&lI.4ROARETE. (1972).

BROWN, J. appt. Bact. 35,443-451.

Plant Growth Substances Produced by Micro-organisms of Soil

and Rhizosphere
MARQARET E. BROWN
Soil illicrobiology Department) Rothamsted Experimental Station, Harpenden,
Hertj'ordshire, England

(Received 10 January 1972)

SUMMUY. Micro-organisms isolated from rhizospheres and rhizoplanes of wheat plants,
and from root-free soil, produced growth regulating substances with the properties
of gibberellins and indolyl-3-acetic acid (IAA). Substances inhibiting extensions of pea
plant internodes and lettuce hypocotyls were also produced, especially by bacteria from
the root region of seedlings 6 days old. Bacteria producing growth promoting substances
were most abundant on roots of older plants. Seedlings grown aseptically with added
fiibberellic acid (GA,) and IAA, or grown with a soil inoculum, developed similarly and
differed in t,heir morphology from those grown aseptically without additives.

IN1004 HILTNER observed that living roots stimulated certain soil micro-organisms,
but little is yet known about effects these organisms have on the plants. There is
evidence that plant morphology, nutrition and physiology are affected (Rovira, 1965))
and Some effects can be attribut'ed to bacteria producing plant growth regulating
substances. There is much evidence that bacteria produce indolyl-3-acetic acid (IAA)
in culture media, especially when tryptophan is present, but gibberellin production is
less frequently reported. Katznelson & Cole (1965) and Sobieszczanski (1966) found
gibberellin-like substances in culture liquids of several micro-organisms, Brown &
Burlingham (1968) that Azotobacter chroococcum produced 3 such substances, and
Eklund (1970) that one strain of P s e u d o m o m showed activity.
This paper reports the results of examining bacteria from soil, rhizosphere and
rhizoplane for their ability t o produce plant growth regulators and suggests how this
property could affect development a t different stages of wheat growth.

Methods
Isolation of bacteria from wheat root regions and plant-free soil
MPdia
Soil extract agar. This contained K,HPO,, 0.2 g ; agar, 15.0 g ; soil extract, 250 ml;
distilled water, 750 ml; pH, 7.0. Soil extract was prepared by autoclaving 1 kg of
allotment soil with 1 1 of water for 30 min and filtering the extract after adding a
trace amount of CaS04. The filtrate was made up t o 1 1.
Holding's (1960)medium. (For Gram negative bacteria.)
Xutrient agar. Nutrient Agar (NA) (Oxoid CM3) plus glucose 10 g/l.

Isolation technique
Wheat (variety Cappelle-Desprez) was grown in a greenhouse in pots of 2 k g
capacity containing soil from the Rothamsted allotment site, a heavy clay-loam rich
[#31
444 Margaret E. Brown

in organic matter, pH 7.0, that had been cultivated with mixed garden crops for 100
years. Plant nutrient solution was added to the soil as required.
Rhizosphere and rhizoplane samples were taken from seedlings 6 days old and
plants, 42 days old that were tillering and 82 days old with ears emerging. Rhizo-
sphere samples were prepared from the complete root system of 4 seedlings. or repre-
sentative parts of root systems of 4 older plants, by shaking the roots in sterile distilled
water after removing all excess soil. Replicate plates of media were inoculated with
suitable dilutions of these samples. The roots were then used t o prepare rhizoplane
samples by the serial washing technique (Harley & Waid, 1955). Roots from the 20th
washing were macerated in sterile distilled water and the macerate diluted and plated
on the media described above.
After incubating a t 25"for 14 days all bacterial colonies were picked from the plates,
or from sectors, to give a minimum of 100 isolates for each sample. These were
cultured on NA to check for purity, and finally stored on NA. The bacteria were then
identified to genus level using Skerman's key (1967).

