1999/2000 FINAL RESEARCH REPORT : WINETECH · 2005. 9. 15. · 1999/2000 FINAL RESEARCH REPORT...
Transcript of 1999/2000 FINAL RESEARCH REPORT : WINETECH · 2005. 9. 15. · 1999/2000 FINAL RESEARCH REPORT...
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1999/2000 FINAL RESEARCH REPORT : WINETECH
FRUIT TYPE : Grapes PROJECT NUMBER: WW18/12
ORGANIZATION : Nietvoorbij RESEARCHER : C.C. Mullins
PROJECT TITLE : An assessment of symbiotic nitrogen fixation by leguminous cover crops and
its contribution to the nitrogen balance in a vineyard-cover crop system.
OBJECTIVE OF PROJECT:
The assessment of the uptake of symbiotically fixed nitrogen by legume cover crops for sustainable
vineyard management.
OBJECTIVES OF CURRENT YEAR:
To evaluate different legume cover crops for their ability to enhance the N nutrition of the vine on a sandy
soil and to determine the nitrogen contribution made by legume cover crops as measured by 15N natural
abundance methodologies.
UPDATED FINDINGS:
Throughout this study it has become evident that, among the legumes, grazing vetch has the greatest
potential to improve the soil N status of the vine in the Olifants River Region as it supplies the highest
aboveground N. Nitrogen supply from legume cover crops are synchronized with vine N demand, yet
adequate moisture has to be supplied in order to gain the full benefit of legumes with regard to N supply.
This research has shown that cover cropping is beneficial to the nitrogen economy of the vine.
ADVANTAGES TO INDUSTRY:
Potential to promote cost-effective, environmentally friendly and sustainable viticultural practices for
resource limited producers. Legume cover crops could supplement nitrogen fertilization and thereby
reduce the application of chemical fertilizers and input costs.
RECOMMENDATIONS FOR THE FOLLOWING YEAR:
The project has been completed and the results will be communicated in article form.
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AGRICULTURAL RESEARCH COUNCIL
LANDBOUNAVORSINGSRAAD
Nietvoorbij Centre for Vine and WineNietvoorbij Sentrum vir Wingerd en Wyn
p/bag p/sak X5026 - Stellenbosch - 7599 - rsa
1999/2000 FINAL RESEARCH REPORT : WINETECH
PROJECT NUMBER WW 18/12
PROJECT TITLE An assessment of symbiotic nitrogen fixation by leguminous cover
crops and its contribution to the nitrogen balance in a vineyard-
cover crop system.
PROJECT LEADER C.C. Mullins
COMMENCEMENT DATE : 1997COMPLETION DATE : 2000
No part of this document may be reproduced or distributed in any form without the express writtenpermission of ARC-lnfruitec/Nietvoorbij.
Telefoon 021 - 809 3100Int. 27 21 809 3100
Telefaks 021 - 809 3002Int. 27 21 809 3002
Telephone
Telefax
021 - 809 3100Int. 27 21 8093100
021 - 809 3002Int. 27 21 809 3002
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1999/2000 FINAL RESEARCH REPORT
PROJECT NUMBER PROJECT LEADER CO-WORKERS
WW18/12 C.C. Mullins P.J.E. Louw
F.D. Dakora
J.C. Fourie
PROJECT TITLE
An assessment of symbiotic nitrogen fixation by leguminous cover crops and its contribution to the
nitrogen balance in a vineyard-cover crop system.
ACKNOWLEDGEMENTS
Winetech is acknowledged for partial funding of this project.
PROBLEM BEING ADDRESSED / AIM OR HYPOTHESIS
It is known that leguminous cover crops can contribute towards the nitrogen balance of a system.
However, how much is contributed and when, are questions that remain unresolved. The aim of the
project is to determine the quantity of nitrogen fixed and at what stage most fixed nitrogen is contributed to
the vine.
RESEARCH NEED BEING ADDRESSED
Winetech Technical Committee, 1996: 3.1 Nitrogen fertilization. (Document in Afrikaans only).
LONG-TERM OBJECTIVES
• Evaluate different legume cover crops for their ability to enhance the N nutrition of the vine on a
sandy soil.
• Determine the nitrogen contribution made by legume cover crops as measured by 15N natural
abundance methodologies.
• Examine the effect of cover cropping on the soil nitrogen status of the vineyard.
• Assess the effect of cover cropping on the nitrogen nutrition of the vine.
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RESULTS AND DISCUSSION
The results of this project have been used for my masters dissertation. All data collected from 1997-
1999 has been presented and discussed in the thesis. Addendum A is a copy of the dissertation.
CONCLUSIONS
As chemical inputs of N fertilizers have to be reduced to limit environmental pollution, it has become
imperative to seek more environmentally friendly and cost-effective alternatives. Legume cover crops
have been known to improve the N status of the adjacent crop. The main objective of this project was to
recommend a legume cover crop species, in terms of its N contribution, for use in vineyards in the Olifants
River region.
For legume cover crops to benefit vineyards in terms of N provision, it is essential that N release by
legumes coincides with vine N demand. This occurs from budbreak to veraison and from harvest to leaf-
fall. This synchrony was observed in this study.
Although N fertilizer application and winter rainfall was reduced in the second season, 515N-values in vine
leaves were lower compared to the previous year. As a low 515N-value denotes a high uptake of
biologically fixed N, this indicated that the proportion of fixed N uptake to fertilizer N uptake gradually
increased in the vine with time. Throughout this study it has become evident that the use of grazing vetch
in vineyards in the Olifants River Region could be successfully used for the provision of N and will result in
the reduction of synthetic fertilizer inputs. The omission of a green cover as described in the control plot
had significantly lower leaf nitrate reductase activity, emphasizing the importance of cover crops in
viticulture.
A high fluctuation in mineral N levels were observed, yet during the decomposition period (January-
February) legume cover crop treatments were higher in nitrogen. A decline in soil N was observed in the
second season due to shortage of irrigation water and low rainfall. Grazing vetch decomposes rapidly
when mechanically tilled at budbreak. This practice can therefore not be recommended to producers. The
die-back treatment had a high dry material on the soil surface than the before budbreak treatments.
Nitrogen fixation proceeded effectively in all treatments for both seasons. Grazing vetch and to a lesser
extent Seradella emena provided an excellent source of aboveground N.
The following conclusions have been drawn from this study:
(i) N supply from legume cover crops are synchronized with vine N demand,
(ii) Grazing vetch is suitable for use as a green cover in vineyards in the Olifants River Region as
it supplies the highest aboveground N.
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(iii) Mechanical tillage of cover crops result in a short-term release of N and this practice is
inefficient,
(iv) Adequate moisture has to be supplied in order to gain the full benefit of legumes with regard
to N supply.
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SUMMARY OF 1999/2000-RESEARCH REPORT
PROJECT NUMBER PROJECT LEADER CO-WORKERS
WW18/12 C.C. Mullins P.J.E. Louw
F.D. Dakora
J.C. Fourie
PROJECT TITLE
An assessment of symbiotic nitrogen fixation by leguminous cover crops and its contribution to the
nitrogen balance in a vineyard-cover crop system.
The aim of this investigation was to evaluate different legume cover crops for their ability to enhance the N
nutrition of the vine on a sandy soil. The study site was located in Lutzville, in the Olifants River region and
was conducted on a six-year old Sauvignon blanc vineyard grafted onto Ramsey rootstock.
The uptake and assimilation of nitrogen by grapevines were monitored monthly in a legume-based cover
cropping system over two seasons during 1997-1999. An in vivo nitrate reductase assay was conducted
on fresh vine leaves during the growing season as it is an indicator of newly absorbed nitrogen. The
uptake of biologically fixed nitrogen was measured by determining the natural abundance of 15N in vine
leaves. The uptake and assimilation of biologically fixed N coincided with the annual demand for nitrogen
by the vine, which occurs from budbreak to the first stage of berry development and after harvest. Nitrate
reductase activity in vines intercropped with legumes was relatively higher than the cereal and control
counterparts, despite the reduction of nitrogen fertilization in these treatments. The omission of a green
cover in the control plot had significantly lower leaf nitrate reductase activity, emphasizing the importance
of cover crops in viticulture. The proportion of fixed N uptake to fertilizer N uptake gradually increased in
the vine with time, as &15N values of vine leaves were lower in the second season. Results from soil
analysis revealed that the soil nitrogen (total and inorganic) was low and differences in soil nitrogen levels
are expected to be more distinct in the longer term.
The N content and biomass of legumes were determined to evaluate various legume cover crop species.
