131
Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated with Mycorrhizal Fungi Khalil Kariman B.Sc. in Plant Protection: Gorgan Uni. Agric. Sci. Nat. Res., Iran M.Sc. in Plant Pathology: Tarbiat Modarres University, Iran This thesis is presented for the degree of Doctor of Philosophy at the University of Western Australia School of Earth and Environment & School of Plant Biology Faculty of Natural and Agricultural Sciences 2013

Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

  • Upload
    others

  • View
    15

  • Download
    0

Embed Size (px)

Citation preview

Page 1: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

Physiological and Molecular Analysis of Tolerance to

Phosphate Toxicity in Jarrah (Eucalyptus marginata)

Seedlings Inoculated with Mycorrhizal Fungi

Khalil Kariman

B.Sc. in Plant Protection: Gorgan Uni. Agric. Sci. Nat. Res., Iran

M.Sc. in Plant Pathology: Tarbiat Modarres University, Iran

This thesis is presented for the degree of Doctor of Philosophy at

the University of Western Australia

School of Earth and Environment

&

School of Plant Biology

Faculty of Natural and Agricultural Sciences

2013

Page 2: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated
Page 3: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

As our circle of knowledge expands, so does the

circumference of darkness surrounding it

Albert Einstein

Page 4: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated
Page 5: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

Dedicated to my father, my mother, my brothers and my

sister for their forever love and constant support during my

entire journey of education

Page 6: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated
Page 7: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

Thesis Abstract

Jarrah (Eucalyptus marginata) is an Australian native tree with the capacity to form

arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations. ECM

symbioses are the main mycorrhizal type for mature eucalypts while AM symbioses are

thought to be transitory event early in eucalypt seedling growth. In previous research,

where eucalypt seedlings of several species have been exposed to isolated strains of

either AM or ECM fungi, plants in an ECM symbiosis had better P nutrition and

growth responses than those in an AM symbiosis. However, little research has been

carried out either exploring ECM and AM associations of jarrah in detail, or the effect

on plant growth and nutrition, of a dual (AM and ECM) symbiosis. In addition, jarrah

is among many Australian native plants that develop toxicity symptoms upon exposure

to high doses of phosphate (Pi). Crucial knowledge gaps exist in understanding the

molecular basis for P sensitivity of native species and how mycorrhizal symbioses

function in the deficiency-toxicity continuum. However, unpublished earlier research

has suggested a correlation between mycorrhizal status and tolerance to artificially high

levels of Pi in soil. To better understand the role of both AM and ECM symbioses in

the plant’s response to Pi and its analogues phosphite (Phi) and arsenate (AsV),

research in controlled conditions has been undertaken. Herbaceous plants in an AM

symbiosis are known to down-regulate the expression of some Pi transporter (class

PHT1) genes in roots. Therefore, I hypothesize that first, ECM fungi are better

symbiotic partners than AM in conferring growth and nutritional benefits to jarrah

plants and second, mycorrhizal associations have the potential to induce tolerance

against Pi, Phi and AsV toxicities, which could be linked with fungal ability to down-

regulate the expression of plant PHT1 genes. Therefore, this research was conducted to

i) investigate AM, ECM and dual (AM & ECM) mycorrhizal associations of jarrah and

their potential growth and P nutritional benefits, ii) clarify if mycorrhizal symbioses

can induce tolerance to jarrah plants exposed to Pi, Phi and AsV toxicity conditions and

iii) reveal any possible relationship between the expression of PHT1 genes in jarrah

roots and either P sensitivity or any mycorrhiza-mediated tolerance to Pi, Phi and AsV

toxicities.

I developed a nurse-pot system to investigate mycorrhizal colonisation of jarrah by

AM and ECM fungal species both separately and in dual AM+ECM culture (Kariman

et al., 2012, Chapter 2). The nurse-pot system was effective at initiating colonisation of

functioning AM (Scutellospora calospora) or putative ECM (Scleroderma sp.) systems.

i

Page 8: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

The presence of S. calospora, Austroboletus occidentalis and Scleroderma sp.

individually significantly increased the shoot biomass of seedlings compared to non-

mycorrhizal (NM) controls whereas no positive response was observed with

Rhizophagus irregularis. In addition, the non-responsive AM fungus R. irregularis

suppressed the ECM symbiosis in a dual pot culture also containing Scleroderma sp.

inoculum. Also noteworthy, despite the positive growth and nutritional responses in the

A. occidentalis treatment, ECM structures were not observed in either nurse or test

seedlings. This suggested that a novel relationship existed between jarrah and A.

occidentalis that was potentially symbiotic and worthy of additional study.

A three-compartment system and 33

P-labelled Pi were used to explore the functional

mechanisms involved in the novel symbiosis observed between jarrah and A.

occidentalis (Chapter 3). The results of this additional analysis showed that the novel

symbiosis with no root colonisation was as beneficial for the host as a typical ECM

association, a finding that challenges the notion that this type of plant-fungal symbiosis

is defined by intraradical structures and an extensive network of extraradical hyphae. A

novel-plant fungus symbiosis was discovered in which i) the fungal partner does not

colonise plant roots, ii) hyphae are limited to the rhizosphere soil and vicinity and

consequently, iii) do not transfer nutrients located much beyond the rhizosphere and

presumably do not transfer nutrients directly. The transcript profile of PHT1 genes in

roots did not change in the presence of A. occidentalis, while the expression of two

genes (EmPHT1;1 and EmPHT1;2) were down-regulated in response to Scleroderma

sp., which formed classic ECM colonisation and transported 33

P to jarrah plants. This

suggests that the Pi uptake route shifts from a direct root pathway to a hyphal pathway

in the ECM plants as it does in the AM symbioses. The high concentrations of citrate

and fumarate measured in the rhizosphere soil indicated that exudation of these

substances to achieve nutrient solubilization and mobilization is a likely mechanism

involved in the proposed novel symbiosis between jarrah and A. occidentalis.

To study the role of AM, ECM and the novel symbioses in the response of jarrah to

toxic levels of P, inoculated and NM nurse plants were exposed to two potentially toxic

pulses of P as Pi (10 and 30 mg P kg-1

soil) (Chapter 4). The results revealed that jarrah

is among the most sensitive species to P fertilization, with toxicity symptoms observed

at a shoot P concentration of no more than 5.5 mg P g-1

DW. All inoculated plants had

significantly lower toxicity symptoms compared to NM controls one week after

addition of the elevated P dose.

ii

Page 9: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

A comprehensive glasshouse experiment was then conducted to investigate the

response of jarrah to highly toxic pulses (1.5 mmol kg-1

soil) of Pi, and its analogues,

Phi and AsV (Chapter 5). The AM colonisation of jarrah plants by S. calospora

declined after 14 weeks growth, which is a common phenomenon occurring in early

stages of AM-eucalypt symbioses. However, addition of Pi and Phi reversed this

natural trend after four weeks compared to untreated AM plants. There was no root

colonisation detected for either Scleroderma sp. or A. occidentalis treatments, however

positive growth responses were detected for both treatments indicating that in this

experiment both fungi may use the novel symbiotic pathway. Four weeks after adding

the Pi pulse, only plants in the AM treatment demonstrated Pi tolerance. Results from

both toxicity experiments (Chapters 4 and 5) showed that the Pi tolerance was due to

lower shoot P concentration in both ECM and novel symbioses whereas Pi tolerance in

the AM symbioses was not always accompanied by lower shoot P concentration than

NM controls. All inoculated plants had Phi tolerance because of lower Phi uptake than

NM controls. For AsV, the most toxic analogue, all plants had died within a week after

the AsV pulse, however inoculated plants had significantly lower shoot As

concentration than NM controls. The expression of plant PHT1 genes was quantified to

see if symbiotic associations result in lower expression of plant PHT1 genes as an

indicator of hyphal P transfer and Pi tolerance. The expression pattern of jarrah PHT1

genes was not altered in response to S. calospora (AM) or either fungus (Scleroderma

sp. and A. occidentalis) that used the novel symbiotic pathway that benefitted the host

without colonisation. However, when jarrah plants were colonised by Scleroderma sp.

under low P conditions, (33

P-labelled experiment, Chapter 3), the expression of two

PHT1 genes was down-regulated. Interestingly, I detected enhanced transcript levels of

four PHT1 genes in roots of NM jarrah plants in response to Pi toxicity and three PHT1

genes in response to Phi and AsV toxicities, which could be linked with jarrah

sensitivity to these analogues.

Overall, the results in this thesis indicate that the range of beneficial fungal

interactions with jarrah is wider than the classic definition of mycorrhizal symbiosis. A

novel symbiosis clearly exists where the fungus does not penetrate the plant root but

has defined beneficial consequences to plant growth and nutrition and tolerance to Pi

and Phi. These benefits are correlated with higher carboxylate concentration in the

rhizosphere soil. Regardless of their colonisation ability, ECM performed better than

AM symbioses in improving jarrah growth and P nutrition, supporting the first

hypothesis of this thesis. However, the induced Pi and Phi tolerance in inoculated

iii

Page 10: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

plants was not correlated with lower expression of plant PHT1 genes, disproving the

second hypothesis concerning PHT1 genes, at least for AM and novel symbioses. In

conclusion, this thesis has demonstrated that ECM and novel symbioses are more

beneficial under P-deficiency conditions while AM symbioses are superior in

protecting jarrah plants against P-toxicity conditions. This research has provided useful

insight to the P nutrition of jarrah in the context of efforts to retain its presence in

disturbed habitats.

iv

Page 11: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

Publications arising from this thesis

Published papers:

- Kariman K, Barker SJ, Finnegan PM, Tibbett M (2012) Dual mycorrhizal

associations of jarrah (Eucalyptus marginata) in a nurse-pot system. Australian

Journal of Botany 60: 661-668

Papers to be submitted:

- Kariman K, Barker SJ, Jost R, Finnegan PM, Tibbett M. A novel plant-fungus

symbiosis benefits the host without forming mycorrhizal structures.

Target journal: Nature

- Kariman K, Barker SJ, Finnegan PM, Tibbett M. Mycorrhizal symbiosis can

induce tolerance in jarrah (Eucalyptus marginata) exposed to toxic pulses of

phosphorus.

Target Journal: Plant and Soil

- Kariman K, Barker SJ, Finnegan PM, Tibbett M. Symbiotic associations and

response of jarrah (Eucalyptus marginata) to phosphate, phosphite and arsenate

toxicities.

Target Journal: Plant Physiology

Conference presentations

- Kariman K, Barker SJ, Jost R, Finnegan PM, Tibbett M (2011). Mycorrhiza-

mediated tolerance to phosphorus toxicity in jarrah (Eucalyptus marginata). The

Rhisosphere 3 International Conference, Perth, Australia.

v

Page 12: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

vi

Page 13: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

Table of contents

Abstract i

Publications arising from this thesis v

Acknowledgements ix

Statement of authorship xi

Chapter 1: General Introduction and Literature Review 1

Chapter 2: Dual mycorrhizal associations of jarrah (Eucalyptus 29

marginata) in a nurse-pot system

Chapter 3: A novel plant-fungus symbiosis benefits the host 37

without forming mycorrhizal structures

Chapter 4: Mycorrhizal symbiosis can induce tolerance in jarrah 59

(Eucalyptus marginata) exposed to toxic pulses of phosphorus

Chapter 5: Symbiotic associations and response of jarrah 79

(Eucalyptus marginata) to phosphate, phosphite

and arsenate toxicities

Chapter 6: General Discussion and Conclusion 105

vii

Page 14: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

viii

Page 15: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

Acknowledgements

I look at my PhD as an amazing journey of my life, a journey that taught me how to

think rationally and how to work scientifically, a journey full of research experience

and a journey filled with unforgettable memories and moments with the UWA staffs

and students.

I would like to express my sincere gratitude to my coordinating supervisor Prof.

Mark Tibbett and my co-supervisors Profs Susan Barker and Patrick Finnegan for their

excellent supervision, constant support, advice and encouragement throughout my

PhD. Mark, Susan and Pat, thanks a lot indeed for teaching me the scientific

rationality, you are such awesome teachers. Susan, I also appreciate your initial

support and encouraging me to apply for international PhD scholarships at the UWA.

I acknowledge the University of Western Australia for the financial support of my

PhD studies by awarding the prestigious Scholarships for International Research Fees

(SIRF) and the University International Stipend (UIS). Financial supports from

Schools of Earth and Environment & Plant Biology are also appreciated.

I would like to thank Tim Morald, Tim Lardner and Evonne Walker in Centre for

Land Rehabilitation for their kind helps. Thanks to John Koch at the Alcoa World

Alumina for his assistance in collecting jarrah seeds from jarrah forest sites. My fellow

students Martha Orozco Aceves, Deborah Lin and Dimanthina Cheong, I really

enjoyed your company and friendship during my PhD.

Special thanks to Ricarda Jost and Hazel Gaza for their support and practical advice

on the molecular aspects of my project. Also, I greatly appreciate technical and

scientific advice from Lindsey Loweth on my 33

P experiment. Many thanks to my

great friends in Pat Finnegan’s group Bahram Mirfakhraei, Fazilah Abd Mannan,

Sandra Kerbler, Robert Pontre and Marina Borges Osorio.

Special thanks to my dear friend Basu Dev Regmi for his technical and scientific

advice throughout my studies. You are such an awesome mate with a unique spirit.

My dear friend Dr. Hossein Khabaz-Saberi, you have always been a great friend and

the person I could trust when I had serious difficulties in my life and studies.

Thanks to my school mates Mansour Ghorbanpour, Daniel Dempster, Leila

Heidarvand, He Chang (Shirely), Anjani Weerasekara, Sanjutha Shanmugam,

Zhengyao Nie, Louis Moir-Barneston, Srinivasan Samineni, John Quealy, George,

Sonja Jakob and Lalith Suriyagoda.

Michal Smirk, I am so grateful for your kind assistance with ICP analysis of my

samples. Many thanks to Gregory Cawthray for his assistance and technical advice for

ix

Page 16: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

carboxylates and sugar analysis of my samples by HPLC. Robert Creasy and Bill

Piasini, I appreciate your positive attitudes and continuous assistance during all my

glasshouse experiments.

I have to express my deepest thanks to my parents, my sister and my brothers for

their countless supports of my education and also special thanks to my brothers for

supporting my initial migration to Australia. Heaps of thanks to my wife for all her

kindness and understanding my situation during the late stages of my PhD. My family

has always been dedicated to provide a better future for me, and my educational

achievements were unfeasible without their kindness and encouragement.

x

Page 17: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated
Page 18: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

1

Chapter 1

General Introduction and Literature Review

General Introduction

Australian soils generally contain low levels of essential nutrients needed for plant

growth. Phosphorus (P) deficiency is considered to be the main critical factor

determining plant productivity and species diversity in Australian terrestrial ecosystems

(Thompson et al., 1983; Adams et al., 1994; Handreck, 1997; Richardson et al., 2009;

Lambers et al., 2010).

Many Australian native plants have adaptive mechanisms to extract as much P as

possible from the low P soils, in which they grow. Mycorrhizal association is an

efficient strategy for increasing P uptake and is estimated to be found in more than 80%

of land plant species (Smith and Read, 2008). There are seven types of mycorrhizal

associations defined by the way in which the plant root interacts with particular fungal

species or genera. Arbuscular mycorrhiza (AM) and ectomycorrhiza (ECM) are the

most abundant types. Arbuscular mycorrhizal associations occur in more than 336 plant

families and probably co-evolved with plant roots over the last 400 million years

(Smith and Read, 2008; Brundrett, 2009). Ectomycorrhizal associations are more

specialized, recently evolved and can form symbiotic associations with many woody

plant species from about 30 families (Wilcox, 1996; Smith and Read, 2008). Besides

the main types, unusual plant-fungus associations with unconventional structures or

without any colonisation also have been documented, which cannot be classified into

any of the already-described mycorrhizal categories (Neumann, 1959; Warcup and

McGee, 1983; Kope and Warcup, 1986; Brundrett, 2009). Generally, the association

can be considered a symbiosis that is mutually beneficial.

Some Australian native plants growing in P-impoverished soils are extremely

sensitive to P fertilization. Phosphorus sensitive species are found in the Proteaceae,

Myrtaceae, Rutaceae, Fabaceae, Mimosaceae, Haemodoraceae and generally are a

feature of heaths and other sclerophyllous plant communities (Specht and Groves,

1966; Grundon, 1972; Heddle and Specht, 1975; Specht et al., 1977; Specht, 1981a,

1981b; Dell et al., 1987; Handreck, 1997; Shane et al., 2004a, 2004b; Thomson and

Leishman, 2004; Hawkins et al., 2008). Jarrah (Eucalyptus marginata) plants are

sensitive to P fertilization presumably as a consequence of their adaptation to P-

deficient soils. Unpublished experiments have shown that jarrah becomes tolerant to P

Page 19: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

2

following mycorrhizal colonisation (Tibbett, unpublished). There is evidence that P

responses may be linked to plant responses to root colonisation by mycorrhizal fungi

but details of this relationship are not clear. The expression of high-affinity Pi

transporter (PHT1) genes in plant roots can be reduced in response to mycorrhizal

colonisation (Karandashov and Bucher, 2005). As a consequence of the mycorrhizal

symbiosis, plant epidermal PHT1 gene expression is reduced in roots. Tolerance of

mycorrhizal jarrah plants to toxic dose of Pi might occur if the symbiotic fungi are able

to regulate the amount of Pi that they supply to the plant and the plant root epidermal Pi

uptake mechanism is reduced or not functional in the symbiotic state. The possibility

that tolerance exists in mycorrhizal jarrah plants and might be due to reduced

abundance of PHT1 gene products is an hypothesis that will be tested in this thesis.

Two phosphate (Pi) analogues phosphite (Phi) and arsenate (AsV) are of significant

importance to environmental scientists and authorities in Western Australia (WA).

Phosphite has been widely used for treatment of the Die Back disease caused by

Phytophthora cinnamomi, which is a destructive pathogen in WA forests and heathland

communities (Dell et al., 2005). Arsenate contamination of soils and underground

water occurs in WA naturally or as a result of anthropogenic activities such as mining

(Smith et al., 2003). Native plant species such as jarrah, therefore, might be exposed to

high levels of these two Pi analogues. Both Phi and AsV can enter plant roots through

the same mechanism as Pi i.e. via Pi transporters (Meharg and Macnair, 1990; Guest

and Grant, 1991; Meharg and Macnair, 1992; Marschner, 1995). Arsenate tolerance in

mycorrhizal plants can be achieved through various mechanisms such as reduction of

AsV to arsenite (AsIII) and the reduced expression of plant PHT1 genes (Sharples et

al., 2000; Gonzalez-Chavez et al., 2002).

According to our hypothesis, altered expression patterns of plant high affinity PHT1

genes are probably always associated with tolerance to Pi, Phi and AsV toxicity in

mycorrhizal plants. However, there is no literature available regarding the PHT1

sequences in jarrah, the response of PHT1 gene expression to ECM symbiosis in any

host, or the role of mycorrhizal associations in tolerance to P toxicity in woody

Australian plant species. Jarrah is an ideal model host to investigate both AM and ECM

symbioses as it has dual symbiotic capacity (Brundrett and Abbott 1991; Howard et al.,

2000) during its lifecycle.

Page 20: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

3

Research Objectives:

1- To determine the effects of selected AM and putative ECM species on jarrah

growth and nutrient acquisition (in particular P) under P-deficient conditions

2- To investigate the symbiosis using locally isolated ECM-type fungi with which

jarrah would be associated in natural settings

3- To investigate the potential role of different fungal symbionts in inducing

tolerance to jarrah plants exposed to toxic pulses of P as Pi

4- To investigate the possibility that mycorrhizal symbioses might induce

tolerance against a toxic pulse of Pi, Phi and AsV and monitor the shoot P and

As concentration after short and long time exposure

5- To clone and sequence the root-expressed high-affinity Pi transporter genes

(PHT1 gene family) from jarrah

6- To quantify the transcript abundance of PHT1 genes in jarrah roots in order to i)

clarify the molecular mechanism behind P sensitivity in jarrah and ii) reveal the

possible correlation with P tolerance in mycorrhizal plants

Page 21: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

4

Structure of the Thesis

Chapter 1: General Introduction and Literature Review

Chapter 2: Dual mycorrhizal associations of jarrah (Eucalyptus marginata) in a nurse-

pot system

Target Journal: Australian Journal of Botany Status: Published (2012, 60: 661-668)

Chapter 3: A novel plant-fungus symbiosis benefits the host without forming

mycorrhizal structures

Target Journal: Nature Status: To be submitted

Chapter 4: Mycorrhizal symbiosis can induce tolerance in jarrah (Eucalyptus

marginata) exposed to toxic pulses of phosphorus

Target Journal: Plant and Soil Status: To be submitted

Chapter 5: Symbiotic associations and response of jarrah (Eucalyptus marginata) to

Pi, Phi and AsV toxicities

Target Journal: Plant Physiology Status: To be submitted

Chapter 6: General Discussion and Conclusion

Page 22: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

5

Literature Review

Australian soils and P status

Australian soils are generally known as nutrient-deficient soils and contain an

average of 300 mg P kg-1

soil, which is regarded as a low P level compared with soils

from other countries (Williams and Raupach, 1983). This is mainly because Australian

soils have derived from low P containing sedimentary rocks and also due to the long

history of geological stability in Australian landscapes (Williams and Raupach, 1983;

Handreck, 1997; Lambers et al., 2011). Some natural processes contribute to P

depletion from soil including weathering (White, 1986, 1994), leaching (Attiwill and

Leeper, 1987) and removing fine soil particles rich in P by wind and water (Beadle,

1962; Raison et al., 1985). The soil total P consists of organic P (Po) and inorganic P

pools and only less than 1% of total P is immediately available (Pi) for plants uptake

(Richardson et al., 2009). Adsorption onto soil particles and formation of AlPO4 or

FePO4 complexes are the main factors limiting the P availability for plants, particularly

in highly weathered soils (Lambers et al., 2008).

Native Australian plants and P nutrition

Many Australian native plants are well-adapted to low nutrient soils and most of them

have evolved mechanisms to capture more P from soil. Mycorrhizal associations and

cluster roots are the most important mechanisms making native plants very effective at

P uptake from low P soil (Handreck, 1997). However, many native species are now

sensitive to P fertilisation and develop phytotoxicity symptoms when exposed to over

moderate P supplies. According to Handreck (1997), an Olsen extractable P

concentration (inorganic plus organic P in the extract) of about 20 mg P kg-1

soil is

lethal to the seedlings of P-sensitive native species.

Groves and Keraitis (1976) showed that P concentrations of up to 100 mg P kg-1

soil

resulted in the mortality of seedlings of Acacia spp, Banksia spp and Eucalyptus spp.

Thomson and Leishman (2004) conducted a set of experiments to investigate the effect

of nutrient addition (various concentrations of NPK as liquid fertilizer) on the response

of some native species from low and high nutrient soils. Seedlings of both plant groups

showed a range of decline in survival after nutrient addition. However, species adapted

to high nutrient soils were less severely affected by nutrient addition. The same trend

was observed for six-month-old plants; species from high nutrient soils were more

tolerant than those adapted to low nutrient soils. Their results also suggested that

Page 23: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

6

seedlings are more sensitive to nutrient fertilization than more mature plants, as

previously shown in other field experiments (Specht, 1963; Heddle and Specht, 1975).

Native Proteaceae species such as Banksia ericifolia and B. grandis were shown to

develop symptoms associated with P toxicity when exposed to P fertilization (Ozanne

and Specht, 1981; Parks et al., 2000; Lambers et al., 2002). P toxicity in Proteaceae is

correlated with an inability to reduce P uptake from the rhizosphere (Shane et al.,

2003). Hakea prostrata, a typical member of the Proteaceae developed P toxicity

symptoms similar to those observed in other species at application rates of more than

100 µmol P d-1

(about 3 ppm) in a hydroponic system (Shane et al., 2004a).

Some heaths and sclerophyllous species are also adversely affected by P fertilization.

The negative effects observed include reduced survival of seedlings, observable

symptoms of P toxicity such as leaf chlorosis and necrosis, hastened life cycle and early

death (Specht, 1963; Specht and Grooves, 1966; Grundon, 1972; Heddle and Specht,

1975; Specht et al., 1977). Application of small quantities of P (2 cwt superphosphate

per acre) resulted in a great reduction in the survival rate of heath seedlings (Specht,

1963). P application rates of more than 11 g m-3

as monocalcium phosphate resulted in

P toxicity symptoms in two cultivars of Caustis blakei and caused a significant

decrease in yield (Gikaara et al., 2005). However, fertilization with rock phosphate

showed no toxicity symptoms or decline in shoot dry weight in the Caustis cultivars

tested (Gikaara et al., 2005). Therefore, the occurrence of P toxicity in plants can also

depend on the type of P fertilisation as various P forms differ in their solubility and P

release into soil (van Straaten 2002).

In conclusion, many Australian native plant species show various degrees of

sensitivity when exposed to high levels of P and the toxicity symptoms and severity

might be different depending on plant species, habitat, P source and growth conditions.

However, there is no literature available on how soil biological components (in

particular, mycorrhizas) behave in native plants exposed to P toxicity conditions. The

capacity to form dual mycorrhizal associations (AM and ECM) makes jarrah an ideal

native species to investigate the function of mycorrizal symbioses under P toxicity

conditions.

Australian native plant species and tolerance to P toxicity

Soil physiochemical and biological properties can play an important role in tolerance

to P toxicity. Two varieties of Caustis blankei grown under leaching conditions showed

reduced symptoms of P toxicity compared with those grown under non-leaching

Page 24: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

7

conditions (Gikaara et al., 2005). Status of other nutrients can also affect the P toxicity

severity. For example, increasing the levels of calcium (Ca) exacerbated the P toxicity

symptoms in Grevillea cv while addition of nitrogen N and potassium (K) reduced

toxicity (Nichols and Beardsell, 1981). Jarrah plants were more tolerant to P

fertilization when supplied with adequate levels of N (Dell et al., 1987). Adequate

supply of iron (Fe) reduced sensitivity to high levels of P in susceptible species

including Acacia spp, Banksia spp, Grevillea spp and Hakea spp (Nichols, 1988;

Goodwin, 1981; Handreck, 1991a; 1991b). Formation of insoluble P (FePO4) from

interaction of iron (Fe+3

) and P, could be a potential mechanism in this case (Brady and

Weil, 1996). The soil pH can also reduce the P availability for plants by making

insoluble complexes such as FePO4 and AlPO4 in acidic soils or CaPO4 in alkaline soils

(Lambers et al., 2008).

Jarrah plants in non-mycorrhizal growth have been shown to be very sensitive to high

levels of P, whereas AM jarrah plants develop less phytotoxicity symptoms (Tibbett,

unpublished). Mycorrhizal plants and non sensitive plants growing in high P conditions

can have reduced PHT1 gene expression and consequently their P uptake is lower,

suggesting that P tolerance in mycorrhizal plants might be correlated with lower

expression of plant PHT1 genes (Karandashov and Bucher, 2005). For example, the

PHT1 gene Mt4 was systemically down-regulated in Medicago truncatula by both P

fertilization and AM colonisation (Burleigh and Harrison, 1999). Also, two high-

affinity PHT1 genes including MtPT1 and MtPT2 were down-regulated following

colonisation of M. truncatula roots by the AM fungus Glomus versiforme (Liu et al.,

1998). In mycorrhizal tomato plants with sufficient P, the PHT1 gene LePT1 was

down-regulated in root hairs, epidermal cells and also non-colonized regions of roots

(Rosewarne et al., 1999). Colonisation of M. truncatula plants by some AM species

including G. mosseae, G. intraradices, G. versiforme, G. caledonium, G. claroideum

and Scutellospora calospora caused the down-regulation of the expression of the high-

affinity PHT1 gene MtPT2 (Burleigh, 2001). However, Gigaspora rosea failed to

reduce PHT1 gene expression in these experiments. In addition, of three AM species

tested, G. versiforme, G. mosseae and G. rosea, only G. mosseae could strongly reduce

the expression of the putative nitrate transporter AI974803. Taken together, many AM

species show the ability to reduce the expression of high-affinity PHT1 genes in order

to alter the P uptake pathway. Mycorrhizal plants take up P mainly via fungal hyphae

rather than plants roots.

Page 25: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

8

Phosphate transporters (PHT)

Phosphorus is a macronutrient that is essential for plant growth, because it is a

structural component of nucleic acids and phospholipids, and is involved in many

cellular functions such as energy transfer and the regulation of enzyme activity.

Phosphate (Pi) is the main form of P taken up by plant roots through membrane-

embedded Pi transporter proteins.

According to the current nomenclature, Pi transporters are classified into three

families called PHT1, PHT2, PHT3 and PHT4 (Bucher et al., 2001; Mudge et al.,

2002; Rausch and Bucher, 2002). The PHT1 family include the Pi transporters involved

in P uptake from the soil solution. Most PHT1 members are induced under P-deficiency

conditions. Most members of this family have high affinity for Pi uptake, and are able

to absorb low concentrations of P (Km 1-10 µm) (Clarkson, 1984). The PHT1 members

have been identified in a variety of plant species (Table 1). Members of the PHT2

family are expected to be mainly involved in the internal Pi transport within plant

tissues (chloroplast Pi transporters) and have been shown to have a low affinity for Pi

uptake that require higher concentrations of P to function (Km 100-1000 µm) such as

Pht2:1 in Arabidopsis (Daram et al., 1999). The PHT3 members are involved in

mitochondrial Pi transport such as ARAth;Pht3;1, ARAth;Pht3;2 and ARAth;Pht3;3 in

Arabidopsis thaliana (Rausch and Bucher, 2002). PHT4 members are thought to be

organellar Pi transporters, mostly plastid-localised and expressed in both leaves and

roots (Guo et al., 2008).

Page 26: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

9

Table. 1. List of plant species with identified PHT1 members

Plant species Reference

Arabidopsis thaliana Muchhal et al., 1996

Potato Leggewie et al., 1997; Rausch et al., 2001

Catharantus roseus Kai et al., 1997

Tomato Liu et al., 1998; Daram et al., 1998

Medicago truncatula Liu et al., 1998; Xiao et al., 2006

Barley Smith et al., 1999

Tobacco Baek et al., 2001

Lupinus albus Liu et al., 2001

Lotus Nakamori et al., 2002

Rice Paszkowski et al., 2002; Ming et al., 2005;

E. camaldulensis Koyama et al., 2006

Maize Nagy et al., 2006

Wheat Tittarelli et al., 2007

Populus trichocarpa Loth-Pereda et al., 2011

A subset of high affinity Pi transporters located in the plasmalemma of root

epidermal cells and root hair cells absorb the Pi directly from the soil solution and are

induced in response to Pi starvation. These include MtPT1 and MtPT2 in M. truncatula

(Chiou et al., 2001), StPT1 and StPT2 in potato (Rausch et al. 2001) and LePT1 in

tomato (Rosewarne et al., 1999). The expression of some of these PHT1 genes has been

shown to decrease following mycorrhizal colonisation or high Pi concentration in soil,

including MtPT2 and LePT1 (Liu et al., 1998; Burleigh and Harrison, 1999; Rosewarne

et al., 1999). The reduced expression of these PHT1 genes is due to the shift in Pi

uptake from a root pathway to a hyphal pathway in mycorrhizal plants or modification

of the Pi uptake in plants exposed to high Pi conditions.

