The effects of cropping systems on selenium and glucosinolate...

The effects of cropping systems on selenium and glucosinolate concentrations

in vegetables

DISSERTATION

Presented in Partial Fulfilment of the Requirements

for the Degree Doctor of Philosophy

in the Faculty of Science and Technology, Aarhus University

By

Eleftheria Stavridou

April, 2011

i

Foreword

This Ph.D.-dissertation has been submitted to Aarhus University in partial fulfilment of

the requirements of the degree of Doctor of Philosophy. Professor Kristian Thorup-

Kristensen, Department of Agricultural Science, University of Copenhagen and Associate

Professor Hanne Lakkenborg Kristensen, Department of Horticulture, Aarhus University

have been my supervisors. The study was conducted during the period March 2008 to March

2011 at the Department of Horticulture, Aarhus University. In January 2010, I stayed at the

Leibniz-Institute of Vegetable and Ornamental Crops Grossbeeren/Erfurt e.V., Germany to

perform glucosinolate analysis under supervision of Prof. Monika Schreiner, to whom I am

very thankful for her valuable advices and collaboration.

The Ph.D. project is mainly focused on the effects of catch crops on the availability of

selenium (Se) in vegetables. During the first half of the PhD, problems in the Se analysis,

which were beyond my control, led to the establishment of an alternative project. Its aim was

to increase glucosinolate concentrations in Brassicas by intercropping.

I am indebted to Scott Young, Associate Professor and Reader in Environmental Science,

Faculty of Science, University of Nottingham for his help with the Se analysis in the later

part of the study. His co-operation ensured that this project was completed on time.

The dissertation addresses theses aims in seven chapters. Chapter 1 is a general

introduction followed by a literature review on Se and glucosinolates (Chapter 2). Chapters 3

and 4 contain the experimental work, which tested the efficiency of catch crops to increase Se

in vegetables. Chapters 5 and 6 include the results from the intercropping experiments and

the root growth studies. Finally, Chapter 7 contains the conclusions and perspectives of this

work.

I owe a debt of gratitude to many people who helped and encouraged me during my Ph.D.

project. First and foremost, I would like to thank Kristian, my supervisor, for his invaluable

guidance throughout the work. Without his enthusiastic encouragement and support this work

would most probably not have been completed. I am grateful to my supervisor Hanne for the

valuable discussions and advice given to me at the last year of my Ph.D. study.

I am very thankful to the technical staff at the Department of Horticulture, Astrid

Bergman, Birthe R. Flyger, Jens Barfod, Marta Gertrud Kristensen and Knud Erik Pedersen

in particular for their valuable work in the field and in the laboratory.

ii

Thanks are also due to all the colleagues at the Department of Horticulture, Aarhus

University for maintaining a pleasant and cheerful environment.

Finally I wish to thank family and friends for their support and encouragement.

iii

Table of Contents

Table of Contents .............................................................................................................. iii

Summary ............................................................................................................................ vii

Summary in Danish ............................................................................................................ ix

Chapter 1 Introduction ....................................................................................................... 1

Chapter 2 Literature review .............................................................................................. 3

1. Selenium ....................................................................................................................... 3

1.1.1. Selenium mineralogy ....................................................................................... 3

1.1.1. Biotic and abiotic processes affecting Se availability in soil ........................... 4

1.1.1.1. Abiotic processes ....................................................................................... 4

1.1.1.2. Biotic transformations ............................................................................... 6

1.2.1. Selenium levels in plants and their effects ....................................................... 8

1.2.2. Selenium uptake and assimilation by plants .................................................... 9

1.2.3. Factors that affect Se uptake by plants ........................................................... 10

1.2.3.1. Selenium form ......................................................................................... 11

1.2.3.2. Competing ions ........................................................................................ 11

1.2.3.3. Organic matter ......................................................................................... 12

1.2.4. Selenium concentrations in vegetables and its bioavailability to humans ..... 12

1.1. Selenium in soils .................................................................................................... 3

1.2. Selenium in plants ................................................................................................. 8

1.3. Selenium essential for humans ............................................................................ 13

1.4. Selenium human intake ....................................................................................... 14

1.5. Strategies to increase Se human intake ................................................................ 15

1.6. Catch crops .......................................................................................................... 16

iv

2. Glucosinolates ............................................................................................................ 17

2.3.1. Genotype ........................................................................................................ 18

2.3.2. Temperature and light .................................................................................... 18

2.3.3. Water availability ........................................................................................... 19

2.3.4. Nutrient supply ............................................................................................... 19

2.3.5. Plant density ................................................................................................... 20

Chapter 3 The effect of catch crop species on selenium and sulphur availability for

the succeeding crops .......................................................................................................... 21

1. Introduction ............................................................................................................... 22

2. Materials and methods .............................................................................................. 24

3. Results ......................................................................................................................... 27

4. Discussion ................................................................................................................... 33

5. Conclusion .................................................................................................................. 36

Chapter 4 Assessment of selenium mineralization and availability from catch crops

............................................................................................................................................. 37

1. Introduction ............................................................................................................... 38

2.1. General ................................................................................................................. 17

2.2. Role in human health ........................................................................................... 17

2.3. Factors affecting plant levels ............................................................................... 18

2.1. Field experiments ................................................................................................ 24

2.1. Plant sampling and analysis ................................................................................. 25

2.2. Soil sampling and analysis .................................................................................. 26

2.3. Data analysis ........................................................................................................ 26

3.1. Soil Se and S ........................................................................................................ 27

3.2. Catch crops .......................................................................................................... 29

3.3. Cash crops ............................................................................................................ 30

v

2. Material and Methods ............................................................................................... 39

3. Results ......................................................................................................................... 43

4. Discussion ................................................................................................................... 45

5. Conclusion .................................................................................................................. 48

Chapter 5 The affect of differential N and S competition in inter- and sole cropping

of Brassica species and lettuce on glucosinolate concentration. ................................... 49

1. Introduction ............................................................................................................... 50


2.1.1. Root measurements ........................................................................................ 53

2.1.2. Harvest and sample preparation ..................................................................... 53

3. Results ......................................................................................................................... 57

3.1.1. Soil N and S ................................................................................................... 57

2.1. Soil and plant material ......................................................................................... 39

2.2. Leaching – tube incubations ................................................................................ 40

2.3. Pot incubations .................................................................................................... 41

2.4. Sample preparation and Se analysis .................................................................... 42

2.5. Calculations and statistical analysis .................................................................... 42

3.1. Composition of catch crops ................................................................................. 43

3.2. Leaching-tube incubations ................................................................................... 43

3.3. Pot incubations .................................................................................................... 44

2.1. Field Experiment ................................................................................................. 51

2.2. Pot experiment ..................................................................................................... 53

2.3. Glucosinolate Analysis ........................................................................................ 54

2.4. The N and S analysis ........................................................................................... 56

2.5. Statistical analysis ................................................................................................ 57

3.1. The field experiment ............................................................................................ 57

vi

3.1.2. Above ground biomass production ................................................................ 58

3.1.3. Root growth .................................................................................................... 58

3.1.4. N and S accumulation .................................................................................... 59

3.1.5. Glucosinolates ................................................................................................ 61

3.2.1. Dry matter production .................................................................................... 63

3.2.2. N and S accumulation .................................................................................... 63

3.2.3. Glucosinolates ................................................................................................ 64

4. Discussion ................................................................................................................... 67

5. Abbreviations Used ................................................................................................... 70

6. Acknowledgment ....................................................................................................... 70

Chapter 6 Effects of N and S fertilization on root growth ............................................ 71

1. Introduction ............................................................................................................... 71


3. Results and Brief Discussion ..................................................................................... 72

Chapter 7 Conclusions and perspectives ......................................................................... 77

Chapter 8 Bibliography .................................................................................................... 79

3.2. Pot experiment ..................................................................................................... 63

4.1. Field experiment .................................................................................................. 67

4.2. Pot experiment ..................................................................................................... 69

vii

Summary

The health benefits of plant phytochemicals, selenium (Se) and glucosinolates (GSLs), as

well as their potential for reducing the risk of several cancer types, have been demonstrated

by several studies. The most common way to increase Se and GSLs in plants is through using

inorganic fertilizers. The resurgent interest in sustainability requires alternative strategies that

are safer for the consumer and less harmful to the environment. Thus the aim of this project

was to evaluate the efficiency of different crop management strategies for increasing the plant

phytochemicals content.

The use of some catch crops has been found to reduce sulphur (S) leaching and increase S

uptake by the succeeding crops considering Se uptake and assimilation in plants follows the

same pathway as S, similar beneficial effects on Se leaching may be expected from the use of

catch crops. In the first study (Chapter 3) three types of catch crops (Italian ryegrass, fodder

radish and hairy vetch) were evaluated under field conditions for their ability to reduce Se

leaching during winter and for increasing Se concentration in vegetables. The catch crops

were found to be unable to reduce Se leaching as their Se uptake was less than 1% of the total

soil soluble Se. Moreover, the incorporation of catch crops in the field seemed to reduce the

recovery of applied Se and its uptake by onions. Although fodder radish was able to take up

high Se concentrations and to utilize native soil Se more efficient than the other species, it did

not succeed to increase Se concentrations in the vegetables probably due to the high S

mineralization.

Synchronization of Se released from decomposing plant residues with crop uptake is

critical to avoid it leaching from the rooting zone before it is taken up by the crop. The

second study (Chapter 4) investigated how different catch crops (Italian ryegrass, fodder

radish, Indian mustard and hairy vetch), containing different amounts of Se, affect the

bioavailable Se pool and how this changes over the growing period. The results showed that

incorporation of enriched plant material increased both Se leaching from soil columns and Se

concentrations in Indian mustard plants indicating Se mineralization. However, incorporation

of non-enriched plant material seem to cause Se immobilization as both Se leaching from soil

columns and Se concentrations in Indian mustard plants were lower than the unamended soil.

The third study (Chapter 5) investigated the potential of intercropping to enhance GSL

concentration in Brassicas by changing the nitrogen (N) to S nutritional balance.

Glucosinolate concentration was not influenced by broccoli and lettuce intercropping, in the

viii

field. Broccoli was the dominant crop and strongly inhibited the growth of lettuce. By

contrast, in the greenhouse experiment, intercropping increased both aliphatic and indole

GSLs in red leaf mustard when the N:S ratio was lower than 8.

From the results presented, it is suggested that crop management strategies may be an

alternative method to increase Se and GSL concentrations in plants but further work is

required to develop efficient cropping systems. Catch crops did not reduce Se leaching but

incorporation of plant materials may increase Se concentrations in plants. In addition

intercropping may increase GSL concentrations but a careful selection of the plant species

and intercropping design is needed to ensure the development of both species otherwise the

effect will be limited.

ix

Summary in Danish

Flere undersøgelser har vist sundhedsmæssige fordele ved de vegetabilske fytokemikalier,

selen (Se) og glucosinolat, og et potentiale for at reducere risikoen for flere kræftformer. Den

mest almindelige måde at tilføre selen og glucosinolater til planter er gennem uorganisk

gødning, men øget interesse for bæredygtighed har ført til et behov for alternative metoder,

der er sundere for forbrugerne og mindre skadelige for miljøet. Målet med dette projekt var

derfor at vurdere effektiviteten af forskellige afgrødestrategier for at øge indholdet af planters

egne fytokemikalier.

Viden om, at brug af visse efterafgrøder reducerer svovludvaskning og samtidig øger

optagelsen af denne hos de efterfølgende afgrøder, samt at selenoptagelse og tilpasning i

planter følger samme mønster som svovl, gør, at det kan antages, at brugen af efterafgrøder

kan have lignende gavnlige virkninger på selenudvaskningen. I det første markforsøg

(Kapitel 3) blev tre typer af efterafgrøder (italiensk rajgræs, olieræddike og vintervikke)

analyseret for deres evne til at reducere selenudvaskningen i vinterhalvåret og øge

selenkoncentrationen i de efterfølgende grøntsager. Efterafgrøderne blev fundet uegnede til at

reducere selenudvaskningen, da selenoptagelsen i efterafgrøderne var mindre end 1 % af den

tilplantede jords opløselige selen. Hertil kommer, at tilplantningen af efterafgrøder på

området syntes at reducere tilgængeligheden af det tilførte selen og selenoptagelsen i løg.

Selvom olieræddike var i stand til at optage højere selenkoncentrationer og udnytte jordens

naturlige selenindhold mere effektivt end de andre arter, kunne brugen af olieræddike som

efterafgrøde imidlertid ikke øge selenkoncentrationen i de efterfølgende grøntsager. Dette

skyldes sandsynligvis den høje svovlmineralisering.

Det er vigtigt, at der er sammenhæng mellem mængden af selen, der frigives fra nedbrudte

planterester, og den mængde som afgrøden optager, for at undgå selentab ved udvaskning fra

jorden omkring rødderne, før afgrøden har mulighed for at optage det. I det andet forsøg

(Kapitel 4) blev det undersøgt, hvordan forskellige efterafgrøder (italiensk rajgræs,

olieræddike, indisk sennep og vintervikke) indeholdende forskellige mængder af selen

påvirker den plantetilgængelige selenmængde, og hvordan dette ændres i løbet af

vækstperioden. Resultaterne viste, at med indarbejdelsen af beriget plantemateriale i jorden

steg selenudvaskningen fra jordsøjlerne, og koncentrationen af selen i indisk sennepsplanter

angav selenmineralisering. På den anden side syntes tilførelsen af ikke-beriget

plantemateriale at medføre selenimmobilisering, da både selenudvaskningen fra

x

jordsøjlerne/kolonnerne og selenkoncentrationerne i planter af indisk sennep var lavere end

fra jord uden tilført plantemateriale.

I den tredje forsøg (Kapitel 5) blev potentialet ved samdyrkning som metode til at forbedre

glukosinolatindholdet (GSL) i kål (Brassica) undersøgt ved at ændre balancen mellem

kvælstof (N) og svovl (S) i gødningen. Markforsøget viste, at GSL-indholdet ikke blev

påvirket ved samdyrkning af broccoli og salat. Broccoli var den dominerende afgrøde og

hæmmede kraftigt væksten af salat. I modsætning til markforsøget, viste potteforsøg, at

samdyrkning påvirkede indholdet af både alifatisk GSL og indol GSL i rød bladsennep, hvis

N:S-forholdet var lavere end 8.

Baseret på de opnåede resultater, foreslås det, at afgrødestyringsstrategier kan være en

alternativ metode til at øge selen- og GSL-koncentrationerne i planter, men yderligere arbejde

er påkrævet for at udvikle effektive dyrkningssystemer. Efterafgrøder reducerede ikke

selenudvaskningen, men tilførsel af plantematerialer kan øge selenkoncentrationen i planter.

Derudover kan samdyrkning øge GSL-koncentrationen, men en omhyggelig udvælgelse af

plantearter og samdyrkningsdesign er nødvendig for at sikre udviklingen af begge arter, ellers

vil effekten være begrænset.

1 Introduction

Chapter 1

Introduction

Awareness about health and environmental issues continues to grow. This goes hand in

hand with an ageing populations and increased risk for lifestyle diseases. Demand for health-

promoting characteristics in food, produced using sustainable methods, is therefore increasing

(Kearney 2010).

Numerous epidemiological studies have demonstrated that phytochemicals in fruit and

vegetables can significantly reduce the risk of cancer and cardiovascular disease. In these

experiments organic selenium (Se) containing compounds and glucosinolates (GSLs) were

tested (Ellis & Salt 2003; Kawasaki et al. 2008; Verkerk et al. 2009).

The most common practice to enhance Se and GSLs in plants is through mineral

fertilization (Lyons et al. 2004; Broadley et al. 2006; Li et al. 2007; Schonhof et al. 2007a;

Omirou et al. 2009). However, concerns over environmental contamination by fertilizers and

pesticides, coupled with questions over the social, economic, and health-related impacts of

conventional agricultural systems, have prompted improvements in agricultural sustainability

(Liebman 1992). As a result there is growing interest in the design and management of agro

ecosystems that rely primarily on the manipulation of ecological interactions rather than the

application of agrochemicals.

Crop management practices have been shown to influence the concentration of

phytochemicals, such as organic Se compounds and GSLs, in crops (Lyons et al. 2004;

Schreiner 2005). Comprehensive understanding of how crop management strategies can be

used to increase phytochemicals in vegetables is important in environments with low nutrient

sources, where improved utilization of limited resources is required and in organic farming

where the use of inorganic fertilizer is restricted. Knowledge of how crop rotation and catch

crops may affect Se leaching or availability is lacking. Catch crops have been used

successfully to reduce sulphur (S) and nitrogen (N) leaching and increase nutrient availability

for the succeeding crop after being incorporated into the soil (Meisinger et al. 1991; Thorup-

Kristensen 1994; Eriksen & Thorup-Kristensen 2002; Eriksen et al. 2004; Thorup-Kristensen

2006b). Based on the chemical similarities between Se and S, selenate is taken up through

high affinity sulphate transporters and follows the same assimilation pathways as S in plants

2 Introduction

(Terry et al. 2000). Similar beneficial effects on Se may be expected from the use of catch

crops.

Intercropping is an old and widespread practice in low input cropping systems in the

tropics (van Noordwijk & Cadisch 2002). However, most studies on intercropping focus on

crop yield and the emphasis in work from temperate regions has been on legume-cereal

intercropping systems and their effect on N dynamics (Hauggaard-Nielsen et al. 2008). Root

system morphology and distribution are fundamental in determining the scale of below

ground interspecific competition and facilitation in intercropping systems (Hauggaard-

Nielsen & Jensen 2005). Nitrogen and S interaction have been found to influence GSL

concentrations in plants (Zhao et al. 1994; Li et al. 2007; Schonhof et al. 2007a; Omirou et al.

2009). Thus, controlled interspecific competition may be a useful tool for manipulating the

balance of nutrient in the soil and enhance GSL concentrations in plants.

The objectives of this research was (1) to evaluate the ability of catch crops to reduce soil

Se content and leaching, (2) to determine if catch crops can increase Se availability and

uptake in vegetables; and (3) to influence the S and N balance in Brassicas’ nutrition by

intercropping to increase GSL concentration.

3 Literature review: Selenium

Chapter 2

Literature review

1. Selenium

1.1. Selenium in soils

1.1.1. Selenium mineralogy

Selenium was discovered by a Swedish chemist, Jöns Jakob Berzelius in 1817 and it ranks

thirty-fourth among elements in the Earth's crust. Selenium is classified in the oxygen group

element (group VIA) of the periodic table. The group VIA also includes the non-metals, S

and oxygen, in the periods above Se; and the metals, tellurium and polonium, in the period

below Se (Combs & Combs 1986; Fordyce 2005). By period, Se lies between the metal

arsenic and the non-metal, bromine. Thus, Se is considered a metalloid, having physical and

chemical properties that are intermediate between those of metals and non-metals. Elemental

Se, like S and tellurium, can exist in either an amorphous state or in one of three crystalline

forms (Combs & Combs 1986). Selenium occurs in nature at six stable isotopes and can exist

in multiple oxidation states (valence states) including -2, 0, +4, and +6 (Combs & Combs

1986; Fordyce 2005).

The chemical and physical properties of Se are very similar to those of S. The two

elements have similar atomic sizes and outer-valence shell electronic configurations. In

addition their bond energies, ionization potentials and electron affinities are practically the

same (Combs & Combs 1986). Despite these similarities, the chemistry of Se and S differ in

two respects which distinguish them in biological systems. Firstly, S compounds tend to

undergo oxidation, while Se compounds are metabolized to more reduced stages. The second

difference is in the acid strengths of their hydrides, H2Se is much more acidic than H2S

(Combs & Combs 1986).

Selenium is a naturally occurring element that is widely distributed in rock and soil. The

main natural sources of Se are from volcanic action and the weathering of sediments and rock

from the Carboniferous, Triassic, Jurassic, Cretacean and Tertiary ages (Girling 1984).

Anthropogenic sources of Se arise from metal processing; fossil fuel combustion (coal and

oil); disposal of sewage sludge; and applications of fertilizer, lime and manure (Fordyce

2005). On average, soil contains from 0.01 to 2 mg Se kg-1, but this concentration can be

4

highly v

the Gre

concent

and thu

United

naturall

1.6 mg

Figure 1from Rei

1

1

Spec

element

selenide

The typ

chemica

(Figure

variable (Fi

eat Plains

trations in t

us these regi

Kingdom,

ly low (Old

kg-1, with a

1-1. Seleniumimann et al. (2

1.1.1. Biotic

1.1.1.1. Abi

ciation of th

t in the soil

e (Se2), elem

pe of Se fou

al factors,

e 1-2) (Gir

igure 1-1)

of the U

he soil are s

ions suppor

Finland an

dfield 1987)

a mean 0.5 m

m in (a) top (02003) cited by

c and abioti

iotic process

he chemical

l and the co

mental Se (S

und is a resu

including p

ling 1984;

(Mayland 1

SA, Canad

sufficiently

rt a unique f

nd some ar

). In Denma

mg kg-1 (Bis

0–25 cm) and y Johnson et al

ic processes

ses

l forms of S

omplexities

Se0) and org

ult of its oxi

pH, adsorb

Dungan &

1994). In so

da, South

y high (30-3

flora. In co

reas of Chi

ark total soi

sbjerg 1972

d (b) bottom (l. (2010)).

s affecting S

Se is difficu

of soil syst

ganic Se are

idation state

bing surface

& Frankenbe

ome regions

America, A

24 mg Se k

ntrast, in co

ina, the ava

il Se concen

2).

(50–75 cm) so

Se availabili

ult due to v

tems. Selen

e the forms

e, which de

e, organic m

erger 1999

Literature

s of the wor

Australia, I

kg-1) to be to

ountries suc

ailability of

ntrations ran

oils from nort

ity in soil

very low co

nate (SeO42-)

in which Se

epends on va

matter and

; Fordyce 2

review: Sel

rld (parts o

India, Rus

oxic to man

ch as New Z

f Se in the

ange betwee

thern Europe.

oncentration

), selenite (

e occurs in

various phys

microbial

2005; Whit

lenium

f China,

sia), Se

ny plants

Zealand,

e soil is

en 0.1 to

. (adapted

ns of the

(SeO32-),

the soil.

sical and

activity

te et al.


2007a). Even so soils with relative high Se content can be deficient if Se is not in an available

form. Hawaiian and Puerto Rican soils, which are produced from Se rich rock under acid and

moist conditions, contain high concentrations of Se but very low Se amounts are water

soluble (Lakin et al. 1938; Combs & Combs 1986). Selenium solubility generally decreases

with decreasing pH and with increasing content of organic matter, clay and iron

oxides/hydroxides (Yamada et al. 1998). Selenate and selenite are the predominant forms of

Se in the soil and are available for plants. Selenate is the major form present under oxidizing

and alkaline soil conditions. In columns filled with fine loamy calcareous soils selenate is

much more mobile than selenite and selenomethionine (Alemi et al. 1991). In acid and

neutral soils, selenite predominates. In a soil column study, the addition of lime increased the

movement and leaching of selenite (Gissel-Nielsen & Hamdy 1977). Selenite is less mobile

than selenate due to the inner-sphere co-ordination of selenite with oxides of iron and

manganese, which are commonly present in soils (Combs & Combs 1986; Tam et al. 1995).

Figure 1-2. Schematic diagram showing the main controls on chemical speciation and bioavailability of selenium in soils. Increasing mobility (adapted from Fordyce (2005)).

Organic matter may act as an electron source facilitating the reduction of selenate to

selenite and hence reduce Se availability in soil (Fordyce 2005). In a leaching incubation

Organic matter, clay, iron oxides

60 128 10 2 4

HSe- Se0 Se is largely

immobile

Se0

SeO32-

SeO32-

SeO42-

Selenate is soluble+

Selenite binds

strongly to Fe-oxides +

clay minerals,

SeO42-

Se0 SeO32-

pH

Oxidizing

Reducing

Redox


experiment, when non-enriched plant material was incorporated into the soil Se

concentrations in leachate were lower than in bare soil, indicating Se immobilization

(Chapter 4, this thesis). Selenate was transformed to reduced and less mobile forms when C

was added to the soil (Neal & Sposito 1991; Alemi et al. 1991). The addition of organic

matter to the soil also decreased the movement of selenite through the column as well as the

amount of leached selenite leading to organically bound Se (Gissel-Nielsen & Hamdy 1977).

Gustafsson and Johnsson (1992) showed that selenite fixation occurred rapidly and it bound

to the top 2 cm of the Oi horizon of a forest floor. Selenium binds, in chelated form, to fulvic

acid, proteins, and other organic compounds, which are constantly produced by soil

microorganisms when organic matter is added (Hamby & Gissel-Nielsen 1976). In a batch

experiment, cattle manure in combination with the addition of selenite and selenate reduced

their adsorption in the soil (Falk Øgaard et al. 2006). Controlling factors may also interact,

e.g. the organic matter content of the soil may affect the pH effect (Falk Øgaard et al. 2006;

Eich-Greatorex et al. 2007).

The presence of ions, such as sulphate or phosphate, can influence the availability of Se by

competing for fixation sites in the soil (Dhillon & Dhillon 2000; Fordyce 2005). Phosphate

may reduce selenite adsorption on soil solid surfaces due to competition for binding sites. It is

more strongly adsorbed than selenite and thus make Se more plant-available (Dhillon &

Dhillon 2000; Eich-Greatorex et al. 2010). The presence of sulphate may decrease the

adsorption of Se in the soil (Dhillon & Dhillon 2000).

1.1.1.2. Biotic transformations

Selenium is predominantly cycled by biological pathways similar to that of S. The

biological transformations of Se which are known to occur are: reduction, oxidation,

methylation and demethylation (Figure 1-3). Microorganisms can use selenate and selenite as

terminal electron acceptors during the respiration of organic carbon and produce elemental Se

(dissimilatory reduction). The dissimilatory reduction of selenate via selenite to elemental Se

has been shown to be a significant and rapid environmental process. Three bacteria species

which are able to respire selenate have been well-characterized, Thauera selenatis, Bacillus

arsenicoselenatis and Sulfurospirillum barnesii (Schröder et al. 1997; Blum et al. 1998; Stolz

& Oremland 1999). On the other hand, Bacillus selenitireducens is a selenite respiring

bacteria (Stolz & Oremland 1999; Oremland et al. 2004). All the above species can reduce Se

oxyanions to elemental Se and accumulations of this element are exogenous, occurring


outside of the cell envelope in the surrounding growth milieu rather than as internalized

precipitates or structures (Stolz & Oremland 1999). Selenate is transported into the

microorganisms’ cell by sulphate permeases, while selenite is transported by distinct

permeases. In the cell both selenate and selenite undergo assimilatory reduction to selenide

ions, which can then be incorporated into cellular proteins (Dungan & Frankenberger 1999).

Very little information is available about the oxidation of elemental Se and other reduced

forms of Se possibly occur in a manner similar to that of S. However, oxidation of Se is

usually considered as a slow phenomenon, so elemental Se appears to be stable in soil.

Except for microbial action, Se is not readily oxidized to a form that can be taken up by

plants (Dungan & Frankenberger 1999).

Figure 1-3. Schematic Se cycle in soil (adapted from Flury et al. (1997)).

Methylation is thought to be a protective mechanism used by microorganisms to detoxify

their surrounding environment. Bacteria and fungi are the predominant groups of Se-

methylating organisms isolated from soil. The volatile Se compound, dimethylselenide

(DMSe), is the major metabolite of Se volatilization (Dungan & Frankenberger 1999). The

removal of a methyl group from the central atom of a methylated compound is defined as

demethylation. Several soil microorganisms are found of be capable to demethylating volatile

Se compounds (Dungan & Frankenberger 1999).

