APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2098-2105 Vol. 53, No.

9
0099-2240/87/092098-08$02.00/0
Copyright © 1987, American Society for Microbiology

Influence of Phosphate on the Growth and Nodulation

Characteristics of Rhizobium trifoliit
KAMTIN LEUNG' AND PETER J. BOTTOMLEYl 2*
Departments of Soil Science' and Microbiology,2 Oregon State University, Corvallis, Oregon 97331-3804
Received 15 April 1987/Accepted 11 June 1987

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external
phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the
rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth
in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over
two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and
cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 ±
0.08 ,uM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 ± 0.04 ,uM
phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to
external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost
viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth.
Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover
(Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphate-
depleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by
including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted
plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating
capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphate-
depleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external
Pi concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05)
higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of
strain 36 at pH 5.5 and 5.0.

Phosphorus is an essential element for the growth of
Rhizobium spp. and is one of the most deficient nutrients in
highly weathered soils containing high proportions of amor-
phous clays and/or iron and aluminum oxides. Since solution
phosphate concentrations of most soils are less than 1 ,uM
(27), it has been hypothesized that Rhizobium strains having
low tolerance to phosphate-deficient environments may have
difficulty in competing with other indigenous soil rhizobia for
nodulation. Surveys have shown, however, that culture
collection representatives of a variety of Rhizobium species
can assimilate Pi at very low solution concentrations (s0.05
,uM), similar to those in soil solutions of phosphate-deficient
soils (11, 31). Strains within Rhizobium species also showed
little variation in their abilities to assimilate phosphate at low
concentrations (s 1 ,uM). Two exceptions were noted:
Bradyrhizobium japonicum USDA 142 (11) and the fast-
growing "Leucaena rhizobia" TAL 582 (5), both of which
had difficulty in growing at phosphate concentrations of si
,uM. It has been proposed that the growth of rhizobia in
phosphate-limited environments either is controlled by the
ability to attain a critical internal phosphate level (31) or is
related to the amount of storage phosphate (12) or to the
efficiency of phosphate uptake and subsequent utilization
(5). One report in the literature indicates that rhizobia might
differ in their phosphate requirements for expressing maxi-
mum nodulation and N2-fixing abilities. Pot experiments in a
high-phosphate-adsorbing, B. japonicum-free soil showed
that the growth response of soybeans to different external
*
Corresponding author.
t Oregon State University Agricultural Experiment Station Tech-
nical Paper no. 8197.
2098
phosphate levels was dependent on the inoculant strain of B.
japonicum used (30). No information has been forthcoming,
however, to suggest that such differences do exist between,
and indeed influence, relative nodulating capabilities by
mixtures of indigenous soil rhizobia.
Recently Almendras and Bottomley (1) found that either
amending Abiqua soil, which is acidic (pH 5.2 ± 0.2) and low
in extractable phosphate (1 to 5 mg of P kg of soil-'), with
monobasic phosphate without changing the soil pH, or
liming the soil to raise the pH to 6.5 enhanced the nodule
occupancy by indigenous serogroup 36 of Rhizobium trifolii
on subterranean clover (Trifolium subterraneum cv. Mt.
Barker). In the study described in this report, experiments
were carried out with the following objectives: (i) to deter-
mine the growth characteristics of representatives of indig-
enous serogroups 6 and 36 under different phosphate con-
centrations typical of soil solution; and (ii) to determine the
influence of phosphate upon the nodulation characteristics of
serogroups 6 and 36 on T. subterraneum cv. Mt. Barker
under nonsoil conditions at pH 5.5 and 6.5.
MATERIALS AND METHODS
Establishing phosphate adsorption and desorption charac-
teristics of synthetic and natural iron oxides. Iron(III) oxide
