ARBUSCULAR MYCORRHIZAL FUNGI - TAXONOMY The fungi which form arbuscular mycorrhizas with plants are all members of the Zygomycete order Glomales. These fungi may colonise roots by: 1. hyphal growth from the soil mycelium or fragments of it; 2. growth from colonised roots; 3. germination of spores. Glomalean spores are typically large (20-)0-500(-800) jum in diameter and multinucleate. A sexual reproductive phase has never been seen Using ontogeny and structure, glomalean spores are classified thus (Morton & Benny, 1990; Morton & Redecker, 2001; see the INVAM website ) KINGDOM Fungi DIVISION Zygomycotina ORDER Glomales SUB-ORDER Glomineae NEW in 2000 FAMILY Paraglomaceae GENUS Paraglomus (2) NEW in 2000 FAMILY Archaeosporaceae GENUS Archaeospora (3) FAMILY Glomaceae GENUS Glomus (89+ incl, Sclerocyctis) FAMILY Acaulosporaceae GENUS Acaulospora (34+) GENUS Entrophospora (4) SUB-ORDER Gigasporinae FAMILY Gigasporaceae GENUS Gigaspora (8+) GENUS Scutellospora (24+)The taxonomy of the Glomales is relatively new and, therefore, incomplete. Not only are there probably many taxa yet to be described, but molecular techniques are revealing that the group may bbe much more diverse than was previously thought. It has proved impossible to classify arbuscular mycorrhizas to species using features of fungi within colonised roots, although some features may indicate to which genus an endophyte may belong. Therefore, classification is normally based upon the morphological characters of spores, and association with the morphology of their mycorrhizas is only possible in pure culture. Because the taxonomy of the glomales is; relatively speaking, in its infancy, there is no satisfactory key to glomalean spores, and the reader is directed towards original descriptions in journals such as Mycotaxon, and to the taxonomists (e.g. Joe Morton, Chris Walker) themselves who will help identify and advise, upon presentation of voucher material Both INVAM (International Collection of Vesicular-Arbuscular Mycorrhizal Fungi) and BEG (Banque Européenne des Glomales) are pleased to receive material ifit is presented in an acceptable form (see Useful Addresses). Generally speaking, the most useful specimens are living pure cultures from which the recipients may produce their own cultures, as well as herbarium material in the form of microscope slides and spores in preservative. The last is rarely 100% satisfactory because microscopic features of taxonomic importance gradually change in all preservatives used to date. Extreme caution should be exercised before naming a spore, spore assemblage or culture. We recommend that the taxonomists at INVAM or BEG should be consulted whenever an arbuscular mycorthiza is to be described EXTRACTION OF GLOMALEAN FUNGAL SPORES FROM SOIL Soils generally contain a large proportion of organic matter, sufficient of which floats to require a particular method of separating spores from the bulk of the soil in which they are being sampled (Sucrose floatation). The quick method detailed below is often highly successfully applied to carefully designed, ‘clean’ pot culture media. SUCROSE FLOTATION (after Walker, 1991) * Collect soil from around host plant roots, * Thoroughly wet a known weight of fresh soil (eg. 100 g) ‘© Weigh a subsample of soil, dry to constant weight at 70°C, and reweigh for calculation of number of spores per unit dry soil ta + (Pass)soil @uspensior) through a 710 um Endecotts sieve, removing stones and roots. «Strain the soil suspension through a fine sieve (32 jm) and transfer the solid matter collected, to four 50 ml centrifuge tubes. Add water, balance the tubes and resuspend the soil sample. © Centrifuge at 1800 rpm for 5 minutes ‘* Discard the supernatant which contains floating organic material, including dead spores. ‘- Resuspend pellets in sucrose solution (440 g L"). Carefully balance the tubes. + Centrifuge up to 1800 rpm and brake immediately. ‘© Rapidly sieve the supernatant (32 um) and wash thoroughly (at least 1 minute) to replace the sucrose and alleviate osmotic stress on spores. © Discard the pellets left in the centrifuge tubes. ‘© Carefully wash all of the solid material from the sieve into a petri dish marked with a grid to facilitate spore counting and collection under a dissecting microscope. 