Trans. Brit. mycol. Soc. 48 (I), 73-80 (1965)Printed in Great Britain

NUTRITION OF SPINELLUS MACROCARPUS

By PAULINE WATSON

Department of Botany, University of Durham

(With 2 Text-figures)

Spinellus macrocarpus has been grown by several workers in pure culture onnatural media and its nutritional requirements have now been investigated.It grew well on media containing glucose, fructose, mannitol or trehalose ascarbon sources but did not utilize gluconate, lactose or galactose. A mixtureof twenty-one amino acids, hydrolysed casein or yeast extract were suitablenitrogen sources, but single amino acids or inorganic nitrogen compounds werenot utilized. S. macrocarpus required thiamin and nicotinic acid for growthbut the latter could be replaced by relatively large amounts of tryptophane.High concentrations of amino acids in the medium inhibited growth especiallyif it contained insufficient nicotinic acid. The results suggest that some othersubstance, 'X', contained in yeast extract but not in hydrolysed casein, isnecessary for growth under some conditions, especially when large amounts ofamino acids are present in the medium.

The parasitic mould Spinellus macrocarpus (Corda) Karsten, which occursin nature on agarics, has been grown in pure culture on natural media byseveral workers in recent years (Ellis & Hesseltine, 1962; Watson, 1962;Leadbeater & Richardson, 1963), and a study has been made of thefactors which influence germination of its sporangiospores (Watson, 1964).The present paper gives an account of an investigation of its nutritionalrequirements, as it was thought that these also might be of importance indetermining its parasitic habit.

MATERIAL AND METHODS

The isolate of S. macrocarpus tested was the same as that used previously(Watson, 1964); it was isolated in 1961 from a fruit-body of Mycenagalopus.

All the media used were variants of a semi-synthetic basal medium onwhich S. macrocarpus grew well. This had the following composition:glucose, 20 g.; KH2P04, I g.; MgS04, 0'5 g.; FeCI3, 0'5 ml. I %solution;ZnS04, 0'5 ml. 0'2 % solution; yeast extract (Difco), 0'5 g.; distilledwater I 1. The details of the variants are given in the accounts of theindividual experiments. Cultures were in flasks (IOO ml.) with loose­fitting glass caps with 20 ml. of medium in each. Vitamins, supplied tosome flasks only, were added in 0'5 ml. water to the 20 ml. of mediumalready in each flask, the resulting slight dilution apparently having noeffect. All the media were sterilized by autodaving for IO min. at 15 lb.;sq. in. Certain constituents were sterilized separately to avoid reactionswith other substances in the media and this is indicated in the accounts ofthe individual experiments.

74 Transactions British Jrfycological SocietyThe flasks were inoculated with uniform 6 mm. diam. disks, cut with a

cork-borer from a culture about 2 weeks old on 2 % malt agar. This wasfound to be the most effective inoculum as the spores require specialconditions for germination and the mycelium is easily killed by excessivemanipulation. All the cultures were incubated at 15° C., the approximateoptimum for growth (Ellis & Hesseltine, 1962).

The initial pH values of the media were between 4 and 5, the approxi­mate optimum (Ellis & Hesseltine, 1962). The final pH values (not given)were almost always slightly lower, apparently due to acid production whichseemed always to be associated with growth.

The average dry weight of mycelium per flask was taken as the measureof growth, except in one experiment where the individual weights ofreplicates were determined. Sintered glass crucibles were used for theharvested mycelia, which were washed, dried at 90° and weighed to thenearest mg. Where starch was used in the medium, the mycelia werefloated out in large volumes of water before filtering, to remove as muchas possible of the unused starch. The mycelia were harvested after 2

weeks' growth, as this was the period for almost the maximum growth onthe most favourable media, but was not long enough to allow autolysis totake place. The weights obtained on identical media in different experi­ments are not always quite comparable, as the incubator temperature waslower than 15° during a part of the duration of some of the experiments,owing to a fault. Five replicate flasks of each medium were used. Thedry weights obtained from replicates were often nearly uniform and thestandard error has been given only where it exceeded 0'3 mg. Preliminaryexperiments showed that no measurable dry weight could be obtained fromflasks where very little growth was visible, even though the agar inoculumwas always harvested with the mycelium, therefore, in later experimentsdry weights for such flasks were recorded as nil, even though the myceliawere not actually harvested.

COMPARISON OF DIFFERENT CARBON SOURCES

Various compounds were substituted for the glucose in the basalmedium, which was otherwise unchanged. These compounds (Table I),all supplied at 2 % concentration included mannitol and trehalose, whichhave both been reported to occur commonly in agarics (Cochrane, 1958).As gluconic acid is effective in making the spores germinate (Watson,1964) it was thought that it might also prove a good C source for growth,and as an adequate concentration of the acid would have made the pHvery low, the sodium salt was used. Because many sugars are known to bechemically changed by autoclaving with phosphate, all the C compoundstested were autoclaved separately in part of the total water and added tothe rest of the medium after sterilization.

