascade Ban Put

5;......................................................................................................................................... 34

List of tables

viii

LIST OF TABLES

Table 1: IRMS results for the first season; isotope values are corrected for blank samples . xiv

Table 2: IRMS results for the second season; isotope values are corrected for blank samples

............................................................................................................................................ xv

Table 3: Results of correlation analysis, first season ........................................................... xvi

Table 4: Results of correlation analysis, second season .................................................... xvii

Introduction

1

A. Introduction

I. An insight into north-west Vietnam’s agriculture

Through the last decades, the agricultural sector of the Socialist Republic of Vietnam has

gone through major changes. With a population growth of over 300 % between 1960 and

1984 and a further doubling until today, the available arable land per person dropped to only

0.2 ha by 1998 and is projected to decline even more. As an effect, the formerly forested

steep hillsides that constitute a big portion of the country‟s surface have been cleared for

agricultural use. Today the share of forested land is only around 17%, compared to 95% in

1943 (Wezel, 2002). Rice is one of the most important agricultural products in Southeast

Asia not only for personal consumption but also for export purposes. Cultivation of paddy

rice (Oryza sativa,L.) occurs in the lowland„s floodplains where the crop is often irrigated or

on rice terraces in the uplands which are rainfed. The lowlands of the two investigated

villages in the north-western district of Yen Chau, Ban Me and Ban Put are mainly character-

ised by irrigated paddy rice (Oryza sativa,L.) production and home gardens for subsistence

purposes. The slopes are cultivated with upland crops like corn (Zea mays L.) and cassava

(Manihot esculenta Crantz) for the generation of income. In the past, slash-and-burn prac-

tices for the clearance of primary forest were very common. After several years of cultivation

leading to a decline in soil fertility, the plots are left fallow, a secondary forest establishes. As

an effect the soil fertility re-establishes by the accumulation of organic matter and nutrients in

the secondary forest. However population growth and socio economical changes result in

land scarcity and a decrease in fallow periods. The region‟s climate is characterised by rela-

tively dry and cold winters and summer monsoons (Le Ba Thao, 1997, cited in Wezel, A. et.

al. 2002). The soils in the area can mainly be classified as Acrisols in the uplands and Gley-

sols in the lowlands, according to the WRB (Deckers et al. 2006). They developed on lime-

stone and schist (Bui Huy Hien et al., 1995, cited in Wezel, A. et. al., 2002). The bare soil is

highly susceptible for erosion, especially during heavy rainfall events in the beginning of the

rainy season, when the crops in the upland fields are freshly planted and do not cover the

soil sufficiently. As an effect, both upland and lowland agriculture suffer from the impacts of

erosion. The already shallow top-soil layer is washed away from the slopes, whilst in the

lowlands irrigation channels and reservoirs are silted and just yet transplanted rice plants

can be buried under the eroded material. The decline in soil fertility and the upcoming land

scarcity will cause a significant pressure on agricultural land which will in the long term lead

to a significant decrease in yield in the uplands as well as in the lowland. Already, environ-

mental stability dropped by the clearing of primary and secondary forests, thereby eliminat-

ing the important environmental services of flood and rain control (Wezel, 2002).

Introduction

2

II. Aim of the study

The research performed by Hertel in 2007 during the first cropping season revealed high

spatial variability of the performance of the rice crop in the lowland terraces. The measured

parameters constitute the basis of this thesis work. In addition, data from the second crop-

ping season will be compared.

The aim of this study is to examine plant responses on environmental conditions and

changes, such as water and nutrient stress by examining the composition of stable isotopes

(13C and 15N) of the rice grains. The focus will be on the use of isotopes for exploring and

understanding the spatial variability of crop performance in the lowland in a dry and a rainy

season. A wide range of data such as crop performance, soil parameters and spatial

data for the two seasons were provided by Hertel (Hertel, 2007) and Schmitter (Schmitter,

2008) and will be used in correlation with the isotope findings. The hypothesis of this study is

that one can infer from the isotopic composition of the paddy rice grains the role of water and

nitrogen as limiting plant resources on the crops‟ performance. By the help of IRMS, in-

sights shall be gained on the competition for nutrients and water in this specific agro ecosys-

tem of paddy fields and cascades.

Literature Review

3

B.Literature Review

I. Deficiencies of nutrients in plant production

All plants depend on the uptake of mineral nutrients. In agriculture, one generally considers

nitrogen (N), phosphorous (P) and potassium (K) as the three major plant nutrients. These

elements are usually incorporated by the help of enzymatic incorporation after transport from

the soil via the root system into the single plant compartments. Two other important ele-

ments, water and carbon dioxide are as vital for plant life like the former three. Carbon diox-

ide is the source for carbon uptake and metabolism via the leaves, whereas water is an es-

sential mediator for all processes in plants. The major part of the water taken up by the plant

roots is lost again through transpiration and only a small fraction belongs to the final fresh

matter of the whole plant. But water not only constitutes an agent for transportation of photo-

synthetic compounds like sugars or plant hormones for signalling purposes, it is also a

transporter of nutrients in the soil, mediating nutrient fluxes through mass-flow and diffusion

(Larcher, 2003). Therefore it also affects the availability of mineral nutrients like N, P and K.

Plants are connected to their environment through the so-called soil-plant-atmosphere-

complex (SPAC), which is the main driver for water uptake by roots and subsequent transpi-

ration. Carbon dioxide uses the same pathway to enter the plant as the water exits, the leaf‟s

stoma. The uptake and loss of the particular two elements is mediated by plant regulators

such as abscisic acid and ethylene, which regulate stomatal aperture and closure

(Wilkinson, 2004). Apparently, the plant has to deal with a trade-off, sometimes even a

vital gamble between carbon assimilation which means growth and water loss that could

lead to desiccation (Chaves, 2004). The plant‟s growth and total water and carbon

balance interacts with the availability of mineral nutrients: Phosphorous is in particular known

for the enhancement of root-growth, thereby increasing the rooting depth and the availability

of other nutrients (Lawlor, 2002). Nitrogen is even more essential and limits plant growth in

most environments. Constituting a big proportion of the leaf enzymes like RUBISCO in C3

plants, the increase of availability of nitrogen for the roots by fertilization with either organic

fertiliser like manure or mineral fertilizers like urea usually increases a plant‟s performance in

growth. This is the main reason for higher biomass accumulation compared to unfertilized

plants and, if not hampered by other stresses, results in higher agricultural yields. The root

can take up mineral nitrogen as nitrate and ammonia, depending on the soil-water status, pH

and microbial community as well as the time of the year. Furthermore, nitrogen can also be

incorporated as organic N-containing compounds such as amino-acids (van Groeningen,

2002). Better N-supply is manifested not only in higher total biomass, but is reflected

by parameters like the leaf nitrogen content (Xue, 2004) and a bigger proportion of plant

Literature Review

4

organs, above all leaves, that contribute to the assimilation of carbon. This parameter is de-

scribed by the leaf area index LAI, which indicates the proportion of a plant stand‟s leaf area

in relation to the ground area it covers (Jing, 2007). If plants suffer water stress, they

need to regulate the aperture of their stoma, which in turn decreases the uptake of carbon

dioxide (at least in the major ~ 90 % of terrestrial plants using the C3 pathway to assimilate

carbon). Another interaction, or rather co-limitation occurs with the scarcity of mineral

nutrients, e.g. lack of nitrogen: As the uptake of nitrate, or ammonia, mainly de-

pends on diffusion to the plant root, low soil water content can lead to nitrogen starvation,

even if the plot received sufficient fertilizer. On the other hand, if water is not primarily limit-

ing, the main cause for stagnation in growth is lack of nitrogen (Hooper, 1999). Nutritional

and environmental stresses limiting plant growth are a very complex issue, the author will

not go into further detail here, but recommend the textbook on physiological plant ecology

(Larcher, 2003). Furthermore it should be pointed out that the aforementioned issues

are a major obstacle to many agricultural production systems and limit the yield in many

parts of the world (Lawlor, 2002).

II. Lowland rice paddies, soils, water and nutrients

Rice (Oryza sativa L.) constitutes, besides winter wheat (Triticum aestivum L.) and corn (Zea

mays L.) one of the world‟s main staples. Cultivation of lowland rice takes place either under

irrigated conditions (79 million ha of land) or under rainfed conditions (54 million ha) fore-

most in Asia and accounts for more than 80% of the world‟s supply with rice (Bouman,

2007). The cultivation practices are numerous around Asia; the most important common

condition is the anoxic soil properties influencing the whole production system. Soils in low-

land rice paddies usually become puddled before the rice seedlings are transplanted from

the nurseries. This procedure reduces the conductivity of pores and enhances a field‟s water

retention capacity (Buresh, 1993). During the rainy season rice is the only crop that can be

grown in many areas due to heavy rainfalls that submerge the paddies (or flood normal fields

with the effect of crop failure). Therefore rice cultivation also has a big environmental impact

(Wade, 1998). Rice is physiologically adapted to these conditions. An important fea-

ture of puddled and flooded or at least water-saturated soil is the nitrogen cycle. Differing

from aerobic (containing O2 to at least 7 Vol-% compared to 21 Vol-% in atmospheric air)

soils, the plant-available nitrogen exists as ammonia instead of nitrate (Brady, 2007), which

affects the uptake pathway and efficiency for the plant (Guo, 2006). Especially in rainfed

paddies, the crop may face severe environmental changes during one season, with soil

properties ranging from aerobic to water-saturated, thereby influencing the soil-N status from

(aerobic) nitrate to (anaerobic) ammonia (Wade, 1998). What is important for the overall

productivity and the efficient use of (fertilizer) nitrogen is the loss of the latter. During aerobic

periods before flooding, nitrate accumulates and can be lost by denitrification or leaching if,

Literature Review

5

as often the case, no catch crop is cultivated. With incipient strong precipitation it will leach

to lower-lying fields and finally to the groundwater (Buresh, 1993). Although rice plants in

their younger stage seem to prefer ammonia nutrition (Guo, 2006), it is reported that the

most efficient rice cultivars in terms of nitrogen-use showing the highest yields, growth per-

formance and N-translocation within the plant are those with a mixed ammonia / nitrate nutri-

tion (Li, 2008). During waterlogged conditions, the pH of the paddy soil solution is

fluctuating around 7, independent from the original soil pH under aerobic conditions. In many

Asian rice soils the toxicity of iron has an adverse effect on plant performance, the same

accounts for the concomitant fixation of phosphorous in Fe-phosphates (Prof. Dr. Thorsten

Müller, lecture notes on the principles of soil chemistry, University of Hohenheim, 2007). The

water-saturated soil is the reason for gaseous losses of ammonia-N by volatilization as NH3

and N2 and the major greenhouse gas N2O. It is as well a major contributor to the atmos-

phere‟s methane (CH4) content by anaerobic respiration of soil microorganisms (Paul, 2006).

The rainfed lowland rice systems depend on sufficient rain during the cropping seasons and

constitute a reservoir for excessive rains during the monsoon, so do the irrigated paddies.