Grazing vetch had the highest above-ground N. &15N values differed according to the various legume plant
components. The roots and shoots were depleted of 15N indicating the active fixation of nitrogen while the
nodules were enriched in 15N. This was due to the inherent characteristic of the species used. The lower
rainfall in the latter season resulted in a decline in legume biomass production. 815N-values of legumes
were lower in the second season indicating that nitrogen fixation proceeded even more successfully
regardless of climatic conditions, proving the long-term benefits of using cover crops. In general grazing
vetch showed the greatest potential for improving the N status of the vine.
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OPSOMMING VAN 1999/2000-NAVORSINGSVERSLAG
PROJEKNOMMER PROJEKLEIER MEDEWERKERS
WW18/12 C.C. Mullins Dr. F. Dakora (UCT)
P.J.E. Louw
J.C. Fourie
PROJEKTITEL
'n Ondersoek na simbiotiese stikstofbinding deur peulplantdekgewasse en die bydrae daarvan tot die
stikstofbalans in 'n wingerd-dekgewassisteem.
Die doel van hierdie projek was om die geskiktheid van verskillende dekgewasbestuurspraktyke te oordeel
op grond van hul vermoe om stikstofvoeding in die wingerd te verbeter op 'n sandgrond te Lutzville.
Die opname en assimilasie van stikstof deur die wingerd is maandeliks gemonitor in 'n
peuldekgewassisteem oor twee seisoene gedurende 1997-1999. 'n In vivo nitraat-reduktase
bepalingstoets is uitgevoer op vars wingerdblare gedurende die groeiseisoen omdat dit 'n aanduiding is
van pasopgeneemde stikstof. Die opname van biologiesgebinde stikstof het oorgeengestem met die
jaarlikse N-aanvraag deur die wingerd, wat plaasvind van bot tot die eerste fase van korrelontwikkeling en
na oes. Nitraat-reduktase aktiwiteit in wingerdblare, tussenverbou met peuldekgewasse, was relatief hoer
as die graan en kontrole behandelings, ten spyte van 'n vermindering in stikstofbemesting in hierdie
behandelings. Die skoonbewerkte kontrole het 'n betekenisvolle laer nitraat-reduktase aktiwiteit getoon.
Dit beklemtoon die rol van dekgewasse in wingerdbou. Die verhouding van gebinde N-opname tot
bemesting N-opname het geleidelik toegeneem in die wingerd want 815N-waardes in wingerdblare het
gedaal in die tweede seisoen. Stikstof in die grond (totaal en anorganies) was laag en verskille word
verwag om meer opvallend te wees op die langtermyn.
Die N-inhoud en biomassa van die peulplant is bepaal om die peulplantdekgewasse te evalueer.
Weiwieke het die hoogste bogrondse N getoon. &15N waardes het verskil volgens die plantkomponente.
Die wortels en lote was laag in 15N - 'n aanduiding van die aktiewe binding van stikstof terwyl die knoppies
verryk was in 15N. Dit was kenmerkend van die spesies onder studie. Die lae reenval in die laaste
seisoen het 'n afname in biomassa produksie veroorsaak. Ten spyte van die lae biomassa produksie het
stikstofbinding wel plaasgevind (&15N waardes van peulplante was laer in die tweede seisoen). Dit was 'n
aanduiding dat stikstofbinding meer suksesvol voortgegaan het ongeag die klimatiese toestande. Dit
bewys die langtermynvoordele van dekgewasse. In die algemeen het weiwieke die grootste potensiaal
om die N status van die wingerd te verbeter. Hierdie dekgewas toon groot potensiaal om as koste-
effektiewe en omgewingsvriendelike alternatief te dien vir die toediening van N-bemesting en sal van groot
nut wees vir hulpbron-beperkte produsente.
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ADDENDUM A
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Contribution of Nitrogen Fixation by Cover Crop Legumes to the
Nitrogen Economy of a Vine-based Cropping System
by
Carmen Mullins
Botany Department
University of Cape Town
Presented for the Degree of Master of Science
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ABSTRACT
The escalating cost of fertilizer N manufacture and concern over soil erosion has renewed
interests in legumes and their role in cropping systems. Winter legume cover crops may provide
significant quantities of fixed N while conserving soil and water resources and sustaining or
improving soil productivity. Poor N content of sandy soils has prompted research into the use of
winter legume cover crops in vineyards. The aim of this investigation was to evaluate different
legume cover crops for their ability to enhance the N nutrition of the vine on a sandy soil. The
study site is located in Lutzville, in the Olifants River region. The study was conducted on a six-
year old Sauvignon blanc vineyard grafted onto Ramsey rootstock. The following legume cover
crops were used: Vicia villosa v. dasycarpa, Medicago truncatula v. Parabinga, Medicago
truncatula v. Paraggio, Ornithopus sativa v. Emena. The cereals used in this study were: Secale
cereale and Avena sativa v. Saia. All treatments were replicated three times in a randomized
block design. A control in which weeds were mechanically controlled in the working row was
included.
The uptake and assimilation of nitrogen by grapevines were monitored monthly in a legume-
based cover cropping system over two seasons during 1997-1999. An in vivo nitrate reductase
assay was conducted on fresh vine leaves during the growing season as it is an indicator of
newly absorbed nitrogen. The uptake of biologically fixed nitrogen was measured by determining
the natural abundance of 15N in vine leaves. The uptake and assimilation of biologically fixed N
coincided with the annual demand for nitrogen by the vine, which occurs from budbreak to the
first stage of berry development and after harvest. Nitrate reductase activity in vines intercropped
with legumes was relatively higher than the cereal and control counterparts, despite the reduction
of nitrogen fertilization in these treatments. The omission of a green cover in the control plot had
significantly lower leaf nitrate reductase activity, emphasizing the importance of cover crops in
viticulture. The proportion of fixed N uptake to fertilizer N uptake gradually increased in the vine
with time as 61SN values of vine leaves were lower in th second season. Results from soil
analysis revealed that the soil nitrogen (total and inorganic) was low and differences in soil
nitrogen levels are expected to be more distinct in the longer term. The N content and biomass of
legumes were determined to evaluate various legume cover crop species. Vicia villosa v.
dasycarpa had the highest above-ground N. &15N values differed according to the varios legume
plant components and nodules were found to be markedly positive. The lower rainfall in the latter
season resulted in a decline in legume biomass production. &15N-values of legumes were lower
in the second season indicating that nitrogen fixation proceeded even more successfully
regardless of climatic conditions, proving the long-term benefits of using cover crops. In general
showed the greatest potential for improving the N status of the vine.
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CONTENTS
1 GENERAL INTRODUCTION 1
1.1 Nitrogen dynamics in agricultural soils 2
1.1.1 Influence of soil type 4
1.2 The assimilation of nitrogen in higher plants 4
1.2.1 Regulation of nitrate reductase activity 6
1.3 Nitrogen nutrition of vines 6
1.4 The role of cover cropping systems in sustainable agriculture 8
1.5 The use of legumes as cover crops 10
1.6 Management of cover cropping systems 14
1.7 References 16
2 GENERAL MATERIALS AND METHODS 23
2.1 Experimental design and fertilizer regimes 23
2.1.1 Nitrogen fertilizer regimes during 199-1997 23
2.2 Plant analysis 26
2.2.1 In vivo nitrate reductase assay 26
2.2.2 15N natural abundance 27
2.3 Soil Analysis 28
2.3.1 Inorganic nitrogen 28
2.3.2 Total soil nitrogen 29
2.3.3 Stastical analysis 29
2.4 References 30
3 VINE UPTAKE AND ASSIMILATION OF NITROGEN IN A COVER
CROPPING SYSTEM IN LUTZVILLE 31
3.1 Introduction 31
3.2 Materials and Methods 33
3.2.1 In vivo nitrate reductase activity of vine leaves 33
3.2.2 Determination of &15N natural abundance of plant material 33
3.3 Results and Discussion 34
3.3.1 Nitrate Reductase Activity 34
3.3.2 &15N natural abundance of vine plant material 35
3.4 Conclusion 48
3.5 References 49
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4 THE INFLUENCE OF COVER CROPPING MANAGEMENT PRACTICES
ON THE SOIL NITROGEN STATUS OF A VINEYARD 51
4.1 Introduction 51
4.2 Materials and Methods 53
4.2.1 Soil analysis 53
4.2.1.1 Inorganic nitrogen 53
4.2.1.2 Total soil nitrogen 53
4.3 Results and Discussion 54
4.4 Conclusion . 72
4.5 References 73
5 THE CONTRIBUTION OF BIOLOGICALLY FIXED NITROGEN BY
LEGUMINOUSCOVER CROPS TO THE NITROGEN STATUS OF A
SAUVIGNON BLANC VINEYARD IN THE OLIFANTS RIVER REGION 75
5.1 Introduction 75
5.2 Materials and Methods 76
5.3 Results and Discussion 78
5.3.1 Biomass production 78
5.3.2 &15N signature of legume components harvested in the field 78
5.3.3 Legume N content 79
5.4 Conclusion 87
5.5 References 88
6 SUMMARY AND CONCLUSIONS 91
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CHAPTER 1
GENERAL INTRODUCTION
At present the terrestrial input of nitrogen from biological nitrogen fixation (BNF) is estimated to
be in the range of 139 to 170 x 106 ton N per year (Marschner, 1997b). The increase in both
the costs of fossil energy and worldwide demand for nitrogen fertilizer used for food production
are major reasons for renewed interest in BNF as an alternative or at least as a supplement to
the use of chemical nitrogen fertilizer. Legumes were an important component of crop rotations
before World War II. In the post-war years, however, inexpensive and abundant fertilizer N
diminished the role of N-fixing plants in cropping systems (Hoyt and Hargrove, 1986). The
escalating cost of fertilizer N manufacture and concern over soil erosion has renewed interests
in legumes and their role in cropping systems. Winter legume cover crops may provide
significant quantities of fixed N while conserving soil and water resources and sustaining or
improving soil productivity.