Production of plant growth regulating sicbstances

Each bacterial isolate was grown for 10-14 days on a rotary shaker a t 26" in 100 nil
of medium containing: sucrose, 10.0 g ; KNO,, 0.5 g ; yeast extract (Difco), 0.5 g ;
solution A, 5 m l ; solution B, 5 ml; soil extract, 250ml; distilled water, 740 ml;
pH, 7-0. Solution A consisted of K,HPO,, 25.0 g; KH,PO,, 25-0g ; distilled water,
250 ml. Solution B consisted of MgSO,. 7H,O, 10.0 g; NaCl, 0.5 g ; FeSO,. 7H,O,
0.5 g; MnS0,.4H20, 0.5 g; distilled water, 250 ml. One hundred g/l of L-tr-yptophan
was sometimes added to this medium.
Cultures were centrifuged a t 2000 g for 20 min and supernatant fluids divided into
2 equal volumes which were then extracted either for gibberellin-like substances or
for indolyl-3-acetic acid (IAA). Analytical grade chemicals were used.

Extraction and chromatography

For gibberellin-like substances the supernatant fluids were extracted as described
by Brown & Burlingham (1968). For IAA, after acidifying with N HC1 t o p H 2.8-3.0,
the supernatant fluids were shaken 3 times with ethyl acetate (10 ml) at 30 min
intervals. Combined ethyl acetate extracts were evaporated to dryness in cold air
and residues dissolved in methanol (0.3 ml). Extracts were examined by paper parti-
tion chromatography using freshly prepared solvent mixture, iso-propanol : aninionia
solution (s.g. 0.88) : water (10 : 1 : 1 by vol), and GA, and IAA were separated and
identified (Brown. & Burlingham, 1968).
Chromatogram portions not treated with reagents were dried for 7 days to remove
solvents and 10 equal cut strips representing the sequence of R f values 0.1-1.0 eluted
separately for bioassays.
Sterile culture medium was also extracted and examined for gibberellins and IAA

Bimsays
Gibberellin-like substances were assayed with 2 tests used by Brown & Burlirighaiii
(1968), elongation of dwarf pea internodes and of lettuce hypocotyls. Amounts
 Growth substances produced by soil bacteria 445

produced by bacteria were finally calculated as GA, equivalents/ml of original

culture.
IAA was assayed using a test in which the length increase of wheat coleoptile
segments (cut 1 mm below the apex of the coleoptile) was measured, after incubating
a t 25" in water containing the appropriate portion of chromatogram. The amount
of L4,4 was calculated from a standard dose response curve and finally given as IAA
equiralents/ml of original culture.

Effect of G A , and I A A on wheat grown aseptically or with a soil inoculum

Grains of wheat were surface sterilized with undiluted Chloros (Boots Ltd.), washed
with sterile water and germinated on NA to detect contamination. Aseptic seedlings
were transferred a t 2 days to sterile tubes of perlite moistened with 20 ml of plant
nutrient solution (Hewitt, 1952). Replicate tubes (8) were inoculated with 10%
(w/v)of soil suspension (1 ml), to provide a balanced inoculum of soil micro-organisms,
or with 2 ml of sterile water containing 0.01 pg of GA, and 0.05 pg of IAA, or were
left untreated to serve as controls. After 18 days in the greenhouse, tubes were checked
for sterility, seedlings were washed, leaves and total root length were measured and
dry weights obtained.

Results
Identijcation of bacteria
The dominant genera in the different samples were Pseudomom, Achromobacter,
Alcaligenes, Flavobacterium, Nocardia and Arthrobacter (all Gram negative), and
Brevibacteriunt (Gram positive). A few Gram negative isolates, each with different
biochemical characters, were not identified. The predominant populations in root
region samples changed quantitatively and qualitatively as plants aged, whereas
those of soil samples remained constant. Pseudomom spp. were stimulated in the
root region of plants 6 days old, but not of older plants; Achromobacter spp. were
stimulated a t 6 and 42 days; Alcaligenes spp. were always stimulated, but especially
a t 42 days; Nocardia spp. were always stimulated, especially a t 82 days, and Arthro-
bacter spp. were stimulated only a t 42 and 82 days. Species of Brevibacterium and
Flavobacteriuwt were fewer in root than in soil samples, and the root samples of plants
6 days old were free from Fhvobacterium.

