The Potential for Vesicular-Arbuscular Mycorrhizal Fungi to Influence The

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1992 The potential for vesicular-arbuscular mycorrhizal fungi to influence the recovery of Hawkesbury sandstone vegetation after disturbance Stanley Edward Bellgard University of Wollongong

Recommended Citation Bellgard, Stanley Edward, The potential for vesicular-arbuscular mycorrhizal fungi to influence the recovery of Hawkesbury sandstone vegetation after disturbance, Doctor of Philosophy thesis, Department of Biology, University of Wollongong, 1992. http://ro.uow.edu.au/theses/1080

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The potential for vesicular-arbuscular mycorrhizal fungi to influence the recovery of Hawkesbury Sandstone vegetation after disturbance.

A thesis submitted in fulfilment of the requirements for the award of the degree of

Doctor of Philosophy

from THE UNIVERSITY OF WOLLONGONG

by

Stanley Edward Bellgard B.Sc. (Hons.) University of Western Australia.

Department of Biology

1992 DECLARATION

This thesis is submitted in accordance with the regulations of the University of

Wollongong in fulfilment of the requirements of Doctor of Philosophy. The work described in this thesis was carried out by me and has not been submitted to any other university or institution.

6 Stanley E. Bellgard June 1992 i

ABSTRACT

Disturbance to plant communities by agricultural and mining practices is an inevitable and

intrinsic part of civilization. Growing public, political and scientific concern about

problems such as land degradation and the greenhouse effect is increasing interest in

revegetation as a counter-measure. One of the primary stresses imposed upon seedlings

colonizing disturbed sites is a lack of nutrients. Plants species colonized by vesicular-

arbuscular mycorrhizal (VAM) fungi have higher tissue levels of some inorganic nutrients

(especially phosphorus), greater biomass yield, and more rapid uptake of water, and they

are often more tolerant of various forms of stress than non-mycorrhizal plants of the same

species. Consequently, it has been hypothesized that the successful revegetation of

disturbed plant communities may be dependant in part on the availability of viable

mycorrhizal inoculum.

The studies described in this thesis were aimed at assessing the potential for VAM fungi

to influence the revegetation of disturbed plant communities. VAM fungi were studied in

detail in two quite different plant communities on Hawkesbury Sandstone soils in

southeastern New South Wales. Although the sites supported vegetation of different

physiognomy, 14 plant species were common to both sites. The following features ofthe

ecology of VAM fungi are presented in this thesis: (i) the mycorrhizal associations of

plant species in the two study sites, (ii) an examination of which part of an intact soil profile represents the major store of potential propagules of VAM fungi, (iii) an investigation of the relationship between the intensity of topsoil disturbance and the infectivity of VAM fungi, and (iv) an examination of which propagules of VAM fungi are capable of initiating VAM after topsoil disturbance.

At both sites, an assessment of the mycorrhizal status of each of the plant species was made. Roots from five representatives of each plant species were collected from random locations within both of the study areas during July-September 1989. Fine feeder roots ii

were assayed for mycorrhizal infection. At the woodland site, 21 of the 32 plant species examined had mycorrhizal associations. At the shrubland site, 31 of the 47 plant species examined were mycorrhizal. Internal hyphae, vesicles, and cortical hyphal coils were

discovered on the roots of two species of Cyperaceae and on the non-proteoid roots of nine species of the Proteaceae. Several species within genera and families previously known to be mycorrhizal were also found for the first time to have associations.

Endomycorrhizal associations predominated at both sites, but several species had both

ecto- and endomycorrhizal associations. The presence or absence of mycorrhizal

associations was consistent in those plant species common to both sites.

The formation of VAM in intact soil profiles was measured in topsoil and subsoil using

bioassay seedlings grown in intact soil cores. VAM most readily developed in the roots

of bioassay seedlings grown in the topsoil. Limited VAM occurred in the roots grown in

subsoil cores. Most colonization of roots by VAM fungi occurred in the soil cores

collected and assayed during Spring and Summer.

Few spores were found in any soil sampled, though at least twice as many spores

occurred in the topsoil than in the subsoil, for all seasons examined. As most of the

propagules that could initiate VAM (i.e. spores, colonized root fragments and fungal

hyphae), were observed in the topsoil, disturbances which involve the removal and

storage of the top 15 cm will adversely affect these fungi.

Removal and storage of the surface layers of soil is known to decrease the infectivity of

VAM fungi. In previous studies investigating VAM fungi and soil disturbance, only two treatments have been examined viz. no disturbance v. profoundly disturbed soil. I investigated the relationship between increasing intensity of topsoil disturbance and the infectivity of VAM fungi. Intact soil blocks were treated with one of four levels of disturbance. Seeds of a bioassay species were sown into the blocks and harvested 14,

21, 28, 35, and 42 days after sowing. Colonization of roots by VAM fungi had Ill

commenced by 14 days in the intact, low, and intermediate disturbance treatments.

Colonization of roots was delayed by up to six weeks for seedlings grown in the most

disturbed of the soil blocks. Although the low and intermediate degrees of soil

disturbance did not cause a delay in the initiation of infection, they did reduce the

proportion of root length colonized by VAM fungi after 21 days. After 21 days, shoot

biomass was significantly less in seedlings grown in the most disturbed of the soil

blocks. The most severe experimental treatment probably disturbed the external hyphal

network and the infected root fragments (containing VAM hyphae and vesicles), which in

turn temporarily reduced the infective potential of the fungus to nil. The observed delay

in the initiation of infection could therefore be explained by the time required for hyphae

to grow from other propagules in the soil.

The propagules of VAM fungi include: (i) spores, (ii) root fragments containing VAM

hyphae and vesicles, and (iii) soil hyphae. The viability of each type of propagule after

soil disturbance will determine in part, the number of infective propagules available to

initiate the VAM association with plants re-colonizing a disturbed site. The aim of this

study was to examine which of the propagules of VAM fungi are capable of initiating

VAM after topsoil disturbance. Soil from the open woodland site was wet-sieved

through a tier of three sieves (1 mm, 250 (lm, and 106 }im), and separated into: (i) root

fragments, (ii) VAM hyphae, (iii) VAM spores. Each of these fractions was assayed to

determine its potential to initiate VAM. Fungal hyphae grew from rootfragments afte r 14

days. VAM hyphal fragments did not produce any VAM infection even after 42 days.

The VAM sporefraction initiate d VAM after 28 days.

Overall, this study showed that: (i) VAM fungi are a component of the soil environment in Hawkesbury Sandstone soils and mycorrhizal associations exist in a high proportion of the indigenous plant species, (ii) most of the propagules that can initiate VAM occur in the top 15 cm of soil, (iii) cutting soil blocks longitudinally into four and nine equal portions has no significant impact upon VAM fungi, but cutting blocks into 25 portions, IV

temporarily can reduce the infective potential of VAM fungi to nil, and (iv) colonized root fragments and spores can be effective propagules initiating VAM after topsoil disturbance. v

ACKNOWLEDGEMENTS

In completing this thesis, I have cause to thank many people. Firstly I thank my

supervising committee; Rob Whelan, Peter McGee (School of Biological Sciences,

University of Sydney), and Ros Muston (Quality Environmental Management Pty. Ltd.) for their judicious supervision and friendship over the last three years.

I extend my thanks to the following people for offering critical comments on the various

drafts of thesis chapters and manuscripts from this research; David Ayre, Mark Brundrett,

Andy Davis, David Jasper, Peter McGee, Ros Muston, Jack Putz and Mandy Reid. My

supervisory committee provided extensive editing and oral comments on drafts of this

thesis for which I am extremely grateful.

I am grateful to members ofthe Biology Department for their warmth and friendship over the last three years. In particular, I thank Jan Fragiacomo, Julie Gray, (Judy Gordon and

Peter Dalmazzo, 1989-1990), Graham Kohler and Dallas Lynch for much needed help in ordering and hiring materials and vehicles.

My gratitude is extended to the Sydney Water Board, especially John Wrigley and David

Hinchley for financial and material support during the project. Additionally, the

Ecological Society of Australia provided some travel funding.

I thank Ken Russell for statistical advice and analysis throughout the project: "Old statisticians never die, they just get broken down by age and sex".

I thank Joy Williams for assistance in the identification of plant species from my study sites and Assoc. Prof. Zeng Liangzhong for help in setting up the experiment described in

Chapters 5 and 6. VI

I thank my friends, and I am especially indebted to the people with whom I have lived;

Bectaun, Craig, Clyde, David, Felicity, Phil, and Reggie: "Thankyou for providing the essential balance".

My love and thanks go to Francine deGruchy. Thankyou for your patience and love during the last year and a half: "It keeps me going".

This thesis is dedicated to my family for their enduring love and support throughout my studies: "If I have been able to see farther than others, it was because I stood on the shoulders of giants".

"All Praises Be To The Almighty" vu

TABLE OF CONTENTS

ABSTRACT i ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF TABLES x LIST OF FIGURES xi

Chapter 1 INTRODUCTION & AIMS 1 1.1 THE NEED FOR REVEGETATION 1

1.2 FACTORS IMPINGING UPON REVEGETATION 2

1.3 THE ROLE OF MYCORRHIZAE 4 1.3.1 Mycorrhizae: definition and classification 4 1.3.2 Mycorrhizae and plant growth 6 1.3.3 Mycorrhizae and the recovery of disturbed plant communities 10 1.4 AIMS OF THE STUDY & FORMAT OF THE THESIS 14

Chapter 2 SITES, SOILS & VEGETATION 16

2.1 INTRODUCTION & AIMS 16

2.2 LOCATION, CLIMATE & GEOLOGY OF SITES 19 2.3 SOILS 21 2.3.1 Materials & Methods 21 2.3.2 Results & Discussion 23

2.4 VEGETATION &RRE HISTORY 23 2.4.1 Floristics & Physiognomy 23 2.4.2 Fire history 26 Vlll

Chapter 3 THE MYCORRHIZAL ASSOCIATIONS OF THE PLANT SPECIES IN THE TWO STUDY SITES 27 3. l INTRODUCTION & AIMS 27

3.2 MATERIALS & METHODS 27

3.3 RESULTS & DISCUSSION 28

Chapter 4 WHICH PART OF AN INTACT SOIL PROFILE REPRESENTS THE MAJOR STORE OF THE POTENTIAL PROPAGULES OF VAM FUNGI? 36 4. l INTRODUCTION & AIMS 36

4.2 MATERIALS & METHODS 37

4.2.1 Procedure of the bioassay 37

4.2.2 Sampling and extraction of spores 38

4.2.3 Statistical analysis 39

4.3 RESULTS 39

4.3.1 Root growth and VAM formation 39 4.3.2 Spore types and abundance 41

4.4 DISCUSSION 41

Chapter 5 THE RELATIONSHIP BETWEEN INTENSITY OF TOPSOIL DISTURBANCE AND INFECTIVITY OF VAM FUNGI 46 5.1 INTRODUCTION & AIMS 46

5.2. MATERIALS & METHODS 47

5.2.1 Design of the experiment 47

5.2.2 Statistical anlysis of results 48

5.3 RESULTS 50

5.3.1 Root growth and VAM formation 50

5.3.2 Shoot dry mass 52

5.4 DISCUSSION 52 Chapter 6 THE PROPAGULES CAPABLE OF INITIATING VAM AFTER TOPSOIL DISTURBANCE 56 6.1 INTRODUCTION & AIMS 56

6.2 MATERIALS & METHODS 58

6.2.1 Experimental design 58

6.2.2 Part I - VAM formation in undisturbed v. disturbed soil blocks 58 6.2.3 Part n - Isolation of VAM inoculum fractions and examination of potential infectivity 5 8 6.2.4 Statistical analysis 61

6.3 RESULTS 62

6.3.1 VAM formation in intact v. disturbed soil blocks 62

6.3.2 Root fragments 62

6.3.3 Hyphal fragments 63 6.3.4 Spore/soil and fines fraction 64

6.4 DISCUSSION 64

Chapter 7 CONCLUDING DISCUSSION 68

7.1 INTRODUCTION 68

7.2 THESIGNMCANCEOPVAMFUNGIlNHAWKESBURY SANDSTONE PLANT COMMUNITIES 69

7.3 THE IMPACT OF SOIL DISTURBANCE ON VAM FUNGI AND THEIR PLANT HOSTS 71

7.4 VAM FUNGI AND REVEGETATION OF DISTURBED LANDSCAPES 73

REFERENCES 77

APPENDIX I-V 100 X

LIST OF TABLES

Table No. Legend Page No.

1.1 Factors impinging upon the revegetation of a disturbed site (adapted from Bradshaw 1983). 3 2.1 Primary climatic data for Wollongong, N.S.W. (Anon. 1990a). 22 2.2 Physical-chemical analysis of sandstone soils from the two study sites. Data given are means (± s.e.) of five replicates taken in each site. 24 3.1 Numbers of plant species found with mycorrhizal associations at Avon and O'Hares sites. 29 3.2 Plant species with previously unreported mycorrhizal associations (Ecto = Ectomycorrhizae, VAM = Vesicular-arbuscular mycorrhizae, V = Vesicular, * = Family not previously reported to be mycorrhizal). 30 4.1 Daily temperature ranges (°C) for an outdoor glasshouse at the Eastern Campus of the Univ. of Wollongong. Data given are means (± s.e.) for 90 measurements made daily during each season using a max./min. thermometer. 38 6.1 Chi-square analysis of root fragments extracted from soil blocks taken from the Avon study site. Data represent the number of rootfragments out of 100 from which hyphae were produced. 63 7.1 Questions addressed in this thesis and a summary of the contributions made. 68 LIST OF FIGURES

Legend Page No.

Extent of Hawkesbury Sandstone lithological formation (adapted from Burrough et al. 1977). 17

Location of study sites within the Metropolitan Catchment Area. 18

Example of disturbance to vegetation in the Metropolitan Catchments - a cleared seismic line. 20

An example of sheet erosion resulting from the removal of the vegetative cover. 20

The Avon study site - open woodland. 25

The O'Hares study site - open sclerophyllous shrubland. 25

V - Vesicle associated with the root of Ptilantheliwn deustem. Scale: 44jj.m. 32

Root of Conospermum longifolium with A = arbuscule. Scale: 17 p.m. 32

Root of Conospermum taxifolium with A = arbuscule. Scale: 17 nm. 33

Root of Telopea speciosissima with multibranched A = arbuscule. Scale: 17 pm. 33

V - Vesicle associated with the root of Persoonia levis. Scale: 44 p.m. 34

V - Vesicle associated with the root of Hakea dactyloides Scale: 44 p.m. 34

Percentage of root length colonized by VAM fungi in 8- and 12-week old seedlings growing in intact soil cores taken from two depths (topsoil = 0-15 cm & subsoil =15- 30 cm) from (a) the Avon and (b) the O'Hares study sites. Each bar represents the mean (± s.e.) of five seedlings extracted from each of 16 replicates. 40 Type I spore extracted from soil samples taken from both the Avon and O'Hares study sites. Scale: 45 [im. 42

Type n spore extracted from soil samples taken from both the Avon and O'Hares study sites. Scale: 45 |im. 42

Density of (a) Type I and (b) Type II spores at two soil depths (topsoil = 0-15 cm & subsoil = 15-30 cm) from the two study sites. Bars represent mean number of fungal spores (± s.e.) for 25 random samples taken at the start of each season. 43

Schematic representation of the increasing degrees of topsoil disturbance. 49

Percentage of root length colonized by VAM fungi in relation to increasing degrees of soil disturbance using intact soil blocks removed from (a) the Avon and (b) the O'Hares study sites. Each bar is the mean (± s.e.) offive seedling s from each of five replicates except the "No dist." treatment which is the mean (± s.e.) of five seedlings from each of 10 replicates. 51 Shoot dry mass accumulation in relation to increasing degrees of soil disturbance using intact soil blocks removed from (a) the Avon and (b) O'Hares study sites. Each bar is the mean (± s.e.) offive seedling s from each offive replicates except the "No dist." treatment which is the mean (± s.e.) offive seedling s from each of 10 replicates. 53 1

Chapter 1

INTRODUCTION & AIMS

1.1 THE NEED FOR REVEGETATION

Destruction of plant communities by mining, agriculture, and other processes is an inevitable and intrinsic part of civilization. Approximately 0.01% of Australia's land surface has been degraded by mining and its associated processes (Anon. 1989). This figure may not seem to represent a significant proportion of Australia's land surface, however, the coincidental geographic location of some mining activities with areas of conservation value have magnified the impacts of the mining industry. Consequently, the activities of the mining industry have now become a focal point of both public and political attention.

A national assessment of the amount of land degraded by agricultural processes revealed that over half of Australia's rural land required treatment to overcome degradation

(Eckersly 1989; Roberts 1989). This is of particular significance if we consider that 68% of Australia (approximately 768 million ha) is currently used for agriculture (Anon.

1987).

One result of mining and agriculture is that native vegetation is removed, and often not returned. This not only changes the visual character and integrity of a landscape, but also reduces the stability of the soil. Three alternatives are often chosen to revegetate denuded sites and stabilize the soil. The first is to allow the vegetation to regenerate naturally. The second is to fabricate a landscape by planting exotic species which germinate and grow quickly, and give the short-term effect of binding the soil surface (Temple & Bungey

1980), and the third is to deliberately re-establish indigenous native species. The choice of revegetation process will be determined largely by the strategic management plan for 2

the area, the actual or proposed use, and its relationship to the adjoining environment

(Law 1984). Construction, establishment, and maintenance costs must also be kept in

balance with the short- and long-term cost benefits (Thames 1977).

Consequently, the overall aim of broad scale revegetation of degraded areas is to produce

economically, a dynamic, self-sustaining plant community which does not require, long-

term maintenance, and can accommodate a continuing use of the natural resources without

irreversible damage (Muston 1987).

1.2 FACTORS IMPINGING UPON REVEGETATION

The post-disturbance remnants of a highly disturbed plant community may be considered

to represent the study of a primary succession (Whelan 1989). The Clementsian view of

primary succession predicts that a plant community and animal associates will re-colonize

the site, given enough time. In addition, thefinal specie s composition will be closely

allied to that found in intact representatives of the general area. Although the absolute

directionality of the process, and the climax community concept proposed by Clement are

both currently considered to be an oversimplification, many ecologists adhere in part to

the Clementsian view (MacMahon 1987).

Clements (1916) went further to categorize the processes responsible for the development

of the succession. According to Clements' simple scheme, ecosystem restoration is

initiated by a number of resilient species which may have survived the disturbance evenL

and a number of nearby or highly mobile species which can gain access to the area.

Upon establishment, "competition" is viewed as the main interaction determining the

subsequent post-disturbance species mixture. It is now widely accepted that a more

complex array of factors impinges upon the regeneration of plant species. However, the

factors can be broadly divided into two classes, similar in essence to those described by Clements i.e., those that influence colonization, and those that act on the subsequent development of the plant community (Table 1.1).

Table 1.1 Factors impinging upon the revegetation of a disturbed site (adapted from Bradshaw 1983).

Colonization Development

Soil conditions Soil development

Environmental factors Environmental factors - availability of water - availability of water - temperature fluctuations - temperature fluctuations - amount of light - amount of light

Lack of nutrients Accumulation of nutrients - mycorrhizae - mycorrhizae - decomposition of leaf litter

Life form - regeneration from seed - regeneration from root-stock

Plant-plant interactions Plant-plant interactions - competition - competition for pollinators - allelopathy

Plant-animal interactions Plant-animal interactions - seed dispersal - herbivory - nutrient cycling - pollination - amelioration of soil conditions - seed dispersal

In a recent treatise on the problems associated with ecosystem reconstruction, Bradshaw

(1983) emphasized that one of the primary stresses imposed on plants colonizing disturbed landscapes is the lack of nutrients. As mycorrhizae have the principal benefit of increasing nutrient uptake (e.g. Mosse 1973; Abbott & Robson 1984), it has been hypothesized that the successful revegetation of disturbed landscapes may be dependant in part on the availability of viable mycorrhizal inoculum (Loree & Williams 1987), and that succession beyond the early stages of recovery may be linked to their presence

(Miller 1987). 4

1.3 THE ROLE OF MYCORRHIZAE

Mycorrhizae are one of several factors which impinge upon colonization and development

of a post-disturbance plant community (Table 1.1). The following discussion expands on

the role of mycorrhizae in plant communities. The discussion begins with a description

of mycorrhizae, and the morphological basis of their classification. Following this, I

discuss how mycorrhizae influence plant growth. Finally, I discuss the role of

mycorrhizae in the revegetation of disturbed plant communities.

1.3.1 Mycorrhizae: definition and classification

The term "mycorrhiza" describes the mutually beneficial association between the hyphae

of a fungus and the roots of a higher plant. The partners in this symbiosis are dependent upon one another for the transfer of material between cells. The mycorrhizal symbiosis usually results in the increase in efficiency of nutrient absorption by the host, in exchange for organic carbon by the fungus (Harley & Smith 1983). The hyphae of mycorrhizal fungi serve as extensions of the hosts' root system (Bethlenfalvay et al. 1984), and are both physiologically and geometrically more effective organs of absorption than the roots themselves (Trappe 1981; Smith & Gianinazzi-Pearson 1989).

Fungi which are known to form ectomycorrhizae occur in 39 genera within three classes of the Eumycota; Basidiomycotina, Ascomycotina, and the Zygomycotina (Miller 1982).

Some 3% of all seed plants are currently known to have ectomycorrhizal associations

(Hudson 1986). One hundred and twenty six species (restricted to a few genera of the

Zygomycetous family Endogonaceae) of endomycorrhizal fungi have been described, and vascular plants of all life-forms form endomycorrhizal associations (Kendrick 1985;

Morton 1988). More recently, Morton & Benny (1990) have revised the classification of arbuscular mycorrhizal fungi and proposed a new order, Glomales, two new suborders, 5

Glominae and Gigasporinae, and two new families, Acaulosporaceae and Gigasporaceae,

with the emendation of Glomaceae.

There is little evidence for specificity between particular fungi and host plants in either

ecto- or endomycorrhizal associations in a given plant community (Harley & Smith

1983). The fact that specificity appears to be generally lacking has profound implications

both for the infection process and for nutrient cycling. The chance that uninfected roots

will make contact with compatible mycelium is high under these circumstances. Also, if

mycorrhizal infection arises from such contacts, the consequence is that roots become

physically incorporated into an established mycelial network. If, in turn, this network

provides functional pathways for the transfer of nutrients between host plants, it will be

of fundamental importance to nutrient cycling processes (Read et al. 1985; Newman

1988).

The traditional dichotomy between ecto- and endomycorrhizae, which aimed to recognize

structural differences between the associations, is no longer popular because it does not

reflect physiological relationships (Smith 1980). However, for the purposes of

convenience, this traditional division will be retained in the following discussion.

In mycorrhizae, the fungus adopts recognizable and characteristic structures upon or

within the host tissue. The morphology of these structures forms the basis of their classification. Roots colonized by ectomycorrhizal fungi are characterized by the presence of a partial or entire fungal sheath of hyphae (septate or aseptate) with or without a Hartig net on the roots of plants (Warcup 1980). A Hartig net is a plexus of fungal hyphae which ramifies between the epidermal and cortical cells.

Five forms of endomycorrhizae have been described. Vesicular-arbuscular mycorrhizae

(VAM) are characterized by the presence of arbuscules and vesicles connected to each other and to the soil by aseptate hyphae. Arbuscules are thought to increase the surface 6

area of the plasmalemma available for exchange by up to threetimes (Co x & Tinker 1976;

Gianinazzi et al. 1983). Vesicles function as storage and reproductive structures (Harley

& Smith 1983). Two variants of the VAM form exist, vesicular associations, and coil

VAM (McGee 1986). Vesicular associations are characterized by intercellular vesicles

occurring within the roots (there are no arbuscules present), while coiling VAM also have

tightly formed coils of hyphae in the epidermal and/or cortical cells of the root. Two

other distinct diagnostic forms exist. Epacrid mycorrhizae have extensive hyphal coils in

the cortical cells of thefine roots of members of the Epacridaceae (McGee 1986). Orchid

mycorrhizae are characterized by the development of coils in the epidermal and cortical

cells of the tubers of members of the Orchidaceae (Harley 1959). Throughout this thesis,

for convenience, "VAM" has been used both as a noun (e.g. VAM are characterized

by...), and as an adjective (e.g. VAM fungi had...).

1.3.2 Mycorrhizae and plant growth

This section addresses some particular aspects of the mycorrhizal symbiosis including:

(1) mechanisms of nutrient uptake, (2) water relations, and (3) energetics of the

mycorrhizal symbiosis.

1. Mechanisms of nutrient uptake

The beneficial aspects of mycorrhizae in mineral uptake are related to increases in the surface area effective in ion absorption. Bowen (1973) and Harley (1975) have reviewed this process for ectomycorrhizae, and Sanders et al. (1975) for endomycorrhizae. In ectomycorrhizae, the increase in the absorptive surface area can be brought about in a number of ways; hyphal and mycelial strand formation, sheath formation, increased branching, increased diameter of root, and increased longevity of the absorbing root. In endomycorrhizae, increases in surface area are primarily related to the extensions of hyphae from the colonized root into the soil (Reid 1984). Tisdall & Oades (1979) 7 calculated that 1 cm of root colonized by VAM fungi had 3 m of soil hyphae associated with it.

