Microbiological Production of Gibberellic Acid in Glucose Media'

A. SANCHEZ-AIARROQUIN
Escuela Nacional de Ciencias Quimicas, Unitersidad de 3lexico, 31exico, D.F., Mllexico
Received for publication 8 July 1963

ABSTRACT MIATERIALS AND ]METHODS

SANCHEZ-M\ARROQUIN, A. (Escuela Nacional de Cien- Strains. The strains of Fusarium (Table 1) were inocu-
cias Quimicas, MIexico, D.F., M\Iexico). -Microbiological lated in potato-glucose-agar slants kept in an incubator at
production of gibberellic acid in glucose media. Appl. 28 C until complete development was obtained. Distilled
Microbiol. 11:523-528. 1963. Gibberellic acid production water (10 ml) was then added, and, from the suspension
from various substrates was studied in 43 strains of Fusa- thus obtained, 1 ml was taken for inoculation in the follow-
rium, among which F. monilhforme straini IOC-3326 was ing medium: glucose, 30 g; magnesium sulfate, 5 g; am-
selected as the best producer. Experiments were carried monium sulfate, 2.7 g; corn steep liquor, 1.5 ml; agar,
out in shaker flasks and pilot plant fermentors. The results 2.0 g; distilled water, 1000 ml.
indicate that the best substrate for gibberellic acid pro- The inoculated flasks, with 50 ml of medium, were
duction with this strain is composed of the following: incubated at 28 C for 24 hr in a mechanical agitator. This
glucose, 20 g; corn steep liquor, 25 g; ammonium nitrate, inoculum was used at a concentration of 1.5 % for both
2.6 g; monopotassium phosphate, 0.5 g; potassium sulfate, flask and pilot plant experiments.
0.2 g; and water, 1000 mnl. Glucose, ammonium nitrate, Fer mentation. The composition of the 11 basic substrates
and corn steep liquor were found to be critical. With this employed for the fermentations is shown in Table 2. Three
medium, maximal yields of 1196 mg per liter in shaker correspond to previously reported formulae (Borrow et al.,
flasks and 997 mg per liter in fermentors were produced. 1955; Stodola et al., 1955; Darken et al., 1959).
The flask fermentations were carried out in 250-ml
In the experimental elongation of rice plants, Kurosawa Erlenmeyer flasks containing 50 ml of media and incubated
(1926) was the first to utilize cell-free filtrates of Fusarium at 30 C in rotatory shakers at 200 rev/min. The fermenta-
moniliforme cultivated on li(uid media, and Yabuta and tions, in general, were allowed to proceed for 7 to 9 days.
Sumiki (1938) succeeded in isolating the two forms of Experiments on a larger scale were carried out in 60-liter
crystalline material called gibberellins A and B. Later, fermentors with regulated aeration and stirring equip-
Curtis and Cross (1954), Stodola et al. (1955), and Taka- ment. The temperature was maintained at 30 C during
hashi et al. (1955) identified gibberellic acid as a third the entire fermentation period. The initial pH was 5.0 to
component obtained from those filtrates. 5.8.
M\lany different substrates have been used in the fer- Added substances. The effects of the following substances,
mentation of gibberellic acid, as summarized by Stodola added to the basic media of Borrow et al. (1955) and
(1958). Recently, Darken, Jensen, and Shu (1959) ob- Darken et al. (1959), were studied: methanol (0.5 to
tained yields of 805 to 880 mg per liter of gibberellic acid. 3.5 %O); thiourea (10 to 50 ,ug/ml); alanine and valine (0.2
With 50-ml flasks and a medium based on corn steep to 0.8 g per liter); sodium molybdate (0.10 to 1.0 mg per
liquor, ammonium sulfate, and monopotassium phos- liter); aluminum sulfate (0.1 to 0.25 mg per liter); ethyl-
phate, to which a mixture in low concentrations of glucose, enediaminetetraacetic acid (EDTA; 5 to 50 ,ug/ml);
glycerol, and lactose was added, the highest yields were ascorbic acid (5 to 50 ,ug/ml); ethanol (0.5 to 4.0 %);