4* Alternatively (if there is little matter other than spores in the sample) transfer to a filter paper via a Bachner funnel. ‘© Lift and transfer spores with ultra-fine forceps (Vomm GmbH - see Usefie? Aaaresses). Though Tequiring practice, this is practical with spores of as litle as SOwm diameter. ‘© [f spores are to be used for culture attempts, allow them to recover from the stress of extraction in axenic water for 24 hours at 4°C. RAPID SPORE EXTRACTION For soils with low organic matter conten and especially culture medium * Take a soil sample and place it in a beaker. © Vigorously add more than twice as much water and allow to soak for a few minutes. + Swirl, allow heavy particles to settle and quickly pour off the resulting supernatant into a 32 um sieve (or coarser mesh size if you are handling large spores). * Repeat about 10 times. Check the remaining soil for spores. There should be none, Carefully wash all of the solid material from the sieve into a petri dish marked with a grid to facilitate spore counting and collection under a dissecting microscope, MAKING MICROSCOPE SLIDES OF GLOMALEAN SPORES Microscope slides are essential for the investigation of spore morphology and as herbarium specimens to represent preserved spores, notes and cultures. A single slide can carry two sets of Spores mounted in PVLG and in PVLG/Melzer’s reagent. + Extract spores according to methods given above and store them in water in a small watch glass. + Take a microscope slide (Chance-Propper 76x26 mm, 1.0/1.2 mm thick, with ground glass panels on both sides at one end for pencil labelling) and place on it two smail drops of PVLG, * To one drop of PVLG add a small drop of Melzer’s Reagent and mix thoroughly with a mounted needle, (If PVLG and Melzer’s reagent have been premixed 1:1 colours may be compared slide to slide) Using ultra-fine forceps, transfer spores from watch glass to drops on slide in equal numbers. If available, use large numbers of spores (>10-50) so that all aspects of them may be seen, With fine forceps, gently lower a cover slip (Chance Propper N*0, 13 mm diameter) onto the drops of mountant. Various treatments may be applied to the spores: i) Do not crush the spores - their gross morphology and dimensions may be observed and ‘measured, but litte else will be seen Carefully apply pressure to the cover slip above the spores with a mounted needle (clean off any stray PVLG) t0.. ) ..crush them gently so that they pop open to resemble pac-men. i) ...crush them further so that inner membranes are disrupted and emerge. Melzer’s reaction, if positive, is often rapid during this stage. Take care not fo break he cover slip! Experience will indicate what are the most useful treatments you can apply to spores. It is essential to reveal every aspect of structure possible, whilst not utterly destroying the spores. 5* Label the slides fully and let them stand for approximately 24 hours, during which the spores will clear. ‘* Place in oven or on slide warming hot-plate at about 60°C for a further 24 hours. The mountant will harden, and the slides become permanent. Excess mountant may be wiped away later with a little water. * Observe, note, draw, photograph and log all details of spore structure. Store safely and, if they are to be used in published descriptions, lodge copies with a relevant, reputable herbarium, Recipes: after Walker, 1991. Also see INVAM WEB Site: http://invam.caf.wvu.egu/ PVLG Polyyny! alcohol-Lacto-Glycerol 1,66g Polyvinyl alcohol (99-100% hydrolysed, 24-32 centipoise viscosity - Kodak) 10 ml water 10 ml lactic acid 1 ml glycerol add PVA to other ingredients & dissolve for 4-5 h in waterbath at 70-80°C Melzer’s reagent 1.5 g potassium iodide 0.5 g iodine 100 g chloral hydrate % Chloral hydrate is dangerous, but Melzer’s reagent will not work without tt 100 mf water CHARACTERISING & DESCRIBING GLOMALEAN SPORES. 1. Measure diameter or length and width of a sample of whole spores to provide average dimensions, 2. Assess the colour of the spores by comparison with a colour chart, INVAM has produced a chart especially for glomalean spores, but any chart will do, even paint colour charts! CAUTION: colour is not diagnostic, but may be a useful guide to identity when associated with other, more objective characteristic. /f you are one of the many who have difficulty with colour vision, please don't be ashamed to seek the assistance of someone who can see colour well 3. Assess the gross morphology of the spores (see @@OO delow) 4. Prepare slides with spores crushed to different degrees in PVLG and Melzer’s PVLG. Described above, 5. Take slides to compound the microscope. Ensure that the microscope is fully adjusted for optimal performance and that the condenser diaphragm is not closed down excessively. A closed diaphragm will certainly improve depth of focus and image contrast, but it will also cause features to be seen which do not exist, especially taminae in non-laminated walls. 6. Examine crushed spores with increasing magnification looking for walls of varying thicknesses and textures. Some will be obvious, others difficult to resolve or interpret. Therefore, many spores must be examined in order to obtain an overall idea of structure detail 7. If Nomarski interference or phase contrast illumination are available they may help with interpretation.8, Interpret what you see with caution and wisdom. Do not see what is not there, but try to understand what truly is there. This can be very difficult - courage. 