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SPORE GERMINATION AND THE PRE-INFECTION PHASE IN ECTOMYCORRHIZAL FUNGI

Thesis submitted to the University of Surrey for the degree of Doctor of Philosophy in the

Department of Microbiology

by

Nahia A. Ali

November 1986

SUMMARY

Spore germination of seven species of ectomycorrhizal fungi was studied under different conditions.

Spores of most species tested did not germinate on laboratory media without stimulators. Various stimulators were tested and the greatest activation was that by Pseudomonas stutzeri which was obtained from fruitbodies of Hebeloma crustuliniforme and stimulated about 21% of spores of that fungus.

Effects of plant roots on spore germination were studied in the laboratory and in soil. In mineral salts medium, spores of Paxillus involutus were stimulated by five of seven tree species and one of four non-tree species tested. Spores of Laccaria laccata and fh_ crustuliniforme were stimulated by birch and pine only, while those of Lactarius turpis and Amanita fulva were only slightly stimulated by birch roots. Birch was more stimulatory and the highest germination (30%) was that of £U_ crustuliniforrne at 0-1mm from the root edge.

In soil, spores of P. involutus only,of the seven species tested ? were stimulated to germinate near roots of birch and spruce in steamed and untreated soils, Birch stimulated up to 96% ofspores to germinate in untreated birch wood soil. Spruce had much less effect on germination in either steamed or untreated soil. The majority of germ tubes grew towards the roots.

Exudate from roots of seedlings of birch previously grown in soil was active in stimulating germination of crustuliniforme, but not of the other six species tested. Activity of the root exudate was associated with the ninhydrin-positive fraction.

The role of the basidiospores of the test fungi ininitiating mycorrhizas in soil was also studied. Spores of P.involutus and laccata established mycorrhizas with birch, butnot spruce or pine roots. Spores of the other five species tested did not establish mycorrhizas with any of the plant tested.

SUMMARY............................................. i

LIST OF TABLES............................... ............. . .V

LIST OF PLATES............................... vii

LIST OF FIGURES ......................................... ix

Chapter 1 LITERATURE SURVEY..............................11.1 Classification of mycorrhizas................... 11.1.1 Myco r r h izas of Orchidaceae............. 11.1.2 Mycorrhizas of Ericales....................... 21.1.3 Vesicular-Arbuscular Mycorrhizas (VAM)..........21.1.4 Ectomycorrhizas.............................. 21 .2 Identification of ectomycorrhiza 1 fungi......... 41.3 Process of infection........................... 61.4 Host fungus specificity....................... 101.5 Importance of mycorrhizal association.......... 111.6 Interaction between mycorrhizas and other

microorganisms.............. 151.7 Basidiospores.................................191.7.1 Production.................................. 191.7.2 Genetics of basidiospores ................. 191.7.3 Spore function............................... 201.7.4 Spore germination ......................221.7.5 Spore dormancy...............................281.7.6 Spore longevity ................... .....301.8 The aims of this study....................... 32

Chapter 2 MATERIALS AND METHODS......................... 332.1 Laboratory Media........................ 332.2 Melzer1 s Reagent............................. 362.3 SV, Sulpho-vanilin........................... 362.4 Formalin-acetic-alcohol.......................36

CONTENTS

P a g e

2.5 Acetic-analine blue stain..................... 362.6 Preparation of saturated salt solution........ ..362.7 Root Exudate.......................... 392.8 Modified Fahraeus cells........................402.9 Procedure for Scanning Electron Microscope

(SEM) study.............................. .....412.10 Seeds............. 422.11 Soil......................................... 432.12 Mycorrhizal fungi............................. 46

Chapter 3 EXPERIMENTS.................................. 523.1 Germination without stimulants................. 523.2 Germination on laboratory medium in the

presence of stimulatory factors........... .563.3 Testing the effect of other microorganisms

on spore germination of the test fungi......... 603.4 Screening a wide range of Pseudomonas

aisolates for stimulatory activity on spore germination of Hebelcma crustulinifprime........ 70

3.5 Study of the nature of stimulatorycompounds produced by Pseudomonas stutzeri......72

3.6 Germination near plant roots in mineralsalts medium................................. 79

3.7 Germination near plant roots in soil............843.8 Response of Paxillus involutus basidiospores

to roots of host and non-host tree seedlingsin soil............................... 97

3.9 Germination in the presence of rootexudate "in vitro"...........................100

3.10 Relationship between number of spores andtheir germinability .................... 103

3.11 Characterization and identification of theactive compounds in the root exudate.......... 104

3.12 Effect of storage conditions on viabilityand germinability of basidiospores............ 116

P a g e

3.13 Testing the role of basidiospores ininitiating ectomycorrhizas................... 125

Chapter 4 DISCUSSION.................................. 1304.1 Spore germination............................ 1304.2 Germination without stimulants........... . 1334.3 Germination in the presence of stimulatory

factors..................... 1344.4 The role of basidiospores in initiating

mycorrhizas................................. 1474.5 Conclusion. ..........................150

ACKNOWLEDGEMENTS........................... 152

REFERENCES................................................153

APPENDIX 1 Germination of Scleroderma citrinumbasidiospores............................... 185

APPENDIX 2 Identification of isolates stimulatingspore germination............................186

APPENDIX 3 Statistical analyses ......................192

P a g e

i v

T ab le

L IS T OF TABLES

Page

1 Soil Analysis........................... 44

2 Species of ectomycorrhizal fungi collected and identified' during the periods (June - December) in 1983 and 1984 beneath birch trees at WhitmoorCommon, Surrey.............. 48

3 Concentration of glucose, malt extract and ammonium tartrate in Fries medium and indifferent combinations tested............... 53

4 Germination of basidiospores on Fries agar withor without living mycelium and activated charcoal.......58

5 Number and sources of isolates of bacteria andfungi that have been tested for stimulating germination........... 61

6 Total number of isolates tested and number of isolates stimulating germination of ectomycorrhizalfungi............................ 64

7 Identity, source, and stimulating activity of thetest bacterial and fungal isolates......... 65

8 Spore germination of crustuliniforme on Friesagar with different supplements ...... 67

9 Effect of iron and vitamins in its growth mediumon the stimulatory activity of stutzeri............. 77

10 Effect of iron and vitamins in the growth mediumon the total numbers of P. stutzeri................... 78

v

11 Germination of basidiospores near roots ofseedlings of different tree and crop species inmineral salts medium (up to eight weeks incubation)....82

12 Germination of Paxillus involutus basidiospores and growth of germ tubes near birch and spruceroots in birch wood soil after two weeks incubation....86

13 Length of germ tubes (urn) produced by Paxillusinvolutus basidiospores within 1mm of the root edge after two weeks being buried in birch wood soil....... 87

14 Germination % of Paxillus involutus basidiospores near roots of birch and spruce in two differentsoils (up to 1mm from the root edge)..................96

15 Effect of roots of host and non-host tree seedlingson germination of spores of E\_ involutus in soil..... ..99

Page

16 Relationship between spore densities and number of spores germinated in FL_ crustuliniforme onFries agar with 10% root exudate.................... 103

17 Germination of spores of Ety involutus following different periods of storage under different conditions................ 124

18 Development of mycorrhizas on seedlings of Betula pubescens grown in birch wood soil supplementedwith fresh spores of two mycorrhizal fungi...........127

v i

PlateL IS T OF PLATES

Page

1 Spores in Petri dishes stored over saturatedCaC12 6H20 in closed jar...... 38

2 Germinated spores of Fty crustuliniforme close toa colony of P. stutzeri (x250)........................ 68

3 Germinated spores of crustuliniforme close toa colony of Micrococcus roseus (x250)....... .......... 69

4 A tube containing Ingestad mineral solution, and Fahraeus cell filled with Ingestad mineral agarand planted with one birch seedling............. 80

5 Germinated spores of laccata and growth ofgerm tubes towards root of birch seedling inmineral salts medium in Fahraeus cell (x500)........... 83

6 Germinated spores of P^ involutus and growth ofgerm tubes towards root of birch seedling (R) ona slide buried in birch wood soil (x300)...............89

7 Spores of PL_ involutus showing germ tubes with two branches, on a slide buried in birch woodsoil (x1200)........................................ 90

8 Fan-shaped appressorium-like structure produced by germinated spore of involutus on a slideburied in birch wood soil (x1200)..... 91

9 Fan-shaped appressorium-like structures produced by germinated spores of |L_ involutus, A: on the surface of a detached cortical cell (CC) (x400),B: on the surface of a root hair (RH) (x1200). Onslides buried in birch wood soil................. 92

v i i

10 Mycorrhiza of birch produced by germinated spores of P^ involutus on a slide buried in birch woodsoil for eight weeks (x135)...........................93

11 Germ tubes from spores of involutus growing towards spores of the same species on a slideburied in birch wood soil (x400)...................... 94

12 Germ tubes from several spores of Eh. involutus growing and encircling the spores of the samespecies (x400).............................. 95

13 Mycelial colonies from spores of Ih_ crustuliniforme germinated on Fries agar plates containing,A: 1%, B: 5%, and C: 10% root exudate/agar............ 102

14 Fluorescein spores of P_j_ involutus stained withfluorescein diacetate (FDA) (x1200)...................119

15 Fluorescein spores of EL_ crustuliniforme stainedwith fluorescein diacetate (FDA) (x1200).............. 120

16 Mycorrhiza of birch with laccata in birch woodsoil (scanning electron microscope).................. 129

Page

17 Enlargement of the above, showing a clampconnection......................................... 129

L IS T OF FIGURES

Figure Page

1 Occurrence and abundancy of the test fungi duringthe season of 1983 and 1984 around birch trees in Whitmoor Common, Surrey..... .........................49

2 Centricon-10, in the concentration mode............... 113

ix

Chapter 1: LITERATURE SURVEY

Mycorrhiza (fungus root): is a symbiotic relationshipbetween the susceptible host root and the mycelium of the fungus.

The mycorrhizal phenomenon was first described by Unger in 1840. He illustrated the mantle but did not recognize its fungal nature. At the same time Hartig (1840) described the mantle and the subsequently termed "Hartig net" without recognizing their fungal nature. The history of mycorrhizas begins with Frank (1885) when the term "Mycorhiza" (with one r) was proposed for the first time. He emphasised that the mycorrhizal rootlets are covered by a mantle of fungal tissue from which hyphae pass inwards between the cells and into the soil. The term mycorrhiza has since been adopted for many other mutualistic associations between soil fungi and below-ground plant organs, seme of which, notably the protocorms of orchids, are not even roots.

1.1 Classification of mycorrhizas

Mycorrhizal associations are usually divided into four main groups: 1). ectomycorrhizas or sheathing mycorrhizas; 2).vesicular-arbuscular(VA) mycorrhizas? 3). mycorrhizas of the Ericales, and 4). mycorrhizas of the Orchidaceae (Lewis, 1973? 1975? 1976? and Read, 1982).

The last three groups were called endamycorrhi zas byPeyronel et al. (1969), and they used the tern ectendamycorrhizasto describe a group of mycorrhizas which share featurescharacteristic of both sheathing mycorrhizas and endcmycorrhizas(Hofsten 1969; Laiho 1965; Wilcox 1971).

1.1.1 Mycorrhizas of Orchidaceae

This kind of mycorrhiza is associated with Basidiomycetes (eg. genus Rhizoctonia) of various affinity which are able to break down cellulose and sometimes lignin. Orchids comprise a range of types, from species containing abundant chlorophyll at maturity to

1

completely achlorophyllous and presumably saprophytic forms. The tissues of the host plant are penetrated by septate hyphae of the causal fungus that often bear clamp connections. The hyphae form intracellular loops and coils known as pelotons. A characteristic feature of the orchid mycorrhiza is degradation of the intracellular fungal structures by the host.

1.1.2 Mycorrhizas of Ericales

Mycorrhizas of Ericales are of two types: (1) ericoid type, which is formed by a penetration of the root cortex by septate hyphae (usually of Ascomycetes eg. Pezizella ericae) which form intracellular coils (Harley, 1959 and 1969); (2) arbutoid typewhich resemble the ectendo type, in which there is an organized fungal sheath as well as inter- and intracellular penetration of the cortex (Lutz and Sjolund, 1973).

1.1.3 Vesicular-Arbuscular Mycorrhizas (VAM)

In this kind of mycorrhiza the primary cortex of the host root is invaded both inter- and intracel lularly by aseptate hyphae of fungi belonging to the family Endogonaceae (irregular septa may occur occasionally). The fungi form branched haustoria (arbuscules) in the cells and swollen vesicles inside and outside the host tissues. Many form spores on mycelia or in sporocarps of complex structure. VAM occur in a wide range of hosts which are economically important, eg. members of the Rosaceae, Gramineae, and Leguminoseae. These include all grain crops and the majority of temperate fruit trees and shrubs. Crops such as citrus, coffee, tea, rubber, and sugar cane also have VA mycorrhizas.

1.1.4 Ectomycorrhizas

The majority of fungi which form ectomycorrhizas with forest trees are Basidicmycetes. Ascomycetes contain few genera only which form ectomycorrhizas, and most of them have hypogeous sporophores,

2

eg. the truffle-forming fungi Tuber, Terfezia and Tirmania. The imperfect fungus Cenococcum graniforme and E - strain fungi belong to the Ascomycetes and also form ectomycorrhizas.

In the ectomycorrhizas the roots are surrounded by a more or less well-developed fungal sheath or mantle. From this sheath hyphae or hyphal cords may extend into the soil and into the base of fruitbodies of the fungi. Hyphae also penetrate intercellularly into the host cortex forming a complex network of hyphae, called the Hartig net, which may completely replace the middle lamella between the cortical cells. Usually there is little or no hyphal penetration into the cells of the host of young mycorrhizas, but in the senescent parts of a mycorrhizal axis, the cortex becomes colonized by hyphae within the cells (Atkinson, 1975). The hyphae never penetrate the endodermal tissues and the stele.

Generally, ectomycorrhizas are formed only on roots with primary tissues. Secondarily thickened roots remain uninfected. Mycorrhizal tree roots are often divided into long roots that grow continuously, and short roots that branch repeatedly and grow little. The majority of ectomycorrhizas are formed on the short roots. Sometimes, a loose sheath and no Hartig net may be formed on the long roots (Harley, 1969). Whether formation of short roots precedes, or is a result of infection is still controversial.

Ectomycorrhizas occur chiefly in temperate tree species. All members of the gymnosperm family Pinaceae eg. Pinus, Picea, Pseudotsuga, Larix, Tsuga, etc., as well as certain angiosperms, eg. Juglandaceae (Juglans, Cary a)? Fagaceae (Quercus, Nothof agus); Betulaceae (Betula); and Myrtaceae (Eucalyptus, whose species are important components of forest and bush in temperate andsub-tropical Australasia) are ectomycorrhizal. Also, some tree species of the family Dipterocarpaceae (belonging to the tropical regions) have been proved to be ectomycorrhizal.

3

1 .2 Id e n t if ic a t io n o f e c to m y c o rrh iza l fu n g i:

Fungi associated with ectomycorrhizas in nature m y be characterized by morphological or anatomical features of the mycorrhizal structure to which they give rise (Dominik, 1969 and Zak, 1971a), such as sheath colour, the shape and size of the mycorrhizas, the presence of surrounding mycelium and rhizcmorphs, sheath texture and thickness, chemical colour reactions andfluorescence, odour and taste, clamp connections, if present, and hyphal form.

As the above characteristics in seme fungi m y change withage and environmental conditions; the following methods are alsouseful for identification of seme ectcmycorrhizal fungi.

1). Tracing the cords and hyphae from a known sporocarp tothe underlying mycorrhizas. This method is applicable only to those mycorrhizal fungi which form sporocarps (Chilvers, 1968; Schramm, 1966; Woodroof, 1933; Zak, 1969; Zak and Marx, 1964).

2). Comparison of the fungus isolated from the mycorrhiza withknown cultures derived from sporocarps (Lamb and Richards, 1970). This method is of course limited to those fungi that will grow on laboratory media.

s3). Mating studies between compatible monoporous mycelia or monokaryons are also useful for identifying mycorrhizal fungi; hyphal fusions, or anastomoses between two compatible monokaryons will lead to dikaryotic hyphae.

Fries and Mueller (1984) examined the utility of employing mating studies to determine the degree of intra- and interspecific genetic isolation in Laccaria. In their study, no clamps were found in pairings between monokaryons from different species or groups.

Mueller and Fries (1985) were able to identify 74 isolates (representing ten Laccaria species) to species or species group by cultural and mating studies. In their study, monokaryotic mycelium was formed by dedikaryotization of dikaryotic mycelium (Kemp, 1974;

4

and Leal-Lara and Eger-Hummel, 1982).

4). Pure culture synthesis of mycorrhizaWith the culturable mycorrhizal fungi, it is possible to

re-form the mycorrhizas on seedlings in pure culture synthesis (Hacskaylo, 1953? Marx and Zak, 1965? Melin,1921; Pachlewski and Pachlewska, 1968), and compare the resulting mycorrhizas with those found in nature. Under artificial conditions the synthesized mycorrhizas may differ in their morphology from those occurring under natural conditions (Zak, 1971b)? this could be due to the effect of the composition of the growth medium on the synthesized mycorrhizas (Thomas and Jackson, 1979). Production of sporocarps by synthesized mycorrhizas will permit a positive identification of the fungus.

5). Another technique successfully used for identification of ectomychorrizal species is immunofluorescence (Schmidt et al.,1974). By this technique protein antisera were prepared against two mycorrhizal fungi (Thelephora terrestris and Pisolithus tinctorius) by injection of the antigens (washed, homogenized mycelia of the test fungi in saline) in rabbits. One week later the blood was collected by cardiac puncture, and the antiserum fractionated and labelled with fluorescein isothiocyanate (Bohlool and Schmidt, 1970? Schmidt et al., 1968).

Schmidt et al. (1974) found that the fluorescent antibodies of each mycorrhizal fungus when tested on mycelial smears reacted strongly with their corresponding fungus antigen.

6). Recently an unambiguous method was used to identifyectmycorrhizal fungi, using dot-blot DNA hybridization, in which the DNA was isolated from repeatedly washed mycelium and/or fruitbodies. By this method Straatsma et al. (1985) proved that the different Cantharellus cibarius cultures, they obtained bydifferent methods, were genetically identical to fruitbodies of C. cibarius found in nature.

5

1.3 Process of infection:

Ectomycorrhizal infections are assumed to be initiated from propagules in soil such as basidiospores; chlamydospores; sclerotia; or from vegetative mycelia from nearby ectomycorrhizal roots.

The means by which the hyphae of mycorrhizal fungi penetrate the host tissue is unclear. However, there is no visible degradation or alteration of cells in ectomycorrhizal root formation (Foster and Marks, 1966 and 1967; Marks and Foster 1973).

The process of intercellular penetration in ectomycorrhizal formation is predominantly mechanical; the walls between the cortical cells of the host root are separated or loosened at the middle lamella due to the effect of the fungal hyphae. The hyphae then wedge themselves into the fissures so formed and advance further by mechanical pressure (Foster and Marks, 1967). Whether enzyme production is also involved is not known; but Ramstedt and Soderhall (1983) indicated that protease and phenoloxidase produced by the fungus may have a role in the penetration process, whereaspectinase activity has never been shown to be significant inmycorrhizal fungi.

Harley and Smith (1983) hypothesized the role of the fungal enzymes in the penetration process as follows1). The fungal enzymes at the hyphal apex or onthe hyphal walls might inhibit or complex with host polymerizing enzymes (which are involved in wall polymer building, Vanderplank, 1978) resulting in deactivation of polymerizing enzymes and alteration of wall formation, thus soluble carbohydrates will be available to the fungus.2). Alternatively, Harley and Smith (1983) suggested that all ectomycorrhizal fungi possess enzymes on their walls capable of hydrolysing the wall material of their hosts.

Evidence to confirm these hypotheses has not yet beenobtained.

After the penetration process is achieved; the hyphae spread longitudinally between the cells of the long root cortex and branch

6

to infect newly emerging short roots. The infection can also spread externally from the sheath by hyphal cords from one short root to another {Robertson, 1954).

1.3.1 Factors affecting infection

The process of infection is influenced by soil factors; the properties of the host root and the interactions between these factors and the environment:

1). Soil nutrient:-Many studies in the literature reported that the intensity

of infection is greater under conditions of low or unbalanced nutrient supply (Hatch, 1936; Marx et al., 1977; Schmidt, 1947; Stahl, 1900); starvation levels of essential nutrients decrease the intensity (number of roots infected) of mycorrhizas (Hatch, 1936 and 1937). Bjorkman (1942) reported that very severe deficienciesof P or N have an adverse effect on mycorrhizal formation. However, when these nutrients are present at low levels the mycorrhizalintensity is increased.

2). Soil pH: -Ectomycorrhizas are present over a wide pH range, but more

abundant in raw humus soils (pH 3.5 - 4.0) than in mull soils(pH5.0 - 5.5) (Mosse et al., 1982). The effect of soil pH onectomycorrhiza formation is difficult to evaluate; changes in soil pH lead to changes in the chemical properties of the soil such as the release of soluble Al, Mn, and Fe under low soil pH (Nye and Ramzan, 1979). Thus the effects on mycorrhiza formation may be due to these metals rather than pH.

3). Soil organic matter:-Although no direct chemical effects of soil organic matter

have been reported; the organic matter affects mycorrhiza formation through its effect on soil structure, water holding capacity, nutrient mineralization, etc. or due to the presence of substanceswhich stimulate the growth of both fungus and host (Melin, 1953).

7

4). Soil moisture and aeration:-Soil with excessive water may affect mycorrhiza formation

because it reduces the availability of 02 (Miller and Laursen, 1978), since this gas diffuses slowly through water, but often soil water contains sufficient dissolved 02 to prevent anaerobiosis (Scott-Russell, 1977).

The lack of 02 affects mycorrhizas, which are strongly aerobic (Harley, 1969), only at the pre-infection phase; since 02 is transported down air-filled spaces within the plant from shoot to root (Greenwood, 1967). Also Read and Armstrong (1972) reported that ectomycorrhizal infection may be initially stimulated by 02 diffusing from host roots.

Soil with low water affects root growth and accelerates suberization, which sometimes leads to changes in the fungal partner (Marks and Foster, 1967? Worley and Hacskaylo, 1959), eg. the fungus Cenococcum graniforme replaced unidentified white fungi on Pinus virginiana under dry conditions (Meyer, 1964).

5). Temperature:-In general ectomycorrhizal fungi grow best around 20C, but

mycorrhizal development has been shown to be strongly temperature dependent (Hacskaylo and Vozzo, 1965; Marx et al., 1970a? Melin, 1925; Mikola, 1948; Moser, 1956). Temperature may affect mycorrhiza formation through its effect on root growth and physiology (Barney, 1951; Lyr and Hoffmann, 1967).

6). Light:-Mycorrhiza formation is strongly affected by light

intensity; at high light intensity large numbers of short roots are colonized with mycorrhizal fungi (Bjorkman, 1942? Hatch, 1937).

The light received by the shoot has indirect effects on root development; reduction in light intensity reduces shoot growth (Anonymous, 1953; Brown, 1955? Daft and El-Ghiahmi, 1978? Elison, 1968? Hayman, 1974? Richardson, 1956) and the number of new roots produced (Hoffmann, 1967). These studies provide strong evidence that root growth is stimulated by substances photosynthesized in

8

the shoot. This interaction between light, shoot, and root may affect mycorrhizal colonization.

7). Growth hormonesGrowth hormones such as auxins, cytokinins, gibberellins

produced by the mycorrhizal fungi (Fortin, 1967 and 1970; Slankis, 1973), may exert a regulatory influence on mycorrhiza formation. There is not enough information about the mechanism through which the fungal hormones influence mycorrhizal formation; but they may have a role in mobilization of carbohydrates in plants (Meyer, 1966) or as found from a study in general plant physiology, fungal hormones (cytokinins) may cause accumulation of amino acids,phosphates, and various other substances in the localized areas to which these hormones are applied (Gunning and Barkley, 1963;Mothes, 1960). Slankis (1971) has described experiments on the relationship between auxin production by the fungus, soil nitrogen level and light level, and their combined effects on the plant root. He found that high nitrogen levels or low light levelsinhibited mycorrhizal formation, but fran additional studies withauxin he speculated that high nitrogen inhibits the synthesis of auxin by the fungus and at low light intensity some factorinterferes with fungal auxin. He interpreted this factor as being either the prevention of a required interaction between fungal auxin and some root metabolite, or possibly the formation of auxin inhibitors in the root.