obtained within 7 to 8 days. The slow addition of glucose morpholine (0.5 to 2 mg/ml); malt (0.2 to 5 g/100
in experimental pilot plant fermentations produced, under ml); malt extract (0.05 to 0.5 g/100 ml); CaCO3 (1 to
the same conditions, more gibberellic acid (620 mg per 7 g per liter); corn, coconut, and sesame oils (5 to 40
liter) than did controls containing the total amount of ,ug/ml); ammonium chloride (1.7 g per liter); ammonium
sugar at the start of the fermentation. sulfate (4.3 g per liter); ammonium phosphate (4.3 g
In the present work, studies are reported on the pro- per liter); ammonium carbonate (3.1 g per liter); and corn
duction of gibberellic acid from various basic substrates steep liquor (0.2 to 35 g per liter). Since ammonium
with total initial addition of glucose or sucrose, as well as nitrate was found to be the most adequate nitrogen source
certain additional substances. A total of 43 strains of at the concentration studied, it was later tested at con-
Fusarium were tested in the preliminary selection. centrations of 1.0 to 4.0 g per liter.
1 This paper was presented at the II Latin-American Congress Chemical determinations. Gibberellic acid was deter-
of Microbiology, San Jose, Costa Rica, 10-16 December 1961. mined by titration with a solution of 0.1 or 0.25 N NaOH
523
5)24 SANCHEZ-AIARROQUIN
as the case required; phenolphthalein was used as indica-
tor, and the results were expressed in terms of milliequiv-
alents of gibberellic acid. In samples that gave high
readings and in the study of the effects of certain com-
pounds on the fermentation yields, the acid was deter-
mined by the fluorometric method of Kavanagh and
Kuzel (1958), by use of a Klett photofluorometer with a
# 54 green filter and a # 3389 filter cell. The samples were
centrifuged at 2000 rev/min for 10 min and purified by
partition chromatography on columns (250 X 20 mm) of
granular potassium carbonate and anhydrous sodium
sulfate mixture, washed with a solution of acetonitrile.
The standard curve was prepared with pure gibberellic
acid from Imperial Chemical Industries. Gibberellic acid
was also identified by chromatography on Schleicher and
Schuell no. 598 paper according to the method of Bird
and Pugh (1958).
Development was for 36 to 37 hr at 22 to 24 C, and
RF values of 0.11 to 0.12 were obtained.
The pH was determined with a Beckmani potentiom-
eter. The total carbohydrates of the dilute centrifuged
mixtures were determined by the method of Shaffer and
Somogyi (1933).
All determinations were run in duplicate, and results
reported are averages of the duplicate values.
The purity of the gibberellic acid after passage through
the purifying column and washing with acetonitrile was
99.8 clc.
RESULTS ANI) DIsCUSSION
The species and strains of Fusav-ium utilized in the
present work are given in Table 1.
All of these strains were tested for the production of
gibberellic acid on 11 media as indicated in Table 2; the
majority had glucose as a source of carbon set at different
concentrations, and only three had saccharose. The other
components were varied to study their influence on yield.
According to the results obtained by paper chromatog-
raphy, which showed RF values of 0.11 and 0.12, appar-
ently only five of the strains indicated in Table 3 produced
gibberellic acid. Nevertheless, a more careful study, and
the results obtained by fluorometric analysis, indicated
that in reality only strains LIMUR-1148 and IOC-3326 of
Fusarium moniliforme actually produced gibberellic acid
in high yields. These were, therefore, selected for more
detailed investigation on the 11 culture media. On the
basis of the preliminary results expressed in Table 3
and those obtained from the check tests in quadruplicate
