P l a n t and Soil 43, 609-619 (1975)
Ms. 2612
SYNTHESIS OF AUXINS, GIBBERELLINS AND
CYTOKININS BY AZOTOBACTER
VINELANDII
AND AZOTOBACTER
BEIJERINCKII
RELATED
T O E F F E C T S P R O D U C E D ON T O M A T O P L A N T S
by ROSARIO AzcON and J. M. BAREA
Microbiology Department, Experimental Station of Zaidln C.S.I.C.,
Granada, Spain
SUMMARY
Culture supernatants of Azotobacter vinelandii and Azotobacter beijerinckii
contain auxins, at least three gibberellin-like substances and three cytokininlike substances. Treating roots of tomato seedlings with these cultures accelerates plant growth and increases yield of fruit, effects probably caused by
activity of the plant hormones. Amounts of hormones produced in these cultures are similar to those produced b y A zotobacter chroococcum and A zotobacter
paspali.
INTRODUCTION
Plant growth regulators of the auxin and gibberellin type are
produced in culture supernatants of Azotobacter chroococcum 5 8,
Azotobacter vinelandii 17 and Azotobacter paspali 2. Cytokinins are
also produced by Azotobacter chroococcum 10 and Azotobacter pa@ali 2.
Responses in plant growth following treatment of seeds or seedling
roots with cultures of A. chroococcum and A. paspali are probably
caused by the gibberellins but cytokinins or auxins may also be
involved.
A. vinelandii and A. beijerinckii used as inoculants for early
vegetables grown in the sub-tropical area of the Mediterranean in
Spain also improved growth probably by producing growth regulators 1. This paper reports experiments to verify presence and activity of growth regulating substances in cultures of A. vinelandii and
A. beijerinckii.
610
ROSARIO AZCON AND J. M. BAREA
M A T E R I A L AND M E T H O D S
Cultures
E l e v e n strains of A z o t o b a c t e r were isolated f r o m t h e rhizospheres of t o m a t o
plants (cultivar Marglobe) growing in a sand: f a r m y a r d m a n u r e m i x t u r e as
used in t h e sub-tropical area of Spain 22. T h e A z o t o b a c t e r were identified b y
t h e m e t h o d of C a l l a o et al. o as A. vinelandii and A. beijerinckii. Strains of
two species A4 (A. vinelandii) and A5 (A. beijerinckii) were selected for s t u d y
because of their effectiveness as bacterial inoculanfs 1. These strains were
g r o w n for 14 days in 70 ml m e d i u m s in 250 ml flasks on a r o t a r y shaker
i n c u b a t e d at 28°C.
Effect o/Azotobacter on tomato growth
R o o t s of seedling t o m a t o (cultivar Marglobe) were dipped in cultures of t h e
A z o t o b a c t e r strains A4 and A5 before t r a n s p l a n t i n g t h e seedlings to pots of a
m i x t u r e of sand : p e a t : f a r m y a r d m a n u r e (1 : 1 : 1 v/v). Control seedlings were
dipped in diluted culture medium. P l a n t s were fed e v e r y t w o weeks w i t h
5 m l / p o t of n u t r i e n t solution 15. One series of plants were grown for 60 days
and d r y weights of shoots t h e n o b t a i n e d and a n o t h e r series was grown until
fruit h a d formed. N u m b e r s of flowers and fruits and w e i g h t of fruit were
recorded.
Establishment o[ Azotobaaer in the tomato rhizosphere
R h i z o s p h e r e soil was sampled a t 14 d a y intervals and A. vinelandii or A.
beijerinckii counted on t h e nitrogen-deficient m e d i u m described b y B r o w n
et al. 6 N u m b e r s were related to 1 g d r y rhizosphere soil.
Extraction o/plant growth regulators
Cultures of A. vinelandii or A. beijerinckii were centrifuged at 2000 X g for
Acidified supernal:ant
I
I
I
fraction A
fraction
Examined for gibbereLLins
B
PartiUoned in ethyl acetate
(Brown and BurLingham's me~hod5)
I
I
I
Ethyl acetate phase
Aqueous phase
I
I
Examined For IAA
(Brown and Walker's metchod8)
]Fig. 1.
I
Examined for eytokinins
(Navarro et al's lg}
D i a g r a m showing m e t h o d s used for e x t r a c t i o n of p l a n t
stances from bacterial cultures.