Insoluble forms Metal Selenides Se0

Microorganisms

Soluble forms SeO4

2-

SeO32-

Immobilized forms Se0

Se2- Organic Se

Oxidation

Reduction

Reduction

Oxidation

Soil

Atmosphere

Methylation

Demethylation

DMSe (CH3)2Se

Leaching

Capillary rise


Many authors have studied the influence of these microbial processes on the Se cycle in

soil, however most of these studies concern soils and sediments with high concentrations of

Se (Stolz & Oremland 1999; Dungan & Frankenberger 1999). Much less is known about the

transformations of Se at lower levels of this element, when both sorption reactions and

biologically mediated redox reactions may be very different.

1.2. Selenium in plants

1.2.1. Selenium levels in plants and their effects

Plants can accumulate a significant amount of Se in their tissues even though it is not

required for their metabolism. Selenium accumulation differs among plant species. According

to their ability to accumulate Se, they can be divided into three categories: “Se-accumulator”;

“Se-indicator”; and “non-accumulator” plants. Several species of the genera Astragalus,

Neptunia, Oonopsis, Morinda, Stanleya and Xylorhiza grow on naturally-occurring

seleniferous soils and can accumulate from hundreds to several thousand milligrams of Se kg-

1 dry weight in their tissues. These plants are referred to as Se accumulators. On the other

hand, Se non-accumulators, which include most of our agricultural forage and arable crop as

well as grasses, contain less than 25 mg Se kg-1 dry weight. The third category of plants,

known as Se-indicators, can grow adequately in both seleniferous and non-seleniferous soils,

and can accumulate up to 1000 mg Se kg-1 dry weight without consequence. Examples of

plants in this group are members of the genera Aster, Astragalus, Atriplex, Brassica,

Castilleja, Comandra, Grayia, Grindelia, Gutierrezia, Machaeranthera, Mentzelia, and

Sideranthus (Terry et al. 2000; White et al. 2004).

Whether Se is essential to higher plants is still a controversial issue. However, there are

indications that Se might be an vital micronutrient for accumulator plants (Trelease &

Trelease 1938). Although there is no evidence for Se requirement in non accumulator plants,

numerous studies report that at low concentrations Se exerts a beneficial effect on growth.

Probably the first positive effect of Se on plant growth was reported by Singh et al. (1980)

who showed that low level applications of Se as selenite stimulated growth and dry-matter

yield of raya. The growth-promoting response to Se was also demonstrated in lettuce (Xue et

al. 2001); ryegrass (Hartikainen et al. 2000); potato (Turakainen et al. 2004); green tea (Hu et

al. 2003); rice (Liu et al. 2004); soybean (Djanaguiraman et al. 2005); and Indian mustard

(Chapter 4, this thesis). Several studies have shown that Se has dual effects. Its protection

against oxidative stress in higher plants coincided with increased GSHPx activity. As pro-


oxidant, it increased the accumulation of lipid peroxidation products (Hartikainen et al. 2000;

Xue et al. 2001).

Selenium supply also alleviates UV-induced oxidative damage in lettuce, strawberry and

ryegrass (Hartikainen et al. 2000; Xue et al. 2001; Valkama et al. 2003); improved the

recovery of chlorophyll from light stress (Seppänen et al. 2003), increased the antioxidative

capacity of senescing lettuce, ryegrass and soybean (Xue et al. 2001; Djanaguiraman et al.

2005); enhanced salt-resistance in sorrel and cucumber seedlings (Kong et al. 2005;

Hawrylak-Nowak 2009); and improved the recovery of potato plants from light and chilling

stress (Seppänen et al. 2003). Moreover, Se supply has been shown to promote growth of

wheat seedlings during drought stress and increase root activity. Likewise it also increases

proline concentration, peroxidase and catalase activity, carotenoids concentration,

chlorophyll concentration and reduced malondialdehyde (Yao et al. 2009). Chu et al. (2010)

and Hawrylak-Nowak et al (2010) reported that plants treated with Se and subjected to low

temperature generally grew better than plants grown without the addition of Se. Similar

results were found by Djanaguiraman et al. (2010) in sorghum grown under high temperature

stress conditions. Low dose of Se as sodium selenite was also associated with a 43% increase

in seed production in fast cycling B. rapa (Lyons et al. 2009). In green tea, application of Se

enhanced total amino acid and vitamin C concentration but decreased polyphenol

concentration (Hu et al. 2003). Nevertheless the exact physiological and molecular

mechanisms that govern the beneficial effects of Se in plants have not yet been fully

explained.

1.2.2. Selenium uptake and assimilation by plants

The physical and chemical similarities of Se and S help explain parallels in their

metabolism in plants. Plant selenate and selenite uptake has been considered analogous to

that of sulphate and sulphite, respectively. Both selenate and sulphate enter root epidermal

cells across the plasma membrane through sulphate transporters against their electrochemical

gradients, with uptake being driven by the co-transport of three protons for each ion.

Assimilation of sulphate from the soil solution occurs through the use of high and low

affinity transporters that are localized in root epidermal and cortical cells. Strong evidence

supports the idea that selenate uptake from the soil is through high-affinity sulphate

transporters in plants. (Terry et al. 2000; Sors et al. 2005).


Unlike selenate, there is no evidence to suggest that selenite uptake is mediated by

membrane transporters. Both selenate and organic Se compound absorption in plants from the

soil solution are active processes, whereas selenite seems to accumulate through passive

diffusion and can be inhibited by phosphate (Terry et al. 2000; Sors et al. 2005). A recent

report suggests that selenite uptake in wheat is also an active process, mediated by proton-

coupled phosphate transporters (Li et al. 2008).

The transport of Se from roots to shoots is considered to occur via the xylem. Plants

transport selenate to leaves where they accumulate substantial amounts, but much less

selenite or selenomethionine is stored. Selenite is rapidly reduced to organic forms of Se

(selenomethionine) in plants which is retained in the roots (Terry et al. 2000; Sors et al.

2005). The distribution of Se in various parts of the plant differs according to species, growth

stage, and the physiological condition of the plant. In Se-accumulator plants, selenate is

concentrated in older leaves whilst organic Se compounds, such as methylselenocysteine is

located in the youngest tissue (Terry et al. 2000; Pickering et al. 2003; Sors et al. 2005). On

the other hand, non-accumulator plants concentrate Se mainly in roots and seeds whilst only

small amounts are found in the stems and leaves (Sors et al. 2005).

The reason that plants differ in their ability to tolerate high tissue concentrations is thought

to be a consequence of variations in their Se metabolism (Terry et al. 2000; Ellis & Salt

2003). Both selenocysteine and selenomethionine can be incorporated into proteins, which

may influence their stability and functional activities. This is thought to account for Se

toxicity in non-accumulator plants. In the Se-tolerant accumulator plants the formation of

selenomethionine and selenocysteine appears to be restricted. Selenium is accumulated in

non-protein amino acids such as Se-methylselenocysteine, selenocystathionine and the

dipeptide γ-glutamyl-Se-methylselenocysteine (Brown & Shrift 1982; Terry et al. 2000).

Selenium enriched garlic contains Se-methylselenocysteine and γ-glutamyl-Se-

methylselenocysteine, which inhibits tumerogenesis. Furthermore, broccoli, onion and radish

grow in soils with high Se concentrations and can convert much of the Se into the amino

acids selenomethionine, Se-methylselenocysteine and selenocysteine (Irion 1999; Finley

2003; Abdulah et al. 2005; Arnault & Auger 2006; Pedrero et al. 2006).

1.2.3. Factors that affect Se uptake by plants

The uptake of Se by plant roots is influenced by the chemical form and concentration of

Se in the soil solution; soil redox conditions; pH of the rhizosphere; and the presence of


competing anions, such as sulphate and phosphate (White et al. 2004; Sors et al. 2005; White

et al. 2007a).

1.2.3.1. Selenium form

Plants acquire Se from the soil predominantly as selenate, as well as selenite and organic

compounds, such as the amino acids selenocysteine and selenomethionine. At the same

concentrations of Se, selenate uptake by plant roots is generally greater than that of selenite

(Zayed et al. 1998; de Souza et al. 1998; Hopper & Parker 1999; Zhao et al. 2005). Total

accumulated Se in Indian mustard (Brassica juncea) roots and shoots was approximately 10

times higher from selenate than from selenite (de Souza et al. 1998). Organic forms of Se

may be more readily available for plant uptake than inorganic forms. Alternatively colloidal

elemental Se and selenide are not available to plants (Kopsell & Randle 1997; Zayed et al.

1998; de Souza et al. 1998; White et al. 2004; Sors et al. 2005; White et al. 2007a).

1.2.3.2. Competing ions

The antagonistic interaction between S and Se for plant uptake has long been noted by

researchers (Girling 1984; Mikkelsen & Wan 1990; White et al. 2004; White et al. 2007a; Li

et al. 2008). Gene expression of the high affinity sulphate transporters is regulated by the S

status of the plant, as well as by the regulators glutathione (GSH) and O-acetylserine (OAS).

Short periods of S starvation, low levels of GSH and high levels of OAS increase

transcription of the high affinity transporter genes as well as sulphate uptake (Terry et al.

2000; Anderson & MeMahon 2001; Sors et al. 2005; Hawkesford & Zhao 2007). Increase of

the high affinity transporter genes can potentially increase selenate uptake (Terry et al. 2000;

Berken et al. 2002; White et al. 2004). The presence of sulphate in the rhizosphere inhibits

selenate uptake and accumulation suggesting direct competition between selenate and

sulphate for transport or the repression of transcription of sulphate transporter genes by

sulphate and its metabolites (Vidmar et al. 2000; White et al. 2004). In contrast to this

antagonistic relationship a synergistic one between S and Se has been reported. Studies in

onions, rice and wheat have shown that low concentrations of Se enhanced S uptake and

accumulation (Mikkelsen & Wan 1990; Kopsell & Randle 1997). Furthermore, the presence

of abundant sulphate can ameliorate the phytotoxic effects of excessive Se and prevent yield

reduction (Mikkelsen & Wan 1990).


Data suggests that during phosphorus starvation selenite uptake is increased. This implies

a role for the phosphate transport pathway in selenite uptake (Li et al. 2008). An antagonistic

effect between phosphorus and Se has been noted (Hopper & Parker 1999; Li et al. 2008).

Ten times more phosphate in the soil than the normal causes a decrease in Se concentration of

about 50% in both roots and shoots in ryegrass and 20% in roots of strawberry clover

(Hopper & Parker 1999). Li et al. (2008) showed that phosphorus starvation resulted in 60%

increase in selenite uptake by wheat, possibly because phosphorus starvation up-regulated the

expression of the phosphate transporter genes.

1.2.3.3. Organic matter

Organic matter affects Se adsorption in the soil and subsequently Se availability and

uptake by plants. Cattle slurry applied with selenate increased Se concentration in wheat

grains at the high pH levels in both peat and loam soils (Falk Øgaard et al. 2006). A trend

towards lower Se concentrations in wheat was observed when Se-rich fish silage was added

compared to the control (Sogn et al. 2007). The incorporation of catch crop plant material,

grown in non-seleniferous soil, decreased Se concentration in Indian mustard plants

compared to unamended soil (Chapter 4, this thesis). Similar results were found by Ajwa et

al. (1998), where the addition of crop residues or animal manure in selenate treated soils

considerably reduced Se uptake by canola and tall fescue.

1.2.4. Selenium concentrations in vegetables and its bioavailability to humans

Vegetables usually contain less than 0.1 mg Se kg-1. However when grown in seleniferous

soil, they can contain up to 6 mg kg-1 (Rayman 2008). In Denmark Se concentrations in

vegetables vary from 0.05 to 6.5 μg per 100 g of fresh weight of the edible part. It is

interesting to note that mushrooms contain the highest Se concentrations, followed by

Cruciferae and Allium species (Danish Food Composition Databank 2008). In order to

promote human health, Se has become the focus of functional food development. Selenium

enriched broccoli, garlic, onions, celery and Brassica sprouts produced by various Se

fertilizations can contain several hundred mg Se kg-1 of dry weight (Kopsell & Randle 1997;

Pyrzynska 2009).

The bioavailability and benefit to human health of dietary Se depends not only on the

amount but also the chemical forms of Se supplied. The dominant organoselenium

compounds differ between plant species. Some vegetables contain high concentrations of


organoselenium compounds that are particularly beneficial to human health. Selenium

displays anti-carcinogenic potential through its incorporation into various selenoenzymes,

which function to reduce free radical injury to cells (Irion 1999). Many Allium (A. cepa L., A.

sativum L., A. schoenoprasum L., etc.) and Cruciferae species (Brassica juncea and B.

oleracea) are able to incorporate high quantities of Se and to produce selenoamino acids,

which are potentially bioactive for nutrition purposes and phytoremediation and are normally

implicated S pathways (Arnault & Auger 2006; Pedrero et al. 2006).

The initial assumption was that the active Se compound against cancer was

selenomethionine, the main Se compound found in cereals. Recent studies have demonstrated

that Se-methylselenocysteine, γ-glutamyl-Se-methylselenocysteine and methylselenic acid

are anti-cancer agents with similar action mechanism (Abdulah et al. 2005). Stable

methylated Se compounds such as selenobetaine or Se-methylselenocysteine serve as

precursors and release methylselenol or methylselenenic acid through the action of cysteine

conjugate β-lyase or related lysases. The monomethylated Se compounds are effective in

vitro at very low concentrations in order to have chemopreventive effects (apoptosis and cell

cycle arrest) in transformed cells (Keck & Finley 2004; Abdulah et al. 2005).

1.3. Selenium essential for humans

Selenium is an essential nutrient for humans, animals and microorganisms. Selenium was

originally considered only its toxic capabilities but the potential health benefits of some Se

compounds have prompted further study of Se (Ellis & Salt 2003).

Selenium is an essential component of more than 30 mammalian selenoproteins or

selenoenzymes. At least fifteen selenoproteins have been characterized for their biological

functions. Selenoproteins can be subdivided into groups based on the location of

selenocysteine in the selenoprotein polypeptides. Such as glutathione peroxidases (GSHPx)

and thioredoxin reductases, which are involved in controlling tissue concentrations of highly

reactive oxygen-containing metabolites and iodothyronine deiodnases types I, II, III that are

involved in the production of active thyroid hormones (Abdulah et al. 2005; Hawkesford &

Zhao 2007). Selenium is associated with reduced risk of cardiovascular disease; optimal

functioning of the immune system; the male fertility; the slower progression of AIDS and a

number of other diseases (Rayman 2000). Increasing evidence points to the anti-carcinogenic

potential of Se-compounds, such as Se-methylselenocysteine and γ-glutamyl-Se-

methylselenocysteine, which have been shown to provide chemo protective effects against


certain types of cancer in humans (Rayman 2000; National Academy of Sciences. Institute of

Medicine. Food and Nutrition Board, 2000; Abdulah et al. 2005; Arnault & Auger 2006).

The first report of Se deficiency in humans occurred in China. Keshan disease is a

cardiomyopathy of children and young women of childbearing age. Another Se-responsive

disease reported in children in China, and less extensively in south-east Siberia, is Kaschin-

Beck disease. It is an osteoarthropathy, characterized by joint necrosis and epiphyseal

degeneration of the arm and leg joints resulting in structural shortening of the fingers and

long bones with consequent growth retardation and stunting (Tinggi 2003).

There is a fine line between the harmful and the beneficial effects of Se in humans.

Selenium toxicity in humans is rare. However the effects of Se toxicity reportedly cause hair

loss; skin lesions; vomiting, nausea; abnormalities in the beds of the fingernails and fingernail

loss; hypo-chronic anaemia and leucopenia (Tinggi 2003).

1.4. Selenium human intake

Geographic differences in the content and availability of Se in soil for food crops and

animal products has a marked effect on the Se status of entire communities (Combs 2001).

Selenium levels in blood and blood plasma and the activities of GSHPx in blood plasma are

common biomarkers used to assess Se status in humans. The American Recommended

Dietary Allowance (RDA), which is based on Se levels considered to be necessary to achieve

plateau concentrations of plasma GSHPx and maximize GSHPx activity, is 55 μg Se day-1 for

both women and men (National Academy of Sciences. Institute of Medicine. Food and

Nutrition Board, 2000). In several EU countries the RDA differs. For example in Nordic

countries it is 40 and 50 μg Se day-1 whilst in UK it is 60 and 70 μg Se day-1 for females and

males, respectively (Nordic Council of Ministers 2004; Broadley et al. 2006). However, there

is growing evidence for further cancer prevention of Se at even higher intake rates. Clark et

al. (1996) demonstrated that dietary supplements of 200 μg of Se day-1 significantly

decreased the incidences of non-skin cancers; carcinomas; prostate; colorectal and lung

cancers; as well as mortality due to lung and total cancers.

According to World Health Organization (WHO) the Tolerable Upper Intake Level for Se

pertains to Se intake from food and supplements is 400 μg day-1 for adults (National

Academy of Sciences.Institute of Medicine.Food and Nutrition Board. 2000). Toxic effects of

Se were observed in people with a blood Se concentration greater than 12,7 μmol L-1. This


corresponds to a Se intake above 850 μg day-1 (National Academy of Sciences.Institute of

Medicine.Food and Nutrition Board. 2000).

Selenium intake in Sweden and Denmark is below Nordic Nutrition Recommendations

2004 (Nordic Council of Ministers 2004; Rayman 2008). In Finland in the mid-1970s, daily

Se intake was 25 μg day-1. However since the introduction of a the nationwide Se fertilization

policy Se has reached a plateau of 110-120 μg day-1 (Varo 1993).

1.5. Strategies to increase Se human intake

Increased human Se intake may be achieved in several ways, with strategies involving

consumption of foods that naturally contain high levels of Se. Brazil nuts, offal, fish and

shellfish are naturally rich food sources of Se, but the content is highly variable. Nevertheless

consumers should be aware that Brazil nuts also contain high amounts of barium. Moreover,

in Western countries, Se supplements are available in both inorganic and organic forms.

However, studies suggest that dietary sources of Se or supplements based on organic forms

are more bioavailable and so effective than inorganic supplements (Rayman 2008).

Direct fortification of food during processing with Se inorganic salts is a resource-saving

way to improve human Se intake. Both inorganic and organic Se forms can be used as food

supplements (Haug et al. 2007). Direct Se supplementation of livestock with inorganic Se or

via Se rich pasture will secure the Se requirement for the animal itself. Thus preventing Se

deficiency disease and also increasing the Se concentration of any animal products (Muñiz-

Naveiro et al. 2006; Haug et al. 2007).

Selenium enriched fertilizers are commonly used to increase plant Se concentrations.

Selenium is added to fertilizer mainly as selenate (Broadley et al. 2006). The best example of

agronomic biofortification of crops comes from Finland. The use of Se enriched multielement

fertilizer has been mandatory there since 1984. Selenium enriched fertilizer raised the Se

content in crops and subsequently the Finland’s Se intake (Varo 1993). Initially, fertilizer was

supplemented on two Se levels: for forage production at 6 mg and for cereal production at 16

mg Se kg-1. In 1990 the Se level was reduced to 6 mg Se kg-1, to avoid the risk of too high Se

intake and excess in the environment (Broadley et al. 2006). Recovery of applied Se is

usually 20-50% (Broadley et al. 2010, Chapter 3, this thesis), but in shallow rooted crops it

can be as little 0.5-4% (Chapter 3, this thesis). The fate of residual Se in the soil is unknown.

It may be leached, volatilized or retained in the soil in reduced forms such as elemental Se or

selenite.


Exploiting the genetic variability in crop plants for Se accumulation may be an effective

method for improving Se intake in humans (Lyons et al. 2005; Broadley et al. 2006).

Breeding plant and crop varieties with enhanced Se-accumulation characteristics to raise Se

levels in the human diet may be an alternative to the Se fertilization.

1.6. Catch crops

In temperate climatic zones during the autumn, after the main crops are harvested

temperature and light conditions allow some plant growth, though not enough to produce

commercial crops. Many attempts have been made to use this period to grow plants to

prevent nutrient leaching; affect nutrient availability; increase soil biological activity and

water content; influence the appearance of pests, pathogens and weeds; and improve soil

physical properties (Thorup-Kristensen et al. 2003).

Recent research in catch crops has focused on their effects on N. It has been demonstrated

that catch crops take up N from the soil and thereby reduce leaching. Incorporating catch

crops into the soil increases N availability for succeeding crops (Thorup-Kristensen 1994).

However, in order to maximize the effects of catch crops the local climate, soil type, main

and catch crop species and farming system must be considered (Thorup-Kristensen 1994;

Thorup-Kristensen 2001; Thorup-Kristensen et al. 2003; Thorup-Kristensen 2006b).

Eriksen and Thorup-Kristensen (2002) demonstrated that catch crops may influence soil

sulphate distribution and reduce sulphate leaching as for N. It has been found that Brassica

species, which usually have a high plant S concentration, can take up 22-36 kg S ha-1, whilst

Italian ryegrass took up only 8 kg S ha-1 (Eriksen & Thorup-Kristensen 2002). This is also

confirmed in the S availability effect on the succeeding crop, S mineralization rates were

higher for Brassicas compared to legumes (Eriksen & Thorup-Kristensen 2002; Eriksen et al.

2004). Selenium behaves similarly to sulphate in the soil system, and it can easily be lost via

leaching in the form of selenate. Catch crops may also exert a significant influence on Se

availability, through Se leaching or its availability for the succeeding crop (Chapter 4, this

thesis).

17 Literature review: Glucosinolates

2. Glucosinolates

2.1. General

Glucosinolates are a group of more than 120 secondary plant metabolites found

throughout several plant families, including Brassicaceae, Capareaceae and Caricaceae

(Fahey et al. 2001). Glucosinolates are S rich, anionic natural products that produce several

different products upon hydrolysis by myrosinases. The breakdown products of GSLs

contribute to plant defence mechanisms, human and livestock health, and the sensory quality

of vegetables (Halkier & Gershenzon 2006). Glucosinolates are classified, depending on their

precursor amino acid, into: aliphatic GSLs, derived from alanine, leucine, isoleucine,

methionine, or valine; aromatic GSLs, derived from phenylalanine or tyrosine; and indole

GSLs, derived from tryptophan (Fahey et al. 2001; Halkier & Gershenzon 2006). Although

GSLs represent a chemically diverse class of plant secondary compounds, the formation of

these compounds consists of three major steps: (a) side chain-elongation of amino acids, (b)

development of the core glucosinolate structure and (c) secondary side-chain modifications of

GSLs (Halkier & Gershenzon 2006).

Glucosinolates occur in all plant parts, but in different concentrations and profiles. Up to

15 different GSLs can be found in the same plant species but usually a maximum of four

different GSLs is present in significant amounts (Verkerk et al. 2009). Glucosinolate

concentration in plants is about 1% of dry weight, although concentrations are highly variable

and can be up to 10% in the seeds of some plants (Kushad et al. 1999; Fahey et al. 2001;

Verkerk et al. 2009).

2.2. Role in human health

Consumption of Brassica vegetables such as broccoli, turnip, cabbage, cauliflower and

kale has been linked to reduced risk of several types of cancer (Verkerk et al. 2009;

Björkman et al. 2011). The anticarcinogenic activity of GSLs is thought to be due to the

ability of certain hydrolysis products to induce phase II detoxification enzymes, such as

quinine reductase, glutathione-S-transferase and glucuronosyl transferased (Halkier &

Gershenzon 2006). Furthermore, of the different dietary derived glucosinolate subgroups,

aliphatic GSLs (like glucoraphanin, sinigrin and glucoiberin) as well as aromatic GSLs

showed the strongest inverse association with cancer risk (Björkman et al. 2011).


2.3. Factors affecting plant levels

2.3.1. Genotype

Genotypic differences in glucosinolate concentrations and profiles between crop species

and cultivars are well documented (Kushad et al. 1999; Verkerk et al. 2009). Work

characterizing the genetic regulation of glucosinolate was initially done to reduce levels in

the seeds of Brassica oil-crops, in an effort to decrease potential toxicants in animal feed

supplements (Halkier & Gershenzon 2006). With this information, breeders have developed

the so-called “single-low’’ and “double-low’’ lines that contain reduced concentrations of

glucosinolate in the seed (Scherer 2001). Moreover, breeding has been used to enhanced the

health promoting glucosinolate in Brassica vegetables (Verkerk et al. 2009).

2.3.2. Temperature and light

A number of studies have shown that growth temperatures clearly influence the

glucosinolate content of many species in the Brassicaceae. Plants exposed to high or low,

rather than optimal intermediate growth, temperatures produce the highest levels (Schreiner

2005; Björkman et al. 2011). Young cabbage plants contain higher glucosinolate

concentrations in their roots and higher diurnal variation at 30 oC than at 20 oC (Rosa &

Rodrigues 1998). However studies of broccoli heads showed that aliphatic GSLs increased

with decreasing temperatures lower than 12 oC (Schonhof et al. 2007b). In contrast, when

exposing greenhouse-grown plants to cold (0–12 oC) night temperatures, Shattuck et al.

(1991) found 29% decrease of the overall glucosinolate concentration of the peel root tissue

of turnip compared to normal growth conditions.

Irradiance and photoperiod also affect glucosinolate concentration in plants. Long

photoperiods typical at high latitudes during summer, have a positive effect on glucosinolate

content (Björkman et al. 2011). In broccoli plants aliphatic GSLs increased at moderated

mean daily radiation (10–13 mol m-2 day-1) (Schonhof et al. 1999; Schonhof et al. 2007b),

whereas indole GSLs were higher at low irradiation (Schonhof et al. 2007b). In five B.

oleracea botanical groups total and indole GSLs had a negative linear but positive quadratic

relationship with temperature and day length; and a positive linear but negative quadratic

relationship with photosynthetic photon flux. Glucoraphanin concentrations were influenced

by average photosynthetic photon flux and day length, but not by temperature (Charron et al.

2005).


2.3.3. Water availability

Many Brassicas grown under water deficiency have higher glucosinolate concentration

than those grown under favourable conditions (Rosa et al. 1996; Schreiner 2005; Radovich et

al. 2005; Zhang et al. 2008; Björkman et al. 2011). Higher glucosinolate concentrations were

found in cabbage when they were not irrigated during head development (Radovich et al.

2005). Ciska et al. (2000) found higher glucosinolate concentrations in cultivars of B.

oleracea, B. rapa and Raphanus sativus in years with hot and dry summers. Zhang et al.

(2008) reported that turnip, which grew during the spring summer season and received 25%

available soil water, had higher levels of total and individual GSLs compared to the 50% and

75% available soil water treatments. Rapeseed glucosinolate concentrations were found to

increase linearly at midday water potential below –1.4 MPa (Jensen et al. 1996). It has been

proposed that increased synthesis of amino acids and sugars, precursors in biosynthesis of

GSLs, during drought and the influence of S uptake are possible the reasons for this response

(Ciska et al. 2000; Zhang et al. 2008).

2.3.4. Nutrient supply

Glucosinolate concentration and profile can generally be influenced by S, N and Se

supply. Sulphur and nitrogen fertilization and the balance between them have a predominant

effect on glucosinolate concentration in Brassicas. An increased S supply results in higher

total glucosinolate concentration in broccoli, turnip, canola and mustard (Krumbein et al.

2001; Rangkadilok et al. 2004; Li et al. 2007; Malhi et al. 2007).

Chen et al. (2006), Stavridou et al. (Chapter 5, this thesis) and Krumbein et. al (2001)

showed that total glucosinolate concentration in pakchoi and broccoli was enhanced at low N

supply. In contrast, Omirou et al. (2009) found that total GSLs responded to N supply, but did

not respond to N applications above 250 kg ha-1. It is clear that individual GSLs respond

differently according to N supply. For example, increased of N supply resulted in raised

indole glucosinolate concentrations in watercress and turnip (Kim et al. 2002; Kopsell et al.

