Danell, E., 1994. Cantharellus cibarius: Mycorrhiza formation and Ecology. Acta Univ. Ups., Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 35. 75 pp. Uppsala. ISBN 91-554-3273-5, ISSN 1104-232 X.

Abstract

Axenic mycelia of Cantharellus cibarius were derived from spores and tissues. Hyphal suspensions were added to sterile pine and spruce seedlings in a culture system called CUS (Culture unit system). Automatic addition of a diluted mineral solution supplemented with glucose and a filtered air flow with 0.2% carbon dioxide was essential for C. cibarius growth and mycorrhiza formation. Successful mycorrhiza formation was repeatedly observed after 8-12 weeks. The need for carbohydrate addition probably reflected a high carbohydrate demand in nature, where new roots are mainly colonized by vegetative hyphae from a mycorrhizal mycelium. Transfer of mycorrhizal plants to a greenhouse led to successful establishment in soil and C. cibarius continued to colonize new roots. The identities of the vegetative mycelia, and the axenic and greenhouse-established mycorrhizae were confirmed by comparing their PCR products of rDNA and their RFLP patterns with those of C. cibarius fruit bodies. Field studies on C. cibarius showed that it has a broad host and biotope range, but in vitro experiments indicated a more narrow range on a strain basis.

The aerobic bacterial population size in C. cibarius was 100-1000 times greater than that of the agarics investigated. The dominating microorganism in C. cibarius fruit bodies was Pseudomonas fluorescens. Based on the inability of P. fluorescens to penetrate mycorrhizae, it was suggested that bacteria were incidently embedded in fruit bodies. This occurrence probably reflects its existence on vegetative hyphae. It was suggested that bacteria are attracted to exudates of the hyphae. C. cibarius did not appear to benefit from this association.

Different analytical methods for determining protein content of fungi were tested. The protein content in C. cibarius, based on total amino acid analysis, was 10% of the dry weight. This was in contrast to the higher values earlier reported. The nutritional value should not be over emphasised.

The results in combination with field observations and a review with comments on physiology, taxonomy, and ecology, make the thesis a comprehensive synthesis of our present knowledge on C. cibarius. The successful transfer to the greenhouse and the increasing understanding of the biology of C. cibarius might lead to a full control over the life cycle, thus allowing genetic, molecular and commercial applications.

Eric Danell, Doctoral dissertation at Uppsala University 1994

Dedication

Maybe, I should have dedicated this thesis to Elias Fries, the father of Swedish mycology, whose bicentenary is celebrated this year. Since I did not know him, I therefore wish to dedicate this thesis to his descendant Nils Fries.

Nils Fries' great knowledge of language, art and literature, field biology, systematics and experimental biology has greatly impressed me. These gentle characters in combination with kind manners and a generous mind, make Nils Fries an excellent scientist.

Articles and manuscripts on which this thesis is based on
This thesis is based on unpublished data and the following articles, which will be referred to in the text by their Roman numerals (I-V):

I. DANELL E & EAKER D (1992) Amino acid and total protein content of the edible mushroom Cantharellus cibarius (Fries). J. Sci. Food. Agric. 60: 333-337.

II. DANELL E & FRIES N (1990) Methods for isolation of Cantharellus species, and the synthesis of ectomycorrhizae with Picea abies. Mycotaxon 38: 141- 148.

III. DANELL E (in press) Formation and growth of the ectomycorrhiza of Cantharellus cibarius. (accepted by Mycorrhiza)

IV. DANELL E, ALSTRöM S, TERNSTRöM A (1993) Pseudomonas fluorescens in association with fruit bodies of the ectomycorrhizal mushroom Cantharellus cibarius. Mycol. Res. 97: 1148-1152.

V. DANELL E (manuscript) Field observations on fruit body growth and management of the edible ectomycorrhizal mushroom Cantharellus cibarius.

Published articles are reproduced with permission from the publishers.

CONTENTS

PREFACE

Aims of the project ........................................... 7

Abbreviations.................................................. 8

Swedish and scientific names of fungi..................... 9

TAXONOMIC INTRODUCTION

Historical notes................................................ 10

Description of the genus Cantharellus...................... 10

Pigments and volatile compounds........................... 12

World distribution............................................. 12

Related genera................................................. 12

Table of European Cantharellaceae.......................... 13

THE EDIBLE CHANTERELLE

Background

Fruit body production and human use...................... 14

Predators and parasites....................................... 14

¹³⁷Cs, medical aspects and heavy metals................... 17

Amino acid and total protein content......................... 18 Paper I

Results and discussion - protein content.................... 18

The AHN method.............................................. 19

Results and discussion - evaluation of methods............ 20

CULTIVATION OF C. CIBARIUS

Background

Evolution and definition of mycorrhiza..................... 23

Earlier efforts to grow C. cibarius........................... 25

Isolation and description of the mycelium Paper II

Tissue culture................................................... 26

Spore germination.............................................. 28

Description of the mycelium.................................. 28

In vitro formation of ectomycorrhiza Paper II-III

Introduction..................................................... 29

Material and methods.......................................... 30

Results........................................................... 34

Discussion....................................................... 38

Transfer to greenhouse Paper III

Methods......................................................... 40

Results and discussion........................................ 40

Description of the mycorrhizae formed...................... 41 Paper II-III

Identification of mycelia & mycorrhizae..................... 41 Paper III

Results and discussion......................................... 41

Protocol for PCR ............................................... 43

Protocol for RFLP.............................................. 44

BACTERIA IN C. CIBARIUS Paper IV

Background...................................................... 47

Methods.......................................................... 47

Results........................................................... 49

Discussion....................................................... 51

FIELD OBSERVATIONS Paper III+V

Biotopes......................................................... 54

Host list......................................................... 56

Fruit body formation

Introduction.................................................... 57

Fruit body formation in other fungi......................... 57

Fruit body formation in C. cibarius......................... 58

Growth of C. cibarius fruit bodies.......................... 59

Decline of C. cibarius in Europe - a review................ 60

Possible causes................................................ 60

CONCLUDING REMARKS................................ 62

ACKNOWLEDGEMENTS.................................. 63

REFERENCES................................................ 64

PREFACE

Aims of the project

The aims of my project was to illuminate the ecology of the edible ectomycorrhizal fungus Cantharellus cibarius. I focused on the following subjects and main questions:

Mycelium. Is it possible to develop a reproducible method for isolating and maintaining
C. cibarius mycelium?

Mycorrhiza. What does C. cibarius mycorrhiza look like, and what is needed to establish the symbiosis? Is C. cibarius only adapted to the physiology of old trees and certain species? Can methods of molecular biology be used to distinguish C. cibarius mycorrhiza from other species?

Bacteria. A major problem when isolating C. cibarius mycelium is the occurrence of bacteria in fruit body tissues, but what kinds of bacteria are there? Are there any benefits for either species in this association?

Field studies. What kinds of biotopes and host trees are associated with C. cibarius? Under what conditions does fruit body formation take place?

If mycologists could control the life cycle of C. cibarius, this would allow us genetical experiments and studies on the molecular biology of the morphogenesis, and a deeper understanding of the physiology and ecology of our important forest fungi. Full control over the life cycle might also open interesting possibilities for commercial production of fruit bodies. However, far more important than eating the chanterelles, is to study the fundamental role of the ectomycorrhizal fungi as essential symbionts to forest trees. The understanding of the ecology and physiology of C. cibarius has become a hot issue also due to the decline of C. cibarius in central Europe.

Due to the widespread interest in C. cibarius, I wanted to write a thesis which is comprehensible not only for mycologists, but also for other biologists and foresters. I have therefore included some reviews needed to understand my results and hypotheses. As a service to experimental mycologists without access to the articles, the summary contains a few detailed descriptions of important methods. I have also gathered some of the scattered literature about C. cibarius into this volume, because many people have found it hard to find literature on the biology of this species. Altogether, this thesis is a first attempt to describe the ecology of C. cibarius. I wish you a pleasent reading.

Eric Danell April 1994

Abbreviations

AAHN (Advanced) Automatic AHN

AHN Acid-catalysed hydrolysis and ninhydrin

BHN Base-catalysed hydrolysis and ninhydrin

cAMP Cyclic adenosine monophosphate

CBS Centraalbureau voor schimmelcultures

CUS Culture unit system

DW Dry weight

FW Fresh weight

ITS Internal transcribed spacer

KBA King's B agar

MFM Modified Fries medium

Mkg Million kg

MS Murashige & Skoog medium

NMR Nuclear magnetic resonance

PCR Polymerase chain reaction

rDNA Ribosomal DNA

RFLP Restriction fragment length polymorphism

SEM Scanning electron microscope

TEM Transmission electron microscope

Swedish and scientific names of fungi

In order to enhance the pleasure of reading, I have excluded authors of scientific names of fungi from the text. Instead, all higher fungi mentioned in the summary, together with their authors, are presented below. In an effort to please biologists with limited knowledge of mycology, I have used the Swedish names according to Lundqvist & Persson (1987).

Agaricales - ordningen skivlingar och soppar

Agaricus bisporus (Lange) Imbach - trädgårdschampinjon

A. langei Møller - blodchampinjon

Amanita muscaria (L.: Fr.) Pers. - röd flugsvamp

Aphyllophorales - ordningen skinn, tickor, kantareller, tagg- och fingersvampar

Apiocrea chrysosperma (Tul.) H. Sydow & Sydow - gul svampsnylting

Boletus edulis Fr. - karljohan

Cantharellus see page 13

Chalciporus rubinellus (Peck) Singer - släkting till pepparsoppen

Clavaria - fingersvampar

Clavariadelphus - klubbsvampar

Coprinus - bläcksvampar

Craterellus see page 13

Entoloma parasiticum (Quél.) Kreisel - en rödskivling

E. pseudoparasiticum Noordeloos

Gomphus clavatus S.F. Gray - klubblik trumpetsvamp

Hebeloma cylindrosporum Romagn. - en fränskivling

Hydnum repandum (L.: Fr.) Fr. - blek taggsvamp

H. rufescens (Pers.: Fr.) Fr. - rödgul taggsvamp

Hypomyces - svampsnyltingar:

H. odoratus G. Arnold

H. lactifluorum (Schw.) Tul.

H. semitranslucens G. Arnold

Laccaria bicolor (Maire) Orton - tvåfärgad laxskivling

L. laccata (Scop.:Fr.) Berk. & Br.- laxskivling

Lactarius rufus Scop.:Fr.- pepparriska

L. pubescens Fr.- blek skäggriska

Leccinum - strävsoppar

Lentinula, L. edodes (Berk.) Pegler - Shii-Take

Piloderma croceum Erikss. & Hjortst. - saffranstråd (gultrådsskinn)

Pisolithus tinctorius (Pers.) Coker & Couch - ärttryffel

(=P. arhizus (Scop.: Pers.) Rauschert

Pleurotus - ostronmusslingar

Pseudocraterellus see page 13

Rhizopogon - hartryfflar

Russula - kremlor

Sarcodon imbricatus (L.:Fr.) Karst. - fjällig taggsvamp

Schizophyllum commune Fr.:Fr. - klyvblad

Sepedonium chrysospermum Tul. - gul svampsnylting (anamorph)

Suillus luteus (L.:Fr.) S.F. Gray - smörsopp

S. variegatus (Sow.: Fr.) O. Kuntze - sandsopp

Stereum hirsutum (Willd.:Fr.) S.F. Gray - raggskinn

Thelephora - vårtöron

Tuber melanosporum Vitt. - périgordtryffel

Verticillium lecanii (Zimm.) Viégas.

TAXONOMIC INTRODUCTION

Historical notes

Cantharellus cibarius, the chanterelle, is an edible mushroom that was described and illustrated back in 1581 by Mathias de l'Obel (Lobelius). In 1601, Charles de l'Escluse (Clusius) wrote the first scientific monograph on fungi in his famous "Fungorum Historia". Clusius used both German and Hungarian names for C. cibarius, which indicates that the chanterelle was well known to ordinary people. Carl von Linné (Linnaeus) described Agaricus chantarellus in e.g. Flora Suecica (1755). After the scientific description he added his personal view, "...that very few fungi are possible to recognize in this mixed up mess" (translation). Elias Fries, influenced by Persoon (1801), developed a comprehensive systematic system for fungi (Systema mycologicum 1821-32). He gave the chanterelle the final name Cantharellus cibarius (from Latin cantharus and Greek kantharos, beaker; and Latin cibus, food). C. cibarius was later placed among gill fungi (Agaricini), separated from relatives such as Craterellus which were placed among fungi with a smooth hymenium (Thelephorei) (Fries 1874). In 1900, Fries' artificial system was revised by Patouillard. He used the term Aphyllophoracés for Hymenomycetes without gills. C. cibarius was grouped with other cantharelloid fungi in Tribu des Cantharellés, still belonging to Agaricacés (gill fungi). The relationship between Clavaria (Aphyllophorales) and Cantharellus was first proposed by Maire (1902), accepted by Smith & Morse (1947) and Heinemann (1961) and further discussed by Petersen (1971). Donk (1964) made a conspectus over the 21 families of Aphyllophorales, including Cantharellaceae. Many other mycologists during the 17th and 18th centuries described the chanterelle as reviewed by Ainsworth (1976) and Persson & Mossberg (in press) among others.

Description of the genus Cantharellus

This description of fruit bodies of the genus Cantharellus (Fig. 1) is mainly based on Corner (1966), Petersen (1971, 1973, 1985) and Smith & Morse (1947):

Cantharellus is a homobasidiomycete genus, which means that spores are formed on unicellular basidia, in contrast to heterobasidiomycetes. The fruit body is gymnocarpic, i.e. naked from the time of its first appearance and not protected by a veil as in Amanita. The pileus (cap) has a sterile top, which distinguishes it from the Clavariaceae. Fruit bodies are fleshy and long lived but not perennial. The hymenium (spore producing layer) is either smooth or folded, with ridges on the stem and pileus. The gill-like ridges (Fig. 1A) differ from true gills of the order Agaricales (Fig. 1B). The Cantharellus hymenium thickens as new basidia develop over the layer of older ones. By contrast, in the Agaricales the basidia form a monolayer. Cantharellus basidia are stichic (Juel 1916) and long, and bear long curved sterigmata (Fig. 1C). Spores are smooth, white or yellow and of variable size. The number of spores per basidium varies between 2 and 8 within the same fruit body's hymenium (nuclear migration studied by Maire 1902). The haploid chromosome number in C. cibarius is 2 (Juel 1916). No cystidia (sterile hyphal ends with characteristic morphologies different from normal sterile hyphal ends) are present. Hyphae are monomitic (i.e. with thin-walled generative hyphae), and the presence of clamp connections (Fig. 1E) separates Cantharellus from Craterellus and Pseudocraterellus (Corner 1957). The characters above indicate that the genus Cantharellus is morphologically less advanced than agaric fungi.

Fig. 1. Schematic illustrations of some morphological characters. A A section of a Cantharellus fruit body and of the hymenium with basidia situated on as well as between the ridges. B A section of an agaric fruit body and of the hymenium with basidia only situated on the gills. C The Cantharellus basidium, in this case with three sterigmata and spores. The stichic immature basidium shows the longitudinal direction of the mitotic spindle used to separate chromosomes. D A schematic agaric basidium with four sterigmata and spores. A chiastic basidium shows the transversal direction of the mitotic spindle. E A hypha with clamp connection, a character only found among certain basidiomycetes, e.g. Cantharellus but not Craterellus.

