Crop residue phosphorus: Speciation and release in

cropping soils

A thesis submitted to the University of Adelaide

in fulfilment of the requirements for the degree of Doctor of Philosophy

SARAH RUTH NOACK

School of Agriculture, Food and Wine

University of Adelaide

12th February 2014

TABLE OF CONTENTS

Abstract……………………………………………..………………..………..…….. iv Declaration………………………………………………………….…………..….... vii Acknowledgements…………………………………………………..………..…….. viii Statement of authorship…………………………………….…………..…..….…… x Structure of this thesis…………………………………..…………………..….…… xii Chapter 1. Review of the literature……………………………………….……...… 1 Introduction………………………………………………………….……..….… 3 Fate of crop residue P in arable soils……………………….………....…..……... 4 Release of soluble P …………………………………………………….….. 5 Microbial processing of crop residues………………………………....…… 6

Immobilisation of P in crop residues by the microbial biomass…..…… 7 Mineralisation of microbial P……………………………………....….. 7 Sorption of P released from crop residues in soils………….…..….….. 9

Crop residue management and P availability…….………………………....…… 10 Timing and quantity of P release from crop residues….…………………… 10 Crop uptake of P from crop residues…………………..…………………… 12 Effect of tillage on release of P from crop residues………………………… 12 Chemistry of phosphorus in crop residues……………………………….…....… 14 Predicting immobilisation and mineralisation from crop residues…....…… 14 Phosphorus species in plant materials….……………………………..….… 16 Inorganic P………………………………………………………..….… 17 Phospholipids………………………………………………….…..…… 18 Nucleic acids.………………………………………………….…..…… 18 Ester P………………………………………………………….….…… 19 Residual P….………………………………………………….…..…… 19 Effect of plant P status on P speciation……………………………..…….… 19 Methods for measuring the release of P and characterising P forms in crop residue………………………………………………………………………….… 22 Sequential chemical P fractionation…………………………………..…..… 23 Solution 31P nuclear magnetic resonance (NMR) spectroscopy…………..… 24 X-ray absorption near edge structure (XANES) spectroscopy………...….… 26 Isotopic labelling……………………………………………………….…… 27 Objectives of this research……………………………...…………………..….… 28 References…………………………………………………………….…….….… 29 Chapter 2. Crop residue phosphorus: Speciation and potential bio-availability 37 Abstract…………………………………………………………………………… 41 Introduction………………….……………………………………………..…..… 42 Materials and methods……………………………………………………..…..… 42 Results……………………………………………………..…………………...… 44 Discussion…………………………………………………………….…….....…. 48

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Conclusions…………………………………………………...………………..… 50 References……………………………………………….………………..……… 50 Chapter 3. Assessing crop residue phosphorus speciation using chemical fractionation and solution 31P nuclear magnetic resonance spectroscopy………….…………… 53 Abstract……………………………………………………..………………….… 57 Introduction……………………………………………………………...………. 58 Methods…………………………………………………………………….….... 60 Results and discussion………………………………………….………..….…… 65 Conclusions………………………………………………………………….…… 78 References……………………………………………………………….….….… 79 Chapter 4. Phosphorus speciation in mature wheat and canola plants as affected by phosphorus supply…………………………………………………………...……… 81 Abstract.…………………………………………………………….………….… 85 Introduction…………………………………………………………….….…...… 86 Materials and methods…………………………………………….…….……...… 87 Results………………………………………………………..…….……..……… 89 Discussion……………………………………….………………………….….… 93 Conclusions……………………………………………………………...….....…. 95 References…………………………………………………………………..….… 96 Chapter 5. Management of crop residues affects the transfer of phosphorus to plant and soil pools: Results from a dual-labelling experiment ……………………...… 99 Abstract…………………….…………………………….……………………..… 103 Introduction…………………………….…………………………………….…… 103 Methods……………………………………………………………….………..…. 104 Results………………………………………………….……………………….… 106 Discussion…………………………………………………….…………........…… 107 Conclusions………………………………………………..……..……..…….…… 110 References………………………………………………………………………… 110 Chapter 6. Summary, conclusions and future research priorities………..……… 113

Crop residue P speciation using 31P NMR………………………….……….…… 116 Comparing and combining solution 31P NMR and chemical fractionation methods……………………………………………………………...…..……..… 119 Relationship between plant P status and P speciation…………………..……..… 120 Residue management affects residue P release………………………...……….. 122 Future research directions/priorities………………………………….………...… 125

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ABSTRACT Crop residues remaining after grain harvest are an important potential source of nutrients,

including phosphorus (P), to the cropping system. Crop residues contain both inorganic

and organic forms of P and these forms may take different pathways into soil P pools. The

rate and quantity of residue P released depends partly on the specific P compounds in the

residues. The most commonly used measure of P in crop residues is total P, followed by

separate measurement of inorganic P and organic P. These measures do not speciate

residue P into specific compounds and consequently, residue P dynamics in soils remains

poorly understood.

This thesis characterises P contained in crop residues using solution 31P nuclear magnetic

resonance (NMR) spectroscopy and compares this technique with conventional chemical

fractionation methods. These initial analytical studies provided the basis for subsequent

investigations of the effect of plant P status and residue management on release of residue

P in a soil-plant system, leading to a better understanding of the potential bioavailability of

residue P in soil.

Inorganic and organic P forms were quantified using 31P NMR spectroscopy in different

plant components (stem, chaff and seed) collected from field grown cereal and legume

crops. The main forms of P detected in stem and chaff were orthophosphate (35-75%) and

the easily degradable organic P forms, phospholipids (10-40%) and RNA (5-30%). The

majority (65-90%) of P in stems was water-extractable, and most of this was detected as

orthophosphate. This indicated that the majority of residue P in aboveground plant residues

has the potential to be delivered to soil in a form readily available to plants and soil

microorganisms.

iv

An integrated approach combining spectroscopic techniques with chemical extraction

assisted with verifying assumptions made when using chemical fractionation methods. The

main assumptions investigated were; the selectivity of chemical extractants for a single P

species, the ability of the extractant to bring all of the P form into solution, and to examine

if other P species were released into solution or if the P speciation was changed with

extraction. The results showed that the orthophosphate concentration in water/acid extracts

was increased due to the hydrolysis of pyrophosphate and organic P species, but decreased

due to incomplete recovery of orthophosphate from the crop residues. These effects largely

cancelled each other out. Treatments widely used to extract phospholipid (extraction with

ethanol:ether and ethanol:ether:chloroform), were found to be selective for phospholipid P,

but were quite ineffective, with only ~10% of the phospholipid P determined by solution

31P NMR extracted in each case. These results strongly suggest that speciation of crop

residue P using chemical fractionation can be compromised by the incomplete recovery of

a given P species and the transformation of other P species during extraction.

As plants approach maturity and start to senesce, the primary sink for phosphorus is the

seed but it is unclear how plant P status affects the resulting P concentration and speciation

in the seed and remaining plant parts, i.e. the residues. Wheat and canola grown in the

glasshouse were supplied three different P rates (5, 30 and 60 kg P ha-1 equivalent)

designed to represent deficient, adequate and luxury levels of P. The speciation of P in

roots, stem, leaves, chaff/pod and grain was examined. Stems and leaves, which contribute

the bulk of post-harvest residue P, were dominated by orthophosphate, regardless of plant

P status. Minor differences were observed in P speciation across the three P application

rates and plant parts. The effect of this on P cycling is likely to be relatively minor in

comparison to the overall contribution of these residues to soil P pools.

v

Release of nutrients, including P from crop residues remaining post-harvest is an important

potential source of nutrients for subsequent crops. The effect of residue size and placement

of field-collected pea residue on subsequent P uptake by wheat, soil hexanol-released P

and resin-extractable P was measured in a glasshouse experiment. On average, > 50% of

residue P was detected in plant, microbial and resin P pools when incorporated in soil

compared to 20% for the two surface-placed residue treatments. When considering how

residue management strategies may influence P supply to crops, incorporating residues

will increase the rate of release and decomposition and therefore the potential for plant

roots (if present) to access this P. The results also indicate that even though residue P takes

longer to break down under no-till management, this system will still provide small but

agronomically significant amounts of P to subsequent crops.

vi

DECLARATION This work contains no material which has previously been accepted for the award of any

degree or diploma in any university or other tertiary institution and, to the best of my

knowledge and belief, contains no material previously published or written by any other

person, except where due reference has been made in text.

I give consent to this copy of my thesis when deposited in the University Library, being

made available for loan and photocopying, subject to the provisions of the Copyright Act

1968.

The author acknowledges that copyright of published work contained within this thesis (as

named in “Publications arising from this thesis”) resides with the copyright holder(s) of

those works.

I also give permission for the digital version of my thesis to be made available on the web,

via the University’s digital research repository, the Library catalogue, the Australian

Digital Theses Program (ADTP) and also through web engines, unless permission has been

granted by the University to restrict access for a period of time.

Sarah Noack Date

vii

ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisors, Mike McLaughlin, Ron Smernik,

Therese McBeath and Roger Armstrong for their guidance, support and encouragement. I

feel incredibly lucky for the opportunities I have been provided with throughout my

candidature. I am very grateful for the considerable effort and time they have spent helping

me complete this project.

Many thanks must also go to Caroline Johnston, Bogumila Tomazk, Colin Rivers, Ashlea

Doolette and Sean Mason for their invaluable help regarding analytical methods.

I would like to acknowledge the generous project funding from the Grains Research and

Development Corporation (GRDC), through project DAV00095 and travel award funding

from the Crop Nutrition Trust, Australian Federation of University Women – South

Australian branch, and the Australian Agronomy Society.

I would also like to express my gratitude to the many other members of the Soils Research

Group who offered advice and assistance during my PhD. In particular, special thanks

must go to Ashlea, Casey, Brooke and Emma for the great times, fond memories and

welcomed distractions from long days of experiments and writing.

Lastly special thanks to Kym, for keeping me on track throughout these last months. Thank

you for your endless support and encouragement.

viii

PUBLICATIONS ARISING FROM THIS THESIS

Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M., Armstrong, R.D., 2012.

Crop residue phosphorus: speciation and potential bio-availability. Plant and Soil

359, 375-385.

Noack, S.R., McBeath, T.M., McLaughlin, M.J., Smernik, R.J., Armstrong, R.D., 2014.

Management of crop residues affects the transfer of phosphorus to plant and soil

pools: Results from a dual-labelling experiment. Soil Biology & Biochemistry (in

press) 71, 31-39.

Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M., Armstrong, R.D., 2014.

Phosphorus speciation in mature wheat and canola plants as affected by phosphorus

supply. Plant and Soil online first.

ix

STATEMENT OF AUTHORSHIP Components of the research described in this thesis have been published or have been

submitted for publication (as listed below). The contribution of each author to these works

is described below.

Chapter 2: Plant and soil; 2012, 359, 375-385.

Chapter 3: Talanta; submitted

Chapter 4: Plant and soil; accepted for publication

Chapter 5: Soil Biology and Biochemistry; accepted for publication

NOACK, S.R. (Candidate)

Experimental development, performed analysis on all samples, data analysis and critical

interpretation, wrote manuscript.

I hereby certify that the statement of contribution is accurate.

Signed Date

McLAUGHLIN, M.J.

Supervised development of work, data analysis and interpretation, reviewed manuscript.

I hereby certify that the statement of contribution is accurate.

Signed Date

SMERNIK, R.J.

Supervised development of work, data analysis and interpretation, reviewed manuscript.

I hereby certify that the statement of contribution is accurate.

Signed Date

x

McBEATH, T.M.

Supervised development of work, data analysis and interpretation, reviewed manuscript.

I hereby certify that the statement of contribution is accurate.

Signed Date

ARMSTRONG, R.D.

Supervised development of work, data analysis and interpretation, reviewed manuscript.

I hereby certify that the statement of contribution is accurate.

Signed Date 04/04/2014

xi

STRUCTURE OF THIS THESIS This thesis is presented as a combination of papers that have been published, accepted or submitted for publication. Chapter 1 provides an overview of the literature on residue P chemistry, fate in cropping soils and methods for the determination of residue P. This chapter also includes the proposed objectives of the research presented in this thesis. Chapter 2 comprises a paper published in Plant and Soil. It describes the application of solution 31P NMR spectroscopy to speciate P forms in various field-collected crop residues. Chapter 3 describes an experiment to compare and combine chemical fractionation methods with solution 31P NMR spectroscopy for the speciation of P in crop residues. This work has been prepared as a manuscript and submitted to Talanta. Chapter 4 comprises a paper that has been accepted for publication in Plant and Soil. It describes a glasshouse experiment used to determine the effect of plant P status on resulting P speciation in mature wheat and canola plant parts. Chapter 5 comprises a paper that has been accepted for publication in Soil Biology and Biochemistry. The experiment used a dual labelling approach (33P and 32P) to measure the effect of size and placement of field-collected pea residue on plant and soil P pools. Chapter 6 provides a synthesis of the findings contained in this thesis and includes recommendations for future work.

xii

CHAPTER 1 REVIEW OF THE LITERATURE

1

2

1. Introduction Phosphorus (P) is an essential nutrient required for plant growth. In soil only a small

proportion of this P is found in the soil solution (e.g., 0.01-3.0 mg P L-1) or in forms

available to crop plants at any given time (Frossard et al. 2000). To improve crop

production, Australian agriculture has long been dependent on the use of P fertilisers

(Donald 1964; McLaughlin et al. 2011). Phosphorus fertiliser application represents a

significant cost for agricultural production and strategies to reduce fertiliser inputs have

important economic benefits for growers.

Release of nutrients, including P from crop residues remaining post-harvest, is an

important potential source of nutrients for subsequent crops (Blair and Boland 1978;

McLaughlin et al. 1988b). The most commonly used measure of P in crop residues is total

P, followed by the distinction between inorganic P and organic P. The carbon (C):P ratio of

crop residues has also been widely used to predict potential P immobilisation or

mineralisation (Fuller et al. 1956; Barrow 1960; Blair and Boland 1978; Kwabiah et al.

