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 ThorupKristensen, 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. Selenium in soils .................................................................................................... 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. Selenium in plants ................................................................................................. 8
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 1.3. 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 2.1. General................................................................................................................. 17
2.2. Role in human health ........................................................................................... 17
2.3. Factors affecting plant levels ............................................................................... 18
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 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. Results ......................................................................................................................... 27 3.1. Soil Se and S ........................................................................................................ 27
3.2. Catch crops .......................................................................................................... 29
3.3. Cash crops............................................................................................................ 30
4. Discussion ................................................................................................................... 33 5. Conclusion .................................................................................................................. 36 Chapter 4 Assessment of selenium mineralization and availability from catch crops
............................................................................................................................................. 37 1. Introduction ............................................................................................................... 38 v
2. Material and Methods ............................................................................................... 39 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. Results ......................................................................................................................... 43 3.1. Composition of catch crops ................................................................................. 43
3.2. Leaching-tube incubations ................................................................................... 43
3.3. Pot incubations .................................................................................................... 44
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. Material and Methods ............................................................................................... 51 2.1. Field Experiment ................................................................................................. 51
2.1.1. Root measurements ........................................................................................ 53 2.1.2. Harvest and sample preparation ..................................................................... 53 2.2. Pot experiment ..................................................................................................... 53
2.3. Glucosinolate Analysis ........................................................................................ 54
2.4. The N and S analysis ........................................................................................... 56
2.5. Statistical analysis................................................................................................ 57
3. Results ......................................................................................................................... 57 3.1. The field experiment ............................................................................................ 57
3.1.1. Soil N and S ................................................................................................... 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. Pot experiment ..................................................................................................... 63
3.2.1. Dry matter production .................................................................................... 63 3.2.2. N and S accumulation .................................................................................... 63 3.2.3. Glucosinolates ................................................................................................ 64 4. Discussion ................................................................................................................... 67 4.1. Field experiment .................................................................................................. 67
4.2. Pot experiment ..................................................................................................... 69
5. Abbreviations Used ................................................................................................... 70 6. Acknowledgment ....................................................................................................... 70 Chapter 6 Effects of N and S fertilization on root growth ............................................ 71 1. Introduction ............................................................................................................... 71 2. Material and Methods ............................................................................................... 71 3. Results and Brief Discussion..................................................................................... 72 Chapter 7 Conclusions and perspectives......................................................................... 77 Chapter 8 Bibliography .................................................................................................... 79 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.
Introduction
1
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 healthpromoting 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; ThorupKristensen 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 (HauggaardNielsen & 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.
Literature review: Selenium
3
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
Literature review: Sellenium
highly vvariable (Fiigure 1-1) (Mayland 11994). In so
ome regionss of the worrld (parts of China,
the Greeat Plains of the USA, Canadda, South America, Australia,
A
IIndia, Russia), Se
concenttrations in the soil are sufficiently
s
y high (30-324 mg Se kg
k -1) to be tooxic to man
ny plants
and thuus these regiions supporrt a unique fflora. In contrast, in co
ountries succh as New Zealand,
Z
United Kingdom, Finland an
nd some arreas of Chiina, the avaailability off Se in thee soil is
naturallly low (Olddfield 1987)). In Denmaark total soiil Se concen
ntrations ran
ange betweeen 0.1 to
1.6 mg kg-1, with a mean 0.5 mg
m kg-1 (Bissbjerg 1972
2).
Figure 11-1. Selenium
m in (a) top (0
0–25 cm) andd (b) bottom (50–75
(
cm) so
oils from nortthern Europe.. (adapted
from Reiimann et al. (22003) cited by
y Johnson et all. (2010)).
11.1.1. Bioticc and abiotiic processess affecting Se
S availabiliity in soil
11.1.1.1. Abiiotic processses
Specciation of thhe chemicall forms of S
Se is difficu
ult due to very
v
low cooncentration
ns of the
2elementt in the soill and the co
omplexities of soil systtems. Selen
nate (SeO42-)), selenite (SeO
(
3 ),
selenidee (Se2), elem
mental Se (S
Se0) and orgganic Se aree the forms in which See occurs in the soil.
The typpe of Se fouund is a resu
ult of its oxiidation statee, which deepends on va
various physsical and
chemicaal factors, including pH,
p adsorbbing surfacee, organic matter
m
and microbial activity
(Figuree 1-2) (Girling 1984; Dungan & Frankenbeerger 1999; Fordyce 22005; Whitte et al.
Literature review: Selenium
5
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).
Organic matter, clay, iron oxides
SeO3
Selenite
binds
strongly to
Fe-oxides +
clay
minerals,
2-
Oxidizing
Se0
Redox
SeO42SeO42Selenate is
soluble+
SeO32-
SeO32-
Se0
Reducing
HSe
0
2
Se is largely
immobile
Se0
-
4
6
8
10
12
pH
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
6
Literature review: Selenium
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
Literature review: Selenium
7
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).