2007), whilst alkenyl GSLs in rape decreased (Zhao et al. 1994).

Increasing N supply decreased seed glucosinolate concentration of oilseed rape when S

was deficient, but increased it when S was applied (Zhao et al. 1993). Similarly, N by S

interaction was found in a greenhouse experiment using pot grown red leaf mustard plants


(Chapter 5, this thesis). In cabbage, total GSLs increased with high S supply and low N rates

(Rosen et al. 2005). Schonhof et al. (2007a), showed that total glucosinolate concentrations

were higher in broccoli plants grown with an insufficient N supply independent of the S level.

Likewise glucosinolate concentration decreased in plants given an insufficient S supply when

combined with an optimal N supply. The balance between N and S supply also played an

important role in regulation of GSLs in turnip and pakchoi (Chen et al. 2006; Li et al. 2007).

In the case of Se the results are contradictory Robbins et al. (2005) showed that increased

Se fertilization decreased glucosinolate concentration in broccoli and this was attributed to

competitive Se and S uptake by plants. However, recently Hsu et al. (2011) found that Se

application did not influence glucosinolate concentration.

2.3.5. Plant density

More space between growing vegetables was found to decrease glucosinolate

concentration of different cabbage cultivars and Brussels sprouts (MacLeod & Nussbaum

1977; MacLeod & Pikk 1978). High planting density (97500 plants ha-1) led to a 37%

increase of glucoraphanin concentration in broccoli (Schonhof et al. 1999). Björkman et al.

(2008) found that intercropping white cabbage with red clover reduced the levels of both

foliar and root GSLs. However, it was also concluded that the response of glucosinolate to

plant competition were greatly influenced by the Delia floralis infestation level. In an

experiment, using pot grown plants, total and individual GSLs in red leaf mustard increased

when intercropped with lettuce (Chapter 5, this thesis).

21 Effect of catch crops on Se and S availability for succeeding crops

Chapter 3

The effect of catch crop species on selenium and sulphur

availability for the succeeding crops‡

Eleftheria Stavridou 1, Kristian Thorup-Kristensen 1,2 and Scott D. Young 3

1 Faculty of Agricultural Sciences, Department of Horticulture, University of Aarhus, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark 2 Present address: Faculty of Life Science, Department of Agriculture and Ecology, University of Copenhagen, Højbakkegård Alle 13, DK-2630 Tåstrup, Denmark 3 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom

Abstract

Catch crops might reduce selenium leaching and thereby increase the overall Se

availability in vegetables. The ability of catch crops (Italian ryegrass (Lolium multiflorum L.),

fodder radish (Raphanus sativus L.) and hairy vetch (Vicia villosa Roth)) to reduce soil

selenium concentration in autumn and make it available to the succeeding crop in spring was

investigated in three experiment during 2007-2010 in Denmark under different fertilizer

regimes. Only in one experiment (no. III), did the catch crops affect the soil Se profile, as

Italian ryegrass and fodder radish increased water-extractable Se content in the 0.25-0.75 m

soil layer. The Se uptake by the catch crops varied from 65 to 3263 mg ha-1, depending on

species, year and fertilization, this corresponded to 0.1-3% of the water-extractable soil Se

content. Fodder radish took up from 3.5 to more than 20 times more Se than the other two

catch crops, depending on year and fertilization. The catch crops took up between 6% and

17% of added Se fertilizer, whereas onions took up only 0.3% to 3%. The influence of catch

crops on Se concentrations and uptake in onions and cabbage was low. A decrease in Se

uptake (non significant) and recovery of applied Se by onions following catch crops was

observed which may indicate Se immobilization during catch crop decomposition in the soil.

Despite its high Se uptake, fodder radish did not increase Se uptake by onions, possibly

because it increased S uptake, which has been shown to reduce Se uptake. Fodder radish and

hairy vetch increased both S and N uptake by onions.

Keywords: cover crops, green manure, mineralization, leaching, onion, cabbage

‡ Submitted to the Plant and Soil


1. Introduction

Selenium is a naturally-occurring element with chemical characteristics similar to S.

Initially, Se in plant products was known for its toxicity to animals but since the late 1950s

has been recognized as an essential nutrient for animals and later for humans. Selenium

concentration in soils is highly variable and mainly depends on the soil parent material. The

concentration ranges between 0.01 and 2 mg Se kg-1 in most soils, with a mean of ~0.4 mg

kg-1; however in seleniferous areas it can be up to 1200 mg Se kg-1 (White et al. 2007b) . The

mean Se intake among Danes is 38-47 μg Se d-1 (Rayman 2008), whereas the European

population reference intake is 55 μg Se d-1 (EC Scientific Committe on Food 2003). Sub-

optimal Se intake and status is associated with cardiovascular disease, myopathy, oxidative

stress-related disorders, increased cancer risk and immune dysfunction (Rayman 2008).

Selenium enriched fertilizers are used to increase Se concentration in crops. Finland was

the first country to establish a nationwide Se biofortification strategy (Eurola et al. 1991).

However, studies showed that only 7 to 35 % of the applied Se is utilized by plants (Eich-

Greatorex et al. 2007; Broadley et al. 2010), the rest might be retained in the soil or lost by

leaching and volatilization. In a simple leaching experiment losses were between 1 and 16 %

of the applied Se (Eich-Greatorex et al. 2007). Wang et al. (1994) showed that Se fertilizers

may have temporarily increased the Se concentration in Finnish river waters and headwater

streams, by surface runoff of the selenate after rainfall. However six years after the

nationwide Se fertilization in Finland started, Se concentrations in natural ground-waters and

wells were below the health-based limit of 10 μg L-1 set for drinking waters (Alfthan et al.

1995). The amount of Se lost by leaching depended on the form of Se present, soil pH, the

presence of competing ions (sulphate, phosphate, oxalate, molybdate), climate and organic

matter (Mayland et al. 1991; Eich-Greatorex et al. 2007). The predominant forms of Se

available to plants are selenate and selenite. Selenate is highly mobile but selenite is sorbed

strongly by hydrous ion oxides, clays and organic matter (Mayland et al. 1991). Selenate

tends to be the predominant form in aerobic and neutral to alkaline environments, whereas

selenite is the major form in acid soils (Mayland et al. 1991).

Although the environmental risk from Se applied as fertilizers at annual rates <10 g ha-1 is

low, there is a need to consider best farming practices that utilize residual Se, after harvest, to

minimize Se leaching. We know little about how Se is affected by farming practices, and to

what extent leaching loss of Se can be reduced by improved plant Se uptake and

recirculation. Catch crops are widely used to improve N management, and they have been

Effect

success

decomp

utilized

The hig

(Eriksen

taken u

pathway

Figur

Underst

or avail

also be

succeed

where w

reduce

organic

Se and

that the

the pres

t of catch cr

sfully used

position of

d by the suc

gh S dema

n & Thorup

up through

ys as S in p

re 3-1. Month

tanding of h

lability for c

effective in

ding crops. B

we want to

adverse en

c farming sy

S, and that

ere will be s

sent work w

rops on Se a

to reduce S

the catch c

cceeding cr

and of Bra

p-Kristensen

high affin

lants (Terry

hly precipitatio

how agrono

crops is lack

n (i) reducin

Better unde

o improve th

nvironmenta

ystems whe

plants with

similar bene

was to test th

and S availa

S leaching.

crop plant

ops (Erikse

assica crop

n 2002). Du

nity sulphat

y et al. 2000

on (bars) and a

omic manag

king, we do

ng Se leachi

erstanding o

he utilizatio

al effects in

ere Se fertil

h high S dem

eficial effec

he hypothes

ability for su

Furthermor

material le

en & Thoru

ps efficient

ue to chem

te transpor

0).

average mont

gement and

o not know t

ing loss and

of this will b

on of the li

n systems w

lization is n

mand also h

cts from catc

ses that 1) c

ucceeding c

re, after be

ed to miner

up-Kristense

tly depleted

ical similar

rters and fo

hly temperatu

crop rotatio

the extent to

d (ii) recycl

be importan

imited Se r

where Se f

not allowed.

have a high

ch crops on

catch crops

crops

ing incorpo

ralization of

en 2002; Er

d soil sulp

rities with s

ollows the

ure (line) durin

on may affe

o which use

ing Se to m

nt both in lo

resource av

fertilization

. Considerin

affinity for

n Se leachin

can reduce

orated into

f its S, wh

riksen et al

phate conce

sulphate, sel

same assim

ng the experim

ect Se leach

e of catch cr

make it avail

ow Se enviro

vailable, in

is practice

ng the simi

r Se, it seem

ng. The obje

soil Se con

the soil,

hich was

l. 2004).

entration

lenate is

milation

ment.

hing loss

rops will

lable for

onments

order to

ed, or in

larity of

ms likely

ective of

ntent and

23


leaching risk, 2) after incorporation catch crops will increase the Se availability for the next

cash crop by mineralization, and 3) that crucifer cover crops will have higher Se uptake and

concentration, and thereby have stronger effects on Se leaching risk and Se availability for

the succeeding crop than other typical grass or legume catch crops.

2. Materials and methods

2.1. Field experiments

Field experiments were established to study the effect of different catch crop species on Se

uptake of vegetables at the Research Centre at Aarslev (1027’E, 5518’N) on an Agrudalf

soil (Table 3-1). Experiments were performed three times, in 2007/08, 2008/09 and 2009/10.

During the experimental period, rainfall and air temperature was recorded daily at a

meteorological station within the Research Center. Average monthly precipitation and

average air temperature during the experimental period are shown in Figure 3-1. Mean

annual precipitation at the site is 624 mm and mean annual air temperature is 7.8 oC.

Table 3-1. Main characteristics of the soil at the experimental site.

Depth (m) Clay (%)

Silt (%)

Sand (%)

C (%) N (%) pHCaCl2

0-0.25 15 27 55 1.8 0.16 7.0 0.25-0.5 18 29 52 0.8 0.07 6.4

0.5-0.75 21 28 50 0.3 0.04 5.1

0.75-1.0 21 27 53 0.2 0.03 5.7

The catch crop species were Italian ryegrass (Lolium multiflorum L.), fodder radish

(Raphanus sativus L.) and hairy vetch (Vicia villosa Roth). A control treatment without catch

crops was included. The experiment had a randomized complete block design with 4

replicates. The catch crop plots were 2.5 by 10 m. Italian ryegrass and fodder radish were

sown at a rate of 20 kg ha-1 and hairy vetch at a rate of 100 kg ha-1 on 02nd, 11th and 06th of

August, respectively in the three years. The catch crops were incorporated by ploughing at

the end of March.

In 2007/08 (experiment I), onions and cabbage were used as cash crop and were transplanted

on 20 April 2008. No fertilization was applied for catch or cash crops. Experiment I showed

that the natural Se concentration was extremely low and in the next two years only onion was

used as cash crop, in order to allow more focus on the effects on of Se and S inputs to the

system. In the 2008/09 (experiment II), fertilization was applied only to the cash crop and


consisted of two S (0 and 65 kg ha-1) and Se (0 and 10 g ha-1) levels in four combinations. In

2009/10 experiment (experiment III), two levels of Se fertilization (0 and 10 g ha-1) were

applied both in catch and cash crop.

Table 3-2. Overview of crops and operations during the experiment

Treatments Experiment I Experiment II Experiment III

Catch crops

Species Italian ryegrass, Hairy vetch, Fodder radish

Italian ryegrass, Hairy vetch, Fodder radish

Italian ryegrass, Hairy vetch, Fodder radish

Fertilization None None 0 g Se ha-1, 10 g Se ha-1

Analysis DM DM, Se, S, N DM, Se, S, N

Cash crops

Species Onions, cabbage Onions Onions

Fertilization None None, 0 kg S ha-1 +10 g Se ha-1, 65 kg S ha-1 +0 g Se ha-1, 65 kg S ha-1 +10 g Se ha-1,

0 g Se ha-1, 10 g Se ha-1

Analysis DM, Se DM, Se, S, N DM, Se, S, N

Soil sampling

Autumn None Se, S (3 layers till 1 m depth) Se, S (3 layers till 1 m depth)

Spring None Se, S (top soil) Se, S (top soil)

where DM: dry matter; Se: selenium; S: sulphur; N: nitrogen.

2.1. Plant sampling and analysis

In each catch crop plot, plant samples from 1 m2 were collected in mid-November (except

in experiment I) by cutting at the soil surface. At harvest, cabbage was sampled from 3 m2,

and onions from 2, 1.2 and 0.72 m2 respectively in the three years. In the experiment I, cash

crops were analyzed only for Se and for this analysis oven air-dried plant material was used.

In cabbage analysis were used only uniform cabbage heads with smooth leaves and the non-

wrapped leaves were removed. Plants with crinkled leaves were excluded from the analysis.

After harvest, the onions were separated into bulbs and leaves; analysis performed only in the

bulbs. Yield, dry matter, N, S and Se accumulation was determined both in catch and cash

crops.


Plant samples were dried at 80 oC in a forced air-drying oven for 20 hours prior to

determination of N and S analysis. Total plant N was determined following dry oxidation by

the Dumas method (Elementar Vario EL. Hanau. Germany) and total S by using an NDIR

(non-dispersive infrared gas analysis) optic to detect the sulphur dioxide formed. Both

measurements were performed in duplicate.

Prior to Se analysis, a subsample of fresh plant material was washed with de-ionized water

to remove the attached soil then deep-frozen and freeze dried. Finely ground material (400

mg) was microwave-digested in pressurized PFA vessels (Anton Paar, ‘Multiwave’) with 3.0

mL of 70% Fisher ‘Trace analysis grade’ (TAG) HNO3, 3 mL water and 2 mL of 30% H2O2.

Digested samples were diluted to 15 mL with milli-Q water (18.2 MΩ cm) and, immediately

prior to analysis, were further diluted 1-in-10 with milli-Q water. Concentrations of Se in

plant samples and leachate were determined using an Inductively Coupled Plasma Mass

Spectrometer (ICP-MS, Thermo-Fisher Scientific X-SeriesII) employing a ‘hexapole

collision-reaction cell’ (with H2 gas) with kinetic energy discrimination (CCT-KED) to

remove polyatomic interferences.

2.2. Soil sampling and analysis

Soil samples were taken in November in soil layers of 0-0.25 m, 0.25-0.75 m and 0.75-1.5

m and in March, prior to catch crop incorporation from the topsoil (0-0.25). Nine distributed

soil samples were taken from each plot with a piston auger (inner diameter 14 mm) and

bulked to provide a single sample for each depth interval from each plot for soil

characterization. The soil samples were frozen at -18 oC within 24 h after sampling. Total

inorganic sulphate was extracted by shaking soil (40 g) with 400 ml CaCl2- solution (0.0125

M) for 60 min. Extracts were filtered and sulphate was measured using inductively coupled

plasma-optical emission spectrometer (ICP-OES). Water-soluble Se was extracted with

deionized water at a water-to-soil ration of 10:1 (W/W); suspensions were shaken for 60 min,

then centrifuged for 20 min at 10000 rpm. The supernatant was filtered to < 0.22 μm,

acidified to 2% HNO3 and stored at 4oC prior to Se analysis by ICP-MS.

2.3. Data analysis

Statistical analysis of the data was performed using the GLM procedure of the SAS

statistical package (version 9.2; SAS Institute Inc, Cary, NC, USA). If the assumption of

normality or homogeneity of variance was not verified, log-transformed data were used.


3. Results

3.1. Soil Se and S

The effects of catch crops on soil water-extractable Se content during their growth in the

autumn and just before their incorporation in the spring was limited and inconsistent. In the

autumn period of experiment II, the catch crops did not influence water-extractable Se

content in soil or Se distribution in the soil profiles (Table 3-3). Although, total soil

extractable sulphate content was unaffected by the catch crops, the amount of sulphate in the

topsoil (0-0.25 m) decreased after Italian ryegrass and fodder radish, but increased after hairy

vetch (Table 3-3). In the 0.25-0.75 m layer, fodder radish significant reduced soil extractable

sulphate content, moreover a reduced soil sulphate content was observed also in the 0.75-1.5

m layer after fodder radish (non significant). In the spring in experiment II, soil soluble Se

content in the topsoil showed a small increase after catch crops (not significant) (Table 3-3).

A non significant increase in sulphate content in the 0-0.25 m soil layer was observed after

fodder radish, while Italian ryegrass appeared to reduce soil sulphate (non significant) (Table

3-3).

In contrast to experiment II, catch crops increased both the soil water-extractable Se

content and affected its distribution in the soil in autumn in experiment III (Table 3-4). Catch

crops influenced soluble Se content mainly in the 0.25-0.75 m soil layer, where, soil Se

content was higher under Italian ryegrass and fodder radish than under bare soil (Table 3-4).

Total water-extractable soil Se content was affected by the catch crops only when Se

fertilization had been applied. Italian ryegrass and fodder radish caused a significant increase

in soil Se content.

In experiment III, the catch crops differed not only in their effect on the amount of soil

extractable sulphate, but also in their effect on its vertical distribution (Table 3-4). Fodder

radish reduced total extractable soil sulphate with 15 to 23 kg S ha-1 as compared to bare soil.

In autumn, the extractable S content in the bare soil was high from 0.25 to 1.5 m depth. In the

topsoil, as in experiment II soil extractable sulphate content was higher under hairy vetch

compared to bare soil, fodder radish and Italian ryegrass. All catch crops reduced extractable

S content in the 0.25-0.75 m layer, especial fodder radish. In the 0.25-0.75 m layer, the


Table 3-3. Soil Se (g ha-1) and S (kg ha-1) content and distribution in the autumn and spring in experiment II under different catch crops.

Catch crops

Soil soluble Se (g ha-1) Soil inorganic S (kg ha-1)

Autumn 2008 Spring 2009 Autumn 2008 Spring 2009

0-0.25m 0.25-0.75m 0.75-1.5m Total 0-0.25 m 0-0.25m 0.25-0.75m 0.75-1.5m Total 0-0.25 m

C 28 a 40 a 25 a 93 a 27 a 10 ab 21 a 20 a 49 a 28 a IR 29 a 44 a 32 a 105 a 31 a 7 c 17 a 26 a 50 a 23 a FR 28 a 39 a 30 a 97 a 28 a 9 c 10 b 15 a 34 a 33 a HV 29 a 37 a 29 a 94 a 27 a 11 a 16 a 26 a 53 a 26 a

where Se: selenium; S: sulphur; C: bare soil; IR: Italian ryegrass; FR: fodder radish; HV: hairy vetch. Means followed by the same letter are not significantly different (n=4).

Table 3-4. Soil Se (g ha-1) and S (kg ha-1) content and distribution in the autumn and spring in experiment III under different catch crops.

Fertilization Catch crops

Soil soluble Se (g ha-1) Soil inorganic S (kg ha-1)

Autumn 2009 Spring 2010 Autumn 2009 Spring 2010

0-0.25m 0.25-0-75m 0.75-1.5m Total 0-0.25 m 0-0.25m 0.25-0-75m 0.75-1.5m Total 0-0.25m

Se0 C 29 a 40 b 38 a 106 a 27 a 8 b 16 a 16 ab 40 a 9 d IR 28 a 45 a 36 a 108 a 26 a 6 b 11 ab 19 a 36 a 11 c FR 28 a 44 a 38 a 109 a 26 a 7 b 4 c 6 c 17 b 22 a HV 28 a 43 ab 32 a 102 a 26 a 12 a 8 bc 13 b 33 a 14 b Average 28 B 43 A 36 A 107 A 26 A 8 A 10 A 14 A 30 A 14 A Se10 C 28 a 40 c 36 a 104 b 27 a 7 b 15 a 10 a 32 a 10 c IR 30 a 45 ab 45 a 119 a 27 ab 5 c 10 b 15 a 29 a 10 c FR 29 a 47 a 39 a 115 a 26 bc 5 c 4 c 9 a 17 b 21 a

HV 29 a 42 bc 39 a 110 ab 25 c 12 a 8 bc 9 a 29 a 15 b Average 29 A 43 A 40 A 112 A 26 A 7 A 9 A 11 A 26 A 14 A

where Se: selenium; S: sulphur; C: bare soil; IR: Italian ryegrass; FR: fodder radish; HV: hairy vetch. Mean values of the three catch crops and bare soil treatments in the same fertilization treatment by different letters (a, b, c) are significantly different. Mean values of fertilization treatment followed by different capital letters (A, B, C) are significant different (n=4).


extractable S content under fodder radish was only 26% of that under the bare soil. Fodder

radish grown without Se fertilization decreased extractable S content also in the 0.75-1.5 m

layer. Extractable soil sulphate content was unaffected by Se fertilization in autumn in

experiment III (Table 3-4).

Selenium fertilization did not influence water soluble Se and extractable S content in the

topsoil in spring 2010 (Table 3-4). Catch crops affected water soluble Se content in the

topsoil only when grown with Se fertilization (Table 3-4). Fodder radish and hairy vetch

reduced water soluble Se content compared to bare soil. Extractable soil S content in the

topsoil was increased under fodder radish and hairy vetch (Table 3-4).

Table 3-5. Yield (Mg DM per ha), Se content (μg kg-1), Se uptake (g ha-1), S- and N-uptake (kg ha-1) in catch crops in experiment II and III.

Fertilization Catch crops Yield (Mg DM ha-1)

Se-content (μg kg-1)

Se-uptake (mg ha-1)

N-uptake (kg ha-1)

S-uptake (kg ha-1)

2008

Se0 IR 3 b 41 c 130 b 83 b 7 b

FR 4 a 212 a 997 a 147 a 26 a

HV 2 c 85 b 177 b 74 b 4 c

Average 3 93 322 101 12

2009

Se0 IR 4 b 30 b 114 b 133 b 8 b

FR 6 a 288 a 1571 a 202 a 28 a

HV 2 c 31 b 65 b 108 b 5 c

Average 4 A 101 B 494 B 148 A 13 A

Se10 IR 4 b 202 c 773 b 136 b 8 b

FR 6 a 514 a 3263 a 223 a 29 a

HV 2 c 316 b 663 b 112 b 4 c

Average 4 A 344 A 1566 A 157 A 14 A


3.2. Catch crops

The plant production in catch crops, from the mid of August to mid-November, were on

average 3, 5, 2 Mg DM per ha for Italian ryegrass, fodder radish and hairy vetch, respectively

(Table 3-5). Fodder radish produced higher yields in experiment III than in experiment I


(data not shown) and experiment II, whereas the yields of Italian ryegrass and hairy vetch

yields were constant. Selenium fertilization did not affect catch crop yields in experiment III.

Highly significant differences in Se concentrations and uptake in catch crops were found

both in experiment II and III (Table 3-5). Selenium concentrations were 2 to 10 times higher

in fodder radish grown both with and without Se fertilizer compared to Italian ryegrass and

hairy vetch and Se uptake was 4 to 24 times higher. Application of 10 g Se ha-1 significantly

increased Se concentrations and uptake by catch crops. The efficiency of Se recovery by

Italian ryegrass, fodder radish and hairy vetch was 7%, 17% and 6%, respectively. Sulphur

and N uptake were higher by fodder radish both in experiments II and III (Table 3-5).

Selenium fertilization did not influence S and N uptake by catch crops in experiment III.

Table 3-6. Yield (Mg DM per ha), Se content (μg kg-1), Se uptake (g ha-1) in onions and cabbage following catch crops in experiment I.

Catch crops

Onions Cabbage

Yield (Mg DM ha-1)

Se content (μg kg-1)

Se-uptake (mg ha-1)

Yield (Mg DM ha-1)

Se content (μg kg-1)

Se-uptake (mg ha-1)

C 9 a 6 a 49 a 4 b 24 a 97 bc IR 9 a 3 c 24 bc 3 c 22 a 71 c FR 10 a 2 bc 21 c 5 a 23 a 117 ab HV 9 a 4 ab 41 bc 6 a 31 a 170 a

where Se: selenium; S: sulphur; C: bare soil; IR: Italian ryegrass; FR: fodder radish; HV: hairy vetch. Means followed by the same letter are not significantly different (n=4).

3.3. Cash crops

In experiment I, none of the catch crops affected onion yield, but higher cabbage yield was

found following fodder radish and hairy vetch (Table 3-6). Selenium concentrations in

onions were reduced following Italian ryegrass and hairy vetch, whereas in cabbage they

were unaffected by the catch crops (Table 3-6). Cabbage contained 4 to 12 times higher Se

concentrations compared to onions and 2 to 6 times higher total uptake. A non significant

decrease in Se uptake by onions following catch crops was observed. Higher cabbage yields

following fodder radish and hairy vetch resulted in higher Se uptake by cabbage grown after

fodder radish and hairy vetch.

In experiment II, catch crops affected the yield of onions only in the Se10S0 treatment where

hairy vetch increased the yield (Table 3-7), while in experiment III higher yield was found

only in onions following fodder radish and hairy vetch where no Se fertilizer was given


(Se0Se0) and where Se fertilizer was added both in the autumn and in the spring (Se10Se10,

Table 3-8). No fertilization treatment influenced onion yields.

Table 3-7. Yield (Mg DM per ha), Se content (μg kg-1), Se-uptake (g ha-1), S- and N-uptake (kg ha-1) in onions following catch crops in experiment II.


Selenium concentration in onions was unaffected by catch crops both in experiment II and

III (Table 3-7, Table 3-8). As in experiment I, catch crops reduced Se uptake by onions (not

significant) grown without Se fertilizer in experiment II (Table 3-6, Table 3-7). In contrast,

the effect of catch crops on Se uptake by onions was not consistent (Table 3-8). Both in

experiments II and III, application of Se to onions at transplanting significantly increased Se

concentrations. However, the average recovery of Se in onions was low, 1-4% and -0.3-0.5%

in experiment II and III, respectively. In experiment III, Se fertilization at the establishment

Fertilization Catch crops Yield (Mg DM ha-1)


Se-uptake (mg ha-1)

N-uptake (kg ha-1)

S-uptake (kg ha-1)

Se0S0 C 4 a 10 a 39 a 32 b 5 c

IR 5 a 7 a 31 a 47 a 4 c

FR 5 a 8 a 31 a 50 a 9 a

HV 5 a 2 a 10 a 53 a 7 b

Average 5 A 7 C 29 C 46 A 6 B

Se0S65 C 4 a 9 a 35 a 31 c 8 b

IR 4 a 5 a 23 a 44 b 10 b

FR 5 a 7 a 33 a 57 a 15 a

HV 5 a 6 a 27 a 55 a 13 a

Average 5 A 7 C 30 C 47 A 11 A

Se10S0 C 4 b 61 a 250 a 30 d 5 bc

IR 4 b 72 a 281 a 40 c 4 c

FR 5 ab 70 a 321 a 52 a 10 a

HV 5 a 87 a 456 a 61 b 7 ab

Average 5 A 72 A 327 A 46 A 7 B

Se10S65 C 4 a 36 a 143 a 31 c 8 c

IR 4 a 40 a 157 a 42 b 10 bc

FR 5 a 48 a 214 a 52 a 10 b

HV 5 a 40 a 194 a 57 a 13 a

Average 4 A 41 B 177 B 45 A 10 A


of catch crops in August was found to increase Se concentration and uptake in onions (not

significant), but less so than direct Se fertilization of the onions. Sulphur fertilization at

transplanting in experiment II decreased Se concentrations in onions up to 54%, when S was

applied with Se, reducing average Se fertilizer recovery by up to 75%. Selenium uptake was

affected by the fertilization treatments similarly to Se concentrations, although a Se

fertilization × catch crop interaction was observed.

