Banksia woodland background · Banksia woodland community (Fig 1.1) is the center of distribution...

Restoration in a postmine environment:

Using ecophysiological techniques to improve the

establishment of framework Banksia woodland seedlings

Stephen M Benigno BSc

This thesis is presented for the degree of Doctor of Philosophy

School of Plant Biology

The University of Western Australia

2012

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DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK

PREPARED FOR PUBLICATION

This thesis contains work prepared for publication, some of which has been co-authored. The publications arising from this thesis are original work undertaken by the student (Stephen Benigno), with guidance from his three supervisors (Jason Stevens, Kingsley Dixon, and Deanna Rokich) and university employee Greg Cawthray. The bibliographical details of the work and where it appears in the thesis are outlined below.

Chapter 2 - Benigno S, Dixon K, Stevens J, Ecophysiological adaptations of three woody

mediterranean tree seedlings to the postmine stresses of drought and soil compaction

In prep, divided into two manuscripts for publication under the titles:

1) Biphasic drought is key to seeding establishment in sandy mediterranean-type soils

2) Interplay of drought and soil compaction lead to catastrophic decline in

phreatophytic species in mediterranean-type environments

Chapter 3 - Benigno S, Dixon K, Stevens J, Increasing soil water retention with native-

sourced mulch improves seedling establishment in postmine mediterranean sandy

soils (abridged version published in Restoration Ecology under the same title,

October 2012 DOI: 10.1111/j.1526-100X.2012.00926.x)

Chapter 4 - Benigno S, Dixon K, Cawthray G, Stevens J, Soil physical strength rather than

excess ethylene reduces root elongation in mechanically impeded sandy soils

(published in Plant Growth Regulation under the same title, April 2012, DOI:

10.1007/s10725-012-9714-2)

Data collection and analysis, presentation, and writing of this thesis are entirely my own. Drs. Stevens, Dixon, and Rokich, as supervisors of this thesis, oversaw the design and implementation of the experiments, and provided reviews and recommendations for the written manuscripts. Greg Cawthray was not a formal supervisor for this project, but provided enough guidance to warrant inclusion with the work undertaken in Chapter 4.

Student Signature Coordinating Supervisor Signature

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Table of Contents Statement of Candidate Contribution ................................................................................... iii Table of Contents ................................................................................................................... v Abstract ................................................................................................................................ vii Acknowledgements ............................................................................................................... ix Abbreviations ......................................................................................................................... x

Chapter One: General Introduction

Perth’s Banksia Woodlands: Ecological Background and Need for Restoration .................. 1 Restoration in Postmine Sites ................................................................................................ 5 The Integration of Ecophysiology and Restoration Ecology ................................................. 8 Study Species ......................................................................................................................... 9 Thesis Aims and Outlines .................................................................................................... 10

Chapter Two: Ecophysiological adaptations of three woody mediterranean tree seedlings

to the postmine stresses of drought and soil compaction Introduction .......................................................................................................................... 13 Materials and Methods......................................................................................................... 15

Experimental Design .................................................................................................. 15 Measurements ............................................................................................................ 17

Results.................................................................................................................................. 21 Discussion ............................................................................................................................ 41 Conclusion ........................................................................................................................... 50

Chapter Three: Increasing soil water retention with native-sourced mulch improves

Banksia seedling establishment in postmine mediterranean sandy soils Introduction .......................................................................................................................... 53 Materials and Methods......................................................................................................... 54

Study Site ................................................................................................................... 54 Experimental Design .................................................................................................. 57 Measurements ............................................................................................................ 59


Chapter Four: Soil physical strength rather than excess ethylene reduces root elongation

of Eucalyptus seedlings in mechanically impeded sandy soils Introduction .......................................................................................................................... 81 Materials and Methods......................................................................................................... 83

Experimental Design .................................................................................................. 83 Measurements ............................................................................................................ 86


Chapter Five: General Discussion and Future Research Seedling Establishment in Postmine Conditions ................................................................. 99 Increasing Seedling Survival through Ecophysiological Analysis .................................... 101 Future Research Directions ................................................................................................ 104 Conclusion ......................................................................................................................... 107

References ............................................................................................................................... 109

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Abstract

Restoration of the diminished Banksia woodland community is necessary to maintain one

of the world’s biodiversity hotspots. In postmine environments, extreme soil compaction

prevents framework overstorey species of this ecosystem from accessing essential

groundwater. Coupled with extreme summer drought, the stresses posed by postmine

environments have prevented the restoration of the Banksia woodland community to its

historic trajectory. This thesis investigates the ecophysiological responses of seedlings to

the abiotic barriers of drought and increased soil compaction to aid restoration in postmine

conditions. In addition, methods to manipulate both the seedling and the postmine

environment are examined, focusing on improving seedling establishment of framework

overstorey species to postmine sites through the use of ecophysiological techniques.

Seedlings of three phreatophytic trees, Banksia attenuata, B. menziesii, and Eucalyptus

todtiana, were subjected to drought, increased soil compaction, and a combination of the

two stresses under a controlled glasshouse study to identify species-specific

ecophysiological adaptations to these commonly encountered stresses. Increased soil

compaction alone did not cause seedling mortality or alter physiological function, but

negatively affected root structure by decreasing root elongation by ~70%. Under drought

stress, the two Banksia species exhibited isohydric characteristics, with a “water-saving”

strategy, whereas E. todtiana can be classified as an anisohydric species and has a more

“drought-tolerant” strategy. The physiology of all species was more severely and promptly

damaged by a second round of water deficit, causing mortality in all seedlings within 48

days of the second drought. Simultaneous drought and soil compaction represented an

environment which severely damaged seedling morphology and physiology. Despite the

discrepancies in physiological mechanisms to cope with drought and/or soil compaction

stresses, no differences in mortality between species were observed.

To alleviate soil compaction and increase soil water retention in situ, an organic and

inorganic soil amendment were incorporated into the top 50 cm of the soil profile in a fresh

restoration site within a sand quarry. The organic amendment increased seedling

establishment of B. attenuata and B. menziesii 24 and 42%, respectively, over two years.

This benefit most likely resulted from a significant increase in moisture within the rooting

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zone. From ecophysiological monitoring, seedlings of B. attenuata and B. menziesii grown

in the organic amendment plots functioned at a significantly higher rate over the dry

summers than seedlings grown in inorganic amendment soil or soil containing no

amendment. Neither the organic nor inorganic amendments displayed an ability to alleviate

the soil compaction associated with reconstructed postmine soils. The belowground

morphology of seedlings in restoration sites was significantly altered from seedlings grown

in native remnant bushland, most notably a restriction of root depth to the top 40 cm of the

soil in restoration sites compared to depths deeper than 100 cm in native bushland.

In compact soils, such as the reconstructed soils of postmine sites, excess ethylene can

accumulate and be absorbed by plant roots. This can stimulate plants to produce thicker and

stunted roots, and has proven to reduce root elongation through soil. An ex situ laboratory

experiment investigated the effects of excess ethylene on E. todtiana seedlings and

uncovered that the physical strength of soils is a greater obstacle to root growth through

compact sands than the presence of ethylene. High amounts of ethylene were able to stunt

root elongation, however compact soil alone also reduced root elongation and did so

without the detection of any excess ethylene in the root-soil system. The physical properties

of the soil should be given higher priority than the presence of ethylene within

reconstructed postmine soils with respect to reduced root elongation. An ethylene inhibitor

tested in this study produced conflicting results on seedling ethylene production, and did

not aid root elongation through compact soil.

The results presented in this thesis expand upon Banksia woodland restoration knowledge

and include previously absent information pertinent to seedling establishment in postmine

sites. The stresses of drought and soil compaction are equally damaging to the survival of

seedlings of the three framework Banksia woodland species, with a biphasic drought

pattern identified as a detrimental factor to seedling health. Site physical conditions must be

manipulated for successful seedling establishment in compact postmine soils, and

increasing soil water retention within the root zone improves seedling establishment during

restoration of postmine mediterranean-type ecosystems.

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Acknowledgements

First and foremost, much appreciation goes towards my supervisors, Kingsley Dixon,

Deanna Rokich, and especially Jason Stevens, whose patience, guidance, and support kept

me focused and challenged.

Greg Cawthray taught me to operate a gas chromatography-mass spectrometer and interpret

the results, a technique I was hopeless with as an undergrad.

Rocla Quarry Products® supplied a blank template for conducting research in the mine

restoration sites at their Gaskell Avenue Sand Quarry. A very accommodating and

progressive company when dealing with the environment, they provided safe working

conditions and all the heavy machinery I could ask for.

The staff and students at Kings Park and Botanic Garden provided many entertaining

distractions from my work. Without all of you this thesis would probably have been

completed about six months earlier, but the entire process would have been much more

monotonous.

To Michael and Brenda Benigno. I always considered it trite to thank your parents in these

circumstances, but now I understand why it’s so popular. Your support in my life could

never be overstated.

And finally, this thesis is dedicated to my time spent in Australia, to all of the incredible

memories and even more incredible friends I made during my time in this country.

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Abbreviations and Symbols

gs - Stomatal conductance rate

A – Photosynthetic rate

E –Transpiration rate

Ci - Intercellular carbon

ε - Carboxylation efficiency

WUE - Water-use efficiency

Fv/Fm - PSII photochemical efficiency

ETR - Electron transport rate

NPQ - Non-photochemical quenching

ΨPD - Predawn xylem water potential

ΨMD - Midday xylem water potential

ΨD - Diurnal range of water potential

KL - Apparent soil-to-leaf hydraulic conductance

SLA - Specific leaf area

SRL - Specific root length

VSW - Volumetric soil water content

EC - Soil electrical conductivity

SOC - Soil organic content

PPFD - Photosynthetic photon flux density

PAR - Photosynthetically active radiation

DIHB - 3,5-diiodo-4-hydroxybenzoic acid

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1

C H A P T E R O N E

General Introduction

Perth’s Banksia Woodlands: Ecological Background and the Need for Restoration

Perth’s Banksia woodland community lies within the Southwest Australian Floristic

Region (SWAFR), one of 25 global biodiversity hotspots (Meyers et al. 2000). Although

encompassing only 13% of the land mass within Western Australia, the SWAFR is home to

7,380, or 75% of the state’s total number of native vascular plant species. Of this amount,

half the species are endemic to the SWAFR, while an additional 34% are of conservation

concern (Hopper and Gioia 2004). Located within the SWAFR, a thin strip of heathland

along the Indian Ocean forms the Swan Coastal Plain (Fig 1.1). This biogeographic region

supports over 2,000 plant species: a diverse mid-storey of woody shrubs and herbaceous

ground cover with relatively few species of overstorey trees (Beard 1995; Dodd 1989).

Fig 1.1 A map of a) the distribution of the Banksia woodland community within the Swan Coastal Plain and

b) Western Australia, depicting the location of the Swan Coastal Plain within the SWAFR (modified from

Beard (1989) and Department of Sustainability, Environment, Water, Population and Communities).

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The landscape of the Swan Coastal Plain is historically ancient (~800,000 years old)

and situated on a well-drained and nutrient-deficient dune system, characteristics that are

unique among other mediterranean-type ecosystems. This anomaly creates the necessity for

species to adapt and fueled the explosive speciation of this region (Bolland 1999; Hopper

and Gioia 2004; McArthur 1991). A dry mediterranean-type climate produces an average

annual rainfall of 800 mm, 86% of which falls between May and October, and temperatures

fluctuate between highs of 35°C during the summer months and lows of 5°C during the

winter (Fig 1.2) (Bureau of Meteorology 2012; Dodd and Heddle 1989).

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Average Monthly R

ainfall (mm

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Avg. Rainfall Max. Temp. Min. Temp.

Fig 1.2 Climate statistics from the Perth regional office. High temperatures of the summer months coincide

with low average precipitation, creating a mediterranean-type climate. Source: Bureau of Meteorology 2012.

As the most common and defining plant community of the Swan Coastal Plain, the

Banksia woodland community (Fig 1.1) is the center of distribution to several large genera

including Banksia trees (Fig 1.3), Leucopogon shrubs, and Caladenia orchids, while the

genus Synaphea and the Banksia subgenus, Dryandra, are completely restricted within its

borders (Beard 1995). The native perennial species that survive in this environment are

reliant on a range of life form characteristics that include rooting morphologies able to

access deep water sources to cope with summer drought (Fig 1.4) (Dodd et al. 1984). Many

trees and shrubs in this region have evolved a dimorphic rooting system: a deep taproot

which draws water from the subsoil, paired with spreading lateral roots able to capture

surface rainwater (Fig 1.4) (Dodd et al. 1984). Phreatophytic trees (deep-rooted species that

obtain a significant portion of water from the phreatic zone of the soil profile) from the

Banksia woodland community are estimated to supply 70 - 90% of average rainfall to the

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Fig 1.3 The inflorescence of a Banksia menziesii cone a) before and b) after pollination. c) The Banksia

woodland community of the Swan Coastal Plain, with an overstorey of B. menziesii and B. attenuata trees.

atmosphere through evapotranspiration (Dodd and Bell 1993; Dodd and Heddle 1989).

Approximately 25 - 61% of this rainfall is attributed to the ability of phreatophytes to

access deep soil moisture sources (Farrington et al. 1989). The recycling of water by

phreatophytic species has a significant effect on groundwater recharge within this system

(Groom 2004), and any decreases in evapotranspiration can be expected to reduce

precipitation and lengthen the dry season (Shukla et al. 1990). Removal of native

vegetation can cause the water table to rise over time, bringing dissolved salts to the surface

and creating a toxic soil environment for native plants (Hunt and Gilkes 1992). If left

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devoid of native vegetation, invasive species can colonize the landscape, potentially

altering above and belowground environmental processes (Reinhart and Callaway 2006;

van der Putten et al. 2007). These biotic and abiotic impediments become permanent if left

untreated, creating an unnaturally fragmented landscape that disrupts dispersal processes

and gene flow for the diverse native species within the Banksia woodland community

(Hercock 1997; van Diggelen 2006).

Fig 1.4 Rooting patterns of plants found in the Banksia woodland community, adapted from Dodd et al.

(1984). Type 4 represents a dimorphic and phreatophytic root system, capable of accessing water at greater

depths with a long taproot, in addition to shallow branching lateral roots.

The return of native flora, and phreatophytes in particular, is necessary to restore

the functioning ecosystem (Bradshaw and Chadwick 1980). Over 80% of the Banksia

woodland community has been developed or cleared for urban use (Hopper and Burbridge

1989; Turner et al. 2006). The extensive destruction of Banksia woodland is due to its

proximity to the metropolitan city of Perth, and the most significant threat to this ecosystem

is the creation of a fragmented landscape through the loss of habitat, while disease, fire,

weeds, and groundwater extraction also pose conservation risks (Gozzard and Mouritz

1989; Lamont et al. 2007). Limited information associated with the conservation and

restoration management of the Banksia woodland community exists (Turner et al. 2006),

yet urban development continues to unabatedly shrink and fragment this threatened

community. Currently, the most economical and reliable method for restoring Banksia

woodland is to respread topsoil to ‘inoculate’ the cleared sites with a native soil seed bank

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(Maher et al. 2008). However, seeds of the most common overstorey phreatophytes,

Banksia attenuata and B. menziesii, are not stored within the topsoil, and are released from

their floral cones after a fire (Fig 1.3) (Turner et al. 2006). Following topsoil replacement,

seeds of these species must be broadcast onsite, but less than 10% have been shown to

germinate and establish, reducing the success of Banksia woodland restoration (Rokich et

al. 2002).

Fig 1.5 A postmine Banksia woodland site, showing the upper ridge with remnant natural woodland, the

degree and depth of sand extraction, and an eight year old restoration site in the foreground. Note the lack of

overstorey trees in the foreground.

Restoration in Postmine Sites

Over 100,000 metric tons of industrial sand is mined globally every year (USGS

2011), and the process is invaluable for many economic development activities such as

road building and concrete production. The development of Perth is dependent on sand

mining for both construction and exportation (Gozzard and Mouritz 1989). After mineral

extraction, these postmine sites provide a useful ‘greenfield’ opportunity for investigating

ecological restoration questions in the Banksia woodlands (Fig 1.5). Human impacts

substantially alter the ecology and sustainability of the woodland environment (Hopper

2004). Understanding the altered environmental conditions of postmine sites can improve

restoration success, re-establish ecosystem function, and provide a guide for future

restoration projects throughout the region (Elmarsdottir et al. 2003). Increased light,

temperature, wind, and predation are introduced aboveground barriers to seedling

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establishment in postmine sites (Hungerford and Babbit 1987; Ronco 1970; Sork 1987), but

the altered belowground conditions are believed to present the most significant barriers to

postmine restoration in the Banksia woodlands (Enright and Lamont 1992; Rokich et al.

2001), and will therefore be the focus of this thesis.

Soil compaction

The postmine conditions in the soil profile are vastly different from undisturbed

sites, as the resulting soil depth is significantly reduced and the soils are reconstructed by

replacement of the topsoil and lower soil profiles (Rokich 1999). Reconstructed postmine

soils are prone to heavy soil compaction, in which both soil strength and bulk density have

been shown to increase, and can disrupt the natural root development and restrict seedling

access to moisture or nutrients during restoration projects (Bradshaw 1997; Kozlowski

1999; Thompson et al. 1987; Wong 2003). Soil-remediating techniques consist of

chemically or physically altering the soil profile to alleviate compaction. The use of soil

amendments involves a specific material or chemical incorporated directly into the soil

profile, and is reliant on the interaction between the properties of the soil and the type and

amount of amendment (Nelson and Oades 1998; Soane 1990; Wong 2003). Physically

ripping or scarifying the soil by mechanical means can alleviate the compaction present in

postmine soils, and must be implemented prior to returning vegetation to the site

(Bradshaw 1997; Craul 1994).

Along the dune systems within the Swan Coastal Plain, the coarse sands of

reconstructed postmine soils have been observed to gradually increase in strength after

mining operations have ceased, despite extensive deep ripping (Fig 1.6) (Rokich 1999;

Rokich et al. 2001). This suggests that soil compaction in this postmine system returns over

time due to natural forces, and a long-term solution such as the incorporation of soil

amendments has not been attempted (McArthur 1991; Rokich 1999). This “cryptic

compaction” phenomenon observed in sandy postmine soils has yet to be investigated in

full detail, and possible causes are believed to be a result of soil particle dispersion, the

wetting and drying cycle of the soils, and/or silica or iron cementation (Enright and Lamont

1992; Horn et al. 1995; Horn et al. 1994; Kozlowski 1999; Rokich 1999). Despite the

unknown cause of this cryptic compaction, the restoration of the soil environment to pre-

mine conditions is essential for natural seedling growth and establishment (Bradshaw 1997;

Cummings et al. 2005; Kozlowski 1999; Wong 2003). This phenomenon could play a large

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role in the high mortality observed in phreatophytic species of the Banksia woodlands by

preventing root penetration and cutting off seedling access to groundwater (Rokich 1999).

Fig 1.6 Evidence for soil-densification in ripped postmine restoration soils along the Swan Coastal Plain,

adapted from Rokich (1999). As time progresses, the maximum depth of penetration by a soil penetrometer in

a 2-year old restoration site becomes shallower and approaches values equal to 4 and 7-year old restoration

sites, indicating soil compaction can increase through time in postmine restoration sites.

Water stress

Increases in soil compaction in postmine settings hinder restoration by disrupting

natural root growth and can place plants at a high risk of drought stress (Marquez 2010;

Thompson et al. 1987; Zisa et al. 1980). Prolonged water stress is recognized as a major

cause of seedling mortality when restoring mediterranean-type ecosystems through

reduction in photosynthetic capacity and reduced growth rates of planted seedlings

(Marañón et al. 2004; Varone et al. 2011). The predicted increase in both the duration and

intensity of summer droughts in mediterranean-type regions due to climate change will

further compromise the health and function of restored postmine Banksia woodland sites

(Fig 1.7) (Giorgi and Lionello 2008). Past research in postmine settings along the Swan

Coastal Plain has shown that the seedlings of the iconic phreatophytes, B. attenuata and B.

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menziesii grown from broadcast seed, are able survive the first summer drought, but

mortality increased to over 90% when drought occurred during the second summer (Rokich

1999). The mechanisms behind this ‘biphasic’ pattern of seedling death are unknown, and

understanding the relationship between drought and seedling mortality could provide

practical implications for managing future restoration success (Burgess 2006; Rokich

1999).

Fig 1.7 Projected changes in temperature and rainfall by the year 2030, under varying levels of CO2

emissions. Source: www.climatechangeinaustralia.gov.au.

The Integration of Ecophysiology and Restoration Ecology

While seedling mortality within postmine sites in the Banksia woodland community

has been attributed to drought stress caused by compact soil and restricted root

development (Burrows 1986; Enright and Lamont 1992; Rokich et al. 2001), studies on the

underlying physiological causes behind seedling mortality and survival have yet to be

undertaken. The investigation of plant ecophysiology has emerged as a valuable tool to

explain the ecological questions that underpin observations such as survival, distribution,

abundance, and other environmental interactions (Lambers et al. 2008a), but despite its

practical uses, ecophysiology has only recently been integrated into the science of

restoration ecology (Cooke and Suski 2008). With the advent of new and portable

technology, ecophysiological assessments have become more manageable and rapid

(Ehleringer and Sandquist 2006). These technological advancements provide fast and direct

connections between site quality and species fitness, allowing for relatively short

monitoring periods indicative of many restoration projects (Cooke and Suski 2008).

Through the integration of laboratory and field studies, ecophysiological traits can be

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examined to assess the tolerance of various species to microclimate conditions and their

capabilities for reintroduction (Ackerly 2000; Pywell et al. 2003; White and Walker 1997).

The knowledge gained from ecophysiological studies not only answers questions about the

physiological function of the species themselves, but also provides significant insights that

can influence the practice of ecological restoration (Ehleringer and Sandquist 2006).

Study Species

Restoration of mediterranean-type regions is most successful when based on late-

successional woody species (Vallejo et al. 2006), and the research presented in this thesis

will focus on the postmine seedling establishment of three dominant overstorey trees of the

Banksia woodland community, B. attenuata, B. menziesii, and Eucalyptus todtiana (Dodd

and Griffin 1989). These woody trees are considered 'framework species', meaning their

successful establishment accelerates restoration and the return of biodiversity in this region

(Blakesley et al. 2001). This effect is achieved through the groundwater recharge

capabilities of these phreatophytes, from both belowground water redistribution and the

recycling of water by evapotranspiration (Dawson 1993; Farrington et al. 1989; Groom

2004; Shukla et al. 1990). The three species occupy similar geographical and

ecohydrological ranges and employ a dimorphic root system to resist water deficit during

summer droughts (Figs 1.4, 1.8) (Dodd et al. 1984). While little is known of the physiology

of E. todtiana, fully-developed adult B. attenuata and B. menziesii are classified as

“drought-avoiders”, utilizing tight stomatal control to cope with drought while maintaining

constant xylem water potential (Froend and Drake 2006; Groom 2004; Levitt 1980;

Veneklass and Poot 2003). The majority of past physiological research performed on these

species focuses on reproductive adult trees in a natural setting. However, water uptake and

root structure are known to differ greatly between seedlings and adults of the same species

(Donovan and Ehleringer 1992), and physiological knowledge from a natural setting may

not pertain to a degraded environment (Ehleringer and Sandquist 2006). Therefore to

develop an ecophysiological blueprint for successful restoration, research must focus on

seedlings subjected to the diverse stress conditions encountered in postmine sites (Shao et

al. 2007).

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Fig 1.8 Geographic range of B. attenuata, B. menziesii and E. todtiana. Source: www.florabase.dec.wa.gov.au.

Thesis Aims and Outline

The aim of this research is to investigate the ecophysiology of B. attenuata, B.

menziesii, and E. todtiana seedlings to guide and improve postmine restoration. Knowledge

of seedling ecophysiology under stress conditions can be utilized to develop novel and

practical restoration techniques (Ehleringer and Sandquist 2006), and approaches to alter

both the environment and the seedlings themselves are explored. The results and

conclusions from this thesis can not only be applied within the Banksia woodland

community, but also along degraded sites where increased soil compaction or drought

present a barrier to seedling recruitment, particularly within a mediterranean-type

ecosystem.

Chapter 2 investigates the physiological and morphological responses of B. attenuata, B.

menziesii, and E. todtiana seedlings to the postmine stresses of drought and soil

compaction under a glasshouse setting.

Under resource-limited conditions, the response to stress in co-occurring species with

similar life forms can differ in both mechanism and magnitude (Austin et al. 2009; Grigg et

al. 2008; Szota et al. 2011). Glasshouse studies examined the response (function and form)

of seedlings to drought and soil compaction and related these findings to survival and

mortality. Subtle changes in morphology such as root and leaf structure can aid in avoiding

drought (Joslin et al. 2000; Marron et al. 2003; Rambal 1993; Turner 1994). Physiological

concepts examined in this chapter include: 1) the enhancement or suppression of seedling

http://www.florabase.dec.wa.gov.au/�

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gas-exchange rates (Flexas and Medrano 2002; Medrano et al. 2002b), 2) increases in

photoprotection during prolonged water deficit through measurement of chlorophyll

fluorescence (Galmés et al. 2007; Medrano et al. 2002a) and 3) xylem water potential and

cavitation avoidance (Davis et al. 1998; Sperry et al. 2002), all of which are known to

differ between species and influence seedling survival during drought. The physiological

mechanisms of damage and recovery over multiple droughts and re-watering were

investigated to explain the high mortality rates that have been observed to occur over a

second period of drought (Rokich 1999). Defining tolerance in each individual species

towards drought and/or soil compaction will be used to recommend approaches for

improving restoration success.