DMSe
(CH3)2Se
Demethylation
Atmosphere
Methylation
Soil
Microorganisms
Insoluble forms
Metal Selenides
Se0
Oxidation
Reduction
Capillary
rise
Soluble forms
SeO42SeO32-
Reduction
Oxidation
Immobilized forms
Se0
Se2Organic Se
Leaching
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 Semethylating 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).
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Literature review: Selenium
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 kg1
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-
Literature review: Selenium
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).
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Literature review: Selenium
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 protoncoupled 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
Literature review: Selenium
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).
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Literature review: Selenium
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
Literature review: Selenium
13
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
14
Literature review: Selenium
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 KaschinBeck 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
Literature review: Selenium
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 (MuñizNaveiro 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.
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Literature review: Selenium
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).
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).
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Literature review: Glucosinolates
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).
Literature review: Glucosinolates
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
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Literature review: Glucosinolates
(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).
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
21
22
Effect of catch crops on Se and S availability for succeeding crops
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). Suboptimal 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 (EichGreatorex 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
a S availaability for su
ucceeding crops
c
Effectt of catch crrops on Se and
successsfully used to reduce S leaching. Furthermorre, after being incorpoorated into the soil,
decompposition of the catch crop
c
plant material leed to minerralization off its S, wh
hich was
utilizedd by the succceeding crops (Erikseen & Thoru
up-Kristenseen 2002; Erriksen et all. 2004).
The higgh S demaand of Bra
assica cropps efficienttly depleted
d soil sulpphate conceentration
(Eriksenn & Thorupp-Kristensen
n 2002). Duue to chemical similarrities with ssulphate, sellenate is
taken uup through high affin
nity sulphatte transporrters and fo
ollows the same assim
milation
pathwayys as S in plants (Terry
y et al. 20000).
Figurre 3-1. Monthhly precipitatio
on (bars) and aaverage monthly temperatu
ure (line) durinng the experim
ment.
Understtanding of how
h
agrono
omic managgement and crop rotatio
on may affeect Se leach
hing loss
or availlability for crops
c
is lack
king, we doo not know the
t extent to
o which usee of catch crrops will
also be effective inn (i) reducin
ng Se leachiing loss and
d (ii) recycling Se to m
make it availlable for
succeedding crops. Better
B
undeerstanding oof this will be
b importan
nt both in loow Se enviro
onments
where w
we want too improve th
he utilizatioon of the liimited Se resource
r
avvailable, in order to
reduce adverse ennvironmentaal effects inn systems where
w
Se fertilization
f
is practiceed, or in
organicc farming syystems wheere Se fertillization is not
n allowed.. Considerinng the similarity of
Se and S, and that plants with
h high S dem
mand also have
h
a high affinity forr Se, it seem
ms likely
that theere will be similar
s
beneeficial effeccts from catcch crops on
n Se leachinng. The objeective of
the pressent work was
w to test th
he hypothesses that 1) catch
c
crops can reduce soil Se con
ntent and
23
24
Effect of catch crops on Se and S availability for succeeding crops
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 (10◦27’E, 55◦18’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)
0-0.25
0.25-0.5
0.5-0.75
0.75-1.0
Clay
(%)
15
18
21
21
Silt
(%)
27
29
28
27
Sand
(%)
55
52
50
53
C (%)
N (%)
pHCaCl2
1.8
0.8
0.3
0.2
0.16
0.07
0.04
0.03
7.0
6.4
5.1
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
Effect of catch crops on Se and S availability for succeeding crops
25
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
Species
Italian ryegrass,
Hairy vetch,
Fodder radish
Italian ryegrass,
Hairy vetch,
Fodder radish
Fertilization
None
None
Italian ryegrass,
Hairy vetch,
Fodder radish
0 g Se ha-1,
10 g Se ha-1
Analysis
DM
DM, Se, S, N
DM, Se, S, N
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
Autumn
None
Se, S (3 layers till 1 m depth)
Spring
None
Se, S (top soil)
Se, S (3 layers till 1 m
depth)
Se, S (top soil)
Catch crops
Cash crops
Soil sampling
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 nonwrapped 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.
26
Effect of catch crops on Se and S availability for succeeding 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.
Effect of catch crops on Se and S availability for succeeding crops
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
27
28
Effect of catch crops on Se and S availability for succeeding crops
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.
Soil soluble Se (g ha-1)
Catch
crops
C
IR
FR
HV
Soil inorganic S (kg ha-1)
Spring 2009
Autumn 2008
0-0.25m
0.25-0.75m
0.75-1.5m
28 a
29 a
28 a
29 a
40 a
44 a
39 a
37 a
25 a
32 a
30 a
29 a
Total
0-0.25 m
93 a
105 a
97 a
94 a
27 a
31 a
28 a
27 a
Spring 2009
Autumn 2008
0-0.25m
10 ab
7c
9c
11 a
0.25-0.75m
0.75-1.5m
21 a
17 a
10 b
16 a
20 a
26 a
15 a
26 a
Total
0-0.25 m
49 a
50 a
34 a
53 a
28 a
23 a
33 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.
