the role of arbuscular mycorrhizal fungi in uptake by

The Role of Arbuscular Mycorrhizal Fungi in Uptake by

Pasture Legumes of Phosphorus Provided as a Pulse

Nazanin K. Nazeri

Plant Pathology (Hons)

Plant Virology (MSc)

This thesis is presented for the degree of

Doctor of Philosophy of

The University of Western Australia

School of Plant Biology

School of Earth and Environment

December 2014

i

Abstract

In the southern Australian cropping zone, large areas of land contain legume-based

pastures; these provide feed for livestock and nitrogen for following crops. Annual

pastures, especially of Trifolium subterraneum (subterranean clover), are widely sown

in Mediterranean climates around the globe and particularly in southern Australia.

However, the predominance of annual pastures and crops in south-western Australia has

caused problems, including dryland salinisation. Consequently, the development of

novel perennial pasture plants, including Australian natives, has been a research priority

over the past 15 years.

Phosphorus fertiliser is a common input to farms in southern Australia and its use has

much increased soil P concentrations. However, rock phosphate is a non-renewable

resource and it is now more important to use it efficiently. Forming a symbiosis with

arbuscular mycorrhizal fungi (AMF) is an important means for plants to enhance their P

uptake, primarily because their external hyphae are able to explore a greater volume of

soil than plant roots can alone. Mycorrhizal experiments are usually conducted under

constant or decreasing P levels due to plant P uptake. However, few studies have

focused on the response of colonised plants to a pulse of P as may occur under field

conditions such as when the first winter rain falls after a hot dry summer or when P

fertilisers are applied to growing plants. To my knowledge, this study is the first to

concentrate on the role of AMF in plant P uptake from a P pulse. Care was taken to

ensure that results were relevant to field conditions and therefore the soil used in all

experiments was a field soil with a moderate concentration of plant available P (18 mg

kg-1), as is common in the cropping areas of Western Australia.

In the first experiment (Chapter 2), the role of AMF in P uptake from a pulse of P was

investigated. Since Trifolium subterraneum is commonly used in mycorrhizal

experiments and its response to varying levels of P in soil has been investigated before,

it was used as the host plant for this experiment. The following hypotheses were

addressed: 1) AMF will enhance uptake of P from a pulse as may occur following

addition of fertiliser or the start of the winter rain; 2) uptake of P is greater when plants

are colonised by a diverse community of AMF (inoculation with field soil), rather than a

single-species inoculum; and 3) presence of microbes in the soil increases P uptake

compared to the control (no microbes). Plants were grown in field soil (+AMF) or in

sterilised field soil (-AMF) with the addition of filtrates (microbes) from the unsterilised

ii

field soil, except for the control (no AMF, no microbes). Plants were harvested at week

11, a day before (day 0), a day after (day 2) and seven days after (day 8) a pulse of P (10

mg kg-1 as KH2PO4). Shoot and root dry weights did not differ among treatments. At

day 0, both shoots and roots of colonised plants had higher P concentrations than did

those of uncolonised plants, as expected. However, after the pulse, the rate of increase

of shoot P concentration in colonised plants was slower than that in uncolonised plants,

and by day 8, colonised and uncolonised plants had a similar shoot P concentration; root

P concentration remained higher in the colonised plants. Thus, the presence of AMF

appeared to restrict the movement of P from the root to the shoot after P was added in

the pulse, i.e. AMF had a “moderating” effect. Little difference in P uptake was found

between plants that were colonised by a diverse community of AMF (originating from

the field soil) and plants that were inoculated by a single species. However, the results

from a commercial DNA test of the field soil showed that more than 97% of AMF

present in the soil belonged to one family. Thus, the field soil may not have represented

a “diverse” treatment. Alternatively, diversity of AMF may not have been of advantage

to plant P uptake due to the moderate soil P concentrations. The effect of the treatment

with soil microbes on plant P uptake was rarely different from that of the control.

Again, this might reflect the moderate concentrations of soil P; experiments that report a

positive effect from the microbe treatments generally have lower concentrations of soil

P.

In the second experiment (Chapter 3), the effect of AMF on the response to a pulse of P

of three (potential) perennial pasture legumes (Kennedia prostrata, Cullen

australasicum, Bituminaria bituminosa), the commonly grown perennial pasture species

Medicago sativa (lucerne) and Trifolium subterraneum was investigated. The following

hypotheses were addressed: 1) AMF will moderate shoot P concentration following a

pulse; 2) the release of carboxylates into the rhizosphere (which is a mechanism used by

plants to mobilise soil P) will decrease following a pulse; and 3) colonisation of plants

by AMF will decrease the release of carboxylates into the rhizosphere. Plants were

grown in the same field soil with a moderate P concentration (+AMF) or in sterilised

field soil (-AMF), all with the addition of filtrates (microbes) from the unsterilised field

soil. After approximately 10 weeks, half of the pots received a pulse of P (15 mg kg-1).

All plants were harvested one week later. Results were consistent with those of

experiment 1; that is, there was a moderating effect of AMF on plant shoot P

concentration following a P pulse. It was also found that the P pulse did not have any

iii

effect on the amount of rhizosphere carboxylates. However, colonisation by AMF

reduced the amount of rhizosphere carboxylates by ~50%, providing strong

confirmation of the concept of “mycorrhizal-mediated resource partitioning” which was

recently presented in the literature.

The main focus of the third experiment (Chapter 4) was to test the hypothesis that

following a significant pulse of P, P movement to shoots is restricted due to storage of

high concentrations of P in intraradical AM hyphae in insoluble forms. In this

experiment, two P pulse levels (150 mg kg-1, 15 mg kg-1 and control (No P pulse)) were

applied a week before harvest to Trifolium subterraneum either inoculated with

Scutellospora calospora (+AMF) or grown in sterilised field soil (-AMF); unsterilised

field soil filtrates were added to all pots. All plants were harvested at week seven. A few

samples were chosen randomly for X-ray microanalysis after being freeze-substituted in

a 10% acrolein in diethyl ether mixture over a 0.3 nm molecular sieve and embedded in

Araldite 502 epoxy resin. Due to low levels of colonisation by AMF, and the ability to

only examine a small number of samples with this technology, samples that were

prepared for X-ray analysis were free of AMF and testing of the hypothesis was not

possible. However, other interesting results were obtained. Plants that received the high

P pulse (150P) had root P concentrations of 20 mg g-1 and granules with extraordinarily

high P concentrations in their cortex (up to 7640 mmol P kg-1). There was a very strong

positive linear relationship between P and K concentrations in the granules. Formation

of granules with high P concentrations might be a plant response to avoid P toxicity

when encountering a sudden availability of high concentrations of P. The structure of

the granules, the relationship between P and K concentrations, and the formation of the

granules in response to a P pulse are all consistent with the formation of polyphosphate

in organisms such as fungi and yeast. However, such high concentrations of

polyphosphate have not previously been credibly reported in plants. Further research is

now needed to confidently ascertain that the granules were not an artefact of the

embedding process and whether polyphosphate is indeed their major constituent.

In summary, this thesis shows that AMF play an additional role to that of enhancing

plant P acquisition when P is limiting, i.e. they also allow plants to maintain leaf P

concentrations within narrower boundaries than the plant could achieve alone when the

plant experiences a pulse of P.

v

Table of Contents

Abstract I

Table of Contents V

Acknowledgments VII

Thesis Declaration and Publication List IX

CHAPTER 1. General Introduction 1

1.1 IMPORTANCE OF PASTURES LEGUMES FOR FARMING SYSTEMS 2

1.1A Bituminaria bituminosa 5

1.1B Cullen australasicum 5

1.1C Kennedia prostrata 6

1.2 PHOSPHORUS IN PLANTS AND SOIL 7

1.2A Phosphorus pulse 8

1.2B Responses of plants to a pulse of P 9

1.3 ARBUSCULAR MYCORRHIZAL FUNGI (AMF) 10

1.3A The growth response of plants to colonisation by AMF 11

1.3B How does AMF help plants to access P? 13

1.3C Phosphate translocation within hyphae and delivery to intraradical interfaces 14

1.3D Transfer of P from AM fungal structures to the root cell 15

1.3E Phosphorus pathways in colonised plants 16

1.3F Role of AMF in uptake of a pulse of P 17

1.3G Which is a better approach in glasshouse experiments? A diverse community of AMF or a single-species inoculum? 17

1.4 GAPS IN KNOWLEDGE 19

1.5 RESEARCH OBJECTIVES 19

1.6 THESIS OUTLINE 20

1.7 REFERENCES 21

CHAPTER 2. Do Arbuscular Mycorrhizas or Heterotrophic Soil Microbes Contribute

toward Plant Acquisition of a Pulse of Mineral Phosphate? 35

2.1 PREFACE 35

2.2 PUBLISHED PAPER 36

CHAPTER 3. Moderating Mycorrhizas: Arbuscular Mycorrhizas Modify Rhizosphere

Chemistry and Maintain Plant Phosphorus Status within Narrow Boundaries 49

3.1 PREFACE 49

3.2 PUBLISHED PAPER 50

CHAPTER 4. A Sudden, High Pulse of Phosphorus Triggers Formation of Granules with

Extraordinary High Concentrations of Phosphorus and Potassium in Cortical Cell

Vacuoles of Trifolium subterraneum 63

4.1 PREFACE 63

4.2 READY FOR SUBMISSION PAPER 64

ABSTRACT 65

INTRODUCTION 66

MATERIALS AND METHODS 67

Glasshouse experiment 67

Sample preparation for X-ray microanalysis 69

Quantitative X-ray microanalysis 71

vi

RESULTS 72

Plant growth 72

Microscopy and quantitative X-ray microanalysis 73

Elemental analysis 75

DISCUSSION 77

A pulse of a high P concentration induces granules high in [P] in the root 77

What is the form of P in the granules? 80

CONCLUSION 81

ACKNOWLEDGEMENTS 82

REFERENCES 82

CHAPTER 5. General Discussion 89

5.1 INTRODUCTION 90

5.2 KEY RESEARCH FINDINGS 90

5.2A Phosphorus uptake from a pulse is enhanced in the roots of colonised plants, but P transport to the shoots is restricted by AMF (after a week) (moderating effect) 91

5.2B Phosphorus uptake did not differ between a diverse community of AMF and a single species culture 93

5.2C Microbes had little effect on uptake of P from a pulse 94

5.2D A pulse of P did not decrease the amount of rhizosphere carboxylates 95

5.2E Colonisation with AMF decreased rhizosphere carboxylates 96

5.2F Formation of granules with high concentrations of P in the cortex area of the roots of Trifolium subterraneum which were treated with a high pulse of P 97

5.3 UNEXPECTED FINDINGS 98

5.3A Inoculation with AMF greatly decreased manganese concentrations 98

5.3B Phosphorus efflux following colonisation with AMF 99

5.3C Colonisation with AMF makes the rhizosphere more alkaline 99

5.4 LIMITATIONS OF THE RESEARCH PRESENTED IN THIS THESIS 100

5.5 FUTURE RESEARCH DIRECTIONS 100

5.6 CONCLUDING REMARKS 100

5.7 REFERENCES 101

vii

Acknowledgments

Four years ago, when I started my PhD journey, having my one and half year old son, I

was not sure if I would be able to reach the end. Having the support and encouragement

of the following people made it all possible.

First of all, I would like to thank my supervisor Associate Professor Megan Ryan who

was not only a supervisor to me but also a friend whom I could talk to when there was

no one else around to listen to my complaints. She will always be my role model in

research. She taught me that doing a PhD is not about doing something impossible, but

it is all about telling an interesting story in science. I also had the honour of having

W/Prof. Hans Lambers as my supervisor. Prof. Lambers’ discipline always amazed and

inspired me. I wish I could send his records of giving precise feedback on my

manuscripts in the shortest time possible to the Guinness Book of Records. I am quite

sure he would be in the top three. I would also like to thank Professor Mark Tibbett, my

third supervisor, for his comments. Finally, thanks to Associate Professor Peta Clode for

guiding me in using X-ray microanalysis and sample preparation and providing

feedback on chapter 4.

I have to declare that I am very lucky to have someone to support me during all hard

times of my life and putting himself behind me without any expectations. My deepest

gratitude to my husband and my son who were always there for me and my life is

meaningless without them. No words can express my gratitude to my life time

supervisors, my mom and dad. Thank you for visualising my dream for me every day of

my life. Being apart from you and your kindness is one of the toughest things I have to

overcome every day.

I want to also thank Evonne Walker and Daniel Kidd for providing me with technical

help and Dion Nicol for collecting soil samples for me from the Newdegate site. Thanks

to Michael Smirk and Elizabeth Halladin for helping me with plant analysis and to Greg

Cawthray for providing help in analysing carboxylates of rhizosphere extracts using

HPLC and for helping me to understand chemistry concepts. Also, thanks to Scott

Glyde for his support. Finally, I also would like to especially thank all my office mates

who made this journey more peaceful by sharing our happy and sad days together.

This research has been financially supported by an Australian Postgraduate Award, the

Future Farm Industries Cooperative Research Centre and a UWA PhD Completion

viii

Scholarship.

I would also like to acknowledge the facilities, and the scientific and technical

assistance of the Australian Microscopy & Microanalysis Research Facility at the

Centre for Microscopy, Characterisation & Analysis, The University of Western

Australia, a facility funded by the University, State and Commonwealth Governments.

ix

Thesis Declaration and Publication List

This thesis is presented as a series of two scientific papers and one manuscript to be

submitted later. The bibliographical details are as follows.

Chapter 2: Nazeri, Nazanin K, Lambers, H; Tibbett, M and Ryan, M. (2013) Do

arbuscular mycorrhizas or heterotrophic soil microbes contribute toward plant

acquisition of a pulse of mineral phosphate? Plant and Soil, 373, 699-710.

Chapter 3: Nazeri, Nazanin K, Lambers, H; Tibbett, M and Ryan, M. (2013)

Moderating mycorrhizas: arbuscular mycorrhizas modify rhizosphere chemistry and

maintain plant phosphorus status within narrow boundaries. Plant, Cell and

Environment 37: 911-921.

Chapter 4: Nazeri, Nazanin K, Clode, P; Lambers, H; Tibbett, M and Ryan, M. (2014)

A sudden high pulse of P triggers formation of granules with extraordinary high

concentrations of phosphorus and potassium in cortical cell vacuoles of Trifolium

subterraneum (in preparation for submission to New Phytologist).

My contribution in each paper is 85% or above. I designed and performed the

experiments, analysed the results and wrote the papers. The contribution of the co-

authors in each paper/chapter is mainly related to advice on the design of the experiment

analysis of results when required, and review of the drafts of the papers/chapters.

Nazanin Nazeri

July 2014

Chapter 1. General Introduction 1

CHAPTER 1.

General Introduction

This chapter introduces the importance of pasture legumes for the productivity and

sustainability of farming systems in Australia and the effect that phosphorus (P)

limitation in Australian soils has on plant growth. The low P concentration of

Australian native soils has resulted in farmers routinely applying P fertilisers. It is

important, if this limited resource is to be used efficiently, that plants are able to quickly

take up the pulse of P that results from application of P fertiliser before the P is sorbed

to become poorly available in the soil. The symbiosis between plants and arbuscular

mycorrhizal fungi (AMF) is common in pasture legumes and is possibly one way that

absorption of a pulse of P could be enhanced. This thesis characterises the role of AMF

in uptake of P delivered as a pulse to pasture legumes.


1.1 Importance of pastures legumes for farming systems

Pasture legumes have historically been important in maintaining productivity in

Mediterranean agricultural systems, which include areas at middle latitudes in all

continents (between 30-40°) and in Australia in south-western and central parts of

southern Australia (Saxena 1988). In 2006, the temperate wheat-sheep zone in southern

Australia contained 39,500 broadacre farms (ABARE 2006; Kirkegaard et al. 2011),

many of which grew both pastures and crops. In addition to grazing livestock,

incorporating pasture legumes into farming systems has the important benefit of fixing

atmospheric nitrogen (N) through the symbiosis formed between legumes and soil

bacteria (Rhizobium/Bradyrhizobium spp.). The majority of N2 fixed in Mediterranean

agriculture is due to N-fixing legumes (Howieson et al. 2000). According to Peoples

and Baldock (2001), Australian pastures fix from 2 to 284 kg N ha-1 yr-1. Moreover,

pasture legumes also break the life cycle of cereal diseases when grown in rotation with

crops (Howieson et al. 2000; Robson 1990) and provide benefits from enterprise

diversification for farmers (Kirkegaard et al. 2011).

Annual pastures have an important role in agriculture in southern Australia. Ninety-two

million ha of land are under self-regenerating annual pastures, most of which are in

rotation with cereal crops (ley-farming) (Reeves and Ewing 1993; Unkovich 2002).

Longer cropping phases require sowing of annual legumes following crops (phase

farming). Ley farming was developed in South Australia in the 1950s based on the use

of Trifolium subterraneum (subterranean clover) and annual medics (Medicago spp.)

(Callaghan 1958; Donald 1965). Because of reliance on N2 fixation from annual pasture

legumes, it is recognised as one of the “advanced and low-input systems” (Robson

1990). Trifolium subterraneum is one of the dominant pasture species in Mediterranean

climates across the globe and is the most widely sown annual pasture in southern

Australia due to its high N-fixation, ease of management including suitability for

continuous grazing, adaptation to a wide range of growing season lengths, a moderate

level of seed dormancy enabling germination after 1-2 years of cropping and high-

quality forage (Hill and Donald 1998; Nichols et al. 2013; Porqueddu and González

2011).

The success of annual legume-based ley pastures in the southern Australian cropping

zone, in conjunction with annual crops such as Triticum aestivum (wheat), Brassica

napus (canola) and Hordeum vulgare (barley), means that Australian dryland cropping


systems are very much dominated by annual plants - crops and pastures. Although these

systems based on annuals have been very successful in the past, they now face a number

of challenges including herbicide resistance in weeds and salinisation (Cocks 2001;

Dear et al. 2008; Powles and Matthews 1992).

The predominance of annual crop and pasture species has contributed to salinisation,

because the annuals are unable to transpire the same amount of water as did the native

perennial vegetation, due to shallower roots and a shorter (winter-spring) growing

season (i.e. 7-8 months) (Lambers 2003). This lower transpiration has caused increased

rates of deep drainage, which, in turn, has raised water tables which has mobilised salt

stores and caused dryland salinity (Dear et al. 2003; Dolling 2001; Loi et al. 2005;

Ridley et al. 1997). Consequently, planting deep-rooted perennial pasture species is one

of several ways now recommended for the control of dryland salinity in southern

Australia and indeed perennial pastures are becoming more popular (Cocks 2001; Dear

and Ewing 2008; Dunin 1970; Small 2003). The need to address dryland salinity was

the primary impetus for a major research focus on development of new perennial

species over the last 15 years (Dear and Ewing 2008). Deep-rooted perennials have the

ability to dry the subsoil during spring and summer, creating a dry soil buffer, which

can subsequently reduce leakage into groundwater by absorbing unused rainfall during

winter when plant demand and evaporation are low. For instance, according to Ward et

al. (2002), in south-western Australia during summer and autumn, soil water content

was reduced by 60 mm more by a perennial pasture (Medicago sativa) than by an

annual pasture (T. subterraneum), with most of the additional water used by the

perennial drawn from soil below a depth of about 60 cm. Such effects could prevent or

considerably delay the onset of secondary salinity when rainfall is sufficient to cause

leakage from a system based on annuals. The perennials that can be utilised in this

manner include a diversity of plant forms ranging from trees and shrubs to herbaceous

forage plants (Cocks 2001).

Perennial legume pastures provide a number of benefits to farming systems in addition

to symbiotically fixing atmospheric N2. These include maintenance of plant cover in

summer/autumn when annual pastures have senesced, thereby reducing wind erosion,

and runoff and leaching of nutrients (Bell et al. 2010; Glover 2005). However, the major

impetus for adoption of new perennial pasture species appears to be an ability to

produce green feed in the summer/autumn when annual pastures have senesced and are

present only as dry residues or a soil seed bank (Finlayson et al. 2012). Summer-green


feed increases farm profits through reducing purchase of supplementary feed and

allowing a high year-round stocking rate (Monjardino et al. 2010; Nicol et al. 2013).

Benefits for animal health and improvements in product quality may also result, for

example, improved wool staple strength (Adams and Briegel 1998).

Currently, the only perennial legume widely grown in the southern cropping zone is the

exotic M. sativa. M. sativa is able to extract water from deeper parts of the soil than

annual pastures do, and can utilise summer/autumn rainfall (Bell et al. 2006). However,

adoption of M. sativa is not particularly high, especially in Western Australia (Cocks

2001). Poor adaptation to acid soils, waterlogging and drought can all contribute to poor

performance (Dolling et al. 2005; Hill 1996; Humphries and Auricht 2001).

In response to the problems associated with M. sativa, the CRC for Plant-Based

Management of Dryland Salinity and its successor, the Future Farm Industries (FFI)

CRC, focused on finding new perennials. Since 2000, about 720 species of legumes,

grasses and herbs have been screened for their potential to be developed as pastures for

temperate southern Australia by researchers associated with these two CRCs (Li et al.

2008; Nichols et al. 2007). Interestingly, this screening included some species native to

Australia (Ryan et al. 2008; Snowball et al. 2010). The potential of Australia’s native

flora for use in agriculture has been remarkably under-explored (Bell et al. 2010;

Hopper and Lambers 2014). Yet, Australia, because of its arid climate and infertile

soils, is an excellent place to search for potential new perennial pasture plants and the

development of deep-rooted perennials adapted to these conditions should be a high

priority for future research in the Australian wheatbelt (Dear et al., 2003).

As a result of the activities of the two CRCs, in 2007, the exotics Bituminaria

bituminosa var. albomarginata and Bituminaria bituminosa var. crassiuscula were

identified as the perennial legumes that showed the greatest potential for domestication

(Real et al. Accessed in 2014). Bituminaria bituminosa then became one of the research

priorities for the FFI-CRC. Native species from the genus Cullen were also found to be

promising (Real et al. 2011).

