Notes and brief articles

and sealed straws are referred to as straw am-poules or simply 'straws'.

The straws are filled with cryoprotectant (10 %aqueous glycerol v/v) to within 1 ern of the topusing a hypodermic syringe and needle, 40 x 0·8mm. Small agar fragments taken from a colonyare immersed in the cryoprotectant using a wireneedle. Alternatively a large agar plug is removedfrom a colony, macerated with cryoprotectant,and appropriate amounts of the macerate intro-duced into each straw with a Pasteur pipette. Theopen ends are then sealed by welding.

Many straws may be sealed simultaneously,depending on the width of the welder. Thestraws can be held together on a ruler by double-sided adhesive tape and sealed in one operation.Each sealed straw is tested for leaks by squeezingfirmly between the fingers. If necessary a strawcan be sealed in a small bunsen flame by holdingit in a pair of forceps and passing the exposed endrapidly through the flame.

Nine replicates of each strain are prepared usingstraws of one colour; the different colours are usedin a fixed sequence. The straws are labelled witha permanent fibre-tip fine-writer pen and arefrozen by suspending in the neck of a liquidnitrogen flask for 1 h before total immersion. Onereplicate is thawed after 24 h and plated out totest for survival. The use of straws does notadversely affect the regrowth of cultures stored inliquid nitrogen. Over 500 stocks of A. bisporushave been successfully stored in this way.

Straw ampoules have considerable advantages.

Polypropylene heat-sealable ampoules have alimited neck diameter and are best used formaterials in suspension. The glass type must besealed individually using a compressed air and gastorch and the polypropylene using a hot-wire in-sert and a pair of pincers. They need to be testedfor leaks by immersion in a dye-bath for 30 min.Leaky glass ampoules may shatter upon removalfrom the liquid nitrogen and neither ampoule isreusable. Screw-cap polypropylene ampoules canbe used for large fragments of materials but arenot suitable for immersion storage or wheresterility is critical, because the liquid nitrogen canleak around the threads of the cap. The use of thistype of ampoule and the necessary storage in thevapour phase reduces the total number that canbe stored. Finally, conventional ampoules costapproximately 5P each and straw ampoules only0'04P each.

The principal advantage of straws is that for agiven volume they permit both the storage of morecultures and greater replication, giving, forexample, at least a ninefold increase in the capacityof the Union Carbide LR-40 vivostat.

Heat-welded lengths of polypropylene drinkingstraws are therefore recommended as a safe,convenient, and cheap alternative to the ampoulescurrently available for the storage of biologicalmaterials in liquid nitrogen.

I am indebted to Miss Joy Hall of the Common-wealth Mycological Institute who introduced meto the techniques of liquid nitrogen storage.

DEMATIACEOUS HYPHOMYCETES AND NARROW DARK

ZONES IN DECAYING WOOD

A. D. M. RAYNER

Department of Biological Sciences, University of Exeter

Decomposing wood, particularly that of hardwoodtrees which has been colonized by fungi causingwhite rot, frequently appears in transverse sectionas a mosaic of pale decay areas separated by

relatively hard, narrow, dark-coloured zones (Fig.1). The latter have often been referred to as zonelines, although the use of this term has beencriticized (e.g. Lopez-Real, 1975), and several

Fig. 1. Transverse section through part of an oak log colonized by Stereum hirsutum. The sapwood isextensively rotted and appears as a mosaic of pale decay areas separated by a network of narrow darkzones.Fig. 2. Longitudinal section through a dark zone in birch wood colonized by Coriolus versicolor. Inaddition to a considerable build up of hyaline hyphae belonging to C. versicolorseveral distinctive darkhyphae are present.Fig. 3. 3%malt agar plate onto which surface-sterilizedchips from decayedbirch wood containing darkzones have been placed. Coriolus versicolor has been produced in all cases, but although some myceliahave merged imperceptibly, others have failed to do so and are separated by narrow dark skin-likezones.

Trans. Br. mycol. Soc. 67 (3), (1976). Printed in Great Britain

Notes and briefarticles

Figs. 1-3. For legends see facing page.

547

Trans. Br. mycol. Soc. 67 (3), (1976). Printed in Great Britain

548 Notes and brief articles

investigations have been made into their nature(e.g, Campbell, 1933, 1934; Hopp, 1938; Lopez-Real, 1975). These studies have all shown suchzones to be of fungal origin, consisting of densesheets of mycelium which often contain swollenand /or pigmented cells, and have been regardedby several workers (e.g, Campbell, 1933, 1934;Lopez-Real, 1975) as forming the outer layers offungal pseudosclerotia within the wood. Apartfrom tho se causing decay no other fungi have, sofar as is known, been described from zone lines.

Evidence that fungi other than those causingdecay may sometimes be associated with narrowdark zones in decaying wood was obtained duringa study of fungal colonization of cut stumps ofbeech (Fagus sylvatica L.), birch (Betula pendulaRoth) and oak (Quercus robur L.) in East Anglia.It was observed that many of these were extensivelydecayed, as a result of natural colonization bywhite-rotting fungi, 2 or more years after felling,especially those which had been treated with a40 % (w/v) solution of ammonium sulphamate(Rayner, 1975). In transverse section the decayedwood often appeared as a mosaic as describedabove . The fungi present in such wood wereidentified either by incubating ca. 2 em thicksections (some of which were surface-sterilized) inmoist conditions for several days, or by plating outsurface-sterilized chips onto 3 % malt agar.Virtually pure mycelia of the decay fungi wereusually readily obtained from decay areas, but thedark zones frequently contained a number ofcharacteristic dematiaceous hyphomycetes. Thisfeature was particularly noticeable in wood decayedby Bjerkandera adusta (Willd. ex Fr.) Karst.,Coriolus versicolor (L. ex Fr.) Quel ., Phlebia meris-moides Fr. and Stereum hirsutum (Willd . ex Fr.)S. F. Gray. However, no dematiaceous hypho-mycetes were present in zone lines associated withrot caused by several other fungi includingHypoxylon multiforme (Fr.) Fr., H. serpens (Pers.ex Fr.) Fr. and Xylaria hypoxylon (L. ex Fr.) Grev.