(Table 4), media B-1 and B-10 were found to give the
highest and most consistent yields at 192 hr of fermenta-
tion with strain IOC-3326. At the time of maximal pro-
duction, which fluctuated from 889 to 1196 mg per liter
according to the substrate used, the final pH of these
media varied from 3.6 to 4.1.
With the same IOC-3326 strain, tests were carried out
to study the influence of the 24 different compounds
APPL. AIICROBIOL.
mentioned above in M\aterials and AMethods, by use of
the two fundamental media of Borrow et al. (1955) and
Darken et al. (1959), both based on glucose as shown in
Table 2. The results are summarized in Table 5, which
gives the optimal concentrations which improved the
process and the minimal concentrations at which inhibi-
tory effects were noted. The substances which gave
slightly increased results were malt extract, methanol,
ethanol, and thiourea, at the concentrations indicated in
Table 5, and the combination of malt with methanol or
TABLE 1. Species of Fusariumi utilized in screening tests
Strain Collection no.*
F. aquaeductum Lagerh........................ IMUR-10
F. aqutaedictunn Lagerh........................ IMUR-651
F. avenaceovn (Fries) Saccardo................. IMUR-1444
F. bulbigenunt Cooke and Massee............... IMUR-800
F. bulbigenum Cooke and Massee var. lycopersici
(Brushi) Wr................................. IMUR-103
F. bulbigenum Cooke and Massee var. lycopersici
(Brushi) Wr................................. IMUR-1416
F. chlamydospo-ruot Wr. and Rkg............... IMUR-930
F. chlautydosporwin Wr. and Rkg............... IMUR-931
F. chlantydosporuitm Wr. and Rkg............... IMUR-1475
F. cutbense E. F. Sm........................... IMUR-9
F. dianthi Prill. and Del....................... IMUR-504
F. equiseti (Corda) Saccardo................... IMUR-805
F. tycopersici (Br. and Wr.) Br................. IMUR-309
F. tycopersici (Br. and Wr.) Br................. IMUR-23
F. 'inoniliformte Scheldon...................... IMUR-909
F. nioniliforime Scheldon ....................... IMUR-1148
F. nivernm E. F. Sm ... IMUR-307
F. orthoceras App. and Wr..................... IMUR-838
F. orthoceras App. and Wr..................... IMUR-836
F. oxysporunr var. cubense (E. and Sm.) Wr.... IMUR-157
F. oxysporumtt Schlech var. cubense (E. and Sm.)
Wr ...... IMUR-267
F. oxysporunm Schlech.......................... IMUR-794
F. roseutit Link.............................. IMUR-782
F. scirpi Lambotte and Fautrey................ IMUR-797
F. solani (Martius) Appel and Wr. var. martii
(App. and Woll.) Wollen..................... IMUR-787
Fusarium sp................................... IMUR-534
Fusarium sp................................... IMUR-345
Fusarium sp................................... IMUR-93
Fusarium sp................................... IMUR-47
Fusarium sp................................... IMUR-22
Fusarium sp................................... IMUR-1255
F. sultphureurn Schlecht........................ IMUR-483
F. vasinfecturn Atk............................. IMUR-1413
F. conglutinans............................... IOC-2142
F. culmorunt.................................. IOC-2143
F. diversisporui............................... IOC-2148
F. poae........................................ IOC-2153
F. semitectum................................. IOC-2162
F. sporotrichoides ............................ IOC-2168
F. bulbigenum ................................ IOC-3149
F. sulphureum................................ IOC-3151
F. orthoceras.................................. IOC-3294
F. moniliforme................................ IOC-3326
*
IMUR = Instituto de Micologia, Universidade do Recife;
IOC = Instituto Oswaldo Cruz.
VOL. 1 l X 1963 V1ICROBIOLOGICAL PRODUCTION OF GIBBERELLIC ACID 525

