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The Role of Arbuscular Mycorrhizal Fungi in Uptake by
Pasture Legumes of Phosphorus Provided as a Pulse
Nazanin K. Nazeri
Plant Pathology (Hons)
Plant Virology (MSc)
This thesis is presented for the degree of
Doctor of Philosophy of
The University of Western Australia
School of Plant Biology
School of Earth and Environment
December 2014
i
Abstract
In the southern Australian cropping zone, large areas of land contain legume-based
pastures; these provide feed for livestock and nitrogen for following crops. Annual
pastures, especially of Trifolium subterraneum (subterranean clover), are widely sown
in Mediterranean climates around the globe and particularly in southern Australia.
However, the predominance of annual pastures and crops in south-western Australia has
caused problems, including dryland salinisation. Consequently, the development of
novel perennial pasture plants, including Australian natives, has been a research priority
over the past 15 years.
Phosphorus fertiliser is a common input to farms in southern Australia and its use has
much increased soil P concentrations. However, rock phosphate is a non-renewable
resource and it is now more important to use it efficiently. Forming a symbiosis with
arbuscular mycorrhizal fungi (AMF) is an important means for plants to enhance their P
uptake, primarily because their external hyphae are able to explore a greater volume of
soil than plant roots can alone. Mycorrhizal experiments are usually conducted under
constant or decreasing P levels due to plant P uptake. However, few studies have
focused on the response of colonised plants to a pulse of P as may occur under field
conditions such as when the first winter rain falls after a hot dry summer or when P
fertilisers are applied to growing plants. To my knowledge, this study is the first to
concentrate on the role of AMF in plant P uptake from a P pulse. Care was taken to
ensure that results were relevant to field conditions and therefore the soil used in all
experiments was a field soil with a moderate concentration of plant available P (18 mg
kg-1), as is common in the cropping areas of Western Australia.
In the first experiment (Chapter 2), the role of AMF in P uptake from a pulse of P was
investigated. Since Trifolium subterraneum is commonly used in mycorrhizal
experiments and its response to varying levels of P in soil has been investigated before,
it was used as the host plant for this experiment. The following hypotheses were
addressed: 1) AMF will enhance uptake of P from a pulse as may occur following
addition of fertiliser or the start of the winter rain; 2) uptake of P is greater when plants
are colonised by a diverse community of AMF (inoculation with field soil), rather than a
single-species inoculum; and 3) presence of microbes in the soil increases P uptake
compared to the control (no microbes). Plants were grown in field soil (+AMF) or in
sterilised field soil (-AMF) with the addition of filtrates (microbes) from the unsterilised
ii
field soil, except for the control (no AMF, no microbes). Plants were harvested at week
11, a day before (day 0), a day after (day 2) and seven days after (day 8) a pulse of P (10
mg kg-1 as KH2PO4). Shoot and root dry weights did not differ among treatments. At
day 0, both shoots and roots of colonised plants had higher P concentrations than did
those of uncolonised plants, as expected. However, after the pulse, the rate of increase
of shoot P concentration in colonised plants was slower than that in uncolonised plants,
and by day 8, colonised and uncolonised plants had a similar shoot P concentration; root
P concentration remained higher in the colonised plants. Thus, the presence of AMF
appeared to restrict the movement of P from the root to the shoot after P was added in
the pulse, i.e. AMF had a “moderating” effect. Little difference in P uptake was found
between plants that were colonised by a diverse community of AMF (originating from
the field soil) and plants that were inoculated by a single species. However, the results
from a commercial DNA test of the field soil showed that more than 97% of AMF
present in the soil belonged to one family. Thus, the field soil may not have represented
a “diverse” treatment. Alternatively, diversity of AMF may not have been of advantage
to plant P uptake due to the moderate soil P concentrations. The effect of the treatment
with soil microbes on plant P uptake was rarely different from that of the control.
Again, this might reflect the moderate concentrations of soil P; experiments that report a
positive effect from the microbe treatments generally have lower concentrations of soil
P.
In the second experiment (Chapter 3), the effect of AMF on the response to a pulse of P
of three (potential) perennial pasture legumes (Kennedia prostrata, Cullen
australasicum, Bituminaria bituminosa), the commonly grown perennial pasture species
Medicago sativa (lucerne) and Trifolium subterraneum was investigated. The following
hypotheses were addressed: 1) AMF will moderate shoot P concentration following a
pulse; 2) the release of carboxylates into the rhizosphere (which is a mechanism used by
plants to mobilise soil P) will decrease following a pulse; and 3) colonisation of plants
by AMF will decrease the release of carboxylates into the rhizosphere. Plants were
grown in the same field soil with a moderate P concentration (+AMF) or in sterilised
field soil (-AMF), all with the addition of filtrates (microbes) from the unsterilised field
soil. After approximately 10 weeks, half of the pots received a pulse of P (15 mg kg-1).
All plants were harvested one week later. Results were consistent with those of
experiment 1; that is, there was a moderating effect of AMF on plant shoot P
concentration following a P pulse. It was also found that the P pulse did not have any
iii
effect on the amount of rhizosphere carboxylates. However, colonisation by AMF
reduced the amount of rhizosphere carboxylates by ~50%, providing strong
confirmation of the concept of “mycorrhizal-mediated resource partitioning” which was
recently presented in the literature.
The main focus of the third experiment (Chapter 4) was to test the hypothesis that
following a significant pulse of P, P movement to shoots is restricted due to storage of
high concentrations of P in intraradical AM hyphae in insoluble forms. In this
experiment, two P pulse levels (150 mg kg-1, 15 mg kg-1 and control (No P pulse)) were
applied a week before harvest to Trifolium subterraneum either inoculated with
Scutellospora calospora (+AMF) or grown in sterilised field soil (-AMF); unsterilised
field soil filtrates were added to all pots. All plants were harvested at week seven. A few
samples were chosen randomly for X-ray microanalysis after being freeze-substituted in
a 10% acrolein in diethyl ether mixture over a 0.3 nm molecular sieve and embedded in
Araldite 502 epoxy resin. Due to low levels of colonisation by AMF, and the ability to
only examine a small number of samples with this technology, samples that were
prepared for X-ray analysis were free of AMF and testing of the hypothesis was not
possible. However, other interesting results were obtained. Plants that received the high
P pulse (150P) had root P concentrations of 20 mg g-1 and granules with extraordinarily
high P concentrations in their cortex (up to 7640 mmol P kg-1). There was a very strong
positive linear relationship between P and K concentrations in the granules. Formation
of granules with high P concentrations might be a plant response to avoid P toxicity
when encountering a sudden availability of high concentrations of P. The structure of
the granules, the relationship between P and K concentrations, and the formation of the
granules in response to a P pulse are all consistent with the formation of polyphosphate
in organisms such as fungi and yeast. However, such high concentrations of
polyphosphate have not previously been credibly reported in plants. Further research is
now needed to confidently ascertain that the granules were not an artefact of the
embedding process and whether polyphosphate is indeed their major constituent.
In summary, this thesis shows that AMF play an additional role to that of enhancing
plant P acquisition when P is limiting, i.e. they also allow plants to maintain leaf P
concentrations within narrower boundaries than the plant could achieve alone when the
plant experiences a pulse of P.
iv
v
Table of Contents
Abstract I
Table of Contents V
Acknowledgments VII
Thesis Declaration and Publication List IX
CHAPTER 1. General Introduction 1
1.1 IMPORTANCE OF PASTURES LEGUMES FOR FARMING SYSTEMS 2
1.1A Bituminaria bituminosa 5
1.1B Cullen australasicum 5
1.1C Kennedia prostrata 6
1.2 PHOSPHORUS IN PLANTS AND SOIL 7
1.2A Phosphorus pulse 8
1.2B Responses of plants to a pulse of P 9
1.3 ARBUSCULAR MYCORRHIZAL FUNGI (AMF) 10
1.3A The growth response of plants to colonisation by AMF 11
1.3B How does AMF help plants to access P? 13
1.3C Phosphate translocation within hyphae and delivery to intraradical interfaces 14
1.3D Transfer of P from AM fungal structures to the root cell 15
1.3E Phosphorus pathways in colonised plants 16
1.3F Role of AMF in uptake of a pulse of P 17
1.3G Which is a better approach in glasshouse experiments? A diverse community of AMF or a single-species inoculum? 17
1.4 GAPS IN KNOWLEDGE 19
1.5 RESEARCH OBJECTIVES 19
1.6 THESIS OUTLINE 20
1.7 REFERENCES 21
CHAPTER 2. Do Arbuscular Mycorrhizas or Heterotrophic Soil Microbes Contribute
toward Plant Acquisition of a Pulse of Mineral Phosphate? 35
2.1 PREFACE 35
2.2 PUBLISHED PAPER 36
CHAPTER 3. Moderating Mycorrhizas: Arbuscular Mycorrhizas Modify Rhizosphere
Chemistry and Maintain Plant Phosphorus Status within Narrow Boundaries 49
3.1 PREFACE 49
3.2 PUBLISHED PAPER 50
CHAPTER 4. A Sudden, High Pulse of Phosphorus Triggers Formation of Granules with
Extraordinary High Concentrations of Phosphorus and Potassium in Cortical Cell
Vacuoles of Trifolium subterraneum 63
4.1 PREFACE 63
4.2 READY FOR SUBMISSION PAPER 64
ABSTRACT 65
INTRODUCTION 66
MATERIALS AND METHODS 67
Glasshouse experiment 67
Sample preparation for X-ray microanalysis 69
Quantitative X-ray microanalysis 71
vi
RESULTS 72
Plant growth 72
Microscopy and quantitative X-ray microanalysis 73
Elemental analysis 75
DISCUSSION 77
A pulse of a high P concentration induces granules high in [P] in the root 77
What is the form of P in the granules? 80
CONCLUSION 81
ACKNOWLEDGEMENTS 82
REFERENCES 82
CHAPTER 5. General Discussion 89
5.1 INTRODUCTION 90
5.2 KEY RESEARCH FINDINGS 90
5.2A Phosphorus uptake from a pulse is enhanced in the roots of colonised plants, but P transport to the shoots is restricted by AMF (after a week) (moderating effect) 91
5.2B Phosphorus uptake did not differ between a diverse community of AMF and a single species culture 93
5.2C Microbes had little effect on uptake of P from a pulse 94
5.2D A pulse of P did not decrease the amount of rhizosphere carboxylates 95
5.2E Colonisation with AMF decreased rhizosphere carboxylates 96
5.2F Formation of granules with high concentrations of P in the cortex area of the roots of Trifolium subterraneum which were treated with a high pulse of P 97
5.3 UNEXPECTED FINDINGS 98
5.3A Inoculation with AMF greatly decreased manganese concentrations 98
5.3B Phosphorus efflux following colonisation with AMF 99
5.3C Colonisation with AMF makes the rhizosphere more alkaline 99
5.4 LIMITATIONS OF THE RESEARCH PRESENTED IN THIS THESIS 100
5.5 FUTURE RESEARCH DIRECTIONS 100
5.6 CONCLUDING REMARKS 100
5.7 REFERENCES 101
vii
Acknowledgments
Four years ago, when I started my PhD journey, having my one and half year old son, I
was not sure if I would be able to reach the end. Having the support and encouragement
of the following people made it all possible.
First of all, I would like to thank my supervisor Associate Professor Megan Ryan who
was not only a supervisor to me but also a friend whom I could talk to when there was
no one else around to listen to my complaints. She will always be my role model in
research. She taught me that doing a PhD is not about doing something impossible, but
it is all about telling an interesting story in science. I also had the honour of having
W/Prof. Hans Lambers as my supervisor. Prof. Lambers’ discipline always amazed and
inspired me. I wish I could send his records of giving precise feedback on my
manuscripts in the shortest time possible to the Guinness Book of Records. I am quite
sure he would be in the top three. I would also like to thank Professor Mark Tibbett, my
third supervisor, for his comments. Finally, thanks to Associate Professor Peta Clode for
guiding me in using X-ray microanalysis and sample preparation and providing
feedback on chapter 4.
I have to declare that I am very lucky to have someone to support me during all hard
times of my life and putting himself behind me without any expectations. My deepest
gratitude to my husband and my son who were always there for me and my life is
meaningless without them. No words can express my gratitude to my life time
supervisors, my mom and dad. Thank you for visualising my dream for me every day of
my life. Being apart from you and your kindness is one of the toughest things I have to
overcome every day.
I want to also thank Evonne Walker and Daniel Kidd for providing me with technical
help and Dion Nicol for collecting soil samples for me from the Newdegate site. Thanks
to Michael Smirk and Elizabeth Halladin for helping me with plant analysis and to Greg
Cawthray for providing help in analysing carboxylates of rhizosphere extracts using
HPLC and for helping me to understand chemistry concepts. Also, thanks to Scott
Glyde for his support. Finally, I also would like to especially thank all my office mates
who made this journey more peaceful by sharing our happy and sad days together.
This research has been financially supported by an Australian Postgraduate Award, the
Future Farm Industries Cooperative Research Centre and a UWA PhD Completion
viii
Scholarship.
I would also like to acknowledge the facilities, and the scientific and technical
assistance of the Australian Microscopy & Microanalysis Research Facility at the
Centre for Microscopy, Characterisation & Analysis, The University of Western
Australia, a facility funded by the University, State and Commonwealth Governments.
ix
Thesis Declaration and Publication List
This thesis is presented as a series of two scientific papers and one manuscript to be
submitted later. The bibliographical details are as follows.
Chapter 2: Nazeri, Nazanin K, Lambers, H; Tibbett, M and Ryan, M. (2013) Do
arbuscular mycorrhizas or heterotrophic soil microbes contribute toward plant
acquisition of a pulse of mineral phosphate? Plant and Soil, 373, 699-710.
Chapter 3: Nazeri, Nazanin K, Lambers, H; Tibbett, M and Ryan, M. (2013)
Moderating mycorrhizas: arbuscular mycorrhizas modify rhizosphere chemistry and
maintain plant phosphorus status within narrow boundaries. Plant, Cell and
Environment 37: 911-921.
Chapter 4: Nazeri, Nazanin K, Clode, P; Lambers, H; Tibbett, M and Ryan, M. (2014)
A sudden high pulse of P triggers formation of granules with extraordinary high
concentrations of phosphorus and potassium in cortical cell vacuoles of Trifolium
subterraneum (in preparation for submission to New Phytologist).
My contribution in each paper is 85% or above. I designed and performed the
experiments, analysed the results and wrote the papers. The contribution of the co-
authors in each paper/chapter is mainly related to advice on the design of the experiment
analysis of results when required, and review of the drafts of the papers/chapters.
Nazanin Nazeri
July 2014
Chapter 1. General Introduction 1
CHAPTER 1.
General Introduction
This chapter introduces the importance of pasture legumes for the productivity and
sustainability of farming systems in Australia and the effect that phosphorus (P)
limitation in Australian soils has on plant growth. The low P concentration of
Australian native soils has resulted in farmers routinely applying P fertilisers. It is
important, if this limited resource is to be used efficiently, that plants are able to quickly
take up the pulse of P that results from application of P fertiliser before the P is sorbed
to become poorly available in the soil. The symbiosis between plants and arbuscular
mycorrhizal fungi (AMF) is common in pasture legumes and is possibly one way that
absorption of a pulse of P could be enhanced. This thesis characterises the role of AMF
in uptake of P delivered as a pulse to pasture legumes.
