ARBUSCULAR MYCORRHIZA

Transcription

1 ARBUSCULAR MYCORRHIZA Dr. Suresh. S.S. Raja Assistant Professor Department of Microbiology Bharathidasan University College Perambalur Prepared

2 Vesicles and Arbuscules Glomus Mycorrhiza is the symbiotic association between plant roots and fungal mycelia Gigaspora

3 CHARACTERISTICS OF ARBUSCULAR MYCORRHIZA Mycorrhiza increase the surface area of the root Absorb nutrients from soil especially phosphorus and micronutrients through hyphae and mobilize into the host cell. Almost 90 % of crop plants are mycorrhizal mostly of arbuscular type (AM) Mycorrhiza possess vesicles and arbuscules. Mycorrhiza species are: Glomus, Gigaspora, Scutellospora, Acaulospora, Entrophosphora and Sclerocystis

4 Gigaspora Scutellospora Glomus Gigaspora Glomus albidum Sclerocystis Gigaspora Glomus mosseae Sclerocystis

5 ROLE OF MYCORRHIZA The benefit of mycorrhizas to plants is mainly attributed to increased uptake of nutrients, especially phosphorus. This increase in uptake may be due to increase surface area of soil contact increased movement of nutrients into mycorrhizae, a modification of the root environment and increased storage. Mycorrhizas can be much more efficient than plant roots at taking up phosphorus. Phosphorus travels to the root or via diffusion and hyphae reduce the distance required for diffusion, thus increasing uptake. The rate of inflow of phosphorus into mycorrhizae can be up to six times that of the root hairs. In some cases, the role of phosphorus uptake can be completely taken over by the mycorrhizal network, and all of the plant s phosphorus may be of hyphal origin

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7 Terminologies APPRESSORIA Hyphal swellings between two adjacent root epidermal cells. These are sites where hyphae first penetrate root cells by exerting pressure and/or enzymatic activity. ARBUSCULES These are intricately branched "haustoria" that form within root cortex cells that look like little trees. Arbuscules are formed by repeated dichotomous branching and reductions in hyphal width from an initial trunk hypha that ends in a proliferation of very fine branch hyphae. They are considered to be the major site of exchange with the host plant. Old arbuscules collapse progressively until only the trunk remains. Collapsed arbuscules are sometimes called peletons. ASEPTATE HYPHAE These are hyphae which are without cross walls (coenocytic hyphae). Cross walls may form as hyphae age.

8 AUXILIARY BODIES These structures, which are also called external vesicles /or accessory bodies, are clustered swellings on external hyphae. These are often ornamented by spines or knobs and are characteristic of Scutellospora and Gigaspora. These apparently do not function as propagules. COLONY Hyphal colonization of a root resulting from one external hypha (these may arise from several adjacent entry points). These are often called infection units. DICHOTOMOUS BRANCHING A symmetrical branching pattern which occurs when two branches arise simultaneously from the tip of a hyphae, plant or fungus organ. Divergent branches grow at the same rate.

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10 INTERNAL HYPHAE, INTRARADICAL HYPHAE Hyphae which grow within the cortex of a root to form a colony and later develop arbuscules and vesicles. These comprise the body (thallus) of a fungus in the root. INTERCELLULAR HYPHAE Hyphae which grow between the walls of adjacent root cells. These are in the root apoplast -- the zone outside the cytoplasm of cells. INTRACELLULAR HYPHAE Hyphae which grow within root cells. These penetrate the walls of cells and grow within them, but are separated from the cytoplasm by the plasma membrane. SOIL HYPHAE These are also known as extraradical or external hyphae and are the filamentous structures which comprise the fungal thallus (body) in the soil. They acquire nutrients, propagate the association, produce spores and other structures. VAM fungi produce thick "runner" or "distributive" hyphae as well as thin, highly branched "absorptive" hyphae.