Soil inorganic S (kg ha-1)
Soil soluble Se (g ha-1)
Fertilization
Se0
Se10
Catch
crops
C
IR
FR
HV
Average
C
IR
FR
HV
Average
Autumn 2009
0-0.25m
0.25-0-75m
0.75-1.5m
29 a
28 a
28 a
28 a
28 B
28 a
30 a
29 a
29 a
29 A
40 b
45 a
44 a
43 ab
43 A
40 c
45 ab
47 a
42 bc
43 A
38 a
36 a
38 a
32 a
36 A
36 a
45 a
39 a
39 a
40 A
Spring 2010
Total
106 a
108 a
109 a
102 a
107 A
104 b
119 a
115 a
110 ab
112 A
0-0.25 m
27 a
26 a
26 a
26 a
26 A
27 a
27 ab
26 bc
25 c
26 A
Autumn 2009
0-0.25m
8b
6b
7b
12 a
8A
7b
5c
5c
12 a
7A
0.25-0-75m
16 a
11 ab
4c
8 bc
10 A
15 a
10 b
4c
8 bc
9A
0.75-1.5m
16 ab
19 a
6c
13 b
14 A
10 a
15 a
9a
9a
11 A
Spring 2010
Total
40 a
36 a
17 b
33 a
30 A
32 a
29 a
17 b
29 a
26 A
0-0.25m
9d
11 c
22 a
14 b
14 A
10 c
10 c
21 a
15 b
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).
Effect of catch crops on Se and S availability for succeeding crops
29
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
3b
41 c
130 b
83 b
7b
FR
4a
212 a
997 a
147 a
26 a
HV
2c
85 b
177 b
74 b
4c
Average
3
93
322
101
12
IR
4b
30 b
114 b
133 b
8b
FR
6a
288 a
1571 a
202 a
28 a
HV
2c
31 b
65 b
108 b
5c
Average
4A
101 B
494 B
148 A
13 A
IR
4b
202 c
773 b
136 b
8b
FR
6a
514 a
3263 a
223 a
29 a
HV
2c
316 b
663 b
112 b
4c
Average
4A
344 A
1566 A
157 A
14 A
2009
Se0
Se10
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).
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
30
Effect of catch crops on Se and S availability for succeeding crops
(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.
Onions
Cabbage
Catch
crops
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
IR
FR
HV
9a
9a
10 a
9a
6a
3c
2 bc
4 ab
49 a
24 bc
21 c
41 bc
4b
3c
5a
6a
24 a
22 a
23 a
31 a
97 bc
71 c
117 ab
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
Effect of catch crops on Se and S availability for succeeding crops
31
(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.
Se-content
(μg kg-1)
Se-uptake
(mg ha-1)
N-uptake
(kg ha-1)
S-uptake
(kg ha-1)
Fertilization
Catch crops
Yield
(Mg DM ha-1)
Se0S0
C
4a
10 a
39 a
32 b
5c
IR
5a
7a
31 a
47 a
4c
FR
5a
8a
31 a
50 a
9a
HV
5a
2a
10 a
53 a
7b
Average
5A
7C
29 C
46 A
6B
C
4a
9a
35 a
31 c
8b
IR
4a
5a
23 a
44 b
10 b
FR
5a
7a
33 a
57 a
15 a
HV
5a
6a
27 a
55 a
13 a
Average
5A
7C
30 C
47 A
11 A
C
4b
61 a
250 a
30 d
5 bc
IR
4b
72 a
281 a
40 c
4c
FR
5 ab
70 a
321 a
52 a
10 a
HV
5a
87 a
456 a
61 b
7 ab
Average
5A
72 A
327 A
46 A
7B
C
4a
36 a
143 a
31 c
8c
IR
4a
40 a
157 a
42 b
10 bc
FR
5a
48 a
214 a
52 a
10 b
HV
5a
40 a
194 a
57 a
13 a
Average
4A
41 B
177 B
45 A
10 A
Se0S65
Se10S0
Se10S65
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).
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
32
Effect of catch crops on Se and S availability for succeeding crops
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.
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)
Se0Se0
C
2c
8a
18 a
19 b
4c
IR
3 bc
12 a
30 a
27 a
4c
FR
3a
9a
28 a
36 a
7a
HV
3b
10 a
28 a
30 ab
5b
Average
3A
10 C
26 B
28 A
5A
C
2a
30 a
66 a
18 ab
3b
IR
3a
31 a
85 a
29 a
4b
FR
3a
22 a
65 a
32 ab
7a
HV
2a
21 a
41 a
21 ab
4b
Average
3A
26 A
64 A
25 A
5A
C
2a
15 ab
35 a
19 b
4b
IR
2a
28 a
65 a
26 a
4b
FR
3a
8b
23 a
30 a
6a
HV
3a
11 b
31 a
29 a
5a
Average
3A
16 B
38 B
26 A
5A
C
2c
29 a
63 a
18 c
4c
IR
2 bc
25 a
58 a
25 b
3c
FR
3a
28 a
79 a
30 a
6a
HV
3 ab
33 a
88 a
30 a
5b
Average
3A
29 A
72 A
26 A
5A
Se0Se10
Se10Se0
Se10Se10
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).