Chapter 1. General Introduction 2
1.1 Importance of pastures legumes for farming systems
Pasture legumes have historically been important in maintaining productivity in
Mediterranean agricultural systems, which include areas at middle latitudes in all
continents (between 30-40°) and in Australia in south-western and central parts of
southern Australia (Saxena 1988). In 2006, the temperate wheat-sheep zone in southern
Australia contained 39,500 broadacre farms (ABARE 2006; Kirkegaard et al. 2011),
many of which grew both pastures and crops. In addition to grazing livestock,
incorporating pasture legumes into farming systems has the important benefit of fixing
atmospheric nitrogen (N) through the symbiosis formed between legumes and soil
bacteria (Rhizobium/Bradyrhizobium spp.). The majority of N2 fixed in Mediterranean
agriculture is due to N-fixing legumes (Howieson et al. 2000). According to Peoples
and Baldock (2001), Australian pastures fix from 2 to 284 kg N ha-1 yr-1. Moreover,
pasture legumes also break the life cycle of cereal diseases when grown in rotation with
crops (Howieson et al. 2000; Robson 1990) and provide benefits from enterprise
diversification for farmers (Kirkegaard et al. 2011).
Annual pastures have an important role in agriculture in southern Australia. Ninety-two
million ha of land are under self-regenerating annual pastures, most of which are in
rotation with cereal crops (ley-farming) (Reeves and Ewing 1993; Unkovich 2002).
Longer cropping phases require sowing of annual legumes following crops (phase
farming). Ley farming was developed in South Australia in the 1950s based on the use
of Trifolium subterraneum (subterranean clover) and annual medics (Medicago spp.)
(Callaghan 1958; Donald 1965). Because of reliance on N2 fixation from annual pasture
legumes, it is recognised as one of the “advanced and low-input systems” (Robson
1990). Trifolium subterraneum is one of the dominant pasture species in Mediterranean
climates across the globe and is the most widely sown annual pasture in southern
Australia due to its high N-fixation, ease of management including suitability for
continuous grazing, adaptation to a wide range of growing season lengths, a moderate
level of seed dormancy enabling germination after 1-2 years of cropping and high-
quality forage (Hill and Donald 1998; Nichols et al. 2013; Porqueddu and González
2011).
The success of annual legume-based ley pastures in the southern Australian cropping
zone, in conjunction with annual crops such as Triticum aestivum (wheat), Brassica
napus (canola) and Hordeum vulgare (barley), means that Australian dryland cropping
Chapter 1. General Introduction 3
systems are very much dominated by annual plants - crops and pastures. Although these
systems based on annuals have been very successful in the past, they now face a number
of challenges including herbicide resistance in weeds and salinisation (Cocks 2001;
Dear et al. 2008; Powles and Matthews 1992).
The predominance of annual crop and pasture species has contributed to salinisation,
because the annuals are unable to transpire the same amount of water as did the native
perennial vegetation, due to shallower roots and a shorter (winter-spring) growing
season (i.e. 7-8 months) (Lambers 2003). This lower transpiration has caused increased
rates of deep drainage, which, in turn, has raised water tables which has mobilised salt
stores and caused dryland salinity (Dear et al. 2003; Dolling 2001; Loi et al. 2005;
Ridley et al. 1997). Consequently, planting deep-rooted perennial pasture species is one
of several ways now recommended for the control of dryland salinity in southern
Australia and indeed perennial pastures are becoming more popular (Cocks 2001; Dear
and Ewing 2008; Dunin 1970; Small 2003). The need to address dryland salinity was
the primary impetus for a major research focus on development of new perennial
species over the last 15 years (Dear and Ewing 2008). Deep-rooted perennials have the
ability to dry the subsoil during spring and summer, creating a dry soil buffer, which
can subsequently reduce leakage into groundwater by absorbing unused rainfall during
winter when plant demand and evaporation are low. For instance, according to Ward et
al. (2002), in south-western Australia during summer and autumn, soil water content
was reduced by 60 mm more by a perennial pasture (Medicago sativa) than by an
annual pasture (T. subterraneum), with most of the additional water used by the
perennial drawn from soil below a depth of about 60 cm. Such effects could prevent or
considerably delay the onset of secondary salinity when rainfall is sufficient to cause
leakage from a system based on annuals. The perennials that can be utilised in this
manner include a diversity of plant forms ranging from trees and shrubs to herbaceous
forage plants (Cocks 2001).
Perennial legume pastures provide a number of benefits to farming systems in addition
to symbiotically fixing atmospheric N2. These include maintenance of plant cover in
summer/autumn when annual pastures have senesced, thereby reducing wind erosion,
and runoff and leaching of nutrients (Bell et al. 2010; Glover 2005). However, the major
impetus for adoption of new perennial pasture species appears to be an ability to
produce green feed in the summer/autumn when annual pastures have senesced and are
present only as dry residues or a soil seed bank (Finlayson et al. 2012). Summer-green
Chapter 1. General Introduction 4
feed increases farm profits through reducing purchase of supplementary feed and
allowing a high year-round stocking rate (Monjardino et al. 2010; Nicol et al. 2013).
Benefits for animal health and improvements in product quality may also result, for
example, improved wool staple strength (Adams and Briegel 1998).
Currently, the only perennial legume widely grown in the southern cropping zone is the
exotic M. sativa. M. sativa is able to extract water from deeper parts of the soil than
annual pastures do, and can utilise summer/autumn rainfall (Bell et al. 2006). However,
adoption of M. sativa is not particularly high, especially in Western Australia (Cocks
2001). Poor adaptation to acid soils, waterlogging and drought can all contribute to poor
performance (Dolling et al. 2005; Hill 1996; Humphries and Auricht 2001).
In response to the problems associated with M. sativa, the CRC for Plant-Based
Management of Dryland Salinity and its successor, the Future Farm Industries (FFI)
CRC, focused on finding new perennials. Since 2000, about 720 species of legumes,
grasses and herbs have been screened for their potential to be developed as pastures for
temperate southern Australia by researchers associated with these two CRCs (Li et al.
2008; Nichols et al. 2007). Interestingly, this screening included some species native to
Australia (Ryan et al. 2008; Snowball et al. 2010). The potential of Australia’s native
flora for use in agriculture has been remarkably under-explored (Bell et al. 2010;
Hopper and Lambers 2014). Yet, Australia, because of its arid climate and infertile
soils, is an excellent place to search for potential new perennial pasture plants and the
development of deep-rooted perennials adapted to these conditions should be a high
priority for future research in the Australian wheatbelt (Dear et al., 2003).
As a result of the activities of the two CRCs, in 2007, the exotics Bituminaria
bituminosa var. albomarginata and Bituminaria bituminosa var. crassiuscula were
identified as the perennial legumes that showed the greatest potential for domestication
(Real et al. Accessed in 2014). Bituminaria bituminosa then became one of the research
priorities for the FFI-CRC. Native species from the genus Cullen were also found to be
promising (Real et al. 2011).
In response to the findings and priorities of the FFI-CRC, I focused my research on the
exotic perennial legumes Bituminaria bituminosa var. albomarginata (tedera) and M.
sativa, along with the native perennial legumes Cullen australasicum and Kennedia
prostrata. The exotic annual pasture legume T. subterraneum was included for
comparative purposes. The perennial species are now introduced in more detail.
Chapter 1. General Introduction 5
1.1A Bituminaria bituminosa
Bituminaria bituminosa (L.) C.H. Stirt. var. albomarginata is a self-pollinated
herbaceous, drought-resistant perennial forage legume with a wide distribution along
both sides of the Mediterranean sea and Macronesian islands. There are three botanical
varieties on the Canary Islands, of which two (albomarginata and crassiuscula) are
deemed eligible to enter into Australia by the Australian and Western Australian
Quarantine and Inspection Services. The albomarginata variety (common name tedera)
is originally from Lanzarote and is the most drought-resistant variety. It can acclimate to
a diverse range of soil types. This variety has the ability to stay green without shedding
its leaves during summer/autumn in dry Mediterranean climates (Méndez et al. 1991).
For example, during two of the driest summers/autumns on record in Western Australia,
B. bituminosa stayed green while M. sativa did not (Real and Verbyla 2010). It also
tolerates grazing and competes well with annual species (Suriyagoda et al. 2013). B.
bituminosa shows potential to be developed as out-of-season forage production across
all rainfall zones in Australia due to its high nutritional value and high biomass
production with low water demand (Foster et al. 2013; Martínez-Fernández et al. 2010;
Real and Verbyla 2010; Ventura et al. 2009). Foster et al. (2012) found significant
differences under water stress in stomatal conductance and also rooting depth of B.
bituminosa seedlings in comparison with seedlings of M. sativa and biserrula (Biserrula
pelecinus L.) and Afghan melon (Citrullus lanatus Thunb.) and concluded B.
bituminosa is the most drought resistant of these species. In field experiments in
Western Australia, Suriyagoda et al. (2013) found that the performance of B. bituminosa
was better than that of C. australasicum in terms of herbage productivity and survival.
1.1B Cullen australasicum
There are a total of 32 species in the Cullen genus and these are found in Africa,
Australia, Spain, Portugal, Sicily, Asia Minor and southern Asia; 25 of these are
endemic to Australia (Grimes 1997). Cullen is distributed widely across a broad range
of climates in Australia, from summer- to winter-dominant rainfall, and can be found in
areas with a wide range of annual rainfall (200 to 1300 mm) (Bennett et al. 2010).
Using an ecogeograpahic analysis, Bennett et al. (2006) identified seven species of
Cullen with potential for development as pastures for the more winter-dominant rainfall
zone of southern Australia. Cullen australasicum is one of the best-performing species
Chapter 1. General Introduction 6
among these seven species and is described as a drought-tolerant perennial for use in
extensive grazing systems (Hayes et al. 2009). Cullen australasicum (Schltdl.) J.W.
Grimes has a number of common names, including tall verbine, native scurf-pea and
native verbine (Cunningham et al. 1992; Dear et al. 2007). Li et al. (2008) studied 23
perennial legumes over three years at field sites across southern Australia. They ranked
Cullen australasicum seventh in persistence and fourth in dry matter yield. In an
evaluation of the productivity and persistence of 103 forage legumes and herbs over
three years, Real et al. (2011) ranked C. australasicum amongst the 11 most promising
species during winter, summer or all year. Ryan et al. (2008) concluded that C.
australasicum was the most promising native perennial legume species for
domestication. Recently, interest in this genus within Australia has increased, with
researchers aiming to release a commercial cultivar (Bennett et al. 2012; Humphries et
al. 2014; Suriyagoda et al. 2010).
1.1C Kennedia prostrata
Little is known of the genus Kennedia. It is widespread across southern Australia in
both woodlands and forests. It has a preference for light, well-drained soils. This
adaptation to light-textured soils suggests tolerance of drought and low soil fertility
(Bell et al. 2010). There are 15 species in the Kennedia genus, all of which are endemic
to Australia. Kennedia prostrata and K. prorepens are the most widely distributed
species. Kennedia species have been considered as legumes with agricultural potential
for a long time (Silsbury and Brittan 1955). Favourable characteristics include large
seeds (up to 45 mg) with good nutritional qualities; some species produce a huge
number of seeds in the first year after establishment (Bell et al. 2010; Rivett et al. 1983).
Kennedia species are well adapted to the low-P soils of Western Australia, although
some species appear to be unable to down-regulate their P-uptake capacity when
exposed to high levels of P (Pang et al. 2009a; Pang et al. 2009b). Due to all these
factors, Kennedia species have been suggested as possible forage plants with
agricultural potential in Western Australia (Bell et al. 2010; Cocks 2001; Silsbury and
Brittan 1955). While a high acid-detergent fibre content and low organic matter
digestibility may result in low voluntary consumption by grazing sheep (Revell et al.
2013), even low intake may benefit livestock health, due to its condensed tannin content
(Bouazza et al. 2012). Consumption of such species at appropriate doses increases the
post-ruminal supply of N (Ben Salem et al. 2010). In an experiment by Kotze et al.
Chapter 1. General Introduction 7
(2009) the development of parasite larvae was inhibited by four out of six Kennedia
species.
Overall, it appears that B. bituminosa, C. australasicum and Kennedia species are all
well-adapted to low-P soils and may perform better than M. sativa under low-P and dry
conditions. In an experiment by Pang et al. (2009b), productivity of tedera when it was
supplied with 0-6 µg P g-1 soil, was better than that of K. prostrata, K. prorepens and
Cullen species.
Figure 1. The three perennial legume species with potential to be developed as pasture species that were used in the present study. Bituminaria bituminosa (a); Cullen australasicum (b) and Kennedia prostrata (c).
1.2 Phosphorus in plants and soil
Phosphorus (P) is an essential plant nutrient. Compared to the other major nutrients, P is
the least mobile and least available in most soils, especially in ancient landscapes. As
soil becomes weathered due to soil erosion and leaching, the plant-available soil P
concentration gradually declines (Hinsinger 2001; Lambers et al. 2010; Raghothama
1999; Vance et al. 2003; Walker and Syers 1976). Ancient landscapes are those that
have been above sea level and have not been glaciated or disrupted by catastrophic
events for millions of years. South-western Australia, the southwest of South Africa and
the Pantepui mountains in Brazil and Venezuela, are all included in this category of
landscapes (Hopper 2009). In south-western Australia, many natural surface soils
contain less than 5 mg kg−1 of bicarbonate-extractable P (McArthur 1991).
Plants take up P in the orthophosphate (Pi) forms H2PO4- and HPO4
2-, which are in the
soil in very low concentrations (0.1-10 µM) (Hinsinger 2001). Also, Pi is very
immobile, unlike nitrate, which can easily move toward the roots via mass flow and
diffusion (Lambers et al. 2006). The contribution of mass flow in maintaining plant P
demand is as little as 1-5% (Lambers et al. 2008).
When crops or pastures are grown on soils with very low P availability, application of
(a) (c)(b)
Chapter 1. General Introduction 8
large quantities of fertilisers containing readily-available P, such as superphosphate, is
needed (Bolland and Gilkes 1997; Dann et al. 1996). Consequently, P concentrations in
agricultural soils in Australia have risen substantially (Vu et al. 2011). However, P is
predominantly derived from phosphate rock, which is a non-renewable resource, and
some predict that resources of inexpensive P will be depleted in 50 to 100 years
(Cordell et al. 2009; Scholz et al. 2013; Vance et al. 2003); some more recent papers
suggest less pessimistic scenarios (Fixen 2009). However, changes to the current
farming systems are necessary, and one option is to increase the use of low-P tolerant
perennial legumes in the rotation (Auricht 1999). In fact, the urgent need to develop
more P-efficient agricultural systems in Australia has been identified in recent reviews
(Richardson et al. 2011; Simpson et al. 2011). One element in such systems could be the
use of pasture plants that require minimal input of P fertiliser. Many Australian plant
species have specific root adaptations for growth in low-P soils (Lambers et al. 2010).
The interception of Pi by these plants can be increased by morphological (root
proliferation, an increase in frequency and length of root hairs, higher root: shoot ratios,
formation of a symbiosis with mycorrhizal fungi, finer roots) and physiological
(increased P-uptake rate and enhanced exudation of low-molecular-weight organic
anions (carboxylates) and phosphohydrolases) mechanisms (Lambers et al. 2006; Ryan
et al. 2012; Smith and Read 2008; Suriyagoda et al. 2012; Vance et al. 2003; Wissuwa
2005).
