Ingoldian Fungi: sorne field and laboratory techniques Enrie DESCALS SHNB Descals, E. 1997. Ingoldian Fungi: some lield and laboratory techniques. BolI. Soco Hist. Nat. Balears, 40: 169-221. ISSN 0212. Palma de Mallorca. So me preparatory techniques lor the taxonomic, chorological and preliminary ecological study 01 Ingoldian lungi (i.e. lungi with conidial shapes adapted lor anchorage in running waters) are discussed and detailed step by step; suggestions are also made lor their improvement. Three introductory topics concerning lield collection are lirst discussed, i.e.: criteria lor selecting sites and timing sampling procedures, the recording 01 complementary lield data and the planning 01 lield trips involving long-distance air trave!. The two main sections cover preliminary studies on species richness (by mean s 01 loa m surveys), and preparatory techniques lor description and herbarium preservation. The core 01 this contribution SOCIETAT O'HISTORIA discusses pure culture: 1- isolation techniques, 2- recording colony characters, and NATURAL DE LES BALEARS 3- sporulation induction techniques. Isolation starts with asexual or sexual spores, either concentrated in stream loa m or produced on natural substrates, Isolation may be done in two basic ways depending on spore size and abundance: 1- very large spores occurring inlrequently are detected and identilied under the dissecting microscope and lifted with the aid 01 mounted hairs or micropipettes; 2- spores 01 any size may be spread on an antibiotic medium, allowed to adhere to agar-based ¡solation media, identilied under a compound microscope, relocated under the dissecting scioe with the aid 01 a linder slide and lifted manually. Anamorph induction is in contact with Iree water, i.e. in/on unchanged water (either standing, aerated or agitated), or in changed water (periodically or continuously) Alternatively, anamorph induction may be by means 01 moist incubation. Herbarium preservation relies mainly on microscope preparations, complemented by the use 01 preserved mycelial portions as well as 01 dried cultures. Keywords: Ingoldian Fungi, field techniques, /aboratory techniques. FONGS INGOLDIANS: ALGUNES TÉCNIQUES DE CAMP I LABORATOR!. Algunes tecniques preparataries per a I'estudi taxonamic, corolagic i ecolagic deis longs ingoldians (Iongs amb formes conidials adaptad es a I'ancoratje en aigües corrents) són detallades i se suggereixen possibles millores d'aquestes. Se comenten tres temes introductoris: criteris per a la selecció de localitats i temporades de recol.lecció, el registre de dades de camp complementaries i la planilicació de campanyes de recol.lecció incloent vols a IIarga distancia. Les dues seccions principals cobreixen I'estudi preliminar de la riquesa especílica en aigües naturals (exploració a base d'escumes), i tecniques preparataries per a la descripció en cultiu pur i herborització. Se discuteixen tres aspectes fonamentals del cultiu pur: 1-tecniques d'a'¡'IIament, 2- caracterització de colanies, 3- tecniques d'inducció de I'esporulació. L'a'illament se fa a partir de espores asexual s o sexuals, ja estiguin concentrades en escumes de rius o produ'ldes sobre substrats naturals. L'a'lllament és de dos tipus depenent de la mida i abundancia de les espores: 1- les espores de gran envergadura i poc Ireqüents són localitzades i identificades sota lupa binocular i a'lllades manualment amb I'ajuda de cabells emmangats o micropipetes; i 2- les espores de qualsevol mida, un cop espargides sobre un medi antibiatic, són incubades per induir la seva adherencia sobre el medi; després són localitzades i identilicades sota el microscopi compost, i tot seguit són relocalitzades sota lupa amb I'ajuda d'un porta localitzador i a'lllades manualment. La inducció de I'anamorl se la posant en contacte el miceli amb aigua, és a dir: en aigua estanca, amb o sense aireació o agitació, o bé amb canvi (periadic o continu) de I'aigua. Alternativament se poden fer incubacions humides, 170 BolI. SOCo Hist. Nat. Balears, 40 (1997) L'herborització se basa en: 1- col.leccions de preparacions microscopiques, suplementades amb porcions de miceli esporulant guardades en conservant; i 2- colonies en cultiu pur dissecades. Paraules elau: fongs ingoldians, tecniques de camp, tecniques de laboratori. Enric DESCALS, Institut Mediterrani d'Estudis Avan9ats (CSIC-UIB), Edifici Mateu Orfi/a, Univ. lIIes Balears, 07071 Palma de Mal/orca, Spain. Recepció del manuscrit: 17-oct-96; revisió acceptada: 6-nov-97. INDEX 1- Introduction .......................................................................................................... 