2003). However, these measures do not identify or take into account the various P species

found within crop residues. Speciation is important for the estimation of P release from

crop residues in cropping soils, as some species of P in residues may be more recalcitrant

than others. Phosphorus is known to occur in several forms in living plants (Bieleski 1973;

Mengel and Kirkby 1982). However, knowledge of how these P forms change as plants

senesce (Batten and Wardlaw 1987a) is limited, with most research in this area focused on

fresh or immature plant materials rather than the senesced materials that are returned to soil

in cereal cropping systems (Miyachi and Tamiya 1961; Barr and Ulrich 1963; Bieleski

1968; Kakie 1969).

Previous studies have demonstrated some effects of residue management on the

distribution of P through the soil profile (Zibilske and Bradford 2003; Bünemann et al.

3

2006). The clearest demonstrations of such effects arise from the comparison of soils under

full tillage (full inversion tillage using a plough) versus no-tillage systems (direct seeding

e.g. using discs and knifepoints). No-till cropping has been adopted by some growers for

many years, but for large parts of Australian cropping regions the uptake of no-till farming

systems is relatively recent and continuing to increase (Llewellyn et al. 2012). With

increasing adoption of no-till methods and a reduction in full tillage and the burning of

residues, it is important for growers to understand the soil fertility and plant nutrition

benefits of retaining residues without incorporation, including for P cycling.

To fully understand the contribution of residue P as a source of plant P for agricultural

production, its chemical nature needs to be understood. This is a prerequisite to further

understand the dynamics of residue P in the soil ecosystem, and its bioavailability to plants

and microorganisms. This review summarises the current knowledge of crop residue P,

factors affecting the P status of crop residues and its biochemical cycling, and outlines the

techniques available for characterisation of P in crop residues. Better identification of P

species in crop residues can improve the understanding of the potential turnover of these P

species in soil, leading to a better assessment of the amount of P that may be provided for

subsequent crops.

2. Fate of crop residue P in arable soils

The above ground or shoot portion of crop residues can remain on the soil surface or be

incorporated after a crop is harvested or grazed, while the roots remain below the surface

in both scenarios. The P within these residues can be released to soil as soluble P,

assimilated by microorganisms, or it can contribute to more chemically stable P pools in

soil (Figure 2.1). Crop residue speciation plays an important role in determining the

partitioning of residue derived P into these three pools. The net effect of crop residues on

4

soil P availability will depend on the balance of these processes and the outcome is often

unclear due to the complexity of P cycling in soil.

Figure 2.1 Role of residue P in plant-soil P cycling. Adapted from Stewart and Tiessen (1987) 2.1 Release of soluble P

In general, 40-80% of P in crop residues is soluble in water and weak acid, although this

varies significantly between studies and residue types (Bromfield 1960; Birch 1961; Barr

and Ulrich 1963; Floate 1970a; Martin and Cunningham 1973; White and Ayoub 1983;

McLaughlin et al. 1988b; Iqbal 2009). The majority of this soluble P is generally assumed

to be inorganic but there has been little work looking into the organic P in this fraction (He

et al. 2009). However, reported inorganic P concentrations in plant material may be

overestimated because some organic P may be hydrolysed in acid extracts, or through the

release of enzymes in water extracts. Plant residues contain enzymes that have the potential

to be released into water extracts. Numerous enzymes have been shown to catalyse the

release of orthophosphate from a wide range of organic P compounds (Crowther and

Westman 1954; Tadano et al. 1993; Bishop et al. 1994).

Residue P

Microbial P

Labile P (Organic)

Plant

Stable Organic P

Soil Solution P (Organic &

Inorganic)

Labile P (Inorganic)

Stable Inorganic P

5

Birch (1961) reported that as much as 50-80% of plant tissue P may be soluble and readily

released into soil solution after incorporation into soil. Similarly, Jones and Bromfield

(1969) reported that 69-78% of the total P in ground phalaris (Phalaris tuberose L. now

known as Phalaris aquatica) and subterranean clover (Trifolium subterranean L.) was

water soluble. Furthermore, they reported up to 90% of the initial inorganic P in these plant

residues was water soluble. Martin and Cunningham (1973) also found a rapid release of P

from plant materials (wheat roots) in which nearly 55% of the total P in fresh roots was

water-soluble. This suggests that the bulk of crop residue P has the potential to be brought

into solution under field conditions after the first significant rainfall. When this soluble P is

released into soil solution, it can be utilised by plant roots and soil microorganisms, sorbed

onto soil particles or precipitated with cations.

2.2 Microbial processing of crop residues

Microbial processes in the soil influence the distribution of P between various inorganic P

and organic P forms and consequently affect the potential availability of P for plant

acquisition (Stewart and Tiessen 1987; McLaughlin et al. 1988b; Magid et al. 1996;

Oberson et al. 2001; Jakobsen et al. 2005). There are comprehensive reviews of microbial

biomass P and factors affecting size and measurement (Richardson 1994; Dalal 1998;

Jakobsen et al. 2005; Oberson and Joner 2005). This section focuses on the incorporation

of crop residue P into the soil microbial pool.

For the soil microbial population to proliferate there needs to be adequate supply of C,

nitrogen (N) and P to assimilate and drive growth. In terms of residue decomposition,

sugars, starch, simple proteins, and cellulose are much easier to decompose compared to

waxes, lignin and phenolic compounds (Parr and Papendick 1978; Collins et al. 1990). As

mentioned above, crop residues contain both inorganic and organic forms of P, suggesting

6

there are a variety of P forms available for microbial uptake and decomposition in crop

residues. A net release of inorganic P to the soil (mineralisation) will occur if microbial

demand for P is less than the quantity of P contained in the residue, but immobilisation will

occur if microbial demand for P exceeds the quantity of P present. Early work by Lockett

(1938) showed that both mineralisation and immobilisation can occur simultaneously.

2.2.1 Immobilisation of P in crop residues by the microbial biomass

Where P is limiting and C is non-limiting, soil microorganisms can immobilise residue P

and soil P during decomposition of low-P plant materials. In a growth chamber study,

McLaughlin and Alston (1986) found that P held in the microbial biomass was

considerably higher in soils which had received medic residues. In a subsequent field

experiment, McLaughlin et al. (1988b) found 29% of medic P was incorporated into

microbial biomass 95 days after residue addition. The authors suggested that much of the P

initially present in decomposing plant material was not available for plant uptake, as the

microbial biomass responded quickly to the change in environmental conditions and

rapidly assimilated the P released from residues. These observations are consistent with a

wide body of literature (Bünemann et al. 2004; Iqbal 2009; Alamgir et al. 2012) that shows

significant amounts of residue P will be immobilised by the microbial biomass. These

studies also show that soil microorganisms are highly efficient in obtaining P to meet their

own requirements and that soil microbial populations are more likely to be limited by the

availability of C rather than P.

2.2.2. Mineralisation of microbial P

The time needed to complete the process of decomposition and mineralisation may range

from days to years, depending on many factors that affect microbial growth, particularly

moisture (Floate 1970b; Srivastava 1992; Van Gestel et al. 1993; He et al. 1997) and

7

temperature (Floate 1970a; Grisi et al. 1998; Soon and Arshad 2002). It appears that there

has been little success in establishing a clear relationship between the turnover of microbial

P and P availability to plants (Fabre et al. 1996; Kwabiah et al. 2003). Radioactive tracer

studies have shown that exchange of P between microbes and the soil solution can occur

without any net change in the size of the microbial P pool, demonstrating the constant flux

of P through this pool (Oehl et al. 2001). It has been suggested that the commonly used

short-term and seasonal experiments are not long enough to measure the relationship

between soil microbial P and P availability (Fabre et al. 1996; Kwabiah et al. 2003).

Using mature wheat, canola and pea residues incorporated into a red-brown Chromosol,

Iqbal (2009) demonstrated that P present in added residues was not available to crop plants

for at least six weeks after addition. Residues were added at a rate of 10 mg P kg-1 soil,

with three crop growth periods of wheat, each lasting for 28 days. Crop period 1 was

grown from day 0 to day 28, crop period 2 from day 28 to day 56 and crop period 3 from

day 56 to day 84. This protocol was chosen to ensure plants of similar growth stage and

thus nutrient demand were growing at different stages of residue decomposition. Microbial

P was much higher for the residue-amended soils and decreased with time as the residue

was broken down. As the microbial P pool turned over, some of this P contributed to an

increase in available P to plants when grown in soil 56-84 days after mature wheat residue

was applied (Table 2.2). It was suggested that the rest of the P released from the microbial

pool was absorbed by the wheat, precipitated, or sorbed to soil particles.

8

Table 2.2 Available and microbial P (mg P kg-1 soil) in soils amended with mature crop residues and an unamended control soil over time (Iqbal 2009).

Treatment Day 0-28 Day 28-56 Day 56-84 Day 0-28 Day 28-56 Day 56-84

Available P mg P kg-1 soila Microbial P mg P kg-1 soilb

Canola 0.2 0.3 0.3 15.6 13.2 12.3 Pea 0.5 1.2 1.2 13. 6 11.0 8.8

Lupin 0.5 1.0 1.0 13.7 12.9 9.9 Wheat 0.2 0.7 1.2 14.2 11.8 8.6 Control 1.3 1.6 1.1 4.5 4.4 4.5

abavailable P (resin extractable P) and microbial P (hexanol-released P) were measured by the method of Kuono et al. (1995) as modified by Bünemann et al. (2004).

2.3 Sorption of P released from crop residues in soils

Phosphorus released from crop residues (through leaching or mineralisation) into soil

solution is rapidly adsorbed onto clay surfaces (Singh and Jones 1976; White and Ayoub

1983; Friesen and Blair 1988; Bah et al. 2006). Umrit and Friessen (1994) found that

residue P has a greater effect on nutrient availability in soils with lower P-sorbing

capacities. In higher P-fixing soils, soluble P released from the residue and from microbial

decomposition was rapidly sorbed to soil rather than remaining available for plants.

Singh and Jones (1976) used several organic materials, incubated them with soil for 150

days and conducted P sorption and desorption measurements. From the sorption isotherms

they showed that in general, organic materials which contained >0.31% P decreased the

amount of P sorbed by the soil and increased the level of P in solution. Hence, if fertiliser

was applied after residue incorporation, more of the fertiliser would be available for plant

uptake as the soil’s P sorbing capacity has been decreased by occupation of P sorption sites

by residue-derived P. Whether this effect is significant would depend on the sorption

capacity of the soil and the concentration of P in the residue.

9

3. Crop residue management and P availability

As outlined above, P in crop residues can be released via a number of different pathways to

the soil solution pool where it is subject to assimilation by plant roots and microorganisms

or sorption onto soil. The maintenance of an adequate supply of P to crops is essential in

the early phases of growth (Batten 1992; Grant et al. 2001). Phosphorus supplied during

later phases of the growth cycle (either from additional fertiliser or soil chemical and

biological processes) can be beneficial (Römer and Schilling 1986), especially in seasons

where there is the potential for increased yield. The most important issues for growers are

whether residue management influences supply of P to crops, how much P residues supply,

and when will this P be available to plants during the growing season.

3.1 Timing and quantity of P release from crop residues

Inconsistencies in the timing and quantities of P released from crop residues in the

literature reflect differences in residue type, placement, moisture supply and rate applied.

Jones and Bromfield (1969) showed that hayed-off pasture (Phalaris tuberose L. now

known as Phalaris aquatica) under sterile conditions, lost 80% of P via leaching (mainly

as inorganic P) in the first two weeks across four different treatments (three leaching

events with 1-4 weeks between leaching events). Little additional P was lost after the third

leaching event. Under non-sterile conditions, where microbial immobilisation was likely to

occur, most P was retained in the residues during decomposition and leaching. Generally

only 10%-20% of the initial residue P was lost using the different leaching intensities. This

data shows that the intensity and duration of the first rainfall affects the amount of

inorganic P released into the soil and the amount left in the crop residues.

Friesen and Blair (1988) found 50% of 32P from labelled oat (Avena Sativa) residues in the

inorganic P soil pool 11 days after addition. In contrast to the previous study, residues in

10

this study were thoroughly incorporated into soil and pots were only watered to field

capacity and not leached. Both studies suggest that the majority of residue P can be

released within the first week of addition. However, both studies represent extreme

conditions unlikely to occur in the field and are likely to have overestimated the release of

P from crop residues. Firstly, in both studies the residues were ground to less than 2 mm,

increasing the surface area available for P leaching or microbial attack, which would have

increased the release of soluble P. In the Friesen and Blair (1988) study, a conventional till

system is simulated. The greater soil-residue contact would have enhanced release of P

from crop residues compared to a no-till system.

More recent studies suggest that the release of residue P can be much slower than the two

previous studies suggest. At the end of a 52-week field study, Lupwayi et al. (2007) found

that mature pea residues released 27% (0.4 kg P ha-1) and mature canola residues released

33% (0.8 kg P ha-1) of total residue P using surface-placed litter bags. Crop residues in the

field are likely to experience more frequent extremes in drying and wetting cycles, as well

as variable temperatures, leading to the slower release of residue P.

Many experiments have studied the release of P from crop residues in both controlled

environments and field experiments. However, few of these studies provide data of direct

relevance to growers, as the residues were not applied at a size, rate and moisture supply

representative of commercial paddock conditions. Using a combination of studies in the

literature, a prediction of the potential P supplied after harvest can be made. For example if

a grower had 4 t dry wt ha-1 of pea stubble after harvest with a P content of 0.1% (Lupwayi

et al. 2004), 4 kg P ha-1 would be incorporated back into the soil annually. The literature

suggests that of this P, approximately 80% is readily soluble (Jones and Bromfield 1969),

which equates to 3.2 kg of readily available P per hectare, annually.

11

3.2. Crop uptake of P from crop residues

The movement of P both to and into plants has been extensively studied and reviewed

(Olsen and Kemper 1968; Bieleski 1973; Mengel and Kirkby 1982; Marschner 2012;

Schachtman et al. 1998). Here the current knowledge of the contribution of crop residue P

to subsequent crop nutrition is summarised.