1.2A Phosphorus pulse
Hot dry summers and cool wet winters are the main characteristics of Mediterranean
environments. Since mobility of P is greatly reduced in dry soil, P becomes especially
limiting to plants over summer (Sardans and Peñuelas 2007). However, the first autumn
rains might release a pulse of P into the soil. This pulse results from the release of P due
to a number of processes including: lysed bacterial cells, microbes rupturing due to
osmotic shock in response to rapid rewetting of the soil and/or sudden fluctuations in
soil matric potential; rapid degradation of dry organic matter on the soil surface, that is,
plant detritus exposed to months of hot, dry weather; and breakdown of large
macroaggregates (Blackwell et al. 2010; Butterly et al. 2009; Grierson et al. 1998;
Turner et al. 2003; Turner et al. 2002; Turner and Gilliam 1976; Wu and Brookes
2005). Up to 58% of the total microbial biomass can be killed by soil drying and rapid
rewetting (Kieft et al. 1987; van Gestel et al. 1993; Wu and Brookes 2005). Qiu et al.
Chapter 1. General Introduction 9
(2004) found that levels of anion-exchange membrane-extractable P in south-western
Australia before winter rainfall were very low (˂2.6 mg P kg–1 dry soil) in surface soils
(just under the litter layer), but that during winter this increased 3.4–56 fold. Thirty
percent of this P increase occurred (from litter) in the first 24 hours. Following opening
rains this pulse of P may be considered analogous to the release of P following addition
of P fertiliser (for example, see Lewis et al. (1987)) and in both cases, quick uptake of P
by plants is desirable. For instance according to Lawton and Vomocil (1954), the
majority of P dissolution of fertiliser occurs within three to six hours of application. For
example, in a soil with field capacity moisture content, 50-80% of soluble P moved
from granules and tablets of fertiliser into the soil surface in 24 hours. This was reduced
to 20-50% for a soil with 2-4% moisture content. The pulse of P that is released into the
soil following rewetting is labile and not wholly accessible for all plants (Bünemann et
al. 2013). A quick uptake of a pulse of P following rainfall or fertiliser application is
important, as a quicker uptake will reduce the loss of P through P sorption,
immobilisation in microbial biomass and leaching (Anderson and McLachlan 1951;
Friesen et al. 1997; Robertson and Nash 2008). Roots are quite capable of rapidly taking
up P after rewetting (Eissenstat et al. 1999).
1.2B Responses of plants to a pulse of P
The capacity of a plant to absorb a pulse of P may depend on its P status. For instance,
when potato plants (Solanum tuberosum) which were grown in aerated nutrient solution
were provided with 1 mol m-3 of phosphate in solution, the absorption rate in P-deficient
plants was three to four-fold greater than that of plants with sufficient P supply
(Cogliatti and Clarkson 1983). Similarly, when barley (Hordeum vulgare) was grown in
hydroponics (and thus presumably non-mycorrhizal) under P limitation for 20 days, the
P influx reached its maximum after 16 days, and then declined gradually, while the
uptake rate for plants treated with a full nutrient solution declined from the start till the
end of the experiment, and reached ~30% of the initial rate at five days after
germination (Lefebvre and Glass 1982).
I surmise that plants that are treated with sufficient P are marginally able to absorb P
from a P pulse, while P-deficient plants respond strongly to a P pulse with a P influx
commencing in the first hours after the pulse is applied and continuing until the plants
reach a high P concentration. After this point, the P influx will decline.
Chapter 1. General Introduction 10
Plants with a low P status are likely to be colonised by AMF. The role of these fungi in
plant uptake of P from a pulse has not been investigated previously.
1.3 Arbuscular mycorrhizal fungi (AMF)
Mycorrhizas are associations between plant roots and certain groups of soil-dwelling
fungi (Brundrett 1991). Their most important role (from a plant perspective) is
enhancing plant uptake of nutrients while, in return, the fungi receive
photosynthetically-derived carbon compounds (Smith and Read 2008). Over 80% of all
vascular plants form a symbiosis with mycorrhizal fungi. There are seven types of
mycorrhizas based on morphology. Arbuscular mycorrhizas are the most prevalent and
are mutualistic symbiotic associations between roots and a small group of fungi in the
phylum Glomeromycota (Bolan 1991; Brundrett 1991; Schüßler et al. 2001; Smith and
Read 2008).
AMF are considered obligate symbionts, which receive carbon (C) from plants and, in
return, supply plants with nutrients. Increase in the uptake of nutrients is thought to
result from the exploration of a greater volume of soil by colonised plants, due to the
growth of external hyphae of the fungi (Grace et al. 2009; Jakobsen et al. 1992a). The
largest effect of AM formation is on plant P nutrition (Smith and Read 2008). Both
plants and AMF acquire P from the soil solution in the form of inorganic
orthophosphate (Bieleski 1973). Phosphorus is required by both symbionts in large
amounts (Smith and Read 2008). In addition, AM symbioses increase host plant
tolerance of diseases and host plant drought resistance, and decrease accumulation of
heavy metals by the host plant (Fitter 2006). Mycorrhizal fungi can also increase host
plant uptake of other elements including sulfur (S), boron (B), potassium (K), calcium
(Ca), magnesium (Mg), sodium (Na), zinc (Zn), copper (Cu), manganese (Mn), iron
(Fe), silicon (Si) and some other trace elements (Clark and Zeto 2000). Depending on
soil type, host plant and other factors, AMF can have a positive, a neutral or a negative
impact on the uptake of these nutrients (Ryan and Tibbett 2008). According to Clark
and Zeto (2000), AMF increase the uptake of P, N, Zn and Cu in most soils, but only
increase uptake of K, Ca and Mg when the plants are grown in acidic soils.
Arbuscular mycorrhizal fungi are called “arbuscular” because they form arbuscules,
structures that consist of finely branched hyphae, which are involved in nutrient
exchange within the cortical cells of plant roots (Peterson et al. 2004; Smith and Read
Chapter 1. General Introduction 11
2008) (Figure 2). Arbuscules, which are located within the cortical cells, but outside the
host cell’s plasma membrane, have long been considered diagnostic for AMF, although
they are not always present (Smith and Read 2008). Presence of intracellular coiled
hyphae is an alternative to identify AMF (Smith and Smith 2011) as are vesicles,
although both are not invariably present (Koide and Mosse 2004). Vesicles start to form
after the first arbuscules are formed and continue to develop when the arbuscules
senesce (Brundrett et al. 1996). Vesicles and fungal spores, both of which may be
intraradical or extraradical, have a role in accumulation of carbon (Olsson et al. 2011)
(Figures 2 and 3).
Figure 2. Distinguishing components of arbuscular mycorrhizal fungi within a colonised root (Brundrett et al. 1996).
Figure 3. Intraradical colonisation, extraradical spores and a very high density of extraradical hyphae in a densely colonised root of Trifolium subterraneum stained with ink (photo by N. Nazeri).
1.3A The growth response of plants to colonisation by AMF
Asai (1944) studied AM colonisation and nodulation in a large number of legumes, and
was the first to recognise the relationship between the development of arbuscular
mycorrhizas and the growth of the host (Asai 1944; Smith and Read 2008). In 1959,
Baylis suggested that increased growth of the host plant was mediated by enhanced P
uptake when arbuscular mycorrhizas were present. Baylis (1959) found that Griselinia
Spores
Extraradical hyphae
Intraradical colonisation
Chapter 1. General Introduction 12
littoralis seedlings, which were grown in a low-P soil and colonised by AMF, took up
3-5 times more P than non-mycorrhizal seedlings (Baylis 1959; Koide and Mosse
2004). It is now generally thought that AMF have the potential to play an important role
in growth of most crops and pasture species (Lekberg and Koide 2005; Ryan and
Tibbett 2008; Smith and Read 2008).
A recent meta-analysis has suggested that AMF may not play a significant role in
growth of annual autumn-sown crops and pastures in temperate southern Australia
(Ryan and Angus 2003; Ryan and Kirkegaard 2012), due, in part, to their relatively low
P demand in cold autumn conditions. However, the authors did speculate that AMF may
play a greater role in growth of summer-active perennials, due to dry soil limiting P
uptake and high summer temperatures enhancing plant P demand (Ryan and Kirkegaard
2012).
In general, the growth response to mycorrhization depends on many factors; from
molecular (e.g., transporter gene expression) to environmental (e.g., soil type, soil
nutrient availability, especially the status of P in the soil and light intensity) and
ecological (the composition of plant and fungus, density and competition) and can be
highly positive, neutral or negative (Smith and Smith 2011). A positive growth response
mostly happens because of an increase in P uptake when P is limiting in soil (Smith and
Read 2008). When the P concentration in soil is low, a non-mycorrhizal root system
cannot absorb P effectively, so the plant will become P deficient and plant growth will
decrease. However, colonised roots show enhanced P uptake and consequently plant
growth may increase. This is the well-known mycorrhizal growth response (Smith and
Read 2008). Suppression of growth of arbuscular mycorrhizal hosts following
colonisation by AMF is much less commonly reported than growth enhancement, but
does occur (Johnson et al. 1997; Smith et al. 2003), especially for seedlings
(Bethlenfalvay et al. 1982). Bethlenfalvay et al. (1982) and Koide (1985) speculated
that growth suppression might be due to drain of carbon from plant to AMF when plant
photosynthetic capacity and shoot-root ratio are low. Moreover, there are reports of
negative growth responses of non-host plants when they are colonised by AMF
(Sanders and Koide 1994; Veiga et al. 2012). For instance in an experiment by Veiga et
al. (2013), when the non-mycorrhizal plant Arabidopsis thaliana was grown together
with the mycorrhizal plant (either Trifolium pratense or Lolium multiflorum) in the
presence of AMF, growth of Arabidopsis thaliana was suppressed by 50% and this was
due to 43% colonisation by AMF. Also, an increase in soil P may lead to a negative
Chapter 1. General Introduction 13
growth response in mycorrhizal plants in comparison to non-mycorrhizal plants (Smith
and Read 2008).
Addition of soluble P has been shown many times to reduce the colonisation level by
AMF (Amijee et al. 1993; Ryan and Ash 1996) and in a study of pastures on 20
Australian dairy farms, colonisation had a negative relationship with soil P
concentration (Ryan et al. 2000). In Western Australia, constant application of fertilisers
in agricultural regions has resulted in a great elevation of soil P and it is possible that
this may now contribute to either a neutral or negative mycorrhizal growth response and
low levels of colonisation by AMF (Ryan and Kirkegaard 2012).
1.3B How does AMF help plants to access P?
Arbuscular mycorrhizal fungi access the same soil P sources that are used by plants
(Gianinazzi Pearson et al. 1981; Smith and Read 2008). Although there has been some
investigation of whether AM roots have the ability to exploit sources of P in soil that are
not available to the plant, such an effect has yet to be confidently shown (Smith and
Read 2008; Yao et al. 2001; Young et al. 1986).
When root exploration is restricted, the external hyphae of the AMF can deliver up to
80% of plant P uptake to the host plant over a distance of more than 10 cm from the root
surface (although it depends on the species of AMF) (Jakobsen et al. 1992b, c; Li et al.
1991). Also, it has been shown that the rate of P uptake by mycorrhizal plants is faster
than that of non-mycorrhizal plants (Sanders and Tinker 1973). Sanders and Tinker
(1973) observed the rate of inflow of P in mycorrhizal roots of onion (Allium cepa) was
much higher (17×10-14 moles cm-1 s-1) than the rate of inflow of P in non-mycorrhizal
roots (3.6×10-14 moles cm-1 s-1). Assuming that the difference in the rate of inflow is
caused by hyphae of the mycorrhiza, they calculated the rate of inflow of P into hyphae
is six times greater than the rate of inflow of P into root hairs. However, according to
Pearson and Jakobsen (1993), the P uptake rate differs with fungal species. Moreover,
colonisation by AMF will change the physiology of the root system and this might have
an influence on the P uptake (Clarkson 1985; Smith and Read 2008).
Forming a symbiosis with AMF and exudation of carboxylates into rhizosphere soil
have been described as “two key plant adaptations for acquisition of phosphorus” (Ryan
et al. 2012). Carboxylates have an important role in mobilising P in soils that contain
very little P or have most of the soil P strongly sorbed to soil particles (Dakora and
Chapter 1. General Introduction 14
Phillips 2002; Jones 1998). Moreover, carboxylates can also increase the solubility of
cations in the soil and, as a result, the concentration of these cations may increase in the
plant (Page et al. 2006; Ryan et al. 2012).
Exudation of carboxylates into rhizosphere soil and forming a symbiosis with AMF are
both carbon-demanding processes (Dinkelaker et al. 1989; Graham 2000) and there
might be trade-off between them. For example, exudation of carboxylates by plants that
are colonised by AMF is significantly less than that of uncolonised plants (Ryan et al.
2012). Also, it has been shown that exudation of carboxylates may become significantly
less with an increase in soil P level (Pearse et al. 2006; Wang et al. 2013). Thus, it has
been speculated that the presence of AMF may increase plant access to Pi at the expense
of access to poorly soluble forms of P – “mycorrhizal-mediated resource partitioning”
(Ryan et al. 2012).
1.3C Phosphate translocation within hyphae and delivery to intraradical
interfaces
The low Pi concentration in the soil solution and the large electrochemical potential
gradient between the soil solution and the plant/fungal cytosol, requires Pi to be taken
by extraradical mycelium using high-affinity Pi-H+ co-transporters, energized by H+-
ATPases (Bucher 2007; Ezawa et al. 2002; Harrison and van Buuren 1995; Smith and
Read 2008; Smith and Smith 2011). The Pi taken up by the fungus is used for cell
functions such as energy generation and biosynthesis of phospholipids, nucleic acids
and the precursors of carbohydrate polymers. Any extra Pi, if available, is transported
into intrahyphal vacuoles and converted to polyphosphate (Ezawa et al. 2002) which
buffers cytoplasmic Pi concentration and stores P for a momentary time as P is
transferred along hyphae (Hijikata et al. 2010; Smith and Smith 2011).
Polyphosphate is a linear polymer of orthophosphate residues, which are linked by high-
energy phosphoanhydride bonds (Harold 1966). Ezawa et al. (2002) hypothesised that
the process involved in polyphosphate synthesis in the AM fungi might be similar to the
process involved in polyphosphate accumulation in yeast (for which four genes were
found to be responsible (Ogawa et al. 2000)).
Temporary storage of Pi seems to be the most important role of polyphosphate in AMF
(Ezawa et al. 2002; Kornberg 1999). Viereck et al. (2004) used in vivo Nuclear
Magnetic Resonance (P-NMR) to characterise polyphosphate and other P pools in
Chapter 1. General Introduction 15
external hyphae and mycorrhizas of Glomus intraradices and suggested that
polyphosphate is the largest form of P storage in the fungi. The chain length of
polyphosphate in AM fungi is variable (Ezawa et al. 1999; Kornberg 1999). According
to Solaiman et al. (1999), most of the polyphosphates in intraradical hyphae are present
as short chains, while in the extraradical hyphae it is present as long chains or granules
(insoluble). The movement of the granules in the vacuoles of extraradical hyphae
toward the intraradical hyphae happens via cytoplasmic streaming (Clarkson 1985).
