Trans. Brit. mycol. Soc. 48 (3),367-37° (1965).Printed in Great Britain

A STUDY OF SOOTY MOULD ON LIME TREES(TILIA X VULGARIS)

By R. J. FRIEND

Department of Botany, University of Exeter, Devon

The isolation and identification of the fungus components of sooty mould onlime were attempted. The most common fungi on the leaves were Aureobasidiumpullulans and Cladosporium herbarum. A number of other common saprophyticfungi were also isolated but they are considered to be only of minor importancein making up the total bulk of the sooty mould. On lime twigs in winter thepredominance of A. pullulans was indicated as well as on the leaves, but twoother fungi isolated from twigs were not found in sooty mould on leaves.No fungus referable to Fumago or to the Capnodiaceae was isolated.

In Britain during late summer and autumn leaves of lime trees (Tiliaspp.) and, in particular, of the common lime (T. x vulgaris), frequentlyhave a conspicuous, black, superficial, fungus growth which is predomi­nantly epiphyllous but also occurs to a minor extent on and near veinson the under surface of many leaves. A similar growth of sooty mouldoccurs on the twigs and branches of these trees, again predominantly onthe upper surface, but it is less conspicuous to the casual observer. As theleaves are deciduous, sooty mould on the leaves must start anew everyyear but that on the branches can be perennial.

It has long been recognized that the growth of sooty mould on limesfollows upon a heavy infestation of the trees by aphids. The 'honeydew'excreted by them has a sugar concentration only slightly different fromthat of the plant sap (Hill, 1962). Under favourable conditions honeydewproduction can be abundant, with some sticking to the leaf from which itoriginated, but much forming a 'copious rain' (Clapham, Tutin & War­burg, 1952) which drifts downwards on to the lower leaves and branchesand also on to other plants in the vicinity. This honeydew is a very suitablesubstratum for the growth of a number of saprophytic micro-organisms.

For many years the opinion that sooty mould on limes consisted chieflyof a growth of Fumago vagans Pers. has prevailed. Also, F. vagans has longbeen considered to be a conidial state of Capnodium sp. (see, for example,Saccardo, 1886). The results recorded in this paper do not support thesetraditional views, but on the contrary indicate that a number of commonsaprophytic hyphomycetes are implicated, while a few slower growing fungiare also present in the perennial sooty mould on twigs and branches.Evidence against the supposed connexion between Fumago and Capnodium,and also for the rejection of F. vagans as a valid binomial, is presentedelsewhere (Friend, 1965).

Transactions British Mycological Society

EXPERIMENTAL

Isolation and identification ofthe fungi present

Isolations from leaves

Leaves from lime trees growing at Exeter, Devon, and bearing aconspicuous growth of sooty mould were gathered in late October 1963and immediately placed in clean Petri dishes. Small portions of the blackdeposit were removed with a sterile scalpel and dropped into sterile waterin a test tube. After vigorous agitation for a few minutes, small fragmentsof the sooty mould were placed on plates of potato dextrose agar andincubated at 20° C.

In a few days there was invariably considerable fungus growth and alsosome growth of bacteria. A number of fungi were isolated, Aureobasidiumpullulans (de Bary) Arn. (= Pullularia pullulans (de Bary) Berkh.), andCladosporium herbarum (Pers.) Link ex Fr., both isolated very frequently;Epicoccum nigrum Link ( = E. purpurascens Ehrenb. ex Schlecht.), Alternariatenuis Nees ex Pers., Fusarium merismoides Corda, F. lateritium Nees ex Fr.and sterile mycelium, were all isolated much less frequently.

Isolations were also attempted from younger leaves in late June 1964.By this date the lime trees were quite heavily infested by aphids and manyleaves had a conspicuous epiphyllous deposit of insect honeydew. Manyof the droplets had turned partly or fully dark in colour owing to thegrowth ofmicro-organisms, whereas at first they had been quite colourlessand transparent. The fungus isolated most commonly from these earlygrowths of sooty mould was A. pullulans, then of much less commonoccurrence was E. nigrum and there was one isolate each of C. herbarum andBotrytis cinerea Fr.

Isolations from twigs in winter

Living twigs bearing a substantial covering of sooty mould on the uppersurface were collected in late winter (3 March 1964) following an over­night minimum temperature of -4°. Only two fungi were isolated. Ofsixteen attempts, five gave A. pullulans and seven gave a dark, verycompact, slow-growing fungus that has produced no certain spore formsyet but may well belong to the genus Septonema. The remaining fourattempts yielded no growth.

On 12 March sixteen samples were taken from twigs from two otherlime trees where again the sooty mould was confined almost exclusively tothe upper surface. Four isolations gave A. pullulans, four? Septonema sp.,two a fungus which would normally be referred to Phoma, one sterilemycelium and the remainder yielded no growth.

Direct microscopical observations of sooty mould

Microscope slides of sooty mould from lime leaves gathered in October1963 were found to have a few to many Cladosporium-like conidia and darkmulticellular bodies which may well have been formed by A. pullulans,which can form such structures (Hoggan, 1924), though they are not

Sooty mould of Tilia. R. J. Friendcharacteristic enough for certain identification. Other components thatcould be identified with fair certainty were conidia of Alternaria and thealga Pleurococcus, both of which occurred much less frequently than theother components mentioned.