Distinct subsets of high affinity Pi transporters are involved in the uptake of Pi

released by mycorrhizal hyphae within root cortical cells and are usually induced

following mycorrhizal colonisation. These include StPT3, StPT4 in potato (Rausch et

Page 27: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

10

al., 2001), MtPT4 in M. truncatula (Harrison et al., 2002; Javot et al., 2007),

HORvu;Pht1;8 (AY187023) in barley, TRIae;Pht1;myc in wheat (AJ830009) and

ZEAma;Pht1;6 (AJ830010) in maize (Glassop et al., 2005), LePT4 in tomato and

StPT4 in potato (Nagy et al., 2005), ORYsa;Pht1;11 and ORYsa;Pht1;13 in rice

(Glassop et al., 2007).

Therefore, the expression of PHT1 genes may be repressed or induced by

mycorrhizal colonisation depending on their location within the plant root and also

their involvement in direct (plant) or indirect (mycorrhizal) pathways of P uptake.

Taken together, the fact that some high affinity Pi transporters (PHT1) are repressed

following mycorrhizal colonisation suggests the possible correlation between the

expression of these genes and Pi tolerance in mycorrhizal jarrah plants.

Mycorrhiza and P uptake

Mycorrhiza is a mutualistic symbiosis between plant roots and some soil borne fungi.

Seven categories of mycorrhizas have been recognized based on the morphological

structures developed and the plant-fungal species involved. These include AM, ECM,

ectendomycorrhiza, orchid mycorrhiza, ericoid mycorrhiza, arbutoid mycorrhiza and

monotropoid mycorrhiza (Smith and Read, 2008). Dark septate endophytes

(Jumpponen, 2001) and Sebacinales (Weiß et al., 2011) can colonize plant tissues and

establish beneficial associations, which are debatably referred to as mycorrhiza or

endophytes.

AM fungi are the most ubiquitous mycorrhizal fungi forming symbiotic associations

with 80% of vascular plants (Smith and Read, 2008). In AM symbiosis, fungal hyphae

penetrate roots and grow between plant epidermal and cortex cells. Some intraradical

hyphae penetrate cortex cells and form tree-like structures called arbuscules, which are

thought to be sites for exchange of metabolites between plant and fungal partners.

Storage structures are also formed within or between root cells called vesicles.

Extraradical hyphae can explore the soil more than 10 cm beyond the rhizosphere (Li et

al., 1991) and transfer mineral nutrients to plants.

ECM fungi are the second most widespread form of mycorrhiza and have an intimate

association with many plant species particularly trees and shrubs belonging to

Betulaceae, Fagaceae, Myrtaceae and Pinaceae (Wilcox, 1996). In ECM symbiosis, the

root surface is covered by fungal sheath, often making rootlets short and stumpy.

Intraradical hyphae grow deep between cortex cells (hartig net) but do not penetrate

cortex cells. ECM fungi form an extensive extraradical hyphal network of up to several

Page 28: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

11

meters and nutrient transfer from up to 40 cm distance from root systems has been

documented (Finaly and Read, 1986).

However, plant-fungus associations with unconventional or undeveloped structures

have also been documented that cannot be classified into any of the above mycorrhizal

categories and are of ambiguous functional status (Warcup and McGee, 1983; Kope

and Warcup, 1986; Brundrett, 2009). A positive growth response has also been reported

for seedlings of E. camaldulensis inoculated with the ECM fungus Pisolithus

tinctorious without the formation of ECM structures (Neumann, 1959). The existing

literature has typically viewed the function of mycorrhizas with respect to the

specialized mycorrhizal structures and hyphal-mediated nutrient uptake, which might

not always reflect the functionally relevant factors of an association. Recent studies,

however, highlighted the role of fungi in biogenic weathering of soils in most forest

ecosystems including mineral dissolution and phosphate (Pi) solubilization, leading to

enhanced nutrient availability for ERH and ECM roots (Landeweert et al., 2001; Finlay

et al., 2009).

Some plant families including Euphorbiaceae, Myrtaceae, Pinaceae, Salicaceae,

Mimosaceae, Fagaceae and Caesalpinaceae are capable of forming symbiotic

associations with both AM and ECM fungi. Dual colonisation has been reported for

certain plant genera such as Pinus (Wagg et al., 2008), Chosenia and Salix (Hashimoto

and Higuchi, 2003), Eucalyptus (Gange et al., 2005), Acacia (Founoune et al., 2002),

Uapaca (Ramanankierana et al., 2007), Quercus (Dickie et al., 2002), Dicymbe

(McGuire et al., 2008), Kunzea (Moyersoen and Beever, 2004), and Leptospermum

(Moyersoen and Beever, 2004; Weijtmans et al., 2007). In these tripartite associations

the AM colonisation is usually replaced by ECM colonisation via several mechanisms

including mechanical barriers by the ECM sheath (Chilvers et al., 1987) and

competition for root carbohydrates (Lodge and Wentworth, 1990). However, limited

research has been carried out to clarify the details about AM/ECM interactions on the

same roots system.

Mycorrhizal symbiosis provide the host plant with several benefits such as enhancing

nutrient and water uptake (Hall et al., 1984; Allen et al., 1991; Marschner and Dell,

1994; Koske et al., 1995; Gemma et al., 1997; Koske et al., 1997), inducing resistance

against soil-inhabiting pathogens (Gianinazzi-Pearson and Gianinazzi, 1983;

Linderman, 1994; Newsham et al., 1995), tolerance against physiological stresses such

as drought and salinity (Ojala et al., 1983; Poss et al., 1985) and tolerance to heavy

Page 29: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

12

metal toxicity (Hildebrandt et al., 2007). However, increasing the P uptake is regarded

as one of the most significant benefits conferred by both AM and ECM fungi (Koide,

1991; Tibbett and Sanders, 2002; Torres Aquino and Plassard, 2004; Smith and Read,

2008).

The positive growth responses in ECM symbiosis are not necessarily correlated with

the formation of ECM structures on the roots. Neumann (1959) observed increased

growth of E. camaldulensis seedlings inoculated with the ECM fungus Pisolithus

tinctorious without formation of mycorrhizal structures and attributed the positive

growth response to the function of fungal hyphae in the rhizosphere soil. Positive

growth responses with partial, discrete patches, or no fungal sheath have been reported

for other ECM species including Peziza whitei, Laccaria ohiensis and the

ascomyceteous WARH 24 isolate in association with certain Asteraceae (Kope and

Warcup, 1986; Warcup and McGee, 1983).

Arbuscular mycorrhizal plants take up P both directly through the Pi transporters

within plant roots and indirectly via extraradical hyphae of mycorrhizal fungi (Smith et

al., 2003). The direct pathway of P uptake can be affected by mycorrhizal colonisation

and the concentration of available P for actively absorbing roots (Smith et al., 2003).

Smith et al. (2003, 2004) showed that there had been a misevaluation of the

contribution of mycorrhizal fungi to P uptake in host plants. Indeed, mycorrhiza-

mediated P uptake could be much higher than previously presumed. Their results

demonstrated that mycorrhizal fungi made a substantial contribution to P uptake in five

plant/fungus combinations. Flax and tomato plants inoculated with G. intraradices

were shown to absorb P exclusively (100 %) through the mycorrhizal pathway. Also,

medic plants inoculated with both G. intraradices and G. caledonium absorbed more

than 60 % of their P via the mycorrhizal pathway. The contribution of the mycorrhizal

pathway to P uptake depends on the plant/fungus combination and the ability of the

fungus to cause the induction of mycorrhiza-specific genes within the host plant, which

are involved in the acquisition of P released into the root cortex by fungal hyphae

(Karandashov and Bucher, 2005). Mycorrhiza-specific plant PHT1 genes such as

OsPT11 in rice, StPT3 in potato, MtPT4 in medic and LePT4 in tomato play a

significant role in the mycorrhizal pathway of P uptake (Paszkowski et al., 2002;

Rausch et al., 2001; Liu et al., 1998; Rosewarne et al., 1999). The extent of P uptake

was demonstrated not to be correlated with the presence or absence of a growth

response in the host (Smith et al., 2003, 2004); this has been the traditional measure of

Page 30: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

13

mycorrhizal ‘effectiveness’ and now must be re-evaluated as it does not necessarily

demonstrate a physiological response to mycorrhization.

ECM symbiosis also markedly contributes to P uptake via extraradical hyphae

exploring the soil beyond the P depletion zone caused by plant roots (Finlay and Read,

1986; Tibbett and Sanders, 2002; Torres Aquino and Plassard, 2004). However, there

has been no study focusing on the relationship between ECM colonisation and the

expression of PHT1 genes in ECM roots. That evaluation will be an important

extension of current understanding about the nutritional consequences of ECM

symbiosis to the host.

Phosphate analogues

Phosphate has several chemical analogues differing in oxidation states or the central

element including phosphite (Phi, HPO3-2

), arsenate (AsV, AsO4-3

), and vanadate (V

[V], VO4-3

) that are acquired by plants via Pi transporters (Guest and Grant, 1991;

Marschner, 1995; Finnegan and Chen 2012). Phosphite is regarded as a plant non-

metabolizable source of P and arsenate is the inorganic form of arsenic (As), both

possessing phytotoxic properties.

Phosphite chemistry and toxicity

Phosphite is a Pi analogue with the chemical formula of HPO3-2

that has been widely

used as a fungicide for controlling fungal pathogens in particular oomycetes mostly

with the generic name of fosetyl-Al or Aliette (Guest and Grant, 1991; McDonald et

al., 2001a). There are two main modes of action for antifungal activity of Phi including

inhibition of phosphorylation reactions (due to accumulation of inorganic

pyrophosphate) and disruption of the metabolic pathways regulated by Pi (Barchietto et

al., 1992; Niere et al., 1994, McDonald et al., 2001b).

From a plant perspective, Phi is generally considered to be a non-metabolizable form

of P as it cannot be assimilated into organic P compounds in plants (Guest and Grant,

1991). Therefore, application of Phi can result in development of phytotoxicity

reactions in plants (Varadarajan et al., 2002, Sukarno et al., 1993; 1996; 1998; Ticconi

et al., 2001). However, there are also reports showing positive nutritional effects of Phi

on plants (Jabahi-Hare and Kendrick, 1987; Lovatt and Mikkelsen, 2006), which are

most likely due to microbe-mediated oxidation of Phi to Pi in soil (Ohtake et al., 1996).

Page 31: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

14

Mycorrhiza and Phi toxicity

Mycorrhizal fungi respond differently upon exposure to Phi. Howard et al., (2000)

did not observe any negative effects on ECM colonisation of Eucalyptus globulus

seedlings by Pisolithus, Scleroderma and Descolea after foliar application of Phi at a

recommended rate of 5 g L-1

. However, doubling the concentration (10 g L-1

) reduced

the ECM colonisation by Descolea. Interestingly, they also reported a four-fold

increase in AM colonisation of Agonis flexuosa after the Phi treatment. Similarly, an

increase in AM colonisation has been reported for leek (Jabahi-Hare and Kendrick,

1987) and lettuce (Clarke, 1978) after foliar application of fosetyl-Al. However, there

are some other reports showing that Phi application decreased AM colonisation in

certain plants such as maize (Seymour et al., 1994) and onion (Sukarno, 1996, 1998).

The negative effects of Phi on mycorrhizal colonisation have been attributed to

accumulation of Phi near root tips (Guest and Grant, 1991) resulting in a damage to

fine roots and consequently reduction of sites for AM and ECM formation (Howard et

al., 2000).

Similar to Pi, many Australian native plants develop toxicity symptoms upon

exposure to high doses of Phi. Barrett (2001) observed the development of toxicity in

plant species from Anarthriaceae, Epacridaceae, Myrtaceae, Papillionaceae and

Proteaceae after spraying Phi at rates from 24 to 144 kg ha-1

. The recommended rate of

5 g L-1

caused mild (less than 25%) damage to shoot of most species in jarrah forest

(Tynan et al., 2001). Howard et al. (2000) observed higher phytotoxicity rate in jarrah

than in E. globulus and A. flexuosa following foliar application of 7.5 and 10 g L-1

Phi.

However, there is not enough information about the function of mycorrhiza and their

potential role in inducing tolerance to native species including jarrah against Phi

toxicity.

Arsenate chemistry and toxicity

Arsenic (As) is a poisonous metalloid belonging to group V of the periodic table and

considered to be a hazardous environmental contaminant with a high carcinogenic

potential. The presence of As in the environment leads to remarkable loss of

biodiversity in affected ecosystems. It is also a serious direct threat to human health, as

people can intake different arsenic species by consumption of contaminated

groundwater and agricultural products (Hadi and Parveen, 2004). Particularly, it has

harmful effects on those organisms directly related to human health, such as plants,

fishes and domestic animals. Arsenic is the cause of many human diseases, such as

Page 32: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

15

fatal cancers (lung, liver and kidney), cirrhosis, gangrene and also the cause of

arsenicosis, which is responsible for thousands of deaths each year (Hadi and Parveen,

2004).

Arsenate and arsenite (AsIII) are the most common oxidation states of As found in

nature. Contamination of groundwater and soil by As compounds is a result of natural

processes such as eruption of volcanoes and erosion of mineral deposits or

anthropogenic activities such as mining, agriculture (application of arsenical pesticides

and herbicides), forestry, drilling and so on (Smith et al., 2003). Arsenate is a Pi

analogue taken into plants via the Pi uptake system. In the cell, As (V) can severely

disrupt vital functions, including the energy production pathway by substituting for P in

the production of ATP, forming ADP-As complex that uncouples the ATP synthesis

pathway (Meharg and Hartley-Whitaker, 2002; Finnegan and Chen, 2012).

Mycorrhiza and AsV toxicity

Compared to chemical methods, biological approaches (bioremediation) are better

options for cleaning up environmental pollutants such as As in sense of both cost-

effectiveness and environmental concerns. Soil-dwelling microorganisms can function

as a key tool to avoid contamination of the food chain by various strategies such as

immobilization, transformation and volatilization of arsenic species in polluted soils.

Arsenate reduction is a widespread strategy to deal with AsV toxicity in

microorganisms such as bacteria (Cervantes et al., 1994), saprophytic fungi such as

Aspergillus and Saccharomyces (Wysocki et al., 2001; Canovas et el., 2003) and

mycorrhizal fungi (Sharples et al., 2000) in which the AsV is reduced to AsIII after

uptake, then the produced AsIII is exported back to soil (Vetterlein et al., 2007). AsIII

is more reactive and more toxic than AsV. The production of the more reactive AsIII

form from AsV means that upon export AsIII will react with soil constituents, probably

with a reduced sulfur group like a thiol, and become unavailable for further uptake. For

example, the ericoid mycorrhizal fungus Hymenoscyphus ericae was shown to reduce

AsV to AsIII inside the fungal cells and then efflux the AsIII into the rhizosphere soil

(Sharples et al., 2000).

Inoculation of Glomus aggregatum onto sunflower (Helianthus annus) alleviated As

toxicity and reduced the As concentrations in the roots and shoots of inoculated plants

(Ultra, 2006). The protective effects were attributed to improved P nutrition of plants

and the AM-mediated transformation of soil inorganic As to less toxic organic forms.

Gonzalez-Chavez et al. (2002) showed that AM symbiosis reduced the As

Page 33: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

16

concentration in shoots of Holcus lanatus through suppression of a plant high-affinity

Pi/AsV transporter. Ahmed et al. (2006) studied the effects of Glomus mosseae on

growth and nutrition of lentil plants irrigated with As-contaminated water. The AM

symbiosis increased plant height, leaf and pod numbers, plant biomass and P content of

shoots and roots and also reduced the As content in plant tissues. Chen et al. (2007)

investigated the effects of G. mosseae on P and As acquisition by Medicago sativa.

Their results showed that mycorrhizal inoculation dramatically increased the dry

weight of plants and also substantially increased the total uptake of P and As. However,

As concentration of shoots were significantly lower than un-inoculated plants because

of dilution effects caused by increased growth of mycorrhizal plants. Taken together,

all the mentioned studies demonstrate that AM fungi could be an important biological

component in bioremediation of As contaminated soils and could reduce the As

concentration in plants tissues.

However, AM fungi were shown to increase metal uptake in some host plants and

therefore might also have a good potential for a phytoextraction strategy. Colonisation

of plants by four AM species including G. intraradices, G. mosseae, G. etunicatum and

Gigaspora gigantean increased the uptake of different heavy metals, such as As,

cadmium (Cd), zinc (Zn), lead (Pb) and selenium (Se) in certain tolerant grass species

(Giasson et al., 2006). G. intraradices showed the highest capacity for heavy metal

uptake in these experiments. The grass species used in this study were a mixture of

Festuca rubra, F. eliator, Agropyron repens and Trifolium repens, all of which are used

for re-vegetation of metal contaminated soils.

In conclusion, AM symbioses might decrease or increase the heavy metal uptake

depending on the sensitivity of the host plant. The function of mycorrhizal association

under heavy metal toxicity conditions seems to be more complicated and depends on

the types of plants being investigated, the AM isolates and the experimental conditions.

AM fungi can reduce the As concentration of plant tissues in As-sensitive plants, which

is particularly important in crop plants with nutritional values. Along with AM

activities in chemical transformation of As molecules or affecting transporter genes, the

dilution effect seems to be a common factor in AM symbiosies leading to lower As

concentration in shoot tissues. As a phytoextraction strategy, AM fungi can increase the

As uptake in plants with a relatively high level of tolerance to heavy metals (Giasson et

al., 2006).

This review of the literature concludes that mycorrhiza can play a substantial role in

plant growth and nutrition. There are unconventional ECM associations with

Page 34: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

17

undeveloped structures where positive growth responses of the host plant are not

correlated with the extent of root colonisation. Mycorrhizas can also function in favour

of plants exposed to toxicity of Pi and its analogues. The induced tolerance could be

due to certain mechanisms such as the reduced expression of PHT1 genes in plant roots

or the dilution effect resulting from higher plant biomass in mycorrhizal plants.

Phosphate transporter genes have been identified in many plant species but there is no

literature available about the PHT1 sequences in jarrah. The outcome of this research

will contribute to better understanding of P nutrition of mycorrhizal and non-

mycorrhizal jarrah seedlings growing under P deficiency and P toxicity conditions.

There will be three hypotheses tested in this thesis regarding the Pi sensitivity and

tolerance in jarrah: i) non-mycorrhizal jarrah plants cannot reduce their Pi uptake under

Pi toxicity conditions, ii) perhaps with mycorrhizal associations jarrah plants can

tolerate high concentrations of Pi in soil and iii) the mechanisms for these traits might

involve regulated or unregulated expression of PHT1 genes.

This PhD thesis aims to i) investigate AM, ECM and dual (AM & ECM) mycorrhizal

associations of jarrah and their potential effects on jarrah growth and nutrient

acquisition under P-deficient conditions, ii) clarify the unconventional symbiotic

association between jarrah and A. occidentalis that was identified in the early stages of

this study, and investigate the underlying mechanisms that result in the host benefit of

this association , and iii) study the role of selected mycorrhizal species in inducing

tolerance against toxic pulses of Pi (PO4-3

), Phi (HPO3-2

) and AsV (AsO4-3

) in jarrah

and reveal any possible correlation with expression of jarrah PHT1 genes.

Literature Cited

Adams MA, Iser J, Keleher AD, Cheal DC (1994) Nitrogen and phosphorus availability

and the role of fire in heathlands at Wilson Promontory. Aust J Bot 42: 269-281

Ahmed FRS, Killham K, Alexander I (2006) Influences of arbuscular mycorrhizal

fungus Glomus mosseae on growth and nutrition of lentil irrigated with arsenic

contaminated water. Plant Soil 283: 33-41

Allen MF (1991) The ecology of mycorrhizae. Cambridge University Press, Cambridge

Attiwill PM, Leeper GW (1987) Forest soils and nutrient cycles. Melbourne University

Press, Melbourne

Baek SH, Chung IM, Yun SJ (2001) Molecular cloning and characterization of a

tobacco leaf cDNA encoding a phosphate transporter. Mol Cells 11: 1–6

Page 35: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

18

Barchietto T, Saindrenan P, Bompeix G (1992) Physiological responses of

Phytophthora citrophthora to a sub-inhibitory concentration of phosphonate.

Pestic Biochem Physiol 42: 151–166

Barrett S (2001) Phytotoxic effects of phosphite on native plant communities in

southern Western Australia. PhD Thesis, Murdoch University

Beadle NCW (1962) An alternative hypothesis to account for the generally low

phosphate content of Australian soils. Aust J Agric Res 13: 434-442

Brady NC, Weil RR (1996) The nature and properties of soils. Prentice Hall

International, London

Brundrett MC, Abbott LK (1991) Roots of jarrah forest plants.1. Mycorrhizal

associations of shrubs and herbaceous plants. Aust J Bot 39: 445-457

Brundrett MC (2009) Mycorrhizal associations and other means of nutrition of vascular

plants: understanding the global diversity of host plants by resolving conflicting

information and developing reliable means of diagnosis. Plant Soil 320: 37-77

Bucher M, Rausch C, Daram P (2001) Molecular and biochemical mechanisms of

phosphorus uptake into plants. J Plant Nutr and Soil Sci 164: 209-217

Burleigh SH, Harrison MJ (1999) The down-regulation of Mt4-like genes by phosphate

fertilization occurs systemically and involves phosphate translocation to the

shoots. Plant Physiol 119: 241–248

Burleigh SH (2001) Relative quantitative RT-PCR to study the expression of plant

nutrient transporters in arbuscular mycorrhizas. Plant Sci 160: 899–904

Canovas D, Duran C, Rodriguez N, Amils R, de Lorenz V (2003) Testing the limits of

biological tolerance to arsenic in a fungus isolated from the River Tinto.

Environ Microbiol 5: 133-138

Cervantes CJiG, Ramirez JL, Silver S (1994) Resistance to arsenic compounds in

microorganisms. FEMS Microbiol Rev 15: 355-367

Chen BD, Xiao XY, Zhu YG, Smith FA, Xie ZM, Smith SE (2007) The arbuscular

mycorrhizal fungus Glomus mosseae gives contradictory effects on phosphorus

and arsenic acquisition by Medicago sativa Linn. Sci Total Environ 379: 226–

234

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

Clarkson DT (1984) Ionic relations. In Wilkins MB ed Advanced Plant Physiology:

Pitman Publishing Ltd, London, UK, 319-353

Clarke CA (1978) Effects of pesticides on VA mycorrhizae. Rothamsted Experimental

Station Report for 1978, Part 1: 236-237

Page 36: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

19

Daram P, Brunner S, Persson BL, Amrhein N, Bucher M (1998) Functional analysis

and cell-specific expression of a phosphate transporter from tomato. Planta 206:

225-233

Daram P, Brunner S, Rausch C, Steiner C, Amrhein N, Bucher M (1999) Pht2;1

encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell 11:

2153–2166

Dell B, Hardy GESJ, Vear K (2005) History of Phytophthora cinnamomi management

in Western Australia In MC Calver, H Bigler-Cole, G Bolton, J Dargavel, A

Gaynor, P Horwitz, J Mills, G Wardell-Johnson, Eds, A forest consciousness:

Proceedings of the 6th

National Conference of the Australian Forest History

Society. Millpress Science Publishers, Rotterdam, 391-406

Dell B, Jones S, Wilson SA (1987) Phosphorus nutrition of jarrah (Eucalyptus

marginata) seedlings. Plant Soil 97: 369-379

Dickie IA, Koide RT, Steiner KC (2002) Influence of established trees on mycorrhizas,

nutrition, and growth of Quercus rubra seedlings. Ecol Monographs 74: 505–

521

Feng M, Qun L, Wei W, Shanshan Z, Bin G, Daleng S (2006) Cloning, expression and

function of phosphate transporter encoded gene in Oryza sativa L. Science in

China Series C: Life Sciences, 49: 409-413

Finlay RD, Read DJ (1986) The structure and function of the vegetative mycelium of

ectomycorrhizal plants. II. The uptake and distribution of phosphorus by

mycelial strands interconnecting host plants. New Phytol 103: 157–165

Finlay RD, Wallander H, Smits M, Holmström S, van Hees PAW, Lian B, Rosling A

(2009) The role of fungi in biogenic weathering in boreal forest soil. Fungal Biol

Rev 23: 101-106.

Finnegan PM, Chen W (2012) Arsenic toxicity: the effects on plant metabolism. Front

Physiol 3: 182

Founoune H, Duponnois R, Bâ AM, Sall S, Branger I, Lorquin J, Neyra M, Chotte JL

(2002) Mycorrhiza helper bacteria stimulate ectomycorrhizal symbiosis of

Acacia holosericea with Psolithus alba. New Phytol 153: 1–9

Gange AC, Gane DRJ, Chen Y, Gong M (2005) Dual colonisation of Eucalyptus

urophylla S.T. Blake by arbuscular and ectomycorrhizal fungi affects levels of

insect herbivore attack. Agric Forest Entomol 7: 253–263

Gemma JN, Koske RE, Roberts EM, Jackson N, De Antonis K (1997) Mycorrhizal

fungi improve drought resistance in creeping bentgrass. J Turfgrass Sci 73: 15-

29

Gianinazzi-Pearson V, and Gianinazzi S (1983) The physiology of vesicular-arbuscular

mycorrhizal roots. Plant Soil 71: 197-209

Page 37: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

20

Giasson P, Jaouich A, Cayer P, Gagné S, Moutoglis P, Massicotte L (2006) Enhanced

phytoremediation: a study of mycorrhizoremediation of heavy metal-

contaminated soil. Remediation 17: 97-110

Gikaara DM, Johnston ME, Edwards DG (2005) Phosphorus management of

Australian native plants. Acta Hort (ISHS) 683: 133-140

Glassop D, Godwin RM, Smith SE, Smith FW (2007) Rice phosphate transporters

associated with phosphate uptake in rice colonized with arbuscular mycorrhizal

fungi. Can J Bot 85: 644-651

Glassop D, Smith SE, Smith FW (2005) Cereal phosphate transporters associated with

the mycorrhizal pathway of phosphate uptake into roots. Planta 222: 688-698

Gonzalez-Chavez C, Harris PJ, Dodd J, Meharg AA (2002) Arbscular mycorrhizal

fungi confer enhanced resistance on Holcus lanatus. New Phytol 155: 163-171

Goodwin PB (1981) Nitrogen, phosphorus, potassium and iron nutrition of Australian

native plants. Unpublished Paper, Department of Agronomy and Horticultural

Science, University of Sydney

Groves RH, Keraitis K (1976) Survival of seedlings of three sclerophyll species at high

levels of phosphorus and nitrogen. Aust J Bot 24: 681-690

Grundon NJ (1972) Mineral nutrition of some Queensland heath plants. J Ecol 60: 171-

181

Guest D, Grant BR (1991) The complex action of phosphonates as antifungal agents.

Biol Rev 66: 159–187

Guo B, Irigoyen S, Fowler TB, Versaw WK (2008) Differential expression and

phylogenetic analysis suggest specialization of plastid-localized members of the

PHT4 phosphate transporter family for photosynthetic and heterotrophic tissues.

Plant Signal Behav 3: 784–790

Hadi A, Parveen R (2004) Arsenicosis in Bangladesh: prevalence and socio-economic

correlates. Pub Health 118: 559–564

Hall IR, Johnstone PD, Dolby R (1984) Interactions between endomycorrhizas and soil

nitrogen and phosphorus on the growth of ryegrass. New Phytol 97: 447-453

Handreck KA (1991a) Available phosphorus in potting media: Extractants and

interpretationof extract data. Commun Soil Sci Plant Anal 22: 529-557

Handreck KA (1991b) Interactions between iron and phosphorus in the nutrition of

Banksia ericifolia L. f. var. ericifolia (Proteaceae) in soil-less potting media.

Aust J Bot 39: 373-384

Handreck KA (1997). Phosphorous requirements of Australian native plants. Aust J

Soil Res 35: 241-289

Page 38: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

21

Harrison MJ, Dewbre GR Liu J (2002) A phosphate transporter from Medicago

truncatula involved in the acquisition of phosphate released by arbuscular

mycorrhizal fungi. Plant Cell 14: 2413–2429

Hashimoto Y, Higuchi R (2003) Ectomycorrhizal and arbuscular mycorrhizal

colonisation of two species of floodplain willows. Mycosci 44: 339–343

Hawkins HJ, Hettasch H, Mesjasz-Przybylowicz J, Przybylowicz W, Craner MD

(2008) Phosphorus toxicity in the Proteaceae: A problem in post-agricultural

lands. Sci Hort 117: 354–365

Heddle EM Specht RL (1975) Dark Island Heath (Ninety-Mile Plain, South Australia)

VIII. The effect of fertilizers on composition and growth, 1950-1972. Aust J

Bot 23: 151-164

Hildebrandt U, Regvar M, Bothe H (2007) Arbuscular mycorrhiza and heavy metal

tolerance. Phytochem 68: 139–146

Howard K, Dell B, Hardy GE (2000) Phosphite and mycorrhizal formation in seedlings

of three Australian Myrtaceae. Aust J Bot 48: 725-729

Jabahi-Hare SH, Kendrick WB (1987) Response of an endomycorrhizal fungus in

Allium porrum L. to different concentrations of the systemic fungicides

metalaxyl (Ridomil) and fosetyl-Al (Aliette). Soil Biol Biochem 19: 95–99

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

truncatula phosphate transporter indispensable for the arbuscular mycorrhizal

symbiosis. Proc Natl Acad Sci U S A 104: 1720-1725

Jumpponen A (2001) Dark septate endophytes – are they mycorrhizal? Mycorrhiza 11:

207-211.

Kai M, Masuda Y, Kikuchi Y, Osaki M, Tadano T (1997) Isolation and

characterization of a cDNA from Catharanthus roseus which is highly

homologous with phosphate transporter. Soil Sci Plant Nut 43: 227-235

Karandashov V, Bucher M (2005) Symbiotic phosphate transport in arbuscular

mycorrhizas. Trends Plant Sci 10: 22-29

Koide RT (1991) Nutrient supply, nutrient demand and plant response to mycorrhizal

infection. New Phytol 117: 365-386

Kope HH, Warcup JH (1986) Synthesised ectomycorrhizal associations of some

Australian herbs and shrubs. New Phytol 104: 591–599

Koske RE, Gemma JN, Jackson N (1997) A preliminary survey of mycorrhizal fungi in

putting greens. J Turfgrass Sci 73: 2-8

Koske RE, Gemma JN, Jackson N (1995) Mycorrhizal fungi benefit putting greens.

USGA Green Sec Rec 33: 12-14

Page 39: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

22

Koyama T, Kato N, Hibino T, Kawazu T, Kimura T, Sakka K (2006) Isolation and

expression analysis of phosphate transporter genes from Eucalyptus

camaldulensis. Plant Biotech 23: 215-218

Lambers H, Finnegan, PM, Laliberté E, Pearse SJ, Ryan MH, Shane MW, Veneklaas

EJ (2011) Phosphorus nutrition of Proteaceae in severely phosphorus-

impoverished soils: are there lessons to be learned for future crops? Plant

Physiol 156: 1058-1066

Lambers H, Raven JA, Shaver GR, Smith SE (2008) Plant nutrient-acquisition

strategies change with soil age. Trends Ecol Evol 23: 95–103

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, Juniper D, Cawthray GR, Veneklass EG, Martinez-Ferri E (2002) The

pattern of carboxylate exudation in Banksia grandis (Proteaceae) is affected by

the form of phosphate added to the soil. Plant Soil 238: 11-122

Landeweert R, Hofflund E, Finlay RD, van Breemen N (2001) Linking plants to rocks:

Ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol 16:

248-254.