In response to the findings and priorities of the FFI-CRC, I focused my research on the

exotic perennial legumes Bituminaria bituminosa var. albomarginata (tedera) and M.

sativa, along with the native perennial legumes Cullen australasicum and Kennedia

prostrata. The exotic annual pasture legume T. subterraneum was included for

comparative purposes. The perennial species are now introduced in more detail.


1.1A Bituminaria bituminosa

Bituminaria bituminosa (L.) C.H. Stirt. var. albomarginata is a self-pollinated

herbaceous, drought-resistant perennial forage legume with a wide distribution along

both sides of the Mediterranean sea and Macronesian islands. There are three botanical

varieties on the Canary Islands, of which two (albomarginata and crassiuscula) are

deemed eligible to enter into Australia by the Australian and Western Australian

Quarantine and Inspection Services. The albomarginata variety (common name tedera)

is originally from Lanzarote and is the most drought-resistant variety. It can acclimate to

a diverse range of soil types. This variety has the ability to stay green without shedding

its leaves during summer/autumn in dry Mediterranean climates (Méndez et al. 1991).

For example, during two of the driest summers/autumns on record in Western Australia,

B. bituminosa stayed green while M. sativa did not (Real and Verbyla 2010). It also

tolerates grazing and competes well with annual species (Suriyagoda et al. 2013). B.

bituminosa shows potential to be developed as out-of-season forage production across

all rainfall zones in Australia due to its high nutritional value and high biomass

production with low water demand (Foster et al. 2013; Martínez-Fernández et al. 2010;

Real and Verbyla 2010; Ventura et al. 2009). Foster et al. (2012) found significant

differences under water stress in stomatal conductance and also rooting depth of B.

bituminosa seedlings in comparison with seedlings of M. sativa and biserrula (Biserrula

pelecinus L.) and Afghan melon (Citrullus lanatus Thunb.) and concluded B.

bituminosa is the most drought resistant of these species. In field experiments in

Western Australia, Suriyagoda et al. (2013) found that the performance of B. bituminosa

was better than that of C. australasicum in terms of herbage productivity and survival.

1.1B Cullen australasicum

There are a total of 32 species in the Cullen genus and these are found in Africa,

Australia, Spain, Portugal, Sicily, Asia Minor and southern Asia; 25 of these are

endemic to Australia (Grimes 1997). Cullen is distributed widely across a broad range

of climates in Australia, from summer- to winter-dominant rainfall, and can be found in

areas with a wide range of annual rainfall (200 to 1300 mm) (Bennett et al. 2010).

Using an ecogeograpahic analysis, Bennett et al. (2006) identified seven species of

Cullen with potential for development as pastures for the more winter-dominant rainfall

zone of southern Australia. Cullen australasicum is one of the best-performing species


among these seven species and is described as a drought-tolerant perennial for use in

extensive grazing systems (Hayes et al. 2009). Cullen australasicum (Schltdl.) J.W.

Grimes has a number of common names, including tall verbine, native scurf-pea and

native verbine (Cunningham et al. 1992; Dear et al. 2007). Li et al. (2008) studied 23

perennial legumes over three years at field sites across southern Australia. They ranked

Cullen australasicum seventh in persistence and fourth in dry matter yield. In an

evaluation of the productivity and persistence of 103 forage legumes and herbs over

three years, Real et al. (2011) ranked C. australasicum amongst the 11 most promising

species during winter, summer or all year. Ryan et al. (2008) concluded that C.

australasicum was the most promising native perennial legume species for

domestication. Recently, interest in this genus within Australia has increased, with

researchers aiming to release a commercial cultivar (Bennett et al. 2012; Humphries et

al. 2014; Suriyagoda et al. 2010).

1.1C Kennedia prostrata

Little is known of the genus Kennedia. It is widespread across southern Australia in

both woodlands and forests. It has a preference for light, well-drained soils. This

adaptation to light-textured soils suggests tolerance of drought and low soil fertility

(Bell et al. 2010). There are 15 species in the Kennedia genus, all of which are endemic

to Australia. Kennedia prostrata and K. prorepens are the most widely distributed

species. Kennedia species have been considered as legumes with agricultural potential

for a long time (Silsbury and Brittan 1955). Favourable characteristics include large

seeds (up to 45 mg) with good nutritional qualities; some species produce a huge

number of seeds in the first year after establishment (Bell et al. 2010; Rivett et al. 1983).

Kennedia species are well adapted to the low-P soils of Western Australia, although

some species appear to be unable to down-regulate their P-uptake capacity when

exposed to high levels of P (Pang et al. 2009a; Pang et al. 2009b). Due to all these

factors, Kennedia species have been suggested as possible forage plants with

agricultural potential in Western Australia (Bell et al. 2010; Cocks 2001; Silsbury and

Brittan 1955). While a high acid-detergent fibre content and low organic matter

digestibility may result in low voluntary consumption by grazing sheep (Revell et al.

2013), even low intake may benefit livestock health, due to its condensed tannin content

(Bouazza et al. 2012). Consumption of such species at appropriate doses increases the

post-ruminal supply of N (Ben Salem et al. 2010). In an experiment by Kotze et al.


(2009) the development of parasite larvae was inhibited by four out of six Kennedia

species.

Overall, it appears that B. bituminosa, C. australasicum and Kennedia species are all

well-adapted to low-P soils and may perform better than M. sativa under low-P and dry

conditions. In an experiment by Pang et al. (2009b), productivity of tedera when it was

supplied with 0-6 µg P g-1 soil, was better than that of K. prostrata, K. prorepens and

Cullen species.

Figure 1. The three perennial legume species with potential to be developed as pasture species that were used in the present study. Bituminaria bituminosa (a); Cullen australasicum (b) and Kennedia prostrata (c).

1.2 Phosphorus in plants and soil

Phosphorus (P) is an essential plant nutrient. Compared to the other major nutrients, P is

the least mobile and least available in most soils, especially in ancient landscapes. As

soil becomes weathered due to soil erosion and leaching, the plant-available soil P

concentration gradually declines (Hinsinger 2001; Lambers et al. 2010; Raghothama

1999; Vance et al. 2003; Walker and Syers 1976). Ancient landscapes are those that

have been above sea level and have not been glaciated or disrupted by catastrophic

events for millions of years. South-western Australia, the southwest of South Africa and

the Pantepui mountains in Brazil and Venezuela, are all included in this category of

landscapes (Hopper 2009). In south-western Australia, many natural surface soils

contain less than 5 mg kg−1 of bicarbonate-extractable P (McArthur 1991).

Plants take up P in the orthophosphate (Pi) forms H2PO4- and HPO4

2-, which are in the

soil in very low concentrations (0.1-10 µM) (Hinsinger 2001). Also, Pi is very

immobile, unlike nitrate, which can easily move toward the roots via mass flow and

diffusion (Lambers et al. 2006). The contribution of mass flow in maintaining plant P

demand is as little as 1-5% (Lambers et al. 2008).

When crops or pastures are grown on soils with very low P availability, application of

(a) (c)(b)


large quantities of fertilisers containing readily-available P, such as superphosphate, is

needed (Bolland and Gilkes 1997; Dann et al. 1996). Consequently, P concentrations in

agricultural soils in Australia have risen substantially (Vu et al. 2011). However, P is

predominantly derived from phosphate rock, which is a non-renewable resource, and

some predict that resources of inexpensive P will be depleted in 50 to 100 years

(Cordell et al. 2009; Scholz et al. 2013; Vance et al. 2003); some more recent papers

suggest less pessimistic scenarios (Fixen 2009). However, changes to the current

farming systems are necessary, and one option is to increase the use of low-P tolerant

perennial legumes in the rotation (Auricht 1999). In fact, the urgent need to develop

more P-efficient agricultural systems in Australia has been identified in recent reviews

(Richardson et al. 2011; Simpson et al. 2011). One element in such systems could be the

use of pasture plants that require minimal input of P fertiliser. Many Australian plant

species have specific root adaptations for growth in low-P soils (Lambers et al. 2010).

The interception of Pi by these plants can be increased by morphological (root

proliferation, an increase in frequency and length of root hairs, higher root: shoot ratios,

formation of a symbiosis with mycorrhizal fungi, finer roots) and physiological

(increased P-uptake rate and enhanced exudation of low-molecular-weight organic

anions (carboxylates) and phosphohydrolases) mechanisms (Lambers et al. 2006; Ryan

et al. 2012; Smith and Read 2008; Suriyagoda et al. 2012; Vance et al. 2003; Wissuwa

2005).

1.2A Phosphorus pulse

Hot dry summers and cool wet winters are the main characteristics of Mediterranean

environments. Since mobility of P is greatly reduced in dry soil, P becomes especially

limiting to plants over summer (Sardans and Peñuelas 2007). However, the first autumn

rains might release a pulse of P into the soil. This pulse results from the release of P due

to a number of processes including: lysed bacterial cells, microbes rupturing due to

osmotic shock in response to rapid rewetting of the soil and/or sudden fluctuations in

soil matric potential; rapid degradation of dry organic matter on the soil surface, that is,

plant detritus exposed to months of hot, dry weather; and breakdown of large

macroaggregates (Blackwell et al. 2010; Butterly et al. 2009; Grierson et al. 1998;

Turner et al. 2003; Turner et al. 2002; Turner and Gilliam 1976; Wu and Brookes

2005). Up to 58% of the total microbial biomass can be killed by soil drying and rapid

rewetting (Kieft et al. 1987; van Gestel et al. 1993; Wu and Brookes 2005). Qiu et al.


(2004) found that levels of anion-exchange membrane-extractable P in south-western

Australia before winter rainfall were very low (˂2.6 mg P kg–1 dry soil) in surface soils

(just under the litter layer), but that during winter this increased 3.4–56 fold. Thirty

percent of this P increase occurred (from litter) in the first 24 hours. Following opening

rains this pulse of P may be considered analogous to the release of P following addition

of P fertiliser (for example, see Lewis et al. (1987)) and in both cases, quick uptake of P

by plants is desirable. For instance according to Lawton and Vomocil (1954), the

majority of P dissolution of fertiliser occurs within three to six hours of application. For

example, in a soil with field capacity moisture content, 50-80% of soluble P moved

from granules and tablets of fertiliser into the soil surface in 24 hours. This was reduced

to 20-50% for a soil with 2-4% moisture content. The pulse of P that is released into the

soil following rewetting is labile and not wholly accessible for all plants (Bünemann et

al. 2013). A quick uptake of a pulse of P following rainfall or fertiliser application is

important, as a quicker uptake will reduce the loss of P through P sorption,

immobilisation in microbial biomass and leaching (Anderson and McLachlan 1951;

Friesen et al. 1997; Robertson and Nash 2008). Roots are quite capable of rapidly taking

up P after rewetting (Eissenstat et al. 1999).

1.2B Responses of plants to a pulse of P

The capacity of a plant to absorb a pulse of P may depend on its P status. For instance,

when potato plants (Solanum tuberosum) which were grown in aerated nutrient solution

were provided with 1 mol m-3 of phosphate in solution, the absorption rate in P-deficient

plants was three to four-fold greater than that of plants with sufficient P supply

(Cogliatti and Clarkson 1983). Similarly, when barley (Hordeum vulgare) was grown in

hydroponics (and thus presumably non-mycorrhizal) under P limitation for 20 days, the

P influx reached its maximum after 16 days, and then declined gradually, while the

uptake rate for plants treated with a full nutrient solution declined from the start till the

end of the experiment, and reached ~30% of the initial rate at five days after

germination (Lefebvre and Glass 1982).

I surmise that plants that are treated with sufficient P are marginally able to absorb P

from a P pulse, while P-deficient plants respond strongly to a P pulse with a P influx

commencing in the first hours after the pulse is applied and continuing until the plants

reach a high P concentration. After this point, the P influx will decline.


Plants with a low P status are likely to be colonised by AMF. The role of these fungi in

plant uptake of P from a pulse has not been investigated previously.

1.3 Arbuscular mycorrhizal fungi (AMF)

Mycorrhizas are associations between plant roots and certain groups of soil-dwelling

fungi (Brundrett 1991). Their most important role (from a plant perspective) is

enhancing plant uptake of nutrients while, in return, the fungi receive

photosynthetically-derived carbon compounds (Smith and Read 2008). Over 80% of all

vascular plants form a symbiosis with mycorrhizal fungi. There are seven types of

mycorrhizas based on morphology. Arbuscular mycorrhizas are the most prevalent and

are mutualistic symbiotic associations between roots and a small group of fungi in the

phylum Glomeromycota (Bolan 1991; Brundrett 1991; Schüßler et al. 2001; Smith and

Read 2008).

AMF are considered obligate symbionts, which receive carbon (C) from plants and, in

return, supply plants with nutrients. Increase in the uptake of nutrients is thought to

result from the exploration of a greater volume of soil by colonised plants, due to the

growth of external hyphae of the fungi (Grace et al. 2009; Jakobsen et al. 1992a). The

largest effect of AM formation is on plant P nutrition (Smith and Read 2008). Both

plants and AMF acquire P from the soil solution in the form of inorganic

orthophosphate (Bieleski 1973). Phosphorus is required by both symbionts in large

amounts (Smith and Read 2008). In addition, AM symbioses increase host plant

tolerance of diseases and host plant drought resistance, and decrease accumulation of

heavy metals by the host plant (Fitter 2006). Mycorrhizal fungi can also increase host

plant uptake of other elements including sulfur (S), boron (B), potassium (K), calcium

(Ca), magnesium (Mg), sodium (Na), zinc (Zn), copper (Cu), manganese (Mn), iron

(Fe), silicon (Si) and some other trace elements (Clark and Zeto 2000). Depending on

soil type, host plant and other factors, AMF can have a positive, a neutral or a negative

impact on the uptake of these nutrients (Ryan and Tibbett 2008). According to Clark

and Zeto (2000), AMF increase the uptake of P, N, Zn and Cu in most soils, but only

increase uptake of K, Ca and Mg when the plants are grown in acidic soils.

Arbuscular mycorrhizal fungi are called “arbuscular” because they form arbuscules,

structures that consist of finely branched hyphae, which are involved in nutrient

exchange within the cortical cells of plant roots (Peterson et al. 2004; Smith and Read


2008) (Figure 2). Arbuscules, which are located within the cortical cells, but outside the

host cell’s plasma membrane, have long been considered diagnostic for AMF, although

they are not always present (Smith and Read 2008). Presence of intracellular coiled

hyphae is an alternative to identify AMF (Smith and Smith 2011) as are vesicles,

although both are not invariably present (Koide and Mosse 2004). Vesicles start to form

after the first arbuscules are formed and continue to develop when the arbuscules

senesce (Brundrett et al. 1996). Vesicles and fungal spores, both of which may be

intraradical or extraradical, have a role in accumulation of carbon (Olsson et al. 2011)

(Figures 2 and 3).

Figure 2. Distinguishing components of arbuscular mycorrhizal fungi within a colonised root (Brundrett et al. 1996).

Figure 3. Intraradical colonisation, extraradical spores and a very high density of extraradical hyphae in a densely colonised root of Trifolium subterraneum stained with ink (photo by N. Nazeri).

1.3A The growth response of plants to colonisation by AMF

Asai (1944) studied AM colonisation and nodulation in a large number of legumes, and

was the first to recognise the relationship between the development of arbuscular

mycorrhizas and the growth of the host (Asai 1944; Smith and Read 2008). In 1959,

Baylis suggested that increased growth of the host plant was mediated by enhanced P

uptake when arbuscular mycorrhizas were present. Baylis (1959) found that Griselinia

Spores

Extraradical hyphae

Intraradical colonisation


littoralis seedlings, which were grown in a low-P soil and colonised by AMF, took up

3-5 times more P than non-mycorrhizal seedlings (Baylis 1959; Koide and Mosse

2004). It is now generally thought that AMF have the potential to play an important role

in growth of most crops and pasture species (Lekberg and Koide 2005; Ryan and

Tibbett 2008; Smith and Read 2008).

A recent meta-analysis has suggested that AMF may not play a significant role in

growth of annual autumn-sown crops and pastures in temperate southern Australia

(Ryan and Angus 2003; Ryan and Kirkegaard 2012), due, in part, to their relatively low

P demand in cold autumn conditions. However, the authors did speculate that AMF may

play a greater role in growth of summer-active perennials, due to dry soil limiting P

uptake and high summer temperatures enhancing plant P demand (Ryan and Kirkegaard

2012).

In general, the growth response to mycorrhization depends on many factors; from

molecular (e.g., transporter gene expression) to environmental (e.g., soil type, soil

nutrient availability, especially the status of P in the soil and light intensity) and

ecological (the composition of plant and fungus, density and competition) and can be

highly positive, neutral or negative (Smith and Smith 2011). A positive growth response

mostly happens because of an increase in P uptake when P is limiting in soil (Smith and

Read 2008). When the P concentration in soil is low, a non-mycorrhizal root system

cannot absorb P effectively, so the plant will become P deficient and plant growth will

decrease. However, colonised roots show enhanced P uptake and consequently plant

growth may increase. This is the well-known mycorrhizal growth response (Smith and

Read 2008). Suppression of growth of arbuscular mycorrhizal hosts following

colonisation by AMF is much less commonly reported than growth enhancement, but

does occur (Johnson et al. 1997; Smith et al. 2003), especially for seedlings

(Bethlenfalvay et al. 1982). Bethlenfalvay et al. (1982) and Koide (1985) speculated

that growth suppression might be due to drain of carbon from plant to AMF when plant

photosynthetic capacity and shoot-root ratio are low. Moreover, there are reports of

negative growth responses of non-host plants when they are colonised by AMF

(Sanders and Koide 1994; Veiga et al. 2012). For instance in an experiment by Veiga et

al. (2013), when the non-mycorrhizal plant Arabidopsis thaliana was grown together

with the mycorrhizal plant (either Trifolium pratense or Lolium multiflorum) in the

presence of AMF, growth of Arabidopsis thaliana was suppressed by 50% and this was

due to 43% colonisation by AMF. Also, an increase in soil P may lead to a negative


growth response in mycorrhizal plants in comparison to non-mycorrhizal plants (Smith

and Read 2008).

Addition of soluble P has been shown many times to reduce the colonisation level by

AMF (Amijee et al. 1993; Ryan and Ash 1996) and in a study of pastures on 20

Australian dairy farms, colonisation had a negative relationship with soil P

concentration (Ryan et al. 2000). In Western Australia, constant application of fertilisers

in agricultural regions has resulted in a great elevation of soil P and it is possible that

this may now contribute to either a neutral or negative mycorrhizal growth response and

low levels of colonisation by AMF (Ryan and Kirkegaard 2012).

1.3B How does AMF help plants to access P?

Arbuscular mycorrhizal fungi access the same soil P sources that are used by plants

(Gianinazzi Pearson et al. 1981; Smith and Read 2008). Although there has been some

investigation of whether AM roots have the ability to exploit sources of P in soil that are

not available to the plant, such an effect has yet to be confidently shown (Smith and

Read 2008; Yao et al. 2001; Young et al. 1986).

When root exploration is restricted, the external hyphae of the AMF can deliver up to

80% of plant P uptake to the host plant over a distance of more than 10 cm from the root

surface (although it depends on the species of AMF) (Jakobsen et al. 1992b, c; Li et al.

1991). Also, it has been shown that the rate of P uptake by mycorrhizal plants is faster

than that of non-mycorrhizal plants (Sanders and Tinker 1973). Sanders and Tinker

(1973) observed the rate of inflow of P in mycorrhizal roots of onion (Allium cepa) was

much higher (17×10-14 moles cm-1 s-1) than the rate of inflow of P in non-mycorrhizal

roots (3.6×10-14 moles cm-1 s-1). Assuming that the difference in the rate of inflow is

caused by hyphae of the mycorrhiza, they calculated the rate of inflow of P into hyphae

is six times greater than the rate of inflow of P into root hairs. However, according to

Pearson and Jakobsen (1993), the P uptake rate differs with fungal species. Moreover,

colonisation by AMF will change the physiology of the root system and this might have

an influence on the P uptake (Clarkson 1985; Smith and Read 2008).

Forming a symbiosis with AMF and exudation of carboxylates into rhizosphere soil

have been described as “two key plant adaptations for acquisition of phosphorus” (Ryan

et al. 2012). Carboxylates have an important role in mobilising P in soils that contain

very little P or have most of the soil P strongly sorbed to soil particles (Dakora and


Phillips 2002; Jones 1998). Moreover, carboxylates can also increase the solubility of

cations in the soil and, as a result, the concentration of these cations may increase in the

plant (Page et al. 2006; Ryan et al. 2012).

Exudation of carboxylates into rhizosphere soil and forming a symbiosis with AMF are

both carbon-demanding processes (Dinkelaker et al. 1989; Graham 2000) and there

might be trade-off between them. For example, exudation of carboxylates by plants that

are colonised by AMF is significantly less than that of uncolonised plants (Ryan et al.

2012). Also, it has been shown that exudation of carboxylates may become significantly

less with an increase in soil P level (Pearse et al. 2006; Wang et al. 2013). Thus, it has

been speculated that the presence of AMF may increase plant access to Pi at the expense

of access to poorly soluble forms of P – “mycorrhizal-mediated resource partitioning”

(Ryan et al. 2012).

1.3C Phosphate translocation within hyphae and delivery to intraradical

interfaces

The low Pi concentration in the soil solution and the large electrochemical potential

gradient between the soil solution and the plant/fungal cytosol, requires Pi to be taken

by extraradical mycelium using high-affinity Pi-H+ co-transporters, energized by H+-

ATPases (Bucher 2007; Ezawa et al. 2002; Harrison and van Buuren 1995; Smith and

Read 2008; Smith and Smith 2011). The Pi taken up by the fungus is used for cell

functions such as energy generation and biosynthesis of phospholipids, nucleic acids

and the precursors of carbohydrate polymers. Any extra Pi, if available, is transported

into intrahyphal vacuoles and converted to polyphosphate (Ezawa et al. 2002) which

buffers cytoplasmic Pi concentration and stores P for a momentary time as P is

transferred along hyphae (Hijikata et al. 2010; Smith and Smith 2011).

