Plant Physiol. (1982) 70, 1564-1567
0032-0889/82/70/ 1564/04/$00.50/0
Short Communication
Screeiing and Selection of Maize to Enhance Associative
Bacterial Nitrogen Fixation'
Received for publication July 8, 1982 and in revised form August 20, 1982
STEPHEN W. ELA, MARY ANN ANDERSON, AND WINSTON J. BRILL
Department of Bacteriology and Centerfor Studies of Nitrogen Fixation, University of Wisconsin,
Madison, Wisconsin 53706
ABSTRACT
The ability of maize (corn, Zea mays L.) to support bacterial nitrogen
fixation in or on maize roots has been increased, through screening and
selection. Isotopic N fixed from "N2 was found on the roots. The nitrogenfixing association was found in germplasm from tropical maize, but this
activity can be transferred to maize currently used in midwestern United
States agriculture.
Intensive research on associations between nitrogen-fixing bacteria and cereal roots has begun only in the last decade (1, 5, 6, 9,
11). If these associations can be induced to contribute significant
amounts of N to cereal crops, grain yields may become less
dependent on industrially produced fertilizer nitrogen. Development of a cereal that can obtain even a minor fraction (10-20%o)
of its nitrogen from such an association can produce an economically important substitution of photosynthetically derived energy
for fossil fuel energy now expended for production of fertilizer
nitrogen. However, some researchers have questioned methods
used to explore these associations (8), and no cereal-N2-fixing
bacterial association is now in commercial use.
If a significant nitrogen-fixing association is to occur, the photosynthetic host must supply one or more reduced carbon compounds to the bacteria to support the energy requirement for
nitrogen fixation. Observations suggest that these associations may
depend on the genetic variability within the host species (5);
however, there has been no attempt to select systematically among
this potential variability for enhancement of a nitrogen-fixing
association. We have used the acetylene reduction assay for nitrogen fixation to test maize plants for the ability to provide on or in
the roots a substrate that can support bacterial nitrogen fixation.
We have found that a few maize lines can show acetylene reduction activity and that this may be increased by selection and
transferred to inactive maize inbreds adapted to midwestern
United States growing conditions. Acetylene reduction can be
immediate and linear; we have confirmed nitrogen fixation on
maize roots with 15N2.
Plants and Bacteria. Seeds (Zea mays L.) were sterilized in a
1:10 dilution of a solution of 5.25% commercial NaOCl plus 0.1%
Triton X-100 for 15 min with frequent agitation. After six rinses
with sterile distilled H20, the seeds were spread on moistened
filter paper in Petri plates at 30°C for 48 h in the dark. Germinated
seeds were placed, one per pot, into moist sterile vermiculite in
400-ml polypropylene beakers, each containing drain holes in the
bottom and a disc of polypropylene mesh screening to retard roots
growing through the drain holes. We grew plants for 27 d under
nonsterile environmentally controlled conditions (16-h days, 2717°C day-night temperatures, 70-50%o RH, 600,uE/m2 lighting).
Each plant was inoculated at planting and 2 weeks after planting
with cells of Azotobacter vinelandii OP suspended in 6.7 mm
phosphate buffer at pH 7.5; we supplied each plant daily with 40
ml of an inorganic nutrient solution low (0.75 mM) in nitrate as
modified from that of Wacek and Brill (12). Controls established
that the plants were nitrogen-limited at the time of assay.
Since we completed our original screening, we have made
several modifications of our growth and inoculation procedures,
none of which appears to effect significantly the frequency or
levels of acetylene reduction activity. Plants can be grown in a
washed sand support on nutrient medium with 1 mm nitrate; they
can receive a daily rinse of distilled H20 to prevent excess salt
accumulation. Inoculation with nitrogen-fixing Azospirillum
strains, an Enterobacter isolated from corn roots by Raju et aL (7)
or a Klebsiella pneumoniae laboratory strain gives results similar
to those obtained from the Azotobacter inoculum.
Nondestructive Acetylene Reduction Assay. The 400-ml polypropylene beaker in which the plant was growing was placed
inside a glass cylinder and a split rubber stopper was fixed around
the stem. Acetylene, with low background ethylene, was added to
7% and the plants incubated under daytime growth conditions for
5 h. Aliquots of the gas phase in the cylinders were analyzed for
ethylene formation on a gas chromatograph fitted with a 1.8-m
Porapak N column.
