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BAREA, MARGARETE. J . uppl. €?act. 37, 583-593.

J. M. & BROWN,

Effects on Plant Growth Produced by Azotobacter


paspali Related to Synthesis of Plant Growth
Regulating Substances
J. M. BARE'\*A K D MARGARETE. BROWN
Soil Microbiology Department, Rothmsted Experimental Station,
Harpenden, Hertfordshire, England

(Received 27 February 1974 and accepted 20 May 1974)

SUMMARY.Treating seedling roots of several plant species with cultures of Azotobucter


pmpali changed plant growth and development and significantly increased weight of
leaves and roots; effects were probably caused by plant growth regulators. Culture
supernatant fluids contained indolyl-3-aceticacid, at least 3 gibberellins and 2 cytokinins.
The added inoculum of A . puspuli survived in plant rhizospheresfor only a few weeks and
no nitrogen was fixed in the root zone of young Paspalurn notatum, the grass with which
A . paapali is associated.

THE NITROGEN FIXING bacterium Azotobacter paspali was found by Dobereiner (1966)
in soils with a pH range of 4.9 to 7.8 and to occur more abundantly in rhizospheres of
Paspalum notatum and P . plicatum than in soil away from roots, but it was absent
from root samples of 81 other pasture plants. Azotobacter paspali improved growth of
P . notatum by fixing atmospheric nitrogen in the rhizosphere (Dobereiner, Day &
Dart, 1972), an ability not known t o be shared by other species of Azotobacter when
associated with roots of any plant species. However, other species of Azotobacter alter
plant growth by producing the growth regulators indolyl-3-acetic acid and gibberellins
(Brown & Burlingham, 1968; Lee, Breckenridge & Knowles, 1970; Azc6n & Barea,
1973). This paper presents the results of examining culture supernatant fractions of
A . paspali for these substances and for cytokinins, and the effects on plant growth
following root treatment with bacterial cultures. Studies on cytokinin production
were made because recently these substances were found in extracts of cells of A .
chroococcum (Coppola, Percuoco, Zoina & Picci, 1971), of Agrobacterium tumefaciens
(Romanow, Chalvignac & Pochon, 1969), and of Corynebacteriumfmciena (Helgeson
& Leonard, 1966; Klambt, Thies & Skoog, 1966), and in RNA preparations from a
wide range of micro-organisms (Skoog & Armstrong, 1970). Cytokinins were also
found in culture supernatant fractions of Arthrobacter sp. (Blondeau, 1970) and
Rhizobium japonicum (Phillips & Torrey, 1970).

Methods
Cultures
Cultures of Azotobacter paspali were grown for 14 days (Brown & Burlingham,
1968) in 100 ml amounts of medium containing (g/l); sucrose, 5.0; K,HPO,, 0.8;
* Present address: Estacion Experimental del Zaidin, Seccion de Microbiologia, Avenida
Cervantes, Granada, Spain.
[5831
584 J. M. Barea and Margaret E. Brown

MgSO,. 7H,O, 0.2; FeS04.7H,0, 0.04; Na,Mo04.2H,0, 0.005; CaCO,, 2.0; pH 7.0,
in 500 ml flasks on a rotary shaker at 28".

Effects of Azotobacter paspali on plant growth


Roots of germinated seedlings of l'aspalum notatunt, Centrosema pubescens, Lyco-
persicum esculenturn cv. Money Maker, Lactuca sativa cv. Tom Thumb, Lolium perenne
and l'riticum cdgare cv. Cappelle-Desprcz were dipped in cultures of Azotobacter
paspali before transplanting either to standard John Innes potting compost number 3,
pH 5.6, or to a light yellow latosol from the cerrado, Brazil. This latosol is a sand with
low organic matter content and little available phosphate [2.4 p/ni of P soluble in
0.5 M NaHCO, a t pH 8.5 (Olsen, Cole, Watanabe & Dean, 1954);pH 4.8; 0.15% of N].
An amount of Ca(H,PO,), (0.1 g ) was added to each pot of Brazilian soil a t the start
of the experiments. Control seedlings were dipped in sterile water or diluted culture
medium without sucrose. Plants were grown in the greenhouse a t a mean day tem-
perature of 21" and night temperature of 13" except for P. notatum and C. pubescens
which were grown a t a day temperature of 30"-35" and night temperature of 30".
Plants were fed with nutrient solution as required. Stem and leaf length and number
of tillers were measured on all plants, except lettuce, a t 14 day intervals. Tomatoes
were grown until fruit developed, and trusses were graded as described by Brown,
Jackson & Burlingham (1968). Dry weights of shoots and roots of all plants, except
tomato, were obtained after 8 weeks.