11 SPORES These are swollen structures with one or more subtending hyphae that form in the soil or in roots. Spores usually develop thick walls, which often have more than one layer. They can function as propagules. Spores of VAM fungi are sometimes called chlamydospores or azygospores. SPOROCARPS Aggregations of spores into groups, which may contain specialised hyphae and can be encased in an outer layer (peridium). Soil particles may be included in the spore mass. This term can be misleading, as the sporocarps produced by most Glomeromycotan fungi are small and relatively unorganised structures compared to those produced by larger fungi. VESICLES Intercalary (-o-) or terminal (-o) hyphal swellings formed on internal hyphae within the root cortex. These may form within or between cells. Vesicles accumulate lipids and may develop thick wall layers in older roots. The production and structure of vesicles varies between different genera of Glomeromycotan fungi. They are storage organs which may also function as propagules.

12 VAM Mycorrhizal associations produced by Glomeromycotan fungi are known as arbuscular mycorrhizas, or vesicular-arbuscular mycorrhizas (formerly also endomycorrhizas, or endotrophic mycorrhizas) and are abbreviated as VAM here. There is disagreement about whether arbuscular mycorrhizas or vesicular-arbuscular mycorrhizas is the most appropriate name to, because some fungi do not produce vesicles, but arbuscules are not consistently used to identify associations (i.e. they are absent in myco-heterotrophs and older roots).

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14 VAM GENERAL STRUCTURE A. Structures in Soil Distributive Hyphae:A network of hyphae forms in the soil with thicker hyphae which function as conduits. Absorptive hyphae: Thin highly branched hyphae which are thought to absorb nutrients. Spores:Large (for a fungus) asexual spherical structures ( µm diameter) formed on hyphae in soil, or in roots. B. Structures in Roots Hyphae:these are non-septate when young and ramify within the cortex. Arbuscules: intricately branched haustoria in cortex cells. Vesicles:storage structures formed by many fungi.

15 Looking at Associations

16 The Dissecting Microscope

17 The Compound Microscope

18 Structures and Developmental Stages 1. Soil Hyphae Mycorrhizal associations may be initiated by spore germination as illustrated here. Hyphae may also originate from fragments of roots. In many cases there already is a pre-existing network of hyphae resulting from previous root activity. Hyphae resulting from spore germination have a limited capacity to grow and will die if they do not encounter a susceptible root within a week or so. Hyphae emerge from a germination shield within the spore in Scutellospora and Acaulospora species. Germinating hyphae which emerged several days after spores were extracted from dry soil. These spores are of Gigaspora decipiens (left) and Scutellospora cerradensis (right). AV = accessory vesicles. GS = germination shield. Bars = 100 µm.

19 Soil Hyphae Soil hyphae, also known as extraradical or external hyphae, are filamentous fungal structures which ramify through the soil. They are responsible for nutrient acquisition, propagation of the association, spore formation, etc. VAM fungi produce different types of soil hyphae including thick "runner" or "distributive" hyphae as well as thin "absorptive" hyphae. The finer hyphae can produce "branched absorptive structures" (BAS) where fine hyphae proliferate Hyphae of Scutellospora and Gigaspora species produce clustered swellings with spines or knobs called auxiliary cells.

20 Mycorrhizal root system washed carefully from coarse sand to reveal the intact network with external hyphae (arrow) with spores (S) produced by Glomus mosseae.

21 Soil hyphae produced by a single germinated spore of Gigaspora (arrow) used to start a mycorrhizal association. The hyphal network has produced accessory vesicles and is spreading through the root system of a clover plant.

22 2. Root Contact and Penetration Mycorrhizal associations start when soil hyphae respond to the presence of a root by growing towards it, establishing contact and growing along its surface. Next, one or more hyphae produce swellings called appressoria between epidermal cells. Root penetration occurs when hyphae from the appressoria penetrate epidermal or cortical cells to enter the root. These hyphae cross the hypodermis (through passage cells if these are present in an exodermis) and start branching in the outer cortex.

23 Soil hyphae have produced 2 appressoria between epidermal cells (arrows). These are seen here in a surface view of a root with attached hyphae.

24 Hyphae at an entry point (E) penetrating cortex cells (arrows) approximately 1 day after contact with the root.

25 Alternating long (L) and short (S) cells in the dimorphic exodermis of a Smilacina racemosa root. Hyphae of VAM fungi have penetrated unsuberised short cells (arrows).