Chapter 3 explores the use of soil amendments to manipulate the postmine environment

and alleviate the stresses of drought and soil compaction on B. attenuata and B. menziesii

seedlings in a field setting.

The physico-chemical properties of reconstructed postmine soils can severely restrict

seedling establishment and must be restored prior to restoration (Bradshaw 1997;

Cummings et al. 2005; Wong 2003). Soil-restoration techniques such as the incorporation

of amendments directly into the soil profile can provide seedlings with more favorable

conditions for establishment by improving soil water retention or reducing soil compaction

(Barzegar et al. 2002; Ekwue and Stone 1995; Larson 1972; Soane 1990, Vallejo et al.

2006). A field experiment performed in reconstructed postmine soils evaluated the benefits

of an organic and inorganic soil amendment on seedling establishment through the

measurement of seedling physiology - a novel approach to determine the effectiveness of

amendments under restoration conditions. Survival was monitored throughout the critical

first two years of restoration, and seedling physiology was integrated with amendment-

specific site conditions to identify any environmental benefits the amendments provided

towards seedling establishment.

Chapter 4 examines the response of E. todtiana seedlings to the chemical and physical

factors which affect reduced root elongation through compact and non-compact sands

under laboratory conditions.

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Seedling roots become stunted in response to both increased soil compaction and excess

soil ethylene (Kozlowski 1999; Lynch 1975; Moss et al. 1988; Stenlid 1982). Given that

ethylene is known to build up in compact soils due to anaerobic conditions, reduced root

elongation in compact soil has been suggested to be the result of chemical factors (Smith

1976; Smith and Robertson 1971; Smith and Russell 1969). Controlling the action of

ethylene on seedling roots reduces its antagonistic effect on root elongation (Robert et al.

1975), and the ethylene inhibitor DIHB (3,5-diiodo-4-hydroxybenzoic acid) was shown to

aid root elongation through compact soils by attenuating ethylene production from roots

(Larqué-Saavedra et al. 1975; Saini 1979; Wain et al. 1968; Wilkins et al. 1977).

Laboratory experiments tested the effect of excess ethylene, soil compaction, and DIHB on

root elongation. Ethylene production and seedling morphology were analyzed to determine

the extent of influence that ethylene asserts over root elongation through compact and non-

compact soils and whether DIHB provides a solution to root stunting in compact soils.

Chapter 5 presents a synthesis of the thesis and discusses areas for future research.

The results from the previous chapters are integrated and discussed with the objective of

raising the success rate of restoration in the biodiversity hotspot of the Banksia woodland

community, as well as restoring postmine soils in other areas of the world, particularly in

the sandy soil environments of mediterranean-type ecosystems. Ecophysiological

knowledge is incorporated with previous research from the Banksia woodland community.

Future research recommendations are made in relation to understanding framework species

physiology in postmine sites with the intent to raise restoration success.

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C H A P T E R T W O

Ecophysiological adaptations of three woody mediterranean tree

seedlings to the postmine stresses of drought and soil compaction

Introduction

Various woody tree species are known to employ unique adaptations to survive the

annual summer drought occurring within mediterranean-type ecosystems (Abril and

Hanano 1998; Beis and Patakas 2010; Galmés et al. 2007; Lo Gullo and Salleo 1988).

Species can differ in their ability to conserve water by regulating stomatal aperture or

maintaining their hydraulic transport system under highly negative water potentials

(Galmés et al. 2007; Pockman and Sperry 2000). Morphological adaptations in root

architecture and root elongation rate allow species to quickly access permanent soil water

sources, while a high degree of leaf sclerophylly aids the maintenance of physiological

function under drought (Schnek and Jackson 2002; Turner 1994). The extent of these

responses is known to be critical for the survival and distribution of species in water-

limiting environments (Davis et al. 1998; Pockman and Sperry 2000). Under disturbed

environmental conditions such as a postmine restoration settings, previous ecophysiological

knowledge from native systems may not be applicable (Ehleringer and Sandquist 2006).

Specific species traits that are best adapted to cope with the environmental restraints of

postmine mediterranean-type ecosystems must be investigated and considered when

restoring degraded lands (Vallejo et al. 2006).

Along the dunes of the southwest Western Australia, three dominant overstorey

trees of the Banksia woodland community, Banksia attenuata (R. Br.), Banksia menziesii

(R. Br.), and Eucalyptus todtiana (F. Muell) rely on deep taproots for groundwater access

(Dodd and Griffin 1989). These facultative phreatophytes can elongate their roots at 3.7 cm

per day up to 12 m in depth, reaching the subsoil water sources necessary for their survival

(Canham 2011; Farrington et al. 1989; Zencich et al. 2002). Fully developed B. menziesii

and B. attenuata trees were shown to possess similar physiological and morphological

attributes under natural environmental conditions: tight stomatal regulation, year-round

transpiration, evergreen leaves, and a dimorphic rooting pattern ensured the growth and

survival of these trees (Groom 2004; Veneklaas and Poot 2003). Despite similarities in

14

ecophysiology (both Banksias are “drought-avoider water-savers”), B. menziesii is thought

to be more drought resistant than B. attenuata, with the ability to tolerate lower water

potentials (Froend and Drake 2006; Groom et al. 2000; Muir 1983). E. todtiana also

possesses a dimorphic rooting morphology, but scant information exists on its physiology

(Dodd et al. 1984). During summer droughts, xylem water potential of adult E.

gomphocephala (a eucalyptus species occupying a similar range and niche) decreases as a

drought-coping mechanism (Drake 2008), which suggests E. todtiana employs a similar

“drought-tolerant” behavior (Levitt 1980). While behavioral information on adult trees can

provide insight into future patterns of survival in natural systems, it is the ecophysiological

adaptations of seedlings under stressed conditions that are of high priority from a

restoration perspective. Seedling and juvenile physiology can differ from that of adult trees,

and the survival and physiology of the seedlings of these three species could differ

significantly in response to drought (Ackerly 2000; Donovan and Ehleringer 1991; Varone

et al. 2011).

The ecophysiological mechanisms that underpin seedling survival in postmine

conditions have yet to be studied and can provide critical insight for the restoration of

postmine systems. The reconstruction of the soil profile in postmine systems increases soil

mechanical impedance and restricts root elongation, potentially obstructing plants from

accessing crucial soil moisture (Bradshaw 1997; Cummings et al. 2005; Thompson et al.

1987). The disruption of the natural root architecture of B. attenuata, B. menziesii, and E.

todtiana by increased soil compaction can place these seedlings under greater drought

stress, and has been postulated as a major driver of seedling mortality, impairing restoration

success in the Banksia woodland community (Burrows 1986; Enright and Lamont 1992;

Rokich et al. 2001). Seedling establishment of 90% has been recorded in postmine soils

after the first dry season, however in the subsequent year survival can drop to 10%,

suggesting that a second period of drought inflicts greater damage to seedling physiology

and is decisive in establishing seedlings to postmine soils (Rokich 1999).

The health and establishment of B. attenuata, B. menziesii, and E. todtiana

seedlings is critical to the successful restoration of the Banksia woodland community, as

these deep-rooted phreatophytes facilitate the redistribution of soil moisture through

evapotranspiration (Burgess 2006; Dodd et al. 1984). The aim of this study is to investigate

the physiological and morphological responses of B. attenuata, B. menziesii, and E.

todtiana seedlings to water deficit and increased soil compaction under controlled

15

conditions with the objective of increasing restoration success in postmine settings within a

mediterranean-type ecosystem. The three species are thought to utilize different drought-

resistant strategies; adjusting their physiology and morphology to cope with drought, soil

compaction, and the combination of the two postmine stresses. The unique responses may

convey specific survival advantages between species, wherein one species outperforms the

other and provides a more successful framework for restoration. A second round of drought

is expected to inflict greater seedling mortality than the first round of drought, even after an

extended period of re-watering and seedling recovery. The physiological mechanisms

behind this ‘biphasic’ pattern of mortality is examined.

Materials and Methods

Experimental design

Local provenance seeds of B. attenuata, B. menziesii, and E. todtiana were obtained

from a commercial seed supplier (Fig 2.1). Seeds of each species were placed on a shallow

tray of potting soil in a cool room at 15̊ C and watered every two days to achieve optimal

germination requirements. After germination, the seedlings were transferred to individual

13 cm tall x 5 cm wide rectangular pots containing a potting mix and placed in an unheated

glasshouse. The seedlings were watered daily from overhead sprinklers and allowed to

grow for six months, simulating standard nursery practice systems.

Fig 2.1 Seeds of a) B. attenuata, b) B. menziesii, and c) E. todtiana.

B. attenuata, B. menziesii, and E. todtiana are dominant overstorey species of the

Swan Coastal Plain and are selected for use in restoration projects as framework species

16

within the Banksia woodland community. To allow for sufficient root growth, white PVC

pipes of 1.0 m length and 9.0 cm diameter (8.6 cm inner diameter) were selected as pots

(552 pots total). Endcaps were fitted at the bottom of the pots with four 1.0 cm diameter

holes drilled into the endcaps to allow for water drainage. The bottom of each pot was

filled with 2.5 cm of blue metal gravel followed by 2.5 cm of pea gravel, and the remaining

95 cm contained soil. The soil used in this study is a coarse white sand (98.6% sand, 1.4%

silt/clay) from the Bassendean dune system, sourced from postmine sites undergoing

restoration in the Swan Coastal Plain (Salama et al. 2005). These dunes are well-draining

and acidic, with very low levels of nutrients, and naturally support a mixed woodland of

both Eucalyptus and Banksia species (McArthur 1991; Salama et al. 2005).

Bulk density of soil in the PVC pots was adjusted to simulate the stress of soil

compaction. Pots representing non-compact soil contained 7.50 kg of soil, creating a bulk

density of 1.55 g cm-3. Pots representing compact soil contained 8.70 kg of soil, creating a

bulk density of 1.80 g cm-3. Using previous soil data from postmine sites in the Swan

Coastal Plain, soil with a bulk density of 1.80 g cm-3 was sufficient to restrict root growth,

while root growth was not impeded in soil with a bulk density of 1.55 g cm-3 (Rokich et al.

2001). To achieve the increased bulk density of 1.80 g cm-3, soil was compacted by hand

using a custom-made flat hammer. Ten centimeter layers of soil were placed in the pots and

hit with the hammer to ensure compaction occurred throughout the profile.

Depending on soil bulk density and water availability, seedlings were subject to

either: 1) drought with non-compact soil (drought treatment), 2) well-watered with compact

soil (compact treatment), 3) simultaneous drought and compact soil (drought compact

treatment), or 4) well-watered with non-compact soil (control). Thirty-two pots, evenly

split between compact and non-compact soil in drought and no drought conditions, did not

contain a seedling to allow for soil impedance measurements. An equal amount of pots

containing compact and non-compact soil were arranged in a completely randomized

design within two separate frames in an unheated glasshouse (Fig 2.2). A single seedling

was transplanted into each pot, and vermiculite was placed over top of the soil surrounding

the seedling to reduce water evaporation from the soil. Seedlings were acclimated in the 1.0

m pots for three months and during this time all pots were watered daily from overhead

sprinklers. Clear plastic sheeting was hung above and along the sides of both frames to

prevent water entry from outside sources. Drought was imposed on half the seedlings by

capping off the overhead sprinklers immediately above one of the frames. This first period

17

of drought lasted for 60 days (first drought phase), after which a re-watering period

occurred for 120 days (recovery phase), and was followed by a second and final 60 days of

drought (second drought phase). The pots in the second frame were watered daily

throughout the entire experiment from overhead sprinklers. At 24 days and 201 days after

seedlings were transferred into the PVC pots, 59 mL of nutrient solution was added to all

seedlings. The nutrient solution was developed specifically to simulate the nutrient poor

soils of southwest Australia, ensuring adequate nutrition was administered in the following

amounts (μM): 400 NO3−, 204 K+, 200 Ca2+, 154 SO4

2−, 54 Mg2+, 40 Fe-EDTA, 4 PO43−,

2.4 BO33−, 0.24 Mn2+, 0.10 Zn2+, 0.030 MoO4

2−, 0.018 Cu2+ (Poot and Lambers 2003).

Measurements

Seedling Survival

Forty-five seedlings from each treatment and species were marked for survival

counts throughout the experiment. Counts were taken five times during the first drought

phase, eight times during the recovery phase, and four times during the second drought

phase. Mortality was defined as a no longer functioning seedling, as derived from

chlorophyll fluorescence values (Fv/Fm) of 0, and if the seedling had completely brown and

brittle stems and leaves.

Physiology and Morphology

Five seedlings from each species and treatment were randomly selected to be

measured non-destructively throughout the entire course of the experiment. Measurements

took place 0, 15, 30, 45, and 60 days after the beginning of the first drought phase and

second drought phase, five days after the start of the recovery phase, and three additional

times throughout the recovery phase. A small plastic tie was secured around a petiole of the

most recently formed, fully expanded and undamaged leaf at the beginning of the

experiment and served as a marker for future gas-exchange and fluorescence

measurements. In the occurrence that one of the original five seedlings died during the

experiment, a random functioning seedling from the same species and treatment was

chosen as a replacement.

18

Fig 2.2 One of two frames containing 1.0 m long PVC pipe pots, each with an individual seedling of B.

attenuata, B. menziesii, or E. todtiana.

A Li-Cor® 6400 gas-exchange analyzer (LI-COR, Inc. Lincoln, NE, USA) assessed

the rates of stomatal conductance (gs), photosynthesis (A), transpiration (E), and

intercellular carbon (Ci). Measurements were taken between 0830 and 1100 h local time,

approximately two hours after the start of the photoperiod. Mid-morning and light-saturated

gas-exchange measurements represent the plant average daily values under drought stress

(Vadell et al. 1995). The Li-Cor was set at a reference level for all measurements taken

throughout the experiment (Flow Rate to the Sample Cell: 300 μmol s-1, Reference Cell

CO2: 400 μmol CO2 mol-1, Artificial Light Source: 6400-02 Red/Blue LED PAR 1,700

μmol m-2s-1). Five measurements on each leaf were performed within ten seconds after the

Li-Cor displayed a steady photosynthetic rate. Carboxylation efficiency (ε) was calculated

as the ratio of photosynthesis to intercellular carbon (A/Ci) (Farquhar and Sharkey 1982),

and has been shown to reflect Rubisco activity within leaves (von Caemmerer 2000).

Using the same recently formed, fully expanded leaf measured by the Li-Cor,

chlorophyll fluorescence was recorded by a PAM Fluorometer (Heinz Walz GmbH,

19

Effeltrich, Germany) (Fig 2.3). Measurements were taken between 1100 and 1400 h local

time. The sample leaf was dark adapted with a lightweight plastic clip for 15 minutes prior

to measurement. The maximum PSII photochemical efficiency (Fv/Fm) was calculated, and

a rapid light curve was performed immediately afterward on the same leaf without

removing the clip. The rapid light curve progressively increased from 0 to 1152 µmol

photons m-2 s-1 over nine light pulses. Maximum electron transport rate (ETR) was

consistently achieved at 822 µmol e- m-2 s-1 for each species, and the ETR and non-

photochemical quenching (NPQ) values were recorded at this PAR intensity.

Fig 2.3 Using a pulse of light to measure the chlorophyll fluorescence of a B. attenuata seedling leaf.

Eight destructive harvests were performed at intervals of 0, 21, 49, 77, 126, 175,

203, and 231 days after the first drought phase was initiated. Five seedlings from each

treatment and species were randomly selected for each harvest. Fluorometry and gas-

exchange measurements were recorded using the same calibrations described above. Xylem

water potential was measured at both predawn (ΨPD, commencing 2 hrs before sunrise) and

midday (ΨMD, 1200-1400 h local time). Water potential was measured from shoots

containing two fully expanded leaves using a Scholander-type pressure chamber (Model

1000 Pressure Chamber Instrument®, PMS Instrument Company Albany, OR, USA). After

removal from the seedling, the shoot was immediately sealed in a zip-lock plastic bag until

the measurement was taken, no longer than 15 minutes after removal (Turner 1988). The

seedling diurnal range of water potential (ΨD) was calculated as ΨD = ΨMD - ΨPD (Whitlow

20

et al. 1992). The regulation of apparent soil-to-leaf hydraulic conductance (KL) was

calculated as KL = -E / (ΨMD - ΨPD) (Aranda et al. 2005). The total area of fresh leaves

along with the total length and average diameter of fresh roots were analyzed on a back-lit

flatbed scanner with a resolution of 0.2 μm using a digital image analyzer (WinRHIZO

Pro®, V. 2007d, Regent Instruments Canada, Inc.). Maximum root depth within the pot was

recorded, and the downward root elongation rate was calculated according to the relative

growth rate equation from McGraw and Garbutt (1990). Specimens were then dried for one

week at 70 C, and the dry weights of leaves and roots recorded. The specific leaf area

(SLA) of seedlings was calculated as the ratio of leaf surface area to leaf dry weight

(Wilson et al. 1999).

Soil properties

On the days of a destructive harvest, soil moisture measurements were recorded in

each pot at depths of 0, 20, 40, 60, 80 and 100 cm, with another moisture reading taken at

the greatest depth of the root in the pot. To maintain soil stratigraphy, tubes were carefully

emptied onto a plastic sheet. An MPM-160-B Moisture Probe Meter® (ICT International

Pty Ltd) was fully inserted into the soil for each measurement. The moisture meter was

previously calibrated to obtain a quadratic polynomial equation (y = -2e-7x2 + 4e-4x; R2 =

0.98) to convert millivolts (x) to gravimetric moisture content (y) (MP406 Moisture Probe

Operation Manual). The gravimetric moisture content was converted to volumetric soil

water content (VSW) using the bulk density of the soil (Black 1965).

Compaction levels in the pots were tested using a soil penetrometer (Rimik CP40II

Cone Penetrometer®, RFM Australia Pty Ltd, QLD, Australia [Cone Diameter 12.83mm;

Area 130 sqmm]) at three times throughout the experiment to a depth of 60 cm (maximum

depth of penetration). The first measurement was taken prior to any water withholding,

while the remaining two measurement rounds were recorded after the first and second

drought phases. Particle density, total porosity and air-filled porosity for each treatment

were calculated for non-compact and compact soils (Table 2.1) (ASTM D854-92 1992).

Statistics

R statistical software (version 2.13.0) was used to perform the statistical tests in this

study. Binomial generalized linear models compared seedling survival between treatments

and species. General linear models compared the physiology and morphology of the

seedlings and the differences in soil moisture and physical properties between treatments,

21

while Tukey’s post hoc test performed pairwise comparisons between treatments when

significant effects were indicated. Data from each phase in the drought treatment (first

drought, recovery, and second drought) was statistically compared against data from the

control treatment measured on the identical dates. ANCOVAs compared differences in the

slopes of the regression lines. SigmaPlot 12 (Version 12.0.0.182, Systat Software, Inc.) was

used to fit regression lines. The homogeneity of the variances was tested by residual plots

and logarithmically or square-root transformed to achieve a normal distribution when

necessary, and all data is presented as untransformed means.

Results

Soil properties

The soil used in this experiment has a particle density of 2.5 g cm-3, and increasing

bulk density reduced the total porosity and air-filled porosity, but increased volumetric soil

water content (Table 2.1). Average volumetric soil water content during the experiment

throughout the entire 100 cm depth of the pot significantly differed between treatments, but

not by species (P < 0.001 treatment, P = 0.965 species). Compared to the control, total

volumetric soil water content significantly increased 16% in the compact treatment and

decreased 52% in the drought treatment (P < 0.001), but did not differ in the drought

compact treatment (P = 0.989) (Table 2.1).

Table 2.1 Characteristics of the soil averaged throughout the entire 100 cm PVC pot for each treatment (n =

106). Compact and drought compact treatments were subjected to a mechanical force to increase bulk

density. Drought and drought compact treatments were subjected to two intervals of water withholding.

Differences in water content are indicated as letters, P < 0.05

Bulk

Density Particle Density

Air-Filled Porosity

Total Porosity

Volumetric Water Content

(g cmˉ³) (g cmˉ³) (%) (%) (cm³ cmˉ³) Control 1.55 2.50 31 40 0.094 a Compacted 1.80 2.50 17 28 0.112 b Drought 1.55 2.50 34 40 0.062 c Drought Compact 1.80 2.50 19 28 0.093 a

Soil water content in the pots containing seedlings was greatest at the depth of 100

cm in every treatment due to the well-drained properties of the soil used in this study (P <

0.001) (Fig 2.4). The drought treatment contained significantly less water than the control

at each 20 cm interval tested throughout the pots (P < 0.001) (Fig 2.4). The drought

22

compact treatment contained less water than the control in the top 20 cm of the soil, but the

amount of soil water gradually increased at deeper intervals within the pots, containing

greater amounts of water than the control at depth of 80 and 100 cm (P < 0.001) (Fig 2.4).

The compact treatment held significantly more water than the control at most depth

intervals throughout the pot (Fig 2.4).

Soil water available to the roots in the drought and drought compact treatments

during the first drought phase was significantly lower than the control (P < 0.001) (Fig 2.5).

During the recovery phase, soil water content in the drought and drought compact

treatments was not significantly different from the control (P ≥ 0.063), but significantly

decreased again during the second drought phase (P < 0.001) (Fig 2.4). The rate and

amount of water lost from the soil in first drought phase was not significantly different to

the second drought phase within the drought treatment (P ≥ 0.209). A 34% decrease in soil

water was recorded 21 days after the beginning of the first drought phase, while a 41%

decrease was recorded 21 days after the beginning of the second drought phase (Fig 2.5). A

47% decrease in soil water content was recorded after 49 days for both the first and second

drought phases (Fig 2.5).

Mechanical impedance of the soil in the drought treatment was not significantly

different from the control at all depths measured (P ≥ 0.733) (Fig 2.6). Soils of the compact

treatment became significantly greater than the control at depths lower than 10 cm (P ≤

0.014), and soils of the drought compact treatment became significantly greater than the

control at depths of 8 cm (P ≤ 0.020) (Fig 2.6). Soils of the compact and drought compact

treatments were unable to record measurements deeper than 22 cm and 20 cm, respectively,

due to high impedance values. Soils in the drought compact treatment had significantly

higher impedance values than the compact treatment at depths of 18 and 20 cm (P ≤ 0.001)

(Fig 2.6).

23

Depth (cm)

0 20 40 60 80 100

Volu

met

ric S

oil W

ater

Con

tent

(cm

3 cm

-3)

0.00

0.05

0.10

0.15

0.20

0.25ControlCompactDroughtDrought Compact

a

b

c c

ab

ca

aab

a ab

cab a

b

c

b

a

b

c

b

Fig 2.4 The average volumetric water content at 20 cm depth intervals within pots. Error bars represent the

standard error of the mean (n = 106). Differences at depth between treatments are indicated as letters, P <

0.05. Soil bulk density in the control and drought treatment = 1.55 g cm-3, compact and drought compact

treatments = 1.80 g cm-3.

Days0 25 50 75 100 125 150 175 200 225

Volu

met

ric

Soil

Wat

er C

onte

nt(c

m3

cm-3

)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Control CompactDroughtDrought Compact

First Drought Recovery Second Drought

*

*

**

*

* **

*

*

*

*

Fig 2.5 The average volumetric soil water content measured to the average depth of roots over 228 days.

Error bars represent standard error of the mean (n = 15), differences from the control are indicated as ‘*’ P <

0.05. The first drought, recovery, and second drought phases are divided by dashed lines. Control and

compact treatments were not subjected to water withholding. Soil bulk density in the control and drought

treatment = 1.55 g cm-3, compact and drought compact treatments = 1.80 g cm-3.

24

MPa0 1 2 3 4 5

Dep

th(c

m)

-60

-50

-40

-30

-20

-10

0 Control Compact Drought Drought Compact

Fig 2.6 The soil penetration resistance within the pots, averaged from three separate intervals during the

experiment. Error bars represent standard error of the mean (n = 9). Soil bulk density in the control and

drought treatment = 1.55 g cm-3, compact and drought compact treatments = 1.80 g cm-3.

Seedling survival

Seedling survival in the drought treatment remained at 100% in all species

throughout the entire first drought phase (Fig 2.7). Seedling deaths in the drought treatment

were first observed during the recovery phase for all species (Fig 2.7). In the drought

treatment, 76% of B. attenuata, 84% B. menziesii and 71% E. todtiana remained alive at

the end of the recovery phase, and mortality was not significantly different from the control

treatment at this point in the experiment (P = 0.366 B. attenuata; 0.579 B. menziesii; 0.276

E. todtiana). Seedling deaths were recorded within 16 days after the commencement of the

second drought phase in all species (Fig 2.7). Within 48 days of the start of the second

drought phase, 100% of seedlings in the drought treatment died (Fig 2.7). No significant

differences in survival were recorded between species throughout the experiment (P ≥

0.860).