Pigments and volatile compounds

Cantharellus cibarius cytoplasm contains bicyclic carotenoid pigments (Arpin & Fiasson 1971, Gill & Steglich 1987) characteristic of the subgenus Cantharellus. The density of pigments of in vitro cultures might however differ within a strain (Paper II). In nature, fruit bodies lacking yellow pigments are sometimes found. They have probably lost the ability of pigment production. C. pallens however, has pale fruit bodies but a pigmented hymenium, and was described as a separate species by Pilát (1959). Carotenoids are rare among agaric fungi (Gill & Steglich 1987). The other subgenus, Phaeocantharellus (Corner 1966), also described as section Leptocantharellus (Donk 1969), is characterized by black or brown aliphatic pigments as in C. tubaeformis. The purpose of most fungal pigments is unknown, but they might be waste products, insect attractive, insect repellent, light protective, light sensitive or act as camouflage. Pyysalo (1976) identified 13 volatile acids and 36 other volatile compounds from C. cibarius. Octenols (causing the characteristic smell of mushrooms in general), caproic acid and acetic acid were found in highest concentrations.

World distribution

The number of described Cantharellus species in the world exceeds 70. The genus is known from every continent except Antarctica (Corner 1966, 1969, Donk 1969, Petersen 1979, Nuhamara 1987, Thoen & Ba 1989, Watling & Abraham 1992). European and Swedish species of the family Cantharellaceae are shown in Table 1. Corner (1966) mentions 17 varieties of the cosmopolitan C. cibarius, of which 14 are found in Europe. Several authors believe that some varieties might actually be different species. (Feibelman et al. in press, Petersen 1979). The definition of a fungal species is either taxonomic or biological (Høiland 1983). Some taxonomists give a fungal variety the rank of species if it has permanent morphological differences from the main species. Two examples are C. pallens and C. amethysteus which are sometimes treated as varieties of C. cibarius, but sometimes considered separate species (Corner 1966). The biological species definition - a group of organisms capable of interbreeding and producing fertile offspring, is hard to prove for basidiomycetes. Fusion of single spore mycelia to heterocaryotic mycelia capable of fruiting must be performed. This is in most cases impossible due to absence of suitable techniques for such culture systems. Methods of molecular biology may reveal differences in certain sequences of DNA of different fungi, but the interpretation of such data is still subjective. This discussion is further illuminated by Dahlberg (1991).

Related genera

In Sweden, close relatives to Cantharellus are Craterellus and Pseudocraterellus, but also Gomphus, Clavariadelphus and Hydnum are sometimes mentioned (Corner 1957, 1966, Donk 1964, Petersen 1971, Persson & Mossberg in press). Reijnders & Stalpers (1992) claimed that Hydnum has a different hymenophoral trama and lacks carotenoid pigments and is therefore not related to Cantharellus. The bases for separation of genera is even more controversial than for species. For example, Pseudocraterellus is separated from Craterellus due to presence of secondary septa. According to Petersen (1973), this character is not permanent in Pseudocraterellus, and might be found in Craterellus, so this separation might not be valid. The difference between Cantharellus and Craterellus is based on occurrence of clamp connections. If this is a character useful to group species of the same evolutionary origin should be further investigated using methods of molecular biology.

Table 1. A list of the European species included in the family Cantharellaceae. Some of the most important synonyms are also given. A Swedish name indicates that the species is found in Sweden.

_____________________________________________________________________

Cantharellus cibarius Fr.: Fr. kantarell

C. pallens Pilát blek kantarell

C. amethysteus Quél.

C. friesii Quél.

C. tubaeformis Fr.: Fr trattkantarell
(= C. infundibuliformis (Scop.) Fr.)

C. lutescens (Pers.: Fr.) Fr. rödgul trumpetsvamp
(= C. aurora (Batsch) Kuyper)
(= C. xanthopus (Pers.) Duby)

C. borealis Petersen & Ryv.

C. melanoxeros Désm. svartnande kantarell
(= C. ianthinoxanthos (Maire) Kühner)
(= C. ciliatus Corner)

Craterellus cornucopioides (L.:Fr.) Pers. svart trumpetsvamp

C. cinereus (Pers.:Fr.) Pers. grå kantarell

C. konradii Maire & Bourdot

Pseudocraterellus undulatus (Pers.:Fr.) Rausch. kruskantarell
(= Pseudocraterellus sinuosus (Fr.:Fr.) Reid)
(= Craterellus crispus (Bull.) Berk.)
(= Cantharellus pusillus Fr.)

P. pertenuis (Skovst.) Reid

THE EDIBLE CHANTERELLE

Background

Fruit body production and human use

Julius von Krombholz, a physician concerned about mushroom poisoning, published one of the first modern illustrated floras on edible and poisonous fungi (1831-1846). C. cibarius and related species were thoroughly treated. Fries (1836) concluded that the prominent C. cibarius was known everywhere in Sweden as one of the few edible mushrooms. Fries (1860-1866) and other mycologists were later supported by their governments to teach people more about edible species (Ainsworth 1976, Kardell et al. 1980, Persson & Mossberg in press). According to Kardell et al. (1980), about 40% of the Swedish population picks wild mushrooms at least once every year, and C. cibarius is considered the most popular of all edible species. Dogs are sometimes trained to find C. cibarius (Hallgren & Hansson-Hallgren 1990).

In Sweden, of the 3 kg (FW, fresh weight) of mushrooms consumed per capita each year, 1 kg is wild mushrooms (Hansson 1990). The wild mushrooms consumed are equivalent to 2-3% of the estimated 300-500 million kg (Mkg) (FW) of edible fungi produced in Swedish forests each year (Kardell et al. 1980). The estimated fresh weight of all wild fruit bodies produced in Swedish forests each year is 2400 Mkg. Average production per hectare (FW) was estimated at 100-550 kg/year (2000-11000 fruit bodies), of which 20% were edible species. Rautavaara (1947) estimated the annual minimum production in Finland to be 100 kg/ha (FW) in humid spruce forests and Gulden et al. (1992) estimated the annual production in Norwegian spruce forests to be 18-405 kg/ha (FW). Oria de Rueda (1989) reported that the annual average production of six common species in the Spanish province of Soria was 544 kg/ha (FW) in the best Pinus sites. The yearly production of C. cibarius in Sweden was estimated at 0.45-2.5 Mkg (FW) (Kardell et al. 1980). According to the authors, this value was probably an underestimate.

According to three Swedish canning companies, at least 60 000 kg salted fruit bodies (=90 000 kg FW) of C. cibarius was imported to Sweden in 1993, most of it originating from Poland and Lithuania. In addition, an estimated 50 000 kg was collected and sold in Sweden in 1993 (Danell unpubl. data). North-American chanterelles are exported to Europe, mainly Germany, where the fungus is declining (Norvell 1992). In 1990, 126 000 kg of C. cibarius was officially collected in Washington, possibly representing 20% of the actual harvest (Molina et al. 1993). American chanterelles (C. subalbidus Smith & Morse and C. cibarius s. lat.) are considered to be of low quality, in terms of taste and texture, by Swedish food companies.

Predators and parasites

Thousands of insect species feed on fruit bodies (Hammond & Lawrence 1989). In Finland, 120 species of dipterans were recorded as fungivorous on agaric fruit bodies (Hackman & Meinander 1979). However, less than 1% of C. cibarius fruit bodies were infested with dipterans, mainly polyphagous limoniid larvae. By contrast, 40-80% of mature agaric fruit bodies are infested by dipterans, mainly mycetophilid larvae. Polyphagous elaterid larvae (Coleoptera) are also occasionally found in C. cibarius,

and in my opinion more frequently than dipterans. Other incidental findings of dipterans and coleopterans are summarized by Hammond & Lawrence (1989) and Hackman & Meinander (1979). I have also observed oribatids (Acarina), opilionids (Arachnida) and Collembola on fruit bodies of C. cibarius. If these are fungivorous, their impacts on the fruit bodies are insignificant. In view of the long life of fruit bodies of C. cibarius (31-84 days according to Paper V, Kälin & Ayer 1983 and Norvell 1992) and the vast number of mycophagous insect species, it is remarkable that C. cibarius is not heavily infested. It should be noted that the protein content is lower in C. cibarius than in other fungi (Paper I). Hostettman in Lausanne found a strong insecticidal effect in extracts of C. cibarius, but the effect was not reproducible (pers. com.). Pang and Sterner (1991) and Pang et al. (1992) have described two metabolites formed in C. cibarius and C. tubaeformis as a response to injury. The effects of these compounds on potential predators have not been tested yet. Selection for insect repelling compounds may explain why fruit bodies of C. cibarius do not need to grow rapidly (Paper V).

Another adaptation to long lived fruit bodies is the ability of C. cibarius fruit bodies to recover from injury. Two decapitated fruit bodies (probably made by a mushroom picker with a knife) had regenerated 1 cm new mycelium on top of the wounded 3 cm stems (Paper V). The new tissue formed a sterile hemisphere with a smooth surface (Fig. 2). The scar on the outside was easily found, but a microscopical study of the inside of the fruit body hardly revealed the regeneration zone.

Fig. 2. A schematic picture of a decapitated fruit body with regenerated tissue on top of the stem.

Slugs and snails (Gastropoda) are polyphagous detritivores and herbivores (Jennings & Barkham 1975). Together with vertebrates they may remove about 50% of the available fruit bodies before they can be used by dipterans (Lacy 1984). However, Worthen (1988) concluded that slugs (Arion spp.) mostly feed on the same mushroom species as generalist mycophagous drosophilids (Diptera), i.e. not C. cibarius. Frömming (1954) listed numerous mushrooms eaten by Limax, Arion and Cochlodina, but C. cibarius was not included. Only the south European snail Helix aperta Born was reported to feed on C. cibarius in the laboratory. However, C. cibarius hardly exists in the natural environment of H. aperta (Walldén pers. com.). My own experience is that gastropods rarely feed on C. cibarius.

Nematodes are known to graze on vegetative hyphae of ectomycorrhizal fungi (Francl 1993). Riffle (1971) reported a repelling effect on nematodes of C. cibarius mycelium in vitro. However, the mycelium used was not described.

Mushrooms, including C. cibarius, are important for squirrels during the mushroom season (Fogel & Trappe 1978, Grönwall 1982). Roe deer eat plenty of fruit bodies (Cederlund et al. 1980), but it is not known whether C. cibarius is used. Rein deer probably eat C. cibarius, sheep have been observed to eat C. cibarius, and wild boar probably eat C. pallens (Danell & Åkerblom unpubl. data). Launchbaugh & Urness (1992) reviewed the mycophagy of cervids, small mammals, birds and turtles, but Cantharellus was not mentioned. According to Miller & Halls (1969), birds like turkeys and grouse feed directly on mushrooms, while e.g. robins tear mushrooms apart in search of larvae. C. cibarius was not specifically mentioned. The strong pepper-like taste of raw C. cibarius is removed through cooking, which might explain why humans are such important predators on C. cibarius.

A rare fungal parasite which forms fruit bodies exclusively on Cantharellus hymenium is the agaric Entoloma pseudoparasiticum. E. parasiticum is another rare C. cibarius parasite which also fruits on other fungal genera (Noordeloos 1992, Overholts 1929 as Claudopus subdepluens Fitzp.). Moulds can also parasitize Cantharellus species. Helfer (1991) reported Hypomyces odoratus (Pyrenomycetes) on C. cibarius, H. semitranslucens on C. tubaeformis and Verticillium lecanii on C. lutescens. I have never observed any parasites on C. cibarius, and these Hypomyces species have not been recorded from Sweden (Eriksson 1992). The orange Hypomyces lactifluorum may resemble Cantharellus when infecting Lactarius or Russula fruit bodies (Arora 1986). Parasitized fruit bodies are highly appreciated in Mexico. Another more familiar parasite is the closely related Apiocrea chrysosperma (Syn. Hypomyces chrysospermus Lk., anamorph Sepedonium chrysospermum), causing the well known yellow rot of Boletus edulis.

There is one report of a suspected viral disease on fruit bodies of C. cibarius and C. tubaeformis (as C. infundibuliformis) (Blattny & Kralik 1968). Symptoms are enations on top of the caps, often affecting a whole cluster of fruit bodies. Virus diseases affecting fruit bodies of Agaricus, Lentinula (as Lentinus), Pleurotus (Pingyan et al. 1990) and Boletus (Pisi et al. 1988) are also known. All fruit bodies of C. cibarius contain large numbers of moulds and bacteria in contrast to most agaric fungi (Paper IV). However, the dominating bacterium Pseudomonas fluorescens Migula was not able to penetrate fungal tissues even though it is motile, and mycelium and most bacterial strains coexisted in vitro without causing significant inhibition (Paper IV). This implies that these contaminating organisms are seldom severely parasitic. Instead they are probably incidentally incorporated during fruit body formation (Paper IV).

¹³⁷Cs, medical aspects and heavy metals

As a consequence of the accident at the nuclear power station in Chernobyl in 1986, large areas in Sweden were contaminated with more than 30 000 Bq/m² of ¹³⁷Cs (Karlén & Johanson 1991). It was soon realized that fungal fruit bodies concentrate caesium (Battiston et al. 1989), especially in cap and gills (Heinrich 1993). It is also believed that mycorrhizae form large reservoirs of radiocaesium in soil (Environmental Radioactivity Special Topic Steering Committee 1993). Fungi are an important source of ¹³⁷Cs in grazing mammals during September - October. (Karlén & Johanson 1991). C. cibarius contains less ¹³⁷Cs than many other fungi (Battiston et al. 1989, Heinrich 1993). However, in some areas of Sweden C. cibarius exceed the Swedish health limit of 1500 Bq/kg (FW) (Nohrstedt 1994). The immobility of caesium in soil, the storage capacity of fungi and continuous studies on the ¹³⁷Cs in mushrooms imply that only time (physical half-life is 30.2 years) will decrease levels of ¹³⁷Cs. Addition of potassium to the soil might decrease levels of ¹³⁷Cs in fungi and plants (Nohrstedt 1994). The Swedish Radiation Protection Institute recommends a maximum dose of ¹³⁷Cs of 1 mSv/year (in addition to natural radionuclide intake). This corresponds to an intake of approximately 80 000 Bq/year (International Commission on Radiological Protection, ICRP, 1989). According to estimates by ICRP (1990), 1 mSv corresponds to a statistical risk of lethal radiation-induced cancer of 0.005%.

The mutagenic capacity of natural C. cibarius extract without ¹³⁷Cs has been tested on bacteria (Grüter et al. 1991). A weak mutagenic capacity was found, higher than for ten other genera tested, but lower than for A. bisporus. However, no separation between the mutagenic capacity of C. cibarius tissues and naturally incorporated microorganisms (Paper IV) was made. The mutagenic capacity was further enhanced by treatment simulating human metabolic activity. On the other hand, C. cibarius extract inhibited the mutagenic activity of e.g. aflatoxin B₁ and benzopyrene on bacteria (Grüter et al. 1990). Another medical aspect is basidiospores as aeroallergens, which are present in the atmosphere in concentrations considerably in excess of pollen grains (Lehrer et al. 1986). Out of 15 basidiomycetes skin tested on adults with respiratory-allergic disease, C. cibarius spores were the least allergenic (5% of the adults were sensitive). Sensitivity to alcohol after consumption of C. tubaeformis has been observed (Persson & Karlson-Stiber 1993).

According to Grzybek & Janczy (1990) and Kuusi et al. (1981), the lead and cadmium contents of C. cibarius was lower than those of other edible species studied at the same site. Fungi like C. cibarius with a low selenium uptake (Piepponen et al. 1983) also have a low uptake of mercury (Kuusi et al. 1981, Lorenz et al. 1978, Stijve and Cardinale 1974). Schellman et al. (1984) showed that cadmium and cupper are strongly bound to the cell walls of fungi. After digestion of A. bisporus, no detectable amounts of heavy metals were released into human blood or urine.

In conclusion, C. cibarius is a harmless species compared to many other edible fungi. The antimutagenic capacity is interesting but needs further investigation.