Polyphosphate is a linear polymer of orthophosphate residues, which are linked by high-

energy phosphoanhydride bonds (Harold 1966). Ezawa et al. (2002) hypothesised that

the process involved in polyphosphate synthesis in the AM fungi might be similar to the

process involved in polyphosphate accumulation in yeast (for which four genes were

found to be responsible (Ogawa et al. 2000)).

Temporary storage of Pi seems to be the most important role of polyphosphate in AMF

(Ezawa et al. 2002; Kornberg 1999). Viereck et al. (2004) used in vivo Nuclear

Magnetic Resonance (P-NMR) to characterise polyphosphate and other P pools in


external hyphae and mycorrhizas of Glomus intraradices and suggested that

polyphosphate is the largest form of P storage in the fungi. The chain length of

polyphosphate in AM fungi is variable (Ezawa et al. 1999; Kornberg 1999). According

to Solaiman et al. (1999), most of the polyphosphates in intraradical hyphae are present

as short chains, while in the extraradical hyphae it is present as long chains or granules

(insoluble). The movement of the granules in the vacuoles of extraradical hyphae

toward the intraradical hyphae happens via cytoplasmic streaming (Clarkson 1985).

Ezawa et al. (2004) demonstrated that the transfer of AM fungus from a Pi-deficient to a

Pi-rich medium resulted in rapid accumulation (less than 3 h) of Pi into large amounts

of polyphosphate. Due to the shorter chain length of polyphosphate in the intraradical

than extraradical hyphae, it is proposed that polyphosphate is hydrolysed in the

intraradical hyphae to release Pi for the plant (Javot et al. 2007b; Ohtomo and Saito

2005; Solaiman et al. 1999). Ryan et al. (2003) found that intraradical hyphae can store

very high concentrations of P (up to 600 mM). Also, it was demonstrated that 30-70%

of the P in the hyphae and 100% of P in the arbuscules was not extractable (insoluble)

(Ryan et al. 2007) and, as a result hyphae could quickly absorb large amounts of P

(which, if left in soil, could be quickly lost (see section 1.2a)). Ryan et al. (2007)

concluded the P in the hyphae was likely to be in the form of polyphosphate.

It seems likely that exo- and endopolyphosphatases (PPX, PPN) and acid phosphatase

(ACPase) would work together to hydrolyze polyphosphate to Pi in AM fungal

symbiosis (Ezawa et al. 2002).

1.3D Transfer of P from AM fungal structures to the root cell

An interesting question is how P is transferred from the AM fungal structures inside the

host root into the root cells. Microscopic examination of roots has focused on

arbuscules in various stages of formation and decomposition (Kinden and Brown 1975a;

Kinden and Brown 1975b), and thus it was initially thought that the decomposition and

deterioration of the arbuscules played an important role in transferring the nutrients. It is

now known that arbuscule breakdown is not necessary for nutrient transfer.

Ultrastructural and physiological evidence, such as very high H+-ATPase activity

associated with the host plasmalemma, indicative of active transport mechanisms,

suggests that most nutrient exchange occurs across the living host-fungus interface

(Koide and Mosse 2004). In other words, Pi that is released from healthy arbuscules is


taken up by plant Pi transporters, which are located on the periarbuscular membrane

(Javot et al. 2007a; Rausch et al. 2001). However, there is also evidence that P “leaks”

from intercellular hyphae into apoplast where, presumably, it can be absorbed by the

plant (Ryan et al. 2003).

1.3E Phosphorus pathways in colonised plants

Recent research has deepened our understanding of the role of arbuscular mycorrhizas

through showing that the fungi can affect the ability of the host plant itself to directly

take up P (Smith and Read 2008). There are two possible pathways for P acquisition in

the colonised plant: the first is a direct pathway, which occurs through plant root hairs

and epidermal cells, and the second is the AM pathway, which occurs through

extraradical mycelium of AMF as described above. Before 1990, it was assumed that

the contribution of the AM pathway and direct pathway were additive (i.e. the AM

pathway supplied mycorrhizal plants with extra P), but more recently, by using

compartmented pots and the tracking of radioactive P to extraradical hyphae and not to

roots, it has become clear that even when there is no positive growth response to

colonisation by AMF, the contribution of the AM pathway to P uptake can be larger

than the direct pathway (Smith and Smith 2011). According to Smith and Read (2008),

colonisation by AMF may lead to a “switch–off” of the direct pathway. For instance, in

an experiment by Smith et al. (2003), three plant species, flax (Linum usitatissimum),

tomato (Solanum lycopersicum) and annual medic (Medicago trancatula) were

colonised by three mycorrhizal fungi (Gigaspora rosae, Glomus intraradices and

Glomus caledonium). The three plant species showed positive to negative growth

responses, but the AM pathway was active in all three plant species. For example in S.

lycopersicum, although there was a consistent negative growth response to inoculation,

G. intraradices delivered 100% of the plant’s P uptake. Thus, even when there is no net

increase in total P uptake recorded as a result of inoculation with AMF, the fungi may

still be playing a major role in P uptake (Smith and Read 2008; Smith and Smith 2011).

In summary, when plants become colonised by AMF, the direct uptake pathway loses

some of its function (Smith et al. 2003). According to Smith et al. (2003), this loss of

function of the direct uptake pathway might result from down-regulation of plant genes

encoding P transporters in the epidermis and root hairs (Chiou et al. 2001; Rosewarne et

al. 1999). The relevance of this finding to the management of AMF in agricultural

systems is still unclear (Ryan and Kirkegaard 2012).


1.3F Role of AMF in uptake of a pulse of P

I hypothesise that plants colonised by AMF will have a greater ability to absorb P from

a pulse of P than uncolonised plants have because having access to a greater volume of

soil than roots alone and having a high capacity to absorb and store P are two main

features of AM hyphae.

There are many reports on the response of AMF to application of P fertilisers which

show a decrease in level of colonisation. Indeed, in a meta-analysis, Treseder (1983)

concluded that AM colonisation level is, on average, reduced by 32% when P fertiliser

is used. It has also been shown that AMF can aid plant access to fertiliser. For instance,

Kapoor et al. (2004) examined Foeniculum vulgare plants, grown with and without

AMF (Glomus macrocarpum or G. fasciculatum) and P-fertiliser (20 kg P ha-1). The

plants that were both colonised and received the fertiliser had significantly more

biomass and a higher shoot P concentration than the plants that were colonised but did

not receive any fertiliser or were fertilised but not colonised. However, as the fertiliser

was added to the soil before growing the plants, the plants did not encounter a pulse of

P.

1.3G Which is a better approach in glasshouse experiments? A diverse

community of AMF or a single-species inoculum?

Many species of AMF are ubiquitous (Öpik et al. 2006), while others seem to be

restricted to specific ecosystems, climates or vegetation (Castillo et al. 2006; Oehl et al.

2007). According to Oehl et al. (2010), land-use intensity and soil type are two

important factors in determining the richness of AM fungal communities and their

composition, but the former has a more important effect. An increase in land-use

intensity may have a negative effect on AM fungal diversity. For instance, Oehl et al.

(2010) found grasslands contained more species of AMF than arable land (AM fungal

species richness in trap cultures after 20 months was 16-26 units for grassland and 13-

21 units for arable land). However, soil types might differ very much in AM fungal

communities and thus can be characterised by their AM fungal communities. For

example, Acaulospora, Scutellospora, Cestraspora and Gigaspora prefer a siliceous

(acidic) grassland as their habitat, while a number of Glomus species prefer calcareous

(alkaline) grasslands (Oehl et al. 2010). However, diversity within a single soil type can

be very high. For instance, in soil from a single field in south-western Australia, more


than 20 species of AMF were present (Tibbett et al. 2008).

In spite of the diversity of AMF commonly reported in natural and agricultural

ecosystems, many glasshouse experiments examining the function of AMF are

performed using a single-species inoculum (pure culture) (Abbott and Robson 1977;

Smith and Read 2008). In an experiment by Wagg et al. (2011), plant productivity was

compared among a mixture of four AM fungal species (Glomus mosseae, G.

intraradices, G. claroideum and Diversispora celata) and the individual inocula of these

species under two soil conditions (low sand content [1:4 ratio of sand to field soil] and

high sand content [4:1 ratio of field soil to sand]). Inoculation with a diverse community

of AMF resulted in significantly higher plant productivity than inoculation with a single

species. Also, the effect of AM fungal diversity and AM fungal species on plant

productivity differed between the two soils. When sand content was high, colonisation

with both the best individual AM fungal species (D. celata), and a mixture of all fungal

species, produced the highest biomass in Trifolium pratense, while with low sand

content, colonisation with the diverse AM species resulted in higher biomass than with

the best individual AM fungal species (D. celata). Wagg et al. (2011) concluded that

diversity in the AM fungal community ensures plant productivity is maintained under

different environmental conditions. Also, according to van der Heijden et al. (1998), an

increase in number of AM fungal species results in an increase in both plant

productivity and biodiversity. Moreover, species of AMF differ in their capacity to

enhance plant uptake of P from soil. This is due to differences in the length of their

extraradical hyphae or P-uptake capacity per unit of hyphal length (Graham et al. 1982;

Jakobsen et al. 1992b, c). In view of these studies, I hypothesise that a diverse

community of AMF will be more efficient in absorbing P from a pulse of P than a single

species.

Using field soil as inoculum is a way to ensure a higher, and more realistic, diversity of

AMF and accompanying microbes in glasshouse experiments. However, field soil

inoculum involves introduction of indigenous AMF and the indigenous microbial

community (heterotrophic soil microbes) simultaneously.

The effect of the microbial community on plant growth might be synergistic or

antagonistic with that of AMF. To avoid confounding effects of AMF and

accompanying microbes when using field soil as inoculum, it has become common

practice to add a filtrate of field soil (which contains no mycorrhizal hyphae or spores,


but contains most other soil microbiota) to non-mycorrhizal controls (Asghari and

Cavagnaro 2011; Lovelock and Miller 2002; Smith and Smith 1981).

1.4 Gaps in knowledge

Arbuscular mycorrhizal fungi are ubiquitous in natural and agricultural systems, but

little is known about their role in temperate pasture systems (with soils that typically

contain moderate to high concentrations of P). In particular, the ability of AMF to

enhance P uptake from a pulse of P as may occur following addition of fertiliser or the

start of the winter rain is unknown.

1.5 Research objectives

The overall aim of this thesis is to enhance understanding of the role of AMF in uptake

of P by pasture plants, when P is delivered as a pulse. In three experimental chapters, I

address the following hypotheses:

Hypothesis 1: AMF will enhance uptake of P from a pulse as may occur following

addition of fertiliser or the start of the winter rain (Chapter 2 – T. subterraneum;

Chapter 3 - perennial legumes).

Hypothesis 2: Uptake of P is greater when plants are colonised by a diverse community

of AMF (inoculation with field soil), rather than a single-species inoculum (Chapter 2).

Hypothesis 3: Presence of microbes in the soil increases P uptake compared to the

control (sterilised field soil) (Chapter 2).

Hypothesis 4: AMF will moderate shoot P concentration following a pulse (based on the

findings of Chapter 2) (Chapter 3).

Hypothesis 5: Release of carboxylates into the rhizosphere will decrease when plants

encounter a pulse of P (Chapter 3).

Hypothesis 6: Colonisation by AMF will decrease the release of carboxylates into the

rhizosphere (Chapter 3).

Hypothesis 7: (developed following results presented in Chapters 2 and 3): Following a

significant pulse of P, P movement to shoots is restricted, due to storage of high

concentrations of P in intraradical AM hyphae in insoluble forms (Chapter 4).


1.6 Thesis outline

This thesis is in agreement with the Postgraduate and Research Scholarship Regulation

1.3.1.33(1) of the University of Western Australia, Australia and is presented as a series

of three scientific papers. Two manuscripts have already been published and the

remaining one is in preparation for submission. All the work presented has resulted

from work done towards this thesis. There are five main chapters in this thesis; a

General Introduction, three Experimental Chapters and a General Discussion. The

General Introduction covers the broad background for the work presented in the thesis

in order to justify the research objectives presented above. A more focussed review of

literature is presented in the introduction of each experimental chapter. The three

Experimental Chapters are presented in the format of scientific papers that can be read

individually or as a part of the whole thesis. Each Experimental Chapter includes the

following sections: Abstract, Introduction, Materials and Methods, Results, Discussion

and References. Each experimental chapter is preceded by a short preface, to link the

chapter to the broader hypotheses addressed by the thesis. This “thesis-as-a-series-of-

papers” format results in some unavoidable repetition, especially in the Materials and

Methods sections of Chapters 2 with 3. The content of each experimental chapter is as

follows:

Chapter 2- This chapter was designed as a preliminary experiment to determine

methodology for further experiments. It investigated the effect of a P pulse (one day

after and seven days after) on uptake of P and other elements in a single species, T.

subterraneum, grown with and without AMF.

The desirability of using field soil as inoculum in order to introduce a diverse

community and of using filtrate to equalise the accompanying soil microbial community

between inoculated and non-inoculated treatments was also investigated.

Chapter 3- This chapter was designed based on the results presented in Chapter 2. It

investigated the effect of a P pulse (after seven days) on uptake of P and other elements

for four perennial legumes (Kennedia prostrata, Bituminaria bituminosa, Cullen

australasicum, M. sativa) and T. subterraneum grown with or without AMF. The effect

of AMF on the rhizosphere pH and carboxylate exudation was also assessed.

Chapter 4- This chapter was designed to investigate the location within mycorrhizal and

non-mycorrhizal roots of P taken up from a pulse using scanning electron microscopy


and quantitative X-ray microanalysis. However, since no AMF were found in the

samples chosen, the chapter focuses on the storage of P from a pulse in uncolonised

areas of the mycorrhizal roots and non-mycorrhizal roots.

The thesis concludes with the General Discussion, which integrates results from all

experimental chapters, evaluates the important issues raised in the research reported in

the thesis, draws together key conclusions, and highlights areas for further research.

1.7 References

ABARE (2006) Australian Farm Survey Report. Canberra.

Abbott LK, Robson AD (1977) Growth stimulation of subterranean clover with vesicular arbuscular mycorrhizas. Aust J Agric Res 28: 639-649.

Adams NR, Briegel JR (1998) Liveweight and wool growth responses to a Mediterranean environment in three strains of Merino sheep. Aust J Agric Res 49: 1187-1193.

Amijee F, Stribley DP, Tinker PB (1993) The development of endomycorrhizal root systems. VIII. Effects of soil phosphorus and fungal colonization on the concentration of soluble carbohydrates in roots. New Phytol: 297-306.

Anderson AJ, McLachlan KD (1951) The residual effect of phosphorus on soil fertility and pasture development on acid soils. Aust J Agric Res 2: 377-400.

Asai T (1944) Uber die Mycorrhizenbildung der leguminosen Pflanzen. Japanese Journal of Botany 13: 463-485.

Asghari HR, Cavagnaro TR (2011) Arbuscular mycorrhizas enhance plant interception of leached nutrients. Funct Plant Biol 38: 219-226.

Auricht GC (1999) Lucerne—an Australian breeding success story. Proceedings 11th Australian Plant Breeding Conference.

Baylis GTS (1959) Effect of vesicular-arbuscular mycorrhizas on growth of Griselinia

littoralis (Cornaceae). New Phytol 58: 274-280.

Bell LW, Bennett RG, Ryan MH, Clarke H (2010) The potential of herbaceous native Australian legumes as grain crops: a review. Renewable Agriculture and Food Systems 26: 72-91.

Bell LW, Ryan MH, Moore GA, Ewing MA (2006) Comparative water use by Dorycnium hirsutum-, lucerne-, and annual-based pastures in the Western Australian wheatbelt. Aust J Agric Res 57: 857-865.

Ben Salem H, Norman HC, Nefzaoui A, Mayberry DE, Pearce KL, Revell DK (2010) Potential use of oldman saltbush (Atriplex nummularia Lindl.) in sheep and goat feeding. Small Ruminant Res 91: 13-28.

Bennett RG, Colmer TD, Real D, Renton M, Ryan MH (2012) Phenotypic variation for productivity and drought tolerance is widespread in germplasm collections of Australian Cullen species. Crop and Pasture Science 63: 656-671.

Bennett RG, Colmer TD, Real D, Ryan MH (2006) Hardy Australians: ecogeography of


Cullen suggests perennial legumes for low rainfall pastures "Ground-breaking Stuff". Proceedings of the 13th Australian Agronomy Conference. Perth: Australian Society of Agronomy.

Bennett RG, Ryan MH, Colmer TD, Real D (2010) Prioritisation of novel pasture species for use in water-limited agriculture: a case study of Cullen in the Western Australian wheatbelt. Genet Resour Crop Evol 58: 83-100.

Bethlenfalvay GJ, Brown MS, Pacovsky RS (1982) Parasitic and mutualistic associations between a mycorrhizal fungus and soybean: development of the host plant. Phytopathology 72: 894-897.

Bieleski RL (1973) Phosphate pools, phosphate transport, and phosphate availability. Annu Rev Plant Phys 24: 225-252.

Blackwell MSA, Brookes PC, de la Fuente-Martinez N, Gordon H, Murray PJ, Snars KE, Williams JK, Bol R, Haygarth PM (2010) Phosphorus solubilization and potential transfer to surface waters from the soil microbial biomass following drying–rewetting and freezing–thawing. Adv Agron 106: 1-35.

Bolan NS (1991) A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant Soil 134: 189-207.

Bolland MDA, Gilkes RJ (1997) The agronomic effectiveness of reactive phosphate rocks. 2. Effect of phosphate rock reactivity. In ‘The role of reactive phosphate rock fertilisers for pastures in Australia’. CSIRO Publishing, Melbourne: pp. 937–946.

Bouazza L, Bodas R, Boufennara S, Bousseboua H, López S (2012) Nutritive evaluation of foliage from fodder trees and shrubs characteristic of Algerian arid and semi-arid areas. J Anim Feed Sci 21: 521-536.

Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N (1996) Working with mycorrhizas in forestry and agriculture. Australian Centre for International Agriculatural Research (ACIAR)

Brundrett MC (1991) Mycorrhizas in natural ecosystems. In: M Begon, A Fitter, A Macfadyen (eds) Advances in Ecological Research. Academic Press.

Bucher M (2007) Functional biology of plant phosphate uptake at root and mycorrhiza interfaces. New Phytol 173: 11-26.

Bünemann EK, Keller B, Hoop D, Jud K, Boivin P, Frossard E (2013) Increased availability of phosphorus after drying and rewetting of a grassland soil: processes and plant use. Plant Soil 370: 511-526.

Butterly CR, Bünemann EK, McNeill AM, Baldock JA, Marschner P (2009) Carbon pulses but not phosphorus pulses are related to decreases in microbial biomass during repeated drying and rewetting of soils. Soil Biol Biochem 41: 1406-1416.

Callaghan AR (1958) The South Australian department of agriculture and its work'. Introducing South Australia, Australia and New Zealand agricultural advisory service, Melbourne: 179-192.

Castillo CG, Borie F, Godoy R, Rubio R, Sieverding E (2006) Diversity of mycorrhizal plant species and arbuscular mycorrhizal fungi in evergreen forest, deciduous forest and grassland ecosystems of Southern Chile. J Appl Bot Food Qual 80: 40-47.

Chiou TJ, Liu H, Harrison MJ (2001) The spatial expression patterns of a phosphate


transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface. Plant J 25: 281-293.

Clark RB, Zeto SK (2000) Mineral acquisition by arbuscular mycorrhizal plants. J Plant Nutr 23: 867-902.

Clarkson DT (1985) Factors affecting mineral nutrient acquisition by plants. Annu Rev Plant Phys 36: 77-115.

Cocks PS (2001) Ecology of herbaceous perennial legumes: a review of characteristics that may provide management options for the control of salinity and waterlogging in dryland cropping system. Aust J Agric Res 52: 137-151.

Cogliatti DH, Clarkson DT (1983) Physiological changes in, and phosphate uptake by potato plants during development of, and recovery from phosphate deficiency. Physiol Plant 58: 287-294.

Cordell D, Drangert JO, White S (2009) The story of phosphorus: Global food security and food for thought. Global Environ Chang 19: 292-305.

Cunningham G, Mulham W, Milthorpe P, Leigh J (1992) Plants of Western New South Wales. CSIRO.

Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245: 35-47.

Dann PR, Derrick JW, Dumaresq DC, Ryan MH (1996) The response of organic and conventionally grown wheat to superphosphate and reactive phosphate rock. Aust J Exp Agric 36: 71-78.

Dear BS, Ewing MA (2008) The search for new pasture plants to achieve more sustainable production systems in southern Australia. Aust J Exp Agric 48: 387-396.

Dear BS, Li GD, Hayes RC, Hughes SJ, Charman N, Ballard RA (2007) Cullen

australasicum (syn. Psoralea australasica): a review and some preliminary studies related to its potential as a low rainfall perennial pasture legume. The Rangeland J 29: 121-132.

Dear BS, Moore GA, Hughes SJ (2003) Adaptation and potential contribution of temperate perennial legumes to the southern Australian wheatbelt: a review. Aust J Exp Agric 43: 1-18.

Dear BS, Reed KM, Craig AD (2008) Outcomes of the search for new perennial and salt tolerant pasture plants for southern Australia. Aust J Exp Agric 48: 578-588.

Dinkelaker B, Rӧmheld V, Marschner H (1989) Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L.). Plant, Cell and Environ 12: 285-292.

Dolling PJ (2001) Water use and drainage under phalaris, annual pasture, and crops on a duplex soil in Western Australia. Aust J Agric Res 52: 305-316.

Dolling PJ, Latta RA, Ward PR, Robertson MJ, Asseng S (2005) Soil water extraction and biomass production by lucerne in the south of Western Australia. Aust J Agric Res 56: 389-404.

Donald CM (1965) The progress of Australian agriculture and the role of pastures in environmental change. Aust J Sci 27: 187-198.

Dunin FX (1970) Changes in water balance components with pasture management in


south-eastern Australia. J Hydrol 10: 90-102.

Eissenstat DM, Whaley EL, Volder A, Wells CE (1999) Recovery of citrus surface roots following prolonged exposure to dry soil. J Exp Bot 50: 1845-1854.

