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Performance and Secondary Chemistry of
Selected Annual Pasture Legumes in
Southeastern Australia
Sajid Latif
DVM, University of Veterinary and Animal Sciences, Lahore Pakistan
A thesis submitted to Charles Sturt University in fulfilment of
the requirement for the degree of Doctor of Philosophy
School of Animal and Veterinary Sciences
Faculty of Science
Charles Sturt University
Wagga Wagga, New South Wales Australia
August 2019
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Table of Contents
Table of Contents ........................................................................................................................... i
Certificate of Authorship ............................................................................................................. vi
List of Abbreviations .................................................................................................................. vii
Acknowledgements .................................................................................................................... viii
Thesis Format................................................................................................................................ x
List of Associated Publications .................................................................................................... xi
Preface ........................................................................................................................................ xv
Abstract .................................................................................................................................... xviii
Thesis Rationale .......................................................................................................................... 20
1.1 Farming Systems in Southeastern Australia ..................................................................... 20
1.2 Choice of Legumes in the Pasture Mix ............................................................................. 22
1.3 Annual Pasture Legumes of Mediterranean Origin .......................................................... 24
1.4 Weed Suppression in Annual Pasture Legumes................................................................ 28
1.5 Secondary Chemistry and Phytoestrogenic Activity of Annual Pasture Legumes on
Grazing Livestock ................................................................................................................... 29
Objectives and Hypotheses ......................................................................................................... 31
2.1 Research Objectives .......................................................................................................... 31
2.2 Hypotheses ........................................................................................................................ 32
2.3 References ......................................................................................................................... 34
Chapter 1. Literature Review ...................................................................................................... 40
Allelopathy and the Role of Allelochemicals in Plant Defense .................................................. 42
1.1 Introduction ....................................................................................................................... 43
1.2 Plant Defense and the Role of Allelochemicals ................................................................ 45
1.2.1. Allelochemical Localization and Release into the Environment .............................. 47
1.3 Classification of Secondary Metabolites ........................................................................... 48
1. 3.1 Phenolic Compounds and their Derivatives .............................................................. 49
1.3.2 Terpenoids .................................................................................................................. 51
1.3.3 Alkaloids .................................................................................................................... 54
1.3.4 Hydroxamic Acids of Benzoxazinoids: A Case Study from the Agronomy .............. 56
1.4 Allelochemical Mode of Action ........................................................................................ 57
1.4.1 Membrane Permeability ............................................................................................. 59
1.4.2 Water and Nutrient Uptake ........................................................................................ 59
1.4.3 Respiration ................................................................................................................. 60
1.4.4 Photosynthesis ............................................................................................................ 61
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1.4.5 Protein and Nucleic Acid Synthesis and Growth Regulation ..................................... 61
1.5. Localization and Transport of Allelochemicals in Donor Plants ...................................... 62
1.5.1 Root Exudation of Allelochemicals ............................................................................ 66
1.5.2 Diffusion ..................................................................................................................... 67
1.5.3 Vesicle Transport ........................................................................................................ 68
1.5.4 Ion Channels ............................................................................................................... 68
1.6. Factors Influencing the Release of Allelochemicals from the Plant ................................. 68
1.7. Role (s) of Allelochemicals in the Rhizosphere, in Neighboring Plants and Other
Organisms ................................................................................................................................ 70
1.7.1 Tolerance to Allelochemicals ..................................................................................... 73
1.8. Metabolic Profiling of Allelochemicals in Complex Plant or Soil Extracts or Mixtures . 74
1.9 Conclusions ....................................................................................................................... 76
1.10 References ....................................................................................................................... 77
Chapter. 2 .................................................................................................................................... 91
Weed Suppressive Potential of Selected Pasture Legumes against Annual Weeds in
Southeastern Australia ................................................................................................................. 91
2.1 Introduction ....................................................................................................................... 94
2.2 Materials and Methods ...................................................................................................... 95
2.3 Results ............................................................................................................................... 97
2.4 Discussion.......................................................................................................................... 99
2.5 Acknowledgments ........................................................................................................... 100
2.6 References ....................................................................................................................... 101
Chapter 3. .................................................................................................................................. 102
Performance and Mechanism of Weed Suppression in Selected Pasture Legumes against Annual
Weeds in Southeastern Australia ............................................................................................... 102
Abstract ................................................................................................................................. 104
3.1 Introduction ..................................................................................................................... 105
3.2 Materials and Methods .................................................................................................... 109
3.2.1 Pasture Establishment ............................................................................................... 109
3.2.2 Data Collection ......................................................................................................... 113
3.2.3 Measurement of Ecophysiological Traits Associated with Competitive Ability ...... 113
3.2.4 Statistical Analysis ................................................................................................... 114
3.3 Results ............................................................................................................................. 115
3.3.1 Climatic Conditions .................................................................................................. 115
3.3.2 Effect of Sowing Date on Pasture and Weed Cover ................................................. 115
3.3.3 Impact of Pasture Mixtures on Weed Infestation ..................................................... 116
3.3.4 Impact of Pasture Monoculture Treatment on Weed Cover ..................................... 118
3.3.5 Effect of Pasture Treatment on Dominant Weed Species ......................................... 119
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3.3.6 Effect of Pasture Biomass on Weed Suppression .................................................... 122
3.3.7 Effect of Competitive Traits on Weed Suppression ................................................. 123
3.4 Discussion ....................................................................................................................... 126
3.5 Conclusions ..................................................................................................................... 131
3.6 Acknowledgments ........................................................................................................... 132
3.7 References ....................................................................................................................... 132
3.8 Supplementary Materials ................................................................................................ 140
Chapter 4 ................................................................................................................................... 142
Metabolomic Approaches for the Identification of Flavonoids Associated with Weed
Suppression in Selected Hardseeded Annual Pasture Legumes ............................................... 142
Abstract ................................................................................................................................. 144
4.1 Introduction ..................................................................................................................... 146
4.2 Materials and Methods .................................................................................................... 150
4.2.1 Field Experimentation .............................................................................................. 150
4.2.2 A Modified Parker Assay for Assessment of Plant Residue Phytotoxicity in Annual
Pasture Legumes ............................................................................................................... 152
4.2.3 Extraction Procedure ................................................................................................ 153
4.2.4 Bioassay of Pasture Legume Extracts for Phytotoxicity .......................................... 153
4.2.5 UHPLC-QTOF-MS Analysis ................................................................................... 154
4.2.6 Data Analysis ........................................................................................................... 156
4.3 Results ............................................................................................................................. 157
4.3.1 Field Experimentation for Assessment of Weed Suppression by Annual Pasture
Legumes ............................................................................................................................ 157
