Inositol. 40 mg. Nicotinic acid. 200,g p-Aminobenzoic acid. 100,g. Pyridoxine* HCl. 50 ,ug. Riboflavine. 5.2 mg. Thiamine* HCI. 100 ,ug. Adenine * SO 4. 40.4 mg.
NUTRITION AND GROWTH OF ARTHROBOTRYS CONOIDES' WALDIMERO COSCARELLI2 AND DAVID PRAMER Department of Agricultural Microbiology, Rutgers, The State University, New Brunswick, New Jersey
Received for publication January 11, 1962 plate is incubated for 2 weeks to 1 month. There will be growth of bacteria, and many of the common fungi will be recognized; but their numbers will be limited by the low nutrient level of the medium. Nematodes present in the inoculum will multiply, and, eventually, fungi which prey on nematodes will be observed. Pure cultures of the fungi are obtained most readily by micromanipulation of spores. By these procedures, a pure culture of the nematodetrapping fungus Arthrobotrys conoides was isolated. A. conoides developed saprophytically on any of a variety of media prepared from complex organic substances and employed commonly for the maintenance of fungus cultures. In pure culture the appearance of the fungus is, in many ways, similar to that of other Moniliales, but the addition of nematodes to a culture causes the mycelium to differentiate into a network of adhesive hyphal loops which are able to capture living worms. Morphogenesis is induced by a metabolic product of nematodes, which was designated nemin (Pramer and Stoll, 1959). Little is known of the nutrition of nematodetrapping fungi, and there is a need at this time for quantitative techniques to be employed in studies of the occurrence, abundance, and distribution of these organisms in soil. A highly selective medium is required on which nematodetrapping fungi can be enumerated, isolated, and identified readily. The nutrition and growth of A. conoides was investigated to provide an experimental basis for the formulation of such a medium.
COSCARELLI, WALDMERO (Rutgers, The State University, New Brunswick, N.J.) AND DAVID PRAMER. Nutrition and growth of Arthrobotrys conoides. J. Bacteriol. 84:60-64. 1962-Despite its ability to capture, kill, and subsist on nematodes, the predacious fungus Arthrobotr'ys conoides does not demonstrate a unique nutritional pattern. Yeast extract, required for growth in a glucose-inorganic salts medium, was replaced by biotin, thiamine, and zinc. Biotin biosynthesis by the fungus is blocked at the desthiobiotin to biotin conversion. A. conoides is unable to synthesize either of the two moieties of thiamine but, when supplied with pyrimidine and thiazole, completes biosynthesis of the vitamin via a coupling reaction. A chemically defined medium that supports rapid and abundant growth of A. conoides is described. Glucose is utilized efficiently (economic coefficient of 31.8 to 42.8), and yields exceeding 9 g (dry wt) of fungus tissue per liter of medium are obtained.
Although fungi that capture, kill, and consume microscopic animals have been known for more than 70 years, they remained a scientific curiosity until Drechsler (1937) described 17 species and presented details of their predacious activity. More than 100 species of fungi that prey on protozoa and nematodes are known today, and there are undoubtedly many more that remain to be described (Duddington, 1955, 1956, 1957). Nematode-trapping fungi are not difficult to isolate. One need only place a quantity of partially decomposed organic material on the surface of a dilute medium such as cornmeal extract agar and observe microscopically the sequence of organisms that develops when the
MATERIALS AND METHODS
The nematode-trapping fungus employed was isolated in this laboratory from decomposing leaves and identified by C. Drechsler as A. conois. In preliminary studies, the influence of type, size, and age of inoculum, concentration of medium constituents, agitation, time of harvest,
1 Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, The State University, New Brunswick, N.J. 2Present address: Bell Telephone Laboratories, Inc., Murray Hill, N.J.