Rainfed lowlands have always been more susceptible to intermittent drought periods,

whereas irrigated lowlands usually do not suffer water shortages (Haefele, 2006). With an

ever growing demand for food staples in Asia and climate change affecting the global water

cycle, water becomes scarcer and its distribution is, also due to inadequate management as

well as erratic rainfalls, constraining lowland rice productivity. Resulting from these unde-

sired conditions, soil nutrient fluxes are altered and influencing upland and lowland produc-

tivity, in particular rice production. Rice may exhibit a rather low efficiency in terms of water

use, compared to other C3 crops adapted to upland or dry conditions (von Caemmerer,

1991). In line with the changing environments for rice plants that often occur during one

cropping season goes the change in soil acidity which might, in turn, amplify Fe-toxicity ef-

fects (Sahrawat, 2004). The incidence of water scarcity does not primarily lead to drought

and, in the worst case desiccation. Even if there is still plant-available water in the bulk soil,

the change in aeration leads to the aforementioned shifts in soil acidity. This can also lead to

a decrease in P-availability and subsequent bad harvest by soil-chemical obstacles as re-

ported in a review by Fukai et al, where several studies dealing with drought-prone rainfed

lowland paddies were compared and different mechanisms resulting in low yields were re-

vealed (Fukai, 1999). There is a co-limitation effect between the major resources

water and nitrogen. This effect may change the distribution patterns of nutrients within a

plant as well as the concomitant resource-use efficiencies like water use efficiency, WUE

and nitrogen use efficiency, NUE (Lawlor, 2002). Besides the effects of N-form on the nutri-

tional status of rice plants, there are general circumstances to be considered: Independent of

the N-form, sufficient supply of nitrogen is only possible with sufficient water for the diffu-

sional transport from the bulk soil to the rhizosphere. Water stress in general reduces the N-

Literature Review

6

uptake as a consequence of reduced transpiration in most plants. In turn, when N is suffi-

ciently available throughout the cropping season, even a drought period can be mitigated in

terms of grain yield. High biomass and thus a high LAI serve as a reservoir for assimilates

that can be re-allocated within the plant to be incorporated into storage proteins and starch

in the grain (Prastersak, 1997). These interactions might vary between cultivars, the magni-

tude to which drought occurred during the trials and of course the timing.

III. Stable isotopes in plant research

The use of stable isotopes as integrators for the understanding of interactions between ele-

ments, plants, their environment and even on an ecosystem-scale for tracing food-webs has

been growing fast during the last two decades (Dawson, 2002). Basic work has helped to

understand the nature and behaviour of stable isotopes in biological systems and also gave

insight to the different pathways in which plants manage to acquire nutrients for growth and

metabolism, the most important ones being C3, C4 and CAM-metabolism of terrestrial plants

(Ehleringer, 1989). Researchers either use the method of natural abundance levels, where a

sample‟s isotope proportion is related to a standard, e.g. the ∂ 13C value of the PeeDee for-

mation, termed PDB, a carbonaceous belemnite rock as a reference for carbon isotope frac-

tionation studies (Dawson, 2002), following Equation 1 on this page. The other method

is the use of enriched material like 15N in significantly higher levels of isotopic composition

than anywhere else in natural compartments. This labelled material can then be used as a

tracer-signal to track and separate the pathways of an element in the environment.

1. Carbon isotopes

a) Theoretical considerations and denotations on 13C

For the purpose of investigating the effects of water scarcity and nutrient starvation in higher

plants, the biological partitioning of the stable 13C isotope using natural abundance levels

has been examined intensively. The fractionation of the two stable isotopes 13C and 12C in

plants today also constitutes an important trait in plant breeding programs for the determina-

tion of a crops‟ water use efficiency, WUE (Farquhar, 1989). The isotopic carbon composi-

tion of a plant sample, ∂13C is specified as (Farquhar, 1982):

Eq. 1,

where Rsample and Rstandard denote the composition ratio of 13C/12C in the sample or in the

standard, respectively (Dawson, 2002). This expression is analogue for every chemical ele-

ment in natural abundance studies. It is important to note here that isotopic 13C/12C composi-

tions (∂) of plants always show negative values. In contrary, the process of isotopic dis-

Literature Review

7

crimination, ∆ is per definition positive. The expression ∆ is important for the separation of

biochemical from physical factors and for the identification of single depletion or enrichment

steps. It is calculated following this equation (Farquhar, 1982):

Eq. 2

Where in the case of plant science studies examining the uptake and fractionation of stable

carbon isotopes, Rproduct and Rsource stand for the abundance ratios of 13C/12C in the exam-

ined compartment, e.g. intracellular space, phloem sap or whole-leaf carbon. The source in

the case of primary carbon-fixation would be the CO2 within the leaf. The product is the first

enzyme involved into fixation, RuBP. Using the definition from Equation 1, the net fractiona-

tion process denoted as ∆ can be described as follows (Farquhar, 1982):

Eq. 3.

The term ∆ refers to the discrimination against a given molecule, e.g. 13CO2, which leads to a

depletion in the 13C content of a sample and thus to lower ∂ values with the transfer from one

compartment to another or a medium like air or water. In contrast to ∂, ∆ is independent of

the isotopic composition of a standard or the atmosphere (Farquhar, 1989b) and shows posi-

tive values. The 13C/12C ratio of air (with respect to the PDB standard) is ~8 ‰, meaning that

normal air contains 8 parts per thousand (“per mil”) of 13CO2 less than the reference V-PDB.

For plant material, there are distinct ranges for the ∂ of the relevant plant species: C3 spe-

cies show ∂ values between -22 and -38 ‰ (mean value ~27 ‰), C4 plants are less depleted

with ∂ values ranging from -8 to -15 (Monneveux, 2007) and CAM plants as intermediaries

also show intermediate ∂ 13C values (Dawson, 2002). The whole process of discrimination of

13C along the C3 carbon fixation pathway from the ambient air would thus, according to

Equation 3 result in a ∆ = ≈ 19 ‰.

In this study the ∂ notation will be used, as the single discrimination steps against 13C are not

the main topic here. Furthermore, the ∂ 13C value of the ambient air in Yen Chau district

would have to be estimated. This is inconvenient as fluctuations in the CO2 content and the

isotopic composition in one specific region may vary with season (Trolier, 1996).

b) Physiological and physical processes leading to carbon isotope

fractionation

The different ranges of ∂13C values are related with interactions between a plant and its envi-

ronment and thus can be used as integrators of the circumstances under which a plant is

Literature Review

8

growing or has been grown. The ∂ of a given plant tissue sample (samples can also derive

from rocks or animal tissue, here the focus is on plants) is a result of physiological - chemical

processes within a plant compartment (Gillon, 1998). The same applies for physical proc-

esses in and in close proximity of the plant, e.g. diffusion processes in and out of the leaf

affected by the leave‟s conductivity and the partial pressure of CO2 (Chaves, 2004). The

total discrimination is the result of two interconnected processes; the first one is the discrimi-

nation against 13CO2 by the acceptors and enzymes involved in the process of carboxylation

(the “initial” step of carbon incorporation and dry matter production). The second are the

physical traits of plant architecture which, through altered stomata conductance and bundle

sheath cells can promote primary carbon fixation.

(1) Fractionation of C isotopes by carbon-fixing enzymes

Photosynthetic discrimination that leads to a depletion in the plant‟s 13C content and thus to

a higher ∆ or a more negative ∂ value has been an indicator in observations that lead to the

identification of the different carbon-fixation pathways (Ehleringer, 1989). From the 1950s

until today, scientists examined many single steps that lead to the so-called net discrimina-

tion. The major process accounting for the bulk of ∆ in C3 plants is the fixation by Ribulose-

1,5-bisphosphate and the subsequent carboxylation by RUBISCO (Larcher, 2003). The iso-

tope effect with respect to the carboxylation reaction lies in the different molar mass of the

13C and thus altered behaviour in enzymatic reactions and bond-forming (Farquhar, 1989b).

The heavier 13C isotope is gradually enriched in the source medium due to lower

reactivity – the “normal” or lighter isotope forms bonds that are easily broken and is thus

more likely to be concentrated in the product (Dawson, 2002). The in-vitro discrimination

(described as b in Equation 4, next page) of enzymatic effects in C3 plants is in general from

27 ‰ (Dawson, 2002) to 28 ‰ (Larcher, 2003) with some deviations due to the fact that

13CO2 is discriminated by the diffusion of dissolved CO2 through water in the mesophyll cells

on its way to the chloroplast‟s stroma (Larcher, 2003). Another factor is the small degree to

which even C3 plants fix carbon by the enzyme PEP-carboxylase, originally being the carbon

fixing enzyme of C4 plants, which could account for up to 5 ‰ of the net discrimination

(Gillon, 1998).

(2) Fractionation by diffusion

The process of discrimination is not only related to fractionation during enzymatic pathways,

but strongly affected by the partial pressure of CO2 inside and outside (ambient air) of the

leaf and this, denoted as pi and pa, respectively, again by the fractionation during diffusion

through the stomata, described as a. The discrimination by diffusion in free air is consistently

reported to have a ∆ of 4,4 ‰ (Gillon, 1998). What can be derived from Equations 1 to 3 is

the use of 13C isotopic discrimination as an estimator for water stress: Under conditions

where plants are sufficiently supplied with water, the stomata are open and thus stomatal

Literature Review

9

conductance is high. Under these conditions RUBISCO preferentially fixes 12CO2 and the

fixed carbon becomes depleted in 13CO2 (Clay, 2001). With increasing water stress, the

leaves‟ stomata are closed to reduce transpirational water loss (Larcher, 2003) and therefore

more 13C is used during enzymatic fixation due to a limited supply through the stoma

(Farquhar, 1993). As already mentioned, the net discrimination against 13C in plant tissues is

the result of different steps, such as enzymatic fractionation, discrimination through lower

diffusivity in air (Berry, 1989) and the chemical framework at the site of carbon fixation, such

as water vapour pressure and pH (Farquhar, 1989b). A theoretical expression with respect

to the partial pressures of CO2 to describe the whole discrimination process for C3 plants

photosynthesis is

Eq. 4,

with a denoting the fractionation during diffusion in the ambient air and the sub-stomatal cav-

ity, respectively (Gillon, 1998), b for the assumed net fractionation during enzymatic carbon

fixation, mainly by RUBISCO, pi and pa the partial pressures inside the leaf and in the ambi-

ent air of CO2. The latter two could also be exchanged with Ci and Ca, the concentrations of

CO2 in a given medium (Brugnoli, 1998). The most important thing to point out here is the

use of Equation 4 (with respect to Equation 1 and Equation 3 and the conclusions drawn

there) as a predictor for C3 plants: with increasing water stress, related to a lower pi/pa ratio,

∆ will decrease (or, using the ∂ notation, the value becomes less negative) (Clay, 2001).

In the single steps during net fractionation the properties of cells as conductors of

CO2 should also be taken into account: Additional resistances occur during the transfer

through the tortuous intercellular spaces (Gillon, 1998). During the transport and the dissolu-

tion in the aqueous media of the chloroplast cell, the ratio of the partial pressure in the

chloroplast to the ambient air concentration drops to (von Caemmerer, 1991).

Finally, the discrimination can be expressed as a result of CO2 being also a limiting factor for

plant growth due to low internal partial pressure:

Eq.5,

where A denotes the molar CO2 flux from the atmosphere into the plant, g is the conduc-

tance of the pores and the stomatal boundary layer, pa and pi denote the partial pressure or

concentrations in the ambient air and in the intercellular spaces, respectively and P stands

for the atmospheric pressure (Farquhar, 1982).

Literature Review

10

(3) Fractionation as a result of environmental conditions

The net composition of 13C/12C in a plant tissue depends on numerous processes, that each

can be affected by environmental and genetic variations and the plant species (Brugnoli,

1998). Concerning environmental factors that alter the isotopic composition of plants and

their single compartments, the different media and connecting parameters that are important

in plant life and growth have to be considered.