Ezawa et al. (2004) demonstrated that the transfer of AM fungus from a Pi-deficient to a
Pi-rich medium resulted in rapid accumulation (less than 3 h) of Pi into large amounts
of polyphosphate. Due to the shorter chain length of polyphosphate in the intraradical
than extraradical hyphae, it is proposed that polyphosphate is hydrolysed in the
intraradical hyphae to release Pi for the plant (Javot et al. 2007b; Ohtomo and Saito
2005; Solaiman et al. 1999). Ryan et al. (2003) found that intraradical hyphae can store
very high concentrations of P (up to 600 mM). Also, it was demonstrated that 30-70%
of the P in the hyphae and 100% of P in the arbuscules was not extractable (insoluble)
(Ryan et al. 2007) and, as a result hyphae could quickly absorb large amounts of P
(which, if left in soil, could be quickly lost (see section 1.2a)). Ryan et al. (2007)
concluded the P in the hyphae was likely to be in the form of polyphosphate.
It seems likely that exo- and endopolyphosphatases (PPX, PPN) and acid phosphatase
(ACPase) would work together to hydrolyze polyphosphate to Pi in AM fungal
symbiosis (Ezawa et al. 2002).
1.3D Transfer of P from AM fungal structures to the root cell
An interesting question is how P is transferred from the AM fungal structures inside the
host root into the root cells. Microscopic examination of roots has focused on
arbuscules in various stages of formation and decomposition (Kinden and Brown 1975a;
Kinden and Brown 1975b), and thus it was initially thought that the decomposition and
deterioration of the arbuscules played an important role in transferring the nutrients. It is
now known that arbuscule breakdown is not necessary for nutrient transfer.
Ultrastructural and physiological evidence, such as very high H+-ATPase activity
associated with the host plasmalemma, indicative of active transport mechanisms,
suggests that most nutrient exchange occurs across the living host-fungus interface
(Koide and Mosse 2004). In other words, Pi that is released from healthy arbuscules is
Chapter 1. General Introduction 16
taken up by plant Pi transporters, which are located on the periarbuscular membrane
(Javot et al. 2007a; Rausch et al. 2001). However, there is also evidence that P “leaks”
from intercellular hyphae into apoplast where, presumably, it can be absorbed by the
plant (Ryan et al. 2003).
1.3E Phosphorus pathways in colonised plants
Recent research has deepened our understanding of the role of arbuscular mycorrhizas
through showing that the fungi can affect the ability of the host plant itself to directly
take up P (Smith and Read 2008). There are two possible pathways for P acquisition in
the colonised plant: the first is a direct pathway, which occurs through plant root hairs
and epidermal cells, and the second is the AM pathway, which occurs through
extraradical mycelium of AMF as described above. Before 1990, it was assumed that
the contribution of the AM pathway and direct pathway were additive (i.e. the AM
pathway supplied mycorrhizal plants with extra P), but more recently, by using
compartmented pots and the tracking of radioactive P to extraradical hyphae and not to
roots, it has become clear that even when there is no positive growth response to
colonisation by AMF, the contribution of the AM pathway to P uptake can be larger
than the direct pathway (Smith and Smith 2011). According to Smith and Read (2008),
colonisation by AMF may lead to a “switch–off” of the direct pathway. For instance, in
an experiment by Smith et al. (2003), three plant species, flax (Linum usitatissimum),
tomato (Solanum lycopersicum) and annual medic (Medicago trancatula) were
colonised by three mycorrhizal fungi (Gigaspora rosae, Glomus intraradices and
Glomus caledonium). The three plant species showed positive to negative growth
responses, but the AM pathway was active in all three plant species. For example in S.
lycopersicum, although there was a consistent negative growth response to inoculation,
G. intraradices delivered 100% of the plant’s P uptake. Thus, even when there is no net
increase in total P uptake recorded as a result of inoculation with AMF, the fungi may
still be playing a major role in P uptake (Smith and Read 2008; Smith and Smith 2011).
In summary, when plants become colonised by AMF, the direct uptake pathway loses
some of its function (Smith et al. 2003). According to Smith et al. (2003), this loss of
function of the direct uptake pathway might result from down-regulation of plant genes
encoding P transporters in the epidermis and root hairs (Chiou et al. 2001; Rosewarne et
al. 1999). The relevance of this finding to the management of AMF in agricultural
systems is still unclear (Ryan and Kirkegaard 2012).
Chapter 1. General Introduction 17
1.3F Role of AMF in uptake of a pulse of P
I hypothesise that plants colonised by AMF will have a greater ability to absorb P from
a pulse of P than uncolonised plants have because having access to a greater volume of
soil than roots alone and having a high capacity to absorb and store P are two main
features of AM hyphae.
There are many reports on the response of AMF to application of P fertilisers which
show a decrease in level of colonisation. Indeed, in a meta-analysis, Treseder (1983)
concluded that AM colonisation level is, on average, reduced by 32% when P fertiliser
is used. It has also been shown that AMF can aid plant access to fertiliser. For instance,
Kapoor et al. (2004) examined Foeniculum vulgare plants, grown with and without
AMF (Glomus macrocarpum or G. fasciculatum) and P-fertiliser (20 kg P ha-1). The
plants that were both colonised and received the fertiliser had significantly more
biomass and a higher shoot P concentration than the plants that were colonised but did
not receive any fertiliser or were fertilised but not colonised. However, as the fertiliser
was added to the soil before growing the plants, the plants did not encounter a pulse of
P.
1.3G Which is a better approach in glasshouse experiments? A diverse
community of AMF or a single-species inoculum?
Many species of AMF are ubiquitous (Öpik et al. 2006), while others seem to be
restricted to specific ecosystems, climates or vegetation (Castillo et al. 2006; Oehl et al.
2007). According to Oehl et al. (2010), land-use intensity and soil type are two
important factors in determining the richness of AM fungal communities and their
composition, but the former has a more important effect. An increase in land-use
intensity may have a negative effect on AM fungal diversity. For instance, Oehl et al.
(2010) found grasslands contained more species of AMF than arable land (AM fungal
species richness in trap cultures after 20 months was 16-26 units for grassland and 13-
21 units for arable land). However, soil types might differ very much in AM fungal
communities and thus can be characterised by their AM fungal communities. For
example, Acaulospora, Scutellospora, Cestraspora and Gigaspora prefer a siliceous
(acidic) grassland as their habitat, while a number of Glomus species prefer calcareous
(alkaline) grasslands (Oehl et al. 2010). However, diversity within a single soil type can
be very high. For instance, in soil from a single field in south-western Australia, more
Chapter 1. General Introduction 18
than 20 species of AMF were present (Tibbett et al. 2008).
In spite of the diversity of AMF commonly reported in natural and agricultural
ecosystems, many glasshouse experiments examining the function of AMF are
performed using a single-species inoculum (pure culture) (Abbott and Robson 1977;
Smith and Read 2008). In an experiment by Wagg et al. (2011), plant productivity was
compared among a mixture of four AM fungal species (Glomus mosseae, G.
intraradices, G. claroideum and Diversispora celata) and the individual inocula of these
species under two soil conditions (low sand content [1:4 ratio of sand to field soil] and
high sand content [4:1 ratio of field soil to sand]). Inoculation with a diverse community
of AMF resulted in significantly higher plant productivity than inoculation with a single
species. Also, the effect of AM fungal diversity and AM fungal species on plant
productivity differed between the two soils. When sand content was high, colonisation
with both the best individual AM fungal species (D. celata), and a mixture of all fungal
species, produced the highest biomass in Trifolium pratense, while with low sand
content, colonisation with the diverse AM species resulted in higher biomass than with
the best individual AM fungal species (D. celata). Wagg et al. (2011) concluded that
diversity in the AM fungal community ensures plant productivity is maintained under
different environmental conditions. Also, according to van der Heijden et al. (1998), an
increase in number of AM fungal species results in an increase in both plant
productivity and biodiversity. Moreover, species of AMF differ in their capacity to
enhance plant uptake of P from soil. This is due to differences in the length of their
extraradical hyphae or P-uptake capacity per unit of hyphal length (Graham et al. 1982;
Jakobsen et al. 1992b, c). In view of these studies, I hypothesise that a diverse
community of AMF will be more efficient in absorbing P from a pulse of P than a single
species.
Using field soil as inoculum is a way to ensure a higher, and more realistic, diversity of
AMF and accompanying microbes in glasshouse experiments. However, field soil
inoculum involves introduction of indigenous AMF and the indigenous microbial
community (heterotrophic soil microbes) simultaneously.
The effect of the microbial community on plant growth might be synergistic or
antagonistic with that of AMF. To avoid confounding effects of AMF and
accompanying microbes when using field soil as inoculum, it has become common
practice to add a filtrate of field soil (which contains no mycorrhizal hyphae or spores,
Chapter 1. General Introduction 19
but contains most other soil microbiota) to non-mycorrhizal controls (Asghari and
Cavagnaro 2011; Lovelock and Miller 2002; Smith and Smith 1981).
1.4 Gaps in knowledge
Arbuscular mycorrhizal fungi are ubiquitous in natural and agricultural systems, but
little is known about their role in temperate pasture systems (with soils that typically
contain moderate to high concentrations of P). In particular, the ability of AMF to
enhance P uptake from a pulse of P as may occur following addition of fertiliser or the
start of the winter rain is unknown.
1.5 Research objectives
The overall aim of this thesis is to enhance understanding of the role of AMF in uptake
of P by pasture plants, when P is delivered as a pulse. In three experimental chapters, I
address the following hypotheses:
Hypothesis 1: AMF will enhance uptake of P from a pulse as may occur following
addition of fertiliser or the start of the winter rain (Chapter 2 – T. subterraneum;
Chapter 3 - perennial legumes).
Hypothesis 2: Uptake of P is greater when plants are colonised by a diverse community
of AMF (inoculation with field soil), rather than a single-species inoculum (Chapter 2).
Hypothesis 3: Presence of microbes in the soil increases P uptake compared to the
control (sterilised field soil) (Chapter 2).
Hypothesis 4: AMF will moderate shoot P concentration following a pulse (based on the
findings of Chapter 2) (Chapter 3).
Hypothesis 5: Release of carboxylates into the rhizosphere will decrease when plants
encounter a pulse of P (Chapter 3).
Hypothesis 6: Colonisation by AMF will decrease the release of carboxylates into the
rhizosphere (Chapter 3).
Hypothesis 7: (developed following results presented in Chapters 2 and 3): Following a
significant pulse of P, P movement to shoots is restricted, due to storage of high
concentrations of P in intraradical AM hyphae in insoluble forms (Chapter 4).
Chapter 1. General Introduction 20
1.6 Thesis outline
This thesis is in agreement with the Postgraduate and Research Scholarship Regulation
1.3.1.33(1) of the University of Western Australia, Australia and is presented as a series
of three scientific papers. Two manuscripts have already been published and the
remaining one is in preparation for submission. All the work presented has resulted
from work done towards this thesis. There are five main chapters in this thesis; a
General Introduction, three Experimental Chapters and a General Discussion. The
General Introduction covers the broad background for the work presented in the thesis
in order to justify the research objectives presented above. A more focussed review of
literature is presented in the introduction of each experimental chapter. The three
Experimental Chapters are presented in the format of scientific papers that can be read
individually or as a part of the whole thesis. Each Experimental Chapter includes the
following sections: Abstract, Introduction, Materials and Methods, Results, Discussion
and References. Each experimental chapter is preceded by a short preface, to link the
chapter to the broader hypotheses addressed by the thesis. This “thesis-as-a-series-of-
papers” format results in some unavoidable repetition, especially in the Materials and
Methods sections of Chapters 2 with 3. The content of each experimental chapter is as
follows:
Chapter 2- This chapter was designed as a preliminary experiment to determine
methodology for further experiments. It investigated the effect of a P pulse (one day
after and seven days after) on uptake of P and other elements in a single species, T.
subterraneum, grown with and without AMF.
The desirability of using field soil as inoculum in order to introduce a diverse
community and of using filtrate to equalise the accompanying soil microbial community
between inoculated and non-inoculated treatments was also investigated.
Chapter 3- This chapter was designed based on the results presented in Chapter 2. It
investigated the effect of a P pulse (after seven days) on uptake of P and other elements
for four perennial legumes (Kennedia prostrata, Bituminaria bituminosa, Cullen
australasicum, M. sativa) and T. subterraneum grown with or without AMF. The effect
of AMF on the rhizosphere pH and carboxylate exudation was also assessed.
Chapter 4- This chapter was designed to investigate the location within mycorrhizal and
non-mycorrhizal roots of P taken up from a pulse using scanning electron microscopy
Chapter 1. General Introduction 21
and quantitative X-ray microanalysis. However, since no AMF were found in the
samples chosen, the chapter focuses on the storage of P from a pulse in uncolonised
areas of the mycorrhizal roots and non-mycorrhizal roots.
The thesis concludes with the General Discussion, which integrates results from all
experimental chapters, evaluates the important issues raised in the research reported in
the thesis, draws together key conclusions, and highlights areas for further research.
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Chapter 1. General Introduction 33
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CHAPTER 2.
Do Arbuscular Mycorrhizas or Heterotrophic Soil Microbes
Contribute Toward Plant Acquisition of a Pulse of Mineral Phosphate?
2.1 Preface
This experiment was established as a preliminary experiment to confirm the
methodology for the next experiment (chapter 3). In this experiment Trifolium
subterraneum was used as the host plant since there have been numerous studies on it
with AMF.
This paper addresses three hypotheses: 1) AMF will enhance uptake of P from a pulse
as may occur following addition of fertiliser or the start of the winter rain; 2) Uptake of
P is greater when plants are colonised by a diverse community of AMF (inoculation
with field soil), rather than a single-species inoculum and 3) Presence of microbes in
the soil increases P uptake compare to the control.
Chapter 2. Arbuscular mycorrhizas and a P pulse 36
2.2 Published paper
Chapter 2. Arbuscular mycorrhizas and a P pulse 37
Chapter 2. Arbuscular mycorrhizas and a P pulse 38
Chapter 2. Arbuscular mycorrhizas and a P pulse 39
Chapter 2. Arbuscular mycorrhizas and a P pulse 40
Chapter 2. Arbuscular mycorrhizas and a P pulse 41
Chapter 2. Arbuscular mycorrhizas and a P pulse 42
Chapter 2. Arbuscular mycorrhizas and a P pulse 43
Chapter 2. Arbuscular mycorrhizas and a P pulse 44
Chapter 2. Arbuscular mycorrhizas and a P pulse 45
Chapter 2. Arbuscular mycorrhizas and a P pulse 46
Chapter 2. Arbuscular mycorrhizas and a P pulse 47
CHAPTER 3.
Moderating Mycorrhizas: Arbuscular Mycorrhizas Modify
Rhizosphere Chemistry and Maintain Plant Phosphorus Status within
Narrow Boundaries
3.1 Preface
According to the results from the preliminary experiment (chapter 2), this experiment
was optimised to have a better understanding of the response of perennial pastures to a
pulse of P when they are colonised by AMF.