Amino acid and total protein content (Paper I)

Levels of carbohydrates (Laub & Lichtenthal 1985), lipids (Aho & Kurkela 1978, Daniewski et al. 1987), minerals (Vetter 1993), vitamins (Leichter & Bandoni 1980, Mäkinen et al. 1978), sterols (Kocór & Schmidt-Szalowska 1972) and some other compounds (Pang 1993) in fruit bodies of C. cibarius are already quantified. However, reported protein levels in fungi, in general, have been strikingly high. The nutritional value has often been over estimated and many reports are conflicting (Table 2).

When determining the total protein content of fungal tissues it is not advisable to use methods based on total nitrogen analysis (Total N), such as the Kjeldahl method (Nordic Committee on Food Analysis 1976). A complication is that fungal cell walls contain chitin, i.e. polymers of N-acetylglucosamine (Deacon 1984), therefore the total nitrogen analyzed is mainly a mixture of protein and chitin (Ofenbeher-Miletic et al. 1984). Other nitrogenous compounds might also interfere (Seelkopf & Schuster 1957). Still, the method is widely used among mycologists (Leichter & Bandoni 1980, Bisaria et al. 1987, El-kattan et al. 1990, Levai 1989, Surinrut et al. 1987, Vetter 1993) and for routine nutrient analysis of mushrooms at the Swedish National Food Administration. Sometimes the chitin content is taken into account by assuming that 70% of the nitrogen originates from proteins (Crisan & Sands 1978, Bano & Rajarathnam 1988).

Another method of protein analysis, used for extracts of mites and pollen, is called BHN (base-catalysed hydrolysis and ninhydrin), and is based on alkaline hydrolysis of proteins and total amino acid analysis with ninhydrin (Richman & Cissel 1988). The automatic and expensive amino acid analysis based on the technique of Spackman et al. (1958) is seldom used for mushrooms (Ogawa 1987, Gerrits 1989). Therefore I wanted to establish a reliable routine method and calibrate it using the automatic amino acid analysis. At the same time, I wanted to publish more reliable protein and amino acid data for C. cibarius. The new method, acid-catalysed hydrolysis and ninhydrin (AHN), is described separately. Parallel tests were Kjeldahl analysis, Bradford analysis (Bradford 1976) and the BHN assay. Automatic (advanced) acid-hydrolysis and ninhydrin analysis (AAHN), using the LKB Model 4151 analyser, served as the reference method.

Results and discussion - protein content

The measured total protein content differed greatly depending on the method used (Table 2). The complete AAHN, which was the reference method, resulted in a value of 9.9% protein (DW). The new AHN method resulted in 8.7% protein (DW), which was the result most similar to the reference among the methods tested. This means that about 10% of the dry weight, or 1% of the fresh weight, of a C. cibarius fruit body is protein. Results of Total N analysis in the literature are therefore 70-200% higher than the AAHN value. The amino acids according to the reference method are shown in Table 3.

The protein content of C. cibarius might vary depending on the genotype and age of the fruit body. Nevertheless, previous values of protein content in mushrooms seem to be overestimated owing to the presence of chitin, urea, nucleotides etc. The nutritional value of C. cibarius and probably other mushrooms as well, should not be over emphasised. However, C. cibarius seems to have a low protein content compared with

The AHN method.

other edible mushrooms (Crisan & Sands 1978, Levai 1989, Lintzel 1943, Seelkopf & Schuster 1957, Singer & Harris 1987). In A. bisporus for example, the protein content was 17% (DW) according to Paper I. Mushrooms in general have a higher protein concentration than many vegetables (Bano & Rajarathnam 1988) and wild plants (Källman 1991), but only on a dry weight basis. For instance, fresh potatoes contain twice as much protein as fresh C. cibarius.

The digestibility of C. cibarius protein was not tested, but the digestibility of Pleurotus proteins is equal to that of most plants, i.e. 90%, whereas meat has a digestibility of 99% (Bano & Rajarathnam 1988). Little is known about the digestibility of carbohydrates or the occurrence of antinutritional substances in C. cibarius, which are important aspects in evaluation of foods. In addition, insects obviously avoid C. cibarius (see previous pages), but it remains to find out if this avoidance is due to the presence of insecticidal or antinutritional substances, or to low nutrient value or a combination of these factors.

Results and discussion - evaluation of methods

With the reference method, all amino acids except for tryptophan were detected in the AHN extract. About 9% of the ninhydrin-detectable nitrogen came from ammonia. AHN produced 220 nmol glucosamine per mg dry tissue, which is 7-fold more than with BHN. According to the reference method, 15% of the ninhydrin-detectable nitrogen came from glucosamine, but half of it was transformed to ammonia.

With the reference method, analysis of BHN extract showed that arginine disappeared but was hydrolysed to ornithine which was still detectable. Histidine and tryptophan were totally lost, as was 85% of the threonine and 60% of the serine. About 15% of the ninhydrin-detectable nitrogen was ammonia.

The amount of amino acids in the AHN extract, was 96% of that in the extract of the reference method. The amount of amino acids detected using AHN was 89% compared with the reference method.

The distribution of nitrogen according to the reference method is shown in Table 4. If the value for glucosamine is multiplied by the conversion factor of 14.5 for chitin (6.89% N in chitin), the result is 4.5% chitin (in accordance with Ofenbeher-Miletic et al. 1984). If the sum of Table 4 is multiplied by the conversion factor of 6.25 for protein (16% N in average protein) as in Kjeldahl analysis, the resulting protein concentration is 13.4% (DW). This theoretical value is somewhat lower than the measured Total N value of 16.6% (DW) obtained using Kjeldahl analysis. This implies that nitrogenous compounds other than chitin might interfere. If the conversion factor of 6.25 is replaced by 4.38 to eliminate chitin-N (Crisan & Sands 1978), the resulting protein concentration is 9.4% (DW) which corresponds better to values determined using the reference method (9.9%). According to Table 4, 77% of the nitrogen originates from amino acids.

For determining total protein content, total amino acid analysis is more exact than Bradford analysis. Bradford analysis is useful for making rough determinations of cytoplasmic proteins. However, cell wall proteins are difficult to extract; the coloured complex is not as stable as the ninhydrin complex, and the standard protein has to be similar in size, amino acid composition and shape compared to the proteins in the sample.

Of the amino acids detected by the reference method, 89% was also detected with AHN. AHN is therefore a good method for those who want to determine total amino acid (total protein) contents of fungi simply and inexpensively. However, since complete hydrolysis might not be obtained and some amino acids are lost with AHN, and since glucosamine is part of the result, it is recommended that AHN should be calibrated with the reference method.

Table 2. Protein percentage of freeze dried C. cibarius measured
with different methods (Brad=bradford, BHN=Base-catalysed Hydrolysis
and Ninhydrin, AHN=Acid-catalysed Hydrolysis and Ninhydrin, 
Reference=automaticAHN, NINP=Ninhydrin and Paper chromatography,
Total N=Total Nitrogen analysis). Amino acid extracts that were used for 
BHN and AHN (BHN-NaOH, AHN-HCl) were also tested with the 
reference method. 
_____________________________________________________________________

Method		Extraction	%protein of		Literature 
		procedure	dry matter
_____________________________________________________________________

Brad		NaOH 1M			3.3			
Brad		Urea 8M			4.5

Brad		0.01% SDS		1.2
Reference	0.01% SDS		7.5

BHN		BHN-NaOH		7.1
Reference	BHN-NaOH		7.6

AHN		AHN-HCl			8.7
Reference	AHN-HCl			9.3
Reference	Reference		9.9

Total N	        Kjeldahl		16.6
NINP		Acid 			5.0 (Seelkopf & Schuster 1957)
Total N					30.9 (Seelkopf & Schuster 1957)
Total N					22.8 (Lintzel 1943)
Total N					21.4 (Crisan & Sands 1978)
Total N					17.7 (Levai 1989)
Total N					2.6 (FW) (Harris & Singer 1987)

Table 3. Amino acid residues of freeze dried C. cibarius measured by the reference method (tryptophan not included). Values are based on one analysis of a sample of eight freeze dried fruit bodies.

_____________________________________________________________________

Amino acid ug/mg dry tissue

_____________________________________________________________________

Aspartic acid^a 9.9

Threonine 5.3

Serine 5.8

Glutamic acid^b 15.6

Proline 4.7

Glycine 4.1

Alanine 5.2

Half-Cysteine 0.9

Valine 5.0

Methionine 1.2

Isoleucine 4.7

Leucine 7.4

Tyrosine 3.8

Phenylalanine 4.8

Histidine 3.0

Lysine^c 8.3

Arginine 8.9

Sum 98.6

_____________________________________________________________________

^a Aspartic acid includes asparagine
^b Glutamic acid includes glutamine
^c Lysine includes ornithine

Table 4. Nitrogen content in C. cibarius analysed with the reference method (AAHN).

_____________________________________________________________________

Nitrogen source Nitrogen ug/mg sample

_____________________________________________________________________

Amino acid N 16.4

Ammonia N 1.9

Glucosamine N 3.1

Sum 21.4

CULTIVATION OF C. CIBARIUS

Background

Evolution and definition of mycorrhiza

It is believed that the conquest of land by green plants in the late Silur was facilitated by symbiotic fungi resembling zygomycetes (Stubblefield & Taylor 1988, Taylor 1990). The ancestors of basidiomycetes could have been zygomycetes since only very few and doubtful fossils of precretaceous ascomycetes exist (Pirozynski 1976, Taylor & Taylor 1993). The hypothesis is also based on cytological comparisons made by Cavalier-Smith (1987). According to 18S ribosomal RNA gene sequence data, the ascomycete lineage split from basidiomycetes in early Devonian (Berbee & Taylor 1993). The fossil record of basidiomycetes (Pirozynski 1976) indicates that a rapid evolution began during the Carboniferous era when dead plants accumulated. Cellulases, ligninases, drought resistance, use of mechanical power by swelling hyphae and high production of sexual spores made basidiomycetes superior saprophytes on most substrates and biotopes. Some basidiomycetes might have lost their saprophytic or parasitic enzymes but still kept their ability to mechanically invade plant roots, mainly those of trees (Malloch 1987, Newman & Reddell 1987, Taylor 1990), thus forming a symbiosis named ectomycorrhiza (Greek: ekto, outside; mykes, fungus and rhiza, root. See Fig. 3). Ectomycorrhizal basidiomycetes probably coevolved with the tree family Pinaceae during the Jurassic era (Allen 1991). However, fossil ectomycorrhizae are only known from the Cenozoic (Taylor 1990). Today, about 5000 fungal species (mainly basidiomycetes, but also fungi imperfecti, zygo- and ascomycetes) of the world flora are considered ectomycorrhizal (Harley 1989). About 830 of these, among them C. cibarius, are found in Sweden (Hallingbäck in press).

The anatomical structure "mycorrhiza" was named and described by Frank (1885) when he studied the possibilities to grow truffles. His assignment did not succeed, but far more important he postulated the mutualistic nature of the mycorrhizal symbiosis. Once Melin had succeeded at forming ectomycorrhiza in vitro (1922), several experiments followed that confirmed Frank's hypothesis (Harley & Smith 1983). The radioactive labelling of CO₂ and mineral nutrients has shown that the fungus relies on simple carbohydrates synthesized by plant photosynthesis. Benefits to the plant include increased levels of mineral, water and phosphorus uptake, due to the enlarged contact with the soil via hyphae, and the ability of the fungus to use organic sources of nitrogen and phosphorus as well as the phosphorus rich mineral apatite (Fig. 3).

Since Frank, five other mycorrhizal types have been discovered, involving most vascular plant families (Harley & Smith 1983), and nearly 12000 articles (Mycolit database) have been published. Mycorrhiza research has for example focused on anatomy (Agerer 1986), colonization (Piché et al. 1988), mineral flow (Dighton 1991), carbon flow and metabolism (Finlay & Read 1986, Ramstedt et al. 1986), nitrogen metabolism (Finlay et al. 1992, Martin & Botton 1993) ecology (Allen 1991, Dahlberg 1991, Read et al. 1992) and hormones (Gay 1988, Nylund 1988, Wallander et al. 1992). Applied research has been focused on e.g. improving yields of vegetables and the survival of micropropagated plants (Lovato et al. 1992), enhancing the survival and growth rate of tree seedlings (Harley & Smith 1983), facilitating the reforestation of polluted sites using the ectomycorrhizal fungus Pisolithus tinctorius (Marx et al. 1992) and increasing the fruit body production of the French Périgord truffle, Tuber melanosporum (Chevalier & Frochot 1981, Rocchia 1992).

Fig. 3.Schematic picture of ectomycorrhiza between Picea abies and C. cibarius. M indicates the hyphal mantle surrounding the short-roots. T shows the transection of an ectomycorrhizal short root. M is mantle and H is Hartig Net, i.e. the net of hyphae between cortex cells of the root that proves the symbiosis.

Earlier efforts to grow C. cibarius

Since C. cibarius is a highly appreciated edible mushroom, many efforts have been made to cultivate the fungus. The main reason for the small number of publications, has been the difficulty to obtain pure mycelium. This problem is due to its highly specific nutrient demands, inability to use complex carbohydrates like cellulose (in contrast to species such as Agaricus bisporus), sensitivity to autoclaved media, slow growth and to the natural contamination of fruit bodies with moulds and bacteria (Paper II and IV).

Verified successful isolations have been made in at least two laboratories. In 1979, Fries succeeded in germinating spores of C. cibarius, using a complex but defined medium. This medium was modified by Straatsma & van Griensven (1986), who repeated Fries experiments and also succeeded in obtaining pure C. cibarius mycelium from fruit bodies (Straatsma et al. 1985). The mycelium was verified by Straatsma et al. (1985) as C. cibarius using dot-blot DNA hybridization. Moore (1989) used the technique of Straatsma in her mycorrhiza experiments. Danell & Fries (Paper II) facilitated the techniques for spore germination and made some additional studies followed by mycorrhiza synthesis. The species identity of my mycelia was confirmed with PCR (polymerase chain reaction) and RFLP analyses (restriction fragment length polymorphism) (Paper III).

The reviews below which should be helpful to experimental mycologists, cover unsuccessful methods and results derived from strains of uncertain species identity. The results below are interesting, but need to be repeated with defined mycelia.

There have been several unsuccessful attempts to obtain pure cultures of C. cibarius: Modess (1941) did not succeed in isolating mycelia from any of the four Cantharellus species tested. Froidevaux (1975) used MMN and PDA medium without any success in obtaining mycelium. Schouten & Waandrager (1979) used several media but did not succeed in obtaining C. cibarius mycelium from spores or fruit body tissues. Danielson (1984) failed to isolate mycelium from fruit bodies using MMN medium. Itävaara & Willberg (1988) failed to isolate mycelium from fruit bodies using the techniques of Straatsma et al. (1986), but established a strain collection from spores based on Fries (1979).

There are some reports of successful isolations of C. cibarius mycelium that have not been verified: Doak (1934) mentioned a successful synthesis of C. cibarius mycorrhiza, but neither the mycorrhiza nor the methods were described. Sugihara & Humfeld (1954) isolated a mycelium from C. cibarius tissues using PDA medium. The strain M83, or NRRL 2370, was the same as ATCC 13228 and CBS 155.69 which was later defined as not being C. cibarius (Stalpers at CBS, pers. com.). Hattula and Gyllenberg (1969) reported that C. cibarius grew very well on 5% malt extract, Modess' medium and Reussers' medium. However, the mycelium was not described and the reported rapid growth on malt extract conflicts with the results of Paper II and Straatsma & van Griensven (1986). Siehr et al. (1969) found that their C. cibarius mycelium was the only one of a large number of basidiomycetes studied, that had the ability to acetylate D-tryptophan. Only moulds had the same character. The C. cibarius mycelium was not described. Torev (1969) reported that C. cibarius and other edible mushrooms were easily grown on simple media without vitamins. In fermentors they budded and formed yeast-like single cells. No description of the C. cibarius mycelium was made, but the yeast-like appearance has not been observed when growing C. cibarius in liquid shaken

cultures (Danell unpubl. data). Colonies were, instead, very dense and pellet-like in accordance with the observations of Moore (pers. com.). If not shaken, the colonies turned woolly. Riffle (1971) studied the impact of nematodes on different fungi, including C. cibarius from New Mexico. The undescribed mycelium was able to grow on PDA medium and resisted the nematodes. Volz (1972) kept stock cultures of C. cibarius on 2% malt agar. Many characters of these mycelia were different from the strains of Straatsma (Stalpers & Moore pers. com.). Ballero et al. (1991) suggested that C. cibarius is saprophytic, since their isolated mycelium grew on malt extract or yeast extract with additional complex carbohydrates, including cellulose. The mycelium was not described; nor was the mycorrhizal status discussed.