9. It is always usefull to draw what you see under the microscope because then you are obliged to interpret what you see, rather than guessing, and conclusions and comparisons may be more confidently made. 10. Compare features spore to spore until structural details become understood. 11, Important features may be photographed to record detail 12, With the various aids available to you (eg. morphology, dimensions, colour, unstained, melzer treated, low-high magnification, bright field, Nomarski, phase contrast) build up a full interpretation of a set of spores and record your findings as notes and illustrations. You now have a well considered description of your unknown spore as observations, notes, diagrams, photographs, Melzer reaction, Qspores of Acaulospora and Archaeospora species have no stalk, but usually a single scar (arrow) may be seen, the spore’s point of attachment to the hypha which originally bore it. The sear is not normally visible until the spore has been crushed and viewed with a compound microscope. When crushed, all can be seen to have a complex internal wall structure which is characteristic of each species. Acaulospora - Spore development spores of Entrophospora species are similar to Acawlospora and have no stalk, but they have two sears (arrows). Scars are not normally visible until the spore has been crushed and viewed with a compound microscope. When crushed, all can be seen to have a complex internal wall structure which is characteristic of each species. Entrophospora - Spore development spores of Glomus, Paraglomus and Sclerocystis species are stalked, but the subtending hyph is attached to the spore without a significant bulbous swelling (see overleaf). Glomus spores may be joined on common hyphae (e.g. G. hoi), aggregated within a woolly peridium of mycelium (eg 7some forms of Glomus mosseae) or in distinct sporocarps (Sclerocystis). All spores in this group have a simple wall structure with no inner walls, and most of them are very difficult to separate. Glomus Spore diversity Qspores ofGigaspora and Scutellospora species are stalked, borne singly on a subtending hypha which has a globose swelling where it is attached to the spore, The two genera in the Gigasporaceae are readily distinguished when crushed on a slide because: a) spores of Gigaspora species have a simple, thin bi-layered wall with a characteristic configuration and no inner walls; b) spores of Scutellospora species have complex groups of inner walls, some which give a strong Melzer reaction. Wall configurations are, as in Acaulosporaceae, used for diagnosis, In addition, Scutellospora spores have a singular germination “shield” which can be seen in whole spores, but is certainly visible when the crushed spore is viewed with a compound microscope. Gigaspora spore ‘Seutellospora spore GLOMALES: SPORE STRUCTURE IN DETAIL Zygospores of fungi in the Glomales may be characterised: 1. according to development (observed in pure pot culture and as reflected by the structure of mature spores); 2. by gross morphology assessed and measured under the dissecting microscope with incident illumination; 3. by finer aspects of their jernal anatomy when crushed and observed with transmitted light on the compound microscope. In the last case walls, progressively laid down within the spore as it matures, take various configurations which are used to characterise different taxa Glomalean zygospores are composed of two classes of wall (Morton, 1995) 1, The “spore wall” is common to all taxa, laid down before flexible inner walls and all layers are continuous with the wall of the subtending hypha. In the field the outermost layer is usually absent. It is composed of polysaccharide material which is readily digested by soil micro-organisms, but it is usually seen in fresh spores from pot culture, The main feature of the spore wall is a rigid, often brittle layer composed of serially deposited laminae. An extremely thin wall, adherent to the inside of the laminate layer is reported to be often or usually present, but this is rarely discernible with light microscopy. Thus, in field gathered material only the laminate wall is likely to be recorded in spore descriptions2, Flexible “inner walls” form independently within the spore wall and are analogised with the concept of “endospore”. These walls form as complete, concentric, spherical envelopes within the spore wall, independent of the subtending hypha. Inner walls may be composed of one or two layers which may be adherent, difficult to separate and, in consequence, difficult to differentiate. Even 50, inner walls are very useful in characterising field gathered spores, despite the loss of outer layers and the difficulties associated with visualising inner walls The application