9

1.4 Host-fungus specificity

In mycorrhizal associations the degree of specificitybetween fungi and their hosts is widely variable. One species of mycorrhizal fungus may form mycorrhizal relationships with more than one species of host, eg. the fungus Cenococcum geophilum (graniforme) forms ectanycorrhizas with the species of more than 20 genera of vascular plant {Harley and Smith, 1983), and the fungus Pisolithus tinctorius infects many hosts (Marx, 1977). A few fungi are reported to be very restricted in their hosts, for example Suillus grevillei (Boletus elegans) which forms mycorrhizas with species of Larix only (Meyer, 1973). Also any one species of host plant may form mycorrhizas with many species of fungi,eg. Douglas fir (Pseudotsuga menziesii) can probably form mycorrhizas with 2000 species (Trappe, 1977), while a hostaccepting only one species is unknown. Meyer (1973) reported that under natural conditions, species of Abies, Larix, Picea, Pirns, Fagus and Quercus are obligate ectcmycorrhizal trees. Whereas others such as Cupressus, Salix, Betula, Alnus and Eucalyptus are more facultative. On the other hand, most ectcmycorrhizal fungi are ecologically obligate-parasites (Garrett, 1960).

Frcm field observations, Molina and Trappe (1982a)recognized what they called 11 sporocarp-specific host associations’*. In these a fungus is only known to fruit either in association with a single genus, a single species, or with a wider taxon, eg. Pinaceae. They recognized that the mycelium of a fungal species may exist ecologically in other associations than those in which the fruitbodies are found. This phenomenon has been described by Harley and Smith (1983) as "ecological specificity".

10

1.5 Importance of the mycorrhizal associationbetween plants and fungi

The mycorrhizal association is the most widespread syrabiosisX and very common under natural soil conditions. There are usually outgrowths of hyphae from the surface of mycorrhizas into the soil, sometimes extending several centimetres from the root surface which, in some ectomycorrhizas, form aggregates, hyphal cords or highly specialized rhizcmorphs (Skinner and Bowen, 1974a,b; Trappe and Fogel, 1977). This may help in binding soil particles and thus increase soil stabilization and perhaps erosion control (Smith,1980). The ability of fungal mycelia to bind sand and soil particles had been observed by Koske et al. (1975) and confirmed by Tisdall and Oades (1979). They reported that hyphae have an important function in increasing stable soil aggregates greater than 2.0mm.

The mycorrhizal relationship is usually beneficial to both partners (fungus and plant). In most ectomycorrhizas; the fungi appear to be dependent on their hosts for carbon and energy sources (Harley, 1978; Last et al., 1979; Melin, 1921; Rcmmell, 1938).Melin and Nilsson (1957) detected 14C-labelled photosynthate in the fungal sheath of Pinus sylvestris mycorrhizas. Also, Lewis and Harley (1965) showed that 14C-labelled sucrose fed to cut stumps of Fagus sylvatica L. was translocated towards the tips of mycorrhizas and converted into trehalose, mannitol and glycogen which cannot be reabsorbed by the host root. From this study and others (Hacskaylo, 1973; Reid and Woods, 1969) it can be deduced that the mycorrhiza acts as a sink in which the carbohydrates are accumulated.

In general, the most important function of the mycorrhizal association is its ability to improve the growth and physiological characteristics of the host plant:

Mycorrhizas improve the host growth by providing an efficient nutrient and water absorbing root system (Hatch, 1937), especially in soils of low or imbalanced nutrient status (Hall et al., 1977; Voigt, 1971).

.Mycorrhizas are more efficient than non-mycorrhizal roots in the absorption of soluble soil phosphate. Different "in vitro"

11

systems have been successfully used to demonstrate the fungal pathway of phosphate uptake in different mycorrhizal associations (Harley and Smith, 1983). The extramatrical hyphae absorb phosphate and translocate it to the host root; in this way they act as an extension of the root and as a bypass for phosphate through the depleted zone which develops around roots. Finlay and Read (1986) reported that labelled phosphorus fed to the distal part of the fungal mycelium frcm infected roots, moves freely through the strands over distances in excess of 40cm within 84 hours.

Nothing is known of the absorbing properties of the external mycelium of mycorrhizas, for instance, where hyphae absorb or whether they have high affinity absorption sites for phosphate. However, much of the detailed experimental work on phosphate absorption by hyphae of ectomycorrhizal fungi has been done with excised mycorrhizas where most of the extramatrical hyphae and also the shoots have been eliminated. Thus the results of theseexperiments may show less phosphate absorption than that which occurs in the intact mycorrhizas, or in nature. As could be expected, uptake by fungal sheaths is an active, metabolically dependent process; being temperature sensitive and requiring oxygen (Harley et al. , 1958). The results obtained with excised mycelialstrands indicate that the uptake by external hyphae is also an active, metabolically dependent process (Skinner and Bowen, 1974).

Hyphae of mycorrhizal fungi can accumulate and store reserve phosphorus as polyphosphate in their vacuoles (Ashford et al., 1975; Chilvers and Harley, 1980). This sequestration ofphosphate into the vacuoles no doubt permits the fungi to accumulate large amounts of phosphorus which would otherwiseinterfere with cell metabolism (Bowen, 1970).

In mycorrhiza, polyphosphate has been implicated in both translocation and storage of phosphorus within fungal hyphae. Asfar as translocation is concerned, most studies have been made with the VA mycorrhizal fungus Glomus mosseae. High phosphorus flux rates in the fungal hyphae show that this fungus transports phosphate rapidly (Cooper and Tinker, 1978; Pearson and Tinker,1975). Cooper and Tinker (1981) reported that the transport process

12

is metabolically dependent; as the translocation rate is reduced by low temperature, and requires oxygen. Also they reported that cytoplasmic streaming is the mechanism which would most likely account for the high phosphorus flux rates in hyphae of mycorrhizalfungi; since translocation of phosphate was stopped by"cytochalasin B" an inhibitor of cytoplasmic streaming. Rapid cytoplasmic current would carry the phosphate down a concentration gradient in the cytoplasm; this concentration gradient would depend on phosphate uptake rates in the external hyphae and phosphate removal rates from the internal hyphae to the host cells, as wellas the efficiency of the stirring by the cytoplasmic streaming along the pathway of translocation.

The other possible mechanism for phosphate translocation could be by the movement of the granule-containing vacuoles in the streaming cytoplasm and, most likely by continuous loading andunloading of their contents to maintain the concentration ofphosphate in the cytoplasm and so ensure its rapid displacement in the cytoplasmic currents (Tinker, 1975).

Polyphosphate formation is usually considered more as asystem for storing large amounts of phosphate than as a transportsystem for phosphorous.

When cytoplasmic orthophosphate levels fall, thepolyphosphate is hydrolysed. This has been demonstrated in a Hebeloma species by Martin et al. (1985), also Harley and Brierley (1954) had shown that under these conditions phosphate moves out from the fungus into the host tissues in ectomycorrhizas. This ability of fungal sheaths in ectomycorrhizas to accumulate large amounts of phosphorus as polyphosphate and rapidly mobilise it when required (Bowen, 1973), makes them a potentially important storage organ for the host plant.

Ectomycorrhizal fungi have also been shown to be active in solubilising organic (insoluble) phosphate such as phytate, in pure culture experiments. Theodorou (1968, 1971a) demonstrated thatmycorrhizal fungi such as strains of Rhizopogon luteolus, Boletus luteus and Cenococcum graniforme (geophilum) are able to hydrolyse phytates (inositol hexaphosphate and lower phosphate esters of

13

myo-inositol) and to use the soluble phosphate for growth. Similarly Ho and Zak (1979) showed that strains of six mycorrhizal fungi, Hebeloma crustuliniforme, Laccaria laccata, Amanita muscaria, Thelephora terrestris, Piloderma bicolor (Co2±icium coroceum), Rhizopogon vinicolor, hydrolyse p-nitrophenyl phosphate by surface phytases. Bousquet et al. (1986) reported that Pisolithus tinctorius can hydrolyse para-nitrophenyl phosphate and Na phytate by surface phosphatase and phytase. Phosphatase activity was found to be increased in conditions of phosphate deficiency (Alexander and Hardy, 1981; Calleja et al., 1980). These findings may explain why, in certain soils, ectomycorrhizal plants sometimes respond more than non-mycorrhizal plants to rock phosphate applications.

Mycorrhizal roots have advantages over non-mycorrhizal roots: they are more resistant to infection by root pathogens(Levisohn, 1954 and 1957; Marx and Bryan, 1969; Marx and Davey,1969a,b). Mycorrhizas protect host roots from attack by pathogens by mechanical and chemical means (Zak, 1964). In most ectomycorrhi zas the thick fungal mantle (sheath) provides a mechanical barrier against the pathogens. Protection may also be afforded by antibiotic production. Marx (1969a,b) reported that some mycorrhizal fungi (Laccaria laccata; Lactarius deliciosus; Leucopaxillus cerealis; Pisolithus tinctorius; and Suillus luteus) produce antibiotics in agar culture which inhibit growth of 44% of the root pathogens tested (eg. Phytophthora cinnamomi and Pythium spp.). Mycorrhizal associations may result in production of metabolites which inhibit growth of root pathogens (Marx, 1972).

Besides protection against pathogens, mycorrhizal associations make the host root more tolerant to frost, high soil temperatures (Cromer, 1935; Harley,1940; Marx and Bryan, 1971; Marx et al., 1970a), drought (Muttiah, 1972; Worley and Hacskaylo,1959), soil phytotoxins (Benson et al., 1980; Bowen, 1973; Zak,1971a) and certain biocides (Voigt, 1954). Mycorrhizas formed byPisolithus tinctorius could partly inactivate high concentrations of toxic metals, such as Zn, resulting from application of sewage sludge (Berry and Marx, 1976).

14

Interaction between mycorrhizas and other microorganisms

Antagonism, competition or synergism between microorganisms in the soil and the rhizoplane are probably the most important, but the least understood, phenomena in rhizosphere biology. These phenomena are extremely difficult to study under natural conditions, especially in mycorrhizas because of their complex ecosystems. However, investigators often extrapolate fromlaboratory experiments on mycorrhizas to the field situations.

The microbial flora in the mycorrhizosphere (zone in the soil surrounding mycorrhizal roots) often differs from that in the rhizosphere of non-mycorrhizal roots, for instance, the pine mycorrhizosphere was reported to contain 9-10 times larger populations of fungi (Tribunskaya, 1955), and that of the yellow birch about a ten times larger bacterial population (Katznelson et al., 1963) than the rhizosphere of non-mycorrhizal roots.

Katznelson et al. (1962) showed that non mycorrhizal birch roots harbour mostly bacteria with simple mineral nutrient requirements, whereas those associated with mycorrizal roots have more complex amino acid requirements and a lower proportion of rapidly growing phosphate-solubilizing bacteria. Also, Foster and Marks (1967) found that different types of microbial populations are associated with different types of mycorrhizas on the same host plant.

Neal et al. (1964) examined the rhizosphere microfloras of three morphologically different mycorrhizas of a Douglas-fir, and compared them with the microflora surrounding adjacent suberized roots and in non-rhizosphere soil. They found that the total population of bacteria surrounding mycorrhizal roots and suberized roots is significantly greater than those found in non-rhizosphere soil. Rhizospheres of yellow and grey mycorrhizas have higher populations of bacteria tnan the rhizosphere of suberized roots. However, the latter contain more bacteria than the rhizosphere of white mycorrhizas. In all the rhizospheres with the exception of the rhizosphere of the white mycorrhiza, the number of streptcmycetes is significantly higher than in the non-rhizosphere

15

soil. Mould counts are lower in mycorrhizal rhizospheres than in the non-rhizospere soil. In all rhizospheres, the predominating moulds are members of the genus Penicillium.

From all these studies, it can be concluded that the microflora surrounding the mycorrhizal roots is influenced quantitatively and qualitatively by the type of mycorrhizal fungi present. This influence may be attributed, in part, to the availability or supply of inorganic and organic nutrients which could favour selective development of specific groups or species of microorganisms around the mycorrhizal roots.

Mycorrhizal establishment may be inhibited or stimulated by the presence of other microorganisms:

Rayner and Neilson-Jones (1944), demonstrated that pine and some other conifer seedlings planted in heathland soil failed to establish ectomycorrhizas even though the soil harboured the ectomycorrhizal fungus Suillus (Boletus) bovinus. This inhibition has been attributed to gliotoxin produced by certain Penicillium species (Brian et al., 1945), and to the antagonistic action of the Calluna endophyte (Handley, 1963).

Levisohn (1957), found that Alternaria tenuis, which is common in arable soil, inhibited growth of Rhizopogon luteolus and four Boletus species in laboratory media, and that in the field only Leccinum scabrum was established, but not the othermycorrhizal fungi that were more susceptible to tenuis in laboratory media. Theodorou (1967) succeeded in establishing symbiosis between Pinus radiata and Rhizopogon luteolus in nursery soil only after elimination of antagonists by partial soilsterilization.

Inoculation with Trichoderma and Azotobacter promotedmycorrhiza formation with oak and pine seedlings (Malyshkin, 1955; Tribunskaya, 1955; Vedenyapina, 1955). Trichoderma lignorum wasmore stimulatory than Azotobacter on pine. Greatest development of oak mycorrhiza was achieved with the above organisms combined with fluorescent bacteria (Shemakhanova, 1962). In nature, Azotobacter chroococcum, Trichoderma lignorum and Pseudomonas fluorescens were found closely associated with oak roots throughout the year(Malyshkin, 1955).

16

Bowen and Theodorou (1979) tested the interactions between eight bacteria isolated from different sources and eightectomycorrhizal fungi on laboratory media and in the rhizoplane of Pinus radiata. They found that different bacteria could depress, have no effect, or even stimulate root colonization by mycorrrhizal fungi and that such effects did not necessarily correspond to their "in vitro" effects on fungal growth. Seme bacteria gave protection against the depressive effects of others. Depressive effects may be based on antibiosis or competition for nutrients. The mostinteresting finding by those authors was that the strain of Bacillus that stimulated growth of Rhizopogon luteolus was isolated from washed mycorrhizas of Pinus radiata roots with R _ luteolus.

Meyer and Lindermann (1986) in their experiments on the inoculation of clover (Trifolium subterraneum) with VAM fungi and a plant growth-promoting bacterium PGPR (Pseudomonas putida), found that the VAM fungus infection in the roots was increasedsignificantly from 7 to 23% by the presence of PGPR at six weeks, also they observed significant increase in root and shoot dry weight when both the PGPR and VAM fungus were present compared to that in the presence of PGPR alone, VAM fungus alone oruninoculated controls.

Although the presence of Pseudomonas spp or other bacteria is not essential for VAM establishment (Mosse and Hepper, 1975), infection by VAM fungi in nature may be aided by the better infection conditions created at or near the root surface by these common rhizosphere bacteria. However, it should be realized that pseudcmonads in the rhizosphere are a diverse group of bacteria and their interactions with the plant and with mycorrhizal fungi will differ between strains of Pseudomonas■ For example, Bowen and Theodorou (1979) observed a fluorescent pseudomonad markedly suppressing the development of several ectomycorrhizal fungal species in pine roots.

Chakraborty et al. (1985), tested the effect of threemycophagous amoebae on the colonization of Pinus radiata roots by the ectcmycorrhizal fungus Rhizopogon luteolus. They found that two

17

mycophagous amoebae (Saccamoebae sp. and Gephyramoeba sp.), reduced the colonization of pine roots in soil by the ectomycorrhizal fungus luteolus when added at the same time as the fungus or two weeks later. The occurrence of mycophagous amoebae as frequent colonizers of the rhizosphere in several countries (Chakraborty, 1983; Old and Patrick, 1979) and their ability to feed on a wide range of fungal species, including vesicular-arbuscular and ectomycorrhizal fungi, has been recorded (Old and Patrick, 1979).

18

1.7.1 Production

In most hymenomycetous ectomycorrhizal fungi, basidiocarps are built up by complicated interwoven hyphae of the filamentous mycelium. The caps of these basidiocarps bear gills or tubes on their lower surfaces. The gills or tubes are also composed of interwoven hyphae. These hyphae produce a series of specialized club-shaped cells, the basidia in a layer termed the hymenium. On the apex of these basidia, basidiospores develop on stalks (sterigmata) from which, as they mature, the spores are discharged in a violent manner. Buller (1958) reported that discharging of spores in hymenomycetous fungi occurs continuously at a fairly consistent rate. They are never all discharged simultaneously or set free in intermittent showers. The process of spore-discharge often requires a considerable period of time. This may be conveniently called "the spore-fall period" (Buller, 1958). So far no report is published about the spore-fall period in ectomycorrhizal fungi.

1.7.2 Genetics of basidiospores

There is little information about the cytology and nuclear behaviour of basidiospores of ectomycorrhizal basidicmycetes, but they conform to the usual pattern of nuclear behaviour in higher basidicmycetes (Harley and Smith, 1983). Generally, in hymenomycetes (eg. Agarics) each hymenial cell, when first formed, contains two nuclei (Buller, 1958; Olah et al.y 1977). In cells destined to become basidia, the nuclei fuse to form a single diploid nucleus (Karyogamy). By means of two successive meiosis divisions the fusion nucleus then divides into four haploid nuclei, whereupon the sterigmata and spores begin their development. When the spores have attained a certain size, the four nuclei each simultaneously approach the four sterigmata, creep through them, and pass into the spores, each of which thus becomes provided with

1.7 Basidiospores

19

a single haploid nucleus. The spores upon gemination, produce a primary mycelium, the cells of which are uninucleate. When compatible pairs of such mycelia are grown together, Plasmogamous fusions occur between certain compatible cells, but nuclear union is delayed until basidia are formed. This plasmogamy initiates the secondary mycelium, with binucleate (n + n) cells from which the basidiocarp is formed.

In the ectcmycorrhiza fungus Hebelcma spp., Bruchet et al.(1986) reported that, as in most members of the Agaricales studied,spores receive one of the four haploid nuclei arising frcm thenuclear divisions which occur in the basidium, and this haploidnucleus divides mitotically in the spore so that the spores arebinucleate. Also they reported that the spores germinate to form

a primary (or hcmokaryotic) mycelium composed of cells all of whichare uninucleate except for certain species (Hebeloma sinapizans) inwhich the growing cells remain pluri- to multi-nucleate. Plasmogamybetween two compatible hcmokaryotic mycelia will initiate thesecondary mycelium which is always made up of binucleate (n + n)cells, the two compatible nuclei forming a dikaryon. oftenSecondary mycelia^display clamp connections.

1.7.3 Spore function

While mycelia of ectomycorrhizal fungi can survive successfully after inoculation into sterile soils (Theodorou, 1967) and in natural soils under ideal conditions, they frequently fail or establish poorly under other field conditions, eg. where soil dries out or where biological antagonisms occur (Theodorou, 1971b). However, Lamb and Richards (1974) have shown that basidiospores of ectomycorrhizal fungi can survive relatively high soil temperatures (c.40t!) and low relative humidity (c.30%) which are not suitable for the hyphae. This has great ecological importance in natural wind-borne dispersion and also has practical importance on inoculation practices (Bowen and Theodorou, 1973).

Basidiospore function in mycorrhizal initiation is little understood. However, it is apparent that the initiation of the

20

symbiosis is dependent upon the availability of susceptible host root and the presence of viable infective propagules of the fungal symbiont. Although mycelium is known to grow internally along with the growing mother roots, thereby infecting newly emerging lateral roots as they move through the cortex (Clowes, 1951; Robertson, 1954; Wilcox, 1968), the initial infection of the root system must be derived from external sources eg. vegetative mycelium from nearly ectomycorrhizal roots or from basidiospores, or other propagules. The frequent occurrence of ectcmycorrhizal infection on trees grown in fumigated nursery beds (Trappe and Strand, 1969) and ectcmycorrhizal synthesis on trees grown in containers of sterile soil (Marx et al., 1970b; Robertson, 1954) have been reported.These studies suggested that the active propagules which initiate infections under these conditions are air-borne basidiospores.

Direct documentation on the role of basidiospores ininitiation of infection is limited for most ectcmycorrhizal fungi.Theodorou (1971b) presents evidence that spores of Rhizopogon luteolus can initiate ectomycorrhizas on Pinus radiata. But theinclusion of some mycelium in the spore inoculum diminishes theclarity of this finding. Using spores as inoculum, Thapar et al. (1967) found that Scleroderma verrucosum formed ectomycorrhizas with Eucalyptus grandis but reisolation to verify the fungal symbiont was not attempted. Marx and co-workers have shown that basidiospores of Pisolithus tinctorius (Marx 1976) and Thelephora terrestris (Marx and Ross, 1970) are efective inoculum for the synthesis of ectomycorrhizas with loblolly pine. Ivory (1983) reported that inoculation with basidiospores of Pisolithus tinctorius, Rhizopoon nigrescens or Scleroderma texense led to the formation of abundant distinctive mycorrhizas at four months on Pinus caribaea.

Fox (1983), in her experiments on inoculation of birch seedlings with basidiospores of seme ectcmycorrhizal fungi, reported that Mearly-stage,f mycorrhizal fungi (Paxillus involutus, Hebelcma sacchariolens, H. leucosa, Inocybe lacera and I. lanuginella) can establish mycorrhizas on seedlings from spores in soil, but that "late-stage" mycorrhizal fungi (Lactarius pubescens

21

and Leccinum roseofracta) cannot do so. Evidence from these studies indicates that spores can serve as functional inoculum.

Because of the difficulty in using pure cultures ofectomycorrhizal mycelium as inoculum (Mikola 1973, Theodorou 1967), Lamb and Richards (1974) recommended basidiospores as analternative. Also Theodorou (1971b) reported that an easy andeffective way of introducing mycorrhizal fungi into soil is by seed inoculation with fresh spores rather than by mixing fruiting bodies, litter or fungal mycelia within the soil.However, the sudden appearance and brief duration of thesporophores of most ectomycorrhizal fungi and the lack ofinformation on the germinability and viability of spores and their survival during storage (Molina and Trappe, 1982b), have hindered the potential of spores as a source of inoculum in commercial practice.

1.7.4 Spore germination

The Hymenomycetes comprise a broad group including coprophilous, lignicolous, litter-decomposing and ectomycorrhizal fungi.

Spores of non-mycorrhizal hymenomycetes, in general, germinate readily on artificial media; many will germinate freely in plain water (Fries, 1966). In contrast, spore germination of the ectomycorrhizal hymenomycetes is very difficult to achieve under laboratory conditions (Benedict et al., 1967; Brefeld, 1908; Fries, 1966, 1978, 1984, 1985; Kneebon, 1950; Marx and Ross, 1970;Rommell, 1921; Stack et al., 1975). Germination is often dependent upon, or stimulated by, the presence of some activating factor, usually of biological origin.

Fries (1941 and 1943), succeeded for the first time in germinating spores of some species of Boletus, Amanita and Tricholcma, when a living yeast (i.e. Torulopsis sanguinea or Rhodotorula glutinis) was used as stimulator. Lamb and Richards (1974) obtained germination of basidiospores of Pisolithus tinctorius (Lycoperdales), Rhizopogon roseolus and Suillus

22

granulatus only in the presence of the yeast Rhodotorula glutinis. However, many mycorrhizal fungi which were tested did not respond at all to the influence of Rhodotorula.

Since seme studies (Fries, 1976 and 1978) indicated the presence of inhibitory factors in the nutrient agar media employed, steps were taken to identify and remove these inhibitors. First it was found in experiments with species of Suillus that germination is very sensitive to the content of ammonium ions in the substrate. A radical reduction of the ammonium concentration considerably improved both rate and percentage of germination in Suillus (Fries,1976). Later it was discovered that germination in various ectomycorrhizal fungi was prevented by an inhibitor in agar which is formed from agarose during autoclaving. Activated charcoal has been used to remove this inhibitor (Fries, 1978). This inhibitor has been identified as a weak organic acid, chiefly active on mycorrhizal-hymenomycetes (Bjurman, 1984). By using activated charcoal in combination with Rhodotorula glutinis as an inducing organism, germination could be induced in additional ectcmycorrhizal species, eg. Laccaria laccata (Fries, 1977), Lactarius helvus, Paxillus involutus, Leccinium scabrum (Fries, 1978) and Cantharellus ciharius (Fries, 1979a).

