JOURNAL OF BACTERIOLOGY Vol. 87, No. 6, pp. 1309-1316 June, 1964 Copyright © 1964 by the American Society for Microbiology Printed in U.S.A.
ANAEROBIC GROWTH OF FUSARIUM OXYSPORUM' H. B. GUNNER2 AND M. ALEXANDER Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York
Received for publication 10 December 1963 ABSTRACT GUNNER, H. B. (Cornell University, Ithaca, N.Y.), AND M. ALEXANDER. Anaerobic growth of Fusarium oxysporum. J. Bacteriol. 87:1309-1316. 1964.-Fusarium oxysporum, an alleged obligate aerobe, was found to be capable of growth in the absence of molecular oxygen, provided the medium contained yeast extract, MnO2 , nitrate, selenite, or ferric ions. The active substance in yeast extract was not identified. The fungus possessed hydrogenase, and was capable of utilizing H12. Under anaerobic conditions, the fungus effected the reduction of nitrate, ceric, ferric, selenite, and tellurite ions, as well as the reduction of several inorganic sulfur compounds and indicators having positive oxidation-reduction potentials. The products of anaerobic nitrate-dependent growth were ethanol, C02, acetic acid, and ammonia. Possible explanations for the apparent inability of obligate aerobes to grow in the absence of 02 are discussed.
cultured aerobically. Similarly, Saccharomyces cerevisiae needs certain sterols or unsaturated fatty acids for good anaerobic growth (Andreasen and Stier, 1954); Mlucor rouxii requires thiamine and nicotinic acid (Bartnicki-Garcia and Nickerson, 1961); and Cytophaga succinicans needs CO2 for development in the absence of 02 (Anderson and Ordal, 1961). The present investigation was designed to determine the basis for the inability of a reportedly obligately aerobic fungus to grow without molecular oxygen. Particular attention was given to the possibility that this inability was associated with the absence of a suitable electron acceptor and the related low oxidationreduction potential.
MATERIALS AND METHODS Fusarium oxysporum f. cubense was grown in a basal medium (medium A) consisting of: glucose, 15.0 g; NH4Cl, 4.0 g; K2HPO4, 0.8 g; KH2PO4, Despite the importance in microbial taxonomy 0.2 g; CaC12, 0.1 g; MgSO4 7H20, 0.2 g; FeCl3* and physiology of 02-dependent growth, little 6H20, 0.03 g; and distilled water, 1,000 ml. All attention has been given to the biochemical basis incubations were performed at 30 C. In experiof the need for molecular oxygen or to the possi- ments made to determine whether the fungus bilities that the 02 requirement of aerobic could couple anaerobic growth to the reduction of microorganisms reflects, not a need for a specific nitrate, selenite, and ferric iron, the medium (B) form of the element, but rather some other had the following composition: glucose, 18.0 g; physiological characteristic or deficiency. The NH4C1, 2.0 g; KH2PO4, 1.5 g; MgSO4 7H20, failure of the so-called strictly aerobic microor- 0.6 g; iron chelate (Jacobson, 1951), 0.025 mg; ganism to grow in the absence of molecular 02 ZnSO4 *7H20, 0.03 g; and distilled water, 1.0 remains largely unexplained. Studies of selected liter. Potassium nitrate, Na2SeO3, and ferric microbial strains, however, indicate that many ammonium citrate were added to final concentramicroorganisms may, indeed, proliferate anaero- tions of 4.0 X 10-2 M 10- M, and 10-3 M, respecbically, but only if suitable growth factors are tively. provided; i.e., the organism is unable to synIn investigations of the inorganic ions susceptithesize the metabolite in 02-free circumstances. ble to reduction by F. oxysporum, 10 ,umoles of For example, Richardson (1936) noted that a the sulfur or nitrogen compounds, 200 ,umoles of strain of Staphylococcus aureus requires uracil glucose, and 400 ,umoles of tris(hydroxymethyl)when grown in the absence of 02, but not when aminomethane (tris) buffer (pH 7.6) were added to the main compartment of a Thunberg tube. 1 Agronomy paper no. 636. In the cap was placed 1.0 ml of fungus suspension 2 Present address: Institute of Agricultural and Industrial Microbiology, University of Mas- from a 36-hr culture grown on a rotary shaker; the tube was evacuated, and the suspension was sachusetts, Amherst. 1309
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GUNNER AND ALEXANDER