Poor N content of sandy soils has prompted research into the use of winter legume cover crops
in vineyards. The aim of this investigation was to evaluate different legume cover crops for
their ability to enhance the N nutrition of the vine on a sandy soil. The study site is located in
Lutzville, in the Olifants River region (Fig 1.1). The study was conducted on a six-year old
Sauvignon blanc vineyard grafted onto Ramsey rootstock. Lutzville is a winter rainfall semi-arid
region. This is a class V climatic region (Winkler, 1962) at 31°36' latitude. Annual precipitation
is less than 200mm (Conradie and Myburgh (unpublished), 1999) of which only 40mm falls
during the growing season, resulting in an irrigation requirement of 675mm for this period (Van
Zyl, 1981). The soil is deep, red, calcareous (Hutton: Maitengwe, according to MacVicar & Soil
Survey Staff, 1977), representative of the so-called 'Karoo-soils' of this area. This study used a
subset of treatments from a larger study designed to measure the effects of several cover crop
management practices on the chemical and physical properties of the soil, including water
usage and vine performance.
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Rietpoort
Nuwerus
,Bitterfontein
Nieuwoudville
Citrusdal
Fig. 1.1: Location of study site
1.1 Nitrogen dynamics in agricuitural soils
Nitrogen entering the soil system is subjected to various chemical and biological
transformations (Table 1.1). Knowledge of N dynamics in soil is therefore essential for
obtaining high use efficiency of fertilizer-N. Results of studies using the stable isotope 15N have
shown that from 20 to 40% of the N applied as fertilizer remains behind in the soil in organic
forms after the first growing season. Only a small portion of this immobilized N (
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Nitrate (N03~) and ammonium (NH4+) are the major sources of inorganic nitrogen taken up by
the roots of higher plants. Levels of exchangeable NH4+ and NO3 vary from day to day and
from one season to another and depend on a variety of environmental factors mentioned
below:
• Seasonal variation: Levels of exchangeable NH4+ and NO 3 are greatly affected by
temperature and rainfall. The amounts found in the surface layer of soils of the temperate
humid climatic zone are lowest in winter because of leaching, rise in spring as
mineralization of organic matter commences, decrease in summer through consumption by
plants, and increase once again in the fall when plant growth ceases and crop residues
start to decay. The level in summer is higher than that in spring.
• Mineralization and immobilization: Biological turnover leads to the interchange of NH4+ and
NO3 with N of the organic matter. Accordingly mineral N levels represent a delicate
balance between mineralization and immobilization and are affected by the activities of soil
microorganisms and the C/N ratios of plant residues.
• Growing plants: Plants exert a depressing effect on mineral N levels in the soil. In addition
to direct uptake, NH4+ and NO3 levels may be altered by immobilization and denitrification
in the root zone, and possibly by inhibition of nitrification by root exudation products
(Harmsen and Kolenbrander, 1965);.
• Leaching of nitrate: Nitrate is the form of N that is most mobile and that is subject to
leaching and movement into water supplies. The magnitude of nitrate is difficult to estimate
and depends on a number of variables, including quantity of nitrate, amount and time of
rainfall, infiltration and percolation rates, evapotranspiration, water-holding capacity of the
soil and presence of growing plants. Leaching is generally greatest during cool seasons
when precipitation exceeds evaporation; downward movement in summer is restricted to
periods of heavy rainfall.
• Volatilization of NH3: Rapid changes in NH4+ can occur as a consequence of chemical
volatilization of NH3. Losses are greatest on saline soils, especially when NH4+-forming
fertilizers are used. Only slight losses occur in soils with pH values less than 7.0, but
losses increased markedly as the pH increases. For any given soil, losses increase when
neutral or alkaline soils containing NH4+ in the surface layer are dried out. The presence of
adequate moisture reduces volatilization, even from alkaline soils.
• Losses of nitrate through denitrification: Significant loss of NO3'-N can and does occur as a
consequence of denitrification. Under anaerobic conditions, such as occur frequently in
soils following a heavy rain, NO3' can be volatilized quantitatively in a comparatively short
time, particularly when energy is available in the form of organic residues.
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• Buildup of NH4 and NO3by fertilizer applications: In soils of humid and semi-humid
regions, any fertilizer N added in excess of plant or microbial needs will be lost through
leaching and/or denitrification. Thus mineral forms of N seldom carry over from one season
to the next. However, where leaching and denitrification are minimal, such as in soils of
arid and semi-arid regions, some carry-over occurs and repeated annual applications of N
fertilizer can lead to a buildup of NO3 in the soil profile.
1.1.1 Influence of soil type
On sandy soils relatively high proportions of fertilizer N may be assimilated by soil microbes
due to the high mass flow rates of nitrate to plant roots (Peschke et a/., 1984). The fertilizer N
thus immobilized in early summer may be mineralized in autumn and may later be leached by
winter rains and will therefore be lost by the system. This may be one reason why sandy soils
are generally poor in organic nitrogen.
In acid soils the mineralization of organic nitrogen is retarded or even blocked (Kuntze and
Bartels, 1979). Soil temperature and soil moisture influence N mineralization (Honeycutt,
1991). Kladiviko and Keeney (1987) found a six to seven fold higher N mineralization rate at
35°C than at 10°C. High mineralization rates are observed in summer and lowest in winter
(Weller, 1983). Dry periods in summer cause a drastic reduction in N mineralization.
Lochmann and co-workers (1989) reported that the net N immobilization (microbial assimilation)
was highest in spring while in summer net mineralization dominated. There is evidence that
soil texture has a strong impact on N mineralization. N mineralization in sandy soils is more
rapid than in soils with a higher clay content. Soils with a higher clay content are able to store
organic nitrogen in the form of adsorbed polypeptides and thus may add to the potential of
mineralizable soil N. In sandy soils in which the proteins of decomposing biomass are hardly
adsorbed, mineralization will occur in late summer followed by nitrate leaching in winter. There
is more organic N in no-till treatments than ploughed treatments. The input of proteins (green
manure) induces a flush of N mineralization within a few weeks, the input of organic C (straw)
an assimilation (immobilization) of inorganic nitrogen.
1.2 The assimilation of nitrogen in higher plants
As mentioned earlier, nitrate and, to a lesser extent, ammonium are the principal sources of
nitrogen that are available to higher plants under normal field conditions; hence the nitrate
assimilation pathway is the major point of entry of inorganic nitrogen into organic compounds.
Most of the ammonium has to be incorporated into organic compounds in the roots, whereas
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nitrate is readily mobile in the xylem and can also be stored in the vacuoles of roots, shoots and
storage organs (Marschner, 1997a). In order to be incorporated into organic structures and to
fulfil its essential functions as a plant nutrient, nitrate has to be reduced to ammonia.
The reduction of nitrate to ammonia is mediated by two enzymes: nitrate reductase and nitrite
reductase. Nitrate reductase (NR) is located in the cytoplasm and regulates the two-electron
reduction of nitrate to nitrite. Nitrite reductase (NiR) is situated in the chloroplast and
transforms nitrate to ammonia in a six electron reduction. Despite the spatial separation of NR
and NiR, nitrite rarely accumulates, in intact plants under normal conditions.
