[ 9 J

T rans. Brit. mycol. Soc. 48 ( I), 9-17 (1965)Printed ill Great Britain

FURTHER OBSERVATIONS ONCALCARISPORIUM ARBUSCULA

By PADUNE WAT SON

Department cifBolan) ', University cifDurham

(With Plat e 1 )

From pr eviou s experiments it seemed likely that Calcarisporium arbuscula Pr euss,which lives as an endophyte in frui t-bodies of Russula and Lactarius spp. , mightbe a specialized parasite with low competitive saprophytic ability. This hypo­thesis is now supported by the observations that it appears to be unable tosurvive for more than a short period in soil and is unable to colonize a sterilesubstra tum in competition with soil fungi. The low competitive saprophyticability is probably due to a low growth rate, and th e ability of Trichodermaviride Link ex Fr. to kill C. arbuscula might be a factor accounting for itsabse nce from soil. C. arbuscula has been shown to produce an antibiotic, calcarin,which inhibits the grow th of many fungi. This is produced both in culture an dwhe n living endophyticall y in aga ric fruit-bodies; such fruit-bod ies are moreresista nt to a ttack by other moulds than those which do not contain the endo­phyte. The sign ifican ce of these results is discussed , with parti cular reference tothe probl em of surviva l during the greater pa rt of th e year when th ere are noagaric sporophores ava ilable.

Calcarisporium arbuscula, a fairly common mould on the fruit-bodies ofsome larger fungi , has been shown to live endophytically in some fruit­bodies of species of Russula and Lactarius (Watson, 1955), so that it isappa rent ly a specia lized parasite. As it is normally found onl y in aut umn,when larger fungi produce their fru it-bodies, the problem of its survivalfrom one season to ano ther is of in terest. The soil is th e most obvious placefor over-wintering, but all a tte mpts to det ect C. arbuscula in soils fromlocalities where it is common in autumn have been un successful and th ereappear to be no published records of its occurrence in soil. This paper givesan account of investigati ons of its relations with soil fungi and otherobservations relevant to the pro blem of its survival from one autumn toanother.

MATERIALS

Vari ous isolates of C. arbuscula were investigated and all gave similarresults, but only two were used for the particular experiments described inth is pap er. Isolate 57/ I , from R. ochroleuca (Pers.) Fr., was used for thetest of compe titive saprophytic ability and 62/5, from R. delica Fr. , for theother experiments. Both isola tes were of the most usual type (Watson,1955), forming numerous conidia and sclerotia.

T he woodland soil used was always collected from th e same experi­mental area, whi ch was chosen because fruit-bodies of Russula spp. occurthere every season, and a proportion of th em has always been found to

10 Transactions British Mycological Societycontain endophytic C. arbuscula. The same locality was also used whentesting survival of spores in the soil.

The composition of the 'BM' agar was as follows: glucose 20 g. ;ammonium tartrate, 3 g.; KH2P04, I g.; MgS04, 0'5 g.; yeast extract,0'5 g.; agar, 10 g.; water, rooo ml.

SURVIVAL IN SOIL

A record of C. arbuscula detected in soil has not been found, thereforetests in which it was added to woodland soil, both in the natural habitatand under more artificial conditions in the laboratory, were made to findif it could be detected if present and to determine for how long it was ableto survive. A typical experiment in the natural habitat is described.

Table I. Survival of Calcarisporium arbuscula in soil

Date ofsoil sampling

(1963)II Apr.22 Apr.26 Apr.9 May

16 May

No. of daysafter addingC. arbuscula

oII

152835

No. ofsoil platesprepared

918799

No. ofplates withC. arbuscula

696oo

Plastic perforated baskets (c. 500 ml. capacity) were used as containersfor inoculated soil in the natural habitat, as it was thought that they wouldallow the maximum amount of contact and interaction with the sur­rounding soil without the identity of the samples being lost. Holes werebored in the bottom to avoid collection of water. They were buried justbeneath the surface litter in the experimental area. Spores of C. arbuscula,in a dense suspension in sterile water, were stirred into the soil sample, andsoil plates (Warcup, 1950) were made, at once and after various intervalsof time. The inocula for the soil plates were transferred directly to Petridishes in the field, using a separately sterilized needle for each (BM agar with0'006% rose bengal added was the medium). The plates were incubatedfor 4 days at 23° C., then the C. arbuscula colonies were counted (TableI), other fungi being ignored.