of Melzer’s reagent to crushed spores may, in many cases, allow further differentiation by colour reaction Spores develop by three separate pathways which define the glomalean families: 1. Glomaceae Borne terminally on unspecialised hyphae. These spores have the simplest internal “organisation with just a rigid, three-layered spore wall (with the same configuration of layers for all species) and no inner walls. To date at least 89 species of Glomus have been described, and some slight differences in spore wall configuration can only be seen during spore development. Field identification relies very much upon other characters, and pot culture is an essential aid to naming Glomus species. Spores usually part from the mycelium bearing a short stalk, a fragment of the subtending hypha along which germination tubes emerge. 2. Acaulosporaceae Develop on or within the neck of the sporiferous saccule which is bore terminally on specialised hyphae, and which withers as spore development proceeds, The spore of the genus Acaulospora (34+ species) is borne on the side of the saccule neck, from which it is released on maturity bearing no stalk, but a single scar of attachment, the cicatrix. The genus Entrophospora (4 species) is characterised by the spore being borne within the neck, from which itis released on maturity, again bearing no stalk, but it has two scars of attachment, at opposite poles. Internal wall structure of spores in both genera is complex with, generally, a rigid, three-layered spore wall and a sequence of flexible, multi-layered inner walls. The outer layer of the innermost wall often appears rough or covered with minute particles - described as “beaded”. In some species inner walls may have little structure and collapse when ruptured. These may be termed ‘amorphous’ and further characterised by their positive reaction to Melzer’s reagent. Germination tubes breach the spore wall, originating at a spiral or, perhaps an alternative form of germination “shield”. 3. Gigasporaceae Borne on a terminal hypha upon a specialised, bulbous sporogenous cell. The walls of the two genera in the Gigasporaceae are markedly different. Gigaspora (which has not yet been found in Europe) has a two-layered spore wall common to all of its 8+ species. The outer layer is rigid without lamina, the inner thick, rigid and laminate, A discrete wall unit, the “germinal wall”, has been described as being adherent to the inner surface of the spore wall, but today it is considered that germ tubes develop on the inner surface of the main laminated wall (Morton, 1996, Pers. Comm.), The 27+ species of Scutellospora, like members of the Acaulosporaceae, have a complex suite of inner walls which aids characterisation, especially in field gathered material. These may be thin and flexible or thicker, leathery to amorphous, with varying degrees of colouration in Melzer’s reagent. Scufellospora also has a characteristic germination shield which provided the generic name. ‘Unfortunately, the germ shield, although apparently characteristic of some species, has defied the taxonomists’ efforts to employ its varied morphology as a diagnostic feature. AT THIS STAGE, THINGS BEGIN To GET A LITTLE TRICKY! The classification of the arbuscular mycorrhizal fungi - the Glomales - has been attempted by several individuals and groups of workers with a reasonable degree of success, but the story is far from being complete. These fungi have no known sexual stage and the intraradical phase has, so far, defied detailed classification, although identification of AM fungi in roots at the genus level ispossible to a limited degree (Abbott, 1982; Brundrett ef al., 1994; Merryweather & Fitter, 1998a,b; Dodd et al, 2000). Spores are the only possible morphological tools we can use with any confidence but their diversity and inter-relationships are, in truth, poorly understood, It is not certain whether spores which look similar may be considered to be the same taxonomic entity and molecular investigations are indicating that a spore may contain nuclei of several different genotypes. and that morphologically similar spore types may be genetically distinct. Ever since spore description began, workers with limited knowledge of the Glomales have published new species based upon occasional field extracted spores which were either degenerate or dead, lacking features of the living fungus. This has confused the taxonomy of the group. Also, more spore types may yet be discovered and described and there are far too few scientists competent or able (funding) to address this essential task BEWARE: Spore assemblages extracted from soils rarely, if ever, represent the fungi which inhabit roots in the same soil in any meaningful way. Spores do not represent the ecologically significant vresent and spores alone cannot be used to characterise or quantify