TABLE 2. Composition of basal test media (grams per liter)

Components Borrow Darken Stodola A B C D B-i B-3 B-4 B-10

Corn steep liquor . .25 25 25 25 25 25 1.25 1.25 25

Sucroseu..- - - 10 20 40
Dextrose .............................. 40 20 15 20 - - - 20 20.00 40.00 30
Ammonium tartrate ................... 9.5 - - - - 9.5
Ammonium sulfate . .1.0 - 1.0 1 1 1.5
Ammonium chloride.- - 3 - -.-.-.-.--- -
Ammonium nitrate ......... ........... - - 2.6 2.6 2.6
Monopotassium phosphate ............. 2.0 0.5 3.0 0.5 0.5 2.0 0.5 0.5 0.5 2.0 2.0
Magnesium sulfate ..................... - 3.0 - - 0.2 0.2
Potassium sulfate ...................... 0.2 - 0.2 0.2 0.2 0.2
Calcium carbonate ..................... - - - 7

TABLE 3. Summary of gibberellic acid production by selected strains TABLE 4. Summary of gibberellic acid yields by strain IOC-8326*
(50-ml flasks)
Acidity as Gibberellic
Medium (phoobeuroetric)ai
ibberellic acid
(photofluorometric)
Time of pH
maximal
production Initial Final
Strain Medium Acidity as
d acid (photo
fluorometric)
mg/liter hr
g/50 ml mg/liter Borrow 472-616 192 5.5 5.2
IMUR-1444 Darken 0.346 0 Darken 457-685 192 5.6 4.6
Borrow 1.557 0 Stodola 213-293 168 5.3 4.7
IMUR-909 Darken 0.259 180 A 660-865 168 5.1 4.3
Borrow 1.297 204 B 206-215 192 5.5 4.6
IMUR-1413 Darken 0.259 0 C 256-260 192 5.0 3.9
Borrow 1.946 0 D 230-245 168 5.4 3.5
IMUR-1148 Darken 0.389 117 B-1 985-1196 192 5.6 3.6
Borrow 0.389 208 B-3 912-1014 192 5.6 4.1
IOC-3326 Darken 0.346 442 B-4 889-1011 192 5.6 4.1
Borrow 0.605 520 B-10 918-1092 192 5.6 3.9
Stodola - 213
A 660
*
Four 50-ml flasks were used for each test. Times of maximal
B - 206 production and pH values are given for the flask with the highest
C 256 yield.
D 190
B-1 - 1112 g per liter (Table 7) were found to be optimal with a yield
B-3 1014 of 598 mg per liter. The beneficial influence of corn steep
B-4 - 1011
B-10 1092 liquor on the production of gibberellic acid in Borrow's
medium with 3.0 g per liter of ammonium nitrate sub-
stituted for ammonium tartrate can be seen in Table 8.
EDTA at the same concentrations. (The latter were in- Course of the fermentations. Figures 1 and 2 show the
ferior with respect to the addition of malt extract alone.) changes in pH and carbohydrate concentration during the
An inhibitory effect was noted in both media with valine, production of gibberellic acid in flasks containing 50 ml of
sodium molybdate, ascorbic acid, and calcium carbonate, the basic Darken and B-1 media by the IOC-3326 strain
at the indicated concentrations, and with mixtures of during 216 hr. As is seen, the consumption of carbohy-
methanol or EDTA and calcium carbonate. Aluminum drates was rapid, these having been almost totally con-
sulfate showed a favorable though weak influence in sumed in both cases within 96 or even 72 hr.
Darken's medium, and an unfavorable one in Borrow's. The pH changes, on the other hand, differed in these
The other materials showed no clear-cut action in either two media. In the basic Darken medium, a drop from 5.8
medium. With regard to the source of carbon, the best to 5.0 was noted in 72 hr; B-1 showed an early rise from
results were obtained with 2 to 3 % glucose, as shown in 5.8 to 7.0 followed by a drop to 5.7 after 48 hr. Also, in
Table 6 which refers to Borrow's medium. both cases, a slight -alkalinization was noted towards the
With regard to the influence of the nitrogen source end of the experiment, between 196 and 216 hr of fermenta-
when ammonium tartrate (the basis of the Borrow me- tion, a consequence, perhaps, of cell autolysis. Acid pro-
dium) was replaced by other organic and inorganic nitro- duction was greater and not as slow in the B-1 medium,
gen compounds in equimolar quantities, and corn steep when compared with that of Darken et al. (1959).
liquor was added at a level of 25 g per liter, it was found The pH curve in the B-1 medium is similar to that re-
that ammonium nitrate gave the best results. Its effect ported by Darken et al. with slow addition of glucose,
at different concentrations was therefore studied, and 3.0 differing only in a delay in the changes produced. Under
526 SANCHEZ-MARROQUIN APPL. MICROBIOL.
TABLE 5. Influence of selected compounds on gibberellic acid conditions of slow addition, the pH diminished in 24 hr,
production by strain IOC-s326* only to rise after 48 hr, and then gradually fell until 168
Final pH Gibberellic acid hr; in our experiments, the pH declined in 72 hr, rose
Compound added to basal media Final (mg/liter) steadily up to 120 hr, and then declined more rapidly up to
B D B D
192 hr.
None (blank) ...................... 4.3 4.1 510 472 Pilot plant production. Gibberellic acid production by
Methanol (3%) .................... 4.5 4.3 525 498 F. moniliforme IOC-3326 was also studied in 60-liter fer-
Thiourea (50,ug/ml) ............... 3.1 4.2 515 494 mentors containing 30 liters of the Borrow, basic Darken,
A12(SO4)3 (0.25 mg/ml) ............. 4.4 5.2 451 478
B-1, and B-3 media (Table 9). The maximal yields were
EDTA (50 ,ug/ml) .................. 5.3 4.5 495 473
Malt extract (0.1%) ............... 3.9 4.7 538 511 obtained from media B-i and B-3 (997 and 975 mg per
Valine (0.2%) ...................... 5.8 5.9 405 260 liter, respectively) at 192 hr of fermentation under the
Sodium molybdate (0.25 mg/ml). 4.7 6.0 390 360 conditions indicated in Table 9. The final pH, in both
Ascorbic acid (50 ,g/ml) ........... 3.9 4.5 486 401 cases, was 3.2 and 3.5, respectively, with an almost total
CaCO3 (7 g/liter) .................. 6.6 6.1 297 109
consumption of carbohydrates. These yields are only
Ethanol (3.5%) .................... 4.3 5.1 520 500
Methanol + EDTA................ 4.8 5.4 456 398 slightly inferior to those obtained in flasks with 50 ml of
Methanol + malt extract ........... 3.1 4.6 570 497 medium (Table 4).
EDTA + malt extract ............. 3.8 4.2 588 510
CaCO3 + EDTA................... 6.5 6.3 293 188
CaCO3 + methanol + malt extract.. 6.2 6.1 196 204
CaCO3 + methanol + EDTA ...... 6.1 6.2 143 216
*
B = Borrow's medium; D = Darken's medium. 8