It thus appears that the spores are unable to survive in soil for more thana short time though they are easily detected if present. In field experiments,especially, it is possible that the spores could become dispersed so that theywould seem to have disappeared, but similar results were obtained withsoil samples in jars in the laboratory, so it seems probable that the sporesare unable to survive.

Spores merely added to soil in this type of experiment would probablybe unable to germinate and colonize substrata there because offungistaticactivity, so they would perhaps be under more of a disadvantage than themycelium in an old fruit-body in the soil, which might be able to grow outand colonize fresh food sources. The spores are also small and though theycan survive for more than 28 days when not subjected to competition, thesclerotia would be a more likely form in which the fungus overwintered.

Calcarisporium arbuscu1a. Pauline Watson II

No experiments have so far been done to test the survival of sclerotia insoil. However, like the spores, they germinate readily in water and on soilplates if added to them, and are probably small enough (250 P or less) tobe picked up in inocula for soil plates. So the lack of records of C. arbus­cula is perhaps most significant, especially as the fungus very readilyproduces its characteristic conidia (Hughes, 195I) in culture and iseasily identified. Presumably, it is absent from soil because, like otherspecialized parasites such as those of roots (Garrett, 1963), it has a lowcompetitive saprophytic ability compared with that of native soil fungi.The following experiment was done to test this hypothesis.

COLONIZATION OF A STERILE SUBSTRATUM IN COMPETITION WITH

SOIL ORGANISMS

A modified version of the 'Cambridge method' (Garrett, 1963) ofmeasuring competitive saprophytic ability was used in this experiment.Autoclaved carrot slices, about 0'5-0'75 em. thick, were chosen forcolonization attempts because, although they have a soft texture like thatof an agaric, they remained reasonably firm and could be sampled quiteeasily. About 10 slices were buried in a sterilized mixture of vermiculiteand maizemeal (vermiculite, 300 g.; maizemeal, 60 g.; water, 750 ml.),inoculated immediately beforehand with spores of C. arbuscula, withwoodland soil (about 0'5 g. per jar), or with both, in sterile jars with loose­fitting lids. Two jars of each variant were prepared and all were kept atroom temperature for 12 days. The carrot slices were then sampled forcolonizing organisms by cutting out pieces of the interior, exposed bybreaking, placing them on plates ofBM agar and incubating for 4 days toenable C. arbuscula or other organisms, if present, to develop sufficientlyfor identification.

Table 2. Numbers of carrot slices colonized in the presenceof soil organisms

Total No. of slices No. of slicesVermiculite mixture no. of colonized by colonized by

inoculated with slices sampled C. arbuscula other moulds

C. arbuscula 24 24 0

Soil 22 0 22C. arbuscula+soil 14 0 14

The results (Table 2) show that C. arbuscula was unable to colonize theslices in competition with soil fungi, at least under the conditions tested,though it could do so in pure culture. This seems to confirm previousevidence (Watson, 1955) that it has low competitive saprophytic ability.As the mould was added to the experimental mixtures as spores, both theirgermination and the colonization of the substratum in competition withsoil fungi were involved. However, as a very low proportion of soil wasused and the vermiculite mixture contained nutrients, there would prob­ably have been no fungistatic activity (Dobbs, Hinson & Bywater, 1960)and the spores should have germinated, as they normally do both in

12 Transactions British Mycological Societywater and on nutrient media. Thus ability to colonize the substratum wasprobably effectively tested.

Garrett (1950) suggested that the characteristics likely to contributetowards a high degree of competitive saprophytic ability would be:(1) high growth rate, (2) good enzyme-producing equipment, (3) pro­duction of antibiotics, and (4) tolerance of antibiotics produced by otherorganisms. Low competitive saprophytic ability might therefore be dueto a lack of one or more of these features. C. arbuscula does not have anyvery exacting nutritional requirements (Watson, 1956) and is able togrow on various media in the laboratory, so it is probably not limited byits enzyme producing capacity, but the other three features were thoughtto be of more importance and have been further studied.

