Trans. Brit. mycol. Soc. 46 (4) 565-571 (1963).

STUDIES ON FUNGI IN COASTAL SOILS

IV. CELLULOSE DECOMPOSING SPECIES IN SAND DUNES

By G. J. F. PUGH, J. P. BLAKEMAN, G. MORGAN-JONES

Department ofBotany, University ofNottingham

AND H. O. W. EGGINS

College ofTechnology, Kingston-upon-Hull

The isolation of cellulose decomposing species from two habitats in each of threedifferent sand-dune systems has shown the widespread occurrence of the speciesisolated. In addition to the common soil fungi Cephalosporium acremonium,Fusarium spp., Penicillium spp. and Trichoderma viride, other isolates includedfive species of Chaetomium, three of Doratomyces, two each of Humicola, Gliomastix,and Sordaria, and among other species not often recorded from the soil, Acrospeiralevis, conidial Apiospora montagnei, Trichocladium asperum and Volutella ciliata. Theresults are compared with reports of previous isolations of cellulose decomposersfrom the soil, from buried organic matter and from coprophilous habitats.Reasons are suggested for the greater number of isolates from the high-watermark than from the dunes.

Recently Eggins & Pugh (1962) have reviewed the necessity for selectivemedia for the isolation of soil fungi with particular requirements. In thepresent investigation cellulose decomposing fungi, isolated from the sanddunes at Gibraltar Point, Lincs., Spurn Head, Yorks., and Ynyslas, Cards.,have been examined. Several methods have been used for studying themicro-organisms associated with the decomposition of cellulose substratain the soil, and Siu (1951) has discussed the role played by micro-organisms,particularly on textiles and military equipment. Tribe (1957) buried stripsof' Cellophane' attached to coverslips so that microbial activity could bestudied in situ. He stated that the advantage of cellulose film over otherforms of cellulose is its transparency, which renders it a perfect substratumformicroscopic 0 bservation. Webley & Duff (1962) devised a method ofburying kaolinite pellets in which various substances, including cellulose,had been mixed, and they suggested that the technique could be used tostudy the development, and in some cases also the physiological activity,of the micro-organisms in soil. Meyers & Reynolds (1959) used pieces ofwood submerged in the sea as substrata which would selectively isolatemarine cellulolytic and lignicolous fungi.

The use of cellulosic substrata for the isolation and cultivation of fungifrom soil inocula appears to have been started by Van Iterson (1904),who used filter-paper soaked in mineral salts to isolate cellulolytic fungifrom the soil. Precipitated cellulose was used by Sanborn (1927) forphysiological studies of bacteria, while Hungate (1950) isolated anaerobiccellulolytic bacteria by the use of ball-milled, acid-treated cotton cellulose.

The selective medium used in the present study, containing ball-milled

566 Transactions British Mycological Society

Whatman's cotton cellulose powder as the main carbon source, has beendescribed (Eggins & Pugh, 1962). The clearing of the opaque whitemedium is the criterion which has been adopted as a measure of the abilityof a fungus to decompose cellulose.

MATERIALS AND METHODS

The areas from which samples were collected are somewhat similar toeach other in that in all three there is a sand-dune system, on the landwardside of which is a salt marsh. In each of the three areas samples werecollected from two habitats: the high-water mark (H.W.M.) on the land­ward side of the dunes, and the top of the dunes. At Gibraltar Point thereis a great amount of organic debris deposited by the tides, and thisblankets the soil at the H.W.M. At Spurn and at Ynyslas there was verylittle drift material at the H.W.M. collecting sites. On the dune top at Gib­raltar Point there is a closed cover of higher plants, mainly Festuca rubra,while .in the other two areas there was an open cover mainly of Ammophilaarenana.

The reaction of the soils at all the sampling sites was near neutral toalkaline. At Gibraltar Point readings have varied between pH 7 and 8,while single readings at Spurn and Ynyslas were 6·8 and 7'2 respectively.

Samples have been obtained from Gibraltar Point on ten differentoccasions, from Spurn twice, and from Ynyslas only once. The sampleswere collected into new polythene bags, and inocula from them wereplated out, using the method and medium described by Eggins & Pugh( I 962). A total of fifty Petri dishes was prepared from the ten samplescollected from each habitat at Gibraltar Point, and a similar number foreach habitat from the two sets of samples from Spurn. Thirty plates wereprepared for each of the Ynyslas habitats. Species which cleared themedium were, when necessary, transferred to the normal diagnostic mediafor identification. Fungal potential was estimated by counting each speciesoccurring on each Petri dish as one isolate. It has been pointed out (Pugh,1963) that potential, in this context, means active mycelium plus pro­pagules, which are the result of previous activity, held dormant by suchfactors as mycostasis.