Hindering leaf conductance and photosynthesis, the exposure to polluted air is reported to

have a decrease in ∆ (Farquhar, 1989b). Important to note is the atmospheric deposition,

especially near intensive livestock feedlots, of atmospheric ammonia on plant canopies and

the uptake by stems and leafs: By alteration of the acid-base balance inside the leave, NH3

affects the enzymatic activity (Yin, 1998). Although difficult to separate from correlating

effects (Farquhar, 1989b), carbon isotope discrimination is also affected by irradiance. The

partial pressure within the leaf depends on stoma conductivity, which again depends on

stoma closure mediated by irradiance and light spectrum (Larcher, 2003). A second

environmental condition that leads to a strong decrease in carbon isotope partitioning due to

stomata closure is salinity, which impacts on the whole plant-water balance (Farquhar,

1989b). Although carbon isotope discrimination has been used as a tool for water

stress assessment in numerous different plants, it has never become a general index for

plant-water relations (Bacon, 2004). Discrimination is, via the pi/pa ratio, also influenced by

any other factor that influences the stomatal behaviour, e.g. nutrient supply, soil compaction

or plant diseases (Clay, 2001). For use in the agricultural context, the effects of nitrogen

are of special interest: Yin and Raven (Yin, 1998) reported that nitrate as the single N-form

available to C3 Triticum aestivum L. plants caused a significantly lower water use efficiency

and thus higher ∆ values for 13C than did ammonia. It can be derived from these studies that

during the interpretation of 13C readings the effect of soil-N status always has to be consid-

ered, as enzymatic discrimination is co-impacted by N-availability and thus cannot always be

seen in line with just the CO2 uptake of the leaf.

c) The use of 13C as an indicator for water use efficiency and compe-

tition in agriculture

The best-examined environmental factor leading to deviations in natural carbon fractionation

patterns is by far the effect of water stress and the relation between assimilation and transpi-

ration, the water use efficiency, WUE. This classic (the efficiency of transpiration, first used

by Maximov in 1929 (Jones, 2004)) ratio, defined either as the instantaneous water use effi-

ciency on the leaf level (A/E, assimilation by evaporative loss, calculated in µmol CO2 mol-1

H2O) or as the integrated or season-long WUE at the whole plant level (calculated as the

Yield of dry mass to the evaporative loss of water over the same time) has become an im-

portant property in breeding crops for dry regions, e.g. Drysdale Wheat (Bacon, 2004).

Literature Review

11

As described before, the discrimination of 13C and its deviations from reference val-

ues depends on plant specific metabolism on the one hand and on the environmental condi-

tions impacting stomata aperture or closure on the other hand (pi/pa ). Therefore, the ∂ 13C

value of a plant enables us to approximate estimations of the net pi/pa in the plant over a

certain growth period or the whole life-cycle (Farquhar, 1989). Referring to the expressions

from Equation 5, we can express the ratio between carbon assimilation A and transpiration E

(or evaporative “loss”, because the water used for assimilation is lost to the atmosphere) as

Eq. 6,

where g denotes the conductivity of carbon dioxide and water respectively, p stands for the

partial pressures of CO2 in the atmosphere and inside the leaf, e for the intercellular and the

ambient vapour pressure and v stands for the difference between vapour pressure inside

and outside of the leaf (Farquhar, 1989b). An approach to describe water use efficiency on

the whole plant level, integrating environmental and plant physiological responses we obtain:

Eq. 7,

with W standing for water use efficiency and denoting the respired proportion of carbon

fixed by day but lost by night through dark respiration and respiration of other plant com-

partments over the whole period (Farquhar, 1989). Equation 6 and Equation 7 describe the

dependency of water vapour loss on the regulation of the partial pressure of carbon dioxide.

They furthermore show the basis for the higher competitiveness of plants using C4-

PEP-carboxylase-pathway, as these plants do not depend on the constant stomatal conduc-

tance for carbon fixation as C3 plants do (Chaves, 2004). The stomatal diffusion pathway,

shared by carbon dioxide and water vapour alike is the main reason for transpirational loss

of water, as the driving gradient v is about 50 times higher than that for the influx of CO2

(Chaves, 2004). It should be pointed out here that although WUE has become an im-

portant trait for breeding dry-area crops, the efficiency exhibited by these plants (to gain

more carbon per unit of water transpired) constitutes a trade-off (Blum, 2005). Especially

under conditions where drought occurs only slightly, crops exhibiting a lower WUE might

obtain a higher yield. In the case of natural ecosystems, plants that risk the gamble between

carbon gain and water efficiency in favour of water might as well be suppressed by their

competitors (Chaves, 2004). The usefulness of 13C patterns for plant breeding shows in the

relationship between the ∂ value and the stomatal conductance: Cultivars having less nega-

tive ∂ values will more likely close their stoma quicker under dry conditions. Thus, combining

the classic traits such as yield or disease resistance with isotopic analysis can help to iden-

tify the breeding lines for harsh conditions (Gregory, 2004). Moreover, there is evi-

Literature Review

12

dence that the carbon-isotope fractionation is also related with nitrogen nutritional conditions.

The photosynthetic capacity is co-limited by the availability of nitrogen for the enzymatic

processes (Kaewpradit, et al., 2008). Therefore, a lower nitrogen supply can impact photo-

synthetic carbon-fixation, resulting in stomata closure and subsequent 13C enrichment in the

plant tissue. This present study is yet another effort to assess limitation effects on crop

development and by this trying to relate the agro ecological conditions to the overall per-

formance. Other studies, like from Pansak et al (2007) showed the possibility of IRMS as a

method to also reveal competitions for resources between different plant species (Pansak,

2007).

2. Nitrogen isotopes

a) Processes leading to nitrogen isotope fractionation

In contrary to carbon dioxide as a plant nutrient, nitrogen enters a plant mainly through the

roots. An exception in this respect is the uptake of atmospheric ammonia, NH3 by the leaves

or stems directly. This pathway is reported to have significant effects on the inner-foliar en-

zymatic activity as the pH in the leaves increases (Yin, 1998) and has a negative impact on

plant growth. The discrimination of the stable isotope 15N is affected by internal partitioning

effects in the plant metabolism as well as by external effects. The latter can show wide varia-

tion: Prior partitioning in soil organisms, as well as plant residues having their own distinct

isotope pattern may make an interpretation difficult (van Groeningen, 2002), at least in stud-

ies working with natural abundance levels of stable nitrogen. However, in recent years

studies have been conducted to understand fractionation processes within the plant follow-

ing the uptake of nitrogen (Evans, 2001). Some studies were also able to relate artificial

laboratory conditions to possible environmental processes. Having a look at intra-plant frac-

tionation processes, the three steps of nitrogen assimilation, uptake, translocation and final

assimilation need to be separately studied in detail. As final assimilation is mediated by en-

zymes, such as nitrate reductase, the 15N signal again depends on the availability of photo-

synthetic energy and water, e.g. the activity of nitrate reductase declines by 20% after only

small water shortages and decreases by up to 50% after longer drought periods (Larcher,

2003). The major enzymes involved in nitrogen assimilation, nitrate reductase and glutamine

synthetase both discriminate against 15N. As nitrogen uptake by the roots is mediated by

high- and low-affinity transporters alike, the external N-concentration seems to have an in-

fluence, but not necessarily the highest (Evans, 2001).

b) Environmental impacts on 15N

There are studies which report a positive correlation between the proportional nitrogen con-

tent (N %) in wheat grains with ∂ 15N (Araus, 2008), although these values both are affected

by N-availability and water stress and therefore affected by intersecting environmental condi-

Literature Review

13

tions. In a study by Yoneyama et al (1991) dealing with rice nutritional effects, ammonia only

against organic nitrogen fertilizers, 15N discrimination in rice seemed to be affected by the

occurrence of ammonia or nitrate: ∂ 15N values in the whole plants showed a significant de-

pletion, increasing with the nutrient solutions‟ ammonia concentration. Therefore, the flux of

ammonia through cell membranes may be the step at which fractionation of nitrogen iso-

topes occurs. Apparently, the smaller size of ammonia molecules may provide a greater op-

portunity for isotopic discrimination than the larger molecules of nitrate. The fractionation

during uptake of nitrate as the only N-form available was considerably lower. Contradictory

to the depletion of 15N in plant samples, the 15N content of grains that were measured in this

context showed enrichment (Yoneyama, 1991). A study assessing the effects of drought

and N-starvation on wild barley (Hordeum spontaneum, C. Koch) points out the change of

the isotopic composition of plant material through enzymatic partitioning and losses of nitro-

gen. Hence, plant compartments like roots, stems and leaves may show strongly different

values (Robinson, 2000).

IV. Review of results and conclusions from former

research

In general, the variability of crop performance-parameters proved to be high in all selections

as within-field variability was examined. As expected, the characteristics assessed showed

significant relationships between the application of fertilizer, the distance to the irrigation

channel and the interaction of the factors: SPAD measurements and LAI showed high sig-

nificance during all growth stages if tested against the factors fertilizer (f), distance to the

channel (dc) and their interaction (f x dc). So, fertilizer application always resulted in higher

grain yields, whereas the distance to the channel of the respective fields and measurement

points had a more diverse effect. In the first and middle fields of the selections grain yields

were significantly higher than in the last fields, highlighting a big influence of the water avail-

ability, the type of channel and the inflow premises of the fields and selections, respectively

(distribution of inlet and outlet, inclination). Taking a closer look on the impacts of different

irrigation situations, the comparison between an earth channel-selection and a concrete

channel-selection showed distinct differences. For the first and middle fields of the two cas-

cades, SPAD and LAI readings were significantly higher in the fertilized parts. This differ-

ence vanished in the last fields; the reason for this should be low water and nutrient avail-

ability due to drought occurrence. More differences can be seen by considering the devel-

opment of the parameters. In the earth-channel cascade which had higher water scarcity,

plant biomass accumulated significantly slower during the single phases as can be derived

from the LAI values. However, the nitrogen content in leaves, denoted by the SPAD readings

Literature Review

14

did not exhibit the same pattern. The contents and the times of the highest accumulation

were very similar along the cascades and in the first and middle fields of both fertilized and

unfertilized compartments of the selections. Only the last fields showed distinct deviations.

As an effect of drought during the early season with only insufficient irrigation water provided

by the channel, nutrient uptake and thus the plant development lagged behind, resulting in a

delay of the harvest. This effect was stronger in the unfertilized part, suggesting that fertiga-

tion led to a higher uptake and storage of nutrients, foremost nitrogen, and a better capability

to regain growth after the water supply was better again with the beginning rainy season. The

hypothesis of Maja Hertel‟s study, that sediment from erosion processes that were flushed

into the paddy fields and distributed unevenly within fields and along the cascade affect the

crop productivity was proved with regard to the unfertilized sections of each selection. There

is an effect of sediment-derived nutrients on the first and middle fields, whereas due to sig-

nificant water stress in the last ones this effect could not be observed. It was concluded that

during plant development the availability of water and nitrogen interact on parameters like

number of panicles as well as panicle length and the amount of grain primordia that all result

in grain yield, size and weight. Difficult to separate though were the trade-offs between water

and nutrients: In fields that suffered from water stress in early growing stages, the LNC was

relatively higher compared to the ones with sufficient water, where more leaf mass shared

the same amount of available fertilizers. As significant drought stress affected almost all

fields during the cropping period, generalizations can hardly be made on the interplay be-

tween sediment-effects, fertilizer-use and water use. In all fertilized cascades however, there

was a clear trend of higher LAI, SPAD value and yield in the middle fields of the selections,

suggesting a positive interrelation between sediment-derived nutrients and mineral fertilizers.