Leggewie G, Willmitzer L, Riesmeier JW (1997) Two cDNAs from potato are able to

complement a phosphate uptake-deficient yeast mutant: Identification of

phosphate transporters from higher plants. Plant Cell 9: 381-392

Li XL, George E, Marschner H (1991) Extension of the phosphorus depletion zone in

VA-mycorrhizal white clover in calcareous soil. Plant Soil 136: 41-48

Linderman RG (1994) Role of VAM fungi in biocontrol. In: Mycorrhizae and Plant

Health. Pfleger FL, Linderman RG, cds., APS Press, St. Paul, MN, 1-26

Liu H, Trieu AT, Blaylock LA, Harrison MJ (1998) Cloning and characterization of

two phosphate transporters from Medicago truncatula roots: Regulation in

response to phosphate and to colonisation by arbuscular mycorrhizal (AM)

fungi. Mol Plant Microbe Interact 11: 14-22

Liu J, Uhde-Stone C, Li A, Vance C, Allan D (2001). A phosphate transporter with

enhanced expression on proteoid roots of white lupin (Lupinus albus L.). Plant

Soil 237: 257–266

Lodge DJ, Wentworth TR (1990) Negative associations among VA-mycorrhizal fungi

and some ectomycorrizal fungi inhabiting the same root system. Oikos 57: 347-

356

Loth-Pereda V, Orsini E, Courty PE, Lota F, Kohler A, Diss L, Blaudez D, Chalot M,

Nehls U, Bucher M, Martin F (2011). Structure and expression profile of the

phosphate Pht1 transporter gene family in mycorrhizal Populus trichocarpa.

Plant Physiol 156: 2141–2154

Page 40: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

23

Lovatt CJ, Mikkelsen RL (2006) Phosphite fertilizers: What are they? Can you use

them? What can they do? Better Crops 90: 11-13

Lu YP, Zhen RG Rea P (1997) AtPT4: A fourth member of the Arabidopsis phosphate

transporter gene family. Plant Physiol 114: 747

Marschner H, Dell B (1994) Nutrient uptake in mycorrhizal symbiosis. Plant Soil 159:

89-102

Marschner H (1995) Mineral nutrition of higher plants, Ed 2. Academic Press, San

Diego

Meharg AA, Hartley-Whitaker J (2002) Arsenic uptake and metabolism in arsenic

resistant and non-resistant plant species. Tansley Review. New Phytol 154: 29-

43

Meharg AA, Macnair MR (1990) An altered phosphate uptake system in arsenate

tolerant Holcus lanatus. New Phytol 116: 29–35

Meharg AA, Macnair MR (1992) Suppression of the high affinity phosphate uptake

system: a mechanism of arsenate tolerance in Holcus lanatus L. J Exp Bot 43:

519–524

McDonald AE, Grant BR, Plaxton WC (2001a) Phosphite (phosphorous acid): its

relevance in the environment and agriculture and influence on plant phosphate

starvation response. J Plant Nutr 24: 1505–1519

McDonald AE, Niere JO, Plaxton WC (2001b) Phosphite disrupts the acclimation of

Saccharomyces cerevisiae to phosphate starvation. Can J Microbiol 47: 969–

978

McGuire KL, Henkel TW, De La Cerda I (2008) Dual mycorrhizal colonisation of

forest-dominating troPcal trees and the mycorrhizal status of non-dominant tree

and liana species. Mycorrhiza 18: 217-22

Ming F, Mi GH, Lu Q, Yin S, Zhang SS, Guo B, Shen DL (2005) Cloning and

characterization of cDNA for the Oryza sativa phosphate transporter. Cell Mol

Biol Lett 10: 401–411

Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S, Shibata D (1997)

Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter

gene in tobacco cultured cells enhances cell growth under phosphate-limited

conditions. Proc Natl Acad Sci U S A 94: 7098–7102

Mosse B (1973) Plant growth responses to vesicular arbuscular mycorrhiza. IV. In soil

given additional phosphate. New Phytol 72: 127-136.

Moyersoen B, Beever RE (2004) Abundance and characteristics of Pisolithus

ectomycorrhizas in New Zealand geothermal areas. Mycolgia 96: 1225-1232

Muchhal US, Pardo JM, Raghothama KG (1996) Phosphate transporters from the

higher plant Arabidopsis thaliana. Proc Natl Acad Sci U S A 93: 10519-10523

Page 41: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

24

Mudge SR, Rae AL, Diatloff E, Smith FW (2002) Expression analysis suggests novel

roles for members of the Pht1 family of phosphate transporters in Arabidopsis.

Plant J 31: 341-353

Nagy R, Karandashov V, Chague V, Kalinkevich K, Tamasloukht M, Xu G, Jakobsen

I, Levy AA, Amrhein N, Bucher M (2005) The characterization of novel

mycorrhiza-specific phosphate transporters from Lycopersicon esculentum and

Solanum tuberosum uncovers functional redundancy in symbiotic phosphate

transport in solanaceous species. Plant J 32: 236-250

Nagy R, Vasconcelos MJ, Zhao S, McElver J, Bruce W, Amrhein N, Raghothama KG,

Bucher M (2006) Differential regulation of five Pht1 phosphate transporters

from maize (Zea mays L.). Plant Biol 8: 186-197

Neumann R (1959) Relationships between Pisolithus tinctorius (Mich. ex. Pers) Coker

et Couch. and Eucalyptus camaldulensis Dehn. Bull. Res. Counc. Zsr. Sect. D

Bot 7: 116-120

Nichols DG, Beardsell DV (1981) Interactions of calcium, nitrogen and potassium with

phosphorus on the symptoms of toxicity in Grevillea cv. Poorinda Firebird.

Plant Soil 61: 437-445.

Niere JO, DeAngelis G, Grant BR (1994) The effect of phosphonate on the acid-soluble

phosphorus components in the genus Phytophthora. Microbiol 140:1661–1670

Newsham KK, Fitter AH, Watkinson AR (1995) Arbuscular mycorrhiza protect an

annual grass from root pathogenic fungi in the field. J Ecol 83: 991-1000

Nichols DG (1988) Nutrition and fertilizer materials. In: Potting Mixes. Australian

Institute of Horticulture, NSW Council, July, 16-30

Nakamori K, Takabatake R, Umehara Y, Kouchi H, Izui K, Hata S (2002). Cloning,

functional expression, and mutational analysis of a cDNA for Lotus japonicus

mitochondrial phosphate transporter. Plant Cell Physiol 43: 1250–1253

Ohtake H, Wu H, Imazu K, Anbe Y, Kato J, Kuroda A (1996) Bacterial phosphonate

degradation, phosphite oxidation and polyphosphate accumulation. Res Cons

Recy 18: 125–134

Ojala JC, Jarrell WM, Menge JA, Johnson LV (1983) Influence of mycorrhizal fungi

on the mineral nutrition and yield of onion in saline soils. Agron J 75: 255–259

Okumura S, Mitsukawa N, Shirano Y, Shibata D (1998) Phosphate transporter gene

family of Arabidopsis thaliana. DNA Res 5: 261-269

Ozanne PG, Specht RL (1981) Mineral nutrition of heathlands: Phosphorus toxicity. In:

Ecosystems of the World, Vol 9A. Heathlands and related shrublands.

Descriptive studies. Ed. R. L. Specht 209-213, Elsevier, Amsterdam, The

Netherlands

Page 42: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

25

Parks SE, Haigh AM, Creswell GC (2000) Stem tissue phosphorus as an index of the

phosphorus status of Banksia ericifolia L. Plant Soil 227: 59-65

Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters

include an evolutionarily divergent gene specifically activated in arbuscular

mycorrhizal symbiosis. Proc Natl Acad Sci U S A 99: 13324-13329

Poss, J.A., Pond, E.C., Menge J.A. and Jarell, W.M. (1985) Effect of salinity on

mycorrhizal onion and tomato in soil with and without additional phosphate.

Plant Soil 88: 307-319

Raison RJ, Khanna PK, Woods PV (1985) Transfer of elements to the atmosphere

during low-intensity prescribed fires in three Australian subalpine eucalypt

forest. Can J For Res 15: 657-664

Ramanankierana N, Ducousso M, Rakotoarimanga N, Prin Y, Thioulouse J,

Randrianjohany E, Ramaroson L, Kisa M, Galiana A, Duponnois R (2007)

Arbuscular mycorrhizas and ectomycorrhizas of Uapaca bojeri L.

(Euphorbiaceae): sporophore diversity, patterns of root colonisation, and effects

on seedling growth and soil microbial catabolic diversity. Mycorrhiza 17: 195-

208

Rausch C, Bucher M (2002) Molecular and biochemical mechanisms of phosphate

transport in plants. Planta 216: 23-37

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

Richardson AE, Hocking PJ, Simpson RJ, George TS (2009) Plant mechanisms to

optimise access to soil phosphorus. Crop Past Sci 60: 124–143

Rosewarne G, Barker S, Smith S, Smith F, Schachtman D (1999) A Lycopersicon

esculentum phosphate transporter (LePT1) involved in phosphorus uptake from

a vesicular-arbuscular mycorrhizal fungus. New Phytol 144: 507-516

Seymour NP, Thompson JP, Fiske ML (1994) Phytotoxicity of fosetyl Al and

phosphonic acid to maize during production of vesicular-arbuscular mycorrhizal

inoculum. Plant Dis 78: 441–446

Shane MW, De Vos M, De Roock S, Cawthray GR, Lambers H (2003) Effect of

external phosphorus supply on internal phosphorus concentration and the

initiation, growth and exudation of cluster roots in Hakea prostrata R. Br. Plant

Soil 248: 209–219

Shane MW, McCully ME, Lambers H (2004a) Tissue and cellular phosphorus storage

during development of phosphorus toxicity in Hakea prostrata (Proteaceae). J

Exp Bot 55: 1033–1044

Shane MW, Szota C, Lambers H (2004b) A root trait accounting for the extreme

phosphorus sensitivity of Hakea prostrata (Proteaceae) from a biodiversity

hotspot. Plant Cell Environ 27: 991–1004

Page 43: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

26

Sharples JM, Meharg AA, Chambers SM, Cairney JWG (2000) Mechanism of

Arsenate Resistance in the Ericoid Mycorrhizal Fungus Hymenoscyphus ericae.

Plant Physiol 124: 1327-1334

Smith FW, Ealing PM, Dong B, Delhaize E (1997) The cloning of two Arabidopsis

genes belonging to a phosphate transporter family. Plant J 11: 83-92

Smith FW, Cybinski DH, Rae AL (1999). Regulations of expression of genes encoding

phosphate transporters in barley roots. Dordrecht: Kluwer Academic Publishers.

Smith E, Smith J, Biswas T, Correll R, Naidu R (2003) Arsenic in Australian

environment: an overview. J Environ Sci Health, Part A 38:223-239

Smith SE, Smith FA, Jakobsen I (2003) Mycorrhizal fungi can dominate phosphate

supply to plants irrespective of growth responses. Plant Physiol 133: 16-20

Smith SE, Smith FA, Jakobsen I (2004). Functional diversity in arbuscular mycorrhizal

(AM) symbiosis: the contribution of the mycorrhizal P uptake pathway is not

correlated with mycorrhizal responses in growth or total P uptake. New Phytol

162: 511-524

Smith SE, Read DJ (2008) Mycorrhizal symbiosis. 3rd

ed Academic Press, London UK

Specht RL (1963) Dark island heath (Ninety-Mile Plain, South Australia) VII. The

effect of fertilizers on composition and growth, 1950-60. Aust J Bot 11: 67-94

Specht RL, Groves RH (1966) A comparison of the phosphorus nutrition of Australian

heath plants and introduced economic plants. Aust J Bot 14: 201-221

Specht RL (1975) The effects of fertilizers on sclerophyll (heath) vegetation-the

problems of revegetation after sand mining of high dunes. Search 6: 459-461

Specht RL, Connor DJ, Cliford HT (1977) The heath-savanna problem: the effect of

fertilizer on sand-heath vegetation of North Stradbroke Island, Queensland.

Aust J Ecol 2: 179-186

Specht RL (1981a) Nutrient release from decomposing leaf litter of Banksia ornata,

Dark Island heathland, South Australia. Aust J Ecol 6: 59-63

Specht RL (1981b) The water relations of heathlands: Morphological adaptions to

drought. In: Ecosystems of the World, Vol 9B. Heathlands and related

shrublands. Analytical Studies. Ed. R. L. Specht, 123-129, Elsevier Scientific,

Amsterdam, The Netherlands

Sukarno N, Smith SE, Scott ES (1993) The effect of fungicides on vesicular- arbuscular

mycorrhizal symbiosis: I. The effects on vesicular-arbuscular mycorrhizal fungi

and plant growth. New Phytol 25: 139–147

Sukarno N, Smith FA, Smith SE, Scott ES (1996) The effects of fungicides on

vesicular-arbuscular mycorrhizal symbiosis. II. The effects on area of interface

and efficiency of P uptake and transfer to plant. New Phytol 132: 583–592

Page 44: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

27

Sukarno N, Smith FA, Scott ES, Jones GP, Smith SE (1998) The effect of fungicides

on vesicular-arbuscular mycorrhizal symbiosis. III. The influence of VA

mycorrhiza on phytotoxic effects following application of fosetyl-Al and

phosphonate. New Phytol 139: 321–330

Tibbett M, Sanders FE (2002) Ectomycorrhizal symbiosis can enhance plant nutrition

through improved access to discrete organic nutrient patches of high resource

quality. Ann Bot 89: 783-789

Ticconi CA, Delatorre CA, Abel S (2001) Attenuation of phosphate starvation

responses by phosphite in Arabidopsis. Plant Physiol 127: 963–972

Thompson CH, Moore AW, Northcote KH (1983) Soils and land use. In: Soils: An

Australian viewpoint. Division of Soils, CSIRO, Melbourne, pp 757-776

Thomson VP, Leishman MR (2004) Survival of native plants of Hawkesbury

Sandstone communities with additional nutrients: effect of plant age and

habitat. Aust J Bot 52: 141-147

Tittarelli A, Milla L, Vargas F, Morales A, Neupert C, Meisel LA, Salvo-G H,

Penaloza E, Munoz G, Corcuera LJ, Silva1 H (2007) Isolation and comparative

analysis of the wheat TaPT2 promoter: identification in silico of new putative

regulatory motifs conserved between monocots and dicots. J Exp Bot 58: 2573–

2582

Torres Aquino M, Plassard C (2004) Dynamics of ectomycorrhizal growth and P

transfer to the host plant in response to low and high soil P availability. FEMS

Microbiol Ecol 48: 149-156

Tynan KM, Wilkinson CJ, Holmes JM, Dell B, Colquhoun IJ, McComb JA, Hardy

GESJ (2001) The long-term ability of phosphite to control Phytophthora

cinnamomi in two native plant communities of Western Australia. Aust J Bot

49: 761-770

Ultra Jr.,VU, Tanaka S, Sakurai K, Iwasaki K (2006) Effects of arbuscular mycorrhiza

and phosphorus application on arsenic toxicity in sunflower (Helianthus annuus

L.) and on the transformation of arsenic in the rhizosphere. Plant Soil 290: 29-

41

Van Straaten P (2002) Rocks for crops: Agrominerals of sub-Saharan Africa. ICRAF,

Nairobi, Kenya, pp, 338

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

Varadarajan DK, Karthikeyan AS, Matilda PD, Raghothama KG (2002) Phosphite, an

analog of phosphate, suppresses the coordinated expression of genes under

phosphate starvation. Plant Physiol 129: 1232–1240

Page 45: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

28

Vetterlein D, Szegedi K, Ackermann J, Mattusch J, Neue HU, Tanneberg H, Jahn R

(2007) Competitive mobilization of phosphate and arsenate associated with

goethite by root activity. J Environ Qual 36: 1811-1820

Wagg C, Pautler M, Massicotte HB Peterson RL (2008) The co-occurrence of

ectomycorrhizal, arbuscular mycorrhizal, and dark septate fungi in seedlings of

four members of the Pinaceae. Mycorrhiza 18: 103–110

Warcup JH, McGee PA (1983) The mycorrhizal associations of some Australian

Asteraceae. New Phytol 95, 667-672

Weiß M, Sykorová Z, Garnica S, Riess K, Martos F, Krause C, Oberwinkler F, Bauer

R, Redecker D (2011) Sebacinales everywhere: previously overlooked ubiquitous

fungal endophytes. PLoS ONE 6: e16793

White ME (1986) The Greening of Gondwana. Reed Australia, Sydney

White ME (1994) After the greening: the browning of Australia. Kangaroo Press,

Sydney

Weijtmans K, Davis M, Clinton P, Kuyper TW, Greenfield L (2007) Occurrence of

arbuscular mycorrhiza and ectomycorrhiza on Leptospermum scoparium from

the Rakaia catchment, Canterbury. N Z J Ecol 31: 255-260

Wilcox H (1996) Mycorrhizae. In: Waisel Y, Eshel A, Kafkafi U (eds) Plant roots —

the hidden half. 2nd edition, New York, Marcel Dekker, pp 149–174

Wild A (1961) A pediological study of phosphorus in 12 soils derived from granite.

Aust J Agric Res 12: 286-299

Williams CH, Raupach M (1983) Plant nutrients in Australian soils. In: Soils: An

Australian Viewpoint. Division of Soils, CSIRO, Melbourne, pp 777-794

Wysocki R, Chery CC, Wawrzycka D, Van Hulle M, Cornelis R, Thevelein J, Tamas

MJ (2001) The glycerol channel Fps1p mediates the uptake of arsenite and

antimonite in Saccharomyces cerevisiae. Mol Microbiol 40: 1391–1401

Xiao K, Liu J, Dewbre G, Harrison M, Wang ZY (2006).Isolation and characterization

of root-specific phosphate transporter promoters from Medicago truncatula.

Plant Biol 8: 1-11

Page 46: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

29

Chapter 2

Page 47: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

30

Page 48: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

31

Page 49: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

32

Page 50: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

33

Page 51: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

34

Page 52: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

35

Page 53: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

36

Page 54: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

37

Chapter 3

A novel plant-fungus symbiosis benefits the host without

forming mycorrhizal structures

Khalil Kariman1,2

, Susan J. Barker2,3

, Ricarda Jost2, Patrick M. Finnegan

2,3, and Mark

Tibbett1,4

1School of Earth and Environment M087, The University of Western Australia,

Crawley, WA 6009, Australia

2School of Plant Biology M084, The University of Western Australia, Crawley, WA

6009, Australia

3Institute of Agriculture M082, The University of Western Australia, Crawley, WA

6009, Australia

4Department of Environmental Science and Technology (B37), School of Applied

Sciences, Cranfield University, Cranfield, Bedfordshire, MK 43 OAL, England

Most terrestrial plants form mutually beneficial symbioses with specific soil-

borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal

hyphae (i) colonise plant roots, (ii) explore the soil beyond the rhizosphere and (iii)

provide host plants with nutrients that might be chemically or physically

inaccessible to root systems1-4

. In contrast to these common features, we report a

plant growth promoting symbiosis between the basidiomycete fungus

Austroboletus occidentalis and jarrah (Eucalyptus marginata) which has quite

different characteristics. By combining nutritional, radioisotopic (33

P) and genetic

approaches, we elucidate the nature of this novel symbiosis. We show (i) the fungal

partner does not colonise plant roots, (ii) hyphae are localised to the rhizosphere

soil (and vicinity) and consequently (iii) do not transfer nutrients located much

beyond the rhizosphere. We argue the fungus does not transfer nutrients directly

to the plant, when compared to a conventional ectomycorrhizal symbiosis

(Scleroderma sp.). Here, transcript analysis of two phosphate transporter genes

(EmPHT1;1 and EmPHT1;2) and hyphal-mediated 33

P uptake suggest that the

PO43-

uptake shifts from epidermal to hyphal pathway in ectomycorrhizal plants

similar to arbuscular mycorrhizal symbioses5 whereas A. occidentalis benefits its

plant symbiont in a more indirect fashion. High concentrations of citrate and

fumarate in rhizosphere soil indicate that nutrient solubilisation and mobilisation

Page 55: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

38

are the likely mechanisms involved in the novel symbiosis. This rhizospheric

association between plant and fungus does not fit into currently described

paradigms of mycorrhizal symbiosis in terms of structure but appears to have

similar benefits to plants. This novel symbiosis challenges our current view of

mycorrhizal associations and rhizosphere ecology and may lead to a reassessment

of ectomycorrhizal relationships. We see this work as a starting point for further

studies on other basidiomycete-woody plant relationships, where a continuum

between heterotrophic rhizosphere fungi and fully mutualistic symbiosis is likely

to exist.

Mycorrhizal symbiosis is a strategy used by more than 80 % of land plant species

4,6

and is considered to be a critical factor in terrestrial ecosystems for sustained

productivity7, nutrient cycling

3,8 and plant soil feedback

9. At the root-fungal interface,

the heterotrophic symbiont colonises plant roots where it receives organic carbon

(mainly glucose and fructose) supplied by the phototrophic partner4. In return,

extraradical hyphae explore the soil beyond the roots for more than 10 cm in arbuscular

mycorrhizal (AM) symbioses10

and up to several meters in ECM symbioses11

, scavenge

nutrients from the soil, and assimilate and subsequently transport them to the host plant.

Seven categories of mycorrhizas have been recognized based on the morphological

structures developed and the plant-fungal species involved. These include arbuscular

mycorrhiza (AM), ectomycorrhiza (ECM), ectendomycorrhiza (ECTENDO), orchid

mycorrhiza (OM), ericoid mycorrhiza (EM), arbutoid mycorrhiza (ARBM) and

monotropoid mycorrhiza (MM)4. However, unusual plant-fungus associations with

unconventional or undeveloped structures have also been documented that cannot be

classified into any of the above mycorrhizal categories and are of ambiguous functional

status6,12,13

. Our previous work showed that A. occidentalis Watling & N.M. Greg.

substantially promoted jarrah growth and nutrient acquisition, without forming

mycorrhizal structures14

. The existing literature has typically viewed the function of

mycorrhizas with respect to the specialised mycorrhizal structures and hyphal-mediated

nutrient uptake, which might not always reflect the functionally relevant factors of an

association. Here, we used radiolabeled phosphate (33

PO43-

) and multiple compartments

to unravel the underlying functional mechanisms for the symbiosis observed between

jarrah and A. occidentalis. In the same experiment we also considered the effects of

conventional ECM formation by Scleroderma sp. on the expression of high affinity Pi

transporter genes (PHT1 gene family) in roots by quantifying the transcript abundance

of three PHT1 genes.

Page 56: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

39

A three-compartment system was used to track 33

PO43-

uptake and hyphal

development in two different jarrah-fungus symbioses (Fig. 1a). A double polyester

mesh bag (with 40 µm pores) was used to separate the root-hyphal compartment

(RHC) from the hyphal only compartment (HC) in which we placed a radiation

compartment (RDC) containing 33

PO43-

-labelled sand. In this system plant roots were

confined to the RHC and only fungal hyphae could penetrate the mesh bag and reach

the 33

PO43-

source located 7 to 12 cm away from the root systems. The treatments were

two fungal species, A. occidentalis and Scleroderma sp. (ECM), and a non-mycorrhizal

(NM) control that received sterilized inoculum. Plants were grown in a mixture of

inoculum (peat-vermiculite substrate cultures) and double-pasteurized washed river

sand (1:10 v/v) in a controlled environment chamber for 16 weeks.

The presence of a fungal partner greatly improved jarrah growth (Fig. 1b). The

positive responses were achieved regardless of the root colonizing ability of the fungus.

There was no sign of root hyphal association or a short root morphology change for

jarrah seedlings grown with A. occidentalis, while the Scleroderma sp. treatment

achieved 77% (± 11, SE) ECM colonisation. However A. occidentalis was as effective

as Scleroderma sp. in enhancing the shoot biomass of plants (Fig. 1b). Furthermore,

jarrah plants inoculated with either fungus had larger root systems than NM controls,

with the largest root system belonging to the Scleroderma sp. treatment.

Both types of symbioses effectively increased the shoot nutrient content of plants

compared to NM controls (Table 1). Shoots of plants inoculated with A. occidentalis

had almost equal contents of P, S, Mg, Cu and even more nitrogen (N) than the

Scleroderma sp. treatment. The root colonizing fungus Scleroderma sp. seems,

however, to be more effective than A. occidentalis at facilitating uptake of the

micronutrients Fe and Zn. Inoculation with A. occidentalis enhanced the shoot P and

Mg concentration and inoculation with Scleroderma sp. enhanced the shoot

concentration of P, S, Mg, Fe and Zn compared with NM plants (Table 1).

Page 57: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

40

Fig.1 Jarrah shoot biomass greatly enhanced by both novel (A. occidentalis) and ECM

(Scleroderma sp.) symbioses. (a) Schematic diagram showing the three-compartment

system used to study the potential functional pathways in mycorrhizal symbioses.

Jarrah roots were confined to the root hyphal compartment (RHC), while extraradical

hyphae (ERH) could pass through the polyester mesh bag (PMB) and colonize both

hyphal (HC) and radiation (RDC) compartments. (b) Shoot and root dry biomass of

jarrah plants. Bars with and are significantly different from NM controls at p <

0.05 and p < 0.10, respectively.

(a) (b)

Dry b

iom

ass

(g p

ot-1

)

ShootRoot

NM control A. occidentalis Scleroderma sp.

0

2

4

6

8

2

4

6

8

Page 58: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

41

Table 1 Shoot nutrient status of jarrah seedlings

Content

Treatments mg pot-1

µg pot-1

P N S Mg Fe Zn Cu

NM Control

0.54

± 0.09

43.14

± 2.86

4.84

± 1.08

8.95

± 2.23

83.68

± 10.55

58.93

± 14.14

19.26

± 1.65

A. occidentalis

1.86**

± 0.56

59.95**

± 1.73

11.49**

± 1.43

21.17 **

± 3.54

165.97**

± 23.32

146.27**

± 13.52

43.52**

± 3.02

Scleroderma sp.

1.91**

± 0.22

44.46

± 0.88

12.61**

± 1.39

20.31**

± 3.41

341.48**

± 26.47

202.10**

± 19.76

48.37**

± 1.71

Concentration

mg g-1

DW

µg g-1

DW

NM Control

0.19

± 0.01

16.26

± 3.49

1.65

± 0.04

3.00

± 0.10

30.38

± 4.18

19.88

± 0.75

7.21

± 1.48

A. occidentalis

0.29*

± 0.03

10.37

± 1.45

1.93

± 0.15

3.49**

± 0.07

27.63

± 1.26

24.80

± 2.29

7.42

± 0.78

Scleroderma sp.

0.31**

± 0.03

7.52**

± 1.21

2.05**

± 0.07

3.26*

± 0.08

56.30**

± 3.92

33.19**

± 2.21

8.10

± 1.04

Data represent mean values (n=3) ± SE. Values of each nutrient followed by ** and * are

significantly different from NM controls at p < 0.05 and p < 0.10, respectively.

Accumulation of 33

P from the RDC was assessed in the shoots of the 16-week-old

plants (Fig. 2a). Scleroderma sp. transported 33

P to jarrah plants, while there was only

background radiation in shoot tissues from the A. occidentalis and NM treatments.

Non-destructive monitoring of shoot radioactivity during the growth period indicated

that the fungal-mediated 33

P uptake in the Scleroderma sp. treatment was detectable

nine weeks after planting (data not shown). Furthermore, the specific activity of

radiolabelled sand remaining after harvest in the Scleroderma sp. treatment was

significantly lower than for the NM and A. occidentalis treatments (Fig. 2a), supporting

the hypothesis that 33

P was taken up by extraradical hyphae. The plant-available P

extracted from the RHC and HC did not contain radioactivity in any of the treatments

showing 33

P did not move or leak from the RDC. Some 33

P labelled molecules were,

however, in transit from the RDC to jarrah shoots within extraradical hyphae and roots

but these organs were not considered for the present study.

Our results are consistent with two possibilities. A. occidentalis hyphae might grow

throughout the compartments, but not transfer 33

P to the RHC, or A occidentalis might

not colonise the RDC. To investigate this we quantified the hyphal length in the

Page 59: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

42

different compartments. Hyphae of A. occidentalis were thinner (1.5 to 4 µm) than

those of Scleroderma sp. (3.5 to 14 µm). Both fungi had significantly higher hyphal

length densities in the RHC compared to the NM controls (Fig. 2b). However, the

hyphal length in both the HC and RDC did not differ significantly between A.

occidentalis and NM treatments. The Scleroderma sp. treatment had significantly

greater hyphal length in both HC and RDC than found for A. occidentalis and NM

treatments.

To ascertain the source of P exploited by plants and fungi and transferred to the

shoots of inoculated plants, we determined the concentration of plant-available P in soil

from the various compartments (Fig. 2c). The A. occidentalis treatment had the lowest

available P in the RHC of all the treatments. The available P in the HC and RDC did

not differ significantly between A. occidentalis and NM control treatments, whereas the

Scleroderma sp. treatment had lower available P in both HC and RDC compared to NM

controls. Thus, the A. occidentalis treated plants did not access P from the HC or RDC

to any greater extent than the NM control plants. Based on the results for 33

P uptake,

shoot P content, hyphal extent and the plant-available P in soil, we conclude that plants

inoculated with A. occidentalis mainly accessed P from the RHC, whereas the plants

inoculated with Scleroderma sp. obtained P from all three compartments (RHC, HC

and RDC).

The transport of PO43-

into plant cells is catalyzed by proteins encoded by members of

the PHOSPHATE TRANSPORTER1 (PHT1) multigene family. The abundance of

transcripts from various PHT1 genes typically increases under low plant P status and

decreases under high plant P status. AM colonisation has been shown to induce a

remodeling of PHT1 transcript profiles; such that there is preferential accumulation of

transcripts from specific PHT1 genes in cells in close proximity to the fungal symbiont

at the expense of transcripts from other PHT1 genes in root epidermal cells not in direct

contact with fungal hyphae5. The outcome of the remodeling is thought to be the

preferential delivery of PO43-

from the mycorrhizal symbiont to root cortex cells instead

of the uptake of PO43-

directly from the bulk soil solution5. A real-time PCR transcript

assay was used to quantify the abundance of transcripts from three jarrah EmPHT1

genes currently available for assay relative to an internal ACTIN reference gene

(EmACT1) in response to the fungal symbionts. The results revealed that there were

significantly less EmPHT1;1 and EmPHT1;2 transcripts in roots from the Scleroderma

sp. treatment compared to the NM control and A. occidentalis treatments (Fig. 2d). The

expression of EmPHT1;5 was similar across treatments. The down-regulation of

Page 60: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

43

transcript abundance for EmPHT1;1 and EmPHT1;2 in plants exposed to Scleroderma

sp. combined with the apparent hyphal delivery of 33

P, indicates that these two PHT1

genes are likely to be responsible for the direct pathway of PO43-

uptake by plant roots.