In addition, a study with commercial interest in fruit body formation of C. cibarius was carried out by Kuhn Champignon in Switzerland, but no data are available (Charlotte Wall pers. com.).

Isolation and description of the mycelium (Paper II)

Tissue culture

A total of 229 fruit bodies of C. cibarius, probably representing at least 70 genotypes, were collected from 13 coniferous forests in the province of Uppland, Sweden. Eight fruit bodies of C. pallens and ten of C. tubaeformis (the latter one was used for spore germination only) were also collected. Under sterile conditions, small samples of tissue from the inside of the fruit bodies were transferred to the following agar media:

1. Murashige & Skoog medium (MS) (Table 5) was supplied with antibiotics (50 ppm streptomycin and ampicillin, 5 ppm benomyl) and then sterile filtered (0.2 um Sartorius cellulose acetate filter). MS was then mixed with an autoclaved agar solution (final agar concentration 1.2%) with 0.05% activated charcoal (to adsorb toxic compounds in autoclaved agar), before addition to 9-cm petri dishes.

2. Modified Fries medium (MFM) (Table 5) was sterile filtered and mixed with autoclaved agar without activated charcoal. As a precaution, activated charcoal was added on top of the agar surface.

3. MFM with C-elements: Tween 80, malic acid, MES and thymine. C-elements substitute CO₂, which is necessary for the production of pyrimidines and fatty acids (Straatsma & Bruinsma 1986).

The petri dishes were incubated in darkness at 20 deg.C. All tissue samples were surrounded or embedded in mucus of bacterial origin within a few days (Paper IV). Growth did not start until after a 17 to 53-day-long lag period (35 days on average). Growth from tissues was only observed on MS, and only if activated charcoal had been embedded in the agar. In this study, 24% of the tissue samples generated mycelia that grew out of the bacterial zone, and peripheral hyphae were reinoculated until sterile. A total of 56 strains of C. cibarius and 4 strains of C. pallens were isolated. Sterile mycelia were later transferred to MFM. Modified Melin-Norkrans medium (MMN) (Molina & Palmer 1982) can also be used, but growth is slower. No growth was observed on 2% malt agar. All 36 tissue samples directly inoculated on MFM died owing to fatal bacterial infections. Repeated trials were never successful.

Table 5.

Composition of Modified Fries Medium (MFM)
based on Straatsma & van Griensven (1986), Eriksson (1965) and Fries 
(1978). Murashige & Skoog medium (MS) from Straatsma et al. (1986).
_____________________________________________________________________
				MFM			MS

D (+) glucose			2.20 g			0
D (-) fructose			2.00 g			0
Sucrose				0			20 g
NH4Cl				0.58 g			0
Succinic acid			0.59 g			0
Meso-inosit			10 mg			100 mg
Macrostock 1a			50 ml			0	
Macrostock 1b			0 ml			100 ml
Eriksson microstock2		1 ml			1 ml
Fries vitamine stock3		5 ml			5 ml

Total volume			1000 ml pH 5.5		1000 ml pH 4.2

Composition of macrostock (g/1000 ml):
1a:
KH2PO4				4.00 
MgSO4 7H2O			2.00 
NaCl				0.40 
CaCl2 2H2O			0.52 

1b:
KH2PO4				1.7
MgSO4 7H2O			3.7
CaCl2 2H2O			4.4
NH4NO3				16.5
KNO3				19.0

2 Eriksson microstock (200 ml):	

EDTA Titriplex III			1.90 g
FeSO4 7H2O				1.40 g

MnSO4 H2O				170 mg
H3BO3					63 mg
ZnSO4 7H2O				287 mg
KI					75 mg
NaMoO4 2H2O				2.50 mg
CuSO4 5H2O				0.25 mg
CoCl2 6H2O				0.25 mg

3 Fries vitaminestock (mg/250 ml):

Thiamine				5.0 
Pyridoxine				5.0
Riboflavine				5.0 
Biotine					1.25 
Nicotinamide				5.0 
P-aminobenzoic acid			5.0 
Panthotenic acid			5.0 

Spore germination

Sections of caps of C. cibarius and C. tubaeformis were adhered to the inside of a petri dish lid using vaseline. Spores fell freely either onto MFM agar or onto the bottom of an empty petri dish. After 18 hours, the spores on the bottom of the empty petri dish were collected and spread out on MFM agar, or stored for a maximum of two months at 4 deg.C. Spore concentrations were 5 x 10⁴ to 10⁶ per petri dish with MFM. Activated charcoal was added to one half of the petri dishes. Activating organisms like the yeast Rhodotorula (Fries 1979) or tomato roots (Straatsma et al. 1985) were not needed. Spores of C. cibarius germinated after 6-12 weeks, but within a petri dish the germinating spores were activated simultaneously within the span of a few days. The best results were obtained when high numbers of spores (10⁶) fell directly on MFM. The incidence of germination in this system was lower than 0.04%. Heterokaryotic mycelia from spores were microscopically similar to mycelia from tissues and responded in the same way to malt agar and C-elements. Spores of C. tubaeformis germinated after 10 months. The clamped, light yellow mycelium grew rapidly (1 mm/day), but long lag periods followed reinoculation, which was only successful five times until the mycelium failed to recover.

Description of the mycelium

Since MS was used to isolate C. cibarius from tissue samples of fruit bodies, it is important to describe mycelial characters. The mycelium on MS grew below the agar surface and was weakly pigmented, giving the colony a greyish appearance. Microscopic characters of diagnostic value included frequent clamp connections (Fig. 1E) and a hyphal thickness of about 3 um. These characters were useful in excluding moulds (often lacking clamp connections and with thinner hyphae) which often occurred in fruit body tissues, probably incidently embedded as spores. Many moulds in the tissue samples were suppressed by the fungicide. Those that were not suppressed appeared within two weeks, well before the expected appearance period of C. cibarius.

After transfer to MFM, activated charcoal was no longer necessary for most strains. The mycelium often turned bright yellow after the first transfer and grew quite slowly (0.2-0.3 mm/day). The mycelium was generally reinoculated after 2 months, but sometimes even after 4 months. Within a few weeks the mycelium spontaneously switched from slow growth and pronounced pigment production to rapid growth (0.5 mm/day) and low pigment production. Most ectomycorrhizal basidiomycetes have a radial growth of 1 mm/day on MFM (data not shown), so C. cibarius is considered a slow-growing species.

The colony on MFM consisted of a white, aerial mycelium with a yellow centre. When such mycelium was transferred to MFM with C-elements, radial growth significantly decreased, but the density of hyphae increased, and the major part was below the agar surface. Mycelia on this medium also produced large amounts of bright orange carotenoid pigments. These mycelia survived for a year in 20 deg.C, but growth ceased after six weeks. If transferred back to the original MFM, pigment production remained high until a shift back to the whitish, rapidly growing surface mycelium occurred spontaneously. This switch might have been a way for the fungus to obtain atmospheric CO₂. In the presence of the C-elements, vegetative growth towards a more suitable atmosphere was no longer acute, and the mycelium could grow within the

nutrient agar where it was protected from drought. At very high concentrations, mycelium from liquid cultures (MFM) had the characteristic smell of C. cibarius. Descriptions based on molecular biology are presented later (Paper III).

I did not find any physiological or microscopical characters that could be used to distinguish between C. pallens and C. cibarius.

Stalpers at CBS found that a Dutch strain claimed to be C. cibarius, was the saprophytic fungus Stereum hirsutum (pers. com.). The mycelium of this species was yellow and fast growing and had multiple clamp connections. Another common yellow ectomycorrhizal basidiomycete is Piloderma croceum. Its mycelium is, however, intensely saffron, and it grows very well on 2% malt agar (Nylund & Unestam 1982).

In vitro formation of ectomycorrhiza (Paper II-III)

Introduction

The soil mycelium generating a C. cibarius fruit body is hard to detect, and it is often mixed with other species. Moreover, before the PCR technique was introduced, no good methods for identifying mycorrhizae were available. Therefore, only a few observations of C. cibarius mycorrhiza have been reported. Doak (1934) mentioned a successful synthesis of mycorrhiza with Pinus spp. and C. cibarius. However, no information on isolation procedures, mycelium or mycorrhiza was given. Froidevaux (1975) described a natural mycorrhiza believed to be C. cibarius and Pseudotsuga menziesii. Froidevaux tried in vain to generate pure mycelium of C. cibarius.

Moore et al. (1989) synthesized mycorrhizae with Betula pubescens and Pinus sylvestris with Hartig nets 1-2 cell layers deep. I synthesized complete mycorrhizae with Picea abies and Pinus sylvestris with fully developed Hartig nets throughout the cortex, limited by the endodermis only (Paper II). The axenic methods used for mycorrhiza formation were based on parafilm-sealed tubes or E-flasks with plant, fungus and nutrient solution (MFM) in perlite.

In addition, the rhizoscope technique of Unestam & Stenström (1989) was tested with C. cibarius spores and mycelium. No mycorrhiza formation or establishment of mycelium was observed. This technique is excellent for rapid mycorrhiza synthesis of fungi with low carbohydrate demands, but for C. cibarius another technique was needed.

The methods of Moore et al. (1989) and Danell & Fries (Paper II) do not allow the plant-fungus system to develop beyond the stage of mycorrhiza formation. The shoots were no more than 5 cm in length after 5 months by which time a few mycorrhizae had formed, and root branching was poor. Poor gas exchange over the sealing film of the tubes (Paper II), limited space, uncontrolled nutrient concentrations and accumulation of excretory products might explain the poor growth and root branching. The same technique resulted in better mycorrhiza formation and root branching when a larger vessel was used (Danell unpubl. data).

Since the pioneer work of Melin (1922, 1925) many other methods for in vitro mycorrhiza formation have been published (e.g. Finlay & Read 1986, Fortin et al. 1980, Kähr & Arveby 1986, Molina & Palmer 1982).

Most of the methods were developed for fast-growing fungi in a nonsterile environment, and few allow further development after the formation of mycorrhizae. Therefore, the purpose of Paper III was to identify and analyse the factors essential to the mycorrhiza formation of C. cibarius and to create a new aseptic system for routine formation of slow-growing fungi. With a technique allowing growth of the double organism beyond the mycorrhiza initiation stage, it would be possible to study the physiology of slow-growing mycorrhizae, e.g. C. cibarius, and to perform experiments on fruit body formation.

Material and methods

The mycorrhiza formation system was based on the principles of McLaughlin's nonmycorrhizal, but aerated and axenic system (1970), and the mycorrhizal adaptations of Jentschke et al. (1991). However, because almost every part was modified this new Culture Unit System (CUS) will be briefly described (Fig. 4).

A single culture unit was made using a specially adapted 2-l glass beaker with a broader beaker of the same volume as a lid. The lid was tightened with silicone tubing (outer diameter 5 mm). In the bottom of the beaker a ceramic lysimeter (Staatliche Porzellanmanufaktur, Berlin, Germany) was connected via silicone tubing (3-mm wall, 3-mm inner diameter) to a 1-l collection bottle. The bottle was connected to a pipeline leading to a vacuum pump which drained the culture unit using a vacuum of -0.8 bar. The drainage bottle was changed every 6 days. The lysimeter was covered with acid- washed (6M HCl for two weeks) quartz sand with an average diameter of 0.9 mm. On top of the sand a 100-ml beaker was placed upside down with a cylinder of filter paper on the outside. The filter paper was used to distribute the nutrient solution. The beaker was also covered with sand, making a total of 1 litre of sand which served as an inert substrate.

The autoclaved nutrient solution (Table 6) was prepared in a 5-l bottle. Heat-sensitive compounds, e.g. glucose, were added to the autoclaved solution after being sterile filtrated (Millipore 0.2 um). Aliprene tubing (Alitea AB, Stockholm, Sweden) which withstands mechanical stress better than silicone, connected the bottle to a peristaltic pump with 20 inlets. The tubing was attached to the inlet of the culture unit. The pump was automatically activated every 90th minute by a microprocessor timer, and 10 ml nutrient solution was added each time. A steel connector on the tubing, placed before the pump made it possible to change autoclaved bottles of nutrient solutions since the end of the steel connector was flamed before connecting the new tubing.

An air pump with triple filters supplied a culture unit with 1 litre of air/minute via aliprene tubing. Additional CO₂ was sometimes added to the air before it entered the system. Gas was released from the culture units passively through a glass wool filter. The increased pressure in the culture unit would also prevent entrance of airborne contaminants.

The whole culture unit was placed in a Fi-totron 600H growth cabinet (Fisons, Loughborough, England) which controls light intensity, photoperiod and temperature. The effects of changes in photoperiod on mycelium would then be studied.

Fig. 4.

Schematic figure of the Culture unit system (CUS). In reality, maximum 20 units were used simultaneously. A air pump for 20 culture units, B beaker with filter paper, C climate cabinet, D drainage bottle, G CO2 gas, L ceramic lysimeter, N nutrient bottle, O passive outlet of gas, P peristaltic pump for 20 culture units, S acid washed quartz sand, T microprocessor timer, J culture unit, V vacuum pump, Black squares indicate filters.

Host plants were derived from seeds of Picea abies and Pinus sylvestris. The seeds were sterilized for 30 seconds in 70% ethanol followed by 20 minutes in 30% H₂O₂. After washing in sterile, demineralized water and drying on a sterile filter paper, the seeds were transferred to agar dishes with MFM. The seedling was used as soon as the cotyledons separated from each other. Under a sterile hood, six to eight seedlings were transferred to each autoclaved and cooled CUS unit. Immediately after this transfer, 10 ml of a suspension of hyphae was added to each root (see below). The culture unit was then sealed and connected to the nutrient, air and drainage pumps in the climate cabinets (Fig. 4).

The host specificity of C. cibarius was investigated by inoculating 60 B. pendula seedlings and 36 Pinus sylvestris seedlings with strain SNGT2 isolated from a fruit body connected to Picea abies. Standard incubation and nutrient procedures were used as described below. The degree of mycorrhiza formation was studied after two months.

Twenty mycelial C. cibarius strains isolated from tissues and spores were tested (Paper III). Also, one strain of Lactarius rufus was used. The protocol for mycorrhiza synthesis is schematically described in Fig. 5. Pieces of MFM agar with mycelium (4-8 weeks old) were axenically transferred to 5-cm petri dishes with sterile filtered (Millipore 0.2 um) liquid MFM. After incubation for 4-6 weeks (20 deg.C, darkness), mycelia from 4-5 petri dishes were fragmented in 100-ml Erlenmeyer flasks containing 50 ml fresh MFM and glass beads. The suspension was filtrated (pore size 1 mm), and an additional 150 ml MFM was added to the suspension. The hyphal suspension was introduced to two CUS units as described above.

Established mycorrhizae were sectioned to confirm the presence of intercellular Hartig nets (Fig. 3). Short-roots with mantles of hyphae were prepared for SEM, TEM and light microscopy according to Nylund (1980) and Paper II.

The nutrient medium was based on Ingestad (1979), with the same proportions of minerals (Table 6). Experiments with addition of glucose or sucrose were carried out using concentrations of 0-0.5%. The standard concentration of glucose during other experiments was 0.2%.