tipped into the main compartment; after 12 hr, the contents of the tube were centrifuged, and the supernatant fluid was examined for reduced products. The reduction of ceric ions was measured by incubation of 1.0 ml of washed F. oxysporum mycelium suspended in 0.05 M phosphate buffer (pH 7.0) with 4.0 ml of a eerie-citrate complex prepared by mixing equal volumes of 10-4 M Ce(HSO4)4 solution and 10-2 M citric acid adjusted to pH 7.0 with NaOH; after centrifugation, the formation of cerous ions was determined at 270 m,u. The reduction of ferric iron to the ferrous form was determined by incubating a washed fungal suspension in a solution containing 5.0 ,ug of ferric iron as ferric ammonium sulfate. Reduction of the higher oxidation states of selenium and tellurium was assayed by incubating 3.0 ml of a washed fungus suspension with 1.0 ml of 1.0% solutions of the various salts, 5.0 ml of 0.1 M glucose solution, and 5.0 ml of 0.05 M tris buffer (pH 7.6). The capacity of the organism to use electron acceptors of various oxidation-reduction potentials was assessed by incubating 3.0 ml of a washed F. oxysporum suspension in 0.05 M phosphate buffer (pH 7.0) with 3.0 ml of a 0.01% solution of the Eh indicator, 5.5 ml of 0.1 M solution of the neutralized substrate, and 5.5 ml of 0.05 M phosphate buffer (pH 7.0). Dye reduction was recorded at hourly intervals. For growth experiments, a culture system that permitted rigorous 02 removal and continuous monitoring with an 02 electrode (Beckman Instruments Inc., model 11098) was devised. In initial studies, 450 ml of medium were placed in wide-mouthed 500-ml Erlenmeyer flasks fitted with rubber stoppers containing inlets for N2, sampling tube, platinum electrode, salt bridge (3.0% agar, 1.0% NaCl), and a vacuum exhaust line. The electrode and salt bridge were connected to a potentiometer by means of a saturated KCl solution in which a calomel electrode was placed. In subsequent studies, the rubber stopper was fitted only with gas inlet and exhaust tubes and a Thunberg tube cap containing the inoculum. In this way, the myeelial suspension could be evacuated and flushed with gas together with the rest of the system prior to addition of the inoculum to the fresh medium. Before entering the culture vessels, the N2 (Seaford grade, rated as 99.99% pure) was passed through a column of reduced copper turnings heated to 600 C, and was then introduced into a
J. BACTERIOL.
gas washing bottle containing a mixture of a chromic salt, zinc, and sulfuric acid; this procedure for removal of traces of 02 was described by Marshall (1960). The entire system was evacuated to 4.0 cm of mercury, and was flushed five times with the O2-free N2 sterilized by passage through a cotton filter. Anaerobiosis was effected within 15 min after the growth medium was removed from the autoclave, while all solutions were still near 100 C, to minimize the amount of dissolved 02 introduced into the system with the growth media. After the 02 electrode gave a continuous zero reading for 1 hr, a time sufficient for the medium to cool, the inoculum was tipped into the medium from the cap, the inlet and outlet tubes of each flask were closed, and the flasks were incubated at 30 C. To determine the products of fermentation, samples of the culture filtrate were removed, the pH was adjusted to 1.0 to 2.0 with concentrated H2SO4 , and the dissolved CO2 was swept into standard Ba(OH)2 by a stream of N2. The fungal suspension in each flask was removed by filtration, washed, dried at 70 C, and weighed. The mycelium and conidia were considered to contain 50% carbon and 5%O nitrogen. Analysis for nitrite was by the method of Kolthoff and Sandell (1938); ammonia was determined by nesslerization (Wilson and Knight, 1952), ferric iron was estimated by the method of Greweling and Peech (1960); glucose determinations were performed as described by Somogyi (1945); CO2 was measured titrimetrically; ethanol and lactic acid were determined by the procedures described by Neish (1952); acetic acid was identified by determination of the Duclaux numbers (McElvain, 1947); and sulfide was estimated by the method of Delwiche (1951). The amount of H2 consumed by F. oxysporum after 7 days was determined manometrically, the culture flasks being attached to mercury manometers. Hydrogenase was assayed quantitatively in a Warburg microrespirometer with the use of 7-day cultures grown anaerobically in 02-free N2 . Washed mycelium suspended in 0.2% KCl was placed in the side arm of the Warburg flasks; the center well contained 0.2 ml of 20% KOH, and the main compartment of the flask contained 50 ,moles of benzyl viologen and 600 ,umoles of phosphate buffer (pH 8.0), in a total volume of 3.2 ml. The assay was performed at 36 C in an atmosphere of H2 purified of 02 by passage through chromous acid. Control flasks were
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ANAEROBIC GROWTH OF F. OXYSPORUM