4.3.2 A Modified Parker Assay for Assessment of Phytotoxicity of Dried Residues of
Annual Pasture Legumes .................................................................................................. 158
4.3.3 Bioassay of Pasture Legume Extracts for Phytotoxicity .......................................... 159
4.3.4 Non-Targeted Metabolic Profiling ........................................................................... 162
4.3.5 Chemometric Analysis ............................................................................................. 162
4.3.6 Quantification of Phytotoxic Flavonoids in Plant Tissue and Rhizosphere Soil ..... 164
4.4 Discussion ....................................................................................................................... 171
4.5 Conclusions ..................................................................................................................... 179
4.6 Acknowledgments ........................................................................................................... 180
4.7 References ....................................................................................................................... 180
4.8 Supplementary Material .................................................................................................. 189
Chapter 5 ................................................................................................................................... 193
Analysis of Coumestans, Flavonoids and Proanthocyanidins in Selected Annual Hardseeded
Pasture Legumes of Mediterranean Origin ............................................................................... 193
Abstract ................................................................................................................................. 195
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5.1 Introduction ..................................................................................................................... 196
5.2. Materials and Methods ................................................................................................... 200
5.2.1 Chemicals. ................................................................................................................ 200
5.2.2 Plant Material ........................................................................................................... 200
5.2.3 UHPLC-QTOF-MS Analysis. .................................................................................. 202
5.2.4 Extraction for Polyphenols. ...................................................................................... 203
5.2.5 Quantification of Total Polyphenol Content (TPC). ................................................. 203
5.2.6 Quantification of Total Proanthocyanidin Content (TPAC) ..................................... 204
5.2.7 Statistical Analysis ................................................................................................... 204
5.3. Results ............................................................................................................................ 205
5.3.1 Quantification of Phytoestrogens ............................................................................. 205
5.3.2 Effect of Cultivars and Growth Stage on Phytoestrogen Levels. ............................. 208
5.3.3 Quantification of Total Polyphenols and Proanthocyanidins. .................................. 210
5.3.4 Abundance of Flavonoids and their Glycosides. ...................................................... 211
5.4. Discussion....................................................................................................................... 214
5.5. Acknowledgements ........................................................................................................ 225
5.6. Conflict of Interest .......................................................................................................... 225
5.7. References ...................................................................................................................... 225
5.8. Supplementary data ........................................................................................................ 235
Chapter 6 ................................................................................................................................... 241
General Discussion .................................................................................................................... 241
6.1.1 Recommendations for Future Studies ........................................................................... 248
6.1.2 References .................................................................................................................... 250
Appendix A –Book Chapter ...................................................................................................... 252
Role of Allelopathy in the Persistence of Weeds ...................................................................... 252
7.1 Introduction ..................................................................................................................... 252
7.2 Classification of Allelochemicals .................................................................................... 254
7.3 Modes of Action of Allelochemicals ............................................................................... 257
7.4 Synthesis, Localization and Release of Allelochemcials from Donor Plants .................. 259
7.5 Factors Affecting Biosynthesis of Allelochemicals ........................................................ 261
7.6 The Role of Microorganisms in the Release and Transformation of Allelochemicals .... 263
7.7 Metabolic Profiling of Allelochemicals .......................................................................... 264
7.8 Case Studies of Invasive Plant Species Exhibiting Allelopathic Interactions ................. 265
7.8.1 Echium plantagineum L. ........................................................................................... 266
7.8.2 Centaurea solstitialis L. ........................................................................................... 267
7.8.3 Sorghum halepense (L.) Pers. ................................................................................... 268
7.8.4 Fallopia japonica (Houtt.) Dcne. ............................................................................. 268
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7.8.5 Parthenium hysterophorus L ................................................................................... 269
7.9 Conclusion ...................................................................................................................... 270
7.10 References ..................................................................................................................... 271
Appedndix B: Media Article ..................................................................................................... 281
Smother Winter Weeds with Annual Legumes ..................................................................... 281
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Certificate of Authorship
I hereby declare that this submission is my own work and to the best of my
knowledge and belief, understand that it contains no material previously published or
written by another person, nor material which to a substantial extent has been accepted
for the award of any other degree or diploma at Charles Sturt University or any other
educational institution, except where due acknowledgement is made in the thesis. Any
contribution made to the research by colleagues with whom I have worked at Charles
Sturt University or elsewhere during my candidature is fully acknowledged.
I agree that this thesis be accessible for the purpose of study and research in
accordance with normal conditions established by the Executive Director, Library
Services, Charles Sturt University or nominee, for the care, loan and reproduction of
thesis, subject to confidentiality provisions as approved by the University.
__________________
Sajid Latif
Charles Sturt University
Date:
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List of Abbreviations
ABC – ATP binding cassette family
ALMT – aluminium activated malate transport family
ATP – adenosine triphosphate
AUD – Australian dollar
BOA – benzoxazolin-2-0ne
DNA – deoxyribonucleic acid
DIMBOA 2, 4-dihydroxy-7-methoxy-2H-1, 4-benzoxazin-3-one
DIBOA 2, 4-dihydroxy-2H-1, 4-benzoxazin-3-one
ESI – electrospray ionisation
GC – gas chromatography
IAS – International Society of Allelopathy
MATE – multidrug and toxic compound extrusion family
MFS – major facilitator superfamily
M – million
MLA – Meat and Livestock Australia
MS – mass spectrometry
N – nitrogen
NMR – nuclear magnetic resonance
NSW – New South Wales
PPB – part per billion
PPM – part per million
PS – photosystem
PSM – plant secondary metabolite
QTOF – quadrupole time of flight
QQQ – triple quadrupole
RNA – ribonucleic acid
SPE – solid phase extraction
UHPLC – ultra-high pressure liquid chromatography
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Acknowledgements
This thesis has been completed under the supervision of Prof. Leslie A. Weston,
Dr. Paul A. Weston, A/Prof. Jane C. Quinn, Dr. Saliya Gurusinghe, Mr. John W. Piltz
and Prof. Peter C. Wynn. First and foremost, praises and thanks to the Allah, the
Almighty, for His showers of blessings throughout my project work to complete the
research successfully.
In addition, I would like to share my deep and sincere gratitude to my principal
supervisor, Prof. Leslie A. Weston for her support, invaluable guidance and her patience
in supervising me during my PhD and helping me learn experimental design, data
collection, metabolic profiling and interpretation of the data and finally presenting my
results as a complete story for journal articles. Her dynamism, vision, sincerity and
motivation have deeply inspired me and I am sincerely grateful for all the opportunities
that I have been given to improve my skills during my PhD. Thanks for challenging me
every day and believing in me. I am also very grateful to Paul A Weston for keeping his
door always open for me and willingness to mentor and assist me in LC-MS, statistics
and beyond. It has been a real privilege to work with such a diverse and supportive
supervisory team
There are many others who have supported me during my PhD. Primarily, I would
like to thank my friends and colleagues in the Plant-Interactions research group for their
continuous support in helping me build my lab skills and encouragement throughout my
journey. I owe a big thanks to Sal for his guidance, patience and optimism. At the same
time, I would like to acknowledge the technical support I received in the National Life
Sciences Hub. I would also like to thank everyone around me who made me smile
throughout my research project.
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Finally, and most importantly, I would like to thank my parents and brothers
especially Dr. Shahzad Latif and Dr. Sarfraz Latif for their unconditional love, advice,
prayers, care and sacrifices and making everything in my life possible and accessible. I
am also very thankful to my wife Zahra Batool Naqvi and my daughter Huda Sajid for
their love, understanding, prayers and continuing support in completing this research
work. I would like to thank all of them who (‘at home and away’), kept in touch and
supported me during the last four years.
Funding Acknowledgement
This PhD research project was supported by the Meat and Livestock Australia
(MLA) project entitled: “Addressing herbicide resistance- options for non-chemical
approaches for the mixed farmers” (B.WEE.0146) awarded to Leslie A. Weston and John
W. Piltz. I would also like to extend my thanks to the Australian Centre for International
Agricultural Research (ACIAR) and the Australia Pakistan Agriculture Sector Linkages
Program for supporting my research with the John Allwright Fellowship, and the Faculty
of Science and the Graham Centre for Agricultural Innovation for providing my operating
funds and travel awards, respectively.
.
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Thesis Format
This PhD thesis is divided into six individual chapters, which include four
manuscripts, all of which have been published or submitted to peer-reviewed journals.
Chapters 1 through 6 are therefore formatted as actual manuscripts as submitted to various
journals and each is preceded by an introductory description.
