[ 4°9 ]

Trans. Brit. mycol. Soc. 38 (4), 409-414 (1955)

CALCAR/SPOR/UM ARBUSCULA LIVING AS ANENDOPHYTE IN APPARENTLY HEALTHY SPORO­

PHORES OF RUSSULA AND LACTAR/US

By PAULINE WATSON*

Department of Botany, Leeds University

Calcarisporium arbuscula can frequently be isolated by tissue-culture methodsfrom the flesh of healthy fruit-bodies of species of Russula and Lactarius. Itappears to live in the growing fruit-bodies as an endophyte, without causing anyapparent disease. When the growth of the infected host is checked, or ceases as itbecomes old, the endophyte reveals its presence by producing its reproductivebodies all over the surface of the agaric.

Calcarisporium arbuscula Preuss was frequently isolated during attempts togrow species of Russula in pure culture, using tissue from the interior ofyoung fruit-bodies as inocula. The only other colonies which developed,apart from obvious contaminants, were slow-growing and produced nospores. These were probably the mycelia of the agarics, although noclamp-connexions could be seen.

Calcarisporium arbuscula has previously been recorded as a parasite onvarious fleshy fungi. There seems to be no report of the isolation of themould from apparently healthy sporophores, and this paper is concernedwith a study of the relationship between C. arbuscula and species of Russulaand Lactarius.

METHODS

The fruit-bodies of Russula and Lactarius were usually wrapped separately inpaper immediately after picking, or were collected individually in sterilecontainers, to prevent cross-infection, although these precautions werelater found to be unnecessary.

Small pieces of flesh from the interior of the sporophore, exposed bybreaking, were cut out with a sterile scalpel and placed on agar.

The earlier isolations were made on 2 %malt agar, but later, after trialswith various media, the following was found to give the best results:glucose, 20 g.; KH2P04, I g.; MgS047H20, 0'5 g.; ammonium tartrate,3 g.; yeast extract (Difco), 0'5 g.; FeCl3 (I % solution), 0'5 ml.; ZnS04solution (I part Zn!500, w!v), 0'5 ml.; agar, 20 g.; distilled water,1000 ml.

After 5-7 days' incubation at 20° C., the inocula from which Calcari­sporium was growing could be clearly distinguished. Usually, each isolatewas then subcultured on to a fresh agar plate to determine the colony typeand, in some cases, single-spore isolates were prepared.

* Now at the Botany Department, University Science Laboratory, Durham CollegesDurham.

410 Transactions British Mycological SocietyInocula, from which no Calcarisporium was produced often gave, after

several weeks, sterile colonies, which are believed to be the mycelia of theagarics. Some of these have been kept in culture, but most soon ceased togrow and did not survive subculturing.

IDENTIFICAnONS

For the identification of the species of Russula and Lactarius the papers byPearson (1948, 1950) proved the most useful.

Russula emetica was considered as four forms, a birchwood form beingdistinguished in addition to the three given by Pearson. This small,fragile, rosy-pink form appeared to be distinct, although it does not seemto have been considered as separate before.

Calcarisporium arbuscula is a very characteristic species which has recentlybeen re-described by Hughes (1951). An isolate made in the presentstudy has been deposited at the Commonwealth Mycological Institute(Herb. I.M.I. 55742).

RESULTS

Calcarisporium has been isolated from many of the species of Russula tested,including species from all the groups into which the genus is usuallydivided (Pearson, 1948). Few attempts have been made to obtain it fromfruit-bodies of Lactarius, but it has been isolated from two species. Theseresults are shown in Table I. The number of infected fruit-bodies mayactually be larger, as bacterial contamination may have preventedCalcarisporium from growing out from some of them.

Table I. Summary ofisolations ofCalcarisporiumfrom Russula and Lactarius

HostR. emetica (oakwood form)R. ochroleucaR. emetica (birchwood form)Ri fragilisRi felleaR. versicolorR. sororiaR.lepidaR. uescaR. nigricansR. clarojlavaOther Russula spp.L. rufusL. torminosus

Totals

No. fruit-bodiessampled(aU ages)

152

350

101666410

244762

4362814

12 64

No. from whichCalcarisporiumwas obtained

3631

2212

9422I

I

I

I772

147

It does not seem to be restricted to a small number oflocalities, havingbeen isolated from nearly all the areas visited, as shown in Table 2.