Examined ['or cytokihins
(Wheeler's method 23)
g r o w t h regulating sub-
PLANT HORMONES PRODUCED BY AZOTOBACTER SPP.
611
40 min. Each supernatant fluid was acidified to p H 3 and divided into two
equal fractions, A and B. These fractions were examined as in figure 1.
Both organic and aqueous phases after partitioning with ethyl acetate were
examined for cytokinins because in model experiments with kinetin it was
found t h a t most of the kinetin partitioned in the organic phase 14 19, with a
small proportion only partitioning ill the aqueous phase.
Paper partition chromatography
The different extracts were examined by descending paper chromatography
using as solvent system freshly mixed isopropanol : ammonia: water (10 : 1 : 1)
by volume. After development chromatograms were examined for fluorescerise under UV light (wavelength, 350 nm) before and after spraying with
chromogelliC reagent (5% conc. H2SO4 in methanol). Re values ranged from
0.3 to 0.4 for IAA (Merck AG Darmstad. Germany) 0.5 to 0.7 for GA3 (Sigma
Chemical Co., St Louis, Missouri, U.S.A.) and 0.7 to 0.8 for kinetin (Sigma)
used as standard authentic substances.
Chromatograms portion not treated with chromogenic reagent were dried
for at least 7 days to remove solvents and 10 equal strips representing the
sequence of Rf values 0.1 to 1.0 used in bioassays.
Bioassays
The yeast bioassay described b y B a r e a et al. ~, was used to screen the extracts for presence of growth regulators. Specific bioassays were then used.
(i) I A A. The portion of chromatogram corresponding in position to the R j
value for authentic IAA was used for the bioassay in which length increase of
wheat coleoptile segments was measured s 2o
(ii) G i b b e r e l l i n s . Extension of lettuce hypocotyls 13 and extension of
cucumber hypocotyls 4 were used as bioassays.
(iii) C y t o k i n i n s . Expansion of radish cotyledons is and measurement of
optical density of chlorophyll retained in the excised three first leaves of
oat ~8 were used as bioassays.
To minimize interference by gibberellins and auxins in cytokinin bioassays
strips of chromatograms representing the sequence of R l values were heated
at 115°C for 20 mill; this destroyed the gibberellins and auxins, b u t not the
cytokinins. These strips were then used in the respective bioassays and responses compared with those from untreated strips.
Amounts of growth regulators were calculated from dose response curves
obtained wittl authentic substances and given as ~g equivalents per ml of
culture supernatant.
612
ROSARIO AZCON A N D J. M. B A R E A
RESULTS
Effects on tomato growth o/treatment with A. vinelandii or A. beijerinckii
Treating roots of seedling tomatoes with cultures of Azotobacter
strains A~ and A5 significantly improved plant growth, and fruits
formed two weeks earlier than those on control plants.
TABLE I
Effects of Azotobacter inoculation on growth of tomato plants
P o t s inoculated with
A. vinelandii
Topics
Stem length (mm)
after 15 days
after 30 days
Shoots dry w e i g h t (g)
after 60 days
n ° of flowers/pot
n ° of fruits/pot
Weight of fruits (g/pot)
A. beijerinckii
22
107
24
109"
2.53 *
31 *
8 **
39.40 ***
Control
LSD (5%)
19
97
2.46 *
32 *
8 **
37.10 ***
3.1
14.2
1.60
25
5
27.37
0.71
5.2
1.5
6.85
* Significance a t 5% level; ** at 2% level; *** at 1% level.
Table 1 shows that dry weight of shoots from plants grown 60
days was significantly increased b y inoculation and these plants
formed more flowers and heavier fruits.
Establishment of A. vinelandii or A. beijerinckii in tomato rhizosphere
Table 2 shows counts of A. vinelandii or A. beijerinckii recovered
from rhizospheres of tomato. The inocula decreased in number
rapidly but some cells were still present after 14 weeks.
TABLE 2
Number of A. vinelandi~ and A. beijrinc#$$ in t o m a t o rhizosphere. (no.s/g d r y rhizosphere
soil)
A zotobacter spp.
inoculated
A. vinelandii
A. beijerinckii
Weeks
2
4
6
8
10
12
14
3700
14000
2300
7500
1200
1500
1600
2300
950
1050
400
500
80
80
PLANT
HORMONES
BY AZOTOBACTER SPP.