This study addressed three hypotheses associated with this scenario for plants grown in
a soil with moderate P levels representative of agricultural systems: 1) AMF will
moderate shoot P concentration following a pulse; 2) Release of carboxylates into the
rhizosphere will decrease when plants encounter a pulse of P and 3) Colonisation by
AMF will decrease the release of carboxylates into the rhizosphere.
Hypotheses 1) and 3) were supported.
Chapter 3. Moderating mycorrhizas 50
3.2 Published paper
Chapter 3. Moderating mycorrhizas 51
Chapter 3. Moderating mycorrhizas 52
Chapter 3. Moderating mycorrhizas 53
Chapter 3. Moderating mycorrhizas 54
Chapter 3. Moderating mycorrhizas 55
Chapter 3. Moderating mycorrhizas 56
Chapter 3. Moderating mycorrhizas 57
Chapter 3. Moderating mycorrhizas 58
Chapter 3. Moderating mycorrhizas 59
Chapter 3. Moderating mycorrhizas 60
CHAPTER 4.
A Sudden, High Pulse of Phosphorus Triggers Formation of Granules
with Extraordinary High Concentrations of Phosphorus and
Potassium in Cortical Cell Vacuoles of Trifolium subterraneum
4.1 Preface
According to the results of the previous experiments (the reduction in P movement to
shoots in colonised plants following the P pulse), in this experiment I intended to
measure the amount of P in the hyphae of AMF using quantitative X-ray microanalysis
since the moderating effect of AMF might be due to storage of high amounts of poorly
soluble P in intraradical hyphae in the roots. The hypothesis for this experiment was:
following a significant pulse of P, P movement to shoots is restricted, due to storage of
high concentrations of P in intraradical AM hyphae in insoluble forms. However,
unfortunately, due to low level of colonisation, none of the samples which were
prepared for analysis contained any hyphae. However, granules were found in roots
and they contained very high concentrations of P. This then became the focus of this
paper.
Chapter 4. Root P accumulation after a P pulse 64
4.2 Ready for submission paper
A sudden, high pulse of phosphorus triggers formation of granules with
extraordinary high concentrations of phosphorus and potassium in cortical cell
vacuoles of Trifolium subterraneum
Nazanin K. Nazeri1, 2, Peta L. Clode5, Hans Lambers1, Mark Tibbett3, Megan H. Ryan1, 2
1School of Plant Biology and Institute of Agriculture, The University of Western Australia, 35
Stirling Highway, Crawley (Perth), WA 6009, Australia
2Future Farm Industries Cooperative Research Centre, The University of Western Australia, 35
Stirling Highway, Crawley (Perth) WA 6009, Australia
3Department of Environmental Science and Technology, School of Applied Sciences, Cranfield
University, Cranfield, MK43 0SZ, England.
5Centre for Microscopy, Characterisation and Analysis, The University of Western Australia,
Crawley (Perth), WA 6009, Australia
Author for correspondence
Nazanin Nazeri
School of Plant Biology, M084
The University of Western Australia, Crawley (Perth), WA 6009, Australia
Ph +61 8 6488 1992
Email: [email protected]
Running title: Root P accumulation after a pulse of P
Chapter 4. Root P accumulation after a P pulse 65
Abstract
Due to the low availability of phosphorus (P) in the soils of across southern Australia,
application of P fertilisers on pastures is common. Addition of P fertiliser causes a pulse
of P to be released into the soil and encountered by plant roots. I intended to measure
the amount of P in the internal hyphae of arbuscular mycorrhizal fungi (AMF)
colonising the roots of Trifolium subterraneum using quantitative X-ray microanalysis
following a large pulse of P (150 mg P kg-1 soil) to soil. Trifolium subterraneum was
grown in a sterilised field soil, containing a moderate amount of P (18 mg kg-1), either
with (+AMF) or without (-AMF) addition of inoculum (Scutellospora calospora). At
week six, plants received either a high pulse of 150 mg P kg-1 soil (150P), a moderate
pulse of 15 mg P kg-1 soil (15P) or no pulse (No-P). One week later, all plants were
harvested. The percentage of root length colonised by AMF, shoot and root dry weight,
and nutrient concentrations in the dry shoots and roots were measured. A small number
of root subsamples were freeze substituted and embedded in resin for examination using
quantitative X-ray microanalysis. The P pulse had no effect on shoot dry weight, but
root dry weight decreased by 28% and 14% when a pulse of 15 mg P kg-1 soil and 150
mg P kg-1 soil, respectively, was applied. Shoot and root P concentrations increased
from No-P to 150P (from 2 to 14 mg P g-1 dry weight (DW) in the shoot and from 2 to
20 mg P g-1 DW in the root). Due to a low level of colonisation, no hyphae were found
in the samples prepared for quantitative X-ray microanalysis. However, large granules
with high concentrations of P and potassium (K) were found in the samples that
received 150P. I conclude that a high pulse of P triggers formation of granules with high
concentrations of P and K in cortical cell vacuoles and suggest that these represent a
means for the plant to avoid P toxicity.
Keywords: Phosphorus pulse, granules, “polyphosphate overplus” phenomenon,
Chapter 4. Root P accumulation after a P pulse 66
Introduction
Plant P supply under field conditions is rarely constant (for example see Hedley et al.
(1982) and Tiessen et al. (1983)), and sometimes plants may encounter a pulse of P.
Such a pulse might occur due to the application of fertilisers (Lewis et al. 1987). The
responses of plants that are colonised by arbuscular mycorrhizal fungi (AMF) to a pulse
of P are different to those of uncolonised plants. For instance, Nazeri et al. (2013)
reported that root P concentration increased to a similar degree in colonised and
uncolonised roots of Trifolium subterraneum L cv. Denmark, a commonly grown
annual pasture legume, but that the rate of increase of P concentration in the shoots was
lower for colonised plants than for uncolonised plants. This result was confirmed in a
second study, where Nazeri et al. (2014) added P in a pulse to colonised and
uncolonised plants of four perennial legumes (Kennedia prostrata, Cullen
australasicum, Bituminaria bituminosa and Medicago sativa) and Trifolium
subterraneum. The authors concluded AMF moderate P uptake when P suddenly
becomes excessive to plant requirements, and thus act to protect the plant. The authors
hypothesised that the reduction in P movement to shoots in colonised plants following
the P pulse might be due to storage of high amounts of poorly soluble P in intraradical
hyphae in the roots (see Ryan et al. (2003)) and thus a reduction in the availability of P
for transport to the shoots. Further confirmation of this concept was recently provided
by Kariman et al. (2014) who found that colonisation of 16-week-old jarrah (Eucalyptus
marginata) seedlings with AMF (as well as ectomycorhizal fungi) resulted in
significantly fewer toxicity symptoms seven days after a high P pulse (30 mg P kg-1
soil) compared to uncolonised plants. There was a positive linear relationship between
shoot P concentration and toxicity symptoms.
To obtain a better understanding of how AMF moderate P uptake following a pulse, it is
essential to know where exactly the P is stored in the root cells or in the fungal tissue.
According to Smith and Smith (2011), when plants are colonised by AMF the direct P-
uptake pathway (through root hairs and epidermal cells) can become inactive, and the
AM uptake pathway becomes the dominant uptake pathway. By mapping the colonised
root system using cryo-scanning electron microscopy, Ryan et al. (2003) showed P
concentrations in the intraradical hyphae were significantly higher (generally 60-170
mM) than P concentrations in uncolonised cortical cell vacuoles (mostly undetectable
Chapter 4. Root P accumulation after a P pulse 67
(i.e. less than 10 mM)). The authors also showed a strong linear relationship between
the concentrations of P, K and Mg. In addition, Ryan et al. (2007) determined the
proportion of insoluble P in the intraradical hyphae (not extracted following destruction
of cell membranes by a variety of methods). The authors found 30-70% of the P in the
hyphae and concluded this P was likely to be in the form of polyphosphate, a commonly
found P storage compound in the Glomeromycota (Hijikata et al. 2010; Karandashov
and Bucher 2005; Viereck et al. 2004).
Polyphosphate is a linear condensed polymer of three to thousands of inorganic
phosphate (Pi) residues, which are linked together by high-energy phospho-anhydride
bonds (Ohtomo and Saito 2005). It is a very widespread molecule and has been
commonly reported in bacteria, fungi, protozoa and mammals (Kulaev et al. 2005).
There are a great number of reports about formation of polyphosphates in AMF (Cox et
al. 1980; Ezawa et al. 2004; Funamoto et al. 2007; Kuga et al. 2008; Rasmussen et al.
2000; Ryan et al. 2007; Solaiman et al. 1999; Viereck 2002). Since 1961, the extraction
of polyphosphate from higher plant tissues has been reported by several authors (Jeffrey
1964; Jeffrey 1968; Miyachi 1961; Nassery 1969). However, recent reports of
polyphosphate in plants are few and the reliability of the methodology in the earlier
reports is questionable (Kulaev et al. 2005).
In the present experiment I grew plants in a field soil with a moderate level of P, with
and without addition of AM fungal inoculum i.e. a similar protocol to that used by
Nazeri et al.(2013; 2014). I used quantitative X-ray microanalysis to determine the
distribution of P in roots. I hypothesised that high concentrations of P, associated with
cations, would be present in the hyphae of fungi. However, due to an unexpectedly low
level of colonisation by AMF, no fungal structures were found in any of the cross
sections and I report instead on the response of uncolonised areas and non-mycorrhizal
roots to a pulse of P.
Materials and Methods
Glasshouse experiment
The experiment was fully randomised with two treatments: arbuscular mycorrhizal
fungi (+AMF and –AMF) and P pulse (0, 15 and 150 mg P kg-1 dry soil added a week
before harvest). Note that the rate of 15 mg P kg-1 dry soil was the same as used by
Chapter 4. Root P accumulation after a P pulse 68
Nazeri et al. (2013; 2014). There were four replicates.
The experiment was conducted using a field soil. The characteristics of the field soil are
described by Nazeri et al. (2013). Soil was collected from the top 10 cm of a weedy
annual pasture paddock at Newdegate (33° 06′ 16″ S, 118° 49′ 50″ E, 333 m elevation)
in the southern cropping zone of Western Australia. Soil analysis was performed by
CSBP Future Farm analytical laboratories, Bibra Lake, Australia. Bicarbonate-
extractable P was 18 mg kg-1 and potassium (K) was 80 mg kg-1 (Colwell 1965).
Available P, measured using the Olsen test, was 11 mg kg-1. There was 6 mg kg-1
ammonium-N and 10 mg kg-1 nitrate-N (Searle 1984). Available sulfur (S) was 9.3 mg
kg-1 (Blair et al. 1991). Soil pH was 5.3 (CaCl2) and 6.0 (H2O). The soil consisted of
76.0% coarse sand, 23.9% fine sand, 0.05% clay and 0.11% silt.
Soil was steam-sterilised at 80°C for one hour on consecutive days, and then dried
overnight in a sterile environment. White non-draining pots were filled with 1.2 kg of
soil as follows. The bottom half of the pots was filled with sterilised field soil (600 g)
and the top half was filled with a 1:4 mixture of sterilised field soil and inoculum from a
pot culture of the AM fungus Scutellospora calospora, which presumably contained
spores, hyphae and colonised root segments. The inoculum had a pH (CaCl2) of 5.0 and
contained 10 mg kg-1 bicarbonate-extractable P and 32 mg kg-1 K (Colwell), 3 mg kg-1
ammonium-N and less than 1 mg kg-1 nitrate-N (Searle 1984).
Seeds of Trifolium subterraneum L., cv. Denmark, were scarified using sand paper and
soaked in water until imbibed. Imbibed seeds were kept on moist filter papers in Petri
dishes for 3-4 days until all were germinated. Two germinated seeds were planted in
each pot. After one week, seedlings were thinned to one plant per pot and a dense
suspension of an appropriate strain of Rhizobium trifolii was added. Pots were watered
to field capacity by weight twice a week. No nutrient solutions were added to the pots.
Pots were arranged randomly in a temperature-controlled glasshouse (mean maximum
temperature ~ 24°C and mean minimum temperature ~ 12°C) at the Crawley campus of
the University of Western Australia, Perth, Australia. The experiment ran from 4 June
2012 until 25 July 2012. At week six, the three P pulse treatments were applied. Twenty
millilitres of 15 (15P) and 150 mg P kg-1 soil (150P) in the form of KH2PO4 were added
to the pots. The control pots did not receive any P (No-P).
All plants were harvested one week later. Shoots were cut at the soil surface, rinsed
once in deionised (DI) water, dried for 72 hours at 70°C and weighed. Shoots were then
Chapter 4. Root P accumulation after a P pulse 69
finely ground and later used for the P digestion. Roots were washed thoroughly, rinsed
once in deionised water (DI), blotted dry with paper towels and fresh weights
determined. A subsample of approximately 3 g was taken from each root system and its
fresh weight recorded. The subsamples were kept in 70% ethanol and later assessed for
mycorrhizal colonisation. The remaining roots were dried at 70°C for 72 hours and
weighed. To assess the mycorrhizal colonisation level, roots were cleared in 10% (w/v)
KOH at room temperature for 3 days and then washed thoroughly with DI water and
stained in 5% (v/v) Shaeffer black ink for 2 hours (Vierheilig et al. 1998). For
destaining, the roots were washed in DI water and stored in lactoglycerol (1:1:2 lactic
acid, deionised water, glycerol) (Vierheilig et al. 1998). The percentage of root length
colonised by AMF was assessed using the line-intersect method for at least 100
intersections per sample (Giovannetti and Mosse 1980).
For determination of tissue P, subsamples of ~ 0.2 g of dried, ground, root and shoot
material were digested in a 3:1 HNO3:HClO4 mixture and analysed using inductively-
coupled plasma (ICP) atomic absorption with a Perkin Elmer Optima 5300 DV optical
emission spectrometer (OES; Shelton, CT, USA).
Data were analysed in Genstat version 14.1 (Lawes Agricultural Trust, Rothamsted
Experimental Station, Harpenden, UK) using one-way ANOVA to assess the effect of
the P-pulse treatment (No-P, 15P and 150P) on response variables in the +AMF pots
(data from the –AMF pots were generally disregarded, see explanation below).
Normality was checked and no transformations were necessary.
Sample preparation for X-ray microanalysis
One plant from each treatment was selected and one to three subsamples (one from –
AMF, 150P; two from +AMF, No-P and three from +AMF, 150P (Table 4)) were
excised from each root. The Subsamples were ~ 1 cm long (excluding root tips) and
immediately plunge-frozen into liquid nitrogen. Frozen subsamples were subsequently
freeze-substituted in a 10% (v/v) acrolein in di-ethyl ether mixture over a 0.3 nm
molecular sieve (Table 1) and embedded in Araldite 502 epoxy resin (Table 2)
following the methods of Marshall (1980).
Chapter 4. Root P accumulation after a P pulse 70
Table 1. Temperature and time at each temperature that frozen root subsamples were freeze-substituted.
Temperature (°C) -100 -90 -70 -20 0
Time (h) 24 168 336 24 hold
All solutions were anhydrous and once embedded, samples were kept desiccated.
Araldite 502 is the preferred epoxy resin, as it contains negligible levels of elements
detectable by energy-dispersive microanalysis (Påsgård et al. 1994), while acrolein is
considered a superior fixative for plants and for use at low temperatures (Echlin 1992).