Some plant PHT1 genes have been shown to reduce their expression in both AM5

and

ECM15

symbioses presumably due to a reduction in P uptake via the direct pathway

(roots). Here, based on the active involvement of Scleroderma sp. hyphae (ECM) in 33

P

uptake and P uptake from both hyphae-accessible compartments (HC and RDC) and

the reduced expression of two plant PHT1 genes, we suggest the PO43-

uptake shifts

from root epidermal to hyphal pathway in ECM plants. This finding may have

profound effects in our understanding of P nutrition of woody plants, which are highly

dependent on ECM associations for their growth, nutrition and survival4,7,8

.

In contrast, there was no difference in the transcript profiles for the three PHT1 genes

in roots of plants from NM and A. occidentalis treatments. This lack of remodeling of

the PHT1 transcript pool in response to A. occidentalis, combined with the observation

that hyphal-mediated 33

P uptake did not occur in plants associated with A. occidentalis

indicates that active PO43-

uptake occurs directly from the soil solution via plant roots in

the novel symbiosis between jarrah and A. occidentalis.

Page 61: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

44

Fig. 2. Functional evidence for root-mediated P uptake in the novel (A.

occidentalis) symbiosis and hyphal-mediated P uptake in the ECM (Scleroderma

sp.) symbiosis. a, Specific activity of 33

P in shoot and 33

P-labelled sand (RDC). b,

Hyphal length density in the three different compartments. c, Plant-available P in

different compartments. d, Expression of three PHT1 genes in jarrah roots relative to an

internal reference gene (actin) determined by real-time PCR. The scale on the vertical

axis is a log2 scale based on ΔCt (the threshold cycle (Ct) of the target PHT1 gene

minus the Ct of the reference gene). A difference of one Ct value corresponds to a 2-

fold difference in transcript abundance. Bars with different letters are significantly

different at p < 0.05. Bars with and within each compartment are significantly

different from NM controls at p < 0.05 and p < 0.10, respectively. Error bars are SE (n

= 3).

b b

a

a a

b

200

100

0

100

200

300

400

500

600

700

Sp

ecif

ic a

ctiv

ity

at

har

ves

t(B

q g

-1D

W)

Shoot P-labelled sand

NM control A. occidentalis Scleroderma sp.

33

a

a

a

a a

a

b

b

a

30

35

40

45

50

EmPHT1;1 EmPHT1;2 EmPHT1;5

Rela

tive e

xpre

ssio

n (

40-∆

Ct)

NM control A. occidentalis Scleroderma sp.

b b b

a

b

b

a

aa

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Root hyphal compartment

Hyphal compartment Radiation compartment

Hyph

al le

ngth

(m

g-1

soil)

NM control A. occidentalis Scleroderma sp.

a b

c d

0

1

2

3

4

5

6

7

8

9

Root hyphal compartment

Hyphal compartment

Radiation compartment

Pla

nt-

av

ailab

le P

g P

g-1

soil)

NM control A. occidentalis Scleroderma sp.

Page 62: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

45

Mannitol, sucrose and fructose were measured in roots by HPLC to identify fungal

presence and nutrition (Fig. 3). Mannitol is a sugar alcohol typically found in fungi

serving various physiological functions such as carbon storage16

and cryoprotection17

.

Plants generally do not synthesize mannitol but there are number of exceptions18

. The

exclusive presence of mannitol in mycorrhizal roots has been attributed to the fungal

structures associated with roots4,19

. Here, mannitol was only found in roots from the

Scleroderma sp. treatment, where hyphae formed typical ECM structures. No mannitol

was found in roots of either NM control or A. occidentalis treatments, both of which

had no sign of fungal colonisation. Moreover, mannitol was present in mycelia from

axenic cultures of both Scleroderma sp. and A. occidentalis growing on solid nutrient

medium (data not shown). Thus, the lack of mannitol in roots from the A. occidentalis

treatment is physiological support for the absence of microscopic fungal structures in

and on the roots here and in our previous work14

supporting our conclusion that A.

occidentalis does not colonise jarrah roots.

Sucrose is the major form of fixed carbon transported in the phloem of higher plants.

Mycorrhizal roots accumulate higher concentrations of sucrose than NM roots. The

sucrose is subsequently hydrolyzed to glucose and fructose by host cell wall invertase

to provide hexoses for fungal consumption4,19

. Roots from both Scleroderma sp. and A.

occidentalis treatments contained higher concentrations of sucrose than NM plants

suggesting that the host plant is a carbon source for both heterotrophic fungal partners

(Fig. 3). ECM plants support the C nutrition of their fungal partners through direct

supply of sucrose (and hexoses) at the root/fungal interface or via repression of their

root monocsaccharide importers to allow C flow from soil towards hyphae, or a

combination of these pathways20-21

. The washed river sand used in this experiment was

very poor in organic matter, and therefore the direct supply of fixed C from plant to the

ECM partner (Scleroderma sp.) is most likely under these experimental conditions,

with high sucrose concentration of ECM roots as preliminary evidence (Fig. 3). The

enhanced sucrose concentration in roots of jarrah plants associated with A. occidentalis

must have required a signal from the fungus as it did not occur in NM roots. However,

as A. occidentalis does not penetrate the roots, we hypothesize that the products of

sucrose breakdown (glucose and fructose) are exuded into the rhizosphere soil, which is

the main fungal habitat (Fig. 2b). The constant amount of fructose in roots across all

three treatments is consistent with this hypothesis. This could be due to the rapid

conversion of fructose to mannitol by hyphae

19 inside roots of the Scleroderma sp.

treatment or hypothetically due to quick exudation into the rhizosphere soil in A.

Page 63: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

46

occidentalis treatment. Carbon flow from roots into the rhizosphere is a key

phenomenon for C cycling in soil and occurs via a variety of processes including C

flow to root-associated symbionts such as mycorrhizas22,23

.

Fig. 3. Jarrah roots contain mannitol only in the Scleroderma sp. treatment where

ECM colonisation occurred. Determinations for each sugar indicated by a different

letter are significantly different at p < 0.05. ND = not detected. Error bars are SE (n =

3).

Nutrient transfer via extraradical hyphae is considered to be the common strategy in

mycorrhizal symbioses1,2

aiding plants to access soil nutrients far away from the root

system. ECM fungi make extensive extraradical hyphal networks in soil and translocate

P over distances of up to at least 40 cm11

. We suggest that “nutrient transfer” is not the

underlying mechanism behind the improved nutrient uptake in the jarrah-A.

occidentalis symbiosis as fungal hyphae are not exploring the soil far from roots and

there was neither a physical contact (colonisation) between the partners nor a change in

the transcript profiles of ECM-responsive PHT1 genes in roots.

Biological weathering of soil to dissolve minerals and release plant nutrients (except

nitrogen) is accomplished mainly by plant roots and soil microbes through the

exudation of metabolites such as carboxylates, protons, respiration-derived CO2,

phenolic compounds and siderophores23,24

. It has been established that ECM fungi

exude carboxylates24

and protons25

to mobilize nutrients from primary silicate minerals,

such as the sand used here.

High performance liquid chromatography (HPLC) analysis showed that citrate was

the main carboxylate present in the rhizosphere soil, along with lower concentrations of

fumarate and shikimate, in all three treatments (Table 2). The rhizosphere from the A.

b

a

ND

a

a

ND

a

a

0

2

4

6

8

10

12

14

Sucrose Fructose Mannitol

Ro

ot

sug

ar c

on

cen

trat

ion

(m

g g

-1D

W)

NM control A. occidentalis Scleroderma sp.

Page 64: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

47

occidentalis treatment had significantly higher concentrations of citrate and fumarate

compared to both Scleroderma sp. and NM treatments. The Scleroderma sp. treatment

had the lowest citrate among the treatments, and fumarate and shikimate concentrations

did not differ from NM controls. Low levels of carboxylates in rhizosphere soil of

ECM plants26-28

have been attributed mainly to high microspatial variability in

concentration and rapid microbial consumption24

. Here, we propose another factor that

might be associated with the low carboxylate concentration in the rhizosphere of ECM

plants. The active nutrient uptake in mycorrhizal symbioses occur via fungal hyphae

rather than plant roots1,2,4

. Carboxylates in ECM symbioses have been shown to

originate primarily from fungal hyphae dissolving nutrients surrounding hyphal tips28

.

In ECM plants, carboxylates are presumably released by extensive hyphae into a much

broader soil volume (hyphosphere) rather than into the immediate rhizosphere soil. Our

data also show that, in a classic ECM symbiosis (Scleroderma sp.), the plant exudation

of citrate is suppressed whereas in the presence of the novel symbiosis, exudation of

citrate is enhanced, although we cannot exclude that some or all of the additional citrate

is of fungal origin. Likewise, the significant enhancement of fumarate in the A.

occidentalis treatment compared to the NM control and Scleroderma sp. treatments

could suggest that this additional fumarate may be of fungal origin. Higher

concentrations of carboxylates in the rhizosphere of NM controls and A. occidentalis

treatments compared to the Scleroderma sp. treatment could be due to the fact that

active nutrient uptake is taking place directly via plant roots in the former two

treatments. We suggest citrate as the main carboxylate associated with nutrient release

and mobilization as observed here and in accordance with other studies23,29,30

correlating citrate production to release of nutrients from primary minerals such as

apatite and biotite.

A. occidentalis enhanced the shoot N content of jarrah plants while no positive effects

were observed with Scleroderma sp. The improved N nutrition is a well-known benefit

in ECM symbiosis, which is mainly associated with the capability of ECM fungi to

exude nutrient-mobilizing enzymes such as acid phosphatases, proteinases, and

polyphenol oxidase to release nutrients from organic sources in soil7,31,32

. The

Scleroderma sp. treatment had the largest root system among treatments but the lowest

shoot N concentration (Table 1) apparently due to dilution of N in a higher shoot

biomass. This is in keeping with the results for 33

P uptake and PHT1 gene expression in

the Scleroderma sp. treatment indicating that the active nutrient uptake does not occur

via roots but extraradical hyphae. However, the ECM symbiosis failed to improve the

Page 65: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

48

N nutrition of jarrah plants probably due to the lack of sufficient substrate (organic

matter) in the river sand used for this study.

The A. occidentalis treatment had relatively higher root biomass than NM controls,

which might have contributed to higher N uptake. Moreover, ECM fungi can increase

bacterial biomass and activity and also modify the soil bacterial community via

exuding carboxylates, particularly citrate30

. The rhizosphere soil in the A. occidentalis

treatment had the highest citrate concentration among the treatments. Accordingly,

modification of rhizosphere soil by exuding citrate could potentially promote beneficial

bacteria including free-living nitrogen fixers leading to higher N availability for host

plants in the novel symbiosis. Consistent with this hypothesis, the Scleroderma sp.

treatment had the lowest citrate concentration in the rhizosphere soil accompanied by

the lowest shoot N concentration.

Table 2- Carboxylate concentration in the rhizosphere soil (µmol g-1

root DW)

Treatments Citrate Fumarate Shikimate

NM control 1234b (±113) 23b (±5) 87a (±13)

A. occidentalis 2525a (±120) 59a (±10) 82a (±17)

Scleroderma sp. 660c (±79) 18b (±3) 78a (±7)

Values within a column followed by different letters are significantly different at p ≤

0.05. Data are mean ± SE (n=3).

The present study shows clear functional differences between the jarrah-A.

occidentalis symbiosis and previously described mycorrhizal associations. Phosphorus

was likely to be the limiting nutrient in this experiment in order to explore the ability of

the fungus to improve P nutrition of host plants and the mechanisms involved. In this

novel symbiosis, fungal hyphae are found and function in the vicinity of root systems,

but do not colonise plant roots. Moreover, the 33

P baiting experiment did not support

the presence of “hyphal-mediated nutrient uptake” which is a well-established strategy

in mycorrhizal symbioses. The enhanced carboxylate release into the rhizosphere soil

correlates with higher shoot nutrient content (P, in particular) in plants harbouring this

novel symbiosis and may be the underlying mechanism. It will be of interest to

determine how much of the enhanced exudation can be attributed to either plant or

fungus. Our results also suggest that the function of mycorrhizas cannot be fully

explained by traditional views that focus on the features of root colonisation and the

Page 66: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

49

contributions hyphae make to nutrient uptake. More investigations are warranted to

unearth other potential strategies that might be involved in this novel symbiosis such as

secretion of protons, nutrient-mobilizing enzymes or other unknown pathways.

Methods Summary

A three-compartment system (Fig. 1a) was used to track the symbiotic 33

P uptake.

Jarrah seedlings were exposed to A. occidentalis (the novel symbiont), Scleroderma sp.

(ab ECM fungus) and a NM control and grown for 16 weeks under controlled

environmental conditions. Non-destructive tracking of 33

P was done using a Geiger

counter to check the presence of radioactivity in shoots of growing plants.

Radioactivity in shoots of harvested plants was determined using a Packard TR 1500

liquid scintillation counter. We used ICP-OES (PerkinElmer, USA) and a combustion

analyzer (Elementar Vario Macro, Germany) to measure the nutrient concentration of

shoot tissues. Hyphae were extracted from soil using a membrane filter technique and

quantified on nitrocellulose gridded filters (0.45 µm HAWG, Millipore). Jarrah PHT1

cDNA sequences were obtained through PCR cloning from total root RNA isolated

using a CTAB-based method and reverse transcribed using the GoScriptTM

reverse

transcriptase kit (Promega). To get longer sequences for the amplified products we used

the SMART™ RACE cDNA Amplification Kit (Clontech) and the Advantage 2 kit

(Clontech). Root-expressed PHT1 cDNAs were amplified using primers

(Supplementary Table 1) designed based on the conserved sequences in PHT1 genes

from E. camaldulensis. The abundance of specific PHT1 gene transcripts was

determined relative to a jarrah actin gene (ACT) as an internal reference using SYBR

Green-based detection on the Applied Biosystems 7500 Fast Real Time PCR system.

Root carbohydrates were extracted using 80% ethanol and quantified by HPLC

analysis. The rhizosphere carboxylates were extracted from roots with the loose sand

removed using 0.2 mM CaCl2, filtered through Acrodisc syringe filters (0.2 µm) and

quantified by HPLC. Full methods and associated references are presented as

Supplementary Information.

Page 67: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

50

References

1-Marschner, H. & Dell, B. Nutrient uptake in mycorrhizal symbiosis. Plant Soil. 159,

89–102 (1994).

2-Cameron, D. D., Leake, J. R. & Read, D. J. Mutualistic mycorrhiza in orchids:

evidence from plant-fungus carbon and nitrogen transfers in the green-leaved

terrestrial orchid Goodyera repens. New Phytol. 171, 405–416 (2006).

3-Finlay, R. D. Ecological aspects of mycorrhizal symbiosis: with special emphasis on

the functional diversity of interactions involving the extraradical mycelium. J.

Exp. Bot. 59, 1115-1126 (2008).

4-Smith, S. E. & Read, D. J. Mycorrhizal Symbiosis. 3rd

ed. Academic Press, London

UK (2008).

5-Karandashov, V. & Bucher, M. Symbiotic phosphate transport in arbuscular

mycorrhizas. Trends Plant Sci. 10, 22-29 (2005).

6-Brundrett, M. C. Mycorrhizal associations and other means of nutrition of vascular

plants: Understanding the global diversity of host plants by resolving conflicting

information and developing reliable means of diagnosis. Plant Soil. 320, 37–77

(2009).

7-van der Heijden, M. G. A., Bardgett, R. D. & Straalen, N. M. The unseen majority:

soil microbes as drivers of plant diversity and productivity in terrestrial

ecosystems. Ecol. Let. 11, 296-310 (2008).

8-Read, D. J. & Perez-Moreno, J. Mycorrhizas and nutrient cycling in ecosystems—a

journey towards relevance?. New Phytol. 157, 475–492 (2003).

9-Bever, J. D. Negative feedback within a mutualism: host specific growth of

mycorrhizal fungi reduces plant benefit. Proc. R. Soc. Lond. 269, 2595-2601

(2002).

10-Li, X. L., George, E. & Marschner, H. Extension of the phosphorus depletion zone

in VA-mycorrhizal white clover in calcareous soil. Plant Soil. 136, 41-48 (1991).

11-Finlay, R. D. & Read, D. J. The structure and function of the vegetative mycelium

of ectomycorrhizal plants. II. The uptake and distribution of phosphorus by

mycelial strands interconnecting host plants. New Phytol. 103, 157–165 (1986).

12-Warcup, J. H. & McGee, P. A. The mycorrhizal associations of some Australian

Asteraceae. New Phytol. 95, 667-672 (1983).

13-Kope, H. H. & Warcup, J. H. Synthesised ectomycorrhizal associations of some

Australian herbs and shrubs. New Phytol. 104, 591–599 (1986).

14-Kariman, K., Barker, S. J., Finnegan, P. M. & Tibbett, M. Dual mycorrhizal

associations of jarrah (Eucalyptus marginata) in a nurse-pot system. Aust. J. Bot.

60, 661-668 (2012).

Page 68: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

51

15-Loth-Pereda, V., Orsini, E., Courty, P. E., Lota, F., Kohler, A., Diss, L., Blaudez,

D., Chalot, M., Nehls, U., Bucher, M. & Martin, F. Structure and expression

profile of the phosphate Pht1 transporter gene family in mycorrhizal Populus

trichocarpa. Plant Physiol. 156, 2141–2154 (2011).

16-Koide, R. T., Shumway, D. L. & Stevens, C. M. Soluble carbohydrates of red pine

(Pinus resinosa) mycorrhizas and mycorrhizal fungi. Mycol. Res. 104, 834–840

(2000).

17-Tibbett, M., Sanders, F. E. & Cairney, J. W. G. Low-temperature induced changes

in trehalose, mannitol and arabitol associated with enhanced tolerance to freezing

in ectomycorrhizal basidiomycetes (Hebeloma spp.). Mycorrhiza 12, 249–255

(2002).

18-Stoop, J. M. H., Williamson, J. D. & Pharr, D. M. Mannitol metabolism in plants: a

method for coping with stress. Trends Plant Sci. 1, 139–144 (1996).

19-Stribley, D. P. & Read, D. J. The biology of mycorrhiza in th e Ericaceae. III.

Movement of carbon-14 from host to fungus. New Phytol. 73,731-741 (1974).

20-Nehls, U., Grunze, N., Willmann, M., Reich, M. & Kuster, H. Sugar for my honey:

carbohydrate partitioning in ectomycorrhizal symbiosis. Phytochem. 68, 82–91

(2007).

21-Nehls, U., Göhringer, F., Wittulsky, S. & Dietz, S. Fungal carbohydrate support in

the ectomycorrhizal symbiosis: a review. Plant Biol. 12, 292–301 (2010).

22-Jones, D. L, Nguyen, C. & Finlay, R. D. Carbon flow in the rhizosphere: carbon

trading at the soil-root interface. Plant Soil 321, 5–33 (2009).

23-Lambers, H., Mougel, C., Jaillard, B. & Hinsinger, P. Plant-microbe-soil

interactions in the rhizosphere: an evolutionary perspective. Plant Soil 321, 83–

115 (2009).

24-Landeweert, R., Hofflund, E., Finlay, R.D. & van Breemen, N. Linking plants to

rocks: Ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol.

Evol. 16, 248-254 (2001).

25-Hoffland, E., Kuyper, T. W., Wallander, H., Plassard, C., Gorbushina, A. A.,

Haselwandter, K., Holmstrom, S., Landeweert, R., Lundstrom, U. S., Rosling, A.,

Sen, R., Smits, M. M., van Hees, P. A. W., van Breemen, N. The role of fungi in

weathering. Front. Ecol. Environ. 2, 258–264 (2004).

26-Leyval, C. & Berthelin, J. Rhizodeposition and net release of soluble organic

compounds by pine and beech seedlings inoculated with rhizobacteria and

ectomycorrhizal fungi. Biol. Fertil. Soils. 15, 259–267 (1993).

27-Wallander, H. Uptake of P from apatite by Pinus sylvestris seedlings colonised by

different ectomycorrhizal fungi. Plant Soil. 218, 249–256 (2000a).

28-Wallander, H. Use of strontium isotopes and foliar K content to estimate weathering

of biotite induced by pine seedlings colonised by ectomycorrhizal fungi from two

different soils. Plant Soil, 222, 215–229 (2000b).

Page 69: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

52

29-Wallander, H., Wickman, T. & Jacks, G. Apatite as a P source in mycorrhizal and

NM Pinus sylvestris seedlings. Plant Soil. 196, 123-131 (1997).

30-Olsson, P. A. & Wallander, H. Interactions between ectomycorrhizal fungi and the

bacterial community in soils amended with various primary minerals. FEMS

Microbiol. Ecol. 27, 195-205 (1998).

31-Bending, G. D, & Read, D. J. The structure and function of the vegetative mycelium

of ectomycorrhizal plants VI. Activities of nutrient mobilising enzymes in birch

litter colonised by Paxillus involutus (Fr.) Fr. New Phytol. 130, 411-417 (1995).

32-Tibbett, M. & Sanders, F. E. Ectomycorrhizal symbiosis can enhance plant nutrition

through improved access to discrete organic nutrient patches of high resource

quality. Ann. Bot. 89, 783-789 (2002).

Supplementary Information (Full Methods)

Plant species and fungal isolates. Jarrah seeds were obtained from a single tree in

Dwellingup, Western Australia. A. occidentalis and Scleroderma sp. isolates were

collected from a jarrah forest rehabilitation site at Langford Park, Western Australia

and banksia woodland at Piney Lakes, Western Australia, respectively. A. occidentalis

is a mushroom belonging to the fungal family of Boletaceae and has wide distribution

across Australia31

. Methods for seed germination, inoculum production and

colonisation studies were previously described32

.

Experimental design. 33

PO43-

was purchased from Perkin Elmer (Wellesley, MA,

USA) and used to investigate the ability of fungi to transport 33

P from the RDC to

jarrah plants. For this purpose, 250 g of sand containing 928 KBq 33

PO43-

was placed in

plastic vials (7 x 7 cm) at the bottom of cylindrical PVC pots (15 x 35 cm). Vials were

topped up with 100 g clean sand (2 cm) before being buried in 3.250 kg of double-

pasteurized washed river sand. A double-thickness polyester mesh bag (40 µm pore

size) was filled with 4.150 kg of a mixture of fungal inoculums (peat-vermiculite

substrate cultures) and double-pasteurized washed river sand (1:10 v/v) and placed

above the radiation compartment. The 33

PO43-

source was located 7 to 12 cm away from

jarrah roots. Three pre-germinated jarrah seeds were planted in each pot and the pot

surface was covered with 3 cm of sterile plastic beads to minimise cross or

environmental contaminations. Plants were grown for 16 weeks in a controlled

environment cabinet with 16 h light (750 µM m-2

s-1

) / 8 h dark, 24/18 ºC temperature

and 60/70 % relative humidity. All plants received the 1 X modified33

Long Ashton

solution minus P once a fortnight started two weeks after planting (10 mL kg-1

soil):

Page 70: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

53

K2SO4 2 mM, MgSO4.7H2O 1.5 mM, CaCl2.2H2O 3 mM, FeEDTA 0.1 mM,

(NH4)2SO4 4 mM, NaNO3 8 mM, H3BO3 46 µM, MnCl2.4H2O 9 µM, ZnSO4.7H2O 8

µM, CuSO4.5H2O 0.3 µM and Na2MoO4.2H2O 0.01 µM.

Measuring the 33

P activity. A Geiger counter (Mini 900 Ratemeter, Thermo

Scientific) was used to non-destructively track the 33

P uptake in shoots of growing

plants. Shoot radioactivity at harvest was determined in tissues oven-dried at 70 ºC for

72 h. A measured quantity (about 200 mg) of dried ground shoot tissue was digested in

a mixture of nitric:perchloric acid solution (4:1 v/v) and diluted to 10 mL with water. A

1.0 mL portion of the diluted digest was mixed with 3.0 mL of scintillation fluid (Irga-

Safe Plus, Perkin Elmer) and counted (TR 1500 liquid scintillation counter, Packard

Instrument Co.). The radioactivity measurement was corrected for decay and

background radiation. To measure 33

P in soil, the plant-available P was extracted using

a NaHCO3-based method34

before measuring the radioactivity as described above.

Radioactivity data were presented as specific activity (SA), which was considered as a

measure of radioactivity per mass of shoot tissues or growth medium.

Nutrient analysis. The acid digested samples from the 33

P measurements were used to

measure the shoot concentration of P, S, Mg, Fe, Zn and Cu using inductively coupled

plasma optical emission spectrometry (ICP-OES; Optima 5300 DV, PerkinElmer,

USA). A measured amount (about 60 mg) of dried ground shoot material was used to

measure the N percentage in a combustion analyzer (Elementar Vario Macro,

Germany). The plant-available P was extracted using a NaHCO3-based method34

before

the P concentration was determined using a Malachite green-based colorimetric assay34

.

Quantification of fungal hyphae in different compartments. Fungal hyphae were

extracted from soil by an aqueous solution and membrane filter technique36

with a

slight modification (5% ink (Black Sheafer) in white vinegar was used to stain hyphae

instead of lactoglycerol-trypan blue). Tennant’s formula37

was used to calculate the

hyphal length on each nitrocellulose gridded filter (0.45 µm Millipore).

Cloning of PHT1 genes and real-time PCR. Total RNA was isolated from jarrah

roots using a cetyltrimethylammonium bromide (CTAB)-based protocol38

with a slight

modification. Sodium D-isoascorbate was added to the extraction buffer just before use

to a final concentration of 100 mM. Total RNA (1.0 µg) was treated with DNase I

(RQ1 RNase-free DNase, Promega, USA) to remove genomic DNA contamination.

Oligo(dT)18 was used to prime cDNA synthesis (GoScriptTM

reverse transcriptase,

Promega) according to the supplier’s instructions. PHT1 cDNA fragments were

amplified by PCR using combinations of two forward primers (EcPT1-F and EcPT2-F)

Page 71: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

54

and two reverse primers (EcPT4-R and EcPT5-R) (Supplementary Table 1) that were

designed based on conserved sequences in the PHT1 genes of E. camaldulensis39

. PCR

amplifications were carried out in 50 µl containing 3 µl of cDNA equivalent to 60 ng of

total RNA, 75 µM dNTP mix, 0.5 µM of each forward and reverse primer, 2 mM

MgCl2, 2.5 U of Taq DNA polymerase (Biotaq, Bioline) in 1X PCR buffer (Bioline).

The PCR program included 1.30 min of initial denaturation at 94 ºC followed by 30

cycles of denaturation at 94 ºC for 40 s, annealing of the primers at 56 ºC for 40 s and

extension at 72 ºC for 2 min. The amplicons were terminated by a final extension at 72

ºC for 10 min. PCR products were purified (Sure Clean Plus, Bioline) and ligated into

pJET1.2 (CloneJET™ PCR Cloning Kit, Fermentas). Plasmids with inserts of the

expected size were submitted to sequencing (Australian Genome Research Facility,

Perth, Australia). PHT1 sequences were confirmed by comparison to publicly available

sequence databases and used to design primers to amplify the 5’ and 3’ ends of PHT1

cDNAs by Rapid Amplification of cDNA Ends (RACE, SMART™ RACE cDNA

Amplification Kit and Advantage 2 Kit, Clontech). RACE products were purified,

ligated into pJET1.2 and sequenced as described above.

Three root-expressed PHT1 cDNAs were identified at the time of performing this

assay: EmPHT1;1 (1052 bp), EmPHT1;2 (980 bp) and EmPHT1;5 (752 bp). Gene

specific primers were designed for RT-PCR (Supplementary Table 1). In addition, a

fragment of a jarrah ACT gene was amplified using Actin-F and Actin-R primers40

and

sequenced to allow the design of gene specific primers to be used as an internal control

for the relative quantification assay (Supplementary Table 1). The SYBR green-based

real-time PCR (q-PCR) was carried out to quantify the transcripts from PHT1 genes

relative to ACT transcripts. Q-PCR reactions were performed in 96-well plates in a 10

µl reaction volume of 0.3 µM of each gene specific primer and 2.5 µl of cDNA

equivalent to 50 ng total RNA in 1x SYBR Green PCR master mix (Applied

Biosystems). All q-PCR experiments were performed on a 7500 FAST Real-time PCR

System (Applied Biosystems).

Quantification of root carbohydrates. Carbohydrates were extracted from freeze-

dried root samples using 80% ethanol41

. High-Performance Liquid Chromatography

(HPLC) was used to determine the concentration of sucrose, fructose and mannitol in

root extracts42,43

. Root extracts were injected into an HPLC separation module (W2695,

Waters, Milford, MA, USA) using acetonitrile/water as the mobile phase and

separation was achieved using a high performance carbohydrate column (5µm x 3.9mm

x 300mm, Waters). Sugars were detected using a refractometer (2414 Refractive Index

Page 72: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

55

(RI) detector, Waters) with individual peak areas quantified. Sugar speciation and

concentration was determined after comparison to primary compound standard sets

covering each analyte.

Measuring the carboxylate concentration in rhizosphere soil. The root system was

shaken carefully to remove the bulk soil. The soil adhering to the root was defined as

the rhizosphere soil44

. The root system was gently shaken in a measured volume of 0.2

mM CaCl2 to wash off the rhizosphere soil. Syringe filters (0.2 µm Supor membrane

(Pall), Acrodisc) were used to filter a subsample of extract into 1 mL HPLC vials. The

filtered extracts were acidified with concentrated phosphoric acid (0.1% v/v) and frozen

at -20 ºC until analysis. The carboxylate concentration of rhizosphere extracts was

determined by HPLC45

using 600E pump, 717 plus autosampler, a 996 photodiode

array detector (Waters) and a C-18 reverse-phase column (Alltima).

Experimental design and statistical analysis. Plants were grown in a completely

randomized design with three replicates. The two fungal treatments were Scleroderma

sp. and A. occidentalis. The NM control received sterilized inoculum to equalize the

amount of nutrients and organic matter among treatments. Another set of controls was

also assessed. These had no 33

P and were used to calculate the background radiation in

soil and jarrah shoot tissues. ANOVA was performed (Statistical Analysis System,

version 9.2, SAS Institute, Inc.; Cary, NC, USA). Means were separated using the least

significant difference at a 5 % significance level unless otherwise stated.

Supplementary References

31-Watling, R. & Gregory, N.M. Observations on the boletes of the Cooloola

sandmass, Queensland and notes on their distribution in Australia. Proc. Royal

Soc. Queensland 97, 97-128 (1986).

32-Kariman, K., Barker, S. J., Finnegan, P. M. & Tibbett, M. Dual mycorrhizal

associations of jarrah (Eucalyptus marginata) in a nurse-pot system. Aust. J. Bot.

60, 661-668 (2012).

33-Cavagnaro, T. R., Smith, F. A., Lorimer, M. F, Haskard, K. A., Ayling, S. M. &

Smith S. E. Quantitative development of Paris type arbuscular mycorrhizas

formed between Asphodelus fistulosus and Glomus coronatum. New Phytol.

149:105–113. (2001).

34-Rayment, G. E. & Higginson, F. R. Australian Handbook of Soil and Water

Chemical Methods. Inkata Press: Melbourne, Australia (1992).