26 3. Hyphal Proliferation in the Cortex Aseptate hyphae spread along the cortex in both directions from the entry point to form a colony. Hyphae within root are initially without cross walls, but these may occur in older roots. two distinctive morphology types - the Arum and Paris series after host plants. These are now known as linear and coiling associations respectively. Both types of associations are important in ecosystems Linear (Arum) series associations where hyphae proliferate in the cortex by growing longitudinally between host cells. This occurs because hyphae grow through longitudinal intercellular air spaces that are present (Brundrett 2004). Coiling (Paris) series where hyphae spread by forming coils within cells because there are no continuous longitudinal air spaces.

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28 Part of a colony of a VAM fungus (Glomus sp.) with hyphae, arbuscules (A) and vesicles (V) growing from an entry point (arrow).

29 (i) Coiling (Paris) Arbuscular Mycorrhizas These are associations where hyphae spread primarily by intracellular growth following a convoluted path through cortex cells. The resulting colonies of VAM fungi generally have a coiled appearance, but may have more digitate branching patterns. Arbuscules may be restricted to a single layer of cells in the inner cortex.

30 Colony of a VAM fungus spreading from the entry point (E) by convoluted hyphae (arrows) in the cortex of an Erythronium americanum root. Hyphal coils tend to occur in roots without prominent air channels. Higher magnification view of two arbuscules (A) growing directly from coils in adjacent root cortex cells of Erythronium americanum. Note how arbuscular branches arise from the same hyphae (arrows) which connect hyphae in adjacent cells. Arbuscules (A) and convoluted hyphae (arrow) in the inner cortex of an Asarum canadense root. Arbuscules only form in the innermost cortex cell layer next to the endodermis in this species

31 (ii) Linear (Arum) Arbuscular Mycorrhizas These are associations where hyphae grow along longitudinal intercellular air channels between the walls of root cells. A relatively rapid parallel spread of intercellular hyphae may occur along these channels. The resulting colonies of VAM fungi have a linear appearance.

32 Intercellular air channels (arrows) in a whole mount of a living leek root (Allium porrum), shown for comparison with mycorrhizal development in the same host. These channels run continuously from the apex to the base of roots. Longitudinal growth of hyphae of a VAM fungus (Glomus versiforme) along cortex air channels in a leek root. Note progressive development of arbuscules with increasing distance from the growing tips of hyphae.

33 4. Arbuscules Arbuscules are intricately branched haustoria that formed within a root cortex cell. They look like little trees. Arbuscules are formed by repeated dichotomous branching and reductions in hyphal width, starting from an initial trunk hypha (5-10 um in diameter) and ending in a proliferation of fine branch hyphae (< 1 um diameter). Arbuscules start to form approximately 2 days after root penetration. They grow inside individual cells of the root cortex, but remain outside their cytoplasm, due to invagination of the plasma membrane. Arbuscules are considered the major site of exchange between the fungus and host. This assumption is based on the large surface area of the arbuscular interface, but has not been confirmed. Arbuscule formation follows hyphal growth, progressing outwards from the entry point. Arbuscules are short-loved and begin to collapse after a few days, but hyphae and vesicles can remain in roots for months or years.

34 Developing arbuscule of Glomus mosseae in a root cell with fine branch hyphae (arrows). The trunk (T) of this arbuscule branched from an intercellular hyphae. An arbuscule of Glomus versiforme in a root cortex cell with branch hyphae densely packed in the cortex cell of the host. Mature arbuscule of Glomus showing trunk (T) and numerous fine branch hyphae (arrows). Arbuscule of Gigaspora margarita with an elongated trunk hypha (T) and tufts of fine branch hyphae (arrows). Note how this arbuscule differs from the Glomus arbuscules above.

35 5. Vesicles Vesicles develop to accumulate storage products in many VAM associations. Vesicles are initiated soon after the first arbuscules, but continue to develop when the arbuscules senesce. Vesicles are hyphal swellings in the root cortex that contain lipids and cytoplasm. These may be interor intracellular. Vesicles can develop thick walls in older roots and may function as propagules. Some fungi produce vesicles which are similar in structure to the spores they produced in soil, but in other cases they are different.