Mycorrhizal modification of the nutrient-uptake properties of roots depends upon: (i) development of extramatrical hyphae in the soil, (ii) hyphal absorption of nutrients, (iii) translocation of nutrients through hyphae over considerable distances, and (iv) transfer of nutrients from the fungus to the root cells (Smith & Gianinazzi-Pearson 1989). The following discussion will highlight the absorption of phosphorus. Research attention has been focussed on phosphorus absorption because it is essential for plant growth, and is also commonly at very low concentrations and lability in the soil solution (Harley &

Smith 1983). Mycorrhizae have been implicated in the enhanced uptake of a number of other nutrients including; zinc (Bowen et al. 1974), copper (Timmer & Leyden 1980), sulphur (Rhodes & Gerdemann 1978a), and calcium (Rhodes & Gerdemann 1978b).

In the case of the eucalypt ectomycorrhizae, it is well established that the absorbing power of roots colonized by mycorrhizae can be much greater that uninfected roots. For example, ectomycorrhizal seedlings of two Eucalyptus species have been shown to be better than uninfected seedlings at absorbing phosphorus applied to soil at low concentrations (Malajczuk etal. 1975; Mulligan & Patrick 1985). It has been suggested, that the absorbing power of endomycorrhizal hyphae (in relation to phosphate uptake) is merely by virtue of their position in the soil, rather than any special uptake property

(Tinker 1975). However, it has been demonstrated that hyphal surfaces absorb greater amounts of 32P than root surfaces (Gray & Gerdemann 1969; Bowen et al. 1975).

It is also important to consider the abundance and distribution of the root and associated fungus from a spatial aspect. The relationship between root length per unit volume of soil and the occurrence of depletion zones of nutrient around absorbing surfaces is paramount to understanding the importance of increases in surface area by mycorrhizae (e.g. Bowen

1983a; Bowen 1983b; Gianinazzi-Pearson 1985). Slow diffusion of phosphate ions in 8 the soil solution as compared with rapid absorption of phosphate by roots and other absorbing organs results in the development of depletion zones around roots (Nye &

Tinker 1977). The longer a segment of root remains actively absorbing from soil at a rate greater than that of movement to it, the wider will be the phosphorus depletion zone

(Harley & Smith 1983). Consequently, an absorbing system which can rapidly colonize undepleted soil will have advantages over other absorption systems. Mycorrhizae and

their extensive external mycelial network impart this advantage upon the associated host,

because the mycelial network ramifies through the soil profile beyond the root rhizosphere (Harley & Smith 1983).

From the above discussion, it is reasonable to accept that an increased rate of phosphorus uptake is due to improved exploration of soil. Apart from this physical extension of the root system, there is another mechanism which results in more efficient uptake by infected roots. It has been suggested that the hyphae of ectomycorrhizal fungi can

solubilize certain nutrients in the soil and can thus exploit sources of nutrients unavailable to uninfected plants (e.g. Cromack 1985). Bartlett & Lewis (1973) suggested that the presence of surface acid phosphatases in ectomycorrhizae and VAM could contribute to the effectiveness of mycorrhizae utilizing complex phosphates. Williamson & Alexander

(1975) found that acid phosphatase activity in beech mycorrhizae exceeded noninfected roots by up to eight times. More recently, Dighton (1983) found that ectomycorrhizae had the capacity to solubilize phosphorus from both inorganic and organic complexes.

Furthermore, the phosphorus released was not incorporated into the fungaltissue, bu t was supplied to the plant host

2. Mycorrhizae and plant water relations

Several observers have suggested that some mycorrhizal fungi are very tolerant of low water potentials, and that mycorrhizal development may be beneficial under drought conditions. It has been proposed that mycorrhizae may be able to benefit the plant by 9

conferring drought tolerance. Parke et al. (1983a) found that seedlings with ectomycorrhizal associations were less affected by desiccation than non-mycorrhizal

seedlings. Net photosynthetic rate of infected seedlings 24 hours following re-watering was seven times that of non-mycorrhizal seedlings. Allen et al. (1981), investigating the

comparative water relations of a grass infected with VAM fungi, found that mycorrhizal plants generated lower leaf water potentials and higher transpiration rates than non- mycorrhizal seedlings at low soil water potentials. From this, and a later work (Allen

1982), it was postulated that the increased water uptake of mycorrhizal plants could be attributed to direct fungal uptake and transport. Similar findings (e.g. Safir & Nelsen

1985) supported this theory in pan, however, the link between nutrient and water uptake, make it difficult to separate improved plant water relations from improved mineral nutrition.

3. The energetics of the mycorrhizal symbiosis

Most benefits ascribed to mycorrhizal associations have been based on observed plant responses. The enhanced growth as a result of the association must therefore be interpreted as a favourable cost:benefit ratio to the host. It is well established that mycorrhizal fungi are almost entirely dependent on soluble carbohydrates produced by the host plant for their carbon source (e.g. Harley & Smith 1983; Parke et al. 1983b).

However, it is not possible to state whether or not this association is an "extra cost" to the plant. Furthermore, the amount of carbon diverted to the fungus is likely to be quite different in ecto- and endomycorrhizal fungi because of their very different anatomies.

Harley (1971,1973,1978) estimated that in ectomycorrhizal trees, approximately 25% of the CO2 respired by the root system is by mycorrhizal fungi. On average, mycorrhizal fungi comprise 4% of the roots system's biomass. Further to this, calculations suggest that this diversion of assimilated carbon is about 10% of that going into timber production. Phillipson et al. (1975) determined that root respiration accounted for 4% of 10

the total soil respiration, but approximately 25% was attributed to mycorrhizal respiration.

These figures were supported by Newman (1978), who calculated that approximately

25% of a tree's total assimilate production was sequestered for the maintenance of an ectomycorrhizal association.

As with ectomycorrhizae, it is not completely clear if there is any significant energy cost

to the host plant in endomycorrhizal associations. However, depression in the rate of

growth of young plants (as measured by dry weight) as they develop mycorrhizae has

been attributed to competition between the symbiont and the plant for limited supplies of

carbohydrate (Harley & Smith 1983). This is because a considerable amount of respiratory energy is required during endomycorrhizal development, both inside and outside the root. It has been calculated that the amount of fungal tissue within a root is

between 4-17% of the total root dry weight (Hepper 1977; Tinker 1978). The amount of

external mycelium is approximately 0.9% of the root dry weight (Bevege et al. 1975), or

36 p.g cm-1 infected root length (Sanders et al. 1975). The total fungal weight estimated by Bethlenfalvay et al. (1982) increased from 2 to 12% of the dry weight of the root during growth. Smith & Gianinazzi-Pearson (1989) reviewed the carbon cost related to the maintenance of an endomycorrhizal association, and put it between 4 and 17%, depending upon the degree of infection.

1.3.3 Mycorrhizae and the recovery of disturbed plant communities

Plant species colonized by mycorrhizal fungi have higher tissue levels of some inorganic nutrients, greater biomass yield, and more rapid uptake of water, and they are often more tolerant of various forms of stress than non-mycorrhizal plants of the same species

(Harley & Smith 1983). Consequently, mycorrhizal plants in natural vegetation might be expected to have an ecological advantage, especially in habitats susceptible to nutrient deficiency and water stress (Read et al. 1976). Highly disturbed plant communities are characterized by soils of low nutrient content, poor water-holding capacity and low 11

organic matter content (Bradshaw 1983; Danielson 1985). It has been proposed that

mycorrhizae may be important in facilitating the establishment and growth of seedlings re-

colonizing disturbed sites. The study and use of mycorrhizae in reclamation is fairly

recent; the first research was published only 20 years ago (Schramm 1966). The final

part of this discussion examines the role of mycorrhizae in revegetation. Three specific

aspects are addressed: (1) soil disturbance and mycorrhizae, (2) mycorrhizae and

colonizing plants, and (3) mycorrhizae and the development of post-disturbance plant

communities.

1. Soil disturbance and mycorrhizal fungi

The propagules of VAM fungi can be severely influenced by damage to vegetation and

soils resulting from topsoil removal and storage during mining (e.g. Rives et al. 1980;

Danielson 1985; Jasper etal. 1987; Stahl etal. 1988), and clear-cut logging (e.g. Parke et al. 1984; Janos 1987; Perry etal. 1987). Agricultural processes such astillage (Evans &

Miller 1988), long fallow periods (Thompson 1987), and biocide application (e.g. Medve

1984; Haas et al. 1987) can also adversely influence mycorrhizae. All of the above- mentioned processes usually result in gready reduced mycorrhizal formation (Brundrett

1991).

Fleming (1984) demonstrated that the inoculum of some ectomycorrhizal fungi rapidly lose their infectivity when separated from their host. However, other species did not exhibit this obligate dependency upon their host. Forestry practices such as clear-cutting, hazard-reduction burning, and scarification have been shown to prevent the survival and re-establishment of ectomycorrhizal fungi (e.g. Parke et al. 1983c, 1984; Amaranthus &

Perry 1987; McAfee & Fortin 1989).

Soil disturbance may have a direct impact on the propagules of VAM fungi or an indirect effect through changes in soil properties (Brundrett 1991). The direct impacts of soil 12

disturbance on VAM fungi may include: (i) a reduction of the number of viable spores,

(ii)fragmentation o f the soil hyphal network, and/or (iii) prevention of the growth of

hyphae from VAM roots (i.e. root pieces containing vesicles and hyphae) to new fine

roots (Evans & Miller 1988; Fairchild & Miller 1988; Jasper etal. 1989a,b,c).

The physical, chemical and biotic characteristics of soil are greatly altered by severe land

disturbance, even if the site is reclaimed and topsoiled (Stahl et al. 1988). In previous work it has been shown that soil moisture, temperature (Daniels & Trappe 1980; Parke et

al. 1983c), soil pH (Black & Tinker 1979), and the concentration of inorganic and

organic nutrients (Slankis 1974) can influence the growth and development of VAM.

Because the efficacy of VAM fungi is closely linked to edaphic factors, the endophytes

present at the site before disturbance may not produce effective mycorrhizae in the

disturbed soil (Mosse et al. 1981).

2. Mycorrhizae and colonizing plants

Mycorrhizae have been implicated in the re-colonization of plants to disturbed sites in a wide range of disturbed systems (e.g. arid: Bethlenfalvay et al. 1984; semi-arid: Loree &

Williams 1987; forest communities in Mediterranean-climatic regions: Gardner &

Malajczuk 1988).

Mycorrhizal infection may also have an important role in the stabilization of the soil in bare and disturbed sites. VAM fungi have been shown to be involved in the formation of soil aggregates in sand-dune soils (e.g. Koske et al. 1975; Sutton & Sheppard 1976;

Forster & Nicolson 1981). This is viewed to be of some considerable significance in the management of beach-dune vegetation (Logan et al. 1989).

Mycorrhizae have been implicated in the success of post-mining rehabilitation of vegetation (e.g. Aldon 1975; Allen & Allen 1980; Rives et al. 1980; Kieman et al. 1983; 13

Jasper et al. 1988). For example, a number of papers describing the revegetation of coal

spoils emphasize the importance of the mycorrhizal association in the survival of the

colonizing plant species (Daft et al. 1974; Daft & Hacskaylo 1976,1977; Khan 1978).

The need of many species of forest trees for ectomycorrhizal associations was initially

observed when attempts to establish plantations of exotic pines routinely failed until the

essential fungi were introduced (Marx 1980). The primary purpose of inoculating with

ectomycorrhizal fungi is to provide seedlings with adequate ectomycorrhizae for planting

in man-made forests. Such treatments have proved essential in forestation of cutover

lands and reclamation of adverse sites such as mining spoils (Binkley 1986).

3. Mycorrhizae and the development of post-disturbance plant communities

After initial colonization, an accumulation of nutrients both in soil and plants is required

to enable development and growth of the plant community (Bradshaw 1983). In natural

plant communities (as opposed to man-made or derelict land), primary mycorrhizal

infection of a new seedling may take place from root to root contact rather than by the

germination of spores (Read etal. 1976; Harley & Smith 1983; Jasper et al. 1989a). A

possible consequence of mycorrhizal infection arising from such contacts is that roots of

plants become physically incorporated into an established mycelial network (Brownlee et

al. 1983; Read et al. 1985). Workers have demonstrated (through the use of labelling

experiments), that there can even be phosphate transport between plants if both

individuals are mycorrhizal (e.g. Heap & Newman 1980; Whittingham & Read 1982).

This suggests the existence of a nutrient-interdependence between individuals, species

and possibly even plant communities. Hirrell & Gerdemann (1979) suggested that carbon compounds may also be transported via mycorrhizal hyphae. These organic molecules could be amino compounds as well as carbohydrates and their movement could well have significance if one host plant was simultaneously symbiotic with a nitrogen- fixing microorganism and a mycorrhizae (Harley & Smith 1983). 14

Another aspect of mycorrhizae and nutrient cycling is related to organic inputs from the

decomposition process. It has been suggested that mycorrhizal fungi remove the major

nutrients, nitrogen and phosphorus from litterfall. Consequently, mycorrhizal fungi have

been identified as important components in the cycling of litterfall (Fogel & Hunt 1979;

Fogel 1980; Parke etal. 1983b).

During the early stages of recovery of vegetation on severely disturbed sites, mycorrhizae

are frequently absent (e.g. Miller 1979; Powell 1982). In addition, the plant species

associated with this early stage of the successional process are either nonmycorrhizal, or

facultatively mycorrhizal (e.g. Reeves et al. 1979; Janos, 1980). Re-inoculation

experiments have shown that the uptake of nutrients are improved following inoculation

(e.g. Jasper et al. 1989b). This tends to suggest that mycorrhizae may play a critical role

in succession, and that succession beyond the early stages of recovery may be linked to

the presence of mycorrhizae (Miller 1987).

The mycorrhizal association is considered to be of particular importance to seedlings

colonizing degraded sites, especially in habitats susceptible to water stress and

phosphorus deficiency (Read et al. 1976). The 0-30 cm section of the Hawkesbury

Sandstone soil profile is characterized by its poor water-holding capacity and low fertility

(Beadle 1954; 1962). Consequently, it is particularly relevant to examine the potential for

mycorrhizae to influence the recovery of plant communities on Hawkesbury Sandstone

soils.

1.4 AIMS OF THE STUDY & FORMAT OF THE THESIS

This thesis examines the potential for VAM fungi to influence the revegetation of disturbed Hawkesbury Sandstone plant communities. A description of the physical characteristics of the study sites is contained in Chapter 2. An assessment of the mycorrhizal associations of the plants within the two study sites is described in Chapter 15

3. An assessment of the part of an intact soil profile where VAM fungi are most infective is described in Chapter 4. An examination of the relationship between the intensity of topsoil disturbance and the infectivity of VAM fungi has been treated in Chapter 5.

Chapter 6 investigates which propagules of VAM fungi are capable of initiating VAM infection after topsoil disturbance. The studies described in Chapters 3 and 6 form the basis of published papers contained in Appendix V. Chapters 4 and 5 are "in press" in the journal Mycorrhiza. Chapter 2

SITES, SOILS & VEGETATION

2.1 INTRODUCTION & AIMS

Hawkesbury Sandstone is a wide-ranging geological formation, extending to the north,

south, and west of the Sydney region (Fig. 2.1). Despite its lithological uniformity, the

Hawkesbury Sandstone country supports a very diverse and varied flora. It has been

suggested that climatic differences control the regional variations of soil and vegetation

(Burrough et al. 1977). Plant communities can range from dry sclerophyllous shrublands

through woodlands to closed forest (Young & Young 1988). In the southern part of the

Sydney Basin the Hawkesbury Sandstone occurs as a west sloping plateau, the

Woronora Plateau. The southern part of the sandstone Plateau encompasses the

Metropolitan Catchment Area ofthe Water Board. This area covers approximately 1200 km2 of plateau inland of Wollongong (Fig. 2.2). Water supply for 250 000 residents and heavy industry is derived primarily from the Avon Reservoir on the Woronora Plateau

(Lambert etal. 1989).

A direct link exists between water quality and the soil and vegetation conditions of the catchment surface relating to runoff (Pigram 1986). Changes in land-use practices affect. the quality of the water as it moves through a catchment. For example, in farming regions, agricultural chemicals such as herbicides, pesticides, fertilizers and sediment may enter surface runoff and groundwater, resulting in a degradation of water quality

(Williams 1980). Sediment from agricultural lands increases turbidity in streams and lakes resulting in loss of aesthetic value and higher costs of water treatment. The management of the native vegetation covering the catchment areas is an important part of the Sydney Water Board's activities. Of particular interest is the rehabilitation of derelict land within the catchment boundaries. One example of disturbance is the clearing of Sydney

HSs HSs

Wollongong

Shellharbour

HSsV Kiama.J - 12

HSs

Nowra .

0 20 km -35 Study area

HSs Hawkesbury Sandstone

Figure 2.1 Extent of the Hawkesbury Sandstone lithological formation Burrough et al. 1977) 7

Wollongong

Lake Illawarra

\ 0

Escarpment

Figure 2.2 Location of study sites within the Metropolitan Catchment Area. 19

vegetation for seismic lines (Fig. 2.3). This results in the removal of the vegetative cover, exposing the friable, poorly formed topsoil. Once the vegetation cover has been removed from these areas, the soil erodes very quickly (Fig. 2.4). The Sydney Water

Board is interested in revegetating these degraded areas in an attempt to conserve the soil and plant species, return the site's natural visual character and integrity, and maintain high water quality.

For this study, two study sites were chosen, one in the Avon Catchment and the other in the O'Hares Catchment (Fig. 2.2). Areas in each catchment were selected for this study, based on the following criteria: (i) the catchments comprised large areas of relatively undisturbed plant communities, isolated from human interference, (ii) although the sites supported different vegetation types, a suite of plant species was common to both, and

(iii) the catchments contained plant communities and plant species which are commonly disturbed by various agencies (e.g. roads, seismic lines, mine shafts, pipelines, and powerlines).

This chapter describes the location, climate, geology, soils, and vegetation of the chosen study sites.

2.2 LOCATION, CLIMATE & GEOLOGY OF SITES

Both catchments are situated on the Woronora plateau. The Avon Catchment is a 14,000 ha catchment located 22.5 km west northwest of Wollongong, 34°22'S, 150°40'E, at an elevation of approximately 400 m. The chosen study site was at the edge of Fire Trail 6A between Fire Trails 6B and 6X (Anon. 1983). The O'Hares catchment is a 7, 500 ha catchment located 23.5 km north of Wollongong, 34°14'S, 150°53'E, at an altitude of approximately 370 m. The chosen study site was adjacent to Fire Trail 10B (Anon.

1983). Figure 2.3 Example of disturbance to vegetation in the Metropolitan Catchments - a cleared seismic line.

Figure 2.4 An example of sheet erosion resulting from the removal of the vegetative cover. The climate of the Wollongong area may be described as "temperate, marine" (Fuller

1980). The long-term average annual precipitation in this region is 1420 mm (Anon.

1988), but this is highly variable across the region. Rain occurs throughout the year, with a slight summer predominance. Average maximum monthly temperatures range from 25.5°C in summer, to 16.7°C in winter. Average minimum monthly temperatures range from 18.6°C in summer, to 8.2°C in winter. Average relative humidity is more or less consistent throughout the year (Table 2.1).

The geology underlying the Woronora Plateau is dominated by the Hawkesbury

Sandstone. This uniform lithological formation is composed of Triassic Quartose sandstone rock (Standard 1961; Herbert & Helby 1980; Branagan 1985).

2.3 SOILS

2.3.1 Materials & Methods

Soil samples were taken from five randomly chosen locations in each of the sites. This was achieved by superimposing a hypothetical grid (10 x 10 m) over the study sites, and obtaining northings and eastings from a table of random numbers. Soil samples were taken from two depths: 0-15 cm and 15-30 cm. Samples were placed in plastic bags and transported back to the laboratory. The soil was passed through a 2 mm sieve before pH analysis, and then air-dried. The following physical and chemical analyses were completed on all soil samples by the Sydney Water Board:

(i) pH using a water to fresh soil ratio of 2:1 (ii) soil particle size distribution using a Bouycos hygrometer (Ball 1986) (iii) weight loss on ignition (Allen et al. 1986) (iv) total phosphorus using a perchloric acid digestion (Hesse 1971) (v) bicarbonate-extractable phosphorus (Colwell 1965) (vi) total nitrogen using Micro-Kjeldahl digestion (Hesse 1971) (vii) soluble sodium, calcium, and magnesium using a 1 M ammonium acetate extraction (pH 7.0) at a soil to extract ratio of 1:25 (Allen et al. 191 A). 22

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The soil at Avon had 30 cm of yellow clayey subsoil overlain by 20 cm of loamy sand topsoil [Yellow Earths (Gn2.21)], while, the soil at O'Hares was a loose shallow sand of 30 cm with abundant ironstone overlying the sandstone surface [Lateritic-Podzolic Soils (Dy3.61)] (Hazelton & Tille 1990). Physical and chemical properties of the two soils differed especially with respect to texture, organic matter content, and in the concentration of some important nutrient elements (Table 2.2). Little difference in the physical or chemical composition was found between the topsoil and the subsoil at either site (Table 2.2).

2.4 VEGETATION & FIRE HISTORY

2.4.1 Floristics & Physiognomy

Foliar projection cover values of the plant species within the two study sites were determined using the point quadrat method (Goldsmith et al. 1986). A hypothetical lxl m grid was overlain on the sites. The points of the grid were sequentially numbered.

One thousand points were selected using a table of random numbers. At each point, the plant species encountered was recorded. These were summed, and divided by ten to give a percentage foliar projection cover value for each of the species.

The Avon site supported an open woodland, dominated by Eucalyptus haemostoma and

E. racemosa, with a shrub understorey (Fig. 2.5). The understorey was dominated by

Leptospermumflavescens, Bossiaea obcordata, Grevillea sphacelata, and Lambertia formosa (Appendix I). The vegetation of the O'Hares study site consisted of an open sclerophyllous shrubland (Fig. 2.6). The shrubland was dominated by Angophora hispida, Lambertia formosa, Pultenaea elliptica, Mirbelia rubiifolia, and Hakea Table 2.2 Physical-chemical analysis of sandstone soils from the two study sites Data given are means (± s.e.) of five replicates taken in each of the study sites.

Avon O'Hares

0-15 cm 15-30 cm 0-15 cm 15-30 cm

pH 5.6 (0.1) 5.7 (0.2) 5.3 (0.1) 5.3 (0.2)

% gravel (>2 mm) 12.6 (2.5) 9.6 (2.2) 32.3 (3.8) 42.0 (10.6)

% sand 61.7 (2.0) 56.9 (3.3) 55.0 (11.3) 42.0 (9.4)

% silt 15.6 (2.0) 20.8 (1.4) 6.7 (0.7) 8.4 (0.7)

%clay 10.1 (0.5) 12.7 (0.8) 6.0 (0.5) 7.6 (0.7)

% organic matter 10.4 (2.1) 8.8 (0.6) 3.1 (0.8) 2.7 (1.2)

Total P (ppm) 30.0 (3.2) 17.0 (3.8) 13.0 (3.0) 14.0 (2.5)

Extr. P (ppm) 0.7 (0.1) 0.5 (0.0) 0.8 (0.3) 1.2 (0.5)

Total N (ppm) 786.0 (83.2) 512.0 (88.3) 468.0 (131.5) 160.0 (6.3)

Sol. Na (ppm) 91.0 (4.4) 92.0 (1.2) 106.0 (15.7) 136.0 (16.3)

Sol. Ca (ppm) 204.0 (17.5) 92.0 (1.2) 160.0 (15.7) 136.0 (16.3)

Sol. Mg (ppm) 139.2 (19.9) 110.4(22.3) 112.8 (6.2) 151.2 (15.7)

NB. Total P = Total phosphorus Extr. P = Bicarbonate-extractable phosphorus Total N = Total nitrogen Sol. Na, Ca, Mg = Soluble sodium, calcium, magnesium. 25

1 jIL .'ll > - Mr*

> v/sfr+'J IC.vl \rtJFJfX *\ i x **: *$Mx%A :

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^r^^»* , /rifc^_ „ ~*~^Jik ™ "^l^**" *^ nr*A g: .^ 1 .!'AJ! ..., •• • / ' :-« SSt"? ••'•• ••'. • A -y W - .-. • v. j,".' • -• '"TS - • A- 3**»ji .t '• p J r ••• i. >- —

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-••. • • #i ~ >.<*. - *'t- *.' Aa& ,.y-.. * - • '' r A .* • ""j.' i B^n^MSOlPM SpS'v^iwi A iy^,th^sc\ i •• fi "&= *[a! l

Figure 2.6 The O'Hares study site - open sclerophyllous shrubland. 26

dactyloides (Appendix I). Although the sites supported vegetation of different physiognomy, 14 plant species were common to both sites, as indicated by asterisks in Appendix I.

2.4.2 Fire history

The fire histories of the two study sites are comparable. Within the Avon study site, a hazard reduction burn was carried out during 1983, while in the O'Hares site, a hazard reduction burn was carried out during 1982 (Anon. 1990b). More recently, since the initiation of this research, the O'Hares site was burned by a wildfire in December of

1990. Chapter 3

THE MYCORRHIZAL ASSOCIATIONS OF THE PLANT SPECIES IN THE TWO STUDY SITES

3.1. INTRODUCTION & AIMS

Before being able to assess the potential for VAM fungi to influence the revegetation of a

disturbed landscape, it is necessary to have some idea of the mycorrhizal status of the

plant species occurring naturally in the undisturbed ecosystem. The only survey of the

mycorrhizae in the plants inhabiting Hawkesbury Sandstone soils was carried out by

Khan (1978). He investigated the incidence of VAM in the plants growing in three

commercial coal tips (comprized of mine dirt and washer discard). All plants examined in

that study (26 species from 11 families) were colonized by VAM fungi, except for four

species in the genera Persoonia and Banksia (Proteaceae). The aims of the present survey

were: (i) to assess the mycorrhizal status of plant species in two examples of

Hawkesbury Sandstone vegetation; and (ii) to observe whether the presence or absence of

mycorrhizae was consistent in plant species common to the two study sites. In addition,

an objective of this survey was to extend the information on the mycorrhizal status of

Australian plant species.

