AN ABSTRACT OF THE THESIS OF
Philip E. Shuler for the degree of Doctor of Philosophy
in Crop Science presented on July 19, 1991.
Title:
The Effect of Preplant Nitrogen Fertilization
and Soil Temperature on Biological Nitrogen
Fixation and Yield of Alfalfa (Medicago sativa
La._
Abstract approved:
Redacted for Privacy
David 5. Hannaway
The usefulness of preplant nitrogen (N) in establishing alfalfa in colder production areas has not been
well characterized.
This study was conducted to deter-
mine the effect of preplant N and soil temperature on
yield, percent N derived from biological nitrogen fixation (PBNF), and shoot N concentration in alfalfa (Medicago sativa L. cv. 'Vernema'.
Field experiments were
conducted in 1987 and 1988 at Powell Butte, OR, to determine the effect of five levels of preplant N (0, 10, 20,
40, 60 kg ha-1) on yield and shoot N concentration of alfalfa.
Growth chamber experiments were conducted from
1989 through 1991 to examine the effect of five levels of
preplant N (0, 10, 20, 40, 80 kg ha-1) and three day/ -
night soil temperatures (18/12°C, 24/16°C, 27/21°C) on
yield, PBNF, and shoot N concentration of alfalfa.
In field experiments, preplant N had no effect on
shoot N concentration in either year.
In 1987 there was
no effect of preplant N on dry matter yield.
Application
of 20-40 kg N ha-1 preplant N increased dry matter yield
in 1988.
In growth chamber experiments, the highest rate
of dry matter accumulation occurred at a soil temperature
of 24/16°C.
At 18/12°C and 24/16°C, 40 kg ha-1 preplant
N resulted in increased shoot and root dry matter yield.
At 18/12°C, 80 kg ha-1 preplant N increased PBNF 14%
relative to the zero N control.
There was no effect of
preplant N on PBNF in plants grown at 24/16°C and 27/The rates of shoot N accumulation were similar at
21°C.
18/12°C and 24/16°C, and were higher than at 27/21°C.
Shoot N concentration was not affected by preplant N
treatments.
The use of 20-40 kg ha-1 preplant N may result in
increased yield without decreasing PBNF when: 1) soil
temperature remains below 15°C for at least two weeks
after planting, and 2) soil nitrate level is less than 16
mg
kg-1.
Proper assessment of the use of preplant N in
alfalfa establishment requires a careful consideration of
both soil temperature and soil N availability.
The Effect of Preplant Nitrogen Fertilization
and Soil Temperature on Biological Nitrogen Fixation
and Yield of Alfalfa (Medicago sativa L.)
by
Philip E. Shuler
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed July 19, 1991
Commencement June 1992
APPROVED:
'Redacted for Privacy
Associate Professor of Crop Science
charge of major
_Redacted for Privacy
Head of Crop and Soil Science Department
Redacted
Dean of
Privacy
_
Date thesis is presented
July 19, 1991
Typed by Philip E. Shuler for
Philip E. Shuler
ACKNOWLEDGEMENTS
The process of earning a doctoral degree in science
is truly an exhaustive one.
To state that the demands
placed on the individual are severe is an understatement.
These stringent demands also have a significant impact on
family, friends, and professional colleagues over a
In this sense the degree program is not
number of years.
in any way a strictly individual effort. Therefore, at
the completion of such a rigorous journey, an expression
of genuine appreciation to my colleagues and friends is
very much in order.
I extend my sincere gratitude to the head of the
Crop and Soil Science Department, Dr. Sheldon Ladd and my
major professor, Dr. David Hannaway, for financial
support in the form of an assistantship.
Dr. Ladd's
concern and willingness to help demonstrated a richness
of character that should be imitated by every
Appreciation is also extended to the
members of my graduate committee: Dr. Dale Moss, Dr. Tim
administrator.
Righetti, Dr. Tom Grigsby, and Dr. Barry Schrumpf, all of
whom have had a unique and positive contribution to my
degree program. A special thanks also goes to the
friendly, ever helpful secretarial staff including Doris
Aponte, Sue Ferdig, Irene Lampert, and Jenelle Post.
Their sharing of clerical skills and personal
encouragement was an essential part of my educational
process.
Friends and family are a priceless treasure and have
provided much essential support during this arduous
quest.
They have given me the freedom to explore a
number of "roads less traveled" with patience and
encouragement.
The St. John family has been a constant
source of support throughout my education process.
Their
faithful encouragement and support for me through many
years have resulted in improved standards of scholarship
and conduct.
To my parents, Woodfin and Ethel Shuler, I
They have
owe an enormous debt of respect and honor.
helped me develop a drive for continuing education, a
concern for integrity in scholarship, and an ability to
persevere through the tough times in life. They have
been a model of hard work and service to all of their
children. The choice fruit of their labors will last us
a lifetime.
Finally, a humble, heartfelt thanks must surely go
to David and Kimberly Hannaway. In them I have seen a
timeless and beautiful principle brought to life: "Do
not forget to do good and to share, for with such
sacrifices God is well pleased".
TABLE OF CONTENTS
Page
I.
II.
INTRODUCTION
1
Objectives
4
REVIEW OF LITERATURE
Introduction
5
BNF in Alfalfa
6
The Effect of Combined Nitrogen on BNF
in Alfalfa
III.
5
10
The Use of Preplant N in Alfalfa
Production
14
The Effect of Temperature on Growth of
Alfalfa
19
The Effect of Temperature on BNF in
Alfalfa
21
Methods of Measuring Biological Nitrogen
Fixation
23
The Difference Method
25
THE EFFECT OF PREPLANT NITROGEN
FERTILIZATION AND SOIL TEMPERATURE ON
BIOLOGICAL NITROGEN FIXATION AND YIELD OF
ALFALFA (MEDICAGO SATIVA L.)
28
Abstract
28
Introduction
30
Materials and Methods
32
Field Experiments
32
Growth Chamber Experiments
35
Statistical Analysis
38
Results and Discussion
39
Field Experiments
39
Growth Chamber Experiments
45
Dry Matter Yield
45
PBNF and N Accumulation
52
TABLE OF CONTENTS (Cont.)
Page
IV.
V.
Conclusions
58
References
62
BIBLIOGRAPHY
66
APPENDIX
76
LIST OF TABLES
Page
Table
II1.1
111.2
111.3
111.4
111.5
111.6
111.7
111.8
111.9
111.10
Soil characteristics of field site at
Powell Butte, OR and washed river sand
used in growth chamber experiments
before treatments applied.
40
Shoot N concentration of alfalfa grown
at five levels of preplant N at Powell
Butte, OR in 1987 and 1988.
41
Dry matter yield of alfalfa grown at
five levels of preplant N at Powell
Butte, OR in 1987 and 1988.
42
Combined analysis of variance of three
day/night soil temperature regimes and
five levels of preplant N in growth
chamber experiments.
46
Shoot dry matter yield at harvest of
alfalfa grown in three soil temperatures
and five levels of preplant N in growth
chamber experiments.
47
Linear regression analysis of shoot dry
matter accumulation (SHDW) in response
to thermal time (GDIDO and preplant N in
growth chamber experiments.
48
Root dry matter yield at harvest of
alfalfa grown in three soil temperatures
and five levels of preplant N in growth
chamber experiments.
50
Percent N derived from biological
nitrogen fixation (PBNF) in alfalfa
grown in three soil temperatures and
five levels of preplant N in growth
chamber experiments.
53
Linear regression analysis of shoot N
accumulation (SHN) in reponse to thermal
time (GDDO and preplant N in growth
chamber experiments.
54
Shoot N concentration at harvest of
alfalfa grown in three soil temperatures
and five levels of preplant N in growth
chamber experiments.
55
LIST OF APPENDIX TABLES
Table
1.
2.
3.
4.
5.
6.
7.
8.
Page
Analysis of variance of dry matter yield
for 1987 and 1988 field experiments at
Powell Butte, OR.
76
Shoot and root dry matter accumulation
of alfalfa grown at a day/night soil
temperature regime of 18/12°C and five
levels of preplant N in growth chamber
experiments.
77
Shoot and root dry matter accumulation
of alfalfa grown at a day/night soil
temperature regime of 24/16°C and five
levels of preplant N in growth chamber
experiments.
78
Shoot and root dry matter accumulation
of alfalfa grown at a day/night soil
temperature regime of 27/21°C and five
levels of preplant N in growth chamber
experiments.
79
Shoot and root N concentration of
alfalfa grown at a day/night soil
temperature regime of 18/12°C and five
levels of preplant N in growth chamber
experiments.
80
Shoot and root N concentration of
alfalfa grown at a day/night soil
temperature regime of 24/16°C and five
levels of preplant N in growth chamber
experiments.
81
Shoot and root N concentration of
alfalfa grown at a day/night soil
temperature regime of 27/21°C and five
levels of preplant N in growth chamber
experiments.
82
Shoot and root N content of alfalfa
grown at a day/night soil temperature
regime of 18/12°C and five levels of
preplant N in growth chamber
experiments.
83
LIST OF APPENDIX TABLES (Cont.)
Table
9.
10.
Page
Shoot and root N content of alfalfa
grown at a day/night soil temperature
regime of 24/16°C and five levels of
preplant N in growth chamber
experiments.
84
Shoot and root N content of alfalfa
grown at a day/night soil temperature
regime of 27/21°C and five levels of
preplant N in growth chamber
experiments.
85
THE EFFECT OF PREPLANT NITROGEN FERTILIZATION
AND SOIL TEMPERATURE ON BIOLOGICAL NITROGEN FIXATION
AND YIELD OF ALFALFA (MEDICAGO SATIVA L.)
I. Introduction
Alfalfa is one of the most important forage crops
cultivated in the U.S. (Barnes et al., 1988).
In Oregon,
it is grown on about 400,000 acres with commercial sales
of over $60,000,000 and a farm-gate value of about $150,000,000 (Oregon Department of Agriculture, 1990).
Proper
management of alfalfa can be a formidable task given the
complexity of the interaction between alfalfa, its microsymbiont Rhizobium meliloti, and the environment.