36 Vesicles (V) produced by a Glomus species in a leek root. This root also contains many intercellular hyphae Lobed vesicles of an Acaulospora species in a clover root Arrows = vesicles, A = arbuscules

37 6. Structural Diversity It is possible to identify individual Glomeromycotan fungi by recognising characteristic root morphology patterns in roots. Identification of endophytes within roots is important for culture quality control, because contaminating fungi can be identified months before they sporulate. This procedure can also be used to determine the mycorrhizal inoculum potential of different fungi by growing trap plants in a soil. It is also possible to identify Glomeromycotan fungi by colonization patterns in roots, but it is difficult to separate species. Morphological features that are important include variations in vesicles (size, shape, wall thickness, wall layers, position and abundance), hyphal branching patterns, the diameter and structure of hyphae (especially near entry points), and the staining intensity of hyphae (dark or faint). Characteristics of genera of Glomeromycotan fungi are listed and illustrated below

38 Mycorrhizas produced by Glomus species: Relatively straight hyphae ramify along the root cortex (if root anatomy permits), often producing "H" branches which result in simultaneous growth in 2 directions. Staining of these hyphae is usually relatively dark. Arbuscules can be dense and compact. Oval vesicles, which usually form between root cortex cells, are present in many cases. These vesicles persist in roots and often develop thickened and/or multilayered walls.

39 Mycorrhizas produced by Scutellospora and Gigaspora species: In Scutellospora VAM looping hyphae are often present near entry points. This genus has similar root colonisation patterns to Acaulospora, but hyphae in the cortex are generally thick-walled and stain darkly. Internal vesicles are not present. Arbuscular trunk hyphae normally are much longer and thicker than those of Glomus. Arbuscules appear wispy due to relatively long curving branches. The root colonization pattern for Gigaspora is very similar to that for Scutellospora, with wide hyphae

40 Mycorrhizas produced by Acaulospora species: Entry point hyphae have characteristic branching patterns. Hyphae in the outer cortex generally are more irregularly branched, looped or coiled than for Glomus. Colonies in roots are often relatively small. Internal hyphae are thin walled, often stain weakly and thus may be very hard to see, but may be visible due to rows of lipid droplets. External hyphae are usually also very hard to see. Intracellular oil-filled vesicles, that are initially rectangular, but often become irregularly lobed due to expansion into adjacent cells, are a characteristic feature. These have thin walls and do not persist in roots.

41 Mycorrhizas produced by fine endophytes: These unusual fungi have been called Glomus tenue, but are substantially different from other Glomus species. Fine endophytes can easily be distinguished by their very narrow hyphae (< 1 um in diameter) and net-like growth pattern in roots. Small hyphal swelling (< 5 um) can occur near entry points and may be analogous to vesicles.

42 7. Spores Spores form as swellings on one or more subtending hypha in the soil or in roots. These structures contain lipids, cytoplasm and many nuclei. Spores usually develop thick walls with more than one layer and can function as propagules. Spores may be aggregated into groups called sporocarps. Sporocarps may contain specialised hyphae and can be encased in an outer layer (peridium). Spores apparently form when nutrients are remobilised from roots where associations are senescing. They function as storage structures, resting stages and propagules. Spores may form specialised germination structures, or hyphae may emerge through the subtending hyphae or grow directly through the wall.

43 Left: Spores separated from soil and sorted into categories based on size and colour. Right: Spores (S) on a piece of filter paper used to start a "pot culture" using pasteurised soil in which a host plant was grown.

44 Spores of Glomus Relatively small white spores of a Glomus species. Spore of Glomus clarum which has a visible inner wall layer (arrow). Sporocarp of Glomus invermaium typical of the dead spores often found in field-collected soil Living spores of Glomus invermaium from a pot culture.

45 Spores of Acaulospora Acaulospora spore with deep pits in the outer wall and inner wall layers stained by Melzer's reagent. Acaulospora spore with several inner wall layers (arrows). One layer has stained darkly with Melzer's reagent. Spores of Scutellospora Species White Scutellospora cerradensis spores with prominent brown germination shields Large black spore with deep pits of Scutellospora reticulata.