All the isolations from sporophore tissue were made from apparently

Calcarisporium arbuscula. Pauline Watson 41 I

perfectly healthy fruit-bodies. The gills of some, which were afterwardsshown to contain the endophyte throughout the fruit-body, were examinedmicroscopically. They appeared to be quite normal, apart from a veryfew conidiophores of Calcarisporium in two individuals. The presence of themould did not seem to be correlated with damage to the sporophore bymaggots or other animals, although inocula from such specimens showedcontamination more frequently than those from undamaged ones.

Table 2. Geographical distribution of isolates of Calcarisporium

Locality

Ruislip Woods, Middx,Clayton Wood, near LeedsBoxhill area, SurreyLeith Hill area, SurreyHampstead Heath, near LondonHayes Common, KentEight other areas

No. fruit-bodiessampled(all ages)

4391332461536889

136

No. from whichCalcarisporiumwas obtained

513216141310II

When several inocula were tried from different parts of a fruit-body, ifCalcarisporium did appear, it usually grew from all of them.

The fruit-bodies studied were classified roughly into groups, accordingto size and stage of development. In species where sufficient numbers werestudied, Calcarisporium could usually be isolated from about 5-15 %of the'young' fruit-bodies, and 10-40 % of the 'young-mature' and 'mature'ones.

Infected fruit-bodies, usually at the' old-mature' stage, could be foundin nature covered with conidiophores and sclerotia of Calcarisporium, butno numerical records of these were kept. An attempt was made to watchthe development in the field of fruit-bodies known to be infected, but thiswas unsuccessful. It was then thought that the effects on the host innature might be similarly produced if they were collected and kept for ashort time. After collection, a small portion of the flesh of each fruit-bodywas removed and used to test for the presence of the endophyte in theusual way. The fruit-bodies were then kept in separate containers in thelaboratory. After several days, the ones containing the endophyte becamecovered with the characteristic conidial apparatus of Calcarisporium, in thefew cases where secondary infection by moulds such as Verticillium did notmask the effects. Verticillium commonly appeared on fruit-bodies kept inthe laboratory irrespective of whether or not they were already infectedwith Calcarisporium.

Calcarisporium seems to be capable of dominating the host without theco-operation of other parasites, when the host's growth is stopped, or itsvigour declines after maturity. This is shown by the fact that inocula fromwithin such fruit-bodies rarely produced any organisms apart fromCalcarisporium.

Attempts to see the mycelium of the endophyte in sections of sporo­ph ores were unsuccessful owing to the complexity of hyphal systems inRussula.

412 Transactions British Mycological SocietyThe isolates of Calcarisporium were of a number of different types. The

clearest distinction was between those producing very numerous blacksclerotia, and a smaller number (about IO % of the isolates) which pro­duced no black sclerotia on the medium used. The larger group can beconsiderably subdivided on the bases of various less well-marked colonycharacters. These different types seem to be distributed in quite a randommanner. In several cases where close groups of the agaric were studied,isolates of different types were obtained from adjacent fruit-bodies.However, inocula from one individual always gave the same type, andsingle spore colonies, if prepared from one of these fresh isolates, alwaysgave colonies identical in appearance. In the fifty-five cases whereCalcarisporium was obtained from more than one inoculum from eachspecimen, there was never any suggestion of more than one type of theendophyte occurring in one sporophore.

Calcarisporium has a relatively low growth rate compared with someparasitic moulds such as species of Verticillium found on agarics. At 23° C.,the approximate optimum temperature, the rate of extension growth onagar was 1-2 mm.jday, A similar rate of advance occurred in previouslyuninfected fruit-bodies of various ages inoculated in the laboratory.

EFFECT ON HOST

The mould appeared to cause no symptoms in the growing fruit-bodies.This suggests that it can live endophytically in the host without causingany obvious disease. An alternative explanation might be that the fruit­bodies studied had only recently been invaded by Calcarisporium which,given time, would soon have produced disease symptoms.

There usually seemed to be a higher proportion of infected specimensamong the older fruit-bodies. This suggests that the young infected onesgrow up normally, and together with some later infected ones, form theinfected part of the population of older fruit-bodies. Another explanationmight be that they become obviously diseased soon after infection, andthat the infected mature ones are the result of recent and rapid invasion bythe Calcarisporium, and have not developed from infected young fruit­bodies. Little evidence could be obtained from the proportion of obviouslydiseased specimens found in nature, as only apparently healthy fruit­bodies were usually collected and no numerical records were kept of thenumbers of diseased specimens.