The use of fertilizer N is an important and controversial issue in alfalfa production.
In a survey of 45
U.S. agronomy departments, Hojjati et al.
(1978) reported
that 17 recommended N in legume establishment, at rates of
22.4-67.3 kg ha-1.
Some alfalfa producers have used pre-
plant nitrogen fertilizer (N), also known as "starter N",
in the establishment of alfalfa stands.
Research results
have varied on the effects of preplant N on yield and
biological nitrogen fixation in alfalfa.
Some results show that starter N does not increase
yield in alfalfa (Eardly et al., 1988; Simson et al.,
1981) and that it reduces stands by increasing weed growth
2
(Peters and Stritzke, 1970).
In field studies, Eardly et
al. (1988) reported that the application of up to 90 kg
ha
- 1
preplant N did not result in significant dry matter
yield increase in the first harvest of the seeding year.
The studies showing no effect of preplant N were conducted
on soils with relatively high N supplying capacity.
Other reports show starter N increases yield in
alfalfa (Peters and Kelling, 1989; Kunelius, 1974; Ward
and Blaser, 1961).
Two factors common to the observation
of a positive response are low soil N and adequate weed
control.
The response in low N soils is explained by the
assertion that alfalfa experiences a period of "N-starvation" in seedling growth.
During this period, demand for
N exceeds the ability of the root to supply N from soil N
or BNF, particularly if BNF capacity has not developed
(MacDuff et al., 1989).
The application of mineral N to legumes affects BNF,
from initial infection to subsequent growth and activity
of nodules (Streeter, 1988).
In general, the relationship
between nodulation and mineral N, particularly nitrate, is
an antagonistic one.
Laboratory studies suggest that a
nitrate concentration in growth media greater than 5 mM
inhibits nodulation in legumes (Streeter, 1988).
The
nitrate concentration of the soil solution, which ranges
3
from 2-400 mg 1-1 in agricultural soils, is therefore
often high enough to inhibit nodulation (Barber, 1984).
Starter N, however, may at times stimulate development of BNF in alfalfa.
Small amounts of combined N
(below inhibitory levels) are required for maximum nodule
growth and BNF in legumes (Mahon and Child, 1979; Allos
and Bartholomew, 1959).
Fishbeck and Phillips (1981)
provide laboratory evidence for a period of N-deficient
growth in alfalfa.
During this period of "N- starvation ",
BNF is not able to meet the N needs of the developing
seedling.
Thus, application of fertilizer N may lead to
increases in BNF and yield in alfalfa in the establishment
year.
The conditions under which N applications will most
likely result in increased yield without adversely affecting BNF are not well defined.
Important factors in the
response appear to be the N supplying ability of soils and
soil temperature (Vance et al., 1988; Barta, 1978).
More
specific information on how these factors affect yield and
BNF is needed to provide a scientific basis for making
recommendations on using starter N in alfalfa establishment in Oregon.
The purpose of this research project was
to determine the effects of soil temperature and preplant
N fertilization on yield, shoot N concentration, and BNF
of 'Vernema' alfalfa in a low N soil.
4
Objectives
Field and growth chamber studies were conducted to
evaluate the effect of soil temperature and preplant N
fertilization on yield, shoot N concentration, and BNF in
alfalfa.
The objective of the field experiments was to
determine the effect of five levels of preplant N on shoot
dry matter production and shoot N concentration of alfalfa
in the establishment year.
The objective of the growth
chamber experiments was to determine the effect of five
levels of preplant N and three soil temperature regimes on
shoot dry matter accumulation, shoot N accumulation, and
the percent N derived from BNF (PBNF).
5
II.
REVIEW OF LITERATURE
Introduction
The relationship of plant growth to soil nitrogen (N)
content is one of the most critical factors to be considered when dealing with the concerns of agricultural development into the next century (Winteringham, 1984).
The
relationship of soil N with alfalfa (Medicago sativa L.)
production presents special challenges.
These challenges
arise primarily because of the ability of alfalfa to
assimilate atmospheric N2 via the process of biological
nitrogen fixation (BNF).
This potentially lessens the
need for fertilizer N applications both to alfalfa, and to
crops following in a rotation.
Significant progress has been made in understanding
the physiology of BNF (Heichel et al., 1989).
Less prog-
ress has occurred in applying this knowledge to the practice of food production (Dreyfus et al., 1988).
Critical
questions remain regarding the contribution of alfalfa to
the N economy of cropping systems through BNF.
A major
reason for this is the complexity of the interaction between alfalfa, its microsymbiont Rhizobium meliloti, and
the environment.
Soil N content, water availability,
temperature, pest levels, and management play key roles in
6
determining seasonal levels of BNF.
Knowledge of how
these factors impact N accumulation and rates of BNF in
alfalfa is incomplete.
This information is needed to
design management practices that will have a positive
impact on N accumulation, BNF, and dry matter production.
BNF in Alfalfa
Alfalfa has a symbiotic association with Rhizobium
meliloti, which provides a means to convert atmospheric N2
It fixes large amounts of atmospheric N2
to ammonia-N.
and derives a large portion of it's N from BNF (Barnes et
al., 1988a).
This biologically assimilated or "fixed" N
is incorporated into the alfalfa plant and contributes to
the high yield and quality of alfalfa.
The use of alfalfa
in animal production and cropping systems as a replacement
for fertilizer N has prompted much research into it's production and N2 fixing abilities.
Research to maximize BNF
in the legume/Rhizobium symbiosis has focused on three
major areas: 1) infection by an effective strain of Rhizo-
bium, 2) investment of N and other nutrients from the
germinating seed to promote nodule formation, and 3) inhibition of nodulation and fixation by soil and fertilizer N
(Atkins, 1986).
Research to select cultivars with higher
BNF has resulted in alfalfa populations with higher yield
7
and quality (Teuber et al., 1984).
Studies of the morphology, physiology, and interac-
tion between alfalfa and various Rhizobium strains show
that BNF in alfalfa is affected by coordinated responses
among many physiological and biochemical traits (Barnes et
al., 1984). The symbiotic association in alfalfa begins
with contact between roots and Rhizobium meliloti.
This
contact is assured when there are high native populations
of effective rhizobia present.
In soils that are acid or
that have low populations of effective bacteria, seed
inoculation is generally effective in insuring contact
between roots and rhizobia (West and Wedin, 1985; Munns,
1977). Numerous studies have demonstrated that the strain
of bacteria which colonizes the root significantly affects
the rate of BNF (Barnes et al., 1984; Burton, 1972).
Therefore, a careful matching of strain and cultivar is
necessary to achieve maximum BNF (Burton and Erdman, 1940;
Gibson, 1962).
In field conditions this is often achieved
by inoculating seed with a mixture of effective strains
(Burton, 1972).
Seasonal variation of BNF in field-grown alfalfa has
been the subject of relatively few reports (Heichel et al.
1984; Henson and Heichel, 1984; Eardly et al., 1988; Wivstad et al., 1987).
Estimates of BNF in alfalfa range
from 50-463 kg N2 ha-1 yr-1, with an average of about 200 kg
8
N2 ha-1 yr-1 (Vance et al., 1988).
Variation in these esti-
mates is due to plant and environmental factors, including
stand age (Heichel et al., 1984), genotype (Tan, 1981),
dormancy characteristics (Heichel et al., 1981), fertility
level (Collins and Duke, 1981), soil N content (Barnes et
al., 1984), and soil moisture (Heichel et al., 1983).
The
effect of soil N on BNF has received much attention since
it is necessary to consider this factor when selecting for
improved alfalfa populations (Teuber and Phillips, 1988).
Laboratory studies suggest that a nitrate concentration in
growth media greater than 5 mM inhibits nodulation in
legumes (Streeter, 1988).
The nitrate concentration of
the soil solution, which ranges from 2-400 mg 1-1 in agri-
cultural soils, is therefore often high enough to inhibit
nodulation (Barber, 1984; Barnes et al., 1984).
BNF contributes less to the N needs of alfalfa in the
beginning of the establishment year than in subsequent
harvests and years.
Heichel et al. (1981) examined BNF
during the seeding year using the 15N isotope dilution
method for BNF determination.
BNF, on a per plant basis,
increased about four-fold from the first to the third
harvest and then declined to almost nil as the plants approached dormancy.
The percent of plant N derived from
BNF ranged from a minimum of 25-30% at first and fourth
harvest to a maximum of 59-69% at second and third har-
9
vests.
In a later study, Heichel et al. (1984) examined
the inter-annual and intra-annual BNF in two cultivars in
The varia-
a four year alfalfa stand on a silt-loam soil.
tion among harvests in the first year was similar to that
observed in the earlier study (Heichel et al., 1981) with
a seasonal average of 59% of crop N derived from BNF (168
kg N ha-1).
In years three and four, BNF did not show
significant intra-annual variation.
This was largely due
to the increased level of BNF at the first harvest, in
contrast to the seeding year where it was significantly
lower at the first harvest than at later harvests.
The
seasonal average portion of crop N derived from BNF increased to 61% (167 kg N ha-1) in year three and 77% (198
kg N ha-1) in year four.
Wivstad et al.
(1987) also reported a lower portion
of N from BNF in the first harvest interval relative to
later harvests.
In a two year study in Sweden, seasonal
BNF in an established alfalfa stand was determined using
15
N and an in situ acetylene reduction technique.
The
proportion of N derived from BNF was 70% (242 kg N ha-1)
in year one and 80% (319 kg N ha-1) in year two.
Lower BNF in the period after establishment reflects
the time required for developing active nodules, which is
typically from two to four weeks (FAO, 1984).
The time
requirement for nodulation and the degree of nodulation
10
are greatly affected by the presence of combined N (nitrate-N or ammonium-N), particularly nitrate.
The level
of nitrate in the soil solution from mineralization is a
function of soil temperature and moisture (Greenwood,
1986).
Thus, soil temperature may interact with soil N
availability in regulating the nodulation of alfalfa
during establishment.
The Effect of Combined Nitrogen on BNF in Alfalfa
A large number of reports have addressed different
aspects of how combined N affects BNF in legumes (Streeter, 1988).