Living mycelium of a mycorrhizal fungus, either alone or in combination with Rhodotorula glutinis and activated charcoal was found to be active in stimulating germination of spores of the same fungus (Birraux and Fries, 1981; Fries, 1978, 1981a and b, 1983a, b; Straatsma et al., 1985)

Even in the presence of such activators, germination percentages of less than 1% during incubation periods of one to four weeks are not uncommon for these ectcmycorrhizal fungi. However, by the use of such activators, Fries (1943, 1966) obtained germination of Suillus luteus in about one week. Without the presence of Rhodotorula, at least three to four weeks were necessary for germination to begin. Other boletes, such as Boletus granulatus, B. variegatus, B. bovinus, B. elegans and viscidus, could be germinated by co-culture with Rhodotorula or a growing

23

Boletus mycelium for one week. Sane of these species would begin germination on synthetic media if incubation was extended to one month or more, but no germination was observed on a malt extract medium. Fries also noted that Amanita rubescens (Fr.) Gray, would germinate without an activator, but the requisite incubation time in the presence of Rhodotorula was reduced to only five to seven days.

The nature of the stimulant produced by the yeast or mycelium is still unknown; several compounds may be involved.Volatile compounds have been found able to induce spore germinationin seme fungi, for example isovaleric acid which induces spore germination of Agaricus bisporus (Losel, 1964; Rast and Stauble, 1970). Fries (1978) tested the effect of isovaleric acid on germination because he suspected that the effects of the associated Rhodotorula or living mycelium were due to a volatile agent, isovaleric acid was without effect on any of the spores of mycorrhizal fungi tested. Oort (1974) found that seme species of Lactarius germinated when exposed to volatile exudations from Ceratocystis fagacearum, a fungus which already has been mentioned as an inducer of gemination in two xylophilous Polyporus species.

Bjurman and Fries (1984) reported that culture filtratesfrom Leccinum aurantiacum could substitute for a living mycelium in triggering spore germination. This indicated the existence of a germination inducing factor (GIF). Although this factor has not yet been identified, it was characterized as: non-volatile; diffusible; soluble in water, methanol and in ethanol; resistant to heat at100°C for fifteen minutes; stable after lyophilization if preserved at -20C and it has a low molecular weight (<8000 dal ton).

Melin (1959, 1962) observed that excised pine rootsstimulated spore germination in species of Russula, Suillus, Amanita, Paxillus, Cortinarius, and Lactarius. These studies were conducted in Melin's "maximum nutrient medium", a substrate containing glucose, various mineral salts, ten B-vitamins and a mixture of nineteen amino acids. The excised roots of Pinus sylvestris used in Melin's studies were grown aseptically in nutrient solution in flask culture for approximately five months

24

prior to use.In earlier work by Melin and co-workers, it was demonstrated

that roots of herbaceous plants (eg. Lycopersicon esculentum; Medicago sativa; Cannabis sativa; Triticum aestivum; and Lepidium sativum) also affected the growth of tree mycorrhizal fungi (Melin, 1954; Melin and Das, 1954). These herbaceous roots were not tested for their ability to stimulate spore germination, but their ability to stimulate vegetative growth parallels, for the most parts, that obtained with pine roots. In later studies, Melin (1963) used filter- or autoclaved-sterilised exudates obtained from Scots pine seedlings grown in pots. The culture-grown roots were used either directly in the test media or, as with the pot-grown seedlings, the exudates were obtained by diffusion into re-distilled water at 4b for six days. Pot-grown and aseptically-cultured roots were similar in their effects upon growth of Boletus variegatus. Also, Melin reported that increased dosages of exudate can exert an inhibitory effect on mycelial growth. This has led to the belief that the exudate contains, in addition to the stimulatory factor, one or more inhibitory principles that can be effective at higher concentration.

By using agar media, seeded with homogenized mycelium of mycorrhizal fungi, usually Boletus (Suillus) variegatus, Melin was able to determine the sites of the release of the stimulatory factor and the inhibitor from the root system of Pinus seedlings which had been raised in aseptic conditions. The growth of the fungus was reduced or inhibited in the neighbourhood of secondarily thickened roots and greatly stimulated around primary rootlets.

Since Melin1s (1963) studies, the effects of plant roots on germination of spores of ectomycorrhizal fungi have been studied only briefly:

Marx and Ross (1970) were unsuccessful in their attempts to germinate basidiospres of Thelephora terrestris (Aphyllophorales) by employing extracts or detached roots of loblolly pine in the media, but the spores germinated and successfully formed ectomycorrhizas with intact roots of aseptic seedlings. Heinnemann and Gaie (1979) observed about 1 % germination of Russula versicolor

25

spores after seven days when a cover glass bearing spores was placed 1 cm deep in a medium of perlite mixed with ground sphagnum in which a seedling of Betula pendula was planted.

Fries and Birraux (1980) found that laying living roots of Pinus sylvestris on agar media seeded with spores of Hebeloma spp. (H. crustuliniforme, H. inqratum and fh_ mesophaeum) increased germination to 1%, whereas in the absence of the roots germination was about 0.1%. In a further study, Birraux and Fries (1981) found an indication of specificity in response of spores to roots of different plant species. They also found that basidiospores of Thelephora terrestris were stimulated by the growing roots of very- young seedlings of Pinus sylvestris, Picea abies, Betula verrucosa and Alnus glutinosa as they grew on agar near the spores. Seedlings of ten species not forming ectomycorrhizas (Pisum sativum, Trigonella foenumgraecum, Medicago sativa, Trifolium repens, Phaseolus aureus, Lycopersicon esculentum, Raphanus sativus, Lepidium sativum, Triticum vulgare and Zea mays) were inactive, and one (Lupinus regalis) very slightly effective in this regard. Also, Bowen and Theodorou (1985) reported that roots of pine (Pinus radiata) stimulated spore germination of Rhizopogon to 60%, whereas roots of Eucalyptus globulus, Medicago truncatula. Trifolium subterraneum and Lolium perenne were inactive. This study was made by dipping roots of sterile seedlings of the test plants in 1% water agar previously seeded with spores of the test fungus. Then the roots were drained and placed in sterile vermiculite at 25/15 °C day/night temperature. Fries and Swedjemark (1986) tested the effect of roots of 5 species of trees and 16 species of herbs and grasses on germination of spores of Hebeloma mesophaeum on synthetic medium N6:5. All five tree species exerted a strong effect on the spores, inducing germination within a week. Seedlings of carrot (Daucus carota) among the sixteen non-tree seedlings tested exerted an effect on the spores similar to that of the tree seedlings. Very little germination was noticed with seedlings of blueberry (Vaccinium myrtillus), white clover (Trifolium repens) or radish (Raphanus sativus) after more than two weeks incubation. The effect of non-tree seedlings in inducing germination was uncertain;

26

since mesophaeum is known to start germination spontaneously after two or three weeks on agar plates (Bruchet 1973, Fries and Birraux 1980).

The results of Melin (1959, 1963)/Fries and Birraux (1980)/ Birraux and Fries (1981) , and of Bowen and Theodorou (1985) would indicate that prior to infection the host can significantly affect the mycelial growth and spore germination of the fungal symbiont in the region external to the root by the release of certain host-produced metabolites.

Melin (1963) has referred to this or these metabolites as nM-factor". Conclusive evidence to the nature of Melins "M-factor" is not available despite efforts directed towards that goal by Melin and his associates. The search has been fraught with difficulties due to the negative effect of inhibitors and the complexity of root exudate (Melin 1963, Rovira 1969). Melin (1963) reported that H. Nilsson had concluded that the M-factor could be replaced in its effect on hyphal growth by nicotinamide adenine dinucleotide (NAD) which is, of course, a hydrogen acceptor essential to many dehydrogenase reactions. No further details have been forthcoming on this report. However, Benedict et al. (1967) tested the effects of exogenous NAD and the exudate from tcmato roots on the mycelial growth of eight species, and spore germination in thirteen additional species of ectomycorrhizal Agaricales. Germination did not occur in any of the thirteen species tested during the incubation period (45-60 days). Only one (Leucopaxi1lus (Clitocybe) amarus f. roseibrunneus sf. majusculus) of the eight fungi tested for mycelial growth was stimulated by NAD. The remaining seven did not respond or were inhibited. Therefore it was concluded that some unidentified component(s) (other than NAD) of pine root exudate was responsible for the stimulatory effects noted by Melin.

Gogola (1970) identified cytokinin activity in the extract of the roots and germinating seeds of Pinus sylvestris. He compared their effect on the growth of Boletus edulis var. pinicolus with pure kinetin, tryptophane, gibberellic acid (GA3) and indoleacetic

27

acid. Kinetin and the extracted cytokinins stimulated growth at low concentrations whereas the other compounds uniformly inhibited

-6 -3 -J.growth between concentrations of 10 and 10 g1 . Both extracted-<S -l -1kinins and kinetin stimulated growth between 10 and 10 g1 but at

higher concentrations sharply inhibited it. Gogola was of the opinion that the M-factor of Melin might be a cytckinin and that both its stimulatory and its inhibitory action at high concentrations might therefore be explained.

Information about the nature of inhibitors noted by Melin (1963) is not available, but Krupa and co-workers in several studies (Krupa and Fries, 1971; Krupa and Nylund, 1972; Krupa et al., 1974; Melin and Krupa, 1971) have demonstrated that roots ofhost plants (Pinus spp) produce volatiles (eg. terpenes) and non-volatiles (eg. phenols) which are effective inhibitors of both mycorrhizal fungi and root pathogens. They postulated that these host-produced inhibitors, at least in part, regulate the symbiosis.

1.7.5 Spore dormancy

Sussman (1965) defined dormancy as "any rest period or reversible interruption of the phenotypic development of the organism".

In general there are two types of dormancy: constitutional dormancy and exogenous or environmental dormancy. Spores in environmental dormancy are inactive because of unfavourable conditions of their environment but will resume development at once if transferred to a favourable situation. Constitutional dormancy is a condition in which a propagule possesses seme innate property such as a wall with a barrier to the penetration of nutrients; a metabolic block, or the production of self-inhibitor s. Where such self-inhibitors of germination are present, their effects are evidenced by reduced germinations in larger populations of spores. Their effects can be overcome either by washing the substances out of the spores, or by adding to the spores some substance which will counteract the inhibitor .A clear distinction between these two types of dormancy is often impossible to make.

28

Dormancy of spores, sporocarps or sclerotia has beenrecorded for fungi from all major taxonomic groups. Many of them are biotrophs (Turian and Hohl, 1981; Weber and Hess, 1976) but little is known about the spores of ectanycorrhizal fungi in this respect. However, the inability of ectomycorrhizal spores to germinate under conditions which otherwise would be favourable for growth, and their requirement for special stimulants to germinate, tend to indicate that ectanycorrhizal spores are in a state of constitutional dormancy (Sussman 1966, Taber and Taber 1982).

Dodd and McCracken (1972) have hypothesized that short chain arnylose molecules in the spore wall of some Russula species prevent oxygen uptake and hence preserve dormancy. They indicated that removal of the starch by washing with water would allow germination to proceed. Although permeability has been implicated as a deciding factor in preserving dormancy in many fungi (Allen, 1965; Merrill, 1970), Blakeslee (1974) provided evidence that permeability is not a limiting factor with the ectomycorrhizal species he studied, as ready penetration of the spore wall by the rather large molecule of nitro-blue tetrazolium chloride (NBT) would indicate that an impenetrable spore wall is not involved. Also, Blakeslee (1974) failed to demonstrate the existance of self-inhibitors in five species of ectomycorrhizal fungi (Amanita citrina; Amanitarubescens; Strobilomyces floccopus; Suillus conthumatus, and Suillus hirtellus), in experiments in which he inoculated agar plates with varying quantities of spores, but noconcentration-related effect could be detected in any instances. Therefore he concluded that the inability of the test basidiospores to germinate was not due to the presence of an impermeable spore wall or some self-inhibitor, but rather due to a deficiency in supply of seme stimulant; since these spores are stimulated in their germination by co-culture with a pink yeast (Rhodotorula sp.) or living mycelium of the same species as the spores and also in the presence of living roots of pine seedlings.

Fries (1978) interpreted the poor germination or inability to germinate of most ectomycorrhizal fungi on laboratory media in various ways, eg. as an expression of dormancy, as a need for a

29

germination-inducing factor, or as the effect of an endo- or exogenous inhibitor. Seme evidence in favour of the two latter possibilities has been obtained in experiments with Boletus (Suillus) species, where amino acids stimulate and ammonium ions inhibit germination (Fries 1976).

1.7.6 Spore longevity

Length of viability of different spores varies tremendously even under nearly optimum conditions (Fries 1943). Information about the longevity of ectomycorrhizal basidiospores in nature is lacking. Under laboratory conditions some information is available for a few ectomycorrhizal fungi; Fries (1966) indicated that spores of Suillus luteus can germinate after six months of storage at low temperature (-10C). Basidiospores of Thelephora terrestris could produce ectomycorrhizas following storage for one month at 5°C (Marx and Ross, 1970) and for 15 months at 4C (Birraux and Fries, 1981). Stack et al. (1975) obtained ectanycorrhizal synthesis from spores of Laccaria laccata, either fresh or rehydrated after lyophilization (in liquid nitrogen) and subsequent storage at room temperature or at -10C for up to seven months. Attempts to germinate these spores on laboratory media were unsuccessful. Basidiospores of Pisolithus tinctorius, kept dry in darkness at 5&, have been successfully used in ectcmycorrhizal synthesis often from one week to 34 months storage (Marx, 1976). Theodorou and Bowen (1973) demonstrated that basidiospores of the fungus Rhizopogon luteolus (mycorrhizal with Pinus radiata) could beused successfully as seed inoculum after freeze-drying and storage for three months at 22b, provided that inoculum level was increased 100-fold by comparison with fresh spores, and that sporesinoculated onto seed could be held dry for at least two days before planting and produce as much infection as fresh spores, provided inoculum was increased ten-fold. Also they reported that drying spores in soil over ten weeks and leaving them for a further two months at 24b day temperature and 16C night temperature did not affect their viability. Lamb and Richards (1974) examined the

30

effect of seme environmental conditions on the storage and germination of various types of propagules of seven mycorrhizal fungi. Chlamydospores of three highly effective unidentified mycorrhizal symbionts, the oidia of Xerocomus subtcmentosus and the basidiospores of Rhizopogon roseolus, Suillus granulatus and Pisolithus tinctorius were tested. They found that chlamydospores had low heat tolerance and lost viability in storage even under optimal conditions of humidity. For germination they showed narrow ranges of tolerance to temperature and pH. Chlamydospores thus appear to have little survival value. Oidia and basidiospores showed high heat tolerance (50-60C) for 48 hours and germinated well after storage for 60 days at relative humidities near 50%. They also germinated over wider ranges of temperature (10-40C) and substrate pH(4.0-7.5) than chlamydospores. Basidiospores of P. tinctorius showed particularly broad tolerances to environmental conditions during both storage and germination. Thus they concluded that basidiospores could be recommended as seed inoculants for direct sowing programs. Fox (1983) reported that storage of soil that had been supplemented with basidiospores of ectcmycorrhizal fungi (Inocybe spp. and Hebeloma spp.) for ten months in outdoor conditions or in a growth room at 18C, did not alter the ability of basidiospores to produce mycorrhizas.

31

1.8 The aims of this study

Frcm the literature survey it can be concluded that the germination of basidiospores of ectcmycorrhizal fungi has been a rather neglected field of study. In most previous work,basidiospore germination has been studied either in simplesolutions, or on synthetic media in the presence of growthsubstances or stimulatory factors such as growing mycelia of filamentous fungi, red yeast or plant roots. Direct observations on basidiospore germination in nature, and their role in theinitiation of infection are still lacking.

In the present study attempts are being made to:

1). Determine conditions for germination of basidiospores of selected ectcmycorrhizal fungi!

2). Study the effect of other microorganisms in stimulating germination of basidiospores of ectcmycorrhizal fungi.

3). Determine whether basidiospores are stimulated by host roots and whether growth frcm basidiospores is directed to host roots.

4). Determine whether there is any specifity in response of basidiospores to roots.

5). Study basidiospore germination under conditions similar to those in nature, that is in the presence of roots and soil.

6). Determine the longevity of basidiospores under different storage conditions.

* The fungal species of ectomycorrhizal fungi studied in this project were selected according to their availability in the studied area and also to include groups of fungi which are different ecologicallyi.e. those occurring early and late in the mycorrhizal succession.

32

Chapter 2 MATERIALS AND METHODS

2.1 Laboratory media

2.1.1 Fries agar (Fries, 1978)

The medium contained:Glucose 4.00gAmmonium tartrate 1.00gKH2P04 0.20gMgS04.7H20 0.1OgNaCl 20mgCaC12. 2H20 26mgZnS04.7H20 0.80mgMnS04.4H20 0.81mgFeCl3.6H20 0.80mgMalt extract (Difco) 1.00gAgar (Difco) 15.00gDistilled water 1000ml

And the following vitamins: thiamin 100ug, pyridoxine 100ug,riboflavin 100ug, biotin 25ug, nicotinamide 100ug, P-amino-benzoic acid 100ug, pantothenic acid 100ug, and inositol 10,000ug, were also added to 1 litre of the above medium. Autoclaved at 120°C for 10 minutes.

In all the germination tests this medium was used with the above ingredients except where otherwise stated.

33

2.1 .2 Modified Melin Norkrans medium (MMN) (Marx, 1969a)

CaCl2 0.05gNaCl 0.025gKH2P04 0.50g(NH4)2HP04 0.25gMgS04.7H20 0.15gFeCl3(1% solution) 1,2mlThiamine (HCl) 100ugMalt extract 3.0gGlucose 10.OgBacto-agar 15.OgDistilled water 1000.0ml

2.1.3 Mineral salts nutrient medium (Ingestad, 1962)

NH4N03 0.40gNaH2P04.2H20 0.50gKC1 0.33gKH2P04 0.43gCaCl2.6H20 0.65gMgS04.7H20 0.49gMgC12.6H20 0.40gNa2S04.10H2O 0.64gFeCl3.6H20 0.014gMnC12.4H20 1,8mgH3B03 2.9mgZnC12 0.12mgCuC12 0.15mgNa2Mo04.2H20 0.022mgDistilled water 1000.0ml

Autoclaved at 120°C for 10 minutes.Quarter strength of the above medium was used.

34

Our tests confirmed Ingestad's (1962) observation that full strength solution was unsuitable for young (c. 3 week-old)seedlings. 1% (w/v) agar was added when solid medium was required.

2.1.4 Nutrient agar

28g/litre Oxoid nutrient agar (CM3).

2.1.5 Potato dextrose agar (PDA)

39g/litre Oxoid PDA (CM139).

2.1.6 Pseudomonas Agar Base CM 559 (Oxoid Ltd.)

24.2g of the agar base, CM559, was suspended in 500ml of distilled water, then 5ml of glycerol was added, boiled to dissolve completely, and autoclaved at 121 °C for 15 minutes. To 500ml of

aagar base cooled to 50 C, the contents of 1 vial of Pseudomonas C-F-C supplement SR103 (rehydrated with 2ml of sterile distilled water) was added, the medium was mixed and poured into sterile Petri dishes.

The antibiotic C-F-C (SR103) contains 0.005g Cetrimide and0.005g Fucidin, both equivalent to 10mg per litre of medium, and0.025g Cephaloridine equivalent to 50mg per litre.

35

2.2 Melzer1s Reagent

The reagent was prepared by dissolving 1,5g iodine, 5g potassium iodide and 100g chloralhydrate in 100ml warm distilled water.

2.3 SV, Sulpho-Vanilin

Prepared by dissolving a few crystals of vanilla in 2mi concentrated sulphuric acid + 2ml distilled water to give a yellow solution.

2.4 Formalin-acetic-alchohol

Consists ofFormaldehyde (40%) = 5mlGlacial acetic acid = 5ml Alchohol (70%) = 90ml

2.5 Acetic-analine blue stain (Jones and Mollison, 1948)

15ml of 5.0% (v/v) aqueous solution of phenol, 1ml of 1.0%(w/v) aqueous solution of water-soluble aniline blue and 4ml ofglacial acetic acid, were mixed and filtered about one hour afterpreparation.

2.6 Preparation of saturated salt solution (Winston and Bates,1960)

The solution was prepared by dissolving solid CaC12.6H20 in boiling water to saturation. This solution was then allowed to stand and cool for a few days to ensure saturation. Then the

36

saturated solution was shaken and distributed in 1 litre jars (100ml/jar) and a wire screen (of c. 5 x 5mm mesh) was placed over the solution and supported by bending its comers downwards to make feet (Plate 1). Plates containing spore prints were placed on the screen and the jars were closed and stored at c. 5°C, this maintained a relative humidity of c. 40% inside the jars.

37

Plate 1 Spores in Petri dishes stored over saturated CaC12 6H20 in closed jar

38

2.7 Root exudate

Root exudate was collected by a modification of the procedure of Ratnayake et al. (1978). Seedlings of birch (Betula pubescens) grown for six weeks in untreated birch wood soil, were carefully lifted and washed to remove sand and debris from the roots. They were then placed in beakers (5 seedlings/beaker) with their root systems completely covered with (200ml) distilled water, aerated by an aquarium aerater for 18-20 hours in a growth chamber under continuous light at 20 °C. The contents of the beakers was collected, passed through Whatman No.1 filter paper, and the volume of the root exudates then reduced by freeze ■ drying (Chemlab Instruments Ltd. SB3 Freeze Dryer) to 4ml/seedling (pH 5.8) before being filter-sterilized by passage through a Millipore filters (HAWP 025, pore size 0.45um) and stored at -20°C. The roots were dried at 65 °C until constant weight, and their dry weights were recorded.

Mean dry weight was 82.0mg/root.

39

2.8 Modified Fahraeus cells

The modified Fahraeus cell is made from a microscope slide and a coverslip separated by coverslip spacing pieces (Nutman, 1970).

To make coverslip spacing pieces; a large coverslip (no. 1 .5, 24 x 50mm) was stuck to gummed paper, 2mm coverslip squares were cut using a diamond pencil. Then the gummed paper with the coverslip squares was placed in water. The small glass squares floated free and were washed in diluted hydrochloric acid, distilled water (several times), acetone and dried.

To make the cell, a standard microscope slide was placed on a sheet of paper on which the positions of spacers and coverslip were marked. Small amounts of freshly made Araldite mixture (epoxy resin) were placed on the slide at the four spacer positions. Then a coverslip square was added at each position and pressed flat. Two, three or four squares of No. 1.5 coverslip were used to give cell depths of 1 -2mm. Then the large coverslip was fixed to the spacer squares with Araldite. The cells were baked for one hour at 100 °C to harden the Araldite. They were then acid washed, followed by several washings in distilled water and placed individually in Petri dishes. They were then sterilized in an oven at 180°C for two hours.

40

2.9 Procedures for Scanning Electron Microscope (S.E.M.) study

Fixation

Small pieces of birch roots bearing mycorrhizas with Paxillus involutus or Laccaria laccata were fixed in 4% (v/v) glutaraldehyde in 0.2M sodium cacodylate (pH 7.2), at 4°C for 3 hours, then washed with the same buffer to remove theglutaraldehyde and post fixed in 1% (w/v) osmium tetroxide in 0.2M sodium cacodylate at 4°C for 20 hours.

Dehydration

After washing with the same buffer, the specimens were dehydrated at 4 °C trough a graded acetone series (20, 40, 60, 80, and 100% v/v in distilled water), being held for 30 minutes at each stage, with three changes for one hour in 100% acetone.

Critical point drying

Specimens in acetone were critical-point dried, for one hour using a Polaron bomb with carbon dioxide as the transitional fluid (Anderson, 1951 , 1966). They were then mounted on aluminium stubsusing double sided Sellotape, sputter coated with gold using a Nanotech unit and examined with a Cambridge S100 scanning electron microscope fitted with a Nikon 35mm camera.

41

2.10 Seeds

The seed-lots of tree species: Birch [Betula pubescens(Ehrh.)] 80 (43); Corsican pine [Pinus nigra (Hoess, Badoux)] 82(4026); Sitka spruce [Picea sitchensis (Bong.) Carr.] 83 (1012); European larch [Larix decidua (Mill*)] 82 (2006); Douglas fir[Pseudotsuga menziesii (Mirb.) Franco] 80 (7974)4; Western hemlock [Tsuga heterophylla (Raf.) Sarg.] 79 (7972); and Eucalyptus[Eucalyptus nitens Maiden] were supplied by the Forestry Commission (Alice Holt).

Seeds of the non-tree species: lettuce (Lactuca sativa L.) Unrivalled; tomato (Lycopersicon esculentum Mill.) Money- maker; onion (Allium cepa L.) Ailsa Craig; cabbage (Brassica oleracea L.) Winnigstadt; and white clover (Trifolium repens L.), (commercial vegetable seeds).

2.10.1 Sterilization of seeds

Seeds were surface sterilized by soaking in 30% (v/v)hydrogen peroxide (30 minutes for the tree seeds and 15 minutes forthe other seeds), followed by one wash in sterile distilled water.