Chapter 1, a review of the literature, was published as a book chapter entitled
“Allelopathy and the Role of Allelochemcials in Plant Defence’’ in Advances in
Botanical Research, Elsevier Press. The results of my research are presented in Chapter
2 through Chapter 5 and are presented as research manuscripts. Chapter 2 is a conference
paper presenting preliminary data on the performance and weed suppressive potential of
annual pasture legumes and was published in the proceedings of the 21st Australasian
Weeds Science Society. Chapter 3 was published in the Journal of Crop and Pasture
Science and presents results obtained from three years of field trials developed to more
closely evaluate the mechanisms of weed suppression in selected annual pasture legumes.
Chapter 4 was published in Plant and Soil as an invited contribution and presents
information on the identification and activity of phytotoxic secondary metabolites
associated with weed suppressive annual pasture legumes. Chapter 5 presents results of
metabolic profiling of pasture legumes for condensed tannins, flavonoids and key
phytoestrogens and was submitted for publication in the Journal of Agriculture and Food
Chemistry. Chapter 6, a final discussion, summarizes the results of my research and
presents my concluding thoughts. Appendix A presents a related book chapter on
allelopathic interference by invasive plants on which I served as co-author, and twelve
abstracts or posters presented at various conferences and symposia from 2015-2019.
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List of Associated Publications
1. Book Chapters
Latif, S., Gurusinghe, S., Weston A., L. (2019). Role of allelopathy in persistence of
weeds’’ in Persistence Strategies for Weeds (Ed. Upadhyaya, M.K., Clements, D., and A.
Shrestha). In preparation.
Latif, S., Chiapusio, G., & Weston, L. (2017). Allelopathy and the role of allelochemicals
in plant defence. In G. Becard (Ed.), Advances in Botanical Research (Vol. 82, pp. 19-
54): Elsevier. NY, USA
2. Journal Articles and Conference Proceedings
Latif, S., Gurusinghe, S., Weston, P. A., Brown, W. B., Quinn, J. C., Piltz, J. W., &
Weston, L. A. (2019). Performance and weed-suppressive potential of selected pasture
legumes against annual weeds in southeastern Australia. Crop and Pasture Science 70(2),
147-158.
Latif, S., Gurusinghe, S., Weston, P. A., Quinn, J. C., Piltz, J. W., & Weston, L. A.
(2019). Metabolomic approaches for the identification of flavonoids associated with weed
suppression in selected hardseeded annual pasture legumes. Plant and Soil.
Latif, S., Weston, P. A., Barrow R. A., Gurusinghe, S., & Weston, L. A. (2019). Analysis
of Coumestans, Flavonoids and Proanthocyanidins in Selected Annual Hardseeded
Pasture Legumes of Mediterranean Origin. Journal of Agriculture and Food Chemistry.
Submitted for review.
Latif, S., Weston, P., Piltz, J., Gurusinghe, S., Brown, W., Quinn, J., & Weston, L. A.
(2018). Weed suppressive potential of selected pasture legumes against annual weeds in
southeastern Australia. Proceedings of the 21st Australasian Weeds Conference (pp. 378-
382) Sydney, NSW, Australia.
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3. Published Abstracts
Latif, S., Gurusinghe, S., Weston, P., Quinn, J., & Weston, L. A. (2019). Metabolomic
approaches for the identification of flavonoids associated with weed suppression in
selected hardseeded annual pasture legumes. 15th Annual Conference of the Metabolomics
Society. The Hague, Netherlands.
Latif, S., Gurusinghe, S., Weston, P., Quinn, J., & Weston, L. A. (2019). Use of non-
targeted metabolic profiling coupled with bioassays for the identification of phytotoxic
secondary metabolites in annual pasture legumes (pp. 21). Australian and New Zealand
Society for Mass Spectrometry Conference. Auckland, New Zealand.
Craig D. Stewart, Tristan A. Reekie, Russell A. Barrow, Sajid Latif, Leslie A. Weston.
(2019). An allelochemical from Festuca species as a lead molecule for development of
pre-emergent herbicides for weed management Gordon Conference on Natural Products
and Bioactive Synthesis, Andover NH, USA.
Latif, S., Weston, P., Gurusinghe, S., Quinn, J., & Weston, L.A. (2018). Metabolic
profiling of bioactive flavonoids in selected annual pasture legumes. 34th Annual Meeting
of the International Society of Chemical Ecology, Budapest, Hungary.
Gurusinghe, S., Latif, S., Brown, W. B., Weston, P. A., & Weston, L. A. (2018). A useful
in vitro bioassay for evaluation of weed suppression provided by cover crop residues. 21st
Australasian Weeds Conference (pp. 366). Sydney, Australia.
Stewart, C., Barrow, R., Latif, S., & Weston, L. A. (2018). An allelopathic compound
from Festuca spp. as a lead molecule for development of pre-emergent herbicides for
weed management. (pp 314). 34th Annual Meeting of the International Society of
Chemical Ecology, Budapest, Hungary.
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Gurusinghe, S., Latif, S., Weston, P. A., Brown, W. B., & Weston, L. A. (2018). Legume
cover crop residues for weed management through chemical suppression of weed
seedling germination and root growth. 10th Annual Symposium of the International
Society for Root Research. Jerusalem, Israel.
Stewart, C., Barrow, R., Latif, S., & Weston, L. A. (2018). A phytotoxic compound from
Festuca species as a lead molecule for herbicide development. Royal Australian Chemical
Institute, Natural Products Chemistry Symposium. Sydney, Australia.
Latif, S., Gurusinghe, S., Weston, P., Quinn, J., & Weston, L. A. (2018). Secondary
metabolites associated with weed suppression in novel annual pasture legumes. Royal
Australian Chemical Institute, Natural Products Chemistry Symposium, Sydney,
Australia.
Latif, S., Weston, P., Gurusinghe, S., Quinn, J., & Weston, L.A. (2018). Targeted analysis
of flavonoids associated with weed suppression in selected annual pasture legumes.
Agilent LC/MS User Symposium. Melbourne, Australia.
Latif, S., Weston, P., Quinn, J., & Weston, L.A. (2017). Secondary metabolites in
Biserrula pelecinus L. associated with photosensitisation in grazing livestock. Royal
Australian Chemical Institute, Natural Products Chemistry symposium. Wollongong,
Australia.
4. Media Articles
Freebairn, B. (2019, 29 April) Smother winter weed with annual legumes. The Land.
Retrieved from https://www.theland.com.au/story/6079996/smother-winter-weeds-with-
annual-legumes/
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Preface
This PhD was supported by the research project entitled: “Addressing herbicide
resistance- options for non-chemical approaches for the mixed farmers” (B.WEE.0146)
funded by Meat and Livestock Australia awarded to Leslie A. Weston and John W Piltz.
Project objectives included surveillance of the frequency of herbicide resistance in key
pasture weeds across VIC, NSW and TAS and development of novel non-chemical
approaches for pasture weed management for mixed farming systems in southern
Australia. My thesis research was supported by this project and therefore, key objectives
of my project included: 1) assessment of the weed suppressive potential of several
recently introduced annual pasture legume species originating from the Mediterranean 2)
evaluation of the fundamental mechanisms associated with weed suppression in these
species and 3) evaluation of the flavonoid and phytoestrogen production in selected
annual pasture legume species under Australian conditions, This was achieved by
performing three years of field and laboratory experimentation with newly introduced and
traditional pasture legumes, developing selected laboratory assays for assessment of
biological activity of crop residues plant extracts and key metabolites as well as metabolic
profiling of plant tissues and rhizosphere soils for determination of key secondary
metabolites associated with weed suppression and reproductive health of livestock. The
role of soil microbiota in transforming plant-produced secondary metabolites in field soil
was also elucidated.