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
Effect of catch crops on Se and S availability for succeeding crops
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.
33
34
Effect of catch crops on Se and S availability for succeeding crops
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
Effect of catch crops on Se and S availability for succeeding crops
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
35
36
Effect of catch crops on Se and S availability for succeeding crops
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.
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
37
38
Selenium mineralization and availability from catch crops
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
Selenium mineralization and availability from catch crops
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
39
40
Selenium mineralization and availability from catch crops
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
Hairy vetch (HV)
Italian ryegrass (IR)
Indian mustard (IM)
Fodder radish (FR)
Hairy vetch-Se enriched
(HV+Se)
Italian ryegrass-Se enriched
(IR+Se)
Indian mustard-Se enriched
(IM+Se)
Fodder radish-Se enriched
(FR+Se)
Sodium selenate (Se)
Sodium selenate + sucrose
(Se+C)
Se content
(mg/kg)
Amount of Se added
0.05
0.07
0.39
1.94
13.12
Leaching
incubation
0.10
0.13
0.69
3.39
22.96
Pot
experiment
0.76
1.03
5.49
26.90
182.35
19.01
33.26
264.21
13.38
23.42
186.00
8.41
14.72
116.96
5.00
5.00
3.98a
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 soilsand 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
Selenium mineralization and availability from catch crops
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 airdried 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.
41
42
Seleniuum mineralization and availabilityy from catch
h crops
2.44. Sample preparation
p
n and Se an alysis
Finely ground material (4
400 mg) waas microwaave-digested
d in pressuurized PFA vessels
(Anton Paar, ‘Mulltiwave’) with 3.0 mL of 70% Fissher ‘Trace analysis grrade’ (TAG)) HNO3,
3 mL w
water and 2 mL of 30%
% H2O2. Diigested sam
mples were diluted
d
to 115 mL with
h milli-Q
water (18.2 MΩ cm)
c
and im
mmediately pprior to an
nalysis, were further diiluted 1-in--10 with
milli-Q water. Conncentrationss of Se in pplant samplees and leach
hate were ddetermined using
u
an
Inductivvely Couplled Plasmaa Mass Speectrometer (ICP-MS, Thermo-Fiisher Scien
ntific XSeriesII) employingg a ‘hexapo
ole collisioon cell’ (H2 gas) with kinetic eneergy discrim
mination
(CCT-K
KED) to rem
move polyattomic interfferences.
2.55. Calculati
tions and sta
atistical anaalysis
As tthe leachatee collected from the lleaching tub
bes did nott represent a full reco
overy of
leachedd Se, the data are onlly used to compare Se
S levels beetween the treatmentss, not to
calculatte total Se recovery
r
fro
om the incubbated materrials. Data from
f
the leaaching-tubee and pot
incubations were log-transfor
l
med to obtaain homogeeneity of vaariance and analysed by 1 way
anova, w
with post-hoc differencces identifieed by the LS
SD procedu
ure. Results with p < 0..05 were
consideered significcant. The sttatistical anaalysis was carried
c
out using SAS (SAS Instittute Inc,
Cary, N
NC, USA, veersion 9.2).
Figure 44-1. Selenium uptake by caatch crops groown in the fieeld. Values arre means of 2 replicates. Horizontal
H
bars represent standardd errors.
Selenium mineralization and availability from catch crops
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.
43
44
Seleniuum mineralization and availabilityy from catch
h crops
Figure 44-2. Concentraations of solub
ble Se (μg/L) in the leachattes released by (a) the enricched plant maaterial, (b)
the non enriched plannt material an
nd (c) the inoorganic treatm
ments during the
t leaching iincubation ex
xperiment.
s
IR+Se: enriched
e
Italiaan ryegrass, FR+Se:
F
enrich
hed fodder raddish, HV+Se:: enriched
Control: unamended soil,
hairy vettch, IM+Se: ennriched Indian
n mustard, IR : non enriched
d Italian ryegrrass, FR: non enriched fodd
der radish,
um selenate, SSe+C: sodium
m selenate
HV: nonn enriched hairy vetch, IM: non enrichedd Indian musttard, Se: sodiu
and sucroose. Values arre means of 2 replicates. Hoorizontal bars represent stan
ndard errors.