The last fields of the unfertilized selections showed higher LAI values, which did not neces-

sarily lead to higher grain yields. The reason for this could be that during irrigation or rainfall

under aerobic soil conditions, nitrate from the upper fields might have been flushed down-

stream and contributing to a better plant-development. Because of water stress in the growth

stages “Panicle Initiation” and “Flowering”, the high leaf mass could not be used by the plant

to convert the leaf-nitrogen and carbon-assimilates into storage proteins and starch in the

grain, moreover the number of grain-primordia is affected adversely by stress in the sensitive

growth-stages (Hertel, 2007).

Material and Methods

15

C. Material and Methods

Data about the crop performance in the first season of 2007 were taken from Hertel (2007)

and details about the area were provided as oral communication by Petra Schmitter.

I. Details about the study area The plant samples that were used for this study come from Chieng Khoi, a commune in Yen

Chau district which lies in the Son La province in Northwest Vietnam and were collected by

Hertel (2007).

a)

b)

c)

Figure 1: a) geographic map of Vietnam showing the Son La province; b) map of Son La province with main roads and water bodies; c) map of the Yen Chau district, depicting the sub-catchment of Chieng Khoi commune and main roads and water bodies (Source: (Hertel, 2007))

Chieng Khoi is one of 13 communities in Yen Chau district; it is divided into six villages

(“Ban”). The soils are derived from silt-fine sandstones, sometimes with lime concretions.

Along the topo-sequence, we find Lixisols and shallow , often anthropogenic Leptosols on

the upper slope, Acrisols on the middle and lower slopes, associated with gleyic Anthrosols

in the depressions due to paddy rice cultivation (Stauss, 2007). The region‟s climate is char-

acterized by tropical monsoon between May and September, when about 70% of the annual

precipitation occurs, resulting in warm (~ 27,9°C) and rainy summers with a humidity fluctu-

ating around 75%. During autumn and winter from October to March, temperatures drop to

an average of ~17,5° C. The total annual precipitation ranges from 1073 mm to 1694 mm.

The latter value is exceptionally high, the overall average is about 1200 mm (Hertel, 2007).

Material and Methods

16

a)

b)

Figure 2: a) Average precipitation and temperature of Yen Chau city recorded from 2000 until 2006; b) Precipitation and temperature during the research period in 2007;

the left y-axis shows the precipitation in mm, the left y-axis shows the temperature in °C; the white arrows in b) show the duration of the two cropping periods; Data provided by Yen Chau weather station, Yen Chau city

II. Agricultural practices and irrigation

The traditional lowlands‟ production system is the cultivation of paddy rice in two cropping

seasons; on the lower slopes there are additional terraces with rainfed rice while most of the

uplands are occupied by maize (Zea mays L.) and cassava (Manihot esculenta Crantz) pro-

duction. Usually situated on the edges of the lower slopes close to the houses, home

gardens show a high variety of crops, mainly for subsistence purposes: Vegetables, fruit

trees, herbs and other crops are cultivated; often there is also animal husbandry. Pigs,

chicken and ducks are kept for the purpose of diet-amendment; water buffaloes serve as

multiple-use animals, usually for work and meat.

In Chieng Khoi district all paddies in the lowland which have good water accessibility are

planted twice a year, one spring-summer crop from February to May/June, and the autumn

crop from July to October during the rainy season. The rice terraces on the foot slopes of the

hills only have an autumn crop. In the first season farmers depend mainly on irrigation water

as the rains are too scarce. There is an artificial lake in Chieng Khoi commune, in the village

of Ban Put. Built in 1968 it serves as a reservoir for irrigation purposes, catching the strong

monsoon rains and the water from two underground springs. The lake is also feeding a river

which is irrigating the paddies of lower-lying villages in the commune. The two irrigation

channels are not only different in their material, but they also show different slopes: The

concrete channel leading to Ban Put has a steeper inclination, by this the water can flush the

water and the sediments contained within with a higher energy into the fields. In contrary, the

earth channel leading to Ban Me is flatter, leading to siltation by sediments because the wa-

ter‟s momentum is not strong enough to flush them further. Moreover, the earth channel has

a high probability of drying out during the first rainfed cropping season as an effect of seep-

10

15

20

25

30

0

50

100

150

200

250

300

J F M A M J J A S O N D

10

15

20

25

30

0

50

100

150

200

250

300

J F M A M J J A S O N D

1st1st 2nd

Material and Methods

17

age in cracks of the soil material. The second season is by far less drought-prone and

the crop is usually sufficiently watered because besides the rainwater there is still irrigation

water coming from the lake. With highly intense rainfall events, the danger in this season is

the erosion of soil material from the slopes which can bury plants, destroy or block irrigation.

Sediments that are mainly delivered from the slopes are often translocated via the channels,

because the latter lie in-between the uplands and the paddies.

Figure 3: a) Erosion sediments in a paddy field after heavy storm event in the beginning of the second season; b) Example of uplands cultivation with steep slopes and a settlement with homegar-den (Source: Schmitter)

As the aim of this study is to figure out if, and to what extent, the changes in the agro eco-

system over two seasons and different fertigation schemes affect the water- and nutrient-

availability within the cascades, only two cascades were selected for in-depth analysis from

Hertel‟s study (Hertel, 2007). A third one is only planted in the rainy season, so a compari-

son between the two seasons will not be possible here. The focus in this study is on the in-

ter-field and inter-toposequence variability. One reason for this is the limited amount of sam-

ples that can be analysed during one batch in the mass spectrometer, a comprehensive

analysis of all available positions was not possible. All three selections were divided in a

fertilized and a non-fertilized section. Fertilizer was applied with regard to the local extension

service‟s recommendations: 3kg of urea, 15 kg NPK and 0.8 kg of potassium per 100m². The

single selections or “terraces” each had a slope gradient of ~1.5 m along a selection length

of ~120m in selections BM1 and BP5 and ~70m in selection BM0. The rice variety was cho-

sen after the farmers preferences: Oryza sativa L., spp. NEP 87, a short duration sticky rice

variety that fits the local conditions and tastes. Out of each selection, the upper, middle and

lower field was chosen, with exception for BP5. There, 4 out of seven fields were chosen

because of the selection length and number of fields.

Material and Methods

18

Figure 4: Top-down view of lower BM0 (bare soil) and BM1 cascade during first season 2007 (Source: Schmitter)

Figure 5: Bottom-top view of BP5 cascade during the first season in 2007 (Source: Schmitter)

III. Development of criteria for sample selection

From each field, measurements were available for 7 different points, one in the middle and

three along the field bunds on each side. The middle- or M-positions should give an average

value of on the one hand, grain yield and general field conditions like soil water availability

and nutrient status on the other. After graphical and statistical analysis of yield plots, addi-

tional samples were selected that either showed large deviations from average yield values

or which were unexpectedly against the total trends produced by Hertel (2007). The single

Material and Methods

19

samples are each marked in the schematic picture in Figure 4. As the amount of samples

that could be tested for isotopic composition during one batch was limited, the author tried to

pick measuring points that are evenly distributed along the cascades.

. 1.0 1.1

2.0 2.1

3.0 3.1

40.1 40.2 41.1

5.0 5.1

Fishpond

1.0 11.1 11.2

2.0 2.1

3.0 3.1

40.1 40.2 41.1 41.2

1.0 1.1

2.0 2.1

3.0 3.1

4.0 4.1

5.0 5.1

6.0 6.1

7.0 7.1

Figure 6: Schematic draft of the cascades BM0, BM1 and BP5.

Green-shaded fields were cropped during 1st and 2

nd season, the blue-shaded BM0 only in the 2

nd

season. Red arrows mark in- and outlets from the irrigation channels through the cascade and into the drainage channel. Only fields with numbers shaded yellow were examined. Blue dots mark points that were selected for IRMS-analysis of the first season; red dots mark points that were selected for IRMS-analysis of the second season. Dots lying close to each other indicate selection of the measur-ing point for both seasons. Source: Hertel, 2007

IRRIGATION CHANNEL BP5

I

R

R

I

G

A

T

I

O

N

C

H

A

N

N

E

L

◄

R

I

V

E

R

BM 0

BM 1

Material and Methods

20

IV. Sample preparation for IRMS

The selected rice grains were prepared for automatic analysis in a Finnigan delta IRMS de-

vice, coupled to a Euro elemental analyzer. The oven-dried and unhusked grains were first

pre-shredded by the use of a knife-mill. Afterwards the husks had to be broken for further

milling in a ball-mill, with which the samples were milled to a fineness of coarse flour. Fur-

thermore, the samples were stored in an air-tight excavator to prevent the sample-flour from

moistening until the final preparation. For the use in the fully-automatic sampling device, the

samples had to be weighed into fine tin capsules. Tin has a lower melting and boiling point

than other elements, which assures sample analyses without significant interference, as the

samples pass a combustion chamber prior to the actual mass spectrometer. Each sample

was weighed assuming a total nitrogen content of each sample of 30µg. For further sample-

details, see Annex 1.

V. Function of mass spectrometers

In general, the samples are combusted in a combustion chamber. The purpose of this step is

to convert the molecules into a gas-phase. These gaseous molecules are in a second step

ionized and fragmented. This occurs under high vacuum, so analysis will not be interfered by

collision events producing analytical noise. The ratio of the mass to the charge of a given

isotope is important for the understanding of the whole theory: It is different (heavier) from

the mass of the elements‟ normal mass. The charge however stays the same in both ele-

ments, leading to a distinct behaviour when ionized. The energy resulting from ionization

leads to distinct patterns in the acceleration of the ions and, if deflected by an energy field

can be directed towards a certain region within a measuring device. There, the number of

the specific isotopes is counted and the ratio gets recorded in a data analyser. Each element

has to be referenced against “normal” gaseous elements, usually derived from normal air

(Dass, 2001). In the case of the mass spectrometer available in Hohenheim University, the

elements that can be detected are carbon and nitrogen, referenced against CO2 and ele-

mental N2. A coupled elemental analyzer analyzes the combusted gases on the total con-

tents of the given elements afterwards.

Results and Discussion

21

D.Results and Discussion

There were two seasons to be analyzed concerning the isotopic composition of 13C and 15N

in the rice grains. The focus in the interpretation will be on carbon isotopic discrimination as

these data will be easier to relate to other factors and the correlations are clearer. Of the first

season, two selections were chosen for further analysis, and three selections from the sec-

ond season. Each selection was again subdivided into one fertilized and one unfertilized

part, resulting in ten different “treatments” that are displayed either grouped by season or

fertigation. Correlation analysis showed significant correlations between the distance of the

measuring points and the IRMS data, see Annex 2 for details. The author will briefly show

the results of yield distribution along the cascades, as yield is one major parameter for fur-

ther interpretation.

I. Distribution of grain yield

1. Yield along the cascades BM1 and BP5 in the first season

Figure 7: Yield in Ban Me 1 cascade, first season

Figure 8: Yield in Ban Put cascade, first season

0

2

4

6

8

10

12

First Middle Last

Yie

ld (

t/ha)

0

2

4

6

8

10

12

First Middle1 Middle2 Last

Yie

ld (

t/ha)

Figures 4 and 5:

Average Yield along the

cascades Ban Me 1

(Fig.4) and Ban Put 5

(Fig.5), First Season:

BM1 shows a significant

fertilizer effect on the

grain yield. There must

have been a nutrient

translocation, probably by

flushing nitrates down the

cascade by either rain or

irrigation water.

In BP5 cascade the

yields are more evenly

distributed. The peak in

the first middle fields

might be an effect of

sediment interactions.

Closed symbols mark

fertilized fields, open

symbols mark unfertilized

fields.