RESULTS

The distribution of the fungi isolated is given in Table I, where thearrangement of the species is based on the number of sampling sites fromwhich each was isolated. Thus the species found at all six sites have beenplaced in group I, and, at the other extreme, species found at only onesite have been placed in group 6. The single sample obtained from eachsite at Ynyslas, and the smaller number of plates prepared, almostcertainly account for the fewer species recorded there. Nevertheless, ascan be seen in groups I and 2, qualitatively, the flora isolated from thedifferent habitats in widely separated sand-dune systems is remarkablysimilar. Quantitatively, however, there are many differences, not onlybetween dunes and H.W.M. in anyone area, but also between each type of

Fungi in coastal soils. IV. G.J. F. Pugh and others 567

habitat in the three areas. Thus, in group I, Cephalosporium acremonium wasisolated much more frequently at Ynyslas H.W.M. than anywhere else,Fusarium spp. (mainly F. culmorum and F. solani) were more common in thetwo Spurn habitats than at Gibraltar Point and Ynyslas. Species whichoccurred relatively more abundantly in the dunes than at the H.W.M,

Table I. Distribution and frequency of occurrence if the fungi

2'0

5"0

6'0

3'0

20'0

6'012'0

10'0

100

Ynyslas

8'0

2'5

2'5

5"1

2'5

1'3

8'9

14'510,86'36'3

25'3

Spurn

1'2

1'2

5'0

4'3

3'7

1,8

0·61'2

('2

0,6

1,8

3'212'0

13'3

6'18'1

g'8

4'9

6'j8'0

3'2

9'4

3'2

9'43'2

3'2

11'0

7'93'2

14'2

Ynyslas

18'g

2'5I'S

4'0

1'5

6'0

13'0

2'5

10'5

24'0

2'52'58'0

Spurn

200

7'52'0

6'03,6

3'3

7'0

3'3

3'0

7'31'54'2

1'5

0,6o'go'g

3'06'02'7

2'7Ij'2

Gibraltarpoint

I. Cephalosporium acremoniumCorda

Doratomyces microsporus (Sacc.)Morton & Smith

Fusarium spp.Penicillium spp,Trichoderma viride Pers. ex Fr.

2, Apiospora montagnei Sacc.(conidial)

Chaetomium funicola CookeGliomastixmurorum (Corda)Hughes var,felina (March.)Hughes

Stachybotrys atra Corda3. Beauveria tenella (Delacr.)

Siem.Volutella ciliata Fr.

4. C, piluliferum Daniels(conidial)

Humicola spp,Nectria gliocladioides Smalley

& Hansen (conidial)Nectria inventa Pethyb.

(conidial)Phomasp.Sordaria tetraspora Wint.Doratomyces nanus (Ehrenb.

ex Link) Morton & Smith5' Alternaria tenuis auct.

C. bostrychodes ZopfScopulariopsis fusca ZachSordaria fimicola (Rab.) Ces.

& de Not,Thermomyces sp.

6, Acrospeira levis Wilts.C. elatum Kunze ex Fr.C, globosum KunzeCylindrocarpon sp,Doratomyces stemonitis (Pers.

ex Fr.) Morton & Smith(Echinobotryum state)

Gliomastixguttuliformis Brown& Kendrick

Pestalotiopsis sp.Pyrenochaeta sp.Sporormia minima Auers,Trichocladium asperum (Corda)Harz

Total number of isolates 331

(Frequency of occurrence is given as a percentage of the total isolations for each sampling site.)

High-water mark Dune topsA \ r- ~ --..

GibraltarPoint

568 Transactions British Mycological Society

included Doratomyces microsporus, Penicillium spp., Trichodermaviride (group I),Beauveria tenella (group 3) and Doratomyces nanus (group 4). Conversely,Fusarium spp. (group I), conidial Apiospora montagnei (Papularia arundinis)(group 2), and, most strikingly, Volutella ciliata (group 3) and conidialNectria gliocladioides (Gliocladium roseum) (group 4) were relatively morefrequently isolated from the H.W.M.