3.2 MATERIALS & METHODS

Roots from five representatives of each plant species were collected from random locations within both of the study areas during July - September 1989. A hypothetical grid (1 x 1 m) was laid over the study sites and northings and eastings were obtained from a table of random numbers. The individual of the targeted plant species lying closest to this random point was then sampled. At least 20 cm of fine feeder root was collected from each perennial plant species by tracing fine lateral roots from the stem to 28 their termini. For annuals, entire plants were excavated, and the roots washed, sorted, and subsequently excised.

Roots were initially fixed in 50% ethanol solution and assayed for mycorrhizal associations after clearing and staining as described by Kormanick etal. (1980).

Following staining, roots were stored in a lactic acid-glycerol solution, and subsequendy examined by both dissecting (10-40x) and compound light (40-400x) microscopes.

Nomenclature of the mycorrhizal associations follows McGee (1986). Where the term

"endomycorrhizae" has been used, it includes both VAM and vesicular associations. In this study, a plant species was classified as mycorrhizal if at least three of the five root samples were colonized by mycorrhizae.

3.3 RESULTS & DISCUSSION

Thirty two plant species representing 13 families were examined for mycorrhizae within the Avon study site (Appendix II). Of these species, 21 (66%) were mycorrhizal (Table

3.1). At the O'Hares site, 31 of the 47 (66%) species (representing 18 families,

Appendix H), were mycorrhizal (Table 3.1).

The proportion of plant species found in this survey to be mycorrhizal was generally lower than the proportions reported in previous surveys carried out in several other plant communities which range from 57 to 91% (Petersen et al. 1985; Mejstrik 1972; Logan et al. 1989; Brockoff & Allaway 1989; McGee 1986; Brundrett & Abbott 1991). However, due to the spatial limitations of this survey and differences in the respective plant species composition ofthe communities surveyed, quantitative comparisons are difficult to make.

Within both sites, ectomycorrhizae were discovered on some plant species not previously reported as having ectomycorrhizal associations (Table 3.2). However, all of the species belonged to either genera or families previously known to contain ectomycorrhizal Table 3.1 Numbers of plant species found with mycorrhizal associations at Avon and O'Hares sites.

Mycorrhizal Numbers of plant species association Avon O'Hares

Ectomycorrhizae 6 9

Vesicular-arbuscular mycorrhizae 7 11

Vesicular association 3 6

Ecto- + Vesicular-arbuscular mycorrhizae 5 5

No mycorrhizae 11 16

Total 32 47

species, viz. Platysace, Apiaceae (Brockoff & Allaway 1989), Poranthera, Euphorbiaceae

(Warcup 1980), Dampiera, Goodeniaceae (McGee 1986), Acacia, Mimosaceae (Warcup 1980), and Eucalyptus, Myrtaceae (Warcup 1980). The presence of ectomycorrhizae on

all of the species of Myrtaceae investigated further supports the suggestion of Warcup

(1980) that all Australian members of the Myrtaceae form ectomycorrhizae.

VAM were also found on some plant species not previously recorded as being

mycorrhizal (Table 3.2) but, in the same way as described for the new ectomycorrhizal

associations discovered, these plant species belonged to genera and families previously

known to have endomycorrhizal associations (e.g. Hibbertia, Dilleniaceae (Khan 1978),

Leucopogon, Epacridaceae (Logan et al. 1989)).

Endomycorrhizae were observed consistently in the roots of members of the Cyperaceae and Proteaceae (Table 3.2). These two families have previously been considered as non- mycorrhizal (e.g. Khan 1978; Malajczuk & Lamont 1981; Malajczuk et al. 1981;

Brundrett & Abbott 1991). 30

Table 3.2 Plant species with previously unreported mycorrhizal associations Ecto = Ectomycorrhizae; VAM = Vesicular-arbuscular mycorrhizae; V = Vesicular associations; * = Family not previously reported to be mycorrhizal.

Family Plant species Mycorrhizal

association

Apiaceae Actinotis helianthi VAM Platysace linearifolia Ecto * Cyperaceae Caustis flexuosa VAM Ptilanthelium deustem V Dilleniaceae Hibbertia serpyllifolia VAM Epacridaceae Brachyloma daphnoides VAM Epacris microphylla VAM Leucopogon juniperinus VAM Euphorbiaceae Amperea xiphoclada Ecto Fabaceae Bossiaea obcordata VAM Daviesia corymbosa VAM Dillwynia parvifolia var. trichopoda Ecto + VAM D. retorta Ecto Gompholobium grandiflorum VAM Pultenaea elliptica Ecto + VAM Goodeniaceae Dampiera stricta Ecto + VAM Mimosaceae Acacia linifolia Ecto + VAM A. obtusifolia Ecto + VAM A. suaveolens Ecto + VAM A. ulicifolia Ecto + VAM Myrtaceae Angophora hispida Ecto Baeckea ramosissima Ecto Eucalyptus haemostoma Ecto E. racemosa Ecto E. stricta Ecto Kunzea capitata Ecto Leptospermum flavescens Ecto L. lanigerum Ecto * Proteaceae Conospermum longifolium VAM C. taxifolium VAM Grevillea buxifolia V G. oleoides V G. sphacelata V Hakea dactyloides V Persoonia levis V P. pinifolia V Tehpea speciosissima VAM Rutaceae Eriostemon australasius VAM Boronia thujona VAM Thymeleaceae Pimelea linifolia VAM 31

For the 11 species of Cyperaceae and Proteaceae identified as being mycorrhizal, all five of the root samples taken possessed internal hyphae and cortical coils. Both vesicles and arbuscules were seen in the roots of Caustis flexuosa but only vesicles in the roots of Ptilanthelium deustem (Fig. 3.1). Within the Proteaceae, Conospermum longifolium

(Fig. 3.2), C. taxifolium (Fig. 3.3), and Telopea speciosissima (Fig. 3.4) each had

VAM, while the remaining six species had vesicular associations (Fig. 3.5 & 3.6). The nature and functional significance of atypical mycorrhizal associations such as vesicular associations remains uncertain (e.g. McGee 1986).

Harley & Smith (1983) suggested that there may be an ecological component which contributes to determination of the specificity and potential host recognition of mycorrhizal fungi. Plant species composition, soil conditions, soil microorganisms, and climatic conditions vary greatiy between individual sites. It is reasonable to expect then, that selection of hosts by fungi may also vary between sites in accordance with the local site conditions. So, the generalization that all Australian members of the Cyperaceae and

Proteaceae are non-mycorrhizal cannot be made until a thorough, broad-scale investigation of a range of Australian plant communities has been carried out The current survey extends the range of Australian plant communities examined for mycorrhizal associations.

Within each of the study sites, plant species with endomycorrhizal associations outnumbered the plants with ectomycorrhizal associations. The predominance of endomycorrhizae agrees with previous surveys carried out in a range of other ecosystems

(e.g. dune systems, Brockoff & Allaway 1989; semi arid, McGee 1986; arid,

Bethlenfalvay etal. 1984; boreal, Malloch & Malloch 1981). At both sites ectomycorrhizae and VAM were found growing on the roots of the same plant. This phenomenon was restricted to five genera belonging to three families viz. Dillwynia,

Mirbelia, and Pultenaea (Fabaceae), Dampiera (Goodeniaceae), and Acacia

(Mimosaceae). In other ecosystems, some members of these genera capable of forming 32

Figure 3.1 V - Vesicle associated with the root of Ptilanthelium deustem. Scale: 44 pm

Figure 3.2 Root of Conospermum longifolium with A = arbuscule. Scale: 17 pm. 33

Figure 3.3 Root of Conospermum taxifolium with A = arbuscule. Scale: 17 pm.

Figure 3.4 Root of Telopea speciosissima with A = arbuscule. Scale: 17 pm. Figure 3.5 V - Vesicle associated with the root of Persoonia levis. Scale: 44 pm.

Figure 3.6 V- Vesicle associated with the root of Hakea dactyloides. Scale: 44 pm. 35

and sustaining both forms of mycorrhizae, have been found possessing just one type of mycorrhizae. This variability may be the result of site-specific seasonal and biotic factors.

The presence or absence of mycorrhizae and the form of the association, i.e. ecto- or endomycorrhizae, was consistent on the roots of the plants common to both sites. The two study sites were approximately 46 km apart and so, even with the spatial limitations of this survey, the consistency of these results suggests some spatial generality in the occurrence of mycorrhizal associations in the vegetation of Hawkesbury Sandstone soils. Chapter 4

WHICH PART OF AN INTACT SOIL PROFILE REPRESENTS THE MAJOR STORE OF POTENTIAL PROPAGULES OF VAM FUNGI?

4.1. INTRODUCTION & AIMS

Disturbance normally involves a mixing of the horizons of the soil profile.

Consequently, the vertical distribution of VAM fungi in the soil profile will determine, in part, the degree to which the VAM fungi are affected by the disturbance. This may in turn influence the amount of viable inoculum of VAM fungi available to initiate infection in the re-colonising plant species (Schwab & Reeves 1981). Numerous studies have shown that the upper soil profile contains the greatest number of fungal spores, and that with increasing depth, there is a decrease in both the number of spores of each species and in the number of species represented (e.g. Sutton & Barron 1972; Wicklow &

Whittingham 1974; Redhead 1977; Smith 1978; Mertz et al. 1979; Koide & Mooney

1987). However, in some arid environments, spores of VAM fungi have been shown to reach their greatest numbers in subsurface profiles (e.g. White et al. 1989). It has been suggested some species of VAM fungi may not even produce spores (e.g. Johnson 1977;

McGee 1989). Consequently, little is known about the distribution of VAM fungi in the soil profiles, despite their importance in plant establishment and growth. Although indications of the distribution of VAM fungi have been obtained using spore counts (e.g.

Porter 1979; Gemma & Koske 1988), infected root fragments (e.g. Powell 1976), hyphal tips (e.g. Buchholtz & Motto 1981), and MPN (McGee 1989), these techniques do not measure the potential of soil to initiate VAM (e.g. Moorman & Reeves 1979; Rives et al.

1980). Because the most important propagules of VAM fungi in soils are generally unknown (Brundrett 1991), a bioassay would give an appropriate measure of the total infectivity of these fungi (Schwab & Reeves 1981). 37

Soil disturbance in the plant communities under investigation usually involves the

removal and storage of the top 15 cm of the soil profile. Although studies investigating

the impacts of topsoil removal and storage on the infectivity of VAM fungi have been

carried out in Western Australia (e.g. Jasper et al. 1987, 1988; Jasper et al. 1989a,b,c,d;

Jasper et al. 1991a), there are no published reports of similar studies in southeastern

Australia. Before investigating the impacts of soil disturbance on VAM fungi, it was

necessary to identify where, in the intact soil profile, VAM fungi were most infective.

This study aimed to: (i) identify the section of the soil profile in which VAM fungi are

most infective, and (ii) investigate the relative importance of spores as a potential source of VAM.

4.2 MATERIALS & METHODS

4.2.1 Procedure of the bioassay

At the start of each season (Autumn, Mar. 1990; Winter, June 1990; Spring, Sep. 1990;

Summer, Dec. 1990), intact soil cores were removed from 16 locations within each site,

chosen at random. A topsoil (0-15 cm) and a subsoil (15-30 cm) core were sequentially

removed, and placed in a 20 cm plastic pot, on gravel to prevent soil washing through.

Seeds of Acacia linifolia (Vent.) Willd. (Mimosaceae), a local plant known to form VAM

(Bellgard 1991) were immersed in boiling water and left until the water had cooled, to

break their enforced dormancy (Cavanagh 1987). The seeds were germinated in

bubbling, sterile, de-ionised water prior to sowing (Jasper et al. 1987). Fifteen pre- germinated seeds were sown into each of the pots. All pots were watered twice daily with tap water, and no additional nutrients were applied. Pots were placed in a naturally lit, outdoor glasshouse for the duration of the bioassay (Table 4.1). To monitor for aerial contamination of VAM fungi in the glasshouse, sixteen pots ofriver san d were sown with 15 pre-germinated seeds of A. linifolia. Table 4.1 Daily temperature ranges (°C) for an outdoor glasshouse at the Eastern Campus of the Univ. of Wollongong Data given are means (± s.e.) for 90 measurements made daily during each season using a max./min. thermometer.

Season Minimum Maximum

Autumn 14.9 (1.8) 23.5 (1.4)

Winter 12.6 (1.3) 19.3 (0.9) Spring 16.6 (1.8) 25.3 (2.2)

Summer 21.0 (2.0) 30.7 (3.3)

Eight and 12 weeks after sowing, five randomly selected seedlings were harvested from each of the 16 topsoil, subsoil, and control pots. Each seedling was carefully extracted from its medium and its roots were washed gently in a 0.4% solution of sodium hexametaphosphate, which dispersed any adhering soil. The roots of each seedling were fixed in 50% ethanol solution, cleared and stained (Kormanick et al. 1980). Total root length and the portion of root colonized by VAM fungi were determined (Ambler &

Young 1977). The length of root colonized by VAM fungi (i.e. VAM length) is a composite index, the sum of the length of root colonized by; vesicles, arbuscules and internal hyphae (NB. % VAM = VAM length/root length x 100).

4.2.2 Sampling and extraction of spores

Soil samples from 0-15 cm and 15-30 cm were collected at 25 randomly chosen locations within each of the sites at the start of each season. Spores of VAM fungi were obtained by wet-sieving and decanting 100 g sub-samples of air-dried soil through a tier of three sieves; 1000 pm, 250 pm, and 106 pm. Turgid spores filled with oil droplets were considered viable (McGee 1989), and counted microscopically. 4.2.3 Statistical analysis

The VAM formation data were analyzed using a three factor "split-plot" Analysis of

Variance (Cochran & Cox 1957) with depths nested within cores, which were nested within sites. The treatment structure was a "season x depth x time" factorial structure, with the factor "season" having four levels (i.e. Autumn, Winter, Spring & Summer), the factor "depth" having two levels (i.e. topsoil = 0-15 cm & subsoil = 15-30 cm), and the factor "time" having two levels (i.e. 8 weeks & 12 weeks).

The analyses of the density of VAM spores was carried out using a two factor "split-plot"

Analysis of Variance with depths nested within "cores", which were nested within sites.

The treatment structure was a "season x depth" factorial structure with the factor "season" having four levels (i.e. Autumn, Winter, Spring & Summer), and the factor "depth" having two levels (i.e. topsoil = 0-15 cm & subsoil = 15-30 cm).

4.3 RESULTS

4.3.1 Root growth and VAM formation

No VAM was found on the roots of the bioassay seedlings grown in the control pots in any season. Roots were of similar length in topsoil and subsoil. The trends observed in the length of root colonized by VAM fungi (i.e. VAM length) and the proportion of root length colonized by VAM fungi (i.e %VAM), were identical. Consequently, only the proportion data are presented. The development of VAM was greater in the roots of plants grown in topsoil than subsoil at both sites, at both sampling dates, and for all seasons (Fig. 4.1a & 4.1b; Appendix Ilia). Limited VAM formed in roots of plants grown in the subsoil cores taken from both sites (Fig. 4.1a & 4.1b; Appendix Ilia).

More VAM formed in the soil cores sampled and assayed during Spring and Summer in both sites and at both soil depths (Fig. 4.1a & 4.1b; Appendix Hla). (a)

Autumn Winter Spring Summer

(b)

I Topsoil-8 weeks E3 Subsoil-8 weeks M Topsoil-12 weeks 0 Subsoil-12weeks

Autumn Winter Spring Summer Season

Percentage of root length colonized by VAM fungi in 8- and 12-week old seedlings growing in intact soil cores taken from two soil depths (topsoil = 0-15 cm & subsoil = 15-30 cm) from (a) the Avon and (b) the O'Hares study sites. Each bar represents the mean (± s.e.) of five seedlings extracted from each of 16 replicates. 41

4.3.2 Spore types and abundance

Three types of spores of VAM fungi were found, and these were present in the soils of both sites, but spore numbers were generally low. Type I spores were globose, hyaline,

160 pm wide with a membranous wall, and no hyphal attachment (Fig. 4.2). Type II spores were roughly oblong in shape, brown, 190 pm wide, with an ornamented wall, and no hyphal attachment (Fig. 4.3). Type lU spores were spherical, amber, 90-120 pm wide with a thin membranous wall, and no hyphal attachment.

Generally, at both sites and in all seasons, more spores were found in the topsoil than in the subsoil. Type I spores were the most abundant. These spores were significantly more numerous in topsoil than in subsoil at both sites and for each season examined (Fig.

4.4a; Appendix Hib). Few Type II spores occurred and they were evenly distributed through the soil during Autumn and Summer. During Spring and Summer, significandy higher numbers were found in the topsoil than in the subsoil (Fig. 4.4b; Appendix Hib).

Type HI spores were uncommon (Appendix IHb).

4.4 DISCUSSION

VAM formed more readily in seedlings grown in topsoil than in subsoil. The difference in VAM formation may be due to differences in the physical/chemical properties of these two soil horizons. Soil moisture (Tommerup 1984), temperature (Daniels & Trappe

1980; Abbott & Robson 1991), soil pH (Black & Tinker 1979), and the concentration of inorganic nutrients (Slankis 1974), have been shown to influence the growth and development of VAM. However, there was little difference observed between the physical/chemical properties of the topsoil and subsoil at each of the sites (see Chapter 2,

Table 2.2). Consequendy, the differences in VAM formation between topsoil and subsoil indicates that most of the propagules that initiate VAM in these soils occurs in the top 15 cm of soil. However, only 11 soil parameters were examined in this study (Table 2.2). Figure 4.2 Type I spore extracted from soil samples taken from both the Avon and O'Hares study sites. Scale: 45 pm.

Figure 4.3 Type n spore extracted from soil samples taken from both the Avon and O'Hares study sites. Scale: 45 pm. (a)

o O K)

-«r

Autumn Winter Spring Summer

(b)

• Avon topsoil 3.0" E3 Avon subsoil H O'Hares topsoil o E3 O'Hares subsoil ST. KI • — 0> La cu.fc c/3 ra

Autumn Winter Spring Summer Season

Figure 4.4 Density of (a) Type I and (b) Type II spores at two soil depths (topsoil = 0-15 cm & subsoil = 15-30 cm) from the two study sites. Bars represent mean number of fungal spores (± s.e.) for 25 random samples taken at the start of each season. 44

It may be possible that some unmeasured factor e.g. toxic levels of metals (Guildon &

Tinker 1983; Koslowsky & Boerner 1989) or dormancy of spores (e.g. Tommerup

1983a) prevented the development and spread of infection of VAM in the subsoil cores, but these scenarios are less likely.

The greatest amount of VAM formation occurred during Spring and Summer. It is

generally accepted that the optimum temperature range for the growth of VAM fungi is

between 18 and 24°C (e.g. Slankis 1974; Daniels & Trappe 1980; Parke et al. 1983c).

In this experiment, the daily range of glasshouse temperatures during Spring and Summer

coincided with the quoted optimal range for VAM formation, and this may explain why

VAM formation peaked during these seasons. During Autumn and Winter, glasshouse

temperatures ranged between 12.6 and 23.5°C. Smith & Bowen (1979) observed that the

onset of VAM was delayed at low temperatures, which suggested that VAM fungal

activity was inhibited at low temperatures. Tommerup (1983b) demonstrated that at low

temperatures, the average rate of hyphal extension of selected species of VAM fungi was

decreased. Additionally, differences occur between VAM fungi in the optimum and lower temperature limits for spore germination (e.g. Schenck et al. 1975; Tommerup

1983a, b; Daniels & Trappe 1980). In the current study, there appears to be a pattern in the infectivity of VAM fungi related to seasonal temperatures, with higher infectivity being associated with those seasons characterized by higher temperatures, namely, Spring and Summer.

The relative importance of spores, old roots and soil hyphae as propagules apparently varies between species of VAM fungi occurring in the same site (McGee 1989). Since the most important propagules of VAM fungi in Hawkesbury Sandstone soils were unknown, a bioassay of intact soil cores, described above, was used to measure the total infectivity of these fungi. This assay did not permit an evaluation of the relative importance of each of the potential propagules of VAM fungi to initiate VAM . The vertical distribution of living spores within the two soil depths was investigated to gain 45

some insight into the relative importance of spores as a source of inoculum. The large soil-borne spores of VAM fungi, are considered by many to be the most important type of

VAM inoculum, but their numbers are often poorly correlated with mycorrhizal formation in soil (Brundrett 1991), and their germination potential varies at different times of the year (e.g. Tommerup 1983a; Gemma & Koske 1988). Few viable spores were found in this study. Low densities of living spores have been found in previous surveys of VAM fungi spore populations in a range of ecosystems (e.g. Mosse & Bowen 1968; Read et al.

1976; Abbott & Robson 1977; Janos 1980; Gay et al. 1982; Visser et al. 1984; Brundrett

& Kendrick 1988; McGee 1989). Although each spore potentially represents an infective unit, because of their low density in the soil profile, it is reasonable to expect that mycorrhizal infectivity will depend more heavily on infective mycorrhizal roots and possibly on the presence of an intact mycelial network (Jasper et al. 1991a).

Disturbances involving the removal and storage of soil have been shown to decrease the infectivity of VAM fungi (Abbott & Robson 1991). In the present study, greater infectivity of VAM fungi was found in the upper layer (i.e. 0-15 cm) of the intact soil profile. Thus, the potential exists for these fungi to be adversely affected by topsoil disturbance, because, most of the "active layer" would be removed in a disturbance. 46

Chapter 5

THE RELATIONSHIP BETWEEN INTENSITY OF TOPSOIL DISTURBANCE AND INFECTIVITY OF VAM FUNGI

5.1 INTRODUCTION & AIMS

Plant roots colonized by VAM fungi carry a loose hyphal network extending into the surrounding soil, providing an extensive surface area for absorption of nutrients and a mechanism by which infection can be spread (Warner & Mosse 1983; Newman 1988).

The external hyphal network can extend for several centimeters into the soil surrounding the plant roots they infect (Heap & Newman 1980a). Sanders & Tinker (1973) found a total of about 80 cm of external hyphae for every centimeter of onion root infected by

VAM fungi. As a source of VAM infection, the external hyphal network is considered to be particularly significant in undisturbed soils containing few living spores (e.g. Read et al. 1976; Read et al. 1985; Jasper et al. 1989a,b; Jasper et al. 1991a). Growing roots are sensitive to VAM colonization for only a short time, and require rapid colonization for an effective association (Brundrett 1991). The external hyphal network provides an extensive source of potentially infective propagules for actively growing roots to intercept.

Jasper et al. (1989a) argued that if the network of hyphae in undisturbed soils is an important inoculum, then observed losses in infectivity after soil disturbance are likely to be because of damage to this network, rather than by damage to the relatively robust structures of spores and remnant root fragments colonized by VAM fungi. The external hyphal network could be disturbed in two ways: (i) the hyphae may be separated from their host roots, and/or (ii) the hyphae may be physically broken up. VAM fungi are considered to be obligate symbionts in that they are dependent upon their host for an organic carbon supply (Harley & Smith 1983; Abbot & Robson 1991). However, it has 47 been demonstrated that the external hyphae of VAM fungi can remain infective even after being separated from their host (e.g. Hepper & Warner 1983; Jasper et al. 1989a). This suggests that physical disruption of the hyphae rather than separation from the host root, may be the cause of the loss of infectivity in disturbed soils.

It has been well documented that soil disturbance can reduce the infectivity of VAM fungi

(e.g. Jasper et al. 1987, 1988; Evans & Miller 1988, 1990; Fairchild & Miller 1988;

Jasper et al. 1989a,b,c; McGonigle et al. 1990; Jasper et al. 1991a). In these previous studies, only two soil disturbance treatments were examined, namely none v. severely disturbed. I investigated the relationship between the intensity of soil disturbance and the infectivity of VAM fungi (as per McGonigle et al. 1990). Additionally, it has been proposed, that if VAM infection is beneficial to plants, and if this benefit is derived from increased nutrient and water uptake, then removal or reduction of VAM infection should result in decreased nutrient uptake, especially in soils with low concentrations of essential plant nutrients (Fitter 1986). The reduction in nutrient uptake could in turn be reflected in a reduction in plant growth. Consequendy, shoot dry mass was measured to assess the impact of any soil disturbance-induced reduction of VAM on the growth of the bioassay seedlings.

5.2 MATERIALS & METHODS

5.2.1 Design of the experiment

Studies described in the previous chapter showed that most of the propagules which may initiate VAM were observed in topsoil. Consequendy, the work described in this chapter concentrated on the 0-15 cm part of the intact soil profile.

At each of the two sites, five different plots were selected at random, and five intact soil blocks (20 x 20 x 15 cm) were taken at each plot. These intact blocks were placed into square plastic containers and removed to the glasshouse. For each plot, two of the

containers were left undisturbed (No dist.), one was divided longitudinally into four

equal portions (Dist. 1), another into nine equal portions (Dist. 2) and the last into 25 equal portions (Dist. 3) (Fig. 5.1).

At least 30 seeds of Trifolium repens L. were sown into each container. All containers

were tap-watered daily, and received no additional nutrients. To test for potential aerial

contamination of containers by VAM fungi in the glasshouse, five containers of river

sand sown with 30 seeds of T. repens were used as a control. All pots were placed in a

naturally lit glasshouse, in which the mean daily temperatures ranged between 20.0°C and

28.7°C for the duration of the bioassay. At 14, 21, 28, 35, and 42 days after planting,

five seedlings were randomly selected from each container, and carefully removed.