2.10.2 Germination of seeds

a - germination on agarFor laboratory experiments, the surface-sterilized seeds

were aseptically transferred to water agar (1.0% w/v) plates and incubated at 20°C under lights.

b - germination on peatFor soil experiments, the surface-sterilized seeds were

stored in sterile distilled water for 24 hours at 4°C to allow imbibition. They were sown in moistened peat in propagation trays, and incubated in the greenhouse (temperature between 12 and 20 °C), in the dark until c. 50% germination had occurred.

42

2.11 Soil

The soils used in this study were of three different types;

a - Birch wood soil, from an area around birch trees at Whitmoor Common.

b - Spruce forest soil, from an area around spruce trees, Alice Holt Forest, Hampshire.

c - Arable soil, University of Surrey.

(See Table 1 for soil descriptions)

2.11.1 Soil analysis

Soil samples were analysed by Reading Soil Services within The Department of Soil Science, The University of Reading.

(See Table 1 for soil analysis)

2.11.2 Collection

The soils were collected from the top 30cm of the soil profile, after removing surface debris and the litter-layer. The soils were air-dried, passed through a 3mm sieve and stored in plastic bags, until used.

2.11.3 Steaming

The soils were steam-treated for three hours on two successive days.

43

Table

1. Soi

l Analysis

sS'u cn. 4->0 * h Cn Eh 2 S0

►,3*H (fi C -H -H Q

lascu3 §44 -Ho 0 0 a) h £<h44 Cn'''

a s g1CD

i io cn 0 cnO Cn CM ga

a a ■a 2a ■§0 aaas g1

1

i PpcS 44

CM

rHsp 044 0

00 rHEh O

cns•H44CM•HUSiarH•HoCO

•HOco

ro

coLD

roro

oNO

ro

NOr-*CN

ooo

E CO

rH

g Sa a•S'V8cn cn

iS <T3■H CO rH • tdQ Q +*E s co

I

r»ro

oON

roro

H1

LD

CNNOCN

OroH1

0 rH CT1cn o <d cn pu 4-) W w co0 +j o cnOr OCO fa

OCO

CNro

oCN

H1CO

"vFH1

O

0 i—II

aja440

!1

•HU44

g-H4J&ocn420O•H

IoOaq•rH

4-)0>423•H

04-J0T5T3

ro

§§•Ha44O0

0440TJ■a0ro§

C•H

844

CM | « | SSI

>rH•H00W

44

Total

Nitrogen: S

oil sam

ple was dig

ested

in concentrated H2S

04. N

itroge

n con

tent

was det

ermined

by

distillation of

ammoni

um fol

lowed

by tit

ration

wit

h 0.0

1M HC1.

2.11.4 Fumigation of soils

The soils were fumigated using a chemical sterilant Dazomet (Basimid). The chemical compound breaks down in soil to a dithiocarbomate which then converts to a volatile toxic compound, methyl isothiocyanate. This was considered to be effective ineliminating mycorrhizal fungal propagules (Iyer and Wojahn, 1976). Dazomet was added to the soil at the rate of 30gm Dazomet to 70litres of soil, and thoroughly mixed in with a fork. The soil was stored in a sealed plastic bag for three weeks, after that the soil was emptied out into trays in a closed bay of the greenhouse andleft for three weeks to allow toxic volatiles to evaporate off.

Cress seeds were used to test the residual phytotoxicity in the soil; cress seeds were sown onto treated soil in Petri dishes. The soil was moistened, the dishes were sealed with P.V.C. tape and the seeds left to germinate for 1-3 days. Successful germination of seeds in comparison to those on control unsterilized peat indicated that the soil was free of significant toxicity.

45

As an initial step in the present study, collection andidentification of mycorrhizal basidiocarps was made on a regular basis (weekly), during the periods June to December in 1983 and 1984 around birch trees at Whitmoor Common in Surrey.

A number of complete specimens of each fungus were collected in cardboard boxes, and field observations were recorded. The specimens were quickly brought to the laboratory, quantitative and qualitative, macroscopic and microscopic characteristics were recorded for each fungus.

Identification was made according to the followingcharacteristics:

1. Size, shape, colour and texture of the cap.

2. Height, width and colour of the stem, also the presence of a ring, volva, root or basal bulb.

3. Colour and texture of the flesh, the exudation of milk, the smell and taste.

4. Colour, shape and the attachment of gills to the stem.

5. Colour and shape of the spores, and presence or absence of warts(spine).

Chemical characterizations were also made when needed such as the production of a distinctive colour on the stem of Russula species with SV (sulpho-vanilin) or FeS04, and the formation of blue-black colour (amyloid reaction), by spores of certain species in Melzer's reagent (see materials, 2.2 and 2.3).

2.12 Mycorrhizal fungi

2.12.1 Collection and identification of basidiocarps

46

The following references were used in the identification of the basidiocarps:

Smith et.al. (1981); Moser (1983); Phillips {1983), (see reference list); and personal assistance was given by R. Jackson; M. Moss; and D. Reid.

Basidiocarps of 28 species of ectomycorrhizal fungi were identified and their identities and taxonomic position aresummarised in Table 2. Of the listed ectomycorrhizal species, seven (Hymenomycetes) were chosen and their basidiospores were used for this study. The occurrence and abundancy of the chosen species are shown in Figure 1.

Basidiospores of one species (Scleroderma citrirku m ) of Gastromycetous ectanycorrhizal fungi were also studied (see appendix 1 ).

47

Table 2 Species of ectanycorrhizal fungi collected and identifiedduring the periods (June-December) in 1983 and 1984 beneath birch trees at Whitmoor Common, Surrey.

Fungi Family OrderAmanita citrina (Schaeff) S.F.Gray Amanitaceae AgaricalesAmanita fulva (Schaeff) Seer m it

Amanita muscaria (L.ex Fr.) Hooker n ii

Amanita rubescens ([Pers.]Fr.) ii ii

S.F.GrayBoletus badius Fr. Boletaceae ii

Boletus chrysenteron it ii

Bull.ex.St.AmansBoletus impolitus Fr. ti ii

Hebeloma crustuliniforme Cortinariaceae ii

(Bull.ex.St.Amans) Quel.Laccaria amethystea Tricholomotaceae it

(Bull.ex Merat) Murr.Laccaria laccata (Scop.ex.Fr.) Cke. ti ti

Laccaria proxima (Boud) Pat ii ii

Lactarius rufus (Scop.ex Fr.) Fr. Russulaceae RussulalesLactarius quietus (Fr.) Fr. ti ii

Lactarius turpis (Weinm.) Fr. ii it

Lactarius vietus (Fr.) Fr. ii ii

Leccinum scabrum (Fr.)S.F.Grav Boletaceae AgaricalesLeccinum versipelle (Fr. & Hok) Snell. ii it

Paxillus involutus (Fr.) Fr. Paxillaceae it

Russula betularum Hora Russulaceae Russulales-Russula,cyanoxantha it ti

(Schaeff.ex.Seer) Fr.Russula' claroflava Grove ii n

Russula nitida (Pers.ex Fr.) Fr. ii ii

Russula ochroleuca (Pers.ex.Seer) Fr. ii it

Russula sororia (Fr.) Romell ii ii

Russula velenovskyi Melzer & Zvara ii ii

Russula violeipes Quel. ti M

Scleroderma citrinum Pers. Sclerodermataceae SclerodermatalesThelephora terrestris (Ehrh.) Fr. Thelephoraceae Aphylophorales

48

Figure 1 Occurrence and abundancy of the test fungi during the season of 1983 and 1984 around birch trees in Whitmoor Common, Surrey.____________________________________

Amanita fulva j_________i -i____

Amanita rubescens________(________ v {____

Hebeloma crustuliniforme* j r t

Laccaria laccata__________________________ I____i i__

Lactarius turpis ( 1

Paxillus involutus________________(_______,— ^

Russula nitida___________________ t________,_______________,

*........... — - ■ I •— ■' | 0 | • i" »... i ■ " " | <Jul Aug Sep Oct Nov Dec

I—--------1 : Total observed season of basidiocarp production.

‘... ~3 : Period of greatest abundance

* : Basidiocarps of crustuliniforme were collected aroundwillow (Salix spp) trees at the University of Surrey.

49

2.12.2 Collection of spores

Clean and mature basidiocarps were selected, the stems were cut off and the caps were surface-sterilized by wiping with tissue soaked in ethanol, and were supported over sterile plastic Petri dishes on two cocktail sticks. Paper towels soaked with water were placed underneath the plates to maintain humidity. The plates were covered with a plastic tray and left (from 1-20 hours) until spore prints were seen in the plates. Then the spores were either used directly or for further study they were stored under differentconditions.

2.12.3 Methods of storing spores

1. Plates with spore prints were placed in polythene bags and storedat 5 or -20°C (under 95 and 45% relative humidity respectively).

2. Plates with spore prints were stored at 5°C and c. 40% relative humidity over a saturated calcium chloride solution in closed containers (see method 2.6).

3. Gills from basidiocarps of the test fungi were wrapped withKleenex tissues, freeze-dried for 24 hours and then stored over anhydrous calcium chloride under vacuum (in a desiccator) at roan temperature.

50

2.12.4 Isolation of ectomycorrhizal fungi from fruitbodies

The caps of young and healthy, freshly collected fruitbodies of the test mycorrhizal fungi were surface-sterilized by wiping with tissue soaked in ethanol, broken open under aseptic conditions, and small portions of the vegetative tissue were dissected out and placed on plates of modified Melin-Norkrans agar (MMN) containing 15 ug/ml aureomycin (chlorotetracycline), and incubated at 20 °C. When hyphae grew out of the fruitbody fragmentsto about 5-10mm, discs (5mm diameters) were cut with sterilecork-borer frcm the margin of the young mycelium and transferred to plates of fresh MMN and maintained on this medium.

Five species of the test fungi were obtained in pure culture by this method, and the other two (Amanita fulva and Russula nitida) did not produce mycelial cultures from fruitbodies.

2.12.5 Isolation of ectcmycorrhizal fungi frcm mycorrhizas

A modification of the procedures described by Chu-Chou (1979) and Chu-Chou and Grace (1981) was used. Freshly collected mycorrhizal roots were washed under running tap water to remove adhering soil particles. Segments of these roots, 5-10 mm long and bearing mycorrhizal tips were transferred to universal bottles containing 15-20ml of Tween 80 (0.003% v/v) and agitated on aGallenkamp wrist action shaker for 15 minutes to remove soildebris, followed by agitation in three changes of distilled water to remove the Tween 80 residue. The roots were thensurface-sterilized by immersion in 30% (v/v) hydrogen peroxide for 10-30 seconds and washed again by agitation for 10 minutes in sterile distilled water. Individual mycorrhizas were then cut off and plated out onto plates of MMN agar containing 15ug/ml aurecmycin, and incubated at 20°C.

51

Chapter 3 EXPERIMENTS

Germination tests were made on a synthetic basic medium of Fries (Fries, 1978) which was shown to be suitable for spore germination of most ectomycorrhizal fungi tested, (Fries, 1978; 1981a; 1983a).

A series of tests was also made in order to find out whether the major components of the basic medium are in concentrations optimal for spore germination of the test fungi; glucose, ammonium ions and malt extract were tested at different concentrations either singly or in combination with other components of the basic medium. Twelve combinations were used as shown in Table 3. The rest of the components were added as described by Fries (1978) (see materials, 2.1.1). The media were then autoclaved at 120°C for 10 minutes. These combinations were used as liquid, or in a solidified medium made by adding 15g/l Difco agar before autoclaving.

Other changes were also made as follows:1. Glucose in some treatments was filter-sterilized separately and added to the autoclaved medium, this is because glucose was shown, occasionally, to be inhibitory to bacteria and fungi, and this was attributed mostly to break-down products of glucose in heat sterilizing processes (Stanier, 1942).2. Since agar media may contain growth-inhibitory substances, (Fries, 1978; Bjurman, 1984), activated charcoal was added because of its capacity to absorb and thus remove inhibitory compounds from the culture medium (Butler and Bolkan, 1973; Day and Anagnostakis, 1971; Fries, 1978).3. For the reason stated above, pure agarose (Electrophoresis Grade - BRL) was used instead of Difco agar to solidify the original medium.

3.1 Germination without stimulants

52

Nil

3.1.1 Germination on solid media

Spore prints of the test ectcmycorrhizal fungi were removed from the plastic Petri dishes (immediately after collection) by dispersing them in sterile distilled water (mixtures of spores from 3-5 fruitbodies of the same species were used). 0.1ml of this suspension (containing c. 500,000 spores) was spread over the surface of the agar plate of 9.5cm diameter (containing 20ml of the test media). Three plates were prepared for each treatment. The plates were left overnight to allow absorption of water by the agar. Half of the agar plate was covered with (c. 7mg/plate) activated charcoal (Darco G60). Then the plates were sealed with P.V.C. tape, incubated in darkness at 20°C, and examined daily under the light microscope for spore germination.

3.1.2 Germination in liquid solution

A piece (2 x 3cm) of sterile cellulose film (boiled in distilled water for 10 minutes before autoclaving to remove any additive material) was placed on a filter pad in a sterile plastic Petri dish of 4.5cm diameter. The filter pad was moistened with the test solution or with sterile distilled water as a control.

0.05ml of the same spore suspension used in (3.1.1) was spread over the cellulose film. Three plates were made for each treatment. The plates were then taped and incubated as above. Examination of the films was made under the light microscope by removing the cellulose films carefully and placing them into another plate without a filter pad. After a quick examination, the films were returned to the plates with filter pads and reincubated. This was repeated every three days, and sterile distilled water was added when needed to avoid drying of the cellulose films.

3.1.3 Results

Spores of Paxillus involutus, Hebeloma crustuliniforme, Amanita fulva, Lactarius turpis and Russula nitida did not

54

germinate in any of the media tested.Spores of Amanita rubescens germinated in all media tested,

except when cellulose films were placed on a filter pad moistened with distilled water, but the germination never exceeded 0.1%, and usually occurred when spores were in clusters. Germination started after two weeks incubation, and macrocolonies were produced on agar plates from germinating spores within 3-4 weeks.

In Laccaria laccata, germination was obtained only when pure agarose was used instead of Difco agar. Germination (<0-1 %) occurred after five weeks incubation and macrocolonies appeared on the surface of the agar plate a week later. Activated charcoal showed no effect in any of the tests.

Incubation was continued for up to four months when solid media were used, but when cellulose film was used the experiment was terminated after three weeks, as all the plates became contaminated due to the frequent opening of the plates forexamination.

55

3.2 Germination on laboratory medium in the presence ofstimulatory factors

3.2.1 Germination in the presence of living mycelium of the same fungus as the spores

Mycelial discs (5mm diameter) obtained from the margin of an actively growing mycelial colony of the test fungi on MMN agar (see method 2.12.4) were transferred to fresh plates of Fries agar, (Fries, 1978, see 2.1.1) previously seeded with spores (as in 3.1.1) of the same fungus.

Because of failure to get mycelial cultures frcmbasidiocarps of Amanita fulva and Russula nitida; mycelium of related fungi, for example, Amanita rubescens and Lactarius turpis were used with spores of A^ fulva and R_j_ nitida respectively. Six plates were made for each fungus, three of them were covered with activated charcoal. The plates were then sealed with P.V.C. tape, incubated in darkness at 20 °C, and examined daily under the light microscope for spore germination.

3.2.2 Gemination in the presence of fragments of basidiocarps of the same species as the spores

Caps of clean and healthy basidiocarps of the test ectcmycorrhizal fungi were surface-sterilized by wiping with tissue soaked in ethanol. Fragments frcm the inside tissues were placed on the surface of Fries agar plates previously seeded with spores of the same fungus. Six plates were made for each fungus, three of them were covered with activated charcoal. The plates were then sealed with P.V.C. tape, incubated in darkness at 20°C and examined daily for spore gemination.

56

3.2.3 Germination in the presence of volatile compounds fromvegetative mycelia or basidiocarps of the same species asthe spores

The inverted dish method (Brown and Merrill, 1973) was used to test for the occurrence of volatile compounds active in stimulating germination of spores of the test fungi.

Plates of Fries agar seeded with spores of the test fungi were taped to plates of growing mycelia on Fries agar or plates containing fragments of clean basidiocarps of the same fungus as the spores. Some of the plates containing the spores were covered with charcoal. Six plates were made for each fungus. Incubation was in darkness at 20°C.

3.2.4 Results

Germination of spores of A^ fulva, L. turpis, and R _ nitida was not stimulated by living mycelium or activated charcoal (Table4) even after a prolonged incubation period of more than four months. Living mycelium of Paxillus involutus in combination with activated charcoal induced germination of spores of the same fungus (5-7 spores out of 500,000) after two weeks incubation, but the germ tubes did not grow longer than three times the spore diameter even after four months incubation. In Hebeloma crustuliniforme, germination (up to 6 spores out of 100,000) started after about four weeks incubation near the mycelial colony and only in the part of the agar plate covered with- charcoal. Germinating spores produced mycelial colonies a week later.

Spores of Laccaria laccata germinated after ten days incubation, but only within 1cm of the growing mycelium and within the area dusted with charcoal. Germinating spores produced mycelial colonies within two weeks. Few spores surrounding the germinating spores were stimulated to germinate. However, germination did not exceed 1% before the growing mycelium covered the whole surface of the plate.

57

Table 4 Germination of basidiospores on Fries agar with or without living mycelium and activated charcoal.

Supplements NoBasidiospores mycelium

+ charcoal mycelium charcoalSupplements

Paxillusinvolutus

+ - - -

Hebelomacrustuliniforme

+ - - -

Laccarialaccata

+++ - - -

Amanitarubescens

++ ++ ++ ++

Amanitafulva

- - - -

Lactarius turpis

- - - -

Russulanitida

- - - -

- : no germination

+ : <0.01% (5 - 7 spores/plate)

++ : 0.01 — 0.1%

+++ : 0.1 — 1 %

58

Germination of A^ rubescens spores occurred in the presence and absence of growing mycelium and activated charcoal, but the time required for the germination to occur was reduced from two weeks to 4-5 days in the presence of its own mycelium. Mycelial colonies were produced from the germinating spores within seven days after germination. No self stimulation was seen.

In all these fungi one gem tube always developed from one of the end points of the spore.

Spores of the test fungi were not stimulated when fragments frcm sporophores of the same species were used as stimulator. Also, no active volatile compound was proved to be produced by growing mycelia or sporophores of the test mycorrhizal fungi.

59

3.3 Testing the effect of other microorganisms on sporegermination of the test fungi

Germination of basidiospores of the test ectomycorrhizal fungi was studied in the presence of different isolates of bacteria and fungi obtained from different sources:

a - bacteria and fungi obtained from birch wood soil, b - bacteria and fungi associated with spores or sporophores of the

test ectanycorrhizal fungi, c - bacteria and fungi associated with the mycorrhizal sheath of

the test ectanycorrhizal fungi, d - bacteria and fungi appearing among the spores, as natural

contaminants on laboratory media.

The number and sources of the isolates of the first three groups are shown in Table 5.

3.3.1 Isolation of bacteria and fungi fran soil

The dilution plating method was used: fresh soil samples were collected, fran the top 30cm of the soil profile in July of 1985 fran twelve different places beneath birch trees at Whitmoor Common, Surrey. The samples were passed through a 3mm sieve and four composite samples were made. 10 grains of each sample were suspended in 90ml sterile distilled water, shaken in a Gallenkamp wrist-action shaker for 15 minutes, and allowed to stand for five minutes. Frcm this suspension serial dilutions (up to 10“^)were made, and 1ml of each dilution was plated (by the poured plate method) with nutrient agar for bacteria, and with potato dextrose agar (PDA) for fungi, three plates were made for each dilution and incubated at 20°C for seven days.

Forty bacterial isolates and thirty fungal isolates were selected and screened for their effect in stimulating germination of spores of the test ectomycorrhizal fungi.

60

Table

5 Num

ber and sou

rces

of iso

lates

of bac

teria

and fungi

that

have

been

tested

for stimulating

gemination.

CN*vF

LD

in

LD

in

0•Hft044Oa

roCN

CN

-oF

in

ro

CN

ro

■8 0 44 0 0 44 -H 0 Ot! . .O 0 4-i 0 0 -H H 0 £

A

0

f0

VDro

CN

VO

VD

0•H&44Oa

CN

in

ro

ro

CN

ro

0 * CS1 •Hu

. . 8 i30 44 O Q >iO

0

2 -3 a 8 .ao0 0 -H H 0 £

o'vF

0•Hft044Oa

oro

00440

r— l r—H •H O O 0 cn h

O 0<0 32 2 2 8ri-, 8am x•H O rH 44 0 ^ VI■g-•s 8

r Ha44 -H0 5X! +48 0 •H 0S 8o ft44 08-9ft £i44ixi0 IrH 0 0 £N ftid »sh e8 ‘rH

0

§ sft o<4-1

1-Hi0ft%0 g 0 344 <4-1 0rH 44O 0 0 0 ■H 44(H 03 5

alc•HtL8

61

3.3.2 Isolation of bacteria and fungi associated with spores or sporophores of ectanycorrhizal fungi

Caps of clean and healthy sporophores of the test ectomycorrhizal fungi were surface-sterilized by wiping with tissue soaked in ethanol, peeled, broken open under aseptic conditions, and small pieces of the inside tissue were placed onto MMN agar (5 pieces per plate). Several plates were made for each fungus and incubated at 20 °C for seven days. Colonies with different morphological characteristics (42 of bacteria and 23 of fungi) were selected.

3.3.3 Isolation of bacteria and fungi associated with mycorrhizal sheaths of the test fungi

Mycorrhizal roots of birch (Betula sp.) or willow (Salix spp.) collected beneath sporophores of the test mycorrhizal fungi, and those having the characteristic morphology of the speciesconcerned were chosen and treated as in method 2.12.5. Individualmycorrhizal tips (c. 40/species) were then plated out onto MMN agar, 5-8 in a plate, and incubated at 20 °C. 36 bacterial colonies and 24 fungal colonies with different morphological characteristics were selected.

All the bacterial isolates were transferred to fresh plates of nutrient agar and maintained on slopes of the same medium.Fungal isolates were transferred to fresh plates of PDA andmaintained on the same medium.

To study the effect of these isolates on spore germination of the test mycorrhizal fungi; a disc of fungal mycelium or streak (c. 4cm long) of bacterial inoculum was placed on the surface of Fries agar plate previously seeded with spores of the testmycorrhizal fungi. Six plates were made for each treatment, three of them covered with activated charcoal. Seme of the plates seeded with spores were left uninoculated with any of the testmicroorganisms as controls. The plates were then sealed with P.V.C. tape and incubated in darkness at 20 °C. Examination was daily under the light microscope.

62

3.3.4 Results

Table 6 shows the total number of the isolates tested and the number of isolates stimulating spore germination of ectomycorrhizal fungi.

Of 42 bacterial isolates associated with sporophores of mycorrhizal fungi, seven were active in stimulating sporegermination of mycorrhizal fungi. All these isolates were obtained from sporophores of Hebeloma crustuliniforme, and stimulated spore germination of that fungus (Table 7), one isolate only stimulated spore germination of Paxillus involutus as well as H.crustuliniforme.

Also, two bacterial isolates, out of 36 (associated with the mycorrhizal sheath) were active in stimulating spore germination (Table 6). These isolates were obtained from mycorrhizas of Salix collected beneath sporophores of crustuliniforme and stimulated spores of the same fungus and of EL involutus (Table 7).

Two bacterial isolates out of 40 (obtained from birch wood soil) were active in stimulating spore germination; one of them, Pseudomonas sp. (S30) stimulated germination of H. crustuliniforme spores and the other, (an unidentified Gram+ coccus S19), stimulated spore germination of crustuliniforme, P. involutus, and Laccaria laccata (Table 7).

One fungus (Tritirachium roseum) and one bacterium(Micrococcus roseus), which appeared on the surface of agar plates, among spores of crustuliniforme, as a natural contaminant (Table7) stimulated spores of Kh_ crustuliniforme around their colonies.

None of the fungal isolates, obtained from sporophores, mycorrhizal sheaths or from soil was active in stimulating spore germination of any of the test ectomycorrhizal fungi up to 15 days incubation; observation for longer was not possible, as the fungal mycelium grew and covered the whole surface of the plate within 15 days.

Pseudomonas stutzeri was the most active bacterium in stimulating germination of crustuliniforme spores, germination started within three days and increased until it was c. 21% up to

63

Table 6 Total number of isolates tested and number of isolates stimulating germination of ectanycorrhizal fungi.