The relative abundance and the ubiquity of a fungus could sometimesbe correlated, but this was not necessarily the case, as can be illustratedby the small number of isolates from the H.W.M. of Penicillium spp. andDoratomyces microsporus in group I, and of Gliomastix murorum var. felina ingroup 2. Conversely, large numbers of isolates of a fungus from one sitedid not necessarily mean that it would be widespread, as can be seen withScopulariopsisfusca and Thermomyces sp. in group 5. The species in groups 5and 6 in general were infrequently isolated, and their occurrence on theisolation plates was probably somewhat fortuitous.

In each area the same number of samples was examined from bothhabitats under study, so it is possible to compare the fungal potential ofthe dunes and H.W.M. in the three sand-dune systems. At Gibraltar Pointthe percentage difference between the two habitats (i.e. the differencebetween the number of isolates from the two habitats expressed as a per­centage of the H.W.M. total) was 51. At Spurn and Ynyslas the percent­age differences were 21 and 19, respectively. While the large differencein potential between the two habitats at Gibraltar Point is probablyto be correlated with the great accumulation of drift material atH.W.M., the smaller differences in the other two areas would reflect therelatively minor amounts of drift material present at the H.W.M. in theselocalities.

DISCUSSION

When organic matter is added to the soil it becomes subjected to aseries of waves of colonization by fungi present in the soil. These wavesoverlap and succeed each other, and it is possible to segregate the initialcolonists from the inhabitants of the residual products of decomposition(Pugh, 1958; Griffin, 1960; Hudson, 1962). Thom & Morrow (1937)pointed out that the distinction between the two groups was to a certainextent theoretical, and evidence to support this view is provided by thefungi in group I, which, with the exception of Doratomyces microsporus, haveall regularly been isolated on non-selective media from the soil, from salt­marsh muds (Elliott, 1930; Pugh, 1962) and from sand dunes (Brown,1958; Pugh, 1963). D. microsporus was isolated in a non-coremial, as wellas its usual coremial form and was regarded as a species of Siysanus untilits taxonomic position was clarified by Morton & Smith (1963). Tricho­derma spp. were the main colonizers of straw buried in the soil by Walker(1941) which demonstrated their ability to compete successfully with otherprimary colonizers. The fungi in group 1 all characteristically producelarge numbers of conidia, so their high frequency of occurrence need notimply that they are more active in decomposing cellulose in the soil thanare the less frequently isolated species, some of which are relatively lightsporers.

Fungi in coastal soils. IV. G.J. F. Pugh and others 569

The majority of the species in groups 2-6 hav e not regularly beenisolated on non-selective media, perhaps because they are unable tocompete with the mor e vigorously growing ' sugar' fungi. J ensen (1931)isolated, from neutral to alkaline soils to which cellulose had been added,Mycogone nigra ( = Humicola grisea White & Downing, 1953) and Coccosporaagricola (shown to be synonymous with Botryotrichum piluliferum by Downing(1953), and its perfect state, Chaetomium piluliferum, was discovered byDaniels (196 I)). J ensen (193I) said that these and some other specieswere present in small numbers or not noticed in isolations from unamendedsoil. On buried strips of ' Cellophane' in neutral to alkaline soils Tribe(1957, 1960) most frequ ently isolated Botryotrichum piluliferum, Humicolagrisea, Rhizoctonia solani, Sporotrichum sp., Stachybotrys atra, and Stysanus sp.Trichoderma uiride occurred only on strips buried in acid litter, while speciesofAspergillus, Fusarium and Penicilliumwere uncommon or absent. Of thesefungi, Aspergillus spp. were not found on the dunes, were infrequentlydetected at H.W.M. when non-selective media were used, and were notisolated on cellulose agar. Tribe (1960) and Daniels (1961) suggested thatBotryotrichum piluliferum may be more common in the soil than the availableevidence suggests. The pr esence of this species in two of the three sand­dune systems examined would support this view. Echinobotryum sp.,Humicola spp. and Stysanus spp. have been recorded by Elliott ( I930) andby Tribe (1960), and these species, as well as Botryotrichum, may be morecommon in soil than evidenc e obtained by non-selective media wouldsuggest. Humicola grisea was found at three sites; H. nigrescens was isolatedonly once (from the dunes at Gibraltar Point). Rhizoctonia solani hasnot been recognized at Gibraltar Point, though it is possible that thesterile white mycelium found on washed roots (Pugh, 1960) may be thisspecies. Garrett (1962) has clearly demonstrated the ability of R. solani tocolonize competitively and to decompose cellulose in the soil. Papulariaarundinis was isolated frequ ently by Brown (1958) on non-selective media,and it was also commonly found on reeds by Ellis, Ellis & Ellis (1951)who said that it sporulated heavily in the summer and early autumn. Ithas been isolated from the soil at Gibraltar Point at all times of the year.Gliomastix murorum var.felina was isolated as Periconiafelinaby Elliott (1930),and as Gliomastix conooluia var. felina by Brown (1958). G. guttuliformis,isolated from the H.W.M. at Ynyslas, agreed compl etely with the descrip­tion of Brown & Kendrick (1958) . They had isolated it from fixed sanddunes in Northumberland and from decaying pine needles in Cheshire.