The contribution of crop residues to the P nutrition of subsequent crops has been studied in

the laboratory (Fuller et al. 1956; Blair and Boland 1978; Dalal 1979), glasshouse/growth

chambers (Till and Blair 1978; McLaughlin and Alston 1986; Nachimuthu et al. 2009) and

the field (McLaughlin et al. 1988a). Comparisons between studies are often difficult due to

the use of different residue loads (both in terms of mass and P concentration), residue size,

experiment length, subsequent crop type, fertiliser addition and soil moisture. Generally,

studies using residues with or without fertiliser have found that 1 – 15% of the shoot P

uptake comes from P in crop residues (Blair and Boland 1978; Till and Blair 1978;

McLaughlin and Alston 1986; McLaughlin et al. 1988a; Nachimuthu et al. 2009). The

majority of these studies have used ground (< 1 mm) residues and incorporated them into

the top 10 cm of soil (representing full tillage). The contribution of P in crop residues to

plant P uptake from these studies is likely to be an over-estimation of that in the field, as

residue size influences release of P from crop residues and the residue size is much smaller

than most field situations. However, what they do show is that crop residues have the

potential to supply a portion of a crop’s total P requirement.

3.3. Effect of tillage on release of P from crop residues

The increasing adoption of no-till systems has implications for the transfer of residue P

into the soil compared with traditional full tillage systems (Shear and Moschler 1969;

Buchanan and King 1993; Deubel et al. 2011). The minimisation of soil mixing in no-till

12

systems results in elevated concentrations of nutrients in the top 0-10 cm, compared to the

rest of the soil profile. This is commonly referred to as nutrient stratification and results

from increased levels of organic matter and fertiliser left on the surface (Crozier et al.

1999; Saavedra et al. 2007). Crop residues left on the soil surface decompose more slowly,

as less surface area is available for microorganisms to attack, compared to residues that are

fully incorporated into soil. Furthermore the moisture and temperature in the surface soil

(e.g. the top 2 mm) are often warmer and drier than for residues incorporated into the top

10 cm of soil.

In no-tillage systems, total and available P concentrations have been reported to be higher

in the topsoil than in the lower horizons due to the lack of mixing of soil and lack of

incorporation of crop residues into soil (Selles et al. 1997; Zibilske and Bradford 2003). A

long-term field trial comparing no-till and conventional till over 21 years found total P

concentrations in the top 5 cm were higher under no-till (1528 mg P kg-1) than

conventional till (776 mg P kg-1) (Saavedra et al. 2007). Zibilske and Bradford (2003)

found higher plant-available P, increased phosphatase activity and greater soil respiration

in soils under no-tillage than full tillage systems. The adoption of no-till resulted in an

enrichment of P in surface soil, and also changed processes affecting P dynamics in soil.

Similar studies have noted this increase in total P or plant-available P in topsoil in no-till

systems (Selles et al. 1997; Crozier et al. 1999). These studies show reduced tillage

increases the concentration of P in surface soil where crop roots acquire it during early

growth; this P is also stored in more accessible forms compared to other parts of the soil

profile (Selles et al. 1997). These findings suggest that when predicting the quantity of P

available to a subsequent crop, the rate of P cycling in this enriched surface layer will

differ from that of P cycled in the remainder of the soil profile.

13

Surface-placed residues experience more frequent fluctuations in wetting and drying events

compared to those residues below the soil surface, as well as larger fluctuations in

temperature. Jones and Bromfield (1969) found that inorganic P was readily leached from

crop residues when microbial activity was inhibited. Wetting events therefore may be the

dominant mechanism contributing to loss of P from surface-placed residues given the

likely lower levels of microbial activity and high solubility of P in crop residues. In an

incubation study using six residue types, Sharpley and Smith (1989) found greater amounts

of inorganic P were leached from surface-placed residues compared with incorporated

residues. Although residue P has been shown to be more readily leached in no-till

compared with full tillage systems, the ability of a subsequent crop to access this residue P

has received little attention. A better understanding of the impacts of stratification and

leaching of nutrients from residues caused by no-till management on plant available P, will

support better fertiliser recommendations.

4. Chemistry of phosphorus in crop residues

4.1 Predicting immobilisation and mineralisation of P from crop residues

During decomposition and mineralisation, different rates of microbial transformation of P

are commonly observed and are assumed to reflect the substrate quality. Total P and the

C:P ratio of crop residues have been widely suggested as indices of residue quality in terms

of its potential to provide P to subsequent crops (Fuller et al. 1956; Enwezor 1976; Singh

and Jones 1976; Kwabiah et al. 2003; Iqbal 2009). Numerous experiments with crop

residues have reported different P concentrations below which P immobilisation occurred.

Some of these are summarised in Table 4.1. It is apparent that a wide range of critical

levels of total P and C:P ratios have been proposed; this is especially the case for C:P ratio,

with critical values ranging from 55:1 – 500:1.

14

Table 4.1. Critical phosphorus concentration (%) and C:P ratio beyond which immobilisation of P occurred.

Authors Residue/Plant Type Residue % P

Residue C:P

Fuller et al. (1956) Wheat, barley, flax straw, tomato, lettuce, alfalfa, clover and bean tops

0.2% 200

Barrow (1959) Phalaris and lucerne 0.2% 55

Floate (1970) Nardus grass and Agrostis-Festuca grass

0.1% Hannapel et al.

(1964) Sucrose

200

Enowzer (1976) Straw and pea

112 - 501

Blair and Boland (1978)

White clover

150-300

White and Ayoub (1983) Faba bean

123 - 251

Kwabiah et al. (2003)

18 plant species

156 – 252

Iqbal (2009) Wheat, canola, lupin, pea 0.2% 253

There are diverging views about the link between C and P cycling (McGill and Cole 1981;

Smeck 1985) and predicting when P from crop residues will be released based on these

measures has proven more difficult than analogous predictions of N release based on N

concentrations or C:N ratio (Kirkby et al. 2011). One difference between P and N is that

there is a substantial amount of inorganic P in crop residues (Birch 1961; Jones and

Bromfield 1969; Martin and Cunningham 1973; McLaughlin et al. 1988b), whereas the

vast majority of N in crop residues is organic. Thus whereas the release of mineral N from

crop residues almost exclusively involves microbial decomposition of organic forms, it is

clear from the literature that release of considerable amounts of mineral P can occur

without microbial decomposition.

A second difference between P and N in crop residues is the diversity of organic forms

present. Most organic N is present in the amide linkages of proteins (Smernik and Baldock

15

2005), whereas P is present in a variety of chemically diverse molecules including

phospholipids, nucleic acids and inositol phosphates (Bieleski 1973). Thus reliable

determination of P speciation in crop residues is needed to better predict their contribution

to P cycling in soil.

4.2 Phosphorus species in plant materials

Various P species have been identified in plant materials, mostly using sequential chemical

fractionation methods. Early research by Bieleski (1963; 1965; 1968; 1969; 1973)

increased our understanding of the physiology and metabolism of P compounds in plants.

Most research on P speciation in plant material has used immature plants (Barr and Ulrich

1963; Kakie 1969; Lee et al. 1976; Chapin and Bieleski 1982; White and Ayoub 1983;

Batten and Wardlaw 1987b), generally grown for 2-6 weeks. There is only a small body of

work that has examined the P speciation of mature plant material (Chapin and Kedrowski

1983; Batten and Wardlaw 1987b; Batten and Wardlaw 1987a). More recently there has

been greater focus on the distribution and location of P forms in plants to improve crop P

uptake and productivity (Veneklaas et al. 2012).

Phosphorus is absorbed by plants from soil solution predominantly as inorganic P (HPO42-

and H2PO4-). After uptake, at physiological pH, H2PO4

- either remains as inorganic P or is

esterified through a hydroxyl group to a C chain (C-O-P) as a simple phosphate ester, or

attached to another phosphate by the energy-rich pyrophosphate bond (Marschner 2012).

Another type of phosphate bond is the relatively stable diester (C-(P)-C). In this asso-

ciation phosphate forms a bridging group connecting units to more complex or

macromolecular structures (Marschner, 2012). The pattern of incorporation of inorganic P

into these other P forms is similar in the shoots and roots. The five major forms of P

identified in fresh plant materials by sequential fractionation are (a) nucleic acids

16

(deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)); (b) phospholipids; (c) P-

esters; (d) inorganic P; and (e) residual P (Bieleski 1968; Chapin and Bieleski 1982).

Bieleski (1973) quoted a typical ratio of DNA:RNA:lipid-P:ester-P of plant tissue (non-

seed portion) as 0.2:2:1.5:1, but noted that these ratios vary depending on the P status of

the plant (described in more detail below).

4.2.1 Inorganic P

Generally around 50% of the P present in growing plant tissues is in the inorganic form as

free inorganic phosphate and located in the cell cytoplasm (Bieleski and Ferguson 1983).

During plant growth, P is temporarily accumulated as inorganic P in the vacuole (Bieleski

1968; 1973). Inorganic P, contained in cellular vacuoles, can be the principal storage form

in vegetative plant parts, whereas phytic acid serves the same role in seeds.

In mature crop residues, the percentage of P present in inorganic forms has been reported

to be 50-80% (Birch 1961; Jones and Bromfield 1969; Martin and Cunningham 1973).

Many studies use water extracts to identify the molybdate reactive P (orthophosphate) in

crop residues (Jones and Bromfield 1969; Martin and Cunningham 1973). However, this

process may overestimate the inorganic P concentration in the original plant material due

to the hydrolysis (including enzymatic hydrolysis) of other P species present in the

material during water extraction and during the acidic colorimetric reaction to determine

orthophosphate (Tarafdar and Claassen 1988; Bishop et al. 1994). Jones and Bromfield

(1969) reported that 40-60% of total P in field and glasshouse grown phalaris (Phalaris

tuberosa L. now known as Phalaris aquatica) and subterranean clover (Trifolium

subterraneum L.) was present as inorganic P. Martin and Cunningham (1973) also found a

rapid release of P (85% of total P) from plant residues (wheat roots, air dried) in which >

90% of the total P released from wheat roots was detected as inorganic P (orthophosphate).

17

4.2.2 Phospholipids

Phospholipids are a class of tri-glycerides, which have at their core glycerol (CH2OH-

CHOH-CH2OH). Phospholipids are lipids in which at least one of the three fatty acid

chains bound to glycerol is replaced by a phosphate-containing group bound to the

glycerol. Harrison (1987) reported phospholipid P comprised 20-40% of total plant P.

Phospholipids form the basic structure of cell membranes, with two hydrophilic outer

surfaces sandwiching an inner hydrophobic region. The P-lipid composition of the various

plant tissues is relatively uniform, the most important factor being whether or not the tissue

is photosynthetic. Chloroplasts are organelles in plant cells and have highly developed

membranes which can contain over 40% of the total lipid P in plant cells (Bieleski 1973).

Phospholipids are, however, abundant in both photosynthetic and senesced tissues, but the

proportion of P present as phospholipid is much higher in photosynthetic tissues (Bieleski

1973; Ashworth et al. 1981).

4.2.3 Nucleic acids

The function of P in plants is most prominent in nucleic acids, which, as units of the DNA

molecule, are carriers of genetic information. As units of RNA, these structures are also

responsible for the translation of genetic information. In both DNA and RNA, phosphate

forms the bridge between ribonucleoside (RNA monomer) units to form macromolecules.

The proportion of P in nucleic acids to total organically bound P differs between tissues

and cells; it is high in meristematic tissues (expanding tissues such as those near the tip of

stem and roots), and low in storage tissues (Bieleski 1973). There are at least two reasons

why DNA concentrations in the cell are not fixed. Firstly, polyploid cells develop quite

easily during cell differentiation and are common in differentiated organs, and secondly

DNA is not confined to the nucleus as the mitochondria and chloroplasts also contain DNA

(Hall and Davies 1979).

18

4.2.4 Ester P

Esters of P, including compounds such as adenosine triphosphate (ATP) and adenosine

diphosphate (ADP), comprise the metabolic machinery of the cell. This is further

complicated by variation across different growth stages and different plant parts. Many

difficulties arise when trying to speciate P esters in biological materials. Firstly, there may

only be trace amounts of P esters to identify. The rapid turnover of P esters in cells makes

it difficult to measure a constant and representative ester P concentration for a given

material (Bieleski and Young 1963).

4.2.5 Residual P

Residual P is the P that cannot be otherwise identified by chemical fractionation. In some

studies this may largely be organic P that has not been extracted (Kakie 1969; Kedrowski

1983; Adu-Gyamfi et al. 1990). The residual P fraction varies in both amount and type,

depending on which forms of P have been extracted in the preceding fractionation steps,

and the capability or extraction efficiency of the chemical fractionation methods used. For

example, in a study by Kakie (1969), 1 – 5% of the total P in tobacco leaves could not be

identified and was designated ‘residual P’.

While many P compounds in plants have been identified, there has been little work to

assess how the concentrations of these P compounds change over the growing season

through to senescence. The reliability of techniques used to extract these fractions is also

problematic (see Section 6.1).

4.3 Effect of plant P status on P speciation

The effect of P status on P forms in plant material has been a focus for determining critical

or optimal P concentrations for plant growth (Kakie 1969; Lee et al. 1976) and for leaf

19

tissue testing (Batten and Wardlaw 1987b; Bollons et al. 1997). When plant P supply is

increased from the deficiency to the sufficiency range, the concentrations of major P forms

(ester, lipids, nucleic acid and inorganic P) in vegetative plant organs increase (Batten and

Wardlaw 1987b; Chapin and Bieleski 1982; Kakie 1969; Lee et al. 1976; Veneklaas et al.

2012; White and Ayoub 1983). Further increases in P supply (i.e. above the sufficiency

level) result in only the inorganic P fraction increasing and it becomes the most abundant

form of P in plants.

In plant cells, the vacuole has many functions including containing waste, maintaining

turgor and also storing small molecules. The vacuole acts as a storage pool of inorganic P

and, when P supply is adequate, ~85-95% of the total inorganic P is located in vacuoles

(Raven 1974; Bieleski and Ferguson 1983). In contrast, in leaves of P-deficient plants,

virtually all inorganic P is found in the cytoplasm and chloroplasts; this is commonly

referred to as the ‘metabolic pool’. This was shown in P-deficient Spirodela plants that

contained no inorganic P in their vacuoles, but had retained inorganic P in both the

cytoplasm and chloroplast (Bieleski 1968). In a more recent study, using in vivo 31P NMR,

it was shown that the cytoplasmic inorganic P content (µM cm-3 root volume) remained

unchanged in maize roots grown in nutrient solutions containing 1µM, 10µM and 0.5mM

inorganic P (Lee and Ratcliffe 1993b; Lee and Ratcliffe 1993a). However, the vacuolar

inorganic P content of the mature maize roots varied ten-fold across these three P

concentrations.