Results and Discussion

22

2. Yield along the cascades BM0, BM1 and BP5 in the second

season

Figure 9: Yield in Ban Me 0 cascade, second season

Figure 10: Yield in Ban Me 1 cascade, second season

Figure 11: Yield in Ban Put 5 cascade, second season

0

2

4

6

8

10

12

First Middle Last

Yie

ld (

t/ha)

0

2

4

6

8

10

12

First Middle Last

Yie

ld (

t/ha)

0

2

4

6

8

10

12

First Middle1 Middle2 Last

Yie

ld (

t/ha)

Figures 6, 7, and 8:

Average Yield along the

cascades Ban Me 0

(Fig.6), Ban Me 1 (Fig.7)

and Ban Put 5 (Fig.8),

Second Season:

BM0 shows a higher yield

in the first fields of the

cascade, especially in the

unfertilized part. Whether

this effect is sediment-

derived or influenced by

human activities (the first

field is close to the set-

tlement, the fields are

also used for washing)

remains unclear.

In BM1 there is a clear

trend to higher yields to-

ward the end of the ter-

race. The incline of the

fertilized section might be

a result of fertilizer-

sediment interactions.

BP5 shows, similar to the

first season, a peak in the

third out of seven fields,

marked by „Middle1‟. The

strong decrease of the

fertilized fields towards

the end might be due to

management failures, as

the unfertilized section

suggests equal water

distribution.

Closed symbols mark

fertilized fields, open

symbols mark unfertilized

fields.

Results and Discussion

23

II. Results and discussion of the IRMS – Carbon

1. Discrimination for 13C along the cascades in the first season

a) Graphical display by selection

Discrimination along BM1 cascade, first season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 12: Selection BM1, unfertilized section marked by empty symbols, fertilized by filled symbols

Discrimination along BP5 cascade, first season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 13: Selection BP5, unfertilized section marked by empty symbols, fertilized by filled symbols

f = -28,3060 + 0,0154 x;R² = 0,9462

f = -28,5956 + 0,0063 x;R² = 0,7248

f = -28,6557 + 0,0153 x ;R² = 0,9581

f = -28,5154 + 0,0080 x ;R² = 0,8622

Results and Discussion

24

From the plots of the composition of 13C/12C in selection BM1 one can see major effects that

have impacted the ∂ values. First, there is a clear difference between the slopes of the two

fits; the discrimination for 13C is different between the fertilized and the unfertilized part of the

selection. Secondly, there is a distinct increase of the ∂ towards the lower end of the cas-

cade in both sections. Comparing the two fits, one can assume that, as in the first season

there has been significant drought, the discrimination in the fertilized section is a result of

mainly water stress. On the other hand, as in the unfertilized part no mineral fertilizer was

added, this section‟s value for ∂ 13C/12C must be caused by both water and nitrogen limita-

tion. Assuming that the water-flow conditions in both sections of this selection were equal, a

strong dependency of the discrimination for 13C during photosynthesis on the nitrogen-

availability can be derived. A range in the difference between the ∂ values of 1,3 ∂ in the

unfertilized part and 0,6 ∂ in the fertilized one shows a clear response on stress caused by

water shortages and combined nitrogen-water limitation, respectively. In general, these ob-

servations match with the distribution of the yield along cascade BM1: A declining and lower

yield level of the unfertilized part all along the cascade and a higher level in grain-yield in the

fertilized part. The contradiction between the isotopic compositions that show clear stress

effects increasing with the distance from the irrigation channel and the above-average yield

in the last field of the fertilized part might be explained by the methodology in sampling and

measuring.

A comparable pattern in the distribution of the isotope values along the cascade can be

made in the selection BP5. Like in selection BM1 that receives the irrigation water from an

earth channel, the fields irrigated by the help of a concrete channel show less discrimination

at the beginning of the cascade and a clear increase of the ∂ values towards the lower ends

of both sections. The deviation of about 2 ∂ in the section without fertilizer addition again

shows a distinct stress-increase in terms of water and nitrogen limitations towards the lower

end. The same applies for the fertilized part, though less severe with the range of ∂ values

from -28,6 to -27,6, still very clear.

Comparing both selections, one can not observe an impact of the two different irrigation

channels during this first season on the stress demonstrated by the ∂ 13C/12C. Both selec-

tions show a similar pattern of increasing stress on the same level with distance to the outlet

of the channels. This is in line with the observations made in the field; there was not enough

water to irrigate sufficiently.

Results and Discussion

25

b) Graphical display by fertigation Discrimination in the unfertilized sections

of BM1 and BP5, first season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 14: Unfertilized sections, circles for BM1 and triangles for BP5

Discrimination in the fertilized sections of BM1 and BP5, first season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 15: Fertilized sections, circles for BM1 and triangles for BP5

f = -28,6557 + 0,0153 ;R² = 0,9581

f = -28.5154 + 0,0080 x ;R² = 0,8622

f = -28,5956 + 0,0063 x ;R² = 0,7248

f = -28,3063 + 0,0154 x ;R² = 0,9462

Results and Discussion

26

Taking a look on the plots of the ∂ 13C/12C values compared by fertilizer-treatments, there are

slight differences between the selections. Near the irrigation channel of both unfertilized sec-

tions the values are lowest, with BP5 having values about 0,3 ∂ lower than BM1. This sug-

gests that close to the concrete channel there was still water available at times when the

points lying further away suffered higher water stress. Furthermore, in fields laying at the

earth-channel the water supply must have been already lower compared to the other. Al-

though there is a physiological maximum up to which 13C is enriched in C3 plant tissues, if

one extrapolates the fit for BM1 the values might be even lower if the selection was longer.

This can be derived from having a look at the BP5 part: At the end of the section, the ∂ val-

ues are even lower (about -27,0) than in BM1. These observations express the conditions

along the cascades: The channels were not transporting enough water to irrigate the crop; it

only reached the upper parts of each section, whereas the lower-lying parts were clearly

drier and thus the grains were enriched in 13C compared to grains from non-stressed plants

or plants that only suffered from a lack of nitrogen.

Although the differences in the distribution of the ∂ 13C/12C values are only slightly different,

there is one subtle distinction and that is the lower levels of ∂ in the fertilized parts of BM1. In

the unfertilized part, this was the opposite way around. As described before, the fertilized

sections in general show lower stress responses as fertigation affects the photosynthetic

capacity of the leaves and thus enhances stomatal conductance. The deviation in the BM1 fit

can be explained by the way the data were plotted. The first measured point in the field was

not 40 meters away from the inlet, but from the irrigation channel. The water first had to flow

through the unfertilized part of the selection. Setting the first point of selection BM1 from 40

meters to zero would show that the deviation between the two sections is negligible.

Summing up the above stated interpretations one can say that for the first season there is a

clear correspondence between the ∂ 13C/12C values and the grain-yields in the 4 sections.

Thus, for this season the IRMS signal of the carbon isotopes is a useful tool to examine co-

herences, water limitations and lack of nitrogen in particular. Even the proportions of the two

first-limiting stresses, water and nitrogen on the yield loss can be estimated. But the exact

attribution of carbon discrimination to a certain percentage of relative yield loss would need

deeper examinations and cultivar-based calibration.

Results and Discussion

27

2. Discrimination for 13C along the cascades in the second sea-

son

a) Graphical display by selection

Discrimination along BM0 cascade, second season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 16: Selection BM0, unfertilized section marked by empty symbols, fertilized by filled symbols

The results of the 13C-isotopic composition of both sections in the BM0 selection were unex-

pected. Judging from the yield levels and distribution, one would have expected the least 13C

enrichment in the unfertilized part, as this one has the highest average yield of the whole

second cropping season. Although the fit is not precise and in the middle field at 35 m the

value drops to -27.6 ‰, the overall trend shows that in the upper part as well as in the lower

part of this section there must have been a limitation to stomatal conductance resulting in

13C enrichment. Contrasting this is the distribution along the fertilized part. Although the

grain-yield declines as well with the distance, the enrichment in the grains of the last field

exhibits limiting effects of some plant resource. As both unfertilized and fertilized part of this

selection have the highest yields in the upper field, this pattern is difficult to explain. One

possibility is the effects of the houses lying near to these both fields. Even if the enrichment

cannot be explained by this, the high yields might be an effect of additional nutrients that

stem from human activities like leftover food, animal manure or human egests. Furthermore

as the points shown in the graphs only denote single points of the whole field, the author of

this study might by coincidence have chosen grains from non-representative corners. Both

trend-lines suggest nitrogen stress in the lower parts of BM0 cascade, even the fertilized

f = -27,3856 + 0,0030 x ;R² = 0,1056

f = -28,2019 + 0,0186 x ;R² = 0,7730

Results and Discussion

28

one. This seems to be a result of multiple causes. One is the uneven distribution due to

manual fertilizer application. This practice might have lead to high within-field yield devia-

tions in nitrogen availability. The other reason could be nitrogen losses, either gaseous or by

leaching through the puddled layer in case of improper field preparations.

Discrimination along BM1 cascade, second season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 17: Selection BM1, unfertilized section marked by empty symbols, fertilized by filled symbols

In selection BM1 the results of the composition in 13C/12C show values one would expect

from the knowledge that during this second season there was sufficient rain and irrigation

water to feed the crop. On average, the ∂ values for the unfertilized part of the selection

range around -27.4. Only the middle field shows a strong difference. The respective yield

trend declines towards the end of the cascade, the overall leaf-carbon dioxide conductance

that is expressed by the carbon-isotopic composition and that is a measure of carbon-

fixation activity shows patterns that go in line. The middle part of the unfertilized section has

a clear incline in both ∂ values of that field. This does not reflect the stable yield inclination

towards the lower parts. Besides these variations, the ∂ 13C/12C values are almost on the

same level for both sections. Like explained for this selection during the first season, if the

reader would shift the plot of the fertilized section to the left in order to match the beginnings

of each field with each other, at least the trend-lines would show the same slope and a range

of ∂ values that reflects less discrimination than in the respective first season.

f = -27,7338 + 0,0056 x ;R² = 0,2326

f = -28,1956 + 0,0057 x ;R² = 0,6773

Results and Discussion

29

Discrimination along BP5 cascade, second season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 18: Selection BP5, unfertilized section marked by empty symbols, fertilized by filled symbols

The average trends of the both sections of selection BP5 show almost the same pattern.

Both have an incline with increasing distance from the irrigation channel. The overall range

of this selection is 1.3 ∂. Especially in the lower parts, the variability in the 13C/12C composi-

tion is large. However, the increase in ∂ values matches with the decline of yields in this se-

lection. Also corresponding to the patterns observed in the yield plots is the stronger de-

creasing trend of the unfertilized part in ∂ 13C/12C values as well as in the grain yield.

f = -27,9183 + 0,0054 x ;R² = 0,5382

f = -28,1057 + 0,0075 x ;R² = 0,7890

Results and Discussion

30

b) Graphical display by fertigation

Discrimination in the unfertilized sections of BM0, BM1 and BP5, second season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 19: Unfertilized sections; diamonds for BM0, circles for BM1 and triangles for BP5

Discrimination in the fertilized sections of BM0, BM1 and BP5, second season

Distance from the irrigation channel (m)

0 20 40 60 80 100 120 140

13C

/12C

(‰

)

-29,0

-28,5

-28,0

-27,5

-27,0

-26,5

Figure 20: Fertilized sections; diamonds for BM0, circles for BM1 and triangles for BP5

f = -27,3856 + 0,0030 x ;R² = 0,1056

f = -27,7338 + 0,0056 x ;R² = 0,2326

f = -28,1057 + 0,0075 x ;R² = 0,7890

f = -28,2019 + 0,0186 x ;R² = 0,7730

f = -27,9183 + 0,0054 x ;R² = 0,5382

f = -28,1956 + 0,0057 x ;R² = 0,6773

Results and Discussion

31

A comparison of all fertilized sections from the second harvest with each other shows com-

parable trends to the comparison of all fertilized parts. What is most striking is the distribu-

tion of the 13C isotopes in the selection BM0. Both fertilized and unfertilized part rank highest

in the enrichment of 13C. Although there is still a lack of clarity with regard to the very high

yields in the upper parts of this selection, in general one has to say that stresses hampering

the unrestricted flux of carbon dioxide into the leaves were highest in this selection. Compar-

ing BM1 and BP5, the differences are less obvious and the values range in the same area.