Establishment of Azotobacter paspali on roots


Roots of young seedlings (7-14 days) of the different plant species were treated with a
14-day culture of A . paspali so that each received c. 7 x 106 cells before trans-
planting. Rhizosphere soils were sampled a t 14 day intervals and A. paspali
counted on a nitrogen-deficient medium containing (g/l); sucrose, 20.0 ; K,HPO,,
0.05; KH,P04, 0.15; MgS04.7H,0, 0.2; CaCl,, 0-02; FeCl,, 0.01; Na2M00,.2H,0,
0.002; CaCO,, 2-0; agar, 15.0; 0.5% alcoholic solution of bromothymol blue, 5 m l ;
pH 7.0. Numbers were related to 1 g of dry rhizosphere soil.

Examination for plant growth regulating substances


Extraction procedures
Cultures were centrifuged at 2000 g for 20 min; each supernatant fluid was then
divided into 2 equal volumes and extracted either for gibberellin-like substances or for
indolyl-3-acetic acid (IAA) using methods described by Brown & Burlingham (1968)
and Brown & Walker (1970), respectively. Cytokinins were extracted from the super-
natant fluid using a method described by Wheeler (1972). After acidifying to pH 3.0
the fluid was extracted 3 times with ethyl acetate. The aqueous fraction was then
separated on a column (10 x 250 rnm) filled with Amberlite resin IR-lBO(H). Sugars
were eluted with 50ml of water, and purines, including cytokinins, with 50ml of
1.0 N ammonia solution. In test experiments with authentic kinetin (Sigma Chemical)
c. 60% was recovered from the aqueous phase and 40% from the ethyl acetate phase.
Hemberg & Westlin (1973) also found that > 90% of authentic kinetin partitioned in
Growth substances produced by A. paspali 585

the ethyl acetata fraction, and that about half the activity of cytokinins from plant
tissues extracted a t pH 3.0 was lost in the ethyl acetate fraction. Therefore, t o guard
against loss of cytokinin from the supernatant fluid extract both the ethyl acetate
fraction and the ammoniacal efluent from the ion-exchange column were concentrated
and used for paper partition chromatography.

Paper partition chromatography


The different extracts and samples of authentic growth regulating substances were
examined on Whatman No. 1 chromatography paper with solvent system freshly
mixed isopropanol : ammonia sp. gr. 0.88 : water, 10 : 1 : 1 by volume. Rf values of
authentic substances differed slightly with each development but all produced fluorcs-
cent spots under UV light (350nm) in the same positions relative to each other after
treating chromatograms with chromogenic reagent, 5% (vlv)conc. H,SO, in methanol.
Rf values ranged from 0.3-0.4 for IAA (Light & Co.),0.5-0.7 for GA, (Light & Co.)
and 0.74.8 for kinetin. To confirm the presence of growth regulators, chromatogram
portions not treated with reagents were dried for 7 days to remove solvents. Ten
equal strips representing the sequence of RI values 0-1-1.0 were eluted separately for
use in bioassays.

B&sa ys
Presence of growth regulators in extracts of the supernatant fractions was first
indicated by their effects on the growth of yeast (Barea, Navarro, Palomares &
Montoya, 1974). Saccharomyces cerevisiae, strain 18, was inoculated into a series of
flasks of liquid medium containing a range of concentrations of IAA, GA, or kinetin
or the eluate of each Rf value. After 24 h a t 28",optical density (o.D.) of each culture
was measured a t 650 nm, and the number of cells in each culture counted. Optical
density and cell number were related linearly to type and concentration of growth
regulator.
Gibbercllin-likesubstances were assayed with 3 tests used by Brown & Burlingham
(1968) as follows: elongation of dwarf pea internodes; of lettuce hypocotyls; and
increased growth of tomato. IAA was assayed by measuring length increase of
wheat coleoptile segments incubated a t 25' in water containing the appropriate
portion of chromatogram (Brown & Walker, 1970). Cytokinins were assayed using the
radish cotyledon expansion test (Letham, 1971) and by determining the O.D. of
chlorophyll retained in excised first leaves of oat (Wheeler, 1972).
Amounts of growth regulators were calculated from dose response curves with
authentic substances and given as pg equivalentslml of culture supernatant fluid.