In an early glasshouse experiment by Kakie (1969), the effect of phosphate concentration

on the distribution of P into various fractions was analysed. The P concentrations in the

nutrient solutions applied to soil ranged from 0-560 mg P L-1. As the P concentration

increased, acid soluble inorganic P (orthophosphate) was the only fraction in tobacco

20

leaves to continually increase, from 35% at 0.08 P % dry wt to 68% of the total P at 1.03 P

% dry wt (Table 4.3). All other P fractions increased in percentage until the nutrient

solution was approximately 0.45 P % dry wt, but then remained almost unchanged up to a

P content of 1.03 P % w/w (Table 4.3).

Table 4.3 Phosphorus fractions in tobacco plants with increasing P fertilisation. Adapted from Kakie (1969).

Kakie’s (1969) study suggests that tobacco plants increase stores of P in all fractions to a

threshold (approximately 0.45 P % w/w) beyond which there is enough P to satisfy the

plant’s needs, and further P is stored exclusively as inorganic P. This can be interpreted as

a saturation phenomenon, and is consistent with observations by Valenzuela et al. (1996)

in melon leaves.

In a similar study, where wheat plants were supplied with either low (0.25 mM P solution)

or high (1 mM) rates of P, at maturity the proportion of inorganic P to other P forms (lipid,

ester and residue P) was three times greater in high-P plants compared to low-P plants

(Batten and Wardlaw 1987b). The leaves of wheat in the low-P treatment had exported

almost all P to the grain and contained a lower concentration of inorganic P.

Conc. of P in solution Total P Acid Soluble

Inorganic P Acid Soluble

Organic P Lipid

P Nucleic Acid

P Residual

P

(mg P L-1) P% dry wt % of total P

0.9 0.2 18.3 20.0 17.8 41.1 2.8 1.7 0.3 20.9 22.0 21.3 39.7 2.1 3.5 0.5 27.0 22.9 19.6 29.2 1.3 8.8 0.7 49.3 15.9 13.3 20.7 0.9 52 0.8 55.8 14.0 11.4 18.0 0.9 560 1.0 67.8 10.9 8.2 12.5 0.6

21

Based on these findings, we expect that crops with higher total P concentrations will leave

more P in crop residues and a larger percentage of that P will be present as inorganic P

(orthophosphate), which represents an addition to soil in a plant-available form. Few

studies have examined the effect of P supply on P speciation in mature plant material (Barr

and Ulrich 1963; Batten and Wardlaw 1987b; Hart and Jessop 1983; Umrit and Friesen

1994). Most of these studies also report that the concentration of inorganic P and its

proportional contribution to total plant P content increased with P nutrition in mature plant

material.

A more detailed study of crop residues with varying P status will increase knowledge about

the distribution of P in plants and may facilitate better predictions of P release and

potential bioavailability of P in crop residues.

5. Methods for measuring the release of P and characterising P forms in crop

residues

The determination of P in crop residues is usually limited to measurement of total P (using

acid digestion). This measurement, however, provides no information on the chemical

forms of P present in crop residues, which is essential to understanding the fate and

mobility of P in crop residues in cropping systems. Our limited knowledge of the

speciation of P forms in crop residues mostly comes from chemical fractionation along

with a few isotope and NMR studies that reveal the distribution in species to some extent.

The speciation of P in crop residues may help to determine the potential for P to be

released from crop residues, which should facilitate calculations of the requirement for

supplemental P fertiliser required for optimal crop yields. To partition total P into more

specific chemical P forms in crop residues, a number of methods can be used, including

22

chemical P fractionation and spectroscopic techniques. Some of these methods are already

being successfully used for speciation of P and other elements in soil and organic

materials; however, there has been little work on crop residues.

5.1 Sequential chemical fractionation

The most commonly used method for characterisation of P in plant materials is sequential

chemical fractionation (Miyachi and Tamiya 1961; Kakie 1969; Hogue et al. 1970;

Kedrowski 1983; Ramon et al. 1990). Interestingly, of all of the fractionation methods

developed, there does not appear to be one that is consistently used in the literature. There

is certainly inconsistency in P fractions identified, which makes it very difficult to compare

results between studies.

Briefly, chemical fractionation is used on solid plant materials and P fractions are extracted

using a suite of different solutions. Some of the early work on P fractionation techniques

(McAuliffe and Peech 1949; Peperzak et al. 1959) designated P forms as (i) acid soluble

(inorganic P) (ii) alcohol-ether soluble (phospholipids) and (iii) residual P (nucleic acids).

These studies formed the basis of more sophisticated chemical fractionation techniques,

and were later adapted and modified by many other studies. For example, in many

fractionation methods, lipid-P is extracted using a mixture of chloroform, ethanol and

water (Bieleski 1968; Kedrowski 1983; Ramon et al. 1990). The lipid P is soluble in the

non-polar phase, which can be separated from the aqueous layer and analysed.

The chemical fractionation method is not ideal because it provides limited information

about organic P forms, does not distinguish between inorganic P forms, and is quite time

consuming. Another concern is that very few studies have validated their fractionation

method by confirming the speciation of P that is removed in each step. Therefore it is not

23

clear what P forms are removed by the different fractionation steps. Future research efforts

should focus on confirming the validity of these methods.

5.2 Solution 31P nuclear magnetic resonance spectroscopy

Solution and solid-state 31P NMR spectroscopy are increasingly used to characterise P

forms in different materials. Solution 31P NMR is most widely used for soil organic P

analysis and has been used to characterise P forms in a diverse range of soils (McDowell et

al. 2005; Turner 2008). The more recent use of 31P NMR spectroscopy for soil, water and

other environmental samples has been reviewed by Cade-Menun (2005). One reason for its

widespread adoption is that it not only enables the characterisation of broad classes of

inorganic and organic P forms (orthophosphate, orthophosphate monoester, orthophosphate

diester and polyphosphates), but also enables the identification of specific P compounds,

such as DNA in the orthophosphate diester region (Turner et al. 2003). A recent review has

detailed the benefits and disadvantages of spectroscopic techniques based on sample

preparation, sensitivity, resolution and quantitation of P species in soils (Doolette and

Smernik 2011).

One disadvantage of solution 31P NMR is the requirement of an extraction step. The

extraction aims to solubilise P in the sample while minimising changes to the P species

present. The most commonly used extractant is a mixture of sodium hydroxide (NaOH)

and ethylenediaminetetraacetic acid (EDTA) (Bowman and Moir 1993). However, any P

that is not solubilised in this solution will not be accounted for in the subsequent solution

NMR analysis. Another problem with solution 31P NMR is that organic P in solution can

be hydrolysed under strongly alkaline conditions. For example it has been shown that

phospholipids are degraded into two orthophosphate monoester compounds (Turner et al.

24

2003). These compounds have now been identified as α– and β–glycerophosphate

(Doolette et al. 2009).

When interpreting NMR spectra, accurate assignment of peaks is also critical. Many

studies rely on comparison to reported literature values to assign peaks to different P

compounds (Makarov et al. 2002; Briceno et al. 2006; McDowell et al. 2006). Problems

can arise, however, as these chemical shifts vary with factors such as pH and concentration

of paramagnetic ions (Crouse et al. 2000; McDowell and Stewart 2005; Doolette and

Smernik 2010). Some species such as pyrophosphate and polyphosphate, have very distinct

chemical shifts. However, others appear in crowded regions (especially orthophosphate

monoesters) making the P species harder to identify (Doolette and Smernik 2010). There

have been several different approaches to overcome this problem. For example Turner et

al. (2003) acquired the spectra from standard P compounds added to NaOH-EDTA soil

extracts. Smernik and Dougherty (2007) spiked samples with low concentrations of model

compounds to correctly identify peaks by measuring the peak height increase.

The speciation of P by NMR in plant material has been primarily been used in

incubation/decomposition studies (Gressel et al. 1996; Miltner et al. 1998; Cheesman et al.

2010) or on immature plant material (Makarov et al. 2002; Makarov et al. 2005). Many of

these studies aim to understand the origins of organic P in soils. Gressel et al. (1996) used

NMR to characterise the P in decomposing forest litter (pine needles, roots and

microorganisms). They reported that of the total P extracted, 12% was in the form of

inorganic P, 62% monoester P, 13% diester P and 13% pyrophosphate. Similarly

Cheesman et al. (2010) reported for field-aged cattail (Typha domingensis Pers.), 39% of

the total plant P measured was inorganic P, 23% monoester P, 4% diester P, 5% DNA, 6 %

25

pyrophosphate and 23% residual. These studies demonstrate the potential to use NMR for

quantifying organic P in plant samples.

Several studies have used NMR to determine P forms in plant material and follow the

transformation of P forms during colonisation and decomposition by microorganisms

(Miltner et al. 1998; Cheesman et al. 2010). Miltner et al. (1998) used NMR to speciate

beech leaf litter before and after 498 days of incubation across a range of soil minerals.

The initial leaf litter contained both monoester and diester P. The most marked changes in

P speciation was an increase in diester P due to accumulation of microbial P and a decrease

in monoester P, attributed to plant P.

From the examples provided above, it is clear NMR spectroscopy has the capability to

identify P species that are removed by each step in sequential fractionation procedures.

This approach has the potential to provide detailed and accurate characterisation of P in

crop residues that can be used to determine labile and non-labile forms of agricultural

relevance.

5.3 X-ray absorption near edge structure (XANES) spectroscopy

The recent adoption of synchrotron-based X-ray absorption near edge structure (XANES)

spectroscopy to analyse soil, soil organic matter and organic wastes (Solomon et al. 2003;

Toor et al. 2005; Negassa et al. 2010) provides an opportunity to characterise P forms in

crop residues. Reference spectral libraries for P L-edge XANES spectra (Kruse et al.

2009) have recently been developed, which will greatly increase the ability to interpret the

spectra of unknown samples. Approximately 50 different inorganic and organic P

compounds have been catalogued to date.

26

However, the ability to differentiate species is the main limitation of P XANES analysis as

in some cases there are only a small number of distinctive features to identify different P

forms (Kruse et al. 2009). The P XANES spectra of organic P compounds are generally

similar, contain little in the way of diagnostic features and are difficult to distinguish from

aqueous or weakly bound phosphate (Shober et al. 2006; Kruse and Leinweber 2008).

5.4 Isotopic labelling

Radioactive 32P and 33P tracing is commonly employed to trace the fate and quantify the

turnover of P from residues, organic amendments, soil, and fertiliser P (Blair and Boland

1978; Till and Blair 1978; White and Ayoub 1983; Friesen and Blair 1988; McLaughlin et

al. 1988a; Oberson et al. 2001; Nachimuthu et al. 2009). Early work by Bieleski (1973)

also used isotopic labelling to investigate the distribution of P compounds within plants.

This was done by measuring concentration of the radioactive tracer 32P in different

chemical fractions in plant cells and isolated organelles. Other information has come from

studies on the rate at which 32P is incorporated into or lost from tissues, a technique

commonly referred to as compartmental analysis.

Isotopic labelling permits a direct assessment of the relative contribution of P derived from

residues or labile soil pools to crops. For example, in a labelling experiment by

McLaughlin et al. (1988a), medic residues and applied fertiliser were labelled with 33P

and 32P, respectively. The percentage contribution of P from each source to a following

wheat crop was then measured. Similar studies using 32P and 33P isotopes to measure the

contribution of crop residues to P uptake by plants, or the microbial P pool have been

previously discussed in Section 3. Once the P chemistry of different residues can be

correctly assessed, isotopic labelling techniques provide a way to compare how residues

with different P chemistry are incorporated in the soil P cycle.

27

6. Objectives of this research

Residue P plays a vital role in soil P cycling. If crop residues are to be better managed as a

source of plant P for agricultural production, there is a need to gain a more comprehensive

understanding of P in crop residues. There is very limited information in the literature

regarding the distribution of P within crop residues (e.g. between roots, stems, leaves etc.),

and even less information regarding the species of P present. Many of the current

analytical techniques are subject to problems with extraction methodologies, including

poor detection, mis-identification and over- or underestimation of specific organic P

species. Generating new information in this field, and improving or expanding currently

available techniques, will improve estimation of potential nutrient availability from crop

residues applied in field crop situations. The main objectives of this research were to:

1) Identify and quantify the P species in a series of field-collected crop residues using

solution 31P NMR spectroscopy to characterise the main P species in crop residues.

2) Compare chemical fractionation and solution 31P NMR spectroscopy for speciation of

crop residue P.

3) Determine the effect of plant P status on the P speciation (both concentration and form)

of mature wheat and canola plant parts.

4) Determine the effect of crop residue management techniques in Australian broad acre

cropping systems on release and uptake of P in crop residues by subsequent crops,

microbes and resin-available P pools.

28

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Bieleski, RL (1968). Effect of phosphorus deficiency on levels of phosphorus compounds in Spirodela. Plant Physiology 43: 1309-1316.

Bieleski, RL (1969). Phosphorus compounds in translocating phloem. Plant Physiology 44: 497-502.

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CHAPTER 2

CROP RESIDUE PHOSPHORUS: SPECIATION AND

POTENTIAL BIO-AVAILABILITY

The work contained in this chapter has been published in Plant and Soil. Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M., Armstrong, R.D., 2012. Crop residue phosphorus: speciation and potential bio-availability. Plant and Soil 359, 375-385.

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Crop residue phosphorus: Speciation and potential bio-availability

Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M., Armstrong, R.D., 2012. Crop residue phosphorus: speciation and potential bio-availability. Plant and Soil 359, 375-385

Sarah Noack

Performed analysis on all samples, interpreted data and wrote the manuscript.

Mike McLaughlin

Supervised development of the work, helped with data interpretation and manuscript evaluation.

Ronald Smernik

Supervised development of the work, helped with data interpretation and manuscript evaluation.

Therese McBeath

Supervised development of the work, helped with data interpretation and manuscript evaluation.