Mortality of ≥ 50% was recorded in all species after 49 days in the drought compact

treatment (Fig 2.7). On the final day of the first drought phase (60 days of withholding

water), a survival rate of 16% for B. attenuata, 24% B. menziesii and 20% E. todtiana was

significantly less than the control (P < 0.001) (Fig 2.7). Five days into the recovery phase

25

100% mortality occurred in B. attenuata and E. todtiana with only 7% B. menziesii

surviving, and the remaining B. menziesii died by day 16 of the recovery period (Fig 2.7).

No deaths occurred in the control, while 4% of B. menziesii and 2% of E. todtiana

died in the compact treatment over the course of the experiment, and did not result in any

significant differences (data not shown).

%

0

50

100

Days

0 25 50 75 100 125 150 175 200 225

a) Banksia attenuata

%

0

50

100

a) Banksia menziesii

Days

0 25 50 75 100 125 150 175 200 225

%

0

50

100

a) Eucalyptus todtiana

b) Banksia attenuata

b) Banksia menziesii

b) Eucalyptus todtiana

Soil WaterPhotosynthesisFv/FmSurvival

Fig 2.7 The photosynthetic rate, effective quantum yield of PSII (Fv/Fm), and volumetric soil water content

expressed as a percentage of the control (n = 5), and the survival percentage (n = 45) for B. attenuata, B.

menziesii, and E. todtiana in the a) drought treatment and b) drought compact treatments. The first drought,

recovery, and second drought phases are highlighted by the increases and decreases of the shaded area. Soil

bulk density in the drought treatment = 1.55 g cm-3, drought compact treatment = 1.80 g cm-3.

26

Control

Physiology

Comparative physiology can determine fitness between species (Ackerly 2000;

Austin et al. 2009), and unstressed seedlings of B. attenuata consistently functioned at

higher physiological rates than B. menziesii and E. todtiana, while B. menziesii functioned

at relatively higher rates than E. todtiana (Table 2.2). B. attenuata photosynthetic rate was

43% greater than B. menziesii (P < 0.001) even though the gs of the two Banksias were not

significantly different, possibly as a result of a 47% increase in ε and a 78% increase in

ETR in B. attenuata (P ≤ 0.002) (Table 2.2). The 358% reduction in average gs in E.

todtiana was most likely responsible for the lower rates of A and E compared to the two

Banksias (P < 0.001) (Table 2.2). Throughout the experiment, E. todtiana maintained an

average 53% and 74% greater NPQ than B. attenuata and B. menziesii, respectively (P <

0.001) (Table 2.2). Average Fv/Fm was 0.823 in B. attenuata, 0.809 in B. menziesii, and

0.735 in E. todtiana. The Fv/Fm of the Banksias were not significantly different from each

other (P = 0.974), but were both significantly greater than E. todtiana (P < 0.001).

Seedling ΨPD of unstressed B. menziesii was more negative than B. attenuata by

16% (P = 0.016) and by 25% than E. todtiana (P = 0.001) (Table 2.3). ΨMD in B. attenuata

and B. menziesii was significantly more negative than E. todtiana by an average of 18% (P

≤ 0.006) (Table 2.3). The ΨD of B. attenuata was significantly greater than B. menziesii by

31% and E. todtiana by 38% (P < 0.001) (Table 2.3). No significant differences were

observed in average KL between the two Banksia species (P = 0.351), and the KL of E.

todtiana decreased by 50% and 40% from B. attenuata and B. menziesii, respectively (P =

0.011 B. attenuata; P < 0.001 B. menziesii) (Table 2.3).

Morphology

Unstressed B. menziesii seedlings had 39% and 72% more leaf surface area than

unstressed B. attenuata and E. todtiana, respectively (P ≤ 0.003), while E. todtiana

exhibited a 38% greater SLA than B. attenuata (P = 0.028) (Fig 2.8). The root to shoot ratio

of E. todtiana was 84% and 71% higher than B. attenuata and B. menziesii, respectively (P

< 0.001 (Fig 2.8) and the total root length of both E. todtiana and B. menziesii was 33%

greater than B. attenuata (P ≤ 0.008) (Fig 2.8). The percentage of fine roots

27

Table 2.2 Seedling physiological values ± standard error of the mean throughout 228 days for the control (n = 60), compact (n = 60) and drought (n = 55) treatments.

Drought compact treatment values were averaged over 60 days during the first drought phase (n ≥ 25). Soil bulk density in the control and drought treatment = 1.55 g

cm-3, compact and drought compact treatments = 1.80 g cm-3. Differences from the control treatment are indicated as ‘*’ P < 0.05, ‘**’ P < 0.01, ‘***’ P < 0.001.

Differences between species within the control treatment are indicated by letter P < 0.05.

Photosynthesis (μmol CO2 m-2 s-1)

Stomatal Conductance (mol H2O m-2 s-1)

Transpiration (mmol H2O m-2 s-1)

Carboxylation Efficiency (mol mol-1)

ETR (μmol e- m-2 s-1)

NPQ

B. attenuata a a a a a a

Control 13.6 (±0.980) 0.352 (±0.086) 4.36 (±0.299) 0.047 (±0.004) 34.1 (±2.27) 1.58 (±0.076)

Compact 13.8 (±1.04) 0.349 (±0.070) 4.91 (±0.266) 0.049 (±0.004) 32.6 (±1.61) 1.46 (±0.087)

Drought 8.36 (±0.875)** 0.132 (±0.005)** 2.99 (±0.361)** 0.031 (±0.003)* 32.3 (±2.57) 1.48 (±0.111)

Drought Compact 3.09 (±0.871)*** 0.024 (±0.004)*** 0.472 (±0.071)*** 0.011 (±0.003)*** 5.19 (±2.77)*** 0.246 (±0.093)***

B. menziesii b a b b b a

Control 9.53 (±0.316) 0.390 (±0.025) 3.54 (±0.132) 0.032 (±0.002) 19.2 (±1.59) 1.39 (±0.063)

Compact 7.97 (±0.388) 0.288 (±0.031) 2.96 (±0.095) 0.025 (±0.001) 24.8 (±1.66) 1.62 (±0.091)

Drought 5.97 (±0.491)*** 0.131 (±0.014)*** 1.90 (±0.221)*** 0.023 (±0.002)*** 27.7 (±2.44) 1.35 (±0.113)

Drought Compact 0.773 (±0.180)*** 0.015 (±0.004)*** 0.364 (±0.091)*** 0.002 (±0.001)*** 2.89 (±1.11)*** 0.288 (±0.084)***

E. todtiana c b c c b b

Control 4.41 (±0.226) 0.081 (±0.005) 1.50 (±0.099) 0.016 (±0.001) 23.7 (±1.40) 2.42 (±0.070)

Compact 4.79 (±0.355) 0.067 (±0.005) 1.23 (±0.082) 0.022 (±0.004) 25.7 (±1.15) 2.35 (±0.056)

Drought 3.30 (±0.349)*** 0.059 (±0.018)*** 1.39 (±0.148) 0.013 (±0.001)* 26.4 (±2.18) 1.9 (±0.149)***

Drought Compact 1.39 (±0.366)*** 0.017 (±0.003)*** 0.425 (±0.081)*** 0.006 (±0.002)*** 12.5 (±3.50)*** 1.01 (±0.259)***

27

28

(roots less than 0.50 mm in diameter) was greater in E. todtiana than both the Banksias (P

< 0.001) (Fig 2.8). The average root diameter was 0.75 mm in B. attenuata, 0.75 mm in B.

menziesii, and 0.70 mm in E. todtiana, and not significantly different between the three

species (P ≥ 0.846) (Fig 2.8). There was no significant difference in the maximum root

depth between the three species 104 days after transplanting into the 1.0 m long pots,

before the roots encountered the bottom of the pot (P ≥ 0.336) (Fig 2.8). The roots of all

species reached the bottom of the pot (1.0 m) within 132 days after transplanting.

Fine

Roo

ts(%

)

0

20

40

60

80

ControlCompactDroughtDrought Compact

Leaf

Sur

face

Are

a(m

2 )

0.000

0.002

0.004

0.006

0.008

Spe

cific

Lea

f Are

a(m

2 kg-1

)

0.000

0.001

0.002

0.003

B. attenuata B. menziesii E. todtiana

Roo

t Sho

ot R

atio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

*

* *

*

* *

*

* *

a

b

c

a

bc

a a

b

*

Tota

l Roo

t Len

gth

(cm

)

0

1000

2000

3000

4000


Max

imum

Roo

t Dep

th a

fter 1

04 d

ays

(cm

)

0

20

40

60

80

100

* * * ** *

**

**

*

*

*a b

c

a a b

a a a

**

Fig 2.8 Seedling morphology in response to drought and/or soil compaction. Soil bulk density in the control

and drought treatment = 1.55 g cm-3, compact and drought compact treatments = 1.80 g cm-3. Error bars

represent standard error of the mean (n ≥ 10), differences from the control within individual species are

indicated as ‘*’ P < 0.05. Differences between species within the control are indicated by letter, P < 0.05.

29

Drought treatment

Physiology

The seedlings in the drought treatment were subjected to two rounds of drought,

with an extended re-watering period occurring between these droughts, simulating the wet

winter and dry summer of a mediterranean-type climate. Although it is referred to as the

drought treatment, physiological and morphological values were averaged over the two

droughts phases as well as the recovery phase to determine how the seedlings respond to

these simulated climatic conditions compared to the unstressed control (Table 2.2).

One of the most sensitive physiological traits in response to drought are leaf

stomata, which regulate the balance of carbon intake (photosynthesis) and water loss

(transpiration) (Crocker et al. 1998). Throughout the entire experiment, significant

decreases in gs were observed in all species (73% in B. attenuata, 66% in B. menziesii, and

27% in E. todtiana (P ≤ 0.001) (Table 2.2). Given the significant decreases in gs,

corresponding drops in averaged A were observed for all species (39% in B. attenuata, 37%

in B. menziesii, and 25% in E. todtiana; P ≤ 0.008), but the averaged rate of E decreased

only in B. attenuata and B. menziesii (31% and 46%, respectively, P ≤ 0.005) (Table 2.2).

Stomatal closure caused by drought can also result in the eventual decrease in internal CO2

concentration and limit carboxylation efficiency (ε) (Sanchez-Rodriguez and Martinez-

Carrasco 1999), and the average ε decreased 34% in B. attenuata (P = 0.039), 28% in B.

menziesii (P < 0.001), and 19% in E. todtiana (P = 0.030) (Table 2.2).

Chlorophyll fluorescence measurements such as ETR and NPQ can provide insight

into how each species copes with drought stress and the extent of damage caused to the

photosystem (Maxwell and Johnson 2000). No significant reductions in the average ETR

were observed throughout the experiment in any of the species (P ≥ 0.075), while a

decrease of 22% in NPQ was recorded only in E. todtiana (P < 0.001) (Table 2.2).

Seedling hydraulic failure can lead to excessive xylem cavitation and mortality, and

the hydraulic conductive properties of all seedlings were shown to be affected by the

drought treatment (Tyree and Sperry 1989; Williams et al. 1997) (Table 2.3). The average

ΨPD decreased 53% in B. attenuata (P = 0.044), 28% in B. menziesii (P = 0.046), and 187%

in E. todtiana (P < 0.001) (Table 2.3). Seedling ΨMD decreased 79% in E. todtiana in the

drought treatment (P < 0.001), but no significant differences in ΨMD were observed in

either B. attenuata or B. menziesii when compared to the control (P ≥ 0.228) (Table 2.3).

30

No significant drops in either averaged ΨD or KL were observed in any of the species in the

drought treatment throughout the experiment (Table 2.3).

Table 2.3 Xylem water potential values ± standard error of the mean for B. attenuata, B. menziesii, and E.

todtiana throughout 228 days for the control (n = 25), compact (n = 25) and drought (n = 20) treatments.

Drought compact treatment values were averaged over 60 days during the first drought phase (n ≥ 5). Soil

bulk density in the control and drought treatment = 1.55 g cm-3, compact and drought compact treatments =

1.80 g cm-3. Differences from the control are indicated as ‘*’ P < 0.05, ‘**’ P < 0.01, ‘***’ P < 0.001,

differences between species in the control are indicated as letters, P < 0.05

ΨPD (MPa)

ΨMD (MPa)

ΨD (MPa)

KL (mmol MPa-1 m-2s-1)

B. attenuata a a a a

Control -0.371 (±0.023) -0.983 (±0.032) 0.612 (±0.39) 8.69 (±1.00)

Compact -0.419 (±0.023) -0.994 (±0.038) 0.575 (±0.042) 7.88 (±0.871)

Drought -0.570 (±0.049)* -1.07 (±0.060) 0.495 (±0.045) 4.81 (±1.17)

Drought Compact -1.93 (±0.222)*** -3.97 (±0.221)*** 2.04 (±0.397)*** 0.292 (±0.110)*

B. menziesii b a b a

Control -0.443 (±0.019) -0.910 (±0.030) 0.467 (±0.029) 7.24 (±0.635)

Compact -0.497 (±0.021) -0.901 ±0.027) 0.404 (±0.032) 9.26 (±0.992)

Drought -0.573 (±0.043)* -0.984 (±0.060) 0.411 (±0.031) 6.59 (±1.60)

Drought Compact -1.05 (±0.097)*** -2.74 (±0.642)*** 1.70 (±0.548)*** 0.452 (±0.075)*

E. todtiana a b b b

Control -0.332 (±0.021) -0.776 (±0.023) 0.444 (±0.026) 4.37 (±0.486)

Compact -0.322 (±0.028) -0.756 (±0.027) 0.427 (±0.030) 3.56 (±0.401)

Drought -0.952 (±0.136)*** -1.39 (±0.155)*** 0.442 (±0.051) 4.11 (±0.519)

Drought Compact -2.80 (±0.611)*** -3.95 (±0.687)*** 1.15 (±0.317)*** 0.610 (±0.213)*

The relationship between seedling physiology and VSW was established through

measurements taken during destructive harvests, and these relationships were compared

between species. The relationship between A and VSW was not significant or normally

31

distributed in any of the three species (data not shown). The rate of gs showed a decreasing

curvilinear relationship with VSW in all three species, and stomatal decrease in response to

drying soil was greater in the two Banksias than in E. todtiana (P ≤ 0.042) (Fig 2.9).

Decreasing VSW was correlated with a decrease in leaf transpiration, with all three species

exhibiting a significant curvilinear relationship between E and VSW. The relationships of

transpiration versus VSW in both B. attenuata and B. menziesii were significantly stronger

than E. todtiana (P ≤ 0.033) (Fig 2.9).

The relationship of ε (y) versus VSW (x) showed a significant decreasing

curvilinear relationship for all three species (B. attenuata y = 1.27x + -0.203, R2 = 0.518, P

< 0.001; B. menziesii y = 2.33x + 1.13, R2 = 0.466, P<0.001; E. todtiana y = 1.00x + -

0.773, R2 = 0.285, P = 0.001) with a significantly stronger response of ε to VSW in the two

Banksias than E. todtiana (P < 0.001). A significant decreasing linear relationship was

observed between ETR (y) and VSW (x) in all species (B. attenuata y = 9.63x+408, R2 =

0.294, P = 0.002; B. menziesii y = 14.5x + 270, R2 = 0.216, P = 0.011; E. todtiana y =

16.1x + 216, R2 = 0.318, P < 0.001), and no differences in the slope existed between

species (P ≥ 0.143) (Fig 2.9). The relationship of both NPQ and Fv/Fm versus VSW was not

normally distributed in all three species (data not shown).

Seedling ΨPD (y) was linearly distributed against VSW (x) in all three species (B.

attenuata y = 0.691 + -3.43x, R2 = 0.401, P < 0.001; B. menziesii y = 0.824 + -4.70x, R2 =

0.275, P = 0.001; E. todtiana y = 1.66 + -13.7x, R2 = 0.718, P < 0.001), with E. todtiana

exhibiting a more negative slope and greater response than both B. attenuata and B.

menziesii (P < 0.001). ΨMD was significantly linearly distributed in relation to VSW in E.

todtiana, while the ΨMD in the two Banksia species did not respond significantly to

decreasing soil water content (Fig 2.9). KL exhibited a significant linear relationship with

drying soil in all species, and this relationship was significantly weaker in E. todtiana than

both Banksia species (P ≤ 0.015) (Fig 2.9).

KL

(mm

ol M

Pa-1

m-2

s-1

)

0

5

10

15

Volumetric Soil Water Content(cm3 cm-3)

0.00 0.02 0.04 0.06 0.08 0.10 0.12

E(m

mol

H2O

m-2

s-1

)

0

2

4

6

8

Volumetric Soil Water Content(cm3 cm-3)

0.00 0.02 0.04 0.06 0.08 0.10 0.12

g s(m

ol H

2O m

-2 s

-1)

0.0

0.1

0.2

0.3

0.4

M

D

(-MPa

)

0

1

2

3

B. attenuata 4.37 0.036 0.665 <0.001B. menziesii 3.97 0.075 0.616 <0.001E. todtiana 2.33 0.075 0.447 <0.001

B. attenuata -3.41 1.21 0.099 0.144B. menziesii -2.94 1.04 0.146 0.072E. todtiana -16.6 2.36 0.772 <0.001

slope y-intercept R2 p valueB. attenuata 16.0 0.332 0.643 <0.001B. menziesii 16.1 0.353 0.588 <0.001E. todtiana 8.76 0.574 0.471 <0.001

B. attenuata 86.1 -1.08 0.629 <0.001B. menziesii 88.4 -0.582 0.420 0.001E. todtiana 40.4 0.923 0.255 0.014

slope y-intercept R2 p value


slope y-intercept R2 p value B. attenuataB. menziesiiE. todtiana

Fig 2.9 The relationship between volumetric soil water content and ΨMD, gs, E, and KL for B. attenuata, B. menziesii, and E. todtiana in the drought

treatment only. The slope, y-intercept, R2 and P values were derived from regression lines transformed to achieve normality when appropriate. Data is

presented as untransformed.

32

33

Correlations between seedling physiologies are able to determine trends and

compare species-specific stomatal or non-stomatal limitations incurred during drought

(Medrano et al. 2002). The common curvilinear relationship between A and gs in droughted

plants was observed in all species (Farquhar and Sharkey 1982; Wong et al. 1985), with a

steeper slope occurring in B. attenuata than B. menziesii and E. todtiana (P ≤ 0.002) (Fig

2.10). A curvilinear relationship also exists between the rates of gs and ε in all species

during drought, and the slope of the curve of B. menziesii was significantly less than E.

todtiana (P = 0.002) and B. attenuata (P = 0.049) (Fig 2.10). The slopes of the curves

between A versus gs and ε versus gs were compared between species to determine the extent

of stomatal and non-stomatal limitations to photosynthesis and how these processes are

affected as drought progresses and gs decreases further (Medrano et al. 2002). In B.

attenuata, the slope of the curve between A versus gs was significantly greater than that of ε

versus gs, suggesting that stomatal limitations to photosynthesis are dominant during

drought before non-stomatal limitations occur (P < 0.001) (Fig 2.10). The slopes of the

curves between A versus gs were not significantly different to ε versus gs in both B.

menziesii and E. todtiana, signifying that stomatal and non-stomatal limitations were taking

place simultaneously throughout drought (P ≥ 0.126) (Fig 2.10).

The rate of A versus E determined the amount of carbon gained per mmol of water

lost through the leaves, depicting seedling water-use-efficiency (WUE) during drought (Fig

2.10). The relationship was significantly curvilinear in all species, and this response was

significantly stronger in B. attenuata than both B. menziesii (P = 0.041) and E. todtiana (P

= 0.016), while B. menziesii had a stronger response than E. todtiana (P = 0.022) (Fig

2.10).

First drought phase

To understand when specific physiological traits were most stressed during times of

drought, the seedling responses to drought have been further explored by specific phase

(first drought, recovery, second drought [Fig 2.5]). During the first drought phase, results

have been analyzed by severity of drought, as determined from average gs according to the

review of the effects of drought on the photosynthesis of C3 plants (Flexas and Medrano

2002; Medrano et al. 2002a). A gs below 0.05 mol H2O m-2 s-1 indicates very severe

drought, and B. attenuata, B. menziesii, and E. todtiana all maintained an average rate of

34

gs

(mol H2O m-2 s-1)

0.0 0.1 0.2 0.3 0.4

A(

mol

CO

2 m-2 s

-1)

0

5

10

15

E(mmol H2O m-2 s-1)

0 2 4 6 8

B. attenuataB. menziesiiE. todtiana

B. attenuata 0.679 0.646 0.753 <0.001B. menziesii 0.504 0.698 0.745 <0.001E. todtiana 0.408 0.618 0.561 <0.001

slope y-intercept R2 p valueB. attenuata 0.676 1.51 0.927 <0.001B. menziesii 0.364 1.18 0.667 <0.001E. todtiana 0.433 1.19 0.523 <0.001


(m

mol

mol

-1)

0.00

0.02

0.04

0.06

0.08

0.10B. attenuata 0.409 -1.18 0.668 <0.001B. menziesii 0.265 -1.33 0.410 <0.001E. todtiana 0.575 -1.04 0.555 <0.001


Fig 2.10 The relationship between A vs E, A vs gs, and gs vs ε in B. attenuata, B. menziesii, and E. todtiana in the drought treatment. The slope, y-intercept,

R2 and P values were derived from regression lines transformed to achieve normality. Data is presented as untransformed

34

35

gs above 0.05 mol H2O m-2 s-1 for the initial 45 days of the first drought phase (Fig

2.11a).The average rate of gs dropped below 0.05 mol H2O m-2 s-1 in all three species when

water was withheld past 45 days in the first drought phase (Fig 2.11b).

Stomatal conductance rate was reduced significantly during the initial 45 days of the

first drought in the two Banksias (P ≥ 0.001) (Fig 2.11a). The decreases in gs in these two

Banksias species corresponded to decreases in E (P < 0.001), and only the rate of A in B.

menziesii was reduced significantly during this time (-31%, P = 0.002) (Fig 2.11a). The gs

of E. todtiana remained constant during the initial 45 days of the first drought phase and

maintained rates of E and A (Fig 2.11a). A 40% increase in ε was observed in E. todtiana

(P = 0.012) and the ETR of all three species increased during the first 45 days of drought

(+29% in B. attenuata P = 0.029; +38% in B. menziesii P = 0.038; +24% in E. todtiana P =

0.003), while no changes in NPQ were recorded (P ≥ 0.421) (Fig 2.11a). After 45 days of

withholding water during the first drought phase the gs of all three species dropped below

the severe drought threshold of 0.05 mol H2O m-2 s-1 (Banksias P < 0.001; E. todtiana P =

0.045) and resulted in the decreases of A (P ≤ 0.033) and E (P ≤ 0.011) (Fig 2.11b).

Significant reductions in ETR, NPQ and ε were recorded after 45 days of the first drought

(Fig 2.11b). Only the ε of B. menziesii was not significantly reduced during this time (P =

0.452) (Fig 2.11b).

Significant changes in hydraulic conductance were observed regardless of drought

severity (gs > or < 0.05 mol H2O m-2 s-1) during the first drought phase in all species (Fig

2.12). The ΨPD decreased in all species (P ≤ 0.001), and the ΨMD of E. todtiana decreased

120% (P < 0.001) while the ΨMD of B. attenuata and B. menziesii was not significantly

affected during the first drought phase (P ≥ 0.058) (Fig 2.12). Significant reductions in KL

were observed in both B. attenuata and B. menziesii (P ≤ 0.023) with no decreases in E.

todtiana recorded (Fig 2.12). The ΨD was unaffected during the first drought phase in all

three species (P ≥ 0.106) (Fig 2.12).

Recovery phase

The physiology of seedlings from the drought treatment were defined to have

recovered when values recorded during the recovery phase were not significantly less than

those from the control treatment (P ≥ 0.05). Recovery was observed in every physiological

trait for B. attenuata, B. menziesii, and E. todtiana, and recovery was found to occur when

values from seedlings in the drought treatment attained values ≥ 70% compared to the

36

control treatment (Table 2.4). Physiological traits from seedlings in the drought treatment

were shown to surpass those of control values during the recovery phase (Table 2.4). The

exact number of days for recovery was dependent on species and specific physiological

trait, and the A, gs, E, ε, ETR, and NPQ of the seedlings in the drought treatment of all three

species had recovered within 17 days after re-watering (Table 2.4). The average KL, ΨPD,

ΨMD, and ΨD of each species attained values significantly similar to the control values

within 17 days after the recovery phase commenced (P ≥ 0.05, data not shown).

Table 2.4 The highest percentage of recovery from seedlings in the drought treatment as compared to the

control treatment, measured during the recovery period. Numbers in parentheses are the maximum days after

re-watering for seedlings in the drought treatment to statistically recovery to control treatment values (n = 5).