Although the removal of bioassay seedlings caused some soil disturbance, less than 5%

of each soil block was disturbed at each sampling occasion. In addition, each soil block

was treated in the same way, and so, the disturbance attributed to the harvesting of the

seedlings was not considered to be a confounding variable. The roots of each seedling

were gentiy washed in a 0.4% sodium hexametaphosphate solution to remove any

adhering soil. Roots were then excised and fixed in 50% ethanol, cleared and stained

(Kormanick et al. 1980), and total root length and root length colonized by VAM fungi

quantified (Ambler & Young 1977). The length of root colonized by VAM fungi (i.e.

VAM length) is a composite index: the sum of the length of root colonized by; vesicles, arbuscules and hyphae (NB. % VAM = VAM length/total root length x 100). The shoots were dried in a 70°C oven for four hours and weighed.

5.2.2 Statistical analysis of results

The data were analyzed by a "split-plot" Analysis of Variance (Cochran & Cox 1957) with seedlings nested within blocks, which were nested within plots. The analysis assumed a factorial relationship between the degree of disturbance and the elapsed time 49

Figure 5.1 Schematic representation of the increasing degrees of topsoil disturbance. 50

until harvesting of the seedlings, since each degree of disturbance occurred in conjunction with each harvest date.

Comparisons among harvest dates are "within container" comparisons, so they are usually more precise than comparisons between different degrees of disturbance, which are "between container" comparisons (and involve more sources of variability). As well, comparisons between degree of disturbance/harvest date combinations (e.g. No dist./21 days v. Dist. 3/35 days) vary in precision depending on whether they have the same degree of disturbance (and are therefore "within container" comparisons) or have different degrees of disturbance (and are therefore "between container" comparisons). A consequence of this is that the Analysis of Variance involves more than one Residual SS, and pairwise comparisons between two means will have different estimates of error depending on the particular comparison.

5.3 RESULTS

5.3.1 Root growth and VAM formation

No VAM was found on the roots of any of the bioassay seedlings grown in the control containers testing for aerial contamination by VAM fungi. The length of roots was not affected by soil disturbance. The trend observed in the length of root colonized by VAM fungi (i.e. VAM length) and the proportion of root length colonized by VAM fungi (i.e.

%VAM) were identical. Consequently, only the proportion data are presented here. The trends observed in the two "No dist." treatments were identical, so the data were grouped. Consequently, there were 10 soil blocks in the "No dist." treatment.

In the undisturbed (No dist.), Dist. 1 and Dist. 2 treatments, VAM formation had commenced by 14 days (Fig. 5.2). In the most disturbed of the soil blocks (Dist. 3), the onset of VAM formation was delayed by between 28 and 35 days in the Avon soil and (a) 40"!

No Dist. Dist. 1 Dist. 2 Dist. 3

(b) • 14 days 30 I E2 21 days M 28 days E3 35 days 20" • 42 days

10"

No Dist. Dist. 1 Dist. 2 Dist. 3 Increasing degrees of disturbance

Percentage of root length colonized by VAM fungi in relation to increasing degrees of soil disturbance using intact soil blocks removed from (a) the Avon and (b) the O'Hares study sites. Each bar is the mean (± s.e.) of five seedlings from each of five replicates except the "No dist." treatment which is the mean (± s.e.) of five seedlings from each of 10 replicates. 52

between 35 and 42 days in the O'Hares soil (Fig. 5.2). Furthermore, even when

colonization commenced in the seedlings growing in the most disturbed blocks, the levels

of VAM were significantly lower than the "No dist." treatment at all stages of the experiment (Fig. 5.2; Appendix IVa).

The low (i.e. Dist. 1) and intermediate (i.e. Dist. 2) degrees of soil disturbance had no effect on the proportion of root colonized by VAM fungi up to day 14 (Fig. 5.2).

However, at 21 days, the low and intermediate degrees of soil disturbance had

significantly reduced the proportion of root length colonized by VAM fungi in both the Avon and O'Hares soils (Fig. 5.2; Appendix IVa).

5.3.2 Shoot dry mass

The low and intermediate degrees of soil disturbance had no impact upon shoot mass. In the Avon soil, Dist. 3 had no affect on shoot mass for the first three weeks. After 21 days, shoot mass was significantly lower in the seedlings growing in the most disturbed soil blocks (Fig. 5.3; Appendix IVb). In the O'Hares soil, this level of disturbance had no impact on shoot mass until five weeks after sowing. After 28 days, shoot mass was significantly lower in the seedlings growing in the most disturbed soil blocks than the undisturbed blocks (Fig. 5.3; Appendix IVb).

5.4 DISCUSSION

A number of authors have suggested that propagules of VAM fungi including infected root fragments (Rives et al. 1980), spores (Gould & Liberta 1981; Jasper et al. 1987,

1988), or hyphal fragments (Jasper et al. 1989a), lose their capacity to initiate infection during soil disturbances because they are (i) physically damaged, and/or (ii) are exposed to post-disturbance soil conditions which are unfavourable for germination or colonization (Stahl et al. 1988). The relative importance of each of these two mechanisms (a)

No Dist. Dist. 1 Dist. 2 Dist. 3

No Dist. Dist. 1 Dist 2 Dist. 3 Increasing degrees of disturbance

Figure 5.3 Shoot dry mass accumulation in relation to increasing degrees of soil disturbance using intact soil blocks removed from (a) the Avon and (b) O'Hares study sites. Each bar is the mean (± s.e.) of five seedlings from each of five replicates except the "No dist." treatment which is the mean (± s.e.) of five seedlings from each of 10 replicates. 54

has not been fully established and may vary in different situations (Brundrett 1991).

Cutting the soil blocks into twentyfifths in the experimental disturbance probably

disrupted the external hyphal network and the root fragments (containing hyphae and

vesicles). This would in turn temporarily reduce the infective potential of the fungus to

nil. This explanation is supported to some extent by the findings of Fairchild & Miller

(1988) and Jasper et al. (1989a,b), who found that disruption ofthe external soil hyphal

network resulting from severe soil disturbance was associated with a reduction in the

infectivity of VAM fungi. The observed delay in the initiation of VAM infection in seedlings growing in the most disturbed blocks may be explained by the time required for

hyphae to grow from propagules in the soil, such as spores and root fragments colonized by VAM fungi.

Evans & Miller (1990) and McGonigle et al. (1990) concluded that, in agricultural soils, disruption of the external hyphal network can reduce nutrient uptake by plants colonized

by VAM fungi in a way that is independent of the amount of root length colonized by VAM fungi. This conclusion is in contrast to the results of my study, in which the most

severe experimental disturbance was associated with a marked decrease in the amount of

root length colonized by VAM fungi. However, the seeming disparity between the

studies may be explained by differences in the number of surviving propagules. Jasper et al. (1991a) demonstrated that colonization of roots by VAM fungi was not decreased after soil from an annual pasture was disturbed. In contrast, in both a forest soil and a heathland soil, the percentage root length colonized by VAM fungi was almost halved if the soils had been disturbed. In the pasture soil, up to 25 times more propagules (i.e. spores and colonized root fragments) survived disturbance than in the forest or heathland soil. Jasper et al. (1991a) concluded that the larger number of surviving propagules in the pasture soil was responsible for the maintenance of the high level of infectivity. The sandstone soils in my study are characterized by very low densities of viable spores

(Chapter 4). This suggests that in these intact soils, the external hyphal network may be the main source of VAM infection. Furthermore, it would appear that if the hyphal 55

network is fragmented in these soils, then the infectivity of VAM fungi decreases dramatically.

The low and intermediate degrees of soil disturbance did not delay the onset of VAM, and had no effect on either the length and proportion of root colonized by VAM for the first

14 days of the bioassay. After this time, however, both the length and the proportion of root colonized by VAM were significantly lower than for the seedlings growing in the intact soil blocks. However, depression in VAM colonization at 21-28 days, may not have any biological significance, because the reduction in VAM colonization over this period was not associated with any reduction in shoot dry mass for seedlings growing in these soil disturbance treatments.

In contrast, the most severe disturbance caused both a delay in the initiation of VAM

infection and also a reduction in shoot biomass of 21-28 day old seedlings of T. repens.

The results of my study reinforce the findings of earlier studies which have demonstrated

that seedlings with a higher proportion of root length colonized by VAM fungi have

greater shoot dry mass than seedlings of the same species with less VAM colonization

(e.g. Evans & Miller 1988; Fairchild & Miller 1988; Jasper et al. 1989c).

Overall, cutting the intact soil blocks longitudinally into four and nine equal portions did

not affect the infectivity of VAM fungi to any great extent. However, cutting the soil

block into 25 equal portions temporarily reduced the infective potential of VAM fungi to

nil. The most severe experimental disturbance described here is considerably more subde

than the usual "bulldozer" facilitated disturbances carried out in the catchments. It is

considered that the scraping, removal and storage of topsoil would have at least the same

impact upon the infectivity of VAM fungi as the most severe experimental disturbance

described here. 56

Chapter 6

THE PROPAGULES CAPABLE OF INITIATING VAM AFTER TOPSOIL DISTURBANCE

6.1 INTRODUCTION & AIMS

From the previous chapter, it was observed that soil disturbance reduces the infectivity of

VAM fungi. The propagules of VAM fungi can consist of blastospores, chlamydospores

or azygospores (resting spores), soil-borne vesicles and mycelia or colonized root fragments containing hyphae and vesicles (Daniels & Skipper 1982). These propagules have been shown to have different susceptibilities to both the direct impacts of topsoil disturbance, and also to the associated changes in the soil environment (e.g. Stahl et al.

1988; Jasper et al. 1989a). The viability of each of these propagules after soil disturbance will determine in part the number of infective propagules available to initiate VAM with plants re-colonizing the disturbed site.

The soil-borne spores of VAM fungi have been considered by many to be the most important type of propagule of VAM fungi (Brundrett 1991). However, soils in a range of ecosystems often contain low numbers of living spores (e.g. Read et al. 1976; Janos

1980; Gay et al. 1982; Visser et al. 1984; Brundrett & Kendrick 1988; Bellgard "Chapter

4"). Some species of VAM fungi apparently do not even produce spores (e.g. Johnson

1977; McGee 1989). While successful germination of VAM spores is dependant upon interactions with a range of soil and environmental factors (e.g. Slankis 1974; Schenck et al. 1975; Black & Tinker 1979; Daniels & Trappe 1980; Tommerup 1983a, 1984; McGee

1989), living spores of VAM fungi will not function as propagules if they are quiescent

(Tommerup 1983b, 1985). Additionally, because of their lipid content and thick walls, blastospores of VAM fungi are considered to be more resistant to adverse environmental conditions than other VAM propagules (Daniels Hetrick 1984; Abbott & Robson 1991). Tommerup & Kidby (1979) demonstrated that some species of Glomus and Gigaspora

can remain infective after lyophilization. In comparison, Scutellospora calospora did not

recover after one wet/dry cycle (McGee 1989). Consequently, it is reasonable to

conclude that the importance of spores as a source of inoculum varies between sites, and

is dependant upon a range of variables including the species of endophyte, the abundance

of the endophyte, and the local soil and environmental conditions.

Non-spore propagules, such as roots colonized by VAM fungi (containing hyphae and

vesicles), can initiate VAM, provided they are in close proximity to an actively growing

root (e.g. Rives etal. 1980; McGee 1987). Tommerup & Abbott (1981) demonstrated that root pieces colonized by several VAM fungi, retained their infective potential even

when stored in dry soil at -50 MPa. It is not known whether storage under moisture

conditions more conducive to root decomposition or to root desiccation might result in

loss of viability for hyphal fragments that are contained in root pieces (Daniels Hetrick 1984; Miller 1987). Thus, the ability of colonized root fragments to function as

propagules of infection may vary between species of VAM fungi, and upon the specific

site and storage conditions (Nemec 1987; McGee 1989).

Plant roots infected with VAM fungi carry a loose hyphal network which extends into the

surrounding soil, providing an extensive surface area for absorption of nutrients and a mechanism by which infection can be spread (Warner & Mosse 1983; Newman 1988).

As a source of infection, the external hyphal network is considered to be particularly significant in undisturbed soils which contain low densities of living spores (e.g. Read et al. 1976; Read et al. 1985; Jasper et al. 1989a). It is clear that the hyphal network can colonize host roots more efficiently than spore inoculum (e.g. Powell 1976; Hall 1976,

1979; Abbott & Robson 1981), however, the hyphal network may be more susceptible to the impacts of soil disturbance than the more robust forms of inoculum, viz., spores and colonized root fragments (Jasper et al. 1989a). 58

From the previous chapter, it was demonstrated that colonization of roots by VAM fungi was delayed by up to six weeks in the roots of seedlings grown in soil blocks which were cut longitudinally into 25 equal sized portions. It was concluded that severe soil fragmentation probably disrupted the external hyphal network and colonized root fragments, thus delaying root infection. The observed delay in the initiation of VAM in the seedlings growing in the most severely disturbed soil blocks was explained by the time required for hyphae to grow to bioassay roots from other propagules remaining in the soil blocks. On the basis of these studies, it was not possible to determine which of the propagules of VAM fungi were responsible for initiating VAM after soil disturbance.

The aim of this study was to identify which propagules of VAM fungi are capable of initiating VAM after topsoil disturbance.

This study was carried out in the Avon site only, because a wildfire burned the O'Hares site in December of 1990.

6.2 MATERIALS & METHODS

6.2.1 Experimental design

From within the site, five different plots were selected at random, and three intact soil blocks (20 x 20 x 15 cm) were taken from each plot. These blocks were placed in square, six litre plastic containers. For each plot, two of the containers were divided longitudinally into 25 equal portions (see Fig. 5.1) and the last was left undisturbed.

6.2.2 Part I - VAM formation in undisturbed v. disturbed soil blocks

Forty seeds of Trifolium repens L. were sown into one of the disturbed soil blocks and the undisturbed block from each of the plots. The remaining disturbed soil block from each couplet was used in Part II of the experiment (Section 6.2.3). The undisturbed block was assayed to ensure that the soil blocks sampled were potentially infective. To

monitor for potential aerial contamination in the glasshouse by VAM fungi, five pots of

washedriver sand sown with 20 seeds of T. repens were used as a control. At each of

14, 28 and 42 days after sowing, ten seedlings were randomly extracted from each

container. The roots of each seedling were washed in a 0.4% sodium hexametaphosphate

solution to remove any adhering soil. Roots were fixed in 50% ethanol, cleared and "

stained (Kormanick et al. 1980), and the total root length, and portion of root length

colonized by VAM fungi measured (Ambler & Young 1977). The length of root length

colonized by VAM fungi (i.e. VAM length) is composite index, the sum of the length of

root colonized by; vesicles, arbuscules, and internal hyphae (NB. % VAM = VAM length/total root length x 100).

6.2.3 Part II - Isolation of VAM inoculum fractions and examination of potential infectivity

The five remaining disturbed soil blocks were separated into their constituent 25 soil

columns. Five columns of the 25 were randomly selected from each block and were

individually washed through a tier of three sieves; (i) 1 mm, (ii) 250 pm, and (iii) 106 pm.

The material caught on the 1 mm, 250, and 106 pm sieves were sprayed with a strong jet

of water in an attempt to remove any hyphae adhering to the root fragments. Cursory

examination of a randomly selected sub-sample of root fragments, confirmed that this

technique successfully removed the majority of adhering soil hyphae from the root

surface. Thesefractions were examined under a dissecting microscope and divided into root fragments and fungal hyphae. The rootfragment fraction and the hyphal fraction from each of the three sieves were bulked. 60

The spore fraction on the 106 pm sieve was not separated from the fine soil, and was termed the "spore/soil" fraction. The fraction caught in a collecting vessel below the tier of three sieves (i.e. <106 pm), was termed the "fines" fraction. The four fractions; root fragments, hyphal fragments, spore/soil, and fines were each tested for potential infectivity.

1. Root fragments

The ability of hyphae to grow from root fragments was tested using a "membrane filter" technique (Tommerup & Kidby 1979). This involves sandwiching at least five root pieces between a pair of 0.45 pm membrane filters (Millipore Corp.) which are inserted between 1 cm layers of steamed river sand. The steamed sand was wetted to field capacity and incubated at 22°C. Three "blanks" containing a clean pair of membrane filters sandwiched between steamed sand were used as a control to monitor for potential contamination. Twelve randomly chosen samples of root fragments (each sample containing five root fragments between 1 and 6 mm long) were selected from each of the soil columns. Four randomly selected filter-sandwiches from each soil column were examined 14, 28, and 42 days after being started. Each pair of filters was recovered and the root fragments stained in place on the filters (Tommerup & Kidby 1979) with 0.01% acid fuchsin (Kormanick et al. 1980).

2. Hyphal fragments

The viability of hyphal fragments was tested using the "soil-funnel" technique (Menge &

Timmer 1982). Here, seedlings are forced to grow in close proximity to VAM fungal inoculum, thus optimizing the chance of initiating an association. At least ten hyphal fragments were used as inoculum in each apparatus. Six randomly chosen samples of

VAM fungal hyphae (each sample containing at least 10 hyphal pieces, 3-6 mm long) were chosen from each soil column. Ten seeds of T. repens were sown into each funnel- 61

setup. These were placed in a naturally lit glasshouse and watered daily with no nutrients applied. The diurnal temperature range in the glasshouse for the duration of this experiment, was between 18.4°C and 25.4°C. To monitor for potential aerial contamination in the glasshouse by VAM fungi, five funnel-setups with 10 seeds of T. repens and no VAM hyphae were used as a control. Plants from two entire funnel-setups from each soil column were harvested 14, 28, and 42 days after sowing. Plants were removed from the funnels and their roots washed in a 0.4 % sodium hexametaphosphate solution to remove any adhering soil. The roots were fixed in 50% ethanol, cleared and stained and the proportion of root length colonized by VAM fungi was quantified.

3. Spore/soil and fines fraction

The ability of the spore/soil and the fines fraction to initiate VAM infection was tested by bioassay. The spore/soil and fines fractions were put into 10 cm plastic pots; on gravel to prevent the fine soil from washing through. Twenty T. repens seeds were sown into the fractions. The pots were placed in the same naturally lit glasshouse as described in the previous section, watered daily, with no nutrients applied. To monitor for potential aerial contamination in the glasshouse by VAM fungi, another five pots of river sand sown with

20 seeds of T. repens were used as a control. Five plants were harvested from each pot

14, 28, and 42 days after being sowed. The plants were removed from the pots and their roots washed in a 0.4% sodium hexametaphosphate solution to remove any adhering soil.

The roots were fixed in 50% ethanol, cleared and stained and the proportion of root length colonized by VAM fungi was quantified.

6.2.4 Statistical analysis

A chi-square statistic (Zar 1984) was used to determine if the number of seedlings colonized by VAM at day 42 was different between the intact and disturbed soil treatments. An unpaired, two-tailed t-test (Zar 1984) was used to determine if the mean proportion of root length colonized by VAM fungi after 42 days differed when grown in intact and disturbed soil blocks.

The data generated by the growth of hyphae from root fragments was either "success"

(i.e. hyphae grew from the rootfragments) o r "failure" (i.e. no hyphae grew from the rootfragments). Chi-squar e contingency tables were used to determine whether the frequencies of "successes" v. "failures" were independent of sampling dates.

6.3 RESULTS

6.3.1 VAM formation in intact v. disturbed soil blocks

No VAM were found in any of the controls monitoring for glasshouse contamination at any of the harvests. VAM formation had commenced by 14 days in the intact soil blocks.

VAM formation commenced in the roots of seedlings growing in the disturbed soil blocks after 42 days. At 42 days, the number of seedlings colonized by VAM in the intact soil blocks was significandy higher than those seedlings growing in the disturbed soil blocks (X2 = 36.97, P < 0.0001). Additionally, a higher proportion of total root length of plants from undisturbed soil (20%) than disturbed soil (7%) were mycorrhizal (t = 9.86, P <

0.001).

6.3.2 Root fragments

Hyphae had grown from the root fragments after 14 days. In all cases, the hyphae produced were straight, thick-walled, aseptate, 4-8 pm, and did not branch. In four out of the five soil blocks sampled, hyphae had grown from a similar number of root fragments at each harvest (Table 6.1). 63

Table 6.1 Chi-square analysis of root fragments extracted from soil blocks taken from the Avon study site Data represent the number of root fragments out of 100from which hyphae were produced.

Time (days since start of assay) 14 28 42

Block 1 Hyphae produced 56 47 68 No hyphae produced 44 53 32 X2 = 9.06, P =•• 0.011 R Block 2 Hyphae produced 46 54 63 No hyphae produced 54 46 37 X2 = 5.83, P =• 0.054 A Block 3 Hyphae produced 52 51 60 No hyphae produced 48 49 40 X2 = 1-96, P = 0.375A Block 4 Hyphae produced 51 48 53 No hyphae produced 49 52 47 X2 = 0.51,P = 0.776A Block 5 Hyphae produced 52 48 56 No hyphae produced 48 52 44 X2 = 1-28, P = 0.527A

NB.R = Reject null hypothesis andA = Accept null hypothesis.

6.3.3 Hyphal fragments

Soil collectedfrom the Avon site contained 13.0±1.5 m of fungal hyphae per gram of soil at field capacity (meanls.e., n=25). This estimate does not include the hyphal fragments smaller than 106 pm because these went through the sieve and were mixed in with the fine soil particulate matter. 64

No VAM was found in the control funnels used to monitor for potential aerial

contamination. At each of the three sampling dates, the hyphal fragments failed to produce any VAM in the roots of the bioassay plants.

6.3.4 Spore/soil and fines fraction

No VAM was found in the controls at any of the sampling times. The spore fraction

produced VAM infection at the 42 day harvest. At 42 days, 53 of the 150 seedlings

sampled were colonized by VAM. The mean proportion of root length colonized by VAM fungi was 4.85±0.62% (meanls.e., n=150).

VAM were not formed in the fines fraction in any of the three sampling occasions.

6.4 DISCUSSION

Soil disturbance can reduce the infective potential of VAM fungi in several ways: (i)

propagules may be physically damaged, i.e. spores may be crushed, and/or the soil

hyphal network and colonized root fragments may be disrupted (Jasper et al. 1989a;

Evans & Miller 1990), (ii) disturbance may alter the physical, chemical, or biological

environment of the soil, which in turn prevents the colonization by or germination of

VAM propagules (Warner 1983; Stahl et al. 1988), and/or (iii) disturbance may eliminate

host plants, leading to changes in the carbon supply available to the fungus (Abbott &

Robson 1991). Brundrett (1991) commented that the relative importance of these

mechanisms has not yet been fully established. Additionally, individual fungal species

may exhibit different responses to both the direct and indirect impacts of soil disturbance

depending upon their own specific host and environmental requirements.

Seedlings growing in the undisturbed topsoil blocks of the first part of the experiment, were rapidly colonized by VAM fungi. This implies that VAM fungi inoculum levels in 65

this soil were high. Read et al. (1976) observed rapid VAM development in various

grassland, shrub and woodland species. The rapid colonization of roots was attributed to

seedling roots intercepting colonized roots of established plants rather than infection from

spores, which were found to occur in low numbers. In a similar way, the infections

which were observed in two-week old seedlings in the present study are believed to be

indicative of infection from roots colonized by VAM fungi and their associated hyphal

network.

Cutting the soil blocks longitudinally into 25 equal sized portions temporarily reduced the

infective potential of VAM fungi to nil. These results reinforce the findings of my previous chapter, in which colonization of roots by VAM fungi was delayed by up to six weeks for seedlings growing in soil blocks which were cut longitudinally into 25 equal portions. By the 42 day harvest, colonization of roots by VAM fungi had commenced.

The delay in the initiation of infection may be due to the time required for hyphae to grow from propagules in the soil which survived both the direct impacts of the disturbance and the associated changes to the soil environment

Root fragments produced hyphae after 14 days. Similarly, McGee (1987) demonstrated that outgrowth of VAM fungi from dried root pieces occurred by 14 days. The studies of

Powell (1976), Warner & Mosse (1980), Tommerup & Abbott (1981) and Biermann &

Linderman (1983), did not identify when hyphae first emerged from colonized root fragments. However, the hyphae produced from the colonized root fragments initiated

VAM infection in bioassay plants within 28-60 days. It is reasonable to conclude that outgrowths of hyphae from colonized root pieces may have been responsible in part for the initiation of infection in the disturbed soil blocks of Part I.

Hyphal fragments failed to initiate VAM infection even after 42 days. A number of authors have demonstrated that disruption of the soil hyphal network results in a decrease in the infectivity of VAM fungi (e.g. Fairchild & Miller 1988; Jasper et al. 1989a,b; 66

Evans & Miller 1990). However, these experimental protocols failed to differentiate

between physical damage to the hyphal network and indirect changes to the soil environment as a result of the disturbance. A number of studies have shown that soil conditions play a critical role in the growth of the hyphae of VAM fungi (e.g. Graham et al. 1982; Abbott et al. 1984; Abbott & Robson 1985). Consequently, the post- disturbance reduction in infectivity of the external hyphal network may be due in part to the redistribution of soil which may expose hyphal fragments to unfavourable conditions for germination and/or colonization.

Another factor which may have contributed to the hyphal fragments failing to produce infection is inoculum density. It has been demonstrated that increased inoculum dosage results in increased percentage root colonization (e.g. Daft & Nicolson 1969; Sanders &

Sheikh 1983; Wilson & Trinick 1983; Wilson 1984). The main effect of increasing the inoculum density appears to be to increase the rate of development of infection. It has been proposed that the increase in the rate of infection results from an increase in the rate of formation of primary points of infection (Wilson & Trinick 1983). It was not known how much hyphal inoculum was required to initiate VAM. Additionally, the hyphae may have included species other than VAM fungi, the hyphae may have been severely battered by the extraction and inoculation process, and even if the hyphae were extracted satisfactorily, not enough hyphae may have been used to initiate infection.