84, 1962 ONUTRITION OF ARTHROBOTRYS CONOIDES
and extent of washing on the yield of fungus tissue was determined. The results enabled a methodology to be established as standard, and in all tests there was strict adherence to the procedures summarized below. Media. Most media had an inorganic salt base of the following percentage composition: NaNO3, 0.37; K2HPO4, 0.1; KCl, 0.05; MgSO4-7H20, 0.05; and FeSO4-7H20, 0.001. This was added in 40-ml quantities to 250-ml Erlenmeyer flasks which were stoppered with nonabsorbent cotton and sterilized by autoclaving for 15 min at 121 C. Upon cooling to room temperature each flask received 1.0 ml of a concentrated glucose solution, which was sterilized separately and added aseptically to give a final sugar concentration of 3%. The reaction of the medium was approximately pH 7.2. When yeast extract was employed, it was included in and sterilized with the basal salt solution, at a concentration of 0.3%. Ash was prepared from yeast extract by incineration in a platinum crucible at 450 C for 15 to 18 hr. The product was solubilized in concentrated HCl and employed as a medium supplement. In later experiments, the ash was replaced with a mineral solution that contained CaCl2 2H20, 0.74 mg; (NH4)6Mo7024 -4H20, 0.37 mg; CuSO4*5H20, 0.80 mg; Mn3O4, 0.27 mg; and ZnO, 0.25 mg; in 1,000 ml of deionized water. The compounds used in experiments to identify the factors in yeast extract required for growth by A. conoides, and the levels at which thev were tested, are listed in Table 1. All but biotin, calcium pantothenate, nicotinic acid, p-aminobenzoic acid, pyridoxine, and thiamine were added to and heat sterilized with the basal salt solution. The forenamed six vitamins and all biotin and thiamine derivatives were sterilized by filtration through sintered glass and added aseptically to the autoclaved medium. Inoculum. Inocula were prepared by homogenizing mycelium harvested from an 11-day-old culture of A. conoides for 1.0 min in an OmniMixer (Ivan Sorval, Inc., Norwalk, Conn.). The resulting suspension was transferred aseptically to a sterile screw-cap centrifuge tube in which the mycelium was washed four times, and then it was suspended in 25 ml of 0.02 M phosphate buffer at pH 7. Samples of the suspension were placed in tared porcelain crucibles, dried at 105 C, and the dry weight of the mycelium per unit volume was determined. The remaining
TABLE 1. Medium supplements Compound
Vitamin-free Casamino acids Biotin Calcium pantothenate Choline chloride Folic acid Inositol Nicotinic acid p-Aminobenzoic acid Pyridoxine * HCl Riboflavine Thiamine* HCI Adenine * SO 4
Cytosine Guanine * HCl
Hypoxanthine Thymine Uracil Xanthine
2g 5 ,g 150 ,g 80 mg 1.2 mg 40 mg 200 ,g 100 ,g 50 ,ug 5.2 mg 100 ,ug 40.4 mg 11.1 mg 20.5 mg 13.6 mg 12.6 mg 11. 2 mg 15.2 mg
suspension was then diluted with sterilized phosphate buffer so that each 1.0 ml contained 2 mg (dry wt) of fungus tissue, and 1.0 ml was added as inoculum to each flask of medium. Inoculated flasks were incubated for 10 days at 28 C without agitation. Harvest and measurement of growth. The content of each flask was harvested on tared Whatman no. 40 paper, washed with 500 ml of distilled water, dried at 105 C for 15 to 18 hr, and stored in a desiccator until weighed. Growth was measured as mg dry weight per 40 ml of medium, and is reported as the mean of four replicates. Chemical procedures. Biotin-L-sulfoxide was prepared by controlled oxidation of biotin, using 30% aqueous hydrogen peroxide as described by Melville (1954). The fine, crystalline product from 1.0 g of biotin was harvested, washed with ethanol, and dried (yield: 236 mg). This was recrystallized twice from water, and the final yield of biotin-L-sulfoxide was 95 mg of polymorphic plates that melted with decomposition at 238 to 241 C. Schoorl's iodometric method using Soxhlet's solutions was employed for the determination of glucose (Browne and Zerban, 1941), and nitrate was measured by the method of Devarda (Rieman, Neuss, and Naiman, 1951). RESULTS AND DISCUSSION
Preliminary studies demonstrated that A. conoides was unable to develop in a glucose-
COSCARELLI AND PRAMER
mineral salts medium but the fungus did grow when the medium was supplemented with yeast extract. To identify the nutritional requisites for growth of A. conoides, a series of experiments was performed having as its objective the replacement of yeast extract by known growth factors. A. conoides developed best in the medium amended with ash, Casamino acids, vitamins, purines, and pyrimidines (Table 2). However, growth on the medium that contained ash, Casamino acids, and vitamins, as well as that TABLE 2. Influence of various medium supplements on growth of Arthrobotrys conoides Medium supplement
Yeast extract Ash + Casamino acids + vitamins + purines + pyrimidines Ash + Casamino acids + vitamins Ash + vitamins Purines + pyrimidines + vitamins + Casamino acids Purines + pyrimidines + vitamins Vitamins Casamino acids + vitamins Ash Ash + Casamino acids + purines + pyrimidines Ash + Casamino acids None
100 120 102 84 28 24 18 16 7 5
* Reported as per cent of that obtained on medium containing yeast extract (245 mg dry
weight/40 ml). TABLE 3.