Photosynthesis (μmol CO2 m-2 s-1)

Stomatal Conductance (mol H2O m-2 s-1)

Transpiration

(mmol H2O m-2 s-1)

B. attenuata 118% (17) 121% (17) 158% (5) B. menziesii 87% (5) 84% (5) 111% (5) E. todtiana 76% (5) 108% (17) 122% (17)

Carboxylation

Efficiency (mol mol-1)

ETR

(μmol e- m-2 s-1)

NPQ

B. attenuata 134% (17) 126% (5) 164% (5) B. menziesii 83% (5) 357% (17) 177% (5) E. todtiana 78% (5) 175% (5) 74% (5)

37

ControlDrought

A(

mol

CO

2 m

-2 s

-1)

0

2

4

6

8

10

12

14

g s

(mol

H2O

m-2

s-1

)

0.0

0.1

0.2

0.3

0.4

ETR

(µm

ol e

- m-2

s-1

)

0

10

20

30

40

50

B. attenuata

B. menziesii

E. todtiana

NPQ

0

1

2

3

(m

ol m

ol-1

)

0.00

0.01

0.02

0.03

0.04

0.05

B. attenuata

B. menziesii

E. todtiana

*

*

*

*

*

*

*

*

*

* **

***

**

*

* *

*

B. attenuata

B. menziesii

E. todtiana

*

**

**

*

*

**

*

*

*

E(m

mol

H2O

m-2

s-1

)

0

1

2

3

4

5

* **

*

**

*

a)

a)

a)

a)

a)

a)

b)

b)

b)

b)

b)

b)

c)

c)

c)

c)

c)

c)

Fig 2.11 Seedling physiology in the control and drought treatment during a) the initial 45 days of the first

drought phase when droughted seedlings experienced moderate to severe drought stress (gs > 0.05 mol H2O

m-2 s-1) (n = 15), b) the last 15 days of the first drought phase when droughted seedlings experienced very

severe drought stress (gs < 0.05 mol H2O m-2 s-1) (n = 5), and c) the initial 45 days of the second drought

phase when droughted seedlings experienced very severe drought stress (gs < 0.05 mol H2O m-2 s-1) (n = 15).

Error bars represent standard error of the mean, differences from the control are indicated as ‘*’ P < 0.05.

38

Second drought phase

During the second drought phase severe drought occurred much quicker, the

average rate of gs dropped below 0.05 mol H2O m-2 s-1 in all three species within 15 days of

withholding water. None of the three species were recorded with gs values greater than 0.05

mol H2O m-2 s-1 during the second drought phase (Fig 2.11c). Physiological traits that were

unaffected during the initial 45 days of the first drought phase were significantly reduced

during the same duration in the second drought phase (Fig 2.11c). Significant decreases in

both gas-exchange and chlorophyll fluorescence was observed in the two Banksia species

(P ≤ 0.018), and only ETR was unaffected during the second drought phase (Fig 2.11c).

The ETR, NPQ and ε of E. todtiana decreased significantly (P ≤ 0.025) while gs, A, E were

unaffected (Fig 2.11c).

The ΨPD and ΨMD of all species decreased significantly during the second drought

phase, (P ≤ 0.010), and ΨD was unaffected in all three species during this time (P ≥ 0.474)

(Fig 2.12). The KL of both Banksias was reduced significantly during the second drought

phase (P ≤ 0.029), but the KL was not affected significantly in E. todtiana (Fig 2.12).


D

(MP

a)

0.0

0.2

0.4

0.6

0.8


M

D(-

MP

a)

0.0

0.5

1.0

1.5

2.0

2.5

*

**

*

P

D(-

MP

a)

0.0

0.5

1.0

1.5

2.0

2.5Control 1st Drought Recovery 2nd Drought

* * **

*

*

KL

(mm

ol H

2O M

Pa-1

m-2

s-1

)

0

2

4

6

8

10

12

14

** *

*

Fig 2.12 ΨPD, ΨMD, ΨD, and KL of B. attenuata, B. menziesii, and E. todtiana seedlings in the control and

drought treatment during the first drought, recovery, and second drought phases. Error bars represent standard

error of the mean (n ≥ 5), differences from the control are indicated as ‘*’ P < 0.05.

39

Morphology

The drought treatment did not cause significant reductions in either leaf surface area

or SLA in any of the three species (P ≥ 0.125) (Fig 2.8). An increase in root to shoot ratio

was observed in B. attenuata (40%) and B. menziesii (36%) (P < 0.001), with no change in

E. todtiana (P = 0.999) (Fig 2.8). Total root length increased 33% in B. attenuata (P =

0.001), with no significant differences in B. menziesii or E. todtiana (P ≥ 0.371). An

increase of 12% was observed in the fine root percentage of both in B. attenuata and B.

menziesii (P ≤ 0.049), and no difference was observed in E. todtiana in the drought

treatment (P = 0.234) (Fig 2.8). Average root diameter was reduced 18% to 0.61 mm in B.

attenuata and 20% to 0.60 mm in B. menziesii (P < 0.001) while the root diameter of E.

todtiana in the drought treatment remained unchanged from the control at an average 0.60

mm (P = 0.084). The roots of all species reached the bottom of the pot (1.0 m) within 132

days after transplanting, and root depth after 104 days was not significantly different from

the control.

Compact treatment

Physiology

Compact soil had no effect on the physiological responses of B. attenuata, B.

menziesii, or E. todtiana, and the average physiological values over the course of the

experiment were not significantly different from the control values (Table 2.2). Seedling

hydraulic conductance (ΨPD, ΨMD, ΨD, and KL) was unaffected in the compact treatment

(Table 2.3).

Morphology

The compact treatment did not significantly affect the aboveground morphology of

any of the three species, with no differences in either leaf surface area or SLA observed (P

≥ 0.540) (Fig 2.8). The root to shoot ratio of seedlings in compact treatment decreased

36%, 33%, and 32% in B. attenuata, B. menziesii, and E. todtiana, respectively (P ≤ 0.003)

(Fig 2.8). The total root length was reduced 29% and 44% in B. attenuata and B. menziesii,

respectively (P ≤ 0.002), but was unaffected in E. todtiana (P = 0.635) (Fig 2.8). Fine root

percentage decreased in B. menziesii by 17% (P < 0.001) and was not significantly different

in B. attenuata or E. todtiana (P ≥ 0.335) (Fig 2.8). The average diameter of roots in the

compact treatment increased 19% to 0.88 mm in B. attenuata, and increased 26% to 0.96

40

mm in B. menziesii (P < 0.001), but the average root diameter of 0.76 mm in E. todtiana

was not significantly different from the control (P = 0.315). Maximum root depth was

reduced in the compact treatment by an average of 71% between all species before the roots

of the control could encounter the bottom of the pot 104 days after transplantation (P <

0.001) (Fig 2.8). After 304 days of growth in the 1.0 m pots, maximum root depth was 34

cm in B. attenuata, 35 cm in B. menziesii, and 42 cm in E. todtiana, with significantly

greater depth recorded in E. todtiana over both Banksias (P ≤ 0.011).

The root elongation rate in all three species decreased over the course of the

experiment in the compact treatment (P < 0.001) (Fig 2.14). From 104 days after

transplantation to 314 days after transplantation, the root elongation rate slowed 50% in B.

attenuata, 44% in B. menziesii, and 52% E. todtiana (Fig 2.13). On day 104, E. todtiana

exhibited a faster downward elongation rate than B. attenuata and B. menziesii (P ≤ 0.005),

and there was no difference between B. attenuata and B. menziesii (P = 0.937) (Fig 2.13).

On day 314, there was no difference in the downward root elongation rate between the

three species in the compact treatment (P ≥ 0.640) (Fig 2.13).


Dow

nwar

d R

oot E

long

atio

n R

ate

(cm

m-1

day

-1)

0.000

0.002

0.004

0.006

0.008

0.010

0.012

After 104 days growthAfter 314 days growth

a a

b

c cc

Fig 2.13 Root elongation rates of B. attenuata, B. menziesii, and E. todtiana seedlings in the compact

treatment (soil bulk density = 1.80 g cm-3) after 104 days and 314 days after planting in 1.0 meter long pots.

Error bars represent standard error of the mean (n = 10), differences between species and treatment are

indicated as letters, P < 0.05.

41

Drought compact treatment

Physiology

All physiological traits during the first drought phase was significantly lower than

the control in all species (P < 0.001) (Table 2.2). Hydraulic conductance (ΨPD, ΨMD, ΨD,

and KL) of all species was significantly less in seedlings subjected to the drought compact

treatment (P < 0.001) (Table 2.3).

Morphology

The leaf surface area of B. attenuata and B. menziesii decreased 39% and 47%,

respectively (P ≤ 0.007), and did not significantly change in E. todtiana (P = 0.925) (Fig

2.8). The SLA of B. menziesii decreased 28% (P < 0.001), and did not significantly differ in

B. attenuata or E. todtiana (P ≥ 0.427) (Fig 2.8). Root to shoot ratio decreased 22% in B.

attenuata (P = 0.024), 29% in B. menziesii (P = 0.007), and 31% in E. todtiana (P = 0.017)

(Fig 2.8). The percentage of fine roots and the average root diameter did not differ from the

control treatment in any of the species (P ≥ 0.390) (Fig 2.8). Total root length decreased

30% in B. attenuata, and 53% in both B. menziesii and E. todtiana (P ≤ 0.001) (Fig 2.8).

Maximum root depth of seedlings in the drought compact treatment was restricted by an

average of 72% between all species before the roots of the control could encounter the

bottom of the pot 104 days after transplantation (P < 0.001) (Fig 2.8).

Discussion

Seedling survival in response to postmine conditions

The species in this study utilized different water-relation strategies to cope with

drought; both B. attenuata and B. menziesii seedlings are “drought-avoider water-savers”

with isohydric behavior, while E. todtiana seedlings are “drought-tolerant” (anisohydric)

(Levitt 1980; Tardieu and Simonneau 1998). There have been conflicting reports as to

whether isohydry or anisohydry is the more favorable strategy to survive drought, and there

is no justification for the environmental or evolutionary importance of possessing one

strategy over the other (Beis and Patakas 2010; Franks et al. 2007; McDowell et al. 2008).

This is supported by the similarities in survival between species and drought-coping

strategies in the drought or drought compact treatments. Reconstructed postmine soils do

not distinguish between isohydric and anisohydric species, and the heavily compact soil

disrupts natural physiological behavior by restricting maximum depth, placing the seedlings

42

at a greater susceptibility to drought stress when grown in compact soil (Kozlowski 1999;

Unger and Kaspar 1994; Zisa et al. 1980). Therefore these different drought-coping

mechanisms did not convey a survival advantage and allow one species to outperform the

others under postmine conditions.

Seedling response to drought

Evidence for species-specific drought-coping strategies

In this study, small decreases in soil moisture resulted in large decreases in both the

stomatal conductance and transpiration rate of leaves in the two Banksias. Immediate

stomatal closure allowed the seedlings to maintain a stable xylem water potential, and has

been described as the first line of defense in response to drought (Sperry and Pockman

1993; Tyree and Sperry 1989; Yordanov et al. 2000). The Banksias therefore exhibit

isohydric behavior to drought, preventing cavitation-induced seedling mortality (Tardieu

and Simonneau 1998). However this behavior can be detrimental to carbon acquisition over

prolonged droughts, given that stomatal conductance is highly correlated with drying soil

(Aranda et al. 2005; Jarvis and Davies 1998; Medrano et al. 2002b). Drought-induced

stomatal closure places the two Banksias at risk of carbon starvation because CO2

absorption and fixation is reduced throughout the entire drought period (Jarvis and Davies

1998; Medrano et al. 2002b). E. todtiana exhibited a different approach to drought by

employing an anisohydric behavior: operating at lower water potentials while maintaining

stomatal conductance rates at or near control levels for a longer period of time during

drought (Tardieu and Simonneau 1998). While anisohydry allows E. todtiana to withstand

lower water potentials, species are known to manage their xylem water potentials near the

point of complete hydraulic failure (Sperry et al. 2002). This places E. todtiana at a greater

risk of hydraulic failure during prolonged drought, but allows the seedlings to maintain

relatively greater carbon gain over an extended amount of time during drought (McDowell

et al. 2008).

Ecophysiological adaptations to drought

The observed relationship between photosynthesis and transpiration provides

evidence that the three species exhibit a range of WUE values, with B. attenuata achieving

the greatest WUE of the three species during water deficit. Higher WUE in dry

environments may be an adaptive trait for seedling survival, but the ecological advantages

43

could be compromised if these seedlings are grown in environments with seasonal

precipitation, where the competition for water is strong during the dry season (Cohen 1970;

DeLucia and Heckathorn 1989). Sands and Nambiar (1984) found that the productivity of

Pinus radiata seedlings transplanted in deep sands was greatly reduced in the first year of

planting because their shallow roots were unable to out-compete weeds for water. After a

second summer, these seedlings had developed a 2 m taproot that was able to access deeper

soil moisture (Sands and Nambiar 1984). Therefore a high WUE is only beneficial if a

conserved source of soil water is available for absorption later in the season (Cohen 1970),

i.e. groundwater access by a deep taproot. Thus there is a need for Banksia and Eucalyptus

seedlings to develop a deep taproot to extract water at depth, otherwise they would be

forced to compete for water in the same root zone as species that do not conserve water use,

placing them at a disadvantage when dry summer occurs.

While photosynthesis was regulated by stomata during drought, non-stomatal

limitations to photosynthesis were also observed in all species. In B. menziesii and E.

todtiana, proportional decreases in both photosynthesis and carboxylation efficiency were

observed as stomata gradually closed during drought, indicating that stomatal and non-

stomatal limitations are of similar importance to photosynthesis when the seedlings are

under moderate drought stress (Medrano et al. 2002b). Stomatal limitations were more

dominant in moderately drought-stressed B. attenuata seedlings, as smaller decreases in the

stomatal conductance of B. attenuata resulted in greater decreases in photosynthesis. Non-

stomatal limitations do not appear to be a dominant factor for B. attenuata until stomatal

conductance drops below 0.05 mol H2O m-2 s-1. Decreases in non-stomatal limitations to

photosynthesis may be a sign of increased water stress, and may indicate that the entire

photosynthetic processes of B. menziesii and E. todtiana are more readily damaged by

drought than that of B. attenuata, where there is integrated down-regulation of

photosynthesis (Medrano et al. 2002b).

Stomatal control over water loss could have allowed seedlings to maintain leaf area

throughout drought (Metcalfe et al. 1990; Rambal 1993). Changes in belowground biomass

in the Banksias, such as a higher root to shoot ratio, a greater percentage of fine roots, and a

higher total root length are advantageous in maintaining water status during droughts

(Ewers et al. 2000; Hacke et al. 2000; Hund et al. 2009). The root plasticity of the two

Banksias species increases the likelihood of survival during drought by maintaining a stable

water status (Tschaplinski et al. 1994). Root structure was maintained in E. todtiana

44

throughout the drought phases, but the morphology of this species appeared to be more

naturally suited to drought conditions, with relatively low leaf area, and a high root to shoot

ratio, total root length, and percentage of fine roots. All three species have relatively low

SLA that was not affected by drought stress, but differs between species, with B. attenuata

possessing the lowest SLA and E. todtiana the highest (Garnier et al. 2001; Li et al. 2005;

Poorter and Jong 1999). A low specific leaf area suggests a higher degree of sclreophylly

that may be beneficial for seedlings under water deficits by making leaves less prone to

wilt, allowing continuous water flow and photosynthetic rates (Hoffmann et al. 2005;

Marron et al. 2003; Turner 1994).

Both the Banksias and E. todtiana maintained a stable ΨD throughout the first and

second drought phases. This strategy has been classified as “isohydrodynamic”, and was

first described in E. gomphocephala, an anisohydric tree species that also inhabits the

geographic range of the three study species (Franks et al. 2007). To continually extract

water, seedlings must endure daily pressure variation in xylem conductance: from highly

stressed (more negative) during the day to relatively relaxed at night (Halvorson and Patten

1974). The isohydrodymanic behavior of both Banksia species and E. todtiana provides

overnight recovery of the seedling hydraulic conductance when evaporative demand is

lower (Gebrekirstos et al. 2006). When the seedlings cannot re-saturate overnight, ΨPD

values would approach ΨMD values, leading to xylem cavitation and a loss of water

absorption (Franks et al. 2007; Gebrekirstos et al. 2006). A larger and more stable ΨD

throughout dry periods could suggest greater daily recovery from drought stress and a

higher tolerance to long-term drought (Halvorson and Patten 1974; Sucoff 1971).

Seedling response during multiple droughts and re-watering

The physiological recovery during the prolonged re-watering period did not

guarantee seedling survival over multiple droughts. Despite the similarities in the rate of

water depletion between the first and second drought phases, B. attenuata, B. menziesii and

E. todtiana seedlings were more susceptible to a second round of drought. The regulation of

seedling physiology during times of drought was observed in all species, albeit to different

degrees, however physiological damage occurred more quickly and intensely during the

second drought phase. Although a full reversal of physiological damage was witnessed

during the recovery phase after the first drought, it is possible that irreversible damage

45

within the seedlings led to the accelerated mortality rates observed during the second

drought phase than the first.

Despite adjustments in gas-exchange, fluorescence, and hydraulic conductivity over

multiple droughts, the specific drought-coping strategies employed by the species in this

study appear be ill-equipped to deal with multiple rounds of drought. The “drought-avoider

water-saver” and “drought-tolerant” strategies are regarded as unsuitable to withstand

multiple rounds of drought during the investigation of drought-preconditioning techniques

for three species of woody Mediterranean seedlings (Vilagrosa et al. 2003). During a

second round of drought, a “drought-avoider water-saver” species (Juniperus oxycedrus)

and “drought-tolerant” species (Quercus coccifera) did not display benefits to

preconditioning, while a “drought-avoider water-spender” (Pistacia lentiscus) exhibited

increases in aboveground biomass, leaf water content, Fv/Fm, and higher water potentials

(Vilagrosa et al. 2003). Similar beneficial behavior was observed in the “water-spender”

carob (Ceratonia siliqua) (Lo Gullo and Salleo 1988), and may be induced by the greater

sensitivity of “water-spenders” to an initial round of drought (Vilagrosa et al. 2003).

However, this strategy requires an adequate soil water source to compensate for

maintaining a constant leaf water content and may not be suitable for soils with a deep, or

inaccessible water table (Lo Gullo and Salleo 1988). Therefore the “drought-avoider water-

saver” species of B. attenuata and B. menziesii, and the “drought-tolerant” E. todtiana,

most likely can only survive multiple droughts by accessing a reliable groundwater source

after the first summer.

Damage to the photosystem

Given that mortality did not begin to occur until water was restored during the

recovery phase, the seedlings appeared to have crossed a damage threshold during the first

drought phase where their inherent physiological capacity to recover is compromised.

Incomplete recovery after re-watering can be reflective of a damaged photosystem

(Miyashita et al. 2005), as the risk of photodamage increases as the stomata close and

excess light is unable to be utilized by the photosynthetic process, over-energizing and

damaging the photosynthetic apparatus (Kitao et al. 2003). Prior to severe drought (gs >

0.05 mmol m-2 s-1), all three species had higher ETR without adjusting NPQ, suggesting

that the dissipation of excess photochemical energy and maintenance of a higher fraction of

open centers in PSII is achieved not through thermal dissipation but through higher electron

46

flow (Cavender-Bares and Bazzaz 2004). However, extending the drought past 45 days

resulted in a breakdown in the mechanisms used to protect the photosystem as CO2

availability was reduced and seedlings shifted towards photorespiration (Medrano et al.

2002a). Photorespiration is not as efficient as photosynthesis in utilizing electrons and

because of this ETR decreases in response to the abundance of energy (Fig 2.11b) (Stryer

1988). At this point during water deficit, NPQ is expected to protect against photoinhibition

and photodamage by thermally dissipating any additional light energy, as low CO2

availability is known to induce trans-thylakoid ΔpH within chloroplasts, promoting an

increase in NPQ (Cavender-Bares and Bazzaz 2004; Cousins et al. 2002; Muller et al.

2001). Instead of increasing, NPQ was negatively affected by the prolonged drought, and

no relationship was observed to exist between drying soil and NPQ in any of the species.

Even without a reliance on NPQ, PSII efficiency (Fv/Fm) remained high in seedlings

of all species that survived over the first drought and recovery phases, even as

photosynthesis fluctuated in response to available water. PSII can be highly drought

resistant, and complete and permanent photoinhibition is not common even during severe

drought (Epron and Dreyer 1993; Medrano et al. 2002a; Yordanov et al. 2000).

Nevertheless, damage to the photosystem was recorded in specific individuals of all species

during the first drought and recovery phases, as seedlings that were defined as dead

measured an Fv/Fm value of 0. Previous reports have stated that recovery of photosynthesis

in some species is not possible if stomatal conductance reach values lower than the severe

drought threshold (gs < 0.05 mmol m-2 s-1) (Flexas et al. 2006; Quick et al. 1992). This was

not the case within our study species, as the recovery of stomatal-regulated physiology was

witnessed in seedlings even when stomatal closure exceeded severe drought values. It is

more likely that once extreme photodamage occurs in these seedlings, recovery of seedling

physiology after re-watering is not possible.

The severe drought experienced within 15 days during the second drought phase

lead to early decreases in photosynthesis, carboxylation efficiency, ETR, and NPQ, while

Fv/Fm approached 0 in all species. Such an intense and rapid decrease in physiology was

sufficient to kill the remaining seedlings of all species 48 days into the second drought

phase. Given Fv/Fm damage was only recorded late in the second drought phase, complete

photoinhibition in these species is most likely a response to an accumulation of damage to

other physiological traits within the seedlings. Nevertheless, the efficiency of PSII remains

intact until the seedling is on the verge of death and therefore should not be used as an early

47

indicator of the health of these specific seedlings under drought (Adams and Demmig-

Adams 2004; Bukhov and Carpentier 2004; Zivcak et al. 2008).

Damage to the hydraulic system

Within five days immediately following the first drought, quick recovery of

transpiration rates to control levels was observed in the two Banksias, while recovery of

transpiration took place within 17 days in E. todtiana. If seedling hydraulic conductance

did not have time to recover prior to an increase in transpiration, this would indicate that

water transpired by the leaves is being released from storage within cavitated xylem vessels

(Sperry and Pockman 1993). This could cause a positive feedback, further increasing the

loss of hydraulic conductivity, creating embolisms and cavitation with death following

soon after, and could explain the deaths recorded during the recovery phase (Sperry et al.

2002). However due to the destructive nature of water potential measurement on seedlings,

hydraulic properties were only able to be recorded 17 days after re-watering, and thus the

recovery of conductance in relation to leaf transpiration immediately following re-watering

could not be determined.

After the first drought phase, increased rates of gas-exchange during the recovery

phase restored KL in the two Banksia species, most likely due to factors such as the growth

of new roots or embolism recovery aided by predawn xylem relaxation (stable ΨD values)

(Franks et al. 2007; Gebrekirstos et al. 2006). However, a greater loss of hydraulic transport

was present in the two Banksia species during the second drought phase as evidenced by

relatively larger decreases in both midday water potential and soil-to-leaf hydraulic

conductance (KL). Hydraulic constraint has been shown to a signal for triggering stomatal

closure (Tyree and Sperry 1989; Sperry et al. 1998), and can also be species specific

mechanism (Aranda et al. 2005). E. todtiana was able to exhibit greater resistance to a loss

of KL throughout both the first and second drought phases and the recovery phase,

potentially due to the maintenance of relatively low transpiration rates compared to xylem

water potential.

Seedling response to simultaneous drought and increased soil compaction

Seedling root elongation was severely restricted by compact soil in all species, and

maximum root depth is generally considered to be an essential trait in determining water

extractive capabilities from the soil (Araki and Iijima 2005; Passioura 1988). The amount

48

of water available to the seedling roots during the first drought phase in this treatment

dropped below 0.03 cm3 cm-3, a value where water becomes unavailable to plants grown in

soil of the type used in this study (Carbon et al. 1982). Even though available water was

located at lower depths of the pots, the seedlings were unable to access this water because

of severely restricted roots. This lack of accessible water caused a complete failure of

hydraulic conductance in all species in a relatively short amount of time, leaving no

potential for recovery.

The isohydric nature of the two Banksia species can place them at risk of carbon

starvation during prolonged drought, while the anisohydric E. todtiana is more likely to

experience xylem cavitation (McDowell et al. 2008). However, it is possible for isohydric

and anisohydric species to experience both carbon starvation and hydraulic collapse, and

both will only occur when the intensity and length of drought is severe (McDowell et al.

2008). It is possible that a combination of both carbon starvation and xylem cavitation was

the cause of seedling mortality, given the rapid and extreme collapse in all physiological

values occurring during the first drought phase. The conditions of increased soil

compaction and drought, and therefore postmine soils, place B. attenuata, B. menziesii, and

E. todtiana seedlings at risk of both carbon starvation and xylem cavitation, when they are

only physiologically designed to accommodate one factor.