Roger Armstrong

Helped with data interpretation and to evaluate and edit the manuscript

39

40

41

A Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M. & Armstrong, R.D. (2012) Crop residue phosphorus: speciation and potential bio-availability. Plant and Soil, v. 359(1-2), pp. 375-385

NOTE:

This publication is included on pages 41-51 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://doi.org/10.1007/s11104-012-1216-5

52

CHAPTER 3 ASSESSING CROP RESIDUE PHOSPHORUS SPECIATION USING

CHEMICAL FRACTIONATION AND SOLUTION 31P NUCLEAR MAGNETIC

RESONANCE SPECTROSCOPY

The work contained in this chapter has been submitted to Talanta.

53

54

Statement of AuthorshipTitle of Paper

Publication Status Published, Accepted for Publication, Submitted for Publication, Publication style

Publication Details

Author ContributionsBy signing the Statement of Authorship, each author certifies that their stated contribution to the publication is accurate and that permission is granted for the publication to be included in the candidate’s thesis.

Name of Principal Author (Candidate)

Contribution to the Paper

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Assessing crop residue phosphorus speciation using chemical fractionation and solution 31P nuclear magnetic resonance spectroscopy

The manuscript has been submitted to Talanta.

Sarah Noack

Performed analysis on all samples, interpreted data and wrote the manuscript.

Ronald Smernik

Supervised development of the work, helped with data interpretation and manuscript evaluation.

Therese McBeath

Helped to evaluate and edit the manuscript.

Roger Armstrong

Helped to evaluate and edit the manuscript.

Mike McLaughlin

Helped to evaluate and edit the manuscript.

55

56

Abstract

At physiological maturity, nutrients in crop residues can be released to the soil where they

are incorporated into different labile and non-labile pools while the remainder is retained

within the residue itself. The chemical speciation of phosphorus (P) in crop residues is an

important determinant of the fate of this P. In this study, we used chemical fractionation

and 31P nuclear magnetic resonance (NMR) spectroscopy, first separately and then

together, to evaluate the P speciation of mature oat (Avena sativa) residue. Two water

extracts (one employing shaking and the other sonication) and two acid extracts (0.2 N

perchloric acid and 10% trichloroacetic acid) of these residues contained similar

concentrations of orthophosphate (molybdate-reactive P determined by colorimetry) as

NaOH-EDTA extracts of whole plant material subsequently analysed by solution 31P NMR

spectroscopy. However, solution 31P NMR analysis of the extracts and residues isolated

during the water/acid extractions indicated that this similarity resulted from a fortuitous

coincidence as the orthophosphate concentration in the water/acid extracts was increased

by the hydrolysis of pyrophosphate and organic P forms while at the same time there was

incomplete extraction of orthophosphate. Confirmation of this was the absence of

pyrophosphate in both water and acid fractions (it was detected in the whole plant

material) and the finding that speciation of organic P in the fractions differed from that in

the whole plant material. Evidence for incomplete extraction of orthophosphate was the

finding that most of the residual P in the crop residues following water/acid extractions

was detected as orthophosphate using 31P NMR. Two methods for isolating and

quantifying phospholipid P were also tested, based on solubility in ethanol:ether and

ethanol:ether:chloroform. While these methods were selective and appeared to extract only

phospholipid P, they did not extract all phospholipid P, as some was detected by NMR in

the crop residue after extraction. These results highlight the need for careful interpretation

of results from chemical fractionation, as separation can be compromised by incomplete

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recovery and side reactions. This study also highlights the benefits of employing a

technique that can simultaneously detect multiple P species (solution 31P NMR) in

combination with chemical fractionation.

Introduction

Phosphorus (P) is a highly mobile element in plants, moving readily between parts of the

plant. The seed becomes the ultimate sink for most P in annual species, while the

remaining P is distributed between roots, stem, leaves and chaff/pod material. In cropping

systems, these latter plant parts (crop residues) remain in the field after grain harvest and

are a potential source of nutrients such as P, for subsequent crops. The speciation of P in

these residues plays an important role in determining its fate, i.e. whether it is released to

soil as soluble P, assimilated by microorganisms, or whether it adds to more chemically

stable P pools in soil. Several methods have been used to characterise P in crop residues,

the two most popular being chemical fractionation [1-5] and 31P nuclear magnetic

resonance (NMR) spectroscopy [6-9].

Chemical fractionation has long been used to characterise P in plant material.

Consequently, an extensive body of data exists in the literature [1-4, 10-12]. Sequential

chemical fractionation characterises P species based on their assumed solubility in, or

reactivity toward, a series of extractants. Generally, crop residue P is separated into (i) P

soluble in weak acid or water (soluble P), (ii) P soluble in non-polar organic solvents

(phospholipid P), (iii) P released by reaction with potassium hydroxide or strong acid

(nucleic acid P), and (iv) residual P.

Solution 31P NMR spectroscopy following sodium hydroxide ethylenediaminetetraacetic

acid (NaOH-EDTA) extraction offers an alternative approach to determining P speciation

58

in environmental and agricultural samples [13-14]. This method provides more detailed P

speciation than is possible with sequential chemical extraction because several organic and

inorganic P species can be identified simultaneously. Sample preparation for solution 31P

NMR involves alkaline extraction, usually with 0.25 mol L-1 NaOH and 0.05 mol L-1

Na2EDTA. The solubility of both organic and inorganic P species is maximised at high pH,

and the additon of EDTA complexes paramagnetic ions which increases the efficiency and

diversity of P species extracted [15]. As with chemical fractionation, there is a portion of P

that was not extractable (residual P). There is also the potential for some organic P

compunds to be hydrolysed under the alkaline extraction conditions [16].

In plant studies, solution 31P NMR spectroscopy has primarily been used to speciate P in

immature material [6, 17] or in material used in incubation/decomposition studies [7-8,

18]. Recently, we used this technique to speciate P in field-collected mature cereal and

legume residues [9] and found that crop residues (stem and leaf material) contained

primarily orthophosphate, along with smaller quantities of phospholipids, RNA, phytate

and pyrophosphate.

Few studies have compared results from these two different approaches. There is a small

body of work which has combined both methods to improve the characterisation of P

species in animal manure using chemical extracts [19-20]. To our knowledge, no study has

correlated the P species obtained from 31P NMR to that from chemical fractionation for

plant material.

The combined use of chemical fractionation and 31P NMR is not limited to comparing

results obtained from each method in isolation. Application of 31P NMR analysis to the

fractionated materials can be used to test basic assumptions that underpin the chemical

59

fractionation approach. Chemical fractionation assumes that each extractant is selective for

the targeted P species and that all of the targeted P species are released to solution during

the treatment. Chemical fractionation methods also assume that other P species present are

not extracted or transformed prior to the step designed to release that particular P species.

Solution 31P NMR provides a means to test these assumptions through determination of the

P speciation of both the extracts and the treated crop residues following each fractionation

step.

The first aim of this study was to compare P speciation of a single plant residue as

determined by chemical fractionation with that determined by solution 31P NMR

spectoscopic analysis. The second aim of this study was to combine chemical fractionation

with solution 31P NMR spectoscopic analysis by analysing the extract and residue fractions

of key steps in chemical fractionation methods (water/acid extraction and lipid extraction)

using 31P NMR spectroscopy. Better identification of P species in crop residues can lead to

improved estimation of the turnover of these P species in soil, leading to a better

assessment of the amount of P that may be provided for subsequent crops.

Methods

Residue properties

The stem and leaf material (hereafter referred to as the residue) of oat (Avena sativa) was

collected from a farm near Truro (139°7'46"E, 34°24'42"S), South Australia, by cutting

mature plants 1 cm above the soil surface at harvest and removing the chaff and seed by

cutting off the heads. The residue was then oven-dried at 60°C. Total P concentration in

the residue (539 mg kg-1) was measured using inductively coupled plasma optical emission

spectroscopy (Perkin-Elmer, Optima 7000DV at 214.97 nm) following digestion of

triplicate ground samples using boiling concentrated HNO3 at 140°C [21].

60

Chemical fractionation

Chemical fractionation was used to quantify (i) soluble P (both orthophosphate

(molybdate-reactive P) and total P) and (ii) phospholipid P using two methods previously

used for the identification of P species in tobacco [2] and tomato plants [5]. Although these

steps would usually be carried out sequentially, in this study the water/weak acid and

phospholipid extractions were all carried out on a fresh residue sample. Figure 1 illustrates

how chemical fraction was combined with 31P NMR spectroscopy for sample analysis.

Fig. 1. Flow diagram of the extraction and analysis procedure for chemical fractionation

and solution 31P NMR spectroscopy.

Four methods that have previously been used to measure water/acid soluble residue P were

tested: (i) water extraction using an end-over-end shaker (WSH); (ii) water extraction with

sonication (WSO) [9]; (iii) extraction with 10% trichloroacetic acid (TCA) [2]; and (iv)

extraction with 0.2 N perchloric acid (PA) [5]. In each case, four aliquots of 2 g of ground

residue were weighed into 50 mL centrifuge tubes and 40 mL of the given extractant was

61

added. All samples except WSO were placed on an end-over-end shaker for 1 hour. The

WSO sample was sonicated (Virtis Virsonic Sonicator®, USA) for 10 minutes at 90 W.

All samples were centrifuged (1400 × g) and filtered through Whatman No. 42 filter paper.

After extraction, the remaining residue was rinsed once with the given extractant and the

filtered rinse combined with the extract. An aliquot of the supernatant was taken from each

replicate tube, digested in HNO3 and analysed as described above [21]. Orthophosphate P

(molybdate reactive) in the soluble extracts was also measured colorimetrically [22]. The

remaining supernatant (10-20 mL) was frozen and subsequently freeze-dried prior to NMR

analysis. The residues of the water and acid extracts (~2 g) were oven-dried at 40°C,

weighed and extracted with 40 mL NaOH-EDTA based on the method of [23] outlined

below.

Two methods that have been previously used to measure the phospholipid concentration in

plant residues were assessed: (i) extraction with ethanol:ether (E:E) [2]; and (ii) extraction

with ethanol:ether:chloroform (E:E:C) [5]. For both extractions, four aliquots of 1 g of

residue were weighed into 50 mL centrifuge tubes and to each tube 25 mL of the selected

extractant was added. The E:E extractions were carried out on an end-over-end shaker for

1 hour; the four E:E:C extractions were carried out in a water bath heated to 50°C for 1

hour. The E:E:C extraction mixtures were shaken manually every 10 minutes.

Once the extraction was complete, all tubes were centrifuged (1400 × g) for 10 minutes

and extracts were filtered using Whatman No.42 filter paper. The remaining residue in

each tube was rinsed twice with an additional 5 mL of the given extractant (E:E or E:E:C)

and these washings were combined with the extract.

62

The supernatant was transferred to Teflon™ beakers and boiled to dryness, leaving a small

pellet. The remaining pellet was digested using concentrated HNO3 at 140°C [21] and the

total P concentration in the digest was determined using ICP-OES. The residues of the E:E

and E:E:C extractions were weighed and extracted with 20 mL NaOH-EDTA based on the

method of Cade-Menun and Preston [23] outlined below.

NaOH-EDTA extraction

The whole oat residue and the residues following treatment with various extractants were

extracted with NaOH-EDTA (in triplicate) using the method of Cade-Menun and Preston

[23], originally developed for soil extraction. This involved shaking approximately 2.0 g of

dried residue with 40 mL of 0.25 mol L-1 NaOH and 0.05 mol L-1 Na2EDTA for 16 hours.

The extracts were centrifuged (1400 × g) for 10 minutes and filtered using Whatman No.42

filter paper. An aliquot (20 mL) from each of the triplicate extracts was immediately frozen

using liquid nitrogen and freeze-dried for NMR analysis.

NMR Analysis of NaOH-EDTA extracts

Freeze-dried NaOH-EDTA extracts were combined for NMR analysis from which a 300-

500 mg subsample was ground, re-dissolved in 5 mL of deionised water, and centrifuged at

1400 × g for 20 minutes. Since the PA and TCA extracts did not attain dryness on freeze-

drying, they were directly dissolved in 0.25 mol L-1 NaOH and 0.05 mol L-1 Na2EDTA to

make a final volume of 5 mL. In each case, the supernatant solution (3.5 mL),

methylenediphosphonic acid (MDP) (0.1 mL at a concentration of 6 g L-1), added as an

internal standard and deuterium oxide (D2O) (0.3 mL) were added to a 10 mm NMR tube

and mixed. The pH of these solutions was tested and where the pH was <13, 100-500 µL

10 M NaOH was added to raise the pH to >13.

63

Solution 31P NMR spectra were acquired at 24°C on a Varian INOVA 400 NMR

spectrometer (Varian, Palo Alto, CA) at a 31P frequency of 161.9 MHz. Recovery delays

ranged from 8 to 50 s and were set to at least five times the T1 (spin lattice relaxation time)

values of the orthophosphate resonance determined in preliminary inversion-recovery

experiments (data not presented). We used a 90° pulse of 30-45 µs, an acquisition time of

1.0 s, and gated broadband 1H decoupling. Between 1600 and 10 000 scans were acquired

for each sample, depending on the P concentration. The spectra presented have a line

broadening of 2 Hz.

Quantification of P species from 31P NMR spectra

The relative concentrations of P species in the NaOH-EDTA extracts were determined

from 31P NMR spectra using integration and, in some cases, deconvolution. The absolute

concentration of each P species (including those determined using integration alone and

those determined using integration and deconvolution combined) was calculated by

integration against a known concentration of the MDP that was added to each NMR tube.

Pyrophosphate (-4.5 to -5.5 ppm) concentrations were determined using integration alone.

For the whole residue samples only, the relative concentrations of P species giving rise to

the numerous individual peaks were quantified by spectral deconvolution, using a method

similar to Bünemann et al. [24]. Each spectrum was fitted with up to twelve peaks as per

Noack et al. [9]. These were identified as orthophosphate, α- and β-glycerophosphate,

phytate (four peaks), and five peaks in the monoester region that can be assigned to RNA

degradation products [25]. Each peak was defined by three parameters: the chemical shift

(frequency), intensity, and the line width, which were allowed to vary in the fit.