Production of plant growth regulating substances

Detection on chromatograms
Chromatograms dipped in chromogenic reagents and examined under UV radiation
showed that the supernatant fluid fractions from the different bacteria contained
unidentified substances with spots fluorescing a t several different Rfvalues, some of
which corresponded to spots produced by sterile culture medium. Many bacteria
produced a substance fluorescing yellow in a position corresponding t o the Rf value
for IAA, and this also gave a pink spot when treated with Gordon & Weber (1951)
reagent. A few bacteria produced a substance fluorescing green in a position corres-
ponding to the Rf value of GA,, but most of the extracts did not.
446 Margaret E. Brown

Detection by bioassay
Gibberellins. Eluates from the chromatograms of each of the bacterial extracts were
tested for growth effects on dwarf peas and classed as those significantly promoting
or inhibiting pea internode extension, or with no effect. Substances promoting
internode extension occurred in 3 zones on the chromatograms, with peaks of activity
a t Rf 0.1, 0.5 and 0.85. The magnitudes of the peaks ranged from (%): 1 5 4 0 , 17-37
and 1 5 4 9 , respectively. There were some slight differences in Rfvalues for each peak
produced by different extracts, but they were within the range shown by authentic
GA, in a series of separate chromatogram developments. Probably, therefore, a
minimum of 3 growth promoting substances were produced by the different bacteria,
but some bacteria formed only 2 or one. Figure 1 is a histogram of the % increase

+
50 -
f
$ 40-
VI
0)
U
30- II

i? 20-
.-
5
0
g-
IG-

0- -
13-
$ 20-
30-
40 -
-
I I I I I

R, value
Fig. 1 . Effects on growth of dwarf pea internodes by components of the supern tant
fluid from a bacterial culture containing 3 growth promoting substances, separated by
chromatography. Shaded portion represents act.ivity significant at the 5 % level.
Horizontal lines at the top of the figure represents position of authentic GA, and
IAA.

in internode extension produced by one bacterial extract containing a t least 3 sub-

stances. The substance with a peak a t Rf 0.5 corresponded in position to authentic
GA, and behaved similarly in the pea bioassay. Calculated from the response curve
of peas to standard amounts of GA,, the original supernatant h i d fractions of the
different bacterial cultures contained 0.001-0.5 pg of GA, equivalents/ml. The 2
plant growth promoting substances with peaks a t Rf0.1 and 0.85 were not identified.
Lettuce hypocotyl bioassay con6rmed the similarity of the substance a t Rf 0.5 t o
GA,. The substance a t Rf 0.1 also significantly st,imulated hypocotyl extension, but
that a t RI 0.85 did not.
Table 1 shows the percentage of isolates producing plant growth promoting sub-
stances. There is a significant increase in the total population of these bacteria around
roots of 42 and 82 days old plants, but not a t 6 days. Bacteria producing 1 or 2
growth promoting substances are stimulated and mostly belong t o the genera Nocardia
 Growth substances produced by soil bacteria 447

TABLE1
Percentage of isolates in root region and soil samples producing plant growth
regulating substances

Number of
&
Rhizo-
sphere
6 days

Rhizo-
plane
&
Rhizo-
sphere
42 days

Rhizo-
plane
-
Percentage of isolates producing plant growth regulating substances in
~~

Rhizo-
sphere
~

82 days
~

Rhizo-
plane
Soil

promoters
PTOdUd
Total 42 35 61* 21 50* 47* 42
3 27 27 21 14 18 6 31
2 5 0 7* 7* 18* 23* 4
1 10* 8 33 * 0 14* 18* 7
Number of
inhibitors
produced
Total 40* 54* 13 21 32 35 35
3 7 19* 7 0 4 0 4
2 25* 23 * 0 14* 25* 35* 4
1 7 11 7 7 4 0 27

* Denotes population significantly different from soil. P=O .05.

and Arthrobacter. Species of Flavobacterium and Brevibacterium, although not stimu-