Table 3-8. Yield (Mg DM per ha), Se content (μg kg-1), Se-uptake (g ha-1), S- and N-uptake (kg ha-1) in onions following catch crops in experiment III.


Catch crops influenced S and N uptake by onions both in experiment II and III (Table 3-7,

Table 3-8). Onions grown after catch crops in all treatments took up more N than onion

grown after bare soil. Fodder radish and hairy vetch increased S uptake by onions

Fertilization Catch crops

Yield (Mg DM ha-1)


Se-uptake (mg ha-1)

N-uptake (kg ha-1)

S-uptake (kg ha-1)

Se0Se0 C 2 c 8 a 18 a 19 b 4 c

IR 3 bc 12 a 30 a 27 a 4 c

FR 3 a 9 a 28 a 36 a 7 a

HV 3 b 10 a 28 a 30 ab 5 b

Average 3 A 10 C 26 B 28 A 5 A

Se0Se10 C 2 a 30 a 66 a 18 ab 3 b

IR 3 a 31 a 85 a 29 a 4 b

FR 3 a 22 a 65 a 32 ab 7 a

HV 2 a 21 a 41 a 21 ab 4 b

Average 3 A 26 A 64 A 25 A 5 A

Se10Se0 C 2 a 15 ab 35 a 19 b 4 b

IR 2 a 28 a 65 a 26 a 4 b

FR 3 a 8 b 23 a 30 a 6 a

HV 3 a 11 b 31 a 29 a 5 a

Average 3 A 16 B 38 B 26 A 5 A

Se10Se10 C 2 c 29 a 63 a 18 c 4 c

IR 2 bc 25 a 58 a 25 b 3 c

FR 3 a 28 a 79 a 30 a 6 a

HV 3 ab 33 a 88 a 30 a 5 b

Average 3 A 29 A 72 A 26 A 5 A


independently of the fertilization treatment. In experiment II, S fertilization of onions at

transplanting increased S uptake, but it did not influence N uptake (Table 3-7). Selenium

fertilization did not affect N or S uptake by onions in either experiment II or III (Table 3-7,

Table 3-8).

4. Discussion

The catch crops did not reduce the soil water-extractable Se content, as Se uptake was

only 0.3-3% of the total water-extractable Se content in the soil. However, the impact of the

catch crops on soil water-extractable Se content was different the two years, which could be

attributed to the differences in precipitation between the two years. The higher precipitation

in 2008 (Figure 3-1) after the establishment of the catch crops compared to 2009 may have

leached Se deeper in the soil profile before the catch crops established a deep root system. It

is interesting to note that soluble Se content in the 0.25-0.75 m soil layer in autumn in

experiment II was higher under fodder radish and Italian ryegrass compared to the control.

The differences among species in subsoil soluble Se in the autumn in experiment II may be

due to the vegetation biomass. Using soil columns Wu et al. (1996) showed that leachate

volumes were greatly influenced by the presence of vegetation. Fodder radish and Italian

ryegrass had greater vegetation biomass and probably higher rate of water use than hairy

vetch. Well established vegetation reduces the amount of the drainage water leaching through

the soil profile and thereby the leaching of Se and other ions in the soil solution (Wu et al.

1996). Nevertheless, under field conditions, the reduced Se loss in the period until mid

November under fodder radish and Italian ryegrass could not secure reduced Se leaching

during the remaining part of the winter season, where vegetation biomass is reduced. Another

explanation for increased Se levels may lie in redox reactions in rhizosphere processes, which

are affected by plant root activity and may increase solubility and oxidation of Se and

subsequently the availability of Se for plant uptake (Blaylock & James 1994).

Selenium fertilization did only increase soil soluble Se content insignificantly in autumn in

experiment III, which is in accordance with Stroud et al. (2010a). This is likely to be caused

by relatively low Se addition through fertilization compared to the extractable Se already

there, and loss of the Se input leaching down to the soil profile, conversion to unextractable

Se fractions or volatilization. The addition of 10 g Se ha-1 represented only c. 10% of the

extractable levels of 102 to 119 g Se ha-1 under the catch crops (Table 3-4) or c. 35% of the

Se in the 0-0.25 m topsoil layer.


Selenium concentrations were higher in fodder radish both in experiment II and III which

may be attributed to the higher S demand of fodder radish. Selenate is taken up by plants

through the high affinity sulphate transporters, as a consequence of the chemical similarity

between S and Se. Several Brassica crops have been shown to accumulate high Se

concentrations (White & Broadley 2009). Moreover, Brassica crops root show higher depth

penetration rates faster and achieve a much higher root density in the subsoil than

monocotyledonous catch crops and hairy vetch (Thorup-Kristensen 2001) allowing them to

take up Se and S from the deeper soil layers.

Although catch crops increased soluble Se concentrations in the subsoil in mid November

this did not influence Se concentrations in cash crops. Onion is a shallow rooted crop, the

estimated root depth at harvest is less than 0.3 m (Thorup-Kristensen 2006b) and catch crops

decreased topsoil Se content in spring. As the content of Se in the catch crops was quite small

compared to the amount of extractable Se in the topsoil layers, it is not surprising that effects

of catch crops on Se availability is dominated by factors other than possible Se release during

catch crop decomposition. The lower Se concentrations in onions in experiment I compared

to the following years may be attributed to Se losses that occurred during the sample

preparation. Studies showed that onions and radish dried at high temperature (>60 oC) can

lose up to 20 % of their Se content through volatilization (Gissel-Nielsen 1970). Moreover,

the lower Se concentration may be the result of dilution, as onion yields in experiment I were

higher than the following years. The higher Se uptake by cabbage following hairy vetch may

be caused by the higher cabbage yield or to the deeper root growth giving cabbage access to

more soil Se. Early harvested cabbage types were found to reach root depths at least 1.1 m

compared to the shallow-rooted onions which reach only 0.3 m (Thorup-Kristensen 2006b),

which increases the efficiency of cabbage to uptake Se from deeper soil layers.

Previously studies have shown that incorporation of catch crops, crop residues and manure

in the soil reduced the availability of the native soil Se or the Se added through fertilization

(Ajwa et al. 1998; Stavridou et al. 2011). In our study, only a non significant reduction of Se

uptake by onions following catch crops was found in experiment I and II, but the results in

experiment III were not consistent. Differences in the effect of catch crops effect on Se

uptake by onions between the experiments may be ascribed to the difference in the organic

matter incorporated in the soil. In 2010 the severe winter reduced catch crop biomass; the

catch crops did not recover in spring and the amount of plant material incorporated in the

field was lower than the previous year. Johnson (1991) found that increase of organic matter


content in the soil from 1.4% to 39% decreased Se uptake by wheat grain and rape. The

decreased Se uptake by onions following catch crops indicates that Se immobilization may

occur when onions are grown without Se fertilization.

The Se fertilizer recovery rate of 6-17% by the catch crops was similar to the range found

in other field trials (Broadley et al. 2010; Stroud et al. 2010b), whereas Se recovery by onions

was lower (-0.3-4%) and differed between the years. While the applied Se fertilizer

represented only a small fraction of the already extractable Se in the soil, it increased Se

concentrations both in catch crops and cash crops even when the recovery was low. The

concentrations of selenite in the topsoil is reported to account for 19-49% of the potassium

dihydrogen phosphate extractable Se (Stroud et al. 2010a) and selenate was not detectable.

Although, in our study only water-extractable Se was measured, it is likely that selenite and

organic Se were the predominant forms present in solution, which explains why the addition

of 10 g selenate ha-1 to a soil already containing c. 100 g Se ha-1 had such a strong effect.

Plants absorb Se from the soil primarily as selenate and plant Se uptake is higher when plants

are treated with selenate compared to selenite (Fordyce 2005; Sharma et al. 2010).

Although the results were not consistent a non significant increase on Se concentrations

and Se uptake in onions was observed when Se fertilizer was applied in August at the

establishment of the catch crops. However, the increase was lower than the increase found

after the direct Se fertilization of onions. These results suggested that selenate was leached

deeper in the soil, volatilized and/or converted to less available Se forms for plants. Stroud et

al. (2010a) found that selenite was the inorganic species in soils sampled before fertilization

and after harvest of wheat, which was fertilized with selenate. Selenate was not detectable in

soil at any sampling date.

Although Se input through fodder radish in experiment III was higher compared to Se

input through Italian ryegrass, hairy vetch and bare soil, it did not influence Se uptake by

onions. The antagonistic interaction between S and Se for plant uptake has long been noted

by researchers (White et al. 2004; White et al. 2007a; Li et al. 2008; Stroud et al. 2010b). In

experiment II, S fertilization decreased Se uptake by onions when Se fertilization was

applied, similar results has obtained in wheat (Stroud et al. 2010b). The regulation of

expression of the high affinity sulphate transporter genes is regulated by the S status of the

plant, where high concentrations of sulphate decrease transcription and potentially decrease

Se uptake by plants (Sors et al. 2005). In 2009, S uptake by onions following fodder radish

grown without S fertilizer was higher than S uptake by onions grown in the S fertilized bare


soil. It is therefore likely that the low Se uptake following fodder radish may have been

caused by the higher S availability from fodder radish.

The findings that subsoil sulphate was lower under fodder radish than Italian ryegrass and

bare soil is consistent with the findings of Eriksen and Thorup-Kristensen (Eriksen &

Thorup-Kristensen 2002), who showed that cruciferous catch crops substantially deplete the

soil available sulphate pool. The higher S uptake by onions following fodder radish reflected

the differences in soil S availability, as S concentrations were higher in fodder radish leading

to increased S mineralization during its decomposition. The high sulphate content in the

topsoil under hairy vetch may be attributed to the rhizosphere acidification typically observed

with N2 fixing legumes, which could promote mobilization of S in the soil (Haynes 1983;

Andersen et al. 2007) and also explain the higher S uptake by onions compared to bare soil.

5. Conclusion

The hypothesis that the use of catch crops reduces Se leaching over winter was not

verified. The Se uptake by catch crops was less than 1% of the total water soluble Se in the

soil. With such low uptake, uptake and mineralization effects on soil Se content will be small,

and other indirect catch crop effects on Se availability, uptake and leaching are likely to

dominate. High rainfall in the early growth stage of the catch crops; can increase Se losses in

the deeper soil layers before plants being able to reduce the excess water drainage. As the

overall Se recovery by the crops was low, special attention should be paid in the fate of

residual Se in the soil. The incorporation of catch crops in the field seems to reduce the

recovery of the applied Se and the uptake by onion. The results showed that the Brassica crop

fodder radish was able to take up much more Se form Se fertilizer and native soil Se than the

other catch crops. However, fodder radish did not succeed to increase Se concentrations in

the succeeding cash crops, probably due to its high S mineralization limiting cash crop Se

uptake.

37 Selenium mineralization and availability from catch crops

Chapter 4

Assessment of selenium mineralization and availability from catch

crops ‡

Eleftheria Stavridou 1, Kristian Thorup-Kristensen 1,2 and Scott D. Young 3

1 Faculty of Agricultural Sciences, Department of Horticulture, University of Aarhus, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark 2 Present address: Faculty of Life Science, Department of Agricultural Science, University of Copenhagen, Højbakkegård Alle 13, DK-2630 Tåstrup, Denmark 3 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom

Abstract

Selenium (Se) release from four plant species (Indian mustard, fodder radish, Italian ryegrass,

and hairy vetch) was measured under controlled leaching conditions and in a pot incubation

experiment as part of a study of the potential for using these plant species as Se catch crops.

Catch crops may reduce Se leaching and, by subsequent release of Se from the plant material

increase the available Se for succeeding crops. Plants grown both without and with Se

addition (250 g Se/ha) were tested. In the leaching experiment frozen plant material was

incorporated into soil columns and incubated at room temperature for up to 19 weeks. The

results showed that Se concentrations in the leachate were higher when Se-enriched plant

material was incorporated in the soil, indicating Se mineralization. When non enriched plant

material was added to the soil Se concentrations in the leachate was generally lower than the

control, indicating Se immobilization. In the pot incubation experiment the results were

consistent with those from the leaching experiment. The addition of enriched plant material

increased Se concentration in Indian mustards plants compared to unamended soil. However,

the addition of plant materials grown without Se significantly decreased Se concentrations on

plant dry matter, again indicating Se immobilization. Fertilization with inorganic Se as

selenate did not affect Se concentrations either in the leachates or in the plants grown in the

pot incubation. Thus, results showed the potential of catch crops to increase Se mineralization

and uptake in succeeding crops.

Keywords catch crops, selenium, mineralization, leaching, incubation.

‡ Accepted by the Soil Use and Management


1. Introduction

Selenium is an essential micronutrient for humans, animals and algae. It mainly reaches

animal and human food chains through plants, after they have absorbed it from the soil. Soils

in some parts of the world have low Se concentrations (0.1-0.6 mg Se/kg), including large

areas of Scandinavia, North America, New Zealand, Australia and China; consequently crops

produced there may not contain sufficient Se to meet human and animal requirements

(Oldfield 2002). There is evidence that Se deficiency is associated with a range of

physiological disorders, such as immune dysfunction, cardiovascular diseases, and an

increased virulence of a range of viruses (Fairweather-Tait et al. 2011). Furthermore,

numerous studies have demonstrated the action of some organic forms of Se against certain

types of cancer (Rayman 2000; Fairweather-Tait et al. 2011). Recommended levels in grain

for human intake are 0.1-0.2 mg Se/kg dry matter (DM), and for livestock 0.2-0.3 mg Se/kg.

Inorganic Se fertilization has proved to be an efficient means of increasing Se concentrations

in food and forage crops. The best example is the Finnish practice where addition of Se in

multielement fertilizers has been mandatory since 1984; this, has resulted in a general

increase of human Se intake (Eurola et al. 1989).

An important consideration in the agronomic bio-fortification of Se is to understand the

residual effect and the fate of Se in the soil. A limited fraction of the applied Se is utilized by

plants (7 to 35%) (Eich-Greatorex et al. 2007; Broadley et al. 2010); thus, if a crop is

amended with 10 g Se/ha the fate of 6.5 to 9.3 g Se/ha is unknown. This might be retained in

the soil, leached or lost to the atmosphere by volatilization and it is inefficient use of a

resource. Changes in management practices or in other soil environmental factors, such as pH

or redox potential may also cause mobilization and leaching of the previously bound Se

(Johnsson 1991). Mobilization of Se may lead to leaching and contamination of groundwater

or drinking water. In Finland, the use of Se fertilizers may have temporarily increased

concentrations of Se in headwater streams and rivers, by surface runoff of soluble selenate

after rainfall (Wang et al. 1994).

Whilst there is little environmental risk from Se applied as fertilizers at annual rates <10

g/ha, there is a need to consider best farming practices that utilize residual Se, after harvest,

to minimize Se leaching. In mineral fertilizers, Se is added mainly as selenate which is highly

mobile in the soil solution and readily available for plant uptake (Broadley et al. 2006). Due

to chemical similarities with sulphate, selenate is taken up through high affinity sulphate

transporters and follows the same assimilation pathways as S in plants (Terry et al. 2000). It


has been demonstrated that catch crops can reduce sulphate leaching and increase S

availability for the following crop (Eriksen et al. 2004). Based on their chemical similarities,

similar beneficial effects on Se and S leaching may be expected from the use of catch crops.

However, before this practice is adopted, more information is needed. In particular, the

synchronization of Se released from decomposing plant residues with crop uptake is critical

to avoid Se loss by leaching from the rooting zone before it can be taken up by the crop. The

aim of the present study was to investigate how different catch crops affect the bioavailable

Se pool and how this changes over the growing period. Plants differ in their ability to

accumulate and assimilate Se; therefore a range of catch crops was tested under controlled

leaching and non-leaching conditions. Brassica species absorb moderate amounts of Se and

convert Se into soluble seleno-amino acids, whereas legumes and ryegrass are non

accumulator species contain that lower Se concentrations as insoluble selenomethionine

(Girling 1984).

The aim of the present study was to investigate a range of catch crop material and their

effect on the bioavailable Se pool in the soil and how this changes over the growing period.

The main hypotheses were: 1) during decomposition catch crop plant materials will release

Se and increase Se availability to the succeeding crop, and 2) Brassica catch crops will

release higher amounts of Se compared to grass and vetch due to higher Se content in the

plant material.

2. Material and Methods

2.1. Soil and plant material

Soil for the two experiments was collected from a non-seleniferous site located at the

Department of Horticulture, Univesity of Aarhus, Aarslev, Denmark (10o27’E, 55o18’N). The

soil, classified as Agrudalf, was collected from the top layer (0-15 cm). Soil was air-dried to a

water content of 3%, sieved (< 5 mm) and mixed with sand (2:1) to ensure good air and water

permeability. The pH value was 7.0 and soil mechanical analysis was: 71% sand, 14% silt,

12% clay and 2% organic matter.

Plant material from Italian ryegrass (Lolium multiflorum L.), fodder radish (Raphanus

sativus L.), hairy vetch (Vicia villosa Roth) and Indian mustard (Brassica juncea L. Czern.)

were used for the incubations. Plants were grown in a non-seleniferous field located at

Aarslev, with, and without, addition of Se (250 g Se/ha). Selenium was applied as sodium


selenate before sowing. The plant material was harvested at the flowering stage, chopped and

stored at -20oC. The material was analyzed for its dry matter (DM) and Se content.

Table 4-1. Selenium (Se) concentrations in plant material and Se added in the leaching and pot experiments by the treatments.

Treatment Se content (mg/kg)

Amount of Se added

Leaching incubation

Pot experiment

Hairy vetch (HV) 0.05 0.10 0.76 Italian ryegrass (IR) 0.07 0.13 1.03 Indian mustard (IM) 0.39 0.69 5.49 Fodder radish (FR) 1.94 3.39 26.90 Hairy vetch-Se enriched (HV+Se)

13.12 22.96 182.35

Italian ryegrass-Se enriched (IR+Se)

19.01 33.26 264.21

Indian mustard-Se enriched (IM+Se)

13.38 23.42 186.00

Fodder radish-Se enriched (FR+Se)

8.41 14.72 116.96

Sodium selenate (Se) 5.00 3.98a

Sodium selenate + sucrose (Se+C)

5.00 3.98a

a as a result of a miscalculation, the inorganic treatment was applied at 10% of the intended rate of 10 g Se/ha in the pot experiment.

2.2. Leaching – tube incubations

A leaching experiment under aerobic conditions was established using a randomised

complete block design with three replicates and 11 treatments including incubation of eight

plant materials, two Se inorganic treatments and a control (Table 4-1). Leaching columns

were constructed from 0.25 m long Perspex glass tubes with an inner diameter of 0.08 m. The

bottom of the tubes was covered with gauze (Lutrasil Thermoselect). Initially, the tubes were

filled up to 0.13 m with soil-sand mixture (1 kg air dried) to a density of 1.53 Mg/m3. The

plant material and the inorganic fertilizer were mixed thoroughly with 0.3 kg air dried soil-

sand mixture and placed on the top of the unammended soil in the tube (up to 0.2 m),

additional a 0.02 m layer of soil mixture (0.05 kg) was placed on top of the plant material

mixture. Plant material was added to provide the equivalent of 3500 kg DM/ha, which

provides approximately 1470 kg C/ha. Sodium selenate was applied corresponding to 10 g

Se/ha in two inorganic treatments. In one of inorganic treatments 1470 kg C/ha was added as


sucrose. The moisture content of the soil-sand mixture was brought to 85% of the water

holding capacity as determined in a pilot experiment. Leaching columns were incubated in

the dark at room temperature and leached after 6, 10, 15 and 19 weeks. Leaching was

performed stepwise by adding two 100 ml aliquots of de-ionized water with leachate from

each step collected separately. Results from a pilot experiment showed that the second aliquot

contained virtually all the soluble Se mineralized from the catch crops material since the

previous leaching. Leachate, was collected in polypropylene tubes, filtered, acidified to 2%

HNO3 and stored at 4oC prior to Se analysis.

2.3. Pot incubations

The treatments in the pot experiment were similar to those of the leaching incubation

(Table 4-1) and used plant material from the same samples. In the two inorganic treatments,

Se was applied at 10% of the intended rate of 10 g Se/ha, as a result of a miscalculation. In

each treatment plant material or inorganic fertilizer was uniformly mixed with 1 kg of air-

dried soil-sand mixture, at the same rates based on area as in the leaching incubations; eight

replicates were used. The amended soil-sand mixtures were then transferred to 4.7 L plastic

pots (upper diameter = 22 cm, height = 16 cm), on top of 3.5 kg of soil-sand mixture. A

further amount (0.3 kg) of the unamended soil-sand mixture was placed over the amended

layer. Pots were positioned in a completely randomized block design in a greenhouse and

pre-incubated for four weeks.

After pre-incubation, two Indian mustard seedlings, grown in trays with commercial

growth medium for two weeks, were planted in each pot. Two set of pots were prepared to

allow two harvests (4 replicates at each harvest). Where required, pre-collected rain water

was added to pots to avoid water stress. Loss of water from the base of the pot was kept at a

minimum; any leachate was re-applied to the pot to avoid Se losses. The plants were grown

in a temperature controlled greenhouse from 27 May to 7 July, 2010. The average day and

night temperatures were 22 and 15oC, respectively; the average day length during the

experiment was 13 h.

The above-ground biomass from the first set of pots (4 replicates) was undertaken 27 days

after transplanting and the second set was harvested 43 days after transplanting. The plants

were freeze-dried, weighted, ground and analyzed for Se content.

442

2.4

Fine

(Anton

3 mL w

water (

milli-Q

Inductiv

SeriesII)

(CCT-K

2.5

As t

leached

calculat

incubat

anova, w

conside

Cary, N

Figure 4bars repr

4. Sample p

ly ground

Paar, ‘Mul

water and 2

18.2 MΩ c

water. Con

vely Coupl

) employing

KED) to rem

5. Calculati

the leachate

d Se, the d

te total Se r

ions were l

with post-h

ered signific

NC, USA, ve

4-1. Selenium esent standard

preparation

material (4

ltiwave’) w

mL of 30%

cm) and im

ncentrations

led Plasma

g a ‘hexapo

move polyat

tions and sta

e collected

ata are onl

recovery fro

log-transfor

oc differenc

cant. The st

ersion 9.2).

uptake by cad errors.

Seleniu

n and Se an

400 mg) wa

ith 3.0 mL

% H2O2. Di

mmediately p

s of Se in p

a Mass Spe

ole collisio

tomic interf

atistical ana

from the l

ly used to

om the incub

med to obta

ces identifie

tatistical ana

atch crops gro

um mineral

alysis

as microwa

of 70% Fis

igested sam

prior to an

plant sample

ectrometer

on cell’ (H2

ferences.

alysis

leaching tub

compare S

bated mater

ain homoge

ed by the LS

alysis was c

own in the fie

ization and

ave-digested

sher ‘Trace

mples were d

nalysis, wer

es and leach

(ICP-MS,

2 gas) with

bes did not

Se levels be

rials. Data f

eneity of va

SD procedu

carried out

eld. Values ar

availability

d in pressu

analysis gr

diluted to 1

e further di

hate were d

Thermo-Fi

kinetic ene

t represent

etween the

from the lea

ariance and

ure. Results

using SAS

re means of 2

y from catch

urized PFA

rade’ (TAG)

15 mL with

iluted 1-in-

determined u

isher Scien

ergy discrim

a full reco

treatments

aching-tube

analysed b

with p < 0.

(SAS Instit

2 replicates. H

h crops

vessels

) HNO3,

h milli-Q

-10 with

using an

ntific X-

mination

overy of

s, not to

e and pot

y 1 way

.05 were

tute Inc,

Horizontal


3. Results

3.1. Composition of catch crops

Selenium concentrations in catch crop material harvested from the field ranged from 0.05

to 19.1 mg Se/kg (Table 4-1); Se addition (250 g/ha) significantly increased Se concentration

and uptake in the catch crops (Figure 4-1). The highest Se uptake was determined in plants

grown with Se, and Se uptake was higher by Italian ryegrass (IR), Indian mustard (IM), and

fodder radish (FR) compared to hairy vetch (HV) (Figure 4-1). Among the plant species

grown without Se no significant differences in Se uptake was found (Figure 4-1).

3.2. Leaching-tube incubations

At the first leaching event, six weeks after the start of the incubation, HV+Se and IR+Se

released significantly more Se than the other treatments (Figure 4-2a). Selenium

concentration in leachate from tubes with IM+Se was also higher than from the control. On

the tenth week of incubation, there was a marked reduction in Se leached from the tubes

containing enriched plant material, but Se concentrations in leachate were significantly higher

where IM+Se had been incorporated in the soil as compared to control. Thereafter,

concentrations of Se in the leachate from Se enriched soils were similar to the control.

The non enriched plant material did not affect Se concentrations in the leachate after six

weeks of incubation compared to the control (Figure 4-2b). From then on, the concentrations

of Se were lower in the leachate with non enriched plant material compared with the control,

indicating Se immobilization, although the differences were not always significant. In

general, Se released from HV+Se and FR+Se in the leachate was not significantly different

from Se released from non enriched HV and FR.

The addition of Se as sodium selenate did not significantly increase Se concentration in

the leachate compared to control soil (Figure 4-2c). At the start of incubation (Week 6), there

was a tendency for a slightly higher Se concentration in the leachate of the columns treated

with inorganic Se than the control, however, the Se leached decreased from Week 10 onward.

Addition of sucrose had little effect on Se leaching, only after 15 weeks of incubation was a

significant decrease observed in the Se concentrations following the addition of sucrose and

the inorganic Se.

444

Figure 4the non Control: hairy vetHV: nonand sucro

3.3

Tota

for firs

increase

was add

inorgan

catch cr

harvest

treatme

approxi

At t

materia

enriche

second

IR+Se w

4-2. Concentraenriched planunamended s

tch, IM+Se: enn enriched haiose. Values ar

3. Pot incub

al above gro

st and seco

ed the biom

ded. There w

nic fertilizat

rops grown

time, and

ents. Howe

imately dou

the first ha

al was incor

d material

harvest, all

was incorpo

ations of solubnt material ansoil, IR+Se: enriched Indianry vetch, IM:

re means of 2

bations

ound bioma

ond harves

mass of IM a

was a trend

tion or as e

in the field

the bioma

ever, the S

ubled at the

arvest, Se u

rporated in t

or the inorg

l treatments

orated into

Seleniu

ble Se (μg/L) nd (c) the inoenriched Italian mustard, IR non enrichedreplicates. Ho

ass producti

t, respectiv

at both harve

towards hig

enriched pla

d. There wa

ass product

Se concent

second harv

uptake by

the soil com

ganic Se fer

s increased

the soil (Ta

um mineral

in the leachatorganic treatman ryegrass, F: non enrichedd Indian mustorizontal bars

on ranged f

vely (Table

ests; the hig

gher bioma

ant materia

as little addi

tion of IM

trations inc

vest.

IM was si

mpared to th

rtilizer did

Se uptake

able 4-2). T

ization and

tes released bments during tFR+Se: enrichd Italian ryegrtard, Se: sodiurepresent stan

from 1.6 to

e 4-2). Pla

ghest yields

ass productio

al, a trend w

itional grow

was the s

creased an

gnificantly

he control, w

not affect

in IM and

The uptake w

availability

y (a) the enricthe leaching ihed fodder radrass, FR: non um selenate, Sndard errors.