Influence of some minerals
A rthrobotrys conoides Medium supplement
Yeast extract Ash of yeast extract + vitamins Calcium + molybdate + copper + manganese + zinc + vitamins Calcium + molybdate + copper + manganese + vitamins Calcium + molybdate + copper +
100 97 90
vitamins Calcium + molybdate + vitamins Calcium + vitamins None
9 13 7
* Reported as per cent of that obtained on medium containing yeast extract (223 mg dry weight/40 ml).
on the medium which contained ash and vitamins only, differed little from that obtained on the medium supplemented with yeast extract. It was apparent that the ability of yeast extract to meet the nutritional demands of A. conoides was due to its mineral and vitamin content. The results indicate that a solution of mineral salts could replace the ash of yeast extract in a medium that contained vitamins; and, of the five elements tested, only zinc was necessary (Table 3). The vitamins essential for development of A. conoides in medium supplemented with zinc were biotin and thiamine (Table 4). When only thiamine was supplied, growth of the fungus was less than one-half that obtained in the presence of both vitamins. In the absence of biotin and thiamine, A. conoides failed to
develop. Despite its ability to capture, kill, and subsist on nematodes, A. conoides does not demonstrate a unique nutiitional pattern. Yeast extract, required for growth of the fungus in a glucoseinorganic salts medium, was replaced by biotin, thiamine, and zinc. Media prepared using reagent grade chemicals frequently need supplemental zinc; and biotin and thiamine are necessary for the growth of many microorganisms. An experiment of central composite design (Box and Hunter, 1957) was performed to determine simultaneously the concentration of each of the three nutrients (biotin, thiamine, and zinc) optimal for proliferation of the fungus. Details of the statistical procedure and analyses of the results will be described in a separate publication (Grant, Coscarelli, and Pramer, in preparation). For the present purpose it is sufficient to state that, for maximal growth of A. conoides in the glucose-inorganic salts medium, biotin, thiamine, and zinc were required at concentrations of 5, 100, and 400 ,ug/liter. The ability of various derivatives of biotin and thiamine to support development of A. conoides was determined to obtain information regarding the specificity of the requirement and the step at which vitamin biosynthesis by the fungus is blocked. The results summarized in Table 5 show that biotin-L-sulfoxide and biocytin satisfy the need by A. conoides for biotin. The yield of fungus tissue from medium supplemented with both the pyrimidine and thiazole moieties did not differ significantly from that obtained when the fungus was supplied with
NUTRITION OF ARTHROBOTRYS CONOIDES
V-OL. 84, 1962
TABLE 4. Influence of some vitamins on growth of Arthrobotrys conoides Medium supplement
Yeast extract Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + thiamine biotin + inositol + riboflavine + folic acid + choline chloride + zinc Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + thiamine biotin + inositol + riboflavine + folic acid + zinc Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + thiamine biotin + inositol + riboflavine + zinc Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + thiamine biotin + inositol + zinc Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + thiamine biotin + zinc Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + thiamine zinc Pantothenate + p-aminobenzoic acid + pyridoxine + nicotinic acid + zinc Pantothenate + p-aminobenzoic acid + pyridoxine + zinc Pantothenate + p-aminobenzoic acid + zinc Pantothenate + zinc None *
11 5 3 7
Reported as per cent of that obtained on medium containing yeast extract (247 mg dry weight/40
TABLE 5. Influence of biotin and thiamine derivatives on growth of A rthrobotrys conoides Medium supplement
Biotin Biotin-L-sulfoxide Biocvtin Desthiobiotin Homobiotin L-Aspartic acid Pimelic acid Norbiotin Thiamine Pyrimidine + thiazole Thiazole Pyrimidine Pyrithiamine None
100 99 89 15 11 9 8 7 100 111 23 13 13 7
The inability of A. conoides to develop on media that contained desthiobiotin and pimelic acid indicates that the pathway of biotin biosynthesis by the fungus is blocked at the desthiobiotin to biotin conversion. Moreover, A. conoides is apparently unable to synthesize either of the two moieties of thiamine but, when supplied with both pyrimidine and thiazole, completes biosynthesis of the vitamin via a coupling reaction. The fact that growth of A. conoides was not inhibited by pyrithiamine is consistent with the concept that this analogue antagonizes only those cells that require intact thiamine (Cochrane,
Since the rate and extent of microbial growth controlled by environmental as well as nutritional factors, the influence of temperature * Reported as per cent of that obtained on and pH on development of A. conoies in a medium containing thiamine (230 mg dry weight/ chemically defined medium was determined. The 40 ml). fungus grew at 20 C, but at 28 C growth was optimal. No significant development occurred intact thiamine, but A. conoides did not develop at 4, 37, and 55 C. Greatest yields were obwhen provided with the pyrimidine or thiazole tained from media with an initial reaction of pH moiety only. Norbiotin and homobiotin did not 5.0, but growth of A. conoides at pH 4.0 and 6.0 interfere with biotin utilization and pyrithiamine did not differ greatly from the maximum. The did not interfere with thiamine utilization by fungus was more tolerant of acid than alkaline A. conoides, when the molar ratio of analogue reactions. to vitamin in the medium was varied from 1 Figure 1 shows the course of growth of A. to 10. conoides at 28 C iia a medium of the following are
COSCARELLI AND PRAMER
coefficient of this magnitude indicates that growth is efficient and accumulation of intermediate metabolic products is minimal (Cochrane, 1958). To our knowledge, the present investigation is the first to describe a chemically defined medium that supports growth of a nematode-trapping fungus. A maximal yield of approximately 9 g (dry wt) of fungus tissue/liter of medium was obtained.