170 A- Criteria lor selecting and timing sampling procedures ........................... 171 B- Complementary lield data ........................................................................... 173 C- Suggestions lor lield trips involving long-distance air travel ................. 173 11- Preliminary lield studies on species richness: loam surveys ..................... 174 111- Preparatory techniques lor lungal identilication, description and preservation ................................................................................................ 177 A- Studying lield material ................................................................................. 177 B- Laboratory sporulation 01 lungi on lield material .................................... 177 C- Pure culture studies ..................................................................................... 179 General isolation media ................................................................................ 182 Instruments lor microtechnique .................................................................... 184 Spore isolation techniques ............................................................................ 184 -Isolation 01 suspended spores ................................................................ 186 -Isolation 01 spores anchored on agar ................................................... 193 Isolating spores Irom teleomorphs on natural substrates ........................ 199 D- Working in the main laboratory Basic lacilities and equipment ..................................................................... 203 Pure culture: 1- The vegetative phase ....................................................... 207 2- Anamorph induction .......................................................... 208 A- Standing water .......................................................... 209 B- Aerated water ............................................................ 212 Concentrating spores in water ......................................................................... 214 IV- Herbarium preservation ................................................................................... 216 1- Introduction The Ingoldian fungi (Descals, 1978), el ose to streams (Iess so in lentic habi inaccurately referred to as the "aquatic tats). Some may also colonize non hyphomycetes" (Ingold, 1942), are a aquatic habitats (forest litter, plant cano loose assemblage of fungi bearing rela pies, etc ... ) and reproduce here as, tively large, modified conidia found in anamorphs and/or teleomorphs. Most are continental waters and humid habi known to be saprotrophic, but some may tats. Such conidia are known to occur be plant parasites. Others may behave especially in the hyphomycetes, but can as endophytes in wood (Fisher & Petrini, also belong to ascomycetes, basidio 1989). The Ingoldian fungi are thus not mycetes, coelomycetes and even am a taxon nor an ecological group, but phibious Entomophthorales (although the they do have in common what is be latter will be left out of this contribution lieved to be a morphological adapta because they are insect parasites and tion of their conidia to colony esta require different techniques for their blishment in fast-running waters study). Ingoldian fungi typically live in or (Webster, 1959). E. Desea/s., /ngo/dian Fungi: fie/d and /aboratory teehniques 171 Conidial morphology will in most number of mycologists are recording and cases allow us to use a short-cut ap isolating them worldwide due to their proach to the study of these fungi, Le.: relevance to stream ecology and, lately, instead of identifying fungi after blind for their possible pharmaceutical applica isolation and culture of large numbers tions. of them (as is generally done for exam Techniques for their isolation, pure pie with soil and other aquatic fUr1gi), culture, in vitro conidiation and preserva Ingoldian conidia are typically species tion are often unique to the group, but diagnostic, Le.: pure culture is needed have so far not been compiled in detail. only in some cases for confirmation of The main purpose here is a description identification to species. Selective iso of techniques which have been tested lation of conidia recognizable in many for a number of years and which may cases to species greatly simplifies pure prove useful to other workers in the culture work, and the implications are field. Many of those techniques have discussed below. been learned or developed in Prof. J. By simply collecting and observing Webster's laboratories at the University conidia in water or foam, or from sub of Exeter (UK). A second aim is to high merged substrates, one can perform light still unsatisfactory techniques and to chorological and ecological studies on suggest means for improving or replac the Ingoldian component in stream com ing them. munities (Barlocher, 1992), Le.: 1- It must be emphasized that many Seasonality of sporulation can be es of the techniques discussed below are timated by identifying conidia trapped in also applicable to hundreds of other foam or in water. 