3.33. Pot incubbations
Totaal above groound biomaass producti on ranged from
f
1.6 to 7.9 g/pot aand 1.8 to 7.6
7 g/pot
for firsst and secoond harvest, respectivvely (Tablee 4-2). Plaant amendm
ments significantly
increaseed the biom
mass of IM at
a both harveests; the hig
ghest yields were foundd where haiiry vetch
was addded. There was
w a trend towards higgher biomaass productio
on when See had been added
a
as
inorgannic fertilizattion or as enriched
e
plaant materiaal, a trend which
w
was also seen with
w the
catch crrops grown in the field
d. There waas little addiitional grow
wth from thee first to thee second
harvest time, and the biomaass producttion of IM was the same
s
at booth harvestss for all
treatmeents. Howeever, the Se
S concenttrations inccreased and the totaal Se uptaake was
approxiimately douubled at the second harvvest.
At tthe first haarvest, Se uptake
u
by IM was significantly greater whhen enricheed plant
materiaal was incorrporated in the
t soil com
mpared to th
he control, while
w
the inncorporation
n of non
enriched material or the inorg
ganic Se ferrtilizer did not affect Se uptake ((Table 4-2)). At the
second harvest, alll treatmentss increased Se uptake in IM and the uptakee was higheest when
IR+Se w
was incorpoorated into the soil (Taable 4-2). The
T uptake was
w greaterr in plants grown
g
in
Selenium mineralization and availability from catch crops
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
45
46
Selenium mineralization and availability from catch crops
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 Seenriched 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
Selenium mineralization and availability from catch crops
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
47
48
Selenium mineralization and availability from catch crops
process, whereby Se anions are reduced to low valence states and then incorporated into lowmolecular-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 highaffinity 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.
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
Krumbein §, and Monika Schreiner §
†,‡
, Hanne L. Kristensen †, Angelika
†
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., TheodorEchtermeyer-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
49
50
Differential N and S competition in intercropping affects glucosinolates
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
Differential N and S competition in intercropping affects glucosinolates
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 (ThorupKristensen 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. Material and Methods
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
51
52
5
Differentiial N and S competition in intercro
opping affeccts glucosin
nolates
precipittation durinng the growtth season arre shown in
n Figure 5-1
1. Mean annnual precipiitation at
the site was 624 mm
m and mean
n annual airr temperaturre 7.8oC.
The experimenttal design was
w a random
mized comp
plete block design withh three repliications.
The brooccoli (Brasssica oleraccea L. var iitalica cv. ‘Tinman’) was
w intercroopped with
h iceberg
lettuce (Lactuca saativa L. var. capitata ccv. ‘Dimantiinas RZ’). Both
B
crops were grown
n also in
pure staands. Each plot
p was 1.6
6 × 3 m, disstance between rows was
w 0.35 m aand within rows
r
0.3
m for both interccropping and
a
sole crropping. Th
he intercro
op design w
was based on the
replacem
ment princiiple, with mixed
m
brocc oli and lettu
uce transplaant in the saame rows. Broccoli
B
seeds w
were sown on
o 26 May, 2009 and l ettuce 2 Jun
ne, 2009 an
nd grown inn a greenhou
use until
transplaanting. Bothh crops werre transplannted on 19 June. The plots were kept weed free by
repeatedd manual weeding.
w
Du
uring the exxperiment, crops
c
receiv
ved 40 mm
m irrigation water
w
to
avoid w
water stress. Irrigation water was applied viaa a moveab
ble irrigatioon boom. The SO4−
and NO
O3− content of
o the irrigation water w
were about 32 mg L-1 and
a 3 mg L--1, respectively.
The four fertilizzer treatmen
nts were: 900 kg ha-1 Ν + 0 kg ha-1 S (N90S0),, 220 kg haa-1 Ν + 0
kg ha-1 S (N220S0),, 90 kg ha-1 Ν + 40 kgg ha-1 S (N900S40), 220 kg
k ha-1 Ν + 40 kg ha-1 S (N220S40). Urrea [(NH2)2CO] was used
u
as thee N source and Kieserrite (MgSO
O4) as the S source.
Nitrogeen and S fertilizers were
w
broaddcast manu
ually on th
he soil surrface 2 day
ys after
transplaanting.
Figure 55-1. Average daily temperaature (line) annd precipitatiion (bars) durring the exper
erimental seasson (JuneSeptembeer, 2009).
Differential N and S competition in intercropping affects glucosinolates
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
53
54
Differential N and S competition in intercropping affects glucosinolates
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
Differential N and S competition in intercropping affects glucosinolates
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 ionexchanger (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.
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
Glucobrassicin
3-indolylmethyl
Neoglucobrassicin
1-methoxy-3-indolylmethyl
4-methoxy-glucobrassicin
4-methoxy-3-indolylmethyl
Indolyl
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 desulfoglucosinolates were performed in duplicate. The desulfo-glucosinolates determinate are
shown in the Table 5-1.
55
56
Differential N and S competition in intercropping affects glucosinolates
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.