Ezawa T, Cavagnaro TR, Smith SE, Smith FA, Ohtomo R (2004) Rapid accumulation of polyphosphate in extraradical hyphae of an arbuscular mycorrhizal fungus as revealed by histochemistry and a polyphosphate kinase/luciferase system. New Phytol 161: 387-392.

Ezawa T, Kuwahara S-y, Sakamoto K, Yoshida T, Saito M (1999) Specific inhibitor and substrate specificity of alkaline phosphatase expressed in the symbiotic phase of the arbuscular mycorrhizal fungus, Glomus etunicatum. Mycologia 91: 636-641.

Ezawa T, Smith SE, Smith FA (2002) P metabolism and transport in AM fungi. Plant Soil 244: 221-230.

Finlayson JD, Lawes RA, Metcalf T, Robertson MJ, Ferris D, Ewing MA (2012) A bio-economic evaluation of the profitability of adopting subtropical grasses and pasture-cropping on crop–livestock farms. Agric Sys 106: 102-112.

Fitter AH (2006) What is the link between carbon and phosphorus fluxes in arbuscular mycorrhizas? A null hypothesis for symbiotic function. New Phytol 172: 3-6.

Fixen PE (2009) World fertilizer nutrient reserves—a view to the future. Better Crops 93: 8-11.

Foster K, Ryan MH, Real D, Ramankutty P, Lambers H (2012) Drought resistance at the seedling stage in the promising fodder plant tedera (Bituminaria bituminosa var. albomarginata). Crop and Pasture Science 63: 1034-1042.

Foster K, Ryan MH, Real D, Ramankutty P, Lambers H (2013) Seasonal and diurnal variation in the stomatal conductance and paraheliotropism of tedera (Bituminaria bituminosa var. albomarginata) in the field. Funct Plant Biol 40: 719-729.

Friesen DK, Rao IM, Thomas RJ, Oberson A, Sanz JI (1997) Phosphorus acquisition and cycling in crop and pasture systems in low fertility tropical soils. Plant Soil 196: 289-294.

Gianinazzi Pearson V, Fardeau JC, Asimi S, Gianinazzi S (1981) Source of additional phosphorus absorbed from soil by vesicular-arbuscular mycorrhizal soybeans [insoluble phosphates, isotopic exchange]. Physiologie végétale 19: 33-43.

Glover JD (2005) The necessity and possibility of perennial grain production systems. Renewable Agriculture and Food Systems 20: 1-4.

Grace EJ, Smith FA, Smith SE (2009) Deciphering the arbuscular mycorrhizal pathway of P uptake in non-responsive plant species. In: C Azcón-Aguilar, JM Barea, S Gianinazzi, V Gianinazzi-Pearson (eds) Mycorrhizas - Functional Processes and Ecological Impact. Springer Berlin Heidelberg.

Graham JH (2000) Assessing costs of arbuscular mycorrhizal symbiosis in agroecosystems. APS Press, St. Paul.

Graham JH, Linderman RG, Menge JA (1982) Development of external hyphae by different isolates of mycorrhizal Glomus spp. in relation to root colonization and growth of Troyer citrange. New Phytol 91: 183-189.


Grierson PF, Comerford NB, Jokela EJ (1998) Phosphorus mineralization kinetics and response of microbial phosphorus to drying and rewetting in a Florida spodosol. Soil Biol Biochem 30: 1323-1331.

Grimes J (1997) A revision of Cullen (Leguminosae: Papilionoideae). Aust Syst Bot 10: 565-648.

Harold FM (1966) Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriological Reviews 30: 772-794.

Harrison MJ, van Buuren ML (1995) A phosphate transporter from the mycorrhizal fungus Glomus versiforme. Nature 378: 626-629.

Hayes RC, Li GD, Dear BS, Humphries AW, Tidd JR (2009) Persistence, productivity, nutrient composition, and aphid tolerance of Cullen spp. Crop and Pasture Science 60: 1184-1192.

Hijikata N, Murase M, Tani C, Ohtomo R, Osaki M, Ezawa T (2010) Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus. New Phytol 186: 285-289.

Hill MJ (1996) Potential adaptation zones for temperate pasture species as constrained by climate: a knowledge-based logical modelling approach. Aust J Agric Res 47: 1095-1117.

Hill MJ, Donald GE (1998) Australian Temperate Pastures Database. National Pasture Improvement Coordinating Committee/CSIRO Division of Animal Production: CD-ROM.

Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237: 173-195.

Hopper SD (2009) OCBIL theory: towards an integrated understanding of the evolution, ecology and conservation of biodiversity on old, climatically buffered, infertile landscapes. Plant Soil 322: 49-86.

Hopper SD, Lambers H (2014) Human relationships with and use of kwongkan plants and lands. In: H Lambers (ed) Plant life on the sandplains in southwest Australia, a global biodiversity hotspot. University of Western Australia, Perth, Australia.

Howieson JG, O’Hara GW, Carr SJ (2000) Changing roles for legumes in Mediterranean agriculture: developments from an Australian perspective. Field Crops Res 65: 107-122.

Humphries AW, Auricht GC (2001) Breeding lucerne for Australia's southern dryland cropping environments. Aust J Agric Res 52: 153-169.

Humphries AW, Hughes SJ, Nair RM, Kobelt E, Sandral G (2014) High levels of diversity for seed and forage production exist in Cullen australasicum, a potential new perennial forage legume for dry environments in southern Australia. Rangeland J 36: 41-51.

Jakobsen I, Abbott LK, Robson AD (1992a) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. New Phytol 120: 509-516.

Jakobsen I, Abbott LK, Robson AD (1992b) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L 2. Hyphal transport


of 32P over defined distances. New Phytol 120: 509-516.

Jakobsen I, Abbott LK, Robson AD (1992c) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L .1. spread of hyphae and phosphorus inflow into roots. New Phytol 120: 371-380.

Javot H, Penmetsa RV, Terzaghi N, Cook DR, Harrison MJ (2007a) A Medicago

truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis. Proceedings of the National Academy of Sciences 104: 1720-1725.

Javot H, Pumplin N, Harrison MJ (2007b) Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles. Plant, Cell Environ 30: 310-322.

Johnson NC, Graham JH, Smith FA (1997) Functioning of mycorrhizal associations along the mutualism–parasitism continuum. New Phytol 135: 575-585.

Jones DL (1998) Organic acids in the rhizosphere–a critical review. Plant Soil 205: 25-44.

Kapoor R, Giri B, Mukerji KG (2004) Improved growth and essential oil yield and quality in Foeniculum vulgare mill on mycorrhizal inoculation supplemented with P-fertilizer. Bioresour Technol 93: 307-311.

Kieft TL, soroker E, firestone MK (1987) Microbial biomass response to a rapid increase in water potential when dry soil is wetted. Soil Biology and Biochemistry 19: 119-126.

Kinden D, Brown M (1975a) Electron microscopy of vesicular-arbuscualr mycorrhizae of yellow poplar. I. Characterization of endophytic structures by scanning electron stereoscopy. Can J Microbiol 21: 989.

Kinden DA, Brown MF (1975b) Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. Host-endophyte interactions during arbuscular development. Can J Microbiol 21: 1930-1939.

Kirkegaard JA, Peoples MB, Angus JF, Unkovich MJ (2011) Diversity and evolution of rainfed farming systems in southern Australia. In: P Tow, I Cooper, I Partridge, C Birch (eds) Rainfed Farming Systems. Springer Netherlands.

Koide R (1985) The nature of growth depression in sunflower caused by vesicular-arbuscular mycorrhizal infection. New Phytol 99: 449-462.

Koide RT, Mosse B (2004) A history of research on arbuscular mycorrhiza. Mycorrhiza 14: 145-163.

Kornberg A (1999) Inorganic polyphosphate: a molecule of many functions. In: H Schröder, WG Müller (eds) Inorganic Polyphosphates. Springer Berlin Heidelberg.

Kotze AC, O’Grady J, Emms J, Toovey AF, Hughes S, Jessop P, Bennell M, Vercoe PE, Revell DK (2009) Exploring the anthelmintic properties of Australian native shrubs with respect to their potential role in livestock grazing systems. Parasitology 136: 1065-1080.

Kulaev IS, Vagabov VM (1983) Polyphosphate metabolism in micro-organisms. Adv Microb Physiol 24: 83-171.

Lambers H (2003) Introduction, dryland salinity: a key environmental issue in southern Australia. Plant Soil 257: 5-7.


Lambers H, Brundrett MC, Raven JA, Hopper SD (2010) Plant mineral nutrition in ancient landscapes: high plant species diversity on infertile soils is linked to functional diversity for nutritional strategies. Plant Soil 334: 11-31.

Lambers H, Chapin III FS, Pons TL (2008) Plant physiological ecology. Springer.

Lambers H, Shane MW, Cramer MD, Pearse SJ, Veneklaas EJ (2006) Root structure and functioning for efficient acquisition of phosphorus: matching morphological and physiological traits. Ann Bot-London 98: 693-713.

Lawton K, Vomocil JA (1954) The dissolution and migration of phosphorus from granular superphosphate in some Michigan soils. Soil Sci Soc Am J 18: 26-32.

Lefebvre DD, Glass ADM (1982) Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate. Physiol Plant 54: 199-206.

Lekberg Y, Koide RT (2005) Is plant performance limited by abundance of arbuscular mycorrhizal fungi? A meta-analysis of studies published between 1988 and 2003. New Phytol 168: 189-204.

Lewis DC, Clarke AL, Hall WB (1987) Accumulation of plant nutrients and changes in soil properties of sandy soils under fertilized pasture in southeastern South-Australia. I. Phosphorus. Aust J Soil Res 25: 193-202.

Li G, Lodge G, Moore G, Craig A, Dear B, Boschma S, Albertsen T, Miller S, Harden S, Hayes R (2008) Evaluation of perennial pasture legumes and herbs to identify species with high herbage production and persistence in mixed farming zones in southern Australia. Aust J Exp Agric 48: 449-466.

Li X-L, George E, Marschner H (1991) Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil. Plant Soil 136: 41-48.

Loi A, Howieson JG, Nutt BJ, Carr SJ (2005) A second generation of annual pasture legumes and their potential for inclusion in Mediterranean-type farming systems. Aust J Exp Agric 45: 289-299.

Lovelock CE, Miller R (2002) Heterogeneity in inoculum potential and effectiveness of arbuscular mycorrhizal fungi. Ecology 83: 823-832.

Martínez-Fernández D, Walker DJ, Romero P, Correal E (2010) The physiology of drought tolerance in Tedera (Bituminaria bituminosa). Options Méditerranéennes 92: 155-159.

McArthur WM (1991) Reference soils of South-Western Australia Perth: Department of Agriculture.

Méndez P, Fernández M, Santos A (1991) Variedades de Bituminaria bituminosa (L.) Stirton (Leguminosae) en el archipiélago canario. Reun cient Soc Esp para el estud de los pastos.

Monjardino M, Revell D, Pannell DJ (2010) The potential contribution of forage shrubs to economic returns and environmental management in Australian dryland agricultural systems. Agric Sys 103: 187-197.

Nichols PGH, Foster KJ, Piano E, Pecetti L, Kaur P, Ghamkhar K, Collins WJ (2013) Genetic improvement of subterranean clover (Trifolium subterraneum L.). 1. Germplasm, traits and future prospects. Crop and Pasture Science 64: 312-346.

Nichols PGH, Loi A, Nutt BJ, Evans PM, Craig AD, Pengelly BC, Dear BS, Lloyd DL,


Revell CK, Nair RM, Ewing MA, Howieson JG, Auricht GA, Howie JH, Sandral GA, Carr SJ, de Koning CT, Hackney BF, Crocker GJ, Snowball R, Hughes SJ, Hall EJ, Foster KJ, Skinner PW, Barbetti MJ, You MP (2007) New annual and short-lived perennial pasture legumes for Australian agriculture—15 years of revolution. Field Crops Res 104: 10-23.

Nicol DL, Finlayson J, Colmer TD, Ryan MH (2013) Opportunistic Mediterranean agriculture – Using ephemeral pasture legumes to utilize summer rainfall. Agric Sys 120: 76-84.

Oehl F, Laczko E, Bogenrieder A, Stahr K, Bösch R, van der Heijden M, Sieverding E (2010) Soil type and land use intensity determine the composition of arbuscular mycorrhizal fungal communities. Soil Biol Biochem 42: 724-738.

Oehl F, Sieverding E, Wiemken A (2007) History and biogeography of arbuscular mycorrhizal fungi–a global view. Micologia–avanços no conhecimento. 5 Congresso Brasileiro de Micologia, Recife,Brazil.

Ogawa N, DeRisi J, Brown PO (2000) New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae

revealed by genomic expression analysis. Mol Biol Cell 11: 4309-4321.

Ohtomo R, Saito M (2005) Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus. New Phytol 167: 571-578.

Olsson PA, Hammer EC, Pallon J, van Aarle IM, Wallander H (2011) Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis. Fungal Biology 115: 643-648.

Öpik M, Moora M, Liira J, Zobel M (2006) Composition of root-colonizing arbuscular mycorrhizal fungal communities in different ecosystems around the globe. J Ecol 94: 778-790.

Page V, Weisskopf L, Feller U (2006) Heavy metals in white lupin: uptake, root‐to‐shoot transfer and redistribution within the plant. New Phytol 171: 329-341.

Pang J, Ryan MH, Tibbett M, Cawthray GR, Siddique KHM, Bolland MDA, Denton MD, Lambers H (2009a) Variation in morphological and physiological parameters in herbaceous perennial legumes in response to phosphorus supply. Plant Soil 331: 241-255.

Pang J, Tibbett M, Denton MD, Lambers H, Seddique KHM, Bolland MDA, Revell CK, Ryan MH (2009b) Variation in seedling growth of 11 perennial legumes in response to phosphorus supply. Plant Soil 328: 133-143.

Pearse SJ, Veneklaas EJ, Cawthray GR, Bolland MDA, Lambers H (2006) Carboxylate release of wheat, canola and 11 grain legume species as affected by phosphorus status. Plant Soil 288: 127-139.

Pearson JN, Jakobsen I (1993) The relative contribution of hyphae and roots to phosphorus uptake by arbuscular mycorrhizal plants, measured by dual labelling with 32P and 33P. New Phytol 124: 489-494.

Peoples MB, Baldock JA (2001) Nitrogen dynamics of pastures: nitrogen fixation inputs, the impact of legumes on soil nitrogen fertility, and the contributions of fixed nitrogen to Australian farming systems. Aust J Exp Agric 41: 327-346.

Peterson RL, Massicotte HB, Melville LH (2004) Mycorrhizas: anatomy and cell


biology. NRC Reseach Press, Ottawa.

Porqueddu C, González F (2011) Role and potential of annual pasture legumes in Mediterranean farming systems. Pastos 36: 125-142.

Powles SB, Matthews JM (1992) Multiple herbicide resistance in annual ryegrass (Lolium rigidum): a driving force for the adoption of integrated weed management. Resistance’91: Achievements and Developments in Combating Pesticide Resistance. Springer.

Qiu S, McComb AJ, Bell RW, Davis JA (2004) Phosphorus dynamics from vegetated catchment to lakebed during seasonal refilling. Wetlands 24: 828-836.

Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Biol 50: 665-693.

Rausch C, Daram P, Brunner S, Jansa J, Laloi M, Leggewie G, Amrhein N, Bucher M (2001) A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 414: 462-470.

Real D, Li GD, Clark S, Albertsen TO, Hayes RC, Denton MD, Mario FDA, Dear BS (2011) Evaluation of perennial forage legumes and herbs in six Mediterranean environments. Chilean Journal of Agricultural Research 71: 357-369.

Real D, Oldham C, Nelson MN, Croser J, Castello M-C, Gherardi S, Finlayson J, Revell C, Pradhan A, O’Hara G (Accessed in 2014) Tedera: from a promising novel species to a commercial pasture option for Mediterranean southern Australia.

Real D, Verbyla A (2010) Maximizing genetic gains using a "plant" model in the Tedera (Bituminaria bituminosa var. albomarginata and var. crassiuscula) breeding program in Australia. Options Méditerranéennes 92: 87-96.

Reeves TG, Ewing MA (1993) Is ley farming in Mediterranean zones just a passing phase. 'Proceedings of the XVII International Grassland Congress’. Rockhampton, Australia.

Revell DK, Norman HC, Vercoe PE, Phillips N, Toovey A, Bickell S, Hulm E, Hughes S, Emms J (2013) Australian perennial shrub species add value to the feed base of grazing livestock in low- to medium-rainfall zones. Animal Production Science 53: 1221-1230.

Richardson AE, Lynch JP, Ryan PR, Delhaize E, Smith FA, Smith SE, Harvey PR, Ryan MH, Veneklaas EJ, Lambers H, Oberson A, Culvenor RA, Simpson RJ (2011) Plant and microbial strategies to improve the phosphorus efficiency of agriculture. Plant Soil 349: 121-156.

Ridley AM, White RE, Simpson RJ, Callinan L (1997) Water use and drainage under phalaris, cocksfoot, and annual ryegrass pastures. Aust J Agric Res 48: 1011-1023.

Rivett DE, Tucker DJ, Jones GP (1983) The chemical composition of seeds from some Australian plants. Aust J Agric Res 34: 427-432.

Robertson FA, Nash DM (2008) Phosphorus and nitrogen in soil, plants, and overland flow from sheep-grazed pastures fertilized with different rates of superphosphate. Agric Ecosyst Environ 126: 195-208.

Robson AD (1990) The role of self-regenerating pasture in rotation with cereals in Mediterranean areas. In: A Osman, M Ibrahim, M Jones (eds) The role of legumes in the farming systems of the Mediterranean areas. Springer, ICARDA, Netherlands.


Rosewarne GM, Barker SJ, Smith SE, Smith FA, Schachtman DP (1999) A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular–arbuscular mycorrhizal fungus. New Phytol 144: 507-516.

Ryan MH, Angus J (2003) Arbuscular mycorrhizae in wheat and field pea crops on a low P soil: increased Zn-uptake but no increase in P-uptake or yield. Plant Soil 250: 225-239.

Ryan MH, Ash JE (1996) Colonisation of wheat in southern New South Wales by vesicular-arbuscular mycorrhizal fungi is significantly reduced by drought. Aust J Exp Agric 36: 563-569.

Ryan MH, Bennett R, Denton M, Hughes S, Mitchell M, Carmody B, Edmonds-Tibbett T, Nicol D, Kroiss L, Snowball R (2008) Searching for native perennial legumes with pasture potential. Proc 14th Aust Agron Conf.

Ryan MH, Kirkegaard JA (2012) The agronomic relevance of arbuscular mycorrhizas in the fertility of Australian extensive cropping systems. Agric Ecosyst Environ 163: 37-53.

Ryan MH, McCully ME, Huang CX (2003) Location and quantification of phosphorus and other elements in fully hydrated, soil-grown arbuscular mycorrhizas: a cryo-analytical scanning electron microscopy study. New Phytol 160: 429-441.

Ryan MH, McCully ME, Huang CX (2007) Relative amounts of soluble and insoluble forms of phosphorus and other elements in intraradical hyphae and arbuscules of arbuscular mycorrhizas. Funct Plant Biol 34: 457-464.

Ryan MH, Small DR, Ash JE (2000) Phosphorus controls the level of colonisation by arbuscular mycorrhizal fungi in conventional and biodynamic irrigated dairy pastures. Aust J Exp Agric 40: 663-670.

Ryan MH, Tibbett M (2008) The role of arbuscular mycorrhizas in organic farming. In: H Kirchmann, L Bergstrom (eds) Organic crop production- ambitions and limitations. Springer Netherlands.

Ryan MH, Tibbett M, Edmonds-Tibbett T, Suriyagoda LDB, Lambers H, Cawthray GR, Pang J (2012) Carbon trading for phosphorus gain: the balance between rhizosphere carboxylates and arbuscular mycorrhizal symbiosis in plant phosphorus acquisition. Plant, Cell Environ 35: 2170-2180.

Sanders F, Tinker P (1973) Phosphate flow into mycorrhizal roots. Pestic Sci 4: 385-395.

Sanders IR, Koide RT (1994) Nutrient acquisition and community structure in co-occurring mycotrophic and non-mycotrophic oldfield annuals. Funct Ecol 8: 77-84.

Sardans J, Peñuelas J (2007) Drought changes phosphorus and potassium accumulation patterns in an evergreen Mediterranean forest. Funct Ecol 21: 191-201.

Saxena M (1988) Food legumes in the Mediterranean type of environment and ICARDA’s efforts in improving their productivity. In: DP Beck, LA Materon (eds) Nitrogen Fixation by Legumes in Mediterranean agriculture. Springer.

Scholz RW, Ulrich AE, Eilittä M, Roy A (2013) Sustainable use of phosphorus: A finite resource. Sci Total Environ 461: 799-803.

Schüßler A, Schwarzott D, Walker C (2001) A new fungal phylum, the


Glomeromycota: phylogeny and evolution. Mycol Res 105: 1414-1421.

Silsbury JH, Brittan NH (1955) Distribution and ecology of the genus Kennedya Vent. in Western Australia. Aust J Bot 3: 113-135.

Simpson RJ, Oberson A, Culvenor RA, Ryan MH, Veneklaas EJ, Lambers H, Lynch JP, Ryan PR, Delhaize E, Smith FA, Smith SE, Harvey PR, Richardson AE (2011) Strategies and agronomic interventions to improve the phosphorus-use efficiency of farming systems. Plant Soil 349: 89-120.

Small E (2003) Distribution of perennial Medicago with particular reference to agronomic potential for the semiarid Mediterranean climate In: SJ Bennett (ed) New perennial legumes. University of Western Australia.

Smith FA, Smith SE (1981) Mycorrhizal infection and growth of Trifolium

subterraneum: use of sterilized soil as a control treatment. New Phytol 88: 299-309.

Smith SE, Read DJ (2008) Mycorrhizal Symbiosis. Academic press, London, UK.

Smith SE, Smith FA (2011) Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales. Annu Rev Plant Biol 62: 227-250.

Smith SE, Smith FA, Jakobsen I (2003) Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. Plant Physiol 133: 16-20.

Snowball R, D'Antuono M, Cohen B, Gajda K, Bennett R (2010) The value of germplasm nurseries in selecting species for field evaluation. Crop and Pasture Science 61: 957-969.