2- A number of eco aquatic as well as terrestrial fungi pro logical parameters can be correlated ducing species-diagnostic conidia. with conidial numbers in stream water. 3- Saprotrophic relationships between Ingoldian fungi and their substrates (e. A-CRITERIA FOR SELECTING ANO g. substrate preference and decomposi TIMING SAMPLlNG PROCEOURES tion rates) can be determined by record ing sporulation levels on the substrates Current aspects of interest in the after controlled submersion (Le. baiting) study of the Ingoldian fungi are: 1- de followed by in vitro induction of scription and classification of anamorphs, conidiation. 4- Fungus-plant-invertebrate 2- studies on life cycles and teleomorph interactions of various kinds may also connections, 3- preliminary studies on be analyzed and quantified at the spe species richness, biogeography and con cies level without culture (e.g.: servation, 4- ecology (substrate decom Suberkropp et a/., 1983). (Some of the position and invertebrate relationships) under 3 and 4 studies, however, may and 5- pharmaceutical and other indus presume a not fully proven direct corre trial uses. lation between conidial numbers and mycelial abundance or activity). 1- Oescription and classification The study of the Ingoldian fungi is of anamorphs: Hundreds of Ingoldian becoming increasingly popular since their anamorphs await description, and a discovery by Ingold (1942). There are number of the close to 300 known ones now well over a thousand publications need redescription because morphologi on these fungi, and a steadily growing cal characters were not properly applied 172 BoII. SOCo Híst. Nat. Balears, 40 (1997) in the past, because new characters object of study in order to justify their based on ontogeny or even .molecular preservation. aspects are being introduced, or be If conidial abundance and species cause herbarium material is scanty, diversity are the aims for studying any of poorly preserved or altogether missing. the three aspects discussed aboye, the In some temperate streams, up to ideal eollecting sites are small streams 80 or more taxa may be collected in a flowing over rocky beds (in mountainous few drops of foam (e.g. Regelsberger et or hilly areas), with foam accumulation, al., 1987). Habitats in warm elimates, upstream from any source of organic however, have been much less studied. urban and agricultural waste. The catch Foam is easier to find in soft or neutral ment area should have a rich and var to mildly acid waters, and possibly for ied, undisturbed native vegetation. In this reason these have been more inten temperate climates, deciduous angios sively explored than hard, alkaline or permous trees are richer sources than saline streams. But the latter may bear conifers, although these usually bear different mycotas. Undisturbed habitats characteristic mycota. A good clue to rich in riparian plant species yield more adequate collecting are as is the pres Ingoldian fungi; but more extreme habi ence of trout and salmon angling sites tats, often with low plant species diver along streams. sity, may support a significant number of Conidial abundance is normally as undescribed fungi. Such is the case with sociated with availability of decaying sub acid moorlands. merged substrates, such as fallen leaves, and therefore the most produc 2- Lite eycles: Only about 27 tive collecting season in streams flowing teleomorphs are known among the through deciduous woods in cold and Ingoldian fungi (Webster 1992, Descals temperate climates tends to be in the ined.), but it is believed that more will autumn, although minor peaks may oc be found if we concentrate on isolating cur at other times. Very few critical from sexual spores. For this purpose, seasonality studies have been carried one should search for streamside habi out in streams flowing exclusively tats with abundant and varied long-Iast through conifers or in tropical latitudes, ing woody substrates (and possibly where leaf fall patterns are less marked. sometimes decomposing leaves) where Casual observations suggest that the teleomorphs can complete their de conidial numbers in stream foams rise velopment. This takes place in most drastically after heavy showers, as