Page 73: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

56

35-Motomizu, S., Wakimoto, T. & Toei, K. Spectrophotometric determination of

phosphate in river waters with molybdate blue and malachite green. Analyst

108, 361–367 (1983).

36-Jakobsen, I., Abbott, L. K. & Robson, A. D. 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 (1992).

37-Tennant, D. A test of a modified line intersect method estimating root length. J.

Ecol. 63, 995–1001 (1975).

38-Korimbocus, J., Coates, D., Barker, I. & Boonham N. Improved detection of

sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR

assay. J. Vir. Methods 103, 109-120 (2002).

39-Koyama, T., Kato, N., Hibino, T., Kawazu, T., Kimura, T. & Sakka, K. Isolation

and expression analysis of phosphate transporter genes from Eucalyptus

camaldulensis. Plant Biotech. 23, 215-218 (2006).

40-Webster, C. G. Characterization of Hardenbergia Mosaic Virus and Development

of Microarrays for Detecting Viruses in Plants. PhD thesis, Murduch

University, Australia (2008).

41-Cononoco, E. A. Improving Yield of Wheat Experiencing Post Anthesis Water

Deficits through the Use of Shoot Carbohydrate Reserves. PhD thesis. The

University of Western Australia, Australia (2002).

42-Smith, J. S., Villalobos, M. C. & Cottemann, C, M. Quantitative determination of

sugars in various food products. J. Food Sci. 51, 1373-1375 (1986).

43-AOAC, Official Methods of Analysis of the Association of Official Agricultural

Chemists. 16th

edition, 5th

revision, AOAC International, Gaithersburg, MD,

USA (1999).

44-Veneklaas, E., Stevens, J., Cawthray, G., Turner, S., Grigg, A. & Lambers, H.

Chickpea and white lupin rhizosphere carboxylates vary with soil properties and

enhance phosphorus uptake. Plant Soil 248, 187-197 (2003).

45-Cawthray, G. R. An improved reversed-phase liquid chromatographic method for

the analysis of low-molecular mass organic acids in plant exudates. J.

Chromatogr. A. 1011, 233–240 (2003).

Page 74: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

57

Acknowledgments: The authors are grateful to Tim Morald who collected an A.

occidentalis mushroom from a jarrah forest rehabilitation site (2007, Langford Park,

Jarrahdale WA) and Neale Bougher who kindly identified the Austroboletus species.

Authors are also thankful for technical advice from Lindsey Loweth, Gregory

Cawthray, Michael Smirk, Hazel Gaza, Stuart Pearse, Megan Ryan, Hossein Khabaz-

Saberi, Basu Regmi and Robert Creasy. We appreciate the University of Western

Australia postgraduate scholarships (SIRF/UIS) awarded to K.K. and also financial

support and grants from the Centre for Land Rehabilitation at the University of Western

Australia (M.T.) and the Australian Research Council (M.T., P.M.F.).

Author Contributions: The experimental work was carried out by K.K. who also

wrote the initial draft of the manuscript. The intellectual input and experimental design

were by K.K., S.J.B., P.M.F and M.T. The gene quantification assay was carried out

with the intellectual and scientific supervision of R.J. All authors contributed to the

manuscript editing.

Author Information: The GenBank accession numbers of jarrah PHT1 genes were as

follows: EmPHT1;1 (KC172372), EmPHT1;2 (KC172373), EmPHT1;5 (KC172376)

and EmACT1 (KC172377).

Page 75: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

58

Supplementary Table 1. Oligonucleotide primer pairs used

Primer Direction Sequence (5’-3’) Product

size (bp)

EcPT1-F Forward CTTACTACTGGCGCATGAAGATGCC 403

EcPT4-R Reverse GGTGAACCAGTACCCAGGGA

EcPT1-F Forward CTTACTACTGGCGCATGAAGATGCC 574

EcPT5-R Reverse CCCAAAGTTGGCGAAGAAGAAGGT

EcPT2-F Forward GGTTCTTGCTGGACATCGCCT 175

EcPT4-R Reverse GGTGAACCAGTACCCAGGGA

EcPT2-F Forward GGTTCTTGCTGGACATCGCCT 346

EcPT5-R Reverse CCCAAAGTTGGCGAAGAAGAAGGT

Primers used for real-time PCR PCR

efficiency

EmPT1-F Forward GAGCCGTCGAGATGGTGTGTAGA 122 1.82

EmPT1-R Reverse CGACTATCTTGCCACTTCCTCCATTGA

EmPT2-F Forward CGATGAGGTGCCCACTGCT 138 1.96

EmPT2-R Reverse CACCTGCTCGACGACTCCGTAAT

EmPT5-F Forward GGACGATGAGGTGTCCACTGCTT 127 2.00

EmPT5-R Reverse TCTGTAATATTCGGCAACACGGGAAGT

Act2-F Forward GGTCCTGTTCCAACCATCCATGATT 136 1.99

Act2-R Reverse GGTAGAACCACCACTGAGGACAATGT

Page 76: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

59

Chapter 4

Mycorrhizal symbiosis can induce tolerance in jarrah

(Eucalyptus marginata) exposed to toxic pulses of phosphorus

Khalil Kariman1,2

, Susan J. Barker2,3

, Patrick M. Finnegan2,3

and Mark Tibbett1,4

1School of Earth and Environment M087, The University of Western Australia,

Crawley, WA 6009, Australia

2School of Plant Biology M084, The University of Western Australia, Crawley, WA

6009, Australia

3Institute of Agriculture M082, The University of Western Australia, Crawley, WA

6009, Australia

4Department of Environmental Science and Technology (B37), School of Applied

Sciences, Cranfield University, Cranfield, Bedfordshire, MK 43 OAL, England

Abstract

Jarrah plants develop toxicity symptoms upon exposure to elevated phosphorus (P)

conditions like many other plants native to Australia and Sub-Saharan Africa. A nurse-

pot system was developed to establish arbuscular mycorrhizal (AM) and

ectomycorrhizal (ECM) associations with jarrah to investigate their potential role in

conferring tolerance against P toxicity. Two AM fungi (Rhizophagus irregularis and

Sutellospora calospora) along with two putative ECM fungi (Austroboletus

occidentalis and Scleroderma sp.) were used. The P transport dynamics of AM, ECM,

dual (AM + ECM) and non-mycorrhizal seedlings were monitored following two

pulses of P as phosphate. Both putative ECM fungi significantly enhanced the shoot P

content of jarrah plants growing under P-deficient conditions. In addition, S. calospora,

A. occidentalis and Scleroderma sp. all stimulated plant growth significantly. The

results revealed that jarrah is among the most sensitive species to P fertilization, with

toxicity symptoms observed at a shoot P concentration of no more than 5.5 mg P g-1

DW. All inoculated plants had significantly lower phytotoxicity symptoms compared to

non-mycorrhizal controls one week after addition of an elevated P dose of 30 mg P kg-1

soil. The reduced toxicity symptoms in all inoculated plants was apparently correlated

with lower shoot P concentration than NM plants even though the differences were not

statistically significant except for R. irregularis and dually inoculated plants. Following

Page 77: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

60

exposure to toxicity-inducing levels of P, the shoot P concentration was 20 to 30%

lower in plants inoculated with R. irregularis, S. calospora, A. occidentalis or

Scleroderma sp., and 40% lower in the dually inoculated plants compared to non-

mycorrhizal controls. The induced P tolerance in inoculated plants correlated with

lower shoot P concentration, but was not associated with symbiosis type or the extent

of root colonisation. The AM fungus R. irregularis was the most effective at reducing

shoot P concentration under toxicity conditions.

Key words: arbuscular mycorrhiza (AM), ectomycorrhiza (ECM), jarrah, phosphorus

(P), tolerance, P pulse

Introduction

Phosphorus is a macronutrient essential for plant growth. It is a structural component

of nucleic acids and phospholipids, and is involved in many cellular functions such as

energy transfer and the regulation of enzyme activity. The availability of P to plants is,

however, limited in many soils. Australia, Sub-Saharan Africa, tropical Asia and South

America are among the main areas that have P-deficient soils (Handreck et al. 1997;

Runge-Metzger 1995, Smaling 2005; Trolove et al. 2003; Sanchez and Buol 1975). P

deficiency is considered to be the main critical factor determining plant productivity

and species diversity in terrestrial ecosystems (Lambers et al. 2010).

Many plant species have mechanisms to enhance the extraction of P from the soil.

Arbuscular mycorrhiza (AM), ectomycorrhiza (ECM) and cluster root formation are

among the main P acquisition strategies available to plants (Lambers and Shane 2007;

Lambers et al. 2009; Smith and Read 2008). AM fungi are found in the majority of

terrestrial ecosystems. In a survey of the literature, they were shown to colonise 74% of

angiosperm species from 336 plant families representing 99% of flowering plants

(Brundrett 2009). ECM fungi establish the second most widespread form of mycorrhiza

and have an intimate association with many woody plant species from about 30

families (Smith and Read 2008).

Plant species that are adapted to P-deficient soils can be exposed to elevated P

conditions via nutrient flushing, soil wetting, fertilization and also when new

plantations are established on previously fertilised soils having high levels of P. Many

low-P adapted plants, particularly those from Australia and South Africa, develop

toxicity symptoms upon exposure to elevated levels of P. The P toxicity response

occurs in at least some species because they cannot down-regulate their net P uptake,

Page 78: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

61

perhaps as a consequence of having evolved on P-impoverished soils over millions of

years (Lambers et al. 2011; Hawkins et al. 2008; Shane et al. 2004a). P sensitive

species are found in the Fabaceae, Haemodoraceae, Mimosaceae Myrtaceae,

Proteaceae, Rutaceae, and generally are a feature of heaths and other sclerophyllous

plant communities (Hawkins et al. 2008; Grundon 1972; Dell et al. 1987, Handreck

1997; Heddle and Specht 1975; Shane et al. 2004b; Specht 1981; Specht et al. 1977;

Specht and Groves 1966; Thomson and Leishman 2004). Depending on the plant

species (Shane et al. 2004b), development of P toxicity symptoms can occur at a shoot

P concentration of less than 1 mg P g-1

DW, such as in Banksia ericifolia (Parks et al.

2000), or more than 40 mg P g-1

DW, as reported for Telopia speciosissima (Grose

1989).

The response of plants to P fertilisation may be linked to their response to root

colonisation by mycorrhizal fungi, but the details of this relationship are not clear.

Although mycorrhizal symbioses are renowned for increasing nutrients uptake in

nutrient-deficient plants, they also function to favour the growth of plants exposed to

nutrient toxicity conditions (Hildebrandt et al. 2007; Smith and Read 2008).

Mycorrhizal fungi can modify the P uptake in the host plant by inducing the plant to

reduce the expression of genes encoding high-affinity phosphate transporter (PHT)

proteins (Burleigh 2001; Burleigh and Harrison 1999; Karandashov and Bucher 2005;

Liu et al. 1998; Rosewarne et al. 1999). Much more information about shoot P

accumulation and toxicity development is required for plants that are commonly used

in combination with P fertiliser in the restoration of native ecosystems (Koch 2007).

This research was carried out to clarify the possible link between mycorrhizal

associations and P tolerance in Eucalyptus marginata (jarrah), an important restoration

species suspected of high P sensitivity. A nurse-pot system was used to establish

mycorrhizal associations of jarrah with the AM species Rhizophagus irregularis

(Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler comb. nov. and

Scutellospora calospora Nicol. & Gerd. and the putative ECM fungi Austroboletus

occidentalis Watling & N.M. Greg. and Scleroderma sp. The main aims were: (i) to

establish the role of the fungal symbionts in stimulating P uptake under P-deficient

conditions, (ii) to determine the ability of the selected fungi to confer tolerance against

toxic pulses of P, (iii) to reveal possible correlations between the induced tolerance and

the shoot P concentration, the mycorrhizal type and the extent of root colonisation, and

(iv) to ascertain the effect of these fungal species on plant growth.

Page 79: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

62

Materials and Methods

Plant materials, fungal isolates and inoculum production

Jarrah capsules were obtained from a single tree near Dwellingup, Western Australia.

Seeds were released from the capsules by incubating at 42 ºC for three days. The four

fungal isolates used were S. calospora, A. occidentalis and Scleroderma sp. from west

Australian habitats and R. irregularis (DAOM197198), an exotic isolate from Pont

Rouge, Québec, Canada (Stockinger et al. 2009). Details about the fungal isolates and

inoculum production were as previously described (Kariman et al. 2012).

Nurse-pot system

A nurse-pot system was developed for the study of mycorrhizal associations of jarrah

seedlings (Kariman et al. 2012). A polyester mesh bag (40 µm pore size) was filled

with double-pasteurized washed river sand and placed in the centre of a plastic pot

containing the same matrix. The matrix outside the mesh bag was filled with a mixture

of double-pasteurized washed river sand and mycorrhizal inoculums. To provide equal

conditions for all treatments, AM and ECM plants were supplied with sterilized ECM

or AM inoculum, respectively. Non-mycorrhizal (NM) plants also received sterilized

AM and ECM inocula. Four pre-germinated jarrah seeds were planted outside the mesh

bag and designated as nurse seedlings. In this nurse-pot system, extraradical hyphae

from the nurse seedlings can penetrate into the mesh bag soil whereas plant roots

cannot pass through it. The experiment was conducted from June to September 2010 in

an unheated glasshouse with the average daytime temperature of 20 °C. All plants

received the 1 X modified Long Ashton solution minus P once a fortnight started two

weeks after planting (10 mL kg-1

soil): K2SO4 2 mM, MgSO4.7H2O 1.5 mM,

CaCl2.2H2O 3 mM, FeEDTA 0.1 mM, (NH4)2SO4 4 mM, NaNO3 8 mM, H3BO3 46

µM, MnCl2.4H2O 9 µM, ZnSO4.7H2O 8 µM, CuSO4.5H2O 0.3 µM and

Na2MoO4.2H2O 0.01 µM (Cavagnaro et al. 2001). We used sealed pots for this

experiment and watered them to field capacity three times a week. After 10 weeks

growth, four non-mycorrhizal test seedlings were transplanted into the mesh bag and

one test seedling was harvested weekly to check the colonisation (see Kariman et al.

2012 for more details).

Page 80: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

63

P addition and toxicity analysis

After harvesting all four test seedlings for colonisation studies, one nurse seedling

was harvested from each pot for P analysis. One day after harvesting the first seedling,

the first P pulse was added to all nurse-pots at the ratio of 10 mg P kg-1

soil (as KH2PO4

in aqueous solution). A nurse seedling was harvested one day after addition of the P

pulse to measure the shoot P concentration. Seven days later, a second P pulse of 30 mg

P kg-1

soil was added. The plant toxicity symptoms were quantified one week after

adding the second P pulse and seedlings were harvested for growth response and P

accumulation under high P conditions. The P toxicity symptoms (including chlorotic

and necrotic areas on leaves) were quantified by ranking plants into six classes from 0

to 5, where 0 corresponded to the absence of toxicity symptoms; 1 from traces to 20 %

of symptomatic leaf tissue area (SLTA); 2 from 20 to 40 % SLTA; 3 from 40 to 60 %

of SLTA; 4 from 60 to 80 % of SLTA and 5 more than 80 % of SLTA.

Measured quantities of ground dried shoot tissues (about 200 mg) were digested in 5

ml nitric-perchloric acid solution (4:1 v/v) and the P concentration was determined

using a vanado-molybdate yellow method (Jackson 1973). The amount of P that

accumulated in the shoot tissues (mg P g-1

DW) following each P pulse was calculated

based on the differences in shoot P concentration between two subsequent harvests and

was designated as the incremental shoot P concentration.

Experimental design and data analysis

The experiment was conducted in a completely randomized design with three

replicates. There were two AM treatments (R. irregularis and S. calospora), two

putative ECM treatments (A. occidentalis and Scleroderma sp.), a dual treatment (R.

irregularis and Scleroderma sp.) and NM controls. All data were analyzed using the

Statistical Analysis System (SAS) version 9.2 (SAS Institute, Inc.; Cary NC, USA)

software package. Means were separated using LSD at 5% significance level unless

otherwise stated.

Page 81: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

64

Results:

ECM fungi enhance P uptake in jarrah under P-deficient conditions

The washed river sand used for plant culture contained less than 6 mg P kg-1

(Colwell

P, data not shown). Therefore, the seedlings grown in the absence of P addition were

grown under P-deficient conditions according to previous reports on eucalypts (Burgess

et al. 1994; Aggangan et al. 1996). We assessed the shoot P content of seedlings prior

to the addition of P to assess the effect of mycorrhizal symbioses on P nutrition under

P-deficient conditions (Fig. 1). Jarrah plants inoculated with either putative ECM

fungus (A. occidentalis and Scleroderma sp.) had significantly higher shoot P content

than the NM controls and the other mycorrhizal treatments. With the AM fungus S.

calospora, jarrah plants had higher shoot P content than NM controls, or plants

inoculated with R. irregularis or the dually inoculated plants, although this difference

was not statistically significant.

Fig. 1. Shoot P content of jarrah seedlings growing under P-deficient conditions after

14 weeks growth. Plants were inoculated with the indicated fungi (except the un-

inoculated control plants). The dual treatment was co-inoculated with R. irregularis

(AM) and Scleroderma sp. (ECM). Bars labelled with different letters are significantly

different at p < 0.05 (n = 3).

bb

b

a

a

b

0

0.5

1

1.5

2

2.5

Sh

oo

t P

co

nte

nt (m

g P

pla

nt-1

)

Page 82: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

65

P sensitivity in jarrah is dependent on mycorrhizal fungi

Fourteen-week-old jarrah seedlings developed mild and patchy symptoms of P

toxicity within three days of being exposed to 10 mg P kg-1

soil. The symptoms were

more severe in plants exposed to a higher dose of 30 mg P kg-1

soil 7 d after the first

dose. Irregular chlorotic spots appeared mainly around the midrib and progressed

toward the leaf margins (Fig. 2). However, the pattern and development of symptoms

differed among individual plants. All plants inoculated with potential mycorrhizal

partners had significantly reduced toxicity symptoms when examined one week after

the second P addition compared to NM control plants (Fig. 3). The extent of the

toxicity symptoms did not differ between plants inoculated with different fungi.

Fig. 2. Development of P toxicity symptoms on an individual leaf from an NM

seedling: a) one day before (rank 0), (b) 3 d after (rank 1) and c) 7 d after the second P

dose (rank 3). Fourteen-week-old jarrah seedlings were exposed to a single dose of 10

mg P kg-1

soil for 7 d before subjecting to a second dose of 30 mg P kg-1

soil for 7 d.

Fig. 3. The average phytotoxicity rank of jarrah plants 7 d after subjecting to the second

dose of P. Plants were inoculated with the indicated fungi and grown as described in

the legends for Fig. 1. Values are the average and standard error (n = 3). Bars labelled

with the same letter are not significantly different at p < 0.05.

a

bb b

b b

0

0.5

1

1.5

2

2.5

3

3.5

4

Ph

yto

tox

icit

y ra

nk

a b c

Page 83: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

66

Mycorrhizal fungi ameliorate the toxic accumulation of P in jarrah shoots

The shoot P concentration of jarrah plants was determined before and after adding

two pulses of P (Fig. 4). Although the P content of plants under ECM treatments was

significantly greater than those in NM control, AM and dual treatment plants (Fig. 1),

the size differences among the plants in the various treatments caused the shoot P

concentration prior to P fertilization to be less variable (Fig. 4, open bars). Generally,

there was a decreasing trend in shoot P concentration for plants inoculated with fungi

compared to NM plants one week after the addition of the second dose of P (Fig. 4,

closed bars). The highest and lowest shoot P concentration belonged to NM control

plants and dually-inoculated plants, respectively, although these plants did not differ

significantly in shoot P concentration prior to the addition of the first P pulse (Fig. 4).

The shoot P concentration was 20 % to 30 % lower in plants inoculated with R.

irregularis (29 %), S. calospora (21%), A. occidentalis (22 %), Scleroderma sp. (19%)

and 42 % lower in the dual treatment (R. irregularis and Scleroderma sp.) compared to

the NM seedlings. There were no toxicity symptoms in any of the treatments one day

after adding the first P pulse. In each case, the shoot tissues had a concentration of less

than 1.8 mg P g-1

DW. The severe P toxicity symptoms in plants one week after adding

the second P pulse (Fig. 3) correlated with shoot P concentrations ranging from 5.5 to

9.5 mg P g-1

DW. Thus, the P toxicity symptoms developed at a shoot P concentration

somewhere between 1.8 and 5.5 mg P g-1

DW. R. irregularis alone and in combination

with Scleroderma sp. was the most effective in preventing shoot P accumulation

following addition of the second P pulse.

Page 84: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

67

Fig. 4 Shoot P concentration of jarrah seedlings one day before first P addition (open

bars), one day after first P addition (checkered bars) and 7 d after the second P addition

(closed bars). Plants were inoculated with fungi and grown as indicated in the legend to

Fig. 1. Bars from each harvest labelled with different letters are significantly different

at the p < 0.05 level (n = 3).

The incremental increase in shoot P concentration was lower in mycorrhizal plants

The incremental increase in shoot P concentration in NM plants after one day

exposure to P fertilization was about 1 mg g-1

DW (Fig. 5). The level of shoot P was

significantly lower in plants inoculated with the putative ECM isolates A. occidentalis

and Scleroderma sp. compared to the NM controls and the other mycorrhizal

treatments. At this sampling time, the NM control plants had the greatest increase in

shoot P concentration, while the plants inoculated with Scleroderma sp. had lowest

increase in shoot P concentration (closed bars, Fig. 5).

One week after the second P addition, shoots of NM control plants had an

incremental increase in P concentration of about 7.5 mg g-1

DW (Fig. 5, open bar).

However, at this sampling point, plants inoculated with R. irregularis and the dual

inoculum had significantly smaller increases in shoot P concentration than the NM

control plants (Fig. 5, open bars). Plants inoculated with S. calospora, A. occidentalis

and Scleroderma sp. also had smaller increases in shoot P concentration than the NM

controls, but these differences were not statistically significant.

ab b abab

aab

a a a aa

a

a

bab ab

ab

b

0

2

4

6

8

10

12

Sh

oo

t P

co

ncen

tra

tio

n (m

g g

-1D

W)

Page 85: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

68

Fig. 5 Incremental increase in shoot P concentration for jarrah seedlings 1 d after the

first P addition (closed bars) and 7 d after the second P addition (open bars). Plants

were inoculated with fungi and grown as indicated in the legend to Fig. 1. Bars of each

type labelled with the same letter are not significantly different at the p <0.05 level

(n=3).

Mycorrhizal fungi enhanced jarrah biomass production

Inoculation of jarrah with S. calospora, A. occidentalis or Scleroderma sp. caused a

significant increase in the root and shoot dry mass of jarrah plants after 16 weeks

growth and two P fertilisations, compared to NM controls (Fig. 6). Jarrah plants

inoculated with R. irregularis alone or in combination with Scleroderma sp. had

significantly lower shoot and root biomass compared to S. calospora, A. occidentalis

and Scleroderma sp. treatments (P < 0.1). A significant depression of root system

growth was observed in seedlings co-inoculated with R. irregularis and Scleroderma

sp.

a ababc

bcc

abc

a

bab ab

ab

b

0

1

2

3

4

5

6

7

8

9

10In

cre

men

tal s

ho

ot P

co

ncen

tra

tio

n

(mg g

-1D

W)

Page 86: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

69

Fig. 6 Shoot and root dry mass of jarrah plants after 16 weeks growth and two P

fertilisations. Plants were inoculated with fungi and grown as indicated in the legend to

Fig. 1. ** and * represent significant differences from the control plants at p < 0.05 and

p < 0.10 (n = 3), respectively.

Discussion

Mycorrhiza and P uptake under P-deficient conditions

The presence of the putative ECM fungi A. occidentalis and Scleroderma sp. caused a

dramatic increase in shoot P content of jarrah plants grown under P-deficient conditions

in keeping with the existing literature on other eucalypts (Bougher et al. 1990, Jones et

al. 1998). As previously described (Kariman et al. 2012), the mycorrhizal colonisation

of jarrah plants were 2.3%, 29%, and 28.3% for R. irregularis (AM), S. calospora

(AM), and Scleroderma sp. (ECM) treatments, respectively. The dual treatment had

less than 1% AM and no ECM colonisation and A. occidentalis did not colonise jarrah

roots. The facilitation of P uptake by A. occidentalis is remarkable as this fungus did

not form mycorrhizal structures with jarrah roots (Kariman et al. 2012). These results

indicate that the P uptake of plants does not necessarily correlate with the root

colonisation ability of ECM isolates. Other potential factors could be behind the

enhanced P uptake by jarrah in the presence of A. occidentalis including the exudation

by the fungus, the plant or both of organic acids to release the P from primary minerals

(Landeweert et al. 2001) or of P-mobilizing enzymes that release P from soil organic

** ** **

*

*

****

0

0.5

1

1.5

2

2.5

3

3.5

Dry

ma

ss (

g p

lan

t-1)

Root Shoot

Page 87: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

70

matter (Bending and Read 1995). In the case of plant exudation, this would require a

fungus-dependent elicitor, as the fungus was required for the enhanced P acquisition.

Neither AM fungus caused an increase in the shoot P content of jarrah plants growing

under P-deficient conditions. This result is in agreement with a report that AM fungi

have a low capacity to improve the P nutrition of E. coccifera (Jones et al. 1998). Even

though S. calospora had higher root colonisation than R. irregularis, the other AM

species examined, the shoot P content did not differ significantly between the two AM

treatments. In the dual treatment, the AM fungus (R. irregularis) seemingly dominated

the outcome, as there was neither an increase in the shoot P content nor a positive

growth response. This outcome suggests that ECM associations may not form and thus

would not provide a growth and nutritional benefits in the presence of non-compatible

AM-eucalypt symbiosis.

Mycorrhiza and tolerance to P toxicity

All four fungal isolates tested induced tolerance to P toxicity in jarrah, as judged by

the reduction in the P toxicity symptoms. The induced tolerance was associated with a

lower shoot P concentration in the inoculated plants. The shoot P concentration of

jarrah plants growing under P-deficient conditions was less than 1.5 mg P g-1

DW in

keeping with previous reports for jarrah (Dell et al. 1987) and E. urophylla (Aggangan

et al. 1996). The symptoms of P toxicity in jarrah had developed at a shoot P

concentration between 1.8 and 5.5 mg P g-1

DW, indicating that jarrah is a highly P-

sensitive species compared to other plants (Shane et al. 2004b and references therein).

A single dose of P fertiliser equivalent to an elemental dose of 40 kg Ha-1

is routinely

applied to jarrah forest rehabilitation sites (Koch and Samsa 2007). While it is

impossible to exactly correlate this field dose with our pot growth experiments,

especially as the soil conditions differ, it is clear from our results that a very low dose

of P can have a harmful effect on jarrah, especially if the plants are not involved in

mycorrhizal associations. This conclusion then produces an important consideration for

the management of jarrah forest restoration.

The observed shoot P concentrations and incremental increases in shoot P

concentration after P fertilisations indicate that jarrah plants had a reduced shoot P

concentration under elevated P conditions in response to all four fungal species in spite

of very low colonisation (R. irregularis and dual treatments) or no colonisation (A.

occidentalis treatment). One potential explanation for these results is that colonisation

caused a down-regulation of P acquisition capacity in jarrah. It is well established in

Page 88: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

71

other plants that PHT proteins located in the plasmalemma of root epidermal cells and

root hairs that form the direct pathway of P uptake are down-regulated following

mycorrhizal colonisation (Burleigh 2001; Burleigh and Harrison 1999; Karandashov

and Bucher 2005; Liu et al. 1998; Rosewarne et al. 1999). Other potential mechanisms

might be involved in the observed induction of P tolerance such as a “dilution effect”

resulting from higher shoot biomass in some inoculated plants (S. calospora, A.

occidentalis and Scleroderma sp. treatments) or the accumulation of nutrients (here, P)

in fungal hyphae, as reported for certain nutrients and heavy metals (Hildebrandt et al.

2007). Different combinations of these potential mechanisms of P tolerance might be

active to various extents in the different symbioses.

The lowest jarrah shoot P concentration after P fertilisation was observed in

association with R. irregularis either alone or in combination with Sclorderma sp.

(ECM). These associations had AM colonisation rates of 2.3 and 0.8 %, respectively.

However, S. calospora, the other AM fungus tested here, had a much higher

colonisation rate of 29 % (Kariman et al., 2012) and this was accompanied by higher

trend for shoot P concentration. This could be primary evidence indicating that the AM

fungus provides a significant indirect pathway for P uptake in jarrah. Smith et al.

(2003; 2004) showed that there had been a misevaluation about the contribution made

by mycorrhizal fungi to P uptake by host plants, such that the mycorrhiza-mediated P

uptake could be much higher than previously presumed. Indeed, P can be almost

exclusively supplied to plants via the mycorrhizal pathway for some plant-fungus

combinations (Smith et al. 2003, 2004).

Our results indicate that the AM fungus R. irregularis is more effective than the

tested ECM isolates at reducing the jarrah shoot P concentration during a long term

exposure to elevated P conditions. Both putative ECM fungi had significantly lower

incremental increases in shoot P concentration one day after adding the first P pulse

compared to other treatments showing that the P has been provided to ECM plants at a

much lower rate during this short time. Conversely, after 7 day exposure to an elevated

P supply, plants associated with R. irregularis alone or together with Scleroderma sp.

had a significantly lower incremental increase in shoot P concentration. These results

suggest that this AM partner may have a low capacity to provide P for the host plant

than the ECM partners.

One implication of our findings is that pre-inoculation of jarrah seedlings in nurseries

with both AM and ECM fungi would be helpful in minimizing the P toxicity response.

This would be applicable where new plantations are being established in soils over-

Page 89: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

72

fertilized with P. However, the AM fungus R. irregularis had no positive effects on

jarrah shoot and root biomass. Furthermore, R. irregularis inhibited ECM colonisation

and function (Kariman et al. 2012) in dually inoculated plants, resulting in the smallest

root system amongst treatments, which would affect the establishment and anchoring of

plants. Therefore, for optimal plantation or restoration success, further work is needed

to select fungal isolates that induce both positive growth responses and P tolerance in

jarrah.

Biomass production

ECM plants produced the largest biomass among the treatments regardless of their

colonisation ability confirming the studies showing substantial effects of ECM fungi on

eucalypt growth (Bougher et al. 1990; Jones et al. 1998; Thomson et al. 1994). To date,

several studies have shown significant contribution of fungi to plant growth without

forming any mycorrhizal structures or by forming non-typical structures (Neumann

1959; Kope and Warcup 1986; Warcup and McGee 1983; Kariman et al. 2012).

Two AM fungi had contrasting effects on jarrah growth and the positive growth

response was only observed with S. calospora. No significant growth response was

observed when plants were inoculated with the non-indigenous AM fungus R.

irregularis alone or in combination with Scleroderma sp. These plants had less than 1

% AM colonisation. Moreover, there was a significant depressive effect on root system

growth in the dually inoculated plants. This could be an example of a parasitic rather

than a mutualistic behaviour of the mycosymbionts (Johnson et al. 1997). The positive

growth response of jarrah to S. calospora was accompanied by a much higher

colonisation rate than observed for R. irregularis and dually inoculated plants.

Nevertheless, the extent of root colonisation does not necessarily have a direct

correlation with positive growth and nutritional responses (Smith et al. 2004; Jakobsen

1995).