GROWTH RATE

The growth rate, known to be fairly low (Watson, 1955), was comparedwith the rates of some common soil fungi, measured as the increase incolony diameter on agar plates at 23° C., which, though above normal soiltemperatures, was not above the optimum for any of the fungi tested andwas assumed therefore to allow a reasonable comparison. Three plates ofBM agar were centrally inoculated with each fungus. Colony diameterswere measured at suitable intervals during the period ofmost rapid growthbefore any signs of staling appeared.

Table 3. Growth rates ofCalcarisporium arbuscula and soilfungi

Average growth rate in mm.jday (t increase in colony diam.),

C. arbuscula

['75

Trichodermaviride

Mucorhiemalis

12'5

M. raman­nianus

,Penicillium

sp.

['5

C. arbuscula has a relatively low growth rate compared with some ofthese fungi (Table 3) but as the soil fungi were chosen as examples of'common' soil fungi on the basis of their occurrence on soil plates this isnot unexpected, as this method of isolating soil fungi is likely to favour thefaster growing sugar fungi. However, such fungi certainly live in the soiland C. arbuscula would need to compete successfully with them to livesaprophytically there, so that its low growth rate might explain itsinability to grow in soil.

INTERACTIONS WITH SOIL FUNGI IN PURE CULTURE

The object of this investigation was to determine whether there was anyevidence of sensitivity to antibiotics, or other reactions which might be ofimportance in determining its saprophytic ability. Plates of BM agarwere inoculated at one side with a soil fungus and at the other withC. arbuscula and incubated at 23° C. The test was repeated several timeswith a number of replicates of each combination and at least ten isolatesof C. arbuscula. Table 4 summarizes the results obtained.

Calcarisporium arbuscula. Pauline Warson 13PI. I, fig. I, shows the inhibition of Mucor hiemalis and PI. I, fig. 2,

illustrates C. arbuscula growing over the inhibited colony of a Mortierella sp.which was dark in colour so that the white mycelium and conidiophoresof C. arbuscula could easily be seen on it. Often the continued growth overthe other mould could only be seen under the microscope, as the darksclerotia, which are the most noticeable feature of C. arbuscula when it isgrowing alone, are not usually produced in this situation. A number ofunidentified soil fungi and several moulds regarded as less specializedparasites of agarics were also tested; all were more or less inhibited and inalmost all tests C. arbuscula grew over them.

Table 4. Interactions between Calcarisporium arbuscula and soilfungi

Trichoderma viride(isolate S/1O)

T. viride(isolate S/ I 2)

M ucor hiemalisAI. ramannianusBotrytis sp.Mortierella sp.

Penicillium (9 sPP·)

Effects of soil funguson C. arbuscula

Grown over and killed

} Apparently none

Apparently none, orslight inhibition

Effects of C. arbusculaon soil fungus

Inhibited at first

Inhibited, later overgrown

Inhibited, later overgrown

The results were rather unexpected and suggested, as did observationsby Gay (I957) and E. M. Turner (personal communication), thatC. arbuscula produced an antibiotic. This has since been confirmed and isbeing investigated. When C. arbuscula grew over other fungi it appearedto be parasitic and details of this parasitism are now being studied.This observation is particularly interesting as C.parasiticum, which has verysimilar conidia and conidiophores, is an obligate parasite in nature onother fungi (Barnett, I 958).

The interactions of C. arbuscula with various contaminants, as well aswith soil fungi, suggest that it is not particularly sensitive to antibiotics,but no specific ones have been tested. The isolate S/ r o of Trichoderma viridedid not appear to produce antibiotics though it was an effective parasite,under experimental conditions, of many soil fungi as well as of C. arbus­cula. This combination of characters has been noted by Brian (I960).T. viride ofthe S/ro type was common in the woodland soil, and its abilityto kill C. arbuscula might help to account for the absence of even restingstructures of the latter, as it has been shown that attack by parasites may beof importance in limiting the survival of dormant structures of plantpathogenic fungi in soil although it may have far less effect on soilinhabiting fungi (Menzies, I963).