much cases out of water. In cold and temper riverbank litter (and to a lesser degree ate climates, teleomorphs seem to be grassland) is also colonized by Ingoldian more abundant in the warmer seasons. fungi (Webster, 1977). Their conidia may be produced aerially but dispersed in 3- Biodiversity and conservation: flood waters. These are hardly explored fields, but Ingoldian fungi may eventually have an 4- Ecology: The relevance of indicator value for monitoring the ef higher fungi capable of underwater fects of Mankind and/or of environmental substrate colonization (among which are (e.g. climatic) changes on freshwater the Ingoldian fungi) and as intermediar habitats. Either endangered or well-pre ies in energy and food webs associated served habitats could be the chosen with running waters is well documented E. Desea/s, /ngo/dian Fungi: fie/d and /aboratory teehniques 173 (Barlocher, 1992). For such studies, the Other valuable information may be choice of site is subject to selected ex obtained from land survey maps (1: perimental criteria rather than those 25,000 to 1: 50,000, and including vege based on fungal species abundance or tation types) as well as from yearly pub variety. lic records kept for larger river basins, 11 the goal is to discover new plant such as patterns of rainfall, water tempe substrates for particular species one rature, chemical parameters and flow should obviously collect in first-order rateo streams, not far from the source of the conidia. c- SUGGESTIONS FOR FIELD TRIPS INVOL VING LONG-DIST ANCE AIR 5- Pharmaceutical and other in TRAVEL dustrial uses: Metabolites produced by the Ingoldian fungi are being studied at Due to the specialized equipment the present by the industry, mainly be needed, which is often not available at cause these fungi are still relatively un destination, long-distance air travel known. By now, most known species presents special problems for mycolo from temperate habitats have been gists wanting to isolate Ingoldian fungi. screened. However, large culture collec What little experience has been gained tions are not often maintained; and, as by the author from such trips may be of techniques and target substances in the use to some readers. search of bioactive compounds keep Ideally one should plan cooperative changing, it is likely that well-known work with a local mycologist, who may sites will have to be repeatedly tapped have much to contribute; e.g.: suggest in the future. The choice of sites here ing collecting sites, arranging for trans depends on the specific needs of the port and accomodation, translating, pro industry. viding necessary basic gear, etc. If time allows, such trips are also an excellent B- COMPLEMENTARV FIELD DATA opportunity for training local postgraduate students. These are geographical, clima The equipment and material must tological, vegetational and physio-chemi be carefully selected and packed. If time cal parameters (Table 1) which should is a limiting factor, or if working condi be recorded for: 1- a more complete tions are inadequate, one should aim for characterization of fungal species, and 2- self-sufficiency. Media, for example, a better understanding of the environ should be prepared prior to travelling. mental conditions needed for in vitro -Hand luggage should be reserved reproduction. for the more delicate and expensive Table 1. Complementary field data. Taula 1. Dades de camp complementa ríes. water temperature riparian vegetation on site " conductivity " " upstream " pH altitude flow characteristics latitude rock type longitude " pH name of stream 174 BolI. Soco Hist. Nat. Balears, 40 (1997) compound and dissecting microscope may serew a foam jaro The ring may equipment (e.g. nosepieces, objeetives, just be a perforated lid of a same-sized condenser lenses, eyepieces, filters), jaro bulbs and cameras, as well as for cul -An inflatable boat may be handy tures on the return trip. for collecting foam in larger bodies of -Flammable items such as alcohol, water. propane cartridges, etc., are not usually -Fixative for foam. A few drops of allowed on airplanes. 