7.9 g/pot a

ant amendm

were found

on when Se

which was

wth from the

same at bo

d the tota

greater wh

while the in

Se uptake (

the uptake

was greater

y from catch

ched plant maincubation exdish, HV+Se:enriched foddSe+C: sodium

and 1.8 to 7

ments sign

d where hai

e had been a

also seen w

e first to the

oth harvests

al Se upta

hen enriche

ncorporation

(Table 4-2)

e was highe

r in plants g

h crops

aterial, (b) xperiment. : enriched der radish, m selenate

7.6 g/pot

ificantly

iry vetch

added as

with the

e second

s for all

ake was

ed plant

n of non

). At the

est when

grown in


soil amended with enriched catch crop material, compared to non-enriched plant material.

There were no significant differences when HV and FR were incorporated into the soil,

although Se input from FR was 35 times higher.

In IM Se concentration and uptake were different. The highest Se concentration at first

harvest was found when IR+Se was incorporated in the soil (Table 4-2). Enriched IM also

increased Se concentrations in plant tissue compared to plants grown on unamended soil. At

the second harvest, Se concentrations in IM were increased 1.5-3.8 times compared with the

control where Se enriched material was incorporated. On the other hand, Se application as an

inorganic salt (at 1.0 g/ha) did not affect Se concentration in IM and had a tendency to lower

concentrations at both harvests. The Se concentrations in plants grown in soil amended with

non enriched plant material were significantly lower than the control at both harvests (Table

4-2). Selenium concentrations in IM were significant different at the two harvests when

enriched plant material was incorporated. Although, Se inputs from HV+Se and IM+Se were

similar, there was a trend for higher Se concentrations in IM following IM+Se incorporation.

Moreover, Se concentrations in plants following FR+Se incorporation in the soil were not

significantly different from Se concentrations following HV+Se application, although the Se

input from FR+Se was lower.

4. Discussion

To produce Se enriched plant material, a selenate addition 25-fold higher than typical field

supplementation levels (c. 10 g Se/ha) was applied to ensure high Se concentrations in plant

tissues (Broadley et al. 2010). No visual abnormalities or growth reduction were caused by

the Se application. Selenium concentrations were lower than the critical level of Se in plants

above which significant decreases in growth would be expected (Wu et al. 1988; Rani et al.

2005); results actually indicated a small yield increase, but this was not significant. As found

previously, the total recovery of applied Se was only 9-20% (Eich-Greatorex et al. 2007;

Broadley et al. 2010). In several cases Se uptake by Brassica species was greater by IR and

HV. These results were expected because of the high affinity for S shown by Brassicas and

their apparent inability to discriminate between Se and S species in soil (Terry et al. 2000).

Increased plant biomass production of IM resulted following incubation of the different

catch crops and especially HV. A similar response was reported by Askegaard & Eriksen

(2007) in the field where legume green manures greatly increased the grain yield of barley as

compare to non-legume species. However, comparing enriched and control amendments, our


results also indicated that Se addition increased plant biomass production, although Se is

generally not considered to be essential for plants. These observations are consistent with

other studies in controlled environments, which have demonstrated that small addition of Se

enhanced yield of potato tubers, lettuce, ryegrass and Brassica rapa seeds (Turakainen et al.

2004; Lyons et al. 2009; Rios et al. 2009).

Table 4-2. Above ground biomass (dry matter, DM), Se concentration and Se uptake of Indian mustard as affected by the different treatments at the first and second harvest.

First harvest Second harvest

Treatment Biomass (g DM/pot)

Selenium concentration (μg/kg)

Selenium uptake (μg/pot)

Biomass (g DM/pot)

Selenium concentration (μg/kg)

Selenium uptake (μg/pot)

Control 1.6 f 49 cd 0.08 c 1.8 e 73d 0.14 f

HV 6.0 ab 21 f 0.13 c 6.4 ab 40 f 0.26 d

IR 4.9 bcd 20 f 0.10 c 5.9 bc 34 f 0.20 e

IM 3.9 d 25 ef 0.10 c 5.2 b 40 f 0.20 e

FR 3.8 d 31 e 0.12 c 5.7 bc 53 e 0.30 d

HV+Se 7.9 a 59 bc 0.48 a 7.6 a 112 c 0.85 b

IR+Se 4.8 bcd 105 a 0.48 a 6.1 bc 276 a 1.66 a

IM+Se 5.5 bc 73 b 0.40 a 6.3 b 162 b 1.05 b

FR+Se 4.3 cd 56 bcd 0.24 b 5.4 bc 110 c 0.60 c

Se+C 2.4 e 46 cd 0.11 c 2.6 d 71 d 0.18 e

Se 2.5 e 42 d 0.10 c 2.9 d 67 d 0.20 e

Different letters within a column indicate significant differences between treatments. Values are means of 4 replicates. Degrees of freedom were 13 (model).

Comparison of the results for the two experiments showed considerable and consistent

differences in Se release from the catch crops. Our results support the hypothesis that Se-

enriched catch crops will provide succeeding crops with Se and increase Se concentration to

levels considered to be adequate for human nutrition (>100 μg Se/kg). In the leaching

experiment, the greatest Se release was observed when HV+Se and IR+Se residues were

incorporated in the soil, followed by IM+Se. However, in the pot experiment, plant Se

accumulation was higher when IR+Se and IM+Se were added to the soil. The comparatively

large Se release in the leachate and plant Se accumulation, are consistent with the relatively

high level of Se addition with the IR+Se treatment. After six weeks of incubation IR+Se had

released only 3% of the added Se in the leachate compare to HV+Se which released 6%. Both

the leaching experiment and the pot incubation experiment showed that Se effects could be


measured shortly after the addition of Se-enriched plant material; especially the leaching

experiment indicated that the effect was transient and disappeared following release of only a

small proportion of the plant Se. In the pot incubation experiment the non-enriched catch

crop materials also slightly increased Se uptake, but this may be due to the relatively short

time period or to the fact that other nutrients released from the plant materials significantly

increased the growth rate of IM.

Our results did not show clear differences in Se release rate between catch crop species

because of differences in Se input. Kahakachchi et al. (2004) found that the major types of Se

in IM treated with selenate was inorganic Se (selenate), followed by selenomethione; only

small proportions of Se-methylselenocysteine and S-(methylseleno)cysteine were found. In

contrast, in plants fed with selenite, the major organoselenium species identified were

selenomethionine Se-oxide hydrate and selenomethionine. Similarly, selenate and

selenomethione were the predominant species found in non accumulator plants (Mazej et al.

2008). In our study, Se was applied as sodium selenate to the catch crops, which means that

the plant residues were likely to have similar forms of Se and differences in Se mineralization

could not be explained by Se speciation. However, our results indicated greater release of Se

from Brassica catch crops compared to the HV+Se treatment. The low Se uptake seen when

HV+Se was incorporated in the soil can be partially attributed to a dilution-concentration

effect. Greater biomass production of IM was found when HV was incorporated in the soil,

probably caused by higher N input. Another explanation may be found in the different

abilities of catch crops to accumulate and provide S to the succeeding crops (Eriksen &

Thorup-Kristensen 2002). The antagonistic effect of sulphate on selenate uptake by plants has

been shown in many studies; sulphate may compete for membrane transporters sites and

regulate the expression of sulphate transporters by internal S status or affect the soil

chemical/biological processes that influence Se availability in plants (Terry et al. 2000;

Stroud et al. 2010b). The addition of sulphate was found to increase Se extractability by

decreasing the retention of Se in soils (Stroud et al. 2010b). Moreover, several studies suggest

that microorganisms transport selenate and sulphate by the same carrier system. The addition

of sulphate was found to inhibit the reduction of selenate by soil bacteria and enhance

sulphate reduction rates (Lindblow-Kull et al. 1985; Zehr & Oremland 1987).

Both experiments indicated immobilization of native Se when non enriched catch crop

materials were incorporated in the soil. Gustafsson and Johnsson (1994) suggest that the

process of Se retention in organic matter is primarily due to a microbially mediated reductive


process, whereby Se anions are reduced to low valence states and then incorporated into low-

molecular-weight humic substances. Reduction in Se accumulation by different plant species

with the addition of crop residues or animal manures to soil has been reported previously

(Ajwa et al. 1998; Dhillon et al. 2010). Amending soil with Se-rich crop residues at levels of

more than 0.4% was found to decrease Se concentrations in sorghum and maize (Dhillon et

al. 2007). Moreover, Se uptake by wheat grain and rape was reduced by up to 88% and 69%,

respectively, when the organic matter content in the plough layer increased from 1.4% to

39% on widely different soil types (Johnsson 1991).

It is interesting to note that the amount of Se leached from soil treated with sodium

selenate and sucrose was lower than the amount of Se leached from the soil treated only with

sodium selenate. This effect has also been described previously by Neal and Sposito (1991).

They found that the addition of dextrose caused immobilization of added selenate by

transforming a large proportion (64-90%) of Se into organic forms. Our results showed lower

plant Se utilization from the inorganic fertilization than previous reports (Bañuelos & Meek

1990), who found that IM up to 36% of the initially added Se. Selenate, as a chemical

analogue of sulphate, is taken up through sulphate transporters and follows S assimilation

pathways in the plant (Terry et al. 2000). The lower plant S requirements, due to growth

inhibition caused by low nitrogen supply, may have decreased the transcription of the high-

affinity sulphate transporters genes and thereby also decreased Se concentration.

5. Conclusion

The results showed that catch crops could be used as an alternative source of Se in crop

production. However in some cases, the addition of non enriched plant material seemed to

cause Se immobilization and decreased Se uptake by IM. The results emphasised the need to

differentiate between the catch crop species when developing recommendations for wider

application, as catch crops have different mineralization rates and mineralization potentials

due to their different chemical composition. The interactions of S and Se in the soil can

determinate Se concentrations in plants, therefore further research is required to ensure that

the catch crops will provide the correct balance between S and Se.

49 Differential N and S competition in intercropping affects glucosinolates

Chapter 5

The affect of differential N and S competition in inter- and sole

cropping of Brassica species and lettuce on glucosinolate

concentration1.

Eleftheria Stavridou †,*, Kristian Thorup-Kristensen †,‡, Hanne L. Kristensen †, Angelika Krumbein §, and Monika Schreiner §

† Faculty of Agricultural Sciences, Department of Horticulture, University of Aarhus, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark § Leibniz-Institute of Vegetable and Ornamental Crops Grossbeeren/Erfurt e. V., Theodor-Echtermeyer-Weg 1, 14979 Grossbeeren, Germany

Abstract

Field and greenhouse pot experiments were conducted to evaluate the potential to use

intercropping as an alternative method to increase glucosinolates in Brassicas by

manipulating nitrogen (N) and sulphur (S) balance by intercropping with lettuce (Lactuca

sativa L. var. capitata). In both experiments, four combinations of N and S fertilization were

used. In the field experiment no effect of intercropping on the total glucosinolates was found

as the growing lettuce was strongly inhibited by the presence of broccoli (Brassica oleracea

L. var italic). The reduction in neoglucobrassicin in broccoli from intercropping was probably

attributed to the lower N concentrations in broccoli florets. In contrast to this, in the pot

experiment both total and individual glucosinolate concentrations in red leaf mustard

(Brassica juncea L.) increased by intercropping. Fertilization treatments influenced

glucosinolate concentrations in both experiments, and an N by S interaction was observed.

Keywords: glucosinolates, intercropping, lettuce, mustard, broccoli, nitrogen, sulphur.

1 To be submitted to Journal of Agricultural and Food Chemistry

‡ Current address: Faculty of Life Science, Department of Agricultural Science, University of Copenhagen, Højbakkegård Alle 13, DK-2630 Tåstrup, Denmark


1. Introduction

The Brassica crops with their high S demand have attracted attention due to the increasing

S deficiency in many parts of the world, caused by intensive crop production, reduced

atmospheric inputs, and soil characteristics (Scherer 2001). Sulphur is found in amino acids,

oligopeptides, vitamins and cofactors, and a variety of secondary compounds in plants.

Glucosinolates (GSLs) are N and S-containing plant secondary metabolites found mainly in

the order Brassicales, and the formation of GSLs is the main reason for the high S demand by

Brassica crops. The enzymatic degradation products of GSLs contribute to the characteristic

flavour of Brassicas, their pathogen defence system or serve as insect attractants (Halkier &

Gershenzon 2006). In relation to human health, hydrolysis products of certain GSLs are

associated with beneficial effects due to their anticarcinogenic properties (Halkier &

Gershenzon 2006). Glucosinolate concentration and profile are influenced both by genetic

and environmental factors (Vallejo et al. 2003; Baik et al. 2003; Verkerk et al. 2009). In most

cases, S supply increases GSL content, which is not surprising, since each GSL molecule

contains two or three S atoms. Sulphur fertilization has not only an impact on the total GSL

content, but also on the accumulation of individual GSLs in different Brassica species, for

example Brassica napus (Zhao et al. 1994), Brassica oleracea var. italic (Krumbein et al.

2001) and Brassica rapa (Li et al. 2007).

Studies have shown contradictory effects of N supply and its interaction with S supply,

GSL concentration, and composition in plants; and it was indicated that to enhance GSL

formation a balanced N and S supply is required as represented by a species specific optimal

N:S (Li et al. 2007; Schonhof et al. 2007a). Chen et al. (2006) and Krumbein et. al (2001)

reported that the total GSL concentration in pakchoi and broccoli was enhanced at low N

supply. In cabbage, total GSLs were increased by high S supply and low N rates (Rosen et al.

2005). Increasing N supply decreased seed GSL concentration of oilseed rape when S was

deficient, but increased it when S was applied (Zhao et al. 1993). Schonhof et al. (2009)

reported that total GSL content in broccoli florets was high at insufficient N supply,

independent of S supply, and low at insufficient S supply in combination with an optimal N

supply. In contrast, a recent study has shown that GSL content in broccoli increased by

increased N supply both at low and high S, but it did not respond to N applications above 250

kg ha-1 (Omirou et al. 2009).

To satisfy the increasing health and environment awareness of consumers, the demand for

vegetables with high amounts of health promoting phytochemicals produced by sustainable


production methods needs to be fulfilled. Efficient utilization of available growth resources is

fundamental in achieving sustainable systems of agricultural production. For the production

of glucosinolate-enriched raw plant material for functional foods or supplements,

intercropping could be used as an alternative strategy to mineral fertilization and

conventional breeding approaches; strategies which have been used so far (Verkerk et al.

2009). There is a resurgence of interest in intercropping because it may increase the efficient

use of natural resources, reduce weed competition, suppress diseases and soil erosion, and

prevents nutrients leaching into deeper soil layers and ground waters, all being significant

factors in soil environment protection (Vandermeer 1989). Intercropping could be used as an

alternative strategy to manipulate N and S balance and hence increase GSLs in Brassicas.

Nitrogen concentration tended to decrease in cauliflower and cabbage when intercropped

with lettuce (Yildirim & Guvenc 2005; Guvenc & Yildirim 2006). In this study lettuce was

selected to be intercropped with Brassicas because it does not have a high S demand, but

requires adequate N. Sulphur concentrations in lettuce grown with less than 4 mM was

approximately 1 mg S g-1 of dry weight (Ríos et al. 2008). In contrast, Brassicas have high S

demand and 3-3.5 mg S g-1 dry matter is the critical S concentration where visible S

deficiency occurs in Brassica napus (Scherer 2001). Moreover, lettuce has similar root

characteristics and root depth penetration as a number of Brassica species (Thorup-

Kristensen 1993; Thorup-Kristensen 2006b). Our hypothesis was that by intercropping

Brassicas with lettuce the availability of S will increase relative to N for the Brassicas, and

that this change of the N to S balance in the nutrition of Brassicas will enhance their

glucosinolate concentration.


2.1. Field Experiment

A field experiment was conducted at The Department of Horticulture, Aarhus University,

Aarslev, Denmark (10o27’E, 55o18’N) on an Agrudalf soil. The upper 0.25 m contains 13%

clay, 15% silt, 35% sand and 1.7% carbon (C). The 0.25-0.50 m layer contain 17% clay, 13%

silt, 34.5% sand and 0.8% C, and the 0.50-1.0 m layer 19.5% clay, 13% silt, 33.5% sand and

0.3% C. The pHCaCl2 is 7.1, 6.8 and 6.4 in the 0-0.25, 0.25-0.50 and 0.50-1.0 m layers,

respectively. During the experimental period, rainfall and air temperature was recorded daily

at a meteorological station at the experimental site. Average air daily temperature and

5

52

precipit

the site

The

The bro

lettuce

pure sta

m for

replacem

seeds w

transpla

repeated

avoid w

and NO

The

kg ha-1

S40). Ur

Nitroge

transpla

Figure 5Septembe

tation durin

was 624 m

experiment

occoli (Bras

(Lactuca sa

ands. Each p

both interc

ment princi

were sown o

anting. Both

d manual w

water stress

O3− content o

four fertiliz

S (N220S0),

rea [(NH2)2

en and S

anting.

5-1. Average er, 2009).

Differenti

ng the growt

mm and mean

tal design w

ssica olerac

ativa L. var

plot was 1.6

cropping a

iple, with m

on 26 May,

h crops wer

weeding. Du

. Irrigation

of the irriga

zer treatmen

, 90 kg ha-1

2CO] was u

fertilizers w

daily tempera

ial N and S

th season ar

n annual air

was a random

cea L. var i

. capitata c

6 × 3 m, dis

and sole cr

mixed brocc

2009 and l

re transplan

uring the ex

water was

tion water w

nts were: 90

Ν + 40 kg

used as the

were broad

ature (line) an

competitio

re shown in

r temperatur

mized comp

italica cv.

cv. ‘Dimanti

stance betw

ropping. Th

oli and lettu

ettuce 2 Jun

nted on 19

xperiment, c

applied via

were about

0 kg ha-1 Ν

g ha-1 S (N90

e N source

dcast manu

nd precipitati

n in intercro

n Figure 5-1

re 7.8oC.

plete block

‘Tinman’) w

inas RZ’). B

een rows w

he intercro

uce transpla

ne, 2009 an

June. The

crops receiv

a a moveab

32 mg L-1 a

+ 0 kg ha-

0S40), 220 k

and Kieser

ually on th

ion (bars) dur

opping affec

1. Mean ann

design with

was intercro

Both crops

was 0.35 m a

op design w

ant in the sa

nd grown in

plots were

ved 40 mm

ble irrigatio

and 3 mg L-

1 S (N90S0),

kg ha-1 Ν +

rite (MgSO

he soil sur

ring the exper

cts glucosin

nual precipi

h three repli

opped with

were grown

and within r

was based

ame rows. B

n a greenhou

kept weed

m irrigation w

on boom. T-1, respectiv

, 220 kg ha

40 kg ha-1

O4) as the S

rface 2 day

erimental seas

nolates

itation at

ications.

h iceberg

n also in

rows 0.3

on the

Broccoli

use until

free by

water to

he SO4−

ely.

a-1 Ν + 0

S (N220-

source.

ys after

son (June-


2.1.1. Root measurements

Root growth of the crops was determined in the pure stands by using minirhizotrons with a

diameter of 70 mm and a total length of 1.5 m installed at an angle of 30o from the vertical

(Thorup-Kristensen 2001). In each plot, two minirhizotrons were installed in the inter-row

area. Roots were observed by lowering a minivideo camera into the minirhizotrons and

recording visible roots on the minirhizotron surface. Root intensity was recorded every two

weeks starting four weeks after transplanting by counting the number of roots crossing lines

painted on the minirhizotron surface. For every 40 mm along each of two 40 mm wide

counting grids on the ‘‘upper’’ surface of each minirhizotron, the number of roots crossing 40

mm of vertical line and 40 mm of horizontal line were counted. As the angle was of 30o from

the vertical, 40 mm along the minirhizotron surface represented a soil layer of 34.6 mm.

From these counts, root intensity was calculated as the number of root intersections m-1 line

in each soil layer.

2.1.2. Harvest and sample preparation

Crops were harvested 50 days after transplanting. Plants were stored at 2oC for one week.

Broccoli and lettuce were separated into edible part (broccoli florets and lettuce heads) and

crop residues (remaining stem and leaves). To determine dry matter (DM) content, three

samples per treatment were placed at 80oC in a forced air-drying oven for 20 hours. The DM

samples were then used for N and S analysis. For glucosinolate analysis, samples of five

broccoli florets from each plot were used. The florets were cut, immediately frozen (-40oC),

freeze dried and ground.

Initial soil mineral N and S were determined in April before the establishment of the

experiment. After harvest, soil samples were analyzed for of N and S content in all

treatments. Soil samples (nine replicates per plot) were taken randomly with a pistol auger

(inner diameter 14 mm). In April, samples were taken of the soil layers 0-0.25 m, 0.25-0.50

m, 0.50 to 0.75 m, and in August 0-0.25 m, 0.25-0.50 m, 0.50 to 1.0 m. The soil samples

were frozen at -18oC within 24 h from sampling.

2.2. Pot experiment

A pot experiment was carried out from 8 May to 8 June, 2010 in a greenhouse at

Department of Horticulture, Aarhus University, Aarslev, Denmark in order to eliminate the

above ground competition which occurred in the field experiment. The soil used was


collected from the top 15 cm of a field located at the Department. The soil was air-dried,

sieved (< 5 mm) and mixed with sand (2:1) to ensure good porosity for air and water. The

mixture of soil was placed in 7 L plastic pots (upper diameter = 0.25 m, height= 0.20 m).

The red leaf mustard (Brassica juncea L. cv ‘Red Giant’) was intercropped with leaf

lettuce (Lactuca sativa L. var. capitata cv. ‘Lugano RZ’). The cultivars were selected

because they were both fast growing. In the intercropping treatment, one side of each pot was

planted with one red leaf mustard seedling and the other side with one lettuce seedling. Both

crops were grown also in pure stands with two plants per pot. In order to minimize the aerial

interaction and competition between crops, plants were separated by a Polystyrene foam

board both in intercropping and sole cropping treatments. Two levels of N were supplied in

the form of urea at 0.9 g pot-1, and 2 g pot-1 corresponded to 203, and 406 kg N ha-1,

respectively. Sulphur was applied at two different rates of 44, and 88 kg ha-1 in the form of

Kieserite corresponding to 0.2, and 0.45 g per pot.

The pots were arranged on a greenhouse bench in a complete randomized block design

with four replicates of each of the 12 treatments. The average day and night greenhouse

temperature were 20 and 14oC, respectively; the average day length during the experiment

was 13 h. The pots were watered daily with pre-collected rain water as needed to avoid water

stress. To avoid leaching losses from the pots a drainage tray was placed under each pot. Any

leachate collected in the trays was re-applied to the pot. Plants were harvested 31 days after

transplanting into the pots. The main midrib from the red leaf mustard leaves was removed

prior to the analysis because it contains low GSL concentration and could lead to a bias

within the leaf sample. The samples of leaf mustard were immediately deep frozen (-40oC),

then freeze-dried, ground and analyzed for GSLs, N and S contents. The lettuce samples were

oven dried at 80oC for 20 hours, ground and analyzed for N and S contents. The dry matter

yield was recorded.

2.3. Glucosinolate Analysis

A modified HPLC method reported by Krumbein et al. (Krumbein et al. 2005) was used to

determine the desulfo-glucosinolate profiles. Duplicates of freeze-dried sample material (0.02

g) were heated to and incubated at 75°C for 1 min, and then extracted with 0.75 ml of 70%

methanol. The extracts were heated for 10 min at 75°C and then, after adding 0.2 ml 0.4 M

barium acetate, centrifuged at 4000 rpm for 5 min. The supernatants were removed, and the

pellets were extracted twice more with 0.5 ml 70% methanol (70°C), shaken vigorously in a


Vortex mixer to dissolve pellets and centrifuged. Just prior the first extraction 100 μl of a 0.5

M stock solution of sinigrin in methanol was added to one of the duplicated as internal

standard. The supernatants were combined and applied to a 250 μl DEA-Sephadex A-25 ion-

exchanger (acetic acid-activated, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and

washed with bi-distilled water. After the application of 100 μl purified aryl sulphatase

solution and 12 h incubation, desulfo-compounds were eluted with 1.5 ml bi-distilled water.

Table 5-1. Glucosinolates determinate by HPLC.

Desulfo-glucosinolate analysis was carried out by HPLC (Merck HPLC pump L-7100,

DAD detector L-7455, automatic sampler AS-7200 and HPLC Manager-Software D-7000)

using Spherisorb ODS2 column (3 μm, 125 x 4 mm). A gradient of 0-20% acetonitrile in

water selected from 2 to 34 min, followed by 20% acetonitrile in water until 40 min, and then

100% acetonitrile for 10 minutes until 50 min. The determination was conducted at a flow of

0.7 mL min-1 and a wavelength of 229 nm. Glucosinolate concentrations were calculated

using sinigrin as internal and external standard and the response factor of each compound

relative to sinigrin (Official Journal of the European Communities, 1990, L 170, 28-34). The

well known desulfo-glucosinolates were identified according to previous work (Zimmermann

et al. 2007) from the protonated molecular ions [M + H]+ and the fragment ions corresponded

to [M + H - glucose]+ by HPLC-ESI–MS2 using Agilent 1100 series (Agilent Technologies,

Waldbronn, Germany) in the positive ionization mode. Determinations of desulfo-

glucosinolates were performed in duplicate. The desulfo-glucosinolates determinate are

shown in the Table 5-1.

Type Common name Chemical structure

Alkene Gluconapin 3-butenyl

Sinigrin 2-propenyl

Hydroxyl alkene Progoitrin 2-hydroxy-3-butenyl

Methylsulphynyl alkene Glucoiberin 3-methysulfinylpropyl

Glucoraphanin 4-methylsulfinlbutyl

Indolyl Glucobrassicin 3-indolylmethyl

Neoglucobrassicin 1-methoxy-3-indolylmethyl

4-methoxy-glucobrassicin 4-methoxy-3-indolylmethyl


2.4. The N and S analysis

In the field experiment, N and S contents were measured both in the edible parts and the

crop residues. Total plant N was determined after dry oxidation by the Dumas method

(Elementar Vario EL. Hanau. Germany) and total sulphur by using NDIR (non-dispersive

infrared gas analysis) optic to detect the sulphur dioxide formed. Finely ground samples were

weighted into quartz boats, and delivered into the hot zone of a multi EA 2000 CS (Analytic

Jena AG. Jena. Germany). Then the samples were pyrolyzed and oxidized at 1300oC in a

stream of oxygen (99.5%). Both measurements were performed in duplicate.

Table 5-2. Effects of cropping system and fertilization treatments of N and S concentrations (kg ha-1) in the soil of the field experiment.

where N (nitrogen), S (sulphur), C (cropping system); a Levels of significance: NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (n=3).

Cropping system Fertilization Soil inorganic N

(0-1.0 m) (kg N ha-1)

Soil inorganic S

(0-1.0 m) (kg S ha-1)

Intercrop N90S0 38 37

N90S40 37 57

N220S0 52 37

N220S40 41 45

Sole lettuce crop N90S0 47 36

N90S40 51 82

N220S0 65 40

N220S40 64 85

Sole broccoli crop N90S0 34 37

N90S40 38 50

N220S0 48 29

N220S40 42 51 Significancea

N *** NS S NS *** C *** *** N×C NS NS S×C NS ** N×S NS NS N×S×C NS NS


2.5. Statistical analysis

Statistical analysis of the data was performed using the GLM procedure of the SAS

statistical package (SAS Institute Inc., Cary, NC, USA, 1990). In the pot experiment,

flowering mustard plants with GSL concentrations significant different from GSL

concentrations in the non flowering plants were excluded from the statistical analysis.

Table 5-3. Effects of cropping system and fertilization treatments on the edible plant part and total above ground dry matter production (kg ha-1).