Controls indicated that the ethylene formed resulted from acetylene reduction. Incubations without acetylene gave insignificant
ethylene production; addition of ethylene at the levels of those
formed at 5 h but at the beginning of incubations without acetylene, showed no loss of ethylene during 5-h incubations, indicating
that microbial oxidation of ethylene was not a complicating factor.
Each of these two types of controls was conducted with plants and
equipment that had not previously been exposed to acetylene. No
acetylene was detected in these controls (4).
Washed Root Assays with Acetylene. Plants were removed from
their beakers and the roots were swirled in 300 ml nutrient solution
with calcium sulfate substituted for calcium nitrate. They were
lifted clear from sand and subjected to a second wash of 30-s
duration in 300 ml of the same solution. The roots were cut from
the shoots below the first nodes and placed in 75-ml polypropylene
bottles which were plugged with rubber bungs fitted with serum
' Supported by the College of Agricultural and Life Sciences, University
of Wisconsin, Madison, Grant DE-AC02-80ER10717 from the Department of Energy, a gift from Cetus Corporation, and Grant AER77-0879
from the National Science Foundation.
1564
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SELECTION OF MAIZE FOR ASSOCIATIVE BACTERIAL N2 FIXATION
1565
Table I. Results of Selection to Enhance Acetylene Reduction Activities
of
Number
Number o
Plant Origin
Plant Code
Plants
Tested
ON33
B1
Wisconsin inbred
ON33 x South American
14
30
Activ- Average Activity,' Active
Active Plants' Average
Plants Oniy
ity,b All Plants
jybAlPat
lnsOl
%
0
50
nmol C2H4/plant h
0.0
0.0
4.3 ± 0.6
2.2 ± 0.5
33
67
98
97
0
61
1.7 ± 0.6
5.1 ±0.7
18 ± 1.5
38 ± 5.0
0.0
7.2± 1.0
IC
ON33 x South American 2
30
BI x B3
125
D6 self-bred
127
J
G self-bred or sib-matedd
158
Wisconsin hybrid
13
ON 18
149
ON18xD6
H
a Equal to or greater than 2 nmol C2H4/plant h.
b Values are means and SE.
c Crosses are female x male.
d
3G plants self-bred; two sib-mated.
B3
D6
G
stoppers and then evacuated three times to half-atmospheric pressure and brought to atmospheric pressure by adding argon. Acetylene was added to 10%o and the roots were incubated at 27°C in
the shade. CM,2 when used, was dissolved in the second wash at
concentrations of 100 mg/I.
Isotopic N Assays. In the first two of three experiments, we
assayed plants by the nondestructive acetylene reduction assay 72
h before performing the isotopic N experiments. In these two
experiments, plants were paired in decreasing rank of acetylene
reduction values. In a third experiment with no prior acetylene
reduction assay, we paired plants in decreasing rank of the sums
of the three longest leaves of each plant.
Each plant was washed and evacuated as described above,
except that, instead of making up with argon and adding acetylene
after the third evacuation, 5N2 or N2 of atmospheric isotopic
abundance was added to the pairs to produce pressures within the
bottles equal to or slightly greater than atmospheric. The 15N2 was
generated from ('5NH4)2SO4 with sodium hypobromite, washed
with alkaline permanganate, 50%o aqueous H2SO4, and stored in
gas-tight syringes shortly before use. In the first experiment involving five pairs of roots, CM was omitted; in the second, also
using five pairs, CM was added to the second wash. The third
experiment, with no previous acetylene reduction assay, also
utilized CM in the second wash. All roots in the three experiments
were incubated for 5 h. Following the incubations, the bottles
were opened, and the roots were promptly dried with forced air at
70°C for 24 h and ground to pass a Wiley mill 40-mesh screen.
Following Kjeldahl digestion and distillation, the ammonium
sulfate was oxidized with sodium hypobromite and the N2 was
analyzed from one member of the pair against the N2 from the
other on a Varian MAT 250 double-inlet mass spectrometer.
Periodic analysis of a standard pair of ammonium sulfate samples
during the analyses of the unknowns showed (17 determinations)
0.00176 + 0.00002 atom % '5N excess.