TABLE 6. Influence of dextrose concentration in Borrow's medium 7

on gibberellic acid production

Dextrose Acidity as gibberellic acid Gibberellic acid 6
(photofluorometric)
glliter g/50 ml mg/liter
5
10 2.07 616
20 2.35 627
30 2.62 623 4
40 4.01 604
3
TABLE 7. Influence on gibberellic acid production of ammonium
nitrate as a substitute for ammonium tartrate in Borrow's 2
medium with 25 g per liter of corn steep liquor
Ammonium nitrate Acidity as gibberellic acid Gibberellic acid
72H 120H 168H 192H 216H
glliter g/SO ml mg/liter
FIG. 1. Gibberellic acid production by Fusarium moniliforme
1.0 0.34 460 IOC-3326 in the medium of Darken et al. (1959) in 50-ml flasks.
1.5 0.52 495
2.0 0.50 536 1300 pH 1
3.0 0.55 598 1200 1:

4.0 0.48 511

TABLE 8. Influence of corn steep liquor concentration on gibberellic

acid production in Borrow's medium with 3.0 g per liter of
ammonium nitrate substituted for ammonium tartrate
Corn steep liquor Acidity as gibberellic acid (photofluorometric)

g/liter g/SO ml mng/liter

0.2 0.12 26
5.0 0.32 416 2,
10.0 0.32 472
15.0 0.34 520 24 48 72 96 120 14t 168 192 216 Hours
25.0 0.44 619
35.0 0.37 612 FIG. 2. Gibberellic acid production by Fusarium moniliforme
IOC-3826 in medium B-i (50-ml flasks).
VOL. 11)1963 V\IICROBIOLOGICAL PRODUCTION OF GIBBERELLIC ACID 527

