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WORKSHOP COURSE ON AQUATIC HYPHOMYCETESNovember 1976

A.R. Perry

About 20 delegates flocked to Exeter to join Professor J. Webster and ProfessorC.T. Ingold in the Department of Biology of the University for their WorkshopCourse on Aquatic Hyphomycetes. Participants came from such distant places asLagos and Ontario; perhaps Professor Ingold's recent excursions into Hyphorny-cete cartoonery (see for example Bull. Brit. Myc. Soc. 8, 32 (1974); 10, 80(1976); Biol. J. Linn. Soc. 7,22 (1975) are the reason for this.

By way of an introduction, on Friday night Professor Ingold gave a talk onthe "Identification of Aquatic Hyphomycetes" and Professor Webster one en-titled "Techniques for studying Aquatic Hyphomycetes".

Aquatic hyphomycetes grow on all sorts of submerged decaying leaves ofbroadleaved trees where the aeration is good, very rarely on those of conifers.The fungus can be identified by its conidia, but more easily by examining thegrowths on the leaf tissue under the low power of a microscope where they willbe seen as little forests often consisting of 4-5 different types growing together.In many species the conidium consists of four long arms diverging from a commonpoint. This so-called tetraradiate conidium is in some species attached to theconidiophore near the point of divergence of the four arms, or in others by thetip of one of the arms. When mature the conidium separates and drifts awayfrom the conidiophore. Any new conidium now to be produced arises from adifferent level of the conidiophore.

A quick method of sampling the conidia in a stream is by examining foamwhich develops on the water surface. It is well known that conidia are readilycaptured by air bubbles which cling together and act as a trap for them.

Most aquatic hyphornycetes can be isolated easily but bacterial contaminationshould be minimized. Submerged leaves are collected and placed in water inpetri dishes (preferably glass) - one leaf to a dish - and incubated at 10-15 0 Cfor 2 days. Using a good low power binocular microscope and a finely drawnPasteur pipette conidia are collected from the culture and transferred to filtered0.1 % malt extract agar (or tap water agar) containing 0.1 % Crystamycin in apetri dish. Transference of the conidia to the agar is facilitated by having previouslydrawn a grid pattern on the back of the petri dish with a glass marker; under themicroscope the grid is observed through the agar and each compartment isinoculated. The lid is replaced and the dish turned upside down and again usingthe microscope the conidia are brought into focus through the agar - hence theneed to use filtered agar. Those compartments containing" only one conidium are"marked then the whole is incubated and the conidia allowed to grow clear of thedrop. The tips of those hyphae arising from single-conidia compartments arecut out and used for re-inoculation. Some conidia get caught up in the meniscusof the water when the leaf is floated out. These can be scooped up on the end ofa fine sewing needle and inoculated onto petri dishes as before. Conidia mayal-so be isolated from foam. This is smeared in the field on to pre-poured agar.Back in the laboratory a special cutter mounted as an objective on the micro-scope may be used to remove any germinating conidium for culturing.

The medium may have to be changed in order to make the fungus sporulate.Forced aeration often does the trick. Strips from 3-4 week-old cultures on agarare put into water in a bottle and vigorously aerated with a stream of sterilecompressed air either through a sintered glass filter or a hypodermic syringe.Spores can be gathered at the tide-line and sub-cultured.

A millipore filter with an 8J.lm pore size may be used in the field to demonstrate

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the concentration of conidia. Water samples are collected and immediately filter-ed using a Buchner Flask and a modified bicycle tyre inflator with the pistonwasher reversed so that a partial vacuum can be created in the flask. A few dropsof cotton blue in lactic acid are added to the filter in the field. Back in the lab.the filter is heated at 60°C for I hour to make it transparent, then conidia onthe filter may be counted under the microscope and by simple arithmetic theirconcentration in the original water sample worked out.