In higher plants NR is a complex enzyme containing two identical subunits (i.e. it exists as a
dimer). Each subunit can function separately in reduction of nitrate and contains 3 prosthetic
groups: flavine adenine dinucleotide (FAD), cytochrome 557 (cytj and a molybdenum cofactor
(MoCo).
In vines, a major part of nitrate is reduced in the leaves (Perez and Kliewer, 1978). The nitrate
content in the different plant organs evolves from nitrate absorption and nitrate transfer
(Mengel, 1986). The following factors determine the nitrate quantity in grapevines or in specific
tissue:
1. absorption by the roots
2. nitrate reductase activity of the roots
3. the transport rate to the next place of reduction
4. nitrate reductase activity in the leaves
5. production of the biomass in the vines and in the individual organs
The translocation of nitrate-N is through the xylem with a partial reduction of nitrate in the roots.
Nitrate is transported in the xylem from the roots, and thus their starting point for amino acid
biosynthesis is glutamine/glutamate (Ireland, 1990). Developing seeds or fruits, which are very
active in amino acid biosynthesis, will receive most of their nitrogen in the form of amino acids
supplied by the phloem. The age of the tissue also affects nitrogen flow: young leaves
consume all of the incoming nitrogen for growth, mature leaves re-export (in the phloem) much
of the nitrogen they receive to the growing apex or developing fruit, as do senescing leaves,
which also convert a lot of their proteins and other nitrogenous molecules to transport
compounds for export.
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Regulation of nitrate reductase activity
NR is regulated by several different modes exerted at different levels, namely: enzyme
synthesis and degradation, reversible inactivation and concentration of substrate and effectors.
Nitrate induces the de novo synthesis of the enzyme. The steady state level of nitrate
reductase is determined by the rate of degradation (turnover) as well as by the rate of
synthesis. A newly synthesized nitrate reductase protein has a half-life of only a few hours in
the cell. Thus when the nitrate is withdrawn, the level of nitrate rapidly decreases. The
enzyme has a half-life of only a few hours (Beevers and Hageman, 1983) and in plants
receiving no nitrate it is merely absent (Li and Gresshof, 1990). NR can be induced within a
few hours by addition of nitrate (Oaks et a/., 1972) and suppressed by certain amino acids
(Bretelerand Smit, 1974; Oaks, 1991).
NR activity in photosynthetic tissues generally varies on a diurnal basis, with peak rates during
the light period and lowest rates at the end of the dark period. Diurnal rhythm in nitrate
reduction may reflect fluctuations in carbohydrate level (Aslam and Huffaker, 1984) and in the
corresponding supply of reducing equivalents and carbon skeletons. However, besides these
coarse regulations various mechanisms of fine regulations exist, on the level of enzyme
modulation in carbon partitioning or direct modulation of the NR by enzyme phosphoryiation
(Kaiser and Spill, 1991). In the light-dark transition this activation of NR occurs within a few
minutes and thus accumulation of nitrite is prevented (Riens and Heldt, 1992). In light,
transpiration increases and nitrate enters the leaf in the xylem stream. Alternatively, light may
cause the release of nitrate from the vacuolar storage pools within the leaf. In photosynthetic
tissues in the light, the light reactions in the chloroplasts are probably the major source of
electrons to nitrate assimilation.
During the ontogenesis of an individual leaf, a typical pattern is observed in NR activity.
Maximum activity occurs when the rate of leaf expansion is maximal. Thereafter, the activity
declines rapidly. Thus in fully expanded leaves, NR activity is usually very low and often the
nitrate levels are correspondingly high (Santoro and Magalhaes, 1983).
1.3 Nitrogen nutrition of vines
Grapevines have a lower fertilization requirement than other horticultural crops (Christensen et
a/., 1978). This is due to the well-distributed root system of vines, facilitating the uptake of
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nutrients from a large soil volume, as well as a long growing season, which stretches over eight
months in a warm climate country like South Africa (Conradie and Saayman, 1989). Under
these climatic conditions, the grapevine utilizes about ca. 3.7kg N for the production of one ton
of fresh grapes, with 1.6kg being in the clusters and 2.1kg in the vegetative growth (Conradie,
1991).
In a study conducted in Germany in the winegrowing area of Rheingau, Lohnertz (1991)
demonstrated that fertilization with amounts ranging from 0 to 70 kgN/ha is sufficient for the
cultivar Riesling. From this study it was evident that increasing nitrate content in different soils,
does not lead to an increase in production. After several years of omitting N fertilization, the
number of bunches per vine were only slightly lower (this was not statistically). This could
indicate that even a visible nitrogen shortage in the variety Riesling has no influence on the
number of influorescences.
Problems created by excess N include excessive vigour, poor bud fruitfulness, excessive berry
drop, bud necrosis, delayed crop maturity, and increased levels of stem necrosis disorder,
bunch rot and leafhopper activity (Peacock et a/., 1991). Luxuriant growth and high N
fertilization are not compatible with quality production. On the other hand, N deficiencies lead
to a reduction in crop size as well as a low N concentration in the must, which can cause
problems such as lagging fermentation in the cellar and consequently inferior wine quality
(Saayman, 1981). It is therefore necessary to adjust the application rates of N fertilizers in order
to ensure optimum efficiency of plant uptake, while at the same time reducing losses of N to the
environment.
Nitrogen uptake of the grapevine from the soil begins after budburst. The internal cycle of N in
the vine makes the plant independent of the N supply of the soil solution, especially in the
period from budburst to unfolding leaves. It sees therefore necessary to omit all actions (tillage,
fertilization), which mobilize soluble nitrogen in the soil before budburst, to prevent leaching
losses. Table 1.2 describes the seasonal uptake of N in vines under South African conditions.
The peak annual demand for nitrogen in grapevines occurs from budbreak through the first
stage of berry development (Peacock et al., 1991). N uptake is greatest from budbreak to
veraison and from harvest to leaf-fall. N applications should be timed to maximize levels of
nitrate to the root zone during these periods. Vines depend heavily on stored forms of N to
support spring growth and post harvest fertilization is more effective in increasing stored N.
Fertilization may not be necessary when high levels of nitrate are present in the irrigation water
or when legume cover crops are grown.
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Table 1.2: Different phases in the N-nutrition cycle of the grapevine (taken from
Conradie, 1991).
Phase in I
nutrition cycle
II
III
IV
Growth Stage
Budbreak to end of bloom
1. End of bloom to end of
rapid shoot growth
2. End of rapid shoot
growth to veraison
Veraison to harvest
Harvest to start of leaf-fall
Period of berry
developmenta
II
III
Specific
characteristics for N-
uptake
New growth partially
dependent on reserve N
accumulated during
previous season(s)
Active root uptake.
Amount of "new" N
sufficient to supply
demand of new growth.
Leaves and bunches
both important sinks for
N.
Root uptake may stop.
Bunches main sink for
N. Redistribution from
roots, shoots and leaves
to bunches.
Active root uptake.
Redistribution from
shoots and leaves to
permanent structure.
According to Winkler et a/., (1974).
1.4 The role of cover cropping systems in sustainable agriculture
Cover crops play a multitude of roles in modern crop management systems (Doran and Smith,
1991):
1. They provide cover and protect the soil from wind and water erosion.
2. They serve as sinks for plant nutrients that might otherwise be lost by volatilization or
leaching.
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3. They provide weed control through competition and allelopathy.
4. They assist in control of disease and insects by increasing crop diversity.
5. They act as a source of supplemental N (legumes) and slow release nutrients.
Legume cover crops fix N symbiotically from the atmosphere and supply it to the succeeding
cover crop. Nonlegume cover crops, in contrast, are effective in reducing nitrate leaching from
the soil during winter months by absorbing large amounts of available N through their extensive
root systems. Cover crops, therefore, affect the cycling of N in the root zone and the N status of
the grapevine (Tan and Crabtree, 1990, Van Huyssteen et al., 1984). Mineralized N from
incorporated cover crop residues can then become available to the grapevine during the
summer months N and contribute to more efficient nitrogen cycling. Shipley and co-workers
(1992) reported a residual level of 94 kg N ha"1 in the soil profile (0-80cm) in autumn following
corn harvest in a cover cropping system. By April of the following spring, cereal rye had
recovered 45% of the residual fertilizer N while crimson clover recovered only 8% of the
fertilizer N, indicating the ability of rye to capture greater amounts of N compared to crimson
clover. Kuo et al. (1997) reported that rye and annual rye grass were ineffective in enhancing
soil inorganic N levels. However, they were more effective than the N-fixers hairy vetch,
Austrian winter pea and canoia in increasing soil organic N accumulation because of a higher
biomass potential and a larger input of biomass C. The two principal elements regulating soil
biological activity, and hence nutrient cycling, are carbon and nitrogen. Winter annual cover
crops can improve the soil C and carbohydrate concentrations due to the magnitude of the C
inputs from the respective cover crops. Large C:N ratios result in N mobilization and reduced N
availability to the succeeding crop. The decomposition rate at which cover crop residues are
incorporated into the soil affects soil organic N and C concentrations. Ladd et al. (1981)
showed that the value of legumes as a source of N was due not so much to their capacity to
provide relatively large amounts of immediately available N, but their ability to maintain or
increase soil organic N levels in the long-term, thereby insuring adequate supply of N by slow
decomposition of the stable organic N.