Results
Effects on plant growth of treatment with Azotobacter paspali
Treating seedling roots with cultures of A . paspali affected development of all plant
species grown in potting compost, but not in Brazilian soil. There was significant
improvement in growth of tomato, lettuce, Centrosema pubescens and Papalum
notaturn, but not of Lolium perenne and wheat, although leaves were slightly longer
586 J. M, Barea and Margaret E. Brown

than those of controls and the number of tillers of wheat was increased significantly.
Tomatoes were altered through to fruit set with most noticeable effects on young
plants, for example, when 4 true leaves had formed, stem length was increased by
145y0 and leaf length by 68% compared with controls treated either with water or
diluted medium (Plate I), but when 12 true leaves had formed differences were still
visible but no longer significant. Figure 1 shows that root treatment shortened
development time of the first and second trusses by 7 and 4 days, respectively, but the
number of flower buds on both trusses was not affected. Two weeks after treatment
the number of leaves of Paspalurn notatum was increased by 18% and their length by
49% (Plate 1); these effects were maintained for 4 weeks before decreasing.

40 t /
.0
-

I I I I I I I I I I I
2 4 6 8 10 12 14 16 18 20 22
Days
Fig. 1. Effect of A . pmpali on development of first and second trusses of tomato. First
truss: 0 , A . paspali; x , control. Second truss: 0,A . p a s p d i ; A, control.

Two sets of P. notaturn plants were harvested 4 weeks after treatment, one set had
received A . paspali and the other set waa of control plants. Roots of both were
checked for presence of A . p p a l i , which was found only on those from the inoculated
set. Roots of both were tested for nitrogenase activity by the acetylene reduction
technique. Whole root systems with closely adhering soil were placed in 28 ml
McCartney vials and assayed as described by Dobereiner et al. (1972). There was no
nitrogenase activity. I n spite of A . paspali being present on inoculated roots it was
not fixing measurable amounts of nitrogen in the rhizosphere of these young plants,
but was affecting growth by another mechanism. Table 1 shows that dry weights of
inoculated plants were significantly greater than those of control plants.

Establishment of Azotobacter paspali in plant rhizospheres


The inoculum of A . p p l i declined rapidly in Brazilian soil, even in the rhizospheres
of P. notaturn where it normally thrives under natural conditions; decline was less
rapid in potting compost (Table 2).
PLATE1. Effect of root treatmcnt with A . pctspnli on ((1) tomato and ( 6 ) Pnspalum
nottiturn.

Bact. f. p. 586
Growth substances produced by A. paspali 587

Production of plant growth regulators by Azotobacter paspali


When examined under UV light, chromatograms of extracts of A . paspali showed
fluorescent spots between Rf0 and 0.16, 0.3 and 0.4, and 0.7 and 0.8. There was no
fluorescence corresponding to the position of authentic GA,. The spot with Rf0.3 to
0.4 corresponded in position to authentic IAA and became coloured pinli by treatment
with Gordon & Weber’s reagent (0.05M FeCl, in 35% (v/v) perchloric acid); authentic
IAA behaved similarly. The spot with Rf 0.7 to 0.8 corresponded in position to
kinetin but the fluorescence differed from that of the authentic substance.

TABLE1
Effect of root treatment with A. paspali on dry weight of plants grown in
potting compost
Dry weight of plants (g)
A
r
Species Shoots Roots
A A
/ I
+ Azotobacter + Azotobacter
SP . Control LSD (5%) SP. Control LSD (5%)

L. perenne 3.37*** 2.92 0.18 4.80 2.76 3.5


Wheat 2.32 1.56 0.93 1.36* 0.65 0.61
Lettuce 0.246** 0.166 0.054 0.022** 0.008 0.009
G . pubeacena 1.62* 0.98 0.58 0.36*** 0.18 0.10
P . notaturn 0.484*** 0.179 0.050 0*058*** 0.023 0.008

Significance at * 6% level, at ** 2% level, at *** 1% level.

