R. W. Weaver

S. F. Wright

Applied and Environmental Microbiology

English

Texas A&M University

Vol. 53, No. 12
Variability in Effectiveness of Rhizobia during Culture
and in Nodules
R. W. WEAVER* AND S. F. WRIGHT
Department of Soil and Crop Sciences, The Texas Agricultural Experiment Station, Texas A & M University,
College Station, Texas 77843
Received 20 April 1987/Accepted 3 September 1987
The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with
siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules.
Seven transfers were made between the initial broth culture and the final broth culture. A total of 40
single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing
effectiveness of the population of one strain did not change on culturing, whereas there was considerable
variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from
individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different
nodules formed by the same Bradyrhizobium strain often differed in their effectiveness.
Rhizobium and Bradyrhizobium spp. are of scientific and
agronomic importance because of their ability to reduce
dinitrogen (effectiveness) in root nodules of leguminous
plants. During laboratory culture, spontaneous mutation of
rhizobia may result in reduced ability to fix dinitrogen. Such
mutants have been readily detected during selection for
resistance to antibiotics (1, 11, 15). The medium used to
culture rhizobia also influences the stability of genes for
dinitrogen fixation and promotes selection for such mutants
(12, 13). Subculturing Bradyrhizobium stock cultures on the
typical yeast extract-mannitol-salts medium (17) used in
most laboratories may also result in loss of effectiveness (6).
Herridge and Roughley (6) tested 17 cultures of Bradyrhizo-
bium sp. (Vigna) strain CB756 obtained from several stock
culture collections. The cultures varied widely in their
effectiveness, and some promoted less than half the plant
dry-matter production promoted by the most effective cul-
tures. Single-colony isolates from one of the most effective
cultures showed wide variability in effectiveness of the
subcultures. Nearly half the subcultures were less than half
as effective as the most effective subcultures.
Variability in effectiveness of Bradyrhizobium sp. (Vigna)
also is present in the population following inoculation into
soil (2). Incubating one strain ofBradyrhizobium sp. in moist
soil at 45°C for 15 days resulted in a large increase in
variability of effectiveness among single-colony isolates.
After the incubation period, 37% of the isolates were less
than 60% as effective as the parent culture, whereas at the
beginning of the incubation period, none of the isolates was
so ineffective. Following the incubation period, 8% of the
isolates were totally ineffective, but all could nodulate the
plant.
Culturing Bradyrhizobium sp. for inoculant production is
particularly challenging because of a generation time of
several hours, the ease with which a faster-growing contam-
inating bacterium can overtake the culture, and the inherent
danger that a large proportion of the population may lose
dinitrogen fixation effectiveness. Single-colony isolates of
bradyrhizobia belonging to the same strain vary consider-
* Corresponding author.
ably in generation time (15, 16). To reduce the time required
for batch production of a culture, a stage-up process is used
that provides for a relatively large initial inoculum that
amounts to 0.1 to 1% of the final cell yield (3, 4, 10). The
large inoculum reduces the lag phase of growth (3), the time
required to reach adequate cell yields, and the danger of a
few fast-growing organisms overtaking the culture.
We report here on the variability in effectiveness of three
strains of Bradyrhizobium sp. (Vigna) following an initial
broth culture and after seven serial transfers in broth.
Variability in effectiveness was also demonstrated for iso-
lates cultured from different nodules and between isolates
from the same nodule.
Three strains of Bradyrhizobium sp. (Vigna) were used to
determine the genetic stability of the dinitrogen fixation
character of these bacteria after culture in broth. These
strains were Thai-201 (obtained from Nantakorn Boonkerd,
Ministry of Agriculture, Bangkok, Thailand), T-1 (from our
collection), and TAL-201 (NifTAL culture collection, Paia,
Hawaii). TAL-309 originally came from Sydney University,
Sydney, New South Wales, Australia, where it was desig-
nated CB756. All strains formed effective nodules on cowpea
(Vigna unguiculata), peanut (Arachis hypogaea), and siratro
(Macroptilium atropurpureum).
The first experiment evaluated the effectiveness of
bradyrhizobia isolated from an initial broth culture of the
parent culture and from a culture developed following seven
serial subcultures. The measure of effectiveness was the
quantity of plant dry matter produced (17). The broth (YMB)
and agar (YMA) media containing yeast extract, mannitol,
and mineral salts have been described previously (17). The
starter culture for broth 1 was from an agar slant of a
Bradyrhizobium culture that was 10 days old. Cells were