lated by roota, were isolated from root region soil samples, and also produced plant
growth promoters. Pseudomom, Flavobacterium and Brevibacterium spp. isolated from
root-free soil produced promoters.
The substances significantly inhibiting pea internode extension developed in 3 zones
on the chromatograms, with peaks of activity a t Rf 0.2, 0.5 and 0.8. The magnitude
of the peaks ranged from (%) 18-33, 19-29 and 1 5 4 3 . There were differences in Rf
values for each peak produced by the extracts and the range of movement was greater
than that of the promoting substances, but was just w i t h that shown by authentic
GA, in separate chromatogram developments. Possibly these substances spread on
the chromatograms rather than forming well d e k e d spots. Probably, therefore, a
minimum of 3 growth inhibiting substances were produced by the different bateria,
but not by all, some produced only 2 or 1. Figure 2 is a histogram of the yodecrease
in pea internode extension produced by one bacterium with active substances in 3
zones on the chromatogram. I n the lettuce bioassay these substances inhibited
hypocotyl extension. Table 1 also shows the percentage of isolates producing inhibi-
tory substances. These bacteria, mostly belonging to the genera P s e u d o m o m and
Achromobacter, were significantly increased in number by roots of 6 day seedlings,
particularly in the rhizoplane, and they produced either 2 or 3 substances. Several
unidentified bacteria from the rhizoplane also produced inhibitors, and one species of
Alculigenes, and one of Brevibacterium, but Nocurdia and Arthrobacter never did.
Filtrates of a few bacteria contained a mixture of promoters and inhibitors, develop-
ing in separate positions on the chromatograms; 13% of soil isolates and of root
448 Margaret E. Brown

0 02 04 06 08 10
R, value
Fig. 2. Effects on growth of dwarf pea internodes by components of the supernatant
fluid from a bacterial culture containing 3 growth inhibiting substances, separated
by chromatography. Conventions as in Fig. 1 .

isolates (7% at 6 days, 30/, at 42 days and 6% at 82 days) produced this mixture. All
other isolates had no effect on pea or lettuce growth.
Sterile culture medium did not contain anything affecting pea or lettuce growth.

Indolyl-3-acetic acid. Table 2 shows that many bacteria from all samples formed
I A A uithout L-tryptophan in the medium, but many of these produced more, especially
TABLE2
Percentage of isoEates from root region and soil samples producing I A A with
and without L-tryptophan in medium
% isolates producing IAA
, \

Without L-tryptophan Producing more IAA with

L-tryptophan
7
* f \

Days Rhizosphere Rhizoplane Soil Rhizosphere Rhizoplane Soil

6 77 84 87 35* 54* 29
42 73 78 86 47* 7 29
82 89 88 87 4 0 28
* Denotes population significantly different from soil. P=O .05.

isolates from the 6 day rhizoplane, when L-tryptophan was present. As plants aged
and the microflora changed, the number of root isolates using L-tryptophan for IAA
synthesis decreased, especially in the rhizoplane, but many still formed IAA without
L-tryptophan. The amounts produced by the different bacteria ranged from 0.02-
0.3 pg/ml of oiiginal culture without L-tryptophan, and from 1.0-10.0 pg/ml with
L-tryptophan. Representatives of each genus produced IAA, and all Pseudomonas
isolates formed up to 10.0 pg of IAA/ml of culture with L-tryptophan present.
 Growth substances produced by soil bacteria 449

Sterile culture medium did not contain IAA or any other substance affecting wheat
coleoptile extension.

Effect of GA, and I A A on wheat grown aseptically compared with a soil inoculum
Tables 3 and 4 show that wheat seedlings grown aseptically for 18 days produced
significantly fewer leaves than those grown with GA, and IAA or a soil inoculum.
Leaves were also shorter and weighed less. Seedlings given plant growth substances
or a soil inoculum produced the same number of leaves of similar weight and length.
When grown with GA, and IAA, seedlings produced more roots with longer laterals
than aseptically grown seedlings, but these roots were shorter and weighed less.
Seedlings grown with a soil inoculum produced fewer roots with longer laterals than
seedlings grown aseptically, but these roots were shorter and weighed less. Roots of

TABLE3
Comparison of esfects on wheat seedling growth of treatments with GA ,+ IAA,
soil or water

, ,.
Effect of treatment on seedling growth
,
Control GAS+IAA Difference SE=* Soil Difference SED*

Leaves
No. 2 2.8 k0.1 2.7 kO.12
Lengt,h (mm)
Weight (mg)
373
2-44
470
2.98
+0.8
+97
+0*54
*34
-1.0.53
435
2.79
+0.7
+
62
+0*35
*34
k0.30