maintained in an atmosphere of N2 freed of 02 by passage through heated copper turnings. Anaerobically grown cultures of F. oxysporum were tested routinely for purity by microscopic examination and by inoculation into nutrient broth containing 100 ,ug/ml of cycloheximide. The tubes were incubated anaerobically under N2. No evidence of contamination was noted. RESULTS F. oxysporum grew in an anaerobic environment, provided that higher oxidation states of certain elements were included in the medium. For example, distinct growth was noted visually after 7 days of anaerobic incubation in an agar medium containing 0.3 % K2S04, 0.3 % Na2SeO3, or 0.3% Na2MoO4 2H20 with ammonium as nitrogen source, or in agar containing 0.3 % KNO3 as sole nitrogen source; no mycelium appeared if the ammonium medium was not supplemented with one of these salts. Aerobically, the fungus developed on all the media tested. The results were identical whether the cultures were incubated in an atmosphere containing N2 or H2 freed of 02 catalytically. Although the amount of residual 02 in the gas phase was not determined, the absence of detectable growth in the ammonium medium containing none of these salts served as a biological test for the existence Of 02 levels insufficient to support aerobic proliferation. Further, plates of Potato Dextrose Agar (Difco) streaked with the aerobic Aspergillus niger (Van Tieghem), Rhizobium trifolii, and Bacillus pantothenticus, and incubated in the same containers, showed no growth. The anaerobic cultures of F. oxysporum consisted of fine hair-like structures made up of short hyphal fragments and many chlamydospores. Upon the return to aerobic conditions, conidia began to bud rapidly from the hyphal surface, and, within 24 hr, the culture assumed the characteristics of the typical F. oxysporum mycelium. Aerobically, by contrast, the dominant spore form was the microconidium, which was somewhat less abundant; chlamydospores appeared abundantly only in old cultures. The fungus also grew anaerobically in the ammonium-containing liquid medium supplemented with 0.1% yeast extract; 6.17 mg/ml of glucose disappeared in 10 days. There was neither growth nor glucose disappearance in the absence of yeast extract. Further, F. oxysporum failed to develop anaerobically in the ammonium medium
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TABLE 1. Reduction of inorganic ions by washed mycelium of Fusarium oxysporum Product
Substrate
concn
Com-
pound
pmoles/inl
K2S03 NaHSO3
K2SO4 Na2S203 Na2S206
K2S208 KNO3 Ce(HSO4)4 FeNH4 (SO4) 2
Boiled mycelium control*
Substrate
1.67 1.67 1.67 1.67 1.67 1.67 1.67 0.04 0.10
Concn
Ag/ml
I
g/m
1.1
H 2S H 2S H 2S H 2S H 2S H 2S
1.5 7.0 5.4 4.9
NO2-
0.3
0.0 0.0 0.0 0.0 0.0 0.0
Ce3+ Fe2+
4.0
0.0
3.9
1.6
+
* Concentration of product in incubation mixtures containing boiled rather than viable mycelium.
containing 0.1% Vitamin Free Casamino Acids, L-arginine HCl, L-glutamic acid, DL-valine, DL-phenylalanine, DL-isoleucine, or DL-tryptophan. The yeast extract effect on growth or glucose disappearance also could not be replaced by thiamine, biotin, pyridoxine, riboflavine, pantothenate, vitamin B12, lipoic acid, folic acid, or ascorbic acid added singly or in various combinations at concentrations ranging from 0.05 to 2.50 mg per liter. The morphology of the fungus grown anaerobically in the presence of yeast extract was similar to that in the nitrate medium, except for the greater abundance of microconidia in the former medium. The data above indicate that growth took place when certain inorganic ions were provided. That the fungus does, indeed, effect a reduction of the inorganic ions is demonstrated by the results of Table 1. All of the sulfur salts and the nitrate, ceric, and ferric ions were reduced. The rate of reduction varied markedly with the specific ion; undoubtedly, reduced products in addition to those listed appeared. The brick-red color of selenium and the black color of tellurium developed in mycelium incubated with Na2SeO3 and K2TeOs for 10 hr, and the hyphae contained red and black inclusions when provided with these salts. There was no selenite or tellurite reduction by boiled mycelium, and the viable fungus showed no activity upon selenate and tellurate. Because one hypothesis to account for the
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GUNNER AND ALEXANDER
TABLE 2. Reduction by Fusar ium oxysporum of dyes of different oxidation-reduction potentials l
Indicator
Substrates used as electron donorst
of inl Eh dicator*
t
I
_
Benzenoneindo-3 'chlorophenol ........ 2,6-Dichlorobenzenoneindophenol .. .... Benzenoneindo-2'methylphenol ....... 2,6-Dichlorobenzenoneindo-3'-methyl phenol .............. 2,6-Dibromobenzenoneindo-3'-methoxy-
0.233 +
+
+
+
0.217 +
+
+
+
0.208 +
+
+
+
0.181 +
+
+
+
phenol .............. 0.159 +
-
+
+
-
+ +
-
+
+
+
-
-
-
-
-
-
Benzenoneindo-3 '-sul-
fonaphthol .......... 0.123 Thionin ............... 0.063 Methylene blue ....... 0.011 + Indigo carmine ....... -0.125 Phenosafranine. -0.252 -0.325 Neutral red .......