Garbaye et al. (1990) showed that fruit body associated bacteria supplied Laccaria mycelia with organic acids until it formed mycorrhiza. I tested the hypothesis that Pseudomonas fluorescens, present in all C. cibarius fruit bodies (Paper IV), could replace the glucose in the medium used for mycorrhiza synthesis (Paper III). In addition to the methods in Paper III, I also inoculated 10 CUS units with whole bacterial communities together with hyphal suspensions. The communities were derived from slurries of C. cibarius fruit bodies. The aim was to determine whether excretory products of the natural bacterial community could replace the glucose addition, thus aiding C. cibarius in mycorrhiza formation.

The gas composition is very important for photosynthesis, normal development of seedlings and fungal growth. According to Straatsma & Bruinsma (1986) C. cibarius is able to assimilate CO₂, a character also found in other fungi (Wainwright 1988). Therefore the effects of CO₂ on mycorrhiza formation were studied (Paper III). The standard concentration of CO₂ in other experiments was 0.2%.

Table. 6. Ingestad mineral solution used for mycorrhiza synthesis (based on Nylund and Wallander 1989).

__________________________________________________________________________

Solution A (g per 4 l):

K₂SO₄ 97.94
KH₂PO₄ 68.58
K₂HPO₄ 80.82
KNO₃ 77.66
NH₄NO₃ 468.46

Solution B (g per 4 l):
Ca (NO₃ )₂ 4H₂O 70.84
Mg (NO₃)₂ 6H₂O 126.66
HNO₃ (4M) 25 ml
Microelements 100 ml

Microelements (g per 2 l):

Fe (NO₃)₃ 9H₂O 202.4
Mn (NO₃)₂ 4H₂O 73.04
Zn(NO₃)₂ 4H₂O 4.81
CuCl₂ 2H₂O 3.22
Na₂MoO₄ 2H₂O 0.71
H₃BO₃ 45.75
HNO₃ (4M) 49.55 ml

Final concentrations (340 ul of each stock solution in 1000 ml):

N 17 mg (60%NH₄, 40%NO₃ w/w, 1:2 mol/mol)

K 11 mg

P 2.6 mg

Ca 1.0 mg

Mg 1.0 mg

S 1.5 mg

Fe 126 ug

Mn 68 ug

B 34 ug

Cu 5.1 ug

Zn 5.1 ug

Cl 5.1 ug

Mo 1.2 ug

Na 0.54 ug

Results

With the standard CUS system (Fig. 6A) mycorrhiza formation with C. cibarius and Picea abies (Fig. 6B) took 8 weeks, compared with the earlier 4-5 months (Paper II, Moore 1989). Sections revealed Hartig nets (Fig. 6C). Complete mycorrhiza formation was established after 10-12 weeks. Mycelial colonization of roots and sand was not deeper than 5-7 cm. The dense and yellow mycelium aggregated sand particles and was interspersed with white airpockets. Numerous white rhizomorphs occurred. Glucose or sucrose was required to obtain mycelial development and mycorrhiza formation during the first 8 weeks. Once the mycorrhiza had become established, the addition of carbohydrates was no longer necessary. Colonization occurred a few weeks faster with glucose than with sucrose. Concentrations higher than 0.2% did not give a faster development. L. rufus did not need extra carbohydrates, and formed a mycorrhiza all the way down to the bottom of the CUS unit (15 cm) in 6 weeks. Within this period, L. rufus sometimes spontaneously formed great numbers of fruit body primordia, as was also observed by Jentschke (1991).

The presence of bacteria or bacterial communities did not stimulate fungal growth when glucose was excluded. A post-harvest study of the bacterial populations in the CUS units showed a complete colonization of the substrate, which was revealed after growth on MFM agar. Mycorrhiza formation normally occurred in the presence of P. fluorescens if glucose was added. P. fluorescens had no observable effects on C. cibarius.

Without extra CO₂ the C. cibarius mycelium developed slowly, and no mycorrhiza had formed after three months. Additional CO₂ was therefore vital for mycorrhiza formation. The CO₂ concentration fluctuated a lot, mainly due to slow gas exchange between the incubation room and the outside temperature. Additional CO₂ was thus minimum 0.2% and occasionally as high as 0.4%. The continuous gas flow also resulted in healthy pine and spruce seedlings. The green shoots remained aseptic and reached 18 cm in 10 weeks.

Differences in host specificity was found. Of the twenty strains tested, all formed mycorrhiza with Picea abies and Pinus sylvestris. One strain originated from a spore collection. However, the density of mycelium and number of mycorrhizal short-roots varied greatly between strains. The C. cibarius strain from Picea abies colonized all units with Pinus sylvestris seedlings, and 20 out of 36 seedlings formed mycorrhiza. However, none of the 60 Betula pendula seedlings formed mycorrhiza, and mycelial growth was slow.

No mycelial responses were observed in connection with changes in photoperiod or ceased shoot growth.

Fig. 5. Schematic protocol for mycorrhiza synthesis and transfer to green house. Tissue samples were taken from the inside of a fruit body and placed on MS medium for 4-6 weeks. Repeated reinocultions were often necessary. When the mycelium became sterile, it was transferred to MFM agar for 4 weeks. Pieces of mycelium was then transferred to liquid MFM for 4 weeks. Mycelia were shaken with fresh liquid MFM in an e-flask with glass beads. Hyphal fragments and seedlings previously grown in sterile conditions were transferred to the culture unit system for 10-12 weeks. Finally, mycorrhizal seedlings were transferred to the greenhouse.

Fig. 6A.Photograph of the culture unit system (CUS).

Fig. 6B.C. cibarius mycorrhiza with Picea abies.

Fig. 6C. Section of a mycorrhizal short root (C. cibarius and P. abies). On top a cluster of hyphae from the mantle. At the bottom two large cortex cells surrounded by hyphae of the Hartig net. Scanning electron micrograph.

Discussion

The C. cibarius demand for additional glucose for growth and mycorrhiza formation was probably due to the artificial environment. In nature, fungi like C. cibarius and Lactarius pubescens colonize new roots mainly through vegetative growth from the mycorrhizal mycelium of a nearby tree (Romell 1938, Fleming 1984). In my study the mycelium was added to the seedlings in the form of suspended hyphal fragments. To recover from fragmentation, C. cibarius hyphae needed simple carbohydrates. An older seedling might have been able to exudate enough carbohydrates to attract the C. cibarius mycelium. It is known that certain fungi have a higher demand for carbohydrates in in vitro culture (Dighton & Mason 1985). Obviously, the second species tested, Lactarius rufus, had a very low carbohydrate demand.

If a simple carbon source is needed, then it is difficult to understand how a young C. cibarius spore mycelium in nature can survive until mycorrhiza formation. My study showed that the closely associated P. fluorescens did not act as a helper bacterium, i.e. excreting organic acids to be used by the young mycelium until mycorrhiza formation (Garbaye et al. 1990). This means that only viable spores that germinate very close to a short-root (possibly of an older plant) in a suitable biotope might succeed. This extremely low probability of mycorrhiza formation (negligible under lab conditions) might be sufficient, considering that the maximum seasonal spore production of a single C. lutescens fruit body is 10⁸ (Kälin & Ayer 1983) and that Cantharellus mycelia can produce fruit bodies for decades (Paper V, Jahn & Jahn 1986). An incidence of successful colonization greater than 1:10¹⁰ is still acceptable if 25 fruit bodies are formed every year for 40 years by the same genotype.

If we consider that 1) spores of C. cibarius did not form mycorrhiza in vitro, 2) the incidence of germination was <0.04%, 3) spore formation is less regulated than in Agaricales (Maire 1902) and 4) that the total spore production in C. lutescens during six weeks is only 1% of the A. bisporus spore production during one week (Kälin & Ayer 1983, Webster 1980), it is evident that vegetative growth is more important than spores for local dispersal. Spores might have another purpose in addition to pioneer colonization. Fries (1981) suggested that ectomycorrhizal Leccinum species produce spores mainly to create mycelia with new genetic combinations which could be fused with the original mycelium. This phenomenon is called homing reaction and might help the original mycelium to achieve the genetic diversity needed to survive environmental changes for decades. Spores of C. cibarius were stimulated to germinate when inoculated near another mycelium. In 1987 Fries suggested that ectomycorrhizal basidiospores are insufficient for long-distance transport.

The long period of establishment, slow growth and dispersal mainly via vegetative growth makes C. cibarius a late colonizer in forest plantations without contact with older trees. The youngest tree I found associated with fruit bodies of C. cibarius was a 28-year-old Picea abies (Paper V). Fungi associated with older forests are sometimes called "late-stage fungi" (Newton 1992).

In contrast to this group, some ectomycorrhizal fungi are considered "early-stage fungi", since they rapidly form mycorrhiza and fruit bodies in young plantations, forest

nurseries or even in vitro. These fungi, e.g. Laccaria, Thelephora and Hebeloma (Debaud & Gay 1987, Godbout & Fortin, 1990) are highly dependent on spore dispersal (Last & Fleming 1985). These fungi are replaced by "late-stage fungi". The reasons for this succession might be:

1. Adaptation to the physiology of either a young or an old host tree.

2. Adaptation to different soils of certain stages during reforestation, i.e. "early-stage fungi" are only competitive when growing in soils with thin litter layers (Finlay et al. 1992, Last et al. 1987).

3. Mode of dispersal, i.e. rapid colonization via spores followed by slow growing long lived species.

The ability of C. cibarius to form a permanent mycorrhiza with young seedlings in the CUS system shows that the term "late-stage fungus" is inappropriate, and that fungal successions in plantations do not depend on the actual age of the tree. The second reason is probably significant, but the importance of dispersal mode was shown by Fleming (1984). He found that seedlings in pots planted near a tree with fruit bodies of "late-stage fungi" were colonized by "early-stage fungi". By contrast, seedlings without pots planted near the same tree were also colonized by "late-stage fungi".

The demand of C. cibarius for high CO₂ levels is not surprising. The normal variation in CO₂ levels at 20 cm depth in pine forest soils is between 0.5 and 2% (Magnusson 1992). Straatsma et al. (1986) showed that the presence of a tomato root could be replaced by 0.5% CO₂ in order to stimulate strong mycelial growth of C. cibarius. However, the oxygen concentration must not be too low. For instance, my field observations and experimental studies indicate that C. cibarius does not survive waterlogging. A similar phenomenon was described by Stenström (1991), who showed that "early-stage fungi" like Hebeloma and Laccaria are not sensitive to waterlogging, whilst "late-stage fungi" like Suillus species are. According to Magnusson (1992) waterlogging in forest soils in spring time might result in a 12% oxygen concentration and a 4% CO₂ concentration. This low oxygen concentration might have favoured the evolution of hydrophobic mycelia able to create air pockets, as described by Unestam (1991). Low oxygen concentrations might also be one reason why C. cibarius is not found in biotopes with poor water drainage, e.g. pits or bogs. Rainfall is not a problem if the soil permits rapid drainage. The occurrence of C. cibarius in the top 5 cm in the CUS units resembles the situation in nature where mycorrhizae are situated where minerals are released from decaying organic material. However, L. rufus grew rapidly down to 15 cm depth suggesting that C. cibarius is largely restricted to the top 5 cm also owing to its higher oxygen demand.

The host specificity of C. cibarius has been considered broad (Mousain 1979). However, the unsuccessful colonization of Betula pendula by a C. cibarius strain from Picea abies suggests that there may be physiological varieties adapted to different groups of host plants. Moore et al. (1989) succeeded in obtaining mycorrhiza with both Betula pubescens and Pinus sylvestris using isolates from Quercus robur. However, the mycelium on P. sylvestris did not colonize the entire cortex. RFLP patterns of ITS of my C. cibarius strains from pine and birch forests did not reveal any differences. It is therefore likely that the ability of a species to colonize a certain host will vary between strains. Strain variations in partner specificity are common among fungi or plants (Lapeyrie & Mendgen 1993, Last et al. 1984).

Transfer to greenhouse (Paper III)

Methods

Beginning in October 1992, pine seedlings with C. cibarius mycorrhiza were transferred from the CUS to greenhouses and a forest nursery. The purpose was to study survival of plant and fungus, and to assess the ability of the fungus to colonize soil and other roots. Each of 10 mycorrhizal seedlings (3 months old) was placed in a separate clay pot. The filter paper from the culture unit was not removed. Twelve other mycorrhizal seedlings were placed in a plastic frame with 64 individual compartments (4.5 x 4.5 x 6.5 cm). The substrate used was a mixture of peat/quartz sand (25%/75% v/v). Water was added automatically once a day on the felt on which the pots were placed. Water entered the pots as a result of both capillary action and root uptake. Later, water was added twice a day. A 20-ml portion of Ingestad nutrient solution (three times stronger than the standard concentration shown in Table 6) was added once a week. The temperature was 18 deg.C (day) and 12 deg.C (night), and the photoperiod was 12 hours. Relative humidity was kept around 80%.

Another set of six seedlings (3 months old) was directly transferred from culture units to plastic pots as a clump. The purpose was to avoid root and rhizomorph damage incurred when separating seedlings from the glass beaker used for nutrient distribution in the CUS. The substrate was peat/quartz sand 50%/50% (v/v). Five pots (30 plants) were placed in another green-house. The temperature was 20 deg.C and the photoperiod 18 hours. Water was added from above when the top-soil became dry. Ingestad nutrient solution (three times stronger than for the culture units) was added once a week.

Six 4-month old mycorrhizal pine seedlings that had been kept in a 20 deg.C culture unit were exposed to 12 deg.C for 24 hours, followed by 10 deg.C for 24 hours and 7 deg.C for three days. Thereafter the seedlings were transferred to a pot, as described above, and placed in a forest nursery tent, kept at approximately 0 deg.C. The purpose was to study resistance to cold and to assess the ability of the mycorrhiza to withstand competition from other mycorrhizal fungi.

Results and discussion

The C. cibarius mycorrhiza was established in the unsterile soil and continued to grow and colonize new roots. Frequent mycorrhizae and rhizomorphs were observed along the walls of the pots. Mycorrhizae were found all the way down to the bottom of the pot, 12 cm from the soil surface. In these cases gas exchange was facilitated by holes in the bottom side. Mycorrhizae sometimes turned whitish after a few weeks in the greenhouse. This reversible pigment change was also observed in vegetative mycelium (Paper II). Species identity was verified using PCR (Paper III). Only two seedlings died after being transferred to pots in the greenhouse. The plastic frames were too small for each individual plant. Consequently the soil became too wet or too dry. The automatic watering from below caused some roots to turn black; however, roots and mycorrhizae in the top soil were not affected in this way. Mycorrhizae prepared for transfer to 0 deg.C survived nights with frost in April.

Increased watering was fatal to C. cibarius mycorrhiza. The pots were always wet, and C. cibarius mycorrhize changed from yellow to brownish before disappearing. Alien mycorrhizae, detected with PCR, occurred especially after increased watering. Probably C. cibarius suffered from water stress and therefore could not resist competition.

Description of the mycorrhiza formed (Paper II-III)

The description is based on Moore et al. (1989) and Paper II-III. C. cibarius and Picea abies formed a mycorrhiza that was initially unramified and later irregularly pinnate (Fig. 6B). The mantle surface appeared smooth to the naked eye but woolly under a microscope. The mantle was yellow but occasionally it turned whitish. The mantle remained 45 (^+/- 3) um thick in the pot cultures. It was described as plectenchymatous by the previous authors, but the inner mantle is rather pseudoparenchymatous (Paper III). Fully developed Hartig nets (Fig. 3 and 6C) colonized the entire cortex.

C. cibarius and Pinus sylvestris formed a dichotomous mycorrhiza. The yellow surface was sometimes strongly woolly in pot cultures (Paper III). Other characters were similar to those of P. abies mycorrhiza.