powder, limonite, and goethite needle ore were obtained
from J. T. Baker Chemical Co., Phillipsburg, N.J., Wards
Natural Science Establishment, Inc., Rochester, N.Y., and
Minerals Unlimited, Ridgecrest, Calif., respectively. Goe-
thite was pulverized and ground to a fine powder before use.
Phosphate adsorption isotherms of the three oxides were
determined at 25°C as described in detail previously (19). In
VOL. 53, 1987
brief, triplicate 3-g samples of oxide were equilibrated with
30-ml portions of 10 mM CaC12 containing 24.3, 48.3, 72.6, or
96.9 ,umol of KH2PO4. The samples were shaken for 1 h daily
over a 14-day period (predetermined to be adequate for
maximum adsorption), and the phosphate remaining in the
supernatants was determined by a modification of the
method of Murphy and Riley (22) described previously (33).
Phosphate desorption characteristics were obtained by shak-
ing intermittently (1 h day-') the air-dried samples from the
adsorption experiment with 30-ml portions of 10 mM CaCl2
at 25°C for 6 days (predetermined to be adequate for achieve-
ment of equilibrium), and the phosphate concentrations in
solution were determined.
Rhizobium strains. The isolates used throughout these
studies, namely strains 6 and 36, are representatives of two
indigenous serogroups, 6 and 36, of R. trifolii, which were
recovered from Abiqua soil and which have been described
previously (17).
Growth of strains 6 and 36 in +PGG and -PGG media.
Bacteria were grown in a previously defined medium (17)
modified as follows. Glucose (10 g liter-') was substituted
for mannitol and autoclaved separately; chelated Fe was
supplied by adding disodium EDTA (27 ,uM) and FeCl3 (14
,uM), and the vitamin mixture of Chakrabarti et al. (13) was
used. 10 mM MES [2-(N-morpholino)ethanesulfonic acid]
was used as a buffering agent, and the medium was adjusted
to pH 5.5 with HCl. Phosphate-supplemented (+PGG) me-
dium contained 2.85 mM phosphate; phosphate-depleted
(-PGG) medium was supplemented with K2SO4 to substi-
tute for the omission of the K+ component of K2HPO4, and
the pH was adjusted to 5.5 with KOH.
Cultures were grown in 30 ml of medium contained in
cotton-stoppered Pyrex (Corning Glass Works, Corning,
N.Y.) test tubes (20 by 2.5 cm) at 30°C in a thermostated
water bath and bubbled continuously with filter-sterilized air
at a rate of approximately 10 ml min-'. To produce phos-
phate-depleted cells the following procedure was routinely
followed. A 0. 1-ml portion of an early-stationary-phase
culture grown under +PGG conditions was used to inoculate
sterile -PGG medium. Growth was monitored until the
culture again reached a constant turbidity (stationary phase).
A 0.1-ml portion of the latter culture was backtransferred
again to -PGG medium, and growth was monitored until the
culture again reached constant turibidity.
Determination of the dry weight, phosphate content, and
morphology of strain 6 and 36 cells grown under different
phosphate regimes. Strains 6 and 36 were grown in +PGG
medium and then phosphate depleted through one or two
consecutive growth cycles in -PGG medium as described
above. The cell densities of the cultures were measured at
stationary phase by plate counting on yeast extract-mannitol
(YEM) agar (35). Cells grown to stationary phase either in
+PGG medium or through one or two consecutive cycles in
-PGG medium were harvested by centrifugation at 25,000 x
g for 15 min, and the cell pellets were washed twice with
distilled water, dried at 55°C to constant weight, and ana-
lyzed for their total phosphate content after digestion by
either MgCl2-perchloric acid (10) or the concentrated H2SO4-
ammonium persulfate (34) method. Phosphate in the digests
was determined as described above. Smears of strain 6 and
36 cells grown under the different phosphate conditions were
prepared, and the cellular dimensions (length and width)
were determined by immunofluorescence microscopy after
the cells had been stained with specific fluorescein-labeled
immunoglobulins raised to somatic antigens as described
previously (14).
INFLUENCE OF PHOSPHATE ON R. TRIFOLII 2099
Growth and viability characteristics of phosphate-depleted
strains 6 and 36 at different phosphate concentrations. Sam-
ples of iron(III) oxide powder containing 0, 8, 13, 16, or 32
iimol of phosphate g of oxide-1 were used to establish
phosphate concentrations ranging from undetectable to 6.54
,uM in growth medium as described previously (11). Samples
(40 ml) of growth medium, each containing a 3-g sample of
iron oxide enclosed in dialysis tubing, were autoclaved for 30
min and incubated at 30°C with continuous aeration for 6
days prior to inoculation. Dissolved phosphate concentra-
tions of the media were analyzed prior to inoculation to