* From Handbook of Indicators, National Aniline Division, Allied Chemical Corp., New York. t Symbols: + denotes reduction was complete within 10 hr; - denotes no detectable reduction at the end of 10 hr.
inability of aerobes to grow without 02 is the need by the organism for an electron acceptor of high oxidation-reduction potential, an examination was made of the coupling of substrate oxidation by the fungus with indicators of different Eh. The substrates included glucose and succinate, one with a low potential, and the other with a high potential in their respective coupled systems. The data are summarized in Table 2. Only substances possessing potentialsof + 0.011 v or greater were reduced by the intact organism. It is apparent that the fungus is not capable of coupling oxidations with externally supplied electron acceptors having a negative Eh , regardless of the initial energy source. In the basal medium, the fungus brought about a moderate drop in Eh and a small disappearance of sugar. If the medium contained yeast extract, however, both the fall in Eh and the disappearance of glucose became appreciably more marked
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(Table 3). The MnO2 included in the basal medium to poise it at a higher initial Eh permitted almost as active a glucose dissimilation (4.6 mg/ ml of glucose disappearance in 72 hr) as did the yeast extract. Parallel nutritional studies failed to reveal any response of the fungus to manganese as such. Thus, maintenance of a high Eh, a treatment which may act physiologically by providing the fungus with oxidized materials that could serve as electron acceptors in the absence of 02, or the provision of factors in yeast extract, permit anaerobic activity and growth of the fungus. Further trials were made to determine whether the fungus could couple anaerobic growth to the reduction of nitrate, selenite, and ferric iron. Although F. oxysporum failed to grow without 02 in the simple medium, anaerobic proliferation did take place if the fungus was provided with nitrate, selenite, or ferric iron (Table 4). Surprisingly, yeast extract alone did not permit anaerobic growth in this instance, although it did stimulate the fungus in the absence of 02 when nitrate, selenite, or ferric ions were present. Selenite allowed the fungus to increase in cell mass in 02-free circumstances, even when, as indicated by the aerobic cultures, the concentration was high enough to be markedly toxic; such anaerobically grown fungi formed considerable quantities of selenium. In nitrate-grown anaerobic TABLE 3. Effect of manganese dioxide and yeast extract on anaerobic activity of Fusarium oxysporum Addition to basal medium
Time hr
None .............
0
24 48
72
Yeast extract (0.5%) .........
0
24 48 72
MnO2 (0.05 M)....