All field research was performed from 2015-2018 in Wagga Wagga NSW at field
research sites managed by the Graham Centre at Charles Sturt University or the Wagga
Wagga Agricultural Institute. All laboratory experimentation was performed at Charles
Sturt University, National Life Science Hub Laboratories (NALSH), Wagga Wagga,
NSW, Australia. In this thesis, both field and laboratory experimentation was conducted,
in association with the afore-mentioned Meat and Livestock Australia research project.
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With reference to the work presented in this thesis, I led and performed the vast majority
of field analyses myself, but garnered assistance when needed with plot design,
establishment and maintenance from Mr. John Piltz and Drs. Leslie Weston and Bill
Brown, and crop and weed biomass evaluation from the technical staff appointed to the
project. In the laboratory, I performed all sample handling, extraction, analytics,
bioassays and bioinformatics analyses myself, but was assisted in statistical analyses,
bioinformatics analyses plus model development by Drs. Paul Weston, Leslie Weston and
Douglas Rutledge. I outlined, prepared and wrote the manuscripts presented in this thesis,
with the assistance of Dr. Leslie Weston, with additional design, data presentation and
editing support provided by my committee members and in Chapter 5, Dr. Russell
Barrow.
The research performed in this PhD thesis on pasture science was novel and
ground-breaking in several important ways:
a) This project evaluated for the first time in replicated experimentation in Australia
the in-field weed suppressive potential of selected annual pasture legumes of
Mediterranean origin over multiple years in contrast to traditional legume pasture
crops.
b) This project also successfully attempted to evaluate key mechanisms of plant
interference (allelopathy and competition for resources) and compared the relative
contribution of selected competitive traits in previously unstudied annual pasture
legumes.
c) This project developed useful and repeatable protocols for both targeted and non-
targeted metabolomics analyses in foliar tissues and associated rhizosphere soils
of various annual pasture legumes.
d) This project successfully identified and quantified a) numerous phytotoxic
flavonoids and their respective glycosides associated with weed suppression in
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selected annual pasture legumes, and b) numerous flavonoids and phytoestrogens
associated with plant defense from herbivory and livestock health and
reproduction.
e) This project developed a comprehensive in-house Personal Compound Database
Library (PCDL) containing advanced spectral data using purified analytical
standards of key flavonoids and phenolics along with other secondary metabolites.
I also believe that this project, which combined analytical chemistry, plant
biochemistry and bioinformatics approaches to study the mechanisms of plant
interference, weed suppression and flavonoid and polyphenolic production in pasture
legumes, has provided greater insight into plant defence systems in pasture legumes as
well as evaluating the role of metabolites produced in the phenylpropanoid biosynthetic
pathway in weed suppression, livestock health, herbivory and plant defence.
xviii
Abstract
This project was designed to assess the performance, secondary chemistry and
weed suppressive potential of annual self-regenerating pasture legumes in an effort to
develop non-chemical weed management strategies for rotational cropping systems in
southeastern Australia. Novel annual pasture legumes were introduced to Australia from
Mediterranean regions of Europe and Africa in recent decades to improve biodiversity of
forage species in Australia while supporting improved pasture production for grazing
livestock. These include Biserrula pelecinus L. (biserrula), Ornithopus sativus Brot.
(French serradella), Ornithopus compressus L. (yellow serradella), Trifolium
glanduliferum Boiss. (gland clover), Trifolium spumosum L. (bladder clover) and
Trifolium vesiculosum Savi. (arrowleaf clover). However, their performance, weed
suppressive potential and capacity to produce phytoestrogens and related flavonoids have
not been fully evaluated under southeastern Australian conditions. Replicated field trials
were conducted over multiple years in Wagga Wagga, NSW with treatments established
as monocultures and mixed stands. Crop and weed growth were assessed by evaluating
several key above-ground competitive traits. Plant tissues (leaf, stem, inflorescence and
root) along with rhizosphere soils were collected for the assessment of phytotoxicity
against weed seedling indicators and to perform metabolic profiling, with an emphasis on
secondary metabolites associated with phytotoxicity and phytoestrogenic activity in
grazing livestock.
Choice of pasture species impacted stand establishment, yearly regeneration and
suppression of common annual weed species in pastures, with arrowleaf clover and
biserrula suppressing annual weeds effectively. Biomass accumulation in pasture species
was found to contribute significantly to reduction of weed biomass for the majority of
species followed by light interception (LI) at the base of the canopy. Interestingly, yellow
serradella cv. Santorini, consistently produced limited crop biomass, while significantly
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reducing weed growth and exhibiting phytotoxicity, suggesting chemical interference as
a potential mechanism of weed suppression.
Foliar tissue extracts and soil incorporated crop residues of selected annual
pasture legumes inhibited the growth of monocot and dicot weed seedling indicators.
Through the development of a comprehensive protocol for non-targeted metabolic
profiling using UHPLC QTOF-MS and chemometric analyses, several phytotoxic
flavonoids were identified. These included quercetin, kaempferol, isoquercetin, and
kaempferol-7-O-glucoside with all present at higher abundance in weed suppressive
annual pasture legumes. In addition, the highest, ecologically significant concentrations
of phytotoxic flavonoids were detected in field collected rhizosphere soils of biserrula cv.
Casbah and serradella cv. Santorini. These results further suggest that plant produced
chemical intereference is one of the key mechamisms of weed suppression in those
species.
Phytoestrogens that impact livestock fertility, mainly coumestrol, 4’-
methoxycoumestrol, daidzein, formononetin and genistein, varied qualitatively and
quantitatively among annual pasture species. Our results demonstrated that
phytoestrogens present in pasture legumes were at concentrations insufficient to pose a
negative impact on livestock reproduction, with the exception of gland clover, bladder
clover and lucerne. Metabolic profiling provided unique insights into flavonoid
biosynthesis through quantification of both pathway intermediates and end products in
selected self-regenerating annual pasture legumes. The results also indicated that most
Trifolium spp. and lucerne upregulated coumestan and flavonoid phytoestrogen
production under southeastern Australian agro-climatic conditions in contrast to hard-
seeded annual legumes.
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Thesis Rationale
1.1 Farming Systems in Southeastern Australia
Mixed farming is practised throughout much of southeastern Australia (Dixon et
al., 2001), and generally enterprises in this region consists of 55% crop and 45% livestock
in rotational cropping systems (Australian Bureau of Statistics, 2018). The percentage
allocated to either crop or pasture is mainly driven by terms of trade, annual rainfall,
weather conditions and cultural factors.
In southeastern Australia, lamb production principally accounts for the major
livestock commodity, followed by beef cattle, and together they contribute over A$ 13
billion to the regional economies (Australian Bureau of Statistics, 2018). Across the
Riverina region of southeastern Australia, the climatic conditions vary from a
Mediterranean to a temperate climate type. Annual rainfall typically ranges from 300 to
650 mm and is frequently winter dominant with irregular rainfall events in the summer
that generally do not support summer cropping. Soils in this region are predominantly red
brown sodosols, grey vertisols or alluvial in nature with typically lower nutrient and
organic matter levels (Page et al. 2018) and variable pH, ranging from 4.5 to 6.5
(Geosciences Australia, 2010).
In the Riverina, pastures are utilised either in ley or phase farming systems to
improve both cereal and livestock profitability on millions of hectares. Traditional ley
farming systems are widely adopted across the grain belt of southeastern Australia, and
usually employ an annual pasture/cereal rotation with less frequent replanting of
perennial pastures. In contrast, phase farming typically involves three to four years of a
cereal production phase followed by a pasture phase requiring re-establishment of
pastures (Angus et al., 2001; Porqueddu et al., 2011). Phase farming is more prevalent on
highly acidic soils across South Australia and NSW; here pasture legumes such as medic
and subterranean clover are frequently poorly adapted and often fail to regenerate after
21
an extended cropping phase (Loi et al., 2005a). In this case, legumes that are well adapted
to variable or acidic soils, prolonged periods of drought, temperature extremes and low
fertility are more likely to succeed.