46 Arbuscular Mycorrhizal Fungi Populations of arbuscular mycorrhizal fungi in the Glomeromycota are thought to have occupied the same soil habitats for millions of years, slowly adapting to changes in site conditions. Many of these fungi have worldwide distribution patterns, but soil factors such as ph restrict the distribution of other taxa. Consequently, habitat information is as important as knowledge of the taxonomic identity of fungi, for comparing the results of experiments, or the selection of isolates for practical use. The classification of the Glomeromycota is based on the structure of their soil-borne spores and DNA sequences. Accurate identification of these fungi often requires them to be isolated in cultures with host plants, to observe developmental stages, avoid the loss of diagnostic features and obtain healthy spores for DNA extraction. There now are many DNA sequences of Glomeromycotan fungi in databases such as Genbank

47 Arbuscular Mycorrhizal Fungi VAM fungi belong to the Glomeromycota. They are primitive fungi at the base of the tree for higher fungi (basidiomycetes). They associated with first land plants and appear to have evolved very slowly since then. They have no known sexual state. They produce microscopic structures, or relatively small sporocarps (truffle-like). Just over 200 species of these fungi are described, yet they are capable of forming mycorrhizal associations with the majority of plants.

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49 Classification scheme for Glomeromycotan taxa

50 Classification scheme for Glomeromycotan taxa Archaeosporales Ambisporaceae Archaeosporaceae Geosiphonaceae Diversisporales Acaulosporaceae Entrophosporaceae Diversisporaceae Gigasporaceae Pacisporaceae Glomerales Glomeraceae Paraglomerales Paraglomeraceae Family Genera Acaulosporaceae Acaulospora, Kuklospora Ambisporaceae Ambispora Archaeosporaceae Archaeospora, Intraspora Diversisporaceae Diversispora Entrophosporaceae Entrophospora Geosiphonaceae Geosiphon (not a mycorrhizal fungus) Gigasporaceae Gigaspora, Scutellospora Glomeraceae Glomus (Sclerocystis) Pacisporaceae Pacispora Paraglomeraceae Paraglomus

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52 SOURCES OF AMF INOCULUM SOIL,CRUDE,ROOT 1.Soil inoculum composed of soil, dried root fragments, and AMF spores, sporocarps, and fragments of hyphae Not a reliable inoculum - last resort Possible transfer of weed seeds and pathogens with the soil is a deterrent Figuring out how much soil to add as inoculum to a growth medium or a field is another challenge, because the abundance and viability of AMF propagules in the soil is often uncertain. Low spore viability or dead spores

53 SOURCES OF AMF INOCULUM soil or root tissue from the site can be taken to start a trap culture to boost the number of viable spore propagules for isolation and further multiplication. These roots and soil are either mixed into the growth medium or applied in a band below the soil surface. Germinated seeds of the indicator plant are then planted and grown long enough for formation of a mixed culture containing mature AMF spores, which are then Extracted, separated into morphological types, identified, and used as starter cultures.

54 SOURCES OF AMF INOCULUM 2. Crude inoculum obtained after a known isolate of AMF and a suitable host are grown together in a medium optimized for AMF development and spore formation. common type available for large-scale crop inoculation. It consists of spores, fragments of infected roots, pieces of AMF hyphae, and the medium Spores can be extracted from such an inoculum by wet-sieving and decanting, and used, alone, before or after surface disinfection spore inocula are known to initiate AMF colonization less rapidly than crude inocula, possibly because crude inocula contain a greater number of different types of infective propagules. 3. Root inoculum Infected roots of a known AMF host separated from a medium in which crude inoculum was produced can also serve as a source of inoculum.

55 MASS PRODUCTION OF VAM BY POT CULTURE TECHNIQUE STERILE POTS SOIL VAM SPORES IN WATCH GLASS SOIL:SAND VAM SPORES HOST PLANT + STERILE SOIL+VAM SPORES KEEP IN GLASS HOUSES YOUNG SEEDLINGS (remove the seedlings after few weeks) SEEDLINGS (check VAM spores microscopically. If present chop the root) CHOPPED ROOTS AS STARTER INOCULUM (Put small amount of starter inoculums one inch below soil layer in the pot) INOCULATED IN LARGER POTS (remove seedlings after 3-4 months. Inoculate seeds in soil) INOCULUM IN BULK (used in field as granular preparation THE PELLETED SEEDS ARE PACKED IN POLYTHENE BAGS