arbuscular mycorrhizal populations. It is possible that if one uses a combination of spore samples, root fungi, trap culture ‘and molecular data a more realistic picture could emerge - but it might still be inaccurate. JAMES'S PERSONAL VIEWS: These fungi are very difficult to name, particularly if spore ‘material has been recovered from field samples. In my first year of sampling I spent hours examining and describing dead spores, non-glomalean spores, moss protonema buds, eel-worm cysts, collembolan eggs etc. and missed loads of stuff I should have recognised. Once I had attended a course dedicated to the subject I was both experienced and informed and ready to begin learning to identify AM fungal spores. Don’t imagine you can go out and sample, extract spores and create a picture of the mycorthizal population in your field site* without an awful lot of tuition and experience (*spores alone are no good anyway) ‘Also, don’t expect to find an identification key for the Glomales - there isn’t one. However, [VAM has published descriptions of 72 common species out of around 155 confidently described to date. One day the BEG expert system on CD might be launched. If it works, and that is some years away, it should be an interactive key to the Glomales as understood at that time. If you've got a copy of the ‘old demo version please do not use it as a definitive key - it contains but 14 partial descriptions and is nowhere near complete for use You may have Schenck & Perez (1990) but beware: it contains a mess of old and incorrect descriptions mixed in with some good, as well as some tired, old and very confusing ‘keys’. It is, nowadays, neither use nor ornament. Throw it energetically into the eco-bin! The most reliable and up-to-date information about the state of the taxonomy of the Glomales (and much more) will be found on INVAM’s World Wide Web site at http://invam.caf.wvu.edu ** Also consider the revised taxonomy by SchiiBler, Schwartzott & Walker (2001) see page 20 . OU can always say sometiieg He Gloais oy toma ibs aeueainescent elaetY _ 10Sterile washed sand is a good base for AM culture, Various clay aggregates have been used, some of which provide an acidic environment, others alkaline, We favour Terragreen™" which we wash thoroughly to reduce its nutrient content, The nutrient supply can then be controlled if added in solution (eg. Hoagland’s, Rorison’s). If additional nutrient is to be incorporated when first making up a growing medium, a slow release formulation is preferable, especially one in which phosphate is not immediately available. Rock phosphate or bone meal fulfil this requirement. Open pots are likely to become infected with unwanted AM fungi (some workers have found that a particular isolate may be extremely invasive), collembola and fungus gnats (which feed on AM fungi), root-feeding fly larvae and fungal leaf pathogens (eg. mildews). Walker & Vestberg (1994) have devised a method of isolating individual cultures in tissue culture bags (Sun-Bags from Sigma) which permits the maintenance of large numbers of cultures in a small space whilst avoiding contamination. However, cultures in bags must be kept cool, for internal temperatures can rise dramatically in hot weather. This method is probably only suitable for glasshouses in temperate climates and controlled environment cabinets. We almost lost our entire collection in Sun-Bags because they became infested with thrips and spider mites which calmly ate the host plants’ leaf epidermis. Some control of soil nematodes may be achieved if Tagetes is used as a host or additional planting. Similarly Pyrethrum will help with the control of soil insects. ‘A most important condition for the initiation and maintenance of AM cultures is light. Since the host provides up to 20% of carbon it produces to its fungal partner, it is essential to ensure that photosynthesis is maximised by supplying powerful illumination. STAINING ARBUSCULAR MYCORRHIZAS Roots are frequently opaque and pigmented, and it is essential to experiment with methods of clearing, bleaching and staining before proceeding with routine staining. The various stains used in mycorrhiza research have subtly different properties and some, such as aniline blue and chlorazol black E, stain all fungal tissue very darkly, sometimes too intensely. The quality of colouration by acid fuchsin varies according to root tissue and fungal species, helping to distinguish different endophytes. Its effectiveness may be greatly enhanced under the Muorescence microscope using green light (Merryweather & Fitter, 1991). Most stains are & extremely hazardous (eg, acid fuchsin, trypan blue, chlorazol black E, especially when dry) and should be used only when safer alternatives, such as aniline blue or methyl blue, are impractical” (refer to appropriate hazard data sheets). Phenol is