In soils, ammonium-N is converted relatively
quickly to nitrate-N by nitrification.
Thus, most re-
search has focused on the effect of nitrate on BNF in
legumes.
Nodulation of alfalfa is highly sensitive to nitrate.
Nitrate application appears to inhibit the infection
process in alfalfa by a decrease in root hair formation, a
decrease in the number of infection threads, and an increase in the number of aborted infection events (Truchet
and Dazzo, 1982; Munns, 1977).
The effect of mineral N on
infection is reflected in changes in nodule number.
In
general, as nitrate concentration in the surrounding media
11
exceeds about 5 mM, the number of nodules per plant decreases in many legumes (Dazzo and Brill, 1978).
In
alfalfa, there was a decline in nodule number at about 11.5 mM nitrate-N relative to a nil-N control (Heichel and
Vance, 1979; MacDowall, 1982).
Most soils have at least 2
mM nitrate in the soil solution (Barber, 1984).
This
suggests most soils contain enough nitrate-N to inhibit
nodulation in alfalfa.
However, most studies to examine
the effects of nitrate-N on nodulation have been conducted
in artificial media and at optimal growth temperatures
(20-25°C).
These results may not be directly applicable
to field conditions where temperature, moisture, and
nitrate levels are more variable.
Other studies show low concentrations of N may increase nodulation of alfalfa (MacDowall, 1982; Fishbeck
and Phillips, 1981; Richardson et al., 1957).
The addi-
tion of 0.3 mM nitrate-N to a vermiculite-gravel mix
resulted in greater nodule number and weight than at nilN.
Alfalfa grown at 2 mM N (as ammonium nitrate) had
greater nodulation than a nil-N control (Fishbeck and
Phillips, 1981).
In a study with three alfalfa varieties,
two of three varieties grown in a 0.03 mM nitrate-N solution had more nodules/plant than nil-N controls (Richardson et al., 1957).
Differences in the effects of low levels of N on
12
nodulation likely reflect differences in rooting media,
form of N, and time of application.
Roots grown in solu-
tion culture may grow and respond differently than roots
developing in a solid media (Greenwood, 1986).
Nitrate in
solution culture may be more readily available for uptake
than in a solid media such as vermiculite or soil.
This
suggests that alfalfa grown in solution culture would be
sensitive to N inhibition of nodulation at a lower concentration than in soil (Heichel et al. 1981; Fishbeck and
Phillips, 1981).
Effects of N on nodulation depend on the form of N
applied.
Alfalfa growing in sterile sand culture supple-
mented with an N solution containing 0.75 mM ammonium-N
resulted in more nodules/plant than with nil-N or nitrateN of a comparable concentration (Richarson et al., 1957).
However, the conversion of ammonium to nitrate by nitrification, which proceeds relatively rapidly in soils, does
not occur in sterile conditions.
Thus, the effects of
nitrate in laboratory studies may be more applicable to
field conditions.
The timing of N application also affects results.
A
stimulative effect of N on nodulation was observed when N
treatments were applied to inoculated, rooted cuttings
(Fishbeck and Phillips, 1981).
When N treatments are
applied at seeding, low concentrations of nitrate general-
13
ly inhibit nodulation (Heichel et al., 1981; Richardson et
al. 1957).
A further consideration in evaluating nitrate effects
on nodules is the indeterminate growth habit of alfalfa
nodules.
Alfalfa nodules can regrow, even after exposure
to nitrate, once the nitrate concentration is lowered
(Becana and Sprent, 1987; Vincent, 1977).
This provides
one explanation as to why established alfalfa has a greater tolerance to higher nitrate levels.
Differing experi-
mental conditions indicate that caution must be used when
extrapolating laboratory results to the behavior of alfalfa in the field.
In contrast to the infection response, the response
of nodule mass per plant is often initially positive.
Low
concentrations of nitrate N (< 5 mM) in nutrient solutions
result in an increase in nodule mass per plant, plant
growth, and N content (Rawsthorne et al., 1985; Das,
1982). In fact, there is substantial evidence that some
combined N is required for maximum growth and nodule
formation of legumes (Rawsthorne et al., 1985; Eaglesham
et al., 1983; Das, 1982).
In certain circumstances,
legumes that have received low amounts of combined N as
nitrate may fix more N2 than plants receiving no additional nitrate (Eaglesham et al., 1983; Gibson, 1974).
This
is explained by the need for small amounts of inorganic N
14
for the formation of functional nodules during early seedling growth.
Although some N is available from seed
reserves, this may not be adequate to meet plant N demands.
For example, Sprent and Raven (1985) estimated
that nearly one-third of the seed N reserves in Phaseolus
vulgaris may be required for nodule formation.
In addi-
tion, the development of functional nodules may require
significantly more N than an equivalent amount of functional roots.
Thus, it is possible that early seedling
growth of some legumes is N limited.
The Use of Preplant N in Alfalfa Production
Preplant N fertilization of alfalfa sometimes increases dry matter yield in the establishment year.
It
appears that the effect depends largely on soil N content.
Weed control and effective inoculation are also important
factors (Peters and Stritzke, 1970).
The rationale behind
the effect is that although nodulated alfalfa meets most
of it's N requirements from BNF, mineral N is required for
early growth and nodule development.
Studies have been
conducted to determine if additional N during this prenodulation phase increases growth and nodulation and leads
to increases in dry matter yield in the establishment
year.
15
Recommendations for the use of preplant N in alfalfa
are based on reports that low levels (<80 kg ha-1) of
preplant N increase yield of first-cut alfalfa (Peters and
Kelling, 1989; Peters and Stritzke, 1970; Ward and Blaser,
1961).
Results from a series of studies in Wisconsin
(Peters and Stritzke, 1970; Simson et al., 1981; Peters
and Kelling, 1987 and 1989) suggest that the N supplying
capacity of the soil, pH, and weed control are important
factors in determining the response of N to preplant N.
Peters and Stritzke (1970) attempted to minimize the
effect of weeds with an application of EPTC (ethyl N,Ndipropylthiocarbamate).
They planted three varieties and
applied 95 kg ha-1 preplant N as ammonium nitrate in each
of three establishment planting experiments.
determined at first bloom.
Yield was
In two out of three years, the
addition of N without EPTC significantly reduced the
density of the alfalfa stand, compared to plots treated
with EPTC with or without N.
The reductions were attrib-
uted to increased weed growth resulting from additional N,
and not to direct fertilizer injury.
Treatment with EPTC
and N increased average alfalfa yields in one of the three
years.
Averaged across varieties the increase in dry
matter was 300 kg ha-1, compared to EPTC with no N.
The
variety 'Vernal' showed the largest individual increase,
about 900 kg ha-1.
One reason given for the ambiguity of
16
the results is that while the EPTC did a reasonable job of
controlling grassy weeds, it may have injured alfalfa in
some plots.
The results suggest that if weeds can be
effectively controlled without injuring alfalfa, yield
increases may result.
Peters and Kelling (1987) applied from 10-40 lbs
acre
-1
-1
(11.2-44.8 kg ha -1
preplant N as either diammonium
sulfate or calcium nitrate.
They reported a first cut
yield response in one year out of two, but only at the
highest rate of diammonium sulfate.
A yield increase in
response to small amounts of N (22.4-44 kg
ha-1)
was sug-
gested when soils contain less than 15-20 tons acre-1
organic matter.
This would be equivalent to less than
0.1% total soil N, assuming that organic matter contains
5% N and that an acre-furrow-slice of soil weighs two
million pounds.
In a later study, Peters and Kelling (1989) examined
the premise that N effects may be greater under acid soil
conditions in low organic matter, light-textured soils.
At the Marshfield, WI location, low, medium, and high pH
treatments were 5.0, 6.2, and 6.9, respectively.
Preplant
N was applied as diammonium sulfate at 0, 20, 40, and 80
lbs N acre
acre
-1
-1
(0, 22.4, 44.8, 89.6 kg ha
(44.8 kg ha
-1
)
ammonium nitrate.
-1
)
and as 40 lbs N
Significant in-
creases in first cut yields were reported for each addi-
17
tional increment of N fertilizer.
Yield increases were
observed within all pH treatments; however, the greatest
proportional increase occurred in the low pH range.
In
the low pH soil, preplant application of 80 lbs N acre-1
(89.6 kg N had) more than doubled the yield, compared to
0 N.
Similarly, yield was increased by preplant applica-
tion of 40 lbs N acre-1 (44.8 kg ha-1) ammonium nitrate
within all pH treatments, with the largest proportional
increase in the low pH treatment.
As in other studies,
there was a greater percentage of weeds in the stand at
the higher rates of preplant N.
Alfalfa yields are not always increased by preplant N
fertilization (Eardly et al., 1985; Simson et al., 1981;
Washko and Price, 1970).
Applying N at the time of seed-
ing did not result in a consistent yield response in
alfalfa grown on a silt loam soil (Simson et al., 1981).
The lack of a response could have been due to the ability
of the soil to supply considerable amounts of mineralized
N for plant growth and nodule development.
Results of
studies on lower-organic matter soils support this concept
(Peters and Kelling, 1987).
An increase in alfalfa dry matter yield in response
to N may be an indication of poor nodulation or ineffective nodules (Lee and Smith, 1972).
In a three year field
experiment, Eardly et al. (1985) examined the effect of
18
preplant N on yield and nitrogen fixation of effectively
and ineffectively nodulated alfalfa in western Oregon.
Preplant N was applied as ammonium nitrate at 0, 45, 90,
134, 179, and 224 kg ha-1 to a silt loam soil (fine-silty,
mixed, mesic Aquultic Argixeroll) that contained an average of 8-12 mg kg
-1
of available soil N.
An increase in
dry matter yield and N concentration was observed only in
ineffectively nodulated plants. In both ineffectively and
effectively nodulated plants, increasing rates of N application resulted in a decrease in nodulation and BNF,
measured by acetylene reduction.
Similar results were
reported by McAuliffe et al. (1958), who established an
alfalfa-tall fescue mixture in a sandy loam soil containing about 1 lb acre-1 (1.1 kg ha-1) inorganic N and 0.8%
organic matter.