Origin of the isolates

Isolates Total number of isolates tested

Number of isolates stimulating germination

1. Sporophore of Bacteria 42 7mycorrhizalfungi Fungi 23 None

2. Mycorrhizal Bacteria 36 2roots

Fungi 24 None

Natural Bacteria (-) * 1contaminant

Fungi (-) 1

Bacteria 40 2Soil

Fungi 30 None

* = (-) uncounted

64

Table 7 Identity, source, and stimulating activity * of the test bacterial and fungal isolates.

Isolatenumber

Identity of the active isolates

Sources Spores stimulated

1 Pseudomonas stutzeri (Lehmann and Neumann).

Sporophores of H. crustuliniforme Ik crustuliniforme

2 Pseudomonas sp. (no.1) ii i i

3 Pseudomonas sp. (no.2) ii ti

4 Pseudomonas sp. (no.3) it ii

5 Pseudomonas sp. (no.4) it i i

6 Corynebacterium sp. (no.1) (Lehmann and Neumann).

ii H. crustuliniforme and P. involutus

7 Isolate of Enterobacteriaceae

i i Ik crustuliniforme

8 Micrococcus roseus (Flugg) Natural contaminant on spores of Ik crustuliniforme

9 Tritirachium roseumI/A'... Fc 7/y'A,

H. crustuliniforme ti

10 Arthrobacter sp. Conn and Dimmick).

Mycorrhizas of Salix sp. with H. crustuliniforme

Ik crustuliniforme

11 Corynebacterium sp. (6a) (Lehmann and Neumann).

ti I f

12 Pseudomonas sp. (S30) Birch wood soil Ik crustuliniforme

13 Unidentified bacterium (S19)

i i H.P.L.

crustuliniforme, involutus, and laccata

* : For the details of identification of these isolates,see appendix No. 2.

65

1cm from the bacterial colony after ten days incubation (Table 8) (spores were counted in ten microscopic filds, chosen randomly in each of three replicate plates). 1-2cm from the bacterial colony germination was c. 1.7%. Numerous spores also germinated underneath the bacterial growth.

Plate No. 2 and 3 shows the germinating spores of H. crustuliniforme on Fries agar plate near a colony of P^ stutzeri and roseus (respectively).

The effect of P _ stutzeri was tested in the presence of activated charcoal and growing mycelium of crustuliniformeeither singly or in combination with each other (Table 8). Charcoal slightly increased germination in the presence of mycelium but not the bacterium. Mycelium had no effect on germination either alone or in combination with the bacterium.

Spores of crustuliniforme were stimulated to germinate by the two isolates of Corynebacterium after two weeks incubation, and after five weeks by M^ roseus and roseum. The exact percentage of germination could not be counted with these isolates, but it was <1% very close to the activator colonies and in the presence and absence of charcoal. Also slight stimulation (<0.1% germination) occurred in the presence of other active bacteria (Pseudomonas No. 1, 2, 3 and 4; Arthrobacter sp. and an isolate belonging to theEnterobacteriaceae).

Spores of P^ involutus were stimulated by the two isolates of Corynebacterium sp., Arthrobacter, and the unidentified soil bacterium (S19) (Table 7) but in the presence of activated charcoal only. The percentage of germination (<0.1 %) was not counted, as most of the spores germinated either very close or underneath the bacterial growth.

Spores of L _ laccata were stimulated by the unidentified soil bacterium (S19) in the presence of activated charcoal. The percentage of germination was very low (<0.01).

In all cases, germinating spores produced mycelial colonies which were then transferred to fresh plates of MMN agar to produce mycelial cultures.

66

Table

8 Spo

re germination

of H.

crustuliniforme

on Fri

es aga

r wit

h diffe

rent

supplements.

u3pmcdco

1a%co

i<DtS5o\0S

P

S•H8

• H p iH O CL) P

CM

8I?

- P O P o o a

•HP

O&

-HI1

§I—I&a,3cn

54-»CO O

• H PQ m

<d•Hp(U-po8 8

oo

*PJ3

IX)*

CM

+

s* H |

r oP ■H •Cl) r H

• P o CNO umtf 6

rH8 r-8 op CMo

CM.

v—<N

i—!•Ha •Hs

IOCMI

tf

•HP0

- PO

E|*8•Hr H01

CM

67

Plate 2 Germinated spores of crustuliniforme close to acolony of EL_ stutzeri (x250)

68

Plate 3 Germinated spores of crustuliniforme close to acolony of Micrococcus roseus (x250)

69

3.4 Screening a wide range of Pseudomonas isolates forstimulatory activity on spore germination of Hebelomacrustuliniforme

Frcm the previous experiment, it was found that all the active isolates of Pseudomonas, irrespective of their origin, stimulated germination of spores of crustulini forme but notspores of any of the other mycorrhizal fungi tested. Therefore it was necessary to test the ability of a wide range of Pseudomonas isolates in stimulating germination of spores of H. crustuliniforme.

Seventy isolates obtained from soil at the University of Surrey were tested.

3.4.1 Isolation

Fresh soil samples were collected around willow (Salix spp.) trees at the University of Surrey, and treated as in (3.3.1). One composite sample was made, and 10 grams were suspended in 90ml sterile distilled water, shaken in a Gallenkamp wrist-action shaker for 15 minutes, and allowed to stand for 5 minutes. From this suspension, serial dilutions, (up to 10-v") were made, and 0.1ml of each dilution was used to inoculate the surface of Pseudomonas Agar Base supplemented with antibiotic C-F-C SR103 (see materials, 2.1.6). Three replicas were made for each dilution, and incubated at 20 for seven days.

3.4.2 Germination test

All the colonies (70 colonies) were used to inoculate plates of Fries agar previously seeded with spores of H^ crustuliniforme. The plates were then sealed with P.V.C. tape and incubated at 20°C.

70

Of 70 isolates, two were active and stimulated germination of spores of the test fungus. Germination was less than 1% close to the bacterial colony.

3.4.3 Results

71

3.5 Study of the nature of stimulatory compounds produced byPseudomonas stutzeri

From the previous experiment (3.3) it has been found that stimulation of spore germination occurred not only very close, but also more than 2cm away from the bacterial colony. This suggests the production of a diffusible compound or compounds by the bacterium causing stimulation.

To learn more of the nature of this or these compounds the following possibilities have been tested.

3.5.1 Test for volatility

The possibility that the active compound produced by the bacterium is volatile was tested by the "inverted dish method" (Brown and Merrill, 1973): Plates of Fries agar were seeded withspores of Ih_ crustuliniforme (as in 3.1.1) and left overnight to allow absorption of water by the agar. Then these plates were taped to plates of two day-old cultures of Ety stutzeri on Fries agar. Four replicates were made and incubated at 20 °C with the plate containing spores on the top, and examined daily under the light microscope for spore germination.

3.5.2 Stable diffusible compounds

To determine whether the stimulation was caused by stable compounds diffusing into the agar medium; Ety stutzeri was grown on Fries agar for 3, 7, or 14 days. Three plates were used for eachperiod. Then the growth was washed, the agar from plates of each treatment was collected separately in 100ml Duran bottles, autoclaved at 120 °C for 10 minutes and re-poured in sterile Petri dishes. The surface of these plates was seeded with spores of H. crustuliniforme. The plates were then taped, incubated at 20°C, and examined daily under the light microscope for spore germination.

3.5.3 Extracellular compounds

Cell-free culture filtrate was used for this test: 200ml of Fries medium in each of four conical flasks was inoculated with 1ml from the same culture of stutzeri (48 hour-old culture) andincubated at 20 °C with continuous shaking using MKV orbital shaker, amplitude 5.

The content of one flask at 1, 2, 3, and 7 days incubation (with bacterial count of 2.0, 3.8, 3.8, 3.7 x 10s respectively)were centrifuged (MSE-Chilspin) at 4500 rpm and 4 *C for 40 minutes. Then the supernatants were collected and half the volume of each supernatant was concentrated 10x by freeze-drying. Both theconcentrated and unconcentrated filtrates were sterilized by passing through Millipore filters (HAWP 025, pore size 0.45um), and used dirctly or, for further study stored at -20°C.

The effect of cell-free culture filtrate on sporegemination was tested as follows:a - 50ul of the concentrated and several dilutions (1, 2.5, 5, 10, 50, 100% v/v in sterile distilled water) of the unconcentratedfiltrate were introduced into 3mm diameter holes in Fries agar plates (4.5cm diameter) previously seeded with spores of the test fungus (Ik crustulinifone). Four holes were made in a plate andfour plates used for each treatment. The plates were then taped andincubated at 20 °C.

b - Filtrate was added to molten and cooled Fries agar in concentrations of 1, 5, 10, and 15% v/v (filtrate/agar) of theunconcentrated filtrate and 1, 2, 4, 8 and 10% v/v (filtrate/agar) of the concentrated filtrate. Small Petri dishes (4.5cm diameter were used for this test).

The surface of these plates was then seeded with spores of the test fungus, the plates sealed with P.V.C. tape and incubated at 20 °C. Four plates were made for each concentration. Examination was daily under the light microscope.

73

3.5.4 Intracellular compourds

Cell-free extracts were used for this test: These wereprepared according to the method used by Garcia-Rodriguez et al. (1984) with some modifications. The cells harvested during the preparation of cell-free culture filtrate (Exp. 3.5.3) were washed twice with 0.1M sodium phosphate buffer at pH 6.8. They were then re-suspended in 5ml of the same buffer and sonicated on ice for three minutes at amplitude 3 using an MES sonicator. The disrupted cell suspension was centrifuged (MSE High Speed 18) at 1500 rpm and 4 °C for 30 minutes. The supernatant was collected and the volume adjusted to 10 ml with distilled water. Then the resulting sample was sterilized by passing through a Millipore filter (HAWP 025, pore size 0.45um) and used directly or, for further study, stored at -20°C.

50ul of the undiluted and 1, 2.5, 5, 10, 20, 50% (v/v in sterile distilled water) dilutions of the prepared cell extract were introduced into 3mm diameter holes in Fries agar plates (4.5cm diameter) previously seeded with spores of the test fungus. Four holes were made in a plate and four plates for each treatment. The plates were then taped and incubated at 20 °C.

3.5.5 Iron-chelating compounds

Under conditions of iron deficiency many bacteria produce specific high-affinity iron-chelating compounds, termed siderophores (Lankford, 1973). A species of Pseudomonas (UV3), isolated frcm leaves of red beetroot by Blakeman and Parbery (1977), produced a siderophore in iron-deficient medium that was shown to be highly stimulatory to the germinaion of Colletotrichum musae (McCracken and Swinburne, 1979, 1980). Also Slade et al.(1986) found that the addition of a suspension of washed cells of Pseudomonas sp. (isolate UV3) to conidial suspension of Colletotrichum acutatum stimulated germination and appressorium formation; filtrate frcm iron-deficient cultures of the Pseudomonas were themselves highly stimulatory. Also they found that a

74

siderophore (SA), purified frcm low-iron culture of the bacterium,stimulated germination and appressorium formation to asignificantly greater extent than the bacterial cells alone.

oPTo test the possibility of production^ siderophore-like compound by Pseudomonas stutzeri; the bacterium was grown in media with no iron and with low and high concentrations of iron. Fries medium was used for this as follows:

a - without iron.b - with 0.8mg/l FeC13.6H20 (representing a normal concentration), c - with 8 mg/1 FeC13.6H20.

Another set of the above three media was made, but emitting the vitamin mixture.

100ml of each of the above media was inoculated with 1ml frcm the same culture of P. stutzeri (48 hour-old culture) and incubated for seven days at 20 °C with continuous shaking [orbital shaker (MKV)] at amplitude 5.

Living bacteria and filtrates frcm the above cutures were tested for their effect on germination of spores of H. crustuliniforme.

The filtrates were prepared as in experiment 3.5.3 and incorporated into molten and cooled agar of the same media (with and without iron or vitamins) in concentrations of 0.1, 0.5, 1, 2, 4, 8, and 10% v/v (filtrate/agar) of the 10X concentrated filtrate.Small Petri dishes (4.5cm diameter) containing 5ml agar were used in this test. Four plates were made for each concentration, and after the agar had set the surface was seeded with spores of H. crustuliniforme.

Germination was also tested on plates of the same media but without filtrate, some of them were inoculated with bacteria frcm broth of the same media, by making a thick streak (c. 2cm long) of the bacterial inoculum on the centre of the plate; some of the plates were left uninoculated as controls.

75

3.5.6 Results

Testing all the possibilities mentioned in this experiment; no germination had occurred in the absence of living growth of P.stutzeri.

In the presence of the vitamin mixture, only living bacteria from media containing 0.8mg/l and 8mg/l FeCl3.6H20 stimulated spore germination on the same media (Table 9), whereas in the absence of the vitamins, stimulation occurred in mediumcontaining 8mg/l but not 0.8mg/l FeC13.6H20, bacteria from iron or vitamin deficient media did not stimulate spore gemination on the same media. This indicates that, for spore gemination the role of vitamins can be replaced by increasing the amount of iron in the medium.

Growth of Eh_ stutzeri (on the basis of cell number) wasaffected by the amount of iron used in the growth medium, and alsoby the presence and absence of vitamins (Table 10). In the presence of vitamins, the best growth was obtained in a medium containing 0.8mg/l FeC13.6H20 in which the total bacterial count was twice that in the medium containing 8mg/l and more than that in medium without iron.

76

Table

9 Eff

ect of

iron

and vit

amins

in its gro

wth medium

on the stimulatory

activi

ty of

P. stutzeri.

1•HEe■H-H■H>

§•H<44O8•H44

54401

•Ho oCN 04E EVD VDt •

E ro ro

jjir-H i—1

fa fai—i r-H

Cn0 S' S'5 CO CO •3•H

o + +

I•H5

8•H440<—)3

440

77

stimulation

Table

10 Eff

ect of

iron

and vit

amins

in the gro

wth medium

on the tot

al num

bers

of P.

stutzeri.

pcd-U0

I

Oo

r - cn aN1 CDID

■HP0N

- P

3cntf I<P0- P

OoI—I3§

4-4X•Hs•H4->•H>

<3* 00CN O CO

© 00 A

o os aID ID

< *oo ooi—I rH

tf tf

S' S'co co o

8•H4-4

I•H5

78

3.6 Germination near plant roots in mineral salts medium

Plants

Seedlings of tree and non-tree species mentioned in thematerials section (2.10) were used.

Procedures

A modification of the Fahraeus slide technique (Nutman,u1970) was used for this study. The modified Fahraes slide is made

from a microscope slide and coverslip separated by glass spacingpieces (see method 2.8).

Basidiospores of the test mycorrhizal fungi were removed frcm the plastic Petri dishes (immediately after collection) by dispersing than in sterile distilled water (a mixture of spores frcm 3-5 fruitbodies was used).

Quarter-strength Ingestad's mineral salts agar (Ingestad, 1962 - see materials, 2.1.3), cooled to c. 40°C after autoclaving,was seeded with the spore suspension, to give a final concentration of c.5 x 10f spores/ml agar. This seeded agar was then used to fill the space between the slide and the coverslip (c. 1 ml/cell). Thecells were planted (immediately after filling with agar medium) with 5 day-old seedlings (frcm aseptically germinated seeds on water agar) of the test plants, by pushing the radicle gently underneath the coverslip, one seedling per cell, control slides had no seedlings. Three replicates were used for each treatment. Each cell was then placed into a boiling tube (32 x 200mm) containing 10ml of quarter strength Ingestad's liquid medium (Plate 4). The plant culture tubes were held in racks and the roots were shaded with black polythene. Incubation was in growth cabinets, the temperature range was 12-20 °C and the light period was 18 hours. Illumination (1000 lux) was by fluorescent lights placed c. 80cmabove the seedlings.

Slides were examined weekly by light microscopy. Sporesgerminating within 1mm frcm the root edge, and germ tubes growing

79

Plate 4

t oL

A tube containing Ingestad mineral solution, andFahraeus cell filled with Ingestad mineral agar andplanted with one birch seedling

80

towards the roots were counted in 10 microscopic fields (in each of the three replicates) under 250.x magnification, photographs were taken of the germinating spores near the roots. Also the maximum distance frcm the root where stimulation occurred was measured.

3.6.1 Results

Roots of five of seven tree species tested and of white clover (Trifolium repens) stimulated germination of Paxillusinvolutus spores. Spores of Laccaria laccata and Hebelomacrustuliniforme were stimulated by birch (Betula pubescens) and pine (Pinus nigra) only, while those of Lactarius turpis andAmanita fulva were slightly stimulated by birch roots. In alltreatments, irrespective of the presence of roots, a few spores (only when they were in clusters), of Amanita rubescens germinated. Spores of Russula nitida did not respond to roots of any of theplants tested (Table 11).

Birch had the greatest effect of the plants tested andgermination of spores of P^ involutus, L. laccata and H. crustuliniforme near its roots started during the second week of incubation. By the end of the second week, growth of germ tubes of these fungi was too dense to permit counting within 1mm of the proximal 5mm of the roots.

The highest countable germination (30%) was that of H. crustuliniforme near the mid-point of the roots. Germination of the three fungi was <0.1% close to root tips and did not occur furtherthan 5mm from root tips. Spores of crustuliniforme germinated upto 8mm frcm the root edge, L _ laccata 5mm and JL involutus 3mm.

Within c.1mm frcm the birch root edge 80% of the germ tubesproduced by P. involutus basidiospores, 82% of L. laccata, and 76%of H^ crustuliniforme grew towards the roots.

The growth of germ tubes of laccata towards birch roots is shown in Plate 5.

81

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82

Plate 5 Germinated spores of laccata and growth of germtubes towards root of birch seedling in mineral salts medium in Fahraeus cell (x500)

83

3.7 Germination near plant roots in soil

Plants

1 - Birch (Betula pubescens)2 - Spruce (Picea sitchensis)

Soils

1 - Birch wood soil2 - Spruce forest soil3 - Arable soil

Procedures

2.0% (w/v) water agar molten and cooled (c. 40°C afterautoclaving) was seeded with spores of the test fungi (as in 3.6). Microscope slides were dipped in alchohol, flamed, cooled, and coated with the seeded agar c. 1mm thick (immediately after seeding with spores). After the agar had set, the slides were buried obliquely in 200cm3 pots of steamed or untreated soils. Birch and spruce seedlings (one week-old from seeds germinated on peat), were planted over the slides so that the roots were likely to make contact with the slides and grow over then, one seedling per pot. Control pots had no seedlings. All the pots were watered to maintain c. 60% water holding capacity. Incubation was in growthcabinets, the temperature was in the range 12-20°C, and the light(1000 lux) was from fluorescent tubes placed c. 80cm above the seedlings, for 18 hours/day.

Three slides frcm each treatment were carefully removed (the pots were unwatered four days before slide recovering) at 2, 4, 6, and 8 weeks after burial. They were air dried and most of the soil was gently brushed off them. Also larger root pieces were carefully removed with the tip of a sharp scalpel. The slides were stained by immersion for 1 hour in acetic aniline blue stain (Jones andMollison, 1948), (see materials and methods 2.5), then they were

84

rapidly washed with water and dehydrated in 95% ethanol for five minutes. Permanent preparations were made by mounting in Euparal (G.B.I. Laboratories Ltd.)

The following observations were made on each slide:

1 - Number of spores germinated, and the direction of germ tubes or hyphae produced by the geminating spores at different distances from the root edge were recorded. Counts were made in 10 microscopic fields (in each of the three replicates) under 250X magnification.

2 - The length of 40 gem tubes of each treatment was measured under the same magnification using an eyepiece micrometer.

3 - Photographs were taken to show the germinating spores anddirection of gem tubes with a 35mm camera.

4 - Other observations were also recorded.

Results

3.7.1 The influence of roots on basidiospore gemination in birch wood soil

Only P^ involutus spores of the seven species tested were stimulated by birch and spruce roots in steamed and untreated soil throughout the duration of the experiment (8 weeks). Birch roots (within 1mm from the edge) stimulated up to 9.6% of spores to geminate in untreated soil, which is significantly more than in steamed soil (88%) (Table 12).

Gemination at 0-1 mm was greater than that at 2-4mm frcm the root edge, and the majority of gem tubes grew towards the root.

Spruce had much less effect on spore gemination in eithersteamed or untreated soil (Table 12) and gemination did not increase near primary roots up to 8 weeks.

Gem tubes grew longer near birch than near spruce roots or in the control slides (Table 13).

85

Table

12 Germination

of Pax

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86

Table 13 Lengths of germ tubes (um)* produced by Paxillusinvolutus basidiospores within 1mm of the root edge after two weeks being buried in birch wood soil.

Seedling root Steamed soil Untreated soil

birch (D65.0 a 72.8 a

spruce 48.2 b 47.8 b

Control 21.1 c 19.7 c

* : Mean lengths of 40 germ tubes

(1) : Figures followed by different letters are significantlydifferent from one another at p = 0.001 (see appendix 3, B.2.).

87

One germ tube only developed from one of the end points of the spore, and usually grew towards the root without branching (Plate 6).

Approximately 9% of germ tubes branched in two immediately after emerging frcm the spore, one branch grew and progressed whereas the other remained very short (c. 5um) and adjacent to the spore (plate 7).

The tips of seme germ tubes branched and formed fan-shaped appressorium-like structures (Plate 8). This was frequently observed near or on the surface of the roots or root hairs of birch (Plate 9), and it may be analogous to the beginning of infection or fungal sheath formation on the roots.

When the incubation period was prolonged to 8 weeks, germ tubes grew longer, a net of hyphae was formed around the root, and short roots when present became mycorrhizas (Plates 10). Unfortunately, the number of mycorrhizas could not be estimated here; as most of the larger pieces of the attached roots were removed and discarded before mounting the slides. Whether monokaryon or dikaryon hyphae caused the infection is unknown, but no clamp formations were seen in the hyphae around the mycorrhizas.

Germ tubes of P_ involutus were shown sometimes to be attracted by spores (usually germinated spore). The germ tube either grew directly towards the spore and formed a fan-shaped structure before apparently becoming attached to the spore, (Plate 11), or encircle the spore in a spiral manner (Plate 12). Contact between spore and gem tube was not clear.

3.7.2 The influence of roots on basidiospore gemination in spruceforest soil and arable soil

Birch seedlings in spruce forest and arable soils grew poorly and had little or no effect on the gemination of JP. involutus basidiospores (Table 14), in contrast to birch wood soil.

Roots of spruce in spruce forest soil had a similar effect to that in birch wood soil, but spruce seedlings died in arable soil.

88

Plate 6 Germinated spores of Ety involutus and growth of germ tubes towards root of birch seedling (R) on a slide buried in birch wood soil (x300)

89

Plate 7 Spores of E\_ involutus showing germ tubes with two branches, on a slide buried in birch wood soil (x1200)

90

Plate 8 Fan-shaped appressorium-like structure produced by germinated spore of Ety involutus on a slide buried in birch wood soil (x1200)

91

Plate 9 Fan-shaped appressorium-like structures produced by germinated spores of involutus, A: on the surfaceof a detached cortical cell (CC) (x400), B: on thesurface of a root hair (RH) (x1200). On slides buried in birch wood soil

92

Plate 10 Mycorrhiza of birch produced by germinated spores of P. involutus on a slide buried in birch wood soil for eight weeks (x135)

93

Plate 1 Germ tubes from spores of EL_ involutus growing towards spores of the same species on a slide buried in birch wood soil (x400).

94

Plate 12 Germ tubes from several spores of I\_ involutus growing and encircling the spores of the same species (x400)

95

Table 14 Germination % of Paxillus involutus basidiospores near roots of birch and spruce in two different soils.(up to 1mm from the root edge).

Seedling RootSpruce Forest Soil ** Arable Soil

Steamed Untreated Steamed Untreated

birch 2.3 2.6 <0.01 <0.01

spruce 2.2 2.5 _ * -

Control <0.01 <0.01 <0.01 <0.01

* : Spruce seedlings died

** : Differences in number of germinated spores near birch andspruce roots in steamed and untreated spruce forest soil arenot significant at p = 0.10 (see appendix 3, A.3.2.).

3.8 Response of Paxillus involutus basidiospores to roots ofhost and non-host tree seedlings in soil

Basidiospores

Basidiospores of Fty involutus were obtained frcm sporophores collected beneath trees of two different species (pine and birch) frcm two different areas (Witley and Whitmoor Common, Surrey). The spores were stored for a month at 5°C and relative humidity c. 40% before use.

SoilUntreated birch wood soil was used for this test.

PlantsSeedlings of birch (Betula pubescens) and pine (Pinus

nigra).

Germination test

Germination of spores frcm both collectionswas tested with seedlings of pine and birch using the buried slide technique, similar to that used in experiment 3.7 and under the same conditions of temperature and light.