Cover crops can also provide a vegetative cover in erosion-prone areas in winter and improve
physical, chemical and biological properties of the soil (Hargrove, 1986). Louw and Bennie
(1991) showed that the presence of a straw mulch or cover crop was an effective way of
preventing crust formation runoff and erosion from vineyard soils. Cover crops are effective in
reducing weeds in strawberry, decreasing insect populations in orchards and vineyards and
curtailing pathogen severity in lettuce. Cover crop growth and C and N contributed by them
depends on species, length of the growing season, climate and soil conditions. The choice of a
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10
cover crop depends on whether the primary purpose is to supply N to the succeeding crop and
reduce fertilizer application, or to improve soil properties.
In a vineyard cover crop trial in Oudtshoorn it was observed that a mulched vineyard soil
conserved water in comparison with a bare soil due to limited evaporation losses (Van
Huyssteen et a/., 1984). Water extraction by weeds on the bare soil also contributed to water
loss. However, cover crops allowed to complete their growth cycle after budburst of the
vineyard wasted so much water - 75mm in 36 days - that this practice could not be
recommended in a dry area with an uncertain water supply. According to the results of this
experiment subtract 100kg N/ha when vetch is used as a cover crop and apply 50kg N/ha
additionally when Wimmera is being sown.
Water conservation in cover crop soils occur due to the following reasons:
1. Reduced evaporation due to a mulch effect
2. Increased infiltration and retention of precipitation
3. Loss of water by transpiration from the cover crop canopy
4. Altered water usage by the summer crop if its growth is affected
5. Mulch serves as barrier to water vapour movement
6. Reduction in solar radiation from shading
7. Insulation of the soil surface
Soil surface structure usually degrades in early spring due to prolonged wet conditions and a
lack of surface cover during winter. Caron et al. (1992) found that cover crops reduce seasonal
variation in aggregate stability when compared to bare fallow. Hermawan and Bomke (1997)
supported this conclusion and showed that improved aggregate stability with cover crops was
related to increasing organic carbon in the soil. Winter cover crops such as annual ryegrass,
may protect aggregate breakdown and surface soil structure during winter rainfall and resulted
in better structure after spring tillage operations when compared to the bare soil. When grown
over winter months, most studies, however, have only found significant benefits after several
years of cover crops (Kuo and Sainju, 1994).
1.5 Use of legumes as cover crops
Legumes normally produce organic matter higher in N content than grasses. As a result of this,
organic matter originating from legumes usually decomposes at a faster rate than that from
grasses. The greatest benefit from green manuring of a legume cover crop is obtained if the
crop is turned under at the stage of 50% flowering or before it produces seed. Grasses usually
-
produce more biomass than legumes under adequate fertility levels. Since grass herbage is
usually lower in N content than legumes, it decomposes more slowly. Crops planted soon after
large amounts of grass herbage have been incorporated into the soil may require additional
nitrogen fertilizer because some nitrogen will be required by soil organisms that are
decomposing the grass cover crop.
Legumes accumulate N from different sources: seed-N, soil-N, fertilizer-N and atmospheric-N.15N natural abundance methodology has been used to estimate N2 fixation by legumes. The N
released from roots is the main route of N-transfer; however, the presence or inoculation of
mycorrhizas (particularly VAM) may stimulate the transfer of N, probably through connection of
mycorrhiza hyphae between component crops (Frey and Schuepp, 1992).
The natural abundance of the stable isotope, 15N, in soils is commonly found to be greater than
in the N2 of the atmosphere (Cheng et al., 1964; Delwiche and Steyn, 1970; Shearer et a/.,
1974) and may increase with depth in the soil profile (Steele et al., 1981). In contrast, the
natural abundance of 15N in plants, which utilize biologically fixed atmospheric N2, is
significantly lower than in the N of the soil in which they grow. This difference has been used
for the detection of N2-fixing plants (Virginia et al., 1981) and for estimating the proportions of
plant N obtained from soil or atmosphere (Amarger et al., 1979).
Under conditions of near-optimal nitrogen supply, the allocation of carbon and nitrogen is
directed towards the shoot, whereas under conditions of extremely low nitrogen supply, it is
towards the root (Van der Werf et al 1993c). In general, fast-growing species produce more
biomass in the short run than slow-growing species, irrespective of the nitrogen supply
(Berendseefa/., 1992).
1.6 Management of cover cropping systems
The capacity of winter cover crops to serve as an effective source of nutrients depends on
climate, growth stage and quality of the cover crop, soil and cropping characteristics and tillage
management practices (Doran and Smith, 1991). With legume cover crops, the potential for
significant N contributions may not be realized unless the legume N becomes available during
the periods of high demand by the subsequent crop. Cover crop management alternatives
aimed at synchronizing N availability to crop demand are important to foster adequate N-use
efficiency relationships in cropping systems. If it is assumed that the C:N ratio of a cover crop
can govern decomposition and N release, then management techniques that modify this
characteristic may provide the flexibility to manage cover crop derived N in a more efficient
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12
manner. If mineralization of organic N does not correspond to plant N growth requirements,
yields will be depressed in the absence of other available N sources (Burket, 1997; Griffin and
Hesterman, 1991).
In the vineyards of the coastal region of the Western Cape, clean cultivation is generally
maintained, consisting of the following: In autumn a cover crop such as oats (Avena spp.),
barley (Hordeum spp.) or Rye (Lolium spp.) is sown in alternate rows. During June/July, a
moderately deep furrow or trench (150-300mm) is ploughed using a trenching plough in the
rows without a cover crop. Vine prunings and manure are ploughed under, and at the same
time the cover crop is disced in. The strip remaining underneath the vines is then ploughed,
while the surviving weeds are hoed by hand towards the middle of the row. Thus a clean hard
soil surface underneath and between the vines is obtained. The inter-row spaces are disced
two or four times during the growing season to keep the vineyard free from weeds. Depending
on the depth of the trenches, this system of cultivation is referred to as the deep (250-300mm)
or shallow (150-200mm) trench furrow system. In cases where a trenchfurrow is not ploughed,
all inter row spaces are sown to a cover crop which is disced into the soil before budburst after
which one to three additional cultivations may be necessary.
Due to the danger of leaching losses, N fertilization is usually applied in three installments -
during budding, after flowering and post-harvest (Saayman, 1982). If a legume cover crop is
grown during winter, about 25kg N/ha (the equivalent of 90kg LAN/ha) is fixed and this quantity
can be omitted from the spring fertilization. A non-leguminous cover crop, on the other hand,
requires about 30 kg N/ha (about 100 kg LAN/ha), half of which should be applied as
topdressing. Cover crops provide cover and protect the soil from wind and water erosion (Doran
and Smith, 1991). Cover cropping is therefore recommended on poor soils and in regions with
insufficient rainfall (Meyer and Cuinier, 1997).
During the growing season of a vineyard from the beginning of September to about the end of
March - growing weeds and/or cover crops compete with the vine for moisture and nutrients
(Fourie, 1988). Van Huyssteen and Weber (1980) showed that a vineyard under dryland
conditions must be free of weed competition during the growing season of the vines. These
weeds must be eliminated as competitors before budburst. Practices in which weeds and/or
cover crops are only controlled, but not killed, will result in a serious decrease in vegetative
growth and yield. Although winter weeds and cover crops in year of normal rainfall are not
regarded as deleterious to the dormant vineyard, the weeds must be destroyed before the
vineyard buds so as to eliminate harmful competition. Weeds that grow in summer are
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13
potentially the most harmful and could compete with the vineyard for nitrogen to such an extent
that fermentation problems could arise.
The following management practices are commonly employed in weed control programs:
• Mechanical control in the working rows and chemical control on the ridges
• Full surface chemical control
• Biological control by means of a cover crop that is destroyed by spraying a post-emergence
herbicide just before budding.