Fungi present in soo0' mould on other plants

Isolations were attempted from sooty mould on certain other plants inExeter, using the same technique.

From Brassica oleracea, Aureobasidium pullulans, C. herbarum and E. nigrumwere isolated regularly. From sooty mould on Prunus sp. and Quercusrobur, A. pullulans was isolated frequently, and E. nigrum less frequently.

Direct microscopical observations of growths of sooty mould on livingplants in Devon and on herbarium specimens from a number of differentplaces were carried out. Frequently there was clear evidence for thepresence of a number of distinct fungi in one specimen of sooty mould. Insooty mould on, for example, Camellia sinensis growing in a heated glass­house there were conidia of Cladosporium and Alternaria, the former beingparticularly abundant. C. herbarum was very widespread in sooty moulds onplants growing in glasshouses and usually it was considered to predominateunder these conditions. In sooty mould on Quercus robur and Prunus sp.many dark multicellular bodies were seen as well as a few Cladosporium-likeconidia. It may well be that these multicellular structures are formed byA. pullulans, as already suggested for the origin of the similar bodies foundin sooty mould on limes.

On herbarium specimens of a variety of plants spores similar to those ofC. herbarum were commonly found in sooty mould. The hosts were distri­buted in both time and place. Dark multicellular bodies, sometimes verynumerous, were again present in most of the specimens examined. It isconsidered most likely that these also were formed by A. pullulans. Othercomponents identifiable with reasonable certamty by direct observationwere Alternaria and Pleurococcus.

DISCUSSION

Cultural work indicated that the chief components of sooty mould onlime leaves were similar to those comprising sooty mould on other plants,with A. pullulans being isolated the most frequently and C. herbarum andE. nigrum occurring commonly. Direct microscopical observations ofsootymoulds also gave evidence that the components of sooty mould on limeleaves were similar to those comprising sooty moulds on many other plantsgrowing under comparable climatic conditions. Conidia of C. herbarumand structures probably referable to A. pullulans were very commonly seen,so there is general agreement between the results from both cultural workand direct observation.

All the evidence gathered from this investigation indicates that sootymould on lime leaves chiefly consists of the mixed mycelium ofa number ofdistinct hyphomycetes which do not include Fumago uagans and for whichno one has yet demonstrated any connexion with any member of theCapnodiaceae. Comparable results have been obtained from studies of

370 Transactions British Mycological Societysooty mould on other plants by a number of investigators (e.g. Fisher,1933; Fraser, 1933; both worked in Australia).

Perennial fungi found in sooty mould on lime twigs and branches areA. pullulans, ?Septonema sp. and, of lesser frequence, Phoma sp. and perhapssometimes C. herbarum. These fungi are able to withstand extremes ofweather conditions. Fungi which do not appear to overwinter are those(other than A. pullulans) which regularly make up the bulk of the sootymould on lime leaves, C. herbarum and E. nigrum. They make rapid growthwhen conditions become favourable. Accidental components on the leavesare considered to be A. tenuis, Fusarium spp. , B. cinerea and a sterilemycelium. None ofthese is considered ever to predominate in sooty mouldon lime leaves in the locality studied.

The primary colonizer of honeydew on lime leaves is A. pullulans. Ampleinoculum is assured by the presence of this fungus in the sooty mould onnearby twigs and branches. Later in the season C. herbarum and E. nigrummake considerable growth. Probably their slow initial growth is at leastpartly due to a paucity of inoculum early in the season. The fungi makingup sooty mould obviously vary considerably both quantitatively andqualitatively according to the availability of inoculum and the environ­mental conditions prevailing. A warmer season would seem to favour thegrowth of C. herbarum,

This report is based on part of an investigation carried out in fulfilmentof the degree of M.Sc. in the University of Exeter. The writer gratefullyacknowledges the assistance rendered by the Commonwealth MycologicalInstitute, Kew, and the Royal Botanic Gardens, Kew, in making availablefor examination herbarium material, and also by the former in identifyingsome fungus isolates, and also by Dr S. A.J. Tarr of the University ofExeter under whose surveillance this project was carried out.

REFERENCES

CLAPHAM, A. R. , TUTIN, T. G. & WARBURG, E. F. ( [952). Flora of the British Isles,p. 367. Cambridge University Press.

FISHER, E. E. (1933). The sooty moulds of some Australian plants. Proc. R. Soc. Viet.,N.S., 45, 171-202.

FRASER, (1933). An investigation of the sooty moulds of N.S.W, I. Proc. Linn. Soc.N.S. W. 58, 375-395.

FRIEND, R.]. (1965). What is Fumago uagans ? Trans. Br. mycol. Soc, 48, 371-375.HILL, G. P. (1962). Exudation from aphid stylets during the period from dormancy to

bud break in Tilia americana (L. ). ]. expo Bot. 13, 144-151.HOGGAN, I. A. (1924). On Dematium pullulans de Bary. Trans. Br. mycol. Soc.9, 100-107.SACCARDO, P. A. (1886). Sylloge Fungorum, IV, p. 547.

(Acceptedfor publication 30 October 1964)