TABLE2
Change in numbers of A . paspali in rhizospheres of diflerent plant species
over a period of 2-8 weeks
Nos/g of dry rhizosphere soil
A,
/ \
Species J. I. Compost Brazilian soil
4
. L
/ >
2 4 6 8 2 4 6 8

L. perenne 182 66 0 58 17600 1300 290 0


Wheat 40 12 22 48 14300 0 0 0
Lettuce 1970 0 0 65 1600 13 155 7
Tomato - - - - 27600 20600 7200 1800
C . pubescena 58 7 0 7 14300 4600 450 180
P . notdurn 4823 219 2079 1325 180 63 63 6

Yeast bioassay
Table 3 shows the O.D. and number of cells in the cultures of Saccharomyces cerevisiae
after incubation with the eluates of each Rfvalue. Increased O.D. and cell number by
eluates from the ethyl acetate fraction a t Rf0.3 to 0.4,0.5 to 0.7 and 0-7 to 0-8 and
by eluates from the aqueous fraction at Rf0 to 0.1 and 0.7 to 0.8 indicated probable
activity of IAA, GA, and cytokinin. Calculated from a dose response curve obtained
42
588 J. M. Barea and Margaret E. Brown

with authentic substances the amounts of hormone equivalents/ml culture were


IAA 0.3 pg, GA, 1.5 pg and cytokinin 1.2 pg.

IAA
In the wheat coleoptile test the eluted substances with Rf 0.3 to 0.4 possessed auxin
activity. In tests with different batches of culture the amount of IAA ranged from
0.01 to 0.15 pg equivallents/ml culture.

TABLE 3
Re-sponses of Saccharomyces cerevisiae to eludes of each Rfvdue from
chromatograms of extracts of A. paspali
Response of Sacchaaromyoes cereviaiae to eluates from chromatograms of
(frmtions)of extrmts of A . paapali
A
r >
Rfvalue Ethyl metete fraction Aqueous fraotion
A A
.
I - , I >
Cell no. Cell no.
O.D. ( x 10e)/ml O.D. ( x 1O6)/ml
. ~~~

0-0-1 0.663 12.7 18.9 *


0-1-0.2 0.600 10.8 16.8)
0-2-0.3 0.428 10-5 0-600 11.3
0.3-0' 4 0.6703 13*3$ 0.6893 13-23
0.4-0.6 0.669 14.0 0.428 10.6
0.6-0.6
0.8-0.7 :::;; 0 * 600
0.662
10.5
12.9
0.7-0.8
0.8-0.9
0.9-1.0
::::;)
0.662
*
17.2
16.8)
13.0
*
::g)*
0.663
18.1 *
17-6)
12.8
Control 0.663 12.8 0.666 12.9
* Response indiocttes presenoe of cytokinine.
t Response indioates presenoe of gibberellins.
1Response indiostes presence of IAA.

Gibberellins
Pea bioassay. Figure 2 shows effects of eluates of each Rf value on pea internode
extension. Significant increases were produced by Substanceswith Rfvalues between
0 and 0.2, 0-4to 0.7 and 0.8 to 0.9; that between Rf 0.4 and 0.7 with maximum
activity at Rf 0.55 corresponded in position to authentic GA,, and waa equivalent to
0.1 pg GA,/ml culture supernatant fluid. Significant internode extension produced
by the other substances also indicated gibberellin-like activity. Substances with Rf
0.2 to 0.3 significantly decreased internode growth.
Lettuce hypomtyl bioassay. Figure 2 also shows effects of eluates on lettuce hypocotyl
extension. Significant increaaes were produced by substances with Rf0 to 0.1, 0.4 to
0.7 (corresponding in position to authentic GA,) and 0.8 to 0.9. The supernatant
fluid fraction waa calculated to contain 0.03 pg CIA, equivalentslml. Significant
hypocotyl extansion by the other substances also indicated gibberellin-like activity.
Growth substances produced by A . paspali 589

Tonmto bioassay
Tomato seedlings roots were treated with extracted but not partitioned super-
natant fluid and with eluates of each Rf value from partitioned extract. Extracts of
supernatant fluid behaved like whole culture in increasing significantly growth of
stems and leaves; when 4 true leaves had formed increases were 40 and 280/, respec-
tively, over controls. Significant increases in leaf growth were produced until 6 true
leaves were formed by eluates of substances with €if 0 to 0.2, 0.4 to 0.7, 0.7 to 0-8 with
peaks of activity of 33, 69 and 69% at Rf 0.15, 0.65 and 0.85, respectively. Stem
height was increased significantly only by substances with Rf 0.5 to 0.7. Truss
development was not affected either by non-partitioned or partitioned extract.