The response of eucalypts to AM inoculation has been a matter of controversy during

the past decades due to inconsistent results. Gomez et al. (1987) observed no growth

stimulation in eight Eucalyptus species three months after inoculation with 30 AM

isolates. Other studies, however, reported positive effects of AM fungi on growth of

different Eucalyptus species (Adjoud et al. 1996; Chen et al. 2000). This study

confirms the inconsistency of growth response in AM-eucalypt symbioses by showing

that two different AM isolates have opposite effects on jarrah. The mentioned studies

suggest that the growth response in AM-eucalypt symbioses is strongly dependent on

Page 90: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

73

the AM fungus (e.g. different fungal nutrient demands or different life history

strategies), the plant species and the experimental conditions.

In conclusion, we demonstrated that both AM and ECM symbionts could induce

tolerance to elevated P in jarrah plants by reducing the shoot P concentration. The

protective effect was independent of the type of fungal association and the extent of

mycorrhizal colonisation. Following exposure to elevated P conditions, inoculated

plants had 20 to 40 % lower shoot P concentrations compared to NM controls.

Moreover, ECM species formed more efficient symbioses with jarrah plants in terms of

plant growth benefits and P nutrition under P-deficient conditions. Finally, the findings

suggest that mycorrhization is a potential strategy to reduce the development of P

toxicity symptoms in P sensitive plant species.

Acknowledgments

The authors appreciate postgraduate SIRF and UIS scholarships awarded to KK by

the University of Western Australia and also the financial support and grants from the

Centre for Land Rehabilitation and Australian Research Council.

Literature Cited

Adjoud D, Plenchette C, Halli-Hargas R, Lapeyrie F (1996) Response of 11 eucalyptus

species to inoculation with three arbuscular mycorrhizal fungi. Mycorrhiza 6:129-

135

Aggangan NS, Dell B, Malajczuk N (1996) Effects of soil pH on the ectomycorrhizal

response of Eucalyptus urophylla seedlings. New Phytol 134: 539-546

Bending GD, Read DJ. (1995) The structure and function of the vegetative mycelium

of ectomycorrhizal plants VI. Activities of nutrient mobilising enzymes in birch

litter colonised by Paxillus involutus (Fr.) Fr. New Phytol 130: 411-417

Bougher NL, Grove TS and Malajczuk N (1990) Growth and phosphorus acquisition of

karri (Eucalyptus diversicolor F. Muell.) seedlings inoculated with ectomycorrhizal

fungi in relation to phosphorus supply. New Phytol 114:77-85

Brundrett MC (2009) Mycorrhizal associations and other means of nutrition of vascular

plants: understanding the global diversity of host plants by resolving conflicting

information and developing reliable means of diagnosis. Plant Soil 320: 37-77

Burgess T, Dell B, Malajczuk N (1994) Variation in mycorrhizal development and

growth stimulation by 20 Pisolithus isolates inoculated on to Eucalyptus grandis W

Hill ex Maiden. New Phytol 127: 731-739

Page 91: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

74

Burleigh SH (2001) Relative quantitative RT-PCR to study the expression of plant

nutrient transporters in arbuscular mycorrhizas. Plant Sci 160: 899–904

Burleigh SH, Harrison MJ (1999) The down-regulation of Mt4-like genes by phosphate

fertilization occurs systemically and involves phosphate translocation to the shoots.

Plant Physiol 119: 241-248

Cavagnaro TR, Smith FA, Lorimer MF, Haskard KA, Ayling SM, Smith SE (2001)

Quantitative development of Paris type arbuscular mycorrhizas formed between

Asphodelus fistulosus and Glomus coronatum. New Phytol 149: 105–113

Chen YL, Brundrett MC, Dell B (2000) Effects of ectomycorrhizas and vesicular–

arbuscular mycorrhizas, alone or in competition, on root colonisation and growth of

Eucalyptus globulus and E. urophylla. New Phytol 146: 545–556

Dell B, Jones S, Wilson SA (1987) Phosphorus nutrition of jarrah (Eucalyptus

marginata) seedlings. Plant Soil 97: 369-379

Gomez TCR, Faria LP, Lin MT (1987) Mycorrhization of eight species of Eucalyptus

with VAM fungi, Durban, South Africa. pp. 86–93

Grose MJ (1989). Phosphorus nutrition of seedlings of Waratah, Telopea speciosissima

(Sm.) R.Br. (Proteaceae). Aust J Bot 37: 313-320

Grundon NJ (1972) Mineral nutrition of some Queensland heath plants. J Ecol 60: 171-

18

Handreck KA (1997) Phosphorous requirements of Australian native plants. Aust J Soil

Res 35: 241-289

Hawkins HJ, Hettasch H, Mesjasz-Przybylowicz J, Przybylowicz W, Craner MD

(2008) Phosphorus toxicity in the Proteaceae: A problem in post-agricultural lands.

Sci Hortic 117: 354–365

Heddle EM, Specht RL (1975) Dark island heath (90-mile plain, South Australia).8.

Effect of fertilizers on composition and growth, 1950-1972. Aust J Bot 23: 151-164

Hildebrandt U, Regvar M, Bothe H (2007). Arbuscular mycorrhiza and heavy metal

tolerance - Phytochem 68: 139-146

Jackson ML (1973) Soil chemical analysis. Parentice Hall India Pvt Ltd, New Delhi

Jakobsen I (1995) Transport of phosphorus and carbon in VA mycorrhizas. In

'Mycorrhiza, structure, function, molecular biology and biotechnology'. (Eds A

Varma and B Hock) pp. 297-324. (Springer Verlag: Berlin, Germany)

Johnson NC, Graham JH, Smith FA (1997) Functioning of mycorrhizal associations

along the mutualism-parasitism continuum. New Phytol 135: 575-586

Jones MD, Durall DM, Tinker PB (1998) Comparison of arbuscular and

ectomycorrhizal Eucalyptus coccifera: growth response, phosphorus uptake

efficiency and external hyphal production. New Phytol 140: 125-134

Page 92: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

75

Karandashov V, Bucher M (2005) Symbiotic phosphate transport in arbuscular

mycorrhizas. Trends Plant Sci 10: 22-29

Kariman K, Barker SJ, Finnegan PM, Tibbett Mark (2012) Dual mycorrhizal

associations of jarrah (Eucalyptus marginata) in a nurse-pot system. Aust J Bot 60:

661-668

Koch JM (2007) Alcoa’s mining and restoration process in South Western Australia.

Rest Ecol 15: S11-S16

Koch JM, Samsa GP (2007) Restoring jarrah forest trees after bauxite mining in

Western Australia. Rest Ecol 15: S17–S25

Kope HH, Warcup JH (1986) Synthesised ectomycorrhizal associations of some

Australian herbs and shrubs. New Phytol 104: 591–599

Lambers H, Shane MW (2007) Role of root clusters in phosphorus acquisition and

increasing biological diversity in agriculture. In: Spiertz JHJ, Struik PC, van Laar

HH (eds) Scale and complexity in plant systems research: Gene-plant-crop

relations. Springer, Dordrecht, Netherlands, pp 237–250

Lambers H, Mougel C, Jaillard B, Hinsinger P (2009) Plant-microbe-soil interactions in

the rhizosphere: an evolutionary perspective. Plant Soil 321: 83-115

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, Finnegan PM, Laliberté E, Pearse SJ, Ryan MH, Shane MW, Veneklaas EJ

(2011) Phosphorus nutrition of Proteaceae in severely phosphorus-impoverished

soils: are there lessons to be learned for future crops? Plant Physiol 156: 1058-1066

Landeweert R, Hofflund E, Finlay RD, Breeman N (2001) Linking plants to rocks:

Ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol 16: 248-

254.

Li HY, Zhu YG, Marschner P, Smith FA, Smith SE (2005) Wheat responses to

arbuscular mycorrhizal fungi in a highly calcareous soil differ from those of

clover, and change with plant development and P supply. Plant Soil 277: 221-232

Liu H, Trieu AT, Blaylock LA, Harrison MJ (1998) Cloning and characterization of

two phosphate transporters from Medicago truncatula roots: Regulation in response

to phosphate and to colonisation by arbuscular mycorrhizal (AM) fungi. Mol Plant

Microbe Interact 11: 14-22

Neumann R (1959) Relationships between Pisolithus tinctorius (Mich. ex. Pers) Coker

et Couch. and Eucalyptus camaldulensis Dehn. Bull Res Counc Zsr Sect D Bot 7:

116-120

Parks SE, Haigh AM, Creswell GC (2000) Stem tissue phosphorus as an index of the

phosphorus status of Banksia ericifolia L. Plant Soil 227: 59-65

Page 93: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

76

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

Runge-Metzger A (1995) Closing The Cycle: Obstacles To Efficient P Management

For Improved Global Food Security. in SCOPE 54 -Phosphorus in the Global

Environment -Transfers, Cycles and Management

Sanchez PA, Buol SW (1975) Soils of the tropics and the world food crisis. Science

188: 598-603

Shane MW, Szota C, Lambers H (2004a) A Root trait accounting for the extreme

phosphorus sensitivity of Hakea prostrata (Proteaceae). Plant Cell and Environ 27:

991-1004

Shane MW, McCully ME, Lambers H (2004b) Tissue and cellular phosphorus storage

during development of phosphorus toxicity in Hakea prostrata (Proteaceae). J Exp

Bot 55: 1033-1044

Smaling E (2005) Harvest for the world, Inaugural address, International Institute for

Geo-Information Science and Earth Observation, 2 November 2005, Enschede, The

Netherlands

Smith SE, Read DJ (2008) Mycorrhizal symbiosis. 3rd

ed Academic Press, London UK

Smith SE, Smith FA, Jakobsen I (2003) Mycorrhizal fungi can dominate phosphate

supply to plants irrespective of growth responses. Plant Physiol 133: 16-20

Smith SE, Smith FA, Jakobsen I (2004) Functional diversity in arbuscular mycorrhizal

(AM) symbioses: the contribution of the mycorrhizal P uptake pathway is not

correlated with mycorrhizal responses in growth or total P uptake. New Phytol 162:

511-524

Specht RL (1981) Nutrient release from decomposing leaf litter of Banksia ornata,

Dark Island heathland, South Australia. Aust J Ecol 6: 59-63

Specht RL, Conner DJ, Cliford HT (1977) The heath-savanna problem: the effect of

fertilizer on sand-heath vegetation of North Stradbroke Island, Queensland. Aust J

Ecol 2: 179-186

Specht RL, Groves RH (1966) A comparison of phosphorus nutrition of Australian

heath and introduced econimic plants Aust J Bot 14: 201-221

Stockinger H, Walker C, Schussler A (2009) 'Glomus intraradices DAOM197198', a

model fungus in arbuscular mycorrhiza research, is not Glomus intraradices. New

Phytol 183: 1176-1187

Thomson BD, Grove TS, Malajczuk N, Hardy GESJ (1994) The effectiveness of

ectomycorrhizal fungi in increasing the growth of Eucalyptus globulus Labill. in

relation to root colonisation and hyphal development in soil. New Phytol 126: 517-

524

Page 94: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

77

Thomson VP, Leishman MR (2004) Survival of native plants of Hawkesbury

Sandstone communities with additional nutrients: effect of plant age and habitat.

Aust J Bot 52: 141-147

Trolove SN, Hedley MJ, Kirk GJD, Bolan NS, Loganathan P (2003) Progress in

selected areas of rhizosphere research on P acquisition. Aust J Soil Sci 41: 471–499

Warcup JH, McGee PA (1983) The mycorrhizal associations of some Australian

Asteraceae. New Phytol 95: 667-672

Page 95: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

78

Page 96: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

79

Chapter 5

Symbiotic associations and response of jarrah (Eucalyptus

marginata) to phosphate, phosphite and arsenate toxicities

Khalil Kariman1,2

, Susan J. Barker2,3

, Ricarda Jost2 Patrick M. Finnegan

2,3and Mark

Tibbett1,4

1School of Earth and Environment, The University of Western Australia, Crawley, WA

6009, Australia

2School of Plant Biology, The University of Western Australia, Crawley, WA 6009,

Australia

3Institute of Agriculture, The University of Western Australia, Crawley, WA 6009,

Australia

4Department of Environmental Science and Technology (B37), School of Applied

Sciences, Cranfield University, Cranfield, Bedfordshire, MK 43 OAL, England

Jarrah (Eucalyptus marginata) is an Australian native tree with the capacity to form

arbuscular mycorrhizal (AM), ectomycorrhizal (ECM) and novel symbiotic

associations during its life cycle. Many plant species adapted to P-impoverished soils

including jarrah develop toxicity symptoms upon exposure to high doses of phosphate

(Pi) and its analogues. The present study was undertaken to investigate the effects of

Scutellospora calospora (AM), Scleroderma sp. (ECM) and Austroboletus occidentalis

(novel symbiosis) on response of jarrah to highly toxic pulses (1.5 mmol kg-1

soil) of

Pi, phosphite (Phi) and arsenate (AsV). The AM colonisation of jarrah plants declined

after 14 weeks growth, which is a common phenomenon occurring in early stages of

AM-eucalypt symbioses. However, addition of Pi and Phi pulses reversed this natural

trend, with the AM colonisation increasing after four weeks by six-fold and two-fold,

respectively, compared to untreated AM plants. There was no root colonisation for

either Scleroderma sp. or A. occidentalis treatments. However, all inoculated plants had

significantly higher shoot and root dry biomass and shoot P content under P-deficient

conditions compared to non-mycorrhizal (NM) plants indicating that both non-AM

fungi used novel symbiotic pathway that did not involve colonisation. Tolerance to Pi

toxicity was only observed for the AM treatment while all fungal treatments could

induce tolerance against the Phi toxicity. The induced Pi tolerance was not correlated

with lower shoot P concentration or content of AM plants while the Phi tolerance was

Page 97: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

80

achieved because of lower Phi uptake in all inoculated plants. All plants were dead a

week after the AsV pulse even though inoculated plants had significantly lower shoot

As concentration than NM controls. The expression profile of plant high affinity

phosphate transporter (PHT1 family) genes was not altered in response to any of the

fungal species tested. Interestingly, the transcript abundance for four PHT1 genes

(EmPHT1;1, EmPHT1;2, EmPHT1;3 and EmPHT1;4) increased in roots of NM plants

in response to the toxic Pi dose and the transcript abundance for three genes

(EmPHT1;1, EmPHT1;3 and EmPHT1;4) increased in response to the toxic Phi and

AsV doses. Our findings support the hypothesis that P-sensitive species are not able to

down-regulate the expression of their PHT1 genes when exposed to high P conditions

and expression of some PHT1 genes actually increases.

Introduction

Phosphorus (P) deficiency is common in many soils around the world including

Australia, Sub-Saharan Africa, tropical regions in Asia and South America (Sanchez

and Buol, 1975; Runge-Metzger, 1995; Handreck et al., 1997; Trolove et al., 2003;

Smaling, 2005). Adaptation to low P soils could potentially be linked to P sensitivity

across native plant communities. Many Australian native species develop phytotoxicity

symptoms when exposed to high levels of phosphate (Pi) and its analogues such as

phosphite (Phi) and arsenate (AsV) (Handreck, 1997; Howard et al., 2000; Barrett,

2001; Tynan et al., 2001; Smith et al., 2003; Shane et al., 2004; Thomson and

Leishman, 2004; Hawkins et al., 2008). reads: According to Handreck (1997), an Olsen

extractable P concentration (inorganic plus organic P in the extract) of about 20 mg P

kg-1

soil is lethal to the seedlings of P-sensitive native species.

Inorganic Pi is the primary source of P that plants uptake. Phosphite, which is

currently the only available treatment to effectively combat Phytophthora cinnamomi

‘dieback’ in plants (Dell et al., 2005), is generally considered to be a non-metabolizable

form of P as it cannot be assimilated into organic P compounds by plants (Guest and

Grant, 1991). Therefore, application of Phi can result in development of phytotoxicity

reactions in plants (Sukarno et al., 1993, 1996, 1998; Ticconi et al., 2001; Varadarajan

et al., 2002). However, there are also reports showing positive nutritional effects of Phi

on plants (Jabahi-Hare and Kendrick, 1987; Lovatt and Mikkelsen, 2006), which are

almost certainly due to microbe-mediated oxidation of Phi to Pi in soil (Ohtake et al.,

1996). Arsenate and arsenite (AsIII) are the most common oxidation states of As found

Page 98: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

81

in nature. Contamination of groundwater and soil by As compounds is a result of

natural processes such as eruption of volcanoes and erosion of mineral deposits or

anthropogenic activities such as mining, agriculture (application of arsenical pesticides

and herbicides), forestry and drilling (Smith et al., 2003). In the cell, AsV can disturb

the energy production through respiration by substituting for P in the production of

ATP, forming a ADP-As complex that uncouples ATP synthesis (Meharg and Hartley-

Whitaker, 2002; Finnegan and Chen 2012). Phosphite and AsV enter plant roots

through the same mechanism as Pi i.e. Pi transporters of the PHT1 family located in the

plasma membrane of epidermal and root hair cells (Guest and Grant, 1991; Meharg and

Macnair, 1992; Finnegan and Chen, 2012).

The toxicity induced by Pi, AsV and Phi might be affected by soil biological

properties such as mycorrhizas. Symbiotic associations have been shown to induce

tolerance against high P conditions in jarrah (Eucalyptus marginata Donn ex Sm.)

(Kariman et al., 2012). Mycorrhizal fungi have the capability to induce the down-

regulation of PHT1 genes transcript levels in roots (Karandashov and Bucher, 2005).

Therefore, there might be a link between tolerance to Pi, Phi and AsV toxicities in

mycorrhizal plants and the expression of PHT1 genes located in root epidermal cells.

Jarrah plants can form arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM)

associations along with a novel symbiosis involving Basidiomycete fungi, in which

root colonisation does not occur (Kariman et al., in preparation: Chapter 3). ECM and

novel symbioses can substantially improve the growth and nutrition of eucalypts (Jones

et al., 1998; Chen et al., 2000, Kariman et al., 2012; Kariman et al., in preparation:

Chapter 3). However, the AM-eucalypt symbiosis is not always accompanied by

growth and nutritional benefits and the positive responses may depend on the plant-

fungus species combination and origin and also on the experimental conditions (Gomez

et al., 1987; Muchovej and Amorim, 1990; Jones et al., 1998; Chen et al., 2000;

Kariman et al., 2012).

Experiments were conducted to investigate whether the toxic effects of Pi, Phi and

AsV on jarrah could be moderated by mycorrhizal symbioses. We hypothesize that the

expression of plant PHT1 genes (at a transcript level) would be affected by symbiotic

associations or toxicity conditions. To answer these questions we grew jarrah plants

alone or in symbiosis with Scutellospora calospora (Nicol. & Gerd.) (AM),

Scleroderma sp. (ECM) or A. occidentalis (Watling & N.M. Greg.) (novel symbiosis,

Chapter 3) to i) determine the growth and P nutrition of non-mycorrhizal (NM) and

inoculated jarrah plants under P-deficient conditions, ii) clarify the role of selected

Page 99: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

82

fungi in inducing tolerance against toxic pulses of Pi, Phi and AsV by monitoring the P

and As uptake after short and long term exposures and iii) quantify the transcript

abundance from five PHT1 genes in jarrah roots in response to different symbiotic

associations or toxic pulses of Pi, Phi and AsV.

Results

Plant biomass

Jarrah seedlings inoculated with each of three fungal species had significantly higher

shoot and root dry biomass compared to NM controls after 18 weeks growth under P-

deficient conditions. There was no significant difference between the shoot or root

biomass among different inoculated treatments.

Fig. 1. Shoot and root dry biomass of jarrah plants after 18 weeks growth under P-

deficient conditions. Bars labelled with different letters are significantly different at p <

0.05. Error bars are SE (n = 3).

Mycorrhizal colonisation

There was no root colonisation for A. occidentalis and Scleroderma sp. treatments

and both fungi used a novel symbiotic pathway as they conferred growth (Fig. 1) and P

nutritional benefits (Fig. 4B, open bars) to the host. For the AM fungus S. calospora,

there was 12 % colonisation after 14 weeks growth, which dropped down to 3 % four

weeks later (Fig. 2). However, four weeks after the Pi and Phi pulses, the AM

colonisation increased by more than six fold (20%) and two fold (7%), respectively,

compared to untreated AM plants. The AsV-treated plants were harvested seven days

after adding the pulse and the colonisation was unchanged from that of untreated AM

plants at 14 weeks (12%). Considering the colonisation results and growth benefits in

b

aa

a

b

a

a a

1.5

1.0

0.5

0.0

0.5

1.0

1.5

Dry

bio

mas

s (g

pla

nt-1

)

Shoot Root

NM Control Scleroderma sp. A. occidentalisS. calospora

Page 100: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

83

all inoculated plants, there were only AM (S. calospora) and novel symbioses (A.

occidentalis and Scleroderma sp.) occurring in this experiment.

Fig. 2. The percentage of mycorrhizal colonisation in the AM (S. calospora) treatment.

The root colonisation was measured for untreated plants after 14 and 18 weeks growth

and also for 18-week-old plants which were exposed to Pi (closed bar) or Phi (grey bar)

pulses for four weeks or 15-week-old plants that had been exposed to AsV (steriped

bar) for one week. Error bars are SE (n = 3).

Tolerance to phosphate, phosphite and arsenate toxicities

There were no toxicity symptoms in the set of NM seedlings, which were not exposed

to Pi, Phi or AsV pulses. Tolerance to a normally toxic pulse of Pi was only observed

for the AM plants, which had significantly lower toxicity ranks compared to NM plants

one week and four weeks after adding the Pi pulse (Fig. 3A and B). When a toxic pulse

of Phi was applied, slight toxicity symptoms were observed in all treatments one week

after the pulse. At that point the phytotoxicity ranks did not differ significantly between

treatments. Four weeks after the Phi pulse, however, all inoculated plants had

significantly lower toxicity symptoms compared to NM plants. There was no tolerance

against the AsV toxicity, regardless of mycorrhizal status, and all plants had died

within a week after the pulse.

0

5

10

15

20

25

Untreated 14 weeks

Untreated 18 weeks

Phosphate 18 weeks

Phosphite 18 weeks

Arsenate 15 weeks

Per

cen

tag

e o

f A

M c

olo

niz

atio

n

Page 101: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

84

Fig 3. Phytotoxicity ranks of jarrah plants exposed to toxic pulses of Pi, Phi and AsV

one week (A) and four weeks (B) after the pulse. For each treatment, bars labelled with

different letters are significantly different at p < 0.05. Error bars are SE (n = 3).

Shoot P concentration and content in the phosphate treatment

AM plants had significantly higher shoot P concentration compared to other

treatments one day before exposure to the toxic Pi pulse (Fig. 4A, open bars). One day

after the pulse, all inoculated plants had significantly lower shoot P concentration than

NM plants (Fig. 4A, checkered bars). Four weeks after the Pi pulse, AM plants had

significantly higher shoot P concentration than NM controls (Fig. 4A, closed bars)

while there was no significant difference in shoot P concentration between both

Scleroderma sp. and A. occidentalis treatments and NM controls (Fig. 4A, closed bars).

All three fungal species could enhance the shoot P content of plants under P-deficient

conditions compared to NM controls (Fig. 4B, open bars). A day after the Pi pulse, all

plants had nearly the same shoot P content (Fig. 4B, checkered bars). All inoculated

plants had significantly higher shoot P content than NM controls four weeks after

adding the Pi pulse (Fig. 4B, closed bars).

Shoot P concentration and content in the phosphite treatment

There was no significant difference between the shoot P concentrations among

treatments one day after the Phi pulse (Fig. 4C, checkered bars). Four weeks after the

pulse, all inoculated plants had significantly lower shoot P concentration than NM

controls (Fig. 4C, closed bars). The results also revealed that the uptake of Phi is much

slower than Pi as the shoot P concentration of NM plants was less than half of the Pi

treatment during the same exposure periods (1 day and 4 weeks after the pulse).

The A. occidentalis inoculated plants had significantly higher shoot P content than

NM controls after one day of exposure to the Phi pulse (Fig. 4D, checkered bars) while

S. calospora and Scleroderma sp. treatments had slightly (not significant) higher P

a

a

a

b

a

a

a

a

aa

a

a

0

1

2

3

4

5

Phosphate Phosphite Arsenate

Phy

toto

xic

ity r

ank

NM Control S. calospora Scleroderma sp. A. occidentalisA

aa

b

b

a

b

a

b

0

1

2

3

4

5

Phosphate Phosphite

Phyto

toxic

ity r

ank

NM Control S. calospora Scleroderma sp. A. occidentalisB

Page 102: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

85

contents than NM plants. After longer exposure to the pulse (4 weeks), the shoot P

content did not differ significantly across treatments (Fig. 4D, closed bars).

Shoot As concentration and content in the arsenate treatment

All inoculated plants had significantly lower shoot As concentration compared to NM

controls one day and one week after addition of the AsV pulse (Fig. 4E). Inoculated

plants also had lower As content one day after adding the AsV pulse compared to NM

controls (Fig. 4F). However, all plants were dead a week later and there was no

significant difference in shoot As content between treatments.

Page 103: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

86

Fig. 4. Phosphorus and As uptake of NM and inoculated jarrah plants. A and B: shoot P

concentration and content of plants one day before (open bars), one day after

(checkered bars) and four weeks after (closed bar) the Pi pulse. C and D: shoot P

concentration and content one d before (open bars), one day after (checkered bars) and

four weeks after (closed bars) the Phi pulse. E and F: shoot As concentration and

content after one day (checkered bars) and one week (closed bars) exposure to the AsV

pulse. Bars labelled with different letters are significantly different at p < 0.05. Error

bars are SE (n = 3).

b a b b

ab b b

b

a ab

ab

0

2

4

6

8

10

12

14

16

18

NM Control S. calospora Scleroderma sp. A. occidentalis

Sh

oo

t P

co

nce

ntr

atio

n

(mg

g-1

DW

)

A

c a b b

a a a a

b

aa

a

0

2

4

6

8

10

12

14

16

18

NM Control S. calospora Scleroderma sp. A. occidentalis

Sh

oo

t P

co

nte

nt (m

g p

lan

t-1)

B

ba

b b

aa a a

a

b

b

b

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

NM Control S. calospora Scleroderma sp. A. occidentalis

Sh

oo

t P

co

nce

ntr

atio

n(m

g g

-1D

W)

C

c

ab b

bab ab

a

a a

a

a

0

0.5

1

1.5

2

2.5

3

3.5

NM Control S. calospora Scleroderma sp. A. occidentalis

Sh

oot P c

on

ten

t (m

g p

lan

t-1)

D

a b b b

a

b bb

0500

100015002000250030003500400045005000

NM Control S. calospora Scleroderma sp. A. occidentalis

Sh

oo

t As

con

cen

trat

ion

(µg

g-1

DW

)

E

ab b b

a

a

a a

0

500

1000

1500

2000

2500

NM Control S. calospora Scleroderma sp. A. occidentalis

Sh

oo

t As

con

ten

t (µ

g p

lan

t-1)

F

Page 104: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

87

Shoot free Pi concentration

The shoot free Pi concentration was measured to reveal any possible correlation with

the Pi and Phi tolerance observed four weeks after the corresponding pulses (Fig. 5).

Similar to the shoot total P results, the AM plants had the highest free Pi concentration

in their shoot tissues four weeks after the Pi pulse (Fig. 5A). The shoot free Pi

concentration in the Scleroderma sp. treatment was also higher than NM controls while

the A. occidentalis treatment had nearly the same free Pi as NM plants. The difference

between shoot total P and free Pi was less than 3 mg P g-1

DW, which could be

attributed to organic P compounds.

Four weeks after the Phi pulse, NM plants had the highest shoot free Pi concentration

similar to the total P concentrations (Fig. 5B). The detection of a relatively high

proportion of free Pi inside the shoot tissues of the Phi-treated plants shows that a

portion of the Phi has been oxidised to Pi in soil or inside the plant tissues.

Response of EmPHT1 transcript abundance to mycorrhiza and toxicity conditions

The transcript abundance from five jarrah EmPHT1 genes was quantified in 14-week-

old plants prior to addition of the toxic pulses (Fig. 6A). Jarrah plants did not down-

regulate the transcript abundance from these five EmPHT1 genes in response to any of

the fungal treatments.

The transcript abundance of the five EmPHT1 genes was quantified in NM plants one

day after addition of the different toxic pulses (Fig. 6B). None of the EmPHT1 gene

transcripts were reduced in abundance in response to addition of Pi, Phi or AsV.

Interestingly, transcript abundance for certain EmPHT1 genes was increased under

toxicity conditions. Jarrah plants accumulated significantly more transcripts for

EmPHT1;1, EmPHT1;2, EmPHT1;3 and EmPHT1;4 in response to Pi and EmPHT1;1,

EmPHT1;3 and EmPHT1;4 in response to Phi and AsV pulses.

Page 105: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

88

Fig. 5. Shoot free Pi concentration of jarrah plants. Concentration of free Pi inside the

shoot tissues four weeks after the Pi (A) and Phi (B) pulses. Bars labelled with different

letters are significantly different at p < 0.05. Error bars are SE (n = 3).

Fig. 6. Transcript abundance from five EmPHT1 genes in jarrah roots relative to an

internal reference gene (EmACT1) determined by real-time PCR. A: transcript

abundance from EmPHT1 genes in response to three different fungal species. B:

transcript abundance from EmPHT1 genes in NM plants under normal conditions

(closed bars) or one day after addition of Pi (checkered bars), Phi (striped bars) and

AsV (gray bars) pulses. The scale on the vertical axis is a log2 scale based on ΔCt (the

threshold cycle (Ct) of the target EmPHT1 gene minus the Ct of the reference gene). A

difference of one Ct value corresponds to a 2-fold difference in transcript abundance.

Bars with different letters are significantly different at p < 0.05. Error bars are SE (n =

3).

c

aab

bc

0

2

4

6

8

10

12

14

16

NM Control S. calospora Scleroderma sp. A. occidentalis

Shoot fr

ee P

concentr

ati

on

(mg P

g-1

DW

)A

a

bb

b

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

NM Control S. calospora Scleroderma sp. A. occidentalis

Shoo

t fr

ee P

concentr

ati

on

(mg P

g-1

DW

)

B

a a

a

a

a

aa

a

a

a

aa

a

a

a

a a

a

a

a

30

35

40

45

50

EmPHT1;1 EmPHT1;2 EmPHT1;3 EmPHT1;4 EmPHT1;5

Rela

tiv

e e

xp

ressio

n (4

0-Δ

Ct)

NM Control S. calospora Scleroderma sp. A. occidentalisA

b b

b

c

a

a a

a

b

a

a

b a

aa

a

b a

ab

a

30

35

40

45

50

EmPHT1;1 EmPHT1;2 EmPHT1;3 EmPHT1;4 EmPHT1;5

Rela

tive e

xpre

ssio

n (4

0-Δ

Ct)

Untreated Phosphate Phosphite ArsenateB

Page 106: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

89

Discussion

Plant biomass

Inoculation of jarrah with S. calospora, Scleroderma sp. or A. occidentalis

significantly enhanced the shoot and root dry biomass compared to NM controls (Fig.