The results (Table I) show that S. macrocarpus, like many other fungi,can utilize glucose, fructose, mannitol and trehalose. It appeared thatsucrose and starch were utilized to some extent, but this cannot be taken asconclusive as these were heat sterilized and some dissociation may havetaken place. S. macrocarpus appears also to resemble many other fungi in

Spinellus macrocarpus. Pauline Watson 75being unable to use lactose or galactose. The lack of growth with gluconatewas interesting, as it did not appear to be due to the high pH which mighthave been expected to develop in the medium and cause staling. It ispossible that sodium inhibits growth or that the compound could not beabsorbed from the medium owing to its ionized state. Spore germinationis not inhibited by sodium gluconate and appears to be stimulated only bygluconic acid lactone and not by the acid (Watson, unpublished), andthis suggests that the lack of growth on media containing gluconate mayby due to an impermeability of the hyphal walls to gluconate ions ratherthan to inhibition by sodium.

Table I. Growth with different carbon sources

n-Fructosen-Galactosen-Gluconate (Na)n-GlucoseLactose

Averagedry wt.jflask

(mg.)

12

oo

13o

n-MannitolStarchSucroseTrehaloseNone

Averagedry wt.jflask

(mg.)

139 (±o'S)6

13o

Additions to vitamin-free medium

Table 2. Growth on media containing various vitamins

Average dry wt.!flask (rng.)

NoneThiaminNicotinic acidOther vitamins*Thiamin+nicotinic acidThiamin+other vitamins*Nicotinic acid +other vitamins*Thiamin +nicotinic acid +other vitamins*

oo5o

18o5

14 (±2)

* Biotin +meso-inositol +pyridoxine+para-amino-benzoic acid+pantothenicacid + folic acid.

VITAMIN REQUIREMENTS

Preliminary experiments showed that S. macrocarpus needed organicnitrogen and vitamins for growth, and this requirement was satisfied byincluding yeast extract in the basal medium. In order to determine thevitamin requirements, vitamin-free acid hydrolysed casein (NutritionalBiochemicals Corporation) was used to replace the yeast extract as anitrogen source and various vitamins were added in different combina­tions. The vitamins tested and the amount of each added to 20 ml. mediumwere as follows: thiamin, 5 flg. nicotinic acid, 50 flg.; biotin, 0'05 flg.;meso-inositol, 25 fig.; pyridoxine, 5 fig.; para-amino-benzoic acid, 5 flg.;pantothenic acid, 5 flg.; folic acid 5 flg·

S. macrocarpus required thiamin and nicotinic acid for good growth andother vitamins did not appear to give any further stimulation (Table 2).As pyridoxine, in some experiments not reported here, inhibited growth,its presence may account for the lower and more variable results when theadditional vitamins were included in the medium. The requirements for

76 Transactions British Mycological Societyboth thiamin and nicotinic acid are believed to be total, but owing tothe method of inoculation, some vitamins are likely to have been trans­ferred with the inoculum, and this would be particularly effective in thecase of thiamin which is required in relatively smaller amounts and couldaccount for the slight growth in the media lacking this vitamin.

20 1--1/' ~icotinicacid

15

5

oL--.,---,---j--,.-------r----,r--50·5 50 500 5000 50.000

p,g.!20 m!'

Fig. I. Growth on media containing different concentrations of nicotinic acid ortryptophane. (Vertical lines represent the standard error of each average mycelialweight).

As ascorbic, arabo-ascorbic and gluconic acids are effective in inducingspore germination, it was thought that they might also affect growth, andthey were subjected to a number of tests not reported fully here. Gluconicacid appeared to have no effect on growth at 0'02-0'2 % and the othertwo acids also had no effect at the low concentrations (c. 0'02 %) suitablefor germination, At c. 0'2 %some inhibition was observed, but this mighthave been due to a pH effect. It appears, therefore, that the substanceswhich have been found to stimulate germination are different from thosewhich are necessary for growth.

REPLACEMENT OF NICOTINIC ACID BY TRYPTOPHANE

In preliminary experiments S. macrocarpus grew in a medium containingvitamin-free casein and thiamin and no other vitamins, but would notgrow when the casein was replaced by acid-hydrolysed casein. Trypto­phane is known to be destroyed by acid hydrolysis and when it was added

Spinellus macrocarpus. Pauline Watson 77to media containing hydrolysed casein good growth was obtained. Theexperiment described was carried out to compare the optimum con­centrations of tryptophane and nicotinic acid for growth, because it hasbeen shown for other organisms which can utilize tryptophane in place ofthe vitamin, that relatively large amounts of it are required.