64

Statistical analyses

Treatment differences were evaluated by least significant difference (LSD, p≤0.05) derived

from analysis of variance (ANOVA) using the GENSTAT version 13 statistical package

(VSN International, Rothamsted, UK). Assumptions of constant error variance

(homogeneity), normality of data distribution and additivity of treatment and replicate

effects were tested for each analysis.

Results and Discussion

Residue P speciation as determined by 31P NMR spectroscopy

The speciation of P in the whole residue was determined by triplicate 31P NMR analysis of

NaOH-EDTA extracts (Fig. 2). Just over half (54%, 294 mg kg-1) of total P in this whole

oat residue was detected as orthophosphate. Phospholipids, detected as their alkaline

degradation products α- and β-glycerophosphate [26] at 5.0 ppm and 4.6 ppm, respectively,

represented the next largest P class, accounting for 11% (58 mg kg-1) of total residue P

(Fig. 2). Ribonucleic acid, again detected as its alkaline degradation products: a set of five

peaks [9, 25] between 4.0 and 4.5 ppm, and pyrophosphate, identified as a peak at -4.5

ppm, each comprised a further 6% (33 and 31 mg kg-1, respectively) of the total residue P.

Phytate, detected as four characteristic signals at 5.7, 4.7, 4.4 and 4.3 ppm in the ratio

1:2:2:1 [25], comprised 5% (27 mg kg-1) of the total residue P (Fig. 2). Consequently 18%

(97 mg kg-1) of the total P was not detected by NMR, (designated as residual P in Fig. 2).

The variation in P species concentrations detected by NMR varied little between analytical

replicates, with coefficients of variation (standard deviation as percentage of mean

concentrations) ranging from 8 to 18%; the exception was residual P (42%).

The presence of these P species is consistent with previous studies of P forms in plant

material [8, 9, 18]. In a study where eight cereal and legume residues were collected from

65

the field, Noack et al. [9] identified orthophosphate, phospholipids, RNA, phytate and

pyrophosphate in stem and leaf material. Orthophosphate was the dominant P form,

representing 25-75% of total residue P, with smaller quantities of lipid P (10-49%), RNA

(5-30%), pyrophosphate (7-14%) and phytate (<1%) detected.

Fig. 2. Concentration (mg kg-1) and standard deviation of P species detected in NaOH-EDTA extracts of the whole oat residues (n=3).

Water and acid extractable P

The extraction of P with either water or acid is the first step in all plant chemical

fractionation methods and is reported to represent the soluble P fraction. The extracted P is

commonly further classified as soluble inorganic (molybdate reactive as determined by

colorimetry) and soluble organic P (determined as the difference between total P and

inorganic P) [2, 5, 27]. However, some studies assume this fraction to consist of inorganic

P only [1]. Here, four different extraction conditions were tested and total soluble P (sum

of inorganic and organic P) and soluble orthophosphate (inorganic P) were determined

(Fig. 3).

Phosphorus species

Orth

opho

spha

te

Phos

phol

ipid

RN

A

Phyt

ate

Pyro

phos

phat

e

Res

idua

l P

Tota

l res

idue

P (m

g kg

-1)

0

50

100

150

200

250

300

350

66

For all extraction conditions tested, the soluble orthophosphate concentration measured by

colorimetry ranged from 235-290 mg kg-1 and were similar to the orthophosphate

concentration determined by NMR analysis (Fig. 3). While the four extraction methods

resulted in similar values for extractable orthophosphate, two extraction methods (TCA

and WSO) extracted higher amounts of P from the residues, apparently due to greater

extraction of soluble organic P concentrations.

Fig. 3. Comparison of total water/acid soluble P (sum of soluble organic and inorganic P) extracted from residues, water/acid soluble inorganic P (soluble inorganic P) and inorganic P (orthophosphate) detected by NMR. Within a P class, measurements appended by a different letter are significantly different (p≤0.05). WHS = water/shake, WSO= water/sonnicated, TCA = trichloroacetic acid and PA = perchloric acid.

Speciation of P in water and acid extracts as determined by 31P NMR spectroscopy

Solution 31P NMR spectroscopy was used to further characterise the P species in the water

and acid extracts. This was achieved by freeze-drying the extracts and re-dissolving them

in NaOH-EDTA, to facilitate comparison of the composition of water/acid extracts with

that of the NaOH-EDTA extract of the whole residue; the resulting 31P NMR spectra are

shown in Fig. 4. Integration of the 31P NMR spectra of the water and acid extracts

confirmed that the majority of signal detected (75-87%) could be ascribed to

67

orthophosphate, with a slightly higher range of values for the weak acid extracts (85-87%)

than the water extracts (75-80%). These values represent a higher percentage of

orthophosphate than obtained by comparing colorimetry values to total P in the extracts

(61-64% and 56-63% for weak acid and water extracts, respectively).

The overall composition of P in the water extracts is in broad agreement with a 31P NMR

study by He et al. [28] on P speciation in water extracts from seven crop residues. Four of

these water extracts (clover, vetch, wheat and lupin) contained P only in the

orthophosphate form [28]. For the remaining three residues (corn, alfalfa and soybean),

orthophosphate was the dominant P form, while monoester P comprised 8-32% of the total

soluble P [28].

Despite its water solubility, pyrophosphate was not detected in any of the water or acid

extracts. The most likely explanation is that pyrophosphate was enzymatically hydrolysed

during the water extractions or hydrolysed during acid extraction [29]. The plant residues

would contain enzymes that would be released from the ground residues into water

extracts. Numerous enzymes have been shown to catalyse the release of orthophosphate

from a wide range of organic P compounds [30-32]. While these enzymes would likely

remain active in the near-neutral pH of water extracts, they would be inactivated at the

high pH of the NaOH-EDTA solutions, which would explain why pyrophosphate is

detected in the NaOH-EDTA extract of the whole residue. Acidic extracts are unlikely to

contain pyrophosphate as this molecule is easily hydrolysed to orthophosphate under acidic

conditions [33].

A close inspection of the monoester region of the 31P NMR spectra of the water and acid

extracts (right side of Fig. 4) reveals some key differences in organic P speciation between

68

the water and acid extracts and the NaOH-EDTA extract of the whole residue. For the two

water extracts, a large peak at 5.0 ppm and a much smaller peak at 4.6 ppm are coincident

with peaks we assigned as α- and β-glycerophosphate in the 31P NMR spectrum of the

whole residue. Spiking subsequently confirmed this assignment for the water extracts. The

presence of glycerophosphate in the 31P NMR spectrum of the whole residue is attributed

to alkaline hydrolysis of phospholipids. Phospholipid, being hydrophobic, should not be

water-extractable, which suggests that some phospholipid present in the whole residues

was hydrolysed during the water and weak acid extraction treatments, releasing

glycerophosphate (which is soluble in water and weak acid) to solution. Similar to

pyrophosphate, this hydrolysis was most likely mediated by enzymes released from the

ground residues into the extract solution [29-32]. This explanation is supported by the very

different proportions of the two glycerophosphate isomers between the whole residue

NaOH-EDTA extracts (approximately equal amounts of α- and β-glycerophosphate) and

the water extracts (predominantly α-glycerophosphate).

The broad peak at 4.4 ppm in 31P NMR spectra of both water extracts provides further

evidence for enzymatic hydrolysis of organic P (Fig 4). This peak corresponds closely with

the chemical shifts in NaOH-EDTA of the four naturally occurring nucleotides: adenosine

5’-monophosphate (AMP), guanosine 5’-monophosphate (GMP), cytidine 5’-

monophosphate (CMP), and uridine 5’-monophosphate (UMP), which all resonate in the

region 4.0-4.5 ppm [9, 25]. These four compounds are the building blocks of RNA. They

differ from the compounds identified as alkaline degradation products of RNA in the NMR

spectrum of the NaOH-EDTA extract of the whole residue in that the phosphate group is

attached at the 5’ position of the ribose unit in naturally occurring nucleotides but attached

at the 2’ or 3’ position of the ribose unit in the alkaline degradation products of RNA. The

presence of the naturally occurring nucleotides in the water extracts suggests that

69

enzymatic hydrolysis of RNA occurred during the water extraction, rather than intact RNA

undergoing alkaline hydrolysis when the freeze-dried extract was re-dissolved in NaOH-

EDTA.

Fig. 4. Solution 31P NMR spectra of NaOH-EDTA whole crop residue and extracts after soluble P fractionation steps. Left wide spectra view and right narrow spectra view. Spectra on the left have been vertically scaled to the maximum intensity of the orthophosphate peak (5.75 ppm). Spectra on the right have been vertically scaled to the maximum intensity of the most intense monoester peak. Peaks assigned as A = orthophosphate, B = monoester P and C= pyrophosphate.

Whole oat residue

Water/shake (WSH)

Trichloroacetic acid (TCA)

Perchloric acid (PCA)

Water/sonicate(WSO)

A

B C

A B

70

The signal-to-noise (S/N) ratio of 31P NMR spectra of the acid extracts is poorer than for

the water extracts (Fig. 4). This is mainly a consequence of an inability to remove the acids

from these extracts by freeze-drying (in contrast to the ease in removing water only from

the water extracts). Thus, the acid extract spectra represent a much smaller total amount of

extract and the samples analysed had proportionally lower P concentrations. Despite the

poor S/N ratios, at least four monoester peaks are apparent. The presence of the conjugate

bases of trichloroacetic acid and perchloric acid in the samples also appears to have

affected the positions of peaks for both orthophosphate and monoesters, such that they do

not align with the peak positions in other samples (Fig. 4). A similar effect of salts on peak

positions has been noted previously [34]. Nonetheless, it is likely that the left-most

monoester peaks are due to α- and β-glycerophosphate; the identity of the other two

monoester peaks is unknown.

Speciation of P in the residue following water and acid extractions as determined by 31P

NMR spectroscopy

Solution 31P NMR spectroscopy was also used to characterise P species in the residues of

the plant material after the water and acid extractions (Fig. 5). An implicit assumption of

chemical fractionation is that these residues remaining after an extraction step do not

contain the same chemical species as are present in the corresponding extract.

Furthermore, the P species in the extract and residue fractions when combined should be

those P forms present in the whole residue.

Crucially, for all water and acid extractions, the majority (64-78%) of P detected in the

residues was orthophosphate (Fig. 4). When orthophosphate detected in the water/acid

extracts and remaining residues is combined, the total is substantially greater than the

concentration of orthophosphate detected by NMR in the NaOH-EDTA extract of the

whole residue. This can be partly attributed to hydrolysis of pyrophosphate during water

71

and acid extraction; pyrophosphate comprised 6% of P in the whole residue but was

virtually absent from all of the water and acid extracts and residues (Fig. 4 and 5).

However, it appears likely that some organic P species were also hydrolysed to release

orthophosphate during water and acid extraction. Again, for the water-based extractions,

this is likely to have occurred through enzymatic hydrolysis, as there would be enzymes

released into solution that are efficient at hydrolysing monoester organic P compounds to

orthophosphate [32]. For the acid extracts there is the potential for weak acid to chemically

hydrolyse some organic P, thereby releasing orthophosphate to solution [35].

The presence of orthophosphate in the crop residues following water and acid extraction is

consistent with the findings of Dou et al. [36]. In a study on the water extractability of

orthophosphate in poultry and dairy manure they found a single extraction did not release

all orthophosphate, with a further 25-30% released in 3-4 subsequent 1 hour extractions

[36].

72

Fig. 5. Solution 31P NMR spectra of NaOH-EDTA whole crop residue remaining after soluble P fractionation steps. Left wide spectra view and right narrow spectra view. Spectra on the left have been vertically scaled to the maximum intensity of the orthophosphate peak (5.75 ppm). Spectra on the right have been vertically scaled to the maximum intensity of the most intense monoester peak.

The main organic P species detected by NMR in the residues following water extraction

were the phospholipid degradation products α- and β-glycerophosphate at 5.0 ppm and 4.6

ppm, respectively (right side of Fig. 5). Interestingly, the ratio of these two peaks

(approximately 1:1) was similar to that seen for the whole residue, indicating that

Whole oat residue

Water/shake (WSH)

Trichloroacetic acid (TCA)

Perchloric acid (PCA)

Water/sonicate(WSO)

73

phospholipid in this fraction remained intact through the water extraction (neutral pH) and

was subsequently converted to glycerophosphate under the alkaline conditions of NaOH-

EDTA extraction [37]. Several organic P species were detected by NMR in the residues

following acid extraction (right side of Fig. 5). We were able to identify the strongest

monoester peaks in the 31P NMR spectrum of the TCA residue as α- and β-

glycerophosphate by spiking. We note that the chemical shift of these species was

approximately 0.2 ppm higher than in the residues following water extraction, probably

reflecting differences in pH and/or ionic strength. The close similarity of the monoester

region of the 31P NMR spectrum of the PCA residue to that of the TCA residue suggests

that α- and β-glycerophosphate were also the major organic P species present.

Interestingly, the α-isomer of glycerophosphate was dominant in the residues following

acid extraction, in common with the water extracts, but in contrast to the acid extracts and

residue following water extraction. This indicates there was some hydrolysis of

phospholipids in these residues prior to extraction with NaOH-EDTA. A short review by

Folch [37] reports under alkaline conditions there is a predominance of β-glycerophosphate

and acid treatment results in predominance of α-glycerophosphate. Other organic P species

in the 31P NMR spectra of the residues following acid extraction were not identified.

Differences in pH and/or ionic strength make it impossible to tell whether or not these

minor organic P compounds are the same as those detected in the NaOH-EDTA extract of

the whole residue.

Implications from NMR analysis of extract and residues fractions of water and acid

extractions

The implications of these findings are far-reaching. Extraction of P with water or acid is

the first step in all chemical fractionation methods [1, 2] and these results demonstrate that

this step does not achieve what it sets out to do, i.e. selectively separate intrinsically water

74

soluble species (recovered in the extract) from those not soluble in water (recovered in the

remaining crop residue). On face-value, the orthophosphate concentrations in the

water/acid extracts aligned well with the orthophosphate concentrations determined by

NMR analysis of the NaOH-EDTA extract of the whole residue. However, detailed

analysis of the extracts and residues following water and acid extraction subsequently

showed this to be a coincidence borne of two flaws in the water and acid fractionation

procedures that cancelled out for this particular material. The incomplete extraction of

orthophosphate meant that the orthophosphate concentrations in the water and acid extracts

were underestimated. This problem was evidenced by the dominance of orthophosphate in

the NaOH-EDTA extracts of residues following water/acid extraction. On the other hand,

enzymatic (in the case of water extracts) or acid (in the case of acid extracts) hydrolysis of

organic P and pyrophosphate meant that some P present as organic or condensed P in the

plant material was detected as orthophosphate in the extracts, erroneously increasing the

orthophosphate concentration. This problem was evidenced by the almost complete

absence of pyrophosphate in any of the water/acid extracts or residues and also the finding

that the organic P composition in the extracts and residues did not align with that of the

whole crop residue.