RESULTS AND DISCUSSION
We screened 1450 corn plants from 180 different seed lots for
the ability to support acetylene reduction. Most of the plants
yielded undetectable rates of acetylene reduction. However, a few
of the plants supported rates greater than 8 nmol/h. These active
plants were concentrated within a few seed lots. Inbred or hybrid
maize bred for high yield in the midwestern United States produced inactive plants (16 lots tested). Seed collections that gave
active plants had at least one parent of Central or South American
2Abbreviation: CM, chloramphenicol.
5.0 ± 1.1
7.6±0.8
18 ± 1.5
39 ± 5.1
0.0
11.8± 1.5
origin; however, most of the plants of tropical origin also did not
yield active plants.
By selecting those plants that showed acetylene reduction, transplanting them, self- or cross-breeding them, and then subjecting
the plants from the resulting kernels to growth and assay conditions similar to those used for screening, we found that we could
increase both the frequency of active plants and the levels of
acetylene reduction associated with their roots. Table I illustrates
these results for one of our selections. Two plants, each a cross
between a Wisconsin inbred (W2207/07 which, itself, showed no
acetylene reduction) and a South American parent, and each of
which gave an acetylene reduction rate of about 8 nmol/h, were
crossed to produce ear D6. Three D6 plants selected for their high
acetylene reduction rates were self-bred to produce G series plants
and five of these, when selected and self-bred or sib-mated, yielded
plants essentially all of which were active and averaged 38 nmol
acetylene reduced per h.
The traits responsible for activity in the D6 line tend toward
dominance as illustrated in Table I. We outcrossed some of the
most active plants of the D6 ear to a number of inbred lines
adapted to midwestern U. S. growing conditions. We observed
that 60 to 70o of the progeny were active, although with lower
acetylene reduction rates when compared to the most active of the
male parents used for these crosses.
Although our success in selecting for increases in acetylene
reduction activity has not yet produced maize lines sufficiently
active to be commercially useful (our most active maize plants
were approximately 0.5% as active as soybean plants inoculated
with Rhizobiumjaponicum, grown and assayed in the same manner
as the maize plants), it provides us with incentive to increase
activities by continued screening and selection. Further, this success (the production of a reproducible positive control) has allowed
us to advance our understanding of the methodological complexities involved in studying associations between cereals and nitrogen-fixing bacteria.
We have used roots of our active plants to probe the characteristics of time courses of acetylene reduction; immediate and linear
acetylene reduction is an important criterion for establishing bona
fide associations between plants and nitrogen-fixing bacteria (10).
Time courses of ethylene formed by active whole plants show two
notable features; lag phases of about 1 h, followed by nonlinear
rates of ethylene formation (Fig. 1). Both the lag and the nonlinearity are results of acetylene addition and not the lack of equilibria of temperature or 02 and CO2 concentrations within the assay
cylinders; if the assays are conducted with a pre-incubation period
of 1 to 3 h without the addition of acetylene and acetylene then
was added, time courses similar to that- illustrated in Figure IA
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1566
ELA ET AL.
Plant Physiol. Vol. 70, 1982
Table II. Nitrogen Fixation on or in Washed Maize Roots
Conditions
160
Prior C2H2 reduction,
-CM
B
0
120
Prior C2H2 reduction,
+CM
C)
u
0
No C2H2, +CM
0
0.
N2 Fixed
atom % x 105
1516 ± 3a
1144 ± 3
528±7
905 ±0
920 1
301 ± 0
199 2
619 4
585 1
165 1
66 2
230 1
nmol
64b
38
15
28
36
11
6
23
22
5
2
8
8
16
6
N2/C2H2
Reduced
Y
33
25
14
27
38
6
4
21
28
7
339±0
447±3
195±0
a The values are the differences of the atom % '5N of root incubated
under enriched '5N2 less the atom % '5N2 of root incubated under N2 of
atmospheric '5N abundance, as determined directly on the mass spectrometer. The errors are those found from at least two introductions of N2 into
the mass spectrometer.
b The atom % '5N of the '5N-enriched N2 in the incubation bottles
averaged 72 by MS.
c)
0
C-
1
3
5
HOURS
FIG. 1. Acetylene reduction assays on typical active maize plants. A,
Nondestructive assay. B, Washed roots minus CM. C, Washed roots with
CM.
observed (data not shown).