Immediately after seeding,
15
N labeled
diammonium sulfate was added at 0, 20, 40, and 80 lbs
acre
.1
N (0, 22.4, 44.8, 89.6 kg ha
-1
).
BNF in seedling
alfalfa, as measured by the 15N isotope dilution technique, decreased in proportion to increasing rates of N.
Dry matter yield of tops was not significantly increased
in either 6-week old or 10-week old seedlings.
The lack of response to preplant N sometimes observed
is often explained by differences in the N-supplying
ability of the soil, pH, extent and effectiveness of
nodulation, and weed control (Peters and Kelling, 1987;
19
Peters and Strizke, 1970).
Consideration of these factors
is essential in comparing results of field studies on the
effect of N on alfalfa yield.
The Effect of Temperature on Growth of Alfalfa
The rate of germination and growth of alfalfa are
affected by temperature.
How, and to what extent, these
processes are affected depends on the interaction of
temperature with the genetic makeup of the plant.
Under
field conditions, temperature effects may be confounded
with high light intensity and moisture stress.
Given the
relative difficulty of controlling these factors in the
field, many studies on the effects of temperature on
alfalfa growth and BNF have been conducted under greenhouse conditions or in growth chambers.
Alfalfa will germinate at temperatures as low as 2°C
and as high as 40°C (Stone et al., 1979; McElgunn, 1973).
The optimum range, assuming other factors are conducive to
germination, is 19-25°C (Fick et al., 1988).
Germination
is very sensitive to moisture stress, and temperature responses in the field can be greatly modified by soil
moisture status (Triplett and Tesar, 1960).
When adequate
water is available, the rate of germination is temperature
dependent.
The rate of germination increases until about
20
27°C and then decreases sharply at about 30°C.
Despite
differences in rate of germination, the percent germination after one week differs little between 5°C and 35°C
Thus, although warmer
(Townsend and McGinnies, 1972).
temperatures may promote earlier germination, with adequate time and water availability, a successful stand may
be established at temperatures higher or lower than the
optimal range.
Temperature also plays a key role in seedling development.
Growth of recently germinated alfalfa seedlings
is most rapid at 20°C to 30°C (Hesterman and Teuber,
1981).
There is a rapid decline in growth rate outside a
temperature range of 10-37°C (Harding and Sheehy, 1980).
Alfalfa development is hastened by increasing temperature
and photoperiod (Fick et al., 1988, Jensen et al., 1967;
Smith, 1970).
Faster early growth may result in higher
yields in first and second year alfalfa (Sibma and Spiertz, 1986).
The relationship of root growth to shoot growth is
also temperature dependent (Lie, 1974; Gist and Mott,
1957; Pearson and Hunt, 1972).
The ratio of the rate of
dry matter accumulation in the roots to shoots is over 50%
higher in alfalfa grown at 15/10°C than plants grown at
35/30°C (Pearson and Hunt, 1972).
In 45 day old alfalfa,
the root/shoot dry weight ratio declined from 0.59 at 16°C
21
to 0.36 at 32°C (Gist and Mott, 1957).
The root/shoot dry
matter ratio of alfalfa varies curvilinearly with respect
to time from emergence.
The exact curvilinear function
varies considerably across the temperature range of 15/10°C to 35/30°C.
As a result, in early growth the
root/shoot dry matter ratio at 15/10°C is smaller than
that at 35/30°C (Pearson and Hunt, 1972).
These results
demonstrate that root/shoot dry weight ratio is a function
of both growth temperature and growth stage.
The Effect of Temperature on BNF in Alfalfa
Temperature affects all aspects of legume infection,
nodule formation, and BNF, with root temperature being
more important than air temperature (Lie, 1974).
There is
a fairly broad temperature range over which these processes will occur, although each process may exhibit a different optimal temperature response (Roughley and Date,
1986). The legume/Rhizobium symbiosis can adapt to a wide
range of soil temperatures.
Nodulated legumes are ob-
served under very cold (Ek-Jander and Fahraeus, 1971) and
very warm (Tadmor et al., 1971) temperature regimes.
Mod-
ulation of temperate legumes may take place, albeit slowly, at temperatures as low as 7°C (Roughley, 1970).
Once
formed, nodules can fix N2 at temperatures as low as 2°C
22
(Day and Dart, 1971). Nevertheless, extremes in tempera-
ture generally inhibit all key aspects of BNF in legumes,
i.e. infection, nodule growth, and N2 fixing activity
(Sprent and Sprent, 1990; Dart et al., 1975).
Nodulation is delayed at cool temperatures.
At
temperatures below 15°C, alfalfa nodulation is inhibited
(Jones and Tisdale, 1921; Duke and Doehlert, 1981).
Maximum acetylene-reduction activity in alfalfa occurs
from 20-25°C (Waughman, 1977).
Nodules generally contain
less bacteroid tissue at low temperatures, with a reduction in efficiency of BNF (Lie, 1974).
In sub-clover, the
greatest amount of bacteroid tissue is formed at 11°C, but
the highest BNF efficiency per unit of bacteroid tissue
occurs at 19°C (Roughley, 1970).
Higher soil temperatures appear to reduce both nodulation and BNF.
In isolated Phaseolus roots, nodulation
was reduced by about 70% at 30°C relative to 25°C.
Howev-
er, a temperature conditioning effect was also observed.
Roots exposed to 25°C for at least 3 days after inoculation and then placed in 30°C nodulated as much as roots
held at a constant 25°C.
Nodulation was completely inhib-
ited at 12°C and 33°C (Barrios et al., 1963).
The impact of temperature stress is modified by the
timing of the stress.
A cultivar of pea (Pisium sativum
cv. Iran) fails to nodulate when exposed to a root temper-
23
ature of 18-20°C.
However, if exposed to a temperature of
25°C for 24 hours within the first 3 days after inoculation, nodulation occurs.
If the temperature is then
lowered to 20°C, nodule growth and BNF proceed normally
(Lie, 1971).
It appears that the first few days after
inoculation is a critical period during which a temperature greater than 20°C is required to initiate infection.
Thereafter growth and activity of nodules is less sensitive to temperature.
It may be that a similar relation-
ship between temperature and nodulation exists in alfalfa,
although apparently this has not been determined.
Methods of Measuring Biological Nitrogen Fixation
Measurement of BNF is controversial (Sprent and Sprent, 1990; Smith et al., 1987).
The difficulty is distin-
guishing the portion of the total plant N that was derived
from atmospheric N2, as opposed to soil or fertilizer derived N.
Most procedures used are modifications of three
general methods: 1) the acetylene reduction method, 2) the
15N isotope dilution technique (IDT), and 3) the difference method (DM).
The relatively simple, rapid, and inex-
pensive nature of the acetylene reduction assay prompted
an large increase in BNF research after it's introduction
(Sprent and Sprent, 1990).
A variety of modifications
24
have been developed in an effort to facilitate it's use
and improve the accuracy of the results obtained (Criswell
et al., 1976; Medereski and Streeter, 1977; Eardly et al.,
1985).
In the basic procedure, attached or detached roots
and/or nodules are exposed to an air-acetylene mixture and
incubated for a period of time ranging from a few minutes
to as long as one or more hours.
A subsample of the
mixture is injected into a gas chromatograph to determine
the quantity of acetylene reduced to ethylene by nitrogenase.
The amount of acetylene reduced is assumed to be
proportional to the rate at which N2 would be reduced to
NH3
The limitations of using detached root systems or
nodules have been widely discussed.
Intact plants exhibit
from two to five times higher rates of acetylene reduction
than detached systems (Mague and Burris, 1972).
In addi-
tion, major problems arise from the usual practice of
employing a closed assay system (Giller, 1987; Minchin et
al., 1986).
Studies with alfalfa, white clover, and peas
demonstrate that there is a large decline in respiration
rate in the presence of acetylene (Minchin et al., 1983;
Haystead, et al., 1980; Minchin et al., 1982).
Attempts
to resolve these difficulties have led to the development
of flow-though systems (Silvester et al., 1989), but
criticisms of the technique persist (Gordon et al., 1989).
25
There appears to be a consensus that BNF is more
accurately estimated with either the IDT or the DM (Heichel et al., 1989; Chalk, 1985; Witty, 1983), although
each method has limitations (Hauch and Weaver, 1986;
Witty, 1983).
the IDT.
15
The DM is simpler and less expensive than
N enriched fertilizer is expensive and the
isotopic analysis of the resulting samples may be costly
if large numbers of samples need to be analyzed.
It has
been reported, however, that the DM tends to underestimate
BNF and is less precise than the IDT (Henson and Heichel,
1984; Ruschel et al., 1979).
The Difference Method
The simplest and most economical way of determining
BNF in legumes is the DM.
This method has become more
attractive to researchers with the release of several nonN2-fixing legume control plants.
The alfalfa BNF research
group at University of Minnesota has registered and released four alfalfa germplasms selected specifically for
use as non-N2-fixing controls in alfalfa BNF research
(Barnes et al., 1988b).
Henson and Heichel (1984) com-
pared the agreement and precision of the 15N isotope dilu-
tion technique (IDT) with the DM for determining BNF in
alfalfa.
An ineffectively nodulating alfalfa strain was
26
used as the non-fixing control plant.
They concluded that
non-fixing alfalfa was an acceptable control for BNF measurement by either the IDT or DM, with the caution that
the DM probably underestimates BNF relative to the IDT.
Therefore, the use of the DM may serve as a less-expensive
substitute for the more expensive IDT, in cultivar evaluation and alfalfa management studies (Henson and Heichel,
1984; Heichel et al., 1989).
The primary assumption underlying the difference
method is that the N2-fixing plant and the non-N2-fixing
plant assimilate identical amounts of soil N and that
fertilizer N applied is also assimilated with equal efficiency (Chalk, 1985; Hauch and Weaver, 1986).
In theory,
the ideal non-N2-fixing plant would be one which is known
to resemble closely the growth and rooting pattern of the
N2-fixing plant.
For this reason a non-nodulating isoline
may be the most ideal non N2-fixing control plant (Heichel
et al., 1989).