The antibiotic produced by C. arbuscula, which it is proposed to call'calcarin', might well be of importance in the host-parasite relations of themould when living endophytically in agarics, and some observations havebeen carried out in this connexion.

14 Transactions British Mycological Society

EVIDENCE OF THE PRESENCE OF CALCARIN IN FRUIT-BODIES

CONTAINING ENDOPHYTIC CALCARISPORIUM ARBUSCULA

As has already been mentioned, various moulds which are regarded asless specialized parasites of agarics, are inhibited by calcarin, and some ofthese were used in an attempt to detect antibiotic activity in fruit-bodies ofRussula ochroleuca containing endophytic C. arbuscula. The moulds testedincluded Apiocrea chrysosperma, Mycogone sp. and Verticillium (?) agaricinum.Plates of BM agar were seeded with spores by mixing a spore suspensionwith the medium just before it had cooled sufficiently to set. Pieces of theinterior flesh of young, healthy fruit-bodies of R. ochroleuca were cut outwith a sterile scalpel and placed on the seeded plates, together with stripsof pure cultures of C. arbuscula as a control. Similar fruit-body pieces wereplaced on unseeded plates to test for the presence of endophytic C.arbuscula, as fruit-bodies containing it are indistinguishable from the otherswhen young (Watson, 1955). The seeded plates of the faster growingmoulds were kept in a refrigerator overnight, before being incubated at23° C., to allow substances time to diffuse out of the pieces of fruit-bodybefore germination started.

The experiment was done on three occasions and altogether six fruit­bodies containing endophytic C. arbuscula and seven without the endophytewere tested. Similar results were obtained with all the moulds andPI. I, fig. 3, illustrates a typical one obtained with Verticillium (?) agari­cinum. Spore germination was clearly inhibited by the slice of C. arbusculacolony and by the piece of fruit-body containing endophytic C. arbuscula.Russula tissue without the endophyte did cause a very slight inhibition butthis does not show on the photograph. These results show that fruit-bodieswith endophytic C. arbuscula probably do contain calcarin and this is ofecological interest as it would explain the observation made by the authorand by Gay (1957) that fruit-bodies infected by C. arbuscula appear to becolonized by other fungi less readily than sound fruit-bodies. The slightinhibition produced by the Russula tissue without endophyte was probablydue to an antibiotic produced by the agaric, as species of Russula areknown to produce antibiotics. Attempts have been made to extractcalcarin from fruit-bodies containing endophytic C. arbuscula so that itcould be identified by paper chromatography but these have not so farbeen successful.

Unfortunately, statistical evidence of protection of fruit-bodies againstattack by other moulds by endophytic C. arbuscula cannot be derived fromthe samples of fruit-bodies of Russula spp. tested. Although large numberswere examined, and inocula from some fruit-bodies did produce othermoulds, the fruit-bodies were not collected in a random manner, so theimpression that they were less easily attacked could be only a subjectiveone. A suitable collection of Russula fruit-bodies necessary to test thevalidity of this impression has not been found in recent seasons thoughendophytic C. arbuscula has been found in some fruit-bodies. In mostpopulations that have been examined the proportion of infected fruit­bodies was low. However, in 1963, old fruit-bodies of Hygrophorus pratensis

Calcarisporium arbuscula. Pauline Watson 15(Pers. ) Fr. found in one small field were covered with conidiophores of themould and tissue-culture methods showed that a very high proportion ofthem contained endophytic C. arbuscula. All the fruit-bodies which couldbe found were collected and tested for the pr esence of C. arbuscula andother moulds by the tissue-culture method, several inocula being takenfrom different parts of each fruit-body. Almost all the inocula werecontaminated with bacteria (apparently the same species in fruit-bodieswith or without C. arbuscula) but these were ignored as they did not appearto have any effect on the fungi or to be inhibited by calcarin. The resultsare given in Table 5.