The presence of Chaetomium spp. and of some species normally consideredto be coprophilous is not unexpected in view of their known ability todecompose cellulose. Tubaki (1954) listed among other Japanese copro­philous species Acrostalagmus cinnabarinus ( = conidial Nectria inventa),Cephalosporium acremonium, Gliocladium roseum ( = conidial Nectria glio­cladioides), Stysanus medius and Trichoderma viride, all of which are normallyconsidered as soil fungi. Their ability to decompose cellulose would enablethem to grow on dung, which is frequently cellulosic, whenever environ­mental conditions were suitable.

The qualitative aspects of this study, together with the results obtainedby other workers, particularly J ensen (1931) and Tribe (1957, 1960),

57° Transactions British Mycological Societyindicate the widespread occurrence ofa number of species previously con­sidered uncommon in the soil. Quantitatively, they show a preference ofsome species for one of the two habitats rather than the other. The totalnumber of isolates obtained from the dunes was about two-thirds that ofthe H.W.M. This difference can be attributed to the organic matter de­posited at the H.W.M. From the results given by Pugh (1963) the per­centage difference in the number of isolates of' sugar' fungi from the twohabitats at Gibraltar Point is approximately 8. This is very much smallerthan the difference of 5 I %in numbers of cellulose decomposers in the sametwo habitats. The partly decomposed state of much of the debris depositedat the H.W.M. by the tides directs attention to the probability that the mosteasily available fraction of the substratum would already have beenutilized, so that there would be no great stimulation of the' sugar' fungi.However, the more resistant parts of the substrata would provide nutri­ment for the cellulose decomposers, and, as the available supply of organicmatter is the main factor in determining the active presence of fungi insoil, it is to be expected that fungi able to utilize the organic materialswill be plentiful where these are abundant. The much smaller differencesbetween the two habitats at Spurn (21 %) and at Ynyslas (19 %) may becorrelated with the relative paucity of drift at the H.W.M. collecting sitesin these areas.

The H.W.M. is an interesting habitat, mycologically, because of thearray of species which occur there on drift material (Jones, 1962) and inthe soil, and also because of the manner in which the change in thephysical environmental conditions imposes limits on the distribution ofsome species which are normally found either above or below the H.W.M.

but only rarely cross it (Pugh, 1963).

Thanks are due to Prof. C. G. C. Chesters for helpful discussions, andone ofus (H. O. W. E) wishes to thank Dr T. A. G. Wells for his encourage­ment.

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BROWN, J. C. & KENDRICK, W. B. (1958). Gliomastix guttulijormis sp.nov. Trans. Brit.mycol. Soc. 41, 499-500.

DANIELS, J. (1961). Chaetomium piluliferum sp.nov., the perfect state of Botryotrichumpiluliferum. Trans. Brit. mycol. Soc. 44, 79-86.

DOWNING, M. H. (1953)' Botryotrichum and Coccospora. Mycologia, 45, 934-g40.EGGINS, H. O. W. & PUGH, G.J. F. (1962). Isolation of cellulose decomposing fungi

from the soil. Nature, Lond., 193, 94-g5.ELLIOTT, J. S. B. (1930)' The soil fungi of the Dovey salt marshes. Ann. appl. BioZ. 17,

284- 30 5.ELLIS, M. B., ELLIS, E. A. & ELLIS,J. P. (1951). British marsh and fen fungi. II. Trans.

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study of cellulose destroyers. J. Bact. 14, 395-397.Sru, R. G. H. (1951). Microbial decomposition ofcellulose. New York: Reinhold.THOM, C. & MORROW, M. B. (1937). Fungous mycelia in the soil. Abstr. in J. Bact. 33,

77-78.TRIBE, H. T. (1957). Ecology of micro-organisms in soils as observed during their

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WALKER, A. G. (1941). The colonization of buried wheat straw by soil fungi, withspecial reference to Fusarium culmorum. Ann. appl. Biol. 28, 333-350.

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(Accepted for publication 9 March 1963)