Pasture performance and persistence are key factors driving grazing operations in
southeastern Australia with respect to optimal livestock productivity. Producers select
pasture species, including legumes, for optimal livestock production, as well as for their
ability to maintain soil organic matter and positive physical soil attributes (e,g, aggregate
stability, bulk density, water infiltration and holding capacity) (Dear et al. 2008).
Livestock rotational grazing is also an important strategy to improve nutrient cycling,
weed and pest management and herbage production.
Weed infestation can seriously impact pasture crop biomass production,
particularly after pasture establishment or in an aged or poorly managed pasture (Dave
and Kirkegaard, 2014). Therefore, effective weed control strategies, particularly towards
the end of the pasture phase, can provide strong opportunities for weed management
before rotational cropping with non-legumes crops predominately wheat, canola and
barely. Effective pasture weed management can be achieved through intense grazing in
established pastures, winter grazing in newly established paddocks, fodder conservation
or hay making, spray topping and/or fallowing, and the site specific use of pasture
herbicides. All of these practices, when used effectively, will significantly reduce weed
propagules in the soil seedbank by up to 70 to 80% (personal communication, L. A.
Weston based on MLA Project Report WEE.0146). Legumes are a key component of
pasture production and provide quality nutrition for grazing livestock but also enhance
soil health. Additionally, use of competitive pasture legumes either in mixed sward or
monoculture can also cost-effectively enhance weed management while improving soil
tilth and fertility, and offering strong nutritional support for grazing livestock.
22
1.2 Choice of Legumes in the Pasture Mix
Numerous factors limit the adoption and sustainability of traditional pastures
containing legumes such as subterranean clover (Trifolium subterranean L.) and lucerne
(Medicago sativa L.) across southern Australia. These include high establishment cost,
poor soils with extreme acidity resulting in reduced stands, unpredictable climatic
conditions leading to ‘false breaks’ or lack of persistence, prevalence of soil erosion in
this region caused by excessive wind and water movement, soil compaction and the recent
emergence of herbicide-resistance in key pasture weeds (Hackney, 2015; Hackney et al.,
2008b; Powles et al., 2010).
Subterranean clover originating from northwestern Europe is the most widely
used annual pasture legume in southeastern Australia and generally suitable to well-
drained or drier soil conditions (Hackney et al. 2008). However, reduced establishment
of sub-clover has been reported following periods of water-logging, loss of soil structure
and compaction due to trampling by livestock and machinery (Dear et al., 2003). In
addition, drought can markedly impact seed set in annual pastures, thereby impacting
rejuvenation in the following year, while summer rainfall events can trigger ‘false
breaks’, e.g. germination followed by lack of persistence due to dry weather conditions.
Subterranean clover generally regenerates by setting seed successfully under drier soil
conditions in southeastern Australia, in contrast to perennial clover species, which do not
persist. The cost of pasture legume establishment can be relatively high at over A $1,500/
hectare. Low subterranean clover persistence in phase farming due to overgrazing,
climate variability and temperature extremes, among other factors, has adversely
impacted its adoption across southeastern Australia (Hackney, 2015; Loi et al., 2005a;
Loi et al., 2001b).
The perennial pasture legume lucerne is the second most widely utilised pasture
legume across all climate zones, if soils are not highly acidic (Hackney et al. 2008). Also
23
of European origin, lucerne is well-adapted to drier soil conditions once established, and
is highly digestible to grazing livestock and when conserved as fodder. It is well adapted
to a variety of soil types, but uses considerable water by foraging through its deep root
system. Lucerne, however, is sensitive to water-logging, certain soil pathogens (resulting
in subsequent disease outbreaks), and is generally intolerant of high stocking density
(Cocks, 1999).
Consistent performance of pastures is required not only for managing livestock
productivity and filling the ‘feed gap’ following winter and spring grazing but also for
improved productivity and yield of rotation crops. Given the recent impacts of drought
on livestock numbers, greater attention to fodder conservation for improved drought
preparedness will be critical to maintain reasonable stock numbers (Hackney et al.,
2008a). Legume breeders have identified that a certain level of hardseededness in some
annual pasture legumes has a significant impact on seed bank persistence leading to
improved regeneration of pasture ground cover, ability to survive ‘false breaks’ and
improved suppression of herbicide-tolerant weeds (Howieson et al., 2000).
Due to climate and soil variability, livestock producers and researchers in NSW
have highlighted the significance of the need for the development of new annual pasture
legumes for crop-pasture rotation systems and strategic use of such legumes for both
grazing and fodder conservation (Henry et al. 2012; Hackney, 2015). Recent findings
have clearly suggested that novel pasture legumes would enhance legume diversity in
existing rotations, providing a number of benefits. High quality herbage production, deep
root systems for procurement of soil water, adaptation to variable soils of low fertility,
tolerance to high stocking rates, low input cost, and hardseededness to escape false
breaks, are critical traits required in a new pasture legume crop for long term sustainability
(Howieson et al., 2000; Loi et al., 2005a). Although it is unlikely that a single legume
species can exhibit all these characteristics, a range of new species possessing many of
24
these attributes will be required to increase options in the pasture toolbox for existing
livestock producers in this region. It has also been suggested that such introductions may
be successfully propagated either as monocultures or mixtures across both phase and ley
rotations.
Legumes have the ability to develop root nodules through the activity of
rhizobacteria that enable legumes to fix atmospheric nitrogen in the plant. Plant-produced
secondary metabolites such as flavonoids have been known to serve as signalling
molecules to initiate development of root nodulation. Specific flavonoids released from
legume seeds as well as roots act as inducers or repressors of nod gene activity in rhizobia
associated with nodulation (Mathesius, 2020; Zhao and Dixon 2010). This mechanism of
attraction was first demonstrated for alfalfa (Medicago sativa; Peters et al. 1986) and
white clover (Trifolium repens; Redmond et al. 1986).
1.3 Annual Pasture Legumes of Mediterranean Origin
Agricultural production systems in southeastern Australia continue to evolve in
regards to climatic, economic, biological, and sustainability constraints. To address the
need for crop adaptation to a changing climate, the Cooperative Research Centre for
Legumes in Mediterranean Agriculture (CLIMA), was formed in 1992 with the aim of
developing alternative and sustainable annual pasture legumes. At this time, a total of 52
annual pasture legume species were selected, evaluated and released for
commercialisation or further testing (Nichols et al. 2007). Table 1 presents a list of
selected pasture legumes that were recently introduced in this region and evaluated in this
PhD project. Factors such as their country of origin, preferred soil pH and rainfall range,
rooting depth and hardseededness are presented. These pasture legumes were selected on
the basis of their agronomic suitability and high rate of adoption in the Riverina region
following consultation with farmers and agronomists.
25
Table 1. Pasture species, including common name and origin, and their respective attributes were evaluated in both field and laboratory studies in
Wagga Wagga, NSW Australia as described subsequently in Chapters 2 through 6 of this thesis.