verify the establishment of constant and specific phosphate
concentrations. Cultures of -PGG strains 6 and 36 were
diluted with sterilized 0.15 M NaCl to 105 cells ml-' and
inoculated into growth medium containing different amounts
of phosphate to give an initial cell density of approximately
103 cells ml-1. Growth was monitored by determining viable
cell counts on YEM agar plates. Phosphate concentrations in
the media were also determined at 24 and 72 h after inocu-
lation to confirm that the oxide samples had adequate
buffering capacity to maintain a constant concentration of
phosphate in solution during the monitoring period. Attach-
ment of strain 6 and 36 cells to the dialysis tubing containing
the iron(III) oxide samples was also assessed at different
time intervals by shaking the sacs in 50 ml of partially
hydrolyzed gelatin in ammonium phosphate (21) for 15 min
and plate counting the solution onto YEM agar plates.
Assessment of the influence of iron(III) oxide on the chem-
ical composition of the bacterial growth medium. Portions of
-PGG medium were treated with samples of iron(III) oxide
containing 0, 12.9, and 23.2 ,umol of phosphate g of oxide-1.
Growth medium without added phosphate and not exposed
to iron(III) oxide was included as a control. After 6 days of
incubation to establish equilibrium of phosphate in solution,
samples of each medium were analyzed for mineral com-
ponents by using a Jarrell-Ash inductively coupled argon
plasma spectrometer.
Evaluation of the nodulating ability of phosphate-depleted
strains 6 and 36 under different external phosphate and pH
conditions. Plant growth medium (+ PGM) used in these
studies was one-half strength of the mineral solution de-
scribed previously (17). The solution was buffered at pH 5.5
or 6.5 with 10 mM MES. K2SO4 (2.5 mM) was added to
substitute for the potassium content of phosphate-depleted
plant growth medium (-PGM). A factorial experiment was
carried out involving -PGG cells of strain 6 or 36, +PGM
(2.85 mM phosphate) or -PGM plant growth medium, and
two pH values (5.5 or 6.5) of the plant growth medium.
(i) Inoculant preparation. -PGG bacteria were grown as
described above, harvested, washed, and diluted to approx-
imately 105 cells ml-' with distilled water.
(ii) Evaluation of the stability of nodulating competence of
strains 6 and 36 after exposure to -PGG conditions. -PGG
cells of strains 6 and 36 were prepared as described above.
Portions of the cultures were plated onto YEM agar and
incubated at 27°C. A total of 25 colonies of each strain were
harvested and inoculated onto subterranean clover seedlings
growing on mineral nutrient agar slants (17). At the same
time, strain 6 and 36 cells grown in normal +PGG medium
(2.85 mM) were used to inoculate subterranean clover seed-
lings in replicates of five to serve as controls. Plants were
grown under greenhouse conditions as described previously
(17), and nodulation was examined 2 weeks after inocula-
tion. Shoot dry weights were determined on plants harvested
6 weeks after inoculation. All of the colonies recovered from
the -PGG cultures of either strain were capable of nodu-
2100 LEUNG AND BOTTOMLEY
nynTd P (kg oxide)-
30F
,.01
--0 D growth as described above. When constant turbidity was
20
VII1. .W
1, le,
0 .,,-
p
- pH 5, 5.5, 6.5, 9, or 11. After the samples had been incubated
0.
i0G
V RESULTS
P In solution (pM)
FIG. 1. Adsorption and desorption isotherms of different iron
oxides. Sorption isotherms ( ) and desorption isotherms (-----) of
synthetic iron(IIl) oxide (-), limonite (0), and goethite (U).
lating and were as symbiotically effective as luxury phos-
phate-grown cells of the same strains (data not shown).
(iii) Preparation of seedlings and inoculation. Measurable
levels of contaminating phosphate were detected in both the
plant growth medium (0.2 RM) and the paper supports (20 ,ug
of P support-1) contained within the commercially available
growth pouches (Northrup King, St. Paul, Minn.). Contam-
inating phosphate in the plant growth medium was removed
effectively by pretreatment with iron(III) oxide powder. The
paper supports were substituted with replicas constructed
from no. 1 chromatography paper (Whatman, Inc., Clifton,
N.J.), which was found to contain neglible quantities of
phosphate or nitrogen. A 7-ml portion of either -PGM or
+PGM plant growth medium (at pH 5.5 or 6.5) was pipetted
into each pouch. The pouches were steam sterilized for 2 h
on two consecutive days prior to use. Surface-sterilized
seeds of T. subterraneum cv. Mt. Barker were germinated as
described previously (9). Vigorous and uniform seedlings
were chosen, and three were transplanted aseptically to each
of the growth pouches. Each seedling was inoculated with 10