56 Producing crude/soil/root inoculums Physical Environment Media- Soil+Sand (Silica not coral) Photosynthate, light intensity, soil air temperature, soil water status Container types plastic bags and pots made of concrete, clay, and plastic. They should have holes in the bottom to ensure adequate drainage. To minimize the amount of light reaching the medium, the containers should not be translucent. If clear material must be used, it should be painted or enclosed by wrapping in an opaque material kg of medium per container Starter culture The culture must be highly infective, contain at least four infective propagules per gram, and be free of pathogenic microorganisms. The aim is to inoculate the inoculum-production medium at a rate of 500 infective AMF propagules per kilogram of medium. Nurse plant species It should grow fast, be adapted to the prevailing growing conditions, be readily colonized by AMF, and produce a large quantity of roots within a relatively short time (45 60 days). It should be resistant to any pests and diseases common in the inoculum-production environment. The best nurse plants are C. dactylon, S. grandiflora, and Z. mays

57 Producing crude/soil/root inoculums Nutrient management P and N should be optimum. Higher or lower limits growth Hogland s solution with or without P to augment growth K 250, Mg 212 (as MgSO4), Zn 10, Cu 5, B 0.1, Mo 0.5. Duration of growth Essential to grow the nurse plant in the inoculum-production medium for weeks. The medium is then allowed to dry slowly by reducing the frequency of watering over a week and then withdrawing water completely for another week. If at the end of the last week the plant is dry, it is removed from the growth medium. The roots of the plant can be chopped into fragments 1 cm long and mixed with the medium, or they can be used separately as root inoculum. The moisture content of the medium at this time should be 5% or lower. If not, the crude inoculum must be spread on a clean surface in an environment with low humidity (RH 65%) and allowed to air-dry until the desired moisture content is reached.

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59 Extracting AMF spores from soil or crude inoculum Wet-sieving and decanting Soil samples from field sites should be taken from the rhizosphere of mycorrhizal native or crop plants at a soil depth where the most root proliferation occurs, usually 0 20 cm. The sample is then passed through a 2-mm sieve. A g soil sample (dry weight) is transferred to a beaker. If the soil is dry at sampling, make sure it is soaked for minutes before attempting to extract spores. Soil aggregates can be crushed with a spatula. Distilled or deionized water is added to obtain a 1-L suspension, and the suspension can be agitated for 1 hour in an electric stirrer. The purpose of these steps is to disperse the soil aggregates and release AMF spores. A 3.5% sodium hexametaphosphate solution can be added to increase soil dispersion.

60 Extracting AMF spores from soil or crude inoculum The soil suspension is poured through a stack of sieves (750, 250, 100, 53, and 37 μm), the finest sieve being at the bottom of the stack. A stream of tap water is added to facilitate the movement of spores. The material that remains in the 37, 100, and 250 μm aperture sieves is suspended in water and transferred to centrifuge tubes and centrifuged for 3 minutes at 2000 g. Spores are sedimented at the bottom of the tube, while organic materials remains in suspension. After removing the supernatant, the sediment is re-suspended in a 50% sucrose solution and centrifuged again for 1 2 minutes at 2000 g. After this, the spores will be in the supernatant or in the sugar-water interface. The supernatant fluid containing the spores is poured onto a 28μm aperture sieve or removed with a syringe and rinsed immediately with water to remove the sucrose. Exposure of spores to high concentration of sugar for too much time can dehydrate them, and therefore they should be transferred to tubes and stored in distilled water at least for 24 hours before mixing them with the growth medium. This will allow them to overcome osmotic shock

61 Extracting AMF spores from soil or crude inoculum The number of AMF spores in a suspension can be determined under a microscope by transferring a small volume of the suspension into a counting chamber such as the type used for counting nematodes. The standard counting chambers used in microbiological laboratories are etched with squares of known area and are constructed so that a film of the suspension of known depth can be introduced between the slide and the cover slip.

62 Extracting AMF spores from soil or crude inoculum Separation into morphotypes Spores of AMF can be transferred to a petri dish for microscopic examination and separation. Spores can be separated into distinct morphological types. Fine-tipped forceps or Pasteur pipettes can be used to transfer spores into vials or micro-dishes with water for subsequent evaluation and identification. Alternatively, spores can be collected on a filter paper and picked up from it singly with forceps or a finetipped instrument such as a dissecting needle or a paint brush. Collection of spores from water suspension is better for avoiding undesired hyphal fragments.