Table 5. Numbers offungi isolatedfromfruit-bodies of Hygrophorus pratensis

Fruit-bodies tested 94With endophytic C. arbuscula 82With C. arbuscula but giving other moulds 5

from at least one inoculumWithout C. arbuscula 12Without C. arbuscula but giving other moulds 7

from at least one inoculum

The conclusion can be drawn that endophytic C. arbuscula helps toprotect the fruit-body in which it is living against attack by less specializedmoulds. As it does not prev ent spore liberation by the agaric, which theless specialized moulds would do if they attacked a sufficiently young fruit­body, this appears to be a ben eficial effect, and the association could beregarded as symbiotic rather than parasitic.

DISCUSSION

The experiments that have been described show that C. arbuscula isapparently not able to grow in soil or survive there as resting structures andthis may be due to its low growth rate, its susceptibility to parasitism byTrichoderma viride or to oth er factors. It is, however, possible that smallnumbers of sclerotia might form an overwintering stage in soil, remainingundetected by soil sampling methods, and their food reserves and hardtexture might contribute to their survival. The inability of the fungus togrow in soil is probably du e to a low competitive saprophytic ability andthis is interesting in view of its ability to produce an antibiotic andapparently parasitize soil fungi in culture.

Antibiotic production by both host and parasite is an interesting aspectof the relations between C. arbuscula and the agarics in which it is able tolive endophytically. The mould is able to enter the fruit-bodies when theyare young,so is presumably insensitive to the antibiotics produced by them;and Russula, Lactarius spp ., and Hygrophorus pratensis have all been shownto produce antibiotics (Brian, 195I). The agarics, similarly, are apparentlyunaffected by calcarin and grow and sporulate normally after infection byC. arbuscula; and they appear to benefit from the association as they areless readily attacked by other less specialized moulds, presumably because

16 Transactions British Mycological Societyof the presence of both antibiotics. The early colonization by C. arbuscula,together with the production of calcarin, probably gives it an advantageover later colonizers; thus it may survive for some time in the remains ofthe host, like some plant pathogenic fungi, and its slow growth rate wouldfavour this (Menzies, 1963). Old, infected fruit-bodies may form anover-wintering substratum, and evidence in support of this was the iso­lation of C. arbuscula from an old fruit-body of H. pratensis as late asJanuary 1964. All other and later attempts to find infected fruit-bodieshave been unsuccessful, but they might become unrecognizable, though theymight then be expected to be present in soil samples if these are collectedfrom near the soil surface.

The host range of C. arbuscula is also of interest. It is apparently able tolive openly on a variety offungi, including ascomycetes and myxomycetes,as well as various agarics (Gay, 1957), but appears to occur endophyticallyonly in species of Russula and Lactarius, and H. pratensis. Its occurrence inH. pratensis was particularly interesting as this species is not generallyregarded as being closely related to the other two genera. It is hoped thatinvestigation of the reasons for this host range may throw more light on therelationship between C. arbuscula and agarics. An unusually high pro­portion of fruit-bodies of H. pratensis were found to be infected in the onefield, but collections of the agaric from other localities showed no infection,nor were a few fruit-bodies of other species of Hygrophorus from the samefield which were examined infected.

The only record of C. arbuscula being found in a situation not obviouslyassociated with larger fungi is that of its occurrence on roots of Fagus(Harley & Waid, 1955)' It occurred on both the lateral mycorrhizal rootsand the long non-mycorrhizal roots. The author has also isolated themould from tree roots (probably Fagus) on several occasions using thesame technique. This would be another possible method of overwintering,and the fungus has been isolated from roots at various times of year.A possible explanation of its occurrence in this habitat is that it may beassociated with the mycelium ofRussula or Lactarius spp. as well as occurringin some of their fruit-bodies. The agaric mycelia, even if present in theroots (and all species of these genera are believed to be mycorrhizal),would be unlikely to grow in culture, whereas C. arbuscula grows readily onagar media. The occurrence of C. arbuscula in long roots as well as in themycorrhiza could be explained by the observation that hyphae ofmycorrhizal fungi in Pinus can be found in long roots as well as in theobvious mycorrhizal ones (Robertson, 1954). Presumably somethingsimilar might occur in Fagus, with C. arbuscula associated with themycelium of Russula or Lactarius both in the long roots and in the mycor­rhizallaterals. This association with roots might be advantageous in thatthe mould would be associated with the agaric fruit-body from thebeginning of its development and would not need to find its way to a newhost through soil, in which it has been shown to be vulnerable.