RI of the appropriate bacterial culture to achieve an
inoculant of about 1,000 cells per plant. Three replicates of
five pouches were prepared for each treatment and placed
into a temperature-controlled growth chamber described
previously (15). The root temperature of the plants was
precisely controlled at 25°C by suspending the pouches in
water baths, and the shoot temperature was maintained
between 23 and 28°C throughout the experiment. Pouches of
seedlings exposed to each external phosphate and pH treat-
ment were included as noninoculated controls and to assess
nodulation by luxury phosphate-grown cells of both strains.
Nodules were enumerated at 2-day intervals up to 20 days
after inoculation. Sterile distilled water was added periodi-
cally in quantities just sufficient to saturate the paper sup-
ports.
(iv) Growth of strains 6 and 36 on the rhizoplane. The
treatments used to investigate growth on the rhizoplane were
identical to those described above, except that four subter-
ranean clover seedlings were grown in each pouch and the
initial inoculant was about 104 cells plant-'. There were
three replicates of each treatment, and samples for rhizo-
plane counts were taken 7 and 21 days after inoculation.
Rhizobia were dissociated from the root systems after re-
APPL. ENVIRON. MICROBIOL.
moval of nodules and enumerated by immunofluorescence as
described previously (15).
Determination of phosphatase activity. Cultures of strains 6
and 36 were grown in +PGG (2.85 mM phosphate) growth
medium at pH 5.5 and subjected to two cycles of -PGG
achieved, 0.5-ml samples of cells were added to dicyclo-
hexylammonium p-nitrophenyl phosphate (8 mg ml-1) in
5.5-ml portions of modified universal buffer (32) adjusted to
at 30°C for 20 to 60 min, 0.5 M NaOH (4 ml) was added to
each sample, and the A400 of the p-nitrophenol produced was
measured. Phosphatase activity was expressed as nano-
moles of p-nitrophenol produced per milligram (dry weight)
per minute.
The phosphate adsorption and desorption isotherms of
synthetic iron(III) oxide and of natural samples of limonite
and goethite were established to compare their phosphate-
buffering capacities (Fig. 1). The synthetic oxide had the
greatest adsorption capacity of the three oxide tested and
under desorption conditions had a greater reserve of phos-
phate than limonite after establishing a range of specific
concentrations of phosphate in solution. This latter charac-
teristic, along with the reproducibility obtained from dif-
ferent batches of the synthetic oxide, and the cost factor
resulted in our using synthetic iron(III) oxide powder as the
phosphate-buffering agent of choice.
Two artifacts of the system were discovered during this
study which resulted in an increase in the Mn2+ concentra-
tion of the bacterial growth medium from 6 to 50 puM after
exposure to samples of phosphate-charged iron(III) oxide. In
addition, the Ca2+ concentration of the growth medium was
raised from 0.6 to 1.3 mM after exposure to phosphate-
charged iron(III) oxide. However, it was determined subse-
quently that neither of these two effects influenced the
growth behavior of strain 6 or 36 in the phosphate-buffered
system (data not shown).
The generation times of strains 6 and 36 were rather
similar (3.4 and 3.5 h, respectively) in +PGG defined bacte-
rial growth medium at pH 5.5 and 30°C (Fig. 2). The
generation times of strains 6 and 36 during the exponential
phase of the first growth cycle in -PGG medium were
similar but longer (5.0 and 4.6 h respectively), and the
terminal cell densities (approximately 3 x 108 cells ml-') of
both strains were similar to but significantly lower than the
final cell density achieved in +PGG medium. During the
second -PGG growth cycle, the generation time of strain 36
was extended to 9.8 h while that of strain 6 remained
unchanged (5 h). Despite this difference, the terminal cell
densities of the second -PGG cultures were similar to but
substantially lower than those observed during the first
-PGG growth cycle.
Despite the different growth rates achieved under the
second cycle of growth under -PGG conditions, the internal
phosphate concentration (on a dry weight basis) of strains 6
and 36 at stationary phase were similar regardless of the
phosphate status on the growth medium. Neither were
differences between the two strains apparent when phos-
phate content was expressed on a single-cell basis (Table 1).
Under -PGG conditions, strains 6 and 36 both changed
morphologically in a similar manner, with both their cell
weights and cell lengths increasing to twice those of cells
grown under +PGG conditions (Table 1). Cells of -PGG
VOL. 53, 1987 INFLUENCE OF PHOSPHATE ON R. TRIFOLII 2101