If the roots of these plants are washed free of sand and then
incubated with acetylene, the rates of ethylene formation are
immediate and linear for the first 2 h but again increase after this
initial period (Fig. IB). If CM is added to the medium used to
wash the roots, acetylene reduction is immediate and linear for 3
to 4 h (Fig. IC). Because the lag observed in the whole plant assay
disappears upon freeing the roots of sand, we ascribe the lag to
slow diffusion of acetylene to and ethylene from the active sites
on or in the roots. Since the presence of CM, an inhibitor of
protein synthesis, produces linear acetylene reduction, we suggest
that the nitrogen-fixing bacteria present, when exposed to acetylene for periods of a few hours, are responding by synthesizing
more nitrogenase (2, 8).
By using the washed root assay, we have determined that the
roots of those plants selected for increased activity in the acetylene
reduction assay are able to reduce N2 directly. In each of the 15
pairs assayed, the root incubated under enriched '5N2 showed a
higher atom % '5N content than the root incubated under N2 of
atmospheric isotopic ratio (Table II).
Of the plants that had previously been assayed with acetylene,
those with roots washed with CM averaged 13 nmol N2 fixed and
those not treated with CM averaged 36 nmol N2 fixed. If we
assume the ratio between acetylene reduced and N2 fixed to be 3,
then values of ethylene formed calculated from N2 fixed averaged
13 and 27%, respectively, of the ethylene formed in the previous
assays. If a significant fraction of the electrons flowing through
nitrogenase produced hydrogen gas when the bacteria reduced N2,
then these percentages would increase. Regardless of this uncerare
15N Excess
A
tainty, nitrogen fixation assayed by '6N2 reduction with washed
roots is in the range expected from previous acetylene reduction
assays of washed and unwashed roots of the same plant (data not
shown). Thus, much of the nitrogen fixation occurs in or on the
roots.
We view our approach to developing an association between
nitrogen-fixing bacteria and maize as both a complementary and
alternative approach to the long range goal of expression of
genetically engineered bacterial nitrogen fixation (nif) in maize.
We can speculate that subsistence farmers with limited supplies of
agricultural nitrogen have unknowingly selected for nitrogen-fixing associations in maize, and in other crops as well (e.g. wheat or
sweet potato). Investigators using our approach to probe associations between economically important plants and nitrogen-fixing
bacteria may require in the future access to a broad cross-section
of the world's germplasm of the particular crops under study (3).
Acknowledgements-We thank Larry Hanson, Ann Wopat, Terry Moen, and the
staff at the University ofWisconsin Biotron for expert technical assistance. Dr. Oliver
E. Nelson and Dr. John H. Lonnquist generously supplied us with seeds and practical
advice on maize growing. Dr. Robert H. Burris contributed invaluable counsel on the
isotopic nitrogen and mass spectrometric portions of this work.
LITERATURE CITED
1. BARBER LE, JD TJEPKEMA, SA RussELL, HJ EVANS 1976 Acetylene reduction
(nitrogen fixation) associated with corn inoculated with spirillum. Appl Environ
Microbiol 32: 108-113
2. DAVID KAV, P FAY 1977 Effects of long-term treatment with acetylene on
nitrogen-fixing microorganisms. Appl Environ Microbiol 34: 640-646
3. HARLAN JR 1975 Our vanishing genetic resources. Science (Wash DC) 188: 618621
4. LETHBRIDGE G, MS DAVIDSON, GP SPARLING 1982 Critical evaluation of the
acetylene reduction test for estimating the activity of nitrogen-fixing bacteria
associated with the roots of wheat and barley. Soil Biol Biochem 14: 27-35
5. NEYRA CA, J DOBEREINER 1977 Nitrogen fixation in grasses. Adv. Agron 29: 138
6. OKON Y, SL ALBRECHT, RH BuRsUS 1977 Methods for growing Spirillum
lipoferum and for counting it in pure culture and in association with plants.
Appl Environ Microbiol 33: 85-88
7. RAJu PN, HJ EVANS, RJ SEIDLER 1972 An asymbiotic nitrogen-fixing bacterium
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8. RIPPKA R, JB WATERBURY 1977 The synthesis of nitrogenase by nonheterocys-
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SELECTION OF MAIZE FOR ASSOCIATIVE BACTERIAL N2 FIXATION
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VAN
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11.
VON
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