One important factor for slightly lower ∂ values in BP5 might be the continuous flow premises

in the irrigation channel. This channel has a steeper inclination than the earth channel;

transporting water with a higher momentum seems very likely. And this could have affected

the availability of water in all corners of the respective fields, leading to a better distribution

of water as well as nutrients. A detailed table with all IRMS results corrected for blank values

is given in Annex 1.

III. Results and discussion of the IRMS – Nitrogen

1. Grain-Nitrogen and 15N, first season

A further method for the examination of environmental impacts on plant performance is the

use of the stable isotope 15N. In the following, the author displays the ∂ 15N/14N values of the

grains examined, additionally the content of nitrogen in the grains will be displayed as this

shows distinct responses to growth conditions as well.

First season, BM1, unfertilized First season, BM1, fertilized

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

Figure 21: Discrimination of 15

N and distribution of grain-nitrogen along the cascade Ban Me 1;

the x-axis shows the distance of the measuring point from the irrigation channel in meters; the left y-axis shows the nitrogen content in grains marked by diamond-symbols. The right y-axis shows the composition of

15N and

14N with respect to standard air in ‰, marked by triangles.

f = 10,1250 - 0,0371 x ;R² = 0,9678

f = 0,8989 + 0,0009 x ;R² = 0,2073

f = 0,9916 + 0,0008 x ;R²= 0,0344

f = 5,0726 + 0,0079 x ;R² = 0,6989

Results and Discussion

32

First season, BP5, unfertilized First season, BP5, fertilized

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

Figure 22: Discrimination of 15

N and distribution of grain-nitrogen along the cascade Ban Put 5;

the x-axis shows the distance of the measuring point from the irrigation channel in meters; the left y-axis shows the nitrogen content in grains marked by diamond-symbols. The right y-axis shows the composition of

15N and

14N with respect to standard air in ‰, marked by triangles.

In the unfertilized section of BM1, there is a depletion of 15N in the grains with increasing

distance to the channel. The proportion of grain-nitrogen increases slightly. The same ap-

plies for the fertilized section, here the grain nitrogen content increases as well with increas-

ing distance. The distribution of the isotopes is different from the unfertilized part. Here we

can observe a slight increase or rather enrichment of 15N towards the lower part of the selec-

tion. Taking the results of the stable carbon analysis and the yields into consideration, the

decrease in 15N goes together with an increase in 13C. The different trend in 15N composition

in the fertilized part must be mainly caused by mineral nitrogen fertilizer. In the un-

fertilized part of selection BP5 the distribution of grain-N content and 15N show the same

trends as in the respective BM1 section, however with different slopes and levels. Grain ni-

trogen contents are constant over the whole cascade. The grains are both depleted in 15N

the farther from the irrigation channel they have been grown; this effect is more extreme in

BM1. The fertilized sections at first sight show contradictory results. The grain-N

content increase is stronger in BP5, although the significantly higher yield in BM1 would

suggest better N-supply there. Clearly differing is the trend in 15N, where the amount is

rather stable along BM1 cascade and a large decrease in BP5. The strongest difference can

be observed looking at fertilized BP5‟s plot: In the last field of the section, grain-N content

increases significantly with distance (and thus with water stress), whereas 15N declines.

f = 1,1212 - 0,0003 x ;R² = 0,0109

f = 8,1390 - 0,0174 x ;R² = 0,2527

f = 1,0065 + 0,0035 x ;R² = 0,4819

f = 8,7703 - 0,0363 x ;R² = 0,3610

Results and Discussion

33

2. Grain-Nitrogen and 15N, second season

Second season, BM0, unfertilized Second season, BM0, fertilized

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

Figure 23: Discrimination of 15

N and distribution of grain-nitrogen along the cascade Ban Me 0;

the x-axis shows the distance of the measuring point from the irrigation channel in meters; the left y-axis shows the nitrogen content in grains marked by diamond-symbols. The right y-axis shows the composition of

15N and

14N with respect to standard air in ‰, marked by triangles.

Second season, BM1, unfertilized Second season, BM1, fertilized

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

15N

/14N

(‰

)

2

4

6

8

10

12

14

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

Figure 24: Discrimination of 15

N and distribution of grain-nitrogen along the cascade Ban Me 1;

the x-axis shows the distance of the measuring point from the irrigation channel in meters; the left y-axis shows the nitrogen content in grains marked by diamond-symbols. The right y-axis shows the composition of

15N and

14N with respect to standard air in ‰, marked by triangles.

f = 10,7125 + 0,0065 x ;R² = 0,0749

f = 1,2470 - 0,0053 x ;R² = 0,5255

f = 1,2986 - 0,0030 x ;R² = 0,3729

f = 8,9577 - 0,0353 x ;R² = 0,4221

f = 10,0480 - 0,0397 x ;R² = 0,5877

f = 1,1427 -0,0029 x ;R² = 0,3005

f = 1,2253 + 0,0004 x ;R² = 0,0193

f = 4,9752 - 0,0087 x ;R² = 0,1039

Results and Discussion

34

Figure 25: Discrimination of 15

N and distribution of grain-nitrogen along the cascade Ban Put 5;

the x-axis shows the distance of the measuring point from the irrigation channel in meters; the left y-axis shows the nitrogen content in grains marked by diamond-symbols. The right y-axis shows the composition of

15N and

14N with respect to standard air in ‰, marked by triangles.

The ∂ values of the unfertilized part of all selections of the second season are generally

higher than in the plots described before. Comparing all fertilized and all unfertilized sections

one can assert that, again with the exception of the unfertilized part of BM0, the ∂ 15N/14N

decreases towards the end of each cascade. The 15N values are higher in the unfertilized

sections, ranging from 6‰ to 12‰, whereas in the fertilized sections the values lie between

3‰ and 9‰ enrichment with respect to the standard air. In the unfertilized sections, the

grain-N content is declining towards the lower lying fields with the exception of BP5, where

at least the trend-line is constant. For the fertilized part of BM1 and BP5 one can observe an

incline in grain-N towards the end of the cascades, although the trend-line for BM1 has a low

goodness of fit. One can conclude by the interpretation of the values for 15N in the fertilized

part that a bigger proportion of fertilizer N accounted for the whole plant performance as the

range of ∂ values is lower. This reflects the signal of mineral fertilizers, which has values

ranging from +0.7 ‰ to -1.9‰ (Choi, 2006). The decrease in grain-N content of the unfertil-

ized part compared to the fertilized sections can be attributed to the application of mineral

fertilizer as well. What needs to be further examined is the rather high increase in the

lower parts of the fertilized BP5 section. There, the nitrogen content in the grains rises by

over one third towards the last field. By comparison with the yield graph, where the respec-

tive field shows the lowest yield of all fields in the whole selection, this seems to be due to

the uneven distribution of fertilizer. It seems like there must have been an external influence

on the lower parts of this section, by looking at the measurements from Maja Hertel (Hertel,

2007) or the results given here, this effect can not be clarified.

Second season, BP5, unfertilized Second season, BP5, fertilized

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

distance from irrigation channel (m)

0 20 40 60 80 100 120 140

gra

in-N

(%

)

0,6

0,8

1,0

1,2

1,4

1,6

d 1

5N

/14N

(‰

)

2

4

6

8

10

12

14

f = 9,7309 - 0,0233 x ;R² = 0,9670

f = 1,0535 - 0,0001 x ;R² = 0,0001

f = 1,0415 + 0,0023 x ;R² = 0,3265

f = 7,1928 - 0,0203 x ;R² = 0,4704

Results and Discussion

35

In general, the results of the analysis of the ∂ 15N/14N show similar patterns in both seasons

as can be seen in the plots of BM1 and BP5. The same applies for the grain-N: Although in

the second season the values for nitrogen content in the grains are on average higher, the

trends are comparable - with exception for BM0, which was anyway only cropped during the

second season and thus cannot be taken into account for comparison. The general trend for

15N depletion along the cascade with only 2 exceptions (unfertilized BM1 first season, unfer-

tilized BM0, second season) can probably be attributed to a spatial gradient.

IV. Summary of results and discussion

The results of the carbon isotope analysis provide helpful insights into the growing conditions

of the rice plants during the two cropping seasons. Especially for the first season, distinct

differences between two treatments (fertilized / unfertilized) are observable. There is a clear

effect on the photosynthetic discrimination for 13C, leading to enrichment of the grain-13C

along the cascades.

The within-field variability in

∂ values might be caused by

random sample selection.

As stated before, there were

7 points available for IRMS

testing (except for the first

season samples of BP5,

where some samples could

not be taken out of Vietnam

because of infestation by

insects). The likelihood of

picking un-representative

samples is thus high. On the other hand, the variability matches with the observations made

by Maja Hertel. The within-field variability of crop-performance parameters was high. So the

fluctuating ∂ values might be another proof of the uneven distribution of plant resources.

Still, on a cascade scale the trends illustrate the varying allocation of resources like nutri-

ents, in particular nitrogen and water. Close to the channels, nitrogen and water stresses

have been lowest, with a clear increase in all treatments and in both seasons towards the

lower parts of the cascades. This issue still has to be clarified. For the first season, lack of

water accounts for a big part of plant stress expressed by the given ∂ 13C/12C composition

values. Especially in rice paddies, water is not only the transport media for the diffu-

sion and mass flow of soil- and fertilizer nutrients. It furthermore constitutes a media that

Results and Discussion

36

accounts for the soil properties and -development. Redox-processes are important factors

leading to gaseous emissions of methane and gaseous nitrogen compounds. The suppres-

sion of weeds and the levelling of soil temperature are two other important effects (Wade,

1998). A further factor that has an impact on water availability and distribution is the

design and the properties of the irrigation channels. In the area, during the monsoon there is

a high leaching and later influx of nutrients by runoff, as well as sedimentation of eroded soil

from the uplands fields into the lowlands paddies (Schmitter, 2008). The channelled water

that transports solute nutrients as well as mineral particles has a yet undefined capacity to

re-allocate these nutrients and sediments. From the knowledge about water‟s properties as a

transport media we know that heavier particles delivered with the irrigation water-flow will

sediment earlier than lighter ones. Solute organic compounds could be transported over very

long distances, but might be as well filtered out by the rice stems. To clarify these issues,

more detailed analyses of soils and water would be needed and the in- and outflows in

gaseous, liquid or solid forms would have to be assessed to have a comprehensive overview

about the balances within fields and along cascades (Schmitter, 2008).