Roots
NO.
Length (mm)
Weight (mg)
6
1379
2.12
6-8
1174
1.69
+0*8
- 205
-0.43
k0.1
k151
k0.19
5.5
1163
1.43
-0.5
- 216
-0.69
*
k0.14
154
k0.22
Measurements are mean of 6 replicates. P = 0.05.
* Standard error of difference.
TABLE4
Comparison between effects of treatment with GA,+ I A A or soil
Effect of treatment on seedling growth
f
A ,
GA +IAA Soil Difference SED*

Leaves
No. 2.8 2.1 -0.1 +O-16
Length (mm) 470 435 - 35 +
37
Weight (mg) 2.98 2.79 -0.19 k0.44

Roots
No. 6.8 5.5 -1.3 0-18
Length (mm)
Weight (mg)
1174
1.69
1163
1.43
- 11
-0.26 -
*52
+0.18
Measurements we mean of 6 replicates. P = O .05.
* Standard error of difference.
450 Margaret E. Brown

seedlings grown with a soil inoculum or GA, and IAA were similar in total length and
lateral root length, but roots were fewer and less heavy in the presence of soil than
GA, and IAA.

Discussion
Many bacteria, especially those found in the rhizosphere and rhizoplane, produce
plant growth regulators of the gibberellin or auxin type when grown in liquid culture.
The gibberellins, separated on chromatograms, are found in 3 positions, and their Rf
values resemble those given for the substances produced by Azotobacter chroococcum
(Brown & Burlingham, 1968). This suggests that a wide range of soil bacteria can
produce 3 and possibly more gibberellins, one of which resembles GA,; some bacteria
form all 3, others fewer. Many soil bacteria also produce substances inhibiting pea
internode and lettuce hypocotyl extension, and nith Rf values close to those of the
gibberellins. However, it is improbable that these growth promoters and inhibitors
are the same substances. The definition of a gibberellin is a substance that induces
shoot elongation of dwarf mutant plants, whose growth responses are thought to be
specific to the gibberellins. Gibberellins do not inhibit shoot growth.
Strzelczyk, Kampert & Krzysko (1971), have shown that in culture Arthrobacter
pascens produced substances that, in the Avena straight growth test, acted both as
plant growth inhibitors and as inhibitors of auxin induced growth, but in the lettuce
hypocotyl test these substances stimulated plant growth but depressed the stimu-
lation produced by authentic GA,. The active principles were located a t Rf 0.8-1-0
(solvent system Go-propanol : ammonia solution : water; 10 : 1 : l), the Rf value
found for some of the plant regulating substances produced by bacteria examined
in my work. However, in my experiments the Arthrobacter spp. tested only produced
substances promoting plant growth. Clearly, activity of plant growth regulators
produced by soil bacteria is very complicated, with interactions between promoting
and inhibiting substances.
The inhibitory substances are produced by many bacteria isolated from the rhizo-
plane, especially of 6 days old wheat seedlings. These bacteria may also produce
inhibitors around roots of wheat seedlings growing in soil. In addition, rhizosphere
bacteria produce IAA,and the organisms isolated from the rhizoplane of seedlings
can use L-tryptophan to synthesize more LAA. Tryptophan is exuded by roots of
some young plants (Vancura & Hovadik, 1965) and is probably also supplied by micro-
bial exudation and autolysis. Roots of wheat seedlings, therefore, may be subject to
the action of plant growth inhibitors and of IAA a t a time of very rapid growth. This
may partly explain why soil-grown seedlings have different root and shoot morphology
from those grown aseptically. At later stages of further rapid growth by wheat,
tillering and earing, more of the micro-organisms stimulated in the root region produce
growth promoters than inhibitors, and uptake by the roots of these promoters may
affect plant development. Bacteria producing small amounts of I A A are still abundant,
but those using L-tryptophan t o synthesize more IAA are few. This may be because
micro-organisms in the root zone of mature plants are dependent more on moribund
cells and root hairs for their food than on exudates, and tryptophan is less readily
available.
 Growth substances produced by soil bacteria 451

The hypothesis that GA, and IAA are among the active substances produced is
supported by results obtained when seedlings grown with an inoculum of soil microbes
or authentic growth regulators had similar morphology. The seedling roots would
selectively stimulate the microbes in the soil suspension to create their own rhizosphere
flora. Presumably this flora then metabolized plant growth regulators that affected
morphology in the same way as did GA, and IAA when added t o aseptically grown
seedlings.

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