TABLE 9. Highest yields of gibberellic acid in 60-liter fermnentors 100 ml of potassium sulfate, and 0.05 % monopotassium
by Fusariumn moniliforime IOC-3326* phosphate) were brought together in medium B-1, higher
yields were obtained. This medium was equally effective
Substrate acbstrateGidberellic Tie Initial pH Final Residual
Gibberellic sugar when applied to tank fermentations. In flasks, as well as in
mg/liter hr
60-liter fermentors, the results were higher than most of
5.8 4.8 0.2
those reported in scientific literature. With this medium,
Borrow .... 565 192
Darkei .. 516 168 5.8 4.6 0.2 maximal yields of 1196 mg per liter of gibberellic acid were
Medium A .. ... 656 168 5.8 3.4 0.1 produced in shaker flasks and 997 mg per liter in fermen-
MIedium B-1 .. ... 997 192 5.6 3.2 0.1 tors. In Kurosawa's (1925) first investigations, extremely
Medium B-3.975 192 5.5 3.5 0.1 low yields were obtained on glucose-based media; Yabuta,
*
Inoculum: 1.5% of a 22-hr growth in sporulation medium. Sumiki, and Uno (1939) obtained
100 mg per liter on
Temnperature: 30 C. Aeration: 0.5 liters per vol per min. Agitation: media base on glycerol and glucose, ammonium chloride,
230 rev/min. and other salts, in a period ranging from 25 to 40 days;
Mitchell and Angel (1950) succeeded in obtaining 200
900 pH
mg per liter in 65 hr in a glucose, ammonium chloride,
8 magnesium sulfate, and monopotassium phosphate me-
800
dium; Stodola et al. (1955) obtained 22 mg per liter in
7007
7 65 hr with strain NRRL-2284; Borrow et al. (1955) ob-
tained 180 mg per liter in 8 to 9 days in the medium al-
600 ready indicated with strain Kew no. 917; and Darken
-~~~~~N et al. (1959) with the same strain obtained up to 880
E500\, mg per liter in flasks with 50 ml of medium containing
5 glucose at 1 %, lactose at 2 %, and glycerol at 2 %. Darken
et al. (1959) also obtained 650 mg per liter in 1000-gal
400 GN~~~~~~~~~~~~~~~~~~
fermentors, using the same medium with glucose at 3 % as
the sole carbon source and a fermentation period of 7 to 8
days, adding the glucose during the course of the fermen-
3 tation.
In the present work, slightly higher yields were obtained
~9RBOHYDRATE(B-1) CARBOHYDRATE (A) in tank fermentations without the necessity of the slow
24 48 72 96 120 144 168 192
addition of the carbohydrate. The changes in pH and the
carbohydrate consumption on the B-1 medium were simi-
FIG. 3. Carbohydrate consumtption, changes in pH, and gibberellic lar to those indicated by Darken et al. (1959) on their
acid produtction by Fusariu.tn moniliforme IOC-3326 in 60-liter media with either glycerol or the slow addition of glucose.
feruientors (media A and B-1). Same conditions as in Table 9.
LITERATURE CITED
The course of fermentation in the B-1 medium, which BIRD, H. L., AND C. T. PUGH. 1958. A paper chromatographic
gave the highest yield, is shown in Fig. 3, in comparison L separation of gibberellic acid and gibberellin A. Plant Physiol.
33 :45-46.
with that obtained on medium A. The fermentation curves BORROW, A., P. W. BRIAN, V. E. CHESTER, P. J. CURTIS, H. G.
are similar to those obtained in flasks. Nevertheless, the HEMMING, C. HENEHAN, E. G. JEFFREYS, P. B. LLOYD, I. S.
time for maximal production in the B-1 medium was re- NIXON, G. L. F. NORRIS, AND M. RADLEY. 1955. Gibberellic
duced to 168 hr as compared with 192 hr required for acid, a metabolic product of the fungus Gibberella fujikuroi:
maximal flask production. some observations on its production and isolation. J. Sci.
Food Agr. 6:340-348.
The main results obtained in the present work indicate CURTIS, P. J., AND B. E. CROSS. 1954. Gibberellic acid. A new
the following: (i) that glucose is better utilized by the metabolite from the culture filtrates of Gibberella fujikuroi.
strains submitted to study than is saccharose in the pro- Chem. Ind., p. 1066.
duction of gibberellic acid, (ii) that ammonium nitrate DARKEN, M. A., A. L. JENSEN, AND P. SHU. 1959. Production of
is a better source of nitrogen in this activity, (iii) that the gibberellic acid by fermentation. Appl. Microbiol. 7:301-303.
KAVANAGH, F., AND N. R. KUZEL. 1958. Fluorometric determina-
addition of corn steep liquor is essential for increased tion of gibberellic and gibberellinic acids in fermentation
yields, and (iv) that none of the 25 additional materials products, commercial formulations, and purified materials.
studied substantially influenced the production of the acid J. Agr. Food Chem. 6:459-463.
when added in variable quantities to the basic Borrow KUROSAWA, E. 1926. Experimental studies on the nature of the
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When those conditions judged optimal for the produc- MITCHELL, J. E., AND C. R. ANGEL. 1950. Plant-growth-regulating
tion of gibberellic acid (2.0 g/100 ml of glucose; 0.26 g/100 substances obtained from cultures of Fusariutm moniliforine.
ml of ammonium nitrate; 2.5 %c corn steep liquor; 0.02 g/ Phytopathology 40:872-873.
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