Soil inorganic N
(0-1.0 m)
(kg N ha-1)
Soil inorganic S
(0-1.0 m)
(kg S ha-1)
Cropping system
Fertilization
Intercrop
N90S0
N90S40
38
37
37
57
N220S0
52
37
N220S40
41
45
N90S0
47
36
N90S40
51
82
N220S0
65
40
N220S40
64
85
N90S0
34
37
N90S40
38
50
N220S0
48
29
N220S40
42
51
N
S
C
N×C
S×C
N×S
N×S×C
***
NS
***
NS
NS
NS
NS
NS
***
***
NS
**
NS
NS
Sole lettuce crop
Sole broccoli crop
Significancea
where N (nitrogen), S (sulphur), C (cropping system);
*, p<0.05; **, p<0.01; ***, p<0.001 (n=3).
a
Levels of significance: NS, not significant;
Differential N and S competition in intercropping affects glucosinolates
57
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
Intercrop
Sole crop
Broccoli
Lettuce
Edible
Total
Edible
Total
N90S0
689
4206
314
450
N90S40
663
4454
266
383
N220S0
726
4558
194
294
N220S40
819
5000
162
232
N90S0
567
5188
2536
3658
N90S40
573
5097
2484
3502
N220S0
742
5433
2337
3538
N220S40
774
5761
2318
3486
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
Significancea
a
where N (nitrogen), S (sulphur), C (cropping system); Levels of significance: NS, not significant; *, p<0.05;
**, p<0.01; ***, p<0.001 (n=3).
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
58
Differential N and S competition in intercropping affects glucosinolates
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.
a
gluco
osinolates
Differrential N and S competition in inteercropping affects
Figure 55-2. Average root intensitty in the 0-11.3 m soil prrofile in the field experim
ment four weeeks after
transplannting (a) and one
o week beffore harvest (bb). where N, nitrogen; S, sulphur; bars represent thee standard
errors (w
where n = 2).
33.1.4. N andd S accumu
ulation
Nitroogen and S accumu
ulation in broccoli and
a
lettuce are show
wn in Tab
ble 5-4.
Intercroopping affeccted N con
ncentrations in broccolli florets an
nd total N uuptake by broccoli.
b
When bbroccoli waas intercropp
ped with letttuce it had
d lower floret N concenntrations an
nd lower
N uptakke comparinng to sole cropped of bbroccoli. Niitrogen conccentrations and N uptaake in all
examined organs of
o broccoli responded
r
too N supply,, but these were
w unaffeected by S leevel and
no signnificant interactions weere found. T
The effect of
o N appliccation on N concentrattion was
strongerr in the broccoli crop residues
r
thaan in the ediible part. Increasing N supply incrreased N
concenttration in broccoli
b
flo
orets by 155-19%, whiile in brocccoli residuees N conceentration
increaseed by 31-622%.
Fertiilization wiith S increaased total S uptake an
nd the S concentratioon both in broccoli
florets and residuees, at both N fertilizattion levels (Table
(
5-4)). As for N concentrattion, the
responsse of S conccentration to
o S fertilizaation was greater in broccoli residdues than in
n florets.
Increasiing the N supply
s
from
m 90 to 2200 kg ha-1 haad no effectt on tissue S concentraations in
broccolli florets or residues. In
ntercroppingg only affeccted S concentrations oof broccoli residues
59
60
Differential N and S competition in intercropping affects glucosinolates
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.
Nitrogen
Cropping
system
Intercrop
Sole crop
Fertilization
Broccoli
Concentration
Sulphur
Lettuce
Florets Residues
Total
uptake
Total
uptake
N90S 0
32.4
20.2
93
N90S 40
31.9
18.9
N220S 0
38.7
N220S 40
Broccoli
Concentration
Lettuce
Florets Residues
Total
uptake
Total
uptake
16
6.2
4.6
20
1
93
14
7.1
7.5
33
1
29.3
141
12
5.2
3.2
16
1
37.6
30.6
158
11
7.4
8.2
41
1
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
N
S
C
N×C
S×C
N×S
N×S×C
***
NS
**
NS
NS
NS
NS
***
NS
NS
NS
NS
NS
NS
***
NS
**
NS
NS
NS
NS
**
NS
***
***
NS
NS
NS
NS
***
NS
NS
NS
NS
NS
NS
***
*
NS
NS
NS
*
NS
***
NS
NS
NS
*
NS
NS
NS
***
**
NS
NS
NS
Significance
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).
petition in inntercroppin
ng affects glucosinolatees
Diffferential N and S comp
under tthe high N and S treatment, witth a 28% increase
i
in S concenttration obseerved in
intercroopping com
mpared to so
ole croppingg of brocco
oli. A significant N suupply × S supply
s
×
croppinng system innteraction fo
or S accumuulation in brroccoli resid
dues was obbserved.
Nitroogen and S uptake by lettuce wass reduced by
y intercropp
ping, as a rresult of thee limited
lettuce growth (Taable 5-4). Nitrogen
N
ferttilization en
nhanced N and
a S uptakke by lettucee only in
the solee cropping trreatment.