Cropping system

Fertilization

Broccoli Lettuce

Edible Total Edible Total

Intercrop

N90S0 689 4206 314 450

N90S40 663 4454 266 383

N220S0 726 4558 194 294

N220S40 819 5000 162 232

Sole crop

N90S0 567 5188 2536 3658

N90S40 573 5097 2484 3502

N220S0 742 5433 2337 3538

N220S40 774 5761 2318 3486

Significancea

N NS *** ** NS

S NS NS NS NS

C NS *** *** ***

N×C NS NS NS NS

S×C NS NS NS NS

N×S NS NS NS NS

N×S×C NS NS NS NS


3. Results

3.1. The field experiment

3.1.1. Soil N and S

Increased N supply increased soil N content after sole cropping of lettuce (Table 5-2). In

intercropping and sole cropping of broccoli, this effect was smaller and only significant when


S fertilizer was not applied together with the N fertilizer. Soil N content did not differ

between intercropping and sole cropping of broccoli, but they were higher in sole cropping of

lettuce.

In the soils where S fertilization was applied, the S concentration was significantly higher

than in the unamended soils (Table 5-2). No differences were found in soil S after

intercropping and sole cropping of broccoli but higher soil S was found after sole cropping of

lettuce where S had been applied.

3.1.2. Above ground biomass production

Intercropping decreased the total above ground biomass production of both broccoli and

lettuce (Table 5-3). The reduction in biomass due to intercropping was greater in lettuce

which reached up to 93%. Increasing the Ν supply from 90 to 220 kg ha-1 increased broccoli

above ground biomass, but had no effect on lettuce biomass. Sulphur fertilization did not

influence broccoli or lettuce above ground production. In contrast, to total above ground

biomass production of broccoli, intercropping and fertilization treatments did not influence

florets biomass production of broccoli.

3.1.3. Root growth

The two vegetables had different root characteristics (Figure 5-2). Lettuce and broccoli

showed quite similar rates of rooting depth penetration, but the root intensity and root

distribution in the soil varied between the crops. The root intensity, 19 days after

transplanting, was comparable in both crops, and they both showed the highest root intensity

in the top 0.25 m soil layer. However, broccoli had established a higher root intensity than

lettuce in the soil layer between 0.25 and 0.5 m. Fertilization affected root growth of the two

crops; higher root intensity was observed when low N and high S were applied. At the final

measurement (one week before harvest), the root intensity of broccoli was much higher than

that of lettuce, but in the top 0.25 m layer lettuce had the highest root intensity. Below this,

the root intensity of lettuce declined gradually, whereas broccoli had its highest root intensity

in the 0.25 and 0.75 m soil layer. Between 0.75 and 1 m, broccoli still had significantly

higher root intensity than lettuce, as lettuce showed practically no roots in this soil layer.

There were some indications that fertilization affected root growth of the two crops; lettuce at

N90S40 showed higher root densities than the other lettuce crops, and broccoli at N220S40

showed lower root intensities, especially compared to broccoli at N220S0.

Differ

Figure 5transplanerrors (w

3

Nitro

Intercro

When b

N uptak

examin

no sign

stronger

concent

increase

Ferti

florets

respons

Increasi

broccol

rential N an

5-2. Average nting (a) and o

where n = 2).

3.1.4. N and

ogen and

opping affec

broccoli wa

ke comparin

ed organs o

nificant inte

r in the bro

tration in b

ed by 31-62

ilization wi

and residue

se of S conc

ing the N s

li florets or

d S compet

root intensitone week bef

d S accumu

S accumu

cted N con

as intercropp

ng to sole c

of broccoli r

ractions we

ccoli crop r

broccoli flo

2%.

ith S increa

es, at both N

centration to

supply from

residues. In

ition in inte

ty in the 0-1fore harvest (b

ulation

ulation in

ncentrations

ped with let

ropped of b

responded to

ere found. T

residues tha

orets by 15

ased total S

N fertilizat

o S fertiliza

m 90 to 220

ntercropping

ercropping a

1.3 m soil prb). where N,

broccoli a

in broccol

ttuce it had

broccoli. Ni

o N supply,

The effect o

an in the edi

5-19%, whi

S uptake an

tion levels (

ation was g

0 kg ha-1 ha

g only affec

affects gluco

rofile in the nitrogen; S,

and lettuce

li florets an

d lower flor

itrogen conc

, but these w

of N applic

ible part. In

ile in brocc

nd the S c

(Table 5-4)

reater in br

ad no effect

cted S conc

osinolates

field experimsulphur; bars

are show

nd total N u

et N concen

centrations

were unaffe

cation on N

creasing N

coli residue

oncentratio

). As for N

occoli resid

t on tissue S

entrations o

ment four werepresent the

wn in Tab

uptake by b

ntrations an

and N upta

ected by S le

N concentrat

supply incr

es N conce

on both in

N concentrat

dues than in

S concentra

of broccoli

eeks after e standard

ble 5-4.

broccoli.

nd lower

ake in all

evel and

tion was

reased N

entration

broccoli

tion, the

n florets.

ations in

residues

59


Table 5-4. Effects of cropping system and fertilization treatments on N and S concentration (mg g-1 DM) and total N and S uptake (kg ha-1) in broccoli and total N and S uptake (kg ha-1) lettuce in the field experiment.

Cropping system

Fertilization

Nitrogen Sulphur

Broccoli Lettuce Broccoli Lettuce

Concentration Total uptake

Total uptake

Concentration Total uptake

Total uptake Florets Residues Florets Residues

Intercrop N90S 0 32.4 20.2 93 16 6.2 4.6 20 1

N90S 40 31.9 18.9 93 14 7.1 7.5 33 1

N220S 0 38.7 29.3 141 12 5.2 3.2 16 1

N220S 40 37.6 30.6 158 11 7.4 8.2 41 1

Sole crop N90S 0 35.3 19.2 109 96 6.3 3.7 21 6

N90S 40 35.7 19.1 107 89 8.0 7.1 36 6

N220S 0 40.5 28.4 162 118 6.2 3.4 20 8

N220S 40 41.0 25.0 156 119 7.5 6.4 37 7

Significance

N *** *** *** ** NS NS NS NS

S NS NS NS NS *** *** *** NS

C ** NS ** *** NS * NS ***

N×C NS NS NS *** NS NS NS **

S×C NS NS NS NS NS NS NS NS

N×S NS NS NS NS NS NS * NS

N×S×C NS NS NS NS NS * NS NS


Dif

under t

intercro

croppin

Nitro

lettuce

the sole

Figure 5concentra

3

Five

indole

quantita

individu

The

cropped

broccol

fertiliza

S. Total

attempt

fferential N

the high N

opping com

ng system in

ogen and S

growth (Ta

e cropping tr

5-3. Influenceation in the fie

3.1.5. Gluco

individual

GSLs gl

atively dete

ual GSLs.

highest tota

d broccoli,

li at N220S

ation, where

l and alipha

t to relate G

and S comp

and S tre

mpared to so

nteraction fo

uptake by

able 5-4). N

reatment.

e of N (a) aneld experimen

osinolates

GSLs, nam

lucobrassici

ermined in b

al GSL leve

while the l

S0 (Table

eas N fertili

atic GSL co

GSL conce

petition in in

atment, wit

ole cropping

or S accumu

lettuce was

Nitrogen fert

nd S (b) concnt.

mely, the ali

in, neoglu

broccoli flor

el (4137 μg

lowest leve

5-5). Total

ization redu

ncentration

ntration to

ntercroppin

th a 28% i

g of brocco

ulation in br

s reduced by

tilization en

centration and

iphatic GSL

ucobrassicin

rets. The to

g-1 DM) wa

el (2458 μg

l GSL con

uced GSL c

n was not aff

the nutritio

ng affects gl

increase in

oli. A signi

roccoli resid

y intercropp

nhanced N a

d N : S ratio

Ls glucorap

n and 4-m

otal GSL wa

as obtained

g g-1 DM)

ncentrations

oncentratio

ffected by in

onal status

ucosinolate

S concent

ficant N su

dues was ob

ping, as a r

and S uptak

o (c) on total

phanin and g

methoxy-glu

as calculate

at high N a

was observ

significant

ns when N

ntercropping

of broccol

es

tration obse

upply × S s

bserved.

result of the

ke by lettuce

l glucosinolat

glucoiberin

ucobrassicin

ed as the sum

and S supply

ved in inter

tly increase

was added

g (Table 5-5

li, the relat

erved in

supply ×

e limited

e only in

te (GSLs)

and the

n were

m of the

y in sole

rcropped

ed by S

without

5). In an

ionships

61


Table 5-5. Effects of cropping system and fertilization treatments on glucosinolate concentration (μg g-1 DM) and N:S ratio in broccoli florets of the field experiment.

Cropping system

Fertilization N:S ratio

Glucosinolatea Total

GSLs

Total

Aliphatic

GSLs

Total

Indole

GSLs GRA GIB GBS NGB MGB

Intercrop N90S 0 5.3 1458 446 252 1246 32 3433 1904 1529

N90S 40 4.5 1425 434 275 1661 30 3824 1859 1966

N220S 0 7.5 777 278 229 1145 29 2458 1055 1403

N220S 40 5.1 1221 370 327 1580 34 3533 1591 1942

Sole crop N90S 0 5.7 1210 430 263 1510 31 3443 1639 1803

N90S 40 4.5 1359 445 277 1754 35 3869 1803 2066

N220S 0 6.6 774 315 260 1561 32 2941 1089 1852

N220S 40 5.5 1197 427 342 2130 40 4137 1625 2513

Significanc

N *** *** *** NS NS NS * *** NS

S *** ** ** NS ** NS *** ** **

C NS NS NS NS * NS NS NS *

N×C NS NS NS NS NS NS NS NS NS

S×C NS NS NS NS NS NS NS NS NS

N×S * ** ** NS NS NS * ** NS

N×S×C * NS NS NS NS NS NS NS NS

where N (nitrogen), S (sulphur), C (cropping system );aGRA: glucoraphanin; GIB: glucoiberin; GBS: glucobrassicin; NGB: neoglucobrassicin; MGB: 4-Methoxy-Glucobrassicin; b Levels of significance: NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (n=3).


between GSLs, N, S and the N:S ratio were evaluated (Figure 5-3). The correlation between

total GSLs and N concentration in broccoli florets was not significant (Figure 5-3a). When S

concentrations in broccoli florets were higher than 6 mg g-1 DM, total GSL concentrations

were around 4 mg g-1 DM, but these decreased to 3 mg g-1 DM when S concentrations were

lower than 6 mg g-1 DM (Figure 5-3b). A significant negative correlation between total

GSLs and N:S ratio was determined in the regression analysis, this was strongest when

broccoli was grown with lettuce (Figure 5-3c).

Total aliphatic GSL concentration responded to S application only under high N

availability. Sulphur fertilizer increased total aliphatic GSLs by 51% in intercropping and by

49% in pure stand. Without simultaneous S fertilization, N fertilization decreased aliphatic

GSLs by 45% and 34% in inter- and sole cropping, respectively. The decrease was lower

when S was also applied. The individual aliphatic GSLs glucoraphanin and glucoiberin

showed similar trends to the total aliphatic GSLs.

Changes in the total indole GSLs were mainly due to variations in neoglucobrassicin and

comparable low concentrations of glucobrassicin and 4-methoxy-glucobrassicin were

detected (Table 5-5). The highest neoglucobrassicin concentrations were observed at high S

supply, regardless of N supply. Neoglucobrassicin content in broccoli florets was reduced in

intercropping as compared to sole cropping.

3.2. Pot experiment

3.2.1. Dry matter production

Red leaf mustard above ground biomass was influenced by intercropping but remained

unaffected by N and S fertilization or any interaction (Table 5-6). When red leaf mustard was

intercropped with lettuce DM production was 1.1-1.5 times higher compared to sole

cropping. Lettuce above ground biomass production was neither affected by intercropping

nor fertilization (Table 5-6). Red leaf mustard DM was 3.9 to 7.8 times higher than that of

lettuce.

3.2.2. N and S accumulation

Nitrogen concentrations in red leaf mustard were affected by the cropping system, the

fertilization treatments, and the S supply by cropping system interaction (Table 5-6).

Increasing N supply from 203 to 406 kg ha-1 increased the N concentrations in mustard leaves


by 15-33%. Sulphur fertilization increased N concentration in red leaf mustard when it was

grown with lettuce. In intercropping, N concentrations in mustard leaves were higher than in

sole cropping. In contrast, intercropping decreased the N concentrations in lettuce (Table 5-

6). Moreover, lettuce N concentrations were affected by the N supply by S supply, N supply

by cropping system, and N supply by S supply by cropping system interactions. Compared to

N, the concentration of S in leaves of mustard was much less affected and only the impact of

S supply was significant (Table 5-6). Sulphur concentrations in lettuce leaves were

unaffected by the cropping system or the fertilization treatments.

Table 5-6. Effects of cropping system and fertilization treatments on above ground biomass production (g plant-

1), N and S concentrations (mg g-1 DM) in red leaf mustard and lettuce in the pot experiment.

Cropping system

Fertilization

Red leaf mustard Lettuce

DM yield (g plant-1)

N content (mg g-1 )

S content (mg g-1 )

DM yield (g plant-1)

N content (mg g-1 )

S content (mg g-1 )

Intercrop N203S44 10.2 42.7 5.6 1.3 30.2 1.9

N203S88 9.2 47.1 6.4 1.2 39.8 2.1

N406S44 9.9 49.0 5.4 1.4 38.2 2.1

N406S88 9.8 57.0 7.6 1.9 25.9 1.8

Sole crop N203S44 6.8 40.3 5.8 1.8 39.4 2.4

N203S88 7.6 37.9 7.1 1.7 37.3 2.4

N406S44 8.3 49.4 5.4 2.1 42.7 2.1

N406S88 8.6 50.3 6.3 1.4 45.4 2.1

Significancea

N NS *** NS NS NS NS

S NS * *** NS NS NS

C ** ** NS NS *** NS

N×C NS NS NS NS * NS

S×C NS * NS NS NS NS

N×S NS NS NS NS * NS

N×S×C NS NS NS NS *** NS

where N (nitrogen), S (sulphur), DM (dry matter), C (cropping system); a Levels of significance: NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (n=4).

3.2.3. Glucosinolates

Individual GSLs, namely the aliphatic GSLs progoitrin, gluconapin and sinigrin, and the

indole GSLs glucobrassicin and 4-methoxy-glucobrassicin were determined in red leaf


mustard (Table 5-7). Aliphatic GSLs were the major fraction of total GSLs in red leaf

mustard at 98-99%.

The most predominant GSL was sinigrin accounting for 70-98% of total GSLs, followed

by gluconapin (3-5% of total GSLs). Total GSL concentrations varied between 8230 and

11571 μg g-1 with different cropping system and fertilization supply (Table 5-7). The highest

GSL concentrations were recorded in pots that received 88 kg S ha-1 and 406 kg N ha-1

irrespective of cropping system. Differences in total GSLs were mainly caused by changes in

the main aliphatic glucosinolate sinigrin. Sinigrin concentration in red leaf mustard was

significantly influenced by intercropping; GSLs were higher in intercropping, independent of

the fertilization levels. The significant N supply by S supply interaction indicated that GSL

concentration, as a response to S supply, is dependent on N supply. Increasing S supply

increased sinigrin concentrations only at the high N level, whereas a slight reduction was

observed at the low N level. The results presented here suggest that N can both increase and

decrease GSL concentrations depending on the S supply. With low S supply, total GSL

concentrations were higher at low N supply. However, at the high S level, increasing N

supply increased total GSLs by 5% in the intercropping and 32% in the sole cropping

systems. The aliphatic GSLs progoitrin and gluconapin were unaffected by either the

intercropping or the fertilization treatments.

Indole GSLs concentrations were generally low in red leaf mustard plants (1-1.5% of the

total GSLs) (Table 5-7). Intercropping increased indole GSLs up to 63%. In addition S

fertilization affected indole GSLs concentrations; in general, increased S fertilization

enhanced indole GSLs with the exception of the low N treatment in the intercropping system.

A significant interaction was observed between N and S fertilization; increasing N when the

S fertilization was high resulted in a significant increase of indole GSLs. Increasing N supply

led to a decrease in indole GSLs when S supply was low.

In intercropping, total and aliphatic GSL concentrations showed a negative correlation

with N:S ratio (r2 = 0.46, p<0.01), whereas the correlation in sole cropping was positive (r2 =

0.12, p>0.05). Similarly correlations were found for indole GSLs but they were not

significant (data not shown).


Table 5-7. Effects of cropping system and fertilization treatments on glucosinolate concentration (μg g-1 DM) and N:S ratio in red leaf mustard in the pot experiment.

Cropping system

Fertilization N:S ratio

Glucosinolatea Total GSLs

Total Aliphatic GSLs

Total Indole GSLs PRO SIN GNA MGB GBS

Intercrop N203S44 7.7 5 10586 452 18 98 11159 11043 116

N203S88 7.4 7 10335 510 18 104 10974 10852 122

N406S44 9.5 11 8478 327 17 82 8915 8816 100

N406S88 7.3 16 10885 498 26 145 11571 11400 171

Sole crop N203S44 7.1 7 8368 364 14 89 8842 8739 103

N203S88 5.4 7 7784 364 9 66 8230 8155 75

N406S44 9.2 2 8218 410 16 73 8720 8630 90

N406S88 8.0 6 10288 458 18 105 10874 10751 123

Significance

N ** NS NS NS * NS NS NS NS

S * NS NS NS NS * NS NS *

C NS NS * NS * ** * * **

N×C NS NS NS NS NS NS NS NS NS

S×C NS NS NS NS NS NS NS NS NS

N×S NS NS * NS NS ** * * **

N×S×C NS NS NS NS NS NS NS NS NS

where N (nitrogen), S (sulphur), C (cropping system); aPRO: progoitrin; GNA: gluconapin; SIN: sinigrin; GBS: glucobrassicin; MGB: 4-methoxy-glucobrassicin; b Levels of

significance: NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (n=4).


4. Discussion

4.1. Field experiment

Variation in the content, as well as in the pattern, of GSL occurred depending on the plant

species, the cultivar and the cropping conditions. In the field experiment total, aliphatic and

indole GSLs were within the ranges reported in previous studies (Verkerk et al. 2009). The

high concentrations of glucoraphanin and neoglucobrassicin determined in broccoli in this

study are consistent with the findings of Baik et al. (2003) and Schonhof et al. ((2004)

Although the present results confirmed that the balance between N and S plays an

important role in the regulation of the synthesis and/or accumulation of GSLs, our hypothesis

that intercropping will increase GSL concentrations was not verified in the field experiment.

Similarly, broccoli dominated and sharply reduced the crop yields of pea and cabbage during

intercropping (Santos et al. 2002). The limited growth of the lettuce mainly attributed to

irradiation competition as broccoli completely shaded the lettuce and broccoli was the

dominant species in the intercrop. The root data showed that lettuce could be able to compete

well with broccoli for nutrients, though broccoli showed higher total root growth, lettuce built

higher root densities in the topsoil and had approximately the same root depth development

as broccoli, in accordance with results of Thorup-Kristensen (Thorup-Kristensen 1993;

Thorup-Kristensen 2006b). Below ground competition possibly also occurred as the root

density of broccoli was higher than that of lettuce. Subsequently, total N and S uptake by

lettuce was limited and intercropping did not influence the balance of inorganic N and S left

in the soil by the crops significantly or the balance between N and S in the broccoli crop.

Increasing the N supply without S fertilization significant decreased the total GSLs. High

N supply have shown to increase protein content in seeds of B. napus and when S was limited

most of the S was incorporated into proteins and therefore less S was available for

glucosinolate synthesis (Asare & Scarisbrick 1995). Total GSLs were positively correlated

with the S concentrations in broccoli florets and the decrease in the total GSLs at high N

supply could be partially explained by the tendency to decrease S concentrations in broccoli

when N fertilization rate increased. Moreover, the N:S ratio increased and a negative

relationship between GSL concentration and N:S ratio was found, as reported before for the

turnip (Li et al. 2007) and broccoli (Schonhof et al. 2007a). Similar results were obtained by

Schonhof et al. (2007) who found that in broccoli florets at high N fertilization the total S


concentrations decreased. They suggested that the phytohormone cytokinin or metabolites

such as cysteine may down regulate S uptake and thus the S assimilation. Only, Omirou et al.

(2009) found that total GSLs increased when N fertilization increased from 50 to 250 kg N

ha-1 irrespectively of S fertilization.

Enhanced S supply increased GSL concentration in several Brassica species (Zhao et al.

1993; Li et al. 2007; Omirou et al. 2009). The low GSL concentration when no S was applied

could be attributed to the fact that the de-novo synthesis of indole GSLs from tryptophan is

limited by the sulphur donor from the thiohydroximate (Nikiforova et al. 2003). Reduced

response of GSLs to S fertilization at low N supply was also observed by Omirou et al.

(2009) in broccoli florets. Glucosinolates are both S and N containing compounds (Mithen

2001) and N limitation may have restricted both aliphatic and indole GSLs synthesis.

Glucoraphanin, is the main aliphatic GSL found in broccoli florets. As for the total GSLs,

at high N levels S fertilization significantly increased aliphatic GSLs, whereas N supply

decreased aliphatic concentrations in broccoli florets. These results agree with those of

Schonhof et al. (2007) who found that broccoli plants grown with low N supply showed no

significant differences in the concentration of glucoraphanin in response to different S

supplies, but this changed when grown with enhanced N. Moreover, they showed that

enhanced N supply decreased aliphatic GSLs. In rape seeds the concentration of S containing

amino acids such as methionine, the precursor amino acid for aliphatic GSLs synthesis,

increased with an increased S supply, and this response was more pronounced at high N

supply resulting in an increasing GSL concentration (Mortensen & Eriksen 1994).

In this study the dominant indole GSL in broccoli florets was neoglucobrassicin. Most

studies (Schonhof et al. 2007a; Omirou et al. 2009) have reported glucobrassicin as the main

indole GSL in broccoli. Differences in results could be due to differences in broccoli cultivars

tested (Baik et al. 2003). Omirou et al. (2009) showed that a lack of N suppressed indole

GSLs in broccoli florets, but in our study N fertilization did not show any clear effect on

indole GSLs. However, the decreased indole GSLs concentrations in broccoli in the

intercropping system might be attributed to the lower N concentrations in broccoli florets

compared to under the sole cropping system. More N is needed to synthesize indole GSLs,

than aliphatic GSLs because two atoms of N instead of one are needed for the biosynthesis of

indole GSLs (Mithen 2001).


In the field experiment, intercropping and N fertilization influenced N concentrations in

broccoli florets. Yildirim and Guvenc (2005) and Guvenc and Yildirim (2006) have found a

non significant tendency of lower N concentration when cauliflower and cabbage was

intercropped with lettuce in an additive design. In our study the lower N concentrations was

not seen as an effect of competition. Although, N concentrations in lettuce were higher in

intercropping the total N uptake was low due to the limited growth of the lettuce.

4.2. Pot experiment

Limited information is available concerning the interactive effects of N and S supply on

the glucosinolate concentrations in B. juncea leaves. Results reported for the seeds of Indian

mustard (Gerendás et al. 2009) where similar to those of our field study in broccoli, where

total GSL concentrations was reduced when N supply increased at low S supply, whereas the

opposite effect was observed at high S supply. In contrast to the field experiments,

intercropping affected total, indole and aliphatic GSL concentrations in red leaf mustard. The

N:S ratio of leaves is frequently used to characterize the nutritional status of a crop, as well as

having a physiological basis through the common presence of these nutrients in proteins.

During early flowering of oilseed rape the critical value of N:S ratio where seed yield losses

occurred due to S deficiency was found to be 9.5 (McGrath & Zhao 1996). Aulakh et al.

(1980) indicated that S supply may not be adequate when the N:S ratio is above 7.5 in

mustard grain. In our research, intercropping increased both aliphatic and indole GSLs in red

leaf mustard when the N:S was lower than 8. Although, we did not observe S deficiency

symptoms or biomass reduction in plants with N:S ratio above 8, S limitation was probably

the reason that intercropping did not affect GSL concentrations. When no S was applied,

protein in mustard grain increased progressively with increasing N supply (Aulakh et al.

1980), therefore less S was available for the GSL synthesis.

Dry weight of individual plants of red leaf mustard increased by intercropping, this was

mainly because lettuce was not competitive relative to red leaf mustard. Both N and S

concentrations in red leaf mustard were influenced by N and S fertilization, respectively. In

our study no clear interaction between N and S fertilizations was observed, an S supply by

cropping system interaction was determined for the N concentration, this indicated that at

high S supplies, S availability increased in intercropping which may have led to the increased

N accumulation observed in mustard plants. Several studies showed that S fertilization

enhanced N uptake by rapeseed mustard (Abdin et al. 2002) and oilseed rape (Zhao et al.


1993). Α combined application of S and N increased the total N accumulation in B. juncea

shoots compared to N application alone (Abdin et al. 2002). Similarly, Gerendás et al. (2009)

found strong interactive effects of S and N supply on N concentration in leaves Indian

mustard.

The present research confirmed that GSL concentrations in Brassicas may be increased by

altering the N:S ratio through appropriate N and S fertilization, and that it may also be

affected by intercropping with non-Brassica crops, though the lettuce plants were too weak

competitors here to achieve a clear test of this hypothesis. If intercropping and fertilization

can be used to increase GSL concentrations it can be used to increase the health benefits

when consuming Brassica vegetables. The effect of intercropping on GSL concentrations was

clearly shown in the pot experiment when the above ground interactions were eliminated. The

results indicated a species specific response to intercropping; therefore further work is

required to develop efficient intercropping systems. A main factor will be through selection

of plant material and intercrop design to develop systems where the non-Brassica species can

develop better than in the present experiments, otherwise their effect will remain limited.

5. Abbreviations Used

N: nitrogen; S: sulphur; DM: dry matter; HPLC, high-performance liquid chromatography;

CFA: continuous flow analysis; GSLs: glucosinolates; PRO: progoitrin; GNA: gluconapin;

SIN: sinigrin; GRA: glucoraphanin; GIB: glucoiberin; GBS: glucobrassicin; NGB:

neoglucobrassicin; MGB: 4-methoxy-glucobrassicin.

6. Acknowledgment

We thank Astrid Bergman and Birthe R. Flyger from the Department of Horticulture,

Aarhus University, Denmark for the skilful technical assistance. From the Leibniz-Institute of

Vegetable and Ornamental Crops Grossbeeren/Erfurt e. V. we thank Kerstin Schmidt for

assistance in N and S analyses and Andrea Jankowsky for help with HPLC analyses.

71 Effects of fertilization on root growth

Chapter 6

Effects of N and S fertilization on root growth

1. Introduction

Intercropping is defined as the growth of two or more crops in proximity in the same field

during a growing season to promote interaction between them. Available growth resources,

such as light, water and nutrients are more completely absorbed and converted to crop

biomass by the intercrop as a result of differences in competitive ability for growth factors

between intercrop components. The more efficient utilization of growth resources leads to

yield advantages and increased stability compared to sole cropping. Interspecific competition

or facilitation may occur in intercropping (Vandermeer 1989). Several studies have been

focused on the spatial structure of above-ground parts of the component crops (Willey &

Reddy 1981). However, component crops also interact with each other underground through

water and nutrient uptake and microbial activities. To improve the utilization efficiency of

soil nutrient resources by intercropping systems, the spatial distribution and activities of roots

requires elucidation. Moreover, the root system is highly responsive to nutrient (nitrogen,

phosphorus) availability and distribution within the soil (Linkohr et al. 2002).