63 Extracting AMF spores from soil or crude inoculum Once spores are isolated and identified, they can be surface-disinfected and used as a starter inoculum for production of inoculum in one of the several ways described already. Spores of AMF are surface-sterilized by exposing them to a solution of liquid detergent (e.g., Tween 20), 0.5% sodium hypochlorite, or 2% Chloramine T, and 0.02% streptomycin sulfate in a filter unit allowing contact for 15 minutes and then rinsing with five changes of water. Alternatively, spores can be exposed to % mercuric chloride for 2 10 minutes and rinsed with three to five changes of sterile distilled or deionized water.

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67 Extracting spores from a crude inoculum and determining their viability Sterilized soil or sand-soil mixture containing a very low concentration of available P is aseptically packed in a petri dish, leveled, and moistened with distilled water or a solution of 0.1% trypan blue to maximum available water holding capacity (Figure 17). The trypan blue solution facilitates the visibility of hyphae. On the surface of the soil, a nylon mesh (pore size 50 μm) is placed. Pieces of membrane filter 10 x 10 mm (cellulose-acetate, pore size 0.45 μm) are placed on the nylon membrane. The nylon mesh and filter squares should be sterilized by immersion for 5 minutes in 70% ethanol and rinsed with sterile deionized or distilled water prior to use. One AMF spore is placed on each filter square. The petri dish is covered and incubated in the dark at 20 C and observed regularly under a stereo microscope for 5 20 days, depending on the AMF species involved. A spore is considered to have germinated when the length of the germ tube exceeds the diameter of the spore. Except during observation for germination, the petri dish must remain closed to avoid desiccation or contamination. Alternatively, spores can be placed on a membrane filter that is folded twice and inserted into moist soil. After a 2-week incubation period, the filter is removed, unfolded, stained, and examined under a microscope

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69 DETECTING AND QUANTIFYING AMF COLONIZATION OF ROOTS Procedure Collecting root samples After the root system is thoroughly washed free of soil, obtain a representative sample by removing four to five portions containing the entire length of the root. Chop the portions into four segments and mix them together. Transfer g (moist weight) portions of the mixture into glass or plastic vials. Rinse the roots with a couple changes of water if needed.

70 DETECTING AND QUANTIFYING AMF COLONIZATION OF ROOTS Clearing roots The aim of clearing is to get rid of nuclear and cytoplasmic materials in order to facilitate maximal penetration of the stain. Clear roots by completely covering them with 10% KOH in deionized water (w/v) for h at ambient temperature. Pour off the KOH solution and rinse the root in at least four changes of water. If roots are dark or pigmented, they can be bleached before they are acidified and stained. The most commonly used bleaching material is alkaline H2O2. It is prepared by mixing 3 ml of NH4OH with 10% H2O2 and 567 ml of tap water. NH4OH may be replaced by the same volume of household ammonia. The duration of bleaching is minutes, after which the roots are rinsed with at least three changes of tap water.

71 DETECTING AND QUANTIFYING AMF COLONIZATION OF ROOTS Acidifying roots Roots must be acidified to facilitate retention of the stain by the target specimen. Cover the roots with 10% HCl for 5 10 minutes. Remove the acid but do not rinse the root after this step. Staining roots Cover roots with an acid fuchsin-lactic acid solution and incubate them at ambient temperature for h. The staining solution is prepared by dissolving 1.5 g of acid fuchsin in a solvent consisting of 63 ml of glycerine, 63 ml of water, and 875 ml of food-grade lactic acid.

72 DETECTING AND QUANTIFYING AMF COLONIZATION OF ROOTS Destaining roots To destain roots, decant the stain from the vials containing the roots and rinse the roots with used but filtered (Whatman #1 filter paper) destaining solution to get rid of the excess stain. Cover the roots with unused destaining solution which consists of the solvent mixture used for dissolving the dye. Incubate the vials at ambient temperature for h. At the end of this period, decant the destaining solution and add unused destaining solution. The roots now should be ready for observation. In each of the above steps in which incubation is involved, the h incubation period can be replaced by heating in a water bath at 90 C for 1 h or autoclaving at 121 C for 15 min, if one has the means for doing so.