Figure 55-3. Influencee of N (a) an
nd S (b) conccentration and
d N : S ratio
o (c) on totall glucosinolatte (GSLs)
concentraation in the fieeld experimen
nt.
33.1.5. Glucoosinolates
Five individual GSLs, nam
mely, the aliiphatic GSL
Ls glucorap
phanin and gglucoiberin and the
indole GSLs gllucobrassiciin, neogluucobrassicin
n and 4-m
methoxy-gluucobrassicin
n were
quantitaatively deteermined in broccoli
b
florrets. The to
otal GSL waas calculateed as the sum
m of the
individuual GSLs.
The highest totaal GSL leveel (4137 μg g-1 DM) waas obtained at high N aand S supply
y in sole
croppedd broccoli, while the lowest
l
leveel (2458 μg
g g-1 DM) was observved in interrcropped
broccolli at N220S
S0 (Table 5-5). Totall GSL con
ncentrations significanttly increaseed by S
fertilizaation, whereeas N fertiliization reduuced GSL concentrations when N was added without
S. Totall and aliphaatic GSL concentrationn was not aff
ffected by in
ntercroppingg (Table 5-5
5). In an
attemptt to relate GSL
G
concentration to the nutritio
onal status of broccolli, the relationships
61
62
Differential N and S competition in intercropping affects glucosinolates
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
Intercrop
N90S 0
Sole crop
N:S
ratio
Glucosinolatea
Total
GSLs
Total
Total
Aliphatic
Indole
GSLs
GSLs
GRA
GIB
GBS
NGB
MGB
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
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
N
S
C
N×C
S×C
N×S
N×S×C
***
***
NS
NS
NS
*
*
***
**
NS
NS
NS
**
NS
***
**
NS
NS
NS
**
NS
NS
NS
NS
NS
NS
NS
NS
NS
**
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
***
NS
NS
NS
*
NS
***
**
NS
NS
NS
**
NS
NS
**
*
NS
NS
NS
NS
Significanc
where N (nitrogen), S (sulphur), C (cropping system );aGRA: glucoraphanin; GIB: glucoiberin; GBS: glucobrassicin; NGB: neoglucobrassicin; MGB: 4Methoxy-Glucobrassicin; b Levels of significance: NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (n=3).
Differential N and S competition in intercropping affects glucosinolates
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
63
64
Differential N and S competition in intercropping affects glucosinolates
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 56). 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 plant1
), N and S concentrations (mg g-1 DM) in red leaf mustard and lettuce in the pot experiment.
Red leaf mustard
Cropping
system
Fertilization
Intercrop
Sole crop
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 )
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
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
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
a
Significance
where N (nitrogen), S (sulphur), DM (dry matter), C (cropping system);
significant; *, p<0.05; **, p<0.01; ***, p<0.001 (n=4).
a
Levels of significance: NS, not
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
Differential N and S competition in intercropping affects glucosinolates
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).
65
66
Differential N and S competition in intercropping affects glucosinolates
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
Intercrop
Sole crop
Fertilization
N:S
ratio
Glucosinolatea
PRO
SIN
GNA
MGB GBS
Total
GSLs
Total
Aliphatic
GSLs
Total
Indole
GSLs
N203S44
N203S88
7.7
7.4
5
7
10586
10335
452
510
18
18
98
104
11159
10974
11043
10852
116
122
N406S44
9.5
11
8478
327
17
82
8915
8816
100
N406S88
7.3
16
10885
498
26
145
11571
11400
171
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
N
S
C
N×C
S×C
N×S
N×S×C
**
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
*
NS
*
NS
NS
NS
NS
NS
*
**
NS
NS
**
NS
NS
NS
*
NS
NS
*
NS
NS
NS
*
NS
NS
*
NS
NS
*
**
NS
NS
**
NS
Significance
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).
Differential N and S competition in intercropping affects glucosinolates
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
67
68
Differential N and S competition in intercropping affects glucosinolates
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).
Differential N and S competition in intercropping affects glucosinolates
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.
69
70
Differential N and S competition in intercropping affects glucosinolates
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.
Effects of fertilization on root growth
71
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).
2. Material and Methods
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.
72
7
Effects of
o fertilizatioon on root growth
g
Figurre 6-1. Minirhhizotron tube with
w two counnting grids.
Directly after planting, min
nirhizotron glass tubess (0.07 m outer diametter and 1.5 m long)
were innserted in thhe soil (Tho
orup-Kristeensen & van
n den Boorrgaard 19988). Two tub
bes were
placed iin the interrrow space, one
o betweenn the first and
a second row
r and thee other one between
b
the third and fourtth row. Thee minirhizottrons were installed
i
at an angle off 30o from vertical,
reaching a depth of
o approxim
mately 1.3 m in the soiil (Figure 6-1).
6
Registtrations were made
he minirhizzotron surfaace (Chapteer 5, this
using a min-video camera to record the roots on th
thesis).