Species Common
name
Cultivar Origin Maturity Release
(year)
pH Min
rainfall
(mm)
Deep
root
system
Hardseeded Reference
Biserrula pelecinus L. biserrula Casbah Morocco Early-mid 1997 4.5-7.5 375-525 + + (Loi et al.,
2001a)
Mauro Sardinia Mid-late 2002 4.5-7.5 + + (Loi et al.,
2006)
Trifolium vesiculosum
Savi.
arrowleaf
clover
Cefalu Mass
selection
Early 1998 4.5-7.5 400-500 + - (Snowball
et al., 1998)
Trifolium glandiferum
Boiss.
gland clover Prima Israel Early 2001 4.5-8.0 350-550 - - (Nutt et al.,
2002)
Trifolium spumosum
L.
bladder
clover
Bartolo Cyprus Early-mid 2008 5.2-8.5 300-700 - + (Loi et al.,
2003)
Trifolium
subterraneum L.
subterranean
clover
Seaton
Park
Adelaide Early-mid 1967 4.2-7.0 350-600 - - (Cocks et
al., 1979)
Ornithopus sativum
Brot.
French
serradella
Margarita Mass
selection
Mid 2003 3.5-6.5 350-400 + + (Loi et al.,
2005a)
Ornithopus
compressus L.
yellow
serradella
Santorini Greece Early-mid 1995 3.5-6.5 400-450 + + (Loi et al.,
1999)
Avila Greece Mid-late 2003 3.5+6.5 + + (Loi et al.,
2005b)
26
Annual hardseeded pasture legumes including Biserrula pelecinus L. (biserrula),
Ornithopus sativus Brot. (French serradella), Ornithopus compressus L. (yellow
serradella), Trifolium glanduliferum Boiss. (gland clover), Trifolium spumosum L.
(bladder clover) and Trifolium vesiculosum Savi. (arrowleaf clover) have been planted
and raised for fodder across New South Wales in recent years (Table 1). In the past, these
species were frequently an important component of naturalised populations of mixed
swards in Mediterranean pasture production systems due to their ability to self-regenerate
and cope with environmental and pedologic variability as a result of complex ecological
and biological mechanisms allowing them to produce higher seed yield and also persist
in local soils (Porqueddu et al., 2011). This adaptation to similar environmental
challenges experienced across southeastern Australia suggests that pasture legumes
originating from the Mediterranean basin are a valuable genetic resource for introduction
in similar bio-climatic zones across both western and southeastern Australia (Nichols et
al. 2007). The initial naturalisation and dispersion of such legumes following successful
breeding and selection programs in Australia has already contributed to more sustainable
livestock production systems in western Australia (Cocks, 1999), but the challenge now
exists to determine if they will successfully adapt to variable southeastern Australian soil
and climate conditions.
To date, a small number of livestock producers (up to 8 %) in southeastern
Australia have embraced the use of newly introduced annual legumes, particularly
biserrula, arrowleaf clover and both French and yellow serradella. An initial survey
conducted in NSW in 2008 revealed that the majority of interested livestock producers
planned to increase pasture cultivation in the future and emphasised the need for
development of new varieties/species with superior production performance (Hackney et
al., 2008a). This survey also identified that only a small fraction of producers relied solely
upon traditional pastures containing subterranean clover. This trend away from the use of
27
traditional pasture legumes is likely to continue given the substantial shift in terms of
trade for livestock production and the recently improved ability to obtain seed by pre-
order for many of these species.
Results also suggested that although an increasing number of producers were
adopting newly introduced annual pasture species, many lacked key technical knowledge
about regional suitability, performance, and weed management. A relative paucity of
information related to their economic benefits following establishment, and difficulty in
obtaining uniform establishment has led to a decline in adoption. In October 2018, in a
recent workshop involving livestock producer and agronomists supporting the Meat and
Livestock Australia project (B.WEE.0146), similar issues were raised by agronomic
consultants and producers when queried about adoption of novel annual pasture legumes
(personal communication). Cost and availability of seed are still factors limiting local
adoption, along with lack of weed management tools for successful establishment. In
addition, animal health issues such as the rapid onset of primary photosensitization in
livestock grazing biserrula has adversely impacted livestock performance and further
impacted its widespread adoption in regional NSW (Quinn et al., 2014).
Traditional legumes including lucerne and subterranean clover are known to cause
bloat and increased protein loss due to rapid fermentation in the rumen and excretion of
nitrogen into the grazing environment (Burggraaf et al., 2008). In addition, ingestion of
these pasture legumes crops may result in acute inflammation of both the small and large
intestine (red gut), sodium deficiency or pregnancy-related toxaemia (Hall et al., 1985;
Humphries, 2013; Mulholland, 1987). Currently, limited information about these
conditions in livestock grazing novel annual pasture legumes is available, but they may
present a reasonable alternative to traditional pasture legumes.
28
1.4 Weed Suppression in Annual Pasture Legumes
Previous research has shown that certain annual pasture species that produce
significant biomass, and have high ground cover or exhibit allelopathic or weed
suppressive activity ( see Chapter 1 for more information) may be useful for sustainable
weed management strategies in established pastures (Fisk et al., 2001). Fisk et. al. 2001
showed that weed biomass and sometimes density were reduced by establishment of both
clovers and medics as cover crops following no-till production of maize by up to 75% in
contrast to the control. Effective pasture management strategies including adequate
nutrition, tactical grazing and good animal husbandry are required for optimal pasture
production and biomass accumulation. Therefore, the appropriate choice of weed
suppressive pasture legumes, well suited to enterprise and environment, in mixed farming
systems offers strong opportunities for cost-effective weed management.
Currently, limited information exists regarding the weed suppressive potential of
novel Mediterranean annual pasture legumes but anecdotal information suggests that
some such as serradella, arrowleaf clover and biserrula may exhibit weed suppressive
properties. It is therefore critical to fill in the existing knowledge gap so that the potential
benefit of novel annual pasture species could be applied to best advantage (Hackney et
al., 2008a). Our own preliminary findings suggested that dense establishment of biserrula,
serradella and numerous clover species would also lead to significant reductions in
volunteer pasture weed establishment, particularly in stands that reseeded successfully
following initial seeding in the previous year (L. Weston, personal communication).
Although Dear, Loi and Howieson allude to the weed suppressive potential of various
pasture legumes when successfully established in Western Australia, quantitative
information about their ability to reduce weed infestation in contrast to standard pasture
species has not yet been obtained.
29
1.5 Secondary Chemistry and Phytoestrogenic Activity of Annual Pasture
Legumes on Grazing Livestock
While the nutritive value of selected annual pasture legumes has been studied in
vivo in South and Western Australia, detailed investigation of the phenolic chemistry of
these species has not been performed with respect to livestock health and reproduction
(Hackney et al., 2015). Plants produce an array of secondary metabolites which are
reported to perform a range of functions, including regulation of symbiotic or pathogenic
plant-microbial interactions, chemical signalling, and associated plant-plant interactions.
One family of secondary plant products, the flavonoids, is frequently noted in reference
to regulation of plant defences against competing organisms, particularly in legumes
(Weston and Mathesius 2013). Some pasture legumes have been noted to produce an
abundance of flavonoids and several in white clover and other legume crops have been
identified as potential nodulation elicitors and phytotoxins (Gomaa et al. 2015) through
one of several mode of actions including alteration of membrane permeability, water and
nutrient uptake, respiration, photosynthesis, protein and nucleic acid synthesis, cell
division (Woo et al. 2005) and growth regulation in susceptible plants (Einhellig, 2004).
Legume-produced phytoestrogens include coumestans and also several flavonoids
which mimic estrogen, an endogenous female sex hormone (Hloucalová et al., 2016;
Reed, 2016). The affinity of these phytoestrogens in binding estrogen receptor β can result
in reproductive abnormalities during embryo development and infertility in both male and
female grazing livestock (Burton and Well, 2002). Currently, only one report related to
the phytochemical profiles of aerial tissues of newly introduced annual pasture legumes
has been reported (Visnevschi et. al., 2015) but this research was conducted in Europe.
Therefore, additional studies on similar legume species are required under typical
Australian climatic conditions.