Belowground morphology was severely negatively affected by simultaneous

drought and soil compaction, with lower root to shoot ratio, total root length, and maximum

root depth recorded in all species. The modification of root morphological traits to aid soil

water uptake during drought (increased root to shoot ratio, total root length, and maximum

root depth) were unable to occur due to the extreme soil compaction (Ewers et al. 2000;

Hacke et al. 2000; Hund et al. 2009). Leaf area in the two Banksias was reduced, possibly

as an extreme strategy to reduce transpiration due to such rapid water loss, or from reduce

leaf growth through a lack of water (Metcalfe et al. 1990; Rambal 1993).

Seedling response to increased soil compaction

Despite the extreme restriction of root elongation and other belowground

morphological traits, seedling physiology was not impeded by compact soil, as increased

soil mechanical impedance by itself does not usually damage seedling function (Kozlowski

1972; Kozlowski 1999; Marquez 2010). Water available to the roots was occasionally

greater than that of the control, as water infiltration is not as rapid in soils with high bulk

49

densities (Kramer and Boyer 1995). Given the high physiological and hydraulic

performance of these seedlings in this treatment, seedlings can become established in

heavily compact soils if sufficient water was supplied (Marquez 2010; Zisa et al. 1980).

However, such water availability would need to occur through adulthood in this restoration

setting.

Fig 2.14 The roots of an E. todtiana seedling growing in a) compact soil (bulk density of 1.80 g cm-3) and b)

non-compact soil (bulk density of 1.55 g cm-3) after 132 days of transplanting in 1.0 m long pots. The

seedling grown in non-compact soil extended to the bottom of the pot.

In response to compact soil, the two Banksia species exhibited an average thicker

root diameter, which is a previously document morphological response to increased soil

compaction (Bengough and Mullins 1990; Materechera et al. 1992; Materechera et al.

1991). This response is thought to aid root elongation through mechanically impeded soil: a

root that expands radially can maneuver soil particles away from the root tip and expand

50

axially into the open space (Atwell 1993). Indeed, differences between the ability of species

to penetrate through compact soil have been related to root thickness, but this principle has

not been confirmed (Bengough and MacKenzie 1994; Bengough and Mullins 1990; Clark

et al. 2003; Materechera et al. 1992; Materechera et al. 1991; Misra et al. 1986). If an

increase in root thickness was the main driver or only reason in aiding elongation through

compact soil, the rate of root elongation of the Banksias would be faster than E. todtiana.

Although E. todtiana initially has a greater root elongation rate over the first 104 days after

transplanting the seedlings in the 1.0 m pots, over time the roots of all three species

elongated at similar rates, most likely due to the increase in soil compaction at greater

depths within the pots.

Root elongation is known to become inhibited at soil strengths of about 2 MPa

(Aggarwal et al. 2006; Allmaras et al. 1988), and this strength was encountered at

approximately the 15 cm depth within the pots. Maximum depth of the seedlings 304 days

after transplanting elongated past 15 cm in all species, and although root elongation rate

slowed throughout the experiment, it is apparent that these species can penetrate this soil

type at strengths greater than 2 MPa. The maximum root depth of E. todtiana, while greater

than both Banksias, is relatively shallow considering the unrestricted roots of these

seedlings in a natural setting can reach depths of ~1.5 m in the first year of growth (Rokich

et al. 2001).

Conclusion

In seasonally dry environments, drought-resistant mechanisms are critical during the

seedling establishment stage, and should be considered in restoration programs. Here we

define specific strategies to cope with drought for Banksia and Eucalyptus from the

biodiverse sandplains of southwest Australia. Ecological evidence from adult trees favors

one of the species in the study in field experiments (B. menziesii) (Groom et al. 2000; Muir

1983). Under drought-stressed conditions the seedlings of these species appear to behave

differently, as witnessed in previous studies (Donovan and Ehleringer 1991; Ehleringer and

Sandquist 2006). B. attenuata seedlings appear to be the most adapted to drought, with

higher WUE, an integrated down-regulation of photosynthesis, a low SLA, and adaptive

root architecture. However, these drought-adapted traits did not convey a survival

advantage. The physiological differences between species during drought were most

apparent in gas-exchange and water relation parameters (isohydric Banksia species versus

51

anisohydric E. todtiana); there was relatively little difference between species with regards

to chlorophyll fluorescence, or energy and heat dissipation values. This suggests that

isohydry and anisohydry, while using different physiological mechanisms, are equally

effective at coping with drought (McDowell 2008). The ability to offset photodamage may

be a more important factor in establishing seedlings under low water conditions.

When focusing strictly on seedling survival, it would be disadvantageous to design

restoration practices around one particular species, as found in this study from two

Banksias and one Eucalyptus species. The framework species method (Blakesley et al.

2001) is not applicable for restoring this particular mediterranean-type ecosystem in a

postmine setting, given there was no apparent survival favorite among these three dominant

overstorey species. A biphasic pattern of drought, as well as imposing simultaneous

drought and soil compaction was shown to be equally antagonistic to survival during the

seedling stage of these three phreatophytic evergreen trees, and must be addressed before

restoring the land from a postmine setting to its historical state. To resist and survive

drought, these species need to have unrestricted access to water during the critical seedling

stage within two years. Methods to supply seedlings with water, or more importantly,

alleviate compact soil and aid root development, are imperative to successfully restore

postmine sites in this biodiverse mediterranean-type environment.

52

53

C H A P T E R T H R E E

Increasing soil water retention with native-sourced mulch improves

Banksia seedling establishment in postmine mediterranean sandy soils

Introduction

In the biodiversity hotspot of southwest Australia, up to 90% seedling mortality in

postmine restoration occurs during a seasonal drought, with the causes behind this decline

unknown (Burrows 1986; Enright & Lamont 1992; Rokich 1999). Soil reconstruction

following mining operations can disrupt structural characteristics and impede seedling root

growth (McSweeney & Jansen 1984). As climate change intensifies droughts, particularly

in mediterranean-type ecosystems such as southwest Australia (Schar et al. 2004), restricted

root development exposes seedlings to greater risk of water stress (Thompson et al. 1987),

and could further exacerbate the high mortality rate (Rokich et al. 2001). Soil restoration to

facilitate natural root development is therefore essential for successful restoration of a

postmine environment (Bradshaw 1997).

Sand quarries are often abandoned with minimal restoration effort, especially sites

mined prior to 1980 (Schroeder 1997). In southwest Australia, the sand quarry extraction

process reduces the overall soil profile from over 30 m to less than 5 m above the season

high groundwater level. Only the top 10 cm of the entire profile is retained for restoration

purposes as this topsoil contains many of the propagules (2621 seeds m-2 (Rokich et al

2000)), supporting organisms (fungal symbionts) and nutritional benefits required for

successful plant establishment. Removal of the natural soil profile has important

ramifications for plant growth in restoration and reconstruction of soil profiles to mimic

natural soil function may be important in optimizing seedling establishment and survival in

restoration.

Reconstructed soils can be manipulated to improve seedling establishment

(Bradshaw 1997), and soils can be mechanically ripped prior to seedling introduction to

reduce soil compaction and enhance root elongation (Ashby 1997). However, compaction

values in postmine sandy soils of the Bassendean dunes in southwest Australia are known

to revert to pre-ripped, root-inhibiting values (Rokich 1999); an uncommon occurrence in

soils that are predominately sand (Harper & Gilkes 1994). Unlike natural hardsetting, the

54

increased soil strength resulting from this “cryptic compaction” is not markedly reduced

when soils are re-wetted (Harper & Gilkes 1994; Rokich 1999), and techniques to alleviate

this phenomenon have not yet been trialed.

The application of soil amendments can be used to restore postmine soil by

overcoming loss of soil structure, restoring hydrological balance and mineral nutritional

capacity. However the effect varies with amendment type and interaction with the soil

environment, dependent on the biotic or abiotic characteristics of the degraded soil (Wong

2003). Broadly speaking, amendments are classified into organic or inorganic amendments.

Organic amendments, such as mulch or manure can resist compaction forces by stabilizing

soil aggregates or diluting the profile with a material of lower bulk density, as well as

increasing soil water retention (Barzegar et al. 2002). Inorganic amendments have been

more specifically designed to meet plant nutritional (eg. fertilizers), or

physical/hydrological (eg. porosity and drainage) requirements (Babalola & Lal 1977).

Here we examine the impacts of soil amendments and their underlying effects on seedling

morphology, physiology, and survival to understand and improve native seedling

establishment past the critical establishment stage in postmine sandy soils. Native mulch

and gravel are utilized as amendments to alleviate seedling response to the seasonal drought

of a mediterranean-type ecosystem and the “cryptic compaction” phenomenon. The

amendments are hypothesized to: 1) reduce soil strength and increase root elongation rate,

2) improve soil water retention and lessen the effects of drought on seedling function, and

3) replace soil nutrients lost during the mining process. Improvements in soil and seedling

health from the amendments will be reflected in greater seedling establishment over two

years, increasing the success of restoring postmine sandy soils in a mediterranean-type

environment.


Study Site

The experiment was conducted over two years (May 2009 – April 2011) within an

operational sand quarry 30 km northeast of Perth, Western Australia (Figs 3.1), where

mining operations removed 20 - 30 m of soil from the profile to 3 m above the water

55

Fig 3.1 Satellite image from 2009 showing an overhead view of the sand quarry where the experiment was conducted. Differently-aged restoration areas, starting from

1999 onwards, are labeled by color. The experimental sites used in this study, both restoration and natural, are depicted on the image.

Fig 3.2 During mining operations within the study site. The scale and extent of soil removal (20-30 meters)

can be seen as mining vehicles extract the siliceous sands.

Fig 3.3 a) Pre- and b) postmine landscape of the Banksia woodlands within the Swan Coastal Plain.

56

57

table prior to the start of the experiment (Figs 3.2 & 3.3). The site is located within the

Bassendean dunes, characterized by low-nutrient, leached acidic podzols with high acidity

and low water holding capacities (Bolland 1999; Dodd & Heddle 1989; McArthur 1991).

Over 80% of the average annual rainfall of 800 mm is recorded between May and October,

and temperatures fluctuate between an average 35°C (February) and 5°C (July) (Bureau of

Meteorology 2012; Dodd & Heddle 1989) (Fig 3.4). Prior to mining, the native flora

consists of a dense shrubby heathland with a diverse mid-storey of woody shrubs and

herbaceous groundcover scattered with relatively few species of trees (Fig 3.3) (Dodd et al.

1984).

Winter Spring Summer Autumn Winter Spring Summer Autumn

Rai

nfal

l(m

m)

0

20

40

60

80

100

120

140

160 Rainfall April 2009 - April 2011Average Yearly Rainfall

Fig 3.4 Average rainfall at Pearce RAAF, 10 km north/northeast of the study site, indicating lower rainfall

during the two years of the experiment. Source: Bureau of Meteorology 2012.

Experimental Design

Three replicate sites (23.5 m x 9.5 m) were located within the area of the quarry

undergoing restoration. These ‘restoration sites’ each contained a control, organic

amendment, and inorganic amendment treatment plot, 6.5 m x 9.5 m, separated by a 2 m

buffer (Fig 3.5b). Three 6.5 m x 9.5 m replicate ‘natural sites’ were located in undisturbed

remnant Banksia woodland, separated at least 50 m by native woodland and cleared of

above-ground vegetation (Fig 3.5c). The organic amendment consisted of native brush

cleared from the quarry prior to soil extraction and crushed to create a mulch (≤ 5 cm size).

58

‘Blue metal’, a crushed basalt gravel of 10 mm size, was used as the inorganic amendment.

Blue metal is commonly used as a drainage medium consisting of magnesium and calcium

silicates with small quantities of potassium, phosphorus, and trace elements (Coventry et al.

2001). A front-end loader deposited 5 m3 of the organic or inorganic amendment over the

corresponding plot (Fig 3.5a). A 4-pronged traxcavator plowed the amendments to a depth

of 0.5 m to achieve a final concentration of 12% amendment to soil volume. This plowing

process was repeated in the control plot to maintain disturbances between treatments at the

restoration sites. Topsoil was then applied using leading practice: 10 cm of fresh topsoil

stripped from an adjacent intact Banksia woodland system after tree removal and was

immediately spread over each restoration site and ripped to a depth of 0.5 m using a

bulldozer operated tyne (Rokich 1999). The topsoil is characterized chemically as being

relatively high in nutrients compared to deeper horizons, although still considered nutrient

impoverished (extractable P < 2 mg/kg and K 15 mg/kg) and physically, as a loamy sand

(94% coarse sand, 1% clay) (McArthur 1991).

Fig 3.5 a) Spreading the organic and inorganic amendment in the restoration sites. b) Labeled restoration sites

prior to mixing the amendments to a depth of 50 cm. c) A natural site cleared of aboveground vegetation.

59

Study Species and Sowing

Banksia attenuata R.Br and B. menziesii R. Br are dominant, phreatophytic, and

evergreen trees of the region, routinely utilized as framework species in local restoration

programs (Dodd et al. 1984). Local provenance seeds were hand-sown 2 cm under the soil

surface in early May 2009. Seeds were sown randomly 0.25m apart, along 14 rows within

each plot. The rows were separated alternatively by 0.25 m and 0.5 m to allow access to

seedlings with minimal disturbance, and a 1-m buffer perimeter surrounded the rows on all

sides of each plot to assure seedlings grew within amended soil. A continuous 2 m × 6.5 m

rectangular area within each plot remained devoid of seeds to allow for soil-specific

measurements without disturbance to the seedlings.

Measurements

Soil Moisture

Soil moisture was recorded at four locations within each plot every two months

using a moisture probe meter (MPM-160-B, ICT International Pty Ltd). At each of the four

locations, four measurements were taken at depths of 0, 25, and 50 cm. A coarse white sand

obtained from the quarry was used to calibrate the moisture probe. The quadratic

polynomial equation (y = -2e-7x2 + 4e-4x; R2 = 0.98) was determined from the calibration

curve and converted the millivolts (x) to gravimetric soil moisture content (y)

(Anonymous).

Soil moisture was matched with rainfall data acquired from the nearest Bureau of

Meteorology site (Pearce RAAF), 10 kilometers north of the study site (Fig 3.4). Rainfall at

this site is characterized by a typical mediterranean-type climate, with 86% of rainfall

occurring during the winter/spring months of May to October (Dodd & Heddle 1989).

Soil Chemical and Physical Properties

The “cryptic compaction” was measured by recording soil impedance every two

months with a Rimik CP20II Cone Penetrometer (RFM Australia Pty Ltd, QLD, Australia,

Cone Diameter 12.83 mm; Area 130 sqmm). A minimum of ten readings were taken in

each plot at random locations, with measurements every 2 cm to a depth of 50 cm. Four

0.25 kg soil samples at depths of 0, 25, and 50 cm were collected at the end of the

experiment and analyzed by CSBP Ltd to test for pH and electrical conductivity (EC) in a

1:5 H2O solution (Rayment & Higginson 1992), soil organic carbon (SOC) (Walkley &

60

Black 1934; Walkley 1947), sulfur (Blair et al. 1991), phosphorus (Colwell 1965; Rayment

& Higginson 1992), and potassium (Rayment & Higginson 1992). Ammonium and nitrate

were determined using flow injection analysis (Lachat Instruments 1992; Searle 1984).

Seedling Survival

To determine the impact of soil amendments on plant survival, survival was

recorded seven months after sowing, before the onset of summer drought. Subsequent

survival was recorded 12 and 24 months after sowing (after the first and second summers).

Of the three restoration sites, one site was subject to extreme predation, (~95%) and

removed from the analysis. Predation at the remaining two restoration sites was minimal

and did not affect the outcome of the experiment.

Seedling Physiology

Ten seedlings of each species from each treatment plot were caged with metal wire

to prevent predation and were used for physiological measurements. Seedling physiology of

each species was determined at eight regular intervals throughout the experiment. The

intervals represented specific times of the year when varying precipitation levels would

have the most effect on seedling physiology. An SC-1 Leaf Porometer® (Decagon Devices,

Inc., Pullman, Washington, USA) recorded the rate of stomatal conductance of seedlings on

the first seven intervals. On the eighth and final interval, a Li-Cor® 6400 gas-exchange

analyzer (LI-COR, Inc. Lincoln, NE, USA) assessed the rates of stomatal conductance,

photosynthesis and transpiration. The Li-Cor was calibrated at: Flow Rate to the Sample

Cell: 300 μmol s-1, Reference Cell CO2: 400 μmol CO2 mol-1, Artificial Light Source:

6400-02 Red/Blue LED PAR 1,700 μmol m-2s-1. Five measurements on each leaf were

performed within ten seconds after the Li-Cor displayed a steady photosynthetic rate.

Water-use efficiency (WUE) was calculated as the ratio of photosynthesis to transpiration.

Measurements were recorded on the abaxial surface (position of stomata) of the youngest

and healthiest leaf available approximately two hours after the start of the photoperiod:

average daily values under drought stress can be determined from mid-morning and light-

saturated gas-exchange measurements (Vadell et al. 1995).

61

Seedling Morphology

At the completion of the experiment in April 2011, four seedlings of each species

from all restoration site plots and five seedlings of each species from the natural site plots

were excavated by hand to a maximum depth of 1.2 m. Seedlings were immediately sealed

in a plastic ziplock bag until root washing. Lateral roots were determined to be the number

of roots extending horizontally from the seedling between the stem/soil interface and 5 cm

down the main tap root. The total area of fresh leaves and root anatomy of the seedlings

were analyzed on a back-lit flatbed scanner with a resolution of 0.2 μm using a digital

image analyzer (WinRHIZO® Pro, V. 2007d, Regent Instruments Canada, Inc.). Specimens

were then oven dried for one week at 70 C (Contherm® Digital Series Oven, Perth

Scientific Pty Ltd) and dry weights of stems, leaves and roots were measured. Specific root

length (SRL) was calculated as the ratio of total root length to mass (Ostonen et al. 2007),

while Specific leaf area (SLA) was calculated as the ratio of leaf surface area to leaf weight

(Wilson et al. 1999).

Seedling Chemical Composition

To understand the impacts of soil amendments on seedling nutrition, healthy leaves

of three seedlings of each species, treatment, and site were oven dried for one week at 70 C.

The foliar concentrations of elements were identified by CSBP Ltd (Bibra Lake, Western

Australia). B, Cu, Zn, Mn, Fe, Ca, Mg, Na, K, P, and S were analyzed on an ICP-AES

(McQuaker 1979), and N was determined using flow injection analysis (Lachat Instruments

1992; Searle 1984).

Statistics

Data was analyzed with R statistical software (version 2.13.0), and transformed

logarithmically to achieve a normal distribution when necessary. All data is presented as

untransformed means. Binomial generalized linear models compared seedling survival

between species, the three restoration treatments, and the first and second year. General

linear models compared the physiology, morphology, and chemical composition of the

seedlings and the differences in soil chemical and physical properties between treatments.

Pairwise comparisons between treatments were performed by Tukey’s post hoc test when

significant effects were indicated. SigmaPlot 12 (Version 12.0.0.182, Systat Software, Inc.)

62

fitted linear regression lines to soil impedance data and ANCOVAs compared differences

in slopes between treatments.

To understand seedling establishment through the first two years after germination,

three points in time for each year were chosen for soil moisture and seedling physiological

data analysis. During the first and second year of the experiment, soil moisture and seedling

stomatal conductance is analyzed during the spring (end of the Wet season), at the

beginning of summer (Early Dry season), and at the end of summer (Late Dry season).

These three seasonal periods over two years represent pivotal points in the life history of a

seedling and relate plant physiology to soil moisture. To determine the effectiveness of

each amendment, soil and seedling data from the organic and inorganic treatments were

compared to the standard restoration procedure (control) and native bushland (natural site),

while differences between the natural site and control are also reported.

Results

Soil Characteristics

Soil Moisture

A significantly higher amount of soil moisture was retained in all three restoration

treatments compared to the natural site throughout the first year of the experiment (P <

0.001) (Fig 3.6a). The amendments had a minimal effect on soil moisture during the first

year compared to the control: the organic treatment increased moisture by 14% during the

Early Dry season (P = 0.019), and the inorganic treatment decreased moisture by 10% in

the Late Dry season (P = 0.014) (Fig 3.6a).

Recorded rainfall was below average during the experiment, particularly during the

second year (May 2010 to April 2011), being 48% lower than average (Fig 3.4). Soil

moisture in the control did not significantly differ from the natural site during the second

year (Fig 3.6a&b). Soil moisture in the organic treatment was higher compared to the

control and natural site throughout the second year: increasing from ~44% in the Wet

season to >70% in the Late Dry season (P < 0.001) (Fig 3.6a). Soil moisture retention in the

inorganic treatment decreased over the second year: in the Wet season soil moisture was

37% and 39% greater than the control and natural site, respectively (P < 0.001), and not

significantly different during the Late Dry season (P ≥ 0.248) (Fig 3.6a).

During the second year, the amount of soil water recorded in the control did not

significantly differ from the natural site at any point in time (Fig 3.6a&b). Soil moisture

63

content in the organic treatment retained an increasing amount of moisture as time

progressed over the second year compared to both the control and natural sites: increasing

from 43% retained moisture in the Wet season to 70% in the Late Dry season compared to

the control (P < 0.001), and an increase of 45% in the Wet season to 100% in the Late Dry

season compared to the natural site (P < 0.001) (Fig 3.6a). Soil moisture content in the

inorganic treatment was significantly higher than the control by 37% in the Wet season and

by 25% in the Early Dry season (P < 0.001) but was not significantly different during the

Late Dry season (Fig 3.6a). The inorganic treatment held significantly more soil moisture

content than the natural site by 39% in the Wet season and by 23% in the Early Dry season

(P ≤ 0.006) but was not significantly different during the Late Dry season (Fig 3.6a). The

increase in soil moisture from the organic amendment occurred at depths of 25 and 50 cm

in the soil profile and became more pronounced as the dry season progressed (Fig 3.6b).

Gra

vim

etric

Soi

l Moi

stur

e C

onte

nt (g

g-1

)

0.02

0.04

0.06

0.08

ControlOrganicInorganicNatural

1

2

3

4

5 6

0.02 0.04 0.06 0.08 0.02 0.04 0.06 0.08 0.02 0.04 0.06 0.08

Gravimetric Soil Moisture Content (g g-1)0.02 0.04 0.06 0.08

2 3 5 60.02 0.04 0.06 0.08

40.02 0.04 0.06 0.08

Dep

th (c

m)

-50

-40

-30

-20

-10

0

1

Winter Spring AutumnSummer Winter Spring AutumnSummer

a

b

Fig 3.6 a) Average gravimetric soil moisture content in each treatment averaged from depths of 0, 25, and 50

cm over two years at the study site, and b) gravimetric soil moisture content by depth at six defined points in

time. Error bars represent the standard error of the mean (2a, n = 12; 2b, n = 4). Numbers 1, 2, and 3 indicate

the Wet, Early Dry, and Late Dry season of the first year, respectively. Numbers 4, 5, and 6 indicate the Wet,

Early Dry, and Late Dry season of the second year, respectively.

64

Soil Physical Properties

Cryptic compaction was observed by locating the depth at which soil impendence

surpassed 2 MPa, the value known to restrict root development (Allmaras et al. 1988). This

value gradually approached shallower depths in all restoration treatments regardless of the

presence or type of amendment (Fig. 3.7). The response of increasing soil compaction was

significantly weaker in the organic treatment than the control (P = 0.003), and no

significant difference was observed between the control and inorganic treatments (P =

0.224) (Fig 3.7). The soil strength of the natural site remained below the 2 MPa threshold

for root impediment in all depths recorded (data not shown). Soil impedance values within

the restoration treatments were similar to that of the natural site until a depth of ~20 cm,

and steadily increased as depth decreased to reach impedance values above 3.5 MPa at 50

cm depth (Fig 3.8). Soil impedance values in the restoration treatments were recorded to

have significantly higher soil impedance values than the native treatment at a depth of 50

cm (P < 0.001) with an increase of 174% in the control, 189% in the organic, and 190% in

the inorganic (Fig 3.8).

Fig 3.7 The average depth at which the soil impedance value of 2 MPa was first encountered in the soil

profile throughout the experiment. Unaveraged data was used to plot the linear regression lines, while the

averaged data points are represented in the graph. Error bars represent the standard error of the mean (n ≥ 25).

65

MPa0 1 2 3 4

Dep

th (c

m)

-50

-40

-30

-20

-10

0Control OrganicInorganicNatural

Fig 3.8 Penetration resistance in the soil by depth in the restoration and natural sites after two years. Error

bars represent the standard error of the mean (n ≥ 30).

Soil Chemical Properties

The average soil pH changed significantly through time in the organic treatment

only, decreasing from 7.63 in December 2009 to 6.37 in April 2011 (P < 0.001) (Fig 3.9).

Soil samples taken at the end of the second summer showed a significantly more acidic soil

in the natural site compared to the control and inorganic treatments (P < 0.001) and a

significantly more acidic soil in the organic treatment compared to the control (P < 0.001)

(Table 3.1). Soil pH was not significantly different between the natural and organic

treatments (P = 0.316) or the control and inorganic treatments (P = 0.548) (Table 3.1). The

pH of soils from all treatments did not differ at depth (P > 0.05).