Root growth of broccoli (Brassica oleracea L. var italica cv. ‘Tinman’) and iceberg

lettuce (Lactuca sativa L. var. capitata cv. ‘Dimantinas RZ’) was measured in the pure stands

of the intercrop field experiment (Chapter 5, this thesis). Roots of both species have similar

morphological characteristics, thus visual discrimination of the root systems in the

intercropped plots would be difficult. Both crops were transplanted on 19 June. The

transplants were grown in peat blocks (4×4×4 cm cubes) and planted in the field with a row

distance of 0.35 m and a planting distance within the rows of 0.30 m. A randomized complete

block design with two replicates was used. Each plot consisted of 4 rows with 10 plants. The

plots were kept weed free by repeated manual weeding.

The four fertilizer treatments were: 90 kg ha-1 Ν + 0 kg ha-1 S (N90S0), 220 kg ha-1 Ν + 0

kg ha-1 S (N220S0), 90 kg ha-1 Ν + 40 kg ha-1 S (N90S40), 220 kg ha-1 Ν + 40 kg ha-1 S

(N220S40). Urea [(NH2)2CO] was used as the N source and Kieserite (MgSO4) as the S

source. Nitrogen and S fertilizers were broadcast manually on the soil surface 2 days after

transplanting.

7

72

Figur

Dire

were in

placed i

the thir

reachin

using a

thesis).

Plan

perform

simple

was est

minirhi

(SAS In

3. Re

Clea

period

depth o

The res

re 6-1. Minirh

ctly after p

nserted in th

in the interr

d and fourt

g a depth o

a min-video

nts were ha

med as desc

averages of

timated as

zotron. Dat

nstitute Inc,

esults and

ar differenc

(Figure 6-2

ne week be

sults for the

hizotron tube w

lanting, min

he soil (Tho

row space, o

th row. The

of approxim

camera to

arvested 50

cribed in th

f registratio

the deepest

ta were ana

Cary, NC,

Brief Disc

es in rootin

2). Broccol

efore harves

e two speci

with two coun

nirhizotron

orup-Kriste

one between

e minirhizot

mately 1.3 m

record the

0 days afte

he Chapter

ons within e

t root obser

alyzed with

USA, versi

cussion

ng depth a

li had fast r

t varied fro

ies concur w

nting grids.

glass tubes

ensen & van

n the first a

trons were i

m in the soi

roots on th

er transplan

5 (this the

each soil lay

rvation on

the GLM

ion 9.2).

among the

root growth

m 0.8 to 1.1

with the co

Effects o

s (0.07 m o

n den Boor

and second r

installed at

il (Figure 6

he minirhiz

nting. Soil

esis). Root

yer for each

each of the

procedure o

crops were

h and grew

1 m for broc

onclusion o

of fertilizatio

uter diamet

rgaard 1998

row and the

an angle of

6-1). Regist

zotron surfa

sampling

intensities

h observatio

e two coun

of the SAS

e obtained

w deeper tha

ccoli and 0.

f Thorup-K

on on root g

ter and 1.5

8). Two tub

e other one b

f 30o from

trations we

ace (Chapte

and analy

were calcu

on date. Ro

nting grids

statistical

during the

an lettuce.

.7-0.9 m for

Kristensen (

growth

m long)

bes were

between

vertical,

re made

er 5, this

ysis was

ulated as

ot depth

on each

package

growth

Rooting

r lettuce.

(Thorup-

Effect

Kristen

with m

broccol

fertiliza

the root

Figur

Also

fertiliza

broccol

DAT u

under b

soil laye

The fer

growth

lettuce.

measure

compar

ts of fertiliz

sen 2001) t

monocots an

li the high N

ation led to

t growth.

re 6-2. Root d

o the root in

ation treatm

li were foun

until the las

broccoli (Fig

er whereas

rtilization tr

period. Br

Nitrogen

ement (Fig

red to the N

ation on roo

that crucifer

nd that lettu

N and S rate

the deepest

depth of lettuc

ntensity and

ments (Figur

nd 19 days a

st measurem

gure 6-3b,

broccoli sh

reatments af

roccoli root

and S fer

gure 6-3a).

220S40 trea

ot growth

rs are chara

uce is a sh

es decreased

t rooting. In

e and broccol

d root distri

re 6-3). No

after transpl

ment (one w

c, d). In the

owed its hig

ffected root

t density w

rtilization in

Lettuce ro

atment.

acterized by

hallow-root

d the root p

n lettuce the

li during grow

ibution in t

differences

lanting (DA

week befor

e lettuce, th

ghest values

t density an

was influenc

nfluenced

oot intensit

y faster root

t species (T

penetration,

e fertilizatio

wing period. B

the soil vari

s in the root

AT) (Figure

re harvest)

he root inten

s in the 0.25

nd distributi

ced by ferti

lettuce roo

ty was high

depth deve

Thorup-Kris

whereas the

on treatment

ars show stand

ied between

t intensity b

6-3a). How

the root in

nsity was hi

5-0.75 m.

ion of crops

lization tre

ot intensity

her at the

elopment co

stensen 200

e high N an

ts did not in

ndard error (n=

n the crops

between lett

wever, from

ntensity was

igher in the

s differently

eatments mo

y only in t

N90S40 tr

ompared

06a). In

nd low S

nfluence

=2).

and the

tuce and

m the 28th

s higher

0-0.5 m

y during

ore than

the first

reatment

73

74

F

Figure 6-3. Averagge root intensity in tthe 0-1.3 m soil pro(d). where N

file in the field expN, nitrogen; S, sulph

eriment 19 days (a)hur; bars represent t

), 28 days (b) 38 daythe standard errors (

Effect

ys (c) and after tran(where n = 2)

ts of fertilization

nsplanting and one w

n on root growth

weeks before harve

h

st

75 Effects of fertilization in root growth

Increasing N fertilization when S was applied decreased root intensity in the topsoil (0-0.25

m soil layer). In the first measurement, fertilization did not significantly influence broccoli

root intensity (Figure 6-3a). In the top 0.50 m of the soil, fertilization treatments influenced

broccoli root density in a similar manner after the 28th DAT (Figure 6-3b, c, d), where

increased N supply decreased root intensity at both S fertilization rates. From the 38th DAT,

fertilization treatments affected broccoli root intensity also in the subsoil (0.5-1 m soil layer)

(Figure 6-3c, d). Application of S reduced root intensity in the subsoil markedly at high N

rate. Moreover, when broccoli was grown without S fertilization the increasing N rate

increased the root intensity, but when S was applied there was a decrease in the root intensity

and the high N and S fertilization led to lower final root intensities than any of the other

treatments.

The results on the effects of N and S fertilization and their interactions to root growth and

distribution to the soil profile under field conditions are, to our knowledge, the first reported

in the literature. In pot experiments, it has found that S fertilization had little effect on root

morphology of ryegrass and sub-clover. Moreover the response was found to be species

specific (Gilbert & Robson 1984). Sulphur application increased root length and root surface

area of alfalfa compared to control (Wang et al. 2003) but decreased total root growth in the

sub- clover (Gilbert & Robson 1984). In Arabidopsis seedlings grown on the surface of agar

plates without sulphate lateral roots are formed closer to the root tip and at increased density.

The increased growth of the root system under sulphate limiting conditions has been related

to the transcriptional activation of the nitrilase 3 gene, which found to have a direct role in

auxin synthesis and root branching (López-Bucio et al. 2003).

In maize a greater root growth and distribution was observed at a moderate N rate than at

zero N or high N (Oikeh et al. 1999). The effect of N supply on root intensity of broccoli is in

contrast with previous results in cauliflower (Thorup-Kristensen & van den Boorgaard 1998),

where little effect of N fertilizer levels on root growth were observed. In spring wheat 67 kg

N ha-1 stimulated root growth within the top 0.3 m of the soil profile but higher N rate (134

kg N ha-1) caused either no change or a decline in root length specially below the 0.3 m soil

layer (Comfort et al. 1988).

Plant root growth and intensity did not influence inorganic S distribution in the soil

(Figure 6-4a). In the topsoil S application increased soil inorganic S under lettuce, which

may be attributed to the low S uptake by lettuce compared to broccoli (Table 5-4). Brassicas

7

76

are high

Thorup

Figur

The

rooting

inorgan

the high

The

between

interact

density

Lettuce

fertiliza

plants o

Tosti &

intercro

conside

root com

h S deman

-Kristensen

re 6-4. Soil in

data for the

intensity a

nic N in the

her root inte

present res

n the two v

tion effect w

was found

e root inten

ation treatm

of different

& Thorup-

opping. How

ered as it ma

mpetition.

nding plants

n 2002).

norganic S (a)

e soil inorga

and soil N

e subsoil co

ensity of bro

sults show t

vegetable c

was found

d in the sub

nsity at harv

ments. Studie

species cou

Kristensen

wever, the

ay influence

s and have

and N (b) pro

anic N after

utilization

mpared to l

occoli in the

that there a

crops, whic

between N

bsoil at bro

vest was lo

es have sho

uld affect te

2010), th

results indi

e root grow

the ability

ofiles after har

r vegetable

n (Figure 6

lettuce. The

e subsoil.

are large dif

h can influ

N and S rate

occoli grow

ower than b

own that be

emporal and

herefore ou

icate that n

wth and distr

Effects o

y to deplete

rvest of crops.

harvest sho

6-4b). Broc

e higher N

fferences in

uence soil N

es in brocco

wn without

broccoli an

lowground

d spatial ro

ur results m

nutrient ava

ribution and

of fertilizatio

e soil inorg

Bars show st

ow further i

ccoli had cl

utilization m

n root grow

N and S ut

oli root inte

S fertilizati

nd was less

interactions

ot distribut

may not g

ailability in

d susbequen

on on root g

ganic S (Er

tandard error (

interaction b

learly redu

may be asc

wth and dist

tilization. A

ensity. Hig

ion at high

s affected f

ns among ne

tion (Li et a

generalized

the soil sh

ntly to influ

growth

iksen &

(n=3).

between

ced soil

cribed to

tribution

A strong

gher root

N rate.

from the

eighbour

al. 2006;

to the

hould be

ence the

77 Conclusion and perspectives

Chapter 7

Conclusions and perspectives

Utilizing the crop production practices could be an efficient way to enhance plant

phytochemical production, such as organic Se compounds and GSLs. A critical aspect in

developing an effective ecological farming system is to manage and organize crops to

achieve the best utilization of the available resources. The studies comprised in the present

dissertation intended to evaluate the efficiency of different cropping systems to increase Se

and GSLs concentrations in plants

From the catch crop studies, it was concluded, that:

Catch crops used in this study were not effective to reduce Se leaching over winter as

the Se uptake by catch crops was less than 1% of the total water soluble Se in the soil.

The incorporation of non-enriched catch crops resulted in reduced Se uptake by

succeeding crops (onions and Indian mustard) indicating immobilization both of the

native soil and applied Se.

The incorporation of Se enriched catch crops increased Se concentration in the plants

and in the leachate, indicating Se mineralization.

Fodder radish was able to take up much more Se form Se fertilizer and native soil Se

than the other catch crops. However, it did not succeed to increase Se concentrations in

the succeeding cash crops.

High rainfall in the early growth stage of the catch crops; can increase Se losses to the

deeper soil layers before plants being able to reduce the excess water drainage.

Fertilization with inorganic Se as selenate did not affect Se concentrations either in the

leachates or in the plants grown in the pot incubation.

The hypothesis that catch crop may reduce Se leaching and Se concentrations in plants

was not verified. As the overall Se recovery both by catch and cash crops was low, special

attention should be paid in the fate of residual Se in the soil. Moreover, the incorporation of

catch crops in the field was found to reduce the recovery of the applied Se and the uptake by

the cash crops. Therefore, careful consideration should be taken when plant residues are

incorporated in the soil. Further research is required to ensure that plant residues incorporated

in the soil will provide the correct balance between S and Se, as the interactions of S and Se

in the soil can determinate Se concentrations in plants

78 Conclusions and perspectives

From the intercropping studies, it was concluded, that:

In the field experiment, the lettuce plants were too weak competitors and the effect of

glucosinolate concentrations in broccoli was limited.

In the field experiment, neoglucobrassicin concentration in broccoli was reduced due to

intercropping.

Higher root density was found in the subsoil at broccoli grown without S fertilization at

high N rate.

Lettuce root intensity at harvest was lower and less affected from the fertilization

treatments than broccoli compared to broccoli.

In the pot experiment, where above-ground competition was eliminated, both total and

individual glucosinolate concentrations in red leaf mustard increased by intercropping.

Different N and S fertilization rates influenced the GSLs concentrations.

Increased knowledge on the competitive interaction between intercrop components in non

Brassica–Brassica intercrops is a basic requirement to better predict and manage the outcome

of competition between components and thus the balance of N and S in the soil. An important

factor in achieving intercrop improvements will be the introduction of better adapted plants.

Another solution could be to introduce some temporal differences using different planting

dates for mixture component plants.

79 Bibliography

Chapter 8

Bibliography

Abdin, M.Z., Ahmad, A., Khan, I., Qureshi, M.I. & Abrol, Y.P. 2002. Effect of S and N nutrition on N-accumulation and N-harvest in rapeseed-niustard (Brassica juncea L. and Brassica campestris L.). In: Plant Nutrition. Food Security and Sustainability of Agro-Ecosystems through Basic and Applied Research, (eds W.J.Horst, M.K.Schenk, A.Bürkert, N.Claassen, H.Flessa, W.B.Frommer, H.Goldbach, H.-W.Olfs, V.Römheld, B.Sattelmacher, U.Schmidhalter, S.Schubert, N.Wirén, & L.Wittenmayer), Kluwer Academic Publishers, Netherlands pp 816-817.

Abdulah, R., Miyazaki, K., Nakazawa, M. & Koyama, H. 2005. Chemical forms of selenium for cancer prevention. Journal of Trace Elements in Medicine and Biology, 19, 141-150.

Ajwa, H.A., Banuelos, G.S. & Mayland, H.F. 1998. Selenium uptake by plants from soils amended with inorganic and organic materials. Journal of Environment Quality, 27, 1218-1227.

Alemi, M.H., Goldhamer, D.A. & Nielsen, D.R. 1991. Modeling selenium transport in steady-state unsaturated columns. Journal of Environmental Quality, 20, 89-95.

Alfthan, G., Wang, D., Aro, A. & Soveri, J. 1995. The geochemistry of selenium in groundwaters in Finland. The Science of the Total Environment, 162, 93-103.

Andersen, M.K., Hauggaard-Nielsen, H., Høgh-Jensen, H. & Jensen, E.S. 2007. Competition for and utilisation of sulfur in sole and intercrops of pea and barley. Nutrient Cycling in Agroecosystems, 77, 143-153.

Anderson, J.W. & MeMahon, P.J. 2001. The role of glutathione in the uptake and metabolism of sulfur and selenium. In: Significance of glutathione to plant adaptation to the environment, (eds D.Grill, M.Tausz, & L.J.E.de Kok), Springer Netherlands, pp 57-99.

Arnault, I. & Auger, J. 2006. Seleno-compounds in garlic and onion. Journal of Chromatogrgraphy A, 1112, 23-30.

Asare, E. & Scarisbrick, D.H. 1995. Rate of nitrogen and sulphur fertilizers on yield, yield components and seed quality of oilseed rape (Brassica napus L.). Field Crops Research, 44, 41-46.

Askegaard, M. & Eriksen, J. 2007. Growth of legume and nonlegume catch crops and residual-N effects in spring barley on coarse sand. Journal of Plant Nutrition and Soil Science, 170, 773-780.

Aulakh, M.S., Pasricha, N.S. & Sahota, N.S. 1980. Yield, nutrient concentration and quality of mustard crops as influenced by nitrogen and sulphur fertilizers. The Journal of Agricultural Science, 94, 545-549.

Baik, H.Y., Juvik, J.A., Jeffery, E.H., Wallig, M.A., Kushad, M. & Klein, B.P. 2003. Relating glucosinolate content and flavor of broccoli cultivars. Journal of Food Science, 68, 1043-1050.

Bañuelos, G.S. & Meek, D.W. 1990. Accumulation of selenium in plants grown on selenium-treated soil. Journal of Environment Quality, 19, 772-777.

Berken, A., Mulholland, M.M., LeDuc, D.L. & Terry, N. 2002. Genetic engineering of plants to enhance selenium phytoremediation. Critical Reviews in Plant Sciences, 21, 567-582.

80 Bibliography

Bisbjerg B . Studies on selenium in plants and soil. 1972. Roskilde, Denmark, Risoe Report, Danish Atomic Energy Commission, Research Establishment Risoe.

Björkman, M., Hopkins, R.J. & Rämert, B. 2008. Combined effect of intercropping and turniproot fly (Delia floralis) larval feeding on the glucosinolate concentrations in cabbage roots and foliage. Journal of Chemical Ecology, 34, 1368-1376.

Björkman, M., Klingen, I., Birch, A.N., Bones, A.M., Bruce, T.J., Johansen, T.J., Meadow, R., Mølmann, J., Seljåsen, R., Smart, L.E. & Stewart, D. 2011. Phytochemicals of Brassicaceae in plant protection and human health. Influences of climate, environment and agronomic practice. Phytochemistry, 72, 538-556.

Blaylock, M.J. & James, B.R. 1994. Redox transformations and plant uptake of selenium resulting from root-soil interactions. Plant and Soil, 158, 1-12.

Blum, J.S., Bindi, A.B., Buzzelli, J., Stolz, J.F. & Oremland, R.S. 1998. Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic. Archives of Microbiology, 171, 19-30.

Broadley, M.R., Alcock, J., Alford, J., Cartwright, P., Foot, I., Fairweather-Tait, S.J., Hart, D.J., Hurst, R., Knott, P. & McGrath, S.P. 2010. Selenium biofortification of high-yielding winter wheat (Triticum aestivum L.) by liquid or granular Se fertilisation. Plant and Soil, 332, 5-18.

Broadley, M.R., White, P.J., Bryson, R.J., Meacham, M.C., Bowen, H.C., Johnson, S.E., Hawkesford, M.J., McGrath, S.P., Zhao, F.J. & Breward, N. 2006. Biofortification of UK food crops with selenium. Proceedings of the Nutrition Society, 65, 169-181.

Brown, T.A. & Shrift, A. 1982. Selenium: Toxicity and tolerance in higher plants. Biological Reviews, 57, 59-84.

Charron, C.S., Saxton, A.M. & Sams, C.M. 2005. Relationship of climate and genotype to seasonal variation in the glucosinolate-myrosinase system. I. Glucosinolate content in ten cultivars of Brassica oleracea grown in fall and spring seasons. Journal of the Science of Food and Agriculture, 85, 671-681.

Chen, X.J., Zhu, Z.J., Ni, X.L. & Qian, Q.Q. 2006. Effect of nitrogen and sulfur supply on glucosinolates in Brassica campestris ssp. Chinensis. Agricultural Sciences in China, 5, 603-608.

Chu, J., Yao, X. & Zhang, Z. 2010. Responses of wheat seedlings to exogenous selenium supply under cold stress. Biological Trace Element Research, 136, 355-363.

Ciska, E., Martyniak-Przybyszewska, B. & Kozlowska, H. 2000. Content of glucosinolates in cruciferous vegetables grown at the same site for two years under different climatic conditions. Journal of Agricultural and Food Chemistry, 48, 2862-2867.

Clark, L.C., Combs, G.F., Turnbull, B.W., Slate, E., Chalker, D.K., Chow, J., Davis, L.S., Glover, R.A., Graham, G.F., Gross, E.G., Krongrad, A., Lesher, J.L., Park, H.K., Sanders, B.B., Smith, C.L. & Taylor, J.R. 1996. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin a randomized controlled trial - A randomized controlled trial. Journal of the American Medical Association, 276, 1957-1963.

Combs, G.F.J. 2001. Selenium in global food systems. British Journal of Nutrition, 85, 517-547.

81 Bibliography

Combs, G.F.J. & Combs, S.B. 1986. The role of selenium in nutrition. Academic press, Orlando, Florida.

Comfort, S.D., Malzer, G.L. & Busch, R.H. 1988. Nitrogen fertilization of spring wheat genotypes: Influence on root growth and soil water depletion. Agronomy Journal, 80, 114-120.

Danish Food Composition Databank 2008. Danish Food Composition Databank, version 7.0.

de Souza, M.P., Pilon-Smits, E.A.H., Lytle, C.M., Hwang, S., Tai, J., Honma, T.S.U., Yeh, L. & Terry, N. 1998. Rate-limiting steps in selenium assimilation and volatilization by Indian mustard. Plant Physiology, 117, 1487-1494.

Dhillon, K.S., Dhillon, S.K. & Dogra, R. 2010. Selenium accumulation by forage and grain crops and volatilization from seleniferous soils amended with different organic materials. Chemosphere, 78, 548-556.

Dhillon, S.K. & Dhillon, K.S. 2000. Selenium adsorption in soils as influenced by different anions. Journal of Plant Nutrition and Soil Science, 163, 577-582.

Dhillon, S.K., Hundal, B.K. & Dhillon, K.S. 2007. Bioavailability of selenium to forage crops in a sandy loam soil amended with Se-rich plant materials. Chemosphere, 66, 1734-1743.

Djanaguiraman, M., Devi, D.D., Shanker, A.K., Sheeba, J.A. & Bangarusamy, U. 2005. Selenium - an antioxidative protectant in soybean during senescence. Plant and Soil, 272, 77-86.

Djanaguiraman, M., Prasad, P.V.V. & Seppanen, M. 2010. Selenium protects sorghum leaves from oxidative damage under high temperature stress by enhancing antioxidant defense system. Plant Physiology and Biochemistry, 48, 999-1007.

Dungan, R.S. & Frankenberger, W.T. 1999. Microbial transformations of selenium and the bioremediation of seleniferous environments. Bioremediation Journal, 3, 171-188.

EC Scientific Committe on Food. Opinion of the scientific committee on food on the revision of reference values for nutrition labelling. 2003. Brussels: Commission of the European Communities.

Eich-Greatorex, S., Krogstad, T. & Sogn, T.A. 2010. Effect of phosphorus status of the soil on selenium availability. Journal of Plant Nutrition and Soil Science, 173, 337-344.

Eich-Greatorex, S., Sogn, T.A., Falk Øgaard, A. & Aasen, I. 2007. Plant availability of inorganic and organic selenium fertiliser as influenced by soil organic matter content and pH. Nutrient Cycling in Agroecosystems, 79, 221-231.

Ellis, D.R. & Salt, D.E. 2003. Plants, selenium and human health. Current Opinion in Plant Biology, 6, 273-279.

Eriksen, J. & Thorup-Kristensen, K. 2002. The effect of catch crops on sulphate leaching and availability of S in the succeeding crop on sandy loam soil in Denmark. Agriculture, Ecosystems and Environment, 90, 247-254.

Eriksen, J., Thorup-Kristensen, K. & Askegaard, M. 2004. Plant availability of catch crop sulfur following spring incorporation. Journal of Plant Nutrition and Soil Science, 167, 609-615.

82 Bibliography

Eurola, M., Ekholm, P., Ylinen, M., Koivistoinen, P. & Varo, P. 1989. Effects of selenium fertilization on the selenium content of selected Finnish fruits and vegetables. Acta Agriculturae Scandinavica, 39, 345-350.

Eurola, M.H., Ekholm, P.I., Ylinen, M.E., Varo, P.T. & Koivistoinen, P.E. 1991. Selenium in Finnish foods after beginning the use of selenate-supplemented fertilisers. Journal of the Science of Food and Agriculture, 56, 57-70.

Fahey, J.W., Zalcmann, A.T. & Talalay, P. 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry, 56, 5-51.

Fairweather-Tait, S.J., Bao, Y., Broadley, M.R., Collings, R., Ford, D., Hesketh, J. & Hurst, R. 2011. Selenium in human health and disease. Antioxidants & Redox Signaling, 14, 1337-1383.

Falk Øgaard, A., Sogn, T.A. & Eich-Greatorex, S. 2006. Effect of cattle manure on selenate and selenite retention in soil. Nutrient Cycling in Agroecosystems, 76, 39-48.

Finley, J.W. 2003. Reduction of cancer risk by consumption of selenium-enriched plants: Enrichment of broccoli with selenium increases the anticarcinogenic properties of broccoli. Journal of Medicinal Food, 6, 19-26.

Flury, M., Frankenberger, W.T. & Jury, W.A. 1997. Long-term depletion of selenium from Kesterson dewatered sediments. Science of the Total Environment, The, 198, 259-270.

Fordyce, F. 2005. Selenium deficiency and toxicity in the environment. In: Essentials of Medical Geology: Impacts of the Natural Environment on Public Health, (eds O.Selinus, B.Alloway, J.A.Centeno, R.B.Finkelman, R.Fuge, U.Lindh, & P.Smedley), Elsevier Academic Press, London pp 373-415.

Gerendás, J., Podestát, J., Stahl, T., Kübler, K., Brückner, H., Mersch-Sundermann, V. & Müling, K.H. 2009. Interactive effects of sulfur and nitrogen supply on the concentration of sinigrin and allyl isothiocyanate in Indian mustard (Brassica juncea L.). Journal of Agricultural and Food Chemistry, 57, 3837-3844.

Gilbert, M.A. & Robson, A.D. 1984. The effect of sulfur supply on the root characteristics of subterranean clover and annual ryegrass. Plant and Soil, 77, 377-380.

Girling, C.A. 1984. Selenium in agriculture and the environment. Agriculture, Ecosystems & Environment, 11, 37-65.

Gissel-Nielsen, G. 1970. Loss of selenium in drying and storage of agronomic plant species. Plant and Soil, 32, 242-245.

Gissel-Nielsen, G. & Hamdy, A.A. 1977. Leaching of added selenium in soils low in native selenium. Z.Pflanzenernaehr.Bodenkd., 140, 193-198.

Gustafsson, J.P. & Johnsson, L. 1992. Selenium retention in the organic matter of Swedish forest soils. European Journal of Soil Science, 43, 461-472.

Gustafsson, J.P. & Johnsson, L. 1994. The association between selenium and humic substances in forested ecosystems - laboratory evidence. Applied Organometallic Chemistry, 8, 141-147.

Guvenc, I. & Yildirim, E. 2006. Increasing productivity with intercropping systems in cabbage production. Journal of Sustainable Agriculture, 28, 29-44.

Halkier, B.A. & Gershenzon, J. 2006. Biology and biochemistry of glucosinolates. Annual Review of Plant Biology, 57, 303-333.

83 Bibliography

Hamby, A.A. & Gissel-Nielsen, G. 1976. Fractionation of soil selenium. Z.Pflanzenernaehr.Bodenkd., 6, 697-703.

Hartikainen, H., Xue, T. & Piironen, V. 2000. Selenium as an anti-oxidant and pro-oxidant in ryegrass. Plant and Soil, 225, 193-200.

Haug, A., Graham, R.D., Christophersen, O.A. & Lyons, G.H. 2007. How to use the world's scarce selenium resources efficiently to increase the selenium concentration in food. Microbial Ecology in Health and Disease, 19, 209-228.

Hauggaard-Nielsen, H. & Jensen, E.S. 2005. Facilitative root interactions in intercrops. Plant and Soil, 274, 237-250.

Hauggaard-Nielsen, H., Jørnsgaard, B., Kinane, J. & Jensen, E.S. 2008. Grain legume–cereal intercropping: The practical application of diversity, competition and facilitation in arable and organic cropping systems. Renewable Agriculture and Food Systems, 23, 3-12.

Hawkesford, M.J. & Zhao, F.J. 2007. Strategies for increasing the selenium content of wheat. Journal of Cereal Science, 46, 282-292.

Hawrylak-Nowak, B. 2009. Beneficial effects of exogenous selenium in cucumber seedlings subjected to salt stress. Biological Trace Element Research, 132, 259-269.