dangerous and un-necessary, and is no longer used in the study of mycorrhiza SEE PAGE 16 Roots may be treated in test-tubes, their contents strained through a small sieve between treatments. ‘A semi-automatic method has been devised in which root samples are placed in plastic tubes perforated at the base or sawn off with mesh glued over the end. The tubes are set in a stainless steel rack (do not use aluminium, which dissolves violently in KOH solutions) which is moved from solution to solution carrying a full set of tubes with it, The treatment solutions are contained in stainless steel reservoirs which contain just enough fluid to cover the roots. In hot staining, these are placed in the water bath. HOT STAINING (after Kormanik & McGraw, 1982; see Brundrett er al., 1994 for alternatives) This method is rapid and, apart from destaining, is completed within a few hours. ‘© Wash root sample and transfer to labelled tube. * Clear in 10% potassium hydroxide solution (caustic) in a water bath at 90°C in a fume cupboard. Clearing removes cytoplasm from host root cells, leaving root structure and fungal elements intact * Obtained from OIL*DRI UK Ltd (01945 581244) > See Vierheilig et al. in References. ir* The length of time required varies greatly between species. White, fleshy roots may take less than 10 minutes, others substantially longer. If roots are feft in hot KOH for too long, the cortex will isintegrate. Some grass species may lose their cortex before clearing adequately at 90°C. Brief treatment in an autoclave (120°C) may help. Darkly pigmented roots may need to be bleached with hydrogen peroxide at room temperature for between 30 minutes and several hours. Both alkaline and acidic HO; (care) have been found to work for different roots (Brundrett et a, 1994) * Drain and wash thoroughly. * Acidify cleared roots by immersion in 1% hydrochloric acid (corrosive) for at least one hour. * Stain for 10-30 minutes in 0.01% acid fuchsin stain (or aniline blue, see cold staining below) in water bath at 90°C in a fume cupboard, Some roots stain better if the rack is lifted and replaced Periodically to refresh stain in the tubes. * Drain and wash thoroughly. * Destain overnight or longer (> 2 days) to remove colouration from empty root cells etc. ‘© Mount roots in destaining solution on a microscope sfide beneath a 50 x 22 mm #1 cover slip. Roots should ideally be mounted as parallel lengths; avoid tangles which make quantification very difficult, Recipes: Stain - 0.01% acid fuchsin dissolved in destaining solution, Destaining solution - 14:1:1 lactic acid: glycerol: water. Food grade lactic acid is cheap, can be bought in butk (pickle factories) and is very effective. COLD STAINING (After Grace & Stribley, 1991, Koske & Gemma, 1989 and Walker & Vestberg, 1994) This method takes longer from start to finish, but requires much less attention than hot staining. Large containers may be used to hold solutions allowing many root semples may be processed at the same time. Aniline blue is recommended because, to date, it has not beer proven hazardous, unlike most other compounds used for staining fungi eg. acid fuchsin, chlorazol black E). * Wash root sample and transfer to labelled tube. © Clear in 20% potassium hydroxide solution for 1-3 days. (As with the hot method, it is necessary to experiment to discover the optimal clearing times). © Drain and wash thoroughly. * Acidify with 1% hydrochloric acid * Stain with 0.05% aniline blue solution (1-3 days). © Destain overnight or longer. * Mount roots in fresh destaining solution on a microscope slide. BUT INK IS SAFEST, READ ON... * Also try acidified sodiuzat hypochlorite solution after Bevege, 1968, 15STAINING FUNGI IN A MYCORRHIZA WITH INK (hot or cold) After Vierheilig ef a/., 1998 and as tried informally by Chis Walker at the York AM course, 2000. We have used it successfully using blue Quink in the York lab. since. It works as well as any other staining method and is cheap and safe. 1. Treat with 10% KOH as usual. 2. Quick rinse in water. acidify oa If He. 3. Drop of ink, not measured, placed with the roots in lactic glycerol. This was just for a few minutes (not timed) 4. Brief destain in lactic glycerol (destaining solution, recipe below). 