PRODUCED
613
Production o/ plant growthregulators by A. vinelandii and A. beijerinckii
(i) Y e a s t b i o a s s a y . Tables 3 and 4 show the optical density
and number of cells in cultures of Saccharomyces cerevisieae incubated with eluated of each Rf value from the different chromatograms.
Eluates from the heated chromatogram of fraction B organic
phase, between R I 0 and 0.4 and 0.6 to 0.7 caused increased O. D. and
cell number, and eluated from the heated chromatogram of fraction
B aqueous phase, between R I 0 and 0.2, 0.3 and 0.5, 0.6 and 0.7,
and 0.8 and 1.0, also caused increases, all indicating cytokinin
activity. This was confirmed by specific bioassays.
(ii) A u x i n s . In the wheat coleoptile bioassay, substances with
R~, 0.3 to 0.4 eluted from chromatograms of extracts from both
cultures possessed auxin activity equivalent to 0.3 to 0.4 #g IAA per
ml culture.
TABLE
3
R e s p o n s e of Saccharomyces cerevisie~e to e l u a t e s of e a c h R f v a l u e f r o m c h r o m a t o g r a m s of
d i f f e r e n t e x t r a c t s of Azotobacter vineIandii
Fraction A
Fraction B
Ethyl acetate phase
Aqueous phase
(Heated chromatograms)
Rf
values
Optical
density
0-0. I
0.1-0.2
0.2-0.3
0.3-0.4
0.4-0.5
0.5-0.6
0.6-0.7
0.7-0.8
0.8-0.9
0.9-1.0
Control
0.62
0.55
0.60
0.61
0.58
0.62
0.$8
0.62
0.57
0.57
0.55
*
*
o
o
Cell
no.
× 106/
ml
Optical
density
17.0
12.4
16.1
16.3
13.8
17.1
13.7
17.5
13.6
13.6
12.3
0.64
0.63
0.62
0.62
0.58
0.60
0.58
0.61
0.57
0.57
0.55
* Indicates cytokinin activity.
x Indicates auxin activity.
° Indicates gibberellin activity.
Cell
no.
x 106/
ml
*
*
x
°
*
°
17.6
17.1
17.1
17.2
13.6
16.2
13.6
16.9
13.5
13.4
12.3
Optical
density
0.64
0.63 *
0.62
0.61 *
0.60
0.60
0.61 *
0.59
0.58
0.57
0.55
Cell
no.
x 10~/
ml
Optical
density
17.5
17.5
17.1
17.0
15.8
18.9
16.7
15.9
13.5
13.8
13.1
0.62
0.66
0.58
0.66
0.64
0.62
0.66
0.57
0.64
0.64
0.57
Cell
no.
x 106/
ml
*
*
*
*
16. t
18.0
14.0
18.1
18.1
17.0
18.2
13.5
17.6
17.6
14.0
614
R O S A R I O A Z C O N A N D J . M. B A R E A
TABLE
4
R e s p o n s e of Saccharomyces cerevisieae to e l u a t e s of e a c h R f v a l u e f r o m c h r o m a t o g r a m s of
d i f f e r e n t e x t r a c t s of Azotobacter beijerinckii
Fraction A
Fraction B
Ethyl acetate phase
Aqueous phase
(Heated chromatograms)
RI
values
Optical
density
0-0.1
0.1-0.2
0.2-0.3
0.3-0.4
0.4-0.5
0.5-0.6
0.6-0.7
0.7-0.8
0.8-0.9
0.9-1.0
Control
0.62
0.64
0.60
0.61
0.61
0.60
0.62
0.64
0.60
0.58
0.55
*
*x
°
*
Cell
no.
x 106/
nil
Optical
density
16.8
17.5
14.7
17.1
17.2
16.8
17.2
17.4
16.1
14.3
12.4
0.64
0.64
0.61
0.62
0.62
0.61
0.63
0.64
0.60
0.59
0.56
*
*x
o
*
Cell
no.
× lOS/
ml
Optical
density
17.5
17.6
15.3
17.5
17.9
16.2
17.5
17.5
16.1
14.4
12.8
0.54
0.65
0.60
0.61
0.58
0.52
0.64
0.64
0.60
0.62
0.55
Cell
no.
x 10~/
ml
*
*
*
*
11.5
18.0
16.1
16.4
14.3
11.2
17.5
17.9
16.2
17.0
12.3
Optical
density
Cell
no.
x lOS/
ml
0.62
0.64 *
0.61
0.56
0.61
0.63 *
0.58
0.64
0.63 *
0.58
0.55
17.1
17.9
16.8
12.8
16.3
17.4
14.0
17.7
17.3
14.1
12.4
* Indicates cytokinin activity.
x Indicates auxin activity.
o Indicates gibberellin activity.