Solaiman MZ, Ezawa T, Kojima T, Saito M (1999) Polyphosphates in intraradical and extraradical hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita. Appl Environ Microbiol 65: 5604-5606.

Suriyagoda LDB, H. RM, Renton M, Lambers H (2010) Multiple adaptive responses of Australian native perennial legumes with pasture potential to grow in phosphorus-and moisture-limited environments. Ann Bot-London 105: 755-767.

Suriyagoda LDB, Lambers H, Renton M, Ryan MH (2012) Growth, carboxylate exudates and nutrient dynamics in three herbaceous perennial plant species under low, moderate and high phosphorus supply. Plant Soil 358: 105-117.

Suriyagoda LDB, Real D, Renton M, Lambers H, Ryan MH (2013) Establishment, survival, and herbage production of novel, summer-active perennial pasture legumes in the low-rainfall cropping zone of Western Australia as affected by plant density and cutting frequency. Crop and Pasture Science 64: 71-85.

Tibbett M, Ryan MH, Barker SJ, Chen Y, Denton MD, Edmonds-Tibbett T, Walker C (2008) The diversity of arbuscular mycorrhizas of selected Australian Fabaceae. Plant Biosyst 142: 420-427.

Turner BL, Driessen JP, Haygarth PM, Mckelvie ID (2003) Potential contribution of lysed bacterial cells to phosphorus solubilisation in two rewetted Australian pasture soils. Soil Biol Biochem 35: 187-189.

Turner BL, McKelvie ID, Haygarth PM (2002) Characterisation of water-extractable soil organic phosphorus by phosphatase hydrolysis. Soil Biol Biochem 34: 27-35.


Turner FT, Gilliam JW (1976) Increased P diffusion as an explanation of increased P availability in flooded rice soils. Plant Soil 45: 365-377.

Unkovich M (2002) David and goliath: symbiotic nitrogen fixation and fertilisers in Australian agriculture. Abstracts 13th Australian Nitrogen Fixation Conference, Adelaide’. Australian Society for Nitrogen Fixation.

van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T, Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396: 69-72.

van Gestel M, Merckx R, Vlassak K (1993) Microbial biomass responses to soil drying and rewetting: The fate of fast- and slow-growing microorganisms in soils from different climates. Soil Biol Biochem 25: 109-123.

Vance CP, Uhde Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157: 423-447.

Veiga RSL, Faccio A, Genre A, Pieterse CMJ, Bonfante P, van der Heijden MGA (2013) Arbuscular mycorrhizal fungi reduce growth and infect roots of the non-host plant Arabidopsis thaliana. Plant, Cell Environ 36: 1926-1937.

Veiga RSL, Howard K, van der Heijden MGA (2012) No evidence for allelopathic effects of arbuscular mycorrhizal fungi on the non-host plant Stellaria media. Plant Soil 360: 319-331.

Ventura MR, Castanon JIR, Mendez P (2009) Effect of season on tedera (Bituminaria

bituminosa) intake by goats. Anim Feed Sci Technol 153: 314-319.

Viereck N, Hansen PE, Jakobsen I (2004) Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo 31P NMR spectroscopy. New Phytol 162: 783-794.

Vu DT, Armstrong RD, Newton PJ, Tang C (2011) Long-term changes in phosphorus fractions in growers’ paddocks in the northern Victorian grain belt. Nutr Cycl Agroecosyst 89: 351-362.

Wagg C, Jansa J, Stadler M, Schmid B, van der Heijden MGA (2011) Mycorrhizal fungal identity and diversity relaxes plant-plant competition. Ecology 92: 1303-1313.

Walker TW, Syers JK (1976) The fate of phosphorus during pedogenesis. Geoderma 15: 1-19.

Wang X, Pearse SJ, Lambers H (2013) Cluster-root formation and carboxylate release in three Lupinus species as dependent on phosphorus supply, internal phosphorus concentration and relative growth rate. Ann Bot-London 112: 1449-1459.

Ward PR, Dunin FX, Micin SF (2002) Water use and root growth by annual and perennial pastures and subsequent crops in a phase rotation. Agric Water Manag 53: 83-97.

Wissuwa M (2005) Combining a modelling with a genetic approach in establishing associations between genetic and physiological effects in relation to phosphorus uptake. Plant Soil 269: 57-68.

Wu J, Brookes PC (2005) The proportional mineralisation of microbial biomass and


organic matter caused by air-drying and rewetting of a grassland soil. Soil Biol Biochem 37: 507-515.

Yao Q, Li X, Feng G, Christie P (2001) Mobilization of sparingly soluble inorganic phosphates by the external mycelium of an abuscular mycorrhizal fungus. Plant Soil 230: 279-285.

Young CC, Juang TC, Guo HY (1986) The effect of inoculation with vesicular-arbuscular mycorrhizal fungi on soybean yield and mineral phosphorus utilization in subtropical-tropical soils. Plant Soil 95: 245-253.

CHAPTER 2.

Do Arbuscular Mycorrhizas or Heterotrophic Soil Microbes

Contribute Toward Plant Acquisition of a Pulse of Mineral Phosphate?

2.1 Preface

This experiment was established as a preliminary experiment to confirm the

methodology for the next experiment (chapter 3). In this experiment Trifolium

subterraneum was used as the host plant since there have been numerous studies on it

with AMF.

This paper addresses three hypotheses: 1) AMF will enhance uptake of P from a pulse

as may occur following addition of fertiliser or the start of the winter rain; 2) Uptake of

P is greater when plants are colonised by a diverse community of AMF (inoculation

with field soil), rather than a single-species inoculum and 3) Presence of microbes in

the soil increases P uptake compare to the control.

Chapter 2. Arbuscular mycorrhizas and a P pulse 36

2.2 Published paper

CHAPTER 3.

Moderating Mycorrhizas: Arbuscular Mycorrhizas Modify

Rhizosphere Chemistry and Maintain Plant Phosphorus Status within

Narrow Boundaries

3.1 Preface

According to the results from the preliminary experiment (chapter 2), this experiment

was optimised to have a better understanding of the response of perennial pastures to a

pulse of P when they are colonised by AMF.

This study addressed three hypotheses associated with this scenario for plants grown in

a soil with moderate P levels representative of agricultural systems: 1) AMF will

moderate shoot P concentration following a pulse; 2) Release of carboxylates into the

rhizosphere will decrease when plants encounter a pulse of P and 3) Colonisation by

AMF will decrease the release of carboxylates into the rhizosphere.

Hypotheses 1) and 3) were supported.

Chapter 3. Moderating mycorrhizas 50

3.2 Published paper

CHAPTER 4.

A Sudden, High Pulse of Phosphorus Triggers Formation of Granules

with Extraordinary High Concentrations of Phosphorus and

Potassium in Cortical Cell Vacuoles of Trifolium subterraneum

4.1 Preface

According to the results of the previous experiments (the reduction in P movement to

shoots in colonised plants following the P pulse), in this experiment I intended to

measure the amount of P in the hyphae of AMF using quantitative X-ray microanalysis

since the moderating effect of AMF might be due to storage of high amounts of poorly

soluble P in intraradical hyphae in the roots. The hypothesis for this experiment was:

following a significant pulse of P, P movement to shoots is restricted, due to storage of

high concentrations of P in intraradical AM hyphae in insoluble forms. However,

unfortunately, due to low level of colonisation, none of the samples which were

prepared for analysis contained any hyphae. However, granules were found in roots

and they contained very high concentrations of P. This then became the focus of this

paper.

Chapter 4. Root P accumulation after a P pulse 64

4.2 Ready for submission paper

A sudden, high pulse of phosphorus triggers formation of granules with

extraordinary high concentrations of phosphorus and potassium in cortical cell

vacuoles of Trifolium subterraneum

Nazanin K. Nazeri1, 2, Peta L. Clode5, Hans Lambers1, Mark Tibbett3, Megan H. Ryan1, 2

1School of Plant Biology and Institute of Agriculture, The University of Western Australia, 35

Stirling Highway, Crawley (Perth), WA 6009, Australia

2Future Farm Industries Cooperative Research Centre, The University of Western Australia, 35

Stirling Highway, Crawley (Perth) WA 6009, Australia

3Department of Environmental Science and Technology, School of Applied Sciences, Cranfield

University, Cranfield, MK43 0SZ, England.

5Centre for Microscopy, Characterisation and Analysis, The University of Western Australia,

Crawley (Perth), WA 6009, Australia

Author for correspondence

Nazanin Nazeri

School of Plant Biology, M084

The University of Western Australia, Crawley (Perth), WA 6009, Australia

Ph +61 8 6488 1992

Email: [email protected]

Running title: Root P accumulation after a pulse of P


Abstract

Due to the low availability of phosphorus (P) in the soils of across southern Australia,

application of P fertilisers on pastures is common. Addition of P fertiliser causes a pulse

of P to be released into the soil and encountered by plant roots. I intended to measure

the amount of P in the internal hyphae of arbuscular mycorrhizal fungi (AMF)

colonising the roots of Trifolium subterraneum using quantitative X-ray microanalysis

following a large pulse of P (150 mg P kg-1 soil) to soil. Trifolium subterraneum was

grown in a sterilised field soil, containing a moderate amount of P (18 mg kg-1), either

with (+AMF) or without (-AMF) addition of inoculum (Scutellospora calospora). At

week six, plants received either a high pulse of 150 mg P kg-1 soil (150P), a moderate

pulse of 15 mg P kg-1 soil (15P) or no pulse (No-P). One week later, all plants were

harvested. The percentage of root length colonised by AMF, shoot and root dry weight,

and nutrient concentrations in the dry shoots and roots were measured. A small number

of root subsamples were freeze substituted and embedded in resin for examination using

quantitative X-ray microanalysis. The P pulse had no effect on shoot dry weight, but

root dry weight decreased by 28% and 14% when a pulse of 15 mg P kg-1 soil and 150

mg P kg-1 soil, respectively, was applied. Shoot and root P concentrations increased

from No-P to 150P (from 2 to 14 mg P g-1 dry weight (DW) in the shoot and from 2 to

20 mg P g-1 DW in the root). Due to a low level of colonisation, no hyphae were found

in the samples prepared for quantitative X-ray microanalysis. However, large granules

with high concentrations of P and potassium (K) were found in the samples that

received 150P. I conclude that a high pulse of P triggers formation of granules with high

concentrations of P and K in cortical cell vacuoles and suggest that these represent a

means for the plant to avoid P toxicity.

Keywords: Phosphorus pulse, granules, “polyphosphate overplus” phenomenon,


Introduction

Plant P supply under field conditions is rarely constant (for example see Hedley et al.

(1982) and Tiessen et al. (1983)), and sometimes plants may encounter a pulse of P.

Such a pulse might occur due to the application of fertilisers (Lewis et al. 1987). The

responses of plants that are colonised by arbuscular mycorrhizal fungi (AMF) to a pulse

of P are different to those of uncolonised plants. For instance, Nazeri et al. (2013)

reported that root P concentration increased to a similar degree in colonised and

uncolonised roots of Trifolium subterraneum L cv. Denmark, a commonly grown

annual pasture legume, but that the rate of increase of P concentration in the shoots was

lower for colonised plants than for uncolonised plants. This result was confirmed in a

second study, where Nazeri et al. (2014) added P in a pulse to colonised and

uncolonised plants of four perennial legumes (Kennedia prostrata, Cullen

australasicum, Bituminaria bituminosa and Medicago sativa) and Trifolium

subterraneum. The authors concluded AMF moderate P uptake when P suddenly

becomes excessive to plant requirements, and thus act to protect the plant. The authors

hypothesised that the reduction in P movement to shoots in colonised plants following

the P pulse might be due to storage of high amounts of poorly soluble P in intraradical

hyphae in the roots (see Ryan et al. (2003)) and thus a reduction in the availability of P

for transport to the shoots. Further confirmation of this concept was recently provided

by Kariman et al. (2014) who found that colonisation of 16-week-old jarrah (Eucalyptus

marginata) seedlings with AMF (as well as ectomycorhizal fungi) resulted in

significantly fewer toxicity symptoms seven days after a high P pulse (30 mg P kg-1

soil) compared to uncolonised plants. There was a positive linear relationship between

shoot P concentration and toxicity symptoms.

To obtain a better understanding of how AMF moderate P uptake following a pulse, it is

essential to know where exactly the P is stored in the root cells or in the fungal tissue.

According to Smith and Smith (2011), when plants are colonised by AMF the direct P-

uptake pathway (through root hairs and epidermal cells) can become inactive, and the

AM uptake pathway becomes the dominant uptake pathway. By mapping the colonised

root system using cryo-scanning electron microscopy, Ryan et al. (2003) showed P

concentrations in the intraradical hyphae were significantly higher (generally 60-170

mM) than P concentrations in uncolonised cortical cell vacuoles (mostly undetectable


(i.e. less than 10 mM)). The authors also showed a strong linear relationship between

the concentrations of P, K and Mg. In addition, Ryan et al. (2007) determined the

proportion of insoluble P in the intraradical hyphae (not extracted following destruction

of cell membranes by a variety of methods). The authors found 30-70% of the P in the

hyphae and concluded this P was likely to be in the form of polyphosphate, a commonly

found P storage compound in the Glomeromycota (Hijikata et al. 2010; Karandashov

and Bucher 2005; Viereck et al. 2004).

Polyphosphate is a linear condensed polymer of three to thousands of inorganic

phosphate (Pi) residues, which are linked together by high-energy phospho-anhydride

bonds (Ohtomo and Saito 2005). It is a very widespread molecule and has been

commonly reported in bacteria, fungi, protozoa and mammals (Kulaev et al. 2005).

There are a great number of reports about formation of polyphosphates in AMF (Cox et

al. 1980; Ezawa et al. 2004; Funamoto et al. 2007; Kuga et al. 2008; Rasmussen et al.

2000; Ryan et al. 2007; Solaiman et al. 1999; Viereck 2002). Since 1961, the extraction

of polyphosphate from higher plant tissues has been reported by several authors (Jeffrey

1964; Jeffrey 1968; Miyachi 1961; Nassery 1969). However, recent reports of

polyphosphate in plants are few and the reliability of the methodology in the earlier

reports is questionable (Kulaev et al. 2005).

In the present experiment I grew plants in a field soil with a moderate level of P, with

and without addition of AM fungal inoculum i.e. a similar protocol to that used by

Nazeri et al.(2013; 2014). I used quantitative X-ray microanalysis to determine the

distribution of P in roots. I hypothesised that high concentrations of P, associated with

cations, would be present in the hyphae of fungi. However, due to an unexpectedly low

level of colonisation by AMF, no fungal structures were found in any of the cross

sections and I report instead on the response of uncolonised areas and non-mycorrhizal

roots to a pulse of P.

Materials and Methods

Glasshouse experiment

The experiment was fully randomised with two treatments: arbuscular mycorrhizal

fungi (+AMF and –AMF) and P pulse (0, 15 and 150 mg P kg-1 dry soil added a week

before harvest). Note that the rate of 15 mg P kg-1 dry soil was the same as used by


Nazeri et al. (2013; 2014). There were four replicates.

The experiment was conducted using a field soil. The characteristics of the field soil are

described by Nazeri et al. (2013). Soil was collected from the top 10 cm of a weedy

annual pasture paddock at Newdegate (33° 06′ 16″ S, 118° 49′ 50″ E, 333 m elevation)

in the southern cropping zone of Western Australia. Soil analysis was performed by

CSBP Future Farm analytical laboratories, Bibra Lake, Australia. Bicarbonate-

extractable P was 18 mg kg-1 and potassium (K) was 80 mg kg-1 (Colwell 1965).

Available P, measured using the Olsen test, was 11 mg kg-1. There was 6 mg kg-1

ammonium-N and 10 mg kg-1 nitrate-N (Searle 1984). Available sulfur (S) was 9.3 mg

kg-1 (Blair et al. 1991). Soil pH was 5.3 (CaCl2) and 6.0 (H2O). The soil consisted of

76.0% coarse sand, 23.9% fine sand, 0.05% clay and 0.11% silt.

Soil was steam-sterilised at 80°C for one hour on consecutive days, and then dried

overnight in a sterile environment. White non-draining pots were filled with 1.2 kg of

soil as follows. The bottom half of the pots was filled with sterilised field soil (600 g)

and the top half was filled with a 1:4 mixture of sterilised field soil and inoculum from a

pot culture of the AM fungus Scutellospora calospora, which presumably contained

spores, hyphae and colonised root segments. The inoculum had a pH (CaCl2) of 5.0 and

contained 10 mg kg-1 bicarbonate-extractable P and 32 mg kg-1 K (Colwell), 3 mg kg-1

ammonium-N and less than 1 mg kg-1 nitrate-N (Searle 1984).

Seeds of Trifolium subterraneum L., cv. Denmark, were scarified using sand paper and

soaked in water until imbibed. Imbibed seeds were kept on moist filter papers in Petri

dishes for 3-4 days until all were germinated. Two germinated seeds were planted in

each pot. After one week, seedlings were thinned to one plant per pot and a dense

suspension of an appropriate strain of Rhizobium trifolii was added. Pots were watered

to field capacity by weight twice a week. No nutrient solutions were added to the pots.

Pots were arranged randomly in a temperature-controlled glasshouse (mean maximum

temperature ~ 24°C and mean minimum temperature ~ 12°C) at the Crawley campus of

the University of Western Australia, Perth, Australia. The experiment ran from 4 June

2012 until 25 July 2012. At week six, the three P pulse treatments were applied. Twenty

millilitres of 15 (15P) and 150 mg P kg-1 soil (150P) in the form of KH2PO4 were added

to the pots. The control pots did not receive any P (No-P).

All plants were harvested one week later. Shoots were cut at the soil surface, rinsed

once in deionised (DI) water, dried for 72 hours at 70°C and weighed. Shoots were then


finely ground and later used for the P digestion. Roots were washed thoroughly, rinsed

once in deionised water (DI), blotted dry with paper towels and fresh weights

determined. A subsample of approximately 3 g was taken from each root system and its

fresh weight recorded. The subsamples were kept in 70% ethanol and later assessed for

mycorrhizal colonisation. The remaining roots were dried at 70°C for 72 hours and

weighed. To assess the mycorrhizal colonisation level, roots were cleared in 10% (w/v)

KOH at room temperature for 3 days and then washed thoroughly with DI water and

stained in 5% (v/v) Shaeffer black ink for 2 hours (Vierheilig et al. 1998). For

destaining, the roots were washed in DI water and stored in lactoglycerol (1:1:2 lactic

acid, deionised water, glycerol) (Vierheilig et al. 1998). The percentage of root length

colonised by AMF was assessed using the line-intersect method for at least 100

intersections per sample (Giovannetti and Mosse 1980).

For determination of tissue P, subsamples of ~ 0.2 g of dried, ground, root and shoot

material were digested in a 3:1 HNO3:HClO4 mixture and analysed using inductively-

coupled plasma (ICP) atomic absorption with a Perkin Elmer Optima 5300 DV optical

emission spectrometer (OES; Shelton, CT, USA).

Data were analysed in Genstat version 14.1 (Lawes Agricultural Trust, Rothamsted

Experimental Station, Harpenden, UK) using one-way ANOVA to assess the effect of

the P-pulse treatment (No-P, 15P and 150P) on response variables in the +AMF pots

(data from the –AMF pots were generally disregarded, see explanation below).

Normality was checked and no transformations were necessary.

Sample preparation for X-ray microanalysis

One plant from each treatment was selected and one to three subsamples (one from –

AMF, 150P; two from +AMF, No-P and three from +AMF, 150P (Table 4)) were

excised from each root. The Subsamples were ~ 1 cm long (excluding root tips) and

immediately plunge-frozen into liquid nitrogen. Frozen subsamples were subsequently

freeze-substituted in a 10% (v/v) acrolein in di-ethyl ether mixture over a 0.3 nm

molecular sieve (Table 1) and embedded in Araldite 502 epoxy resin (Table 2)

following the methods of Marshall (1980).


Table 1. Temperature and time at each temperature that frozen root subsamples were freeze-substituted.

Temperature (°C) -100 -90 -70 -20 0

Time (h) 24 168 336 24 hold

All solutions were anhydrous and once embedded, samples were kept desiccated.

Araldite 502 is the preferred epoxy resin, as it contains negligible levels of elements

detectable by energy-dispersive microanalysis (Påsgård et al. 1994), while acrolein is

considered a superior fixative for plants and for use at low temperatures (Echlin 1992).

The substitution method, when performed under anhydrous conditions, maximises the

retention and immobilisation of diffusible ions (Condron and Marshall 1990; Marshall

1980; Orlovich and Ashford 1995; Påsgård et al. 1994). This substitution method has

been reliably used to prepare plant tissues (Altus and Canny 1985; Bidwell et al. 2004;

Crawford et al. 1998; De Filippis 1978; Orlovich and Ashford 1995; Ross et al. 1983),

algal samples (Mostaert et al. 1996), optic nerves (Wells et al. 2012), coral epithelia

(Clode and Marshall 2002; Clode and Marshall 2003; Clode and Marshall 2004;

Marshall et al. 2007; Marshall and Wright 1991, 1995), and Malpighian (Hyatt and

Marshall 1985) and renal tubules (Marshall et al. 1994), for elemental analysis.

Table 2. The process of embedding freeze-substituted subsamples in Araldite 502 epoxy resin.

Resin-embedded mounts were prepared so as to create a transverse section of the root in

the block face. All cutting was done on a Leica EM UC6 ultramicrotome using an

anhydrous glass knife. Anhydrous -cut 1 µm-thick sections were initially collected and

placed on glass slides for toluidine blue staining to check the quality of the sample and

to identify suitable regions of the root for analysis. Optical images from stained sections

were collected using a Zeiss Axioskop microscope fitted with a digital camera. Once a

suitable region was identified, the block face was finely planed using an anhydrous

Step Process Time 1 Rinse substituted samples in anhydrous diethyl ether 10 mins × 2 2 Infiltrate in anhydrous resin:ether 1:3 24 h 3 Infiltrate in anhydrous resin:ether 1:1 24 h 4 Infiltrate in anhydrous resin:ether 3:1 24 h 5 Infiltrate in 100% anhydrous resin under vacuum Few hours 6 Mount in final moulds in 100% anhydrous resin and place under

vacuum 24 h

7 Cure samples in moulds at 60°C under vacuum 2 days 8 Keep resin blocks dessicated Indefinitely


knife to create a perfectly flat, cross-sectional surface. The block was then mounted on

an aluminium microscope stub with double-sided carbon tape, and the edges painted

with conductive carbon paint, before being coated with 20 nm of carbon.