under the same circumstances (Fig. 4). At phosphate con-

centrations of -0.56 ,uM, strain 36 lost viability within 48 h,
and it commenced proliferation only after a 48-h lag phase
when the phosphate concentration was 0.89 + 0.04 ,uM. No
evidence was found for cells of either strain 6 or strain 36
attaching to the walls of the dialysis sac, which might have
been a factor affecting the number of viable cells of strain 36
recovered from the growth medium (data not shown).
Phosphatase activity was found to be high in cultures of
-PGG strains 6 and 36 (Table 2) and was up to 200-fold
higher after two cycles of -PGG growth than in +PGG
medium. pH also influenced the activity of the phosphatase
CO 5 of both strains, which was significantly greater (P = 0.05) at
0
Strain 36 pH 6.5 than at pH 5.5 after one cycle of -PGG growth and
significantly greater (P = 0.05) at pH 5.5 than at pH 5.0 after
two cycles of depletion. The phosphatase activity of -PGG
strain 6 was significantly greater (P = 0.05) than that of strain
36 at pH 5.0 and 5.5.
Both -PGG strains 6 and 36 were able to nodulate T.
subterraneum cv. Mt. Barker regardless of the phosphate
status of the plant growth medium or the pH (Fig. 5).
Although the numbers of nodules formed by day 20
postinoculation were similar for all treatments, significant
differences were observed in the time of appearance of
nodules for strain 36 subjected to different treatments and
between strains 6 and 36 exposed to seedlings in -PGM
0 20 40 60 80 100 plant growth medium at pH 5.5. For example, at pH 5.5 in
Time (h)
-PGM plant growth medium, nodules became visible on
FIG. 2. Growth of R. trifolii 6 and 36 under +PGG and -PGG plants inoculated with -PGG strain 36 at 10 to 12 days
conditions. Symbols: 0, growth in 2.85 mM phosphate; *, growth
after transfer from 2.85 mM phosphate to -PGG conditions; O,
growth in second phosphate depletion cycle under -PGG condi-
tions. Values on each curve represent the mean of three separate
determinations. All values for standard deviations (95% confidence
level) are less than the size of the symbols.

strain 6 were observed on occasion to express pleiomorphic

morphology.
-PGG strains 6 and 36 showed different responses when
exposed to growth medium containing different phosphate
concentrations (Fig. 3 and 4). The strain 6 inoculum did not
proliferate, but remained viable for at least 72 h when
solution concentrations of phosphate were s0.28 ,uM. At a
phosphate concentration of 0.51 ± 0.08 ,uM, strain 6 showed -4 C
ability to grow after an extended lag phase of 24 h. At
solution phosphate concentrations of .6.45 ,uM, -PGG
strain 6 commenced proliferation without any lag period at a
growth rate equivalent to that achieved when -PGG strain 6
was reintroduced into normal +PGG (2.85 mM P) growth 2
medium. The behavior of -PGG strain 36 was quite different
TABLE 1. Characteristics of R. trifolii 6 and 36 after growth
under + PGG and -PGG conditionsa b
Phosphate contentc
Dry wt (cell basis)
Dry wt basis (mg of Cell basis (10-13 g cell-')
Strain P g [dry wtL-') (10-15 g cell-')
0 20 40 60 80
B, Bo B, Bo B, Bo Time (h)
6 7.7 1.3* 4.3 1.4* 5.6 11.0* FIG. 3. Growth of R. trifolii 6 in different concentrations of Pi at
36 6.9 1.7* 3.2 1.4* - 4.6 8.0* pH 5.5. The following phosphate concentrations represent the mean
a See Materials and Methods for details of experimental procedures.
values achieved in two separate experiments ± 1 standard devia-
b t test comparisons were made on the pairs of specific parameter values
tion. (a) *, 2.85 mM; O, 6.50 + 0.3 zM. (b) 0, 0.89 + 0.04 ,uM; 0,
obtained for each strain grown under the two phosphate conditions (*, P = 0.51 + 0.08 ,uM. (c) *, 0.28 pM;O, zero. Vertical bars represent
0.05). No paired comparison made between strains was significantly different. the standard deviations at the 959o confidence level. Absence of
C B, and Bo represent cells after growth under + PGG and after two cycles vertical bar indicates that the standard deviation is smaller than the
of batch culture growth under -PGG conditions, respectively. size of the symbol.
2102 LEUNG AND BOTTOMLEY APPL. ENVIRON. MICROBIOL.