Hawrylak-Nowak, B., Matraszek, R. & Szymanska, M. 2010. Selenium modifies the effect of short-term chilling stress on cucumber plants. Biological Trace Element Research, 138, 307-315.

Haynes, R.J. 1983. Soil acidification induced by leguminous crops. Grass and Forage Science, 38, 1-11.

Hopper, J.L. & Parker, D.R. 1999. Plant availability of selenite and selenate as influenced by the competing ions phosphate and sulfate. Plant and Soil, 210, 199-207.

Hsu, F.C., Wirtz, M., Heppel, S., Bogs, J., Krämer, U., Khan, M.S., Bub, A., Hell, R. & Rausch, T. 2011. Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism. Plant, Cell & Environment, 34, 192-207.

Hu, Q., Xu, J. & Pang, G. 2003. Effect of selenium on the yield and quality of green tea leaves harvested in early spring. Journal of Agricultural and Food Chemistry, 51, 3379-3381.

Irion, C.W. 1999. Growing alliums and brassicas in selenium-enriched soils increases their anticarcinogenic potentials. Medical Hypotheses, 53, 232-235.

Jensen, C.R., Mogensen, V.O., Mortensen, G., Fieldsend, J.K., Milford, G.F.J., Andersen, M.N. & Thage, J.H. 1996. Seed glucosinolate, oil and protein contents of field-grown rape (Brassica napus L.) affected by soil drying and evaporative demand. Field Crops Research, 47, 93-105.

Johnson, C.C., Fordyce, F.M. & Rayman, M.P. 2010. Symposium on 'Geographical and geological influences on nutrition' Factors controlling the distribution of selenium in the environment and their impact on health and nutrition. Proceedings of the Nutrition Society, 69, 119-132.

Johnsson, L. 1991. Selenium uptake by plants as a function of soil type, organic matter content and pH. Plant and Soil, 133, 57-64.

84 Bibliography

Kahakachchi, C., Boakye, H.T., Uden, P.C. & Tyson, J.F. 2004. Chromatographic speciation of anionic and neutral selenium compounds in Se-accumulating Brassica juncea (Indian mustard) and in selenized yeast. Journal of Chromatography A, 1054, 303-312.

Kawasaki, B.T., Hurt, E.M., Mistree, T. & Farrar, W.L. 2008. Targeting Cancer Stem Cells with Phytochemicals. Molecular Interventions, 8, 174-184.

Kearney, J. 2010. Food consumption trends and drivers. Philosophical Transactions of the Royal Society B: Biological Sciences, 365, 2793-2807.

Keck, A.S. & Finley, J.W. 2004. Cruciferous vegetables: Cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Integrative Cancer Therapies, 3, 5-12.

Kim, S.J., Matsuo, T., Watanabe, M. & Watanabe, Y. 2002. Effect of nitrogen and sulphur application on the glucosinolate content in vegetable turnip rape (Brassica rapa L.). Soil Science and Plant Nutrition, 48, 43-49.

Kong, L.A., Wang, M. & Bi, D.L. 2005. Selenium modulates the activities of antioxidant enzymes, osmotic homeostasis and promotes the growth of sorrel seedlings under salt stress. Plant Growth Regulation, 45, 155-163.

Kopsell, D.A., Barickman, T.C., Sams, C.E. & McErloy, J.S. 2007. Influence of nitrogen and sulfur on biomass production and caratenoid and glucosinolate concentration in watercress (Nasturtium officinale R.Br.). Journal of Agricultural and Food Chemistry, 55, 10628-10634.

Kopsell, D.A. & Randle, W.M. 1997. Selenate concentration affects selenium and sulfur uptake and accumulation by "Granex 33"onions. Journal of the American Society for Horticultural Science, 122, 721-726.

Krumbein, A., Schonhof, I., Rühlmann, J. & Widell, S. 2001. Influence of sulphur and nitrogen supply on flavour and health-affecting compounds in Brassicacea. In: Plant Nutrition. Food Security and Sustainability of Agro-Ecosystems Through Basic and Applied Research, (eds W.J.Horst, M.K.Schenk, A.Bürkert, N.Claassen, H.Flessa, W.B.Frommer, H.Goldbach, H.-W.Olfs, S.Schubert, & L.Wittenmayer), Kluwer Academic Publishers, Netherlands pp 294-295.

Krumbein, A., Schonhof, I. & Schreiner, M. 2005. Composition and contents of phytochemicals (glucosinolates, caratenoids and chlorophylls) and absorbic acid in selected Brassica species (B. juncea, B. rapa subsp. nipposinica var. chinoleifera, B. rapa subsp. chinensis, B. rapa subsp. rapa). Journal of Applied Botany and Food Quality, 79, 168-174.

Kushad, M.M., Brown, A.F., Kurilich, A.C., Juvik, J.A., Klein, B.P., Wallig, M.A. & Jeffery, E.H. 1999. Variation of glucosinolates in vegetable crops of Brassica oleracea. Journal of Agricultural and Food Chemistry, 47, 1541-1548.

Lakin, H.W., William, K.T. & Byers, H.G. 1938. "Nontoxic" seleniferous soils. Industrial and Engineering Chemistry, 30, 599-600.

Li, H.F., McGrath, S.P. & Zhao, F.J. 2008. Selenium uptake, translocation and speciation in wheat supplied with selenate or selenite. New Phytologist, 178, 92-102.

Li, L., Sun, J., Zhang, F., Guo, T., Bao, X., Smith, F.A. & Smith, S.E. 2006. Root distribution and interactions between intercropped species. Oecologia, 147, 280-290.

85 Bibliography

Li, S., Schonhof, I., Krumbein, A., Li, L., Stützel, H. & Schreiner, M. 2007. Glucosinolate concentration in turnip (Brassica rapa ssp. rapifera L.) roots as affected by nitrogen and sulfur supply. Journal of Agricultural and Food Chemistry, 55, 8452-8457.

Liebman, M. 1992. Research and extension efforts for improving agricultural sustainability in the north central and northeastern United States. Agriculture, Ecosystems & Environment, 39, 101-122.

Lindblow-Kull, C., Kull, F.J. & Shrift, A. 1985. Single transporter for sulfate, selenate, and selenite in Escherichia coli K-12. Journal of Bacteriology, 163, 1267-1269.

Linkohr, B.I., Williamson, L.C., Fitter, A.H. & Leyser, H.M.O. 2002. Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis. Plant Journal, 29, 751-760.

Liu, Q., Wang, D.J., Jiang, X.J. & Cao, Z.H. 2004. Effects of the interactions between selenium and phosphorus on the growth and selenium accumulation in rice (Oryza Sativa). Environmental Chemistry and Health, 26, 325-330.

López-Bucio, L., Cruz-Ramírez, A. & Herrera-Estrella, L. 2003. The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology, 6, 280-287.

Lyons, G., Ortiz-Monasterio, I., Stangoulis, J. & Graham, R. 2005. Selenium concentration in wheat grain: Is there sufficient genotypic variation to use in breeding? Plant and Soil, 269, 369-380.

Lyons, G.H., Genc, Y., Soole, K., Stangoulis, J.C.R., Liu, F. & Graham, R.D. 2009. Selenium increases seed production in Brassica. Plant and Soil, 318, 73-80.

Lyons, G.H., Lewis, J., Lorimer, M.F., Holloway, R.E., Brace, D.M., Stangoulis, J.C.R. & Graham, R.D. 2004. High-selenium wheat: agronomic biofortification strategies to improve human nutrition. Journal of Food, Agriculture & Environment, 2, 171-178.

MacLeod, A.J. & Nussbaum, M.L. 1977. The effects of different horticultural practices on the chemical flavour composition of some cabbage cultivars. Phytochemistry, 16, 861-865.

MacLeod, A.J. & Pikk, H.E. 1978. A comparison of the chemical flavour composition of some Brussels sprouts cultivars grown at different crop spacings. Phytochemistry, 17, 1029-1032.

Malhi, S.S., Gan, Y. & Raney, J.P. 2007. Yield, seed quality, and sulfur uptake of Brassica oilseed crops in response to sulfur fertilization. Agronomy Journal, 99, 570-577.

Mayland, H.F. 1994. Selenium in plant and animal nutrition. In: Selenium in the enviroment, (ed B.S.Frankenberger Jr WT ), Marcel Dekker, New York pp 29-46.

Mayland, H.F., Gough, L.P. & Stewart, K.C. 1991. Selenium mobility in soils and its absorption, translocation, and metabolism in plants. Selenium in Arid and Semiarid Environments, Western United States.US Geol.Sur.Cir, 1064, 55-64.

Mazej, D., Osvald, J. & Stibilj, V. 2008. Selenium species in leaves of chicory, dandelion, lambós lettuce and parsley. Food Chemistry, 107, 75-83.

McGrath, S.P. & Zhao, F.J. 1996. Sulphur uptake, yield responses and the interactions between nitrogen and sulphur in winter oilseed rape (Brassica napus). The Journal of Agricultural Science, 126, 53-62.

86 Bibliography

Meisinger, J.J., Hargove, W.L., Mikkelsen, R.L., Williams, J.R. & Benson, V.M. 1991. Effects of cover crops on groundwater quality. ed WL Hargrove, Soil and Water Conservation Society, Ankeny,Iowa pp 57-68.

Mikkelsen, R.L. & Wan, H.F. 1990. The effect of selenium on sulfur uptake by barley and rice. Plant and Soil, 122, 151-153.

Mithen, R. 2001. Glucosinolates-biochemistry, genetics and biological activity. Plant Growth Regulation, 34, 91-103.

Mortensen, J. & Eriksen, J. 1994. Effect of sulphur deficiency on amino acid composition. Norwegian Journal of Agricultural Sciences, 15, 135-142.

Muñiz-Naveiro, Ó., Domínguez-González, R., Bermejo-Barrera, A., Bermejo-Barrera, P., Cocho, J.A. & Fraga, J.M. 2006. Study of the bioavailability of selenium in cows' milk after a supplementation of cow feed with different forms of selenium. Analytical and Bioanalytical Chemistry, 385, 189-196.

National Academy of Sciences.Institute of Medicine.Food and Nutrition Board. 2000. Selenium. In: Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, (ed W.D.National Academy Press), pp 284-324.

Neal, R.H. & Sposito, G. 1991. Selenium mobility in irrigated soil columns as affected by organic carbon amendment. Journal of Environmental Quality, 20, 808-814.

Nikiforova, V., Freitag, J., Kempa, S., Adamik, M., Hesse, H. & Hoefgen, R. 2003. Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic pathways provides response specificity. The Plant Journal, 33, 633-650.

Nordic Council of Ministers 2004. Nordic Nutrition Recommendations 2004: integrating nutrition and physical activity. Nord: Copenhagen.

Oikeh, S.O., Kling, J.G., Horst, W.J., Chude, V.O. & Carsky, R.J. 1999. Growth and distribution of maize roots under nitrogen fertilization in plinthite soil. Field Crops Research, 62, 1-13.

Oldfield, J.E. 1987. The two faces of selenium. Journal of Nutrition, 117, 2002-2008.

Oldfield, J.E. 2002. Selenium world atlas: updated edition. Selenium-Tellurium Development Association, Grimbergen (Belgium).

Omirou, M.D., Papadopoulou, K.K., Papastylianou, I., Constantinou, M., Karpouzas, D.G., Asimakopoulos, I. & Ehaliotis, C. 2009. Impact of nitrogen and sulfur fertilization on the composition of glucosinolates in relation to sulfur assimilation in different plant organs of broccoli. Journal of Agricultural and Food Chemistry, 57, 9408-9417.

Oremland, R.S., Herbel, M.J., Blum, J.S., Langley, S., Beverdige, T.J., Ajayan, P.M., Sutto, T., Ellis, A.V. & Curran, S. 2004. Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria. Applied and Environmental Microbiology, 70, 52-60.

Pedrero, Z., Madrid, Y. & Camara, C. 2006. Selenium species bioaccessibility in enriched radish (Raphanus sativus): a potential dietary source of selenium. Journal of Agricultural and Food Chemistry, 54, 2412-2417.

Pickering, I.J., Wright, C., Bubner, B., Ellis, D., Persans, M.W., Yu, E.Y., George, G.N., Prince, R.C. & Salt, D.E. 2003. Chemical form and distribution of selenium and sulfur in the selenium hyperaccumulator Astragalus bisulcatus. Plant Physiology, 131, 1-8.

87 Bibliography

Pyrzynska, K. 2009. Selenium speciation in enriched vegetables. Food Chemistry, 114, 1183-1191.

Radovich, T.J.K., Kleinhenz, M.D. & Streeter, J.G. 2005. Irrigation timing relative to head development influences yield components, sugar levels, and glucosinolate concentrations in cabbage. Journal of the American Society for Horticultural Science, 130, 943-949.

Rangkadilok, N., Nicolas, M.E., Bennett, R.N., Eagling, D.R., Premier, R.R. & Taylor, P.W.J. 2004. The effect of sulfur fertilizer on glucoraphanin levels in broccoli (B. oleracea L. var. italica) at different growth stages. Journal of Agricultural and Food Chemistry, 52, 2632-2639.

Rani, N., Dhillon, K. & Dhillon, S. 2005. Critical levels of selenium in different crops grown in an alkaline silty loam soil treated with selenite-Se. Plant and Soil, 277, 367-374.

Rayman, M.P. 2000. The importance of selenium to human health. The Lancet, 356, 233-241.

Rayman, M.P. 2008. Food-chain selenium and human health: emphasis on intake. British Journal of Nutrition, 100, 254-268.

Reimann, C., Siewers, U., Tarvainen, T., Bityukova, L., Eriksson, J., Gilucis, A., Gregorauskiene, V., Lukashev, V.K., Matinian, N.N. & Pasieczna, A. 2003. Agricultural soils in Northern Europe: a geochemical atlas. Schweizerbart'sche Verlagsbuchhandlung, Stuttgart, Germany.

Rios, J.J., Blasco, B., Cervilla, L.M., Rosales, M.A., Sanchez-Rodriguez, E., Romero, L. & Ruiz, J.M. 2009. Production and detoxification of H2O2 in lettuce plants exposed to selenium. Annals of Applied Biology, 154, 107-116.

Ríos, J.J., Blasco, B., Cervilla, L.M., Rubio-Wilhelmi, M.M., Ruiz, J.M. & Romero, L. 2008. Regulation of sulphur assimilation in lettuce plants in the presence of selenium. Plant Growth Regulation, 56, 43-51.

Robbins, R.J., Keck, A.S., Banuelos, G. & Finley, J.W. 2005. Cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate, and sulforaphane content of broccoli. Journal of Medicinal Food, 8, 204-214.

Rosa, E.A.S., Heaney, K., Portas, A.M. & Roger, G. 1996. Changes in glucosinolate concentrations in Brassica crops (B. oleracea and B. napus) throughout growing seasons. Journal of the Science of Food and Agriculture, 71, 237-244.

Rosa, E.A.S. & Rodrigues, P.M.F. 1998. The effect of light and temperature on glucosinolate concentration in the leaves and roots of cabbage seedlings. Journal of the Science of Food and Agriculture, 78, 208-212.

Rosen, C.J., Fritz, V.A., Gardner, G.M., Hecht, S.S., Carmella, S.G. & Kenney, P.M. 2005. Cabbage yield and glucosinolate concentrations as affected by nitrogen and sulfur fertility. HortScience, 40, 1493-1498.

Santos, R.H.S., Gliessman, S.R. & Cecon, P.R. 2002. Crop interactions in broccoli intercropping. Biological Agriculture and Horticulture, 20, 51-75.

Scherer, H.W. 2001. Sulphur in crop production-invited paper. European Journal of Agronomy, 14, 81-111.

Schonhof, I., Blankenburg, D., Muller, S. & Krumbein, A. 2007a. Sulfur and nitrogen supply influence growth, product appearance, and glucosinolate concentration of broccoli. Journal of Plant Nutrition and Soil Science., 170, 65-72.

88 Bibliography

Schonhof, I., Kläring, H.P., Krumbein, A., Claußen, W. & Schreiner, M. 2007b. The effect of light and temperature on glucosinolate concentration in the leaves and roots of cabbage seedlings. Agriculture, Ecosystems & Environment, 119, 103-111.

Schonhof, I., Krumbein, A. & Brnckner, B. 2004. Genotypic effects on glucosinolates and sensory properties of broccoli and cauliflower. Nahrung/Food, 48, 25-33.

Schonhof, I., Krumbein, A., Schreiner, M. & Gutezeit, B. 1999. Bioactive substances in cruciferous products. In: Agri-food quality II: quality management of fruits and vegetables, (eds M.Hägg, R.Ahvenainen, A.M.Evers, & K.Tiilikkala), The Royal Society of Chemistry, Cambridge, UK pp 222-226.

Schreiner, M. 2005. Vegetable crop management strategies to increase the quantity of phytochemicals. European Journal of Nutrition, 44, 85-94.

Schröder, I., Rech, S., Krafft, T. & Macy, J.M. 1997. Purification and characterization of the selenate reductase from Thauera selenatis. Journal of Biological Chemistry, 272, 23765-23768.

Seppänen, M., Turakainen, M. & Hartikainen, H. 2003. Selenium effects on oxidative stress in potato. Plant Science, 165, 311-319.

Sharma, S., Bansal, A., Dhillon, S.K. & Dhillon, K.S. 2010. Comparative effects of selenate and selenite on growth and biochemical composition of rapeseed (Brassica napus L.). Plant and Soil, 329, 339-348.

Shattuck, V.I., Kakuda, Y. & Shelp, B.J. 1991. Effect of low temperature on the sugar and glucosinolate content of rutabaga. Scientia Horticulturae, 48, 9-19.

Singh, M., Singh, N. & Banhdari, D.K. 1980. Iinteraction of selenium and sulfur on the growth and chemical-composition of raya. Soil Science, 129, 238-244.

Sogn, T.A., Govasmark, E. & Eich-Greatorex, S. 2007. Assessment of seafood processing wastes as alternative sources of selenium in plant production. Acta Agriculturae Scandinavica, Section B-Plant Soil Science, 57, 173-181.

Sors, T.G., Ellis, D.R. & Salt, D.E. 2005. Selenium uptake, translocation, assimilation and metabolic fate in plants. Photosynthesis Research, 86, 373-389.

Stavridou, E., Thorup-Kristensen, K. & Young, S. 2011. Assesment of selenium mineralization and availability from catch crops. Soil Use and Management, in press.

Stolz, J.F. & Oremland, R.S. 1999. Bacterial respiration of arsenic and selenium. FEMS Microbiology Reviews, 23, 615-627.

Stroud, J.L., Broadley, M.R., Foot, I., Fairweather-Tait, S.J., Hart, D.J., Hurst, R., Knott, P., Mowat, H., Norman, K. & Scott, P. 2010a. Soil factors affecting selenium concentration in wheat grain and the fate and speciation of Se fertilisers applied to soil. Plant and Soil 1-12.

Stroud, J.L., Li, H.F., Lopez-Bellido, F.J., Broadley, M.R., Foot, I., Fairweather-Tait, S.J., Hart, D.J., Hurst, R., Knott, P. & Mowat, H. 2010b. Impact of sulphur fertilisation on crop response to selenium fertilisation. Plant and Soil, 332, 31-40.

Tam, S., Chow, A. & Hadley, D. 1995. Effects of organic component on the immobilization of selenium on iron oxyhydroxide. The Science of the Total Environment, 164, 1-7.

Terry, N., Zayed, A.M., de Souza, M.P. & Tarun, A.S. 2000. Selenium in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology, 51, 401-432.

89 Bibliography

Thorup-Kristensen, K. 1993. Root development of nitrogen catch crops and of a succeeding crop of broccoli. Acta Agriculturae Scandinavica, Section B Plant Soil Science, 43, 58-64.

Thorup-Kristensen, K. 1994. The effect of nitrogen catch crop species on the nitrogen nutrition of succeeding crops. Nutrient Cycling in Agroecosystems, 37, 227-234.

Thorup-Kristensen, K. 2001. Are differences in root growth of nitrogen catch crops important for their ability to reduce soil nitrate-N content, and how can this be measured? Plant and Soil, 230, 185-195.

Thorup-Kristensen, K. 2006a. Effect of deep and shallow root systems on the dynamics of soil inorganic N during 3-year crop rotations. Plant and Soil, 288, 233-248.

Thorup-Kristensen, K. 2006b. Root growth and nitrogen uptake of carrot, early cabbage, onion and lettuce following a range of green manures. Soil Use and Management, 22, 29-38.

Thorup-Kristensen, K., Magid, J. & Jensen, L.S. 2003. Catch crops and green manures as biological tools in nitrogen management in temperate zones. Advances in Agronomy, 79, 227-302.

Thorup-Kristensen, K. & van den Boorgaard, R. 1998. Temporal and spatial root development of cauliflower (Brassica oleracea L. var. botrytis L.). Plant and Soil, 201, 37-47.

Tinggi, U. 2003. Essentiality and toxicity of selenium and its status in Australia: a review. Toxicology Letters, 137, 103-110.

Tosti, G. & Thorup-Kristensen, K. 2010. Using coloured roots to study root interaction and competition in intercropped legumes and non-legumes. Journal of Plant Ecology, 3, 191-199.

Trelease, S.F. & Trelease, H.M. 1938. Selenium as a stimulating and possibly essential element for indicator plants. American Journal of Botany, 25, 372-380.

Turakainen, M., Hartikainen, H. & Seppänen, M.M. 2004. Effects of selenium treatments on potato (Solanum tuberosum L.) growth and concentrations of soluble sugars and starch. Journal of Agricultural and Food Chemistry, 52, 5378-5382.

Valkama, E., Kivimäenpää, M., Hartikainen, H. & Wulff, A. 2003. The combined effects of enhanced UV-B radiation and selenium on growth, chlorophyll fluorescence and ultrastructure in strawberry (Fragaria × ananassa) and barley (Hordeum vulgare) treated in the field. Agricultural and Forest Meteorology, 120, 267-278.

Vallejo, F., Tomas-Barberan, F.A., Benavente-Garcia, A.G. & Garcia-Viguera, C. 2003. Total and individual glucosinolate contents in inflorescences of eight broccoli cultivars grown under various climatic and fertilisation conditions. Journal of the Science of Food and Agriculture, 83, 307-313.

van Noordwijk, M. & Cadisch, G. 2002. Access and excess problems in plant nutrition. Plant and Soil, 247, 25-39.

Vandermeer, J.H. 1989. The ecology of intercropping. Cambridge University Press, Cambridge, UK.

Varo, P. 1993. Selenium fertilization in Finland: selenium content in feed and foods. Norwegian Journal of Agricultural Sciences, 11, 151-158.

90 Bibliography

Verkerk, R., Schreiner, M., Krumbein, A., Ciska, E., Holst, B., Rowland, I., De Schrijver, R., Hansen, M., user, C. & Mithen, R. 2009. Glucosinolates in Brassica vegetables: The influence of the food supply chain on intake, bioavailability and human health. Molecular Nutrition & Food Research, 53, S219-S265.

Vidmar, J.J., Tagmount, A., Cathala, N., Touraine, B. & Davidian, J.C.E. 2000. Cloning and characterization of a root specific high-affinity sulfate transporter from Arabidopsis thaliana. FEBS Letters, 475, 65-69.

Wang, D., Alfthan, G., Aro, A., Lahermo, P. & Väänänen, P. 1994. The impact of selenium fertilisation on the distribution of selenium in rivers in Finland. Agriculture, Ecosystems and Environment, 50, 133-149.

White, P.J., Bowen, H.C., Parmaguru, P., Fritz, M., Spracklen, W.P., Spiby, R.E., Meacham, M.C., Mead, A., Harriman, M. & Trueman, L.J. 2004. Interactions between selenium and sulphur nutrition in Arabidopsis thaliana. Journal of Experimental Botany, 55, 1927-1937.

White, P.J. & Broadley, M.R. 2009. Biofortification of crops with seven mineral elements often lacking in human diets-iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytologist, 182, 49-84.

White, P.J., Broadley, M.R., Bowen, H.C. & Johnson, S.E. 2007a. Selenium and its relationship with sulfur. In: Sulfur in plants an ecological perspective, (eds M.J.Hawkesford & L.J.de Kok), Dordrecht: Springer, The Netherlands pp 225-252.

White, P.J., Broadley, M.R., Bowen, H.C. & Johnson, S.E. 2007b. Selenium and its relationship with sulfur. In: Sulfur in plants: an ecological perspective, pp 225-252.

Willey, R.W. & Reddy, M.S. 1981. A field technique for separating above- and below-ground interactions in intercropping: an experiment with pearl millet/groundnut. Experimental Agriculture, 17, 257-264.

Wu, L., Huang, Z.Z. & Burau, R.G. 1988. Selenium accumulation and selenium-salt cotolerance in 5 grass species. Crop Science, 28, 517-522.

Wu, L., Mantgem, P.J. & Guo, X. 1996. Effects of forage plant and field legume species on soil selenium redistribution, leaching, and bioextraction in soils contaminated by agricultural drain water sediment. Archives of Environmental Contamination and Toxicology, 31, 329-338.

Xue, T.L., Hartikainen, H. & Piironen, V. 2001. Antioxidative and growth-promoting effect of selenium on senescing lettuce. Plant and Soil, 237, 55-61.

Yamada, H., Kang, Y., Aso, T., Uesugi, H., Fujimura, T. & Yonebayashi, K. 1998. Chemical forms and stability of selenium in soil. Soil Science and Plant Nutrition, 44, 385-391.

Yao, X., Chu, J. & Wang, G. 2009. Effects of selenium on wheat seedlings under drought stress. Biological Trace Element Research, 130, 283-290.

Yildirim, E. & Guvenc, I. 2005. Intercropping based on cauliflower: more productive, profitable and highly sustainable. European Journal of Agronomy, 22, 11-18.

Zayed, A., Lytle, C.M. & Terry, N. 1998. Accumulation and volatilization of different chemical species of selenium by plants. Planta, 206, 284-292.

Zehr, J.P. & Oremland, R.S. 1987. Reduction of selenate to selenide by sulfate-respiring bacteria: Experiments with cell suspensions and Estuarine sediments. Applied and Environmental Microbiology, 53, 1365-1369.

91 Bibliography

Zhang, H., Schonhof, I., Krumbein, A., Gutezeit, B., Li, L., Stützel, H. & Schreiner, M. 2008. Water supply and growing season influence glucosinolate concentration and composition in turnip root (Brassica rapa ssp. rapifera L.). Journal of Plant Nutrition, 171, 255-265.

Zhao, C., Ren, J., Xue, C. & Lin, E. 2005. Study on the relationship between soil selenium and plant selenium uptake. Plant and Soil, 277, 197-206.

Zhao, F., Evans, E.J., Bilsborrow, P.E. & Syers, J.K. 1993. Influence of sulphur and nitrogen on seed yield and quality of low glucosinolate oilseed rape (Brassica napus L). Journal of the Science of Food and Agriculture, 63, 29-37.

Zhao, F., Evans, E.J., Bilsborrow, P.E. & Syers, J.K. 1994. Influence of nitrogen and sulphur on the glucosinolate profile of rapeseed (Brassica napus L). Journal of the Science of Food and Agriculture, 64, 295-304.

Zimmermann, N.S., Gerendás, J. & Krumbein, A. 2007. Identification of desulphoglucosinolates in Brassicaceae by LC/MS/MS: Comparison of ESI and atmospheric pressure chemical ionisation-MS. Molecular Nutrition & Food Research, 51, 1537-1546.

The effects of cropping systems on selenium and glucosinolate...

Documents

Transcript of The effects of cropping systems on selenium and glucosinolate...