Grbic and Zorsic (1963) and also Nedelthcev (1965), compared yield and berry quality of vines
from differently cultivated soils. They found that when using herbicides, summer cultivation of
the vineyards can be reduced to absolute minimum or even completely omitted, with no
adverse effects on quality and yield. Quidet and co-workers (1967) could also not find any
differences in yield between mechanically clean cultivated and herbicide treated vineyards on
different soil types in France. These examples indicate that mechanically clean cultivation may
be redundant.
The N fixed by the nodules of legume cover crops is transported to the stems and leaves of the
growing legume to form proteins, chlorophyll and other N-conaining compounds. The fixed
nitrogen only becomes available to the next crop until the legume decomposes. Annual
legumes that are allowed to flower and mature will transport a large portion of their biomass
nitrogen into the seeds or beans. Once the legume has stopped actively growing, the N-fixing
symbiosis ends. In annual legumes this occurs at the time of flowering; no additional N gain will
occur after that point. Unless it is intended for a legume to reseed itself, it is a general practice
to kill the legume cover crop in the early- to mid-blossom stage. At this point, the maximum N
would have been obtained and residue composition can commence.
The management and climatic events following the death of the legume greatly affects the
amount and timing of N release from the legume to the soil. Bacteria immediately decompose
the cover crop. The result can be a very rapid and large release of nitrate into the soil within a
week of the green manure's demise. This N release is more rapid when covers are ploughed
down than when left on the surface. As much as 140 Ib. N/A has been measured 7-10 days
after plough down of hairy vetch (Sarrantonio and Scott, 1988). Green manures that are less
protein rich (N-rich) will take longer to release N. Those that are old and fibrous or woody are
generally left for hard-working but somewhat sluggish fungi to convert slowly to humus over the
years, gradually releasing small amounts of nutrients. Temperature, soil moisture, soil pH and
soil fertility affect microbial decomposers.
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14
1.6.1 Effect of tillage on soil N status
In agriculture, the purposes of tillage operations are to prepare seedbeds and rootbeds,
incorporate amendments, control weeds and pests, enhance infiltration, and control erosion
(Schafer and Johnson, 1982). Tillage leads to a rapid decline in soil organic nitrogen (Campbell
and Souster, 1982; Collins et al., 1992) and a decline occurs when a soil is left fallow
(Campbell and Souster, 1982; Elliot, 1986). Van Huyssteen and Weber (1980) showed that in
vineyards, soil parameters such as pore volume distribution, compaction indices, activity of
microorganisms and availability of plant nutrients, were found to be more favourable in
minimum tillage treatments than on conventionally tilled plots. Previous studies have shown
that higher levels of organic C, nitrate N and extractable Ca, Mg and K are observed in the soil
when crop residues remain on the surface (no-till) as compared with situations in which
residues are ploughed under (Lai, 1976). Fields with winter cover crops had higher soil organic
C, total N and exchangeable Ca and Mg than fields without cover crops (Lewis and Hunter,
1940; Wilson et al., 1982). They also found the inclusion of winter legumes and covers resulted
in higher soil pH, organic C, total N and exchangeable cations than those cropped to winter
grasses.
Tillage speeds up organic matter decomposition by exposing more surface area to oxygen,
warming and drying the soil and breaking residue into smaller pieces with more surfaces that
can be attacked by decomposers. The resulting loss of organic matter causes the breakdown
of soil aggregates and the poor structure often seen in overtilled soil. Frequent tillage can
result in the deterioration of the soil structure. However, when conducted in early spring, tillage
could increase surface evaporation (Slowifiska-Jurkiewicz, 1994). This reduces the water
content and improves soil aggregation. (Perfect et al., 1990). The effects of tillage on soil
aggregation may also be controlled by the presence of cover crops.
For many decades shoot prunings and winter cover crops were buried in vineyards as a
cultivation practice. In this process the soils have been regularly tilled in order to provide a
bare surface of pulverized soil (Van Huyssteen, 1987). Although meant to create favourable
surface soil conditions, research has showed that under South African conditions excessive
mechanical cultivation can damage pore continuity to the deeper layers. Conservation tillage
and cover crops are important management tools for improving short-term erosion control, and
increasing long-term soil N reserves and N mineralization and enhancing soil productivity
(Ebelhar et al., 1984; Frye et al., 1985; Wood ef al., 1991). However, a long-term study
conducted in southern Brazil (Amado et al., 1998) showed that the use of legumes in corn
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15
cropping systems increased N soil reserves, regardless of tillage system. Increased soil N from
legume cover crops induced increase in corn yield. It was evident from this study that
combined use of legume cover crops and no-tillage, by promoting increased soil N and crop N
uptake, is and efficient management practice to promote soil sustainability.
Conventional ploughing and aggressive disking can cause a rapid decomposition of green
manures, which could provide a premature release of N in the cropping season. No-till systems
will have a reduced and more gradual release of N, but some of that N may be vulnerable to
gaseous loss, either by ammonia volatilization or by denitrification. Studies have shown that,
regardless of the tillage system employed, decomposing legumes release a pulse of available
mineral N at two to five weeks after killing of the cover crop in Spring (with herbicide or tillage),
followed by a gradual decline over the growing season. Thus, depending on management, soil
and climatic conditions, N from legume cover crops may not be more efficiently used than N
from legume cover crops may not be more efficiently used than N from fertilizer.
Legume cover crops play an important role in sustainable agricultural practices. Its use can
lead to a potential reduction in N fertilizer costs for small-scale farmers. It is therefore the aim
of this study to determine the most effective cultivation practice for managing the legume cover
crop that can contribute the maximal amount of nitrogen at the most suitable time, i.e. the
release of nitrogen by the cover crop is synchronized with peak periods of nutrient demand by
the vine.
Chapter 3 assesses the effect of cover cropping on the nitrogen nutrition of the vine.
Chapter 4 examines the effect of cover cropping on the soil nitrogen status of the vineyard.
Chapter 5 determines the nitrogen contribution made by legume cover crops as measured by15N natural abundance methodologies.
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16
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Saayman D. 1981. Wingervoeding. In: Wingerdbou in Suid-Afrika (J. Burger and J. Deist, eds.), pp.
343-383. ARC Infruitec-Nietvoorbij, Stellenbosch, South Africa.
Saayman D. 1982. Fertilization of Wine Grapes. Farming in South Africa. Viticultural and Oenological
series, No. E.3/1982, 4pp.
Santoro LG, Magalhaes ACN. 1983. Changes in nitrate reductase activity during development of
soybean leaf. Zeitschrift fuer Pflanzenphysiologie 112, 113-121.
Schafer RL, Johnson CE. 1982. Changing soil condition - the soil dynamics of tillage. In: Predicting
Tillage Effects on Soil Physical Properties and Processes. ASA Special Publication No. 44,
(P.W. Unger and D.M. Van Doren Jr. eds.),pp. 13-28.
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Sarrantonio M, Scott TW. 1988. Tillage effects on availability of nitrogen to corn following a winter
green manure crop. Soil Science Society of America Journal 52, 1661-1668.
Shipley PR, Meisinger JJ, Decker AM. 1992. Conserving residual corn fertilizer nitrogen with winter
cover crops. Agronomy Journal 84, 869-876.
Shearer GB, Khol DH, Commoner B. 1974. The precision of determinations of nitrogen-15 in soils,
fertilizers and shelf chemicals. SoU Science 118, 308-316.
Slowifiska-Jurkiewicz A. 1994. Changes in the structure and physical properties of soil during spring
tillage operations. Soil Tillage Research 29, 397-407.
Steele KW, Wilson AT, Saunders WMH. 1981. Nitrogen isotope ratios in surface and sub-surface
horizons of New Zealand improved grassland soils. New Zealand Journal of Agricultural
Research 24, 167-170.
Tan S, Crabtree GD. 1990. Competition between perrennial ryegrass sod and 'Chardonnay" wine
grapes for mineral nutrients. Horticultural Science 25, 533-535.
Van der Werf A, Enserink T, Smit B, Booij R. 1993. Allocation of carbon and nitrogen as a function of
the internal nitrogen status of a plant: modeling allocation under non-steady-state conditions.
Plant and Soil 155/156, 183-186.
Van Huyssteen L. 1987. Benefits of minimum tillage of vineyards,. In Research highlights 1987: Plant
Production (L.L. Lotter ed.). Dept. of Agric & Water Supply, Pretoria, pp. 115-116.
Van Huyssteen L, Weber HW. 1980. The effect of conventional and minimum tillage practices on some
soil properties in a dryland vineyard. South African Journal of Enology and Viticulture 1, 35-45.