Pea bioassay
40
30
20
10
L S.D
0
P=0.05
- 10
I I I I I
0.2 0 4 0.6 0.0 1.0

Leltuce bioassay
6 40
30
C
g 20
g
-
10
L.S D.
s o P=O 05
p -10

'
ae -20 0.2 0.4 06
I?,values
00 1.0

Fig. 2. Effects of eluates of eeoh RIvalue from chromatograms of extra& of A . papdi.

Gytokinina
Table 4 shows results from one of 3 assays determining the optical density of
chlorophyll retained in 5 excised first leaves of oat using aqueous and ethyl acetate
fractions. All assays of the aqueous fraction gave similar results with responses
between Rf 0 to 0.1 corresponding to an unidentified cytokinin, and between Rf 0.6
and 0.8 coresponding in position to zeatin (Wheeler 1972). The original culture was
calculated to contain 0.02 pg cytokinin equivalenta/ml. There was also a response
between R, 0.2 and 0.4 that could not be identified. Assays of the ethyl acetate
fraction indicated a cytokinin between Rf 0-7and 0.9 and a gibberellin a t R, 0-5 t o 0.6.
Similar responses by the 2 fractions to those obtained from the oat leaf bioassay
590 J. M. Barea and Margaret E. Brown

were found in 3 bioassays using excised radish cotyledons. Table 5 shows results from
one bioassay and that the aqueous fraction gave responses a t Rf 0 to 0.1, 0.3 to 0.4,
and 0-7 to 0.8. The original culture supernatant liquid was calculated to contain0-02 pg
cytokinin equivalents/ml. The ethyl acetate fraction gave a cytokinin response a t
Rf0.7 to 0.8, and a possible gibberellin response between Rf0.6 to 0.7 ; an unidentified
response was also obtained from Rf0.1 to 0.2.
The amounts of each growth regulator estimated by spccific plant bioassays were
less than those indicated by the yeafit bioassay.
TABLE4
Cytokinin bioassay by measuring optical density of chlorophyll retained in
5 excised first leaves of oat
Assay results from chromatogram eluates
A
< > Assay results from standards
O.D. >

R f value I , Authentic
Aqueous Ethyl acetato substance Conc. (p/m) O.D.
fraction fraction

&O* 1 0.367 0.244 '0.01 0.347


0.1-0.2 0.260 0 * 244 Kinetin s0.1 0.595
0.2-0.3 0.331 0.268 1.0 0.898
0.3-0.4 0.371 0.287 '0.01 0.319
0.4-0.5 0.293 0,284 GA 3 0-1 0.337
0.6-0.6 0.297 0.294 1.0 0.398
0*6-0*7 0.340 0.297 '0.01 0.296
0.7-0.8 0-370 0.331 IAA 0.1 0,322
0.8-0.9 0.276 0.321 ,1*0 0.357
0.9-1.0 0,284 0.279 0.284
Control 0.284 0.284

TABLE5
Cytolcinin bioassay using excised radish cotyledons
Assay results from chromatogram eluates
, A
Assay results from standards
Mean fresh weight (mg) of A
I -l
cotyledon (6 reps)
Authentic Conc. (p/m) Wt*
Rf value
' Aqueous Ethyl acetate ' substance (mg)
friction fimtion
~

0-0.1 16.40 12.90 0.01 14.16


0.1-0.2 12.75 14.97 Kinetin 0.1 17-00
0.2-0.3 12.80 12.50 1.0 21.00
0.3-0.4 13.05 12.90 0.01 13.00
0.4-0.5 12.90 12.75 GAS 0.1 14-90
0.6-0.6 12.75 12.80 1.0 15.40
0.6-0.7 12.97 14.90
0.7-0.8 16.20 14.33
0.8-0.9 14.10 12.80
0.9-1.0 13.40 12.70
Control 12.80 12.80
*Mean fresh weight of radish cotyledons ( 5 replicates).
Growth substances produced by A. paspali 591