The dematiaceous fungi formed a very charac-teristic and constant assemblage of differentspecies, most of which occurred in all three treespecies, although in varying proportions. It hasnot been possible as yet to identify all of them withcertainty, but the most important included threespecies of Rhinocladiella Nannf., a species ofCladosporium Link ex Fr., the Catenularia Grovestate of Chaetosphaeria myriocarpa (Fr.) Booth anda species which has been tentatively assigned toEndophragmia uniseptata ElIis.

The problem was further investigated in decay-ing birch and oak logs which were extensively

colonized by Coriolus versicolor and Stereumhirsutum respectively at a site in Stoke Woods,near Exeter, Devon. Once again the rotted woodhad the mosaic appearance described previously,and it was shown by incubation or isolation thatthe decay areas contained mycelium of C. versicoloror S. hirsutum, whereas the dark-coloured zonescontained dematiaceous fungi many of which wereidentical to those found in East Anglia. Thepresence of these latter was further confirmed bysectioning freshly cut fragments of wood by meansof a sledge microtome. Microscopic examinationof the resulting sections revealed a considerablebuild up of mycelium of the decay fungi in thedark zones, especially in the medullary rays,which often contained contorted, encrusted orpigmented cells, and was accompanied by pigmentproduction in the wood. In this respect theappearance of the dark zones did not differ signi-ficantly from previous descriptions of zone linesor pseudosclerotial plates (e.g. Lopez-Real, 1975).However, the zones also frequently containedcharacteristic dark, septate hyphae without clamp-connexions running along the length of some of thexylem elements (Fig. 2). These hyphae readilygrew and produced conidia when sections wereplaced on moist filter paper in Petri dishes andincubated for several days at room temperature(ca. 18 0q .

A number of mechanisms have been suggestedfor the way in which zone lines originate (e.g.Campbell, 1933, 1934; Lopez-Real & Swift, 1975)including interaction between different species ofdecay fungi and response to various physicalfactors such as desiccation or fluctuations in themoisture content of the wood. Some evidence wasobtained that the zones observed in the presentstudy were produced as a result of antagonisticinteraction between different mycelia, often be-longing to the same species, of decay fungi. Thuswhen chips of wood containing dark zonesremoved from logs colonized by Stereum hirsutumor Coriolus versicolor were placed several at a timeon to plates of 3 %malt agar the pattern of growthof mycelia of the decay fungi was such that somemerged freely, whilst others, although similar oridentical in appearance, failed to do so, showingvarying degrees of antagonism towards oneanother, often accompanied by yellowish toochreous pigment production by S. hirsutum anddevelopment of brown skin-like zones by C.versicolor (Fig. 3). As a result the plates oftenacquired a reticulate appearance, areas of vigorousmycelium being separated by narrow zones whereantagonism occurred, a pattern which clearly

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549

REFERENCES

CAMPBELL, A. H. (1933). Zone lines in plant tissues. I.The black lines formed by Xylaria polymorpha(Pers .) Grev. in hardwoods. Annals of AppliedBiology 20, 123-145.

CAMPBELL, A. H. (1934). Zone lines in plant tissues.II. The black lines formed by Armillaria mellea(Vahl.) Quel . Annals of Applied Biology 21, 1-22.

Hors, H. (1938). The formation of coloured zones bywood-destroying fungi in culture. Phytopathology28,601-620.

LOPEZ-REAr., J. M . (1975). Formation of pseudo-sclerot ia (' zone lines ') in wood decayed by Armillariamellea and Stereum hirsutum. I. Morphologicalaspects. Transactions of the British MycologicalSociety 64, 465-471.

LOPEZ-REAL, J. M. & SWIFT, M. J. (1975). Formationof pseudosclerotia (' zone lines ') in wood decayed byArmillaria mellea and Stereum hirsutum. II. For-mation in relation to the moisture content of thewood. Transactions of the British MycologicalSociety 64,473-481.

RAYNER, A. D. M. (1975). Fungal colonization ofhardwood tree stumps. Ph.D. Thesis, Univers ity ofCambridge.Part of this work was carried out during the

tenure of an N.E.R.C . research studentship.

Notes and brief articles

reflected the appearance of the decayed wood.Furthermore antagonism only occurred betweenisolates from those decay areas which were sep-arated by dark zones. Further evidence in thisconnexion was provided by the appearance ofincubated segments of decayed wood: mycelia ofC. versicolor or S. hirsutum produced on thesedid not fuse in the region of the dark zones, butinstead frequently produced crustose or skin-liketissues, probably corresponding to pseudosclerotialplates .

It would seem then that the dark zones represen-ted sites where separate mycelia of decay fungi hadfailed to merge, producing pseudosclerotial tissuesinstead, and leaving a zone of relatively undecayedwood in which the dematiaceous hyphomycetescould grow freely. It should be stated that theantagonism observed between separate isolates ofdecay fungi could not be directly explained interms of incompatibility between monokaryons,since all those examined contained hyphae withclamp connexions.

Trans. Br, mycol. Soc. 67 (3), (1976). Printed in Great Britain