The substitution method, when performed under anhydrous conditions, maximises the
retention and immobilisation of diffusible ions (Condron and Marshall 1990; Marshall
1980; Orlovich and Ashford 1995; Påsgård et al. 1994). This substitution method has
been reliably used to prepare plant tissues (Altus and Canny 1985; Bidwell et al. 2004;
Crawford et al. 1998; De Filippis 1978; Orlovich and Ashford 1995; Ross et al. 1983),
algal samples (Mostaert et al. 1996), optic nerves (Wells et al. 2012), coral epithelia
(Clode and Marshall 2002; Clode and Marshall 2003; Clode and Marshall 2004;
Marshall et al. 2007; Marshall and Wright 1991, 1995), and Malpighian (Hyatt and
Marshall 1985) and renal tubules (Marshall et al. 1994), for elemental analysis.
Table 2. The process of embedding freeze-substituted subsamples in Araldite 502 epoxy resin.
Resin-embedded mounts were prepared so as to create a transverse section of the root in
the block face. All cutting was done on a Leica EM UC6 ultramicrotome using an
anhydrous glass knife. Anhydrous -cut 1 µm-thick sections were initially collected and
placed on glass slides for toluidine blue staining to check the quality of the sample and
to identify suitable regions of the root for analysis. Optical images from stained sections
were collected using a Zeiss Axioskop microscope fitted with a digital camera. Once a
suitable region was identified, the block face was finely planed using an anhydrous
Step Process Time 1 Rinse substituted samples in anhydrous diethyl ether 10 mins × 2 2 Infiltrate in anhydrous resin:ether 1:3 24 h 3 Infiltrate in anhydrous resin:ether 1:1 24 h 4 Infiltrate in anhydrous resin:ether 3:1 24 h 5 Infiltrate in 100% anhydrous resin under vacuum Few hours 6 Mount in final moulds in 100% anhydrous resin and place under
vacuum 24 h
7 Cure samples in moulds at 60°C under vacuum 2 days 8 Keep resin blocks dessicated Indefinitely
Chapter 4. Root P accumulation after a P pulse 71
knife to create a perfectly flat, cross-sectional surface. The block was then mounted on
an aluminium microscope stub with double-sided carbon tape, and the edges painted
with conductive carbon paint, before being coated with 20 nm of carbon.
Quantitative X-ray microanalysis
Two transverse planed sections from each root were examined. In most cross sections,
regions of interest from three structures (granules in cortex cells, cortex cell vacuoles
and vascular tissue) were measured multiple times. Root blocks were analysed in a
Zeiss Supra 55 field emission scanning electron microscope (SEM) fitted with an
Oxford X-Max80 SDD X-ray detector (80 mm2) interfaced to Oxford Instruments
AZtecEnergy software. The microscope was operated at 15 kV in high current mode.
Immediately prior to each map acquisition, the instrument was calibrated and the beam
current measured and recorded using a pure copper standard. Elemental maps were
acquired at a resolution of 1024 × 768 pixels, for > 400 frames, with a dwell time of 50-
100 µs per pixel. Drift correction and pulse-pile up correction were activated. For such
analyses of bulk samples, the analytical resolution approximates a 2-3 µm sphere, with
detection limits around a few mmol kg-1 (Roomans and Dragomir 2007).
Using the Oxford Instruments Aztec Energy software, quantitative numerical data were
extracted from regions of interest drawn on the element maps, with individual spectra
from each pixel summed and processed to yield concentration data. Summed spectra
from regions of interest were quantified using the AZtec XPP model for matrix
corrections. Standards comprised polished microprobe standards of pure elements or
minerals of well-defined composition.
Granules were within the cell vacuole of some cells in the root cortex and the analysis
of them was discrete. In some samples granules were formed in between cells and not
inside a cell individually and as a result, for the analysis of cell vacuoles, vacuoles both
with and without any granules inside were selected. Vascular tissue analysis was
performed by selecting random areas of vascular tissue; the small sizes of these cells
meant that it was not possible to analyse cell vacuoles separately from surrounding cell
walls.
All elemental concentrations are given in mmol kg−1 embedded tissue which, if the resin
fully occupies the space previously occupied by water in the living tissue, closely
reflects the concentration on a wet weight basis (i.e. mM). All data are presented as
Chapter 4. Root P accumulation after a P pulse 72
mean ± standard error of the mean, where n = the number of regions analysed.
Results
Plant growth
At harvest, symptoms of P toxicity were visible (chlorosis and necrosis around the
margins (Rossiter 1952)) in a few leaves of the uncolonised plants at 150P; plants
colonised by AMF had no P toxicity symptoms. The level of colonisation by AMF was
low-moderate in colonised plants, varying between 7-37%. No colonisation was found
in uninoculated plants. The P pulse did not affect the percentage of root length
colonised by AMF. AMF did not affect shoot dry weight. From here on, this paper
reports primarily on the plants colonised by AMF.
Table 3. Measurements of Trifolium subterraneum growth and nutrient concentrations in the +AMF treatment with three levels of phosphorus (P) pulse (No-P, 15 P and 150 P) (ns, no significant difference).
Treatments No-P 15P 150P LSD at P=0.05 P-value Shoot dry weight (g) 0.65 0.55 0.66 ns - Root dry weight (g) 0.27 0.20 0.23 0.05 0.029 Shoot [P] (mg g-1) 2.63 7.40 14.29 1.82 ˂0.001 Root [P] (mg g-1) 2.84 10.29 20.13 4.75 ˂0.001 Plant P content (mg) 2.49 6.00 14.05 1.46 ˂0.001 Shoot [K] (mg g-1) 23.20 27.78 38.13 3.1 ˂0.001 Root [K] (mg g-1) 19.05 26.71 37.97 6.42 ˂0.001 Shoot [Na] (mg g-1) 8.8 10.3 8.3 1.46 0.013 Root [Na] (mg g-1) 21.5 17 11 3.36 ˂0.001
The P pulse did not affect shoot dry weight, but root dry weight was ~26% and ~ 14%
less in the 15P and 150P treatments, respectively, than in the No-P treatment (Table 3).
Shoot and root P concentrations increased substantially from the No-P to the 150P
treatment, reaching 14 and 20 mg g-1 in shoot and root, respectively, in the 150P
treatment. Plant P content increased from the No-P to the 15P treatment by 141% and
then from the 15P to the 150P treatment by ~134% (Table 3). This large increase in
plant P concentration presumably reflects to some extent the low P adsorption capacity
of the soil.
Shoot and root K concentrations also increased from the 0P to 150P treatment, albeit
less drastically than did P. Shoot K concentration increased by ~20% from the No-P to
15P treatment and then increased ~37% from the 15P to 150P treatment. Root K
Chapter 4. Root P accumulation after a P pulse 73
concentration increased ~40% from the No-P treatment to the 15P treatment, and then
increased ~42% between the 15P and 150P treatments. Shoot Na concentration
increased 42% from the No-P treatment to the 15P treatment and then decreased 19% to
the 150P treatment. Root Na concentration decreased ~21% from No-P to 15P treatment
and decreased 35% from 15P to 150P treatment (Table 3).
Microscopy and quantitative X-ray microanalysis
Structural features
The perfectly flat surface of the block face which is required for accurate quantitative
analyses (unlike the cryo-analytical SEM used by Ryan et al. (2003)) meant that it was
hard to see clearly inside the planed transverse sections of roots. However, structures
that could be confidently identified as mycorrhizal were not present. The low level of
colonisation meant that the likelihood of encountering mycorrhizal structures was low.
However, unexpected structures were observed in the root cortex and, rarely, in vascular
tissue. These were round granules (Fig. 1). In the 150P treatment, there was a granule in
nearly every root cortex cell, while in the No-P treatment granules were rare (Fig.1).
Also, the diameter of granules in the 150P treatment was much larger (average of 5.5
µm in sample one and 11 µm in sample 2) than in the No-P treatment (average 2.5 µm
in sample 1 and 5.1 µm in sample 2) (Table 4). These granules were visible in the
optical microscope when the cross sections of the acrolein-treated roots were stained
using toluidine blue.
Chapter 4. Root P accumulation after a P pulse 74
Figure 1. Electron microscopy images of transverse sections of a root from A) No-P treatment (+AMF, subsample 2 from Table 4) and B) the 150P treatment (subsample 2 from Table 4) (granules evident throughout). Beneath each image is the corresponding phosphorus (P) map (green) (II) and potassium map (pink) (III). Lighter colours indicate higher concentrations. (Granules are labelled G, the epidermis is shown with an arrow, and the cortex and the vascular tissues are indicated.)
Vascular tissue
Cortex
Epidermis
100 µm
G
G
VascularTissue Cortex
Epidermis
Epidermis
100 µm
(I)
(III)
(II)
G
G
(I)
(II)
(III)
(A)
(B)
Chapter 4. Root P accumulation after a P pulse 75
Table 4. Number of analyses (granule, cell vacuole and vascular tissue) from each root subsample and average diameter of granules.
+AMF -AMF
No-P 150P 150P
Subsample 1 2 1 2 3 1
Granules in cortex cells 2 3 8 6 5 16
Cortex cell vacuoles 2 3 3 7 2 10
Vascular tissue 2 4 2 7 0 6
Average granule diameter (µm) 5.1 2.5 5.5 11 3.6 5.6
Elemental analysis
Greatly elevated P was found in the granules in the 150P treatment. In the 150P
colonised treatment, the concentration was 185-7642 mmol kg-1 and in the 150P
uncolonised treatment, 10-4036 mmol kg-1. Vascular tissue in one colonised 150P
treatment was similarly elevated (1411-2747 mmol kg-1) (Fig. 2). The elevated P
concentration in the granules was accompanied by elevated K, Mg, Na and, sometimes,
Cl concentrations. Elevated concentrations of some nutrients occurred in other samples,
but were not consistent with treatment.
For the granules in the 150P treatment, there was a strong correlation between the
concentrations of P and K (r2=0.97), but no correlation between those of P and Na, or
Mg and Ca (Fig. 3).
Chapter 4. Root P accumulation after a P pulse 76
Cl (m
mol kg
-1)
0
500
1000
1500
2000
4000
6000
Ca
(m
mol kg
-1)
0
100
200
300
2000
4000
6000
S (
mm
ol kg
-1)
0
200
400
600
800
2000
4000
6000
Mg
(m
mol kg
-1)
0
200
400
600
2000
4000
6000
Cell vacuole
Na
(m
mol kg
-1)
0
1000
2000
3000
4000
6000
K (
mm
ol kg
-1)
0
200
2000
4000
6000
Granule
P (
mm
ol kg
-1)
0
500
1000
2000
4000
6000
Cell vacuole Vascular tissue
No-P+AMF
150P+AMF
150P-AMF
No-P+AMF
150P+AMF
150P-AMF
No-P+AMF
150P+AMF
150P-AMF
Chapter 4. Root P accumulation after a P pulse 77
Figure 2. Nutrient concentrations (phosphorus (P), potassium (K), sodium (Na), magnesium (Mg), sulfur (S), calcium (Ca) and chloride (Cl)) in granules, the vacuole of cortex cells that did not contain granules and vascular tissue in the No-P (no pulse of P was added to the plants) and 150P (plants received 150 mg P kg-1 soil seven days before the harvest). Most data are from the +AMF treatment, but data from a single –AMF cross section are included in the interests of greater sample size. Data are summarised in box plots where the boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. The median of the nutrient concentration is shown as solid line. Individual points are outliers.
Discussion
Plants respond to stress in many ways. In this study the stress consisted of plants
encountering a pulse of a high P concentration. I hypothesised that P supplied in excess
to a plant’s needs would be restricted from movement to shoots due to storage in poorly
soluble forms in the intraradical hyphae of AMF. This study has shown, for the first
time, formation of granules with extraordinarily high concentrations of P and K in the
cortical cells of T. subterraneum exposed to a pulse of high P concentration. Assuming
the granules are not an artefact of sample preparation, such as the possible precipitation
and coagulation of stress-related latex-like proteins by acrolein fixation (Condon and
Fineran 1989), the implications arising from this finding are discussed below.
A pulse of a high P concentration induces granules high in [P] in the root
Large granules were formed in abundance and were spread widely in the cortex area of
the roots treated with 150P, while granules that were formed in the No-P treatment were
small and rarely easily noticed. Whilst roots were colonised by AMF, I do not consider
these granules to be transverse sections of AM fungal hyphae, because their
morphology was not consistent with that shown by Ryan et al. (2003).
The presence of granules with greatly elevated [P] in the 150P treatment is in
accordance with the shoot and root P concentrations. The root and shoot P
concentrations in plants in the 150P treatment were eight and five times higher,
respectively, than those in plants that did not receive any P in a pulse.
Chapter 4. Root P accumulation after a P pulse 78
(c)
Figure 3. Correlation between phosphorus (P) concentration in the granules treated with 150P with (a) Potassium (K) (r2=0.97, P<0.0001), (b) Sodium (Na) (r2=0.04, P=0.41), (c) Magnesium (Mg) (r2=0.05, P=0.35) and (d) Calcium (Ca) (r2= 6.681E-004, P= 0.91).
P (mmol kg-1
)
0 2000 4000 6000 8000 10000
Ca
(m
mo
l kg
-1)
0
20
40
60
80
100
120
140
Mg (
mm
ol kg
-1)
0
200
400
600
800
1000
1200
1400
Na (
mm
ol kg
-1)
0
500
1000
1500
2000
2500
K (
mm
ol kg-1
)
0
2000
4000
6000
8000
(a)
(d)
(b)
(c)
Chapter 4. Root P accumulation after a P pulse 79
Except under severe P limitation, the concentration of Pi in the cytosol of plants is
maintained at constant concentrations (i.e. 5-10 mM) (Lee and Ratcliffe 1993; Lee et al.
1990). The Pi concentrations in the vacuole varies considerably more than that in the
cytosol. This vacuolar P concentration may be undetectable under P starvation, but
generally does not increase above 25 mM unless P toxicity is expressed (Lee and
Ratcliffe 1993; Lee et al. 1990; Mimura 1995; Ryan et al. 2003). For instance, when P
toxicity was induced in Hakea prostrata R.Br. (Proteaceae) by Shane et al. (2004) the
concentration of P in vacuoles of mesophyll cells was greatly elevated (~150-250 mM),
but still much below vacuolar P concentrations in the present experiment where P
concentration reached to up to 7642 mmol kg-1 in the granules and up to 959 mmol kg-1
in the vacuole of cortex cells – values much higher than ever previously reported.