scraped from the slant, suspended in YMB, and added to 20
ml ofYMB contained in 125-ml Erlenmeyer flasks stoppered
with cotton plugs. The starter inoculum provided approxi-
mately 5 x 106 cells per ml of broth, an inoculum anticipated
to provide approximately 1% of the final cell yield. Cultures
were grown for 5 to 7 days (29°C at 100 rpm), by which time
they had reached the early stationary phase of growth.
Inoculum (0.2 ml) was removed and used to inoculate broth
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1987, p. 2972-2974
0099-2240/87/122972-03$02.00/0
Copyright © 1987, American Society for Microbiology
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NOTES 2973
Ut)
-5.-
U)0
0
U)
n
U)
4-0
0
(n
U)
40-
301
204
10~
40*
30+
20+
10-
40-
30-
204+
10l
_
Initial culture s - 120 * 2.8.s29 310
Seventh culture i = 109 * 3.9 2 615
Thai-201
n O lnl I n
40 65 90 115 140 165 190
Plant Dry Weight (mg)
FIG. 1. Effectiveness of 40 single-colony isolates of three strains
of bradyrhizobia from initial broth cultures and after seven serial
subcultures into broth. Isolates were grouped according to dry-
matter production of shoots of siratro.
culture 2. Samples (0.1 ml) were also removed, serially
diluted in YMB containing 0.01% (vol/vol) Tween 40 (8), and
spread plated on YMA for isolation of single colonies. After
1 week, 40 well-spaced colonies were isolated and subcul-
tured on YMA slants. Broth culture 2 was grown to early
stationary phase, and 0.2 ml of culture was used as a starter
for broth 3. Culture 2 was then discarded. The same proce-
dure was followed until broth culture 7 had been grown.
When broth culture 7 was grown, a sample was removed for
isolation of 40 colonies as was done with broth 1. Before
plating for isolation, wet mounts of broth cultures were
examined by phase microscopy. Cells were observed to be
predominately single, with only rare clumps of two or three.
Subcultures on slants were allowed to grow for 2 weeks,
after which they were stored at 5°C.
To test the effectiveness of bradyrhizobia, siratro plants
were grown in cone-tainers filled with sterilized vermiculite
(5). Siratro was chosen as the test plant instead of peanut or
cowpea because of its small size. Nutrient solution (17) was
added to each cone-tainer with an automatic pipette. Solu-
tion in excess of that needed to saturate the vermiculite
drained from the bottom of the cone-tainer. Plants were
grown for 5 weeks under high-intensity fluorescent lights
providing 650 microeinsteins/m2 per s at the top of the plants
for 14 h/day. The temperature was 31°C during the light
period and 21°C during the dark period. Before being
planted, seeds were surface sterilized by being soaked for 5
min in a dilute solution of household bleach (0.525 g of
sodium hypochlorite per 100 ml of water) and rinsed 10 times
in distilled water. Two seeds were planted in each cone-
tainer, and seedlings were thinned to one per cone-tainer.
For inoculum preparation, subcultures of the sets of 40
isolates were made on to YMA slants and allowed to grow
for 8 days at 28°C. The developed cultures were suspended
in sterilized plant nutrient solution at a density of approxi-
mately 107 cells per ml. Each isolate was used to inoculate
three plants by slowly adding 1 ml of inoculum to each
cone-tainer. Several cone-tainers did not receive inoculum;
these plants served as uninoculated controls to test for
cross-contamination.
Parent culture (Thai-201 and TAL-309) controls were six
replicate plants inoculated with 107 cells from a broth culture
grown from a stock slant. Plants were grown for 5 weeks,
and plant tops were then removed for dry-matter determina-
tion. Roots were examined for nodulation.
Two additional investigations were undertaken to deter-
mine the effectiveness of isolates from nodules formed on
siratro plants inoculated with our stock culture of TAL-309.
Five plants were inoculated with approximately 106 cells
which had been cultured in YMB, diluted in plant nutrient
solution, and grown as previously described. Nodules were
collected from all of the plants at 5 weeks after planting, and
the nodules were combined. Ten nodules were chosen at
random and used to obtain single-colony isolates. The nod-
ules were surface sterilized with HgCI2 (17) and macerated in
YMB containing 0.01% (vol/vol) Tween 40. The suspension
was streaked on YMA, and five isolated colonies from each
nodule were subcultured. In addition, 20 single-colony iso-
lates were obtained from each of two nodules and cultured
on YMA slants. Inoculum was prepared and effectiveness
tests were conducted with siratro as described above for the
previous experiment. There were six replications (cone-
tainers) per isolate. Controls were 24 replicate plants inocu-
lated with 107 cells of the parent culture of TAL-309 and 10
uninoculated plants. Uninoculated plants were dispersed
among the treatments to check for cross-contamination.
Plants were grown for 6 weeks and harvested, and the dry
weight of the shoots was measured.
The effectiveness of 40 single-colony isolates from broth
culture varied considerably for the three strains (Fig. 1).
Isolates from subcultures 1 and 7 of T-1 were the least