The spore fraction initiated VAM infection at the 42 day harvest. This coincided with the observed onset of VAM infection in the roots of bioassay seedlings growing in the disturbed soil blocks of Part I. It must be noted that Part I and II of the experiment were not running concurrently. However, the observed coincidental onset of VAM infection suggests that spores may have been responsible in part for the initiation of VAM infection in the disturbed soil blocks of Part I. 67

Both colonized root pieces and spores can be effective propagules initiating VAM in host plants after topsoil disturbance. Thus, the ability of VAM fungi to persist in soil after disturbance may depend partly on the type of propagules formed. Studies investigating the relationship between soil disturbance and the colonization and sporulation of VAM fungi species are required before we can fully understand the processes by which VAM fungi persist after topsoil disturbance and storage. 68

Chapter 7

CONCLUDING DISCUSSION

7.1 INTRODUCTION

The principal aim of the studies described in this thesis was to evaluate the potential for vesicular-arbuscular mycorrhizal (VAM) fungi to influence the recovery of disturbed

Hawkesbury Sandstone plant communities. In order to address this question, it was necessary to understand how common VAM were in plants and how VAM fungi were affected by soil disturbance in these plant communities. A number of questions have been addressed in this thesis. These questions and a brief summary of the contributions made by this study are presented in Table 7.1.

Table 7.1 Questions addressed in this thesis and a summary of the contributions made.

Question Contribution to question

Are mycorrhizae present in Yes. 66% of the plant species Hawkesbury Sandstone plant in each of the two sites examined communities? were mycorrhizal (Chapter 3). Where in the intact soil profile The 0-15 cm section of the intact are VAM fungi most infective? soil profile represents the major store of the potential propagules of VAM fungi (Chapter 4). 3. Does soil disturbance reduce Yes. The initiation of VAM the infectivity of VAM fungi? was delayed by up to six weeks (Chapter 5). 4. Which propagules of VAM fungi Hyphae of VAM fungi grew from are capable of initiating VAM root fragments after 14 days. VAM infection following disturbance? spores initiated infection after 28 days. Hyphal fragments failed to initiate infection after 42 days (Chapter 6). 69

7.2 THE SIGNIFICANCE OF VAM FUNGI IN HAWKESBURY SANDSTONE PLANT COMMUNITIES

Plants colonized by VAM fungi have higher tissue levels of some inorganic nutrients, greater biomass yield, and more rapid uptake of water, and they are often more tolerant of various forms of stress than non-mycorrhizal plants of the same species (Harley & Smith

1983). Within the examples of Hawkesbury Sandstone vegetation I examined, approximately half of the plant species were colonized by VAM fungi (Table 3.1). The fact that the proportion of plants with VAM was consistent in both plant communities examined is of particular significance. Although the two sites had 14 plant species in common, the sites were at different elevations, on different soils, supported vegetation of markedly different physiognomies, and were approximately 46 km apart. The consistency in the proportion of species possessing VAM in the plant communities examined suggests some spatial generality in the distribution of VAM fungal species in

Hawkesbury Sandstone soils and vegetation.

The soils in the current study are low in essential plant nutrients, especially extractable phosphorus (see Table 2.2). Previous surveys examining the mycorrhizal status of plant species inhabiting a range of low-nutrient soils, have also revealed that a majority of the plant species possess VAM (see Section 3.3). Consequendy, the prevalence of VAM associations in the current study may be a response to the low nutrient content of

Hawkesbury Sandstone soils. It seems reasonable to expect that other plant communities in New South Wales inhabiting soils of low-nutrient status e.g. the mallee belt region

(western N.S.W.), the mulga region (northwestern N.S.W.), and the alpine grasslands/shrublands of the Snowy Mountain National Park (A.L. O'Neill pers. comm.), may also be characterized by a majority of plant species possessing VAM.

Species of Dillwynia, Mirbelia, Pultenaea (Family: Fabaceae), Dampiera (Family:

Goodeniaceae), and Acacia (Family: Mimosaceae), were found to possess both VAM 70

and Ectomycorrhizae (see Appendix II). Additionally, species of Mirbelia, Pultenaea and

Acacia also possessed nitrogen fixing nodules. The formation and maintenance of dual and tripartite associations may represent a major energy investment on the part of the plant. It seems likely that each of the associations provides some benefit under particular environmental conditions. The relative importance of VAM, Ectomycorrhizae and nitrogen fixing nodules to plants which can readily form all three associations could be examined by experiments which use one, both or all three sources of inoculum.

Cost:benefit analyses of each, both, or all three of these associations to host plants could be determined by growing the plants under varying levels of inoculum, soil and environmental conditions, and monitoring plant performance under each regime.

Plants with specialized nutrient uptake strategies often do not form VAM (Brundrett &

Abbott 1991). Within the Proteaceae, species of Banksia, Isopogon, Lambertia and

Lomatia did not have any VAM, while species of Conospermum Grevillea, Hakea,

Persoonia and Telopea possessed VAM. In the root samples of those species that did not form VAM, I found well developed proteoid roots. These were first described by Purnell

(1960), and are dense mats of intense, lateral root production formed in the surface soil and in pockets of humus-rich soil. The overall geometry and positioning of proteoid roots in the soil profile appear to make them ideal for enhancing absorption of poorly mobile ions from soil and organic matter and for uptake of nutrients from leachates of decomposing litter and leaves (Bowen 1981). Drosera pygmaea DC. (Family:

Droseraceae), also did not have VAM, but possesses stalked mucilaginous glands which serve to attract, capture, digest and absorb nutrients from insect prey (Lamont 1984).

It has been suggested that seasonal variation in root growth impinges directly upon mycorrhizal activity and may be substantial enough to change the apparent mycorrhizal status of plants (e.g. Allen 1983; Anderson et al. 1984; Giovannetti 1985; Brundrett &

Kendrick 1988). My survey was carried out only once, during 1989. Further studies documenting the phenology, root anatomy, and mycorrhizal colonization of the plants in 71

the two study sites would be valuable contributions to understanding the distribution,

ecology and activity of VAM fungi in Hawkesbury Sandstone plant communities.

Additionally, quantitative and structural aspects of the morphology of the VAM occurring

in members of the Cyperaceae and Proteaceae may reveal atypical morphologies diagnostic of these two families.

7.3 THE IMPACT OF SOIL DISTURBANCE ON VAM FUNGI AND THEIR PLANT HOSTS

It is now widely accepted that soil disturbance can reduce the infectivity of VAM fungi, and that removal or reduction of VAM may result in decreased nutrient uptake, especially

in soils of low concentrations of essential plant nutrients (Fitter 1988). Because VAM

fungi are very common and frequently beneficial to plant growth, they have recently

received some attention. Research into the function and significance of VAM fungi in

natural and disturbed ecosystems has been stimulated by the acceleration of severe land

disturbance resulting from population pressures, agriculture and the mining industry (Loree & Williams 1984).

In the current study, the topsoil represented the major store of the potential propagules of

VAM fungi. Because of the low density of living spores in the soil profile, mycorrhizal

infectivity may be dependant more heavily on infective mycorrhizal roots and possibly on

the presence of an intact mycelial network. Land disturbances usually involve

disturbance to the topsoil. I demonstrated that the colonization of roots was delayed by

up to six weeks for seedlings growing in disturbed topsoil blocks. Associated with this

delay in infection, the seedlings growing in the most disturbed topsoil blocks had

significantly less shoot dry mass than those growing in the intact topsoil blocks.

A number of authors have observed that the successful pioneers in disturbed sites are

non-mycorrhizal (e.g. Reeves etal. 1979; Miller 1979; Allen & Allen 1988). It has been 72

hypothesized that such plants are successful because they do not require a VAM

association (e.g. Marx 1975; Miller 1987). It appears that disturbance temporarily

reduces the infective potential of VAM fungi to nil. The reduced post-disturbance

infectivity of VAM fungi has serious implications for both the fungi and potential plant

hosts. VAM fungi may not be regenerating rapidly enough to keep pace with plant

regeneration. VAM fungi are biotrophic, and hence, colonization of roots is essential for

their continued occurrence in soils (Abbott & Robson 1991). Consequently, post-

disturbance hyphae growing from colonized root fragments and/or spores need to

intercept actively growing roots to produce an effective association, and thus in turn,

perpetuate the fungus. If contact with host roots does not occur when the hyphae are

infective, then the fungus will perish. The death of the VAM fungal propagules will

cause a rapid decline in the infective potential of the soil, and reduce the possibility of any

future associations being initiated. Re-colonizing plants may be disadvantaged, because

they will be forced to grow without their associated VAM symbionts.

The autecology of the plant species comprising Hawkesbury Sandstone plant

communities may well exacerbate the situation I have propounded in the preceding

scenario. A number of the important perennial plant species are very slow growing, and

more importantiy, they require highly specific cues to initiate and facilitate seed release,

and germination. As a consequence, the delay in the onset of VAM infection I identified

from my study (see Chapter 5) may not even be an issue in the field, because there may

not be a viable seed resource available. Studies investigating the attenuation of mycorrhizal infectivity in topsoil stockpiles have shown that infectivity declines

substantially after the initial disturbance. After 10 to 12 weeks, the infectivity of VAM fungi in Hawkesbury Sandstone soils may have declined appreciably (I have not tested this as such, but it offers an interesting avenue of research). Seedlings may start to appear after 12 weeks (depending upon season). If, in the ensuingtime, th e infectivity of

VAM fungi has declined appreciably, then seedlings which were usually mycorrhizal in the undisturbedfield situation , may be forced to grow with less VAM (or no VAM). This in turn may inhibit establishment. These seedlings may be less tolerant of the post- disturbance soil environment, and may eventually be eliminated

Topsoil removal and storage are a common precursor in today's site development

practices. Although a number of authors have demonstrated that prolonged storage of

topsoil greatiy reduces its infectivity (e.g. Rives et al. 1980; Gould & Liberta 1981;

Liberta 1981; Warner 1983; Miller et al. 1985), no viable alternatives have been

proposed. It may be envisaged that VAM fungal propagules in stripped/stored topsoil

will be without their living plant hosts. Being separated from their living hosts will mean

that the fungi will "die-off* and the infective potential of the soil will be gready reduced.

The sowing of an appropriate "nurse crop" over the stockpile would provide infective

VAM fungal propagules with a living host. In this way, at least the surface layers of the

topsoil will retain some degree of mycorrhizal infectivity. Further research may also

reveal that stockpiling conditions, i.e. dry v. wet / shallow v. deep, could play an

important part in the preservation of mycorrhizal infectivity (e.g. Abdul-Kareem & McRae 1984; Harris et al. 1987).

Finally, VAM fungi contained in returned topsoil may not be suited to the physical and

chemical edaphic conditions of the disturbed surface. Mosse et al. (1981) and Stahl et al.

(1988) suggested that the endophytes at the site before disturbance may not produce

effective mycorrhizae in the disturbed soil, due to the altered post-disturbance soil

conditions. Experimentation investigating the suitability and adaptability of VAM fungal

strains to a variety of post-disturbance edaphic regimes is also essential.

7.4 VAM FUNGI AND REVEGETATION OF DISTURBED LANDSCAPES

Research into the potential for VAM fungi to influence revegetation commenced in 1967, when Nicolson (1967) suggested that plant growth could be improved by incorporating 74

VAM fungi into soil. Since then, Daft & Nicolson (1974), Daft et al. (1975), and Daft &

Hacskaylo (1976), observed extensive infection of most plants colonizing coal wastes, and proposed that colonization of plant roots by VAM fungi was essential for successful colonization and subsequent growth of plants. Inoculation experiments using VAM fungi and/or soil containing VAM fungi have demonstrated that the incorporation of the VAM symbiosis can improve the growth and survival of desirable revegetation species (e.g.

Cundell 1977; Daft & Hacskaylo 1977; Aldon 1978; Lambert & Cole 1980; Khan 1981; Jasper et al. 1989d).

Implementation of sound management practices can help to ensure successful development of mycorrhizae after disturbance. Stockpiling and storage of topsoil should be imnimized to help retain soil fertility (e.g. Powell 1980), plant propagule viability (e.g.

Stark & Redente 1987), as well as populations of mycorrhizal fungi (e.g. Rives et al. 1980; Gould & Liberta 1981; Liberta 1981; Warner 1983; Miller et al. 1985). The findings of Chapter 6 suggested that relativelyfresh inoculu m retains its infectivity for at least six weeks. This supports the findings of previous work, and stresses the importance of the use of relatively new topsoil as a source of the inoculum of VAM fungi.

The first step in a management programme would be to re-contour the site. Careful consideration must be given to the final drainage pattern, degree of slope and elevation of the site. Topsoil removed from any subsequendy disturbed site should be direcdy spread onto the contoured areas of the initially disturbed site. Topsoil stockpiling can sometimes be avoided altogether by transferring salvaged soil directly onto a site currently undergoing reclamation (Williams & Stahl 1987). The addition of fresh topsoil to a disturbed site aims to ensure that a full suite of infective propagules of all the microbial symbionts, that were present in the soil before the disturbance event, are present after the disturbance event (Jasper et al. 1991b). Seeding of native species may follow, but this is dependent upon the state of the native soil seed bank. The second disturbed site can then be re-contoured in preparation for topsoiling from a site yet to be disturbed. 75

It was mentioned earlier that some authors have found that endophytes at the site before disturbance may not produce effective mycorrhizae in the disturbed soil, due to the altered post-disturbance soil conditions. Such results illustrate the importance of the selection of suitable, efficient strains of VAM fungi for re-introduction to post-disturbance soils. The fungi selected must be capable of widespread colonization of hosts roots in the soil to be revegetated (Powell 1982). While initial selection of suitable VAM fungal strains should be conducted under controlled conditions (i.e. in greenhouse and growth cabinets), field tests are essential to ensure that the selected endophytes are indeed beneficial to the host plants (Abbott & Robson 1982).

Consequently, research oriented towards reclamation should emphasize methods of maintaining inoculum levels in soil, as well as techniques for introducing suitable endophytes to disturbed sites (Allen & Allen 1980). However, if the removal of fresh topsoil to disturbed sites cannot be achieved, then topsoil storage seems inevitable. I have already suggested that a "nurse crop" may help perpetuate the infectivity of VAM fungi during topsoil storage. It may be envisaged that the re-spreading of topsoil after the storage period, again subjects the soil to disruption. Any or all of the mycelial network developed by residual VAM fungal propagules and the sown "nurse crop" within the stockpile, may be disrupted during the re-spreading procedure. So, in effect, we have another disturbance associated with re-spreading topsoil, and again, the problem of ensuring that VAM fungal propagules have actively growing roots to intercept

A mechanism must be developed which will delay the introduction of native seed until a mycelial network has been developed. To address this particular situation, another "nurse crop" may be sown. A sterile "nurse crop" should be utilized. For a topsoil stockpile, it is important to ensure that the stockpile is covered for the duration of the storage period to restrict soil erosion. Consequendy, sowing of a "nurse crop" and allowing it to freely seed and spread over the stockpile is not of any consequence. In fact, it may be actively encouraged to ensure a self-sustaining cover-crop develops. However, once the topsoil 76

has been re-spread over the disturbed area, it would not be ecologically responsible to

allow an exotic crop species to completely dominate the soil surface. The exotic crop may

prove to be an aggressive root-space competitor, and thus not give any other plant

species, especially native seed, the chance of getting established. In comparison, a sterile

"nurse crop" (e.g. barley or wheat treated with gamma radiation), would provide a quick-

growing crop which would cover the newly spread topsoil, and provide the necessary

living roots for the infective propagules of VAM fungi (contained within the re-spread

topsoil) to colonize. At the end of the growing season, the crop would die, and not

return. However, the mycelial network developed in association with the roots of the

sterile "nurse crop" would remain. At this stage of the protocol, seed of selected native

species could be sown. The native seedlings would have the opportunity to link into the

already established VAM mycelial network, and obtain the associated benefits of

increased nutrient and water uptake. Further research is required to determine, if an

established mycelial network developed during a previous plant growth cycle, can retain

the capacity to absorb or at least translocate nutrients to an establishing plant that has

become colonized by the mycelium, or, must a new mycelium be developed.

Serious problems in inoculum production and inoculation technology must be overcome

before commercial application of VAM fungi in the rehabilitation of disturbed lands can

take place (Loree & Williams 1984). Even if commercial production of inoculum of

VAM fungi is achieved, VAM fungi will not address all of the problems associated with

the reclamation of disturbed lands. VAM fungi are one important component of the

below-ground microbial population. I have shown that VAM fungi are adversely affected

by soil disturbance. Land managers must consider modifying their rehabilitation techniques, and aim to re-establish VAM fungal populations to disturbed soils. The presence of VAM fungi will help ensure the successful development of plant communities on disturbed Hawkesbury Sandstone sites. REFERENCES

Abbott, L.K. & Robson, A.D. (1977). The distribution and abundance of vesicular-

arbuscular endophytes in some Western Australian soils. Australian Journal of Botany, 25, 515-522.

Abbott, L.K. & Robson, A.D. (1981). Infectivity and effectiveness of vesicular-

arbuscular mycorrhizal fungi: effect of inoculum type. Australian Journal of

Agricultural Research, 32, 631-639.

Abbott, L.K. & Robson, A.D. (1982). The role of vesicular-arbuscular mycorrhizal

fungi in agriculture and the selection of fungi for inoculation. Australian Journal

of Agricultural Research, 33, 389-408.

Abbott, L.K. & Robson, A.D. (1984). The effect of VA mycorrhizae on plant growth.

VA Mycorrhizae (Ed. by C.L1. Powell & D.J. Bagyaraj), pp. 113-130. CRC

Press, Inc., Boca Raton, Florida.

Abbott, L.K & Robson, AD. (1985). Formation of external hyphae in soil by four

species of VAM fungi. New Phytologist, 99, 245-255.

Abbott, L.K. & Robson, A.D. (1991). Factors influencing the occurrence of vesicular-

arbuscular mycorrhizas. Agriculture, Ecosystems and Environment, 35,121-

150.

Abbott, L.K., Robson, A.D. & de Boer, G. (1984). The effect of phosphorus on the

formation of hyphae in soil by the vesicular-arbuscular mycorrhizal fungus,

Glomus fasciculatum. New Phytologist, 97, 437-446.

Abdul-Kareem, A.W. & McRae, S.G. (1984). The effects of topsoil of long-term storage

in stockpiles. Plant and Soil, 76, 357-363. Aldon, E.F. (1975). Endomycorrhizae enhance the survival and growth of fourwing

saltbush on coal spoils. USD A Forestry Service Research Note RM, 294,1-

2. 78

Aldon, E.F. (1978). Endomycorrhizae enhance shrub growth and survival on mine

spoils. The Reclamation of Disturbed Arid Lands (Ed. by R.A. Wright), pp.

174-179. University of New Mexico Press, Albuquerque.

Allen, E.B. & Allen, M.F. (1980). Natural re-establishment of vesicular-arbuscular

mycorrhizae following strip mining reclamation in Wyoming. Journal of Applied

Ecology, 17, 139-147.

Allen, E.B. & Allen, M.F. (1988). Facilitation of succession by nonmycotrophic

colonizer Salsola kali (Chenopodiaceae) on a harsh site: effects of mycorrhizal

fungi. American Journal of Botany, 75, 257-266.

Allen, M.F. (1982). Influence of vesicular-arbuscular mycorrhizae on water movement

through Bouteloua gracilis. New Phytologist, 91, 191-196.

Allen, M.F. (1983). Formation of vesicular-arbuscular mycorrhizae in Atriplex gardneri

(Chenopodiaceae): seasonal response to a cool desert. Mycologia, 75,773-776.

Allen, M.F., Smith, W.K., Moore, S.Jr. & Christensen, M. (1981). Comparitive water

relations and photosy thesis of mycorrhizal and non-mycorrhizal Bouteloua

gracilis HBK Lag ex Steud. New Phytologist, 88, 683-693.

Allen, S.E., Grimshaw, H.M., Parkinson, J.A. & Quarmby, C. (1974). Chemical

Analysis of Ecological Materials. Blackwells Scientific Publications, Oxford.

Allen, S.E., Grimshaw, H.M. & Rowland, A.P. (1986). Chemical analysis. Methods in

Plant Ecology (Ed. by P.D. Moore & S.B. Chapman), pp. 285-344. Blackwell

Scientific Publications, Oxford. Amaranthus, M.P. & Perry, D.A. (1987). Effect of soil transfer on ectomycorrhiza

formation and the survival and growth of conifer seedlings on old, nonreforested clear-cuts. Canadian Journal of Forest Research, 17,944-950.

Ambler, J.R. & Young, J.L. (1977). Techniques for determining root length infected by

vesicular-arbuscular mycorrhizae. Soil Science Society of America Journal, 41,

551-556 Anon. (1987). Agricultural Land Use and Selected Inputs, Australia. Australian Bureau

of Statistics, Canberra.

Anon. (1989). Australian Mining Industry Council. Canberra.

Anon. (1990a). Climatic Average Australia. Australian Government Publishing Service,

CanberraV

Anon. (1990b). Hazard Control Burning Schedules. Sydney Water Board, Sydney.

Ball, D.F. (1986). Site an(l\ils. Methods in Plant Ecology (Ed. by P.D. Moore &

S.B. Chapman), pp. 215-184. Blackwell Scientific Publications, Oxford.

Bartlett, E.M. & Lewis, H.D. (1973)/Surface phosphatase activity of mycorrhizal roots

of beech. Soil Biology and Biochemistry, 5, 249-257.

Beadle, N.C.W. (1954). Soil phosphate and thesdelimitation of plant communities in

Eastern Australia. Ecology, 35, 370-375.X

Beadle, N.C.W. (1962). Soil phosphate and the delimitation of plant communities in

Eastern Australia II. Ecology, 43, 281-288. \

Bellgard, S.E. (1991). Mycorrhizal associations of plant species in Hawkesbury

sandstone vegetation. Australian Journal of Botany, 39,^57-364.

Bethlenfalvay, G.J., Brown, M.S. & Pacovsky, R.S. (1982). Relationships between

host and endophyte development in mycorrhizas of soybeans. Nqw Phytologist,

90, 537-543. \

Bethlenfalvay, G.J., Dakessian, S. & Pacovsky, R.S. (1984). Mycorrhizae in\southern

California desert: ecological implications. Canadian Journal of Botany, 6^.519- 524. \

Bevenge, D.L, Bowen, G.D. & Skinner, M.F. (1975). Comparative carbohydrate \

physiology of ecto- and endomycorrhizas. Endomycorrhizas (Ed. by S.E.

Sanders, B. Mosse & P.B. Tinker), pp. 149-174. Academic Press, London.

Biermann, B. & Linderman, R.G. (1983). Use of vesicular-arbuscular mycorrhizal

roots, intraradial vesicles and extraradial vesicles as inoculum. New Phytologist,

95, 97-105.

Binkley, D. (1986). Forest Nutrition Management. John Wiley & Sons, New York. Anderson, R.C., Liberta, A.E. & Dickman, L.A. (1984). Interaction of vascular plants

and vesicular-arbuscular mycorrhizal fungi across a soil moisture-nutrient gradient. Oecologia, 64, 111-117.

Anon. (1983). Bushfire Contol Map for the Metropolitan Catchments. Water Board, Sydney.

Anon. (1987). Agricultural Land Use and Selected Inputs, Australia. Australian Bureau of Statistics, Canberra.

Anon. (1989). Australian Mining Industry Council. Canberra.

Anon. (1990a). Climatic Average Australia. Australian Government Publishing Service, Canberra.

Anon. (1990b). Hazard Control Burning Schedules. Sydney Water Board, Sydney.

Ball, D.F. (1986). Site and soils. Methods in Plant Ecology (Ed by P.D. Moore &

S.B. Chapman), pp. 215-284. Blackwell Scientific Publications, Oxford.

Baitlett, E.M. & Lewis, H.D. (1973). Surface phosphatase activity of mycorrhizal roots

of beech. Soil Biology and Biochemistry, 5, 249-257.

Beadle, N.C.W. (1954). Soil phosphate and the delimitation of plant communities in

Eastern Australia. Ecology, 35, 370-375.

Beadle, N.C.W. (1962), Soil phosphate and the delimitation of plant communities in

Eastern Australia II. Ecology, 43,281-288.

Bellgard, S.E. (1991). Mycorrhizal associations of plant species in Hawkesbury

sandstone vegetation. Australian Journal of Botany, 39, 357-364.

Bethlenfalvay, G.J., Brown, M.S. & Pacovsky, R.S. (1982). Relationships between

host and endophyte development in mycorrhizas of soybeans. New Phytologist,

90, 537-543.

Bethlenfalvay, G.J., Dakessian, S. & Pacovsky, R.S. (1984). Mycorrhizae in a southern

California desert: ecological implications. Canadian Journal of Botany, 62,519-

524. Bevege, D.L, Bowen, G.D. & Skinner, M.F. (1975). Comparitive carbohydrate

physiology of ecto- and endomycorrhizas. Endomycorrhizas (Ed. by S.E.

Sanders, B. Mosse & P.B. Tinker), pp. 149-174. Academic Press, London.

Biermann, B. & Lindeiman, R.G. (1983). Use of vesicular-arbuscular mycorrhizal

roots, intraradial vesicles and extraradial vesicles as inoculum. New Phytologist, 95, 97-105.

Binkley, D. (1986). Forest Nutrition Management. John Wiley & Sons, New York.

Black, R. & Tinker, P.B. (1979). The development of endomycorrhizal root systems.

n. Effect of agronomic factors and soil conditions on the development of

vesicular-arbuscular mycorrhizal infection in barley and on the endophyte spore density. New Phytologist, 83, 401-413.