According to Chalk (1985), 15N isotope
studies support this assumption.
The percent of the plant
N that is derived from the atmosphere (% Ndfa) may be
calculated from the following three equations in which DRM
refers to whole plant or shoot dry matter, fp refers to
the N2-fixing plant and nfp refers to the non-N2-fixing
plant:
(1)
N yield = DRM x % N
27
(2)
BNF = N yield (fp) - N yield (nfp)
(3)
% Ndfa = [BNF / N yield (fp)] x 100
It is preferable to use total DM (root + shoot dry
matter yield) in the calculation, however given the difficulty of recovering root systems in the field, herbage
samples are usually used to estimate N yield.
The DM
technique gives similar results in either low or high N
soils (Henson and Heichel, 1984).
However, if fertilizer
N is applied it is recommended that rates be kept low due
to the sensitivity of nodulation and BNF to soil nitrate
(Ruschel et al., 1979).
The relative simplicity and
economy of the DM may make measurement of BNF more affordable in future studies.
28
III.
THE EFFECT OF PREPLANT NITROGEN FERTILIZATION AND
SOIL TEMPERATURE ON BIOLOGICAL NITROGEN FIXATION
AND YIELD OF ALFALFA (Medicago sativa L.)
Abstract
The usefulness of preplant nitrogen (N) in establishing alfalfa under different environmental conditions has
not been well characterized.
This study was conducted to
determine whether preplant N applications to alfalfa
(Medicago sativa L. cv. 'Vernema') established in colder
production areas significantly affects yield, percent N
derived from biological nitrogen fixation (PBNF), and
shoot N concentration in first cut alfalfa.
Field experi-
ments were conducted at Powell Butte, OR, on an Ayres
sandy loam to determine the effect of preplant N on yield
and shoot N concentration of first cut alfalfa.
Growth
chamber experiments were conducted to examine the effect
of preplant N and soil temperature on yield, PBNF, and
shoot N concentration of first cut alfalfa.
In field
experiments, preplant N application had no effect on shoot
N concentration in either year.
In 1987 there was no
effect of preplant N on dry matter yield.
Application of
20-40 kg ha-1 preplant N resulted in increased dry matter
yields in 1988.
In growth chamber experiments, at 18/12°C
29
and 24/16°C, 40 kg ha-1 preplant N resulted in increased
shoot and root dry matter yield.
Increasing rates of
preplant N did not result in lower PBNF at any temperature.
Shoot N concentration was not affected by preplant
N treatments at any temperature.
Preplant N may increase
first cut yield under conditions of low soil temperature
(<15°C) and low soil N availability (<15 ppm nitrate)
without decreasing PBNF.
Assessment of both soil tempera-
ture and soil N availability is essential in determining
the potential for a yield response to preplant N.
30
Introduction
Alfalfa (Medicago sativa L.) is one of the most
important forage crops cultivated in the U.S. (Barnes et
al., 1988).
The use of nitrogen (N) in the establishment
of pure stands of alfalfa and other legumes is controverHojjati et al.
sial.
(1978) reported that 21 of 45 U.S.
universities recommended N in establishment of pure legume
stands.
Three states had no recommendations and two had
indefinite recommendations.
The state of Oregon does not
have a definite recommendation for the use of preplant N,
also known as "starter N", in alfalfa establishment.
Some results suggest that starter N does not result
in increased yield (Eardly et al., 1985; Simson et al.,
1981).
In addition, starter N increases weed growth and
may reduce stands (Ward and Blaser, 1961; Peters and
Stritzke, 1970).
In field studies, Eardly et al.
(1988)
reported that the application of up to 90 kg ha-1 preplant
N did not result in significant dry matter yield increase
in the first harvest of the seeding year.
Other reports
suggest that, if weeds are controlled, starter N may
increase yield in the establishment year (Peters and
Stritzke, 1970; Peters and Kelling, 1987; 1989).
N sup-
plying capacity of the soil, pH, inoculation, and weed
control are important factors in determining the response
31
of alfalfa to preplant N.
Differences in these factors
may explain the different responses to preplant N.
The application of combined N to legumes affects
infection, growth, and activity of legume nodules (StreetThe relationship between BNF and mineral N,
er, 1988).
particularly nitrate, is often antagonistic.
Some studies
indicate, however, that combined N is required for maximum
nodule growth and maximum BNF in legumes (Mahon and Child,
1979; Allos and Bartholomew, 1959).
The specific conditions under which N applications
will result in an increase in alfalfa yield are not well
defined.
The N-content of the soil and soil temperature
are important factors (Minchin and Sprent, 1983; Barta,
1978).
Specific information on how these factors affect
yield and BNF is needed. The objective of these studies
was to determine the effect of preplant N fertilization
and soil temperature on the yield, shoot N concentration,
and percent of plant N derived from BNF (PBNF) of first
cut alfalfa.
32
Materials and Methods
Field Experiments
Field experiments were conducted in 1987 and 1988 at
the Central Oregon Agricultural Experiment Station, Powell
Butte, OR, on an Ayres sandy loam (loamy, mixed, mesic,
shallow Xerollic Durargid), with 0-2% slope.
This site
was chosen because it is a low N soil and the soil temperature is cool (10-15°C) at planting.
Twenty pre-treatment
soil samples (five from each of four replicates) were
taken on March 19, 1987 and March 20, 1988.
These were
then bulked into four samples for analysis.
Soil NH4-N
and NO3-N were extracted with 2 M KC1.
NO3 and NH4
Concentration of
in extracts was determined by a colorimetric
procedure (Keeney and Nelson, 1982) in an Alpkem RFA 300
Autoanalyzer.
Soil temperatures at 10 cm and 20 cm depth
were obtained from records of the National Weather Service
weather station located at Redmond, OR, about 12 km from
the Powell Butte Experiment Station.
A randomized complete block design consisting of four
replications of five rates of NH4NO3 was used to provide N
levels of 0, 10, 20, 40, and 60 kg ha-1.
were 10 m X 2 m.
Individual plots
In 1987, 80 kg ha-1 P2O5 and 112 kg ha-1 S
were applied pre-plant.
In 1988, 154 kg ha-1 P2O5 and 100
33
kg ha-1 S were applied.
Shortly before planting, an aque-
ous solution of NH4NO3 was sprayed onto the plots using a
small sprayer fitted with a 1 m wide boom.
'Vernema' alfalfa (Round Butte Seed Growers, Culver,
OR), inoculated with a commerical peat based Rhizobium
inoculum (Nitragin Co., Milwaukee, WI), using a sucrose
solution sticker, was broadcast onto plots at 22 kg
on June 18, 1987 and 25 kg
ha-1
on May 27, 1988.
ha-1
Follow-
ing planting, the plots were irrigated and managed according to commercial practices for the area.
ous weed infestation occurred.
In 1987, seri-
On July 31 a mixture of
0.2 kg a.i. ha-1 Buctril and 0.6 kg a.i. ha-1 2,4-DB was
applied.
Thereafter the plots were hand-weeded for con-
trol of lambsquarter and redroot pigweed.
In 1988, an
unusually warm period in late July, combined with problems
with the irrigation system, resulted in significant water
stress on some plots, which hastened maturation.
There-
fore, the 1988 harvest occurred several weeks earlier than
normally expected.
Randomly selected shoot samples were taken three
times during each growing season.
In 1987 samples were
taken on July 16, August 13, and August 27, and in 1988 on
July 14, July 29, and August 16.
The samples were dried
at 70°C and ground in either a small spice grinder or a
Wiley Mill (Arthur H. Thomas, Philadephia, PA) to pass a
34
40 mesh screen.
Shoot N concentration was determined by a
modified Kjeldahl method (Bremner and Mulvaney, 1982).
Samples were digested using a CuSO4- Se -K2SO4 catalyst.
Total NH4
in the digest was determined by a colorimetric
procedure in an Alpkem RFA 300 Autoanalyzer (Alpkem Co.,
Portland, OR).
Plots were harvested at the early bloom stage, as
determined by visual inspection.
This occurred on Septem-
ber 10, 1987 and August 16, 1988.
A self-propelled small
plot forage harvester was used to cut a 6 m2 swath within
each plot.
A swing-arm mounted electronic balance enabled
whole plot fresh weight to be determined immediately after
cutting.
Subsamples of herbage were collected, sealed in
plastic bags, and removed to the experiment station where
they were weighed and then placed in a 70°C drying oven.
The dry samples were reweighed and the calculated percent
moisture used to determine the dry matter yield for each
plot.
35
Growth Chamber Experiments
Growth chamber experiments were conducted in 1990-91
in growth chambers located on the Oregon State University
campus, Corvallis, OR.
Washed river sand (Corvallis Land-
scape Supply, Corvallis, OR) was used to fill 10 cm diameter X 1 m long, capped PVC tubes.
Seven 0.3 cm diameter
holes were drilled in the end caps and covered with 2 cm
gravel, to allow drainage. The river sand was analyzed
m2
m-2
and 0.4 g
B as H3B03, 4 g in S as CaSO4, 6 g
P as
CaHPO4, and 12.5 g m-2 K as KC1 were added to assure proper
mineral nutrition.
A mix of incandescent and fluorescent
tubes provided an average PPFD of 350-450
s
.
mole photon
M-2
The photoperiod was 16h day.
A split-plot design was used for the growth chamber
experiments with three day/night temperature regimes (27/21°C, 24/16°C, and 18/12°C) as the main plots, and five
preplant N rates (0,
subplots.
1,
2,
4, and 8 g m-2 N as NH4NO3) as
Four replicates (two tubes per replicate) were
used to insure adequate material for sampling.
N treat-
ments were applied as 50 ml aqueous solutions.
After
applying N treatments, 100 mg per pot of certified 'Verne-
ma' alfalfa seeds were pressed into the soil surface and
covered with a thin layer of a peat based Rhizobium inoculum and soil.
36
Seeds of ineffectively nodulating 'Saranac' alfalfa
(courtesy of D.K. Barnes, USDA-ARS, Univ. of Minnesota,
St. Paul, MN) were planted in separate tubes as non -N2fixing controls.
Earlier experiments had demonstrated
that these plants would require additional N to grow in
the river sand.