Quantitative X-ray microanalysis

Two transverse planed sections from each root were examined. In most cross sections,

regions of interest from three structures (granules in cortex cells, cortex cell vacuoles

and vascular tissue) were measured multiple times. Root blocks were analysed in a

Zeiss Supra 55 field emission scanning electron microscope (SEM) fitted with an

Oxford X-Max80 SDD X-ray detector (80 mm2) interfaced to Oxford Instruments

AZtecEnergy software. The microscope was operated at 15 kV in high current mode.

Immediately prior to each map acquisition, the instrument was calibrated and the beam

current measured and recorded using a pure copper standard. Elemental maps were

acquired at a resolution of 1024 × 768 pixels, for > 400 frames, with a dwell time of 50-

100 µs per pixel. Drift correction and pulse-pile up correction were activated. For such

analyses of bulk samples, the analytical resolution approximates a 2-3 µm sphere, with

detection limits around a few mmol kg-1 (Roomans and Dragomir 2007).

Using the Oxford Instruments Aztec Energy software, quantitative numerical data were

extracted from regions of interest drawn on the element maps, with individual spectra

from each pixel summed and processed to yield concentration data. Summed spectra

from regions of interest were quantified using the AZtec XPP model for matrix

corrections. Standards comprised polished microprobe standards of pure elements or

minerals of well-defined composition.

Granules were within the cell vacuole of some cells in the root cortex and the analysis

of them was discrete. In some samples granules were formed in between cells and not

inside a cell individually and as a result, for the analysis of cell vacuoles, vacuoles both

with and without any granules inside were selected. Vascular tissue analysis was

performed by selecting random areas of vascular tissue; the small sizes of these cells

meant that it was not possible to analyse cell vacuoles separately from surrounding cell

walls.

All elemental concentrations are given in mmol kg−1 embedded tissue which, if the resin

fully occupies the space previously occupied by water in the living tissue, closely

reflects the concentration on a wet weight basis (i.e. mM). All data are presented as


mean ± standard error of the mean, where n = the number of regions analysed.

Results

Plant growth

At harvest, symptoms of P toxicity were visible (chlorosis and necrosis around the

margins (Rossiter 1952)) in a few leaves of the uncolonised plants at 150P; plants

colonised by AMF had no P toxicity symptoms. The level of colonisation by AMF was

low-moderate in colonised plants, varying between 7-37%. No colonisation was found

in uninoculated plants. The P pulse did not affect the percentage of root length

colonised by AMF. AMF did not affect shoot dry weight. From here on, this paper

reports primarily on the plants colonised by AMF.

Table 3. Measurements of Trifolium subterraneum growth and nutrient concentrations in the +AMF treatment with three levels of phosphorus (P) pulse (No-P, 15 P and 150 P) (ns, no significant difference).

Treatments No-P 15P 150P LSD at P=0.05 P-value Shoot dry weight (g) 0.65 0.55 0.66 ns - Root dry weight (g) 0.27 0.20 0.23 0.05 0.029 Shoot [P] (mg g-1) 2.63 7.40 14.29 1.82 ˂0.001 Root [P] (mg g-1) 2.84 10.29 20.13 4.75 ˂0.001 Plant P content (mg) 2.49 6.00 14.05 1.46 ˂0.001 Shoot [K] (mg g-1) 23.20 27.78 38.13 3.1 ˂0.001 Root [K] (mg g-1) 19.05 26.71 37.97 6.42 ˂0.001 Shoot [Na] (mg g-1) 8.8 10.3 8.3 1.46 0.013 Root [Na] (mg g-1) 21.5 17 11 3.36 ˂0.001

The P pulse did not affect shoot dry weight, but root dry weight was ~26% and ~ 14%

less in the 15P and 150P treatments, respectively, than in the No-P treatment (Table 3).

Shoot and root P concentrations increased substantially from the No-P to the 150P

treatment, reaching 14 and 20 mg g-1 in shoot and root, respectively, in the 150P

treatment. Plant P content increased from the No-P to the 15P treatment by 141% and

then from the 15P to the 150P treatment by ~134% (Table 3). This large increase in

plant P concentration presumably reflects to some extent the low P adsorption capacity

of the soil.

Shoot and root K concentrations also increased from the 0P to 150P treatment, albeit

less drastically than did P. Shoot K concentration increased by ~20% from the No-P to

15P treatment and then increased ~37% from the 15P to 150P treatment. Root K


concentration increased ~40% from the No-P treatment to the 15P treatment, and then

increased ~42% between the 15P and 150P treatments. Shoot Na concentration

increased 42% from the No-P treatment to the 15P treatment and then decreased 19% to

the 150P treatment. Root Na concentration decreased ~21% from No-P to 15P treatment

and decreased 35% from 15P to 150P treatment (Table 3).

Microscopy and quantitative X-ray microanalysis

Structural features

The perfectly flat surface of the block face which is required for accurate quantitative

analyses (unlike the cryo-analytical SEM used by Ryan et al. (2003)) meant that it was

hard to see clearly inside the planed transverse sections of roots. However, structures

that could be confidently identified as mycorrhizal were not present. The low level of

colonisation meant that the likelihood of encountering mycorrhizal structures was low.

However, unexpected structures were observed in the root cortex and, rarely, in vascular

tissue. These were round granules (Fig. 1). In the 150P treatment, there was a granule in

nearly every root cortex cell, while in the No-P treatment granules were rare (Fig.1).

Also, the diameter of granules in the 150P treatment was much larger (average of 5.5

µm in sample one and 11 µm in sample 2) than in the No-P treatment (average 2.5 µm

in sample 1 and 5.1 µm in sample 2) (Table 4). These granules were visible in the

optical microscope when the cross sections of the acrolein-treated roots were stained

using toluidine blue.


Figure 1. Electron microscopy images of transverse sections of a root from A) No-P treatment (+AMF, subsample 2 from Table 4) and B) the 150P treatment (subsample 2 from Table 4) (granules evident throughout). Beneath each image is the corresponding phosphorus (P) map (green) (II) and potassium map (pink) (III). Lighter colours indicate higher concentrations. (Granules are labelled G, the epidermis is shown with an arrow, and the cortex and the vascular tissues are indicated.)

Vascular tissue

Cortex

Epidermis

100 µm

G

G

VascularTissue Cortex

Epidermis

Epidermis

100 µm

(I)

(III)

(II)

G

G

(I)

(II)

(III)

(A)

(B)


Table 4. Number of analyses (granule, cell vacuole and vascular tissue) from each root subsample and average diameter of granules.

+AMF -AMF

No-P 150P 150P

Subsample 1 2 1 2 3 1

Granules in cortex cells 2 3 8 6 5 16

Cortex cell vacuoles 2 3 3 7 2 10

Vascular tissue 2 4 2 7 0 6

Average granule diameter (µm) 5.1 2.5 5.5 11 3.6 5.6

Elemental analysis

Greatly elevated P was found in the granules in the 150P treatment. In the 150P

colonised treatment, the concentration was 185-7642 mmol kg-1 and in the 150P

uncolonised treatment, 10-4036 mmol kg-1. Vascular tissue in one colonised 150P

treatment was similarly elevated (1411-2747 mmol kg-1) (Fig. 2). The elevated P

concentration in the granules was accompanied by elevated K, Mg, Na and, sometimes,

Cl concentrations. Elevated concentrations of some nutrients occurred in other samples,

but were not consistent with treatment.

For the granules in the 150P treatment, there was a strong correlation between the

concentrations of P and K (r2=0.97), but no correlation between those of P and Na, or

Mg and Ca (Fig. 3).


Cl (m

mol kg

-1)

0

500

1000

1500

2000

4000

6000

Ca

(m

mol kg

-1)

0

100

200

300

2000

4000

6000

S (

mm

ol kg

-1)

0

200

400

600

800

2000

4000

6000

Mg

(m

mol kg

-1)

0

200

400

600

2000

4000

6000

Cell vacuole

Na

(m

mol kg

-1)

0

1000

2000

3000

4000

6000

K (

mm

ol kg

-1)

0

200

2000

4000

6000

Granule

P (

mm

ol kg

-1)

0

500

1000

2000

4000

6000

Cell vacuole Vascular tissue

No-P+AMF

150P+AMF

150P-AMF

No-P+AMF

150P+AMF

150P-AMF

No-P+AMF

150P+AMF

150P-AMF


Figure 2. Nutrient concentrations (phosphorus (P), potassium (K), sodium (Na), magnesium (Mg), sulfur (S), calcium (Ca) and chloride (Cl)) in granules, the vacuole of cortex cells that did not contain granules and vascular tissue in the No-P (no pulse of P was added to the plants) and 150P (plants received 150 mg P kg-1 soil seven days before the harvest). Most data are from the +AMF treatment, but data from a single –AMF cross section are included in the interests of greater sample size. Data are summarised in box plots where the boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. The median of the nutrient concentration is shown as solid line. Individual points are outliers.

Discussion

Plants respond to stress in many ways. In this study the stress consisted of plants

encountering a pulse of a high P concentration. I hypothesised that P supplied in excess

to a plant’s needs would be restricted from movement to shoots due to storage in poorly

soluble forms in the intraradical hyphae of AMF. This study has shown, for the first

time, formation of granules with extraordinarily high concentrations of P and K in the

cortical cells of T. subterraneum exposed to a pulse of high P concentration. Assuming

the granules are not an artefact of sample preparation, such as the possible precipitation

and coagulation of stress-related latex-like proteins by acrolein fixation (Condon and

Fineran 1989), the implications arising from this finding are discussed below.

A pulse of a high P concentration induces granules high in [P] in the root

Large granules were formed in abundance and were spread widely in the cortex area of

the roots treated with 150P, while granules that were formed in the No-P treatment were

small and rarely easily noticed. Whilst roots were colonised by AMF, I do not consider

these granules to be transverse sections of AM fungal hyphae, because their

morphology was not consistent with that shown by Ryan et al. (2003).

The presence of granules with greatly elevated [P] in the 150P treatment is in

accordance with the shoot and root P concentrations. The root and shoot P

concentrations in plants in the 150P treatment were eight and five times higher,

respectively, than those in plants that did not receive any P in a pulse.


(c)

Figure 3. Correlation between phosphorus (P) concentration in the granules treated with 150P with (a) Potassium (K) (r2=0.97, P<0.0001), (b) Sodium (Na) (r2=0.04, P=0.41), (c) Magnesium (Mg) (r2=0.05, P=0.35) and (d) Calcium (Ca) (r2= 6.681E-004, P= 0.91).

P (mmol kg-1

)

0 2000 4000 6000 8000 10000

Ca

(m

mo

l kg

-1)

0

20

40

60

80

100

120

140

Mg (

mm

ol kg

-1)

0

200

400

600

800

1000

1200

1400

Na (

mm

ol kg

-1)

0

500

1000

1500

2000

2500

K (

mm

ol kg-1

)

0

2000

4000

6000

8000

(a)

(d)

(b)

(c)


Except under severe P limitation, the concentration of Pi in the cytosol of plants is

maintained at constant concentrations (i.e. 5-10 mM) (Lee and Ratcliffe 1993; Lee et al.

1990). The Pi concentrations in the vacuole varies considerably more than that in the

cytosol. This vacuolar P concentration may be undetectable under P starvation, but

generally does not increase above 25 mM unless P toxicity is expressed (Lee and

Ratcliffe 1993; Lee et al. 1990; Mimura 1995; Ryan et al. 2003). For instance, when P

toxicity was induced in Hakea prostrata R.Br. (Proteaceae) by Shane et al. (2004) the

concentration of P in vacuoles of mesophyll cells was greatly elevated (~150-250 mM),

but still much below vacuolar P concentrations in the present experiment where P

concentration reached to up to 7642 mmol kg-1 in the granules and up to 959 mmol kg-1

in the vacuole of cortex cells – values much higher than ever previously reported.

Thus the presence of distinct granules in the root cells of T. subterraneum is presumably

a strategy to reduce P toxicity in response to the sudden supply of a high concentration

of P. Similar to other species, T. subterraneum is typically reported to down-regulate P

uptake when grown with a high P supply. For example, in an experiment by Asher and

Loneragan (1967), 8 to 13 day - old T. subterraneum seedlings were grown in nutrient

solutions with five P levels (0.04, 0.2, 1, 5 and 25 µM) applied as phosphate. Plants

were harvested after 4 weeks of growth in nutrient solutions. Both shoot and root P

concentrations had reached a plateau by 5 µM (when dry weight was maximum) being

~ 10 and 12.5 mg g-1, respectively. Hence, it seems the shoot P concentration in our

experiment of 14 mg g-1 in the 150P treatment and, in particular, the root P

concentration of 20 mg g-1, is extraordinarily high (Table 3). I surmise these

unexpectedly high values represent the plant response to a pulse of a high P

concentration. In other words, when a pulse of P is applied to the plant, there is no time

for the plant to down-regulate its P-uptake capacity and, as a result, excessive uptake of

P occurs. This interpretation is consistent with that of Cogliatti and Clarkson (1983),

who found the P-uptake rate in P-starved potato plants (Solanum tuberosum L.) was

three- to four-fold higher than in P-sufficient controls after a pulse of P. Moreover, high

P concentrations may also occur if Zn is deficient (Cakmak and Marschner 1986);

however the Zn concentration in the plants in this experiment (63-100 mg kg-1); data not

presented) showed this was not the case. Based on these reports I hypothesise that when

P supply becomes suddenly excessive to plant requirements, the transport of at least

some of the excess P to the shoot is restricted by the formation of granules in the root.

This phenomenon has not previously been reported.


What is the form of P in the granules?

I surmise that the P in the granules must be in poorly soluble forms as the plants,

particularly in the +AMF treatment, did not show signs of toxicity, and there were no

signs of cells suffering high osmotic pressure (i.e. swelling) as reported elsewhere when

leaves in high-P treatments were examined with cryo-SEM (Ryan et al. 2009; Shane et

al. 2004).

Formation of P-rich granules has not previously been reported for plants. However,

there is substantial literature showing their presence in the hyphae of AMF and

ectomycorrhizas (Ashford et al. 1986; Cox et al. 1980; Lapeyrie et al. 1984; Ling-Lee et

al. 1975). The reaction of these granules to cytochemical tests resulted in the authors

concluding they were polyphosphate. In an experiment by Lapeyrie et al. (1984),

metachromatic granules were observed in the mycelial hyphae of Paxillus involutus

when grown in a phosphate-rich solution, while no granules were observed when no

phosphate was added. The size and the number of these granules increased during

incubation. Lapeyrie et al. (1984) presumed these granules contained polyphosphates.

Transmission electron microscopy studies suggest that polyphosphates in the hyphae of

AMF are mostly present in the form of granules (Cox et al. 1975; Peterson 1991). On

the other hand, Orlovich and Ashford (1993) concluded that these were an artefact of

the fixation process for specimen preparation, however, this had been disputed. For

instance, Bücking and Heyser (1999) reported vacuolar granules were present in living

hyphae of various ectomycorrhizal fungi (Suillus bovines, Paxillus involutus, Pisolithus

tinctorius and Laccaria laccata). These granules were analysed for elemental

composition by X-ray spectroscopy either after chemical preparation or cryo-fixation,

freeze-drying and pressure-infiltration, and it was concluded that they were not an

artefact of specimen preparation and can be referred to as polyphosphate granules. The

size of the granules was between 100 to 3000 nm which is one of the lowest ranges in

our study, but since these granules were formed in hyphae with a diameter significantly

less than that of root cortical cells, this is not surprising.

Formation of granules with high P concentration in response to a sudden pulse of high P

concentration is also in accordance with the “polyphosphate overplus” phenomenon

reported for bacteria and yeast. For instance, when P-starved yeast are transferred to a

medium with phosphate, glucose and certain cations (K and Mg), cells may accumulate

polyphosphates up to 20% of the cell dry weight (van Wazer 1961). According to


Sicko-Goad and Jensen (1976), transferring phosphate-starved cells of cyanobacteria

(Plectonema boryanum) to a medium containing 10 mg phosphate litre-1 caused an

“overplus” in the cells within 1 h of transfer, with the most dramatic increase found in

both acid-soluble and acid-insoluble polyphosphates. Moreover, high concentrations of

P were always accompanied by high concentrations of K, Na and Cl.

Further support for the granules in our study being composed of polyphosphate comes

from the presence of countering cations. In our study, K was the main counter-ion. K

was abundant as the P was supplied as KH2PO4. However many other studies report K

as the main counter-ion. Bücking and Heyser (1999) reported K and Mg as the cations

involved in charge balance of granules in ectomycorrhizas and mentioned them as a

“general character of fungal polyphosphate granules”. Moreover, Ryan et al. (2003)

found a strong linear relationship between P and K in the intraradical hyphae of AMF,

albeit with a highest concentration of K of 350 mM, which is negligible compared to the

K concentration in the granules (6881 mM). The positive relationship between P and K

is not surprising as polyphosphates are highly charged anions which form strong bonds

with cations (van Wazer 1961). For instance, Peverly et al. (1978) found formation of

polyphosphate bodies in P-starved Chlorella pyrenoidosa was positively and strongly

related to the presence of K in the medium. Transferring P-starved cells into a complete

nutrient solution containing 100 µM P resulted in accumulation of high concentrations

of P and K within several hours. Omitting K from the medium, even in the presence of

other cations such as Ca and Mg, prevented accumulation of high concentrations of P

and formation of polyphosphate. However, absence of Ca or Mg did not have a similar

effect.

Based on the present results, I surmise that it is possible that in our study polyphosphate

granules were formed in the cortical cell vacuoles of T. subterraneum in response to a

pulse of a high P concentration. These granules might degrade over time and become

accessible (toxic) to the cell and the plant. However, I cannot at all be confident that our

granules were composed of polyphosphate, as recent reports of polyphosphate in plants

are few (Denton et al. 2007; van Voorthuysen et al. 2000). Based on this literature, I did

not expectto find polyphosphate.

Conclusion

Although the roots were colonised, no fungal tissue was found in the samples prepared


for X-ray microanalysis and our hypothesis of finding high concentrations of P in the

hyphae of AMF could not be addressed. However, the formation of high-P granules in

response to a P pulse and also the presence of high concentrations of K was consistent

with the formation of polyphosphate. This is the first suggestion that high

concentrations of P in the form of polyphosphate occur in higher plants. It would be

useful to know: how long do these granules persist in the cells after stress; do the

granules form in the shoots as well the roots; does granule formation differ between

species; and, are polyphosphate granules quickly dispersed as the plant down-regulates

its P-uptake capacity; In addition if the granules do represent a means for plants to

temporarily store high concentrations of P, what are the implications for efficient use of

P fertiliser? Further experiments are required to address these questions and to clarify

that granules are not an artefact of sample preparation.

Acknowledgements

The authors acknowledge the facilities, and the scientific and technical assistance of the

Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy,

Characterisation & Analysis, The University of Western Australia, a facility funded by

the University, and State and Commonwealth Governments. This study was supported

by the School of Plant Biology, UWA, and the Future Farm Industries Cooperative

Research Centre. NK Nazeri also appreciates the APA scholarship awarded by the

University of Western Australia. I would like to thank Lyn Kirilak, Peter Duncan,

Evonne Walker and Daniel Kidd for technical help.

References

Altus DI, Canny MJ (1985) Loading of assimilates in wheat leaves. II. The path from chloroplast to vein. Plant, Cell Environ 8: 275-285.

Asher CJ, Loneragan JF (1967) Response of plants to phosphate concentration in solution culture: I. Growth and phosphorus content. Soil Sci 103: 225-233.

Ashford AE, Peterson RL, Dwarte D, Chilvers GA (1986) Polyphosphate granules in eucalypt mycorrhizas: determination by energy dispersive x-ray microanalysis. Can J Bot 64: 677-687.

Bidwell SD, Crawford SA, Woodrow IE, Sommer-Knudsen J, Marshall AT (2004) Sub-cellular localization of Ni in the hyperaccumulator, Hybanthus floribundus (Lindley) F. Muell. Plant, Cell Environ 27: 705-716.

Blair GJ, Chinoim N, Lefroy RDB, Anderson G, Crocker G (1991) A soil sulfur test for


pastures and crops. Aust J Soil Res 29: 619-626.

Cakmak I, Marschner H (1986) Mechanism of phosphorus-induced zinc deficiency in cotton. I. Zinc deficiency-enhanced uptake rate of phosphorus. Physiol Plant 68: 483-490.

Clode PL, Marshall AT (2002) Kalisomes in corals: a novel KCl concentrating organelle? Tissue Cell 34: 199-209.

Clode PL, Marshall AT (2003) Calcium associated with a fibrillar organic matrix in the scleractinian coral Galaxea fascicularis. Protoplasma 220: 153-161.

Clode PL, Marshall AT (2004) Calcium localisation by X-ray microanalysis and fluorescence microscopy in larvae of zooxanthellate and azooxanthellate corals. Tissue Cell 36: 379-390.

Cogliatti DH, Clarkson DT (1983) Physiological changes in, and phosphate uptake by potato plants during development of, and recovery from phosphate deficiency. Physiol Plant 58: 287-294.

Colwell JD (1965) An automatic procedure for the determination of phosphorus in sodium hydrogen carbonate extracts of soils. Chem Ind-London 10: 893-895.

Condon JM, Fineran BA (1989) The effect of chemical fixation and dehydration on the preservation of latex in Calystegia silvatica (Convolvulaceae). Examination of exudate and latex in situ by light and scanning electron microscopy. J Exp Bot 40: 925-939.

Condron RJ, Marshall AT (1990) A comparison of three low temperature techniques of specimen preparation for X-ray microanalysis. Scanning Microsc 4: 439-447.

Cox G, Moran KJ, Sanders F, Nockolds C, Tinker PB (1980) Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. III. polyphosphate granules and phosphorus translocation. New Phytol 84: 649-659.