Three slides from each treatment were recovered after four weeks incubation, air-dried and most of the soil and large root pieces gently removed (as in 3.7). The slides were then stained and mounted in Euparal (as in 3.7).

Numbers of spore germinated and the direction of germ tubes produced by the germinating spores were estimated in 10 microscopic fields (under 250X magnification) near the root (0-1 mm from the edge) in each of the three replicates.

97

Results

The results in Table 15 show no specificity in response of spores of E\_ involutus to seedling roots of the host plant. The number of spores germinated near roots of the non-host seedlings is slightly higher than that near roots of host seedlings. On the other hand the number of germ tubes growing towards roots of birch, when the origin of spores was birch, was higher than when the spores originated fran pine. However, the numbers of germ tubes growing towards pine were nearly the same whether the spores originated fran pine or birch.

Roots of birch had a greater effect on spore germination and attraction of germ tubes than those of pine in all cases.

98

Table 15 Effect of roots of host and non-host tree seedlings on germination of spores of P. involutus in soil.

Origin of P. involutus

Germination test seedlings

Germination % (within 1mm frcm the root edge)

Germ tubes directed towards root %

a; cubirch birch 84 a 98 a

pine birch 86 a 94 b

birch pine • 20 b 75 c

pine pine 18 b 74 c

birch control (no seedling)

<0.01 -

pine control (no seedling)

<0.01 -

(1) : Differences between figures followed by the same letter aresignificant at p = 0.05 but not at p = 0.01, and those followed by different letters are significant at p = 0.001.

(2) : Differences between figures followed by different letters aresignificant at p = 0.001, and those followed by the same letters are not significant at p =0.10 (see appendix 3, A.4.2.).

99

3.9 Germination in the presence of root exudate "in vitro*'

Exudations collected in distilled water for 18-20 hours from roots of birch seedlings, previously grown for 6 weeks in birch wood soil (see method 2.7) were tested for their effect on spore germination of the test fungi.

Various methods were tested as follows:

1 - 50ul of the undiluted and dilutions (10, 5 and 2.5% v/v indeionized water) of the root exudate preparation were introduced into 3mm diameter holes (4 holes/plate) in Fries agar plates (4.5cm diameter) previously seeded with spores of the test fungi (c. 2.0 x 1O^spores/plate).

2 - Undiluted exudate was incorporated into molten and cooled Fries agar in the proportion of 1, 5, 10, and 20% (v/v) exudate/agar. The agar surface was then seeded with spores of the test fungi.

3 - Spores of the test fungi were soaked in root exudate (undiluted and 10% (v/v) in deionized water) for one hour and 24 hours at 5°C. Then 0.1ml of the soaked spores was spread over the surface of the Fries agar.

Six plates were made for each concentration using the above methods; in three of them the surface was dusted with activated charcoal. The plates were then sealed with P.V.C. tape and incubated in darkness at 20°C.

4 - Spores of the test fungi were suspended in root exudate (undiluted and 10% v/v in deionized water) 2mm deep in glass rings cemented to glass slides, in sterile Petri dishes, and incubated at 20 °C.

Results

Root exudates did not stimulate germination of Paxillus involutus, Laccaria laccata, Amanita fulva, Amanita rubescens,

100

lactarius turpis, or Russula nitida spores in any concentration used up to three months incubation. Spores of Hebeloma crustuliniforme were stimulated after three days incubation by root exudate introduced in holes -or incorporated in Fries agar in concentrations of 5, 10, and 20% (v/v) but not 1% (Plate 13),either in the presence or absence of activated charcoal. Also, spores germinated when they were incubated directly in root exudate (undiluted and 10% (v/v) in deionized water).

101

Plate 13 Mycelial colonies from spores of H _ crustuliniforme germinated on Fries agar plates containing, A: 1%,B: 5%, and C: 10% root exudate/agar

102

3.10 Relationship between number of spores and their germinability

In order to find out whether there is any relationship between numbers of spores and their germinability; different densities of spores of Hebeloma crustuliniforme were tested on Fries agar plates containing 10% root exudate (see exp. 3.9).

3Three different spore concentrations between 1.0 x 10 to 1.0x 10 spores/plates were used. Spores used in this test were stored(for three months before use) at 5 °C and relative humidity c. 40% (see method 2.12.3).

The results in Table 16 show the number of spores used and the number of spores germinated after 10 days incubation at 20 °C.

Fran this table it can be seen that the number of sporesgerminated is approximately proportional to the number of sporesused.

Table 16 Relationship between spore densities and number of spores germinated in H. crustuliniforme on Fries agar with 10% root exudate.

Total number of spores/plate

Number of spores * germinated

Germination %

1.23 x 'I05' 2820 2.3

1.23 x 10* 200 1 .6

1 .23 x 103 15 1 .2

* : mean of two replicates

103

3.11 Characterization and identification of the active compoundsin the root exudate

For characterization and identification of the active compound(s) in the root exudate, the following tests were made:

3.11.1 Volatility test

This test was made by the inverted dish method (Brown and Merrill, 1973). Plates of Fries agar seeded with spores of H. crustuliniforme were taped to plates containing root exudate or filter paper soaked with root exudate.

The plates were incubated at 20°C in darkness.

Result

No active volatile compounds were demonstrated to be present in the root exudate, since no germinating spores were seen up tothree months incubation.

3.11.2 Ion exchange experiment

Cationic, anionic and neutral compounds of the root exudate were separated using cation- and anion-exchange resins (see flow diagram).

Cation-exchange resin Dowex 50 x 8, hydrogen form, mesh100-200, and anion-exchange resin Dowex 1x8, chloride form, mesh100-200 (Dow chemical USA), were packed (separately) in columns (3cm by 15cm), and washed with deionized water until the pH of thewashing water became c. pH 7. 40ml of the root exudate preparation were passed slowly through the cation-exchange column which was then washed by passing about 500ml deionized distilled water through it. The resulting sample (which contains the neutral and the anionic compounds), was passed through the anion-exchange column and washed with about 500ml deionized distilled water. This final effluent was presumed to contain the neutral substances e.g. sugars.

104

Flow diagram: Fractionation of root exudates

Root exudates in distilled water 1

Freeze-driedI

Made up to 4ml/root with H20 (pH5.8)

40ml (of the above)\y

Cation-exchange column

500ml H20

EluateIAnion-exchange column

Absorbed

1L. 5N. H000HiFreeze-dried

500ml H20

Eluate

n/Freeze-dried

Made up to 20ml with H20 (pH4) and adjusted to pH5.8 with 10N.KOH

Filter Sterilized

M/Made up to 20ml with H20 (pH5.6) and adjusted to pH5.8 with with 10N.KOH

IFilter Sterilized

IBioassay * Bioassay *

AbsorbedI1L. 4N. NH40HI

Eluate1Freeze-driedI

Made up to 20ml with H20 (pH5) and adjusted to pH5.8 with 1ON.KOH

vFilter sterilized

Bioassay *

* Bioassays were made by incorporating fractions to be tested into molten and cooled Fries agar in the proportions of 5, 10, 20% v/v, in small Petri dishes (4.5cm diameter). The surface of these plates was seeded with spores of the test fungus.

105

The cationic substances were eluted from the cation-exchange column with 1 litre 4N.NH40H, and the anionic substances with 1 litre 5N.HC00H frcm the anion-exchange column.

The three fractions were evaporated to dryness (freeze-drying) and dissolved in 20ml deionized water.

The pH of the three fractions was respectively 5.6, 5.0, and 4.0 and all were adjusted with 10N.KOH to pH5.8, that of the original root exudate. The fractions were sterilized by passing through a Millipore filter (HAWP 025, pore size 0.45um) and stored at -20°C. These fractions were incorporated into molten and cooled Fries agar (at c. 40°C after autoclaving) in the proportions of 5, 10, and 20% (v/v). Small Petri dishes (4.5cm diameter) were used.After the agar had set the surface was seeded with spores of H. crustuliniforme (c. 200,000 spores/plate). Four plates were madefor each concentration, sealed with P.V.C. tape, and incubated at 20 °C in darkness.

Results

Spores of H. crustuliniforme germinated on plates containing the fraction of the root exudate eluted from the cation-exchange column, at all concentrations tested, after three days incubation. The other fractions were inactive.

Stimulation by the active fraction was about half that of the unfractionated root exudate, this may be because some of the active compound was still held on the resin particles either because the volume of the eluent (NH40H) was not enough to extract all- the active compounds from the resin, or because of the presence of seme other compounds which could not be eluted by NH40H.

3.11 .3 Ninhydrin test

About 0.1ml of the unfractionated exudate and the active fraction was spotted on strips of filter paper, air dried, sprayed with ninhydrin solution (0.2% in acetone); air-dried again and heated over a hot plate for two minutes.

106

Results

Spots of both the unfractionated and active fraction of the root exudate gave a positive reaction to ninhydrin (purple colour).

3.11.4 Paper chromatography

a - Separation of the active compound by descending paper chromatogrophy.

Samples:1 - crude root exudate (unfractionated).2 - active fraction of the root exudate (eluted from

cation-exchange column).

These samples were used either in water (i.e. without further treatment) or 10ml of each sample was freeze-dried and the powder dissolved in 1ml diethyl ether.

Standard amino acids:Fourteen amino acids were used as standards. These were: Glycine(L), Alanine(L), Valine(L), Leucine(L), Isoleucine,

Aspartate, Glutamate(L), Asparagine, Glutamine, Lysine(L), Arginine(L), Histidine(L), Serine, and Threonine(L).

10mM solutions of each of these amino acids were prepared in 20% ethanol in water.

Solvent system:Ethanol: water: ammonia soln. 0.880.(80 : 10 : 10 by volume)

Procedure:On Whatman No. 1 paper (56 x 46cm) a pencil line was drawn

10cm from one of the narrow ends. The origins of the samples were marked on this line.

Different amounts (20, 30, 50ul) of the crude root exudate

107

and the active fraction in water or in diethyl ether were spotted on the marked places (using a 10ul capillary tube). One sheet was used for the sample in water and one for the sample in diethyl ether.

20ul of each of the 14 standard amino acids were spotted between the samples on each of the two sheets. The spots were dried rapidly with a hair dryer. Then the prepared papers were hung in the tank with the end closest to the origin of the samples sitting in the trough. The solvent was poured carefully into the trough and into the bottom of the tank an hour before hanging the paper to allow saturation of the environment with the solvent.

After eight hours running (solvent moved c. 30cm), the chromatograms were lifted from the solvent, air-dried and sprayed with ninhydrin solution (0.2% in acetone).

Results

The spots of the standard amino acids gave positivereactions with ninhydrin, and each amino acid moved to a distance different from that of the other amino acids.

Samples did not show any spots reacting positively with ninhydrin at any concentration tested, but a streak of fluorescentsubstances was observed when the paper was examined under UV light. When the crude exudate was used the fluorescent streak extended 2cm from the origin, whereas the fluorescent streak of the activefraction was 12cm long but the brightest part of it was between 0.5and 1.5cm from the origin.

b - Bioassay of the chromatogram

For bioassay; 50 and 100ul of the crude exudate or the active fraction (in water or in diethyl ether) were spotted on preparative (Whatman 3MM) filter paper (56 x 46cm). Two sheets were used for the sample in water and two for the sample in diethyl ether.

Spots on one of the two sheets were dried with hot air,

108

whereas spots on the other one were left to dry at room temperature. Four spots, on each sheet were made for eachquantity.Then the chromatograms were run in the same solvent for the same time as in the previous experiment (a).

At the end of the running period (eight hours) the chromatograms were lifted from the solvent, air-dried under aseptic conditions and cut into strips (1 x 2cm) from the origin to the solvent front.

The bioassay test was performed as follows:

1 - The strips were eluted by placing them in 5ml of cooled and molten Fries agar in small Petri dishes (one strip/plate), and after the agar had set the surface was seeded with spores of H. crustuliniforme.

2 - The strips were placed on the surface of Fries agar plates (one strip/plate) previously seeded with spores of the test fungus.

3 - The strips were placed on sterile slides (one strip/slide), in Petri dishes and covered with about 1ml of Fries agar previously seeded with spores of the test fungus.

4 - The strips were placed in small Petri dishes (one strip/plate), moistened with sterile distilled water and covered with sterile dialysis membrane. The spores of the test fungus were placed on the dialysis membrane.

All the plates were sealed with P.V.C. tape and incubated at20 °C.

Results

No germinating spores were seen in any of the above tests up to three months incubation compared with more than 1% germination on plates containing 10% root exudate. This might be due to the low concentration of the active compounds on the strips tested.

109

c - Testing the effect of synthetic amino acids on sporegermination of H. crustuliniforme

The fourteen amino acids which were used as standard samples in the paper chromatography, were tested for their effect on spore germination of the test fungus, either singly or in combinations of "three" (chosen randomly) as follows:

Glycine + Alanine + Valine Glycine + Leucine + Aspartate Glycine + Isoleucine + Glutamate Glycine + Serine + Threonine Glycine + Isoleucine + Histidine Alanine + Leucine + Glutamate Alanine + Histidine + Lysine Valine + Isoleucine + Asparagine Leucine + Arginine + Aspartate Aspartate + Serine + Lysine Asparagine + Arginine + Threonine Glutamate + Asparagine + Histidine Glutamine + Lysine + Serine Lysine + Arginine + Histidine

and Casamino acids (acid hydrolysed casein) Gxoid code L41

20mM solutions in water were prepared from each ofthe above amino acids and from casamino acids. To test the effect of these amino acids on spore gemination; two different methods were used; firstly: 50ul of each amino acid or casamino acids were introduced into 3mm diameter holes (3 holes) in Fries agar plate, the surface of which had previously been seeded with spores of the test fungus (spores were stored for 4 months at 5°C and c. 40% relative humidity, and a spore suspension was made in sterile distilled water), and ; secondly: each amino acid was incorporated into the agar medium in a concentration of 10% (v/v) amino acid solution/agar.

When a combination of three amino acids was tested; 50ul of each of them was introduced into one of the three holes in the same

110

plate. Also equal amounts of the three amino acids were mixed, and the mixture was incorporated into Fries agar in a concentration of 10% (v/v) mixture/agar, and after the agar had set the surface wasseeded with spores of the test fungus (as above).

Three plates were made for each treatment,. taped, and incubated at 20°C in darkness.

ResultsIn all plates containing amino acids, either singly or in

combination of three, and also casamino acids, few spores (5-10 out of 200,000 spores) germinated after 10 days incubation, andproduced mycelial colonies a week later, compared with nogermination at all in the control plates (without amino acids).

It is possible that an increase in the concentrations tested, and the use of combinations other than those used herecould lead to better germination.

Fries (1976) reported that spores of Boletus luteus were stimulated to germinate when a mixture of 18 amino acids (AAM) (with a composition resembling that of hydrolysed casein) was added to the nutrient medium (Fries agar) in a concentration of about 1% (10mM). The effect of AAM increased when the nutrients of the agar medium were diluted to a quarter of the original concentration.Also, Fries (1976) reported that glutamic acid at a concentrationof about 10mM in nutrient medium exhibited a stronger germination inducing activity than any of the other amino acids tested.

3.11.5 Molecular weight determination

3.11.5.1 By ultrafiltration

A microconcentrator (centricon-10, Amicon, Figure 2), with membrane of 10,000MW cut off was used.

The principle of this method is, that the compound with molecular weight (< 10,000) smaller than the membrane pores emerges as filtrate and is collected in the filtrate cup. At the same time, macromolecular compounds larger than the membrane pores are

111

retained and become concentrated in the sample reservoir.

Procedure

2ml of the root exudate preparation were placed in the sample reservoir, and centrifuged (MSE-High Speed 18) at SOOOrpm and 4 °C for 90 minutes or until c. 50ul of the sample remained in the sample reservoir. Them 1ml of distilled water was added to the remaining 50ul and recentrifuged for 20 minutes. The volume of the concentrated sample was adjusted to that of the filtrate to avoid the concentration effect.

These two fractions were incorporated within Fries agar at a concentration of 10% v/v, and after the agar had set, the surface was seeded with spores of crustuliniforme, sealed with P.V.C. tape and incubated at 20°C in darkness.

Results

Spores germinated in plates containing the fraction with molecular weight <10,000 but not in plates containing compounds with molecular weight larger than 10,000.

112

(X

113

Figure

2

Centrioon-10,

in

the concentration

mode

3.11.5.2 By Dialysis membrane

Dialysis tubing (Visking) was used for this test.This tube is permaeble to water and the average pore radius

is 24 angstrom units. Thus the tubing will permit the diffusion oflow molecular weight (12-14,000) compounds in aqueous solutionthrough its walls, but not allow that of the higher molecular weight compounds.

This tube is made from a regenerated cellulose and containsglycerine and about 0.1% sulphur. Strips were cut frcm this tube, boiled for ten minutes in water to remove glycerine and sulphur, and autoclaved for 10 minutes at 121 °C. Then these strips were placed on the surface of Fries agar plates containing 10% v/v root exudate or on strips of filter paper soaked with root exudate. Spores of Ety crustuliniforme suspended in sterile distilled water were spread over the dialysis membrane strips. The plates were taped and incubated at 20°C in darkness.

Results

Spores on the dialysis membrane were stimulated to germinate, this indicates that the active compounds of the root exudate passed through this membrane and stimulated spore germination.

3.11.6 Testing the thermo-stability of the active compound

Eppendorf tubes containing 1ml of the unfractionated and ofthe active fraction of root exudate were placed in boiling waterfor 20 minutes. Then the treated root exudates were incorporated inFries agar in a concentration of 10% v/v, and after the agar had set the surface was seeded with spores of HL_ crustuliniforme. Control plates containing unboiled exudate were also made. The plates were then taped and incubated at 20°C.

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Result:

Gemination on plates containing boiled and unboiled exudate was similar, this indicates that boiling has no effect on the activity of the exudate.

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3.12 Effect of storage conditions on viability and germinabilityof basidiospores

Storage conditions can affect the viability and germinability of fungal spores. Optimum storage conditions for basidiospores of ectomycorrhizal fungi are not known. However, in this study, basidiospores of the test ectcmycorrhizal fungi were stored under different humidities at different temperatures (see methods of storing spores, 2.12.3).

3.12.1 Viability Test

To test the viability of fungal spores and hyphae;biological assays, eg. germination or growth, are commonly used. Such assays are suitable for cells that germinate or grow rapidly. Spores of many fungi (eg. mycorrhizal fungi), however, are difficult to germinate in a reasonable period of time (Dewey and Tyler, 1958; Fries, 1978; Meiners and Waldher, 1959), and although viable, they may require months to germinate. Therefore rapid viability tests are needed,

Blakeslee (1974) used the ability of spores, of somemycorrhizal fungi he studied to reduce nitro-blue tetrazolium chloride as a quick test for viable and physiologically active spores. This test was adapted from that generally used in the testing of seed viability (Moore, 1962).

In the present study, fluorescein diacetate (FDA) stain was used as a vital stain for spores of the test fungi.

FDA is a fluorogenic substrate, which is non-fluorescentuntil it is enzymatically hydrolysed. This stain was used by Soderstrcm (1977) for the vital staining of fungi in pure cultures and in soil. He obtained a good correlation between relative staining efficiency, growth rate, and respiration.

Staining Procedure:

A stock solution of fluorescein diacetate (Koch-Light

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Laboratories Ltd, Colnbrook, England) containing 2mg.ml acetone was prepared and kept at -20 6C. This solution was diluted to 10ug/ml in phosphate buffer (60mM, pH 7.4) shortly before use.

Basidiospores to be tested were suspended in sterile distilled water. As controls, spores were autoclaved for ten minutes at ^O^C to kill them.

One or two drops from the prepared spore suspensions were added to 2ml' of the diluted stain solution and left for five minutes. Then the spores were collected by filtration through a non-fluorescent membrane (Millipore MF Black filter 0.8um) in a Pyrex microanalysis filter holder. The filter was gently sucked off and placed on a glass slide. A drop of non-fluorescent immersion oil was added to the topside of the filter, which was then covered with a coverglass.

The preparation thus obtained was immediately (within one hour) studied microscopically. The result was recorded as very bright-green fluorescence, bright green, faint and non-fluorescent.

The microscope used was a Leitz Dialux 20, fitted with a Ploenpak Epifluorescence illumination system (Mercury lamp, HBO 50N). Filter Block for F.I.T.C. Objectives used were NP2 Fluotar2 40/0.70, and 100/1.32 oil. Microphotographs were taken with an Olympus OM2.

Results

By this test; dead spores (autoclaved) of all fungi tested gave negative results (non-fluorescent) with FDA, Freeze-dried spores of the test fungi showed a very low percentage of viability. Less than 0.1% of the spores gave bright green fluorescence , and the remainder were either very faint or non-fluorescent, this observation was seen with unstored and stored spores for up to 20 months.

The majority of untreated spores of the test fungi were viable for up to 20 months storage under different humidities at different temperatures. More than 90% of spores of Paxillus involutus and Hebeloma crustuliniforme (plates 14 and 15), gave

-1

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very bright-green fluorescence when they were fresh or stored for up to 20 months. Spores of laccaria laccata, Amanita fulva, Amanita rubescens, Lactarius turpis and Russula nitida were not tested for their viability (with FDA) when they were fresh, but after 20 months storage, most of the spores when aggregated in clusters and less than 1% of spores not in clusters were very bright-green fluorescent.

Exact percentages could not be counted; as the stained spores fade quickly (within seconds) when exposed to the fluorescent light during examination.

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Plate 14 Fluorescein spores of Eh_ involutus stainedfluorescein diacetate (FDA) (x1200)

with

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Plate 15 Fluorescein spores of rh_ crustuliniforme stained withfluorescein diacetate (FDA) (x1200)

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3.12.2 Germinability test

One must distinguish between viability and germinability; not all viable spores will germinate, especially those ofectanycorrhizal fungi, and this may be attributed to the fact that the optimum conditions for gemination have not yet been found.

In the present study the ability of spores to geminate differed with different methods of testing.

Basidiospores of nitida did not geminate (either freshor stored for up to 20 months under different conditions) in any gemination tests. Those of turpis and fulva geminated very poorly when they were fresh (within 24 hours of collection) near birch roots grown in mineral salts medium but not in any other conditions tested. Spores of A^ rubescens geminated on laboratory media when they were fresh or stored for up to one month at different humidities and temperatures. However, freeze-dried spores could not be tested on laboratory media because of fungal andbacterial contamination which covered the surface of the agar plates within a few days. This was because the spore inoculum was prepared by maceration of freeze-dried gills which normally contained many contaminants.

Fresh spores of L _ laccata geminated near birch and pine roots grown in mineral salts medium, and on the surface of Fries agar solidified with pure agarose or in the presence of the active bacteria (S19). Stored spores did not geminate under these conditions.

Germinability of spores of fh_ crustulinifome was tested monthly for up to 18 months storage under different conditions.

Two methods were used:

1 - On the surface of Fries agar plates supplemented with birchroot exudate (10% v/v exudate/agar): Gemination of fresh andstored spores started within three days incubation, and increased until the surface of the agar plates were covered with mycelial colonies produced by the geminating spores.

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2 - Germination on Fries agar plates inoculated with a stimulatorybacterium (Pseudomonas stutzeri):

Germination of stored spores differed frcm that of fresh spores; the time required for germination to occur increased from 3 days for fresh spores to 15 days for spores stored for up to 8 months, and 25 days for up to 18 months under any of the conditions studied. Also the germination percentage decreasd with the age of the spores until it became <0.01 very close to the bacterial colony after 18 months storage.

Stored spores of EL_ involutus did not germinate on laboratory media, even in the presence of growing mycelium of the same species, or in the presence of the active bacteria(Corynebacterium spp., Arthrobacter sp. or S19), which stimulated fresh spores to germinate.

Stored spores of E\_ involutus were stimulated to germinate near birch roots grown in soil, but not in mineral salts medium following 20 months storage.

The buried slide technique and birch wood soil were used for this test.

After four weeks burial the slides were recovered,air-dried, and permanent preparations were made (as in experiment 3.7). Spores germinating near roots (0-1 mm frcm the edge) were counted in 10 microscope fields in three replicates (slides) of each treatment.

Table 17 shows the germination percentages of P. involutus spores following different storage times under different conditions. Germination percentage of spores stored for 10 months at 5 °C and a relative humidity of c. 40% was higher than that of spores stored under other conditions for the same period of time. Whereas after 20 months storage all spores stored under different humidities at 5 °C, and at -20 °C gave similar percentages of germination (c. 40%).