(iii) G i b b e r e l l i n s . Lettuce hypocotyls bioassay: Figures 2 and 3
show the activity of eluates from chromatograms of fraction A on
lettuce hypocotyl extension.
Significant increases were produced by substances extracted
from cultures of A. vinelandii with Rf values between 0.4 and 1.0,
giving a peak of activity at R~ 0.55 (corresponding in position to
authentic GA3). This was equivalent to 0.05 ~g per ml culture
supernatant fluid.
Significant hypocotyl extension was produced by substances
extracted from cultures of A. beijerinckii with R I values between 0.2
and 0.3, and 0.5 and 0.7 giving a peak of activity at R/, 0.55. This
was equivalent to 0.04 [xg GA3 per ml culture supernatant fluid.
Substances with R I 0.9 to 1.0 significantly decreased hypocotyl
growth.
Cucumber hypocotyl bioassay: Figures 2 and 3 also show the effects
P L A N T HORMONES P R O D U C E D BY A Z O T O B A C T E R SPP.
615
Azotobacter vinelandii:
Letct uce
bioassay
40(+)
30-
20-
= ~
0,2
R
0,4
0,6
0,8
RF values
20 °
1'o/
[
1o-
ga3.., Cucumber
bioassay
(+) 4030-
~3
O~
lOI
o° c-~
LSD
o,z
0,4
o,6
0,8
11o/
1
Rf values
20
Fig. 2. Effects on extension of lettuce and cucumber hypocotyls by components of fraction A of the supernatant fluid of Azotobacter vimlandii cultures separated by chromatography. Shaded portion represents activity significant at 5% level. Horizontal lille at the top of the figure represents position
of authentic GAs.
of eluates from chromatograms of fraction A on cucumber hypocotyl extension. Results are similar to those obtained in the lettuce
bioassay. Eluates from cultures of A. vinelandii also show activity
at R I 0.1 to 0.2, and those from cultures of A. bei/erinckii activity at Rf 0 to 0.1 and 0.9. to 0.3.
The supernatant fluid fraction was calculate to contain 0.06 ~g
(A. vinelandii) and 0.05 ~g (A. beiierinckii ) of GAs equivalent per
ml.
(iv) C y t o k i n i n s . Heated portions of chromatograms from extracts of fraction B organic and aqueous phases were used.
Chlorophyll retention test: Table 5 show results from tests based on
determining the optical density of chlorophyll retained in three
excised first leaves of oat. A. vinelandii and A. bei/erinckii gave
similar results with peaks of activity shown by eluates with R I
616
ROSARIO AZC6N AND J. M. BAREA
AzOtobae~er beijerincki[:
~ga3 ~Lel:'~uee bioass~y
40
(+)
30
20
~o
10
L
.
.
.
1.0 ]LSD
.
(-) 10
2(3
Rf
values
~a3 ~Cucumber bioassay
~,
(+)
30
~
20
~,
10
= ~"
•
T LSD
-i
(")20
|
qf values
Fig. 3. Effects on extension of lettuce and cucumber hypocotyls by components of fraction A of the supernatant fluid of Azotobacter beijerinckii cultures separated by chromatography. Conventions as in Fig. 2.
TABLE
5
C y t o k i n i n b i o a s s a y b y m e a s u r i n g o p t i c a l d e n s i t y of c h l o r o p h y l l
r e t a i n e d in t h r e e e x c i s e d f i r s t l e a v e s of o a t
Heated chromatograms from
Azotobacter vinetandii
Azotobacter beijerinclcii
values
Ethyl
acetate
phase
Aqueous
phase
Ethyl
acetate
phase
Aqueous
phase
0-0.1
0.1-0.2
0.2-0.3
0.3-0.4
0.4-0.5
0.5-0.6
0.6-0.7
0.7-0.8
0.8-0.9
0.9-1.0
Control
0.13 *
0.12
0.12
0.13 *
0.11
0.13 *
0.12
0.12 *
0.1 1
0.10
0.10
0.12 *
0.10
0.1 1
0.12 *
0.12
0.13 *
0.12
0.12
0.12 *
0.10
0.10
0.10
0.131"
0.10
0.10
0.11
0.11
0,11
0,12 *
0.10
0.13 *
0.10
0.12 *
0.10
0.12
0.12 *
0.12
0.14 *
0.12
0.12
0.1 1
0,12 *
0.10
RI
* Indicates cytokinin activity.