Identification of mycelia and mycorrhizae (Paper III)

There is always a risk of ending up with contaminating fungi in the culture instead of C. cibarius. Thus it is important to verify the species identity by comparing fruit body mycelium with culture mycelium and mycorrhiza. It would also be valuable to distinguish the mycorrhiza of C. cibarius from those of other ectomycorrhizal species when no fruit bodies are present. In addition to morphological studies, methods of molecular biology provide important means of comparing fruit body material, root tips in the soil and axenic material. Straatsma et al. (1985) used dot-blot DNA hybridization to compare vegetative and fruit body mycelia. However, the method is laborious and not applicable for mycorrhizal material where the amount of DNA is very low.

I compared ribosomal DNA (rDNA) using PCR (polymerase chain reaction) followed by RFLP (restriction fragment length polymorphism), as described according to the protocol and Fig. 7. The internal transcribed spacer (ITS) region of rDNA was chosen as a target for the PCR since inter-species variations have been recorded by Gardes et al. (1991). The ITS1 and ITS4 primers are designed for fungi, which is important since the C. cibarius fruit body tissue (i.e. the reference mycelium) is not sterile (Paper IV), and mycorrhizal samples contain plant DNA. RAPD (random amplified polymorphic DNA) was not used since the small primers (about 10 bases versus 20 in ITS4) would have annealed to alien DNA. Sterility during DNA preparation was therefore not crucial. Another set of primers, ITS4-B and ITS1-F, was initially tested since Gardes & Bruns (1993) showed that they were superior in some cases. Since I did not get any amplification with these, I used ITS1 and ITS4 for all my experiments.

Results and discussion

The molecular identification proved that C. cibarius had been isolated and successfully formed mycorrhiza. The size of the characteristic ITS from rDNA of vegetative mycelium corresponded with that of the C. cibarius fruit body ITS (Fig. 8). The PCR-product of C. cibarius and C. pallens was much longer (1400 base pairs) than those in other basidiomycetes and members of Cantarellaceae tested (between 6-800 bp) (Fig. 9).

Fig. 7. Schematic protocol for PCR and RFLP analysis.

DNA is extracted from Cantharellus fruitbodies, mycorrhizae and mycelium. DNA from another fruit body species is also extracted. After PCR, a great number of copies from a particular DNA sequence is achieved. These copies are distinguished from the copies of the other species by electrophoresis in a gel (first square). After enzymatic degradation of the DNA fragments (RFLP), a higher resolution is shown in the bottom gel.

Protocol for DNA extraction and PCR.

Protocol for RFLP.

After digestion with the three restriction enzymes, C. cibarius and C. pallens produced identical RFLP patterns (Fig. 10). This suggests a close relationship between these two species since other Cantharellus spp. are easily distinguished. Sequencing of the amplified ITS regions could be used to more precisely determine how closely related they are. Efforts to produce heterothallic mycelia from monocaryotic mycelia of the two species should also be made (i.e. to check interspecies barriers of genetic flow). These results should, of course, be considered together with morphological and ecological data on the two species. Results on American Cantharellus spp. (Feibelman et al. in press) show that the ITS length varies even within a species. The Swedish PCR-products representing strains from different biotopes and geographical sites were similar. It is possible that the American C. cibarius is different from the European one, and that some of the varieties described by Corner (1966) might be separate species.

The identity of mycorrhizae from greenhouse seedlings inoculated with C. cibarius was verified using a sterile C. cibarius mycorrhiza as a reference (Fig. 11). Alien mycorrhizae were clearly distinguished from the C. cibarius patterns (data not shown). As few as one or two mycorrhizal root tips were required for PCR and RFLP (DNA extract diluted 1:5 before PCR).

Foster et al. (1993) reviewed the applications of PCR in mycology. As a complement I should like to mention some applications concerning ectomycorrhizal fungi. PCR not only can be used to check the identity of an isolated mycelium, or be another tool for taxonomists, but it can also reveal the hidden world below ground. Interspecies competition can now be followed for several years, independently of fruit body production, since PCR can be applied to individual mycorrhizae. The impact of fertilizers and acid deposits, etc., can be thoroughly investigated since the mycorrhizae can be identified. Earlier, fruit body production was the only way to measure decline in C. cibarius. Furthermore, if proper primers are used, individual clones can be identified as a control or alternative to somatic incompatibility (Dahlberg & Stenlid 1990). Such studies offer clues as to the age and size of individual C. cibarius mycelia and allow the mycorrhizal establishment of a certain C. cibarius strain to be monitored after transferring it from the laboratory to the field. Laccaria strains, used to improve survival and growth of seedlings, can already be identified using PCR (Henrion et al. 1992).

Fig.8.Gel electrophoresis of amplified ITS of different C. cibarius/C. pallens strains. M: fragment size marker (5615SA/SB, BRL); Vegetative C. cibarius mycelia from tissues: lane 1 SNET1; lane 2 SNCT1; lane 3 SNGT2; lane 4 SVAT7; lane 5 NTGT4; lane 6 NTGT1; Vegetative C. cibarius mycelia from spores: lane 7 NTGS1; lane 8 GöHa; Fruit bodies of C. cibarius: lane 9 Pinus sylvestris biotope, long. 18deg.23' E, lat. 60deg.31' N; lane 10 Betula pubescens ssp. tortuosa biotope, long 13deg. 42' E, 64deg.36' N; lane 11 Betula pendula biotope 14deg.05' E, lat. 58deg.05' N Fruit body of C. pallens: lane 12 LVT9; Vegetative C. pallens mycelia from tissues: lane 13 LVT3; lane 14 LVT15; lane 15 LVT16, lane 16 LVT17; M: fragment size marker.

Fig.9.Gel electrophoresis of amplified ITS of different basidiomycetes. M: fragment size marker (see Fig. 1.); lane 1: C. cibarius; lane 2: C. pallens; lane 3: C. tubaeformis ; lane 4: C. lutescens ; lane 5: C. melanoxeros ; lane 6: Gomphus clavatus; lane 7: Hydnum repandum; lane 8 Sarcodon imbricatus ; lane 9 Suillus variegatus; M: fragment size marker (see Fig. 8).

Photograph of gels (Figs 8 & 9), not originaly included in the thesis..

Fig. 10.RFLP analysis of the amplified ITS from C. cibarius and C. pallens, fruit bodies and axenic mycelia (no difference between strains). Each strain occupy a set of three lanes. Left lane: Restriction enzyme (RE) MBO I, Middle lane: (RE) HINF I, right lane: (RE) HAE III.

Photograph of gel (Fig 10), not originally included in the thesis .

Fig. 11. RFLP analysis of the amplified ITS from C. cibarius mycorrhizae. Lane 1: M: fragment size marker (see Fig 8.); lanes 1-3 ITS of axenic mycorrhiza; lane 1 uncut ITS; lane 2 Restriction enzyme (RE) MBO I; lane 3 (RE) HINF I; lanes 4-6 ITS of pot mycorrhiza; lane 4 uncut ITS; lane 5 (RE) MBO I, lane 6 HINF I; lanes 7-9 and 10-12 ITS of pot mycorrhiza as described above.

BACTERIA IN C. CIBARIUS (Paper IV)

Background

In 1929, Swartz found unidentified bacteria in unbroken puffballs, but he could not explain why or how the bacteria colonized the fruit bodies. Li & Castellano (1987) isolated three strains of azospirilla from fruit bodies of the ectomycorrhizal fungi Hebeloma, Laccaria and Rhizopogon. These strains belonged to nitrogen-fixing bacteria (Tilak et al. 1988). Richter et al. (1989) demonstrated that some actinomycetes could stimulate growth of some ectomycorrhizal fungi. Bacteria isolated from roots of Pinus sylvestris stimulated growth of ectomycorrhizal fungi (Strzelczyk & Kampert 1987). Ali & Jackson (1989) showed that corynebacteria and Pseudomonas sp. stimulated germination of basidiomycete spores. The mechanisms were, however, not explained. Chard et al. (1987) described the polysaccharides of some Pseudomonas-like bacteria in Laccaria and Lactarius. Garbaye et al. (1990) isolated several fungi and bacteria from fruit bodies of Laccaria laccata. Some of these, especially fluorescent pseudomonads, enhanced mycorrhiza formation (Duponnois & Garbaye, 1991).

According to Rainey et al. (1990) Pseudomonas putida (Trevisan) Migula removes a fruit body formation inhibitor produced by the common white mushroom A. bisporus. P. putida might therefore be partly responsible for the fruit body inducing effect of the casing soil, a hypothesis in accordance with Eger (1972).

During attempts to isolate mycelium of C. cibarius, bacteria repeatedly grew out on the tissue cultures from fruit bodies (Paper II, Ballero et al. 1991, Itävaara & Willberg 1988 Schouten & Waandrager 1979 Straatsma et al. 1985). Straatsma et al. (1985) listed alien organisms recovered from C. cibarius, but no further studies have been made. I therefore wanted to isolate and identify the most common of these bacteria. Using TEM, I wanted to reveal the sites of the bacteria in fruit bodies and also see whether they were actively growing. Mycelium coinoculated with bacterial strains was observed in an effort to assess their effect on fungal growth. Axenic mycorrhizae were also inoculated with bacterial strains to study the mode of entrance. I also wanted to determine whether bacterial populations differ between species of basidiomycetes.

Methods

Bacteria were isolated from fruit bodies of Cantharellus cibarius collected at 12 localities in Sweden. Seven other species were collected for the sake of comparison (Table 7). Soon after picking, the outer layer of each fruit body was peeled off with a sterile scalpel, leaving about 50% of the tissue. Batches of fruit bodies (5-10 g peeled fresh weight) were aseptically minced in sterile water with an omnimixer. Appropriate dilutions of each suspension were inoculated in duplicate on different media (Paper IV).

All agar plates were incubated for 48 hours at 15 deg.C and 25 deg.C to search for aerobic bacteria. Bacterial colonies on King's B agar (KBA) (King et al. 1954) were observed under UV-light (350 nm) to distinguish fluorescent pseudomonads from nonfluorescent ones.

Identification and purification were concentrated on 64 bacterial strains representing the most commonly occurring bacterial colonies in all C. cibarius samples. A first identification of nine fluorescent strains was made with the API 20 NE system (Peladan & Monteil 1988), with additional tests of growth on 0.5% trehalose or sorbitol agar according to Doudoroff & Palleroni (1975). These tests were complemented by an independent survey with 96 additional oxidative tests according to Biolog GN Microplate (Biolog Inc.).

The growth of P. fluorescens was studied in King's B broth over a pH range of 2-10. Both total and viable counts were recorded every third hour for 12 hours, and finally after 24 hours.

TEM was used to study the attachment sites and growth of bacteria inside the fruit bodies of C. cibarius (Paper IV).

The screening of bacteria affecting mycelial growth in vitro was concentrated on the 30 most representative strains. Two isolates of C. cibarius were pure cultured on the rich MFM. Four replicates of each fungal isolate were inoculated in dual cultures with each bacterial strain in a 15-cm petri dish when the fungal colonies were about 15 mm in diameter. Petri dishes with only C. cibarius mycelium were used as controls. Stunted fungal growth or lysed hyphae were considered as strong inhibition; fungal overgrowth of the bacteria or visually simple coexistence was considered non-inhibition.

Interactions between bacteria and mycorrhizae were studied using two strains of fluorescent bacteria, classified as non-inhibitory based on the results of the above screening. About 5 x 10⁹ bacteria were aseptically added to living axenic mycorrhizae in the CUS system. Three to six weeks after bacterial inoculation, pieces of mycorrhizae and sand grains were placed on agar plates with MFM to check the vitality of mycelia and bacteria. Mycorrhizal short-roots were also prepared for TEM studies. Since the limited number of CUS units only allowed tests on a few bacterial strains, whole bacterial communities were inoculated into 5 CUS units. The bacterial community was derived from a slurry of a minced fruit body and was added to established mycorrhizae after 9 weeks.

Phosphate is considered to be one of the most important nutrients transported from mycorrhizal fungi to their host plants (Harley & Smith 1983). If P. fluorescens had phytase activity for mineralization of organic phosphorus, this might explain why C. cibarius tolerates its presence. To test this hypothesis, CUS units were inoculated according to the schedule below (unpubl. experiment):

4 CUS units with Picea abies

4 CUS units with P. abies and C. cibarius mycorrhiza

4 CUS units with P. abies and P. fluorescens

4 CUS units with all three organisms

The phosphorus source of the Ingestad solution (13 mg P/5000 ml) was replaced by sterile filtered inositol hexaphosphate (calcium salt from SIGMA) containing equal amounts of phosphorus. The aim was to study differences in plant growth and phosphate content in drainage bottles (using Spectroquant, MERCK).

To test the hypothesis that C. cibarius exudate compounds that could be used by bacteria, studies on exudates of axenic C. cibarius mycelium were done with NMR analysis (nuclear magnetic resonance, Chang 1981) at the Department of Organic Chemistry 2, University of Lund. The mycelia were grown in five 5-cm petri dishes containing 4 ml liquid MFM each. After four weeks the medium was removed, mixed and freeze-dried. The NMR analysis was made with a Bruker 500 MHz cryomagnet. NMR spectra, showing numbers and positions of hydrogen protons, were compared with a fresh reference MFM medium and NMR spectra from the catalogue of Pouchert & Behnke (1993).

Results

The bacterial population in C. cibarius varied from 3 x 10⁵ to 7 x 10⁶ colony forming units (cfu) per gram fresh weight (Table 7). The mean value of 2.4 x 10⁶ was about 100-1000 times greater than the bacterial population size for various species in the order Agaricales (Table 7). In the agarics, the population was probably even lower since the few bacterial colonies observed may have resulted from contaminations occurring during the preparation procedure. Boletus edulis, Amanita muscaria and Agaricus langei appeared to be bacteria free. Among the Aphyllophorales that were studied, Hydnum repandum and C. pallens contain approximately 10% of the amount of bacteria found in C. cibarius. In Hydnum rufescens the bacterial population was about equal to that in C. cibarius. In Sarcodon imbricatus the population was less than 5% of that in C. cibarius.

No correlation between the stage of development of the fruit body and the number of indigenous bacteria was found (data not given), with the exception of specimens damaged by freezing in late October. In these cases the populations exceeded 4 x 10⁷ bacteria per gram fresh weight. These values were not included in the calculation of the mean value. The population of fluorescent Pseudomonas in non-sterile podzolic soil at the growing site was estimated at only 12% of the total bacterial population, while in fruit bodies of C. cibarius the fluorescent Pseudomonas population constituted 78% of the total.

Of the nine fluorescent Pseudomonas strains tested with API 20 NE, six were determined as Pseudomonas fluorescens Migula biovariety I. Five of these were able to digest trehalose and sorbitol in accordance with Doudoroff & Palleroni (1975). The parallel identification of three strains with the Biolog system confirmed them to be P. fluorescens, although one strain was biovar II. Of the remaining three trehalose and sorbitol negative strains, one seemed to be P. putida, and the other two would not be identified.

Zygomycetes and Penicillium sp. (Paper II) and other moulds (Straatsma et al. 1985) as well as Streptomyces sp., Xanthomonas sp. and Bacillus sp. were sometimes isolated.

The pH range over which P. fluorescens grew was pH 5-10. The generation time was about three hours throughout the pH-interval. At pH 2 and 3 the bacteria died within six hours. At pH 4 the bacteria survived at least 24 hours but no growth was recorded.

Table 7.