+
-,4
E
3
2
0
0)
0

i!6
0
4) Time (d)
0
FIG. 5. Nodulation characteristics of -PGG R. trifolii 6 (-) and
L__ 36 (OI). (a) Nodulation at pH 5.5 in -PGM plant growth medium. (b)
Nodulation at pH 5.5 in plant growth medium containing 2.85 mM
phosphate. (c) Nodulation at pH 6.5 in -PGM plant growth me-
dium. (d) Nodulation at pH 6.5 in plant growth medium containing
2.85 mM phosphate. Vertical bars represent standard deviations at
0 20 40 60 80 the 95% confidence level. Absence of vertical bar indicates that the
Time (h) standard deviation is smaller than the size of the symbol.
FIG. 4. Growth of R. trifolii 36 in different concentrations of
phosphate at pH 5.5. The following phosphate concentrations rep-
resent the mean values achieved in two separate experiments ± 1 when -PGG bacteria were inoculated onto seedlings at pH
standard deviation. (a) *, 2.85 mM; L, 6.50 ± 0.03 ~LM; *, 0.28 5.5 in -PGM plant growth medium (Fig. 5a). Strain 6
,uM; 0, zero. (b) 0, 0.89 + 0.04 ,uM; 0, 0.51 ± 0.08 p.M. Vertical
bars represent the standard deviations at the 95% confidence level. showed superior nodulating ability over strain 36 in both the
Absence of vertical bar indicates that the standard deviation is time to first nodule appearance and the number of nodules
smaller than the size of the symbol. which had developed on five of the seven sampling dates (P
= 0.05) (Fig. 5a).
At pH 6.5, both the time of nodule appearance and the rate
postinoculation (Fig. 5a). However, when cells of -PGG of nodule formation by both organisms were similar (Fig. 5c
strain 36 were exposed to seedlings developing in +PGM and d). An influence of pH was apparent with regard to
plant growth medium at pH 5.5 (Fig. Sb), nodules were -PGG strain 36, which nodulated 2 days earlier at pH 6.5
clearly visible 8 to 10 days postinoculation, and the number (Fig. 5c) than at pH 5.5 under -PGM plant growth condi-
of nodules continued to accumulate at a rate significantly tions (Fig. Sa).
greater (P = 0.05) than for the same strain under -PGM Despite the stringently low phosphate status of the sys-
conditions (Fig. Sa). Significant (P = 0.05 differences be- tem, the magnitude of rhizoplane populations indicated that
tween strains given the same treatment were observed only strains 6 and 36 had both proliferated to a similar extent
during the 7-day postinoculation period (ranging from 5.4 ±
2.2 to 8.4 ± 1.0 generations), regardless of external phos-
TABLE 2. Influence of pH on the phosphatase activities of phate or pH treatments (Table 3). Despite acceleration of
R. trifolii 6 and 36 grown under + PGG and -PGG conditionsa nodulation by -PGG strain 36 as a result of external
Phosphatase activity (nmol of p-nitrophenol phosphate at pH 5.5 or pH 6.5, no significant influences of
mg [dry wt]-1 min-') for strain: either of those treatments were observed when rhizoplane
densities of strain 36 were compared for treatments at either
pH 6 36 7 or 21 days postinoculation (Fig. 6). Rhizoplane enumera-
+ PGGb 1st 2nd G 1 st 2nd tions at 21 days revealed that there had been a sufficient
-PGGc -PGGd +PGG _PGG -PGG increase in the populations of bacteria on the rhizoplanes
5.0 1 42 54 1 30 30 between 7 and 21 days, which (with the exception of strain 6
5.5 1 54 178 1 38 76 at pH 6.5) had allowed their densities to be maintained on the
6.5 1 235 230 1 190 255 root systems. No effects of treatment on seedling growth
9.0 0 22 57 0 44 58 were apparent over the 21-day period. Plant dry weights
11.0 0 16 15 0 26 33 were similar, and no visible symptoms of phosphate defi-
ciency were present (data not shown).
LSDo.05 0.1 53 51 0.4 42 15
a See Materials and Methods for details of experimental procedures.
b Glucose-glutamate-defined medium with 2.85 mM phosphate. DISCUSSION
c Cell transferred from +PGG to -PGG growth medium (contained 0.58
The findings reported here can be discussed in relation to
,uM contaminating phosphate) and grown until constant cell density was those in the accompanying paper (2) on the effects of soil
reached.
d Cells from first -PGG cycle were transferred to another batch of -PGG acidity and phosphate on nodulation behavior by indigenous
medium and grown until constant cell density was reached. serogroups 6 and 36 directly in soil. Furthermore, they are of
VOL. 53, 1987 INFLUENCE OF PHOSPHATE ON R. TRIFOLII 2103