Bowen, G.D. (1973). Mineral nutrition of ectomycorrhizae. Ectomycorrhizae: their

Ecology and Physiology (Ed. by G.D. Marks &T.T. Kozlowski), pp. 155-201. Academic Press, London.

Bowen, G.D. (1981). Coping with low nutrients. The Biology of Australian Plants

(Ed. by J.S. Pate & A.J. McComb), pp. 33-64. University of Western Australia Press, Nedlands, Western Australia.

Bowen, G.D. (1983a). Micro-organisms and tree growth. Research for Forest

Management (Ed. by J.J. Landsberg & J. Parsons), pp 180-201. CSIRO,

Canberra.

Bowen, G.D. (1983b). Microbial determinants of plant nutrient uptake. Soils: an

Australian Viewpoint, pp. 693-710. Academic Press Inc., London.

Bowen, G.D., Bevege, D.L & Mosse, B. (1975). Phosphate physiology of vesicular-

arbuscular mycorrhizas. Endomycorrhizas (Ed. by S.E.Sanders, B. Mosse &

P.B. Tinker), pp. 242-260. Academic Press, London.

Bowen, G.D., Skinner, M.F. & Bevenge, D.L (1974). Zinc uptale by mycorrhizal and

uninfected roots of Pinus radiata and Auracaria cunninghamii. Soil Biology and

Biochemistry, 6, 141-144. 81

Bradshaw, A.D. (1983). The reconstruction of ecosystems. Journal of Applied Ecology, 20, 1-17.

Branagan, D. (1985). An overview of the geology of the Sydney region. Engineering

Geology ofthe Sydney Region (Ed. by P.J. Pells), pp. 3-48. Balkema, Rotterdam.

Brockoff, J.O. & Allaway, W.G. (1989). Vesicular-arbuscular mycorrhizal fungi in

natural vegetation and sand-mined dunes at Bridge Hill, NSW. Wetlands, 8,47- 54.

Brownlee, C, Duddridge, J.A., Malibari, A. & Read, DJ. (1983). The structure and

function of mycelial systems of ectomycorrhizal roots with special reference to

their role in forming inter-plant connections and providing pathways for assimilate

and water transport. Plant and Soil, 71,433-443.

Brundrett, M.C. (1991). Mycorrhizas in natural ecosystems. Advances in Ecological

Research,!!, 171-313. Brundrett, M.C. & Kendrick, B. (1988). The mycorrhizal status, root anatomy, and

phenology of plants in a sugar maple forest. Canadian Journal of Botany, 66,

1153-1173. Brundrett, M.C. & Abbott, L.K. (1991). Roots of jarrah forest plants. I. Mycorrhizal

associations. Australian Journal of Botany, 39,445-457.

Buchholtz, K. & Motto, H. (1981). Abundance and vertical distributions of mycorrhizae

in Plains and Barrens forest soil from the New Jersey Pine Barrens. Bulletin of

the Torrey Botanical Club, 108, 268-271. Burrough, P.A., Brown, L. & Morris, E.C. (1977). Variations in vegetation and soil

patterns across the Hawkesbury Sandstone plateau from Barren Grounds to Fitzroy Falls, New South Wales. Australian Journal of Ecology, 2,137-159.

Cavanagh, T. (1987). Germination of hard-seeded species (order Fabales). Germination

of Australian Native Plant Seed (Ed. by P. Langkamp), pp. 58-201. Inkata Press,

Melbourne. 82

Clements, F.E. (1916). Plant Succession: an Analysis ofthe Development of Vegetation. Carnegie Institute, Washington.

Cochran, W.G. & Cox, G.M. (1957). Experimental Designs, 2 Ed. Wiley, New York.

Colwell, J.D. (1965). The estimation of phosphorus fertilizer requirements of wheat in

southern N.S.W. by soil analysis. Australian Journal of Experimental

Agriculture and Animal Husbandry, 3,190-197.

Cox, C. & Tinker, P.B. (1976). Translocation and transfer of nutrients in vesicular-

arbuscular mycorrhizas. I. The arbuscule and phosphorus transfer: a quatitative

and ulttastructural study. New Phytologist, 77, 371-378.

Cromack, KJr. (1985). Mycorrhizal fungi and nutrient mobilization. Proceedings ofthe

6th NACOM (Ed. by R. Molina), pp. 114-116. Forest Research Laboratory,

Oregon.

Cundell, A.M. (1977). The role of microorganisms in the revegetation of ship-mined

land in the western United States. Journal of Range Management, 30,299-305.

Daft, M.J. & Hacskaylo, E. (1976). Arbuscular mycorrhizas in the anthracite and

bituminous coal wastes of Pennsylvania. Journal of Applied Ecology, 13, 523-

528. Daft M.J. & Hacskaylo, E. (1977). Growth of endomycorrhizal and nonmycorrhizal red

maple seedlings in sand and anthracite spoil. Forest Science, 23,207-216.

Daft, M.J. & Nicolson, T.H. (1969). Effect of Endogone mycorrhiza on plant growth.

HI. Influence of inoculum concentration on growth and infection in tomato. New

Phytologist, 68, 953-958 Daft M.J. & Nicolson, T.H. (1974). Arbuscular mycorrhizas in plants colonizing coal

wastes in Scotiand. New Phytologist, 73, 1129-1138.

Daft, M.J., Hacskaylo, E. & Nicolson, T.H. (1975). Arbuscular mycorrhizas in plants

colonizing coal spoils in Scotiand and Pennsylvania. Endomycorrhizas (Ed. by

F.E. Sanders, B. Mosse & P.B. Tinker), pp. 561-580. Academic Press,

London. 83

Daniels Hetrick, B.A. (1984). Ecology of VA mycoiThizal fungi. VA Mycorrhiza (Ed

by CLL Powell & D.J. Bagyaraj), pp. 35-55. CRC Press, Inc., Boca Raton, Florida.

Daniels, B.A. & Skipper, H.D. (1982). Methods for the recovery and quantitative

estimation of propagules from soil. Methods and Principles of Mycorrhizal

Research (Ed by N.C. Schenck), pp. 23-35. The American Phytopathological

Society: St. Paul, Minnesota.

Daniels, B.A. & Trappe, J.M. (1980). Factors affecting spore germination ofthe

vesicular-arbuscular mycorrhizal fungus, Glomus epigaeus. Mycologia, 72,

457-471. Danielson, R.M. (1985). Mycorrhizae and the reclamation of stressed terrestrial

environments. Soil Reclamation Processes - Microbiological Analyses

Applications (Ed. by R.L. Tate & D.A. Klein), pp. 173-201. Marcel Dekker,

New York. Dighton, J. (1983). Phosphatase production by mycorrhizal fungi. Plant and Soil, 71,

455-462. Eckersly, R. (1989). Regreening Australia: the Environmental, Economic, and Social

benefits of Reforestation. CSIRO, Canberra. Evans, D.G. & Miller, M.H. (1988). Vesicular-arbuscular mycorrhizas and the soil-

diturbance-inducedreduction of nutrient absorption in maize. I. Causal

relationships. New Phytologist, 110, 67-74. Evans, D.G. & Miller, M.H. (1990). The role of the external mycelial network in the

effect of soil disturbance upon vesicular-arbuscular colonization in maize. New

Phytologist, 114, 65-71. Fairchild, G.L. & Miller, M.H. (1988). Vesicular-arbuscular mycorrhizas and the soil-

disturbance-induced reduction of nutrient absorption in maize. IL Development of

the effect. New Phytologist, 110, 75-84.

Fitter, A.H. (1986). Effect of benomyl on leaf phosphorus concentration in Alpine

Grasslands: a test of the mycorrhizal benefit New Phytologist, 103,767-776. Fleming, L.V. (1984). Effects of trenching and coring on the formation of

ectomycorrhizas on birch seedlings grown aroung mature trees. New Phytologist, 98, 143-153.

Fogel, M. & Hunt G. (1979). Fungal and arboreal biomass in a wester Oregon Douglas-

fir ecosystem: distribution patterns and turnover. Canadian Journal of Forest

Research, 9, 245-256.

Fogel, R. (1980). Mycorrhizae and nutrient cycling in natural forest ecosystems. New Phytologist, 86, 199-212.

Forster, S.M. & Nicolson, T.H. (1981). Microbial aggrgation of sand in a martime dune

succession. Soil Biology and Biochemistry, 13, 205-208.

Fuller, L. (1980). Wollongong's Native Trees. Weston and Co. Publishers Pty. Ltd.,

Kiama, NSW.

Gardner, J.H. & Malajczuk, N. (1988). Recolonization ofrehabilitated bauxit e mine sites

in Western Australia by mycorrhizal fungi. Forest Ecology and Management, 24,

27-42.

Gay, P.A., Grubb, P.J. & Hudson, H.J. (1982). Seasonal changes in the concentration

of nitrogen, phosphorus and potassium, and in the density of mycorrhiza in

biennial and matrix-forming perennial species of closed chalkland turf. Ecology,

70, 571-593.

Gemma, J.N. & Koske, R.E. (1988). Seasonal variation in spore abundance and

dormancy of Gigaspora gigantea and in mycorrhizal inoculum potential of a dune

soil. Mycologia, 80, 211-216.

Gianinazzi, S., Dexheimer, J., Gianinazzi-Pearson, V. & Marx, C. (1983). Role ofthe

host-arbuscule interface in the VA mycorrhizal symbiosis: ultracytological studies

of processes involved in phoshate and carbohydrate exchange. Plant and Soil,

71, 211-215. Gianinazzi-Pearson, V. (1985). Mycorrhizal effectiveness in phosphate nutrition: how,

when, and where? Proceedings ofthe 6th NACOM (Ed. by R. Molina), pp. 150-

154. Forest Research Laboratory, Oregon. Giovannetti, M. (1985). Seasonal variations of vesicular-arbuscular mycorrhizas and

endogonaceous spores in a maritime sand dune. Transactions ofthe British Mycological Society, 84, 679-684.

Goldsmith, F.B., Harrison, CM. & Morton, A.J. (1986). Description and analysis of

vegetation. Methods in Plant Ecology (Ed by P.D. Moore & S.B. Chapman), pp. 437-524. Blackwell Scientific Publications, Oxford.

Gould, A.B. & Liberta, A.E. (1981). Effects of topsoil storage during surface mining on

the viability of vesicular-arbuscular mycorrhiza. Mycologia, 73,914-922.

Graham, J.H., Linderman, R.G. Menge, J.A. (1982). Development of external hyphae

by different isolates of mycorrhizal Glomus spp. inrelation to root colonization

and growth of troyer citrange. New Phytologist, 91, 183-189.

Gray, L.E. & Gerdemann, J.W. (1969). Uptake of 32P by vesicular-arbuscular fungi. Plant and Soil, 30, 415-422.

Guildon, A. & Tinker, P.B. (1983). Interactions of vesicular-arbuscular mycorrhizal

infection and heavy metals in plants. I. The effect of heavy metals on the

development of vesicular-arbuscular mycorrhizas. New Phytologist, 95,247- 261.

Haas, J.A., Bar-Yosef, B., Krikun, J., Markovitz, T. & Kramer, S. (1987). Vesicular-

arbuscular mycorrhizal fungus infestation and phosphorus fertilization to

overcome pepper stunting after methyl bromide fumigation. Agronomy Journal,

79, 905-910.

Hall, I.R. (1976). Response of Coprosma robusta to different forms of endomycorrhizal

inoculum. Transactions ofthe British Mycological Society, 67,409-411

Hall, I.R. (1979). Soil pellets to introduce vesicular-arbuscular mycorrhizal fungi into

soil. Soil Biology and Biochemistry, 11, 85-91

Harley, J.L. & Smith, S.E. (1983). Mycorrhizal Symbiosis. Academic Press, London.

Harley, J.L. (1959). The Biology of Mycorrhizae. Leonard Hill, London.

Harley, J.L. (1971). Fungi in ecosystems. Journal of Ecology, 59, 653-668. Harley, J.L. (1973). Symbiosis in the ecosystem. Journal of the National Science Council of Sri Lanka, 1, 31-48.

Harley, J.L. (1975). Problems of mycotrophy. Endomycorrhizas (Ed. by S.E. Sanders, B. Mosse & P.B. Tinker), pp. 1-24. Academic Press, London.

Harley, J.L. (1978). Ectomycorrhizas as nutrient absorbing organs. Proceeding ofthe Royal Society of London B, 203, 1-21.

Harris, J.A., Hunter, D. & Birch, P. (1987). Vesicular-arbuscular mycorrhizal

populations in stored topsoil. Transactions ofthe British Mycological Society, 89, 600-603.

Harvey, A.E., Jurgensen, M.F. & Larsen, M.J. (1980). Clearcut harvesting and

ectomycorrhizae: survival of activity on residual roots and influence on a

bordering forest stand in western Montana. Canadian Journal of Forest Research, 10, 300-303.

Hazelton, P. A. & Tille, P.J. (1990). Soil Landscapes ofthe Wollongong-Port Hacking 1:100 000 Sheet. Soil Conservation Service of NSW, Sydney.

Heap, A.J. & Newman, E.I. (1980a). Links betweenroots b y hyphae of vesicular-

arbuscular mycorrhizas. New Phytologist, 85, 169-171.

Heap, A.J. & Newman, E.I. (1980b). The influence of vesicular-arbuscular mycorrhizas

on phosphorus transfer between plants. New Phytologist, 85, 173-179.

Hepper, CM. (1977). A colorimetric method for estimating vesicular-arbuscular

mycorrhizal infection in roots. Soil Biology and Biochemistry, 9,15-18.

Hepper, CM. & Warner, A. (1983). Role of organic matter in the growth of a vesicular-

arbuscular mycorrhizal fungus in soil. Transactions ofthe British Mycological

Society, SI, 155-156.

Herbert, C & Helby, C (1980). Guide to the Sydney Basin. Geological Survey of

NSW, Sydney.

Hesse, P.R. (1971). A Textbook of Soil Chemical Analysis. John Murray, London. Hirrell, M.C. & Gerdemann, J.W. (1979). Enhanced carbon transfer between onions

infected with a vesicular-arbuscular mycorrhizal fungus. New Phytologist 83, 731-738.

Hudson, H.J. (1986). Fungal Biology. Edward Arnold, London.

Janos, D.P. (1980). Mycorrhizae influence tropical succession. Biotropica 12, 56-64.

Janos, D.P., (1987). VA mycorrhizas in humid tropical ecosystems. Ecophysiology of

VAmycorrhizal plants (Ed. by G.R. Safir), pp. 107-134. CRC Press, Boca Raton, Florida.

Jasper, D.A., Abbott, L.K. & Robson, A.D. (1989a). Soil disturbance reduces the

infectivity of external hyphae of vesicular-arbuscular mycorrhizal fungi. New Phytologist, 111, 93-99.

Jasper, D.A., Abbott, L.K. & Robson, A.D. (1989b). Hyphae of a vesicular-arbuscular

mycorrhizal fungus maintain infectivity in dry soil, except when the soil is

disturbed. New Phytologist, 112, 101-107.

Jasper, D.A., Abbott, L.K. & Robson, A.D. (1989c). The loss of VA mycorrhizal

infectivity during bauxite mining may limit the growth of Acacia pulchella R.Br.

Australian Journal of Botany, 37, 33-42.

Jasper, D.A., Abbott, L.K. & Robson, A.D. (1989d). Acacias respond to additions of

phosphorus and to inoculation with VA mycorrhizal fungi in soils stockpiled

during mineral sand mining. Plant and Soil, 115, 99-108.

Jasper, D.A., Abbott, L.K. & Robson, A.D. (1991a). The effect of soil disturbance on

vesicular-arbuscular mycorrhizal fungi in soilsfrom differen t vegetation types.

New Phytologist, 118, 471-476. Jasper, D.A., Brundrett, M.C., Abbott, L.K. & Robson, A.D. (1991b). Ensuring

diversity and growth in revegetation - a program to develop techniques for

inoculation with VA mycorrhizal fungi. Proceedings ofthe AM J.C.

Environmental Workshop Vol. I. pp. 172-174. Australian Mining Industry

Council, ACT. Jasper, D.A., Robson, A.D. & Abbott, L.K. (1987). The effect of surface mining on the

infectivity of vesicular-arbuscular mycorrhizal fungi. Australian Journal of Botany, 35, 641-652.

Jasper, D.A., Robson, A.D. & Abbott, L.K. (1988). Revegetation in an Iron-ore mine -

nutrient requirements for plant growth and the potential role of vesicular-

arbuscular (VA) mycorrhizal fungi. Australian Journal of Soil Research, 26, 497-507.

Johnson, P.N. (1977). Mycorrhizal Endogonaceae in a New Zealand forest New Phytologist, 78, 161-170.

Kendrick, B. (1985). The Fifth Kingdom. Mycologue Publications, Ontario.

Khan, A.G. (1978). Vesicular-arbuscular mycorrhizas in plants colonizing black wastes

from bituminous coal mining in the Illawarra region of New South Wales. New

Phytologist, 81, 53-63.

Khan, A.G. (1981). Growth of endomycorrhizal onions in unsterilized coal waste. New

Phytologist, 87, 363-370. Kiernan, J.M., Hendrix, J.W. & Maronek, D.M. (1983). Endomycorrhizal fungi

occurring on orphan strip mines in Kentucky. Canadian Journal of Botany, 61,

1798-1803. Koide, R.T. & Mooney, H.A. (1987). Spatial variation in inoculum potential of

vesicular-arbuscular mycorrhizal fungi caused by the formation of gopher

mounds. New Phytologist, 107, 173-182.

Kormanick, P.P., Bryan, W.C. & Schultz, R.C. (1980). Procedures and equipment for

staining large numbers of plant root samples for endomycorrhizal assay.

Canadian Journal of Microbiology, 26, 536-538. Koske, R.E., Sutton, J.C. & Sheppard, B.R. (1975). Ecology of Endogone in Lake

Huron sand dunes. Canadian Journal of Botany, 59, 87-93

Koslowsky, S.D. & Boerner, R.E.J. (1989). Interactive effects of aluminium,

phosphorus and mycorrhizae on growth and nutrient uptake of Panicium virgatum

L. (Poaceae). Environmental Pollution, 61,107-125. 89

Kostkova, J. & Cudin, P. (1989). Vesicular-arbuscular mycorrhizae in plant succession

on colliery spoil-tips from strip mining. Agriculture, Ecosystems and

Environment, 29, 211-215.

Lambert, D.H. & Cole, H.Jr. (1980). Effects of mycorrhizae on establishment and

performance of forage species in mine spoil. Agronomy Journal, 72,257-260.

Lambert, J.H., Muston, R. M. & Heath, C (1989). The advantages of integrated

planning of utility developments, pp. 158-163. Planning for Environmental

Change - National Environmental Engineering Conference. The Institute of

Engineers, Australia. Lamont, B.B. (1984). Specialized modes of nutrition. Kwongan: Plant Life ofthe

Sandplain (Ed. by J.S. Pate & J.S. Beard), pp. 236-245. University of

Western Australia Press, Nedlands, Western Australia. Law, D.L. (1984). Mined-Land Rehabilitation. Van Nostrand Reinhold, New York.

Liberta, A.E. (1981). Effects of topsoil-storage duration on inoculum potential of vesicular-arbuscular mycorrhizae. Symposium on Surface Mining Hydrology,

Sedimentology and Reclamation, pp. 45-48. University of Kentucky,

Lexington, Kentucky. Logan, V.S., Clarke, P.J. & Allaway, W.G. (1989). Mycorrhizas androot attribute s of plants of coastal sand-dunes NSW. Australian Journal of Plant Physiology, 16,

141-146. Loree, M.A.J. & Williams, S.E. (1984). Vesicular-arbuscular mycorrhizae and severe

land disturbance. VA Mycorrhizae and Reclamation of Arid and Semiarid Lands.

pp. 1-14. University of Wyoming, Laramie, Wyoming.

Loree, M.A.J. & Williams, S.E. (1987). Colonization of western wheatgrass (Agropyron smithii Rydb.) by vesicular-arbuscular mycorrhizal fungi during the

revegetation of a surface mine. New Phytologist, 106, 735-744.

MacMahon, J.A. (1987). Disturbed land and ecological theory. Restoration Ecology: a

Synthetic Approach to Ecological Research (Ed. by W.R.Jordan, M.E. Gilpin &

J.D. Aber), pp. 221-238. Cambridge University Press, Cambridge. Malajczuk, N. & Lamont, B.B. (1981). Specialized roots of symbiotic origin in

heathlands. Mediterranean Type Ecosystems ofthe World Vol II (Ed. by F. Di

Castri, D.W. Goodall & R.L. Specht.) pp. 165-186. Elsevier, New York.

Malajczuk, N., Linderman, R.G., Kough, J. & Trappe, J.M. (1981). Presence of

vesicular-arbuscular mycorrhizae in Eucalyptus spp. and Acacia sp., and their

absence in Banksia sp. after inoculation with Glomus fasciculatum. New Phytologist, 87, 567-572.

Malajczuk, N., McComb, A.J. & Loneragan, J.F. (1975). Phosphorus uptake and

growth of mycorrhizal and uninfected seedlings of Eucalyptus calophylla R. Br. Australian Journal of Botany, 23, 231-238.

Malloch, D. & Malloch, B. (1981). The mycorrhizal status of boreal plant species from

north-eastern Ontario. Canadian Journal of Botany, 59, 2167-2172.

Marx, D.H. (1975). Mycorrhizae and establishment of trees on strip-mined land. Ohio

Journal of Science, 75, 288-297.

Marx, D.H. (1980). Ectomycorrhizal fungus inoculations: a tool for improving forestation practices. Tropical Mycorrhiza Research (Ed. by P. Mikola), pp. 13-

71. Clarendon Press, Oxford.

McAfee, B.J. & Fortin, J.A. (1989). Ectomycorrhizal colonization on black spruce and

jack pine seedlings outplanted inreforestation sites. Plant and Soil, 166,9-17.

McGee, P. (1986). Mycorrhizal associations of plant species in a semiarid community.

Australian Journal of Botany, 34,585-593.

McGee, P. A. (1987). Alteration of growth of Solanum opacum and Plantago

drummondii and inhibition of regrowth of hyphae of vesicular-arbuscular

mycorrhizal fungi from dried root pieces by manganese. Plant and Soil, 101,

227-233. McGee, P.A. (1989). Variation in propagule numbers of vesicular-arbuscular

mycorrhizal fungi in a semi-arid soil. Mycological Research, 92,28-33. McGonigle, T.P., Evans, D.G. & Miller, UM. (1990). Effect of degree of soil

disturbance on mycorrhizal colonization and phosphorus absorption by maize in

growth chamber andfield experiments. New Phytologist, 116, 629-636.

Medve, RJ. (1984). The mycorrhizae of pioneer species in disturbed ecosystems in

Western Pennsylvania. American Journal of Botany, 71,787-794.

Mejstrik, V.K. (1972). Vesicular-arbuscular mycorrhizas ofthe species of a Molinietum

coerulae L.I. association: the ecology. New Phytologist, 71, 883-890.

Menge, J.A. & Timmer, L.W. (1982). Procedures for inoculation of plants with

vesicular-arbuscular mycorrhizae in the laboratory, greenhouse and field.

Methods and Principles of Mycorrhizal Research (Ed. by N.C Schenck), pp. 59-

68. The American Phytopathological Society, St. Paul, Minnesota.

Mertz, S.M., Heithaus, J.J.ITI. & Bush, R.L. (1979). Mass production of axenic spores of the endomycorrhizal fungi Gigaspora margarita. Transactions ofthe British

Mycological Society, 72, 167-169.

Meyer Schoenberger, M. & Perry, D.A. (1982). The effect of soil disturbance on growth

and ectomycorrhizae of Douglas-fir and western hemlock seedlings: a greenhouse

bioassay. Canadian Journal of Forest Research, 12, 343-353.

Miller, O.K. Jr. (1982). Taxonomy of ecto- and ectendomycorrhizal fungi. Methods and

Priciples of Mycorrhizal Research (Ed. by N.C. Schenck), pp. 91-101. The

American Phytopathological Society, St Paul, Minnesota.

Miller, R.M. (1979). Some occurrences of vesicular arbuscular mycorrhizae in natural

and disturbed ecosystems of the Red Desert. Canadian Journal of Botany, 57,

619-623. Miller, R.M. (1987). Mycorrhizae and succession. Restoration Ecology: A Synthetic

Approach to Ecological Research (Ed. by W.R. Jordan, M.E. Gilpin & J.D.

Aber.) pp. 205-220. Cambridge University Press, Cambridge.

Miller, R.M., Carnes, B.A. & Moorman, T.B. (1985). Factors influencing survival of

vesicular-arbuscular mycorrhiza propagules during topsoil storage. Journal of

Applied Ecology, 22, 259-266. 92

Moorman, T. & Reeves, F.B. (1979). The role of endomycorrhizae in revegetation

practices in the semi-arid West. n. A bioassay to determine the effect of land

disturbance on endomycorrhizal populations. American Journal of Botany, 66, 14-18.

Morton, J.B. (1988). Taxonomy of VA mycorrhizal fungi: classification, nomenclature,

and identification. Mycotaxon, 32,267-224.

Mosse, B. (1973). Advances in the study of vesicular-arbuscular mycorrhiza. Annual

Review of Phytopathology, 11, 171-196.

Mosse, B. & Bowen, G.D. (1968). The distribution of Endogone spores in some

Australian and New Zealand soils, and in an experimentalfield soil at

Rothamsted. Transactions ofthe British Mycological Society, 51,485-492.