73 DETECTING AND QUANTIFYING AMF COLONIZATION OF ROOTS Observing stained roots and estimating AMF colonization level Stained root fragments can be spread in petri plates or mounted on microscope slides and examined for the occurrence of typical AMF structures. The most accurate method of determining the level of infection is the grid line intersect method. In this method, stained root preparations are spread on petri plates with grid lines on the bottom. The roots are then examined under a stereo microscope at 40x magnification. Each intersection of root and gridline is checked for the presence or absence of AMF structure(s) and scored as colonized or not colonized by AMF. Using these values the percentage of AMF colonization can be calculated. In this technique, the grid lines simply serve to systematically locate points of observation. For best accuracy, at least 200 root-gridline intersects must be tallied, although 100 root-gridline intersects are acceptable in most instances. The method can also be used to estimate the proportion of the root length that is colonized by AMF.

74 DETECTING AND QUANTIFYING AMF COLONIZATION OF ROOTS The number of root-gridline intersects to the total length of root spread is related by the formula, R = πan/2h where R = the total length of root π =.1 16 A = the area in which roots are distributed n = the number of root-gridline intersections H = the total length of straight lines.

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76 A gridline intersection example using a 8.5 cm diameter round Petri dish with a 1/2 inch (14/11 cm) grid, and a 1 m test sample of thread cut into fragments and randomly redistributed 10 times (Figure 4.3 in Brundrett et al. 1996). Row and column totals are summarised in the table below. Redistributi on Intersects (cm) Average cm ± 2.5 (standard error)

77 DETERMINING THE ABUNDANCE OF INFECTIVE PROPAGULES IN CRUDE INOCULUM AND IN SOIL Procedures The technique is based on determining the presence or absence of microorganisms in several individual aliquots of each of several consecutive dilutions of a sample of soil or other materials containing microbial propagules. A serial dilution, usually 10-fold, of a soil or crude inoculum sample is prepared using sterile sand, soil, or sand-soil mixture as the diluent. From each dilution, a predetermined amount of material, say 20 g, is used to inoculate each of five cups containing g of sterile soil or sand-soil mixture optimized for mycorrhizal activity with a soil-solution P concentration of 0.02 mg/l. Germinated seeds or seedlings of a suitable mycorrhizal plant (onion, clover, leucaena, etc.) are sown in these cups, which are placed in a reservoir containing water or P-free nutrient solution.

78 DETERMINING THE ABUNDANCE OF INFECTIVE PROPAGULES IN CRUDE INOCULUM AND IN SOIL the indicator plant of choice for MPN determination is Leucaena leucocephala, and it is grown on a 1:1 mansand:soil mixture. The P concentration of the medium is 0.02 mg/l and its ph is 6.2. The medium is supplemented weekly with 100 ml of P-free Hoagland s solution. The plants are then allowed to grow in the greenhouse or growth chamber for four weeks. At the end of the growth period, the roots are excised, washed and cleared. The stained roots are spread in a petri dish and scored for the presence or absence of AMF colonization. Do not count detached hyphae or germinated spores.

79 DETERMINING THE ABUNDANCE OF INFECTIVE PROPAGULES IN CRUDE INOCULUM AND IN SOIL To calculate the most probable number of infective propagules in a sample, the statistical table developed by Cochran is essential. In the table, p1 stands for the number of positive replicates in the least concentrated dilution, and p2 and p3 represent the numbers of positive replicates in the next two higher dilutions. The most probable number of infective propagules in the quantity of the original sample is obtained by multiplying the reciprocal of the middle dilution by the number in the table located at the point of intersection of the experimentally observed values corresponding to p1, p2, and p3. The value represents the most probable number of infective propagules for the quantity of soil used to inoculate test plants (20 g in the current example). The number of infective propagules per gram of soil can be obtained by dividing the number of infective propagules observed by the quantity of soil.

80 DETERMINING THE ABUNDANCE OF INFECTIVE PROPAGULES IN CRUDE INOCULUM AND IN SOIL Suppose the following number of positive replicates are obtained for the following dilutions: 10 1 = = = = = 0 In this series, p1 = 5, p2 = 4, and p3 = 1 For this combination of p1, p2, and p3, Co hran s ta le gives 1.7 as the most probable number of infective propagules applied in the 10 2 dilution. Multiplying this value by the dilution factor 102 gives 107 as the number of infective propagules in the original sample. The number of infective propagules per gram of soil is calculated (107 / 20 = 5.35) to be approximately five.

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