Therefore, 80 kg ha-1 N was added to the
ineffective 'Saranac' pots before planting.
Four replicates of plant samples were collected by
removing 6-10 whole plants at approximately the early
vegetative, early bud, late bud, and early flowering
growth stages (final harvest) as determined by the mean
stage by count (MSC) method (Fick and Mueller, 1989).
Thermal time in growing degree days (GDD) was calculated
according to the equation
GDD = E ([Tum + Tmin / 2) - Tb)
where Tom is the daily maximum temperature, Trnib is the
minimum daily temperature, and Tb is the base temperature.
The most common Tb used for alfalfa is 5°C (Sharratt et
al., 1989) and was used for GDD calculations.
Whole plant
samples were collected by carefully digging out individual
plants with a small spoon and washing roots with water
periodically to loosen and clean them.
By digging down
around the roots about 12-16 cm and moving the root back
and forth, the large tap root could be removed largely
intact from the pot.
The weight of the large tap root is
37
assumed to account for most (>90%) of the total root
weight.
Roots and shoots were dried at 70°C.
The dry
samples were weighed and ground in either a small spice
grinder or a Wiley mill to pass a 40-mesh screen.
Total N
concentration was determined by a modified Kjeldahl N
procedure as described under field experiments.
Total N yield was calculated as the product of dry
weight and N concentration.
The percent of plant N de-
rived from the atmosphere (PBNF) was determined by the
difference method as described by Henson and Heichel
(1984) and Heichel et al. (1989), according to the equations:
(1) BNF = N yield (fp)
N yield (nfp)
(2) PBNF = [BNF / N yield (fp)] x 100
in which fp refers to the N2-fixing plant and nfp refers
to the non-N2-fixing plant.
This procedure assumes that
the N2-fixing plant and the non-N2-fixing crop assimilate
equal amounts of soil and fertilizer N and that the nonN2-fixing plant develops root and shoot systems similar to
the N2-fixing line.
In our experiments, the fixing and
non-fixing lines were not identical in growth habit.
Also, as noted above, additional fertilizer N was required
to maintain vigorous growth of the non-fixing 'Saranac'.
Therefore, the calculations of PBNF presented herein are
subject to error to the degree that the non-fixing line
38
departed from the pattern of N-uptake in the fixing line.
Thus, the data presented is to be used for treatment
comparisons rather than quantitatively.
Statistical Analysis
Data were analyzed using the SAS microcomputer software system (SAS, 1990).
In the field experiments a one
way analysis of variance (ANOVA) for combined years data
showed significant differences between years with respect
to shoot N, but not for dry matter yield at harvest.
Therefore an ANOVA for shoot N was conducted separately
for years.
Significance of differences between treatment
means were computed by Duncan's multiple range test (SAS,
1990).
In the growth chamber experiments the PROC ANOVA
and PROC GLM procedures were used to examine differences
between temperature treatments, N treatments and temperature X N interaction effects with respect to shoot and
root dry weight at harvest, shoot N concentration, and
PBNF.
The PROC STEPWISE and PROC REG procedures were used
to determine the best fitting linear regression relating
shoot dry matter and N accumulation to cumulative thermal
time.
39
Results and Discussion
Field Experiments
The physical and chemical characteristics of the soil
at the Powell Butte experiment site are summarized in
Table III.1.
Relative to agricultural soils in the U.S.
the values for nitrate are low (Barber, 1984).
The aver-
age 10 cm soil temperatures during the 2 weeks following
planting was 18°C in 1987 and 10°C in 1988.
Shoot N concentration declined in both years as the
plants matured, probably reflecting dry matter dilution
(Table 111.2).
Shoot N concentration within sampling
dates was not affected by N treatments.
et al.
Similarly, Eardly
(1988) reported no increases in N concentration in
10 week old field grown as preplant N rates increased from
zero to 90 kg ha-1.
In an earlier experiment (Eardly et
al., 1985) shoot N concentration increased only at N rates
from 90-224 kg ha-1.
There were no differences in dry matter yield among N
treatments in 1987 (Table 111.3).
In 1988 the yield of
the highest preplant N treatment was significantly lower
than at 20 and 40 kg ha-1 N.
Visual inspection of the
field plots about one week before harvest revealed serious
water stress on a number of plots due to difficulties with
Table 111.1.
Soil characteristics of field site at Powell Butte, OR and
washed river sand used in growth chamber experiments before treatments applied.
Soil
pH
P
K
B
mg
/4144.
NO3
Mg
Ca
cmol kg
kg-1
-1
1987 Field t
6.6
14
261
0.4
4.2
5.3
10.1
4.0
1988 Field
6.6
17
288
0.5
2.4
17.0
10.8
4.4
River Sand t
6.7
22
238
0.3
2.2
6.1
6.9
3.4
t Mean of four samples taken to a depth of approximately 15 cm.
t Mean of six samples.
Table 111.2. Shoot N concentration of alfalfa grown at five levels of
preplant N at Powell Butte, OR in 1987 and 1988.
Preplant N
1987
1988
Days after planting
Days after planting
28
56
70
48
63
81
g N kg-
kg ha-1
41t
37
38
39
32
27
10
43
38
40
37
32
27
20
45
35
36
34
26
23
40
45
35
36
36
31
27
60
45
34
36
38
33
26
44a1
36b
36b
37a
30b
26c
0
Means
t Means were not significantly different within dates at P=0.05.
: Date means followed by different letters are significantly different
at the 0.01 level as determined by Duncan's Multiple Range Test.
42
Dry matter yield of alfalfa grown at five
Table 111.3.
levels of preplant N at Powell Butte, OR in 1987 and
1988.
Preplant N
1988
1987
Mg ha-1
kg ha-1
0
7.1
3.0
10
6.0
6.4
20
6.5
14.8
40
6.6
8.6
60
6.6
2.6
N.S.
4.3
LSD(0.05)
43
irrigation.
Because of the uneven slope of the plots, the
effects were not localized entirely in one part of the
field or in a distinct pattern that would facilitate
covariance analysis. Thus, it is possible that the low
yield at the highest N level reflects the effects of water
stress and not a negative effect of preplant N.
It seems
unlikely, though, that the large increases in dry matter
yield observed at 20-40 kg N ha-1 are an artifact of irrigation effects.
The range in dry matter yield of replicates at 20 kg N ha-1 was 11.6-18.1 Mg ha-1 compared to
0.7-12.9 Mg ha-1 for all other N treatments.
Therefore,
an increase in yield was obtained at 20 and 40 kg ha-1 N
relative to zero N.
The 1987 and 1988 experiments were conducted in adjacent fields and the soil nitrogen content was similar in
both years. Therefore, aside from the difficulties with
the irrigation system previously mentioned, the only measured factor that may explain the differences in yield
response between years is the difference in soil temperature during seedling development. The average 10 cm soil
temperature during the 2 weeks following planting was 18°C
in 1987 and 10°C in 1988. The lower soil temperature in
1988 was well below the optimal temperature for nodulation
of alfalfa (18°C) as reported by Jones and Tisdale (1921).
N available from mineralization would also be reduced
(Greenwood, 1986).
In 1988, the readily available fertilizer N may have provided additional N during a period when
mineralizable N and BNF could not meet the plant N demand.
Other researchers have proposed that an N deficit may
develop during the period of nodule development in alfalfa
when the N demand of the plant can not be met by either
BNF or soil N (Peters and Kelling, 1987; Fishbeck and
Phillips, 1981).
The deficit would be exacerbated by
44
soils with low N-supplying capacity (Peters and Kelling,
The increase in dry matter yield in 1988 implies
that the potential for this N deficit is increased when
1989).
soil temperatures are below optimum for BNF and soil N
mineralization.
An additional concern with the use of preplant N is
the promotion of weed growth. In both years of the field
study, the addition of preplant N increased weed growth
and necessitated herbicide application.
However, in 1988
weeds were controlled earlier and the weed infestation was
much smaller, which may have contributed to higher yields
at 20-40 kg ha-1. Peters and Strizke (1970) noted that
when weeds were controlled without injury to alfalfa, N
application resulted in yield increases in two out of
three cultivars.
Lee and Smith (1972) suggested that a yield response
of legumes to fertilizer N may simply reflect poor nodulation or ineffective nodules. This apparently assumes that
most of the plant N demand during early growth is provided
by BNF.
In field experiments with alfalfa, however,
Heichel et al.
(1981) observed that about 25-30% of plant
N was derived from BNF during the period preceding the
first harvest. Thus, even with effective nodulation,
considerable amounts of mineralized or fertilizer N is
required to meet the plant demand for N in the period
preceding first harvest. In the 1988 experiment, visual
inspection of plants demonstrated the presence of nodules
at all N levels within five weeks after planting. In
addition, neither shoot N concentration (Table 111.2) nor
plant color suggested N stress at any time during the
season.
Thus, the use of preplant N may result in in-
creased yield in low N soils when soil temperature is cool
and weeds are controlled.
45
Growth Chamber Experiments
Dry Matter Yield
Combined analysis of variance of the growth chamber
experiments indicated that temperature, N, and the temperature X N interaction had significant effects on shoot
dry matter yield (Table 111.4).
Dry weight declined
slightly as root temperature increased from 18/12°C to
24/16°C, and then decreased sharply at 27/21°C (Table
111.5), possibly as a consequence of increased respiration
relative to photosynthesis (Fick et al., 1988).
N treat-
ments within temperatures had the largest effect on dry
weight at 18/12°C.
The largest dry matter yield was
observed at 40 kg ha-1 in both the 18/12°C and 24/16°C
soil temperature treatments.
At 24/16°C and 27/21°C, the
yield of the control was equal to the yield at the highest
rate of preplant N (80 kg ha-1).
Linear regression of seasonal shoot dry matter accu-
mulation versus thermal time (GDDO is presented in Table
111.6.
At 24/16°C (r2=0.75) and 27/21°C (r2=0.91) soil
temperatures, GDD5 was a reasonably good predictor of
shoot dry matter accumulation independent of preplant N
rate.