4% formalin or 90% methyl alcohol ap -Agar media should not be ex pear to be adequate for short-term pres posed to freezing. Large, modern ervation, and formalin-acetic-alcohol airplanes normally have temperature-con (FAA, Anon. 1968) may be used if stor trolled luggage compartments, and age is for longer periods. The fixative poured agar media can be packed with should not affect later treatments in slide the check-in luggage. But this may not preparation. be so in smaller aireraft on inland flights. -Waders: thigh-high gum boots. A -For check-in luggage, can vas repair kit for punctures should be kept travelbags with an additional thick, poly handy. styrene foam lining should be more -Rucksack with general purpose shock-proof than hard-walled, Samsonite gear (see below). type suitcases. Water tightness is a fur -A eouple of buckets. ther aspeet to considero -A container (e.g. a large tin can), with the bottom cut off. 11- PRELlMINARV STUDIES ON SPE -Field notebook. CIES RICHNESS: FOAM SURVEVS PROCEDURE FOR FOAM SAMPLlNG An idea of species richness of -Label jars with a felt pen befo re waterborne fungi may be obtained by wetting. examining natural foams, although the -Look for elean, thiek scum below technique is not quantitative, as waterfalls, along rapids, on the down Ingoldian conidia are trapped with higher stream side of any obstacles (e.g. boul efficiency than others (Webster, 1959; ders and woodpiles), along lakeshores Iqbal & Webster, 1973a) and information where drift collects through wave and on foam trapping dynamics is lacking. wind aetion, ete. Foam surveys fulti! an added useful Foam that breaks down quiekly in purpose: selecting sites and seasons the jar will have trapped few eonidia. for later isolation of interesting species. Muddy scums harbour too many bacte rial eontaminants and debris. RECOMMENDED GEAR -Scoop up the foam with the jar -Foam jars: wide-mouthed (ea. 5 itself or with the lid or spoon, and IM cm diam.) screw-capped jars. Plastic is MEDIATELV DECANT ALL EXCESS preferred for lightness and strength, but WATER, as the conidial coneentration in conidia may adhere less to glass. water is mueh lower than in foam and -A spoon or kitehen skimmer. this will have a strong diluting effeet. -Extensible rod for foam jars: foam Foam is often scaree (especially is sometimes in awkward spots out of when submerged leaves are not abun arm's reach. It is relatively easy to fit a dant) and several subsamples per site ring at the end of a rod, onto which one may be needed. Some of the foam will E. Descals, Ingoldian Fungi: field and laboratory techniques 175 have liquefied between scoops, and The use of phenol is discouraged when decanting, spores in this water by safety regulations in some countries from previous scoops will be lost. There as it is carcinogenic, but contaminant fore use a fresh jar for every scoop fungi seem to be able to grow in lactic and compound the samples after the acid within microscope preparations, and last scoop for the site. a general biocide will have to be added. For collecting foam along lake Cotton blue is claimed to often shores, where it is often scanty and result in serious crystal precipitation. trapped among pebbles and vegetation, The synthetic resins DPX 8711 one may slightly dig an open-ended can (Difco) or Merckoglas (Merck code 11-20 into the ground and then pour some UN-1866/3.2 IMDG WGK2) have not lake water into the can. This will raise proven satisfactory in recent trials, as the water level long enough for the foam the spores are severely distorted and to be collected. The use of an inverted standard stains are not readily mixed. funnel may even help concentrate the (Although a stain may not be needed for foam within. phase or DIC optics). PVA is currently being used in certain laboratories tor -Do not Iiquefy the foam (by shak mounting myxomycete and other spores ing the jar) if it is meant for spore iso (Pando, pers. comm.), and maybe lation. worth trying. When you have returned to the -Sealant: vehicle: Semipermanent liquid mounts need -Fix sorne foam in a labelled vial to be sealed, but there is so far no as a voucher specimen. satisfactory sealant in the market. The -Note the collection number and commercial resin "Glyceel", which had complementary field data in the field become of widespread use (but no notebook. longer produced) will eventually shrink, and cracks appear precisely at the mar PREPARING SPORE DEPOSITS ON gin of the coverslip, allowing the evapo MICROSCOPE SUDES ration of the mountant. The preparation will then need repairing, which is time MATERIALS consuming and often damages the con -Mountant: conc. lactic acid with tents. To correct this, Gams (ined., sorne acid fuchsin (with or without phe Ananet Newsletters 10:3, 17:7, accessi nol) are of standard use. However, semi ble through Internet) suggests applying a permanent mountants may cause a sig . second layer of Glyceel soon after the nificant loss of optical resolution. This is first. But if this is done too late, it will especially noticeable with differential in set unevenly and wrinkle severely. terference contrast(DIC) optics. It is It isclaimed that more .elastic nail therefore preferable to do all the de polish brands containing nylon are more scriptive work from temporary water satisfactory. mounts, and only add chemicals after Volkmann-Kohlmeyer & Kohlmeyer wards for preservation. On the other (1996, and in /itt.) recommend the hand, if one is doing large batches time preparation of permanent slides by is short, and there may be no alternative means of a "double-coverglass" tech but to first fix the specimens. nique, which might be adapted for lngoldian fungi. 