30
In summary, limited information is currently available on performance, weed
suppression and secondary metabolism on annual hardseeded pasture legumes under
climatic conditions experienced in southeastern Australia. Therefore, this project
represents an opportunity to address the paucity of information related to pasture crop
competition and defense chemistry impacting weeds and livestock health. To avoid
duplication in this section, the first chapter in this thesis summarises the literature on
allelopathy and production of allelochemicals in higher plants and provides a further
review on fundamental information on secondary metabolism related to plant defence and
use of metabolic profiling for the study of plant interactions. Following are the key
objectives and hypothesis addressed in this thesis.
31
Objectives and Hypotheses
2.1 Research Objectives
Reliance on existing pasture legume species in recent decades has adversely
impacted livestock production due to inconsistent establishment, lack of persistence and
limited competitiveness against annual weeds. In addition, rising costs associated with
pasture establishment, weed management and loss of pasture quality due to weed
incursion, limited herbicides choices for pasture weed management options and the recent
increase in numbers and distribution of herbicide-resistant weeds suggest that
complementary non-chemical weed management strategies for mixed farming systems in
southeastern Australia are necessary for successful rotational grazing systems. Although
the introduction of novel annual pasture legumes of the Mediterranean origin first
occurred in 2007, limited information exists with respect to regional establishment and
performance in southern Australia, total biomass accumulation, subsequent crop
regeneration and weed suppressive potential, in contrast to traditional lucerne and
subterranean clover standards. More particularly, the exact mechanisms linked to weed
suppression in annual pasture legumes are unknown and are thought to be associated with
1) competition for resources, 2) allelopathic interference or 3) microbially-mediated
interactions arising from legume residue decomposition in soils see Chapter 1 for a review
on these mechanisms).
To address these questions, research trials were designed with both field and
laboratory experimentation to: 1) assess both the performance and weed suppressive
potential of selected annual pasture legumes of the Mediterranean origin in replicated
experimentation in southeastern Australia, 2) evaluate the underlying mechanisms of in-
crop weed suppression associated with annual pasture legume establishment, 3) profile
and quantify key secondary metabolites associated with weed suppression in residues and
soils of annual pasture legumes using both non-targeted and targeted metabolic profiling
32
approaches, and 4) evaluate annual pasture legumes for the production of coumestans and
other secondary metabolites associated with livestock health and reproductive
performance.
2.2 Hypotheses
Hypothesis 1: Performance and weed suppressive potential varies among newly
introduced annual and traditional pasture legumes such as subterranean clover and
lucerne.
Limited knowledge exists regarding persistence and weed suppressive potential
of annual pasture legumes in southeastern Australia. To address this questions, replicated
field trials were conducted at Wagga Wagga NSW over a three year period (2014 to 2017)
to evaluate: (a) the suppressive potential of eight selected pasture legume monocultures
and pasture mixtures against annual weeds; and (b) the impact of autumn (March) and
winter (June) sowing on stand establishment and performance. The findings from
experiments addressing these questions are presented in Chapter 2.
Hypothesis 2: Competition for resources is a dominant mechanism impacting weed
suppression in annual pasture legumes.
Field trials were performed over three years (2015 to 2017) at two different
locations to evaluate the suppressive potential of selected pasture legumes as
monocultures and in mixed stands against dominant annual pasture weeds. Pasture
canopy cover was assessed by determination of crop biomass, normalized difference
vegetation index (NDVI), leaf area index (LAI), visual crop cover ratings and
photosynthetically active radiation (PAR) above and below the crop canopy. Crop and
weed biomass, NDVI, LAI and PAR measurements data provided supporting information
on crop competitiveness against weeds. Partial least square analysis was performed to
33
assess the relative effect of canopy traits on weed suppression and crop biomass. The
findings from experiments addressing these questions are presented in Chapter 3.
Hypothesis 3: Weed suppression in pasture legumes is influenced by chemical
interference through the release of allelochemicals by weed suppressive pasture
legumes and their residues.
Certain pasture legumes may exude phytotoxins (also known as allelochemicals)
in their rhizospheres, thereby interfering with weed species either de novo or through
subsequent microbial transformation of residues and associated metabolites. In
association with field observations of weed suppression, metabolic profiling of foliar
tissues of pasture legumes, roots tissue and their rhizosphere soils was performed to
further evaluate the potential for allelochemical interference with weed establishment.
This hypothesis was evaluated with experiments described in Chapter 4.
Hypothesis 4: The production of bioactive secondary metabolites, including key
phytoestrogens and condensed tannins, varies among selected annual pasture legumes.
Significant concentrations of phytoestrogens in lucerne and subterranean clover
foliage are associated with reduced herd reproductive efficiency and other reproductive
and production issues including bloat, red gut, pregnancy toxaemia and sodium
deficiency. While the nutritive value of pasture legumes has been studied in vivo in
southeastern and Western Australia, the chemistry of these species has not been closely
evaluated. Non-targeted and targeted metabolic profiling of selected annual pasture
legumes was performed to reveal the actual concentrations of phytoestrogens, flavonoids
and condensed tannins under field conditions experienced in southern Australia, in an
attempt to determine their potential to impact livestock production. The findings from
experiments addressing these questions are presented in Chapter 5.
34
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40
Chapter 1. Literature Review
Allelopathy and the Role of Allelochemicals in Plant Defense
This manuscript presents a comprehensive introduction to allelopathy and the role
of allelochemicals in plant defence, and serves as a literature review to the succeeding
chapters in this thesis. It also describes the classification of allelochemicals based on their
chemical structure and mode of action, describes the localisation and release of such
metabolites from donor plants, and discusses the recent advances in profiling
allelochemcials in foliar tissue and rhizosphere soils.
I, Sajid Latif, developed this chapter and wrote over 80% of its content under the
mentorship of Prof. Leslie A Weston and Dr. Genevieve Chiapusio who have reviewed,
commented and edited the chapter before submission for publication. This was published
as Chapter 2 in the Elsevier book entitled Advances in Botanical Research series (volume
82) “How Plants Communicate With Their Biotic Environment” edited by Guillaume
Becard.
Latif, S., Chiapusio, G., & Weston, L. A. (2017). Chapter Two - Allelopathy and the Role
of Allelochemicals in Plant Defence. In G. Becard (Ed.), Advances in Botanical Research
(Vol. 82, pp. 19-54): Academic Press. CA USA.
https://www.sciencedirect.com/bookseries/advances-in-botanical-research/vol/82
41
42
Allelopathy and the Role of Allelochemicals in Plant Defense
Sajid Latif1, Genevieve Chiapusio2 and Leslie A. Weston1
1Graham Centre for Agricultural Innovation, School of Agriculture and Wine Science, Charles
Sturt University, Wagga Wagga NSW 2678 Australia
2Université de Bourgogne Franche-Comté, Laboratoire Chrono-environment UMR CNRS 6249,
BP 71427 25211 Montbéliard Cedex France
Contents
1. Introduction
2. Plant Defence and the Role of Allelochemicals
3. Classification of Secondary Metabolites
4. Allelochemical Mode of Action
5. Localization and Transport of Allelochemicals in Donor Plants
6. Factors Influencing the Release of Allelochemicals From the Plant
7. Role(s) of Allelochemicals in the Rhizosphere, in Neighbouring Plants and
Other organisms
8. Metabolic Profiling of Allelochemicals in Complex Plant or Soil Extracts or
Mixtures
9. Conclusions
Abstract
Allelopathy is described as the interference to plant growth resulting from chemical
interactions among plants and other organisms mediated through release of plant-
produced bioactive secondary metabolites referred to as allelochemicals. A number of
mechanisms have been studied for the release of allelochemicals from various plant
tissues including volatilization or leaching from aerial parts, exudation from roots and
decomposition of plant residues in soil. Despite differences in biological activity and
mode of action, related compounds commonly share similar biosynthetic pathways while
some classes of metabolites can be produced using diverse biosynthetic pathways.