The slow growth-rate of the endophyte supports the first hypothesis.Mature fruit-bodies, with caps of about 5 cm. diameter, have several timesproved to contain it in all the inocula tried from various parts of thesporophore and seem to be systemically infected. The endophyte wouldtake about 20 days to permeate an entire large mature sporophore at itsnormal growth rate even at the optimum temperature, if it can becomesystemic as a result of a single infection. Precise times of development ofRussula fruit-bodies have not been recorded, but this time seems consider­ably longer than the time taken for a 'young' sporophore to develop tothe 'young-mature' stage, or the latter to become 'mature'. However, ifthey were infected while very young there would probably be time to

Calcarisporium arbuscula. Pauline Watson 413produce systemic infection by maturity. Therefore it seems that the largeinfected sporophores might either have developed from young infectedones, or have been entered at a number of different points by the endo­phyte, as this would enable it to permeate the entire fruit-body in a muchshorter time. A study of the isolates indicates that the former hypothesis isprobably correct and that a single infection is probably usual. Althoughthe isolates were often distinguishable in appearance, and the typesseemed to be randomly distributed, no indication of more than one typeoccurring in one fruit-body was obtained.

All this evidence strongly suggests that a single infection is sufficient toenable the endophyte to permeate the entire fruit-body. Probablyinfection takes place while the host is quite young, and it grows upnormally containing the endophyte. Finally, when the host's vigour isreduced after maturity, or after being collected, the endophyte becomesapparent by producing its reproductive structures all over the surface ofthe host.

DISCUSSION

Parasites of higher plants, such as Epichloe and members of the Usti­laginales, are known which can infect their hosts systemically, withoutproducing disease symptoms for at least part of their association. How­ever, there seem to be no previous records of a similar relationshipbetween a fleshy fungus and its parasite.

This relationship is also interesting from the aspect of colonization ofmoribund organisms by soil scavengers. Calcarisporium, by entering thesporophores while they are still vigorous, gains an advantage over lessspecialized parasites when the agaric becomes old or damaged, and morevulnerable to attack from scavenging fungi. Then it produces its repro­ductive bodies in large numbers all over the dying host. Because of itsmuch lower growth rate, it seems ill-adapted to colonize the host incompetition with less specialized parasites such as Verticillium. These,however, seem unable to attack healthy growing fruit-bodies.

This is similar to the colonization of other dead and dying plant tissuein the soil, where root-inhabiting fungi are unable to colonize dead plantmaterial in competition with obligate saprophytes, but can continue tolive in the dead tissue if already established in it as a parasite. Calcari­sporium could be considered as an 'ecologically obligate parasite' like someroot-inhabiting fungi (Garrett, 1950). It resembles them in showingspecialized parasitic ability combined with easy growth in culture andprobably low competitive saprophytic ability.

Calcarisporium has previously been recorded as growing parasitically onvarious fungi, including Ascomycetes as well as agarics (Hughes, 195I).It seems to have developed a subtle parasitic arrangement with the twogenera of agarics studied, and it would be very interesting to know moreof its relationship with the other very varied fungi on which it has beenrecorded as growing openly. It would also be possible to grow the hostand parasite in mixed cultures to study their relationship, which might beinteresting from the nutritional and antibiotic aspects, as well as that ofparasitism.

414 Transactions British Mycological SocietyThanks are due to Prof. C. T. Ingold, who supervised this work, and to

Mr A. D. Greenwood and others for criticizing the manuscript. I also wishto thank Birkbeck College for the award of a Research Studentship, duringthe tenure of which most of the work was done.

REFERENCES

GARRETI, S. D. (1950). Ecology of the root-inhabiting fungi. Biol. Rev. 25, 220-254.HUGHES, S. J. (1951). Studies of Microfungi. IX. Calcarisporium, Verticicladium and

Hansfordia. Mycol. Pap., no. 43.PEARSON, A. A. (1948). The genus Russula. Naturalist, Lond., pp. 85-108.PEARSON, A. A. (1950). The genus Lactarius. Naturalist, Lond., pp. 81-99.

(Accepted for publication 25 February 1955)