The yeast extract in the basal medium was replaced by the same con­centration of acid-hydrolysed casein as nitrogen source and thiamin(4 Jlg./20 mI.). In addition various concentrations of DL-tryptophane ornicotinic acid were added.

Relatively large amounts of tryptophane were needed to replace nico­tinic acid (Fig. I) as in some other fungi as well as in animals. Trypto­phane appeared to inhibit growth at higher concentrations and nevergave as much growth as optimum concentrations of the vitamin. Theseobservations and the variability of the weights of replicates of many ofthe media will be discussed later.

COMPARISON OF DIFFERENT NITROGEN SOURCES

Preliminary experiments suggested that S. macrocarpus required organicN and the determination of its vitamin requirements made it possible toinvestigate this further. Various N sources, at a total concentration ofI %, were substituted for the yeast extract in the basal medium and ade­quate thiamin and nicotinic acid were added.

The only N sources tested which gave any appreciable growth wereacid-hydrolysed casein and a mixture of approximately equal amountsof twenty-one amino acids (alanine, arginine, aspartic acid, cystine,glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine,lysine, methionine, norleucine, ornithine, phenyl alanine, proline, serine,threonine, tryptophane, tyrosine and valine). The dry weights were notdetermined but were estimated to be about 10-20 mg. per flask, and thereappeared to be little variation among the replicates. All the other com­pounds tested-potassium nitrate, ammonium chloride, ammoniumtartrate and glycine, gave almost no growth and were indistinguishablefrom the control flasks without nitrogen. In other experiments variousother amino acids were tested singly and these also failed to supportgrowth and often inhibited it completely.

It therefore appears that S. macrocarpus requires a mixture of amino acidsand is unable to utilize inorganic N compounds, at least as the sole Nsource. Many experiments were done in an attempt to detect specificamino acid requirements but no one acid has ever been shown to beessential and the rate of growth on different mixtures of amino acidsappeared to be very approximately proportional to the number of differentacids supplied. Relatively large amounts ofanyone acid, even in an other­wise favourable medium, inhibited growth.

Transactions British Mycological Society

GROWTH ON MEDIA CONTAINING DIFFERENT CONCENTRATIONS

OF HYDROLYSED CASEIN AND NICOTINIC ACID

High concentrations of amino acids, either singly or in some mixturessuch as hydrolysed casein, were found in preliminary experiments toinhibit growth especially when nicotinic acid was present at suboptimalconcentrations. A comparison was made therefore of the growth on mediacontaining three concentrations of hydrolysed casein and optimal or sub­optimal concentrations of nicotinic acid. All the variants contained

80

60

20

•••••Hydrolysed 2·5 2-5 0·25 0·25

caseinNicotinic 500 5 500 5

acid

0-025 0·025 %

500 5 /lg./20 mI.

Fig. 2. Growth on media containing three concentrations of hydrolysed casein attwo levels of nicotinic acid. (Individual weights from five replicates of each medium).

thiamin and the other constituents of the basal medium with the exceptionof yeast extract. Mycelial weights from individual flasks of each variantare given in Fig. 2, as their variability is thought to be particularlyinteresting.

The results confirm those of previous experiments, the inhibition ofgrowth being very marked with the highest concentration of hydrolysedcasein and suboptimal nicotinic acid. The variability of many of thereplicates is thought to be due to a requirement for another substance' X',contained in varying amounts in the inocula, and replaceable to some

Spinellus macrocarpus. Pauline Watson 79extent by nicotinic acid, necessary for growth on media containing highconcentrations of amino acids; this will be further discussed later.

GENERAL DISCUSSION

Like a number of other parasitic fungi, Spinellus macrocarpus appears tohave rather specialized nutritional requirements. Its apparently lowtransaminating ability, if innate, and not an artifact produced by un­favourable cultural conditions, might explain its restriction in nature to aparasitic habit. It is interesting that several other fungi such as Sac­charomycopsis guttulata (Shifrine & Phaff, 1959) and Trichophyton menta­grophytes (Robbins & Ma, 1945) which require a mixture of amino acidsfor growth are also parasites. Another parasite, Phytophthora fragariae,which was at one time believed to require a complex nitrogen source hasnow been shown to grow with single amino acids, if a suitable concentra­tion of calcium is added to the medium (Davies, 1959). It is possib le thatS. macrocarpus also requires some addition to the medium to enable it toutilize single amino acids, but there is no evidence so far that calcium orother ions have any effect. Few fungi are known to require a supply ofnicotinic acid (Cochrane, 1958), and it is interesting therefore to findanother fungus with this deficiency.