Van Huyssteen L, Van Zyl JL, Koen P. 1984. The effect of cover crop management on soil conditions
and weed control in a Colombar vineyard in Oudtshoorn. South African Journal of Enology and
Viticulture 5, 7-17.
Vance CP, Griffith S.M. 1990. The molecular biology of N metabolism. In: Plant Physiology,
Biochemistry and Molecular Biology, ed. D.T. Dennis and D.H. Turpin. Addison-Wesley
Longman, London, pp. 373-388.
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Virginia RA, Jarell WM, Kohl DH, Shearer G.B. 1981. Symbiotic nitrogen fixation in Prosopis
(Legumiosae)-dominated desert ecosystems. In: Curernt Perspectives in Nitrogen Fixation
(A.H. Gibson and W.E. Newton, Eds)p. 483. Australian Academy of Science Canberra.
Weller F. 1983. Stickstoffumsatz in einigen obstbaulich genutzten Boden Sudwestdeutschlands.
Zeitschrift fuer Pfianzenernaehrung und Bodenkunde 146, 261-270.
Wilson GF, Lai R, Okigbo BN. 1982. Effect of cover crops on soil structure and on yield of subsequent
arable crops grown under strip tillage on an eroded Alfisol. Soil Tillage Research. 2, 233-250.
WinklerAJ, Cook JA, Kliewer WM, Lider LA. 1974. General Viticulture. 710pp. University of California
Press, Berkeley and Los Angeles.
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Chapter 2
GENERAL MATERIALS AND METHODS
The following techniques were used to achieve the objectives of this study:
(i) Quantification of the contribution of fixed nitrogen by legume cover crops:
• 15N natural abundance of field- and glasshouse-grown legumes and
reference crop
• Growth measurement of legume plant components
(ii) Determination of the effect of cover cropping on the nutritional status of the vine:
• In vivo nitrate reductase assay of leaf blades
• Total amino acid content of vines
• 15N natural abundance of vine leaves
(iii) Monitoring the effect of cereal and legume cover crops on the nitrogen levels of the soil:
• Soil inorganic nitrogen levels
• Total soil nitrogen determination by Kjeldahl method
2.1 Experimental design and fertilizer regimes
The study was conducted on a six-year old Sauvignon blanc vineyard grafted onto Ramsey
rootstock. The trial was situated at the ARC-Nietvoorbij experimental farm near Lutzville. Six
leguminous and cereal cover crops, with a total of fifteen treatments, were replicated three times
in a randomised block design (See Table 2.1). Vines were spaced one and a half metres apart
within rows, and three metres apart between rows. A control in which weeds were mechanically
controlled in the working row was included. Each treatment row consisted of 6-8 vines and was
surrounded by a buffer row intercropped with Secale cereale L. All sample collections were taken
from these 6-8 vines.
2.1.1. Nitrogen fertilizer regimes during 1997-1999
All non-legume treatments, including the control, received a topdressing of N fertilizer (Fig. 2.1).
Vines in all legume treatments received a berm application of fertilizer and soil samples were
always taken in the middle of the cover crop row where no N was applied. In 1997 all treatments
received 42 kg N/ha after harvest and after budbreak (near flowering), except the two grazing
vetch treatments which were mechanically tilled and chemically controlled at budburst including
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Seradella Emena which was chemically controlled before budburst received 21 kg N/ha after
harvest. In 1998 all treatments received 42 kg N/ha in April and 28 kg N/ha in May.
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Table 2.1 : Cultivation management on a vineyard-cover crop system in Lutzville
Cover Crops* Cultivation management of cover crops
Secale cereale L.
(Rye)
Chemically controlled before vine budburst
Allowed to die-back
Avena sativa v. Saia
(Oats)
Chemically controlled before vine budburst
Allowed to die-back
Mechanically tilled before vine budburst
Vicia villosa v. dasycarpa
(Grazing vetch)
Mechanically tilled before vine budburst
Chemically controlled before vine budburst
Allowed to die-back
Medicago truncatula v. Parabinga Chemically controlled before vine budburst
(Parabinga medic) Allowed to die-back
Medicago truncatula v. Paraggio
(Paraggio medic)
Chemically controlled before vine budburst
Allowed to die-back
Ornithopus sativa v. Emena
(Seradella Emena)
Chemically controlled before vine budburst
Allowed to die-back
Control - no cover crop Mechanically tilled in row, chemical control under vine
"Common names are indicated in parentheses
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vine
legume cover crop stand
cereal cover crop stand
fertilizer placement
Fig 2.1 : Diagrammatic representation of fertilizer placement in a cover cropping trial in
Lutzville
2.2 Plant analysis
2.2.1 In vivo Nitrate Reductase Assay
The nitrate reductase assay was conducted monthly during the growing season to assess the
assimilation of biologically fixed and synthetic nitrate by the vine. Approximately six mature vine
leaves from each treatment were collected, placed on ice and analyzed immediately. Each
replicate was sampled separately (due to the diurnal variation of nitrate reductase activity) and on
the same side of the vine. The leaves were chopped and the exact weight of 0.5-1.0g was
recorded. Each treatment was duplicated. The method as described by Hageman and Hucklesby
(1971) was followed and adapted for field conditions as the laboratory was situated 440km from
the trial site and stability of the nitrate reductase enzyme would therefore not be feasible. The
leaves were transferred to a McCartney bottle which contained 10ml of phosphate buffer (0.1 M
KH2PO4/K2HPO4 at pH 7.7, 0.1M KNO3 and 1% v/v isopropanol) and was wrapped in foil in order
to exclude light. The bottle was shaken well for 2-3 minutes and flushed with nitrogen gas for 1
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minute to displace oxygen in order to prevent deactivation of the enzyme. The mixture was
incubated for 30 minutes at room temperature. One ml of the extract, 2ml of 1% sulphaniiamide
in 1M HCI and 2ml 0.01% NED (napthyl-ethylene-diamine-dihydrochloride) was added to a
cuvette. The absorbance was measured at 540nm on a Spectronic 20 spectrophotometer.
Sodium nitrite concentrations of 0.01, 0.02, 0.04, 0.06 and 0.08 µmol were used as standards.
Activity of the enzyme was expressed as |imol/g fresh weight/hr.
2.2.2 15N Natural abundance
All 15N methods used for field studies depend upon the sources of nitrogen for plant growth, viz.
soil nitrogen, fertilizer nitrogen and atmospheric N2 being of different isotopic composition
(Bergersen and Turner, 1983). The determination of 15N required the use of a mass
spectrometer, which separates ions in a highly evacuated atmosphere on the basis of their
mass/charge ratios and determines their relative proportions. This reaction is done in an
evacuated container and the N2 produced is introduced into the mass spectrometer. Double-
collector instruments, which have high precision, are used when studying variations in natural 15N
abundance.
All plant material prepared for isotope ratio analysis was oven-dried to constant weight at 75°C
and milled to a fine uniform powder. A subsample was weighed (1mg for legume material and
2.5mg for non-legume material) and transferred to tin capsules, whereafter each sample was
analysed on-line in a NA 1500 NC (CHN analyser) connected to a Conflo device MAT 252.
Vines: Vine leaf material was collected from each treatment monthly during the growing season of
the vine to obtain evidence for the transfer of legume-fixed N2 to vines.
Legumes: Nitrogen fixation by legume cover crops was studied by using the natural abundance of15N. Legume cover crop material (roots, shoots and nodules) was sampled annually from the field
during winter to quantify the contribution of legumes to the N status of the vineyard. Four
replicates of the four cover crop species were grown in the glass-house. The plants were
watered with distilled water thrice a week prior to germination. Thereafter the plants were fed
300ml of half-strength Hoagland's N-free nutrient solution thrice a week and received a flushing of
distilled water after every tenth feeding. All potted legumes were harvested prior to flowering and
divided into its various components (i.e. roots, shoots and nodules). All plant parts were oven-
dried, weighed to determine biomass production and ground for 15N determination.