1) as observed in previous experiments (Kariman et al., 2012; Kariman et al., in

preparation: Chapter 3). In our previous study, both basidiomycete treatments

(Scleroderma sp. or A. occidentalis) had significantly higher shoot biomass than the

AM treatment S. calospora (Kariman et al., 2012). However, in the present work there

was no significant difference between the shoot biomass in different inoculated

treatments. Plants colonised by the AM fungus S. calospora performed much better

than in our previous work in terms of both biomass production and P nutrition under P-

deficient conditions (see below). Variable results including positive, neutral or negative

physiological responses have been documented for eucalypts associated with different

AM fungi (Gomez et al., 1987; Muchovej and Amorim, 1990; Adjoud et al., 1996;

Kariman et al., 2012). However, in our previous work (Kariman et al., 2012) there were

four seedlings in each nurse-pot, which might have caused a competition between

seedlings leading to a slighter positive growth response compared to the present work

having one seedling per pot.

Mycorrhizal colonisation

The A. occidentalis isolate established a novel symbiotic association with jarrah

plants where a growth benefit occurred without root colonisation, as was also observed

previously (Kariman et al., 2012; Kariman et al., in preparation: Chapter 3). In the

earlier study, the ECM isolate Scleroderma sp. formed a classic ECM colonisation only

in one replicate. Two other replicates did not colonise roots and behaved like the A.

occidentalis treatment (Kariman et al., 2012). Here again, in the present study,

Scleroderma sp. behaved like A. occidentalis and did not form mycorrhizal structures

in any of three replicates. These two fungi always provided clear growth and nutritional

benefits regardless of their colonisation ability (Kariman et al., 2012; Kariman et al., in

preparation: Chapter 3). We accordingly suggest Scleroderma sp. as a fungus with dual

functional capacity, sometime behaving like a typical ECM fungus and sometimes

establishing the novel symbiotic association like A. occidentalis.

Our experiments were carried out in an unheated glasshouse. The temperature

dropped down to 6 and 7 ºC in the night time in our nurse-pot study (Kariman et al.,

2012; Chapter 2) and the present work, respectively, and only one replicate (out of 6)

Page 107: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

90

formed ECM colonisation with jarrah roots. However, in our preliminary experiment

(data not shown) and 33

P experiment (Kariman et al., in preparation: Chapter 3) the

minimum temperatures were 14 and 18 ºC, and under those conditions Scleroderma sp.

formed ECM colonisation in all six replicates. Hence, there might be a correlation

between temperature and the dual behaviour of Scleroderma sp. The temperature drop

to below 10 ºC could be a potential signal for the fungus to establish a novel symbiotic

association. Regardless of what drives this dual behaviour, the colonisation results from

our current and previous experiments (Kariman et al., 2012, Chapter 2; Kariman et al.,

in preparation: Chapter 3) revealed that the novel symbiotic association is an alternative

pathway for ECM fungi to provide a benefit to the host plant. In other words, putative

ECM fungi such as Scleroderma sp. have the potential to establish both novel

symbiotic and ECM associations during their symbiotic life cycle. It will be interesting

to determine if similar benefits are gained by other so-called ‘non-mycorrhizal’ plant

species when grown in the company of such fungi.

The AM colonisation of jarrah plants by S. calospora declined after 14 weeks growth

(Fig. 2), which is in line with the existing literature about AM-eucalypt symbioses,

which shows that AM colonisation decreases after seedlings have grown for a while

and is replaced by ECM colonisation (Chen et al., 2000, Adams et al., 2006). Addition

of Pi often leads to a decrease in mycorrhizal colonisation (Jasper and Davy, 1993;

Bobbink, 1998; Cornwell et al., 2001) although other studies show that AM

colonisation was enhanced (Bolan et al., 1984) or it was not affected in higher Pi

conditions (Duke et al., 1994; Kabir et al., 1997). However, in the present study, four

weeks after the addition of the Pi and Phi pulses, the AM colonisation increased by six-

fold and two-fold, respectively, compared to untreated AM plants. Both P sources (Pi

and Phi) somehow reduced the drop in colonisation. Accordingly, we hypothesize that

three-month-old seedling eucalypts do not suppress their AM relationships if they

receive enough or even toxic levels of P.

Inconsistent results have been reported for the effects of Phi treatment on AM

colonisation. Similar to our results, an increase in AM colonisation has been reported

for plant species such as Agonis flexuosa (Howard et al., 2000), leek (Jabahi-Hare and

Kendrick, 1987) and lettuce (Clarke, 1978) after Phi application. The increased AM

colonisation in Phi-treated plants has been attributed to differences in host nutritional

uptake or altered root metabolism (Jabahi-Hare and Kendrick, 1987; Howard et al.,

2000). However, there are some other reports showing that Phi application decreased

AM colonisation in plants such as maize (Seymour et al., 1994) and onion (Sukarno,

Page 108: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

91

1996, 1998). The negative effects of Phi on mycorrhizal colonisation have been

attributed to accumulation of Phi near root tips (Guest and Grant, 1991) resulting in

damage to fine roots and consequently reduction of sites for AM and ECM formation

(Howard et al., 2000). Clearly more research is required to better understand the

responses of plants to Phi, especially the consequence of this treatment to plants

growing in natural habitats.

P uptake under P-deficient conditions

All inoculated plants had significantly higher shoot P content than NM plants under

P-deficient conditions (Fig. 4 B, open bars). In our previous study, the AM jarrah plants

(S. calospora) had slightly higher shoot P content than NM plants but the difference

was not statistically significant (Kariman et al., 2012). Here, S. calospora behaved

extraordinary well compared to our previous study by having the highest shoot P

content among treatments. The EmPHT1 transcript results might clarify the high

performance of S. calospora in terms of P uptake as this AM fungus did not reduce the

expression of any jarrah EmPHT1 genes. Therefore, the shoot P content of the AM

plants might have come from both direct (plant roots) and indirect (mycorrhizal)

pathways but the actual contribution of each pathway is not clear and warrants

additional study.

Tolerance to phosphate toxicity and Pi uptake dynamics

The Pi pulse applied was 1.5 mmol kg-1

soil (equal to 46 mg P kg-1

soil) and Pi

tolerance was only observed for the AM treatment (Fig. 3 A and B). The other two

fungal treatments (Scleroderma sp. and A. occidentalis) did not induce Pi tolerance in

the present study while they were able to induce tolerance against a lower Pi pulse of

30 mg P kg-1

soil (Kariman et al., in preparation: Chapter 4). All inoculated plants in

the present study had significantly lower shoot P concentration than NM plants one day

after the Pi pulse. This was correlated with the higher shoot biomass of inoculated

plants resulting in P dilution within shoot tissues designated as the “dilution effect”.

Interestingly, AM plants had the highest shoot P concentration (about 15 mg P g-1

DW)

four weeks after adding the Pi pulse and still showed the lowest toxicity symptoms

among treatments. Therefore, the Pi tolerance in AM plants is not necessarily

correlated with lower shoot P concentration.

Plants inoculated with Scleroderma sp. and A. occidentalis had lower toxicity

symptoms upon exposure to a lower Pi pulse of 30 mg P kg-1

soil (Kariman et al., in

Page 109: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

92

preparation: Chapter 4), probably because their shoot P concentrations (less than 7.5

mg P g-1

DW) were about 20 % less than in NM plants. In the present experiment,

however, there was no Pi tolerance for these treatments. Plants received a higher pulse

(46 mg P kg-1

soil) and the shoot P concentration was more than 12 mg P g-1

DW. We

thus conclude that the Pi tolerance in both Scleroderma sp. and A. occidentalis

treatments (ECM and novel symbioses) that was previously reported (Kariman et al., in

preparation: Chapter 4) was due to dilution effects and can be seen at Pi pulses of less

than 30 mg P kg-1

under these experimental conditions. Moreover, all the inoculated

plants had significantly higher shoot P content than NM controls four weeks after the Pi

pulse indicating that symbiotic associations do not cause jarrah plants to reduce their

net P uptake under Pi toxicity conditions.

One possible mechanism for the induced P tolerance could be that AM plants have

the ability to assimilate inorganic P into organic pools and therefore do not accumulate

free Pi, which causes toxicity symptoms. Therefore, the shoot free Pi concentration of

plants was measured to test this hypothesis. The results were almost similar to those of

shoot total P and AM plants had the highest free Pi concentration within their shoot

tissues among treatments (Fig. 5 A). The difference between shoot total P and free P

was less than 3 mg P g-1

DW, which presumably derive from organic P compounds.

Therefore, the Pi tolerance in AM jarrah plants is not linked with the plants ability to

quickly assimilate inorganic P into organic P pools. Plant species have been shown to

reduce the expression of their PHT1 genes in response to AM (Karandashov and

Bucher, 2005) and ECM (Loth-Pereda et al., 2011) colonisation. However, this is not

apparently the mechanism for the induced Pi tolerance in AM jarrah plants as we did

not observe the reduced transcript abundance for any of the five PHT1 genes tested.

Other potential mechanisms could be involved such as accumulation of inorganic P in

the stem tissues or sequestration of Pi molecules in vacuoles to avoid their interference

with normal cell functions. However, it is not clear how such a difference in

mechanisms of nutrient storage might be determined given that the symbiotic fungi are

contained in the root system.

Tolerance to phosphite toxicity and Phi uptake dynamics

From a plant perspective, Phi is a non-metabolizable form of P (Guest and Grant,

1991) and the positive nutritional effects of Phi on plants (Jabahi-Hare and Kendrick,

1987; Lovatt and Mikkelsen, 2006) are most likely due to microbe-mediated oxidation

of Phi to Pi in soil (Ohtake et al., 1996) and/or suppression of plant diseases (Thao and

Page 110: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

93

Yamakawa, 2009). Here, there were slight phytotoxicity symptoms in all plants a week

after the Phi pulse (Fig. 3 A) and the shoot P concentrations did not differ significantly

between treatments (Fig. 4 C). Four weeks after the pulse, the shoot P concentration of

jarrah plants was significantly less in all the fungal species treatments than the NM

control (Fig. 4 C), which is apparently correlated with the reduced phytotoxicity

symptoms in all inoculated plants (Fig. 3 B). The results also showed that the uptake of

Phi is much slower than Pi in NM plants as the P concentration was nearly half that of

the comparable Pi treatment. Furthermore, there was no significant difference between

the shoot P content of plants 4 weeks after the Phi pulse. This means that inoculated

plants with higher shoot biomass had the same shoot P content as NM plants indicating

lower net Phi uptake in inoculated plants. Interestingly, this reduction of uptake did not

occur in the Pi treatment and all inoculated plants had significantly higher shoot P

content than NM plants. This suggests that both AM and novel symbiotic associations

can differentiate Phi (a toxic P source) from Pi (metabolizable P source) and

consequently reduce the Phi uptake of plants. It is possible that the PHT1 gene

expression difference observed for EmPHT1;2 between these two treatments (Figure

6B) may provide an explanation for the differences observed. Further analysis of the

temporal and spatial expression of this particular PHT1 gene family member is

warranted.

The shoot free Pi concentration was also determined in plants exposed to the Phi

pulse for four weeks. This was done to see if mycorrhizal fungi can oxidise Phi to Pi in

the soil as a potential mechanism for the Phi tolerance observed. The results did not

support this hypothesis as NM plants had the highest free Pi concentration similar to

the shoot total P results (Fig. 5 B). This might mean that these fungal species are not

involved in oxidation of Phi to Pi in the soil. The free Pi concentration of plants (mg P

g-1

DW) was about 20% of shoot total P. Accordingly, about 80% of shoot total P

comes from Phi molecules (and organic P compounds) and about 20% comes from

shoot free Pi suggesting that plants exposed to the Phi pulse took up more intact

molecules of Phi than the oxidised form (Pi) from soil.

There was a relatively high proportion of Pi (about 20% of shoot total P) in shoots of

all Phi-treated plants, which was higher than shoot total P in untreated plants (Fig. 4A,

open bars). This suggests that the Phi addition has somehow increased the Pi pool in

plants. One possible mechanism is oxidation of Phi to Pi either within plant tissues or

in the soil. All inoculated treatments had significantly lower concentrations of shoot

total P (mainly originated from Phi molecules) and free Pi than NM controls suggesting

Page 111: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

94

that this trend might be correlated with the uptake feature and soil is the primary source

of Pi molecules where the Phi oxidation occurred.

Tolerance to arsenate toxicity and AsV uptake dynamics

Mycorrhizal fungi can induce tolerance against the AsV toxicity by mechanisms such

as reducing AsV to AsIII, binding of phytochelatins to As to make peptide-metal

complexes, accumulation inside fungal vacuoles and down-regulation of plant high

affinity PHT1 genes (Meharg and Macnair, 1992; Sharples et al., 2000; Hildebrandt et

al., 2007). Although the induced tolerance to As toxicity can be due to the improved

growth and Pi uptake (Ahmed et al., 2006; Xu et al., 2008; Christopherson et al., 2009),

the As tolerance have also been observed where AM symbioses do not enhance growth

or Pi nutrition of host plants (Li et al., 2006; Grace et al., 2009). In the present study,

there was no tolerance against the AsV toxicity in terms of reducing the phytotoxicity

symptoms and all plants were dead a week after adding the pulse (Fig. 3 A), apparently

due to higher toxicity of AsV than Pi and Phi molecules. The average As toxicity

threshold for crop plants is about 40 µg g-1

DW (Sheppard et al., 1992), which is far

below the concentrations observed in this experiment and the apparent reason for death

of all treated jarrah plants. The results, however, showed that different fungal

treatments can reduce the shoot As concentration upon exposure to AsV toxicity.

Although one week later, the shoot As content of plants did not differ significantly

across treatments, inoculated plants had lower shoot As content one day after adding

the AsV pulse compared to NM controls (Fig. 4 B). It seems that inoculated plants

reduce their net AsV uptake similar to the other toxic analogue (Phi) as they had higher

shoot biomass but nearly the same shoot As content as NM plants. Here, we did not

observe the reduced transcript abundance of plant PHT1 genes under AsV toxicity

conditions so the reduced As uptake in inoculated plants might be due to one of the

other mechanisms mentioned above, or the changed function of additional PHT1 gene

family members that were not identified in this study. Jarrah plants received the same

concentration (1.5 mmol kg-1

soil) of Pi, Phi and AsV but developed different extents

of phytotoxicity symptoms. Our results demonstrate that AsV is the most toxic

analogue and Pi and Phi are the second and third most toxic analogues, respectively.

AM symbioses can reduce sen

Page 112: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

95

The transcript abundance of EmPHT1 genes in non-mycorrhizal and inoculated plants

We are aware of five PHT1 genes in E. marginata, although there are likely to be

additional genes, based on the situation in other plants. The abundance of transcripts

from several jarrah EmPHT1 genes was quantified by real time PCR for 14-week old

plants prior to addition of different pulses, to determine if there was a correlation with

symbiotic associations. Down-regulation of PHT1 genes can occur in mycorrhizal roots

regardless of the soil P status (Karandashov and Bucher, 2005) and we therefore did not

quantify PHT1 transcripts in inoculated plants subjected to the pulses. Jarrah plants did

not reduce the expression of their PHT1 genes in response to any of fungal species

tested which were representatives of AM and novel symbioses (Fig. 6 A).

The expression profile of PHT1 genes is not altered in roots of plants harbouring the

novel symbiosis (Kariman et al, in preparation: Chapter 3). In our previous study,

Scleroderma sp. behaved like a typical ECM fungus having a high colonisation rate of

77 % and two PHT1 genes (EmPHT1;1 and EmPHT1;2) had significantly lower

transcripts in ECM roots than NM controls. Recently, Loth-Pereda et al. (2011) have

shown that poplar plants reduce the expression of two PHT1 genes in response to both

AM and ECM symbioses. However, in the present study Scleroderma sp. used the

novel symbiotic pathway (no colonisation) and the expression of PHT1 genes was not

affected.

Surprisingly, jarrah plants did not down-regulate their PHT1 gene transcripts in

response to the AM fungus S. calospora. Many plants species down-regulate the

expression of their PHT1 genes in response to AM colonisation (Karandashov and

Bucher, 2005). However, there are exceptions where gene expression was not affected.

Burleigh reported (2001) the reduced expression of the PHT1 gene MtPT2 in Medicago

trancatula following colonisation with the AM fungi Glomus versiforme, G.

intraradices, G. caledonium and G. claroideum, G. mosseae and S. calospora but not

with Gigaspora rosea. This lack of down-regulation response in certain AM symbioses

has been attributed to the inability of the fungal partner to supply P for the host plant

(Burleigh, 2001). Here, S. calospora was quite effective in improving the P nutrition of

jarrah plants under these experimental conditions. Therefore, the lack of down-

regulation response for plant PHT1 genes is not necessarily correlated with the inability

of the AM fungi to supply P for their host plants. However, the PHT1 family can

contain a large number of genes including 9 PHT1 genes in Arabidopsis thaliana

(Muchhal et al., 1996) and 13 genes in Oryza sativa (Paszkowski et al., 2002; Glassop et

al., 2007) and 12 PHT1 genes in Populus trichocarpa (Loth-Pereda et al., 2001).

Page 113: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

96

Therefore, there might be some unidentified PHT1 gene members in jarrah roots with

different responses to AM symbiosis.

Expression of PHT1 genes in response to phosphate, phosphite and arsenate toxicities

Plant species that are not P sensitive reduce the expression of PHT1 genes in their

roots in response to high P conditions (Burleigh and Harrison, 1999; Rausch and

Bucher, 2002; Grunwald et al., 2009). Conversely, none of the jarrah PHT1 genes that

we examined were reduced in their expression in response to any of the three analogues

tested (Fig. 6 B). In fact, the transcript abundances of some PHT1 genes were increased

under toxicity conditions.

The enhanced transcript abundance of PHT1 genes could have occurred to i) take up

more of these three analogue molecules from soil solution or ii) to facilitate the internal

translocation of the additional target molecules inside plant tissues. As previously

described, the transcripts of EmPHT1;1 and EmPHT1;2 were down-regulated

following the ECM colonisation (Kariman et al., in preparation: Chapter 3), which

could be evidence for involvement of these two genes in the direct pathway (via root

epidermal cells) of P uptake from the soil. Thus, enhanced expression of these two

genes after adding the Pi pulse might mean that jarrah plants actually increase their Pi

uptake from the soil solution under P toxicity conditions. Nevertheless, changed

transcript abundance may not necessarily reflect a change in the activity of the

respective transporter. We therefore suggest that native plants regulate the expression

of their PHT1 genes in response to the external P concentration (soil) rather than the

plant internal P status. Although the P sensitivity of Australian native plants has been

studied well during the past decades, this is the first report showing that native plants

cannot reduce expression of their PHT1 genes upon exposure to a toxic dose of Pi.

Conclusion

Our results showed that the addition of P sources (Pi and Phi) can enhance AM

colonisation, which declined in untreated jarrah plants. All fungal species could

enhance jarrah growth and shoot P content under P-deficient conditions. Symbiotic

associations have the potential to induce tolerance against toxicity caused by Pi, Phi

and probably lower pulses of AsV. Inoculated jarrah plants did not decrease their net Pi

uptake under toxicity conditions, whereas they reduced the uptake of both toxic

analogues (Phi and AsV). The induced tolerance for the plant toxic Pi analogue (Phi)

could be due to the reduced uptake combined with a dilution effect. However, the Pi

Page 114: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

97

tolerance is not correlated with lower P uptake in AM plants. Finally, we suggest that

the expression of PHT1 genes in P-sensitive species is regulated by external P

concentration in soil rather than the internal plant P status as jarrah plants up-regulated

some PHT1 transcripts under Pi toxicity.

Materials and Methods

Plant materials, fungal isolates and inoculum production

Jarrah seed capsules were obtained from a single tree near Dwellingup, Western

Australia. Capsules were incubated at 42 ºC for three days to release the seeds. The

three fungal isolates used were S. calospora WUM 12 (3) (AM), Scleroderma sp.

(ECM) and A. occidentalis (novel symbiosis). Details about the fungal isolates,

inoculum production, root staining and colonisation studies were previously described

(Kariman et al., 2012). The AM colonisation was an average value of more than 300

grid intersect observations on three subsamples.

Experimental design and treatments

Jarrah plants were grown alone or in symbiosis with the three fungal isolates.

Fourteen weeks after planting, three replicates from all NM and inoculated treatments

were harvested to analyze the mycorrhizal colonisation and shoot P concentration of

plants. The washed river sand used for plant growth contained less than 6 mg P kg-1

soil

(data not shown) and therefore these plants were considered to have grown under P-

deficient conditions. Plants subsequently received a toxic pulse (1.5 mmol kg-1

soil) of

Pi (KH2PO4), Phi (KH2PO3) or AsV (Na2HAsO4) in aqueous solution. Three replicates

were harvested one day after adding the pulses to monitor the P and As uptake during

the short term exposure. The remaining plants were harvested four weeks after the

pulse except the AsV-treated plants, which were harvested one week after the pulse.

Growth conditions

To prepare the growth medium, double-pasteurized washed river sand was mixed

with AM inoculum (10:1 w/w) and subsequently mixed with ECM inoculum (10:1 v/v).

To provide equal conditions for all treatments, AM and ECM plants were supplied with

sterilized ECM or AM inoculum, respectively. Non-mycorrhizal plants also received

sterilized AM and ECM inocula. Square plastic pots (8 x 8 x 18 cm) were lined with

double plastic bags and filled with 1 kg of the growth medium prepared as described

above. One pre-germinated jarrah seed (Kariman et al., 2012) was planted in each pot

Page 115: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

98

and the pot surface was covered with 3 cm of sterile plastic beads to minimise cross or

environmental contaminations. All plants received 1 X modified Long Ashton solution

minus P once a fortnight starting two weeks after planting (10 mL kg-1

soil): K2SO4 2

mM, MgSO4.7H2O 1.5 mM, CaCl2.2H2O 3 mM, FeEDTA 0.1 mM, (NH4)2SO4 4 mM,

NaNO3 8 mM, H3BO3 46 µM, MnCl2.4H2O 9 µM, ZnSO4.7H2O 8 µM, CuSO4.5H2O

0.3 µM and Na2MoO4.2H2O 0.01 µM (Cavagnaro et al., 2001). We used sealed pots for

this experiment and watered them to field capacity three times a week. The experiment

was conducted from February to June 2012 in an unheated glasshouse with the average

daytime temperature of 22 °C.

Nutrient analysis and phytotoxicity assessment

Measured quantities of ground dried shoot tissues (about 200 mg) were digested in

5 ml nitric-perchloric acid solution (4:1 v/v). The shoot total P and As concentrations

were determined using inductively coupled plasma optical emission spectrometry (ICP-

OES; Optima 5300 DV, PerkinElmer, USA). A measured amount (about 40 mg) of

dried ground tissues was homogenized in 1 mL acetic acid and used to measure the

shoot free Pi concentration using the ammonium molybdate method (Ames, 1966).

Phytotoxicity symptoms (including chlorotic and necrotic areas on leaves) were

quantified by ranking plants into six classes from 0 to 5, where 0 corresponded to the

absence of toxicity symptoms; 1 from traces to 20 % of symptomatic leaf tissue area

(SLTA); 2 from 20 to 40 % SLTA; 3 from 40 to 60 % of SLTA; 4 from 60 to 80 % of

SLTA and 5 more than 80 % of SLTA.

Phosphate transporter (PHT) genes and real time PCR

Complementary cDNAs from five EmPHT1 genes were cloned and sequenced from

jarrah roots as previously described (Kariman et al., in preparation: Chapter 3). Total

root RNA was isolated using a CTAB-based method (Korimbocus et al., 2002) with a

slight modification. Sodium D-isoascorbate was added to the extraction buffer just

before use to a final concentration of 100 mM. Total RNA (1.0 µg) was treated with

DNase I (RQ1 RNase-free DNase, Promega, USA) and subsequently reverse

transcribed using the GoScriptTM

reverse transcriptase kit (Promega). Gene specific

primers were designed for five PHT1 genes and a jarrah actin gene (internal control) for

the gene quantification assay (Table 1). The SYBR green-based real-time PCR (q-PCR)

was carried out to quantify the transcripts from PHT1 genes relative to EmACT1

transcripts. Q-PCR reactions were performed in 96-well plates in a 10 µl reaction

Page 116: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

99

volume of 0.3 µM of each gene specific primer and 2.5 µl of cDNA equivalent to 50 ng

total RNA in 1x SYBR Green PCR master mix (Applied Biosystems). All q-PCR

experiments were performed on a 7500 FAST Real-time PCR System (Applied

Biosystems).

Statistical analyses

The experiment was conducted in a completely randomized design with three

replicates. All data were analyzed using the Statistical Analysis System (SAS) version

9.2 (SAS Institute, Inc.; Cary NC, USA) software package. Means were separated using

LSD at 5% significance level.

Table 1. Gene specific primers used for real time PCR

Primer Direction Sequence (5’-3’) Product

size (bp)

EmPT1-F Forward GAGCCGTCGAGATGGTGTGTAGA 122

EmPT1-R Reverse CGACTATCTTGCCACTTCCTCCATTGA

EmPT2-F Forward CGATGAGGTGCCCACTGCT 138

EmPT2-R Reverse CACCTGCTCGACGACTCCGTAAT

EmPT3-F Forward CAACAACTTCGGCTTGTTCAGCAGA 183

EmPT3-R Reverse ACTTCCTCAATTGCGTTCATGGTGTC

EmPT4-F Forward TGGACATCGCCTTCTATAGCCAGAATCTT 113

EmPT4-R Reverse GCCCAATCCGGTACACCTCTTCTATG

EmPT5-F Forward GGACGATGAGGTGTCCACTGCTT 127

EmPT5-R Reverse TCTGTAATATTCGGCAACACGGGAAGT

Act2-F Forward GGTCCTGTTCCAACCATCCATGATT 136

Act2-R Reverse GGTAGAACCACCACTGAGGACAATGT

Acknowledgments: We appreciate the University of Western Australia postgraduate

scholarships (SIRF/UIS) awarded to K.K. and also financial support and grants from

the Centre for Land Rehabilitation at the University of Western Australia (M.T.) and

the Australian Research Council (M.T., P.M.F.).

Page 117: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

100

Literature Cited

Adams F, Reddell P, Webb MJ, Shipton WA (2006) Arbuscular mycorrhizas and

ectomycorrhizas on Eucalyptus grandis (Myrtaceae) trees and seedlings in native

forests of tropical north-eastern Australia. Aust J Bot 54: 271-281

Adjoud D, Plenchette C, HalliHargas R, Lapeyrie F (1996) Response of 11 eucalyptus

species to inoculation with three arbuscular mycorrhizal fungi. Mycorrhiza 6:129-

135

Ahmed FRS, Killham K, Alexander I (2006) Influences of arbuscular mycorrhizal

fungus Glomus mosseae on growth and nutrition of lentil irrigated with arsenic

contaminated water. Plant Soil 283: 33-41

Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatises.

Methods Enzymol 8: 115-118

Barrett S (2001) Phytotoxic effects of phosphite on native plant communities in

southern Western Australia. PhD Thesis. Murdoch University, Perth

Bobbink R (1998) Impacts of tropospheric ozone and airborne nitrogenous pollutants

on natural and semi-natural ecosystems: a commentary. New Phytol 139: 161-168

Bolan NS, Robson AD, Barrow NJ (1984) Increasing phosphorus supply can

increase the infection of plant roots by vesicular-arbuscular mycorrhizal fungi.

Soil Biol Biochem 16: 419-420.

Burleigh SH, Harrison MJ (1999) The down-regulation of Mt4-like genes by phosphate

fertilization occurs systemically and involves phosphate translocation to the shoots.

Plant Physiol 119: 241–248

Burleigh SH (2001) Relative quantitative RT-PCR to study the expression of plant

nutrient transporters in arbuscular mycorrhizas. Plant Sci 160: 899–904

Cavagnaro, TR, Smith FA, Lorimer MF, Haskard KA, Ayling SM, Smith SE (2001)

Quantitative development of Paris type arbuscular mycorrhizas formed between

Asphodelus fistulosus and Glomus coronatum. New Phytol 149:105–113

Chen YL, Brundrett MC, Dell B (2000) Effects of ectomycorrhizas and vesicular–

arbuscular mycorrhizas, alone or in competition, on root colonisation and growth of

Eucalyptus globulus and E. urophylla. New Phytol 146: 545–556

Christophersen HM, Smith SE, Pope S, Smith FA (2009) No evidence for competition

between arsenate and phosphate for uptake from soil by medic or barley. Environ

Int 35: 485–490.

Clarke CA (1978) Effects of pesticides on VA mycorrhizae. Rothamsted Experimental

Station Report for 1978, Part 1: 236-237

Cornwell WK, Bedford BL, Chapin CT (2001) Occurrence of arbuscular mycorrhizal

fungi in a phosphorus-poor wetland and mycorrhizal response to phosphorus

fertilization. Am J Bot 88: 1824–1829

Page 118: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

101

Dell B, Hardy GESJ, Vear K (2005) History of Phytophthora cinnamomi management

in Western Australia In MC Calver, H Bigler-Cole, G Bolton, J Dargavel, A

Gaynor, P Horwitz, J Mills, G Wardell-Johnson, Eds, A forest consciousness:

Proceedings of the 6th

National Conference of the Australian Forest History

Society. Millpress Science Publishers, Rotterdam, 391-406

Duke SE, Jackson RB, Caldwell MM (1994) Local reduction of mycorrhizal arbuscle

frequency in enriched soil microsites. Can J Bot 72: 998-1001

Finnegan PM, Chen W (2012) Arsenic toxicity: the effects on plant metabolism. Front

Physiol 3: 182

Glassop D, Godwin RM, Smith SE, Smith FW (2007) Rice phosphate transporters

associated with phosphate uptake in rice colonised with arbuscular mycorrhizal

fungi. Can J Bot 85: 644-651

Gomez TCR, Faria LP, Lin MT (1987) Mycorrhization of eight species of eucalypts

with VAM fungi. In: DM Sylvia, LL Hung, JH Graham (eds) Mycorrhizae in the

next decade: practical applications and research priorities (7th NACOM).

Gainesville University, Florida, p 125

Grace EJ, Smith FA, Smith SE. (2009) Deciphering the arbuscular mycorrhizal

pathway of P uptake in non-responsive hosts. In: Azco´n-Aguilar C, Barea JM,

Gianinazzi S, Gianinazzi-Pearson V, eds. Mycorrhizas: functional processes and

ecological impact. Springer-Verlag, Berlin Heidelberg, 89–106.

Grunwald U, Guo W, Fischer K, Isayenkov S, Ludwig-Müller J, Hause B, Yan X,

Kuester H, Franken P (2009) Overlapping expression patterns and differential

transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-

fertilised and phytohormone-treated Medicago truncatula roots. Planta 229: 1023–

1034

Guest D, Grant BR (1991) The complex action of phosphonates as antifungal agents.

Biol Rev 66: 159–187

Handreck KA (1997) Phosphorous requirements of Australian native plants. Aust J Soil

Res 35: 241-289

Hawkins HJ, Hettasch H, Mesjasz-Przybylowicz J, Przybylowicz W, Craner MD

(2008) Phosphorus toxicity in the Proteaceae: A problem in post-agricultural lands.