Freeze-dried spores germinated very poorly (< 1 %) following 10 or 20 months storage at room temperature. Also (see Table 17), spores of Ety involutus lost a third of their germinability after 10

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omonths storage at 5 C and c. 40% relative humidity compared with 96% germination of fresh spores (experiment 3.7) and more than half following storage at 5 and -20 °C under 95% and 45% relative humidities (respectively) for the same period of time.

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Table 17 Germination of spores of P. involutus following different periods of storage under different conditions.

Storage ConditionsGermination 1)

o,'o

Temperature (tl) Relative Humidity (%)

period of storage (months)

5 40 10 * 67 ***5 40 20 ** 40

5 95 10 455 95 20 39

-20 45 10 43-20 45 20 40

Freeze-dried over anhydrous 10 <1spores at room CaC12 under 20 <1temperature vacuum

(1) : near birch root (0-1mm from the root edge), in all casesgermination in the control slide (without root) was less than 0.01%.

* : Number of spores germinated after 10 months storage (exceptfreeze-dried spores) are significantly different (p = 0.001) from those stored for 20 months under the same conditions.

** : After 20 months storage under different conditions, numbersof germinated spores are not different frcm one another at p a 0.10.

*** : Number of germinated spores after 10 months storage at 5Cand 40% r.h. is significantly different (p = 0.001) from those stored under other conditions for the same period (10 months) (see appendix 3, A.5.2.).

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3.13 Testing the role of basidiospores in initiatingectomycorrhizas

Basidiospores

Basidiospores of Paxillus involutus, Laccaria laccata,Hebeloma crustuliniforme, Amanita fulva, Amanita rubescens,Lactarius turpis and Russula nitida.

Plants

Seedlings of birch (Betula pubescens), spruce (piceasitchensis) and pine (Pinus nigra).

Soils

1 - Birch wood soil.2 - Spruce forest soil.

Procedure

Three week-old seedlings (from sterile seeds germinated on peat) were sown in 500cm3 pots containing soil (previously fumigated with Dazomet, see method 2.11.4); birch seedlings were sown in pots of birch wood soil, and those of spruce and pine in pots of spruce forest soil. One seedling was planted in each pot. All the potswere watered with sterile tap water, to maintain c. 60% of waterholding capacity. One week later basidiospores of the test fungi were added to the pots in the following way; the stipes were partly removed and the caps were surface-sterilized with ethanol and suspended close to the surface of the soil (cocktail sticks were used to support the caps). Four f ruitbodies of L^ laccata and only one of the other fungi were used for each pot. After 24 hours, the fruitbodies were removed and discarded, a small volume of water (30ml) was added to each pot to wash the spores below the surface.Control pots had no added spores.

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Three pots were used for each treatment and distributed randomly in a growth cabinet. The temperature ranged frcm 12-20°C and the light period was 18 hours/day. Illumination (1000 lux) was fran fluorescent lights placed c. 80cm above the seedlings. Watering was with sterile tap water fran below the pots (the pots were stood in saucers).

Seedlings frcm all the pots were harvested after three months growth, and the roots carefully removed from the soil.

For assessment of mycorrhizal development, the root systems of three seedlings were washed free of soil, random samples (about a third of the root size) were taken and the total number of short roots and those with mycorrhizas were counted under the binocular microscope.

To isolate the causal fungus, short roots with mycorrhizas were collected, washed, sterilized, and plated out on MMN agar containing 15ug/ml aurecmycin (as in method 2.12.5).

Short roots with mycorrhizas were fixed in formalin-acetic alchohol (see 2.4), and the freezing microtome was used to cut 6um thick transverse sections, then these sections were'mounted with lactophenol, and examined under the light microscope to observe fungal sheath and Hartig net formations.

For scanning electron microscope study, the mycorrhizalshort roots were fixed and prepared as in method (2.9) andphotographed.

Results

Basidiospores of crustuliniforme, A. fulva, A. rubescens, L. turpis and R _ nitida did not produce mycorrhizas with roots of any of the test plants. Also no mycorrhizas were formed on seedling roots in the control pots (unsupplemented with spores).

Spores of Ek_ involutus and laccata produced mycorrhizas with roots of birch, but not spruce or pine roots.

Mycorrhizas were established in all the three seedlings ofbirch inoculated with spores of laccata, and only in one out ofthree inoculated with Ek_ involutus (Table 18), and nearly half the

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Table 18 Development of mycorrhizas on seedlings of Betulapubescens grown in birch wood soil supplemented with fresh spores of two mycorrhizal fungi.

Basidiospore inoculum

P. involutus L. laccata

i ... ... .

None - Control

No. of seedlings inoculated

3 3 3

No. of seedlings with mycorrhizas

1 3 Nil

No. of root tips counted

1292 2813 -

No. of root tips with mycorrhizas

587 1224 -

% of root tips with mycorrhizas

45.4 43.5

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number of the root tips was infected with the inoculum spores.Attempts to isolate the causal fungus from the sheath were

unsuccessful; because the mycorrhizas were either contaminated because of insufficient sterilization, or killed when treated with hydrogen peroxide for 10-30 seconds. But microscopical examination confirmed the presence of fungal sheath and Hartig net in the transverse sections of the short roots. Also photographs by SEM show the hyphae of mycorrhizal fungi with clamp connections (Plates 16 and 17).

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Plate 16 Mycorrhiza of birch with laccata in birch wood soil (scanning electron microscope)

Plate 17 Enlargement of the above, showing a clamp connection.

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Chapter 4 DISCUSSION

Sussman (1966) defined germination as the first irreversible stage which is recognizably different frcm the dormant organism.For this discussion gemination will be considered in threedistinct though relatively arbitrary phases viz.1). The physiological and morphological changes which must occur

within the spore before g e m tube emergence can take place.2). The act of protrusion of the g e m tube frcm the spore wall.

This constitutes the major visible criterion of gemination although frcm a metabolic standpoint gemination is well advanced by this time.

3). The elongation of the g e m tube and the establishment ofpolar growth giving rise to the characteristic growth f o m of the fungus.

The biology of spore gemination within the ectomycorrhizal Hymenomycetes is distinct frcm that of non-mycorrhizal Hymenomycetes. In general, spores of non-mycorrhizal fungi geminate readily on artificial media; or in plain water (Fries, 1966). In contrast, spore germination of the ectomycorrhizal fungiis very difficult to achieve under laboratory conditions, and often dependent upon, or stimulated by the presence of sane activation factor, usually of biological origin.

The failure of basidiospores of ectomycorrhizal fungi to geminate or poor gemination may be due, at least in part, to a type of dormancy (Fries, 1978; Merrill, 1970; Taber and Taber,1982).

For most ectomycorrhizal fungi studied, a period of rest (or a maturation period) is not necessary before a spore can geminate (Kneebon, 1950). Buller (1909 - 1931) in his studies on sporulation in the Agarics, reported that a discharged spore is probably mature; such spores were used in the present study.

4.1 Spore germination

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So far, no condition among ectcmycorrhizal fungi has been found which could be considered analogous to morphological immaturity in seeds. That is, no species are known in which the spores require a period of dormancy to attain morphological maturity.

G erm in ation o f spores o f th e te s t e cto m y co rrh iza l fu n g i d id

n o t im prove fo llo w in g s to ra g e under c o ld c o n d itio n s fo r p e rio d s up

to tw enty m onths.

Endogenous or self-inhibitors could possibly be a cause of the inability of ectomycorrhizal spores to germinate on laboratory media (Fries, 1978). In the present study there is no indication that endogenous or self-inhibitors are produced by spores of the fungi studied within the limits of the spore densities tested. In the literature, there are no reported studies of these fungi in this respect, except that of Blakeslee (1974) who tested the possibility of production of self-inhibitors by the spores of Amanita rubescens and four other species of ectomycorrhizal fungi (Amanita citrina; Strobilcmyces floccopus; Suillus conthumatus and Suillus hirtellus), by inoculation of MHM (modified Hagem medium) with varying quantities of spores, but no concentration-related effect was detected in any instances.

Spores of the test species were not shown to have an impermeable wall. The rapid uptake (three to five minutes) of fluorescein diacetate by spores of all the fungi tested indicates the permeability of the spore wall.

However, gemination of spores of all the fungi tested except one (Russula nitida) were stimulated at least by one treatment as shown in the following sections.

These observations might support the idea that spores of ectomycorrhizal fungi are in a state of constitutional dormancy (Sussman, 1966), in which they are unable to geminate under conditions that normally allow vegetative growth to proceed and required an external activator to geminate. This may be due to an innate property of the spore itself which is not yet known.

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The mechanism by which the external activator stimulates dormant spores of ectomycorrhizal fungi is still unknown. In general spores contain reserve foods, sufficient for thegermination process, but they are unable to break down these reserve foods to utilizable respiratory substrates (Kneebon, 1950; Rast and Stauble, 1970; Taber and Taber, 1982) until activation occurs. Therefore, the role of the activator could be to provide a suitable stimulus which might activate the dormant enzymes of the spores and increase the respiratory rate of the spores.

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4.2 Germination without stimulants

Germination tests were performed on Fries medium which was shown to be suitable for spore germination of most ectomycorrhizal fungi (Fries, 1978, 1983b). Also several modifications of thismedium were tested in order to find out whether the components of the basic medium were present in concentrations optimal for spore germination of the test fungi, and whether they have any inhibitory or stimulatory effect on germination.

With all modifications tested, spores of Amanita rubescens were induced to germinate within two weeks without addition of any stimulatory factors, but the germination never exceeded 0.1%. Germination of spores of Laccaria laccata was obtained only when pure agarose was used instead of Difco agar. This suggests the presence of compounds in Difco agar which inhibited sporegermination of L^ laccata, and the absence or small concentrations of such inhibitors in pure agarose. Spores of Paxillus involutus, Hebeloma crustuliniforme, Amanita fulva, Lactarius turpis and Russula nitida did not germinate in any of the media tested without activators.

Only very few ectomycorrhi zal-Hymenomycetes have been found which are capable of germinating on synthetic nutrient media without any activator organisms, or without any special organic supplements, eg. Tricholoma sp; Amanita rubescens (Fries, 1943, 1966) and Hebeloma mesophaeum (Bruchet, 1973; Fries and Birraux, 1980).

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4.3 Germination in the presence of stimulatory factors

Spores of all ectomycorrhizal fungi tested, except those of Russula nitida, were stimulated by at least one method used during this study.

4.3.1 Stimulation by growing mycelium of the same fungus as the spores.

Spores of Paxillus involutus, Laccaria laccata and Hebeloma crustuliniforme were slightly stimulated by growing mycelium of the same species as the spores (Table 4) and in the presence of activated charcoal only. Germination of Amanita rubescens sporeswas accelerated in the presence of its own mycelium; as the time required for germination to occur decreased from two weeks in the absence of the mycelium to 4-5 days with the mycelium. For thisfungus the same thing has been observed by Fries (1966) andBlakeslee (1974).

The response of spores to living mycelium has been reported in a number of mycorrhizal fungi, including Jh involutus, Leccinum spp. amd L^ laccata (Fries, 1979a and b, 1981b, 1983b). Thereaction between spores and vegetative hyphae which leads to spore germination is called an "inductor reaction", and it is mediated by substance or substances exuded fran the hyphae which induce the spores to germinate.

The nature of the substance or substances by which the mycelium stimulate spore germination was not determined in thepresent study, but they are most likely to be non-volatile compounds, as no active volatile compound was proved to be produced by growing mycelia of the test fungi.

Rast and Stauble (1970), demonstrated that the triggering agent for spore germination in Agaricus bisporus is isovaleric acid which is produced not only by mycelium of this species, but also by several other fungi, including yeast, and may be of relatively widespread occurrence. However, when Fries (1978) tested the effect of isovaleric acid on germination, it was without effect on the

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spores of any of the mycorrhizal fungi tested. Bjurman and Fries (1984) found that culture filtrates from Leccinum aurantiacum were active in stimulating germination of spores of the same fungus as was the growing mycelium. The active compound has not been identified, but it is non-volatile; diffusible; soluble in water; methanol and in ethanol; resistant to heat at 100°C for fifteen minutes; stable after lyophilization if preserved at -20 °C and has a low molecular weight (<8000 dalton).

4.3.2 Stimulation by other microorganisms

In the present study seven isolates (out of 42) of bacteria obtained frcm sporophores of ectcmycorrhizal fungi, two (out of 36) obtained from mycorrhizal roots and two (out of 40) obtained from soil (Table 6) were found to be active in stimulating germination of certain mycorrhizal fungi. None of the fungal isolates obtained frcm the same sources was active in stimulating germination of any of the test fungi.

One fungus (Tritirachium roseum) and one bacterium (Micrococcus roseus) which appeared among the spores of H. crustuliniforme on the surface of agar plates as natural contaminants were also active in stimulating gemination of spores near their colonies. The most active isolate was that of Pseudomonas stutzeri which was obtained from sporophores of H. crustuliniforme and induced c.21% of spores of the same fungus to germinate (Table 8) within 1cm of its colony. This bacterium and all the active isolates of Pseudomonas spp. which were obtainedfran sporophores of crustuliniforme (four isolates) or from soil (one isolate) were specific in that they stimulated spores of H. crustuliniforme but not those of any of the other six fungi tested (Table 7).

Isolates of Corynebacterium spp. obtained from sporophores of crustuliniforme, and mycorrhizal roots of willow (Salix spp.) with crustuliniforme were active in stimulating spores of H.Crustuliniforme and of Paxillus involutus. Spores of Laccaria laccata were stimulated by one isolate (unidentified soil bacterium

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S19) only. This isolate also stimulated spores of P. involutus andH. crustuliniforme.

Spores of Amanita fulva, Amanita rubescens, Lactarius turpis and Russula nitida did not respond to any of the bacterial and fungal isolates tested in this study.

These findings indicate that only certain species of microorganisms can stimulate spore germination■of certain species of ectomycorrhizal fungi.

Information about the effect of other microorganisms on spore germination of ectanycorrhizal fungi is very limited; forexample, the fungus Ceratocystis faqacearum was used to germinatespores of Lactarius species by Oort (1974). Also red yeasts ( i.e. Torulopsis sanguinea and Rhodotorula glutinis) obtained from sporophores of ectomycorrhizal fungi were reported to be active in stimulating spore germination of some ectanycorrhizal fungi (Fries, 1941, 1943, 1966; Lamb and Richards, 1974). However, thegermination induced by the red yeast was usually characterized by being slow and sparse, the percentage seldom exceeding 1%. Fries (unpublished data, cited by Fries, 1984) claimed that no newgermination inducers more powerful than the rather inefficient Rhodotorula were discovered during his extensive testings ofvarious sorts of microorganisms isolated fran forest soil.

So far, no named species of bacteria has been mentioned to be effective in stimulating spore germination of ectomycorrhizal fungi. However, Bowen and Theodorou (1979) studied the interactions between eight bacteria isolated fran different sources and eight ectanycorrhizal fungi on laboratory media, and in the rhizoplane of Pinus radiata. They found that only one bacterium "Bacillus sp. WR1" (out of two isolated from washed mycorrhizas of P. radiata roots with Rhizopogon luteolus) significantly increased colonisation of pine roots by four of the mycorrhizal fungi tested, and also stimulated growth of R^ luteolus on agar media. Whereas other bacteria, isolated from nursery soil, either showed no effect or inhibited growth of the mycorrhizal fungi tested.

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Azcon et al. (1986) tested the effect of a number of soil microorganisms (bacteria and fungi) on spore germination and development of the VAM fungus Glomus mosseae. They found that the gemination rate of surface-sterilized chlamydospores and development of hyphae was significantly hastened by the presence of un-named free-living fungi tested. Daniels and Trappe (1980) noted that spore gemination of the VAM fungus Glomus epigaeus was inhibited in the absence of microorganisms. Meyer and Linderman (1986), in their experiments on the inoculation of clover (Trifolium subterraneum) with VAM fungi and a plant growth-promoting bacterium PGPR (Pseudomonas putida) found that VAM fungus infection in the roots was increased significantly from 7 to 23% by the presence of the PGPR. Also they observed significant increase in root and shoot dry weight when both the PGPR and VAM fungus were present compared to that in the presence of PGPR alone, VAM fungus alone, or uninoculated controls.

The active substances by which microorganisms stimulate spore gemination of mycorrhizal fungi is still unknown. However, Fries (1966 and 1976) reported that yeast could stimulate gemination by production of diffusible substance(s) (eg. amino acids), and volatile compounds (unknown), or by removing inhibitory compounds (eg. NH4) from the medium.

In the case of Pseudomonas stutzeri reported in this study; volatile compounds, cell-free culture-filtrate and cell extracts were inactive in stimulating gemination of the test ectomycorrhizal fungus (H. crustulinifome). Also, gemination did not occur on agar plates on which Eh_ stutzeri was allowed to grow for sometime before the growth was washed off and the agar autoclaved, poured in Petri dishes and re-used for gemination tests without the bacterium. Gemination occurred only in the presence of growing bacteria close to the spores. This indicates that the stimulatory compound(s) produced by Ek_ stutzeri are chemically labile and inactivated immediately after exudation into the medium. Fries (1966) reached the same conclusion.

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4.3.3 Stimulation by plant roots

There is experimental evidence that the basidiospores of sane• species of mycorrhizal fungi do have the potential to initiate mycorrhizas (Fox, 1983? Ivory, 1983; Marx, 1976). However, little is known of the details of this process.

The fact that few or no basidiospores of most ectomycorrhizal species tested germinate on common laboratory media suggests that in nature special conditions occur, presumably near roots, that result in germination. Such conditions and their effects constitute pre-infection interactions which appear to regulate the initial infection, and hence the occurrence of mycorrhizas, and they differ from post-infection interactions which appear to be responsible for such phenomena as development, abundance, and maintenance of the mycorrhiza (Blakeslee, 1974).

The pre-infection stages, as described by Barea (1986) and Nylund and Unestam (1982), consist of four phases; 1 - activation of mycorrhizal propagules in soil until germination is initiated; 2 - hyphal growth through soil; 3 - fungal growth in the rhizosphere? and 4 - establishment of hyphal envelope on the root from which the intercellular penetration will occur.

In the pre-infection phase, the prevailing direction of theinfluence (especially when basidiospores are used as inoculum) isfran host root to the fungus (Nylund and Unestam, 1982), since the interaction is between active (host roots) and dormant (basidiospores) organisms.

The exudation of various organic and inorganic substances is the primary mechanism through which plant roots can exert an influence on their external environment.

In fact, the effects of plant roots in inducing sporegermination of ectomycorrhizal fungi have been observed by several authors. Melin (1959) reported observation of germinating spores of species of Suillus, Amanita, Lactarius and Paxillus when exposed to exudates from pine and tomato roots. Also germination in Hebeloma spp. was strongly stimulated by pine roots (Fries and Birraux,1980), and germination in Thelephora terrestris by pine and birch

138

roots (Birraux and Fries, 1981). Heinnemann and Gaie (1979) observed stimulation of spores of Russula versicolor by roots of Betula pendula seedlings. Bowen and Theodorou (1985) reported that roots of pine stimulated germination of 67% of spores of Rhizopogon s p .

All these experiments, and others, were performed under laboratory conditions, either in solution or on agar plates. The artificial conditions of these experiments have little relation to the natural conditions to which basidiospores are subjected in the rhizosphere in soil. However, in the present study, germination of basidiospores and their behaviour were studied near plant roots in the laboratory situation (in mineral solution) and undersemi-natural conditions; that is in the presence of roots and soil.

Spores of the test fungi, except those of Russula nitida, were stimulated at least near roots of one species of the testplants grown in mineral solution (Table 11), but only Paxillus involutus spores of the seven species tested were stimulated near roots of the test plants in soil.

The best germination rate (96% of P_j_ involutus spores within 1mm of the root edge) was observed in (untreated) birch wood soil close to birch roots (Table 12), and this was about ten times greater than that near birch roots in mineral salt medium. This is probably related to the interaction between roots, soil, and rhizosphere microorganisms which might modify and increase root exudation as shown by Barber and Martin (1976). They demonstrated that exudation fran roots grown in soil is much greater than hadbeen estimated previously fran solution culture studies, and theyrelated this, at least in part, to the presence of microorganisms; since soil microorganisms are able to produce compounds which increase root cell-permeability. Microorganisms also influence plant growth by increasing the availability of nutrients.

Ayers and Thornton (1968) had demonstrated that wheat roots growing in sand consistently exuded greater amounts of ninyhydrin-reacting compounds than did roots growing in nutrient solution. They attributed the difference to abrasive injury caused by the sand particles.

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Steamed soil is not sterile, depending on the duration of treatment and temperature reached. Most fungi and vegetative cells of bacteria are likely to be killed, while bacterial spores will survive. Following steaming, bacteria multiply rapidly, usually to numbers exceeding those in untreated soil; this may be due to decomposition and release of the nutrients from the dead cells (Warcup, 1957). The poorer germination of spores of P. involutus near birch roots in steamed rather than in untreated soil, might not be only related to the effect of microorganisms, but also to some other factors caused by steaming. Soil steaming may result in the release of substances which may be toxic to the fungal spores and plants. Also, steaming alters the physical properties of soil, for example, its water holding capacity (Warcup, 1957).

The germination percentage of involutus spores near birch roots in birch wood soil was higher than that in spruce and arable soil. This is most likely attributed to the effect of soil components on growth and root exudation of birch seedlings, and also possibly on the spore germinability. Also, these soils probably contain different populations of microorganisms which might compete differently with spores of ectanycorrhizal fungi.

Spores of Ek_ involutus respond differently to roots of diferent plant species grown in mineral salt medium or in soil; birch was usually most stimulatory. However, comparison between the effects of different plants is difficult, particularly because the size and quantity of roots of different species were not equivalent.

In contrast to spores of P_j_ involutus, those of Laccaria laccata and Hebeloma crustuliniforme were stimulated by birch roots in mineral salts medium but not in soil. This suggests different requirements for gemination, or differences in competitiveness.

The failure of basidiospores of Amanita fulva, Amanita rubescens, Lactarius turpis and Russula nitida to geminate in soil near roots, and lack of gemination (R. nitida) or poor gemination (A. fulva, A. rubescens, L^ turpis) in mineral salts medium near

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roots may be understandable if these are regarded as "late-stage" mycorrhizal fungi. Fox (1983) suggested that "early-stage" mycorrhizal fungi of birch (P. involutus, Hebeloma sacchariolens,H. leucosa, Inocybe lacera and lannginella) can establishmycorrhizas on seedlings frcm spores in soil, but that "late-stage" fungi (Lactarius pubescens and Leccinum roseofracta) are unable to do so. "Late-stage" fungi appear to depend on a continuing supply of photosynthate frcm a mature tree to colonize seedling roots (Fleming, 1984).

4.3.3.1 Growth of germ tubes

Growth o f Germ tubes tow ards ro o ts , most apparent in th e

response o f P^ in v o lu tu s spores to b ir c h r o o ts , p ro b a b ly rep re se n ts

a chem otropic response to a g ra d ie n t o f one o r more components o f

th e ro o t exudate , n o t n e c e s s a r ily th ose s tim u la tin g g erm in atio n

(Tommerup and K id b y , 1980). I t is analogous to th e observed

beh aviou r o f o th e r fu n g i, in c lu d in g pathogens, in s o i l near ro o ts

(Jackson , 1957, 1960), and o f obvious advantage to th e fungus

p a r t ic u la r ly i f i t s spores rem ain v ia b le in s o i l fo r q u ite lon g

p e rio d s (Fox, 1983).Such responses have not been seen in VAM fungi. Studies by

Mosse and Hepper (1975) showed that the initial direction of the germ tubes produced by germinating VA spores was not influenced by the presence of host roots. The hyphae appear to spread out radially, and the mycelia, with low hyphal density, tend to occupy a spherical region surrounding the spore (Sanders and Sheikh,1983). As these authors pointed out, the rate of this radial spread of the hyphae can be related to the probability of a hypha encountering a suitable rhizosphere. Obviously, the possibility of chance contact between VA mycelia and roots must exist. The risk of failure to make contact with the root appears to be minimized in Gigaspora species, which produce aerial germ-tubes able to locate the roots. This could be a response to a volatile attractant released by the plant (Koske, 1982). Also, Powell (1976) showed that the hyphae frcm Endogone spores on slides buried in sterilized

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soil did not grow towards the root until about 1.6mm from them, where the main hypha (diameter 20-30um) then gave rise to characteristic fan-shaped complexes of septate lateral branches (hyphal diameter 2-7 um) and infection of the root usually occurred from these narrow lateral hyphae (pre-infection fans).