176 BoII. Soco Hist. Nat. Balears, 40 (1997) -Large coverslips (22x22, or 20x20 the dropper because this might get con mm), grease-free slides in slide boxes taminated and transfer the spores to and slide labels. slides with other samples. -Invert the coverslip and, to avoid PROCEDURE FOR PREPARING trapping air, rest one side on the SPORE DEPOSITS preparation with the aid of a needle and slowly lower over the dried spot. -Air-dry 3-4 drops of foam placed -Heat gently over the Bunsen on the centre of a slide. This may be burner and, if necessary, apply slight done in the laboratory, where gentle pressure with a needle to release heat may be applied for faster drying (e. trapped gas bubbles. g. under atable lamp or with a fan -Seal and label the slide, noting heater or hot plate). down the collection code, date and -Drying a known volume of lique mountant used. The latter record will be fied foam on each slide may allow the useful for reflooding if the slide needs information to be at least partially com repairing. parable with other foam samples. -If the stream water is alkaline or LlQUEFYING FOAM saline, salt crystallization will take If an even spore concentration is place, and when an acid mountant is needed in the foam, for example for added (e.g. lactic acid), alkaline deposits quantitative studies, or for spreading will release abundant CO under the conidia on agar media for isolation, li 2 coverslip, spoiling the preparation. quefy it by: In this case, add a drop of the -Shaking the closed foam jar vio mountant to the deposit and heat gently lently for a few times. to release the gas befo re covering. AI Shaking is not always effective, ternatively, the water in the spore sus especially with some very thick scums. pension may be acidified prior to mount Gentle heat (applied close to the foam, ing. Another option may be to test for example with a hot metal) is effective Waterman's ink diluted to approx. 1/10 but it may be detrimental to spore viabil (an aqueous solution used by Ii ity. Applying alcohol, or freezing, as well chenologists for observing asci, and suc as anti-foaming agents, may be worth cessfullY used by us for staining conidia testing. on membrane filters). This would not have an acid reaction. The preparation ARTIFICIAL FOAM would not be permanent, however. If stream foam cannot be readily -For a very thin preparation, and found, it may be obtained artificially by al so to reduce gas release, remove the reducing the surface tension of stream larger debris (sand grains, organic water with commercial detergents or matter, insect parts, etc.) with a needle wetting agents (e.g. Tween-80), resulting or forceps under the dissecting micro in foam production. A technique involving scope prior to applying the coverslip. digging up a small reservoir under a -Add a small drop of mountant to waterfall is detailed in Iqbal (1983; a coverslip. 1995). The drop is not placed on the slide An alternative (but only briefly to avoid touching any fungal material tested) way of concentrating spores in (including spores in foam) with the tip of artificial foam would be the following: E. Desea/s, /ngo/dían Fungí: fíe/d and /aboratory techníques 177 Sample a known volume of stream wa ately after sampling, comparing fungal ter in a bucket, add a drop of di/. morphology with that produced in the Tween-80 to a second bucket, pour the laboratory, and, if differences are signifi water into this from a certain height (to cant, illustrating and preparing voucher create turbulence and hence foam), and specimens. then back into the first. Do this several Conidiophores are normal/y pro times with the same water, scooping up duced over the entire surface of leaves the foam as it builds up and placing it or wood, but these are usual/y opaque, in ajar, where it may then be proc and leaf clearing techniques, for exam essed (either for isolation or for preser pie, are too severe on such delicate vation). fungal material. Conidiophores must then If the number of conidia of specific be seen along the leaf margins and on Ingoldian species trapped in the artificial the sides of woody tissues and petioles, foam in relation to