Of the three Calcarisporium species which have been described, bothC. arbuscula and C. parasiticum appear to be highly specialized parasites ondifferent types of hosts; thus it would be interesting to know more aboutthe ecology of the third species, C. pallidum Tubaki (1955)' This was found

Trans. Brit. mycol. Soc. Vol. 48, Plate I

(Facingp. 17)

Calcarisporium arbuscula. Pauline Watson 17on agarics but its parasitic status does not seem to have been fully investi­gated. Neither of these two latter species seems to have been recordedfrom Great Britain but, as C. arbuscula occurs in Japan (Tubaki, 1955) andin North America (Barnett, 1960), they also may well not be restricted toone part of the world.

Thanks are due to Dr J. Webster for identifying the isolates of Tricho­derma, to Mr J. S. Redhead for the photographs and to my colleagues formany helpful discussions.

REFEENCES

BARNETT, H. L. (1958). A new Calcarisporium parasitic on other fungi. Mycologia, 50,497-500.

BARNETT, H. L. (1960). Illustrated genera of imperfect fungi. Minneapolis: BurgessPublishing Co.

BRIAN, P. W. (1951). Antibiotics produced by fungi. Bot. Rev. 17,357-430.BRIAN, P. W. (1960). The ecology ofsoilfungi. Liverpool University Press. (In discussion,

p. In)DOBBS, C. G., HINSON, W. H. & BYWATER,]. (1960). Inhibition of fungal growth in

soils. The ecology of soilfungi, pp. 130-147. Liverpool University Press.GARRETT, S. D. (1950). Ecology of the root inhabiting fungi. Bioi. Rev. 25, 220-254.GARRETT, S. D. (1963). Soilfungi and soilfertility. Oxford: Pergamon Press.GAY, ]. L. (1957). Biological studies on microfungi associated with hymenomycetes.

Ph.D. Thesis, University of Nottingham.HARLEY,]. L. & WAID,]. S. (1955)' A method ofstudying active mycelia on living roots

and other surfaces in the soil. Trans. Brit. mycol. Soc. 38, 104-118.HUGHES, S.]. (195 I). Studies of microfungi. IX. Calcarisporium, Verticicladium and

Hansfordia. Mycol. Pap. 43, 1-6.MENZIES, ]. D. (1963). Survival of microbial plant pathogens in soil. Bot. Rev. 29,

79- 122.ROBERTSO)l, :\f. F. (1954). Studies on the mycorrhiza of Pinus syloestris. New Phytol. 53,

253-283.TUBAKI, K. (1955). Studies on Japanese Hyphomycetes. II. Fungicolous group.

Nagaoa, 5, II-40.WARCUP, J. H. (1950). The soil-plate method for isolation of fungi from soil. Nature,

Land., 166, 117-118.WATSON, P. (1955)' Calcarisporium arbuscula living as an endophyte in apparently

healthy sporophores of Russula and Lactarius. Trans. Brit. mycol. Soc. 38, 4°9-414.WATSON, P. (1956). Some studies of a mould isolated by tissue culture from species of

Russula. Ph.D. Thesis, University of London.

EXPLANATION OF PLATE

(All x i.)Fig. I. Plate inoculated with Calcarisporium arbuscula (smaller colony) and Mucor hiemalis,viewed from underside.Fig. 2. Plate inoculated with C. arbuscula (light colour) and Mortierella sp., viewed from above.Fig. 3. Plate seeded with spores of Verticillium (?) agaricinum, with the addition of, in clockwisedirection starting from the label' A', I and 2, pieces of tissue from Russula fruit-body; 3, piece ofRussula fruit-body containing C. arbuscula; 4, strip of colony of C. arbuscula.

(Accepted for publication 6 May 1964)

Myc'48