66

Summer Autumn Winter Summer Autumn

pH

6

7

8

9 Control Organic Inorganic Natural

Fig 3.9 The pH of soils averaged from depths of 0, 25, and 50 cm over two years. Error bars represent the

standard error of the mean (n = 12).

Soil sulfur and electrical conductivity (EC) did not significantly differ by depth in

any of the treatments (P = 0.173 and 0.112, respectively). A significant increase in both

sulfur and EC values was seen in the inorganic treatment when compared with the

remaining treatments (sulfur P = 0.013, EC P = 0.006) (Table 3.1). Soil organic carbon

percentage was not significantly different between the treatments (Table 3.1) (P = 0.22).

Within the natural site, organic carbon was 117% higher at the surface than at 50 cm (P <

0.001). Significant differences in organic carbon percentage at depth did not exist in the

restoration treatments. Other soil nutrients from the restoration and natural sites did not

differ by treatment or depth (phosphorus < 2 mg kg-1, potassium < 15 mg kg-1, ammonium

< 1 mg kg-1, and nitrate < 1 mg kg-1).

Table 3.1 Soil properties averaged at depths of 0, 25 and 50 cm at the end of two years. Values are the mean

(n = 12), ± standard error. ‘†’ symbol indicates a significant difference from the natural site, ‘*’ symbol

indicates a significant difference from the control (P < 0.05).

Organic Carbon EC pH Sulfur (%) (dS mˉ¹) (mg kgˉ¹)

Natural 0.473 (±0.098) 0.015 (±0.003) 6.05 (±0.103)* 1.26 (±0.229)

Control 0.365 (±0.071) 0.017 (±0.002) 7.36† (±0.135) 1.01 (±0.198)

Organic 0.368 (±0.059) 0.014 (±0.002) 6.37 (±0.120)* 1.30 (±0.264)

Inorganic 0.348 (±0.069) 0.032 (±0.007)†* 7.6† (±0.120) 3.19 (±0.947)†*

67

Fig 3.10 Soil profile of the a) standard restoration procedure (control) and b) the natural site.

Seedling Survival

Seedling survival was strongly dependent on year and treatment within the quarry

restoration area (Table 3.2). After the first summer, no significant differences were

observed between treatment and species (P = 0.623 and 0.157, respectively) (Table 3.2).

After the second year survival significantly decreased in all treatments and species (P <

0.001 control, inorganic, B. attenuata, B. menziesii; P = 0.031 organic) (Table 3.2). While

there were no significant differences in seedling survival between species (P = 0.970), a

significant interaction existed within treatments (P < 0.001). Seedling survival in the

organic treatment was significantly higher than the control (B. menziesii P < 0.001; B.

attenuata P = 0.002). Survival in the inorganic treatment did not differ from the control (B.

menziesii P = 0.062, B. attenuata P = 0.631). Seedling survival in the natural site was not

reported or considered for the purposes of this experiment, given all un-caged seedlings

died from herbivore predation. No deaths were observed in caged seedlings within the

natural site.

68

Table 3.2 Seedling survival percentage after the first and second years of the experiment. Each year spanned

a summer drought period. ‘†’ symbol indicates a significant difference from the first year. ‘*’ symbol

indicates a significant difference from the control (P < 0.05).

Seedling Physiology

Stomatal conductance rate is an accurate predictor of plant drought stress, and

differences in stomatal conductance between treatments were used to determine seedling

health over two years (Flexas and Medrano 2002; Medrano et al. 2002). Seedlings grown in

the natural site were not measured until the winter of 2010 due to small leaf size and

possible damage caused by attaching the equipment onto the leaf. Therefore no

comparisons were made between restoration sites and the natural site in the first year of the

experiment. During the first year of seedling growth in the restoration sites, average rates of

stomatal conductance remained above 0.13 mol H2O m-2 s-1 for both species in all

treatments, signifying that these seedlings were not severely drought stressed throughout

this time (Fig 3.11) (Flexas and Medrano 2002). The stomatal conductance of both species

were relatively higher in the organic treatment during the Late Dry season (> 0.20 mol H2O

m-2 s-1), significantly greater than the control for B. attenuata (P < 0.001), but not B.

menziesii (Fig 3.11).

During the second year of seedling growth, the stomatal conductance rate in all

treatments remained above 0.15 mol H2O m-2 s-1 during the Wet season for both B.

attenuata and B. menziesii. As summer progressed, seedlings of both species in the control

Species 1st year 2nd year

Natural B. attenuata 100% 100%

B. menziesii 100% 100%

Control B. attenuata 91% 55%†

B. menziesii 85% 42%†

Organic B. attenuata 88% 79%†*

B. menziesii 92% 84%†*

Inorganic B. attenuata 81% 52%†

B. menziesii 92% 57%†

69

and inorganic treatment rapidly approached values of ~0.05 mol H2O m-2 s-1 during the

Early Dry and Late Dry seasons, the value that plants become severely water stressed

(Flexas and Medrano 2002) (Fig 3.11). The stomata of seedlings of both species in the

natural site closed gradually and did not decrease lower than 0.09 mol H2O m-2 s-1 during

Early Dry or Late Dry seasons of the second year (Fig 3.11). The stomata of both species

growing in the organic treatment remained open throughout the summer of the second year,

and did not reach values lower than 0.18 mol H2O m-2 s-1 (Fig 3.11). The high stomatal

conductance rates recorded in the organic treatment were significantly greater than those of

the control during the Early Dry and Late Dry seasons (P < 0.001) (Fig 3.11).

Banksia menziesii

Stom

atal

Con

duct

ance

( mol

H2O

m-2

s-1)

0.0

0.1

0.2

0.3

0.4

0.5

ControlOrganicInorganicNatural

Banksia attenuata

Spring Summer Autumn Winter Spring Summer Autumn

Stom

atal

Con

duct

ance

(mol

H2O

m-2

s-1)

0.0

0.1

0.2

0.3

0.4

0.5

*

*

1 2 3 4 5 6

1 2 3 4 5 6

†

*

*

*

*†

†

†

Fig 3.11 Average stomatal conductance of B. attenuata and B. menziesii seedlings over two years. Error bars

represent the standard error of the mean (n = 10). Numbers 1, 2, and 3 indicate the Wet, Early Dry, and Late

Dry season of the first year, respectively. Numbers 4, 5, and 6 indicate the Wet, Early Dry, and Late Dry

season of the second year, respectively. ‘†’ symbol indicates a significant difference from the natural site, ‘*’

symbol indicates a significant difference from the control (P < 0.05).

70

The maintenance of high stomatal conductance rates allows greater carbon uptake

into the leaf but can also facilitate the loss of water (Crocker et al. 1998). Given the high

stomatal conductance rates in the organic treatment of both Banksia species, greater rates of

photosynthesis and transpiration were observed during the Late Dry season of the second

year (Fig 3.12). B. menziesii seedlings in the organic treatment exhibited a 131% increase

in photosynthesis compared to the control, while B. attenuata seedlings increased their

photosynthetic rate by 97% (P < 0.001) (Fig 3.12). Compared to the natural site, the

photosynthetic rate of B. menziesii in the organic treatment was not significantly different,

but was 83% higher in B. attenuata (P = 0.012) (Fig 3.12). B. menziesii seedlings

significantly increased transpiration rates by 311% in the organic treatment over the control

(P < 0.001) and 139% over the natural site (P = 0.008), while B. attenuata seedlings

increased transpiration by 247% over the control (P < 0.001) and by 216% compared to the

natural site (P < 0.001) (Fig 3.12). The proportionally greater rates of transpiration

compared to photosynthesis decreased WUE in B. menziesii seedlings by 37% in the

organic treatment compared to the control (P < 0.001), and no significant difference in

WUE between the organic and natural sites was recorded (Fig 3.12). In B. attenuata

seedlings, the organic treatment decreased WUE by 37% compared to both the control and

natural sites (P < 0.001) (Fig 3.12).

No significant differences in photosynthesis or transpiration were observed in the

inorganic treatment compared to the control and natural sites in both species during the

Late Dry season of the second year (Fig 3.12). B. menziesii seedlings exhibited a significant

decrease in WUE in the control and natural sites by 45% and 40%, respectively (P ≤ 0.005),

and B. attenuata seedlings exhibited a 37% decrease in WUE compared to both control and

natural sites (control P < 0.001, natural P = 0.041) (Fig 3.12). The WUE and the

photosynthetic and transpiration rates of seedlings in the control did not differ from the

natural site in both B. menziesii and B. attenuata during the Late Dry season of the second

year (Fig 3.12).

71

Natural Control Organic Inorganic

Pho

tosy

nthe

sis

( m

ol C

O2

m-2

s-1

)

1

6

11

16

21 B. attenuataB. menziesii


Wat

er U

se E

ffici

ency

( m

ol C

O2

mm

ol-1

H2O

)

1

3

5

7

*

* **

†

*

† * † †


Tra

nspi

ratio

n(m

mol

H2O

m-2

s-1

)

1

3

5

7 *

*†

†

Fig 3.12 Photosynthesis, transpiration, and water use efficiency of B. attenuata and B. menziesii seedlings

during the Late Dry season of the second year. Error bars represent the standard error of the mean (n = 10).

‘†’ symbol indicates a significant difference from the natural site, ‘*’ symbol indicates a significant difference

from the control (P < 0.05).

Seedling Morphology

No significant differences in root morphology were observed in the organic and

inorganic treatments compared to the control (Table 3.3). The changes observed in

belowground morphological traits were uniform across the three restoration treatments

when compared to the natural site (Table 3.3). The seedlings of both species in the natural

site consistently formed a single taproot, while the seedlings in the restoration treatments

produced a branching root architecture confined in the top 40 cm of the soil profile (Fig

3.14). The most discernable effect of the restoration treatments on root architecture was an

average decrease in root depth by 57% in B. menziesii and by 55% in B. attenuata

compared to the natural site (P < 0.001) (Table 3.3). Root excavation at the natural site was

halted at a depth of 1.2 m because of the limits of manual extraction by hand in loose soil,

though it is most likely that seedlings growing in the natural site extended their roots to

72

even greater depths than those recorded. The total number of root forks in the restoration

treatment increased significantly by an average of 358% in B. menziesii (P ≤ 0.048) and

609% in B. attenuata (P ≤ 0.015), and the total length of roots increased by an average of

239% in B. menziesii (P ≤ 0.029) and 367% in B. attenuata (P ≤ 0.005) (Table 3.3). There

were no differences in the number of lateral roots, total root weight, root weight of cluster

roots, average root diameter, or SRL observed between the restoration and natural sites

within B. attenuata and B. menziesii (Table 3.3).

Table 3.3 Morphological traits of B. attenuata and B. menziesii seedlings after a growth period of two years.

Values are the mean (n ≥ 4), ± standard error. ‘†’ symbol indicates a significant difference from the natural

site, ‘*’ symbol indicates a significant difference from the control (P < 0.05).

Natural Control

B. attenuata B. menziesii B. attenuata B. menziesii

Lareral Roots (#) 9.60 (±2.29) 11.2 (±1.38) 9.63 (±1.31) 12.5 (±1.07)

Root Forks (#) 961.2 (±155.4)* 1629 (±265.0)* 5274 (±1535)† 7473 (±2684)†

Root Depth (cm) 78.6 (±11.5)* 73.6 (±8.39)* 41.5 (±4.17)† 37.7 (±3.60)†

Total Root Weight (g) 4.26 (±2.25) 3.40 (±1.04) 9.02 (±2.45) 6.79 (±1.84)

Proteoid Root Weight (g) 0.667 (±0.323) 1.73 (±0.675) 3.53 (±2.21) 1.99 (±0.930)

Total Root Length (cm) 215.6 (±30.81)* 384.0 (±38.77)* 846.0 (±185.2)† 1361 (±351.7)†

Root Diameter (mm) 1.97 (±0.447) 1.32 (±0.158) 1.52 (±0.138) 1.31 (±0.177)

SRL (m gˉ¹) 0.961 (±0.184) 2.33 (±0.068) 1.11 (±0.169) 2.75 (±0.553)

Leaf Weight (g) 1.59 (±0.206)* 3.41 (±0.548) 7.35 (±1.20)† 6.20 (±1.12)

Leaf SA (cm²) 248.1 (±26.97)* 565.0 (±85.50) 867.2 (±128.1)† 784.5 (±135.9)

SLA (cm² gˉ¹) 159.4 (±5.381)* 167.4 (±3.594)* 121.5 (±3.644)† 128.7 (±2.341)†

Organic Inorganic

B. attenuata B. menziesii B. attenuata B. menziesii

Lareral Roots (#) 10.0 (±1.45) 14.25 (±0.818) 9.63 (±1.31) 13.5 (±1.60)

Root Forks (#) 5041 (±1311)† 7544 (±3714)† 9664 (±4706)† 7366 (±2576)†

Root Depth (cm) 35.4 (±1.97)† 34.7 (±6.59)† 37.1 (±3.71)† 32.0 (±2.56)†

Total Root Weight (g) 7.57 (±2.36) 5.81 (±3.36) 8.74 (±2.20) 4.93 (±1.23)

Proteoid Root Weight (g) 1.92 (±0.956) 1.96 (±1.21) 3.14 (±1.19) 1.84 (±0.880)

Total Root Length (cm) 869.9 (±100.1)† 1146 (±531.8)† 1304 (±501.9)† 1092 (±266.0)†

Root Diameter (mm) 1.32 (±0.214) 1.33 (±0.317) 1.27 (±0.124) 1.05 (±0.138)

SRL (m gˉ¹) 1.74 (±0.234) 2.56 (±0.228) 2.28 (±0.575) 3.73 (±0.810)

Leaf Weight (g) 9.93 (±1.70)† 10.04 (±2.38)† 6.37 (±0.80)† 5.51 (±0.355)

Leaf SA (cm²) 1268 (±190.4)† 1207 (±215.7)† 769.2 (±90.94)† 654.3 (±33.68)

SLA (cm² gˉ¹) 130.2 (±3.216)† 133.2 (±11.22)† 122.1 (±2.662)† 119.7 (±3.470)†

73

The organic and inorganic amendments had no significant effects on the

aboveground morphology of both B. attenuata and B. menziesii when compared to the

control (Table 3.3). B. menziesii seedlings growing in the organic treatment increased their

leaf biomass by 194% and their leaf surface area by 114% over the natural site (P ≤ 0.030),

but no differences were observed in these leaf traits when comparing the control and

inorganic treatments to the natural site (Table 3.3). B. menziesii seedlings from all three

restoration treatments exhibited a 24% average decrease in SLA when compared to the

natural site (P ≤ 0.026) (Table 3.3). When compared to the natural site, leaf biomass of B.

attenuata increased 362% in the control, 525% in the organic, and 301% in the inorganic

treatments (P < 0.001), and leaf surface area was increased by 250%, 411%, and 210% in

the control, organic, and inorganic treatments, respectively (P ≤ 0.002) (Table 3.3). An

average 22% lower SLA was observed in B. attenuata seedlings in the restoration

treatments when compared to the natural site (P < 0.001) (Table 3.3).

Leaf Nutrient Composition

Nutrient analysis of seedling foliage varied between B. attenuata and B. menziesii,

with few consistent patterns among treatments. B. attenuata and B. menziesii were revealed

to contain low amounts of foliar nitrogen, phosphorus, potassium, copper, and sodium in

their leaves, while seedlings from the natural site contained higher than average levels of

zinc (Fig 3.13) (Epstein and Bloom 2005). The amount of zinc in leaves from the natural

site were an average eight-times greater in B. menziesii and seven-times greater in B.

attenuata than levels found in the restoration treatments (P < 0.001) (Fig 3.13). The leaves

of both species from the organic and inorganic treatments had higher amounts of copper

than the natural site, being double in the organic treatment with an 85% increase in the

inorganic treatment (P < 0.001) (Fig 3.13). A large spike in manganese concentration was

observed in B. menziesii leaves from the organic treatment, which increased 135% and

193% compared to the control and natural sites, respectively (P < 0.001) (Fig 3.13).

Significant increases in potassium and magnesium were also observed in seedlings in the

organic treatment, and increases in potassium, sulfur, calcium, and magnesium were shown

in the leaves of seedlings from the inorganic treatment (Fig 3.13).

74

Natural

Control

Organ

ic

Inorgan

ic

Referen

ce

Pota

ssiu

m%

0.0

0.2

0.4

0.6

0.8

1.0

Sulfu

r%

0.0

0.1

0.2

0.3

Mag

nesi

um%

0.0

0.1

0.2

0.3

Phos

phor

us%

0.00

0.05

0.10

0.15

0.20

Cal

cium

%

0.0

0.5

1.0

1.5

Nitr

ogen

%

0.2

0.7

1.2

1.7

Natural

Control

Organ

ic

Inorgan

ic

Referen

ce

Boro

nm

g kg

-1

0

5

10

15

20

25

Man

gane

sem

g kg

-1

0

100

200

300

Sodi

umm

g kg

-1

0.0

0.5

1.0

1.5

Zinc

mg

kg-1

0

50

100

150

200

Iron

mg

kg-1

0

50

100

150

200

Cop

per

mg

kg-1

0

2

4

6

†

*

*

* *

*

*

†

† †

†

†

†

†

† † † † † †

†

†

††

Fig 3.13 Average foliar nutrient concentration of seedling leaves after two years of growth, with B. attenuata

(black), B. menziesii (grey), and a reference (white) regarded as sufficient nutrition by Epstein and Bloom

(2005). Error bars represent the standard error of the mean (n = 3). ‘†’ symbol indicates a significant

difference from the natural site, ‘*’ symbol indicates a significant difference from the control (P < 0.05).

75

Discussion

This experiment aimed to test the effects of an organic and inorganic amendment on

seedling establishment in reconstructed postmine soils, investigating any benefits these

amendments might provide to seedling survival over two years. Previous observations in

mediterranean-type postmine restoration have recorded mortality rates of > 90% after two

years (Rokich 1999). The incorporation of a native-sourced mulch (organic treatment) into

the soil resulted in an ~80% survival rate from both species over two years, an increase of

24-42% over standard restoration practices or addition of blue metal gravel (control or

inorganic treatment).

The survival of seedlings growing in organically amended soils was most likely

enhanced by access to higher amounts of soil moisture stored in the shallow root zone (50

cm depth). During the intense drought of the second year, seedlings growing in the

organically amended soil maintained stomatal conductance rates > 0.18 mol H2O m-2 s-1,

indicating seedlings were not drought stressed (Flexas & Medrano 2002). Both B. attenuata

and B. menziesii employ a “drought-avoider water-saver” strategy, relying on tight stomatal

regulation during times of low water availability (Groom, 2004). Given these Banksia

species do not leaf-shed or reach low tissue water potentials in response to drought

(Veneklaas & Poot 2003), the sustained high rates of stomatal conductance over an

extended dry period could cause xylem cavitation and eventual seedling death without a

sufficient source of soil moisture (Williams et al. 1997). In comparison, the scarce amount

of moisture located in the soils of the control and inorganic treatment during the second

summer (< 0.03 g g-1) is considered unavailable to plants in soils of this region (Carbon et

al. 1982), and is most likely responsible for the chronic water stress (stomatal conductance

< 0.05 mol H2O m-2 s-1) (Flexas & Medrano 2002) and mortality observed during this time.

Seedling water stress in the control and inorganic treatment was most likely an

indirect result of reduced root elongation. Adult B. attenuata and B. menziesii cope with

summer drought conditions by accessing groundwater reserves to depths over 7 m, and this

ability to access stored groundwater is necessary for survival (Zencich et al. 2002). Given

the soil amendments could not alleviate the cryptic soil compaction, seedlings in restoration

sites formed a branching network of roots restricted to the top 40 cm of the soil profile,

while seedlings in the natural Banksia woodland constructed a single taproot extending

deeper than 70 cm. Non-predated seedlings in the natural sites most likely survived due to

this greater root depth. Access to groundwater > 50 cm could explain the less severe drop in

76

stomatal conductance in the natural site, compared to the seedlings in the control and

inorganic treatment. However, a disruption of water-seeking root morphology does not

necessarily influence seedling survival, as the roots of seedlings in the organically amended

soil were also restricted but had access to retained moisture within the root zone. While

average soil moisture and stomatal conductance in the organic treatment was higher than

the natural site throughout the majority of the experiment, the ~20% mortality in the

organic treatment could be explained by the possible spatial variability of the amendment in

the soil, or the limited capacity of superficial water to sustain plants.

The cryptic compaction observed in all restoration sites suggests this phenomenon

is stronger than the ability of the organic and inorganic amendments to stabilize postmine

soils. The extent of compaction was reduced by the addition of organic matter, but the

difference was negligible on soil strength as it affects root access to the water table. A

higher amendment-to-soil ratio may be required to ease compaction and aid downward root

elongation; however the volume of amendment was comparable to previous experiments in

which organic matter reduced soil impedance (Barzegar et al. 2002; Larson 1972). These

experiments tested more conventional types of organic amendments (crop waste, manure)

in an agricultural setting. Given the present trial was undertaken in a biodiverse ecosystem

currently under restoration, only native mulch was considered suitable due to phytohygiene

issues. Additionally, the amendments may not have been distributed to a sufficient depth.

Root-inhibiting compaction occurred in the restoration sites at depths of 40-50 cm,

corresponding with the depth limit of amendment addition. Incorporating amendments

deeper into the profile might provide greater initial alleviation to compaction; however this

remains technically difficult for large scale restoration.

The shallow, branching network of roots observed in reconstructed postmine soils is

most likely a direct result of high impedance (Kozlowski 1999). This type of root

architecture can result in greater root-soil contact, and could be responsible for the

increased aboveground biomass witnessed in most of the restoration treatments (Arvidsson

1999; Kemper et al. 1971). However the relatively low specific root length suggests that B.

attenuata and B. menziesii expend considerable effort in developing their roots (Ostonen

2007). If this shallow, branching root architecture was unable to access available water, it

could be considered a waste of resource investment, regardless of any short-term benefit to

aboveground biomass this root structure may provide. When observed in a natural setting,

B. attenuata has the capacity to elongate their roots at a rate that maintains contact with a

77

declining water table (3.7 cm day-1) (Canham 2011). When the elongation rate of roots was

reduced by the reconstructed soils of the restoration treatments, the seedlings experienced

water stress (control and inorganic treatment only).

Fig 3.14 Excavated and washed roots of B. menziesii seedlings from the a) natural site and b) standard

restoration procedure (control) treatment within the restoration site. Note the shallow maximum depth

(<40cm) and branching architecture of the roots growing within the control.

A large investment in leaf production, shown by higher leaf surface area and lower

specific leaf area in the restoration treatments, is not the natural phenotypic state for B.

attenuata or B. menziesii. A greater total leaf surface area signifies a larger ratio of leaf

transpiring area to available water, and due to a strain on the plant water relations and

hydraulics, saplings possessing a large leaf surface area are more prone to drought damage

than seedlings that have a small leaf surface area (Varone et al. 2011). In this study,

seedlings of both species in the restoration treatments had greater leaf surface area with

equal or greater transpiration rates compared to the natural site. This may have caused the

seedlings to suffer from greater water deficits and xylem cavitation caused by higher

evaporative demand and low water availability, in addition to the carbon deficit as a result

of reduced stomatal conductance (McDowell et al. 2008). Seedlings in the natural site were

able to preserve a lower total evaporative demand by retaining a relatively small leaf

surface area. The larger leaf surface area of the restoration treatments was possibly caused

by either the greater root-soil contact, as previously mentioned, or the greater amount of

soil moisture available in the first year of growth.

78

Soil or foliar nutrient concentration amongst treatments did not explain the pattern

of seedling mortality. It is unlikely that the amendments were beneficial or detrimental to

seedling growth and survival through the exchange of nutrients, as no differences existed in

soil P, K, ammonia or nitrate between treatments. The macronutrients N, P, and K as well

as the micronutrients Na and Cu identified in seedling leaves were sufficiently lower in all

treatments than the concentrations considered ‘adequate’ for plant growth by Epstein and

Bloom (2005). B. attenuata and B. menziesii characteristically grow in regions with

leached, nutrient-deficient sands, and reduced amounts of these nutrients were also

recorded in the natural site (Salama et al. 2005). An increase in soil salinity in the

inorganically amended soil was observed by an electrical conductivity twice that of the

remaining treatments and average salinity values of the Bassendean dune system

(McArthur 1991), although the average value of 0.032 dS m-1 is relatively low and should

not adversely affect seedling health (Hunt and Gilkes 1992). The higher leaf concentrations

of Ca2+ in seedlings growing in the inorganic soil were most likely a result of the blue metal

gravel mineral components and higher electrical conductivity values (Bernstein 1975;

Coventry et al. 2001).

The majority of nutrient elements within the Bassendean dune system are associated

with organic matter (Bolland 1999), so it was expected that a greater concentration of

nutrients would be found in the leaves of seedlings from the organically amended soils.