Summing up the impacts of water that could have lead to the observed patterns in 13C distri-

bution, the author distinguishes between to major processes. This is not necessary for the

first season, where these impacts can be clearly attributed to specific factors, but for the

second season. On the one hand, water might have been lost in the lower lying parts due to

different textural soil conditions. This would explain why, even in the second season with

usually sufficient water supply and in fertilized sections, there is still a notable increase in ∂

values in the last fields. On the other hand there might be a combined effect of water,

mineral fertilizers and sediments affecting the ∂ 13C values. For the first season water is, as

stated before, the main cause for isotopic enrichment along the cascades. For the second

season it is likely that sediments had a strong impact. Shortly after transplanting of seed-

lings, highly intense precipitation caused strong erosion. At least in the upper parts of each

field close to the slopes, these sediments buried the seedlings and this definitely accounts

for some part of the yield loss. This is one factor that could describe why in the upper parts

of selections BM1 and BP5 the average yield was lower than in subsequent ones. And as

coarse sediments with a low SSA and a low water holding capacity promote plant stress, the

lower yield levels in the beginning of each section might be caused by this issue. These ob-

servations match with other studies from Thailand (Haefele, 2006). This development has to

be seen as a cumulative process over many seasons, finally leading to an alteration in soil

properties like texture and SSA. In the long run due to sedimentation, the upper parts of the

cascades will exhibit yield distributions similar to the ones observable in BP5. But these as-

sumptions are, if at all, only valid for the drier first cropping season. For an overview

about correlations between the texture and the values displayed, please see Annex 2.

Results and Discussion

37

There, the soil analyses from the beginning of the first season, before fertilizer application,

are displayed in relation to ∂ values. As in the second cropping season enough water

is there to irrigate the crop in addition to the rains, deviations in the water holding capacity of

a field might not have a large impact. What seems more likely to affect both yields and the

isotopic compositions of the grains are the impacts of water movement. During the rainy

season, the flushes of flowing water carry large amounts of the three major mineral plant

nutrients N, P and K (Singh, 2008). Provided there was the same amount of fertilizer added

per field and a similar planting density, the steeper slope of the concrete channel leading to

Ban Put might have caused a bigger momentum on the irrigation water. This could be the

cause for a peak in yield in the third out of seven fields, because this rather small field could

have acted as a resistor or filter to nutrient percolation by retarding the mass flow of water.

Therefore, it is likely that the stress responses of the ∂ 13C/12C composition in rice grains are

based on nutrient limitations. The hampered stomatal conductivity resulting from a lack of

nitrogen does not necessarily have to result in lower yields. Partial stresses during some

phases of the growing season might have caused temporal reduction in the photosynthesis

activity. In a later phase, already fixed nitrogen could still have been re-allocated within the

plant to contribute to a good or average grain yield.

Besides the usual gaseous nitrogen losses in the paddy fields caused by the biochemical

turnover of Urea causing low levels of NUE in lowland rice production systems (Buresh,

1993), the flow premises might be also a factor impacting the 15N composition.

In order to interpret the results for the nitrogen-content and -composition, one has to take

environmental conditions and management practices into account again. The most obvious

difference here is the application of mineral fertilizers. The other is the distribution of water

and the impacts of the variability of the former. Usually, lowland paddy soils have low oxygen

contents. Biological nitrogen turnover to nitrate is thereby restricted and the most abundant

form of nitrogen available for the plants is ammonia. With increasing seepage or drying of

the soil, the proportion of nitrate of the whole plant-available nitrogen increases. Given the

rather high temperatures in the north-western uplands of Vietnam, the gaseous losses

caused by denitrification after the water saturation declined are probably big. So, the signal

for ∂ 15N/14N we observe here could be caused by two processes. One might be the deple-

tion of 15N during nitrification, leading also to depletion in the plant tissues nurtured under

these conditions. Högberg reports in his review (1997) that the isotopic fractionation occur-

ring during nitrification is larger than the fractionation caused by mineralization from organic-

N compounds to ammonia (Högberg, 1997). Taking gaseous N-emissions into account, the

signal from the ∂ 15N/14N measurements might have again been altered as the isotope effect

of the denitrification process is enrichment of 15N in the leftover nitrogen (Blackmer, 1977). In

Results and Discussion

38

paddy soils, depending on the timing and the amount of fertilizer application and the N-form,

there are high gaseous losses by the volatilization of NH3. Högberg (1997) reports that dur-

ing the volatilization of soil-ammonia to gaseous ammonia the discrimination effect can be

up to -40‰ compared to the N-source. Comparing these studies with the results obtained

here, it is likely that there has been a fractionation during nitrogen turnover in the paddy soil.

Nitrate, depleted in 15N might have been the abundant N-form particularly in the last fields,

leading to decreasing grain-15N values except fertilized BM1, where yield and comparatively

low 13C discrimination signify higher water saturation of the soil.

A further interpretation for the results given here is of course the effect of the fertilizer‟s iso-

topic composition. As there was no fertilizer-sample available for the IRMS, the author relies

on other studies and interpretation. In a study conducted by Choi et al (2006), grain 15N val-

ues were significantly lower in the treatment with application of mineral fertilizer. They stated

∂ values of +0.7 ‰ for urea and -1.9‰ for ammonia phosphate (Choi, 2006). Organic nitro-

gen compounds often show higher 15N values than mineral fertilizers. As soil-turnover of

nitrogen would need deeper analysis and the parameters for inherent soil fertility of the re-

spective fields are only available for the beginning of the first season before fertilizer was

applied, this issue can not be examined in further detail here. The fact that the trend-

lines of ∂ 15N values towards the end of unfertilized sections have a slightly stronger decline

supports the interpretation that 15N-depleted nitrate might have played a role in the plant

nutrition there and that nitrification must have taken place. Otherwise, one would rather ex-

pect a constant trend over the whole cascade. But as the readings lie in the same range as

the fertilized sections with an approximated low 15N-signal of the mineral fertilizers, this ap-

praisal could be true. One possible interpretation of the 15N trends compared to

grain-N could be the role of mineral fertilizers. The extent to which it contributes to grain-N

under water stress conditions rises with increasing stress, because the lowest values can be

found in the last fields of fertilized sections. But there might as well be a plant-physiological

effect included in the readings, because under water stress conditions uptake and metabo-

lism of nitrogen can differ (Larcher, 2003). A final integrated interpretation of yield, carbon

and nitrogen isotope distribution along the cascade might be environmental feedback on the

composition and ∂ values of the grains: Depending on temperature and water content as well

as water velocity, this fluid factor could have caused multiple interactions. Referring to Yin

and Raven (1998), plant performance and stoma closure are also affected by the deposition

of gaseous ammonia in the leaves. By changing the internal pH and thus the enzymatic ac-

tivity, the ∂ 13C will be enriched. This could explain why, in all seasons and sections, even

with high water availability, 13C has been enriched. The extent to which volatile loss of am-

monia took place can only hardly be estimated. But taking up the thread of water movement

influencing nutrient fluxes, it might as well have influenced the oxygen status of the paddy

Results and Discussion

39

soil: Assuming that water in paddy soils is rather depleted in oxygen compared to free-

flowing water, aeration of the bulk soil and the root-zone in particular by the rice plants‟

aerenchyma could have lead to the observed results. Directly beneath the roots, oxygen is

relatively “enriched” (Larcher, 2003), which could have lead to nitrification and nitrogen loss

that even affected yield, grain-N and 15N values. Again quoting Yin and Raven (1998), the

effects of the N-form on different C3 grasses show unclear effects on ∂ 13C. In their study,

plants that received ammonia only showed better WUE than those that received nitrate. To

my knowledge, no comparable study about N-cycling with respect to WUE in rice consider-

ing gaseous influences as well has been carried through. So, there are some constraints

for the thorough interpretation of ∂ 15N/14N values. As concluded by other authors dealing

with 15N natural abundance levels in plant ecological studies, the whole plant environment

would have to be examined (van Kessel, 1994). The multiple pathways during N-uptake,

fractionation and turnover processes in plant or soil and environmental conditions like leach-

ing and volatilization make it difficult to derive general principles in the context of this limited

study.

The choice of the cultivar is another factor that shows and changes responses to environ-

mental conditions as shown in studies from Wade et al (1998) or Fukai et al (1999) who re-

ported a wide range of genotype-based plant responses on environmental constraints to

their performance (Fukai, 1999). As an effect the variety used in this study, Oryza sativa L.

spp. NEP87 might not be the best choice in terms of resource use efficiency. It could be that

this genotype was actually bred for a wide range of environments and that other, autochtho-

nous cultivars would perform better with limited resource availability.

V. Conclusion

The aim of this study was to assess the limitation-effects of the two important plant re-

sources water and nitrogen on the overall performance of paddy rice. There are various in-

terconnections to respect while interpreting data on plant growth and crop development.

The hypothesis, that one can identify the role of nitrogen and water on growth performance

parameters was partially proved. In particular the analysis of the proportion of 13C isotopes in

the grains could be clearly related to other performance values like grain yield and nitrogen

uptake. This method is a consistent integrator of plant responses to environmental limita-

tions. However, there are several factors that impact on the net discrimination effect for or

Conclusion

40

against 13C during photosynthetic fixation of carbon. As pointed out in the literature review,

soil properties like salinity, compaction or nitrogen availability affect this process. Further-

more, environmental and seasonal fluctuations altering the composition of the plants‟ ambi-

ent air cause a change in the ∂ signal. This is the reason why the IRMS method never could

be integrated as a generalized measure of crops to water limitations. But in the framework of

this study and the ability to put the isotope-readings in a context, these analyses provide an

insight into the overall limiting effects. It could be shown that in dry conditions the major part

of the stress response by stomatal closure is caused by limited water availability. A second

factor, as stated above, is the availability of nitrogen to the plants‟ photosynthetic system.

Though not completely clarified, there is reference suggesting that even under well-watered

conditions and application of mineral fertilizers, the net availability of nitrogen to the plants

can be constrained by either gaseous losses and the percolation or the passage of solute

nutrients with irrigation water. The analyses of the ∂ 15N/14N values showed more difficulties.

Because there are various interactions of nitrogen compounds in the biosphere, natural

abundance 15N studies are more demanding to perform. What could be shown is a hint on

the proportion of the contribution of fertilizer-N to the overall nitrogen uptake. A quantification

of the relative yield loss because of both factors, 13C and 15N is not possible in this context.

There are some methodological limits to the interpretation of the given results as well. For

the interpretation of the 15N results a sample of the fertilizer applied in the field would have

been a helpful reference. A second, maybe more severe factor is the preparation of the grain

samples. The unhusked grains were milled by the help of a knife mill first, and then ground

to coarse flour in a ball mill. During this preparation process, there was an unequal com-

minution of the grains and the husks. Thus, the samples did not receive a homogenous mix-

ture of husk and grain components. As husks usually have totally different elemental compo-

sitions, this might be a cause of large isotopic deviations within one examined section.

Lastly, in order to receive a better overview over the spatial distribution of isotope effects, a

higher number of samples per field, if not all seven corners, would have been helpful for the

interpretation of spatial variability as well.

References

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Annex 1

xiv

Annex 1

Identifier 1 Sea-son

Selec-tion

Dis-tance

Fert.