Mosse, B., Stribley, D.P. & LeTacon, F. (1981). Ecology of mycorrhizae and

mycorrhizal fungi. Advances in Microbial Ecology, 5,137-210.

Mulligan, D.R. & Patrick, J.W. (1985). Phosphorus and carbon economies of ectomycorrhizal seedlings of Eucalyptus pitularis Smith. Australian Journal of

Plant Physiology, 12, 669-679. Muston, R. (1987). Fabricate orrehabilitate: an ecological approach to the revegetation

of degraded ecosystems. Xeric Australia: Landscape Design and Management.

pp. 57-67 Australian Institute of Landscape Architects, Adelaide.

Nemec, S. (1987). Effect of storage temperature and moisture on Glomus species and

their subsequent effect on citrus root stock seedling growth and mycorrhiza

development Transactions ofthe British Mycological Society, 89, 205-212.

Newman, E.I. (1978). Root microorganisms - their significance in the ecosystem.

Biological Reviews, 53, 511-554. Newman, E.I. (1988). Mycorrhizal links between plants: their functioning and

ecological significance. Advances in Ecological Research, 18,243-270.

Nicolson, T.H. (1967). Vesicular-arbuscular mycorrhiza - a universal plant symbiosis.

Scientific Progress, Oxford, 55, 561-581. Nye, P.H. & Tinker, P.B. (1977). Solute Movement in the Soil-Root System. Blackwell Scientific Publications, Oxford.

Parke, J.L., Linderman, R.G. & Black, CH. (1983a). Therole of ectomycorrhizas in

drought tolerance of Douglas-fir seedlings. New Phytologist, 95, 83-95.

Parke, J.L., Linderman, R.G. & Trappe, J.M. (1983b). Effects of forest litter on

mycorrhizal development and growth of Douglas-fir and western red cedar

seedlings. Canadian Journal of Forest Research, 13, 666-671

Parke, J.L., Linderman, R.G. & Trappe, J.M. (1983c). Effect of root zone temperature

on ectomycorrhiza and vesicular-arbuscular mycorrhiza formation in disturbed

and undisturbed soils of southwest Oregon. Canadian Journal of Forest

Research, 13, 657-665.

Parke, J.L.,Linderman, R.G. & Trappe, J.M. (1984). Inoculum potential of

ectomycorrhizal fungi in forest soils of southwest Oregon and northern

California. Forest Science, 30, 300-304.

Perry, D.A., Molina, R & Amaranthus, M.P. (1987). Mycorrhizae, mycorrhizospheres and reforestation: current knowledge and research needs. Canadian Journal of

Forest Research, 17, 929-940.

Peterson, R.L., Ashford, A.E. & Allaway, W.G. (1985). Vesicular-arbuscular

mycorrhizal associations of vascular plants on Heron Island, a Great Barrier Reef

coral cay. Australian Journal of Botany, 33, 677-685.

Phillipson, J., Putman, R.J., Steel, J. & Woodell, S.R.J. (1975). Litter input, Utter

decomposition, and the evolution of carbon dioxide in a beech woodland.

Wytham Woods, Oxford. Oecologia, 20, 203-217.

Pigram, J J. (1986). Issues in the Management ofAusralia's Water Resources.

Longman Cheshire, Melbourne. Porter, W.M. (1979). The "Most Probable Number" method for enumerating infective

propagules of vesicular-arbuscular mycorrhizal fungi in soil. Australian Journal

of Soil Research, 17,515-519. 94

Powell, CLL (1976). Development of mycorrhizal infections from Endogone spores

and infected root segments. Transactions of the British Mycological Society, 66, 439-445.

Powell, Cll (1980). Mycorrhizal infectivity of eroded soils. Soil Biology and Biochemistry, 12, 247-250.

Powell, CLL (1982). Selection of efficient VA mycorrhizal fungi. Plant and Soil, 68, 3-9.

Purnell, H.M. (1960). Studies of the family Proteacea. I. Anatomy and morphology of

the roots of some Victorian species. Australian Journal of Botany, 8, 38-50.

Read, D J., Francis, R., Finlay, R.D. (1985). Mycorrhizal mycelia and nutrient cycling

in plant communities. Ecological Interactions in Soil (Ed. by A. H. Fitter, D.

Atkinson, D J. Read & M.B. Usher), pp. 193-217. Blackwell Scientific Publications, Oxford.

Read, D J., Koucheki, H.K. & Hodgson, J. (1976). Vesicular-arbuscular mycorrhizae

in natural vegetation systems. I. The occurrence of infection. New Phytologist,

77, 641-653.

Redhead, J.F. (1977). Endotrophic mycorrhizas in Nigeria: species of the Endogonaceae and their distribution. Transactions ofthe British Mycological Society, 69,275-

280. Reeves, F.B., Wagner, D., Moorman, T. & Kiel, J. (1979). Therole o f

endomycorrhizae in the revegetation practices in the semi-arid west. I. A

comparison of incidence of mycorrhizae in severely disturbed versus natural

environments. American Journal of Botany, 66, 6-13.

Reid, CCP. (1984). Mycorrhizae: A root-soil interface in plant nutrition. Microbial -

Plant Interactions, pp. 29-50. Soil Science Society of America, Wisconsin.

Rhodes, L.H. & Gerdemann, J.W. (1978a). Hyphal translocation and uptake of sulphur

by vesicular-arbuscular mycorrhizae of onions. Soil Biology and Biochemistry,

10, 335-360. Rhodes, L.H. & Gerdemann, J.W. (1978b). Translocation of calcium and phosphate by

external hyphae of vesicular-arbuscular mycorrhizae. Soil Science, 126,125- 126.

Rives, C.S., Bajwa, Ml, Liberta, A.E. & Miller, R.M. (1980). Effects of topsoil

storage during surface mining on the viability of VA mycorrhiza. Soil Science, 129, 253-257.

Roberts, R.W. (1989). Land Conservation in Australia: a 200 Year Stocktake. Soil Conservation Society of NSW, Sydney.

Safir, G.R. & Nelsen, CE. (1985). VA mycorrhizas: plant and fungal water relations.

Proceedings ofthe 6th NACOM (Ed. by R. Molina), pp. 161-164. Forest Research Laboratory, Oregon.

Sanders, F.E. & Sheikh, N.A. (1983). The development of vesicular-arbuscular

mycorrhizal infection in plant root systems. Plant and Soil, 71, 223-246.

Sanders, F.E. & Tinker, P.B.H. (1973). Phosphate flow into mycorrhizal roots.

Pesticide Science, 4, 385-395.

Sanders, F.E., Mosse, B. & Tinker, P.B. (Eds) (1975) Endomycorrhizas. Academic

Press, London.

Schenck, N.C, Graham, S.O. & Green, N.E. (1975). Temperature and light effect on

contamination and spore germination of vesicular-arbuscular mycorrhizal fungi.

Mycologia, 67, 1189-1192.

Schramm, J.E. (1966). Plant colonization studies on black wastes from anthracite mining

in Pennsylvania. American Philosophical Society Transactions N.S., 56, 1-194.

Schwab, S. & Reeves, F.B. (1981). Therole of endomycorrhizae in the revegetation

practices in the semi-arid West. EI. Vertical distribution of vesicular-arbuscular

mycorrhiza inoculum potential. American Journal of Botany, 68,1293-1297.

Slankis, V. (1974). Soil factors influencing formation of mycorrhizae. Annual Review

of Phytopathology, 12, 437-457.

Smith, S.E. (1980). Mycorrhizas of autotrophic higher plants. Biological Reviews, 55,

475-510. 96

Smith, S.E. & Bowen, G.D. (1979). Soil temperature, mycorrhizal infection and

nodulation of Medicago trunculata and Trifolium subterraneum. Soil Biology and Biochemistry, 11, 469-473.

Smith, S.E. & Gianinazzi-Pearson, V. (1989). Physiological interactions between

symbionts in vesicular-arbuscular mycorrhizal plants. Annual Review of Plant Physiology, 39, 221-244.

Smith, T.F. (1978). A note on the effect of soil tillage on thefrequency and vertical

distribution of spores of vesicular-arbuscular endophytes. Australian Journal of

Soil Research, 16, 359-361.

Stahl, P.D., Williams, S.E. & Christensen, M. (1988). Efficacy of native vesicular-

arbuscular mycorrhizal fungi after severe soil disturbance. New Phytologist,

110, 347-353.

Standard, J.C. (1961). A new study of the Hawkesbury Sandstone: preliminary

findings. Journal ofthe Royal Society New South Wales, 95, 145-146.

Stark, J.M. & Redente, E.F. (1987). Production potential of stockpiled topsoil. Soil

Science, 144, 72-76. Sutton, J. C & Sheppard, B.R. (1976). Aggregation of sand-dune soil by

endomycorrhizal fungi. Canadian Journal of Botany, 54, 326-333.

Sutton, J.C & Barron, G.L. (1972). Population dynamics of Endogone spores in soils.

Canadian Journal of Botany, 50,1909-1914. Temple, J.M. & Bungey, D. (1980). Revegetation: Methods and Management. State

Pollution Control Commission, NSW. Thames, J.L. (1977). Reclamation and Use of Disturbed Lands. University of Arizona

Press, Arizona. Thompson, J.P. (1987). Decline of vesicular-arbuscular mycorrhizae in long fallow

disorder offield crops and its expression in phosphorus deficiency of sunflower.

Australian Journal of Agricultural Research, 38, 847-867. Timmer, L.W. & Leyden, R.F. (1980). The relationship of mycorrhizal infection to

phosphurus-induced copper deficiency in sour orange seedlings. New Phytologist, 85, 15-23.

Tinker, P.B. (1975). Effects of vesicular-arbuscular mycorrhizae on higher plants.

Symposium ofthe Society of Experimental Biology, 29, 325-349.

Tinker, P.B. (1978). Effects of VA mycorrhizae on plant nutrition and plant growth.

Physiological Vegetation, 16, 743-751.

Tisdall, J.M. & Oades, J.M. (1979). Stabilization of soil aggregates by the root systems

of ryegrass. Australian Journal of Soil Research, 17,429-441.

Tommerup, LC. (1983a). Spore dormancy in vesicular-arbuscular mycorrhizal fungi.

Transactions of the British Mycological Society, 81, 37-45.

Tommerup, LC (1983b). Temperature relations of spore germination and hyphal growth

of vesicular-arbuscular mycorrhizal fungi in soil. Transactions ofthe British

Mycological Society, 81, 381-387.

Tommerup, LC (1984). Effect of soil water potential on spore germination by vesicular- arbuscular mycorrhizal fungi. Transactions ofthe British Mycological Society,

83, 193-202. Tommerup, LC (1985). Inhibition of spore germination of vesicular-arbuscular

mycorrhizal fungi in soil. Transactions ofthe British Mycological Society, 85,

267-278. Tommerup, LC & Abbott, L.K. (1981). Prolonged survival and viability of VA

mycorrhizal hyphae after root death. Soil Biology and Biochemistry, 13,431-

433. Tommerup, LC & Kidby, D.K. (1979). Preservation of spores of vesicular-arbuscular

endophytes by L-drying. Applied and Environmental Microbiology, 37, 831-

835. Trappe, J.M. (1981). Mycorrhizae and productivity of arid and semiarid rangelands.

Advances in Food Producing Systems for Arid and Semiarid Lands (Ed by J.T.

Manassah & EJ. Brishey), pp. 93-124. Academic Press, New York. Visser, S, Griffiths, CL. & Parkinson, D. (1984). Topsoil storage effects on primary

production and rates of vesicular-arbuscular mycorrhizal development in

Agropyron trachycaulum. Plant and Soil, 82, 51-60.

Warcup, J.H. (1980). Ectomycorrhizal associations of Australian indigenous plants. New Phytologist, 85, 531-535.

Warner, A. (1983). Re-establishment of indigenous vesicular-arbuscular mycorrhizal fungi after topsoil storage. Plant and Soil, 73, 387-394.

Warner, A. & Mosse, B. (1980). Independent spread of VAM fungi in soil.

Transactions ofthe British Mycological Society, IA, 407-410.

Warner, A. & Mosse, B. (1983). Spread of veiscular-arbuscular mycorrhizal fungi

between separate root systems. Transactions ofthe British Mycological Society, 80, 353-380.

Whelan, RJ. (1989). The influence of fauna on plant species composition. Animals in

Primary Succession - The Role of Fauna in Reclaimed Lands (Ed. by J.D.

Majer), pp. 107-142. Cambridge University Press, Cambridge.

White, J.A., Munn, L.C. & Williams, S.E. (1989). Edaphic and reclamation aspects of

vesicular-arbuscular mycorrhizae in Wyoming Red Desert soils. Soil Science

Society of America Journal, 53, 86-90.

Whittingham, J. & Read, D J. (1982). Vesicular-arbsucular mycorrhiza in natural

vegetation systems. HL Nutrient transfer between plants with mycorrhizal

interconnections. New Phytologist, 90, 277-284.

Wicklow, D.T. & Whittingham, W.F. (1974). Soil microfungal changes among the

profiles of disturbed conifer-hardwood forests. Ecology, 55, 3-16.

Williams, S.E. & Stahl, P.D. (1987). Importance of mycorrhizal fungi in land

revegetation. Proceedings ofthe 39th Annual Meeting ofthe Great Plains

Agricultural Council, Forestry Committee, pp. 71-88. GPAC Publication #122.

Williams, W.D. (1980). Catchment Management - an Ecological Basis for Water

Resource Management. (Ed. by W.D. Williams), pp. 100-116. Australian

National University, Canberra. 99

Williamson, B. & Alexander, LJ. (1975). Acid phosphatase localized in the sheath of

beech mycorrhiza. Soil Biology and Biochemistry, 7, 195-198.

Wilson, J.M. (1984). Comparitive development of infection by three vesicular-arbuscular

mycorrhizal fungi. New Phytologist, 97, 413-426.

Wilson, J.M. & Trinick, M.J. (1983). Infection development and interactions between

vesicular-arbuscular mycorrhizal fungi. New Phytologist, 93, 543-553.

Young, R.W. & Young, A.R.M. (1988). "Altogether barren, perculiarlyromantic": th e

sandstone lands around Sydney. Australian Geographer, 19, 9-25.

Zar, J.H. (1984). Biostatistical Analysis 2 ed. Prentice-Hall Inc., New Jersey. Appendix I Percentage foliar projection cover values of the plant species from within the Avon and O'Hares study sites (* = plant species common to both sites).

Family Plant species % f.p.c.

Avon Apiaceae Platysace linearifolia (Cav.) C* 0.8 Cyperaceae Ptilanthelium deustem (R. Br.) Kiithenth* 2.8 Euphorbiaceae Amperea xiphoclada (Sieb. ex Spreng.) Druce 1.0 Poranthera corymbosa Brongn. 1.6 Fabaceae Bossiaea obcordata (Vent) Druce 4.4 Dillwyinia parvifolia var. tricopoda Blakely 0.4 Goodeniaceae Gompholobium grandiflorum Sm. 2.6 Mdaceae Dampiera stricta (Sm.) R. Br. * 0.8 Lindsaeaceae Patersonia sericea R. Br. 2.8 Mimosaceae Lindsaea linearis Sw.* 2.0 Acacia linifolia (Vent.) Willd 2.8 A. obtusifolia A. Cunn 3.0 Myrtaceae A. ulicifoila (Salisb.) Court 2.4 Eucalyptus haemastoma Sm. 19.6 E. racemosaQss. 10.2 Proteaceae Leptospermumflavescens Sm. 4.2 Banksia spinulosa Sm.* 3.2 Grevillea sphacelata R. Br. 3.4 Hakea dactyloides (Gaertn.)Cav. * 2.4 Isopogon anemonifolius (Salisb.) Knight * 2.6 /. anethifolius (Salisb.) Knight 1.2 Lambertia formosa Sm. * 4.2 Lomatia silaifolia (Sm.) R. Br. * 1.6 Rutaceae Persoonia levis (Cav.) Domin. * 2.8 Telopea speciosissima R. Br. 2.4 Thymelaeaceae Eriostemon australasius Pers. * 1.6 Xanthorrhoeaceae Boronia thujona Penfold et Welch 1.4 Pimelea linifolia Sm. 0.2 Lomandra confertifolia (F.M. Bailey) Fahn * 4.4 L. glauca (R. Br.) Ewart * 1.4 O'Hares L. obliqua (Thunb.) MacBride * 2.6 Xanthorrhoea arbor ea R. Br. 3.2 Apiaceae Actinotis helianthi Labil. 1.4 Platysace linearifolia (Cav.) C 1.4 Cyperaceae Caustis flexuosa R. Br. 1.6 Ptilanthelium deustem (R. Br.) Kiithenth 2.2 Dilleniaceae Hibbertia serpyllifolia R. Br. ex DC. 2.1 Droseraceae Drosera pygmaea DC 1.7 Epacridaceae Brachyloma daphnoides (Sm.) Benth. 2.8 Epacris microphylla R. Br. 1.7 2.2 Fabaceae Leucopogon juniperinus R. Br. Daviesia corymbosa Sm. 2.2 Dillwyinia retorta (Wendl.) Druce 1.4 Mirbelia rubiifolia (Andr.) G. Don. 3.7 Pultenaea elliptica Sm. 4.4 Goodeniaceae Dampiera stricta (Sm.) R Br. 1.4 Mdaceae Patersonia sericea R. Br. 1.4 Lindsaeaceae Lindsaea linearis Sw. 2.2 Mimosaceae Acacia myrtifolia (Sm.) Willd. 1.4 Myrtaceae A. suaveolens (Sm.) Willd. 1.4 Angophora hispida (Sm.) Blaxell 9.3 Baeckea ramosissima A. Cunn. 2.9 Eucalyptus haemastoma Sm. 3.2 E. stricta Sieb. ex Spreng. 3.1 Kunzea capitata Reichb. 3.8 Leptospermum juniperinum Sm. 0.9 Olacaceae L. lanigerum (Ait) Sm. 2.0 Proteaceae Otec .srric&z R. Br. 0.7 Banksia ericifolia L. f. 1.3 B. paludosa R. Br. 1.0 B. serrata L. f. 0.7 5. spinulosa Sm. 1.0 Conospermum longifolium Sm. 2.0 C. taxifolium Sm. 1.0 Grevillea buxifolia (Sm.) R. Br. 1.3 G. oleoides Sieb. ex Schultz et f 2.0 //afcea dactyloides (Gaeitn.)Cav. 3.3 Hakea teretifolia (Salisb.) J. Britten 1.7 Isopogon anemonifolius (Salisb.) Knight 1.0 Lambertia formosa Sm. 4.2 Lomatia silaifolia R. Br. 1.7 Persoonia levis (Cav.) Domin. 1.7 Rutaceae P. pinifoliaR.Br. 1.9 Xanthorrhoeaceae P. lamina Pers. 1.4 Petrophilepulchella (Schrad.) R. Br. 2.8 Eriostemon australasius Pers. 2.2 Lomandra conifertifolia (F.M. Bailey) Fahn 2.7 L. g/awaz (R. Br.) Ewart 2.0 NB. % f.p.c. = PercentagL. obliquae folia (Thunb.r projectio) MacBridn covere. 0.6 102 cu

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Appendix Ilia Details of the analysis carried out on the %VAM data from the Avon and O'Hares sites Significant synergistic interactions occurred in all cells. A posteriori comparisons were carried out using the appropriate L.S.D.'s to identify differences between means. Data given are means for five samples taken from each of 16 replicates harvested eight and 12 weeks after sowing in each of four seasons.

Autumn* Winter Spring Summer

Eight weeks

1 2 3 4 0-15 cmt 8.8a 4.8a 40.7a 42.6a 1 1 2 3 15-30 cm 2.2b 1.9b 10.9b 12.5b 12 weeks**

2 3 3 0-15 cmt 14.9al 9.2a 41.6a 41.4a 2 3 3 15-30 cm 5.8bl 4.1b 9.1b 9.3b

NB. * Entries in vertical columns followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 1.1, P < 0.01). t Entries in horizontal rows followed by the same superscripted number are not significantly different as indicated by analysis of variance (L.S.D. = 1.2, P < 0.01). ** Entries in "bold" are significantly different to the corresponding season x depth entries at eight weeks as indicated by analysis of variance (L.S.D. = 0.9, P < 0.01).

Appendix Illb Details of the analysis carried out on the density of VAM spores of each type from the Avon and O'Hares sites Entries given are mean number of fungal spores. Twenty five random samples were taken at the start at each of the seasons. Type I

Autumn* Winter Spring Summer

1 2 3 3 0-15 cmt 6.46a 7.02a 9.72a 9.20a 15-30 cm 1.96b1 2.08b1 ^Ob1 2.28b1

NB. * Entries in vertical columns followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 1.02, P < 0.01). t Entries in horizontal rows followed by the same superscripted number are not significantly different as indicated by analysis of variance (L.S.D. = 1.04, P < 0.01). Type n

Autumn* Winter Spring Summer

1 1 2 2 0-15 cmt 1.00a 1.24a 2.54a 1.98a 1 2 2 1 15-30 cm 1.40a 0.90a 0.96b 1.36b

NB. * Entries in vertical columns followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 0.42, P < 0.01). t Entries in horizontal rows followed by the same superscripted number are not significandy different as indicated by analysis of variance (L.S.D. = 0.43, P < 0.01).

Type m

Autumn* Winter Spring Summer

0-15 cmt 0.04a1 0.08a1 0.14a1 0.12a1 15-30 cm 0.00a1 0.00a1 0.10a1 0.10a1

NB. * Entries in vertical columns followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 0.13, P < 0.01). t Entries in horizontal rows followed by the same superscripted number are not significandy different as indicated by analysis of variance (L.S.D. = 0.12, P < 0.01). Appendix IVa Details of the analysis carried out on % VAM data from the Avon and O'Hares sites Significant synergistic "treatment x time" interactions occurred in all cells. A posteriori comparisons using the appropriate Residual SS were carried out to identify differences between means. Data presented are mean % VAM (n=25 in all cases except * where n=50). Avon •

14 dayst 21 days 28 days 35 days 42 days

No dist. * 6.80a1 14.29b1 26.46c1 32.99c!1 29.22c1

1 1 3 2 3 2 3 2 3 Dist. 1 ** 5.24a 13.98b ' 18.24c ' 22.65d ' 22.78d '

1 2 4 2 3 2 4 2 3 Dist. 2** 4.88a 6.55a ' 18.44b ' 17.60b ' 23.15c '

2 2 5 2 4 2 5 2 4 Dist. 3** 0.00a 0.00a ' 0.00a ' 4.85b ' 7.16b '

O'Hares •

14 dayst 21 days 28 days 35 days 42 days

No dist. * 5.98a1 12.09b1 27.77c1 28.29c1 28.55c1

1 3 2 3 2 3 2 3 2 3 Dist. 1 ** 4.39a ' 6.78a ' 22.33b ' 19.42b ' 19.57b '

1 3 2 3 2 3 2 3 2 3 Dist. 2** 3.80a ' 5.17a ' 19.49b ' 18.79b ' 21.08b '

2 4 2 4 2 4 2 4 2 4 Dist. 3** 0.00a ' 0.00a > 0.00a > 0.00a ' 2.07a '

• * Entries in horizontal row followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 2.67, P < 0.01). ** Entries in horizontal rows followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 3.78, P < 0.01). t Entries in vertical columns followed by same number are not significantly different as indicated by analysis of variance (L.S.D.No dist v. Dist 1,2,3 = 3.93, L.S.D.Dist 1 v. Dist 2 v. Dist 3 = 4-54, P < 0.01).

• * Entries in horizontal row followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 2.46, P < 0.01). ** Entries in horizontal rows followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 3.48, P < 0.01). t Entries in vertical columns followed by same number are not significantly different as indicated by analysis of variance (L.S.D.No dist v. Dist 1,2,3 = 3-27, L.S.D.Dist 1 v. Dist 2 v. Dist 3 = 3-77, P ^ 0.01). Appendix IVb. Details of the analysis carried out on shoot dry mass data from the Avon and O'Hares sites Significant synergistic "treatment x time" interactions occurred in all cells. A posteriori comparisons using the appropriate Residual SS were carried out to identify differences between means. Data presented are mean shoot dry mass (mg) (n=25 in all cases except * where n=50).

Avon •

14 dayst 21 days 28 days 35 days 42 days

1 1 1 1 1 No dist. * 24a 51b 69c 76d 77 d

Dist. 1 ** 25a1 52b1 71c1'3 72c1'3 74c1'3

Dist. 2 ** 29a1 49b1 69c1'3 75d!'3 75d1'3

1 1 57c2,4 2 4 2 4 Dist. 3 ** 25a 49b 61d ' 61d '

O'Hares •

14 dayst 21 days 28 days 35 days 42 days

No dist. * 35a1 60b1 74c1 78c1 79c1

Dist. 1 ** 29a1 60b1 70c1 75c1 77c1

Dist. 2 ** 26a1 57b1 73c1 76c1 79c1

1 1 1 2 2 Dist. 3 ** 28a 53b 73c 70c 70c

• * Entries in horizontal row followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 7, P < 0.01). ** Entries in horizontal rows followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 9, P < 0.01). t Entries in vertical columns followed by same number are not significantly different as indicated by analysis of variance (L.S.D.No dist v. Dist 1,2,3 = 10, L.S.D.Dist 1 v. Dist 2 v. Dist 3 = 10'p ^ °-01)- -__

• * Entries in horizontal row followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 6, P < 0.01). ** Entries in horizontal rows followed by the same letter are not significantly different as indicated by analysis of variance (L.S.D. = 9, P < 0.01). t Entries in vertical columns followed by same number are not significantly different as indicated by analysis of variance (L.S.D.No dist v. Dist 1,23 = 8> L.S.D.Dist 1 v. Dist 2 v. Dist 3 = 9,P < 0.01). Appendix V. Published papers

Aust. J. Bot.. 1991,39,357-64

Mycorrhizal Associations of Plant Species in Hawkesbury Sandstone Vegetation

S. E. Beilgard

Department of Biology, University of Wollongong, P.O. Box 1144, Wollongong, NSW 2500, Australia.