Cox G, Sanders F, Tinker P, Wild J (1975) Ultrastructural evidence relating to host-endophyte transfer in a vesicular-arbuscular mycorrhiza. In: FE Sanders, B Mosse, PB Tinker (eds) Endomycorrhizas. Academic Press, London and New York.

Crawford SA, Marshall AT, Wilkens S (1998) Localisation of aluminium in root apex cells of two Australian perennial grasses by X-ray microanalysis. Funct Plant Biol 25: 427-435.

De Filippis LF (1978) Localization of organomercurials in plant cells. Zeitschrift für Pflanzenphysiologie 88: 133-146.

Denton MD, Veneklaas EJ, Freimoser FM, Lambers H (2007) Banksia species (Proteaceae) from severely phosphorus‐impoverished soils exhibit extreme efficiency in the use and re‐mobilization of phosphorus. Plant, Cell Environ 30: 1557-1565.

Echlin P (1992) Low-temperature microscopy and analysis. Springer, New York.

Ezawa T, Cavagnaro TR, Smith SE, Smith FA, Ohtomo R (2004) Rapid accumulation of polyphosphate in extraradical hyphae of an arbuscular mycorrhizal fungus as revealed by histochemistry and a polyphosphate kinase/luciferase system. New Phytol 161: 387-392.

Funamoto R, Saito K, Oyaizu H, Saito M, Aono T (2007) Simultaneous in situ detection


of alkaline phosphatase activity and polyphosphate in arbuscules within arbuscular mycorrhizal roots. Funct Plant Biol 34: 803-810.

Giovannetti M, Mosse B (1980) An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol 84: 489-500.

Hedley M, Stewart J, Chauhan B (1982) Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Sci Soc Am J 46: 970-976.

Hijikata N, Murase M, Tani C, Ohtomo R, Osaki M, Ezawa T (2010) Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus. New Phytol 186: 285-289.

Hyatt AD, Marshall AT (1985) X-ray microanalysis of cockroach fat body in relation to ion and water regulation. J Insect Physiol 31: 495-508.

Jeffrey DW (1964) The formation of polyphosphate in Banksia ornata, an Australian heath plant. Aust J Biol Sci 17: 845-854.

Jeffrey DW (1968) Phosphate nutrition of Australian heath plants. II. The formation of polyphosphate by five heath species. Aust J Bot 16: 603-613.

Karandashov V, Bucher M (2005) Symbiotic phosphate transport in arbuscular mycorrhizas. Trends Plant Sci 10: 22-29.

Kariman K, Barker SJ, Finnegan PM, Tibbett M (2014) Ecto-and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings. Mycorrhiza: 1-9.

Kuga Y, Saito K, Nayuki K, Peterson RL, Saito M (2008) Ultrastructure of rapidly frozen and freeze-substituted germ tubes of an arbuscular mycorrhizal fungus and localization of polyphosphate. New Phytol 178: 189-200.

Kulaev IS, Vagabov V, Kulakovskaya T (2005) The biochemistry of inorganic polyphosphates. Wiley, England.

Lapeyrie FF, Chilvers GA, Douglass PA (1984) Formation of metachromatic granules following phosphate uptake by mycelial hyphae of an ectomycorrhizal fungus. New Phytol 98: 345-360.

Lee RB, Ratcliffe RG (1993) Subcellular distribution of inorganic phosphate, and levels of nucleoside triphosphate, in mature maize roots at low external phosphate concentrations: measurements with 31P-NMR. J Exp Bot 44: 587-598.

Lee RB, Ratcliffe RG, Southon TE (1990) 31P NMR measurements of the cytoplasmic and vacuolar Pi content of mature maize roots: relationships with phosphorus status and phosphate fluxes. J Exp Bot 41: 1063-1078.

Lewis DC, Clarke AL, Hall WB (1987) Accumulation of plant nutrients and changes in soil properties of sandy soils under fertilized pasture in southeastern South-Australia. I. Phosphorus. Aust J Soil Res 25: 193-202.

Ling-Lee M, Chilvers GA, Ashford AE (1975) Polyphosphate granules in three different kinds of tree mycorrhiza. New Phytol 75: 551-554.

Marshall AT (1980) Freeze-substitution as a preparation technique for biological X-ray microanalysis. Scan electron micros Pt 2: 395-408.

Marshall AT, Clode PL, Russell R, Prince K, Stern R (2007) Electron and ion


microprobe analysis of calcium distribution and transport in coral tissues. J Exp Biol 210: 2453-2463.

Marshall AT, Schroen C, Condron RJ (1994) X-ray microanalysis of renal proximal tubules in cadmium-treated rats. J Submicr Cytol Path 26: 59-66.

Marshall AT, Wright OP (1991) Freeze‐substitution of scleractinian coral for confocal scanning laser microscopy and X‐ray microanalysis. J micros 162: 341-354.

Marshall AT, Wright OP (1995) X-ray microanalysis of coral mucus. Microbeam Anal 4: 305-315.

Mimura T (1995) Homeostasis and transport of inorganic phosphate in plants. Plant Cell Physiol 36: 1-7.

Miyachi S (1961) Inorganic polyphosphate in spinach leaves. J Biochem(Tokyo) 50: 367-371.

Mostaert AS, Orlovich DA, King RJ (1996) Ion compartmentation in the red alga Caloglossa leprieurii in response to salinity changes: freeze‐substitution and X‐ray microanalysis. New Phytol 132: 513-519.

Nassery H (1969) Polyphosphate formation in the roots of Deschampsia flexuosa and Urtica dioica. New Phytol 68: 21-23.

Nazeri NK, Lambers H, Tibbett M, Ryan MH (2013) Do arbuscular mycorrhizas or heterotrophic soil microbes contribute toward plant acquisition of a pulse of mineral phosphate? Plant Soil 373: 699-710.

Nazeri NK, Lambers H, Tibbett M, Ryan MH (2014) Moderating mycorrhizas: arbuscular mycorrhizas modify rhizosphere chemistry and maintain plant phosphorus status within narrow boundaries. Plant, Cell Environ 37: 911-921.

Ohtomo R, Saito M (2005) Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus. New Phytol 167: 571-578.

Orlovich DA, Ashford AE (1993) Polyphosphate granules are an artefact of specimen preparation in the ectomycorrhizal fungus Pisolithus tinctorius. Protoplasma 173: 91-102.

Orlovich DA, Ashford AE (1995) X‐ray microanalysis of ion distribution in frozen salt/dextran droplets after freeze‐substitution and embedding in anhydrous conditions. J Micros 180: 117-126.

Påsgård E, Lindh U, Roomans GM (1994) Comparative study of freeze-substitution techniques for x-ray microanalysis of biological tissue. Micros Res Tech 28: 254-258.

Peterson RL (1991) Morphometric and energy dispersive X-ray analysis of polyphosphate distribution in the VAM fungus Glomus versiforme associated with leek. Symbiosis 11: 63-72.

Peverly JH, Adamec J, Parthasarathy MV (1978) Association of potassium and some other monovalent cations with occurrence of polyphosphate bodies in Chlorella

pyrenoidosa. Plant physiol 62: 120-126.

Rasmussen N, Lloyd DC, Ratcliffe RG, Hansen PE, Jakobsen I (2000) 31P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi. Plant Soil 226: 245-253.

Roomans GM, Dragomir A (2007) X-ray microanalysis in the scanning electron


microscope. In: J Kuo (ed) Electron Microscopy. Humana Press.

Ross GD, Morrison GH, Sacher RF, Staples RC (1983) Freeze substitution sample preparation for ion microscopy of plant tissue. J Micros 129: 221-228.

Rossiter RC (1952) Phosphorus toxicity in subterranean clover and oats grown on Muchea sand, and the modifying effects of lime and nitrate-nitrogen. Aust J Agric Res 3: 227-243.

Ryan MH, Ehrenberg S, Bennett RG, Tibbett M (2009) Putting the P in Ptilotus: a phosphorus-accumulating herb native to Australia. Ann Bot-London 103: 901-911.



Searle PL (1984) The berthelot or indophenol reaction and its use in the analytical chemistry of nitrogen. A review. Analyst 109: 549-568.

Shane MW, McCully ME, Lambers H (2004) Tissue and cellular phosphorus storage during development of phosphorus toxicity in Hakea prostrata (Proteaceae). J Exp Bot 55: 1033.

Sicko-Goad L, Jensen TE (1976) Phosphate metabolism in blue-green algae. II. Changes in phosphate distribution during starvation and the "polyphosphate overplus" phenomenon in Plectonema boryanum. Am J Bot 63: 183-188.

Smith SE, Smith FA (2011) Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales. Annu Rev Plant Biol 62: 227-250.


Tiessen H, Stewart JWB, Moir JO (1983) Changes in organic and inorganic phosphorus composition of two grassland soils and their particle size fractions during 60–90 years of cultivation. J Soil Sci 34: 815-823.

van Voorthuysen T, Regierer B, Springer F, Dijkema C, Vreugdenhil D, Kossmann J (2000) Introduction of polyphosphate as a novel phosphate pool in the chloroplast of transgenic potato plants modifies carbohydrate partitioning. J Biotechnol 77: 65-80.

van Wazer JR (1961) Phosphorus and its compounds. I. Chemistry. Interscience Publishers, New York

Viereck N (2002) In vivo 31P NMR spectroscopy for the study of P pools and their dynamics in arbuscular mycorrhizal fungi. PhD Thesis. Roskilde University. Denmark. pp.123.

Viereck N, Hansen PE, Jakobsen I (2004) Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo 31P NMR spectroscopy. New Phytol 162: 783-794.


Vierheilig H, Coughlan AP, Wyss U, Piche Y (1998) Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl Environ Microbiol 64: 5004-5007.

Wells J, Kilburn MR, Shaw JA, Bartlett CA, Harvey AR, Dunlop SA, Fitzgerald M (2012) Early in vivo changes in calcium ions, oxidative stress markers, and ion channel immunoreactivity following partial injury to the optic nerve. J Neurosci Res 90: 606-618.

CHAPTER 5.

General Discussion

In this discussion I address the hypotheses which were tested in chapters two, three and

four. Unexpected results from each experiment are also discussed and results from all

results chapters are considered together.

Chapter 5. General Discussion 90

5.1 Introduction

Incorporating pasture legumes in agricultural systems in Australia not only maintains

feed for grazing livestock, but also fixes atmospheric nitrogen, thus increasing soil

nitrogen availability, and breaking the disease life cycle when the pastures are grown in

rotation with crops (Howieson et al. 2000; Kirkegaard et al. 2011; Peoples and Baldock

2001; Robson 1990). The annual legume Trifolium subterraneum is widely grown

across southern Australia due to its good adaptation to climate, soils and agricultural

systems including grazing and crop rotations (Porqueddu and González 2011; Puckridge

and French 1983). Due to problems that have arisen from reliance on annual pastures,

such as dryland salinity and leaching of nutrients, for the last 15 years researchers have

focused on identifying and developing new perennial pasture legumes adapted to

Western Australian conditions; conditions for which there are currently few or no

perennial legume options (for instance, waterlogging, acidic soils, salinity and drought)

(Li et al. 2008; Nichols et al. 2007). Bituminaria bituminosa, Cullen australasicum and

Kennedia prostrata are promising perennial legumes for the area of the Western

Australia cropping zone with low to moderate rainfall and poor soils (Real et al. 2011).

As perennial pastures, in contrast to annual pastures, are present when the first autumn

rains occur, they also present an opportunity to capture the pulse of P that may be

released into the soil solution at this time (Espigares and Peco 1995; Sardans and

Peñuelas 2007); a pulse similar to that which occurs when P fertiliser is applied. As

arbuscular mycorrhizal fungi (AMF) are well known to enhance plant P uptake (Smith

and Read 2008), the primary objective of this project was to study whether AMF will

enhance uptake of P from a pulse. Soil with moderate levels of P, that is, levels

commonly found in soils in the cropping areas of Western Australia (Scanlon 2011;

Weaver and Reed 1998) were used in all experiments.

5.2 Key research findings

Table 5.1 lists all the hypotheses addressed in previous experimental chapters. All

results are then discussed individually.


Table 5.1. The hypotheses that were addressed in previous chapters and whether they were accepted or rejected.

Hypothesis Chapter Accepted or

rejected

1 AMF will enhance uptake of P from a pulse as might occur

following fertiliser application or the first winter rain 2 and 3

Partly

accepted

2

Uptake of P is greater in plants colonised by a diverse

community of AMF (inoculation with field soil), rather than a

single-species inoculum

2 Rejected

3 Presence of microbes in the soil increases P uptake compare to

the control 2 Rejected

4 AMF will moderate shoot P concentration following a pulse 3 Accepted

5 Release of carboxylates into the rhizosphere will decrease when

plants encounter a pulse of P 3 Rejected

6 Colonisation by AMF will decrease the release of carboxylates

into the rhizosphere 3 Accepted

7

Following a significant pulse of P, P movement to shoots is

restricted, due to storage of high concentrations of P in

intraradical AM hyphae in insoluble forms

4 Unable to

test

5.2A Phosphorus uptake from a pulse is enhanced in the roots of colonised

plants, but P transport to the shoots is restricted by AMF (after a week)

(moderating effect)

In Chapter 2, the annual pasture legume Trifolium subterraneum was used to develop

methodology for the experiment presented in Chapter 3. However, in doing so,

Hypotheses 1 and 4 were also addressed (see Table 5.1).

It has previously been shown that plant P uptake will increase when Trifolium

subterraneum forms a symbiosis with AMF (Abbott and Robson 1977; Bolan et al.

1987; Smith 1982). In my first experiment I, therefore, expected to see a higher P

concentration in both shoots and roots of colonised plants than in those of uncolonised

plants, both before and, to a greater degree, after a pulse of P. A day before (day 0) and

a day after (day 2) the pulse, as expected, both shoot and root P concentrations in

colonised plants were higher than those in uncolonised plants. However, unexpectedly,


from day 2 to day 8, the rate of increase in shoot P concentration in colonised plants was

lower than that in uncolonised plants. This resulted in quite similar shoot P

concentrations in colonised and uncolonised plants by day 8. In contrast, the rate of

increase in root P concentration did not change from day 2 to day 8, and thus P

concentrations remained higher in colonised plants.

Remarkably consistent results were found in my second experiment (Chapter 3), where

a similar experiment was performed using Trifolium subterraneum and three perennial

pasture legumes. In this experiment, I found a 53% increase in shoot P concentration in

colonised plants following a pulse, but a 143% increase for uncolonised plants, while

the root P concentration was raised by the pulse by a similar amount for both colonised

and uncolonised plants.

The results can be explained based on the findings of Smith and Read (2008), who

reported that when plants are colonised by AMF, the “mycorrhizal uptake pathway”

(through hyphae of the AMF) is the dominant pathway for nutrient uptake regardless of

plant growth response and the “direct uptake pathway” (through root hairs and

epidermal cells) becomes “switched-off” or inactivated. In my experiment, therefore,

the lower rate of P uptake in the shoots of colonised plants is likely due to the primary

path of uptake being through the hyphae of AMF, with large amounts of the P then

stored in the intraradical hyphae of AMF in forms such as polyphosphate, while in the

uncolonised plants, P uptake was through the direct uptake pathway (root hairs and

epidermal cells) which allowed transport of P to the shoot without any barrier. In

accordance with this, there are also several reports of the presence of high

concentrations of P, probably polyphosphate, in the hyphae of AMF (Ryan et al. 2003;

Ryan et al. 2007; Solaiman et al. 1999). Such an effect, that is, the reduced transport of

P to the shoot in mycorrhizal plants following a P pulse, has recently also been reported

by Kariman et al. (2014). According to these authors, seven days after a high pulse of P

(30 mg P kg-1 soil), phytotoxicity symptoms in jarrah tree seedlings (Eucalyptus

marginata) colonised by AM (as well as ectomycorrhizal) fungi, were significantly

lower in colonised than in uncolonised plants. Moreover, colonised plants had lower

levels of shoot P concentrations than uncolonised plants, and there was a positive linear

relationship between shoot P concentration and toxicity symptoms.

My results, along with those of Kariman et al. (2014), suggest that AMF can, to some

degree, protect plants from a pulse of P; that is, AMF moderate the effect of a pulse of


P.

5.2B Phosphorus uptake did not differ between a diverse community of AMF

and a single species culture

In order to optimise the methodology for Chapter 3, in Chapter 2 the P uptake by a

diverse community of AMF (i.e. the community naturally present in the field soil in

which the experiment was undertaken) was compared with an inoculum containing a

single species (as is routinely used in mycorrhizal experiments) to examine whether a

diverse community of AMF is more efficient for a plant to absorb P following a pulse

(hypothesis 2).

It is known that AM fungal species differ in their ability to affect P uptake and growth

of their host plant. For example, Jansa et al. (2005) reported that accessibility of P

differs among AM fungal species and is related to the distance that hyphae can develop

from the root and acquire P, and also the efficiency of fungi in extracting P from the

soil. The authors stated that Glomus mosseae and G. intraradices can take up P up to 10

cm from the root, but for G. claroideum this distance is only 6 cm. On the other hand,

G. mosseae did not provide any net P-uptake benefit for maize (Zea mays L. cv. Corso)

while net P uptake was significantly improved by G. intraradices due to its more

efficient P extraction. Thus, it can be expected that if a soil contains a number of

species, all differing slightly in their hyphal characteristics, then the result might be a

higher plant productivity than if only one species of AMF is present. Indeed, both van

der Heijden et al. (1998) and Maherali and Klironomos (2007) found AM fungal species

richness had a positive effect on plant productivity. Similarly, Wagg et al. (2011) found

plant biomass and productivity in heterogeneous environments is increased by greater

diversity of AM fungal species. As a result, I expected to find greater plant biomass and

P uptake when the inoculum contained a diverse mycorrhizal community (AMF from

the field) compared to a single species inoculum. However, little difference in host plant

shoot and root dry weight, or P uptake, was found. The failure of inoculation with a

diverse community of AMF to promote plant P uptake and growth more than the single

species inoculum may be explained by the findings of Bainard et al. (2013) and

Maherali and Klironomos (2007). Maherali and Klironomos (2007) found that plant

biomass and productivity is increased only when taxa from two families (for instance

Gigasporaceae and Glomeraceae) were present and having only one family of AMF in


the soil will not stimulate plant productivity. Also, the authors predicted that

coexistence of species within each family is less likely, since they all have the same

spatial niche requirements, while taxa from different lineages coexist since their spatial

niche requirements are different. In support of this finding, Bainard et al. (2013) did not

find any significant differences in crop growth in response to AM fungal richness and

explained this might be due to the lack of different families of AM fungal species in the

soil and presence of only one family of AMF (Glomeraceae) in the soil. In our

experiment more than 97% of the DNA detected belonged to the Glomus family.

5.2C Microbes had little effect on uptake of P from a pulse

In Chapter 2, I also examined whether inoculation of microbes into the soil increases P

uptake compared to a sterilised control (hypothesis 3). Using field soil as inoculum for

AMF will introduce not only a diverse community of AMF, but will also introduce a

diverse microbial community, which might act synergistically to enhance plant growth

or inhibit the activity of AMF and thus reduce the positive impact of AMF on plant

growth (Azcón et al. 1976; Hetrick and Wilson 1988; Singh and Kapoor 1998).

Microbes have an important role in making soil P available for plants (Richardson 2001;

Whitelaw 1999). Inorganic and organic pools of total soil P can become accessible (via

solubilisation and mineralisation or their impact on root structure and function) or

inaccessible (by immobilising available sources of P) through the activities of soil

microbes (Richardson 2001). Plant growth-promoting microorganisms are of particular

importance and include a wide diversity of bacteria and fungi, which are found in the

rhizosphere and can increase plant growth by increasing supply of nutrients (Richardson

et al. 2009).

In mycorrhizal experiments, filtrates are commonly used to equalise soil microbes

between non-mycorrhizal controls and AM fungal treatments when field soil (or even

AM fungal inoculum) has been added (Asghari and Cavagnaro 2011). The filtrate is

made by passing a mixture of deionised water and soil through series of sieves from

1000 µm to 50 µm. Filtrate of the field soil is assumed to contain the soil microbial

community, but no mycorrhizal hyphae or spores (Hetrick and Wilson 1988). Addition

of filtrates, or non–sterile soil sievings, to sterilised soil was found to increase growth of

uncolonised plants by up to 7-fold (Hetrick and Wilson 1988). The authors explained,

an increase in growth of plants receiving filtrates or non-sterile soil sievings compared


to the plants grown in pasteurised soil might reflect an increase in soil organic matter

and fertility due to the presence of microbes in the soil. Accordingly, I expected to find

an increase in P uptake by plants that had received field soil filtrates compared with

control plants (no AMF, no microbes). However, I did not find any effect of the

microbes on plant P uptake before or after the pulse of P and, overall, the effect of the

microbe treatments rarely differed from the control (Chapter 2). The main reason for the

lack of effect of the microbes treatment in my experiment might be the moderate level

of P (18 mg kg-1) in the soil, compared with the lower level of P in the soil used by

Hetrick and Wilson (1988) (5 mg kg-1). According to Richardson (2001) and Krey et al.

(2011) an increase in the level of soil P may decrease the activity of soil

microorganisms involved in P mobilisation.

Since there was no impact from the microbe treatment on plant growth or P uptake, it

can be assumed that the major impact of the field soil inoculum resulted from the

actions of the AMF it contained. I therefore chose to use the field soil inoculum in the

next chapter (Chapter 3) (again with the addition of the filtrates to the control.)

5.2D A pulse of P did not decrease the amount of rhizosphere carboxylates

Plants employ different mechanisms, depending on species, under P-deficiency

conditions. Increased root-hair formation, symbioses with AMF, formation of cluster

roots, and exudation of carboxylates are some of these mechanisms (Abel et al. 2002;

Lambers et al. 2006). The major component of root exudates under P deficiency

conditions can be carboxylates, which are low-molecular-weight organic anions,

released from roots into the soil; these increase P availability through mobilising both

organic and inorganic P through complexing metal cations that bind phosphate and

thereby displacing the phosphate from the soil matrix by ligand exchange (Gardner et al.