The following day dawned fine for our field excursion. The first stop was atthe River Teign at Steps Bridge near Dunsford (SX 805882). Here foam sampleswere taken and various species of partially decomposed leaves were collectedinto polythene bags from the river to be taken back to the lab. and examinedfor conidia. A sample of water was filtered through a millipore filter using thetechnique previously described. Later on, back in the lab., the concentration ofconidia in the river was worked out to be 33,739 conidia per litre of water: therewas no shortage of material! Coffee was taken at the nearby Steps Bridge Cafe,then we were taken to our second destination, Becky Falls near Manaton(SX 758800). Here more specimens were collected from Becka Brook;a streamflowing through deciduous woodland. On its way to Becka Falls the coach be-came entangled with the vehicular followers of a fox hunt among the narrowcountry lanes. Whilst it was being extricated a few of us were privileged toglimpse the uneatable loping off in the opposite direction from the unspeakable.After Becky the coach took us to Hay Tor and we walked up to the Rocks toenjoy the view and our lunch in glorious sunshine. Back in Exeter that afternoonconidia were identified and techniques tried out. Species collected during the daywere as follows (B =Becky Falls; S =Steps Bridge):Alatospora acuminata BSAnguillospora crassa BS; longissima BArticulospora tetracladia BSCentrospora acerina BClavariopsis aquatica BSClavatospora longibrachiata BS; stellata BSCulicidospora aquatica B; gravida BDactylella aquatica BSDendrospora erecta BSFlagellospora curvula BSHeliscus lugdunensis BSLateriramulosa uni-inflata BLemonniera filiforme B; terrestris BSMargaritispora aquatica BSTetrachaetum elegans BSTetracladium marchalianum BS; setigerum BTricladium chaetocladium B; eccentricum B; patulum BS; splendens BSVaricosporum elodeae BVolucrispora graminea B

After dinner Mr E. Descals gave a talk on "Perfect States of Aquatic Hypho-mycetes", outlining the present knowledge of aquatic hyphomycetes and theirperfect states which are such diverse genera as Nectria, Massarina and Lepta-sporomyces. Much of the work reported had been done in Professor Webster'sdepartment and some was not yet published. Afterwards we were treated to ashowing of their film on spore development in aquatic hyphomycetes.

The following morning Mr P.I. Fisher spoke on "Aero-aquatic Hyphomycetes"!These fungi grow particularly well on submerged decaying leaves under semi-aerobic conditions which tend to make the leaves black. Leaves are collectedfrom the lower layers of submerged litter - especially under still water as in

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ponds - using a rake. They are washed and each is placed in a petri dish with amoist filter paper. All surplus water is drained off and the plates are incubatedat 10-IS°C. Spores eventually appear on the surface and may be plucked offwith a needle and plated out onto 0.1 % malt agar.

These fungi may also be cultivated in a leaf decoction (15g living leaves in1 litre water, boiled, filtered) in a flask then inoculated onto quartz sand inpetri dishes.

Superb living material and photographs were available for study. After lunchwhich most of us took in a local pub we re-assembled to cram in yet again asmuch information as was available until it was time to leave.

I left Exeter with a feeling of great achievement, and my memory of theweekend is one of excitement. This was without doubt engendered by theenthusiasm of Professor Webster and his colleagues, and of Professor Ingold.I thank them sincerely for a weekend well spent.

ON THE EXCEPTIONAL ABUNDANCE OF VERPA CONICAWITH NOTES ON UNUSUAL OCCURRENCE OF OTHER FUNGI

Derek Reid

Verpa is a genus of Discomycetes belonging in the Morchellaceae, which includesthose fungi popularly known as "morels". The fruit-bodies are characterized byhaving a campanulate or cylindric, smooth or at most slightly and irregularlylongitudinally wrinkled, olive-brown or dark brown fertile head, which is pendu-lous from the apex of a whitish or cream-coloured hollow stalk. The head,between 2.5-4.0 em tall, is never ornamented with deep polygonal pits and raisedribs as in Morchella or Mitrophora, The smooth stalk, up to .13 cm high and 1 ernthick, is covered with closely crowded horizontal bands of brown granules. Theascospores are elliptical, 20-24 x 12-14p.m, and lack internal oil drops but have anumber of minute granules adhering at either end.

Specific distinctions are difficult. Dennis (1968) in British Ascomycetes writ-ing of Verpa conica Swartz ex Pers. noted "Specimens with the fertile head morecylindrical and fitting closely round the apex of the stalk are often distinguishedas V. digitaliformis Pers.", The latter are often more robust.

One of the difficulties in deciding how many species are involved, is that thesefungi occur so rarely, and then either singly or in twos or threes. It is seldompossible to make comparison between ample collections during the same shortseason. However Dr Philip Jones of Llanelli has had the good fortune to collectboth forms this spring and is convinced that they represent two species. He writesthat in specimens referable to V. conica the apex of the stipe is rounded like acone with the campanulate flared cap having a narrow attachment. In contrastall the 10-12 specimens referable to V. digitaliformis had stipes which were muchmore robust with a cylindrical apex and caps which were more or less parallelsided resembling inverted flower-pots. Dr Jones's illustration showing these dis-tinctions is reproduced below.

An unusual variant was received from Cornwall in which the cap, about3 em diam., had exactly the form of Gyromitra esculenta (Pers.) Fr., borne on ashort stalk 2 em high. This has been referred to V. krombholzii var, morchelloidesGrelet, but again is probably only an extreme variant of V. conica.

For the purposes of this paper all records are treated under Verpa conica.