At 18/12°C, a linear regression equation containing
both GDD5 and preplant N was a better predictor (r2=0.70)
Table 111.4.
Combined analysis of variance of three day/night soil temperature
regimes and five levels of preplant N in growth chamber experiments.
Mean squares
PBNF
% Shoot N
135.6
0.01
304.8**
0.04
0.6
24.8
0.02
0.7**
1.2**
83.2t
0.02
0.6**
1.0**
76.1
0.03
0.2
0.3
35.0
0.02
Source of variation
df
Total
58
Block
3
0.3
Temperature
2
7.2**
Block*Temperature
6
0.2
N level
4
Temperature*N level
8
Error
35
Shoot DM
Root DM
0.9
28.7**
*, ** Significant at the 0.05 and 0.01 probability levels, respectively.
t Significant at the 0.07 probability level.
47
Table 111.5.
Shoot dry matter yield at harvest of alfal-
fa grown in three soil temperatures and five levels
of preplant N in growth chamber experiments.
Day/night soil temperature
Preplant N
18/12°C
kg hdl
24/16°C
27/21°C
g plant-1
1.6±0.2t
1.4±0.3
0.8±0.1
10
1.2±0.3
1.6±0.1
0.8±0.1
20
1.8±0.4
1.4±0.1
0.9±0.2
40
2.7±0.8
2.0±0.7
0.7±0.1
80
2.5±1.1
1.5±0.4
0.8±0.1
0
t Values are means of four replicates ± standard deviation except for the 0 N treatment for 18/12°C which
is the mean of three replicates.
Table 111.6.
Linear regression analysis of shoot dry matter accumulation (SHDW)
in response to thermal time (GDD5) and preplant N in growth chamber
experiments.
Soil
Regression equation
2
F:GDD5t
F:N
12.9**
2.3*t
0.70
r
temperature
18/12°C
SHDW = 0.9 X 10
-3
GDD5 + 9.4 X 10
24/16°C
SHDW = 1.1 x 10
-3
GDD5 - 0.97
15.1**
NS
0.75
27/21°C
SHDW = 5.0 X 10
-4
GDD5 - 0.60
28.4**
NS
0.91
-2
N - 0.79
t GDD5 is the cumulative growing degree days with a base temperature of 5°C.
49
of shoot dry matter accumulation than GDD5 alone.
Shoot
dry matter accumulation rate was similar at 18/12°C (1.1
X 10
-3
g GDD5
-.1
)
and 24/16°C (0.9 X 10
-3
-1
g GDD5 ).
At
27/21°C, shoot dry matter accumulation rate (5.0 X 104 g
GDD5 -1) was reduced by 55%.
Smith (1970) also reported a
decline in shoot dry weight in alfalfa when growth temperature was increased from 15/10°C to 27/21°C.
More
rapid early growth at cooler temperatures, i.e. less than
24/16°C for 'Vernema', would deplete N from seed reserves
more rapidly.
The result would be higher demand for soil
available N than at warmer temperatures. This may explain
the yield response to preplant N at the coolest soil
temperature.
Temperature, preplant N, and temperature X N interaction had significant effects on root dry weight (Table
111.4 and 111.7).
The largest effects were observed at
18/12°C, in which root dry weight increased with increasing rates of preplant N application, with the maximum
yield at 80 kg ha-1 (Table 111.7).
Similar results were
reported by MacDowell (1982) with alfalfa grown at 25/20°C, in which root dry weight increased in response to
added N up to 15 mM.
Root growth at 27/21°C was reduced
and confirms that the highest soil temperature regime was
above the optimum for root dry matter accumulation for
'Vernema' alfalfa.
50
Table 111.7.
Root dry matter yield at harvest of alfalfa
grown in three soil temperatures and five levels of
preplant N in growth chamber experiments.
Day/night soil temperature
Preplant N
18/12°C
27/21°C
g plant-1
kg ha-1
2.5±0.3t
1.3±0.3
0.6±0.2
10
2.1±0.3
1.4±0.2
0.7±0.1
20
2.7±0.6
1.3±0.1
0.7±0.2
40
3.4±1.0
1.6±0.3
0.7±0.1
80
4.3±2.0
1.4±0.2
0.6±0.1
0
t
24/16°C
Values are means of four replicates ± standard deviation except for the 0 N treatment for 18/12°C which
is the mean of three replicates.
51
Combined N has been shown to increase dry matter
yield in alfalfa depending on the amount and time of
application in greenhouse and growth chamber studies
(Fishbeck and Phillips, 1981).
Heichel and Vance (1979)
noted an increase in dry weight in response to added N in
nodulated alfalfa grown at 27/19°C in solution culture
for 14 days.
1
)
Increasing N from zero to 3.5 mM (50 mg kg
in their experiments resulted in a 60% increase in
average whole plant dry weight.
Nodule number, however,
was reduced by about 40% over the same range of N.
In
this relatively short growth period, the availability of
higher amounts of inorganic N resulted in greater dry
matter accumulation than when BNF was the sole source of
N.
Available N, soil type, and legume species are
important factors in determining the dry matter accumulation response of legumes to added N (Peters and Kelling,
1989; Fishbeck and Phillips, 1981; Hojjati et al., 1978).
In addition, the interactive effect of temperature and
nitrate level on dry matter accumulation has been reported for white clover (MacDuff et al., 1989).
The strong
significance of the temperature and preplant N interaction (Table 111.4) indicates this is also true for dry
matter accumulation in alfalfa grown in a low N soil.
52
PBNF and N Accumulation
Both temperature and the temperature X N interaction
had significant effects on PBNF (Table 111.4).
Average
PBNF was similar at 24/16°C and 27/21°C, but generally
higher at 18/12°C (Table 111.8).
PBNF was not reduced by
increasing preplant N at any temperature.
At 18/12°C, 80
kg N ha-1 resulted in a 14% increase in PBNF.
BNF is
profoundly affected by root temperature and is closely
related to changes in dry matter accumulation (Gordon et
al., 1989; MacDuff et al., 1989). Changes in PBNF in
response to temperature have been shown to parallel
changes in dry matter accumulation in alfalfa (Wery et
al., 1986) and other legumes (Gates and Silsbury, 1987;
Date and Roughley, 1986).
This relationship was con-
firmed in our growth chamber experiments (Tables 111.5,
111.8).
Linear regression of shoot N accumulation in response to GDD5 and preplant N is presented in Table
111.9.
The rate of N accumulation was similar at root
temperatures of 18/12°C (2.1 X 10-3 g N GDD5-1) and 24/16°C
(2.6 X 10-3 g N GDD5-1)
g N GDD5-1).
,
but reduced at 27/21°C (1.1 X 10 -3
At 18/12°C, the best fitting linear regres-
sion included a preplant N component. Shoot N concentration was unaffected by N treatments (Table 111.10).
53
Table 111.8.
Percent N derived from biological nitrogen
fixation (PBNF) in alfalfa grown in three soil
temperatures and five nitrogen levels in growth
chamber experiments.
Day/night soil temperature
Preplant N
18/12°C
24/16°C
27/21°C
PBNF
kg ha-1
73±2t
64±10
67±9
10
68±6
71±3
72±3
20
75±5
66±5
72±6
40
81±7
73±5
70±5
80
83±6
65±11
71±5
Means
76a4
68b
70b
0
t Values are means of four replicates ± standard deviation except for the 0 N treatment for 18/12°C which
is the mean of three replicates.
: Temperature means followed by different letters are
significantly different at the 0.01 level as determined by Duncan's Multiple Range Test.
Table 111.9. Linear regression analysis of shoot N accumulation (SHN) in
response to thermal time (GDD5) and preplant N in growth chamber
experiments.
Soil
Regression equation
2
F:GDD5t
F:N
r
11.8**
3.2*
0.67
2.15
14.9**
NS
0.74
GDD5 - 1.38
26.8**
NS
0.90
temperature
18/12°C
SHN = 2.1 X 10
24/16°C
SHN = 2.6 x 10
-3
27/21°C
SHN = 1.1 X 10
-3
GDD 5 + 3.1 X 10
GDD5
N - 2.43
t GDD5 is the cumulative growing degree days with a base temperature of 5°C.
55
Table 111.10.
Shoot N concentration at harvest of alfal-
fa grown in three soil temperatures and five levels
of preplant N in growth chamber experiments.
Day/night soil temperature
Preplant N
g m
18/12°C
-2
24/16°C
g N
27/21°C
kg-1
21±1t
20±1
22±1
10
20±1
22±2
21±1
20
20±1
23±2
22±2
40
21±2
22±2
22±1
80
22±1
21±1
23±2
0
t The effects of temperature, N level, and temperature X
N level interaction were not significant at P=0.05.
Values are means of four replicates ± standard
deviation except for the 0 N treatment for 18/12°C
which is the mean of three replicates.
56
In white clover grown in solution culture with 10 mM
nitrate, PBNF decreased from 29% at a root temperature of
17°C to 11% at 25°C (MacDuff et al., 1989).
In our
study, PBNF also declined at higher temperatures, from
64% at 18/12°C to 45% at 27/21°C (Table 111.8).
PBNF was
highest at 18/12°C, despite reports that alfalfa nodulation is inhibited at soil temperatures below 15°C
and Tisdale, 1921).
(Jones
The warmer daytime temperatures may
have eliminated negative effects of the cool night temperatures.
At 18/12°C, the addition of fertilizer N up to 80 kg
ha
-1
.
did not result in a decline in PBNF, despite the
well documented negative effects of combined N on BNF
(Streeter, 1988).
Most of those studies, however, have
been conducted at average day/night soil temperatures
above 20°C.
Studies with field-grown alfalfa by Wery et
al. (1986) suggested that when a lack of N was limiting
growth, the addition of a small amount of N (35 kg
ha-1
N) favored fixation instead of nitrate assimilation.
The
additional N also resulted in a greater accumulation of N
and dry matter.
In the field, root temperature in the spring will
often remain below shoot temperature during early plant
development.
During this stage of relatively rapid
growth, the shoot demand for N may exceed the N available
57
from the soil and/or BNF.
Under these conditions, the
addition of small amounts of readily available fertilizer
N could result in increased dry matter accumulation.