Cor-ner

Yield (Kg/m²)

Amount

Amt%

N-Amt%

C-Amt%

d 15N/14N

AT% 15N/14N

d 13C/12C

AT% 13C/12C

G1_BM11.0BH 1 BM1 2,00 0 B 0,519 2,68 0,944 0,944 40,237 10,287 0,370228 -28,223 1,074789

G1_BM111.0MH 1 BM1 7,50 0 M 0,346 2,75 0,868 0,868 38,087 9,53 0,369952 -28,145 1,074874

G1_BM13.0BH 1 BM1 37,25 0 B 0,308 2,66 0,900 0,900 38,009 8,896 0,36972 -27,787 1,075266

G1_BM13.0MH 1 BM1 37,08 0 M 0,285 2,93 0,949 0,949 38,259 8,717 0,369655 -27,88 1,075164

G1_BM140.2B-CH 1 BM1 68,16 0 B-C 0,174 3,13 0,903 0,903 35,314 7,542 0,369226 -27,304 1,075794

G1_BM140.2MH 1 BM1 68,16 0 M 0,270 2,98 1,035 1,035 38,481 7,621 0,369255 -27,103 1,076014

G1_BP51.0A-DH 1 BP5 4,40 0 A-D 0,325 2,83 1,041 1,041 38,325 9,448 0,369922 -28,74 1,074224

G1_BP51.0MH 1 BP5 4,40 0 M 0,344 2,99 1,093 1,093 38,336 8,214 0,369471 -28,492 1,074495

G1_BP53.0A-DH 1 BP5 53,55 0 A-D 0,894 2,91 1,104 1,104 38,625 5,432 0,368455 -27,688 1,075374

G1_BP53.0MH 1 BP5 53,55 0 M 0,398 2,96 1,335 1,335 38,075 5,742 0,368569 -27,832 1,075217

G1_BP55.0MH 1 BP5 91,30 0 M 0,413 2,79 1,047 1,047 38,040 7,608 0,36925 -27,428 1,075659

G1_BP57.0BH 1 BP5 103,30 0 B 0,179 2,68 0,985 0,985 39,271 5,804 0,368591 -26,91 1,076226

G1_BP57.0MH 1 BP5 106,18 0 M 0,387 2,84 1,131 1,131 37,938 7,48 0,369203 -27,106 1,076011

G1_BM111.2MH 1 BM1 42,20 1 M 0,635 2,73 0,894 0,894 34,996 5,544 0,368496 -28,456 1,074534

G1_BM13.1CH 1 BM1 81,65 1 C 0,347 3,1 0,980 0,980 39,445 5,547 0,368497 -27,966 1,07507

G1_BM13.1MH 1 BM1 63,99 1 M 0,728 3,1 1,268 1,268 38,516 5,58 0,368509 -28,059 1,074968

G1_BM141.1DH 1 BM1 125,45 1 D 1,346 2,65 1,057 1,057 39,769 6,252 0,368755 -27,821 1,075228

G1_BM141.1MH 1 BM1 99,08 1 M 0,834 2,86 1,094 1,094 38,404 5,694 0,368551 -28,089 1,074936

G1_BP51.1MH 1 BP5 4,40 1 M 0,528 2,97 1,100 1,100 38,039 6,128 0,368709 -28,546 1,074436

G1_BP53.1BH 1 BP5 49,30 1 B 0,843 2,96 1,198 1,198 37,389 9,499 0,36994 -28,13 1,07489

G1_BP53.1MH 1 BP5 53,55 1 M 0,723 2,84 1,103 1,103 38,345 8,239 0,36948 -27,999 1,075034

G1_BP55.1MH 1 BP5 91,30 1 M 0,529 3,21 1,050 1,050 39,434 7,077 0,369056 -27,721 1,075338

G1_BP57.1AH 1 BP5 103,30 1 A 0,379 2,74 1,456 1,456 39,491 4,764 0,368211 -27,498 1,075583

G1_BP57.1DH 1 BP5 111,80 1 D 0,321 2,9 1,431 1,431 40,070 2,604 0,367423 -27,753 1,075303

G1_BP57.1MH 1 BP5 106,18 1 M 0,674 3,15 1,544 1,544 39,155 4,221 0,368013 -27,827 1,075222

Table 1: IRMS results for the first season; isotope values are corrected for blank samples

Annex 1

xv

Identifier 1 Season Selection Distance Fert. Corner Yield (Kg/m²) Amount Amt% N-Amt% C-Amt% d 15N/14N AT% 15N/14N d 13C/12C AT% 13C/12C

G2_BM01.0DH 2 BM0 16,50 0 D 1,441 2,83 1,062 1,062 39,168 10,564 0,370329 -27,344 1,075751

G2_BM01.0MH 2 BM0 9,00 0 M 0,855 3,1 1,329 1,329 39,868 10,665 0,370366 -27,192 1,075917

G2_BM03.0MH 2 BM0 32,85 0 M 0,587 2,75 0,963 0,963 39,715 11,414 0,370639 -27,606 1,075464

G2_BM05.0BH 2 BM0 56,25 0 B 0,748 2,74 1,075 1,075 39,742 11,835 0,370793 -27,197 1,075911

G2_BM05.0DH 2 BM0 61,15 0 D 0,388 2,9 0,827 0,827 36,213 10,452 0,370288 -27,305 1,075793

G2_BM05.0MH 2 BM0 58,70 0 M 0,795 2,67 0,987 0,987 39,287 10,871 0,370441 -26,966 1,076164

G2_BM11.0DH 2 BM1 21,00 0 D 0,332 2,88 0,929 0,929 38,289 9,869 0,370075 -27,829 1,07522

G2_BM11.0MH 2 BM1 7,50 0 M 0,444 2,68 1,279 1,279 50,903 8,524 0,369584 -27,849 1,075198

G2_BM13.0A-DH 2 BM1 37,08 0 A-D 0,587 2,79 0,991 0,991 39,097 9,197 0,36983 -27,277 1,075824

G2_BM13.0MH 2 BM1 37,08 0 M 0,623 2,8 1,002 1,002 39,040 9,354 0,369887 -27,147 1,075966

G2_BM140.2B-CH 2 BM1 68,16 0 B-C 0,752 2,86 1,032 1,032 39,839 7,12 0,369072 -27,511 1,075568

G2_BM140.2MH 2 BM1 68,16 0 M 0,637 2,8 0,930 0,930 38,552 6,735 0,368931 -27,454 1,075631

G2_BP51.0BH 2 BP5 2,00 0 B 0,257 2,82 1,108 1,108 40,260 9,371 0,369894 -28,254 1,074755

G2_BP51.0MH 2 BP5 4,40 0 M 0,568 2,93 0,967 0,967 38,415 9,869 0,370076 -27,96 1,075077

G2_BP53.0A-DH 2 BP5 53,55 0 A-D 0,531 3,1 1,160 1,160 40,195 8,482 0,369569 -27,744 1,075313

G2_BP53.0MH 2 BP5 53,55 0 M 0,546 2,89 1,046 1,046 39,293 8,635 0,369625 -27,641 1,075426

G2_BP55.0AH 2 BP5 88,30 0 A 0,660 2,89 0,975 0,975 40,100 7,734 0,369296 -27,359 1,075734

G2_BP55.0MH 2 BP5 91,30 0 M 0,569 2,9 0,990 0,990 39,443 7,543 0,369226 -27,216 1,075891

G2_BP57.0MH 2 BP5 106,18 0 M 0,366 3,02 1,120 1,120 38,207 7,177 0,369093 -27,561 1,075514

G2_BM01.1MH 2 BM0 9,00 1 M 0,775 2,85 1,323 1,323 39,991 9,364 0,369891 -28,17 1,074846

G2_BM03.1DH 2 BM0 33,26 1 D 1,023 2,87 1,080 1,080 39,446 6,919 0,368998 -27,631 1,075437

G2_BM03.1MH 2 BM0 32,75 1 M 0,901 3,03 1,218 1,218 38,426 7,224 0,36911 -27,275 1,075826

G2_BM05.1AH 2 BM0 53,05 1 A 0,580 2,89 1,133 1,133 38,999 6,893 0,368989 -27,182 1,075928

G2_BM05.1MH 2 BM0 56,40 1 M 0,605 3,02 1,194 1,194 38,856 7,878 0,369349 -27,312 1,075785

G2_BM111.2MH 2 BM1 42,20 1 M 0,507 3 1,200 1,200 38,202 4,395 0,368077 -28,114 1,074909

G2_BM13.1AH 2 BM1 57,10 1 A 0,522 2,71 1,420 1,420 39,748 3,505 0,367752 -27,78 1,075274

G2_BM13.1MH 2 BM1 63,99 1 M 0,770 2,73 1,131 1,131 39,412 5,49 0,368477 -27,781 1,075273

G2_BM141.1CH 2 BM1 125,45 1 C 0,941 2,71 1,293 1,293 38,539 3,297 0,367676 -27,609 1,075461

G2_BM141.1MH 2 BM1 99,08 1 M 1,136 3,06 1,255 1,255 39,480 4,825 0,368234 -27,496 1,075584

G2_BP51.1MH 2 BP5 4,40 1 M 0,604 2,8 0,968 0,968 39,599 7,729 0,369294 -27,889 1,075154

G2_BP53.1CH 2 BP5 63,30 1 C 0,880 3,04 1,374 1,374 40,158 4,762 0,368211 -27,628 1,07544

G2_BP53.1MH 2 BP5 53,55 1 M 0,735 2,69 1,236 1,236 38,572 5,571 0,368506 -27,688 1,075375

G2_BP55.1MH 2 BP5 91,30 1 M 0,338 2,96 1,054 1,054 39,507 6,24 0,36875 -27,083 1,076036

G2_BP57.1B-CH 2 BP5 106,18 1 B-C 0,266 3 1,308 1,308 39,342 4,49 0,368111 -27,61 1,07546

G2_BP57.1MH 2 BP5 106,18 1 M 0,328 2,77 1,302 1,302 38,908 5,727 0,368563 -27,297 1,075802

Table 2: IRMS results for the second season; isotope values are corrected for blank samples

xvi

Annex 2

Tables of correlations between isotopic composition and plant parameters

First season, unfertilized sections of BM1 and BP5

Yield

Plant Density

Height MT/PI

Height F

Distance Channel

Total Carbon

Total Organic Carbon

Total Nitrogen

K Clay Silt Sand SSA

∂ 15N/14N -0,23 -0,84°° -0,31 0,11 -0,68° -0,66° -0,28 -0,75°° 0,78** -0,59° -0,26 0,36 -0,57°

∂ 13C/12C -0,20 0,36 -0,35 -0,44 0,93°° 0,26 0,37 0,34 -0,72°° -0,07 0,30 -0,24 0,00

First season, fertilized sections of BM1 and BP5

Yield

Plant Density

Height MT/PI

Height F

Distance Channel

Total Carbon

Total Organic Carbon

Total Nitrogen

K Clay Silt Sand SSA

∂ 15N/14N 0,42 0,32 0,35 0,42 -0,46 0,92** 0,58 0,80** 0,26 0,93** 0,74** -0,81°° 0,93**

∂ 13C/12C -0,11 -0,73°° -0,27 -0,61° 0,84** 0,01 -0,11 0,05 -0,40 -0,22 -0,35 0,34 -0,26

Table 3: Results of correlation analysis, first season

Results that correlate positively are marked with * and ** for p ≤ 0,05 and p ≤ 0,01;

Results that correlate negatively are marked with ° and °° for p ≤ 0,05 and p ≤ 0,01.

Annex 2

xvii

Tables of correlations between isotopic composition and plant parameters

Second season, unfertilized sections of BM0, BM1 and BP5

Yield

Plant Density

Height MT/PI

Height F

Distance Channel

∂ 15N/14N 0,27 0,01 -0,51° 0,32 -0,53°

∂ 13C/12C 0,52* 0,43 -0,51° 0,03 0,44

Second season, fertilized sections of BM0, BM1 and BP5

Yield

Plant Density

Height MT/PI

Height F

Distance Channel

∂ 15N/14N 0,05 0,14 -0,76°° 0,11 -0,67°°

∂ 13C/12C -0,15 0,25 -0,14 0,02 0,46

Table 4: Results of correlation analysis, second season

Results that correlate positively are marked with * and ** for p ≤ 0,05 and p ≤ 0,01;

Results that correlate negatively are marked with ° and °° for p ≤ 0,05 and p ≤ 0,01.