TABLE 3. Number of generations completed by R. trifolii 6 and 36 on subterranean clover rhizoplanes after 7 and 21 days"
No. of generations' for strain":
Time (days) 6 36
postinoculation
pH 5.5, pH 5.5, pH 6.5, pH 6.5, pH 5.5, pH 5.5, pH 6.5, pH 6.5,
-PGM + PGMd -PGM + PGM -PGM + PGM -PGM + PGM
7 7.5 (1.7)d 7.1 (0.5) 8.4 (1.0) 8.1 (0.5) 5.7 (0.9) 6.4 (0.3) 5.4 (2.2) 5.7 (2.3)
21 10.1 (1.4) 9.5 (0.6)* 8.2 (0.2) 9.2 (1.5) 9.0 (1.3)* 8.6 (1.0)* 8.3 (0.9) 7.2 (0.7)
a
See Materials and Methods for details of experimental design and analytical procedures.
b Number of generations determined from the differences between the initial inoculant sizes and the populations on the rhizoplanes at the specified times.
Numbers in parentheses represent standard deviations at the 95% confidence level.
c Within a column the 21-day mean significantly (*, P = 0.05) exceeds the 7-day mean as tested by paired t comparisons.
d + PGM, 2.85 mM phosphate.

significance to the concepts being established in the litera-
ture regarding phosphate metabolism on Rhizobium spp. in
general.
Strains 6 and 36 exhibited differences in phosphate metab-
olism (i) during the preparation of phosphate-depleted cells,
(ii) in terms of their critical external phosphate requirements
for growth, (iii) in terms of their viability under extremely
low external phosphate conditions, and (iv) in their
nodulating capabilities at pH 5.5 under low external-
phosphate conditions. These differences were observed al-
though both organisms achieved similar cell densities, cell
morphology, and similar cellular phosphate contents in
either +PGG or -PGG media. Although strain 6 had a lower
critical external phosphate requirement (0.51 ± 0.08 ,uM)
than strain 36 (0.89 + 0.04 ,uM), both values are higher than
those determined for the majority of other Rhizobium spp.
tested, which included two strains of R. trifolii (5, 11). Our
values, however, can be related to the phosphate character-
istics of the Abiqua soil from which the organisms were
originally isolated. This soil has a high capacity to adsorb Pi
(650 mg of P kg-' produces a soil solution of 5 ,uM), and soil
solution Pi concentrations of the unamended soil range
between the limits of detection (0.1 ,uM) and 0.7 ,uM.
Addition of 25 to 55 mg of P kg of soil-', which stimulates
nodulation of soil-grown plants by strain 36 (2), raised soil
solution phosphate to 0.8 to 1.0 ,uM: a concentration similar
to the critical level required for growth of strain 36.
The critical values we obtained are similar to levels
established for ammonium (0.6 F.M)- and nitrite (0.3 p,M)-
oxidizing bacteria residing in highly weathered, high-
phosphate-adsorbing Ultisols (20). The critical levels for
-W
--6
A
these two representatives of R. trifolii are lower than solu-
tion phosphate values considered to be optimum for growth
of the host plant, subterranean clover, which range between
1 and 10 ,uM (3, 24).
The loss of viability of strain 36 in growth medium
containing phosphate concentrations of '0.51 + 0.08 ,uM
bears some resemblance to findings of others (5, 6). Several
strains of Rhizobium meliloti were observed to lose viability
in low-phosphate medium containing less than 1.5 mM Ca2 .
Ca2+ has also been shown to improve phosphate uptake by
other microorganisms such as Caulobacter spp. (25) and
cyanobacteria (28). In this regard, we had determined that
soil solutions of Abiqua soil before and after liming with
Ca(OH)2 contained 1.0 and 3.5 to 4.0 mM Ca2+ respectively
(A. Almendras and P. J. Bottomley, unpublished observa-
tions). However, we determined that raising the Ca2+ con-
centration of the growth medium from 0.6 to 3.5 mM had no
effect on either the viability of -PGG strain 36 under
subcritical phosphate levels or the critical phosphate level
needed to initiate cell proliferation (data not shown).
The physiological reasons for the different tolerances and
phosphate-sequestering abilities of R. trifolii 6 and 36 under
low-phosphate conditions remain unknown. Two possibili-
ties can be proposed on the basis of the literature. Poole and
Hancock (25) have observed variation in the ability of
different Pseudomonas spp. to synthesize inducible mem-
brane proteins in response to phosphate starvation, which in
some cases correlated with their inability to grow under low
(0.1 mM)-phosphate conditions. Evidence has also been
obtained for leakage of Pi and phosphorus-containing metab-
olites from a marine strain of Rhodotorula rubra when
growing under phosphate-limited conditions (29). Further
studies are required to elucidate whether these are plausible