Thus the presence of distinct granules in the root cells of T. subterraneum is presumably
a strategy to reduce P toxicity in response to the sudden supply of a high concentration
of P. Similar to other species, T. subterraneum is typically reported to down-regulate P
uptake when grown with a high P supply. For example, in an experiment by Asher and
Loneragan (1967), 8 to 13 day - old T. subterraneum seedlings were grown in nutrient
solutions with five P levels (0.04, 0.2, 1, 5 and 25 µM) applied as phosphate. Plants
were harvested after 4 weeks of growth in nutrient solutions. Both shoot and root P
concentrations had reached a plateau by 5 µM (when dry weight was maximum) being
~ 10 and 12.5 mg g-1, respectively. Hence, it seems the shoot P concentration in our
experiment of 14 mg g-1 in the 150P treatment and, in particular, the root P
concentration of 20 mg g-1, is extraordinarily high (Table 3). I surmise these
unexpectedly high values represent the plant response to a pulse of a high P
concentration. In other words, when a pulse of P is applied to the plant, there is no time
for the plant to down-regulate its P-uptake capacity and, as a result, excessive uptake of
P occurs. This interpretation is consistent with that of Cogliatti and Clarkson (1983),
who found the P-uptake rate in P-starved potato plants (Solanum tuberosum L.) was
three- to four-fold higher than in P-sufficient controls after a pulse of P. Moreover, high
P concentrations may also occur if Zn is deficient (Cakmak and Marschner 1986);
however the Zn concentration in the plants in this experiment (63-100 mg kg-1); data not
presented) showed this was not the case. Based on these reports I hypothesise that when
P supply becomes suddenly excessive to plant requirements, the transport of at least
some of the excess P to the shoot is restricted by the formation of granules in the root.
This phenomenon has not previously been reported.
Chapter 4. Root P accumulation after a P pulse 80
What is the form of P in the granules?
I surmise that the P in the granules must be in poorly soluble forms as the plants,
particularly in the +AMF treatment, did not show signs of toxicity, and there were no
signs of cells suffering high osmotic pressure (i.e. swelling) as reported elsewhere when
leaves in high-P treatments were examined with cryo-SEM (Ryan et al. 2009; Shane et
al. 2004).
Formation of P-rich granules has not previously been reported for plants. However,
there is substantial literature showing their presence in the hyphae of AMF and
ectomycorrhizas (Ashford et al. 1986; Cox et al. 1980; Lapeyrie et al. 1984; Ling-Lee et
al. 1975). The reaction of these granules to cytochemical tests resulted in the authors
concluding they were polyphosphate. In an experiment by Lapeyrie et al. (1984),
metachromatic granules were observed in the mycelial hyphae of Paxillus involutus
when grown in a phosphate-rich solution, while no granules were observed when no
phosphate was added. The size and the number of these granules increased during
incubation. Lapeyrie et al. (1984) presumed these granules contained polyphosphates.
Transmission electron microscopy studies suggest that polyphosphates in the hyphae of
AMF are mostly present in the form of granules (Cox et al. 1975; Peterson 1991). On
the other hand, Orlovich and Ashford (1993) concluded that these were an artefact of
the fixation process for specimen preparation, however, this had been disputed. For
instance, Bücking and Heyser (1999) reported vacuolar granules were present in living
hyphae of various ectomycorrhizal fungi (Suillus bovines, Paxillus involutus, Pisolithus
tinctorius and Laccaria laccata). These granules were analysed for elemental
composition by X-ray spectroscopy either after chemical preparation or cryo-fixation,
freeze-drying and pressure-infiltration, and it was concluded that they were not an
artefact of specimen preparation and can be referred to as polyphosphate granules. The
size of the granules was between 100 to 3000 nm which is one of the lowest ranges in
our study, but since these granules were formed in hyphae with a diameter significantly
less than that of root cortical cells, this is not surprising.
Formation of granules with high P concentration in response to a sudden pulse of high P
concentration is also in accordance with the “polyphosphate overplus” phenomenon
reported for bacteria and yeast. For instance, when P-starved yeast are transferred to a
medium with phosphate, glucose and certain cations (K and Mg), cells may accumulate
polyphosphates up to 20% of the cell dry weight (van Wazer 1961). According to
Chapter 4. Root P accumulation after a P pulse 81
Sicko-Goad and Jensen (1976), transferring phosphate-starved cells of cyanobacteria
(Plectonema boryanum) to a medium containing 10 mg phosphate litre-1 caused an
“overplus” in the cells within 1 h of transfer, with the most dramatic increase found in
both acid-soluble and acid-insoluble polyphosphates. Moreover, high concentrations of
P were always accompanied by high concentrations of K, Na and Cl.
Further support for the granules in our study being composed of polyphosphate comes
from the presence of countering cations. In our study, K was the main counter-ion. K
was abundant as the P was supplied as KH2PO4. However many other studies report K
as the main counter-ion. Bücking and Heyser (1999) reported K and Mg as the cations
involved in charge balance of granules in ectomycorrhizas and mentioned them as a
“general character of fungal polyphosphate granules”. Moreover, Ryan et al. (2003)
found a strong linear relationship between P and K in the intraradical hyphae of AMF,
albeit with a highest concentration of K of 350 mM, which is negligible compared to the
K concentration in the granules (6881 mM). The positive relationship between P and K
is not surprising as polyphosphates are highly charged anions which form strong bonds
with cations (van Wazer 1961). For instance, Peverly et al. (1978) found formation of
polyphosphate bodies in P-starved Chlorella pyrenoidosa was positively and strongly
related to the presence of K in the medium. Transferring P-starved cells into a complete
nutrient solution containing 100 µM P resulted in accumulation of high concentrations
of P and K within several hours. Omitting K from the medium, even in the presence of
other cations such as Ca and Mg, prevented accumulation of high concentrations of P
and formation of polyphosphate. However, absence of Ca or Mg did not have a similar
effect.
Based on the present results, I surmise that it is possible that in our study polyphosphate
granules were formed in the cortical cell vacuoles of T. subterraneum in response to a
pulse of a high P concentration. These granules might degrade over time and become
accessible (toxic) to the cell and the plant. However, I cannot at all be confident that our
granules were composed of polyphosphate, as recent reports of polyphosphate in plants
are few (Denton et al. 2007; van Voorthuysen et al. 2000). Based on this literature, I did
not expectto find polyphosphate.
Conclusion
Although the roots were colonised, no fungal tissue was found in the samples prepared
Chapter 4. Root P accumulation after a P pulse 82
for X-ray microanalysis and our hypothesis of finding high concentrations of P in the
hyphae of AMF could not be addressed. However, the formation of high-P granules in
response to a P pulse and also the presence of high concentrations of K was consistent
with the formation of polyphosphate. This is the first suggestion that high
concentrations of P in the form of polyphosphate occur in higher plants. It would be
useful to know: how long do these granules persist in the cells after stress; do the
granules form in the shoots as well the roots; does granule formation differ between
species; and, are polyphosphate granules quickly dispersed as the plant down-regulates
its P-uptake capacity; In addition if the granules do represent a means for plants to
temporarily store high concentrations of P, what are the implications for efficient use of
P fertiliser? Further experiments are required to address these questions and to clarify
that granules are not an artefact of sample preparation.
Acknowledgements
The authors acknowledge the facilities, and the scientific and technical assistance of the
Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy,
Characterisation & Analysis, The University of Western Australia, a facility funded by
the University, and State and Commonwealth Governments. This study was supported
by the School of Plant Biology, UWA, and the Future Farm Industries Cooperative
Research Centre. NK Nazeri also appreciates the APA scholarship awarded by the
University of Western Australia. I would like to thank Lyn Kirilak, Peter Duncan,
Evonne Walker and Daniel Kidd for technical help.
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CHAPTER 5.
General Discussion
In this discussion I address the hypotheses which were tested in chapters two, three and
four. Unexpected results from each experiment are also discussed and results from all
results chapters are considered together.
Chapter 5. General Discussion 90
5.1 Introduction
Incorporating pasture legumes in agricultural systems in Australia not only maintains
feed for grazing livestock, but also fixes atmospheric nitrogen, thus increasing soil
nitrogen availability, and breaking the disease life cycle when the pastures are grown in
rotation with crops (Howieson et al. 2000; Kirkegaard et al. 2011; Peoples and Baldock
2001; Robson 1990). The annual legume Trifolium subterraneum is widely grown
across southern Australia due to its good adaptation to climate, soils and agricultural
systems including grazing and crop rotations (Porqueddu and González 2011; Puckridge
and French 1983). Due to problems that have arisen from reliance on annual pastures,
such as dryland salinity and leaching of nutrients, for the last 15 years researchers have
focused on identifying and developing new perennial pasture legumes adapted to
Western Australian conditions; conditions for which there are currently few or no
perennial legume options (for instance, waterlogging, acidic soils, salinity and drought)
(Li et al. 2008; Nichols et al. 2007). Bituminaria bituminosa, Cullen australasicum and
Kennedia prostrata are promising perennial legumes for the area of the Western
Australia cropping zone with low to moderate rainfall and poor soils (Real et al. 2011).
As perennial pastures, in contrast to annual pastures, are present when the first autumn
rains occur, they also present an opportunity to capture the pulse of P that may be
released into the soil solution at this time (Espigares and Peco 1995; Sardans and
Peñuelas 2007); a pulse similar to that which occurs when P fertiliser is applied. As
arbuscular mycorrhizal fungi (AMF) are well known to enhance plant P uptake (Smith
and Read 2008), the primary objective of this project was to study whether AMF will
enhance uptake of P from a pulse. Soil with moderate levels of P, that is, levels
commonly found in soils in the cropping areas of Western Australia (Scanlon 2011;
Weaver and Reed 1998) were used in all experiments.
5.2 Key research findings
Table 5.1 lists all the hypotheses addressed in previous experimental chapters. All
results are then discussed individually.
Chapter 5. General Discussion 91
Table 5.1. The hypotheses that were addressed in previous chapters and whether they were accepted or rejected.
Hypothesis Chapter Accepted or
rejected
1 AMF will enhance uptake of P from a pulse as might occur
following fertiliser application or the first winter rain 2 and 3
Partly
accepted
2
Uptake of P is greater in plants colonised by a diverse
community of AMF (inoculation with field soil), rather than a
single-species inoculum
2 Rejected
3 Presence of microbes in the soil increases P uptake compare to
the control 2 Rejected
4 AMF will moderate shoot P concentration following a pulse 3 Accepted
5 Release of carboxylates into the rhizosphere will decrease when
plants encounter a pulse of P 3 Rejected
6 Colonisation by AMF will decrease the release of carboxylates
into the rhizosphere 3 Accepted
7
Following a significant pulse of P, P movement to shoots is
restricted, due to storage of high concentrations of P in
intraradical AM hyphae in insoluble forms
4 Unable to
test
5.2A Phosphorus uptake from a pulse is enhanced in the roots of colonised
plants, but P transport to the shoots is restricted by AMF (after a week)
(moderating effect)
In Chapter 2, the annual pasture legume Trifolium subterraneum was used to develop
methodology for the experiment presented in Chapter 3. However, in doing so,
Hypotheses 1 and 4 were also addressed (see Table 5.1).
It has previously been shown that plant P uptake will increase when Trifolium
subterraneum forms a symbiosis with AMF (Abbott and Robson 1977; Bolan et al.
1987; Smith 1982). In my first experiment I, therefore, expected to see a higher P
concentration in both shoots and roots of colonised plants than in those of uncolonised
plants, both before and, to a greater degree, after a pulse of P. A day before (day 0) and
a day after (day 2) the pulse, as expected, both shoot and root P concentrations in
colonised plants were higher than those in uncolonised plants. However, unexpectedly,
Chapter 5. General Discussion 92
from day 2 to day 8, the rate of increase in shoot P concentration in colonised plants was
lower than that in uncolonised plants. This resulted in quite similar shoot P
concentrations in colonised and uncolonised plants by day 8. In contrast, the rate of
increase in root P concentration did not change from day 2 to day 8, and thus P
concentrations remained higher in colonised plants.
Remarkably consistent results were found in my second experiment (Chapter 3), where
a similar experiment was performed using Trifolium subterraneum and three perennial
pasture legumes. In this experiment, I found a 53% increase in shoot P concentration in
colonised plants following a pulse, but a 143% increase for uncolonised plants, while
the root P concentration was raised by the pulse by a similar amount for both colonised
and uncolonised plants.
The results can be explained based on the findings of Smith and Read (2008), who
reported that when plants are colonised by AMF, the “mycorrhizal uptake pathway”
(through hyphae of the AMF) is the dominant pathway for nutrient uptake regardless of
plant growth response and the “direct uptake pathway” (through root hairs and
epidermal cells) becomes “switched-off” or inactivated. In my experiment, therefore,
the lower rate of P uptake in the shoots of colonised plants is likely due to the primary
path of uptake being through the hyphae of AMF, with large amounts of the P then
stored in the intraradical hyphae of AMF in forms such as polyphosphate, while in the
uncolonised plants, P uptake was through the direct uptake pathway (root hairs and
epidermal cells) which allowed transport of P to the shoot without any barrier. In
accordance with this, there are also several reports of the presence of high
concentrations of P, probably polyphosphate, in the hyphae of AMF (Ryan et al. 2003;
Ryan et al. 2007; Solaiman et al. 1999). Such an effect, that is, the reduced transport of
P to the shoot in mycorrhizal plants following a P pulse, has recently also been reported
by Kariman et al. (2014). According to these authors, seven days after a high pulse of P
(30 mg P kg-1 soil), phytotoxicity symptoms in jarrah tree seedlings (Eucalyptus
marginata) colonised by AM (as well as ectomycorrhizal) fungi, were significantly
lower in colonised than in uncolonised plants. Moreover, colonised plants had lower
levels of shoot P concentrations than uncolonised plants, and there was a positive linear
relationship between shoot P concentration and toxicity symptoms.
My results, along with those of Kariman et al. (2014), suggest that AMF can, to some
degree, protect plants from a pulse of P; that is, AMF moderate the effect of a pulse of
Chapter 5. General Discussion 93
P.
5.2B Phosphorus uptake did not differ between a diverse community of AMF
and a single species culture
In order to optimise the methodology for Chapter 3, in Chapter 2 the P uptake by a
diverse community of AMF (i.e. the community naturally present in the field soil in
which the experiment was undertaken) was compared with an inoculum containing a
single species (as is routinely used in mycorrhizal experiments) to examine whether a
diverse community of AMF is more efficient for a plant to absorb P following a pulse
(hypothesis 2).
It is known that AM fungal species differ in their ability to affect P uptake and growth
of their host plant. For example, Jansa et al. (2005) reported that accessibility of P
differs among AM fungal species and is related to the distance that hyphae can develop
from the root and acquire P, and also the efficiency of fungi in extracting P from the
soil. The authors stated that Glomus mosseae and G. intraradices can take up P up to 10
cm from the root, but for G. claroideum this distance is only 6 cm. On the other hand,
G. mosseae did not provide any net P-uptake benefit for maize (Zea mays L. cv. Corso)
while net P uptake was significantly improved by G. intraradices due to its more
efficient P extraction. Thus, it can be expected that if a soil contains a number of
species, all differing slightly in their hyphal characteristics, then the result might be a
higher plant productivity than if only one species of AMF is present. Indeed, both van
der Heijden et al. (1998) and Maherali and Klironomos (2007) found AM fungal species
richness had a positive effect on plant productivity. Similarly, Wagg et al. (2011) found
plant biomass and productivity in heterogeneous environments is increased by greater
diversity of AM fungal species. As a result, I expected to find greater plant biomass and
P uptake when the inoculum contained a diverse mycorrhizal community (AMF from
the field) compared to a single species inoculum. However, little difference in host plant
shoot and root dry weight, or P uptake, was found. The failure of inoculation with a
diverse community of AMF to promote plant P uptake and growth more than the single
species inoculum may be explained by the findings of Bainard et al. (2013) and
Maherali and Klironomos (2007). Maherali and Klironomos (2007) found that plant
biomass and productivity is increased only when taxa from two families (for instance
Gigasporaceae and Glomeraceae) were present and having only one family of AMF in
Chapter 5. General Discussion 94
the soil will not stimulate plant productivity. Also, the authors predicted that
coexistence of species within each family is less likely, since they all have the same
spatial niche requirements, while taxa from different lineages coexist since their spatial
niche requirements are different. In support of this finding, Bainard et al. (2013) did not
find any significant differences in crop growth in response to AM fungal richness and
explained this might be due to the lack of different families of AM fungal species in the
soil and presence of only one family of AMF (Glomeraceae) in the soil. In our
experiment more than 97% of the DNA detected belonged to the Glomus family.