Number of aerobic bacteria found in different basidiomycetes. 
Counts are made on King's B agar. 
_______________________________________________________________________________
		     	     							
Species	           	       M cfu / g F.W. ± S.E.          %fluorescent Pseudomonas 
								mean/range
_______________________________________________________________________________

Cantharelllus cibarius		2.4 	±1.4	n=35, f=88	78 / 18-98
Forest soil (depth 1-5 cm)	1.8	±0.92	n=9		12 / 9-22	
Sarcodon imbricatus		0.083	±0.03	n=2, f=4	      0
Hydnum repandum			0.30	±0.03	n=6, f=14	      0
H. rufescens			2.5	±1.1	n=9, f=26	85 / 65-99
C. pallens			0.26	±0.06	n=6, f=12	15 / 10-29
Boletus edulis			0.004	±0.008	n=10, f=10	     <1
Amanita muscaria 		0.013	±0.004	n=12, f=12	      0
Agaricus langei			0.0012 	±0.0027  n=5, f=5	      0
_______________________________________________________________________________

cfu = colony forming units
F.W.= fresh weight
S.E.= standard error
n = number of samples (5-10 g of peeled fruitbodies or soil/sample)
f = total number of fruitbodies of all samples 
			

Table 8. Mushroom associated bacteria and their effect on growth of
C. cibarius mycelium
_______________________________________________________________________________
	
Group	Bacterial features   	Influence on 	No of strains
				fungal growth
_______________________________________________________________________________	
	
1	F+, T+ 			No inhibition		4
   	F+, T+			Minor inhibition	1
   	F+, T+			Strong inhibition	0

2	F+, T-			No inhibition		1
   	F+, T-			Minor inhibition	4
   	F+, T-			Strong inhibition	3

3	F-, T-			No inhibition		6
   	F-, T-			Minor inhibition	8
   	F-, T-			Strong inhibition	3
_______________________________________________________________________________

F+ = fluorescent Pseudomonas
T+ = ability to digest trehalose 
Group 1: Pseudomonas fluorescens  
	(52% of the average bacterial population in C. cibarius)
Group 2: Fluorescent Pseudomonas spp.
	(26% of the average bacterial population in C. cibarius) 
Group 3: mixed bacteria (22% of the average bacterial population in C. cibarius)
	

TEM studies of fruit bodies of C. cibarius collected in the field revealed scattered bacteria, single or in pairs, most often surrounded by mucus and sometimes dividing (Fig. 12). This mucus layer is typical for the isolated Pseudomonas. The bacteria were often found in the interhyphal space, seldom directly in contact with single hyphae. In contrast, the TEM studies of mycorrhizal short-roots inoculated with P. fluorescens did not reveal many bacteria inside the mantles. Bacteria were mostly found on the surfaces (Fig. 13). Both fungus and bacteria grew out from the mycorrhizae when inoculated on MFM.

The results of the in vitro screening are shown in Table 8. Six out of thirty strains strongly inhibited the mycelium on the rich MFM. No strongly inhibitory strains were found among P. fluorescens biovar. I. Non-inhibitory strains did not affect morphology, but slightly increased pigmentation of the hyphae. The radial growth rate of the control mycelium was 0.6 mm/day and 0.55-0.6 mm/day for non-inhibited mycelia.

The test of the phytase hypothesis did not reveal any significant differences between units inoculated with mycorrhiza and bacteria and those inoculated with mycorrhiza alone (unpubl. data). The addition of bacteria did therefore not increase levels of phosphates from organic phosphorus sources. Phosphorus contamination was negligible.

According to NMR, no traces of the originally added glucose or fructose were still present in the medium after four weeks. The few peaks that appeared did not differ from those found with fresh MFM, so no excretion of C. cibarius was detected (unpubl. data).

Discussion

The total population of aerobic bacteria inside a fruit body of C. cibarius (Paper IV) was about the same as that in the upper part of podzol soil (Richards 1987). By contrast, fluorescent Pseudomonas represented only 12% of the aerobic bacterial population in soil but 78% of the population in fruit bodies of C. cibarius. The mycorrhiza experiment indicated that the bacteria would not actively penetrate the fungal tissues, even though they are motile. Other, probably randomly occurring, fungi and bacteria, were also found in fruit bodies. Therefore, it is likely that C. cibarius and related species such as H. rufescens incidentally interweave microbes during development and growth of the fruit bodies. If the source of bacteria is wind and water, the population of Pseudomonas in fruit bodies would have been more similar to the original soil population, which was not the case here. Fluorescent Pseudomonas are thus the dominating bacteria in fruit bodies of C. cibarius. If C. cibarius incidentally incorporates all bacteria surrounding the vegetative hyphae, then my findings would indicate that fluorescent Pseudomonas are the most abundant bacteria in vicinity of the vegetative hyphae in soil. So then, what attracts P. fluorescens in the mycosphere?

It is known that Pseudomonas predominates in the rhizosphere (Alström, 1987) and is more favoured by roots compared with species such as Bacillus (Bowen & Rovira 1976). C. cibarius must either actively or passively, favour Pseudomonas, or Pseudomonas must inhibit the growth of other bacteria. It is unlikely that bacteria feed directly on hyphae since I have shown growth of bacteria inside C. cibarius fruit bodies, which usually live five times longer than agaric fruit bodies. However, large

amounts of endophytic bacteria may live directly on plant tissues without having any significant detrimental impact on the host (Gerhardsson pers. com.). The ability of P. fluorescens to use fungal trehalose is rare among bacteria (Doudoroff & Palleroni, 1975). If this bacterium can establish itself on the vegetative mycelium or the mycorrhiza it might have access to a carbohydrate that most other microbes can not digest. However, according to NMR analysis neither trehalose nor any other soluble organic compound was excreted into the medium. Still, the NMR analysis was performed on an artificial medium developed to facilitate nutrient uptake. In nature, it is likely that excretory products other than volatiles (Pyysalo 1976) are produced, which might serve as a nutrient source for bacteria. Oxalic acid is sometimes excreted by ectomycorrhizal fungi to liberate phosphorus from apatite rock (Dighton 1991, Lapeyrie 1988). Since phosphorus is essential in the nutrient exchange between fungus and host plant, scavengers like bacteria may proliferate in the vicinity of excreting hyphae. Masaphy et al. (1987) suggested that Pseudomonas spp., which were found attached to A. bisporus hyphae, use oxalic acid and other excretory products. Crystals of calcium oxalate are sometimes found on other agaric hyphae and mycorrhizae (Lapeyrie 1988).

Since P. fluorescens did not grow at pH 4, although it was able to survive, it seems remarkable that it is found in acid podzols. In Uppsala county, the mean pH (H₂O) of the upper soil horizon H-A2 is 4.1 (Johansson 1991). However, this value is the average pH of a large volume of soil. Microhabitats, like rhizospheres or mycospheres, may have different pH values. Cantharellus extract had a pH of 5.8-6.2, indicating that the pH near the hyphae and in the fungal mantles of the mycorrhizae is favourable for bacterial growth. The natural generation time of Pseudomonas is about 77 hours in soil, and 5-6 hours on roots (Richards 1987), which indicates that even though pH 4.0 is not optimal, the bacteria may proliferate slowly. The pH of the media used to isolate bacteria was about 6, thus there may have been selection for bacteria unable to tolerate a low pH.

It remains to be determined whether the association with P. fluorescens benefits the fungus. Fluorescent pseudomonads produce 2-ketogluconic acid, which can release phosphate from calcium phosphate and apatite rock in weakly acid soils, and phytases which can mineralize inositol polyphosphates (Richards 1987). However, since mycorrhizal fungi produce oxalic acid and phytases (Dighton 1991), which are used to mineralize inorganic and organic phosphorus in acid soils, the contribution from bacteria may be insignificant. In fact, when inositol hexaphosphate was the only source of phosphorus, my mycorrhizal seedlings did not grow significantly better with Pseudomonas than in their absence. The small differences in phosphate concentrations between drainage bottles also indicate that the contribution of phytases from bacteria was of minor importance.

Garbaye et al. (1990) suggested that Pseudomonas can help young mycelia of Laccaria laccata by excreting organic acids which can be used as a carbon source until the mycorrhiza colonisation is established. My studies on Cantharellus and P. fluorescens did not confirm such a relationship (Paper III).

Through in vitro screening for bacteria influencing fungal growth some strongly inhibitory strains were identified. However, since the test medium was rich in nutrients these strains are not necessarily inhibitory in nature. The inhibition might have been caused by toxic exudates or by nutrient depletion. As a precaution non-inhibitory strains were chosen for mycorrhizal experiments in nutrient-poor environments.

TEM photographs of naturally occurring bacteria in the tissues of a fruitbody of Cantharellus cibarius.Fig.12a A bacterium prepared for division with enlarged chromosome.Fig.12b Two bacteria embedded in polysaccharides (mucus).

Fig.13 TEM photograph of a mycorrhizal mantle with inoculated Pseudomonas fluorescens colonizing the surface. B=bacteria, H=fungal hypha. Bar=1.7micrometer.

FIELD OBSERVATIONS (Paper III+V)

Biotopes

C. cibarius is common in all forested parts of Sweden except for central Lapland (Ryman & Holmåsen 1984), but coordinated reports from northern Sweden are lacking (Fig. 14). It is difficult to describe the preferred biotope of C. cibarius since I have found it in many different environments. In rainy years, rich fruit body production has been observed in usually dry and rocky localities with Pinus sylvestris and Cladina lichens. Fruit bodies occurred more constant in mixed forests of mainly Picea abies (Paper II). Rich production areas are also grazing fields with old Betula pendula, and south-facing slopes in the mountains together with B. pubescens ssp. tortuosa (Paper V). Fruit bodies have also been found in open forests of Quercus and Corylus. C. cibarius is hardly found in forests younger than 18 years, preferably in forests 30-400 years old (Paper II, O'Dell et al. 1992, Oria de Rueda 1989). One reason why C. cibarius is so abundant in the Swedish archipelago and in the mountains might be that many such forests are old and therefore contain vast areas of individual mycelia. Although C. cibarius was not found in areas without trees, it sometimes flourishes immediately after a clear-cutting. C. cibarius was rarely found in wet soils where water occasionally accumulates and was seldom found together with Sphagnum moss. In dry environments fruit bodies may flourish near edges of bogs and ponds as described by Smith & Morse (1947). This hydrophobicity is in accordance with in vitro studies (Paper III). Well-drained soils or slopes (Paper III and V) with a low content of organic matter (less than 10%, Jansen & van Dobben 1987) seem to be highly productive sites. Acidity should be pH 4.5-5.5 (Straatsma 1986, Jansen & van Dobben 1987), but tolerance levels are strain-specific; one out of sixty strains grew on MFM pH 3.0 (Danell unpubl. data). Due to the slow development of fruit bodies (Paper V, Smith & Morse 1947), fruit body formation is dependent on high humidity. Drought will disrupt further development (Danell unpubl. data). The absence of a closed canopy, water holding logs and moss layers can be compensated for by intensive precipitation, as in Pinus-Cladina sites.

The optimal growth of C. cibarius mycelia in vitro was 0.5 mm/day (Paper II-III). If natural growth occurs during April-September and at the same rate as under lab conditions, radial growth should not exceed 9 cm/season (considering the lower temperatures, probably less). However, field observations, based on occurrence of fruit bodies in sediment from a pond where no mycelium or roots had existed before, indicated that vegetative growth was 14 cm/season (Paper V). This rate is similar to growth rates of other ectomycorrhizal fungi (Last & Fleming 1985).

C. cibarius is considered to have a broad host range (Table 9). However, we may actually be dealing with a group of separate species (Feibelman et al. in press, Petersen 1979) with different host affinities. In addition, within C. cibarius sensu stricto there may be strains differing in their host affinities (Paper III). Most of the data in Table 9 are based on field observations. My field data confirm all Swedish hosts except for Carpinus betulus. In vitro experiments have confirmed at least B. pubescens, Picea abies and Pinus sylvestris (Paper II and Moore 1989). Juniperus communis is often mentioned by laymen as an associate to C. cibarius. However, J. communis forms arbuscular mycorrhiza rather than ectomycorrhiza (Reinsvold & Reeves 1986), and I have never observed C. cibarius growing exclusively with J. communis. It is more likely that Pinus or Betula, which are often found together with J. communis, are the host trees in such cases.

Fig.14. The map shows findings (1972-1993) of C. cibarius reported to the Swedish Mycological Society. It should be noted that white areas do not necessarily indicate absence of C. cibarius, but rather absence of reports. Each dot has a radius of 2.5 km. Data were processed by the Swedish Threatened Species Unit in Uppsala.

Tab. 9. Listed host species of C. cibarius. If not stated, based on literature review of Mousain (1979). Swedish hosts are written in Swedish.

_____________________________________________________________________

Abies alba Mill.

Betula pendula Roth. vårtbjörk

B. pubescens Ehrh. glasbjörk

B. pubescens ssp. tortuosa (Ledeb.) Nyman, (Paper V) fjällbjörk

Carpinus betulus L. avenbok

Castanea sativa Mill.

Corylus avellana L. hassel

Eucalyptus sp. (Oria de Rueda 1989)

Fagus sylvatica L. bok

Picea abies (L.) Karst. gran

P. sitchensis (Bong.) Carr

P. smithiana (Wall.) Boiss. (Watling & Abraham 1992)

Pinus hartwegii Lindl.(Valdes-Ramirez 1972)

P. montezumae Lambert (Valdes-Ramirez 1972)

P. strobus L.

P. sylvestris L. tall

Populus tremula L. asp

Pseudotsuga menziesii (Mirb.) Franco

Q. ilex L.

Q. petraea Liebl. bergek

Q. pubescens Willd.

Quercus robur L. ek

Shorea javanica K. & V. (Nuhamara 1987)

Tsuga heterophylla (Raf.) Sang

Fruit body formation

Introduction

Primordial development in C. cibarius and other species was described by Reijnders (1963). Two important questions in this regard are: What triggers fruit body formation, and is it possible to induce fruit body formation in C. cibarius under controlled conditions? I define a trigger as a stimulus (or a combination of stimuli) that cause vegetative mycelium to form hyphal aggregations and dense primordia. This process is called initiation (Manachère 1980) or carpogenesis (Reijnders 1963). The development of primordia into fruit bodies is a second morphogenetic step, highly dependent on water which is needed for cell elongation (Hammond 1985). For mushroom growers, the "key" to successful fruit body formation is often the creation of an environment suitable for normal primordial development.

Fruit body formation in other fungi

In the saprophytic A. bisporus, bacteria are needed to remove an initiation-inhibiting substance produced by the mycelium itself (Rainey et al. 1990, Rainey 1991). After initiation of primordia, normal development is dependent on high humidity and gas exchange, but light is not needed. It is often recommended that the temperature should be lowered to allow rapid fruiting (Manachère 1980), but fruit bodies will appear anyway (Danell unpubl. data). The biochemical background seems to be a storage of trehalose and glycogen in mycelium and primordia. Once a certain level of stored carbohydrates is reached, morphogenesis begins with synthesis of mannitol which attracts water and starts cell elongation (Hammond 1985).

In the saprophytic Pleurotus spp., my experience is in accordance with Rajarathnam and Bano (1987): mycelium spontaneously initiate primordia when grown in a constant environment. Continued normal development is dependent on light, high humidity and gas exchange.

For the saprophytic Schizophyllum commune, gas exchange and light are important for initiation and normal development, but the addition of cerebrosides may also induce initiation (Kawai 1987). In the saprophytic Coprinus spp., periods of both darkness and light are important. Initiation is induced by the increase in cAMP levels that occurs as substrate is depleted. Glucose will therefore inhibit initiation. Humidity is important. (Manachère 1980).

The facultative ectomycorrhizal fungus Chalciporus rubinellus (as Boletus rubinellus Peck.) needs light, gas exchange and high humidity for host independent in vitro fruiting (McLaughlin 1970).

The ectomycorrhizal Hebeloma cylindrosporum spontaneously formed fruit bodies after six weeks in vitro with host plants under constant conditions. Temperature had no effects on the yield of fruit bodies (Debaud & Gay 1987). The fruiting period was 9.5 months. Another ectomycorrhizal fungus, Laccaria bicolor, needed a host and low levels of nitrogen and phosphorus for fruit body formation. Fruiting began 16 weeks after inoculation. A decrease in temperature inhibited fruit body formation. A shorter photoperiod resulted in earlier fruiting. It was suggested that a pool of carbohydrates in the mycelium might be closely related to fruiting. (Godbout & Fortin 1990,1992).