1983; Jones 1998; Lambers et al. 2006; Pang et al. 2009a; Veneklaas et al. 2003). It has

previously been found that the amount of rhizosphere carboxylates increases under low-

P conditions (Lambers et al. 2006; Pearse et al. 2006; Suriyagoda et al. 2012). For

instance, Pang et al. (2009a) compared the amount of rhizosphere carboxylates between

two levels of KH2PO4 (6 and 48 µg P g-1 soil) for 11 legume species (four species -

Medicago sativa, B. bituminosa, C. australasicum and K. prostrata –were used in

Chapter 3). It was found that all these species had higher amounts of carboxylates at the

lower P level. However, this is not supported in all studies. For instance, in an


experiment by Suriyagoda et al. (2012) the effect of 5, 20 and 80 mg P kg-1 soil on the

amount of rhizosphere carboxylates was measured for two native perennials (K.

nigricans and Ptilotus polystachyus) and M. sativa. An increase in soil P decreased the

amount of carboxylates in the rhizosphere only in M. sativa and K. nigricans (only from

P20 to P80). Also, Pearse et al. (2006) demonstrated the response of 13 crop species to

soil P in terms of carboxylates was variable.

In Chapter 3, no effect of the P pulse on the amount of rhizosphere carboxylates was

found, which supports findings of Pearse et al. (2006) and Suriyagoda et al. (2012).

However, the results are not consistent with those of Pang et al. (2009a). The failure of

the P pulse to affect the amount of rhizosphere carboxylates may reflect the initial

moderate level of soil P in my experiment, or the short period of time (1 week) during

which plants were exposed to the P pulse.

5.2E Colonisation with AMF decreased rhizosphere carboxylates

Chapter 3 also examined whether colonisation with AMF decreases the release of

carboxylates into the rhizosphere (hypothesis 6). This effect was previously, and first,

reported by Ryan et al. (2012). According to Ryan et al. (2012) since exudation of

carboxylates and forming a symbiosis with AMF are both carbon demanding processes

for the plants (Graham 2000), it is not surprising that there will be trade-offs between

the two processes (Lynch and Ho 2005). Ryan et al. (2012) found up to a 50% decrease

in carboxylates when 12 species of Kennedia were colonised by AMF. My experiment

supports the findings of Ryan et al. (2012), as colonisation by AMF decreased the

amount of rhizosphere carboxylates by 52% (Table 5.2). The similarities between the

results of these two studies conducted under quite contrasting conditions (field soil with

moderate levels of P [18 mg kg-1] in my experiment and washed river sand with low

levels of P [3 mg kg-1] in Ryan et. al (2012)) are corroborated by the similar results from

K. prostrata from both studies, as the same accession of this species was used in both

experiments (Table 5.2).


Table 5.2. Comparison between the amount of rhizosphere carboxylates in Kennedia prostrata in studies by Ryan et al. (2012) (in low-P washed river sand) and Chapter 3 (in moderate-P field soil) and the level of colonisation by AMF in each study.

-AMF +AMF Reference

Carboxylates (µmol per g root DW) 91 41 Ryan et al. (2012)

70 38 Nazeri et al. (2014)

Colonisation by AMF (%) 0 35 Ryan et al. (2012)

0 23 Nazeri et al. (2014)

Thus, it seems that colonisation by AMF can greatly decrease the amount of

carboxylates in the rhizosphere soil and, thereby, as suggested by Ryan et al. (2013),

potentially change the mixture of soil P pools utilised by the plant – mycorrhizal-

mediated resource partitioning.

5.2F Formation of granules with high concentrations of P in the cortex area of

the roots of Trifolium subterraneum which were treated with a high pulse of P

In response to the moderating effect of AMF on plant uptake of P from a pulse, which

was found in Chapters 2 and 3, I decided to measure the amount of P which was stored

in the intraradical hyphae of the mycorrhiza and compare this with the amount of P in

the root cells of Trifolium subterraneum. The hypothesis of this experiment was:

following a large pulse of P, AMF restrict movement of P to the shoots due to the

storage of high concentrations of P in insoluble forms in hyphae. Due to low level of

colonisation and inability to determine where exactly the colonisation was present in the

root, I could not find any hyphae in any of the samples that were prepared for

quantitative X-ray microanalysis. Thus, the main hypothesis of this experiment was not

addressed. However, other findings from this experiment were unexpected, and very

interesting.

Large granules, which according to quantitative X-ray microanalysis contained very

high concentrations of P and K, were observed in the cortex area of the roots of plants

that had received a pulse of a high P concentration (150 mg P kg-1 soil), no matter

whether colonised or uncolonised. These granules were hardly visible in samples that

received 15 mg P kg-1 soil. Such granules have not been reported previously. A number


of factors suggest they may consist of polyphosphate: granular nature, strong linear

relationship between P and K concentrations, and formation after a P pulse. However,

polyphosphate is not commonly reported in plants and thus more research is required.

Moreover, there is also the possibility of these granules being artefacts due to the

experimental procedure I followed. But even if the granular nature is an artefact, the

high concentrations are undoubtedly correct, as is their association with K, even if these

high concentrations may not be associated with granules before harvest.

5.3 Unexpected findings

5.3A Inoculation with AMF greatly decreased manganese concentrations

Inoculation with AMF resulted in a decrease in shoot (Chapters 2 and 3) and root

(Chapter 2) manganese (Mn) concentration. However root Mn concentration did not

decrease in all species in colonised plants (Chapter 3). The finding that AMF caused a

reduction in host plant Mn concentration is in accordance with many studies (Fageria et

al. 2002; Kothari et al. 1991; Liu et al. 2000; Posta et al. 1994; Ryan and Angus 2003).

Changes in soil microbial community might be responsible for the difference in Mn

concentration between colonised and uncolonised plants. In an experiment by Posta et

al. (1994), although the total microbial population was higher in colonised plants (Zea

mays L. cv. Tau), the proportion of Mn-reducing microbial populations in uncolonised

plants was five-fold higher than that in colonised plants, resulting in a higher shoot Mn

concentration in the uncolonised plants than in the colonised plants. Also, the amount of

Mn-mobilising root exudates in colonised plants was less than that in uncolonised

plants. However, my findings presented in Chapter 3 suggest that the explanation may

be more complicated. In Chapter 3, colonisation by AMF caused a higher rhizosphere

pH, lower shoot Mn concentration and a lower amount of rhizosphere carboxylates.

Integrating all these results, I concluded that plants that are colonised by AMF exude

less carboxylates into the rhizosphere which changes the rhizosphere chemistry (making

it more alkaline) and this results in less Mn uptake by the plant. This explanation is

consistent with Marschner (1991), who stated that plants grown under acidic conditions

might encounter Mn toxicity. Overall, both processes (i.e. changes in the soil microbial

community and changes in rhizosphere chemistry) might play a role in reducing the Mn

concentration in colonised plants.


5.3B Phosphorus efflux following colonisation with AMF

One of the interesting, unexpected, findings in Chapter 3 was great elevation in

rhizosphere Colwell P in colonised K. prostrata and C. australasicum following a P

pulse.

There are several studies that show P efflux when plants are supplied with high

concentrations of P (Elliott et al. 1984; McPharlin and Bieleski 1989; Schjørring and

Jensen 1984). For instance, in an experiment by Cogliatti and Santa Maria (1990), P

efflux was increased when wheat (Triticum aestivum cv. Klein Atalaysa) was grown at

higher P concentrations.

Elevation of rhizosphere Colwell P following a P pulse in colonised plants might be due

to one or both of the following: 1) P efflux from the roots of colonised plants, since they

acquire more P (through mycorrhizal hyphae) than they need for growth and transport to

the shoot; and 2) P efflux from the mycorrhizal hyphae (which has been reported by

Ryan et al. (2003) and Solaiman and Saito (2001).

In my experiment, high concentrations of Colwell P in the rhizosphere might be due to

both factors. More research is now required to better understand this response (and why

it did not occur in the other three species in the experiment).

5.3C Colonisation with AMF makes the rhizosphere more alkaline

The second unexpected result from Chapter 3 was higher rhizosphere pH in both

colonised and uncolonised plants compared to bulk soil. According to Raven and Smith

(1976), the increase in rhizosphere pH compared to bulk soil is due to absorption of

nitrate. Also, Bago et al. (1996) found supplying nitrate to colonised plants increases

rhizosphere pH while supplying ammonium drops pH. In my experiment, plants were

treated with similar amounts of nitrate and ammonium during the experiment (3.3 mg N

kg-1 soil) and since nitrate is more mobile than ammonium in the soil (Binkley 1984),

the rise in rhizosphere pH was expected. On the other hand, an increase in rhizosphere

pH of colonised plants can be explained by a reduction in the amount of rhizosphere

carboxylates. The strong negative correlation between the amount of rhizosphere

carboxylates and rhizosphere pH also supports this explanation.


5.4 Limitations of the research presented in this thesis

Although the experiments were designed to be as close to natural (field) conditions as

possible (using field soil as inoculum, growing plants in the field soil with moderate

levels of P, and adding filtrates to the non-inoculated plants to maintain the microbial

community), there are some limitations of this research. One of the most important

factors that might limit applicability of my experiments to field conditions is the

glasshouse conditions under which the experiments were conducted. My experiments

were conducted in a temperature-controlled environment, whereas in the field plants

encounter a greater range of temperatures. Also, in the field other factors such as pests

and diseases and neighbouring plants and the competition between them might result in

different P-uptake responses from plants (Collis George and Davey 1960; De Vries and

Tiller 1978).

One of the outstanding findings of this study was the moderating effect of mycorrhiza.

This effect was shown during the first week after adding the pulse of P. A longer period

of time might hide such an effect, and the results might be different.

Soil P levels also might change the results presented in the thesis. All the experiments

were performed under a moderate soil P level. Using soil with lower or higher P levels

might eliminate the moderating effect of AMF.

5.5 Implications for future research

To address the limitations discussed above in the thesis and to have a better

understanding of how AMF moderate plant P uptake following a P pulse, performing

experiments over a longer period of time is to be recommended. Many root physiology

experiments overlook or remove the effect of AMF. Often, they are conducted in

sterilised soil. According to the results that came from this thesis, I believe that AMF

are not only important from the perspective of nutrient uptake, but also because of their

role in changing rhizosphere chemistry and microbial community.

5.6 Concluding remarks

The main focus of this thesis was to investigate the role of AMF in plant uptake of P

from a pulse of P by perennial pasture plants.


According to the results in Chapters 2 and 3, colonisation by AMF does not necessarily

enhance plant P uptake from a P pulse, but may actually protect plants from P toxicity.

This could be particularly important for native plants such as Kennedia species which

suffer from P toxicity at relatively low P availability (Pang et al. 2009b).

Based on the results presented in this thesis, I suggest that the traditional view of AMF

as a beneficial biological “fertiliser” to enhance crop uptake of P and other poorly

mobile nutrients is too simplistic. My work indicates that AMF also play a moderating

role in P nutrition and affect plant nutrition in a complex manner through their effect on

rhizosphere pH, carboxylates and P content, and, undoubtedly, other means.

5.7 References

Abbott LK, Robson AD (1977) Growth stimulation of subterranean clover with vesicular-arbuscular mycorrhizas. Aust J Agric Res 28: 639-649.

Abel S, Ticconi CA, Delatorre CA (2002) Phosphate sensing in higher plants. Physiol Plant 115: 1-8.

Asghari HR, Cavagnaro TR (2011) Arbuscular mycorrhizas enhance plant interception of leached nutrients. Funct Plant Biol 38: 219-226.

Azcón R, Barea JM, Hayman DS (1976) Utilization of rock phosphate in alkaline soils by plants inoculated with mycorrhizal fungi and phosphate-solubilizing bacteria. Soil Biol Biochem 8: 135-138.

Bago B, Vierheilig H, Piché Y, Azcón-Aguilar C (1996) Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture. New Phytol 133: 273-280.

Bainard LD, Koch AM, Gordon AM, Klironomos JN (2013) Growth response of crops to soil microbial communities from conventional monocropping and tree-based intercropping systems. Plant Soil 363: 345-356.

Binkley D (1984) Ion exchange resin bags: Factors affecting estimates of nitrogen availability. Soil Sci Soc Am J 48: 1181-1184.

Bolan NS, Robson AD, Barrow NJ (1987) Effects of vesicular-arbuscular mycorrhiza on the availability of iron phosphates to plants. Plant Soil 99: 401-410.

Cogliatti DH, Santa Maria GE (1990) Influx and efflux of phosphorus in roots of wheat plants in non-growth-limiting concentrations of phosphorus. J Exp Bot 41: 601-607.

Collis George N, Davey BG (1960) The doubtful utility of present-day field experimentation and other determinations involving soil-plant interactions. Soils and Fertilizers 23: 307-310.

De Vries MPC, Tiller KG (1978) Sewage sludge as a soil amendment, with special reference to Cd, Cu, Mn, Ni, Pb and Zn-comparison of results from experiments conducted inside and outside a glasshouse. Environ Pollut 16: 231-240.


Elliott GC, Lynch J, Läuchli A (1984) Influx and efflux of P in roots of intact maize plants : Double-labeling with 32P and 33P. Plant Physiol 76: 336-341.

Espigares T, Peco B (1995) Mediterranean annual pasture dynamics: impact of autumn drought. J Ecol 83: 135-142.

Fageria NK, Baligar C, Clark RB (2002) Micronutrients in crop production. Adv Agron Volume 77: 185-268.

Gardner WK, Barber DA, Parbery DG (1983) The acquisition of phosphorus by Lupinus

albus L. Plant Soil 70: 107-124.

Graham JH (2000) Assessing costs of arbuscular mycorrhizal symbiosis in agroecosystems. APS Press, St. Paul.

Hetrick B, Wilson GT (1988) Effects of soil microorganisms on mycorrhizal contribution to growth of big bluestem grass in non-sterile soil. Soil Biol Biochem 20: 501-507.

Howieson JG, O’Hara GW, Carr SJ (2000) Changing roles for legumes in Mediterranean agriculture: developments from an Australian perspective. Field Crops Res 65: 107-122.

Jansa J, Mozafar A, Frossard E (2005) Phosphorus acquisition strategies within arbuscular mycorrhizal fungal community of a single field site. Plant Soil 276: 163-176.

Jones DL (1998) Organic acids in the rhizosphere–a critical review. Plant Soil 205: 25-44.

Kariman K, Barker SJ, Finnegan PM, Tibbett M (2014) Ecto-and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings. Mycorrhiza: 1-9.

Kirkegaard JA, Peoples MB, Angus JF, Unkovich MJ (2011) Diversity and evolution of rainfed farming systems in southern Australia. In: P Tow, I Cooper, I Partridge, C Birch (eds) Rainfed Farming Systems. Springer Netherlands.

Kothari SK, Marschner H, Rӧmheld V (1991) Effect of a vesicular–arbuscular mycorrhizal fungus and rhizosphere micro-organisms on manganese reduction in the rhizosphere and manganese concentrations in maize (Zea mays L.). New Phytol 117: 649-655.

Krey T, Caus M, Baum C, Ruppel S, Eichler-Löbermann B (2011) Interactive effects of plant growth–promoting rhizobacteria and organic fertilization on P nutrition of Zea mays L. and Brassica napus L. J Plant Nutr Soil Sci 174: 602-613.

Lambers H, Shane MW, Cramer MD, Pearse SJ, Veneklaas EJ (2006) Root structure and functioning for efficient acquisition of phosphorus: matching morphological and physiological traits. Ann Bot-London 98: 693-713.

Li G, Lodge G, Moore G, Craig A, Dear B, Boschma S, Albertsen T, Miller S, Harden S, Hayes R (2008) Evaluation of perennial pasture legumes and herbs to identify species with high herbage production and persistence in mixed farming zones in southern Australia. Aust J Exp Agric 48: 449-466.

Liu A, Hamel C, Hamilton RI, Ma BL, Smith DL (2000) Acquisition of Cu, Zn, Mn and Fe by mycorrhizal maize (Zea mays L.) grown in soil at different P and micronutrient levels. Mycorrhiza 9: 331-336.


Lynch JP, Ho MD (2005) Rhizoeconomics: carbon costs of phosphorus acquisition. Plant Soil 269: 45-56.

Maherali H, Klironomos JN (2007) Influence of phylogeny on fungal community assembly and ecosystem functioning. Science 316: 1746-1748.

Marschner H (1991) Mechanisms of adaptation of plants to acid soils. Plant Soil 134: 1-20.

McPharlin IR, Bieleski RL (1989) Pi efflux and influx by P-adequate and P-deficient Spirodela and Lemna. Aust J Plant Physiol 16: 391-399.

Nichols PGH, Loi A, Nutt BJ, Evans PM, Craig AD, Pengelly BC, Dear BS, Lloyd DL, Revell CK, Nair RM, Ewing MA, Howieson JG, Auricht GA, Howie JH, Sandral GA, Carr SJ, de Koning CT, Hackney BF, Crocker GJ, Snowball R, Hughes SJ, Hall EJ, Foster KJ, Skinner PW, Barbetti MJ, You MP (2007) New annual and short-lived perennial pasture legumes for Australian agriculture—15 years of revolution. Field Crops Res 104: 10-23.

Pang J, Ryan MH, Tibbett M, Cawthray GR, Siddique KHM, Bolland MDA, Denton MD, Lambers H (2009a) Variation in morphological and physiological parameters in herbaceous perennial legumes in response to phosphorus supply. Plant Soil 331: 241-255.

Pang J, Tibbett M, Denton MD, Lambers H, Seddique KHM, Bolland MDA, Revell CK, Ryan MH (2009b) Variation in seedling growth of 11 perennial legumes in response to phosphorus supply. Plant Soil 328: 133-143.

Pearse SJ, Veneklaas EJ, Cawthray GR, Bolland MDA, Lambers H (2006) Carboxylate release of wheat, canola and 11 grain legume species as affected by phosphorus status. Plant Soil 288: 127-139.

Peoples MB, Baldock JA (2001) Nitrogen dynamics of pastures: nitrogen fixation inputs, the impact of legumes on soil nitrogen fertility, and the contributions of fixed nitrogen to Australian farming systems. Aust J Exp Agric 41: 327-346.

Porqueddu C, González F (2011) Role and potential of annual pasture legumes in Mediterranean farming systems. Pastos 36: 125-142.

Posta K, Marschner H, Römheld V (1994) Manganese reduction in the rhizosphere of mycorrhizal and nonmycorrhizal maize. Mycorrhiza 5: 119-124.

Puckridge DW, French RJ (1983) The annual legume pasture in cereal—Ley farming systems of southern Australia: A review. Agric Ecosyst Environ 9: 229-267.

Raven JA, Smith FA (1976) Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytol 76: 415-431.

Real D, Li GD, Clark S, Albertsen TO, Hayes RC, Denton MD, Mario FDA, Dear BS (2011) Evaluation of perennial forage legumes and herbs in six Mediterranean environments. Chilean Journal of Agricultural Research 71: 357-369.

Richardson AE (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Funct Plant Biol 28: 897-906.

Richardson AE, Barea JM, McNeill AM, Prigent-Combaret C (2009) Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil 321: 305-339.

Robson AD (1990) The role of self-regenerating pasture in rotation with cereals in


Mediterranean areas. In: A Osman, M Ibrahim, M Jones (eds) The role of legumes in the farming systems of the Mediterranean areas. Springer, ICARDA, Netherlands.

Ryan MH, Angus J (2003) Arbuscular mycorrhizae in wheat and field pea crops on a low P soil: increased Zn-uptake but no increase in P-uptake or yield. Plant Soil 250: 225-239.



Ryan MH, Tibbett M, Edmonds-Tibbett T, Suriyagoda LDB, Lambers H, Cawthray GR, Pang J (2012) Carbon trading for phosphorus gain: the balance between rhizosphere carboxylates and arbuscular mycorrhizal symbiosis in plant phosphorus acquisition. Plant, Cell Environ 35: 2170-2180.

Sardans J, Peñuelas J (2007) Drought changes phosphorus and potassium accumulation patterns in an evergreen Mediterranean forest. Funct Ecol 21: 191-201.

Scanlon T (2011) Decline in subterranean clover (Trifolium subterraneum L.)-based pasture systems: causes and solutions. M.Sc. Thesis. University of Western Australia. pp.260.

Schjørring JK, Jensen P (1984) Phosphorus nutrition of barley, buckwheat and rape seedlings. II. Influx and efflux of phosphorus by intact roots of different P status. Physiol Plant 61: 584-590.

Singh S, Kapoor KK (1998) Effects of inoculation of phosphate-solubilizing microorganisms and an arbuscular mycorrhizal fungus on mungbean grown under natural soil conditions. Mycorrhiza 7: 249-253.

Smith SE (1982) Inflow of phosphate into mycorrhizal and non-mycorrhizal plants of Trifolium subterraneum at different levels of soil phosphate. New Phytol 90: 293-303.

Smith SE, Read DJ (2008) Mycorrhizal Symbiosis. Academic press, London, UK.


Solaiman MZ, Saito M (2001) Phosphate efflux from intraradical hyphae of Gigaspora

margarita in vitro and its implication for phosphorus translocation. New Phytol 151: 525-533.

Suriyagoda LDB, Lambers H, Renton M, Ryan MH (2012) Growth, carboxylate exudates and nutrient dynamics in three herbaceous perennial plant species under low, moderate and high phosphorus supply. Plant Soil 358: 105-117.

van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T, Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396: 69-72.


Veneklaas EJ, Stevens J, Cawthray GR, Turner S, Grigg AM, Lambers H (2003) Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake. Plant Soil 248: 187-197.

Wagg C, Jansa J, Stadler M, Schmid B, van der Heijden MGA (2011) Mycorrhizal fungal identity and diversity relaxes plant-plant competition. Ecology 92: 1303-1313.

Weaver DM, Reed AEG (1998) Patterns of nutrient status and fertiliser practice on soils of the south coast of Western Australia. Agric Ecosyst Environ 67: 37-53.

Whitelaw MA (1999) Growth promotion of plants inoculated with phosphate-solubilizing fungi. Adv Agron 69: 99-151.

the role of arbuscular mycorrhizal fungi in uptake by

Documents