In the literature, there are no reports of the production of pre-infection structures by hyphae or basidiospores of ectanycorrhizal fungi; but observations reported in this studydemonstrated the formation of fan-shaped structures by germ tubesfrom spores of P. involutus, (Plate 8). This may be analogous to the beginning of the infection or sheath formation; since seme ofthe fans were formed very close to or on the surface of the rootsor root hairs.

Mycorrhizas were formed from hyphae produced by germinating spores of EL_ involutus, but it was not clear whether the casual hyphae were monokaryon or dikaryon.

Another sort of cheraotropism was occasionally observed between germ tubes and spores (usually germinated spores) of P. involutus in soil. This is an indication of the production of substances by spores which attract hyphae nearby to grow towards the spore. This mode of reaction was first observed by Morton and French (1970) in Polyporus dryophilus and by Bitis (1970) in Clitocybe trunciola. Similar reactions were observed between oidia and hyphae in many species of Coprinus (Kemp, 1970, 1975, 1977). Kemp described the reaction as "honing". Fusion between oidia and hyphae was also observed, and this is followed by a lethal or non-lethal cytoplasmic reaction depending on the taxonomic relationship of the partners. Homing reaction also occurs among ectomycorrhizal-Hymenomycetes, such as laccaria, (Fries, 1983b), and Leccinum, (Fries, 1981b). In Laccaria homing takes place between germinated spores and hyphae of monosporous or tissue culture mycelium. In Leccinum, spores usually produce vesicles when induced to germinate by mycelium of the same fungus (Fries, 1978). Such spores (with vesicles), were attractive to the hyphae of the inducer mycelium. Ungerminated spores (without vesicles), were never attractive. The response involved growing of the hypha

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towards a vesicle and attachment to it by the hyphal tip (Fries, 1981b), Fries called this hypha "responsive hypha".

Although no evidence is available that honing reactions lead to fusion between hyphal tips and spores of ectomycorrhizal fungi sane observations by Fries (1981b) may support this idea. Friesobserved that homing reactions between hyphae of Leccinumaurantiacum and spore vesicles of Leccinum versipelle or Leccinum vulpinum lead to the death of the attached hyphal tip cell and probably also of the vesicle, whereas, when mycelia and spores both belonged to L. aurantiacum, no lethal reaction was observed. Fries, discussing this further, stated that if the spore and the hypha belong to the same species and no lethal reaction occurs, a transfer of nuclei fran the spore vesicle to the hypha could take place. This will lead to dikaryotization if the mycelium ishonokaryotic and, in heterothallic species, is of a compatiblemating type.

A l l re sp o n s iv e m yce lia o f Leccinum sp e c ie s were o b ta in ed v ia

spore g e rm in a tio n , and thus presumed to be m onosporous, except one

m ycelium (n o .668 L _ au ran tiacu m ) , w hich was o b ta in ed as a

h e te ro k a ry o tic b a s id io c a rp t is s u e c u ltu re .

From his results, Fries (1981b) speculated that theoccurrence of neutral mycelia of Leccinum sp., which do not respond to the spore vesicles of the same species, may be a consequence of senescence. However, when he (Fries, 1983c) tested new tissue cultures for honing capacity immediately after isolation from basidiocarps, no case of homing was observed. On the other hand, when further monosporous mycelia of aurantiacum and L.versipelle were isolated, most of them turned out to be responsive. However, if homokaryotic, but not heterokaryotic, mycelia are responsive to the chemotropically active substances produced frcm the spore vesicles, one would expect all monosporous (homokaryotic) mycelia to be responsive, and the tissue culture (heterokaryotic) mycelia non-responsive. Fries (1983c) attributed the negative results with some monosporous mycelia probably to the technical shortcomings of the isolation procedure. That is, the presence of potentially slow-germinating spores among the hyphae of developing

143

monosporous mycelium could lead to undetected matings, which would change the mycelium from a homo- to a heterokaryotic one, i.e. from responsive to non-responsive. Also, he attributed the responsiveness of the heterokaryotic tissue culture of the strain no. 668, aurantiacum, to the probability of a spontaneousdedikaryotization.

All these are speculative interpretations, and moreinvestigation is needed to explain the genetic and physiological mechanisms which control these different modes of reaction.

Hcming reactions between spores and hyphae of JL_ involutus have not been reported before. However, observations in this study showed that homing reactions occurred only between seme spores and hyphae, but not between others (possibly because ofincompatibility). More experimental work is needed to confirm this finding.

4.3.4 Stimulation by root exudate "in vitro"

Evidence published by several authors (eg. Birraux andFries, 1981; Bowen and Theodorou, 1985; Fries and Birraux, 1980; Fries and Swedjemark, 1986; Melin, 1959) shows that one or more compounds in root exudates have the ability to promote germination of spores of ectomycorrhizal fungi.

In the present study, exudate frcm birch roots was active in stimulating spore germination of Hebeloma crustuliniforme. Although several methods were used to identify the active substances in the exudate, exact identification could not be made. However, theactive substances are non-volatile; diffusible; soluble in water; resistant to heat treatment at 100 °C for at least 20 minutes, stable at -20°C. Also, the active substances are ninhydrin positive and seemed to possess ionizable groups because of retention on cation-exchange resin (Dowex 50). Frcm this resin the activesubstances were eluted with 4N NH40H (pH> 14). The active substanceshave a relatively low molecular weight; they passed through a membrane with 10,000MW cut off, and diffused through a dialysis

144

membrane of 12-14 x 10 MW cut off. Attempts to separate the active substances and to determine the Rf using paper chromatography were unsuccessful. More attempts should be made with different quantities of sample, different solvent systems and different location reagents.

Although no specific stimulant of spore germination in rootexudates has been identified so far, there are some indications inthe literatures of the nature of compounds having the ability tostimulate germination, for example, Nilsson (cited by Melin, 1963)reported that the stimulatory effect of the M-factor on hyphalgrowth could be replaced by nicotinamide adenine dinucleotide(NAD), but the results of Benedict et al. (1967) do not fully agreewith this. They tested the effect of exogenous NAD on mycelialgrowth of eight species, and spore germination in thirteenadditional species of ectomycorrhizal Agaricales. Germination wasnot obtained in any of the thirteen species tested. Only one of thee ig h t fu n g i te s te d fo r m y c e lia l grow th, was s tim u la te d by NAD, th e

remaining seven were without response, or showed inhibition.Therefore, it was concluded that some unidentified component(s)(other than NAD) of pine roots was responsible for the stimulatoryeffects noted by Melin. Also, Gogala's (1970) results disagreedwith those of Nilsson. He identified cytokinin activity in anextract of the roots and germinating seeds of Pinus sylvestris, andcompared their activity on the growth of Boletus edulis with purekinitin, tryptophane, gibberllic acid (GA3), and indoleacetic acid.Kinetin and the extracted cytokinins stimulated growth at lowconcentrations, whereas the other compounds uniformly inhibitedgrowth between concentrations of 10“ eand 10_:g1“1. Both extracted

6 —1 1kinins and kinetin stimulated growth between 10 and 10 g1 , but at higher concentrations they inhibited it. Gogala was of the opinion that the M-factor of Melin might be a cytokinin and that both its stimulatory and its inhibitory action at high concentration might therefore be explained.

Blakeslee (1974) reported that basidiospore germination of Amanita rubescens was stimulated by exogenously supplied

3

145

cytokinins (kinetin). In his study, he interpreted the effect of Pinus taeda short roots in stimulating germination as being partially due to the production of cytokinin-like compounds. His attempt to test the ability of the cytokinin-active fraction of P. taeda root extract to stimulate germination of A^ rubescens was unsuccessful for unknown reasons.

Fries et al. (1985) found that the vegetative growth of two ectomycorrhizal fungi was stimulated by lipids frcm root exudate of Pinus sylvestris. Recently, Fries and Swedjemark (1986) reported that the lipidic phase but not the water phase frcm pine root extract was active in stimulating germination of spores of Hebelcma mesophaeum. Also, they reported that the lipidic phase was still active, even when the free fatty acids had been removed by alkali treatment.

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4.4 The role of basidiospores in initiating mycorrhizas

Numerous experiments on the growth of young coniferous and broadleaved trees in fumigated nursery beds and in containers of sterile soil have shown that they became mycorrhizal by apparently air-borne sources of infection, eg. basidiospores (Marx et al., 1970b; Robertson, 1954; Trappe and Strand, 1969).

Direct documentation on the role of basidiospores in initiation of infection is available only for a limited number of ectomycorrhizal fungi: Theodorou (1971b) presents evidence thatspores of Rhizopogon luteolus can initiate ectomycorrhizas on Pinus radiata. But the inclusion of some mycelium in the spore inoculum diminishes the clarity of this finding. Using spores as inoculum, Thapar et al. (1967) found that Scleroderma verrucosum formed ectomycorrhizas with Eucalyptus grandis, but reisolation to verify the fungal symbiont was not attempted. Also basidiospores of Pisolithus tinctorius (Marx, 1976) and Thelephora terrestris (Marx and Ross, 1970) were shown to be effective inoculum for the synthesis of ectomycorrhizas with loblolly pine. Ivory (1983) reported that inoculation with basidiospores of Pisolithus tinctorius, Rhizopogon nigrescens or Scleroderma texense led to the formaton of abundant distinctive mycorrhizas on Pinus caribaea.

However, there is little, if any, information in this respect about the ectcmycorrhizal species tested in this study.

In the greenhouse pot experiment using soil fumigated with Dazomet, basidiospores of Hebeloma crustuliniforme, Amanita fulva, Amanita rubescens, Lactarius turpis and Russula nitida did not produce mycorrhizas with roots of any- of the test plants (birch - grown in birch wood soil; pine and spruce - grown in spruce forest soil). Spores of Paxillus involutus and Laccaria laccata produced mycorrhizas with roots of birch, but not of spruce or pine.

Species of Amanita, Lactarius and Russula have been shown or presumed to be "late-stage", and those of Paxillus, Hebeloma and Laccaria have been shown to be "early-stage" mycorrhizal fungi (Deacon et al., 1983).

These authors established that a broad distinction can be

147

made between early- and late-stage fungi (i.e. those occurring early and late in the mycorrhizal succession). Early-stage fungi readily infect seedlings from resident or introduced inocula insterile soil whereas late-stage fungi cannot (Danielson et al., 1984; Deacon et al., 1983; Last et al., 1985). Late-stage fungihave also been shown to be apparently dependent on a continuing supply of photosynthate from a mature tree to colonize seedling roots (Fleming, 1984).

The factors responsible for the observed differences in the distributions of mycorrhizal fungi with respect to age of the host are not known, but presumably changes in the rhizosphere induced by the host are of major importance. Smith (1970) reported that in sugar maple, root exudates of young (three week-old) seedlings contain a diverse range of carbohydrates, whereas mature trees,(fifty five year-old) predominantly exude amino acids, amides and organic acids. It may be that "early-stage" and "late-stage fungidiffer in their nutrient requirements during the initial phases ofroot colonisation, host age thus directly influencing theircapacity to establish mycorrhizas; alternatively, competitive interactions involving rhizosphere organisms may prevent seme mycorrhizal fungi frcm establishing infections. The failure of basidiospores of A _ fulva, rubescens, L. turpis and FL nitida to establish mycorrhizas in this study could be explained by the above finding if these fungi are regarded as "late-stage" mycorrhizal fungi.

The reason for the failure of basidiospores of H. crustuliniforme (which is presumed to be an "early-stage" fungus) in establishing mycorrhizas is unknown, but it might be related to unsuitability of the soil that was used in this study.

Birch wood and spruce forest soils (with pH of 4.05 and 4.3 respectively) differ frcm the soil frcm which f ruitbodies of Hebeloma were collected (pH 7.5). In a study by Last et al. (1985) the colonization of birch roots by Hebeloma sacchariolens was tested in four different soils (two mineral soils pH 5.4 and 5.1 ; Sedge peat pH 4.7; and Sphagnum peat pH 3.4). They found that the persistence of fty sacchariolens was very strongly related to soil

148

type. In the first season inoculated seedlings growing in Sphagnum peat had few (25%) mycorrhizas attributable to sacchariolens compared with 100% in the other three soils. This fungus continued to predominate during the second season on seedlings growing in three of the four soils tested, but it failed in the most acid, viz. Sphagnum peat. Therefore they suggested that H. sacchariolens is able to colonise birch roots growing in unsterile substrates with a pH of 4.7 but not in more acidic (pH 3.4) conditions.

Basidiospores of Jk_ involutus and laccata failed toproduce mycorrhizas on roots of pine and spruce during the three months duration of the experiment. This may be because, during this time, pine or spruce did not produce enough root material which would cause significant stimulation of spore germination.

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4.5 Conclusion

In the present study, investigations were made into the role of basidiospores in initiating mycorrhizas. In soil, most of Paxillus involutus basidiospores germinated very close to the root, and the majority of germ tubes grew towards it, this resulted in the mycorrhizal formation. 4mm beyond the roots, the majority of the spores did not germinate (<0.1% germinated). This would be of selective advantage to both partners (fungus and root) if the population of spores in soil could lie in wait for new roots to grow and reach them.

It is not known for how long the spores of ectomycorrhizal fungi will survive in soil, but under controlled laboratory conditions tested in this study, spores remained viable for up to twenty months, and they might survive longer in soil. Fox (1983) reported that spores of Inocybe spp. and Hebeloma spp. remained infective for at least ten months in soil stored at 18°C.

However, th e fu n c t io n o f b a s id io sp o re s as m y co rrh iza l

in it ia t o r s i s n o t understood i f th e fa ilu r e o f some e c ta n y c o rrh iz a l

fu n g i to produce fr u itb o d ie s i s co n s id e re d .

In their field work, R.Jackson and D.Rogers (personal communication) observed the occurrence of mycorrhizas of sane species of ectomycorrhizal fungi, but without production of fruitbodies. Also, Molina and Trappe (1982a) reported that some species of ectomycorrhizal fungi which are known to produce fruitbodies with sane species of the host plants, do not do so with others. They called this as "sporocarp-specific host associations", in which the mycelium may exist ecologically in other associations than those in which the fruitbodies are found. In such cases infection of newly formed roots will be by hyphae fran the infected ones. But it is possible that primary infection may happen byspores carried in air or by any other means.

Basidiospores may have a further function in addition tothat of dispersal and initiation of mycorrhizas. The honingreaction observed by Fries (1981b) between hyphae and spores might provide a potential mechanism of variation; for new dikaryotic

150

combinations might arise when basidiospores germinate in the vicinity of existing dikaryons.

However, in view of both the ecological and the economic importance of spores as a source of mycorrhizal infection of seedlings, more knowledge of the physiology of those of mycorrhizal fungi is needed.

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ACKNOWLEDGEMENTS

I am especially grateful to Dr. R.M. Jackson for supervision, guidance, and endless help during the preparation of this project and writing up this thesis.

Appreciation is expressed to Dr. P.J. Whitney for his helpful discussion, Dr. M.O. Moss and Mrs. T. Amor for their assistance in the identification of the bacterial isolates.

Special thanks are due to Dr. M. Crowder for his help in statistical analysis of the data.

I must also thank Mrs. S. Luff, Dr. A. Chamberlain, Dr. M. Al-Rubeai, Mr. I. Baldwin, Mr. T. Baker, and all the staff members in the Department of Microbiology at the University of Surrey for invaluable assistance.

I also wish to thank Mr. and Mrs. S. Foxley for typing this thesis.

Finally, I must thank every member of my family for their continued patience and encouragement during this project.

This project was financed by the Ministry of Higher Education and Scientific Research / IRAQ.

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REID, C .P .P . and WOODS, F.W. (1969). T ra n s lo c a tio n o f 1 4 C -la b e lle d

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RICHARDSON, S .D . (1956). S tu d ie s on ro o t grow th o f A cer

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P roceed ings K o n in k lijk e N ederlandse Akademie van W etenschappen

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ROBERTSON, N .F . (1954). S tu d ie s on th e m ycorrh iza o f P inus

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183

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184

APPENDIX 1

Basidiocarps of Scleroderma citrinum were collected beneath birch trees at Whitmoor Common, Surrey.

Mature basidiocarps were surface sterilized by wiping with tissue soaked in ethanol, broken open under aseptic conditions and the spores were placed in a sterile plastic Petri dish and kept at 5°C and 40% relative humidity.

Spore germination on the surface of agar plates and near plant roots in mineral salts medium (using the Fahraeus slide technique) was unsuccessful because of heavy contamination of the spores with bacteria and fungi which covered the surface of the agar plates and killed the seedlings of the test plants within a few days. In birch wood soil, using the buried slide technique similar to that used in experiment 3.7 and under the same conditions a few spores (c. 1%) germinated. Two germ tubes were produced by most of the germinated spores, as shown in the photograph below.

Germination of Scleroderma citrinum basidiospores.

Plant roots (birch) had no significant effect on the percentage of germination but germ tubes grew towards the root when germinationoccurred near the root surface.<ai

A spore of Scleroderma citrin~urn with two germ tubes on a slide buried in birch wood soil (x1500).

185

APPENDIX 2

Identification of isolates stimulating spore germination.

1, Bacterial IsolatesInitially the isolates were identified to genus using the

criterion shown in the following table.Table 1. Initial characteristics of the isolates

CharacteristicsNo . of isolates as listed in Table 7 on Page

1 2 3 4 5 6 7 8 10 11 12 13

Shape R R R R R R R S S/R R R SGram stain + - + + + — +Spore formation -Motility + + + + + - + - - + —Growth in air + + + + 4* + + + + + +Growthunaerobically

- - - - - (+) ( + ) - - ( + ) -

Catalase + + + + + + 4 4 + + + +Oxidase 4 + + + + + +Glucose (acid) 4 + 4 + + - 4 - - +Carbohydrates0/F/-

0 0 0 0 0 - F - - 0

Temperature 5 °C20 °C + + + + + + + + + + + +25 °C + + 4 + + + + + + + + +30 °C ( + ) ( + ) ( + ) ( + ) ( + ) + + + + ( + ) (+) (+)37 °C

R = Rod-shaped (+) = Very slightly positiveS = Sphere - = Negative reaction

S/R ss Variable 0 = Oxidation+ = Positive reaction F = Fermentation

186

The isolates could then be ascribed to the following genera:

1). Pseudomonas: isolates No. 1, 2, 3, 4, 5 and 12.

2). Corynebacterium: isolates No. 6 and 11.

3). Micrococcus: isolate No. 8.

4). Isolate No. 7 belongs to the family Enterobacteriaceae.

5). Isolate No. 13 could not be identified.

6). Isolate No. 10 was very close to the genus Arthrobacter: thisbacterium as described by Skerman (1967), when grown in a complexmedium, undergoes a marked change in form during the growth cycle. Older cultures (2-7 days) are composed entirely or largely of coccoid cells. In sane strains the coccoid cells are uniform in size, and spherical, and resemble micrococci. In others they are spherical to ovoid or slightly elongate. Gram-positive, however the rods may be readily decolorized and may show only Gram-positive granules in otherwise Gram-negative cells. Coccoid cells are Gram-positive but may be weakly so.

Chemoorgantrophs, metabolism respiratory never fermentative, little or no acid is produced from glucose in peptone medium, catalase positive, strict aerobes, optimum temperature 20-30°C; most strains grow at 10°C but usually not at 37°C.

The second stage is to identify these isolates to species. Isolate No.1 (the most active isolate of the genus

Pseudomonas), isolates belonging to the genus Corynebacterium (No. 6 and 11), Micrococcus (No. 8) and isolates belonging to the family Enterobacteriaceae (No. 7) were studied further in order to identify them to species.

Several tests were made as shown in Table 2. According to this

187

table, isolate No. 1 was identified as Pseudomonas stutzeri,isolates No. 6 and 11 of the genus Corynebacterium could not be identified to the species but they are very close to theCorynebacterium hofmannii, isolate No. 8 was identified toMicrococcus roseus. Isolate No. 7 of Enterobacteriaceae could not be identified to species but it is very close to Cedecea lapagei.

The following references were used in the identification:

1). COWAN, S.T. (1979). Cowan and Steel's Manual for theidentification of Medical Bacteria. Second Edition, Cambridge University Press, Cambridge, London, New York, Melbourne.

2). KRIEG, N.R., HOLT, J.G. (1984). Bergey's Manual of Systematic Bacteriology, Vol. 1. Williams and Wilkins, Baltimore, London.

3). SKERMAN, V.B.D. (1967). A Guide to the Identification of the Genera of Bacteria. Second Edition. The Williams and Wilkins Company, Baltimore.

188

Table

2 Further

characterization of

the

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tested.

2. Fungal Isolate

The active fungus (isolate No. 9, Table 7) was identified as Tritirachium roseum VAN-BEYMA according to the characteristic features described by Hughes (1953) 1 and Van-Beyma (1942)(2>as follows:

Slow growing, colonies 5-1Omm diameter, cushion-like, pink-violet coloured, no odour, agar not stained.

Conidia are small, sub-spherical to cylindrical, mainly1.74-2.32 x 2.32-2.9um, produced regularly to the left and to the right and the fertile region of the sporogenous cell resembles the rachis of a wheat ear and may be conspicuously zig-zag (Fig. 1). The sporogenous cells are verticillate branches of the main conidiophore.

(1). HUGHES, S.J . (1953). Conidiophores, conidia, andclassification. Contribution No. 1283 from the Division of Botany and Plant Pathology, Science Service, Department of Agriculture, Ottawa, Canada.

(2). VAN-BEYMA, T.K.F.H. (1942). Beschreibung Einiger Neuer Pilzarten aus dem Centraalbureau Voor Schimmelcultures, Baam (Nederland). Antonie Van Leeu Wenhock 8P, 105-122.

190

Figure 1 . Tritirac±Liuin roseum, conidiophore and conidia frcm five weeks culture on Fries agar.

191

APPENDIX 3Statistical Analyses

2 (1)A - The chi-squared (X ) test (Clarke, 1982) was used to analysethe data in tables 11, 12, 14, 15 and 17. In all these tables,comparison of the treatments with the control was not made becauseof very low (<0.01 %) germination percentages in the control.

In each table, for the comparison between replicates (3 replicates) the level of significance was calculated according to the chi-square 3x2 contingency table (the calculations were made by computer) and for the comparison between treatments (2 treatments) the 2x2 contingency table was used as shown below:

Observednumbers

With characteristic (i.e. germinated)

Without characteristic (i.e. non-germinated)

Total

Sample 1 a b (a + b)Sample 2 c d (c + d)

a + c b + d N

N = a + b + c + dX*= N(ad-bc f ____________________

(a + b) (a + c) (b + d) (c + d)Abbreviations and levels of significance used in this testAbbreviations:

g: Number of spores germinated ng: Number of spores non-germinated g%: Germination percentages d: Number of spores with germ tubes directed towards rootsnd: Number of spores with germ tubes non-directed towards rootsd%: Percentage of germ tubes directed towards roots

Levels of significance:*: significant at p = 0.05**: significant at p = 0.005***: significant at p = 0.001

Figures without asterisk non-significant at p = 0.10

(1): CLARKE, G.M. (1982). Statistics and Experimental Design (Second Edition). Edward Arnold, London.

192

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A.5.2 Comparison between treatments

TreatmentGermination o f spores

numberg ng X

1 3350 1616 67***

7.80

2 2145 3216 40

1 3350 1616 67***580

3 2800 3468 45

2 2145 3216 40 2.16

4 2545 4033 39

2 2145 3216 40 0.049

6 2832 4281 40

3 2800 3468 45***

47.27

4 2545 4033 39

4 2545 4033 39 1.81

6 2832 4281 40

5 2273 2972 43***

15.44

6 2832 4281 40

202

B. The level of significance for the data in Table 13 (page 87) was calculated according to Student’s t-test as follows:

t = Difference between means (r'r"1> /Standard error of differences'

B.1 : Statistical analysis of the lengths of germ tubes produced by P. involutus near birch and spruce roots in steamed and untreated soil

Treatment Soil Plant Length of germ tubes SD SENumber treatment (mean of 40) um

1 steamed birch 65.0 22.9 3.622 untreated birch 72.8 24.63 3.893 steamed spruce 48.2 21 .99 3.504 untreated spruce 47.8 23.60 3.745 steamed control 21.1 5.53 0.876 untreated control 19.7 4.03 0.64

SD: Standard deviationSE: Standard error of differences

B.2 Comparison between treatments

Treatment number t (df = 78)

1 and 2 1.501 and 3 3.34 *1 and 5 11.50 **2 and 4 4.60 **2 and 6 13.21 **3 and 4 0.0783 and 5 7.51 **4 and 6 7.41 **5 and 6 1 .26

*: significant at p = 0.002 **: significant at p = 0.001

figures without asterisk non-significant at p = 0.10

203