O.D.
Authentic kinetin:
0.1 lag = 0 . 1 2
i
0.D.
O.D.
1.0/~g = 0.40
10.0/~g = 0.68
P L A N T HORMONES P R O D U T E D BY A Z O T O B A C T E R SPP.
617
TABLE 6
Cytokinin bioassay using excised radish cotyledons. (Mean fresh
weight (rag) of cotyledon (5 reps))
H e a t e d c h r o m a t o g r a m s from
R2
values
A zotobacter vinelandii
Ethyl
acetate
Aqueous
phase
phase
0-0.1
0.1-0.2
0.2-0.3
0.3-0.4
0.4-0.5
0.5-0.6
0.6-0.7
0.7-0.8
0.8-0.9
0.9-1.0
Control
17.40
16.10 *
13.60
13.00 *
11.60
13.10
13.00 *
12.10
11.20
12.00
12.10
16.20
11.30
11.60
14.40
12.00
12.50
16.20
11.20
17.20
13.00
12.10
*
*
*
*
Azotobacter beijerinckii
Ethyl
acetate
Aqueous
phase
phase
12.00
11.20
12.10
15.70 *
12.40
12.20
17.00 *
12.60
12.80 *
12.10
12.10
15.70
16.30
12.00
14.75
13.00
12.20
11.80
15.00
12.60
12.00
12.10
* In dicates c y t o k i n i n activity.
A u t h e n t i c k i n e t i n : 0.02/,g = 13.87 mg
0.20/zg = 18.08 mg
2.00/zg = 22.35 rag.
values 0 to 0.2, and 0.5 to 0.8, this latter corresponding in position
to zeatin. A. vinetandii also gave activity at Rf 0.2 to 0.4 and A.
beijerinckii activity at 0.9 to 1.0.
Radish cotyledons expansion test: Table 6 shows that this test gave
similar responses to those obtained from the oat leaf bioassay.
The original culture supernatant was calculated to contain
0.05 ~g kinetin equivalent per ml. The cytokinins produced by the
two cultures of Azotobacter partitioned equally in the organic and
aqueous phases.
DISCUSSION
Culture supernatants of A. vinelandii and A. beijerinckii contain
at least three gibberellin-like substances, indolyl-3-acetic acid and
at least three substances possessing cytokinin activity. All behave
in bioassays similarly to the substances found in cultures of A.
chroococcum and A. paspali, and were produced in similar quantities.
618
ROSARIO AZCON AND J. M. BAREA
Like inocula of A. chroococcum 7, inocula of A. vinelandii and A.
beijerinckii survive in rhizospheres of treated plants until harvest,
but do decrease in number. Despite this decline, the effect on tomato
growth continues until fruit set. Unlike A. chroococum 16 A. vinelandii and A. beijerinckii increase tile number and yield of fruit
It may be that because conditions in the greenhouse experiments
resembled those of the subtropical area of Spain from where the
bacteria were originally isolated, that activity of these cultures was
particularly favoured.
Similarity of plant growth production by A. chroococcum, A.
vinelandii, A. beiierinckii, and A. paspali supports the evidence of a
close affinity between the species as suggested several authors 11 12 21
These authors showed that those species had closely related DNA
base composition and should be grouped together being the only
species to bear the generic name Azotobacter.
ACKNOWLEDGEMENTS
The a u t h o r s w i s h t o t h a n k D r M a r g a r e t E. B r o w n , R o t h a m s t e d E x p e r i m e n t a l S t a t i o n , H a r p e n d e n , E n g l a n d for h e l p f u l a d v i c e a n d criticism.
Received August 20, 1974
REFERENCES
1 Azc6n, R., Barea, J. M. and Callao, V., Selection of phosphate-solubilizing and
nitrogen-fixing bacteria for using as biological fertilizers. (Spanish with an English
summary). Cuad. C. Biol. 2.1, 23-30 (1973).
2 Barea, J. M. and Brown, M. E., Effect on plant growth produced by Azotobacter
paspali related to synthesis of plant growth regulating substances. J. Applied
Bacteriol. 37, 583-593 (1974).