Sci Hortic 117: 354–365

Hildebrandt U, Regvar M, Bothe H (2007) Arbuscular mycorrhiza and heavy metal

tolerance. Phytochem 68: 139–146

Howard K, Dell B, Hardy GE (2000) Phosphite and mycorrhizal formation in seedlings

of three Australian Myrtaceae. Aust J Bot 48: 725-729

Jabahi-Hare SH, Kendrick WB (1987) Response of an endomycorrhizal fungus in

Allium porrum L. to different concentrations of the systemic fungicides metalaxyl

(Ridomil) and fosetyl-Al (Aliette). Soil Biol Biochem 19: 95–99

Page 119: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

102

Jasper DA, Davy JA (1993) Root characteristics of native plant species in relation to the

benefit of mycorrhizal colonisation for phosphorus uptake. Plant Soil 155/156: 281-

284.

Jones MD, Durall DM, Tinker PB (1998) Comparison of arbuscular and

ectomycorrhizal Eucalyptus coccifera: growth response, phosphorus uptake

efficiency and external hyphal production. New Phytol 140: 125-134

Kabir ZIP, O'Halloran IP, Fyles JW, Hamel C (1997) Seasonal changes of arbuscular

mycorrhizal fungi as affected by tillage practices and fertilization: hyphal density

and mycorrhizal root colonisation. Plant Soil 193: 285-293

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 (2012) Dual mycorrhizal associations

of jarrah (Eucalyptus marginata) in a nurse-pot system. Aust J Bot 60: 661-668

Kariman K, Barker SJ, Jost R, Finnegan PM, Tibbett M (in preparation: Chapter 3) A

novel plant-fungus symbiosis benefits the host without forming mycorrhizal

structures.

Kariman K, Barker SJ, Finnegan PM, Tibbett M (in preparation: Chapter 4)

Mycorrhizal symbiosis can induce tolerance in jarrah (Eucalyptus marginata)

exposed to toxic pulses of phosphorus

Korimbocus J, Coates D, Barker I, Boonham N (2002) Improved detection of

sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR

assay. J Vir Methods 103: 109-120 (2002)

Li HY, Smith SE, Holloway R, Zhu YG, Smith FA (2006) Arbuscular mycorrhizal

fungi contribute to phosphorus uptake by wheat grown in a phophorus-fixing soil

even in the absence of positive growth response. New Phytol 172: 536–543.

Loth-Pereda V, Orsini E, Courty PE, Lota F, Kohler A, Diss L, Blaudez D, Chalot M,

Nehls U, Bucher M, Martin F (2011) Structure and expression profile of the

phosphate Pht1 transporter gene family in mycorrhizal Populus trichocarpa. Plant

Physiol 156: 2141–2154

Lovatt CJ, Mikkelsen RL (2006) Phosphite fertilizers: What are they? Can you use

them? What can they do? Better Crops 90: 11-13

Meharg AA, Macnair MR (1990) An altered phosphate uptake system in arsenate

tolerant Holcus lanatus. New Phytol 116: 29–35

Meharg AA, Macnair MR (1992) Suppression of the high affinity phosphate uptake

system: a mechanism of arsenate tolerance in Holcus lanatus L. J Exp Bot 43: 519–

524

Meharg AA, Hartley-Whitaker J (2002) Arsenic uptake and metabolism in arsenic

resistant and non-resistant plant species. Tansley Review. New Phytol 154: 29-43

Page 120: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

103

Muchhal US, Pardo JM, Raghothama KG (1996) Phosphate transporters from the

higher plant Arabidopsis thaliana. Proc Natl Acad Sci USA 93: 10519–10523

Muchovej RMC, Amorim EFC (1990) Development and effect of endo- and

ectomycorrhizal fungi on seedlings of Eucalyptus grandis. In: Abstracts of the 8th

NACOM, Jackson, Wyo, p 251

Ohtake H, Wu H, Imazu K, Anbe Y, Kato J, Kuroda A (1996) Bacterial phosphonate

degradation, phosphite oxidation and polyphosphate accumulation. Res Cons Recy

18: 125–134

Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters

include an evolutionarily divergent gene specifically activated in arbuscular

mycorrhizal symbiosis. Proc Natl Acad Sci U S A 99: 13324-13329

Rausch C, Bucher M (2002) Molecular mechanisms of phosphate transport in plants.

Planta 216: 23–37

Runge-Metzger, A (1995) Closing the cycle: obstacles to efficient P management for

improved global food security. In: H Tiessen (Ed.). Phosphorus in the global

environment. John Wiley & Sons Ltd, NewYork

Sanchez PA, Buol SW (1975) Soils of the tropics and the world food crisis. Science

188: 598-603

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: 1033-1044

Sharples JM, Meharg AA, Chambers SM, Cairney JWG (2000) Mechanism of Arsenate

Resistance in the Ericoid Mycorrhizal Fungus Hymenoscyphus ericae. Plant Physiol

124: 1327-1334

Sheppard BS, Caruso JA, Heitkemper DT, Wolnik KA (1992) Arsenic speciation by ion

chroma- tography with induced coupled plasma mass spectrometry detection.

Analyst 117: 971-975

Smaling E (2005) Harvest for the world, inaugural address, International Institute for

Geo-Information Science and Earth Observation, Enschede, The Netherlands

Smith E, Smith J, Biswas T, Correll R, Naidu R (2003) Arsenic in Australian

environment: an overview. J Environ Sci Health, Part A 38: 223-239

Sukarno N, Smith SE, Scott ES (1993) The effect of fungicides on vesicular- arbuscular

mycorrhizal symbiosis: I. The effects on vesicular-arbuscular mycorrhizal fungi and

plant growth. New Phytol 25: 139–147

Sukarno N, Smith FA, Smith SE, Scott ES (1996) The effects of fungicides on vesicular-

arbuscular mycorrhizal symbiosis. II. The effects on area of interface and efficiency

of P uptake and transfer to plant. New Phytol 132: 583–592

Sukarno N, Smith FA, Scott ES, Jones GP, Smith SE (1998) The effect of fungicides on

vesicular-arbuscular mycorrhizal symbiosis. III. The influence of VA mycorrhiza

Page 121: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

104

on phytotoxic effects following application of fosetyl-Al and phosphonate. New

Phytol 139: 321–330

Thao HTB, Yamakawa T (2009) Phosphite (phosphorous acid): fungicide, fertilizer or

bio-stimulator? Soil Sci Plant Nutr 55: 228–234.

Ticconi CA, Delatorre CA, Abel S (2001) Attenuation of phosphate starvation responses

by phosphite in Arabidopsis. Plant Physiol 127: 963–972

Thomson VP, Leishman MR (2004) Survival of native plants of Hawkesbury

Sandstone communities with additional nutrients: effect of plant age and habitat.

Aust J Bot 52: 141-147

Trolove SN, Hedley MJ, Kirk GJD, Bolan NS, Loganathan P (2003) Progress in

selected areas of rhizosphere research on P acquisition. Aust J Soil Sci 41: 471–499

Tynan KM, Wilkinson CJ, Holmes JM, Dell B, Colquhoun IJ, McComb JA, Hardy

GESJ (2001) The long-term ability of phosphite to control Phytophthora

cinnamomi in two native plant communities of Western Australia. Aust J Bot 49:

761-770

Varadarajan DK, Karthikeyan AS, Matilda PD, Raghothama KG (2002) Phosphite, an

analog of phosphate, suppresses the coordinated expression of genes under

phosphate starvation. Plant Physiol 129: 1232–1240

Xu P, Christie P, Liu Y, Zhang J, Li X (2008) The arbuscular mycorrhizal fungus

Glomus mosseae can enhance arsenic tolerance in Medicago truncatula by

increasing plant phosphorus status and restricting arsenate uptake. Environ Poll

156: 215–220

Page 122: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

105

Chapter 6

General Discussion and Conclusion

This PhD thesis has provided fundamental insights on fungal symbiotic associations

of jarrah. In the first glasshouse experiment (Chapter 2), I developed a nurse-pot system

to explore the extent and temporal colonisation dynamics of AM, ECM and dual (AM

& ECM) mycorrhizal associations of jarrah and their potential growth and nutritional

benefits. The results also uncovered an unusual plant-fungus association in which

benefits were achieved without root colonisation.

I was compelled by curiosity to answer this question: How a fungus could be as

beneficial for the host plant as a mycorrhiza even without a physical contact

(colonisation) between the two symbiotic partners? Therefore, I developed a

radioisotope tracer experiment (Chapter 3) using 33

P and a multi-compartment system

to shed more light on this new symbiosis. Mycorrhiza have been defined and classified

according to the type of symbiotic structures formed between plant roots and fungal

hyphae. However, the presented results open up a new perspective of plant-fungus

relationships, indicating that the benefits rendered to the host plants are not necessarily

correlated with formation of specialised ECM symbiotic structures. Indeed, in the novel

symbiosis the rhizosphere biochemistry is modified in order to achieve higher nutrient

availability for plant uptake rather than direct nutrient transfer via hyphae. Recent

studies have highlighted the key role of ECM fungi in soil biodegradation and nutrient

cycling (Landeweert et al., 2001; Pritsch and Garbaye, 2011). However, the existing

literature has focused on the fungal hyphae involved in ECM associations or their

saprophytic stages rather than the hyphae involved in a completely novel form of

symbiosis (Chapter 3).

During the recent decades and along with the green revolution, P fertilisers have been

added to many P-impoverished soils to achieve higher yields for commercial crop

plants. Thus, the soil P levels have risen beyond the tolerance of many low-P adapted

species in agricultural and forest environments, which is considered a serious threat for

biodiversity and function of native communities (Handreck et al., 1997; Lambers et al.,

2010). Many Australian native species are sensitive to P fertilization and finding

sustainable ways to address this issue is of environmental and ecological importance. I

focused on mycorrhizal associations as a potential biological approach to deal with P

sensitivity. In this regard, I studied symbiotic associations of jarrah and their effect on

the expression of plant PHT1 genes and P uptake across the P deficiency-toxicity

Page 123: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

106

continuum. The toxicity experiments (Chapters 4 and 5) provided new insights into the

P sensitivity of jarrah plants. P-sensitive native plants are thought to be unable to

reduce the expression of their PHT1 genes under P toxicity conditions (Shane et al.,

2004; Hawkins et al., 2008) but this has not been investigated previously. This is the

first study showing the molecular mechanism behind the P sensitivity in a low-P

adapted plant, which is apparently the inability to reduce the expression of PHT1 genes

in roots when exposed to toxic doses of Pi. AM, ECM and novel symbioses showed a

good potential to induce tolerance against Pi and its analogues, suggesting the

mycosymbionts as functional biological components to deal with toxicity caused by

these analogues.

There were two main hypotheses for this research i) ECM fungi are better symbiotic

partners for jarrah compared to AM species in terms of positive growth and nutritional

responses and ii) fungal symbionts have the potential to induce tolerance of jarrah to

toxic pulses of Pi, Phi and AsV and the tolerance might be linked with the expression

of PHT1 genes in roots. The main findings of this study are briefly discussed with

regard to these hypotheses and the existing knowledge of this field. Here, I integrate the

findings of all experiments and the implications and future research directions of this

area:

1- Jarrah growth and nutrition

For AM symbioses, a positive growth response was only observed with S. calospora

while R. irregularis had no effects or sometimes depressive effects on jarrah growth

(Chapters 2 and 5). The variable results observed in jarrah-AM symbioses support the

reports from research on other eucalypts (Gomez et al., 1987; Adjoud et al., 1996;

Chen et al., 2000). There are likely to be major differences between these two AM

fungal isolates due to their taxonomic separation (Kruger et al., 2012) and original

habitat (Western Australia vs Canada). However, a clear understanding of neutral or

negative jarrah responses to different AM fungi requires further investigations. Besides,

the positive growth and P nutritional responses in plants inoculated with S. calospora

(Chapter 5) was achieved with relatively low AM colonisation, supporting the notion

that positive responses are not necessarily linked with the extent of root colonisation in

AM symbiosis (Jakobsen, 1995; Smith et al., 2004).

The colonisation results for Scleroderma sp. and A. occidentalis indicated that the

ECM structures and hyphal nutrient uptake are not the only factors reflecting the fungal

contribution to better growth and nutrition of the host plants. Acidifying the

Page 124: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

107

rhizosphere soil by increasing the concentration of carboxylates (Chapter 3), is

apparently a significant factor leading to the enhanced nutrient availability in the novel

symbiosis where the root colonisation does not occur. However, there might be other

functional pathways in the novel symbiosis such as proton exudation (Hoffland et al.,

2004), mineralisation of organic matter (Bending and Read, 1995; Tibbett and Sanders,

2002) or other unknown mechanisms. The two ECM isolates were collected from forest

habitats, where ECM fungi play a major role in decomposing soil organic matter and

nutrient cycling (Pritsch and Garbaye, 2011). Therefore, it would be worthwhile to

examine the fungal ability to decompose organic matter (via secretion of nutrient

mobilising enzymes) while having a novel symbiotic relationship.

The washed river sand used in this study was fairly poor in organic matter, which is

the primary N source for ECM hyphae (Bending and Read, 1995; Tibbett and Sanders,

2002). The improved N nutrition in the A. occidentalis treatment (Chapter 3) was

surprising as the ECM symbiosis (Scleroderma sp.) failed to do so. The highest citrate

concentration was observed in the rhizosphere of jarrah plants associated with A.

occidentalis, which could potentially modify the soil bacterial communities and

promote free living N2 fixers (Olsson and Wallander, 1998). The N2 fixing bacteria

might have been endemic to the sterile fungal cultures or recruited during the

experiment. However, this is a speculative hypothesis but one that warrants testing.

2- Mycorrhizal relationships

Jarrah plants had a very low AM colonisation with both R. irregularis and S.

calospora in keeping with the existing literature about AM colonisation of jarrah and

other eucalypts (Brundrett and Abbott, 1991; Howard et al., 2000). The AM

colonisation of jarrah seedlings was not increased by using a nurse-pot system (Chapter

2). Apparently, the established network of extraradical hyphae does not increase the

naturally low AM colonisation of jarrah whereas nurse-pot systems have been

successfully used to enhance the AM colonisation of certain crops including tomato

and barley (Brundrett et al., 1985; Finlay et al., 1989; Arnebrant and Finlay, 1993;

Rosewarne et al., 1997). However, in classic nurse-pot systems test plants are

transplanted into hyphal networks originating from roots of highly colonised species

such as leek, not from the same plant species. I used jarrah as both test and nurse plant

to be able to study both AM and ECM associations, simultaneously. It would be

interesting to see if the same results would be achieved for AM colonisation if test

Page 125: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

108

jarrah seedlings were transplanted into hyphal networks associated with highly

colonised nurse plants such as leek.

Addition of two P sources (Pi and Phi) inhibited the decline of AM colonisation,

which naturally happens after 2-3 months in AM-eucalypt symbioses for unidentified

reasons (Chen et al., 2000, Adams et al., 2006). AM fungi do not always have positive

effects on P nutrition of eucalypts (Jones et al., 1998; Chapter 2), which could be

potentially linked with decline or suppression of the AM symbiosis shortly after

establishment. Therefore, adequate or even toxic supplies of Pi might be able to extend

the AM symbiosis in jarrah (and other eucalypts), which is an interesting hypothesis to

be tested.

Three individual experiments (Chapters 2, 3 and 5) showed that Scleroderma sp. is a

fungus with dual behaviour capable of establishing both ECM and novel symbioses.

This suggests that the novel symbiosis is an alternative pathway for at least some ECM

fungi. There was preliminary evidence for a possible correlation between minimum

growth temperature and this dual behaviour. Scleroderma sp. mostly established the

novel symbiosis when the temperature dropped below 7 ºC at night during the growth

season. A. occidentalis, however, seems to be a typical fungus for the novel symbiosis,

always establishing the novel symbiosis with jarrah plants. The 33

P experiment

(Chapter 3) showed that hyphae do not extend beyond the rhizosphere in the novel

symbiosis, which was accompanied by the lack of hyphal 33

P transfer to jarrah. In the

ECM association, however, the direct hyphal connection with roots was accompanied

by hyphal networks extended well beyond the rhizosphere. Therefore, lack of ECM

structures and less extensive hyphal networks (bounded to rhizosphere and vicinity)

could be considered to be two simple factors that differentiate novel from ECM

symbiosis where clear positive growth and nutritional benefits exist for the plant host.

Another important investigation would be to establish the mutuality of the interaction

between jarrah and both these fungal species when they are in the novel symbiosis

form.

The novel symbiosis was observed for A. occidentalis and Scleroderma sp. isolates,

which belong to fungal families of Boletaceae and Sclerodremataceae, respectively.

Considering the diverse basidiomycete communities associated with forest habitats, it

would be interesting to determine how widespread this novel symbiosis is across plant

and fungal communities.

Page 126: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

109

3- Symbiotic associations and tolerance to Pi, Phi and AsV toxicities

AM fungi showed greater potential to induce tolerance against Pi toxicity than the

ECM or novel symbiotic fungi, and the tolerance was not correlated with lower shoot P

concentration (Chapters 4 and 5). This shows that the induced tolerance is due to some

unknown mechanisms other than lower Pi uptake or the dilution effect caused by higher

shoot biomass in mycorrhizal plants. Although both S. calospora and R. iregularis

induced Pi tolerance, only the former stimulated jarrah growth significantly (Chapters 2

and 5). I accordingly propose that AM fungi with positive responses such as S.

calospora could be promising potential candidates to deal with Pi toxicity in jarrah. For

example, pre-inoculation of jarrah seedlings with AM fungi in nurseries prior

transferring to natural (or disturbed) habitats could be an application of this finding.

However, in ECM and novel symbioses the Pi tolerance was only achieved when

symbiotic plants had lower shoot P concentration than NM controls (Chapter 4),

suggesting the dilution effect as the main mechanism for Pi tolerance in these cases.

Both AM and novel symbioses could induce tolerance against Phi toxicity via

reduced Phi uptake in inoculated plants. In the last experiment (Chapter 5), all

inoculated plants had significantly higher shoot P content than NM controls four weeks

after the Pi pulse whereas the shoot P content did not differ significantly across

inoculated treatments four weeks after the Phi pulse. It seems that the symbiotic

associations somehow differentiate Phi from Pi as a toxic P form and consequently

reduce the Phi uptake of host plants. Accumulation of Phi inside fungal mycelia could

be one potential mechanism here, as has been observed for other toxic molecules in

AM symbioses (Hildebrandt et al., 2007). Furthermore, the expression of EmPHT1;2

was unchanged in plants receiving the Phi pulse whereas the Pi-treated plants had

higher expression of EmPHT1;2 than untreated NM controls, which might be

correlated with lower uptake of Phi than Pi in NM plants (Chapter 5). Finally, the

dilution effect could always be considered as a mechanism of tolerance in all types of

symbioses with positive growth responses.

Although none of the fungi could help jarrah plants survive the AsV toxicity, they

were quite effective at reducing the shoot As concentration (Chapter 5). Reduction of

AsV to AsIII inside fungal hyphae and exporting the produced AsIII into soil, As

accumulation inside fungal vacuoles and the growth dilution effect are among the

potential mechanisms behind the reduced shoot As concentration in inoculated jarrah

plants (Sharples et al., 2000; Hildebrandt et al., 2007). Lower expression of plant PHT1

genes has also been reported as a mechanism for AsV tolerance in AM symbiosis

Page 127: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

110

(Meharg and Macnair, 1992). This is not apparently the underlying mechanism for the

present study as neither AM nor novel symbioses caused plants to reduce the

expression of their PHT1 genes in roots. Similar to the Phi results, the expression of

EmPHT1;2 was not affected by the AsV pulse while the Pi-treated plants had higher

transcript abundance than untreated NM controls, suggesting that this PHT1 gene might

not be involved in transport of Phi and AsV in jarrah plants. Future detailed assessment

of the spatial and nutritional expression patterns of this particular PHT1 gene family

member are justified by these observations.

4- Symbiotic associations and expression of PHT1 genes in roots

Many plant species reduce the expression of their PHT1 genes in response to AM

symbiosis (Karandashov and Bucher, 2005) but there is only one study on an ECM

symbiosis (Loth-Pereda et al., 2011) showing that two PHT1 genes had reduced

expression in poplar plants inoculated with both AM and ECM fungi. This is the

second report showing that ECM plants (Scleroderma sp.) reduced the expression of

some PHT1 genes in roots, which was evidently linked with hyphal 33

P uptake (Chapter

3). However, when ECM fungi do not colonise plant roots and establish a novel

symbiosis, the expression of PHT1 genes is not affected as observed for A. occidentalis

(Chapters 3 and 5) and Scleroderma sp. (Chapter 5). This is presumably due to the lack

of hyphal nutrient uptake in the proposed novel symbiosis.

The AM fungus S. calospora failed to repress the expression of jarrah PHT1 genes,

which was an observation in contrast to the reports on other plant species (Karandashov

and Bucher, 2005). Nonetheless, there is a study showing that the AM fungus

Gigaspora rosea was not able to affect the expression of the PHT1 gene MtPT2 in

Medicago truncatula, while this gene had lower transcript abundances in plants

colonised by other AM species tested (Burleigh, 2001). However, it is also relevant that

jarrah was not inclined to reduce the expression of most of the assessed PHT1 gene

family members, even following exposure to highly toxic concentration of Pi, Pi and

AsV (Chapter 5). In addition, AM-eucalypt symbioses are different from AM

relationships in other plant species in terms of being transitory events (Chen et al.,

2000; Adams et al., 2006; Chapter 5). Accordingly, I hypothesize that the lack of

change in the expression of PHT1 genes in AM jarrah roots could be related with either

the genetic characteristics of jarrah as a low-P adapted species or the transitory nature

of AM-eucalypt symbioses. It will be important to ensure that all members of the PHT1

gene family have been identified before these additional experiments are conducted.

Page 128: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

111

5- Conclusion and future research directions

This research has contributed to understanding the dual mycorrhizal associations of

jarrah and led to the discovery of a new plant-fungus symbiosis. The observations of

the novel symbiosis indicate that basidiomycete fungi can establish beneficial

symbioses without colonising plant roots apparently via their modifying functions in

the rhizosphere soil. This new symbiosis could be a widespread phenomenon across

plant/fungal communities. Together, the results of this thesis show that ECM fungi,

regardless of their colonisation ability, are superior symbiotic partners for jarrah to AM

fungi in terms of conferring growth and nutritional benefits. Positive growth responses

in AM-jarrah symbioses might depend on the fungal taxonomic position, origin of

isolates or experimental conditions. Finally, although all ECM, novel and AM

symbioses can have protective effects against Pi toxicity, AM symbioses function

better upon exposure to higher Pi pulses.

I propose the following research directions to address the hypotheses and questions

arising from this thesis:

- Determine the distribution of novel symbiosis among plant/fungal communities.

Representative woody plant species and basidiomycete fungi from different

families can be used to find out how widespread could be this novel symbiosis.

- Clarify if the dual behaviour of some ECM fungi such as Scleroderma sp. is

correlated with low minimum temperature during the growth season.

- Explore the molecular basis in the ECM-novel continuum to provide

fundamental insights into plant-fungus signalling pathways. For example,

expression of genes associated with nutrient uptake and certain secondary

metabolites such as carboxylates could only be down-regulated in ECM plants,

not in the novel symbiosis.

- Determine if the high carboxylate concentration in the rhizosphere soil in the

novel symbiosis has a plant or fungal origin.

- Shed more light on the mutuality of the novel symbiosis by showing how the

plant partner actually feeds the fungus. The hypothesis is that host plants exude

the hexoses such as glucose and fructose into the rhizosphere soil to feed the

fungal partner.

- Investigate how the ECM fungi involved in the novel symbiosis can decompose

the soil organic matter to release nutrients, by focusing on certain enzymes such

as acid phosphatases, proteinases or polyphenol oxidases.

Page 129: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

112

- Study the effects of ECM colonisation on the expression of nutrient transporters

other than P, in particular N. There was preliminary evidence for the reduced N

uptake by roots of ECM plants.

- Investigate the contrasting physiological effects of different AM fungi on

eucalypts in more detail, focusing on endemic fungi.

- Clarify if adequate or even toxic supplies of Pi can extend the duration of

symbiotic relationships between eucalypts and AM fungi, which decline

naturally under low P conditions.

- Elucidate the mechanism of Pi tolerance in AM jarrah plants. The tolerance was

neither correlated with lower shoot P concentration nor the expression of PHT1

genes. Localisation of P in different plant organs could be helpful in

understanding the tolerance mechanism.

- Study the mechanism of tolerance to toxic Pi analogues (Phi and AsV) in all

ECM, novel and AM symbioses. Accumulation of these toxic molecules inside

fungal mycelia could be a potential mechanism here.

- Clarify if the inability to reduce the PHT1 gene expression under Pi toxicity

conditions is linked with P sensitivity in other Australian native plant species.

- Determine if eucalypts generally do not alter the expression of their PHT1

genes in AM symbioses, which would be opposite of the existing literature for

other plant species.

Finis

Literature Cited

Adams F, Reddell P, Webb MJ, Shipton WA (2006) Arbuscular mycorrhizas and

ectomycorrhizas on Eucalyptus grandis (Myrtaceae) trees and seedlings in native

forests of tropical north-eastern Australia. Aust J Bot 54: 271-281

Adjoud D, Plenchette C, HalliHargas R and Lapeyrie F (1996) Response of 11

eucalyptus species to inoculation with three arbuscular mycorrhizal fungi.

Mycorrhiza 6: 129-135

Arnebrant KEH, Finlay RD (1993) Nitrogen translocation between Alnus glutinosa (L.)

Gaertn. seedlings inoculated with Frankia sp. and Pinus contorta Doug. ex Loud

seedlings connected by a common mycelium. New Phytol 124: 213-242

Bending GD, Read DJ (1995) The structure and function of the vegetative mycelium of

ectomycorrhizal plants VI. Activities of nutrient mobilising enzymes in birch

litter colonized by Paxillus involutus (Fr.) Fr. New Phytol 130: 411-417

Page 130: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

113

Brundrett MC, Abbott LK (1991) Roots of jarrah forest plants.1. Mycorrhizal

associations of shrubs and herbaceous plants. Aust J Bot 39: 445-457

Brundrett MC, Pichel Y, Peterson RL (1985) A developmental study of the early stages

in vesicular-arbuscular mycorrhiza formation. Can J Bot 63: 184-194

Burleigh SH (2001) Relative quantitative RT-PCR to study the expression of plant

nutrient transporters in arbuscular mycorrhizas. Plant Sci 160: 899–904

Chen YL, Dell B, Brundrett MC (2000) Effects of ectomycorrhizas and vesicular-

arbuscular mycorrhizas, alone or in competition, on root colonisation and growth

of Eucalyptus globulus and E. urophylla. New Phytol 146: 545-556

Finlay RD, Odham HEG, Soderstrom B (1989) Uptake, translocation and assimilation

of nitrogen from 15N-labelled ammonium and nitrate sources by intact

ectomycorrhizal systems of Fagus sylvatica infected with Paxillus involutus. New

Phytol 113: 47-55

Gomez TCR, Faria LP and Lin MT (1987) Mycorrhization of eight species of

Eucalyptus with VAM fungi, Durban, South Africa. pp. 86–93

Hoffland E, Kuyper TW, Wallander H, Plassard C, Gorbushina AA, Haselwandter K,

Holmstrom S, Landeweert R, Lundstrom US, Rosling A, Sen R, Smits MM, van

Hees PAW, van Breemen N (2004) The role of fungi in weathering. Front Ecol

Enviro 2: 258–264

Handreck KA (1997). Phosphorous requirements of Australian native plants. Aust J

Soil Res 35: 241-289

Hawkins HJ, Hettasch H, Mesjasz-Przybylowicz J, Przybylowicz W, Craner MD

(2008) Phosphorus toxicity in the Proteaceae: A problem in post-agricultural lands.

Sci Hortic 117: 354–365

Hildebrandt U., Regvar M., Bothe H. (2007). Arbuscular mycorrhiza and heavy metal

tolerance - Phytochem 68: 139-146

Howard K, Dell B, Hardy GE (2000) Phosphite and mycorrhizal formation in seedlings

of three Australian Myrtaceae. Aust J Bot 48: 725-729

Jakobsen I (1995) Transport of phosphorus and carbon in VA mycorrhizas. In

'Mycorrhiza, structure, function, molecular biology and biotechnology'. (Eds A

Varma and B Hock) pp. 297-324. (Springer Verlag: Berlin, Germany)

Jones MD, Durall DM, Tinker PB (1998) Comparison of arbuscular and

ectomycorrhizal Eucalyptus coccifera: growth response, phosphorus uptake

efficiency and external hyphal production. New Phytol 140: 125-134

Karandashov V, Bucher M (2005) Symbiotic phosphate transport in arbuscular

mycorrhizas. Trends Plant Sci 10: 22-29

Page 131: Physiological and Molecular Analysis of Tolerance …...Physiological and Molecular Analysis of Tolerance to Phosphate Toxicity in Jarrah (Eucalyptus marginata) Seedlings Inoculated

114

Krüger M, Krüger C, Walker C, Stockinger H, Schüßler A (2012) Phylogenetic

reference data for systematics and phylotaxonomy of arbuscular mycorrhizal

fungi from phylum to species-level. New Phytol 193: 970-984

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

Landeweert R, Hofflund E, Finlay RD, van Breemen N (2001) Linking plants to rocks:

Ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol 16:

248-254

Loth-Pereda V, Orsini E, Courty PE, Lota F, Kohler A, Diss L, Blaudez D, Chalot M,

Nehls U, Bucher M, Martin F (2011) Structure and expression profile of the

phosphate Pht1 transporter gene family in mycorrhizal Populus trichocarpa. Plant

Physiol 156: 2141–2154

Meharg AA, Macnair MR (1992) Suppression of the high affinity phosphate uptake

system: a mechanism of arsenate tolerance in Holcus lanatus L. J Exp Bot 43: 519–

524

Olsson PA, Wallander H (1998) Interactions between ectomycorrhizal fungi and the

bacterial community in soils amended with various primary minerals. FEMS

Microbiol Ecol 27: 195-205

Pritsch K, Garbaye J (2011) Enzyme secretion by ECM fungi and exploitation of

mineral nutrients from soil organic matter. Ann Forest Sci 68: 25-33

Rosewarne GM, Barker SJ, Smith SE (1997) Production of near-synchronous fungal

colonisation in tomato for developmental and molecular analyses of mycorrhiza.

Mycol Res 101:l 966-970

Shane MW, Szota C, Lambers H (2004) A root trait accounting for the extreme

phosphorus sensitivity of Hakea prostrata (Proteaceae). Plant Cell Environ 27: 991-

1004

Sharples JM, Meharg AA, Chambers SM, Cairney JWG (2000) Mechanism of Arsenate

Resistance in the Ericoid Mycorrhizal Fungus Hymenoscyphus ericae. Plant Physiol

124: 1327-1334

Smith SE, Smith FA, Jakobsen I (2004) Functional diversity in arbuscular mycorrhizal

(AM) symbioses: the contribution of the mycorrhizal P uptake pathway is not

correlated with mycorrhizal responses in growth or total P uptake. New Phytol 162:

511-524

Tibbett M, Sanders FE (2002) Ectomycorrhizal symbiosis can enhance plant nutrition

through improved access to discrete organic nutrient patches of high resource

quality. Ann Bot 89: 783-789