TIME (DAYS) FIG. 1. Assimilation of glucose and nitrate during growth of Arthrobotrys conoides on a chemically defined medium. Symbols: 0 = growth; @ = glucose; A = nitrate.
composition: glucose, 30 g; NaNO3, 3.7 g; KH2PO4, 3.4 g; KCl, 0.5 g; MgSO4 7H20, 0.5 g; PeSO4 *7H20, 10 mg; ZnO, 0.5 mg; thiamine, 0.1 mg; and biotin, 5 ,g; in 1,000 ml of deionized water. The initial reaction of the medium was adjusted with HCI to pH 5.0. The results of periodic analyses medium for periodic analyses of of the the medium for residual residual sugar and nitrate are also illustrated in Fig. 1. After a lag period of 3 days there was a phase of rapid growth during which the dry weight of fungus tissue increased linearly with time. Development of the fungus reached a maximum on the 13th day of incubation and was constant until the 15th day, when the experiment was terminated. During the phase of rapid growth, the fungus assimilated glucose and nitrate at constant rates. Residual concentrations of glucose and nitrate were inverse functions of growth, and the onset of the stationary phase was associated with simultaneous depletion of the medium glucose and nitrate. The reaction of the medium changed from an initial value of pH 5 to pH 7, but this did not appear to influence growth adversely. An approximation of the efficiency of carbon utilization by A. conoides was obtained by
calculationo an series of enomic coefficies from the analytical data in Fig. 1. The values varied from a maximum of 42.8 at the 4-day period to a minimum of 31.8 in 15 days; the mean of 13 measurements was 36.8. An economic
research grants G5949 and G9749 from the National Science Foundation. The authors are grateful to D. Hendlin of Merck, Sharp and Dohme for the generous quantities of biocytin, desthiobiotin, pyrithiamine, and the pyrimidine and thiazole moieties of thiamine; to W. E. Scott of Hoffmann-La Roche, Inc. for supplying norbiotin and homobiotin in ample amounts; and to C. L. Grant of the University of New Hampshire, for preparation and spectrochemical analysis of ash from yeast extract. LITERATURE CITED B
E . for designs responseexperimental surfaces. Ann. Math. Stat.exploring 28:195241. BROWNE, C. A., AND F. W. ZERBAN. 1941. Physical and chemical methods of sugar analysis. John Wiley and Sons, Inc., New York. COCHRANE, V. W. 1958. Physiology of fungi. John Wiley and Sons, Inc., New York. DRECHSLER, C. 1937. Some hyphomycetes that prey on free-living terricolous nematodes. Mycologia 29:447-552. attack microC. L. 1955. DUDDINGTON, Botan.Fungi Rev.that 21:377-439. scopic animals. DUDDINGTON, C. L. 1956. The predacious fungi: Zoopagales and Moniliales. Biol. Revs. Cambridge Phil. Soc. 31:152-193. DUDDINGTON, C. L. 1957. Predacious fungi, p. 218-237. In R. E. 0. Williams and C. C. Spicer [ed.], Microbial ecology. Cambridge University Press, New York. MELVILLE, D. B. 1954. Biotin sulfoxide. J. Biol. Chem. 208:495-501. Nemin: N. R. STOLL. 1959.trap D., AND substance PRAMER, forma-a causing morphogenic tion by predacious fungi. Science 129:966-967. RIEMAN, W., J. D. NEUSS, AND B. NAIMAN. 1942. Quantitative analysis. McGraw-Hill Book Co., Inc., New York. factor