58
Conclusions
Under field conditions, the N nutrition of alfalfa
involves differing contributions of soil N and BNF.
The
contribution that each N pool makes to plant N requirements is strongly influenced by the environment, particularly the efficiency of the legume-Rhizobium symbiosis,
soil nitrate levels, and soil temperature.
Because of
the generally antagonistic effect of nitrate on BNF, as
well as it's tendency to increase weed growth, it is not
normally desirable to apply fertilizer N in legume establishment (Eardly et al., 1985).
Most studies have shown
that the yield of properly established, effectively nodulated alfalfa does not respond to N fertilizer.
For the
same reason, the application of manures or other N sources is not generally recommended as a desirable alfalfa
production practice.
However, studies with alfalfa (Peters and Kelling,
1989; Fishbeck and Phillips, 1981) and other legumes
(Schomberg and Weaver, 1990) suggest that in low N soils,
some yield response to starter N fertilization may be
observed.
When alfalfa is established in a low N envi-
ronment and shoot demand for N exceeds the quantity of N
available from soil N or BNF, an N deficit will develop
(MacDuff et al., 1989).
Our studies suggest that this N
59
deficit is exacerbated by low soil temperatures during
the development of the symbiosis.
Alfalfa dry matter
yield and PBNF exhibited the greatest response to small
amounts of preplant N (20-40 kg ha-1) when the day/night
soil temperature was 18/12°C.
An accurate prediction of the N-fertilizer requirements of a non-N2-fixing crop involves a consideration of
the quantity and distribution of inorganic N in the soil,
the demand of the crop for N, and the ability of the root
system to extract inorganic N from soil (Greenwood,
1986).
In legumes, the ability of the legume-Rhizobium
symbiosis to supply N over time also must be considered.
Some general guidelines for the use of preplant N in
alfalfa may be derived from these experiments.
The pre-
treatment 2 M KC1 extractable nitrate concentration in
the field experiments at Powell Butte in 1988, when a
yield response to 20 kg ha-1 preplant N was observed,
ranged from 13-20 mg kg-1.
The average daily soil tem-
perature at 10 cm for the two weeks after planting was
10°C.
In growth chamber experiments, the greatest dry
matter and PBNF response to fertilizer N was at a soil
temperature of 18/12°C.
level was about 6 mg kg-1.
The average initial soil nitrate
Statistical analysis of
growth chamber studies indicated that temperature and
temperature X N interaction effects are more important
60
than N effects.
This may explain why a response to N in
the field was observed even though the initial nitrate
level was higher than in the soil used in the growth
chamber study.
Based on the field conditions in these
experiments, 20-40 kg ha-1 preplant N would result in
greater yield at first harvest only if the average daily
soil temperature for at least two weeks after planting is
less than 15°C and the soil nitrate level before planting
is less than 16 mg kg-1.
In any production practice, growers must consider
the cost/benefit ratio in regard to production goals.
To
correctly assess the need for preplant N in alfalfa
establishment, both soil available N and soil temperature
at planting should be considered.
To establish a repre-
sentative profile of soil temperature flucuations, weather records of nearby weather stations have records of
daily 10 cm (4") and 20 cm (8") soil temperature.
If
daily average soil temperatures are below 15°C for at
least two weeks after normal planting time, it may be
advisable to plant later in the season.
If this is not
feasible, and if both soil nitrate and soil temperature
profiles indicate a potential N deficit, a small application of preplant N (20-40 kg ha-1) may provide an in-
crease in first harvest yield.
If there is an adequate
population of Rhizobium meliloti present, this amount of
61
N should not result in reduced seasonal PBNF and thus BNF
will eventually be able to supply most of the plant N
needs.
62
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V. APPENDIX
Table 1. Analysis of variance of dry matter yield for 1987 and 1988 field
experiments at Powell Butte, OR.
Source
DF
Sum of squares
Total
38
551936040.38
Year
1
Replicate
N Level
Error
Mean square
F value
P > F
6433662.57
6433662.57
0.53
0.4728
3
1609333.51
536444.50
0.04
0.9875
4
178729622.07
44682405.52
3.67
0.0151
30
365163422.23
Table 2. Shoot and root dry matter accumulation of alfalfa grown
at a day/night soil temperature regime of 18/12°C and five
levels of preplant N in growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
47
123
219
kg ha-1
252
47
123
219
252
g plant.1
0
0.1
0.5
1.8
1.6
0.1
0.5
1.9
3.4
10
0.1
0.4
1.1
1.2
0.1
0.4
1.2
2.1
20
0.1
0.4
1.4
1.8
0.1
0.4
1.8
2.7
40
0.1
0.4
1.1
2.7
0.1
0.4
1.5
3.4
80
0.1
0.4
2.0
2.5
0.1
0.4
1.9
4.3
Table 3. Shoot and root dry matter accumulation of alfalfa
grown at a day/night soil temperature regime of 24/16°C
and five levels of preplant N in growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
44
76
95
kg ha-1
134
44
76
95
134
g plant-1
0
0.1
0.2
0.3
1.4
0.1
0.2
0.3
1.3
10
0.1
0.3
0.4
1.6
0.1
0.3
0.4
1.4
20
0.1
0.2
0.4
1.4
0.1
0.3
0.4
1.3
40
0.1
0.2
0.4
2.0
0.1
0.2
0.4
1.6
80
0.1
0.3
0.5
1.5
0.1
0.3
0.5
1.4
Table 4. Shoot and root dry matter accumulation of alfalfa grown at
a day/night soil temperature regime of 27/21°C and five levels of
preplant N in growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
73
85
107
kg ha-1
145
73
85
107
145
g plant-1
0
0.1
0.2
0.3
0.8
0.1
0.2
0.2
0.6
10
0.1
0.2
0.3
0.8
0.1
0.1
0.2
0.7
20
0.1
0.2
0.3
0.8
0.1
0.2
0.2
0.7
40
0.1
0.2
0.4
0.7
0.1
0.2
0.3
0.7
80
0.1
0.2
0.3
0.8
0.1
0.2
0.2
0.6
Table 5.
Shoot and root N concentration of alfalfa grown at a
day/night soil temperature regime of 18/12°C and five levels
of preplant N in growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
47
123
219
kg ha-1
252
g N
47
123
219
252
kg-1
0
8.8
22.0
19.8
20.5
29.3
29.8
25.5
22.8
10
8.8
21.5
19.2
20.0
29.1
30.0
27.5
25.8
20
6.2
20.8
18.8
20.0
29.0
28.8
24.5
25.2
40
4.6
21.2
24.2
20.5
30.0
28.8
28.0
24.8
80
2.6
20.2
23.0
22.0
29.2
29.2
26.5
25.0
Table 6. Shoot and root N concentration of alfalfa grown at a day/night
soil temperature regime of 24/16°C and five levels of preplant N in
growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
44
76
95
134
g N
kg ha-1
44
76
95
134
kg-1
0
29.7
30.2
22.2
25.3
17.9
19.6
26.0
20.0
10
31.9
29.6
23.5
25.6
20.0
19.0
23.2
21.6
20
30.0
29.8
22.8
24.2
18.5
17.1
15.2
22.6
40
29.3
29.7
24.5
23.9
18.0
18.1
25.5
21.5
80
29.0
29.2
22.8
23.1
17.9
19.2
24.5
20.6
Table 7. Shoot and root N concentration of alfalfa grown at a
day/night soil temperature regime of 27/21°C and five levels
of preplant N in growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
73
85
107
kg ha-1
145
g N
73
85
107
145
kg-1
0
20.6
19.1
22.4
21.9
32.2
28.6
31.8
26.0
10
20.8
20.9
21.3
21.3
31.7
31.5
28.6
28.9
20
20.3
21.6
21.1
21.7
32.7
29.6
31.3
29.7
40
20.4
19.0
21.4
21.8
32.0
27.2
29.1
27.9
80
19.2
19.0
20.4
22.6
31.5
29.4
31.4
29.2
Table 8. Shoot and root N content of alfalfa shoots grown at a day/night soil
temperature regime of 18/12°C and five levels of preplant N in growth
chamber experiments.
Preplant N
47
Shoots
Roots
Days after planting
Days after planting
123
219
kg ha-1
252
47
123
219
252
g N plant-1
0
0.1
1.0
1.0
3.2
0.1
1.4
1.2
5.9
10
0.1
0.8
1.2
2.5
0.1
1.1
1.7
5.4
20
0.1
0.7
1.1
3.5
0.1
1.0
1.4
6.8
40
0.1
0.8
1.6
5.7
0.1
1.2
2.0
8.5
80
0.1
0.7
3.3
5.5
0.1
1.0
3.8
10.9
Table 9. Shoot and root N content of alfalfa grown at a day/night soil
temperature regime of 24/16°C and five levels of preplant N in
growth chamber experiments.
Preplant N
44
Shoots
Roots
Days after planting
Days after planting
76
95
kg ha-1
134
44
76
95
134
g N plant-1
0
0.2
0.7
0.7
3.5
0.1
0.5
0.8
3.5
10
0.2
0.8
1.0
4.2
0.1
0.5
0.8
4.2
20
0.3
0.7
0.8
3.4
0.1
0.4
0.5
3.4
40
0.4
0.7
1.1
4.5
0.1
0.5
1.0
4.5
80
0.3
0.8
1.1
3.4
0.1
0.5
1.2
3.4
Table 10. Shoot and root N content of alfalfa grown at a day/night
soil temperature regime of 27/21°C and five levels of preplant N
in growth chamber experiments.
Preplant N
Shoots
Roots
Days after planting
Days after planting
73
85
107
kg ha-1
145
73
85
107
145
g N plant-1
0
0.3
0.4
0.7
1.7
0.4
0.4
0.8
1.6
10
0.2
0.4
0.7
1.8
0.2
0.4
0.6
1.9
20
0.2
0.4
0.7
1.9
0.4
0.4
0.7
2.0
40
0.3
0.4
0.8
1.6
0.3
0.5
0.8
1.9
80
0.2
0.4
0.6
1.8
0.3
0.4
0.8
1.8