Obviously, the problems identified here for water and acid extractions would compromise

the remaining steps of sequential fractionation schemes. The incomplete recovery of

orthophosphate would result in an overestimation of P species in subsequent fractionation

steps (e.g. nucleic acid or residual P) and the transformation of organic P species

invalidates the implicit assumption that unextracted P species are unaffected by preceding

steps.

75

Phosphorus species detected in organic solvent treatments

For both organic solvent treatments, extraction with ethanol:ether (E:E) and extraction

with ethanol:ether:chloroform (E:E:C), only a small proportion (1.2% and 1.4%,

respectively) of total P (as determined by acid digestion of plant residue) was detected by

ICP-AES in the extract (after acid digestion). It is possible that these values may be an

underestimation of P in these extracts as the lipid material isolated following removal of

the organic solvents was very hydrophobic and may have resisted acid digestion. These

apparent phospholipid contents were certainly much lower than the 11% of total P

determined to be phospholipid by NMR analysis of the whole residue (Fig. 2). It was not

possible to determine the P speciation of the organic solvent extracts because they could

not be dissolved in NaOH-EDTA, again due to their waxy, hydrophobic nature. However,

we were able to analyse the crop residues following the organic solvent extractions by 31P

NMR after extraction in NaOH-EDTA (Fig. 6). The spectra obtained are similar in

appearance to the 31P NMR spectrum of the whole residue NaOH-EDTA extract, except

that the relative size of peaks for α- and β-glycerophosphate (the alkaline degradation

products of phospholipids) was diminished for the E:E and E:E:C residues.

76

Fig. 6. Solution 31P NMR spectra of NaOH-EDTA whole crop residue extracts and remaining residue after extraction with different phospholipid fraction steps. Peaks assigned as A = orthophosphate, B = α-glycerophosphate, C = and β-glycerophosphate.

These results indicate that while both E:E and E:E:C treatments were selective for

extracting phospholipid P, they were not exhaustive in that they did not extract all

phospholipids originally present. In contrast to the water and acid extractions, the organic

solvent extractions did not appear to transform organic P in the remaining residue, nor

would they be expected to, as any released enzymes would be inactivated in such solvents

and these treatments do not involve a major shift in pH that would result in chemical

hydrolysis of organic P species. The apparent incomplete extraction of phospholipids

under the treatment conditions reported here would result in an underestimation of this P

form by chemical fractionation and, when used as a step in sequential fractionation, an

Whole residue

E:E (ethanol:ether)

E:E:C (ethanol:ether:chloroform)

A B C

A

BC

A

BC

77

overestimation of one or more P species identified in subsequent steps (i.e. nucleic acid or

residual P).

Conclusions

A simple comparison of two alternative approaches for measuring the orthophosphate

concentration in mature oat residue – 31P NMR spectroscopy following NaOH-EDTA

extraction and colorimetry following water or acid extraction – showed they produced

similar values. However, this apparent consistency hid serious deficiencies in the latter

approach because an overestimation of orthophosphate due to hydrolysis of organic P and

pyrophosphate was balanced by underestimation of orthophosphate due to incomplete

extraction. This fortuitous coincidence cannot be relied on, and a re-think of chemical

fractionation approaches, including sequential fractionation, to determine P speciation of

plant and further more soil material is required. In particular, there is a need to further

investigate the impact that hydrolysis of organic and condensed (e.g. pyrophosphate) P

forms during fractionation procedures may have on the interpretation of fractionation

results, as well as the degree to which incomplete extraction modifies results. In both

instances, the use of 31P NMR spectroscopy in combination with chemical fractionation

appears to offer a way forward. These results can aid in the characterisation of P species in

sequential chemical extracts of plant material and provide a better understanding of the fate

of crop residue P post-harvest.

Acknowledgements

The authors thank the Grains Research and Development Corporation (GRDC) for

providing funding to support this research (DAV00095) and the University of Adelaide for

the James Frederick Sandoz Scholarship. We thank Waite Analytical Services for their

help with total P analysis and Janine Guy for technical assistance.

78

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[24] E.K. Bünemann, R. Smernik, A.L. Doolette, P. Marschner, R. Stonor, S.A. Wakelin,

A.M. McNeill, Soil Biol Biochem, 40 (2008) 1908-1915.

[25] B.L. Turner, N. Mahieu, L.M. Condron, Soil Sci Am J, 67 (2003) 497-510.

[26] A.L. Doolette, R.J. Smernik, W.J. Dougherty, Soil Sci Am J, 73 (2009) 919-927.

[27] C.E. Barr, A. Ulrich, J Agric Food Chem, 11 (1963) 313-316.

[28] Z.Q. He, J.D. Mao, C.W. Honeycutt, T. Ohno, J.F. Hunt, B.J. Cade-Menun, Biol Fert

Soils, 45 (2009) 609-616.

[29] C.A. Shand, S. Smith, Biol Fert Soils, 24 (1997) 183-187.

[30] J.C. Tarafdar, N. Claassen, Biol Fert Soils, 5 (1988) 308-312.

[31] T. Tadano, K. Ozawa, H. Sakai, M. Osaki, H. Matsui, Plant Soil, 155 (1993) 95-98.

[32] M.L. Bishop, A.C. Chang, R.W.K. Lee, Soil Sci, 157 (1994) 238-243.

[33] J.P. Crowther, A.E.R. Westman, Canadian Journal of Chemistry-Revue Canadienne

De Chimie, 32 (1954) 42-48.

[34] R.J. Smernik, W.J. Dougherty, Soil Sci Am J, 71 (2007) 1045-1050.

[35] P. Masson, C. Morel, E. Martin, A. Oberson, D. Friesen, Comm Soil Sci Plant Anal,

32 (2001) 2241-2253.

[36] Z. Dou, J.D. Toth, D.T. Galligan, C.F. Ramberg, J.D. Ferguson, J Environ Qual, 29

(2000) 508-514.

[37] J. Folch, Journal of Biological Chemistry, 146 (1942) 31-33.

80

CHAPTER 4

PHOSPHORUS SPECIATION IN MATURE WHEAT AND

CANOLA PLANTS AS AFFECTED BY PHOSPHORUS SUPPLY

The work contained in this chapter has been accepted for publication in Plant and Soil. Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M., Armstrong, R.D., 2014. Phosphorus speciation in mature wheat and canola plants as affected by phosphorus supply. Plant and Soil online first.

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82

Statement of AuthorshipTitle of Paper

Publication Status Published, Accepted for Publication, Submitted for Publication, Publication style

Publication Details

Author ContributionsBy signing the Statement of Authorship, each author certifies that their stated contribution to the publication is accurate and that permission is granted for the publication to be included in the candidate’s thesis.

Name of Principal Author (Candidate)

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Phosphorus speciation in mature wheat and canola plants as affected by phosphorus supply

This manuscript has been accepted for publication in Plant and Soil.

Sarah Noack

Performed analysis on all samples, interpreted data, wrote manuscript and acting as corresponding author

Mike McLaughlin

Helped with data interpretation and manuscript evaluation.

Ronald Smernik

Helped with analysis of samples, data interpretation and manuscript evaluation.

Therese McBeath

Helped with data interpretation and manuscript evaluation.

Roger Armstrong

Helped to evaluate and edit the manuscript.

83

84

85

NOTE:

This publication is included on pages 85-97 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://doi.org/10.1007/s11104-013-2015-3

A Noack, S.R., McLaughlin, M.J., Smernik, R.J., McBeath, T.M. & Armstrong, R.D. (2014) Phosphorus speciation in mature wheat and canola plants as affected by phosphorus supply. Plant and Soil, v. 378(1-2), pp. 125-137

98

CHAPTER 5

MANAGEMENT OF CROP RESIDUES AFFECTS THE

TRANSFER OF PHOSPHORUS TO PLANT AND SOIL POOLS:

RESULTS FROM A DUAL-LABELLING EXPERIMENT

The work contained in this chapter has been accepted for publication in Soil Biology & Biochemistry. Noack, S.R., McBeath, T.M., McLaughlin, M.J., Smernik, R.J., Armstrong, R.D., 2014. Management of crop residues affects the transfer of phosphorus to plant and soil pools: Results from a dual-labelling experiment. Soil Biology & Biochemistry (in press) 71, 31-39.

99

100

Statement of AuthorshipTitle of Paper

Publication Status Published, Accepted for Publication, Submitted for Publication, Publication style

Publication Details

Author ContributionsBy signing the Statement of Authorship, each author certifies that their stated contribution to the publication is accurate and that permission is granted for the publication to be included in the candidate’s thesis.

Name of Principal Author (Candidate)

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Name of Co-Author

Contribution to the Paper

Signature Date

Management of crop residues affects the transfer of phosphorus to plant and soil pools: Results from a dual-labelling experiment

This manuscript has been accepted for publication in Soil Biology & Biochemistry.

Sarah Noack

Performed analysis on all samples, interpreted data, wrote manuscript and acting as corresponding author.

Therese McBeath

Supervised development of work, helped in data interpretation and manuscript evaluation.

Mike McLaughlin

Helped with data interpretation and manuscript evaluation.

Ronald Smernik

Helped with data interpretation and manuscript evaluation.

Roger Armstrong

Helped with data interpretation and to evaluate and edit the manuscript.

101

102

lts from a

A Noack, S.R., McBeath, T.M., McLaughlin, M.J., Smernik, R.J. & Armstrong, R.D. (2014) Management of crop residues affects the transfer of phosphorus to plant and soil pools: resudual-labelling experiment. Soil Biology and Biochemistry, v. 71(April), pp. 31-39

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This puof t

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NOTE: tion is included on pages 103-111 in the print copy esis held in the University of Adelaide Library.

also available online to authorised users at:

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CHAPTER 6

SUMMARY, CONCLUSIONS AND FUTURE RESEARCH

PRIORITIES

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Nutrients including phosphorus (P) released from crop residues remaining in the field post-

harvest are potentially an important source of nutrients for subsequent crops. The P within

these crop residues can be released to soil as soluble P, assimilated by microorganisms, or

contribute to more chemically stable P pools in soil. Crop residue speciation plays an

important role in determining the partitioning of residue-derived P into these three pools. The

net effect of crop residues on soil P availability will depend on the balance of these processes.

To fully understand the contribution of residue P as a source of P for agricultural production,

its chemical nature needs to be understood. Better identification of P species in crop residues

can improve our understanding of the potential turnover of these P species in soil. Phosphorus-

31 nuclear magnetic resonance (NMR) spectroscopy has been shown to be a good method for

the analysis of P species in soil and manures with a smaller body of work applying this

technique to plant material.

The studies outlined in this thesis focused on the speciation of crop residue P using

solution 31P NMR spectroscopy techniques, and applying this technique to validate the P

species extracted in well-established chemical fractionation methods. This methodology

provided the basis for investigating the fate of residue P in soil-plant systems by measuring the

effect of plant P status on residue P concentration and speciation and subsequently the

contribution of residue P release to soil-plant systems with different residue management.

Speciation of crop residue P using 31P NMR spectroscopy showed:

1. Orthophosphate will be the dominant form of P in most crop residues that will be

returned to soil in the field;

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2. Chemical fractionation techniques only partially extract the intended P classes and the

extractants selected caused conversion of P species, clouding interpretation of plant P

speciation;

3. Plant P status of mature wheat and canola residues had little effect on the composition

of P species returned to soil; and

4. Crop residues contributed only a small proportion of P to subsequent crop growth and

this contribution differed with residue management practice.

As a consequence of these four main outcomes, the research contained in this thesis

demonstrated the importance of accurate speciation of residue P and the relationship between

P speciation in residues and subsequent bioavailability of P from residues. This research

provides evidence that overturns previous research recommending the use of carbon (C):P

ratios as an accurate predictor of the rate of release of P from crop residues.

Crop residue P speciation using 31P NMR

Differences in the chemical composition of P in crop residues plays an important role in

residue-soil P cycling and the processes involved. Analysis of a series of crop residues

collected from commercial paddocks revealed a significant percentage of P in the stems and

chaff of crops was in the form of orthophosphate. Water-extractable P represented the

majority of total P (average 85%, of which 93% was detected as orthophosphate (molybdate

reactive)) in stem and leaf residues, which are the bulk of plant material returned to soil post-

harvest. This suggests that the majority of residue P in a field has the potential to be released

into solution after the first significant rainfall. This P will be returned to the soil in a readily

available form, which would be available for assimilation by plants (via root uptake) and

microorganisms, as well as sorption onto soil minerals.

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The identity and quantity of P species detected in crop residues was different to the initial

expectations based on the literature. A key finding was the greater concentration of

orthophosphate in the residue material than originally anticipated. This result highlights the

need to consider that P cycling from residues to soil is different to nitrogen (N) cycling. The

release of mineral N from crop residues almost exclusively involves microbial decomposition

of organic forms. For the release of crop residue P, it is evident that large amounts of water-

soluble P can be lost from crop residues without involving microbial decomposition at all.

Hence, the movement of inorganic P (orthophosphate) out of crop residues via leaching in

rainfall or irrigation water assumes a much greater importance than has previously been

recognised.

Another major advance of this work is clearer identification of the nature of organic P species

in plant material. The main organic P species identified were phospholipids and RNA, both of

which are regarded as highly labile. There was an unidentified fraction of P that was not

recovered using NaOH-EDTA (on average 12%), however there was no other presence of

recalcitrant organic P species, which would be expected to directly contribute to a stable

organic soil P pool. The identification of these organic P species again highlights the labile

nature of P in crop residues, which also detracts emphasis from the importance of

mineralisation as the sole process releasing residue P into soil solution.