r-6--36
6 I36I reasons for the differences in behavior of strains 6 and 36.
Very little work has been done on the nodulation of roots
00%
C0) by phosphate-depleted rhizobia under conditions of phos-
4) 8 phate-depleted plant nutrient solution. Our data suggest that
0
neither the internal phosphate status of the bacteria nor the
external Pi concentration is critical for nodulation to occur.
Although the concept of the rhizosphere as a zone extremely
C I depleted in Pi is well established (7, 8, 16), the ability of
strains 6 and 36 to proliferate on the rhizoplane implies that
they were obtaining organic phosphate from the roots on the
uV
Pi Po
I I

Pi Po P1
subclover plants by utilizing their acid phosphatase capabil-
Po P Po

P status of plant growth medium
FIG. 6. Rhizoplane densities established by -PGG R. trifolii 6
and 36 at 7 days (O) and 21 days (@) after inoculation onto subclover
seedings in plant growth mediuni at pH 5.5 (A) and pH 6.5 (B). P0
and P1 represent plant growth medium containing either zero or 2.85
mM phosphate, respectively. Vertical bars represent the standard
deviations at the 95% confidence level.
ities. The observations of Barber and Loughman (4) should
not be overlooked, however, with the possibility that strains
6 and 36 might also have sequestered Pi leaking from the
plant roots under the extremely low external solution phos-
phate concentrations.
The improved speed of nodulation by -PGG strain 36 at
pH 5.5 as a result of the presence of Pi in the plant growth
2104 LEUNG AND BOTTOMLEY
medium can be related to its inferior survivability and
phosphate-sequestering abilities under low-phosphate condi-
tions. However, it should be stressed that -PGG strain 36
did accomplish similar numbers of generations and achieve
similar rhizoplane densities, regardless of the phosphate
status of the plant growth medium over the first 7-day
period. The improvement in nodulation by -PGG strain 36
after the pH of the plant growth medium was raised to pH 6.5
is in agreement with our previous soil studies (2, 18). We can
only speculate at this time upon a possible role for phospha-
tase. For -PGG strain 36, the activity of phosphatase was
significantly higher at pH 6.5 than at pH 5.5 or 5.0. Further-
more, it is well established that the pH in the zone immedi-
ately adjacent to plant roots can be 1 to 1.5 pH units lower
than the value in the solution some distance from the roots
(23). Perhaps the higher pH facilitated the ability of strain 36
to hydrolyze organic phosphate from the rhizoplane-
rhizosphere zone, whereas the low pH at the rhizoplane of
the roots in the acid solution made this a less effective
mechanism of sequestering phosphate.
In summary, very few studies have been carried out under
controlled environmental conditions in an attempt to better
characterize observations on Rhizobium-legume interactions
made initially under soil conditions. This study establishes a
link between differences in phosphate-sequestering ability
and differences in nodulation capabilities by members of
indigenous serogroups 6 and 36 and can be related to findings
made on the influence of lime and phosphate on nodulation
of subclover by indigenous R. trifolii in soil (2).
ACKNOWLEDGMENTS
This research was supported by funds from the U.S. Department
of Agriculture (Science and Education competitive grant 85-CRCR-
1-1704) and the Oregon Agricultural Experiment Station.
We thank T. Righetti for assistance with mineral analyses and C.
Pelroy for typing the manuscript.
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