variable. The statistical measure of variability, the F test for
the ratio of variances (14), indicated no significant difference
(at the P = 0.05 level) between the two cultures. However,
the mean effectiveness for culture 7 (F test, P = 0.05) was
higher than for culture 1. Even though the difference was
slight (3%), it indicated that the population was shifting to
higher effectiveness with development of new generations.
Populations of the other two strains were considerably more
variable with respect to effectiveness than was T-1 (Fig. 1),
and continued subculturing significantly increased variability
and reduced the mean effectiveness of the populations by
approximately 9%.
Parent culture controls of T-1 and TAL-309 produced
plants with dry-matter contents in shoots of 101 (+4.5) and
111 (+6.5) mg, respectively. The quantity of dry matter
produced by plants treated with the parent cultures was not
significantly different from the mean of the population
treated with T-1 (Fig. 1), but was significantly higher for
plants treated with TAL-309 (Fig. 1). Strain TAL-309
(CB756) has a history of being a variable strain (6). These
_ Initial culture i = 97 ± 1.4, s2=I Seventh culture i = 100 ± 1.3. S- 68
Tl
mlI.m
_
Initial culture S - 89 ± 2.2, s2 187
= Seventh culture i - 81 ± 3.1, S 337
TAL-309
n n. n 1S b 101I
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APPL. ENVIRON. MICROBIOL.
TABLE 1. Ability of 20 single-colony isolates of Bradyrhizobium
sp. (Vigna) strain TAL-309 from two siratro nodules
to stimulate dry-matter production in shoots of siratro
grown in a nitrogen-deficient medium
Isolate Dry
wt (mg/plant)a
Nodule lb Nodule 2c
1 193 273
2 181 259
3 177 253
4 171 250
5 155 245
6 155 244
7 154 244
8 153 242
9 150 239
10 148 237
11 148 232
12 143 232
13 143 228
14 135 227
15 128 225
16 125 222
17 118 220
18 114 215
19 112 193
20 111 185
aEach value is the mean of results for six plants.
b Least significant difference, 45; coefficient of variation, 28. Average dry
weight was 145 mg. per plant.
cLeast significant difference, not significant, coefficient of variation, 24.
Average dry weight was 233 mg. per plant.
results confirm those of Boonkerd and Weaver (2), who
found that plants treated with the parent culture tend to yield
the same amount of or slightly more dry matter than a mean
of the amount produced by plants treated with individual
isolates.
The mean effectiveness of five isolates of TAL-309 from
each of 10 nodules ranged from 122 to 241 mg of dry matter
produced per plant. Isolates from half the nodules were
considerably less effective than the most effective isolates,
indicating that both highly effective and relatively ineffective
bradyrhizobia were forming nodules on the plant (data not
shown). Single-colony isolates from individual nodules were
generally of similar effectiveness. However, there was one
instance with strain TAL-309 of a statistically significant
difference (at the P = 0.05 level) between isolates from a
single nodule (data not shown).
To provide a better probability of detecting differences in
the population of TAL-309 within individual nodules, 20
single-colony isolates were obtained from each of two nod-
ules. Differences in effectiveness among the 20 isolates were
apparent (Table 1), and there was a large difference in the
mean effectiveness of isolates from the two nodules.
It is possible that the differences in effectiveness within
nodules was due to mixed infection. However, given the low
probability of having mixed infections at our inoculum
density (9), it seems unlikely that this occurred. Also, when
mixed infections occur, one strain is usually dominant (7).
With the high degree of variability expressed by 40 isolates
from the parent culture in the first experiment, it seems more
probable that as a population developed in a nodule, some
variability in effectiveness developed.
The results of our investigation indicate that populations
of bradyrhizobia in cultures and in nodules may be highly
variable with respect to the ability to promote dinitrogen
fixation. It does not appear that continuous subculturing of
bradyrhizobia results in large shifts in effectiveness. It may
be convenient to use cells from a broth subculture to serve as
a starter for another culture, and our results indicate that this
may not be detrimental to production of a quality inoculant.
Individual strains should be examined for stability of effec-
tiveness before stability in serial subcultures is assumed.
This work was supported by United States Department of Agri-
culture-United States Agency for International Development BNF
Limiting Factors Program grant 84-CRSR-2-2521 and Texas Agri-
cultural Experiment Station project H-3701.
We thank Lisa Lucia and Linda Adair for technical assistance.
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2974 NOTES
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Variability in Effectiveness of Rhizobia during Culture and in Nodules

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Variability in Effectiveness of Rhizobia during Culture and in Nodules

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