However, only slight increases in select nutrients were identified in these seedlings. The

native organic matter used in this study might require more time for decomposition to

release nutrients, or the seedlings themselves did not accumulate more nutrients than what

was required for growth and survival. Evidence of organic matter decomposition can be

seen in the gradual acidification of organically amended soils over two years. Organic

matter is known to acidify soils by releasing H+ ions from the nitrification of NH4+ (Ritchie

and Dolling 1985), and has been show to be effective in reducing pH in postmine sands

(Jones et al. 2010). The soils of this region are naturally slightly acidic (McArthur 1991)

and the organic amendment returns soil pH to pre-mine levels that could benefit the

seedlings long-term by aiding nutrient acquisition (Sims 1986). Micronutrient deficiencies

develop in seedlings at pH levels > 8.0 due to low adsorption or precipitation reactions, and

the higher pH of reconstructed soils both in the control and inorganic treatments places

seedlings at a greater risk of inadequate nutrient acquisition (Jones et al. 2010; Sims 1986).

79

Species of the family Proteaceae, including B. attenuata and B. menziesii, produce

cluster (proteiod) roots to more efficiently ‘mine’ nutrients from leached soils (Lambers et

al. 2008b). The amount of foliar zinc has previously been found to positively correlate with

the number of proteoid roots in Hakea prostrata (Shane and Lambers 2005). However, the

amount of proteiod roots in B. attenuata and B. menziesii did not differ between natural and

restoration treatments while the foliar concentrations of zinc in the restoration treatments

were on average 89% lower than the levels found in the leaves of seedlings in the natural

site. The high zinc concentration in leaves of the natural site could be due to more efficient

zinc uptake by proteiod roots in natural soils, or a higher zinc source located within the

natural soils themselves. Proteiod roots also aid the accumulation of manganese in leaf

tissue (Foulds 1993; Shane and Lambers 2005), and may be an explanation for the

relatively high manganese values of B. menziesii in the organic treatment. Due to the

similar proteiod root biomass between treatments, it is unclear why high levels of

manganese appeared in only B. menziesii in the organic treatment.

Conclusion

From the seedling morpho-physiological and pedological results of this study, the

use of a native-sourced mulch can improve seedling function and establishment in postmine

Mediterranean-type sandy soils. The practical application of an amendment for postmine

restoration depends upon its influence on soil water retention (Jones et al., 2011), as

supported in this study, and the rhizological restrictions of the compact soil are overcome

by increasing soil moisture in the active root zone during summer droughts. The lack of

observable trends in soil or foliar nutrient concentration amongst treatments suggests that

the pattern of seedling mortality observed in this study is not due to an amendment effect

on nutrient cycling. The recurring summer droughts of mediterranean-type ecosystems are

the main cause of decline in plant function, therefore timing restoration projects

appropriately could improve their success. Synchronization with ENSO events can provide

a reliable means of maximizing precipitation to increase seedling health and establishment

(Sitters et al. 2012).

80

81

C H A P T E R F O U R

Soil physical strength rather than excess ethylene reduces root elongation

in mechanically impeded sandy soils

Introduction

Mechanically impeded (compact) soils inhibit seedling growth and development by

confining root exploration to shallow soil depths, thus significantly reducing access to

essential water and nutrients (Unger and Kaspar 1994). In addition to greater physical

strength, mechanically impeded soils can contain a higher amount of ethylene than non-

compact soils as gaseous diffusion out of the soil profile is constrained by tightly packed

soil particles (Smith et al. 1974). The entrapment of gasses other than oxygen causes

anaerobiosis, which prevents ethylene breakdown and enhances its retention (Lynch 1975).

Concentrations of ethylene found in field soils can be greater than 30 ppm which exceeds

concentrations known to regulate the growth of plants, and those known to reduce root

growth (Smith and Restall 1971; Smith and Robertson 1971; Smith 1976; Goodwin 1978).

Given an increase in soil mechanical impedance can reduce root growth as well as

increase the ethylene concentration within the soil environment, it has been speculated that

ethylene may play a role in root growth regulation in mechanically impeded soils

(Hettiaratchi et al. 1990; Atwell 1993). This assertion arises from the similarities in growth

responses of roots in both mechanically impeded soils and soils containing high amounts of

ethylene (Moss et al. 1998). In addition, Konings and Jackson (1979) found that only low

concentrations of ethylene are needed to suppress root elongation in Oryza sativa. Seedling

roots of Vicia faba and Zea mays have been shown to decrease root length and increase

endogenous ethylene production when encountering a mechanical barrier (Kays et al. 1974;

Whalen 1988; Sarquis et al. 1991) suggesting that the increase in ethylene synthesis by

plant roots grown in mechanically impeded soil is adequate to reduce root elongation,

despite the physical attributes of the soil (Konings and Jackson 1979). Furthermore, the use

of ethylene inhibitors has been shown to reverse the effects of mechanical impedance on

root elongation (Wilkins et al. 1976; Saini 1979; Moss et al. 1988).

82

A dihalobenzoic acid, 3,5-diiodo-4-hydroxybenzoic acid (DIHB), known to inhibit

endogenous ethylene production and exogenous ethylene absorption, was shown to

promote root growth of seedlings grown in high ethylene conditions (Robert et al. 1975;

Robert et al. 1976a; Jackson et al. 1984). The mode of action of DIHB most likely involves

blocking the transport of indol-3yl-acetic acid in the biosynthesis of ethylene in root tissue

(Wain 1984; Beffa et al. 1987). DIHB appears to act by modifying the cell wall

extensibility factor of roots, a process influenced by ethylene, and treated cells extend to a

greater length than untreated cells (Wain et al. 1968; Goss et al. 1987). When seedlings

grown in compact soils were treated with DIHB, increases in root elongation up to 54%

were observed without compromising the shoot growth of the seedlings (Wilkins et al.

1977; Saini 1979).

The link between excess soil ethylene, compact soils, and DIHB presents an

opportunity to investigate the amount of root inhibition that can be attributed to either

physical or chemical factors or a combination of both. Such information is vital in devising

soil reconstruction procedures during restoration programs, particularly for mining

companies who are required to create growing medium (soil) for plant establishment. No

studies have yet attempted to grow seedlings on impeded soil: previous research has used

artificial substrates to mimic mechanically impeded soils to examine the relationship

between ethylene and reduced root elongation. The aim of this study is to determine the

extent ethylene (chemical) and compact soil (physical) play in regulating root elongation,

and whether the negative consequences from these conditions can be ameliorated by

chemical manipulation using DIHB. Roots exposed to either excess soil ethylene or

compact soils are expected to increase their endogenous ethylene production rate and

decrease their root elongation. The inclusion of DIHB is expected to counteract the

negative effects of compact soil or excess ethylene on seedling roots by inhibiting or

reducing the ability of the seedling to produce and/or absorb ethylene. Here we present the

outcomes of a study into ethylene and soil compaction using the common tree, Eucalyptus

todtiana (F.Muell.), which is utilized in postmine restoration in the biodiversity hotspot of

southwest Australia.

83


Experimental Design

The study soils were sourced from postmine sites undergoing restoration and are

comprised of highly weathered siliceous sands (98.6% sand, 1.4% silt/clay) from the

Bassendean dune system (Salama et al. 2005). These dunes support a mixed woodland of

Eucalyptus and Banksia species, and are well-drained and acidic, with very low levels of

nutrients (McArthur 1991; Salama et al. 2005). The soil properties were analyzed by CSBP

Ltd. (Bibra Lake, Western Australia) to test for pH (Rayment and Higginson 1992),

electrical conductivity (EC) (Rayment and Higginson 1992), organic carbon % (Walkley

1947; Walkley and Black 1934), sulfur (Blair et al. 1991), phosphorus (Colwell 1965;

Rayment and Higginson 1992), potassium (Rayment and Higginson 1992), ammonium and

nitrate (Lachat 1992; Searle 1984) (Table 4.1).

Fig 4.1 a) The custom hammer used to achieve soil bulk densities of 1.8 g cm-3. b) An experimental tube

containing three E. todtiana seedlings.

The study species, Eucalyptus todtiana (F. Muell.), is a co-dominant framework

overstorey species used for restoration of sandplain environments in southwest Western

Australia. Seeds of E. todtiana were sterilized in 2% hypochlorite bleach, washed in sterile

water, and germinated on moistened filter paper in Petri dishes in an illuminated

refrigerated incubator set at temperature and light conditions consistent with field

germination requirements for E. todtiana (Thermoline Scientific, Australia, 15 C, PPFD

23.2 ± 1.32 μmol m-2 s-1, 12 h light/12 h dark cycle). After germination, seedlings with

84

radicles less than 5 mm long were transferred to tubes (polycarbonate, 120 ml, 108 mm

height, 44 mm diameter, Techno-Plas Pty Ltd.) for the remainder of the experiment (Fig

4.1b). The tubes contained 100 ml of autoclaved soil with bulk densities of 1.6 g cm-3 (non-

compact) or 1.8 g cm-3 (compact) (Table 4.1). In field conditions, this soil type with a bulk

density of 1.8 g cm-3 restricted root length up to 83%, while a bulk density of 1.6 g cm-3

was not restrictive to root growth (Rokich et al. 2001). Bulk densities for the compact

treatment were created by hand, using a custom hammer to compress the sand by 20 g

layers into the tubes (Fig 4.1a). To ensure the precise and repeatable construction of soil

bulk density, exactly 160 g of soil was fit into 100 mL tubes to achieve a bulk density of

1.6 g cm-3, while exactly 180 g of soil was used for a bulk density of 1.8 g cm-3.

Table 4.1 Soil physical properties and volumetric soil water content in non-compact and compact soil.

Bulk Density (g cm-3)

Particle Density (g cm-3)

Volumetric Soil Water

(cm³ cmˉ³)

Total Porosity

(%)

Air-Filled Porosity

(%)

Non-compact 1.6 2.5 0.15 36 21 Compact 1.8 2.5 0.10 28 18

A preliminary study determined the differences in the strength and root-restricting

properties of the study soil with bulk densities of 1.6 g cm-3 and 1.8 g cm-3 (Fig 4.1). Given

the small size of the experimental tubes, larger and similar-shaped pots (40 cm height x 9

cm diameter) were used to accommodate a Rimik CP20II Cone Penetrometer® (RFM

Australia Pty Ltd, QLD, Australia, Cone Diameter 12.83mm; Area 130sqmm). Ten pots

each were filled with the study soil in bulk densities of either 1.6 g cm-3 or 1.8 g cm-3,

wetted to field capacity and measured to a depth of 32 cm using the penetrometer. Seven

additional separate pots for each soil bulk density were used to grow one E. todtiana from

seed over 120 days, at which point each seedling was harvested and root depth recorded.

After the addition of soil, the tubes were moistened to field capacity with de-ionized

water. To achieve field capacity, 15 g of water was added to non-compact soils and 10 g to

the compact soil (Saxton and Rawls 2006) (Table 4.1). Particle density, total porosity and

air-filled porosity for each treatment were calculated for non-compact and compact soil

(ASTM D854-92 1992) (Table 2). Four seedlings were added to each tube, spaced 2 cm

apart, and covered with a light layer of soil (2 cm) to facilitate downward root growth.

After seven days, the number of seedlings in the tubes was reduced to three of similar shoot

85

size and leaf number, and grown for a total of 44 days in an incubator under the same

conditions as above.

Ethylene treatments

A range of ethephon concentrations, 1 μM, 10 μM, and 100 μM (Graham and

Linderman 1981; Kawase 1974), were added to tubes with non-compact soil to test the

effects of varying amounts of ethylene on root growth (Ethrel®, Bayer CropScience,

contains ethephon at 480 g l-1). Ethephon is an ethylene-releasing compound used as a

means to distribute exogenous ethylene into the soil and plant tissue (Amchem 1969), and

is absorbed by the plant where it is then catabolized to ethylene (Kawase 1974). A solution

of 0.1 μM DIHB (Alfa Aesar) was prepared according to a dose-response curve

representing the minimum concentration required to achieve a positive effect on seedling

root growth in compact soil (0.1 μM DIHB produced +15% root length, +16% root weight,

and +31% root to shoot ratio; effects of additional DIHB concentrations not shown). To test

the effects of DIHB on plant growth and ethylene production, DIHB was added to

individual identical tubes containing compact soils, or non-compact soils with or without

ethephon. Treatments consisted of 10 experimental tubes and were arranged in a

completely randomized design within the incubator.

The experimental tubes were weighed every 3-5 days and returned to field capacity

by replacing the appropriate chemical solution(s) for each treatment (< 4 mL solution per

re-watering). When the volumetric soil water content of these soils falls below 0.03 cm3

cm-3, water becomes unavailable to roots (Carbon et al 1982). Volumetric soil water

content during the experiment was always greater than 0.06 cm3 cm-3, ensuring seedlings

were neither waterlogged nor under water deficit during the study. Ethephon and DIHB

solutions were added nine times over 44 days from freshly made solutions. As ethephon is

most stable at pH < 4, each watering solution (1 μM, 10 μM, and 100 μM ethephon, 0.1 μM

DIHB, or de-ionized water) was adjusted to pH 4 with 1 M HCl. The solutions were added

carefully by syringe around the seedlings. No nutrients were added to the tubes and

seedlings utilized their endosperm (average seed mass of 6.2 mg) and residual soil nutrients

to sustain growth (Table 4.2).

86

Table 4.2 The average concentration of nutrients, organic carbon, electrical conductivity and pH of the study

soil (n = 12) with standard error in parentheses.

Nitrate

(mg kg-1)

Ammonium

(mg kg-1)

Phosphorus

(mg kg-1)

Potassium (mg kg-1)

Sulphur (mg kg-1)

Organic Carbon

(%)

Electrical Conductivity

(dS m-1)

pH

(H2O 1:5) 1.50

(±0.258) <1 <1 12.3 (±0.947) <1 0.381

(±0.104) 0.016

(±0.003) 5.77

(±0.123)

Measurements

Ethylene Detection

After 44 days of growth, endogenous root ethylene production was determined

using the method from Atwell et al. (1988). Seedlings were carefully removed from the

tubes and the soil surrounding the roots was washed off with de-ionized water. Immediately

after weighing, the entire root system from two seedlings of the same treatment were

excised and transferred to a 2.0 ml plastic vial and sealed with a new rubber #13 Suba-

Seal®. After transfer to vials, roots were maintained at 22 C in the dark during which time

ethylene was measured over 60 hours. Gas samples of 1.0 ml were withdrawn to assay

endogenous ethylene by gas chromatography, using a Shimadzu® (Kyoto, Japan) GC-8A

gas chromatograph equipped with a flame-ionization detector and a Porapak N (100-200

mesh) column, with injector and detector temperatures at 120 C. Column length was 100

cm with internal diameter of 2.0 mm and column temperature was held isothermally at 120

C. High purity nitrogen at 60 cm3 min-1 was used as the carrier gas. Ethylene was identified

by co-chromatography with an ethylene-in-air standard. The minimum ethylene detection

limit during this experiment was 3.47 nl in a 1.0 ml air injection sample. Chromatography

measurements were made at intervals of 3, 12, 24, 36 and 60 hours after root systems were

sealed within the vials. Immediately before the extraction of the gas sample, 1 ml of

ethylene free air was injected into the vials to keep the volume of gas in the vials at

equilibrium and also to reduce the likelihood of forming a vacuum in the vial. Ethylene

production values were adjusted by fresh weight of the root sample per hour and are

presented accordingly. Plastic vials not containing seedling roots (control vials) were sealed

in the same manner and tested using the same methods to determine the presence of

residual ethylene in the system. Damage to plant tissue (excising seedling roots) can

potentially create wound ethylene (Saltviet and Dilley 1979), however given that ethylene

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production was measured on a per mass basis and compared between distinct treatments,

any production of wound ethylene was negligible to the results of this study.

To detect exogenous (soil) ethylene, ‘blank’ tubes without seedlings of E. todtiana

were prepared and watered with the same treatment solutions as the seedling tubes to

quantify the amount of ethylene released by the seedling roots into the surrounding soil

(Graham and Linderman 1981). A 29-gauge syringe was inserted into the middle of the

blank tubes and adjacent to the roots of seedlings in the seedling tubes to extract gas. Ten

1.0 ml samples were extracted from each treatment and analyzed by gas chromatography in

the same manner as described above. Tubes were tested 48 hours after the most recent

chemical application and immediately prior to seedling removal.

Seedling Morphology

The fresh root and shoot masses and root lengths were measured immediately after

soil was washed from the roots and before transfer to the 2.0 ml plastic vials. For

microscopy and the measurement of cell lengths, tissue up to 10 mm behind the root tip

was fixed in 5% buffered glutaraldehyde (Feder and O’Brien 1968), then dehydrated and

embedded in JB-4 Plus plastic resin. Longitudinal sections 3 μm thick were cut on a Leica

RM 2045 microtome, stained in 0.1% toluidine blue, and mounted on glass slides.

Measurements of cortex cell lengths were made 2 – 10 mm behind the root tip using a

microscope equipped with a Nikon Coolpix 4500 Digital Camera and NIS-Elements

imaging software (Coherent Scientific Pty. Ltd. South Australia, Australia).

Data Analysis

Analysis of variances were used to compare the morphology, anatomy, and ethylene

production of the control to treatments without the addition of DIHB, and treatments with

DIHB were compared to their non-DIHB counterpart (Genstat 10th Edition, VSN

International LTD). Homogeneity of the variances was tested by residual plots. All data are

presented as untransformed means. Fisher’s unprotected LSD test was used to compare

means at the 0.05 significance level.

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Results

Soil Bulk Density

In the preliminary study to determine the effects between the two soil bulk densities,

maximum root depth of E. todtiana seedlings was reduced by 44% when the study soils

were compacted to a bulk density of 1.8 g cm-3 (P < 0.001) (Fig 4.2a). At depths lower than

2 cm, the strength of soils with a bulk density of 1.8 g cm-3 was always significantly greater

than soils with a bulk density of 1.6 g cm-3 (P < 0.001) (Fig 4.2b). Measurements deeper

than 20 cm were unable to be recorded in the 1.8 g cm-3 bulk density soils due to the

extreme soil strength (Fig 4.2b).

Ethylene Production

Exogenous ethylene was only detectable in soils from the 100 μM ethephon

treatment without DIHB present, and the amount of ethylene detected in tubes containing

seedlings (5.12 nl) was not significantly different from the amount of ethylene in the

‘blank’ tubes without seedlings (4.55 nl) (P = 0.420). Exogenous ethylene was not

detectable in any tubes of remaining treatments, regardless of DIHB addition, soil bulk

density, or seedling presence.

Fig 4.2 Comparison of a) E. todtiana seedling maximum root depth after 120 days of growth (n = 7) and b)

soil strength of the study soil (n = 10) in bulk densities of 1.6 g cm-3 (Non-compact) and 1.8 g cm-3 (Compact)

in 40 cm height pots.

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Table 4.3 Endogenous ethylene production of two freshly excised E. todtiana seedling roots measured by gas

chromatography after 12 hours, with and without the incorporation of 0.1 μM DIHB in the soil. Treatments

indicate the amount of soil ethephon; control and compact treatments contain no soil ethephon. Compact

treatment soil bulk density = 1.8 g cm-3; all other treatments soil bulk density = 1.6 g cm-3. Values are

averages (n = 9) with standard error in parentheses. Treatments without DIHB are statistically compared to

the control; treatments with DIHB are compared to non-DIHB counterpart (* P < 0.05; ** P < 0.01; *** P <

0.001).

Ethylene Production (nl/g FW/hr)

Without DIHB With DIHB

Control 18.1 (±4.40) 3.80 (±1.91)** Compact 3.60 (±2.65)* 2.00 (±2.02) 1 μM ethephon 8.80 (±1.96) 2.70 (±1.38)* 10 μM ethephon 19.6 (±1.63) 25.6 (±2.75) 100 μM ethephon 189 (±22.9)*** 363 (±164)*

The amount of endogenous ethylene produced from seedling roots was detectable in

all treatments after 12 hours of incubation within the vials, the exception being the 100 μM

ethephon treatment, where endogenous ethylene was detectable three hours after

incubation. No residual ethylene was detected in the control vials. All treatments

maintained ethylene production up to 60 hours post-incubation with maximum ethylene

production occurring at 12 hours, and declining thereafter. There was no significant

difference in maximum ethylene production in roots from the 1 μM and 10 μM ethephon

treatments compared to the control (P = 0.072 and 0.757, respectively) (Table 4.3).

Ethylene production in roots from the 100 μM ethephon treatment increased 944% (P <

0.001) and roots growing in soils with a bulk density of 1.8 g cm-3 in the compact treatment

showed an 80% decrease in ethylene production (P = 0.012) against the control (Table 4.3).

DIHB reduced the amount of ethylene production from roots in the control treatment by

~80% (P = 0.009) and the 1 μM ethephon treatment by ~70% (P = 0.021) (Table 4.3). The

addition of DIHB into the 100 μM ethephon treatment resulted in a ~90% increase in

ethylene production (P = 0.030) (Table 4.3). DIHB had no significant effect on the amount

of ethylene production in the compact or 10 μM ethephon treatments (P = 0.649 and 0.082

respectively) (Table 4.3).

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Fig 4.3 a) Average maximum root length and b) average fresh weight root to shoot ratio of E. todtiana with

or without the inclusion of 0.1 μM DIHB. Treatments indicate the amount of soil ethephon; control and

compact treatments contain no added soil ethephon. Compact treatment soil bulk density = 1.8 g cm-3; all

other treatments soil bulk density = 1.6 g cm-3. Data are presented as means (n > 20) with standard error bars.

Letters represent significant differences (P < 0.05).

Seedling Morphology

Root length decreased ~50% in seedlings grown in the 100 μM ethephon treatment

(P < 0.001) whereas the 1 μM and 10 μM ethephon treatments did not affect root length

(Fig 4.3a). The increased soil bulk density in the compact treatment decreased root length

by 44% (P < 0.001) (Fig 4.3a). Root fresh weight significantly increased by 55% in the 10

μM ethephon treatment (P < 0.001) (Table 4.4). Root cortex cell lengths were not affected

in any of the ethephon treatments (Table 4.4). Root cortex cell lengths in the compact

treatment were significantly longer by 21% (P < 0.001) (Table 4.4). Shoot weight was

significantly reduced by 20% in the 100 μM ethephon treatment (P = 0.01). Adding

ethephon to the soil, regardless of concentration, caused a significant increase in the root to

shoot ratio (P ≤ 0.007) (Fig 4.3b). The root to shoot ratio, root weight, and shoot weight of

seedlings growing in compact soil were not affected.

The addition of DIHB did not alter root length in any of the treatments imposed on

the seedlings, regardless of ethephon concentration or soil bulk density (Fig 4.3a). Root

cortex cells exposed to DIHB significantly increased by 26% in the control (P < 0.001) and

47% in the 1 μM ethephon treatment (P < 0.001) over their non-DIHB counterparts (Table

4.4). The addition of DIHB to the 100 μM ethephon treatment significantly decreased root

cell length by 18% (P < 0.001) (Table 4.4). The addition of DIHB reduced shoot weight by

16% in the 100 μM ethephon treatment (P = 0.03) (Table 4.4). DIHB significantly

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increased the root to shoot ratio compared to its non-DIHB counterpart in every treatment

with the exception of the 100 μM ethephon treatment (P: Control = 0.012, Compact =

0.015, 1 μM = 0.045, 10 μM = 0.017, 100 μM = 0.844) (Fig 4.3b).

Table 4.4 Root and shoot fresh weight and root cortex cell length of E. todtiana, with and without the

inclusion of 0.1 μM DIHB into the soil. Values are averages (n > 20) with standard error in parentheses.

Treatments indicate the amount of soil ethephon; control and compact treatments contain no soil ethephon.

Compact treatment soil bulk density = 1.8 g cm-3; all other treatments soil bulk density = 1.6 g cm-3.

Treatments without DIHB are statistically compared against the control; treatments with DIHB are compared

against their non-DIHB counterpart (* P < 0.05; ** P < 0.01; *** P < 0.001)

without DIHB with DIHB

Treatment Root Fresh Weight (mg)

Control 19.7 (±1.89) 26.9 (±3.25) Compact 18.1 (±1.35) 20.6 (±1.96) 1 μM ethephon 26.4 (±3.11) 22.9 (±2.27) 10 μM ethephon 30.5 (±2.34)*** 34.9 (±2.99) 100 μM ethephon 21.4 (±1.89) 18.2 (±2.46)

Shoot Fresh Weight (mg)

Control 64.2 (±3.70) 63.2 (±2.99) Compact 57.4 (±3.72) 48.6 (±3.06) 1 μM ethephon 61.4 (±4.64) 55.3 (±4.10) 10 μM ethephon 57.9 (±2.33) 56.2 (±2.32) 100 μM ethephon 51.7 (±2.90)** 43.4 (±2.64)*

Root Cortical Cell Length (μm)

Control 58.8 (±2.73) 73.9 (±2.66)*** Compact 71.1 (±3.62)** 71.2 (±2.27) 1 μM ethephon 64.2 (±1.91) 94.1 (±2.65)*** 10 μM ethephon 63.6 (±2.70) 67.3 (±2.46) 100 μM ethephon 53.1 (±1.85) 43.5 (±1.65)***

Discussion

Ethylene Production vs Root and Cell Length

Regardless of DIHB presence, root length in both compact and 100 μM ethephon

treatments was significantly less than the control, a result that supports previous research

(Smith and Robertson 1971; Kozlowski 1999). Unexpectedly, these two treatments differed

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from each other in ethylene production. The 100 μM ethephon treatment, simulating a high

ethylene soil, stimulated ethylene production while compact soil reduced ethylene

production. Despite these differences, both treatments created an environment that stunted

root length.