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Weeds: In order to calculate the P-value it was necessary to identify suitable reference plants and
to determine the &15N-values of glass-house grown legume cover crops. Weeds growing in and
around the vineyard and which were present during cover crop growth were identified and
evaluated for use as a reference plant. Dimorphotheca pluvialis, Senecio arenarius and
Raphanus raphanistrum were the predominant weeds growing with the cover crops. The above-
mentioned weeds were also collected during August 1998. Assumptions inherent in 15N methods
are that: (i) the reference plant lacks the ability to fix N2 and the 15N/14N ratio measured in its
products of growth is the same as plant-available soil mineral N and (ii) the legume and non-N2-
fixing reference plant explore a soil N pool of identical 15N/14N abundance (Peoples and Herridge,
1990)
All 15N data was expressed in terms of parts per thousand (515N or %o):
&15N %o = 1000 x (atom% 15Nsample - atom% 15Nstandard) / atom%
15Nstandard,
where the standard is usually atmospheric N2 (0.3663 atom%). The proportion (P) of plant N
derived from N2fixation was calculated using the expression (Bergersen et al., 1985):
P = (&15 N ref. - &15N Ieg.)/(&15N ref. - B)
in which it is assumed that the 15N composition of the non-fixing reference plant (ref.) integrates
the isotopic composition of plant-available soil N and that the 15N composition of the legume (leg.)
is due to N assimilated from the same soil source plus the proportion derived from atmospheric
N2. The value B is the &15N of the plants grown in glasshouse culture with atmospheric N2 as the
sole source of nitrogen.
2.3 Soil analysis
Soil samples were collected monthly throughout 1997-1999 from the middle of the cover crop row
at each fourth vine at depths 0-300mm, 300-600mm and 600-900mm. All soil samples were air-
dried, ground and sieved prior to analysis.
2.3.1 Inorganic nitrogen
Exchangeable NH4+ and NO3 were extracted from soil by adding 60ml 2N KCI to 10g of soil. The
mixture was shaken for 1 hour and then filtered. The inorganic N content of the KCI extract was
measured on an autoanalyser. The filtrate was analyzed for NH4+ by the indophenol blue
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reaction. The indophenol blue method of determining NH4+ depends on the fact that the phenolic
compound, salicylate, reacts with NH3 in the presence of an oxidising agent, in this case,
hypochlorite to form a coloured complex under alkaline pH conditions. The addition of sodium
nitroprusside as a catalyst in the reaction between salicylate and NH3 increased the sensitivity of
the method about 10-fold. The maximum sensitivity and accuracy of the indophenol blue method
is attained when spectrometric measurements of the intensity of the coloured complex are made
at 636 nm. NO3 in the extract was reduced to NO2 by passage through a column of copperized
Cd in an NH4CI matrix, and the resulting NO2" was quantified by a modified Griess-llosvay
method.
2.3.2 Total Soil N
The Kjeidahl procedure was employed for the determination of total nitrogen in the soil. Total
Kjeldahl N includes ammonium, amines, and other organic forms which can be converted to
ammonium forms during the digestion. This procedure does not measure all of the nitrates in the
tissue. The method of Bremner and Mulvaney (1982) was adhered to with a few minor
adjustments. The digestion was performed by heating 10g of soil with 30ml of concentrated H2S04
and selenium. Digestion of the sample promoted the oxidation of organic matter to NH4+-N. The
NH4+-N in the digest was determined by collecting the NH3 liberated by distillation of the digest
with 10N NaOH. The distillate was collected in 4% boric acid indicator solution and 0.1 M HCI.
The total N content was expressed in parts per million.
2.4 Statistical Analysis
Statistical analysis was performed by SAS and the differences between means were determined
by LSD at 0.05.
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30
REFERENCES
Bergersen FJ, Turner GL. 1983. An evaluation of 15N methods for estimating nitrogen fixation in
a subterranean clover - perennial ryegrass sward. Australian Journal of Agricultural Research
34,391-401.
Bergersen FJ, Turner GL, Gault RR, Chase DL. 1985. The natural abundance of 15N in an
irrigated soybean crop and its use for the calculation of nitrogen fixation. Australian Journal of
Agricultural Research 36,411-423.
Bremner JM, Mulvaney CS. Nitrogen - Total. In: Methods of Soil Analysis, Agro. Monogr. 9, pt.
2, Second Edition (A.L Page, ed.), pp. 595-624. American Society of Agronomy, Madison,
Wisconsin.
Hageman RH, Huclesby DP. 1971. Nitrate reductase from higher plants. Methods in Enzymology
23, 491-503.
Peoples MB, Herridge DF. 1990. Nitrogen fixation by legumes in tropical and subtropical
agriculture. Advances in Agronomy 44, 155-223
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31
CHAPTER 3
VINE UPTAKE AND ASSIMILATION OF NITROGEN IN A COVER CROPPING SYSTEM IN
LUTZVILLE
3.1 Introduction
In contrast with annual crops, grapevines have a relatively low nutritional requirement for nitrogen
(Saayman, 1982). This is attributed to:
(i) the small quantities of nitrogen that are naturally taken up by the vine;
(ii) the vine's well developed rooting system that can readily obtain its required nutrients,
even in poor soil;
(iii) a period of about 8 months during which nutrients can be assimilated;
(iv) the fact that approximately 90% of the annual growth is returned to the soil as leaves and
shoots and because 90% of that portion which is removed annually (the crop) consists of
carbon dioxide and water.
Conradie (1980) showed that the aerial growth of the vine requires ca. 3.7kg N for the production
of one ton of fresh grapes, with 1.6kg being in the clusters and 2.1kg in the vegetative growth.
Nitrogen uptake is greatest from budbreak to veraison and from harvest to leaf-fall. Nitrogen
fertilizer applications are therefore usually timed to maximize levels of nitrate in the root zone
during these periods. However, with the escalating cost of fertilizer, there is a need to examine
alternative sources of mineral nutrients for use in vineyards. A safe and beneficial alternative is
the use of legume cover crops.
Legume cover crops fix N symbiotically from the atmosphere and supply it to the succeeding
crop. Nonlegume cover crops, in contrast, are effective in reducing nitrate leaching from the soil
during winter months by absorbing large amounts of available N through their extensive rooting
systems. Cover crops, therefore, affect the cycling of N in the root zone and the N status of the
grapevine (Tan and Crabtree, 1990; Van Huyssteen et a/., 1984). Mineralized N from
incorporated cover crop residues can then become available to the grapevine during the summer
months and contribute to more efficient nitrogen cycling.
The N fixed by the nodules of legume cover crops is transported to the stems and leaves of the
growing legume to form proteins, chlorophyll and other N-containing compounds. The fixed
nitrogen only becomes available to the next crop until the legume decomposes. Annual legumes
that are allowed to flower and mature will transport a large portion of their biomass nitrogen into
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32
the seeds or beans. Once the legume has stopped actively growing, the N-fixing symbiosis ends.
In annual legumes this occurs at the time of flowering. Unless it is intended for a legume to
reseed itself, it is a general practice to kill the legume cover crop in the early- to mid-blossom
stage. At this point, the maximum N would have been obtained and residue decomposition can
commence.
The management and climatic events following the death of the legume greatly affects the
amount and timing of N release from the legume to the soil. With legume cover crops, the
potential for significant N contributions may not be realized unless the legume N becomes
available during the periods of high demand by the subsequent crop. Cover crop management
alternatives aimed at synchronizing N availability to crop demand are important to foster
adequate N-use efficiency relationships in cropping systems. If mineralization of organic N does
not correspond to plant N growth requirements, yields will be depressed in the absence of other
available N sources (Burket, 1997; Griffin and Hesterman, 1991).
Although the idea of mulch tillage is common amongst producers and consultants, there are
problems in selecting a suitable species. The aim of this investigation was to evaluate different
legume cover crops for their ability to enhance the N nutrition of the vine on a sandy soil and to
determine whether the release of nitrogen by the cover crop is synchronized with peak periods of
nutrient demand by the vine.
The real criterion of success or failure of a soil cultivation measure is not the change
accomplished in the soil per se but the response of the crop (Van Huyssteen and Weber, 1980).
The 15N natural abundance method has been used in this study to determine the fate of fixed
nitrogen from the soil and its consumption by the vine. There are two stable nitrogen isotopes of
nitrogen, 15N and 14N. Soil nitrogen frequently contains a slightly higher percentage of 15N than
does nitrogen in the atmosphere. The&15N signature of fixed N is distinctly different from that of
soil N (Unkovich, 1996), and can therefore be used to differentiate between soil N and biologically
fixed N in vine plants. In most biochemical reactions, through isotope discrimination, the lighter of
the two isotopes is favoured slightly over the heavier. Thus, during N2 fixation these two
phenomena result in the fixed nitrogen having a slightly lower 15N.
Seasonal fluctuations of nitrate reductase activity in grapevine leaves and roots correspond to
periods of maximal root growth (Hunter and Ruffner, 1997). The determination of leaf nitrate
reductase activity therefore provides valuable information of nitrate assimilation in vines and was
used as an indicato