that in the sample of or by examining scrapings mounted on stream water is proven to be constant, slides. (Preparation of free conidia, e.g. the aboye technique might al/ow for in foam, has already been discussed). quantification. There is a need for developing an Preliminary trials carried out in this efficient, preferably quantitative micro laboratory by A. Díaz (unpub/.) on technique for sampling conidiophores conidia from pure culture suggest that from natural substrates. Col/odion peels Tween- 80 does not affect viability. have proven successful with plant mate rial and are worth testing. /11- PREPARATORV TECHNIQUES FOR FUNGAl IOENTIFICATION, OESCRIP /l1-B: lABORATORV SPORUlATION OF TION ANO PRESERVATION FUNGI ON FIElO MATERIAL The source of the material to be -Rinse field material in abundant studied may be: a- fungi natural/y colo water. nizing and sporulating on the substrates, Tapwater wil/ have fungal contami either as conidiogenous structures or as nants (occasional/y even Ingoldian free conidia in water or foam; b- the conidia) but may be used for short same fungi, but after having induced incubations if the aim is induction of their sporulation in the laboratory; c conidiogenous structures. fungi grown on artificial media but in The basic steps for submerged in duced to sporulate either in the field cubation are explained under anamorph (never done) or under control/ed condi induction below. tions in the laboratory. So far, natural substrates have been submerged in standing or aerated, /l1-A: STUOVING FIElO MATERIAL often unchanged distil/ed water (DW). These conditions may not be ideal, as Especial/y when describing new there is presumably a buildup of staling species, one should test that the mor compounds interrupting or distorting phology of conidiogenous structures and sporulation processes. More natural con of free conidia produced in the labora tinuous water flow or drip systems such tory (cases 2 and 3 aboye) is no! differ as those applied to pure culture (see ent from that in nature. This may be below) are now being tested. done by fixing field material immedi- 178 Boll. Soco Hist. Nat. Balears, 40 (1997) Incubation in water contain ing antibacterial antibiotic solu tions has not been tested, but may improve sporulation. -Cool temperatures around 15°C for cold-temperate species should be used to slow down bacterial growth. -Incubating in glass Petri dishes may reduce the number of conidia adhering to the dish walls, but does not allow for near- ultraviolet (NUV) irradiation, unless the glass is Pyrex. The effect of NUV on fun Fig. 1. Harvesting surface spores from moist-incubated gal sporulation on natural substrates: the container is slowly flooded wlth dlstllled substrates has not been critically water and the water surface touched with a broad tested. loop, 'which may then be spread on a slide or on -Check for sporulation alter antibiotic agar. . one day, and then every 2-3 Fig. 1. RecoUecció d'espores superficials a partir de substrats incubats amb humitat; /'aigua destd.lada es days for at least 10 days. deixa entrar en el recipient lentament i per la The maximum time for incu superfície de /'aigua es passa una ansa de sembr~ bation to allow all resident spe ampla que després es frega sobre una preparaclO cies to sporulate under the d'agar antibiótico aboye conditions is not known, but certainly two days, as re ported in many publications, may not be Field material (as well as pure cul sufficient. tures) should always be subjected to moist incubation too, as not all AERATED WATER: Ingoldian fungi are aquatic sporulators -Place substrate in a glass con (Fig. 1). tainer, e.g. a measuring cylinder or coni cal flask. 1- STANDING WATER in Petri dishes: -Add DW without filling up (as tur -Use a low substrate/water ratio bulence may cause some spilling). because of rapid build-up of bacterial -Force air into the system, to just and yeast populations, as well as of create gentle mixing of the water. An staling compounds from both fungi and aquarium pump system such as de bacteria. For example, place one small scribed under pure culture, but ignoring leaf, or portions of a larger one, in a 9 precautions for sterile technique, is suit cm diam. Petri dish containing 30 mi able (Fig. 2). water. Excessive bubbling will thrust -Filtered stream water may in spores out of the suspension and these some cases induce more sporulation will be lost into the air or remain tapped than DW, and should be tested. on the walls. -Changing the water regularly -Incubate and check for sporulation should increase spore production and/or as for standing water. lengthen the sporulation phase.