0
24 48 72
Eh
pH
Glucose metabolized mg/ml
mv
-208 -281 -289 -299
7.25 7.60 7.49 7.57
0.0 1.9 2.1 2.0
-238 -339 -380 -400
7.65 7.70 7.68 7.60
0.0 0.8 5.2 5.1
-105 -165 -162 -190
7.25 7.80 7.57 7.70
0.0
0.8 4.7 4.6
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ANAEROBIC GROWTH OF F. OXYSPORUM11 1313
cultures, 35.0 and 19.0 ,ug/ml of ammonium nitrogen were found in the presence and absence of yeast extract at the end of 7 days, but the nitrite-nitrogen levels were less than 0.1 ,ug/ml. Ferric-grown anaerobic cultures had, at the end of 21 days, 22.8 and 12.6 ,ug/ml of ferrous iron in the presence and absence of yeast extract. No ferrous iron was found in any of the aerobic cultures, but 1.3 ,ug/ml of ferrous iron were found in the anaerobic vessels receiving heat-inactivated mycelium. Approximately 0.47 mg of ferrous iron was produced per mg of mycelium formed; assuming the mycelium to contain 5% nitrogen derived from the ammonium produced by nitrate reduction, about 0.6 mg of ammonium-nitrogen was produced from nitrate per mg of mycelium. The respective oxidized ions, ferric and nitrate, were not the sole electron acceptors for anaerobic growth, however. For example, after 7 days in a nitrate-containing medium, nitrate and 100.8 mg of glucose-C had disappeared, and 4.5 mg of cell-C, 11.0 mg of C02-C, 66.0 mg of ethanol-C, and 18.0 mg of lactic acid-C appeared; little or none of these products was found in nitrate-free solutions, the fermentation of glucose generating electron acceptors utilizable by the fungus. Although an oxidation-reduction balance was not made in this instance because of the complexity of the medium (1% peptone, 1 % glucose, 1 % KNO3, 0.01 % yeast extract), the molar ratios of the products suggested an anomalous fermentation, namely the formation of 6 moles of ethanol, 2 moles of C02, and 1 mole of lactic acid in the course of disappearance of 3.0 moles of glucose. A possible explanation for the small yield of CO2 compared to ethanol, assuming a typical alcoholic fermentation, is the reassimilation of a portion of the evolved gas. To test the possibility of an effect of CO2 on growth of the fungus, various concentrations of NaHCO3 were added to medium B (450 ml per flask) containing either 0.4% KNO3 or 0.2% NH4Cl as sole nitrogen source. Yeast extract (5.0 mg per liter) was included in the medium, the pH was adjusted to 6.5 to 6.8, and the flasks were incubated anaerobically for 7 days. No detectable growth took place in the NH4Cl series, regardless of the bicarbonate concentration. The results of the nitrate series (Table 5) indicate a clear stimulation by bicarbonate with an optimal response at about 20 mmoles per 450 ml of medium. Higher
TABLE 4. Growth of Fusar-ium oxysporum in presence of nitrate, selenite, or
ferric iiron Fungus (mg, dry wt)
Addition to medium
Incubation No yeast extract Yeast extract (days) _ Aero- Anaero- Aero- Anaerobic bic bic bic
None Nitratet Selenite None Ferric iron Ferric iron t
7
119.2 68.3 30.4
1.0 252.8 16.1 139.8 4.6 34.3
1.7 28.5 6.0
21
103.5 81.5 0.0
1.6 158.3 11.5 187.6 0.0 0.0
2.2 23.4 0.0
* Added at a rate of 0.5% in cultures grown for 7 days and 0.1% in those grown for 21 days.
t No ammonium in the medium. t Inoculum inactivated by heating. TABLE 5. Anaerobic growth of Fusarium oxysporum in a nitrate medium containing various concentrations of bicarbonate NaHCO, concn*
Growtht (dry wt)
Final pH
0 10 20 50
3.7 7.8 15.7 10.9
6.8 7.7 8.0 8.6
* Expressed as mmoles per 450 ml. t Expressed as mg per 450 ml.
bicarbonate concentrations appeared to inhibit the fungus. A stimulation of F. oxysporum multiplication in soil and a specific incorporation of C14-CO2 into the mycelium of the fungus were reported by Stover and Freiberg (1958). The observed response to NaHCO3 does not result from a pH effect, because the fungus grew readily from pH 3.0 to 9.0. To test the possibility that an additional oxidizable substrate may have been provided to the fungus in the form of the H2 generated in the chromous acid deoxygenating treatment, cultures were incubated under N2 purified of 02 only by passage over the heated reduced copper and under H2 freed of 02 by passage through the chromous acid scrubbing bottle. Medium B was used, the nitrogen source being either KNO3 or NH4C1 (0.2%). In cultures incubated under N2 , growth was noted in solutions containing nitrate
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GUNNER AND ALEXANDER
TABLE 6. Produzcts of anaerobic growth of Fusariuinm oxysporum in atmospheres of hydrogen
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The presence of hydrogenase in the fungus was demonstrated with washed cell suspensions that and nitrogen had been grown aerobically and anaerobically in medium B amended with nitrate. Mycelium N2 atmosphere H2 atmosphere 1.6 mg of dry weight was added to representing Determination the caps of Thunberg tubes, and 50 ,moles of AnmoNitrate Anmmo Nitrate nium* nium benzyl viologen and 600 /Amoles of phosphate (pH 8.0) were placed in the tube. The total Gas consumed, was 4.0 ml. The tubes were evacuated, volume mmoles ........... O. 0.0 0.96 1.26 flushed five times with H2 purified of 02 by the Growth, mg (dry wt). 3.8 7.8 O. 5 6.6 chromous acid method, mixed, and incubated at Glucose utilized, 30 C. By this procedure, fungal suspensions mmoles ...........