Plannts were haarvested 50
0 days afteer transplan
nting. Soil sampling and analy
ysis was
perform
med as desccribed in th
he Chapter 5 (this theesis). Root intensities were calcu
ulated as
simple averages off registratio
ons within eeach soil lay
yer for each
h observatioon date. Root depth
was esttimated as the deepestt root obserrvation on each of thee two counnting grids on each
minirhizotron. Datta were anaalyzed with the GLM procedure of
o the SAS statistical package
(SAS Innstitute Inc, Cary, NC, USA, versiion 9.2).
3. Reesults and Brief Disccussion
Cleaar differences in rootin
ng depth aamong the crops weree obtained during the growth
period (Figure 6-22). Broccolli had fast rroot growth
h and grew
w deeper thaan lettuce. Rooting
depth one week beefore harvest varied fro m 0.8 to 1.1
1 m for brocccoli and 0..7-0.9 m forr lettuce.
The ressults for thee two speciies concur w
with the co
onclusion of Thorup-K
Kristensen (Thorup(
ot growth
Effectts of fertilization on roo
Kristensen 2001) that
t
cruciferrs are charaacterized by
y faster root depth deveelopment co
ompared
monocots annd that lettu
uce is a shhallow-roott species (T
Thorup-Krisstensen 200
06a). In
with m
broccolli the high N and S ratees decreasedd the root penetration,
p
whereas thee high N an
nd low S
fertilizaation led to the deepestt rooting. Inn lettuce thee fertilizatio
on treatmentts did not in
nfluence
the roott growth.
Figurre 6-2. Root depth
d
of lettuce and broccolli during grow
wing period. Bars show stand
ndard error (n=
=2).
Alsoo the root inntensity and
d root distriibution in the
t soil variied betweenn the crops and the
fertilizaation treatm
ments (Figurre 6-3). No differencess in the roott intensity bbetween letttuce and
broccolli were founnd 19 days after
a
transpllanting (DA
AT) (Figure 6-3a). How
wever, from
m the 28th
DAT uuntil the lasst measurem
ment (one w
week beforre harvest) the root inntensity wass higher
under bbroccoli (Figgure 6-3b, c, d). In thee lettuce, th
he root inten
nsity was hiigher in the 0-0.5 m
soil layeer whereas broccoli showed its higghest valuess in the 0.25
5-0.75 m.
The ferrtilization trreatments afffected roott density an
nd distributiion of cropss differently
y during
growth period. Brroccoli roott density w
was influencced by fertilization treeatments mo
ore than
lettuce. Nitrogen and S ferrtilization innfluenced lettuce roo
ot intensityy only in the
t
first
measureement (Figgure 6-3a). Lettuce rooot intensitty was high
her at the N90S40 trreatment
comparred to the N220S40 treaatment.
73
74
Effectts of fertilization
n on root growth
h
F
Figure 6-3. Averagge root intensity in the 0-1.3 m soil profile in the field experiment 19 days (a)), 28 days (b) 38 day
ys (c) and after tran
nsplanting and one weeks
w
before harvest
N, nitrogen; S, sulphhur; bars represent the
t standard errors (where
(
n = 2)
(d). where N
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
75
76
7
Effects of
o fertilizatioon on root growth
g
are highh S demannding plantss and have the ability
y to depletee soil inorgganic S (Eriksen &
Thorup-Kristensenn 2002).
Figurre 6-4. Soil innorganic S (a) and N (b) proofiles after harrvest of crops. Bars show sttandard error (n=3).
(
The data for thee soil inorgaanic N afterr vegetable harvest sho
ow further iinteraction between
b
rooting intensity and
a soil N utilizationn (Figure 6-4b).
6
Brocccoli had cllearly reduced soil
inorgannic N in thee subsoil compared to llettuce. Thee higher N utilization m
may be asccribed to
the highher root inteensity of bro
occoli in thee subsoil.
The present ressults show that
t
there aare large diffferences in
n root grow
wth and disttribution
betweenn the two vegetable
v
crops,
c
whic h can influ
uence soil N and S uttilization. A strong
interacttion effect was
w found between N and S ratees in brocco
oli root inteensity. Hig
gher root
density was foundd in the sub
bsoil at brooccoli grow
wn without S fertilizatiion at high N rate.
Lettucee root intennsity at harv
vest was loower than broccoli
b
an
nd was lesss affected from
f
the
fertilizaation treatm
ments. Studiees have shoown that belowground interactions
ns among neeighbour
plants oof different species cou
uld affect teemporal and
d spatial root distributtion (Li et al.
a 2006;
Tosti & Thorup-Kristensen 2010), thherefore ou
ur results may
m
not ggeneralized to the
intercroopping. How
wever, the results indiicate that nutrient
n
avaailability in the soil sh
hould be
consideered as it maay influencee root grow
wth and distrribution and
d susbequenntly to influence the
root com
mpetition.
Conclusion and perspectives
77
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.
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79
Chapter 8
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