The detection of phytate in some non-seed plant parts (chaff) was also an interesting finding.

Based on recent work, it was suggested that these plant parts were no longer storing P as

orthophosphate due to high cell P concentrations inducing P storage as phytate. The portion of

phytate returned to soil in non-seed plant parts is small however, and this is highly dependent

on seed losses during harvesting processes (as up to 90% of seed P is phytate). There is still

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much debate surrounding the stability of phytate, which will impact the fate of this residue P

form in soil. Current literature suggests that phytate returned to soil from crop residues has the

potential to be mineralised by microorganisms or become stabilised with time (Celi et al.

1999; He et al. 2006; Hill and Richardson 2007).

The use of 31P NMR to speciate P in crop residues provides improved understanding of

residue P species returned to soil and their potential fate. The commonly used C:P ratio does

not distinguish between P present as inorganic P (orthophosphate) and organic P. This ratio

assumes that all residue P must be decomposed by microorganisms, and released as

orthophosphate through mineralisation, to be plant available. However, speciation of crop

residues suggests that, on average, half of the P in residues is already in a form readily

available to both plants and microorganisms. As a result, this potentially contributes to

explanation of why there is such a large range of C:P ratios currently used to predict

mineralisation.

Lastly, in this investigation, there was also an apparent contradiction between the high

orthophosphate content in the water extracts compared to the NaOH-EDTA extracts used

for 31P NMR analysis. This suggests that during water extraction there was conversion of

organic P to inorganic P, overestimating the orthophosphate concentration in the original plant

material. Water or weak acid extraction of soluble P is the first step in any chemical

fractionation procedure. The conclusions drawn in this study suggested that further work was

necessary to assess the P species extracted by chemical fractionation methods, which was the

focus of Chapter 3.

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Comparing and combining solution 31P NMR spectroscopy and chemical fractionation

methods

Chapter 3 outlined a combined approach for identification of P species in crop residues using

chemical fractionation and solution 31P NMR spectroscopy. Results from the Chapter 2

showed there was a large difference between the orthophosphate concentration determined in

initial crop residues by 31P NMR and water-soluble residue P (a measure of orthophosphate

plus some soluble organic P). This finding was the basis for a study in which P extracted by

chemical fractionation methods was analysed using solution 31P NMR to test key assumptions

that underpin the chemical fractionation approach, namely that (i) chemical extractants are

selective for the intended P species; (ii) the extractant releases all of the intended P form into

solution; and (iii) non-targeted P species are not transformed during extraction.

The results showed that the orthophosphate concentrations measured in four water and acid

extracts (water shake or sonicate, 2% perchloric acid and 10% trichloroacetic acid) were

similar to the orthophosphate concentration determined on the initial plant material by

solution 31P NMR spectroscopy. However, solution 31P NMR analysis of the extracts and

residue following water/acid extraction showed this result occurred by coincidence, as two

biases effectively cancelled each other out. Firstly, the orthophosphate concentration in the

water/acid extracts increased through the hydrolysis of pyrophosphate and organic P species.

Secondly, there was incomplete recovery of orthophosphate from the crop residues, resulting

in an underestimation of this P form. For two extractants designed to extract phospholipid

(extraction with ethanol:ether and ethanol:ether:cholorform) the extractants were selective for

phospholipid P but only ~10% of the phospholipid P determined by solution 31P NMR was

extracted.

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Chemical fractionation methods are inexpensive compared to solution 31P NMR, and can

provide rapid information about the solubility and potential lability of residue P species. A

large body of work in the literature has employed chemical fractionation to determine

operationally defined P forms (e.g. soluble P) and/or specific P compounds (e.g.

phospholipids, nucleic acids). The results here provide evidence that speciation of residue P

can be compromised by the incomplete recovery of the intended P species and the

transformation of other P species during extraction. Therefore interpreting the chemical

composition of plant material using chemical fractionation is problematic. The terminology

used to describe the P forms extracted by specific fractionation steps should rather be based on

the extractant (e.g. ethanol:ether soluble P) rather than a specific P species (e.g.

phospholipids).

An integrated approach using chemical fractionation and 31P NMR provides a method to

overcome some of the issues with chemical fractionation. Solution 31P NMR spectroscopy

provides precise knowledge of extracted P forms available and allows accurate interpretation

of plant fractionation procedures.

Relationship between plant P status and P speciation

Chapter 4 further explored the effect of P status on the P composition of crop residues. Results

in Chapter 2 suggested that the large variation in orthophosphate concentrations found in stem

residues may be related to their total P concentrations, in that for crop residues with higher

total P concentrations, a greater proportion was present as orthophosphate. This finding was

consistent with previous studies showing that as the supply of P to the plant increases from the

deficiency to the sufficiency range, the concentrations of major P fractions (ester, lipids,

nucleic acid and inorganic P) in vegetative plant organs also increases. It is commonly

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assumed that further increases in P supply to luxury levels result in only the inorganic P

concentration increasing as the major P storage compound. However, much of this work was

based on chemical fractionation methods and most studies did not use mature plant residues.

Both wheat and canola provided with three different P rates accumulated different P species in

various plant parts (roots, stem, leaves, chaff/pods and seed). However, the relative

proportions of P species was relatively unaffected by plant P status for the majority of wheat

and canola plant parts. This contrasts with many previous studies, which have reported that

increases in P status increase inorganic P concentrations and increase inorganic P as a

proportion of total P. Even at deficient P concentration in this study, the primary form of P in

crop residues was orthophosphate.

The non-seed plant parts (root, stem, leaves and chaff/pods) constitute the residue pool

returned to soil after the seed is harvested and removed from the field during harvesting

operations. The percentage of each P species that would be returned to soil in the residue P

pool was calculated from the total P content and speciation for each plant part. Although P

status caused differences in P speciation in some plant parts, these differences tended to cancel

out when speciation was expressed on a whole plant residue content basis. Orthophosphate

was the dominant P species in crop residues, followed by phytate (wheat) or phospholipid

(canola) with RNA and pyrophosphate present in only very small amounts. As plant P status

increased, the percentage of these P forms remained relatively unchanged for whole plant

residues of canola and wheat.

Although minor differences were observed in P speciation across the varying P application

rates and plant parts, the effect of this on P cycling is likely to be minor in comparison to the

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overall contribution of these residues to soil P pools. This glasshouse experiment showed that

the dominant P form in crop residues that is returned to soil after harvest is orthophosphate,

regardless of plant P status. Further characterisation of residue material with different total P

or C:P ratios is not necessary as the bulk of residue P will be returned as orthophosphate.

It appears from the results above that the placement of residues in the soil profile (e.g.

standing or laying on the soil surface or incorporated) will have a greater impact on residue P

release as the P species in crop residues remain relatively constant. Since the bulk of residue P

is orthophosphate, different residue management strategies (and therefore placement) may

favour P release more than other strategies. Residue management factors that will be

important are soil-residue contact, moisture (both soil moisture and interception of rainfall)

and temperature. The effect of different residue management on residue P release was the

focus of the final experimental chapter.

Residue management effects residue P release

In the final experimental chapter (Chapter 5), the effect of common residue management

techniques (surface placed or soil incorporated) and residue size (ground and 5 cm lengths) on

the fate of residue P in soil was measured. The experiment used a dual-labelling approach (33P

and 32P) to measure the contribution of residue, soil and fertiliser P to the growing plants and

to different soil pools. The majority of the P in surface-placed residues remained in the residue

itself or another soil P pool that was not measured in this study. Approximately 25% of P

added in surface-placed residues was detected in the resin-extractable, microbial and plant P

pools (where residues supplied equivalent to 0.6-0.7 kg P ha-1 to plants). The low amount of

residue P released in surface-applied treatments suggests P will be released from crop residues

slowly under no-till residue management compared to conventionally cultivated systems.

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Incorporating residues in the top 10 cm of soil can result in moisture and temperature

conditions that favour the release of soluble P from crop residues for plant and microbial

uptake. Transfer of residue P into soil and plant P pools was greater when residues were

incorporated, as it is in conventionally cultivated systems. On average, 80% of the P added in

ground incorporated residue and 60% of the P added in large incorporated residue was

detected in plant, microbial and resin P pools. The small and large residue pieces supplied

wheat plants with the equivalent of 2.0 and 1.1 kg P ha-1when incorporated.

Following harvest, crop residues will start to decompose and release P via leaching of

orthophosphate in residues, enzymatic breakdown of organic P in residues and microbial

decomposition and turnover. Based on the experiments discussed in this thesis, the processes

of leaching and enzymatic breakdown of organic P are likely to dominate so that the fate of

residue P is linked to the fate of leached orthophosphate P. When considering residue

management strategies based on their potential for influencing P supply to crops,

incorporating residues will increase the rate of release and decomposition and therefore the

potential for plant roots (if present) to access this P. The results also show that even though

residue P takes longer to break down under no-till management, this system was still able to

provide a small proportion of the subsequent crop’s P uptake.

As shown above, the subsequent wheat crop accessed 15% (0.2 – 0.8 kg P ha-1) of the residue

P added in commonly practised no-till systems. In the glasshouse experiment residue

treatments were able to provide similar amounts of P to the subsequent crop as fertiliser P,

however there is the need to validate this under field conditions. A field survey of the amount

of residue P remaining post harvest was undertaken as part of this thesis. The survey revealed

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that the residue P content ranged from 1-5 kg P ha-1in the field. This is in comparison to the

addition of 10-20 kg P ha-1 as fertiliser in the same environments sampled. Based on total P

alone, crop residues contain 10-25% of the amount of fertiliser P. It is likely that under field

conditions the contribution of residue P will be reduced compared to the 15% measured in the

glasshouse experiment due to less favourable moisture and temperature conditions.

Not only is the quantity of residue P released important, but also the timing during the season

when the P is released. It has been shown that the majority of residue P has the potential to be

released during the first significant rainfall. However, depending on the season, this P may

occur during summer where weeds will be the only plants to benefit from this released P. In an

ideal scenario, residue P release would coincide with crop demand. Accurate prediction of the

release of residue P is difficult due to the dependence on environmental conditions and the

multiple pathways (leaching, enzymatic breakdown and microbial decomposition) for release

and transport into soil.

Ultimately, when growers are planning their fertiliser management for the season, it is

unlikely that an estimate of residue P needs to be taken into consideration. While residue P

cycling is an important process for replenishing soil P pools, difficulties in trying to predict

the quantity and timing of release currently is not accurate enough for growers to use.

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Future research directions/priorities

To address some of the issues raised in this study, and in order to further develop our

understanding of residue P cycling in soils, further research in the following areas in

recommended.

1. This study demonstrated the variation in the amount of residue P in field samples at

maturity. Assessment of a greater number of field-collected residues will provide

greater knowledge of the amount of residue P remaining post-harvest. In the range of

environments sampled, 1-5 kg P ha-1 remained but, a more defined range for both

cereal and legumes would be useful to allow growers to make informed estimates of

the amount of residue P remaining post-harvest. Also, it should be noted that plants

drop leaves prior to harvest (when sampled in this study), especially for pulse crops,

which would increase the total concentration of P originating from crop residues.

So far, only the amount of residue P in the above-ground biomass has been measured,

and the concentration of P in crop roots needs further investigation. Roots are located

deeper in the soil profile, positioning the P released from these residues in a better

location to be intercepted by crop roots. While the glasshouse study in Chapter 4

indicated there was little below-ground residue, this is likely due to incomplete

recovery of roots and the fact that plants were grown in pots which markedly affect

root growth and distribution compared to field-grown plants.

2. Following harvest, crop residues will start to decompose and release P via leaching of

orthophosphate from residues, enzymatic breakdown of organic P in residues and

microbial decomposition and turnover. The work in this thesis has demonstrated that

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the former two processes dominate, so that the fate of residue P is linked to the fate of

leached orthophosphate. Further leaching and incubation studies using sterile and non-

sterile residues could enable the distinction between residue P that is released via

leaching or immobilised by microorganisms. In addition, 31P NMR could potentially be

used to track changes in P speciation in the initial residue following

leaching/incubation with soil. As shown in this thesis, residue P forms are dominated

by orthophosphate and monoester P while microbial P species have been shown to

primarily consist of diester P. While there is some overlap between plant and microbial

P species, there is the potential to use 31P NMR to observed the disappearance of

residue P forms (leaching) and appearance on new P forms (microbial origin).

3. The glasshouse experiment showed the release of residue P and uptake by a subsequent

crop was significant enough to be measured in both conventional and no-till systems.

However, this was performed at constant temperature and optimal soil moisture

conditions, which favoured faster residue P release compared to some field conditions.

Field evaluation is necessary to investigate the effect of temperature and rainfall on

residue P release in cropping soils. Without field evaluation of these processes, this

information is of little benefit to growers.

4. In recent years, a number of new technologies and management options for residues

post-harvest have been explored. The focus for much of this has been around no-till

systems. This thesis does not explore residue P release from standing residues for

which there is minimal contact with the soil. Investigating long-term release of residue

P in different no-till systems is important as it is likely to be slower, but still has the

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potential to provide useful amounts of P to crops over the growing season, and

potentially subsequent seasons.

While the use of no-till has become widespread in Australia, residue burning is still

practised in high rainfall zones with large residue loads and for the control of weed

seeds and pests (e.g. snails). Research into the implication of residue burning on P

speciation in the resulting ash is important for understanding the fate and availability

of this P. Furthermore, another consideration for the fate of this P is that the resulting

ash is lighter than straw and will be more susceptible to wind and water erosion. An

understanding of how residue burning effects P speciation and fate in soil will assist

with assessing the implications for these scenarios.

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References

Celi L, Lamacchia S, Marsan F A and Barberis E (1999) Interaction of inositol hexaphosphate

on clays: Adsorption and charging phenomena. Soil Sci 164, 574-585.

He Z, Honeycutt C W, Zhang T and Bertsch P M (2006) Preparation and FT-IR

characterization of metal phytate compounds. J Environ Qual 35, 1319-1328.

Hill J and Richardson A E (2007) Isolation and assessment of microorganisms that utilize

phytate. In Inositol phosphates: Linking agriculture and the environment. Eds. B L

Turner, A E Richardson and E J Mullaney. CAB International, London, UK.

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