The effects of different concentrations of amino acids and nicotinic acidon the growth of S. macrocarpus are interesting because they appear toresemble effects observed in animals, where various unbalanced mixturesof amino acids and hydrolysed casein may inhibit growth of chicks(Anderson, Combs, Groschke & Briggs, 1951) and rats (Bonner & Yanofsky,1951), especially if nicotinic acid is limiting. S. macrocarpus also resemblesanimals, as well as fungi such as Trichophyton equinum (Georg, 1949) andsome mutants of Neurospora (Bonner & Yanofsky, 1951), in being appar­ently able to synthesize nicotinic acid if supplied with tryptophane. Inanimals, however, tryptophane is an essential amino acid, and though itcan be converted to nicotinic acid, the reverse substitution in the dietis presumably not possible. In T. equinum and some Neurospora mutants,growth can take place if either nicotinic acid or tryptophane is supplied,and it has been suggested for Neurospora that this might be explained by'leakage' of tryptophane, which can be synthesized by the mutant whennicotinic acid is supplied, but not otherwise. The same explanation couldpresumably be used to explain the observations on T. equinum and S.macrocarpus.

The extreme variability in dry weights of mycelia from replicate flasksof some media was characteristic of many experiments with S. macro­carpus. The most likely explanation seems to be that such media werelimiting in some factor, which will be referred to as 'X', that is essentialfor growth, and that the mycelial inocula carry different amounts of it.Where uniform dry weights were obtained, X was presumably present insufficient amount in the media or the conditions permitted its synthesisby the fungus, and the lack ofuniformity of the inocula was not noticeable.Yeast extract appears to contain adequate amounts of X, whereas thevariable dry weights and inhibition of growth caused by high concentrations

80 Transactions British Mycological Societyof hydrolysed casein could be explained as being due to a deficiency of X.If this explanation is correct, relatively larger amounts of X must berequired in media containing high concentrations of hydrolysed casein,especially if only suboptimal amounts of nicotinic acid are present, andX is probably replaceable by nicotinic acid in media containing lowerconcentrations of hydrolysed casein. This would suggest that X is relatedto nicotinic acid and is perhaps some compound to which the vitamin isconverted in the cell, such as DPN (diphosphopyridine nucleotide), and thatthis reaction is inhibited by high concentrations of amino acids. The effectsofDPN and related compounds on S. macrocarpus are now being investigated.

The effects on the growth of S. macrocarpus of the concentrations ofnicotinic acid, amino acids and probably also other compounds mayexplain its narrow host range, which cannot be explained by the factorsinfluencing spore germination (Watson, 1964). The nicotinic acid contentsof different plants vary considerably and, though data are not availablefor species of agarics it is possible that they might show a similar varia­bility. This, associated with the large amounts of amino acids which canreadily be demonstrated in them, might make growth possible on only a fewspecies which contain suitable concentrations of the effective compounds.It is hoped to investigate this aspect of host-parasite relations further.

All the work described in this paper has been done with a single isolate,for practical reasons, but several other isolates now cultured will beinvestigated and compared, as in some fungi it is known that particularvitamin deficiencies may only occur in some strains, and if the nutrition ofS. macrocarpus defines its parasitism, it is obviously of great importanceto study a number of isolates.

Thanks are due to Mr R. W. Stuart for efficient technical assistance.

REFERENCES

ANDERSON,]. 0., COMBS, G. F., GROSCHKE, A. C. & BRIGGS, G. M. (1951). Effects onchick growth of amino acid imbalances in diets containing low and adequatelevels of niacin and pyridoxine. ]. Nutr. 45, 345-360.

BONNER, D. M. & YANOFSKY, C. (1951). Editorial review. The biosyntheses of trypto-phane and niacin and their relationships. ]. Nutr. 44, 603-616.

COCHRANE, V. W. (1958). Physiology offungi. New York: Wiley.DAVIES, M. E. (1959). The nutrition of Phytopthora fragariae. Trans. Brit. mycol. Soc.

\ 42, 193-200.'ELLIS, ].]. & HESSELTINE, C. W. (1962). Rhopalomyces and Spinellus in pure culture.

Nature, Land., 193, 699.GEORG, L. K. (1949). Conversion of tryptophane to nicotinic acid by Trichophyton

equinum. Proc. Soc. expo Biol. N.Y., 72, 653-655.LEADBEATER, G. & RICHARDSON, M. (1963). Spinellus in pure culture. Nature, Land.,

198, 1015.ROBBINS, W.]. & MA, R. (1945). Growth factors for Trichophyton mentagrophytes. Amer.

]. Bot. 32, 509-523.SHIFRINE, M. & PHAFF, H.]. (1959). Nutritional requirements of Saccharomycopsis

guttulata (Robin) Schionning, Mycologia, 51, 318-328.WATSON, P. (1962). Culture of Spinellus. Nature, Land., 195, 1018.WATSON, P. (1964). Spore germination in Spinellus macrocarpus. Trans. Brit. mycol. Soc.

47, 239-245.

(Accepted for publication 28 June 1964)