Compact soil can slow the release of ethylene gas from the soil (Smith and Restall

1971), therefore it is possible that ethylene, derived from the root and trapped in close

proximity, can accumulate and cause the stunted root length observed in the compact

treatment. The roots of seedlings growing in compact soil in the present study were

restricted to the top 30 to 35 mm of the soil profile, and the retention of a morphologically-

altering amount of ethylene in such a small area seems unlikely. Tests of exogenous

ethylene around the roots support these data, as no detectable ethylene was observed in the

soil of the compact treatment, while ethylene was observed in soils of the 100 μM ethephon

treatment. It is therefore more likely that the physical strength of the soil itself is the

explanation for the stunted root growth in the compact treatment, by physically impeding

downward root growth.

Addition of DIHB produced conflicting results in response to added ethylene.

Previous studies have shown DIHB to have an inhibitory effect on endogenous ethylene

production in cress seedlings (Lepidum sativum) (Larqué-Saavedra et al. 1975; Robert et al.

1976a; Robert et al. 1976b). This was confirmed in the present study in the low ethylene

environments such as the control and 1 μM ethephon treatments. However in high ethylene

environments (100 μM ethephon treatment), DIHB greatly increased the amount of

ethylene production from the roots, and the mechanisms for these actions are discussed in

the subsequent section. Despite the varying effects DIHB has on endogenous ethylene

production, the predicted increase in root length following the addition of this chemical was

not observed.

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Fig 4.4 Root cortex cell length vs ethylene production of two excised E. todtiana seedling roots after an

incubation time of 12 hours. Closed symbols represent treatments without DIHB, open symbols represent

treatments with 0.1 μM DIHB. Treatments indicate the amount of soil ethephon; control and compact

treatments contain no added soil ethephon. Compact treatment soil bulk density = 1.8 g cm-3; all other

treatments soil bulk density = 1.6 g cm-3. Values represent means (n > 20) with standard error bars.

E. todtiana roots that produced greater amounts of ethylene appeared to shorten root

cortical cell length, similar to the results from Le et al. (2001) that found the length of root

cells in Arabidopsis was reduced in a dose-dependent manner and reached a threshold as

seedlings were exposed to increases in the ethylene precursor 1-aminocyclopropane-1-

carboxylic acid (ACC). However results from this experiment demonstrate that ethylene

production alone should not be viewed as the main driver of root cell length; 1) ethylene

production in the 100 μM ethephon treatment was substantially higher than in the control,

yet root cell lengths between these two treatments were not significantly different, 2) cell

length increased when DIHB reduced endogenous ethylene production (control and 1 μM

ethephon treatments), 3) cell length decreased when ethylene production increased through

DIHB exposure (100 μM ethephon treatment). Atwell (1988) found that root cell lengths

decreased when grown in compact soil; however this study showed roots growing in the

compact treatment exhibited longer root cell lengths than those growing in the control. This

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increase in cell length did not contribute to overall root length in the compact treatment,

and it is possible that the compact soil decreased root cell production in these seedlings.

DIHB Efficacy and Mode of Action

The differences in ethylene production and morphological effects of DIHB raise

questions about its mode of action in vivo. DIHB is known to have an antagonizing effect

on ethylene production in soil and plant tissues (Wain et al. 1968; Jackson et al. 1984). In

the present study, both the inhibition and enhancement of ethylene production were

observed in response to DIHB. The inclusion of 0.1 μM DIHB increased ethylene

production in higher concentrations of soil ethephon, suggesting the ethylene-inhibiting

action of DIHB becomes ethylene-promoting at a soil ethephon concentration between 10

μM and 100 μM. One possible explanation is the interplay between ethylene and other plant

growth regulating hormones. Both auxin synthesis and signaling are necessary for ethylene

to impose its effect on root growth and are linked in their production, transport, and action

with the possibility of a feedback mechanism existing between the two (Burg and Burg

1966; Dugardeyn and Van Der Straeten 2008; Alarcon et al. 2009). DIHB could interact

with auxin transport (Beffa et al. 1987), thereby reducing ethylene production in low

ethylene environments (control and 1 μM ethephon treatments). However, DIHB could

facilitate a feedback loop when large amounts of ethylene are encountered exogenously

(100 μM ethephon treatment). Cytokinins also affect ethylene production in roots and could

influence the mechanism of DIHB as a plant growth regulator (Bertell and Eliasson 1992).

These plant hormones were not measured for the purposes of this study, and it remains

unclear how DIHB affects ethylene production based on our results. Reactions involving

ethylene are species-specific and depend on internal and environmental factors, creating a

complex array of interactions that suggest ethylene should be viewed as growth

modulating, rather than a stimulating or inhibiting chemical (Dugardeyn and Van Der

Straeten 2008).

Seedling Morphology

Small amounts of ethylene can naturally enhance growth in seedlings (Pratt and

Goeschl 1969), while higher doses are known to decrease the shoot growth of certain plants

(Abeles 1986), supporting observations of decreased shoot growth within the 100 μM

ethephon treatment. This reduction in shoot growth could be attributed to lower root-soil

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interaction (Kozlowski 1999), however given that reduced shoot growth was not observed

in the compact treatment, high amounts of ethephon may have caused this reduction.

Therefore, early seedling shoot growth appears to be more detrimentally influenced by the

chemical, and not physical, composition of the soil.

An increase in the root to shoot ratio was consistently observed after the addition of

ethylene, DIHB, or both, predominantly caused by a reduction in shoot weight. These

results indicate that ethephon, especially when paired with the chemical DIHB, increases

biomass allocation to belowground root production possibly through the ability of ethylene

to stimulate the production of root-growth hormones such as gibberellic acid and cytokinin

(Gaspar et al. 1996; Lorbiecke and Sauter 1999). This chemical pairing can be useful in

situations where rapid root growth or uptake of nutrients is important for seedling

establishment.

Fig 4.5 Micrographs of root cortex cells (20x) from the a) control and b) compact treatments (bulk densities

1.6 and 1.8 g cm-3, respectively). Despite a reduction in root length, root cells in the compact treatment were

on average significantly longer than the control.

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Fig 4.6 Seedlings of E. todtiana, showing root length a) without DIHB and b) with 0.1 μM DIHB. Treatments

indicate the amount of soil ethephon; control and compact treatments contain no added soil ethephon.

Compact treatment soil bulk density = 1.8 g cm-3; all other treatments soil bulk density = 1.6 g cm-3.

Exogenous Ethylene Production

Ethylene was only detectable in soils with the highest amount of added ethephon

(100 μM ethephon treatment), but differences in ethylene production and root morphology

in the 1 μM and 10 μM treatments indicate that roots of E. todtiana seedlings were sensitive

to the amounts of applied exogenous ethephon. Given the presence of seedlings did not

influence exogenous ethylene detection, any amount of endogenous ethylene released from

seedling roots was not sufficient to significantly affect the amount of ethylene in the soil.

Kinetics of ethylene released from ethephon into soil through time has been previously

documented (Graham and Linderman 1981), but attempts to replicate the data with this

sandy soil type were unsuccessful for this study. Graham and Linderman (1981) tested

ethylene release from different soil types, and observed a more rapid dissipation of ethylene

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from a peat/sand soil mix compared with peat alone. The inability to detect exogenous

ethylene in the present study is possibly due to the coarse sandy soil that could have

facilitated a more rapid diffusion of gasses.

Conclusion

The correlation of endogenous ethylene production and poor root growth in

compact soils has led to speculation that these processes are linked (Kays et al. 1974;

Sarquis et al. 1991; Okamoto et al. 2008). Moss et al. (1988) discuss the risks of using

correlative evidence to predict or assume the role of ethylene in root length under

mechanical impedance, citing the use of artificially mimicked substrates in place of

compact soils and the inability of certain ethylene inhibitors to overcome stunted root

elongation. The action and effectiveness of DIHB in inhibiting ethylene production and

restoring root length should be revisited following the contrary results shown here.

While high concentrations of exogenous ethylene stunted root growth markedly,

soil mechanical impedance induced a similar response without increasing endogenous

ethylene production from the E. todtiana seedlings or exogenous ethylene in the coarse

sandy soil. An increase in ethylene was not solely necessary to reduce root length, and the

physical properties of the sand substrate (high bulk density) were sufficiently and

independently able to inhibit root elongation. While ethylene plays a complex role in

regulating root growth, this phytohormone may not be as predominant of a factor for poor

root development in compact sandy soils as previously thought. It appears the physical

strength of the soil, and therefore the amount of force the root exerts, ultimately dictates the

rate at which roots elongate through mechanically impeded sand.

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C H A P T E R F I V E

General Discussion and Future Research

Seedling Establishment in Postmine Conditions

In Chapters 2 and 3, seedling mortality was shown to be a result of root stunting and

an inability to access sufficient soil moisture. During the establishment stage, the

morphological priority of the phreatophytic Banksia and Eucalyptus seedlings is the

development of a deep taproot, and in postmine conditions this morphological lifeline was

disrupted: roots growing through heavily compact soils were unable to elongate at a rate

sufficient to maintain contact with the retreating wetting front during the hot and dry

mediterranean-type summers. Seedlings of B. attenuata, B. menziesii and E. todtiana

suffered from intense physiological damage indicative of extreme drought stress when

subjected to postmine conditions in either glasshouse or field conditions, which

consequently had a severe negative effect on seedling survival.

The biphasic pattern of mortality published in Rokich (1999) was subsequently

observed in Chapters 2 and 3 of the present thesis: a greater percentage of seedling death

occurred after a second period of drought. In both experiments the downward elongation of

seedling roots was prevented, from either the 1.0 m depth limitation of the pots in the

glasshouse experiment, or from the heavily compact field soil within the restoration sites of

the sand quarry. This root restriction prevented access to deep soil moisture over both the

first and second dry periods, and the negative effects on seedling survival were more

apparent during a second drought. In a natural setting with non-compact soil, it is doubtful

that seedlings of these species are able reach a reliable water source within their first year,

as water table depths can range from 2.5 to 30 m and seedling roots grow ~1.5 m in their

first year (Fig 5.1) (Rokich et al. 2001; Zencich et al. 2002). The seedlings of these study

species appear to have evolved a reliance on physiological mechanisms to survive during

the first water deficit, such as high degrees of photoprotection and the isohydric properties

of the two Banksias and anisohydric properties of E. todtiana, which allows sufficient time

for roots to access soil moisture reserves (Figs 5.1, 5.2). Despite recovery after the first

drought, these drought-resistant strategies are not functional over an extended period of

time during a second drought, unless seedlings have access to adequate soil moisture (Fig

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5.2). Thus when dealing with a biphasic, or multiple, drought pattern, the survival of these

tree seedlings, and consequently the restoration of Bankia woodland, is dependent on a

consistent soil water source after the first year of establishment.

Fig 5.1 Illustration of root growth during the first two years of seedling life for B. attenuata, B. menziesii, and

E. todtiana, grown in compact or non-compact soil. Seedling roots in compact soil are impeded at a shallow

depth, and create a network of roots in the top layer of soil. Winter rains give seedlings access to surface

water. During the First Summer, the depth of soil water decreases out of reach of seedling roots, regardless of

soil type. During the Second Summer, seedlings that are unable to access deeper soil moisture reserves are at

a greater risk of water deficit and death. Fig 5.2 presents a detailed description of seedling physiology over

the same time period.

Despite the differences in ecophysiological mechanisms of drought resistance

between species, such as a down-regulated photosystem in B. attenuata seedlings or a

reduction in water potential by E. todtiana, no differences in survival were observed

between the three species under glasshouse conditions or between the two Banksias grown

in postmine sites in the field. Drought stress and the restriction of seedling roots by natural

or artificial means generated an equally negative response to seedling survival between the

species of B. attenuata, B. menziesii, and E. todtiana. Therefore restoration must aim for

the re-introduction of all three species to re-create the Banksia woodlands. To do this, the

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construction of favorable site conditions are required to effectively establish B. attenuata,

B. menziesii, and E. todtiana seedlings, and techniques to resolve and alleviate the stresses

associated with postmine sites must be priority in restoration programs within the Banksia

woodlands.

Fig 5.2 Physiological responses to a biphasic drought pattern in a) the isohydric B. attenuata and B. menziesii,

and b) the anisohydric E. todtiana. As drought progresses, each box represents a hydration threshold where

specific physiological responses or damage were observed, dependent on first or second water deficit.

Increasing Seedling Survival through Ecophysiological Analysis

Raising water availability within the root zone

When sufficient water was retained within the root zone, seedlings growing in

compact soil exhibited increased rates of physiological function and a large percentage of

seedlings survived past the second dry period. Root depth was still severely restricted to the

top 40 cm of the soil profile, but sustaining amounts of moisture were recorded in the root

zone of the well-watered compact soil treatment in the glasshouse trial, and in the organic

treatment in the field trial. This indicates that the constraints of compact soil on root

morphology are therefore only indirectly responsible for the decreases in seedling health

and establishment. In the short term, the most important environmental factor affecting

seedling survival in postmine soils within mediterranean-type ecosystems is not the rapid

development of a deep taproot, but the presence of a sufficient amount of water in the root

zone of the soil profile.

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In the field, an increase in the water retention of postmine soils was achieved

through the addition of an organic soil amendment at depth. While no benefits were

observed by using the inorganic gravel as an amendment, finely mulched organic matter

improved seedling establishment by 24 – 42%, not by aiding downward root elongation,

but by raising the water holding capacity of the reconstructed postmine soils. By

monitoring gas-exchange rates, seedlings growing in the organically amended soil were

shown to consistently outperform seedlings from the control and inorganically amended

restoration treatments that exhibited gas-exchange rates distinctive of high drought stress

during the dry summers. Given the organic matter in this experiment consisted of a native-

sourced mulch cleared prior to mining, this technique presents a practical and successful

use for a resource that might otherwise be wasted.

Fig 5.3 A visual account of understorey species suppression in the organic and inorganic soil amendment

treatment plots from Chapter 3. The boundary and name of each treatment is labeled in red.

Compact soil forces phreatophytic species to share a similar root zone, and therefore

access the same water sources, as understorey Banksia woodland species that do not

naturally develop a deep taproot. Many of these species do not function with the same

water-saving adaptations that the phreatophytic study species employ. In particular, fast-

growing weeds and native annual species are ‘drought-escapers’, completing their entire

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life-cycle before the most intense period of drought (Ludlow 1989). Without the ability to

regulate water loss, these drought-escapers could potentially squander soil moisture

essential to Banksia and Eucalyptus seedling survival (Cohen 1970). To ensure maximum

soil moisture is available to the seedlings over the first two summers during the

establishment stage, invasive weeds, and initially all understorey species, should be

excluded from postmine restoration sites that experience seasonal drought. The organic and

inorganic amendments used in Chapter 3 could initially suppress the colonization of

invasive weeds and understorey species in postmine restoration, as all treatment plots

containing these amendments were visually observed with less plant cover (Fig 5.3).

However due to time and technique constraints, any possible benefits of secondary species

suppression through the use of soil amendments was unable to be confirmed for the

purposes of this thesis. Weedy species are a known major impediment to seedling

colonization within restoration sites along the Swan Coastal Plain, and if left alone can

prevent native seedling establishment in postmine sites (Fig 5.4). The use of herbicides or

surface mulching could also exclude these species from restoration sites, increasing the

amount of soil moisture in the root zone that is critical for seedling establishment (Ashby

1997; Rokich 1999).

Fig 5.4 A fresh postmine Banksia woodland site on the left, with an older postmine site colonized by weedy

invasive species on the right.

Protection of seedling photosystem

A stable and resilient PSII observed in all three species helped prevent

photoinhibition through the dispersion of excess energy by increasing leaf electron

transport rate, but this protection mechanism appeared to breakdown during the second

drought phase. Photoprotection is necessary in high-light and high-temperature

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environments such as the woodland floor of the sparse-canopy Banksia woodland

community (Beard 1989; Demmig-Adams and Adams 1992), and would be especially

beneficial in postmine restoration conditions where a forest canopy is unavailable to shield

seedlings from the sun.

Over two summers in the non-compact soil of an urban bushland site, Close et al.

(2009) observed favorable trends in survival between transplanted seedlings of B. attenuata

and B. menziesii grown under shade conditions compared to seedlings in direct sunlight.

The seedlings grown under shadecloth also exhibited greater photosystem efficiency during

the second summer, suggesting that the high light and heat conditions of the Banksia

woodland floor can cause photoinhibition in these seedlings (Close et al. 2009). The use of

shadecloth in Banksia woodland restoration is feasible, as the optimum tree density is one

tree species per 4.5 m2 (Rocla Quarry Products® pers. Comm.), making installation an

affordable restoration technique in this ecosystem. The lower intensity light provided by the

shadecloth will create a more favorable microclimate and offer additional photosystem

protection, especially during the second dry period when the seedlings of these species

were shown to experience a greater vulnerability to intense photodamage.

Future Research Directions

Seedling ecophysiology

The research set forth in this thesis poses further ecophysiological questions

pertaining to drought and soil compaction. The exact physiological mechanism(s) behind

the increased mortality during a second dry period is still unclear. One theory lies in

accurate measurement of hydraulic failure within seedlings over multiple droughts. Critical

leaf water potential was shown to occur between 20 and 50 percent loss of conductance in

B. attenuata and B. menziesii adults through the creation of percent loss of conductance

(PLC) curves (Froend and Drake 2006). Inter- and intraspecific differences in PLC curves

can occur under drought stress in the two Banksias (Canham et al. 2009), so it seems

probable that PLC curves in seedlings would differ from that of adults, and also between

periods of multiple droughts. However, given the destructive nature of xylem water

potential measurements in seedlings, measurement of hydraulic failure was not the main

focus of this study. A controlled experiment investigating the water potential characteristics

of the seedlings of these species could hold clues about how xylem water transport shapes

the biphasic pattern of seedling mortality observed in this system.

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Understanding the depth of seedling water acquisition within the soil profile could

have profound effects on the success of restoration under postmine conditions and also in

areas where drought presents a barrier to seedling establishment. If the roots of Banksia and

Eucalyptus seedlings growing in a natural woodland do not elongate rapidly enough to

make contact with the water table before the first summer, it would be pertinent to identify

the depth from which they are able to draw water during this initial drought (Fig 5.1). A

similar question can be asked of seedlings growing in compact soil at a postmine

restoration site, with or without water-retaining soil amendments (Fig 5.1). Finally, does

the depth from which seedlings access water differ between the first and second summers in

both natural and postmine restoration environments (Fig 5.1)? Through the analysis of

hydrogen isotope ratio in xylem water, Zencich et al. (2002) was able to demonstrate the

soil depths where adult B. attenuata trees accessed groundwater throughout the different

seasons of the year. Deep groundwater was mainly utilized during the dry summers when

surface water is unavailable, but water uptake from shallow depths was more prevalent

after winter rains recharged the soil (Zencich et al. 2002). Given root zones differ between

seedlings and adults (Donovan and Ehleringer 1992), as well as seedlings growing in

compact and non-compact soils, investigating the depth at which seedlings access water

could be useful in understanding the basic physiological function of these species and aid in

postmine restoration (Ehleringer and Sandquist 2006).

Postmine soil enhancement

Seedling establishment was improved during the critical second year of growth

through the benefits of an organic amendment on soil water potential. However due to the

time limitations of this study, monitoring past the second year was not possible, and the

long-term effects of an organic soil amendment on seedling establishment is still unknown.

Successful restoration is an on-going endeavor, and to provide definitive proof of the

effectiveness of organic amendments in postmine restoration, seedling physiology, root

growth, and survival must be investigated over a longer period of time.

The organic amendment reduced the effects of drought stress on seedlings, but the

cryptic soil compaction phenomenon was unable to be prevented by the soil amendments

and severely restricted root growth, potentially presenting a long-term negative barrier to

seedling survival (Zisa et al. 1980). Thus the underlying chemical and physical mechanisms

behind seedling root elongation were explored through the use of ethylene and the ethylene

106

inhibitor, DIHB, in an effort to manipulate seedling physiology to aid root elongation

through compact soil. Results from this study confirmed that the physical properties of soil

are independently sufficient to severely stunt root elongation, and further investigation into

the causes of cryptic soil compaction are needed. Soil particle re-arrangement can occur

due to postmine soil reconstruction and/or alternating wet-dry cycles of a mediterranean-

type climate, which can re-deposit clay particles between the pores of larger sand grains,

and is a known contributing factor in raising soil bulk density and limiting root growth

(Arunachalam et al. 2004; Dikinya et al. 2006a; Dikinya et al. 2006b). Detailed

investigation of pedological processes was beyond the scope of this thesis, but knowledge

of the causes behind cryptic soil compaction will provide useful insights into soil

remediation opportunities in postmine conditions.

While more soil-based research is needed to comprehend the cryptic soil

compaction observed in postmine restoration, there appears to be potential in the use of

organic amendments to combat this phenomenon. A concentration of 12% organic material

to soil ratio reduced the rate at which the soil strength increased, and a higher volume of

organic matter could raise the resistance of soils to compactive forces even further

(Barzegar et al. 2002). The physical structure of the soil must be altered to mitigate

compaction in sandy soils, and the incorporation of a greater percentage of organic matter

has potential to become a useful technique to alleviate soil-densification in reconstructed

postmine soil. Additionally, research involving the development of techniques to

incorporate amendments deeper into the subsoil profile by deep tillage or other means

would enhance soil quality by providing a larger volume of organically amended soil for

seedlings to explore (Chong and Cowsert 1997; Dunker et al. 1995).

The restoration of soil through the use of plants presents an alternative method to

alleviate soil compaction through the initial introduction of a pioneer species immediately

following mining operations. The introduction of a deep-rooted and fast-growing species

can loosen the soil by creating channels and pores for the roots of native seedling to

explore, a process known as biotillage (Drury et al. 1991; Kayombo and Lal 1993; Rautaray

2011). Selection of an appropriate species depends on the specific restoration goals (Dua et

al. 2002), and the genus Austrostipa is a short-lived native grass that could provide soil

bioremediation qualities for postmine restoration. Immediately prior to topsoil replacement

and seed broadcasting for native revegetation, the aboveground biomass of these biotillage

species can be plowed into the ground, effectively ripping the soil, an already standard

107

procedure at many postmine sites. This replaces any organic matter in the soil profile lost

due to mining activity, thereby increasing soil water retention for native seedlings,

lessening soil-densification, and creating a more favorable environment for native species

establishment.

Conclusion

This thesis demonstrates the first comprehensive study of seedling ecophysiology in

response to postmine environments, and utilizes the results to develop novel techniques to

guide the restoration of the Banksia woodland community. Previous studies observed

seedling mortality in postmine environments, however there was little understanding of the

physiological mechanisms behind these deaths. The monitoring of seedling ecophysiology

under postmine conditions has increased our knowledge of Banksia woodland ecology and

provided strategies to manage the restoration and conservation of this diverse and

threatened biome. While this thesis focuses on three specific species in southwest Australia,

soil compaction and drought pose major threats to the seedling establishment of many

different species in postmine sites throughout the world, especially those found within

mediterranean-type ecosystems. The ecophysiological results and restoration techniques

explored in this thesis can be used as a template for degraded sites in other ecosystems

experiencing seasonal drought or soil compaction that impact upon the natural

establishment of seedlings.

Key findings from this study include:

1. Cryptic soil compaction is a measurable occurrence within sand quarries along the

Swan Coastal Plain of southwest Western Australia. This phenomenon has the

ability to impede the root elongation of phreatophytic tree species during postmine

restoration projects, and subject them to water stress and early death.

2. The postmine stresses of drought and soil compaction occurring simultaneously

cause greater damage to seedling health and survival than each stress acting alone.

3. Isohydric and anisohydric species rely on different physiological mechanisms to

resist drought, but there is no distinguishable survival advantage between each

108

strategy throughout two rounds of water withholding (biphasic drought), or

simultaneous drought and soil compaction.

4. The seedlings of species in this study, B. attenuata, B. menziesii, and E. todtiana,

are detrimentally and equally affected by the postmine stresses of drought and soil

compaction. The framework method is therefore not applicable with these species in

this restoration setting, and site conditions must be manipulated beforehand.

5. Despite reducing root elongation, excess ethylene is not the cause of stunted roots

growing in compact sand. The physical strength of soil is sufficient to impeded root

growth, and must be alleviated when restoring postmine compact sand.

6. Native trees and brush can be cleared prior to mining activity and used as a much

for amending postmine soil. Although the 10% v/v trialed in this study will not

alleviate cryptic soil compaction in sand quarry restoration projects, it will increase

soil water retention sufficiently to raise the health and establishment of seedlings

during summer droughts.

Fig 5.5 The emerging cotyledons of a recently germinated B. menziesii seedling, growing in a postmine

restoration site.

109

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Banksia woodland background · Banksia woodland community (Fig 1.1) is the center of distribution...

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