Abstract The mycorrhizal associations of plant species in an open woodland and heathland on Hawkesbury Sandstone soils were examined. The two geographically disjunct sites supported vegetation of differing physiognomy, but possessed many species common to both sites. At the woodland site, 21 of the 32 plant species examined had mycorrhizal associations. At the heath site. 31 of the 47 plant species examined were mycorrhizal. Mycorrhizal associations were found on representatives of the Cyperaceae and Proteaceae, families not previously thought to be mycorrhizal. Internal hyphae, vesicles, and cortical hyphal coils were discovered on the roots of two species of Cyperaceae and on the non-proteoid roots of nine species of the Proteaceae. Several species within genera and families previously known to be mycorrhizal were also found for the first time to have associations. Endomycorrhizal associations predominated at both sites, but several species had both ecto- and endomycorrhizal associations. The presence or absence of mycorrhizal associations was consistent on the roots of those plant species common to both sites examined.

Introduction Mycorrhizal associations are generally considered to be a mutualistic symbiosis, the principal benefit to the plant being increased nutrient uptake (Mosse 1973; Smith 1980; Schenck 1982; Bowen 1983; McGonigle and Fitter 1988). While the existence of these associations has been known for many years, their ecological significance in the life of plants growing in the field has only recently begun to be realised (Miller 1987). Currently, there is an appreciable amount of research being conducted on the potential role of mycorrhizae in the revegetation of disturbed landscapes (e.g. Daft and Nicolson 1974; Reeves et al. 1979; Allen and Allen 1980; Zak et al. 1982; Jasper et al. 1987; Stahl et al. 1988). It has been proposed that, because one of the primary stresses imposed on plants recolonising disturbed landscapes is the lack of nutrients (Bradshaw 1983), successful revegetation of disturbed landscapes may be dependent upon the availability of mycorrhizal inoculum (Loree and Williams 1987). This paper describes part of a large-scale investigation of the significance of mycor­ rhizae in the post-disturbance revegetation of Hawkesbury Sandstone vegetation. Before the significance of mycorrhizae in the revegetation of a disturbed landscape can be evaluated, it is necessary to have some idea of the mycorrhizal status of the plant species occurring naturally in the undisturbed ecosystem. The aims of the present survey were to: assess the mycorrhizal status of plant species in two examples of Hawkesbury Sandstone vegetation; and to observe whether the presence or absence of mycorrhizae was consistent in plant species common to the two study sites. In addition, this survey will extend the information on the mycorrhizal status of Australian plant species.

0067-1924/91/040357S05.00 358 S. E. Bellgard

Materials and Methods The southern part of the Hawkesbury Sandstone plateau, located 90 km south of Sydney and to the west of the city of Wollongong, covers an area of about 1200 km2. The long-term average annual precipitation in this region is 1420 mm, with a slight summer predominance in distribution (Anon. 1988). The study sites were located within the Avon (150°40'E., 34°22'S.) and O'Hares (150°53'E., 34° 14'S.) catchments. The vegetation of the Avon site was a woodland with an overstorey of Eucalyptus haemostoma Sm. and E. racemosa Cav. (Myrtaceae), and a shrub understorey. The soil at Avon had 30 cm of yellow clayey subsoil overlain by 20 cm of loamy sand topsoil (yellow earths, Gn2.21) (Hazelton and Tille 1990). The O'Hares site supported a sclerophyllous open heath dominated by Angophora hispida (Sm.) Blaxell (Myrtaceae). The soil at O'Hares was a loose shallow sand of 30 cm with abundant ironstone overlying the sandstone surface (lateritic-podzolic soils, Dy3.61) (Hazelton and Tille 1990). An initial vegetation survey was carried out in April 1989 to determine the plant species composition and the relative abundance of each species. Although the two sites supported vegetation of differing physiognomy, 14 plant species were common to both sites, as indicated by asterisks in Appendix 1. Voucher specimens are held by the herbarium at the Department of Biology, University of Wollongong. Using the point quadrat method, percentage foliar projection cover values were estimated (Goldsmith et al. 1986). Roots from five representatives of each plant species were collected from random locations within both of the study areas during July-September 1989. A hypothetical grid (IX 1 m) was laid over the study sites and 'northings' and 'eastings' were obtained from a table of random numbers. The individual of the targeted plant species lying closest to this random point was then sampled. At least 20 cm of fine feeder root was collected from each perennial plant species by tracing fine lateral roots from the stem to their termini. For annuals, entire plants were excavated, and the roots washed, sorted, and subsequently excised. Roots were initially fixed in 50% ethanol solution and assayed for mycorrhizal associations after clearing and staining as described by Kormanick et al. (1980). Following staining, roots were stored in a lactic acid-glycerol solution, and subsequently examined by both dissecting (10—tOx) and compound light (40-400X) microscopes. Nomenclature of the mycorrhizal associations follows McGee (1986). Where the term 'endomycorrhizae' has been used, it includes both vesicular-arbuscular mycorrhizae (VAM) and vesicular associations. In this study, a plant species was classified as mycorrhizal if at least three of the five root samples were colonised by mycorrhizae.

Results and Discussion Thirty-two plant species representing 13 families were examined for mycorrhizae within the Avon study site (Appendix 1). Of these species, 21 (66%) were mycorrhizal (Table 1). At the O'Hares site, 31 of the 47 (66%) species (representing 18 families, Appendix 1), were mycorrhizal (Table 1). Table 1. Numbers of plant species found with mycorrhizal associations at Avon (open woodland) and O'Hares (open heath) sites in the Hawkesbury Sandstone plateau Ecto = ectomycorrhizae; VAM = vesicular-arbuscular mycorrhizae; V = vesicular association

Mycorrhizal Numbers of plant species association Avon O'Hares

Ecto 6 9 VAM 7 11 V 3 6 Ecto + VAM 5 5 No mycorrhizae 11 16 Total 32 47

The proportion of plant species found in this survey to be mycorrhizal was generally lower than the proportions reported in previous surveys carried out in several other plant communities which range from 57 to 91% (Mejstrik 1972; Petersen et al. 1985; Ill

Mycorrhizae in Hawkesbury Sandstone Vegetation 359

Table 2. Plant species with previously unreported mycorrhizal associations Ecto = ectomycorrhizae; VAM — vesicular-arbuscular mycorrhizae; V = vesicular associations; * = family not previously reported to be mycorrhizal

Family Plant specie] Mycorrhizal Family Plant species Mycorrhizal association association

Apiaceae Actinoas htiumiki VAM Myrtaceae Angophora hispula Ecto Plaiysace linearifolia Ecto Baeckea ramoassima Ecto Cyperaceae* Caustis flexuosa VAM Eucalyptus haemostoma Ecto Ptilanthelium deustem V £ racemosa Ecto Dilleniaceae Hibbenia serpyilifolia VAM L stricta Ecto Epacridaceae Brachvioma daphnoida VAM Kunzea capitata Ecto Epochs microphylla VAM Leptospermum fhnescens Ecto Leucopogon jumperinus VAM L lanigerwn Ecto Euphorbiaceac Amperta xiphockda Ecto Proteaeceae' Conospermum longifolhm VAM Fabaceae Bossiaea obcordata VAM C taxi folium VAM Daviesia corymbosa VAM Grevtilea buxifoha V Dillwyinia parvifoiia var. Ecio + VAM G.oieoides V mcopoaa G. sphacelata V D. retorta Ecto Hakea daciyloides V Gompholobium grandiflorum VAM Persoonia levis V Pultenaea clliptica Ecto • VAM P. pinifolia V Goodeniaceae Dampiera stricta Ecto + VAM Telopea speciosissima VAM Mimosaceae Acacia linifolia Ecto • VAM Ruiaceae Eriostemon australasius VAM A. obmsifolia Ecto + VAM Boronia ihuiona VAM A.suaveoiens Ecto + VAM Thymeiaeaceae Pimelea linifolia VAM A. ulicifolia Ecio + VAM

McGee 1986; Brockoff and Allaway 1989; Logan et al. 1989). However, due to the spatial limitations of this survey and differences in the respective plant species composition of the communities surveyed, quantitative comparisons are difficult to make. Within both sites, ectomycorrhizae were discovered on some plant species not pre­ viously reported as having ectomycorrhizal associations (Table 2). However, all of the species belonged to either genera or families previously known to contain ectomycorrhizal species, i.e. Platysace, Apiaceae (Brockoff and Allaway 1989), Poranthera, Euphorbiaceac (Warcup 1980), Dampiera, Goodeniaceae (McGee 1986), Acacia, Mimosaceae (Warcup 1980), and Eucalyptus, Myrtaceae (Warcup 1980). The presence of ectomycorrhizae on all of the species of Myrtaceae investigated further supports the suggestion of Warcup (1980) that all Australian members of the Myrtaceae form ectomycorrhizae. VAM were also found on some plant species not previously recorded as being mycorrhizal (Table 2) but, in the same way as described for the new ectomycorrhizal associations discovered, these plant species belonged to genera and families previously known to have endomycorrhizal associations (e.g. Hibbertia, Dilleniaceae [Khan 1978], Leucopogon, Epacridaceae [Logan et al. 1989]). Endomycorrhizae were observed consistently in the roots of members of the Cyper­ aceae and Proteaceae (Table 2). These two families have previously been considered as non-mycorrhizal (e.g. Khan 1978; Malajczuk and Lamont 1981; Malajczuk et al. 1981). For the 11 species of Cyperaceae and Proteaceae identified as being mycorrhizal, all five of the root samples taken possessed internal hyphae and cortical coils. Both vesicles and arbuscules were seen in the roots of Caustis flexuosa but only vesicles in the roots of Ptilanthelium deustem. Within the Proteaceae, Conospermum longifolium, C. taxifolium and Telopea speciosissima (Fig. la) each had VAM, while the remaining six species had vesicular associations (Fig. lb). The nature and functional significance of atypical mycorrhizal associations such as vesicular associations remain uncertain (e.g. McGee 1986). 360 S. E. Beilgard

1 8«.l!,«rJ I T. %-.q, Fig. la. Root of Telopea speciosissima with multibranched arbuscie (,4). Scale: 17 ^m.

Harley and Smith (1983) suggested that there may be an ecological component which contributes to determination of the specificity and potential host recognition of mycor­ rhizal fungi. Plant species composition, soil conditions, soil microorganisms, and climatic conditions vary greatly between individual sites. It is reasonable to expect then, that selection of hosts by fungi may also vary between sites in accordance with the local site conditions. The current survey extends the range of Australian plant communities examined for mycorrhizal associations. Within each of the study sites, plant species with endomycorrhizal associations outnumbered the plants with ectomycorrhizal associations. The predominance of endo­ mycorrhizae agrees with previous surveys carried out in a range of other ecosystems (e.g. dune systems, Brockoff and Allaway 1989; semi arid, McGee 1986; arid, Beth­ lenfalvay et al. 1984; boreal, Malloch and Malloch 1981). At both sites ectomycorrhizae and VAM were found growing on the roots of the same plant. This phenomenon was restricted to five genera belonging to three families, i.e. Dillwynia. Mirbelia and Pultenaea (Fabaceae), Dampiera (Goodeniaceae) and Acacia (Mimosaceae). In other ecosystems, some members of these genera capable of forming and sustaining both forms of mycor­ rhizae have been found possessing just one type of mycorrhizae. This variability may be the result of site-specific seasonal and biotic factors. A study is currently underway investigating seasonal and spatial variation in the formation of mycorrhizae in the intact soil profile of these sites. 113

Mycorrhizae in Hawkesbury Sandstone Vegetation

Fig. lb. V = vesicle associated with the root of Persoonia levis. Scale: 44 rm.

The presence or absence of mycorrhizae and the form of the association, i.e. ecto- or endomycorrhizae, was consistent on the roots of the plants common to both sites. The two study sites were about 46 km apart and so, even with the spatial limitations of this survey, the consistency of these results suggests some spatial generality in the occurrence of mycorrhizal associations in the vegetation of Hawkesbury Sandstone soils.

Acknowledgments This research was supported both by a University of Wollongong Special Research Studentship and by the Sydney Water Board. My thanks go to Assoc. Profs R. J. Whelan and F. E. Putz, and Drs P. A. McGee, R. M. Muston and A. R. Davis, for commenting on the manuscript; Mrs J. Williams for assistance in identifying plant species; and Mr D. Lynch for assistance in the preparation and production of the photomicrographs. This is contribution Number 79 from the Ecology and Genetics Grou£ at the University of Wollongong. References Allen. E. B., and Allen, M. F. (1980). Natural re-establishment of vesicular-arbuscular mycorrhizae following strip mining reclamation in Wyoming. Journal of Applied Ecology 17, 139-47. Anon. (1988). 'Climatic Average Australia.' (Australian Government Publishing Service: Canberra.) Bethlenfalvay, G. J., Dakessian, S., and Pakovsky, R. S. (1984). Mycorrhizae in a southern Californian desert. Canadian Journal of Botany 62. 519-24. Bowen, G. D. (1983). Microbial determinants of plant nutrient uptake. In 'Soils — An Australian Viewpoint.'pp. 693-710. (CSIRO: Melbourne/Academic Press: Sydney.) Bradshaw, A. D. (1983). The reconstruction of ecosystems. Journal of Applied Ecology 20. 1-17. 362 S. E. Bellgard

Brockoff, J. O., and Allaway, W. G. (1989). Vesicular-arbuscular mycorrhizal fungi in natural vegetation and sand-mined dunes at Bridge Hill, New South Wales. Wetlands 8(2), 47-54. Daft, M. J., and Nicolson, T. H. (1974). Arbuscular mycorrhizas in plants colonizing coal wastes in Scotland. New Phytologist 73, 1129-38. Goldsmith, F. B., Harrison, C. M., and Morton, A. J. (1986). Description and analysis of vegetation. In 'Methods in Plant Ecology.' (Eds P. D. Moore and S. B. Chapman.) pp. 437-524. (Blackwell Scientific Publications: Oxford.) Harley, J. L., and Smith, S. E. (1983). 'Mycorrhizal Symbiosis.'(Academic Press: London.) Hazelton, P. A., and Tille, P. J. (1990). 'Soil Landscapes of the Wollongong-Port Hacking 1:100000 Sheet.* (Soil Conservation Service of NSW: Sydney.) Jasper, D. A., Robson. A. D., and Abbott. L. K. (1987). The effect of surface mining on the infectivity of vesicular-arbuscular mycorrhizal fungi. Australian Journal of Botany 35, 641-52. Khan. A. G. (1978). Vesicular-arbuscular mycorrhizas in plants colonizing black wastes from bituminous coal mining in the Illawarra region of New South Wales. Sew Phytologist 81, 53-63. fCormanick, P. P., Bryan, W. C, and Schultz. R. C. (1980). Procedures and equipment for staining large numbers of plant root samples for endomycorrhizal assay. Canadian Journal of Microbiology 26, 536-8. Logan, V. S., Clarke, P. J., and Allaway, W. G. (1989). Mycorrhizas and root attributes of plants of coastal sand-dunes of New South Wales. Australian Journal of Plant Physiology 16. 141-6. Loree, M. A. J., and Williams, S. E. (1987). Colonization of western wheatgrass (Agropyron smithii Rydb.) by vesicular-arbuscular mycorrhizal fungi during the revegetation of a surface mine. New Phytologist 106, 735-44. Malajczuk, N., and Lamont, B. B. (1981). Specialized roots of symbiotic origin in heathlands. In 'Mediterranean Type Ecosystems of the World Vol II'. (Eds F. Di Castri, D. W. Goodall and R. L. Specht.) pp. 165-86. (Elsevier New York.) Malajczuk. N., Linderman, R. G., Kough, J., and Trappe. J. M. (1981). Presence of vesicular-arbuscular mycorrhizae in Eucalyptus spp. and Acacia sp.. and their absence in Banksia sp. after inoculation with Glomus fasciculatus. New Phytologist 87, 567-72. Malloch, D., and Malloch, B. (1981). The mycorrhizal status of boreal plant species from north-eastern Ontario. Canadian Journal of Botany 59. 2167-72. McGee, P. A. (1986). Mycorrhizal associations of plant species in a semiarid community. Australian Journal of Botany 34. 585-93. McGonigle, T. P., and Fitter, A. H. (1988). Growth and phosphorus inflows of Trifolium repens L. with a range of indigenous vesicuiar-arbuscular mycorrhizal infection levels under field conditions. New Phytologist 108, 59-65. Mejstrik, V. K. (1972). Vesicular-arbuscular mycorrhizas of the species of a Molinietum coerulae L.I. association: the ecology. New Phytologist 71, 883-90. Miller, R. M. (1987). Mycorrhizae and succession. In 'Restoration Ecology: A Synthetic Approach to Ecological Research'. (Eds W. R. Jordan III, M. E. Gilpin and J. D. Aber.) pp. 205-20. (Cambridge University Press: Cambridge.) Mosse, B. (1973). Advances in the study of vesicular-arbuscular mycorrhizas. Annual Review of Phy­ topathology 11, 170-96. Peterson, R. L., Ashford, A. E., and Allaway, W. G. (1985). Vesicular-arbuscular mycorrhizal associations of vascular plants on Heron Island, a Great Barrier Reef coral cay. Australian Journal of Botany 33. 677-85. Reeves, F. B., Wagner, D., Moorman, T., and Kiel, J. (1979). The role of endomycorrhizae in revegetation practices in the semi-arid west.- I. A comparison of incidence of mycorrhizae in severely disturbed vs. natural environments. American Journal of Botany 66. 6-13. Schenck, N. C. (1982). Introduction. In 'Methods and Principles of Mycorrhizal Research'. (Ed. N. Schenck.) pp. ix-x. (The American Phytopathological Society: St. Paul, Minnesota.) Smith, S. E. (1980). Mycorrhizas of autotrophic higher plants. Biological Reviews 55, 475-510. Stahl, P. D., Williams. S. E., and Christensen, M. (1988). Efficacy of native vesicular-arbuscular mycorrhizal fungi after severe soil disturbance. New Phytologist 110, 347-54. Warcup, J. H. (1980). Ectomycorrhizal associations of Australian indigenous plants. New Phytologist 85,531-5. Zak. J. C, Danielson, R. M.. and Parkinson, D. (1982). Mycorrhizal fungal spore numbers and species occurrence in two amended mine spoils in Alberta. Canada. Mycologia 74, 785-92. Mycorrhizae in Hawkesbury Sandstone Vegetation 363

Appendix 1. Mycorrhizal associations of plants from dry sclerophyllous Hawkesbury Sandstone vegetation within the Avon and O'Hares Catchments, New South Wales The values represent the number of root samples out of five possessing an association (% f.c. = % foliar projection coven Rank in % f.c. - ranking based on % foliar projection cover, Ecto = ectomy­ corrhizal association: I.h. = internal hyphae: V = vesicles: A = arbuscules; Cc. = cortical coils; * = plant species common to each of the sites)

Family Plant species

Avon Apiaceae Platysace iineanfolia I Cav.) C. Norman * 0-8 16 5 0 0 0 0 Cyperaceae Ptilanthelium deustem (R. Br.) Kukenth.* :•! 8 0 5 3 0 5 Euphorbiaceae Ampereaxiphoclada (Sieb. ex Spreng.) Druce 1-0 15 4 0 0 0 0 Poranthera corymbosa Brongn. 16 12 4 0 0 0 0 Fabaceae Bossiaea obcordaia 1 Vent.) Druce 44 3 0 5 5 5 5 Diilrvyma parnfoiia var. incopoda Blakely 0-4 [7 5 5 5 5 5 Gompholobium grandiflorum Sm. 26 9 0 4 5 5 S Goodeniaceae Dampiera stricta (Sm.) R. 3r." 0-8 16 5 5 5 5 5 Ihdaceae Paursonus sencea R. Br. i-t 3 0 0 0 0 0 Lindsaeaceae Lindsaea linearis Sw.* :o II 0 0 0 0 0 Mimosaceae Acacia linifolia (VenU Willd. 28 3 5 4 5 5 3 A. obiusifolia A. Cunn. 3-0 7 5 5 5 5 5 A. ulicifolia I Salisb.) Court :-4 10 5 5 5 5 5 Myrtaceae Eucalyptus haemastoma Sm. 19-6 I 5 0 0 0 0 £ racemosa Cav. 10-2 2 5 0 0 0 0 Leptospermum flavescens Sm. 4-2 5 0 0 0 0 Proteaceae Banksia spinulosa Sm.' 3-2 0 0 0 0 0 Grcvtilea sphacelaia R. Br. 3-4 0 5 3 0 5 Hakeadactyloides (Gaertn.) Cav.* 14 10 0 5 3 0 5

Isopogon antmomfotius (Salisb.) Knight' :•« a 0 0 0 0 1. anethifolius iSalisb.) Knight 1-2 14 0 0 0 0 0 Lambertia formosa Sm.* 4-2 0 0 0 0 0 Lomatia silaifolia \Sm.) R. Br.* 1-6 12 0 0 0 0 0 Persoonia levis (Cav. 1 Domin* 2-8 0 0 Telopea speciosissima R. Br. 24 10 0 5 Rutaceae Eriostemon ausiraiasms Pen.' 1-6 12 0 5 Boronia thu/ona Penloid et Welch 14 13 0 5 Thyraeiaeaceae Pimelea linifolia Sm. 0-2 18 0 5 Xanthorrhoeaceae Lomandraconfenifolia[F. M. Bailey) Farm* 44 0 0 0 0 0 L glauca (R. Br.) Ewart* 14 13 0 0 0 0 0 L obliqua (Thunb.) MacBride* 26 9 0 0 0 0 0 Xanthorrhoea arborea R. Br. 3-2 6 0 0 0 0 0 O'Hares Apiaceae Actinolis heiianthi LabilL 1-4 18 0 5 5 5 5 Platysace linearifolia (Cav.) C. Norman 14 18 5 0 0 0 0 Cyperaceae Causta flexuosa R. Br. 1-6 17 0 5 4 3 5 Ptilanthelium deustem IR. Br.) Kukenth. 2-2 12 0 5 4 0 5 Dilleniaceae Hibbertia serphytlifolia R. Br. ex DC. 21 13 0 5 5 5 5 Droseraceae Drosera pygmaea DC. 1-7 16 0 0 0 0 0 Epacndaceae BrachylomadaphnotdesiSm.) Benin. 2-8 10 0 5 4 5 5 Epochs microphylla R. Br. 1-7 16 0 5 5 4 5 Leucopogon juntpennus R. Br. 2-2 12 0 4 5 5 5 Fabaceae Daviesia corymbosa Sm. 2-2 12 0 5 5 5 5 Dillwvnta retorta (WendU Druce 14 18 5 0 0 0 0 Mirbetia rubiifolia

Appendix 1 (continued)

Family Plant species ^ f.c. Ranic Ecio l.h. V A Cc. in % f.c.

Myrtaceae Angophorahlspida(Sm.) Blaxell 9-3 1 5 0 0 0 0 Baeckea ramosissima A. Cunn. 29 9 5 0 0 0 0 Eucalyptus haemasioma Sm. 3-2 7 5 0 0 0 0 £ smcta Sieb. ex Spreng. 3-1 8 5 0 0 0 0 Kunzea capuata Reichb. 3-8 4 5 0 0 0 0 Lepiospermum /umpennum Sm. 0-9 21 5 0 0 0 0 L lamgerum i Ait.) Sm. 20 14 5 0 0 0 0 Olacaceae Olax smcta R. Br. 0-7 -*-> 0 0 0 0 0 Proteaceae Banksia ericifolia L I. [-3 19 0 0 0 0 0 8. paludosa R. Br. 1-0 20 0 0 0 0 0 8. serrata L. f. 0-7 *n 0 0 0 0 0 3. spinulosa Sm. 10 20 0 0 0 0 0 Conospermum longifolium Sm. 20 14 0 5 5 - C. laxifolium Sm. 10 20 0 5 5 5 Grevtllea buxifolia (Sm.) R. Br. 1-3 19 0 5 0 5 G. oteoides Sieb. ex Schult ei i. 20 14 0 5 0 ^ ffafaa dactylotdes (Gaenn.i Cav. 3-3 6 0 3 0 5 ff. teretifolla I Salisb.) J. Bntten 1-7 16 0 0 0 0 0 Isopogon anemomfolius (Salisb.) Knight 10 20 0 0 J 0 0 Lambertia formosa Sm. 4-2 3 0 0 0 0 0 Lomatia silaifolia (Sm.) R. Br. 1-7 16 0 0 0 0 0 Persoonia levis (Cav.) Domin 1-7 16 0 5 4 0 5 P. pmifolia R. Br. 1-9 15 0 5. 3 0 5 P. /aunna Pers. 1-4 18 0 0 0 0 0 Peirophilepulchella iSctimi.) R. Br. 28 10 0 0 0 0 0 Rutaceae Eriostemon austratasius Pers. ri 12 0 5 5 5 5 Xanthorrhoeaceae Lomandraconferttfolia[F. M. Bailey) Farm 2-7 11 0 0 a 0 0 i. ^/aucj 1R. Br.) Ewart 20 14 0 0 0 0 0 i. oM/aua(Thunb.) MacBnde 0-6 23 0 0 0 0 0

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Bellgard, S.E. 1992, The propagules of vesicular-arbuscular mycorrhizal (VAM) fungi capable of initiating VAM infection after topsoil disturbance, Mycorrhiza, 1:147-152.