3 B a r e a , J. M., N a v a r r o , E., P a l o m a r e s , A. and M o n t o y a , E. A., rapid microbiological assay method for auxins, gibbercllic acid and kinetin using yeast. J.
Applied Bacteriol. 37, 171-174 (1974).
4 Brian, P. W. and H e m m i n g , H. G., Promotion of cucumber hypocotyl growth by
two new gibberellins. Nature 189, 74 (1961).
5 Brown, M. E. and B u r l i n g h a n , S. K., Production of plant growth substances by
Azotobacter chroococcum. J. Gem Microbiol. 53, 135-144 (1968).
6 B r o w n , M. E., B u r l i n g h a n , S. K. and J a c k s o n , R. M., Studies on Azotobacter
species in soil I. Plant and Soil 17, 309-319 (1962).
7 Brown, M. E., B u r l i n g h a n , S. K. and J a c k s o n , R. M., Studies on Azotobacter
species in soil II. Plant and Soil 17, 320-331 (1962).
8 B r o w n , M. E. and W a l k e r , N., Indolyl-3-acetic acid formation by Azotobacter
chroococcum. Plant and Soil 32, 250-253 (1970).
9 Callao, V., M o n t o y a , 1~. and H e r n a n d e z , E., A rapid identification method for
P L A N T HORMOMES P R O D U C T E D BY A Z O T O B A C T E R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
SPP.
619
Azofobaeter. (Spanish wi~h English, French and German summaries). Agrochimica 6,
51-55 (1961).
C o p p o l a , S., P e r e u o c o , G., Z o i n a , A. and P i e e i , G., Citochinine ill germi terrieoli
e relafivo signifieato Ilei rapporti piante-mieroorganismi. Allnali Microbiol. 21, 45-53
(1971).
De L e y , J., D N A base composition and classification of some more free-living
nitrogen-fixing bacteria. Antonie v a n Leeuwenhoek J. Microbiol. Serol. 34, 66-70
(1968).
De L e y , J. and P a r k , I. W., Molecular biological t a x o n o m y of some free-living
bacteria. Antonie v a n Leeuwenhoek J. Microbiol. Serol. 32, 6-16 (1966).
F r a n k l a n d , B. and W a r e i n g , P. F., Effect of gibberellic acid on hypocotyl growth
of lettuce seedlings. Nature 183, 255-256 (1960).
H e m b e r g , T. a n d W e s t l i n , P. E., The quantitative yield in purification of cytokinins, model experiments with kinetin. Physiol. P l a n t a r u m 28, 228-231 (1973).
H e w i t t , E. J., Sand and water culture m e t h o d used in the s t u d y of plant nutrition.
Technical Communication 22. F a r n h a n Royal, Bucks: Commonwealth Agricultural
bureau (1952).
J a c k s o n , R. M., B r o w n , M. E. and B u r l i n g h a n , S. K., Similar effects on tomato
plants of Azotobacter inoculation and application of gibberellins. Nature 203, 851852 (1964).
Lee, M., Breckenridge, C. and Knowles, R., Effect of some culture conditions
on the production of indole-3-acetie acid and a gibberellin-likesubstance by Azotobaeter vinelandii. Can. J. Microbiol. 16, 1325-1330 (1970).
Lethan, D. S., Regulators of cell division in plant tissues XII. Physiol. Plantarum
25, 391-396 (1971).
Navarro, E., Barea, J. M. and Molltoya, E., A method for cytokinins extraction
from bacterial cultures. (Spanish). Abstracts IV National Congress of Microbiology.
Granada, Spain (1973).
N i t s c h , J. P. and N i t s c h , C., Studies on the growth of eoleoptile and first internode
sections. A new, sensitive, straight-growth test for auxins. Plant Physiol. 31, 94-111
(1956).
N o r r i s , J. R. and C h a p m a n , H. M., in Identification Methods for Microbiologists,
Part. B ( G i b b s , B. M. and S h a p t o n , D. A. ed.). Academic Press, London, 19-27
(1968).
R u e d a , E. and R u e d a , J. M., Cultivos enarenados de hortalizas extratempranas.
Mundiprensa ed. Madrid, Spain (1965).
W h e e l e r , A. W., Changes in growth-substances contents during growth of wheat
grains. Ann. Applied Biol. 72, 327-334 (1972).