Discussion
Changes in plant growth and development following treatment of roots with cultures
of A . paspali indicate activity of plant growth regulating substances, and are very
similar to those produced by treating roots with A . chroococcum. Culture supernatants
of A . paspali contain a t least 3 gibberellin-like substances, one of which is closely
related to GA, or GA,, and all of which behave in bioassays like the gibberellins
found in cultures of A . chroococcum,and are produced in similar quantities. Indolyl-3
acetic acid is present in cultures. Responses to bioassays based on chlorophyll
retention in oat leaves and radish cotyledon expansion suggest 2 cytokinin type of
regulators are also formed with Rf values between 0 and 0.1 and 0.7 and 0.8 ; positive
identification of the latter with zeatin has not yet been obtained. The cytokinin with
RI 0.7 to 0.8 partitions close to one of the gibberellins and contamination with this
gibberellin may explain why the fluorescence observed under UV light does not
match that of authentic kinetin.
Work on cytokinin production by other bacteria suggests this matmerialis found in
young cultures, but to obtain most of the active material, extraction procedures may
need modifying, and that cytokinins may have structures not previously described.
I n the present experiments 14 day old cultures were extracted, but younger cultures
should be examined ; extraction procedures ought to be improved and eluates tested
with more sensitive bioassays such as effects on growth of soybean callus (Miller,
1960).
Unlike A . chroococcum which survive in rhizospheres of treated plants until harvest
(Brown, Burlingham & Jackson, 1962), numbers of A . paspali decline rapidly, even
around roots of their associated plant, Paspalum notatum. The decline is less rapid in
potting compost which is richer in organic matter and supports better plant growth
than the Brazilian soil. A . paspali differs in morphology from A. chroococcum by
producing long filamentous cells in young cultures that are not involution forms,
many of which persist in old cultures together with typical cysts. Decline of the
added inoculum may be related to the presence of these filamentous cells, because
experiments with A . chroococcum showed that vegetative cells neither establish well,
nor survive long in the rhizosphere, and that establishment is related to inoculum
size (Brown et al., 1962, 1964). Establishment of A . paspali may, therefore, depend
upon the limited number of cysts in the inoculum and these cannot compete success-
fully with the natural rhizosphere microflora. Dobereiner & Campelo (1971) also
found that large populations of A . pwpali did not establish in the rhizosphere of
transplanted P. notatum for several months, suggesting that young roots did not
provide a suitable habitat. They thought that A . pwpali might need to penetrate the
root cortex to develop the specific association with P. notatunt.
I n our experiments rapid disappearance of the inoculum suggests that effects on
plant growth are mainly due to activity of the growth regulators present in the
inoculum applied to the seedling roots, any further production of growth regulators
to boost the initial stimulus can only occur in the brief period following treatment
when cells are still relatively numerous. Previous observations on effects of A .
chroococcum on tomatoes (Jackson, Brown & Burlingham, 1964) suggest that 4 .
paspali acts similarly and alters morphology principally by gibberellin production ;
592 J. M. Barea and Margaret E. Brown

IAA and cytokinin may also be involved, interacting with the gibberellins and inde-
pendently affecting different phases of development. This explanation is greatly
strengthened by the fact that A . p p a l i is not fixing nitrogen in the rhizoaphere of
the young P.notatum plants, so the observed improvement in growth cannot be due
to nitrogen fixation.
The failure of A . pa-spdi to fix nitrogen is of interest when compared with the
considerable amounts fixed in root zones of mature P. notatum taken from long
established lawns (Dobereiner et al., 1972). Together wit,h the decline of A . paspali
inoculuni around young roots this finding further emphasizes the unfavourable
habitat provided by young roots for multiplication and nitrogen fixation. The young
roots, however, absorb the growth regulators produced by A . pa-spali which then
influence plant differentiation and development.

We wish to thank members of the Botany department, Rothamsted Experimental


Station, €or their advice.

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