Fruit body formation in C. cibarius

In the province of Uppland, Sweden, I have sporadically observed C. cibarius fruiting in late June, but July - early October is the main period. However, occasional fruiting has been observed in southern Sweden in December as well as in springtime during mild winters at protected sites. It seems that frost ends the fruiting season of C. cibarius.

If fruit bodies are collected, up to three harvests per season can be expected at a given site (Paper V). It takes six to eight weeks to produce a new harvest, provided that there is no drought. It is important that the fruit bodies are collected before they become too mature, i.e. before the pileus becomes flattened. Otherwise the following flush can be delayed, a phenomenon also described by Cooke & Flegg (1962) who studied A. bisporus. Irrigation was effective in ensuring fruit body formation at one site (Paper V). At this highly productive, irrigated site (15 x 35 m), 17 kg (FW) of C. cibarius was harvested in 1992. In highly productive, non-irrigated Spanish pine forests, up to 18 kg/ha might be harvested (Oria de Rueda 1989). Slee (1991) estimated the annual maximum C. cibarius production at 50 kg/ha in some Scottish forests. Removal of the litter layer, especially in polluted areas, appears to enhance fruit body production in C. cibarius (Arnolds 1991). C. cibarius fruit body primordia form close to the roots, but the vegetative mycelium is seldom visible.

In the CUS, only compact knots of hyphae without visible stipes were observed in C. cibarius. No visible changes in morphology occurred in response to photoperiod or temperature changes. The distinct formation of pointed primordia of L. rufus appeared rapidly without any changes in the CUS system (Paper III). Field observations on the occurrence of fruit bodies (see above) and experiments in the CUS indicate that the decrease in photoperiod after midsummer, which leads to changes in the hormonal balance of the host plant and a flush of carbohydrates to the roots (Wardlaw 1990), does not trigger fruit body formation (in accordance with Goodbout & Fortin 1992).

Temperature can hardly be a trigger due to the long possible period of morphogenesis and variations between day and night. There are very few examples of temperature change as an essential inducing factor, even though fruit body formation may begin earlier after a cold shock (Manachère 1980).

Agerer (1985) suggested that the level of fruit body production in general is mainly determined by the weekly mean temperature during the first half year, on condition that there is no drought. Dahlberg (1991) showed that the number of rainy days and precipitation were the most important factors explaining variations in fruit body production. Too much rain 7-14 days prior to fruit body formation can reduce production (Eveling et al. 1990). Eveling et al. (1990) also found a high statistical correlation between fruit body production and the average daily temperature 2-4 months prior to the fruiting season (possibly due to early starting point for fungal growth, higher metabolic/anabolic activity of hyphae and a higher production of plant assimilates). This has recently been found to apply to C. cibarius as well (Norvell 1992). Fruit bodies appear early if a hot May is followed by a rainy June, as was true in Uppsala in 1993, when C. cibarius fruit bodies appeared before midsummer. Fruit body production will thereafter continue until it is terminated by drought or frost. An extreme example is the unlimited fruit body production of the ectomycorrhizal Suillus luteus observed in a pine plantation on Mount Cotopaxi in Ecuador (Hedger 1986). At this site, temperature is always 8-16 deg.C during daytime, and fruit bodies of all stages are found throughout the year.

The final hypothesis, yet to be tested but based on literature data and supported by my field observations and results in the CUS units, is:

Once a critical mycelial biomass level is reached, primordial initiation begin. The time needed to build up the required carbohydrate stores after winter is mainly dependent on the mean temperature in spring. The soil humidity is a crucial factor determining the rate at which primordia develop into fruit bodies. Differences in the appearance of ectomycorrhizal fruit bodies during the year might reflect interspecific variation in the carbohydrate demand and rate of biomass formation. If this hypothesis is true, then it is only a matter of time before C. cibarius fruit bodies can be produced under optimal green house conditions.

Growth of C. cibarius fruit bodies

I also measured the rate of fruit body development and the age of fruit bodies (Paper V). One 3 x 3 m plot in each of three forests were studied during the same period. Diameter growth of a total of 71 fruit bodies was measured every fourth day until zero growth. Only fruit bodies with a minimum diameter of 5 mm were followed.

The growth rate varied greatly both within and between plots, and none of the fruit bodies grew at a constant rate. Since fruit body "growth" is partly a cell elongation process demanding water, differences in humidity due to variation in the degree of sun exposure, etc., might partly explain differences in growth rate. Clusters of fruit bodies grew slower than solitary fruit bodies. The most rapid growth recorded for one fruit body was 19 mm/4 days. The average growth rate during the same period for the three plots was 5.2 mm (^+/- 2.0), 9.6 mm (^+/- 4.8) and 7.2 mm (^+/- 2.8). In comparison, an agaric like Pleurotus grows 4-5 times faster, but most agarics are not older than six days.

The average age of C. cibarius fruit bodies was not calculated since all fruit bodies disappeared before actually degenerating, owing either to drought or unexpected picking. The oldest fruit body (31 days of age) was 58 mm in diameter (84- and 55-day-old specimens were recorded by Kälin & Ayer 1983 and Norvell 1992). Under more favourable conditions, Swedish C. cibarius fruit bodies may reach 11 cm in diameter and a fresh weight of 60 g (Danell unpubl. data).

Maybe the predatory pressure on most agarics has selected for rapid fruit body formation. Since C. cibarius is not attractive (see "Predators and parasites"), the same selective force has not favoured rapid growth, or it favoured repellent compounds instead.

Decline of C. cibarius in Europe

- a review

The decline in fruit body production among numerous species in Europe is serious (Arnolds 1988, 1991, Gulden et al. 1992, Elfström 1993, Jansen 1990, Kirby 1988, Ohenoja 1988). In Germany, C. cibarius is vanishing and is included on the German red list, and even other Cantharellus species are endangered (Naturschutzbund Deutschland, 1992). Restricted picking has been suggested (Ebert 1992, Jansen 1990), but according to Paper V, Arnolds (1991), Egli et al. (1990) and Norvell (1992), picking has no negative impact on successive fruit body formation of Cantharellus spp. For example, a C. pallens site was harvested each year between 1941 and 1980 without any decrease in fruit body production (Jahn & Jahn 1986). The impact of picking on long distance dispersal is unknown. Trampling in the area might have some negative influence however (Egli et al. 1990).

The decline in the Netherlands is even more striking (Arnolds 1988, 1991, Jansen and van Dobben 1987). Between 1960 and 1980, the number of localities with C. cibarius decreased by 60%. Since the decline of C. cibarius was studied for decades, variation due to seasonal variations is minimized. The strongest decline was observed in coniferous forests. Related species such as Craterellus cornucopioides and Hydnum rufescens may already be extinct in the Netherlands (Arnolds 1988).

It is difficult to determine whether or not C. cibarius is declining in Sweden. According to experienced field mycologists there has been a decline (Nitare 1988). C. melanoxeros and Craterellus cinereus are included on the Swedish red list of endangered and rare species (Databanken för hotade arter och Naturvårdsverket 1990). Threats against these originally rare species are mainly due to destruction of open Quercus and Corylus biotopes.

It has to be emphasised, that the decline of C. cibarius is based on observations of fruit bodies. Whether soil mycelia and mycorrhizae are declining as well is not known. Since the PCR technique is adapted to C. cibarius (Paper III), it could be used as a tool to monitor changes in mycelial and mycorrhizal frequencies.

Possible causes

Biotope destruction is especially important for mycorrhizal species that spread mainly vegetatively, such as C. cibarius (Paper V). A clear-cutting will eliminate large C. cibarius clones by removing their carbohydrate supply and destroying the drought- protective mosslayers. Also the liberation of nitrogen following a clear-cutting might have toxic effects on some fungi (see below). Field observations indicate that recolonization through spores might take decades since fruit bodies of C. cibarius are associated with old trees only. The youngest host tree that I have found was a 28-year- old Picea abies (Paper V).

Removal of logs and other water-holding substrates (Paper V, Molina et al. 1993, Norvell 1992) in forests might decrease fruit body production.

Nitrogen deposits probably influence C. cibarius in several complex ways. Since nitrogen is a limiting element in many undisturbed ecosystems, it is believed that most fungi have no uptake limits (Wallander 1992). Since C. cibarius mycelium does not grow deep below the surface of the soil (5-10 cm according to Paper III), it is strongly exposed to any deposits. Natural nitrogen fixation in soil is 1-3 kg/(ha^.year), but due to anthropogenic atmospheric N-deposition, input levels can increase to 25 kg/(ha^.year) as in southern Sweden (Dahlberg 1991) and 48-100 kg/(ha^.year) as in the Netherlands (Arnolds 1991). A great fraction of the fungal carbohydrates might therefore be lost owing to the uptake of nitrogen and consequent formation of glutamine (review by Wallander 1992). In vitro experiments on other ectomycorrhizal fungi have shown that an excess supply of nitrogen can lead to a strong decrease in fungal biomass (Arnebrant in press, Wallander & Nylund 1992). A decrease of fruit bodies of C. cibarius due to the addition of fertilizers was noted by Nohrstedt (1994) and Menge & Grand (1978). Raised levels of nitrogen will also favour certain plant (e.g. Deschampsia flexuosa) and mushroom species that compete with remaining species such as C. cibarius (Agerer 1989, Gulden et al. 1992, Kellner & Mårshagen 1991, Kellner 1993). Certain nitrogenous (and sulphur) compounds lower the pH of the soil, thereby altering the mobility of numerous toxic and essential elements (Arnolds 1988). The optimum pH for C. cibarius is 4.5-5.5 (field and in vitro data from Jansen & van Dobben 1987, Straatsma 1986). Heavy metals are only toxic locally in extreme concentrations (Arnolds 1988, Rühling et al. 1984). Removal of the litter layer containing high amounts of nitrogen stimulated a partial recovery of C. cibarius fruit body production (Arnolds 1991).

Liming decreases ectomycorrhizal fruit body formation according to Agerer (1989), even in sites where the pH has been artificially lowered. Erland (1990) found that liming induced a change in the mycorrhizal types in soil without altering the number of mycorrhizae. Drastic changes in soil chemistry should therefore be avoided. However, one study indicated that the single addition of 2000 kg of 50% CaO/ha enhanced the formation of C. cibarius fruit bodies. (Olle Eriksson pers. com.).

CONCLUDING REMARKS

* This thesis is the first comprehensive summary on the ecology of C. cibarius.

* The protein content of C. cibarius fruit bodies was 1% (FW), which was in contrast to the higher values earlier reported. The protein content of fruit bodies is comparable to that of some vegetables, but the nutritional value of C. cibarius should not be over emphasised. AHN is a rapid and less expensive alternative to automatic amino acid analysis. Further studies on antinutritional compounds and insect repellents might explain why C. cibarius is not infested by snails and insects.

* A mycelial collection of C. cibarius strains has been established from spores and tissues. Care and patience is needed to follow Straatsma and Fries.

* Mycorrhiza formation of C. cibarius can be made routinely using the CUS system. High demand for carbon dioxide by the fungus reflects its adaptation to the soil atmosphere. Its high carbohydrate demand compared with that of other ectomycorrhizal species, may reflect the importance of vegetative dispersal. Late appearance of fruit bodies in plantations is not due to an inability to colonize young seedlings. The term "late-stage fungus" is therefore inappropriate.

* C. cibarius fruit bodies contained 100-1000 times more aerobic bacteria than the agarics tested. The dominating bacterium was Pseudomonas fluorescens. Bacteria are probably incidently embedded in the primordium, but are harmless despite their ability to proliferate. They may feed on organic compounds excreted by the fungus or maybe directly on hyphae.

* PCR and RFLP can be used effectively to distinguish between C. cibarius mycorrhiza and vegetative mycelium from those of other species. However, C. pallens resembled C. cibarius in terms of the molecular and microscopical characters studied thus far. The affinity of C. pallens for Quercus and Corylus, and its paler pigmentation are the only characters different from C. cibarius. Since PCR can be used for C. cibarius mycorrhiza, mycelial age, genotype size and sensitivity to pollutants can be thoroughly investigated.

* For the first time, the life cycle of C. cibarius from spores over vegetative mycelium and mycorrhiza formation until final establishment in greenhouse is possible. The last stage, i.e. fruit body formation, should be possible to reach under optimal conditions in the greenhouse if the fungal biomass is large enough. Cultivation of C. cibarius might also be possible through the outplanting of colonized seedlings, as is the case with the black truffle Tuber melanosporum. However, additional field data on the ecology of C. cibarius is needed.

* Future research should be focused on determining optimal greenhouse conditions and competition between different ectomycorrhizal species (using PCR and RFLP). Genetical and biochemical research on the morphogenesis can only be made when all stages of the life cycle of a few strains are under control.

* This is the completion of four years work, but not the completion of my research.....

ACKNOWLEDGEMENTS

During the past few years I have spent a most exciting but tough period. Encouragement and valuable discussions have involved many people, and I have made a lot of new friends. To all of you I wish to express my cordial appreciation and gratitude. However, I wish to mention a few people of great importance to my project:

Kenneth Söderhäll, head of the Department of Physiological Botany, supported me with lab facilities. I thank him especially for arranging the only comprehensive mycology course in Sweden, it was essential for my training. Nils Fries, my supervisor to whom this thesis is dedicated, played a vital role in teaching me to become a combined field and experimental biologist. Tage Eriksson, my supervisor, took a strong interest in the project. His financial support during development of experimental techniques, his encouragement and sincere interest in mycology, especially after his retirement, meant a lot to me. Kind support and understanding was also given by Peter Engström. I thank all other people at our department for nice discussions and kind help.

Torgny Unestam, my supervisor and leader of the mycorrhiza group at the Department of Forest Mycology and Pathology, I thank you for accepting me as one of your disciples. The brainstorm sessions with Anders Dahlberg, Gösta Lindeberg and others have been most valuable - I thank you all! Karin Grip and Nisse Högberg played important roles during our amusing but sometimes frustrating efforts to successfully perform PCR. I am also happy for the assistance and friendship of Sun Yu-Ping.

I am most greatful to Svengunnar Ryman and his colleagues at the Department of Systematic Botany and the Botanical Museum. The cooperation with Olle Persson and Bo Mossberg who made the book of European chanterelles has been mutually fruitful. Svampklubben, Svampfrämjandet and Svampkonsulenterna were also important for my education in mycology and ecology. Sadhna Alström and her colleagues at the Department of Plant and Forest Protection are greeted for their cooperation in bacteriology.

Lynn Moore and Gerben Straatsma at Horst Mushroom Experimental Station are highly appreciated for their hospitality and pioneer research. Francis Martin and his colleagues in Nancy were essential for my PCR training. The kind invitation by Georg Jentschke, Göttingen, finally resulted in my successful mycorrhiza synthesis - thank you! Jim Trappe and his collaborators in Corvallis taught me fungal ecology, and I am most thankful for fruitful brainstorms and your kind Post-Doc invitation.

Monika, my fiancée who reads French articles much better than me, supported me with good advice when writing the thesis. I am also grateful to you for nursing and still loving me during periods of intensive work. I also want to thank Kaarina, Börje, Anders, Ann-Sofie, Kerstin, Urban, Katarina and all my relatives for being there.

This project would never have been started without the generous scholarship from the Foundations of Anders Wall. I am very thankful to Anders and Charlotte Wall, who took a personal interest in my project and supported me in all ways. The financial support is also gratefully acknowledged from the Swedish Natural Science Research Council, Swedish Council for Forestry and Agricultural Research, the fund of Knut and Alice Wallenberg, the fund of Hildur and Sven Wingquist (skogssällskapet), the Eliasson fund, Anna and Gunnar Vidfelt's fund, Fonden för skogsvetenskaplig forskning and the Lennander and Lundell scholarships.

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