5.2C Microbes had little effect on uptake of P from a pulse
In Chapter 2, I also examined whether inoculation of microbes into the soil increases P
uptake compared to a sterilised control (hypothesis 3). Using field soil as inoculum for
AMF will introduce not only a diverse community of AMF, but will also introduce a
diverse microbial community, which might act synergistically to enhance plant growth
or inhibit the activity of AMF and thus reduce the positive impact of AMF on plant
growth (Azcón et al. 1976; Hetrick and Wilson 1988; Singh and Kapoor 1998).
Microbes have an important role in making soil P available for plants (Richardson 2001;
Whitelaw 1999). Inorganic and organic pools of total soil P can become accessible (via
solubilisation and mineralisation or their impact on root structure and function) or
inaccessible (by immobilising available sources of P) through the activities of soil
microbes (Richardson 2001). Plant growth-promoting microorganisms are of particular
importance and include a wide diversity of bacteria and fungi, which are found in the
rhizosphere and can increase plant growth by increasing supply of nutrients (Richardson
et al. 2009).
In mycorrhizal experiments, filtrates are commonly used to equalise soil microbes
between non-mycorrhizal controls and AM fungal treatments when field soil (or even
AM fungal inoculum) has been added (Asghari and Cavagnaro 2011). The filtrate is
made by passing a mixture of deionised water and soil through series of sieves from
1000 µm to 50 µm. Filtrate of the field soil is assumed to contain the soil microbial
community, but no mycorrhizal hyphae or spores (Hetrick and Wilson 1988). Addition
of filtrates, or non–sterile soil sievings, to sterilised soil was found to increase growth of
uncolonised plants by up to 7-fold (Hetrick and Wilson 1988). The authors explained,
an increase in growth of plants receiving filtrates or non-sterile soil sievings compared
Chapter 5. General Discussion 95
to the plants grown in pasteurised soil might reflect an increase in soil organic matter
and fertility due to the presence of microbes in the soil. Accordingly, I expected to find
an increase in P uptake by plants that had received field soil filtrates compared with
control plants (no AMF, no microbes). However, I did not find any effect of the
microbes on plant P uptake before or after the pulse of P and, overall, the effect of the
microbe treatments rarely differed from the control (Chapter 2). The main reason for the
lack of effect of the microbes treatment in my experiment might be the moderate level
of P (18 mg kg-1) in the soil, compared with the lower level of P in the soil used by
Hetrick and Wilson (1988) (5 mg kg-1). According to Richardson (2001) and Krey et al.
(2011) an increase in the level of soil P may decrease the activity of soil
microorganisms involved in P mobilisation.
Since there was no impact from the microbe treatment on plant growth or P uptake, it
can be assumed that the major impact of the field soil inoculum resulted from the
actions of the AMF it contained. I therefore chose to use the field soil inoculum in the
next chapter (Chapter 3) (again with the addition of the filtrates to the control.)
5.2D A pulse of P did not decrease the amount of rhizosphere carboxylates
Plants employ different mechanisms, depending on species, under P-deficiency
conditions. Increased root-hair formation, symbioses with AMF, formation of cluster
roots, and exudation of carboxylates are some of these mechanisms (Abel et al. 2002;
Lambers et al. 2006). The major component of root exudates under P deficiency
conditions can be carboxylates, which are low-molecular-weight organic anions,
released from roots into the soil; these increase P availability through mobilising both
organic and inorganic P through complexing metal cations that bind phosphate and
thereby displacing the phosphate from the soil matrix by ligand exchange (Gardner et al.
1983; Jones 1998; Lambers et al. 2006; Pang et al. 2009a; Veneklaas et al. 2003). It has
previously been found that the amount of rhizosphere carboxylates increases under low-
P conditions (Lambers et al. 2006; Pearse et al. 2006; Suriyagoda et al. 2012). For
instance, Pang et al. (2009a) compared the amount of rhizosphere carboxylates between
two levels of KH2PO4 (6 and 48 µg P g-1 soil) for 11 legume species (four species -
Medicago sativa, B. bituminosa, C. australasicum and K. prostrata –were used in
Chapter 3). It was found that all these species had higher amounts of carboxylates at the
lower P level. However, this is not supported in all studies. For instance, in an
Chapter 5. General Discussion 96
experiment by Suriyagoda et al. (2012) the effect of 5, 20 and 80 mg P kg-1 soil on the
amount of rhizosphere carboxylates was measured for two native perennials (K.
nigricans and Ptilotus polystachyus) and M. sativa. An increase in soil P decreased the
amount of carboxylates in the rhizosphere only in M. sativa and K. nigricans (only from
P20 to P80). Also, Pearse et al. (2006) demonstrated the response of 13 crop species to
soil P in terms of carboxylates was variable.
In Chapter 3, no effect of the P pulse on the amount of rhizosphere carboxylates was
found, which supports findings of Pearse et al. (2006) and Suriyagoda et al. (2012).
However, the results are not consistent with those of Pang et al. (2009a). The failure of
the P pulse to affect the amount of rhizosphere carboxylates may reflect the initial
moderate level of soil P in my experiment, or the short period of time (1 week) during
which plants were exposed to the P pulse.
5.2E Colonisation with AMF decreased rhizosphere carboxylates
Chapter 3 also examined whether colonisation with AMF decreases the release of
carboxylates into the rhizosphere (hypothesis 6). This effect was previously, and first,
reported by Ryan et al. (2012). According to Ryan et al. (2012) since exudation of
carboxylates and forming a symbiosis with AMF are both carbon demanding processes
for the plants (Graham 2000), it is not surprising that there will be trade-offs between
the two processes (Lynch and Ho 2005). Ryan et al. (2012) found up to a 50% decrease
in carboxylates when 12 species of Kennedia were colonised by AMF. My experiment
supports the findings of Ryan et al. (2012), as colonisation by AMF decreased the
amount of rhizosphere carboxylates by 52% (Table 5.2). The similarities between the
results of these two studies conducted under quite contrasting conditions (field soil with
moderate levels of P [18 mg kg-1] in my experiment and washed river sand with low
levels of P [3 mg kg-1] in Ryan et. al (2012)) are corroborated by the similar results from
K. prostrata from both studies, as the same accession of this species was used in both
experiments (Table 5.2).
Chapter 5. General Discussion 97
Table 5.2. Comparison between the amount of rhizosphere carboxylates in Kennedia prostrata in studies by Ryan et al. (2012) (in low-P washed river sand) and Chapter 3 (in moderate-P field soil) and the level of colonisation by AMF in each study.
-AMF +AMF Reference
Carboxylates (µmol per g root DW) 91 41 Ryan et al. (2012)
70 38 Nazeri et al. (2014)
Colonisation by AMF (%) 0 35 Ryan et al. (2012)
0 23 Nazeri et al. (2014)
Thus, it seems that colonisation by AMF can greatly decrease the amount of
carboxylates in the rhizosphere soil and, thereby, as suggested by Ryan et al. (2013),
potentially change the mixture of soil P pools utilised by the plant – mycorrhizal-
mediated resource partitioning.
5.2F Formation of granules with high concentrations of P in the cortex area of
the roots of Trifolium subterraneum which were treated with a high pulse of P
In response to the moderating effect of AMF on plant uptake of P from a pulse, which
was found in Chapters 2 and 3, I decided to measure the amount of P which was stored
in the intraradical hyphae of the mycorrhiza and compare this with the amount of P in
the root cells of Trifolium subterraneum. The hypothesis of this experiment was:
following a large pulse of P, AMF restrict movement of P to the shoots due to the
storage of high concentrations of P in insoluble forms in hyphae. Due to low level of
colonisation and inability to determine where exactly the colonisation was present in the
root, I could not find any hyphae in any of the samples that were prepared for
quantitative X-ray microanalysis. Thus, the main hypothesis of this experiment was not
addressed. However, other findings from this experiment were unexpected, and very
interesting.
Large granules, which according to quantitative X-ray microanalysis contained very
high concentrations of P and K, were observed in the cortex area of the roots of plants
that had received a pulse of a high P concentration (150 mg P kg-1 soil), no matter
whether colonised or uncolonised. These granules were hardly visible in samples that
received 15 mg P kg-1 soil. Such granules have not been reported previously. A number
Chapter 5. General Discussion 98
of factors suggest they may consist of polyphosphate: granular nature, strong linear
relationship between P and K concentrations, and formation after a P pulse. However,
polyphosphate is not commonly reported in plants and thus more research is required.
Moreover, there is also the possibility of these granules being artefacts due to the
experimental procedure I followed. But even if the granular nature is an artefact, the
high concentrations are undoubtedly correct, as is their association with K, even if these
high concentrations may not be associated with granules before harvest.
5.3 Unexpected findings
5.3A Inoculation with AMF greatly decreased manganese concentrations
Inoculation with AMF resulted in a decrease in shoot (Chapters 2 and 3) and root
(Chapter 2) manganese (Mn) concentration. However root Mn concentration did not
decrease in all species in colonised plants (Chapter 3). The finding that AMF caused a
reduction in host plant Mn concentration is in accordance with many studies (Fageria et
al. 2002; Kothari et al. 1991; Liu et al. 2000; Posta et al. 1994; Ryan and Angus 2003).
Changes in soil microbial community might be responsible for the difference in Mn
concentration between colonised and uncolonised plants. In an experiment by Posta et
al. (1994), although the total microbial population was higher in colonised plants (Zea
mays L. cv. Tau), the proportion of Mn-reducing microbial populations in uncolonised
plants was five-fold higher than that in colonised plants, resulting in a higher shoot Mn
concentration in the uncolonised plants than in the colonised plants. Also, the amount of
Mn-mobilising root exudates in colonised plants was less than that in uncolonised
plants. However, my findings presented in Chapter 3 suggest that the explanation may
be more complicated. In Chapter 3, colonisation by AMF caused a higher rhizosphere
pH, lower shoot Mn concentration and a lower amount of rhizosphere carboxylates.
Integrating all these results, I concluded that plants that are colonised by AMF exude
less carboxylates into the rhizosphere which changes the rhizosphere chemistry (making
it more alkaline) and this results in less Mn uptake by the plant. This explanation is
consistent with Marschner (1991), who stated that plants grown under acidic conditions
might encounter Mn toxicity. Overall, both processes (i.e. changes in the soil microbial
community and changes in rhizosphere chemistry) might play a role in reducing the Mn
concentration in colonised plants.
Chapter 5. General Discussion 99
5.3B Phosphorus efflux following colonisation with AMF
One of the interesting, unexpected, findings in Chapter 3 was great elevation in
rhizosphere Colwell P in colonised K. prostrata and C. australasicum following a P
pulse.
There are several studies that show P efflux when plants are supplied with high
concentrations of P (Elliott et al. 1984; McPharlin and Bieleski 1989; Schjørring and
Jensen 1984). For instance, in an experiment by Cogliatti and Santa Maria (1990), P
efflux was increased when wheat (Triticum aestivum cv. Klein Atalaysa) was grown at
higher P concentrations.
Elevation of rhizosphere Colwell P following a P pulse in colonised plants might be due
to one or both of the following: 1) P efflux from the roots of colonised plants, since they
acquire more P (through mycorrhizal hyphae) than they need for growth and transport to
the shoot; and 2) P efflux from the mycorrhizal hyphae (which has been reported by
Ryan et al. (2003) and Solaiman and Saito (2001).
In my experiment, high concentrations of Colwell P in the rhizosphere might be due to
both factors. More research is now required to better understand this response (and why
it did not occur in the other three species in the experiment).
5.3C Colonisation with AMF makes the rhizosphere more alkaline
The second unexpected result from Chapter 3 was higher rhizosphere pH in both
colonised and uncolonised plants compared to bulk soil. According to Raven and Smith
(1976), the increase in rhizosphere pH compared to bulk soil is due to absorption of
nitrate. Also, Bago et al. (1996) found supplying nitrate to colonised plants increases
rhizosphere pH while supplying ammonium drops pH. In my experiment, plants were
treated with similar amounts of nitrate and ammonium during the experiment (3.3 mg N
kg-1 soil) and since nitrate is more mobile than ammonium in the soil (Binkley 1984),
the rise in rhizosphere pH was expected. On the other hand, an increase in rhizosphere
pH of colonised plants can be explained by a reduction in the amount of rhizosphere
carboxylates. The strong negative correlation between the amount of rhizosphere
carboxylates and rhizosphere pH also supports this explanation.
Chapter 5. General Discussion 100
5.4 Limitations of the research presented in this thesis
Although the experiments were designed to be as close to natural (field) conditions as
possible (using field soil as inoculum, growing plants in the field soil with moderate
levels of P, and adding filtrates to the non-inoculated plants to maintain the microbial
community), there are some limitations of this research. One of the most important
factors that might limit applicability of my experiments to field conditions is the
glasshouse conditions under which the experiments were conducted. My experiments
were conducted in a temperature-controlled environment, whereas in the field plants
encounter a greater range of temperatures. Also, in the field other factors such as pests
and diseases and neighbouring plants and the competition between them might result in
different P-uptake responses from plants (Collis George and Davey 1960; De Vries and
Tiller 1978).
One of the outstanding findings of this study was the moderating effect of mycorrhiza.
This effect was shown during the first week after adding the pulse of P. A longer period
of time might hide such an effect, and the results might be different.
Soil P levels also might change the results presented in the thesis. All the experiments
were performed under a moderate soil P level. Using soil with lower or higher P levels
might eliminate the moderating effect of AMF.
5.5 Implications for future research
To address the limitations discussed above in the thesis and to have a better
understanding of how AMF moderate plant P uptake following a P pulse, performing
experiments over a longer period of time is to be recommended. Many root physiology
experiments overlook or remove the effect of AMF. Often, they are conducted in
sterilised soil. According to the results that came from this thesis, I believe that AMF
are not only important from the perspective of nutrient uptake, but also because of their
role in changing rhizosphere chemistry and microbial community.
5.6 Concluding remarks
The main focus of this thesis was to investigate the role of AMF in plant uptake of P
from a pulse of P by perennial pasture plants.
Chapter 5. General Discussion 101
According to the results in Chapters 2 and 3, colonisation by AMF does not necessarily
enhance plant P uptake from a P pulse, but may actually protect plants from P toxicity.
This could be particularly important for native plants such as Kennedia species which
suffer from P toxicity at relatively low P availability (Pang et al. 2009b).
Based on the results presented in this thesis, I suggest that the traditional view of AMF
as a beneficial biological “fertiliser” to enhance crop uptake of P and other poorly
mobile nutrients is too simplistic. My work indicates that AMF also play a moderating
role in P nutrition and affect plant nutrition in a complex manner through their effect on
rhizosphere pH, carboxylates and P content, and, undoubtedly, other means.
5.7 References
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