5. “Bob's your uncle, Fanny's your aunt”. [The published method (Vierheilig ef al.) uses vinegar, but I (Chris Walker) don't like the smell of acetic acid, so I changed it when I did it at home to 1% HCI. However, at your labs, lactic glycerol worked just as well.) STAINING WITH INK: Method refined by Claire Cowie Bsc. for her 3rd year research project on snowdrop mycorrhiza (Galanthus nivalis) at the University of York 2000-2001 The AM fungi in the roots were stained using a method adapted from Vierheilig et al. (1998). The roots were first cleared in 10% potassium hydroxide at 90°C for 15 minutes. After rinsing in tap water, the roots were acidified in 16% hydrochloric acid for 30 seconds, and then rinsed with tap water again. The stain used was a 1% black Parker Quink solution with lactic acid and glycerol, in which the roots were placed at 90°C for three minutes, The roots were destained for over 24 hours in lactic glycerol, and also stored in the solution, The destaining solution was again used in mounting the roots on microscope slides. QUANTIFICATION OF ARBUSCULAR MYCORRHIZAS (the magnified intersection method - MeGonigle ef al, 1990) * Place the slide on the stage of a compound microscope equipped with a cross-lines eyepiece graticule. Assessment of mycorrhizal colonisation using an old dissecting microscope may lead to inaccuracy due to the difficulty of differentiating mycorrhizal fungi and others at low magnifications. It is essential to confirm that fungi are arbuscular to obtain meaningful results. * Scan the slide methodically, one axis of the graticule aligned with the long axis of each root encountered. * For a simple count of percentage of root length colonised by mycorrhizal endophyte (%RLC), score presence or absence of arbuscular endophyte touched by the graticule axis which crosses the root each time a root is encountered. * You will get no better than a reasonable estimate of %RLC from 25 intersections per slide. Accuracy is achieved only if 100 or more counts are recorded * Counts are recorded as percentages: %RLC = 100 x Number of intersections with arbuscular hyphae Total number of intersections counted * Quantification can be refined by counting intersection ‘hits’ on hyphae, entry points, arbusules, vesicles or other structures considered individually. Remember that Glomales is an order of great 16diversity. Some species (e.g. all of the Gigasporaceae) never form vesicles and others might lack them simply because they have not yet formed them or they have been laid down in the roots of another plant at that time of year * Ifyou can distinguish different AM fungal morphotypes in roots you might like to try quantifying them individually (see Merryweather & Fitter, 1998b), Specimen score sheets (originally designed for bluebell roots and not necessarily applicable to others without considered modification) are given towards the back of this manual. ‘+ The more you refine your scoring the more information you will obtain from your data. THE EXTERNAL MYCELIUM EXTRACTION Membrane Filter Method (also see Sylvia, 1992; Brundrett ef al., 1994) Equipment and materials (5-30 g sample) © 500 ml glass beaker 200 ml glass beaker (tall & thin if possible) * 5S ml syringe * Millipore filter holder/fiunnel * Side-arm Buchner-type flask to take filter holder * Filter membranes: diameter 15 mm, pore sizal4S ym * Magnetic stirrer and follower (’flea’) © Forceps ‘Flat stirring rod (e.g. plastic plant pot label) * Plastic pasteur pipettes ‘© Glass microscope slides and cover slips * 0.01% Acid Fuchsin staining solution (see ‘Recipes’ p. 13) + Destaining solution (see ‘Recipes’ p. 13) Method * Place 5 - 30 g (fresh weight - experiment to discover the optimal sample weight for your soil) of growth medium in a beaker and add water to make up to 500 ml. Ifusing ‘real’ soil use Calgon (a polyphosphate detergent) to disaggregate. + Stir at full speed with magnetic stirrer for 1 - 2 min. ‘* Remove 200 ml to tall beaker. (for real soils you might need to use another dilution). * Stir again briefly at full speed, then reduce to 40% speed; stop stirring and counter-mix with flat stirring rod + Take a5 ml sample with the syringe, centrally at about 1 cm depth * Set up the Millipore filtering apparatus. ”* Vacuum on Transfer the sample in the syringe to the filter funnel, allowing all the liquid to pass through the filter. * Vacuum off When the filter is dry, remove it carefully with forceps. Set the filter funnel aside leaving the filter in place. * Vacuum on Carefully add a few drops of acid Fuchsin solution. Wait until dry, ~30 seconds. © Iffilter retains too much colour: Vacuum on Replace funnel, rinse with water and drain © Vacuum off Transfer filter to a clean, labelled slide. ‘© Mount whole filter in destaining solution under a coverslip. QUANTIFICATION Gridline intercept method (see Miller & Jastrow, 1992; Brundrett ef al., 1994) Equipment: * Compound microscope set for magnification around x100 or x125 ‘Eyepiece graticule marked with a grid (1 cm? divided into 100 parts) © Hand tally counter You need to know. * Soil fresh weight:dry weight ratio (subsample of the same medium) * Sample amount (i. dilution) * Grid size (microscope eyepiece graticule marked with grid) © Magnification * Cylinder diameter ‘+ Number of grids assessed (you need two tally counters: one for the number of grids and one for the hyphal intersects) Hyphal length calculation: see Brundrett et al., 1994 p.32. 18