Recently considerable research has also been undertaken to critically understand the role
43
of allelochemicals in plant succession and plant invasion in native and non-native
ecosystem. In addition, numerous studies have been performed on the selection and
utilization of weed suppressive crops and their residues for weed management in
sustainable agriculture systems. A better understanding of allelochemical production with
respect to plant defense strategies, both physical and chemical, may also allow us to better
protect and manage developing crops, limit the spread of invasive weeds, preserve native
plant stands, and create strategies for allelochemical development and application as
novel pesticides. The use of sensitive analytical techniques associated with performance
of metabolomics in concert with other omics technologies has led to new advances in the
identification of unique allelochemicals, the biosynthetic pathways associated with their
production, their complex role(s) in the soil rhizosphere, and their production as impacted
by a changing climate. Identification of novel plant metabolites, including
allelochemicals, may result in a source of biologically based pesticides through the
provision of complementary structures for future synthesis and as an aid in the
development of new molecular target sites.
Keywords: Allelopathy, allelochemicals, rhizosphere, metabolomics, plant invasion,
ecological succession, secondary metabolites
1.1 Introduction
Plants and associated microflora collectively produce a vast assortment of compounds
of diverse chemical nature, the majority of which do not appear to have a role in primary
metabolism necessary for growth and development of the plant. These low organic weight
compounds are referred to as plant secondary products or secondary metabolites. Certain
classes of secondary metabolites called allelochemicals have stimulatory and/or
inhibitory effects on the growth, health and behavior of neighboring plants (Rice, 1974).
This phenomenon is referred to as allelopathy, a term first applied by Hans Molisch
(1937), broadly defined by some researchers as direct or indirect biochemical interactions
44
among plants, and potentially also microorganisms, mediated through release of
allelochemicals by plants (Weston & Duke, 2003).
The International Society of Allelopathy (IAS) defined allelopathy as chemical
interactions among plants and other organisms, excluding herbivores (Weir et al., 2004).
Given that most researchers studying allelopathy have focused on interactions among
plants mediated by allelochemicals, we have chosen to describe plant-plant interactions
in this chapter. However, it is important to note that microbes can both degrade and also
activate secondary metabolites, in some cases rendering them more biologically active as
plant growth inhibitors (Cippolini, 2012). Secondary products exhibiting allelopathic
potential are referred to as “allelochemicals” (Whittaker and Feeny (1971), however, both
terms are not interchangeable. Moreover, allelopathic interference due to the action of
allelochemicals differs from interference due to competition in which plants compete with
neighboring plants for soil resources including, water, space, light, gases and macro- and
micro-nutrients necessary for growth (Weston & Duke, 2003). However, both
competition and allelopathy generally result in growth reduction or interference with
plant growth over time.
Allelopathy is not a new phenomenon and has been described for over 2000 years in the
literature; in ancient manuscripts plants were frequently reported to “sicken the soil” and
produce toxins that adversely affected the growth of other plants (Weston & Duke, 2003).
The Greek botanist Theophrastus (300 BC) first noted that chickpeas exhausted the soil
and inhibited weeds. Later, the Roman scholar Pliny described the toxicity of the walnut
tree to neighboring plants (Weir et al., 2004). In 1832, De Candolle performed the first
well-described experiment to study toxicity associated with root exudates of allelopathic
species (Singh et al., 2001). In recent years, following on from these foundational studies,
research into the mechanisms associated with allelopathic interactions has increased
exponentially. The latest studies on allelopathy have been critical in advancing our
45
understanding of plant ecosystems and their drivers and have focused on the following:
the impact of allelochemicals on successful invasion by plant invaders in the non-native
range; the role of allelochemicals in plant succession in dynamic ecosystems; the
selection and utilization of weed suppressive crops and their residues for enhanced weed
management; and the role of root exudates in driving rhizosphere interactions with plants
and/or microbial symbionts.
This chapter aims to present a general overview of allelopathic interactions among plants
and plant defense mechanisms, the biochemical classification of allelochemicals,
allelochemical mode(s) of action and key allelochemical interactions occurring in the
rhizosphere or plant-soil interface.
1.2 Plant Defense and the Role of Allelochemicals
Plants are sessile and therefore cannot easily move away from potential threats or
towards beneficial entities. During the course of evolution, plants have developed both
physical and chemical mechanisms of defense from pests and pathogens (Bernards,
2010). Traditionally resource competition has been considered as the single most
important factor that influences the patterning of plant communities (Niklas & Hammond,
2013). However, recent research has described allelopathy as an important aspect of plant
defense that impacts plant community diversity (Fernandez et al., 2013). In this process,
plants release a diverse repertoire of low molecular weight secondary metabolites that are
considered to interact with the surrounding environment by inhibiting the germination or
growth of neighboring plants (Ben et al., 2006; Fernandez et al., 2016). The majority of
allelochemicals in the plant kingdom are found in vascular plants, but our knowledge of
secondary metabolites in ancient terrestrial non-vascular plants such as mosses or
liverworts has also increased in recent years. Allelochemicals can, therefore, play an
important role in plant succession through their release by pioneer plants (i.e.
Bryophytes), which contribute substantially to the accumulation of aboveground biomass,
46
particularly in cold temperate biomes including boreal forests and peatlands (Chiapusio
et al., 2013; Michel et al., 2011).
Approximately 100,000 secondary metabolites have been identified to date in plants
(Afendi et al., 2012; Croteau et al., 2000). A smaller number of these are described as
bioactive allelochemicals and are generally classified as members of specific chemical
families that include phenolics, terpenoids, glycosteroids and alkaloids (Ahuja et al.,
2012). Plants can allocate large investments in carbon resources to synthesize, regulate
and store secondary metabolites including allelochemicals involved in plant defense.
However, many plants have evolved cost-effective strategies for production and recycling
of these bioactive metabolites; the conversion of one product to another through plant
metabolism may result in a completely different biological function. In some cases, a
single compound or related family of compounds can have multiple functions from an
ecological context. For example, strigolactones are stimulants of germination of parasitic
plants while other lactones are potent germination inhibitors. Despite differences in
biological activity and mode of action, related compounds commonly share similar
biosynthetic pathways while some classes of metabolites including phenolics can be
produced using diverse biosynthetic pathways and precursors (Neilson et al., 2013).
Plants have further evolved protection from potential predators, including herbivores and
pathogens, through specific physical and chemical defense mechanisms. Plants possess
specialized morphological structures including trichomes, spines, and hairs that provide
generalized physical defense. Specialized defense mechanisms include the production
and release of bioactive metabolites, some of which are constitutive and found in almost
every class of plants while others are synthesized or activated in response to biotic and
abiotic stressors (Bartwal et al., 2013). Precursors, which are formed immediately after
pathogen attack from pre-existing constituents are referred as phytoanticipins or more
commonly as phytoalexins (Pedras et al., 2011; VanEtten et al., 1994). These low
47
molecular mass metabolites are produced in host plants under diverse stressors and
exhibit broad-spectrum inhibitory activity and extensive chemical diversity.
The development of microbial symbiosis is also crucial for the survival and growth of
vascular plants. For example, mycorrhizal fungi form symbiotic associations with 90%
of vascular plants and are also ubiquitously distributed in a wide range of ecosystems
(Finlay, 2008; Read et al., 2004). These organisms often play fundamental roles in
belowground processes because they are crucial for the mobilization of nitrogen and
phosphorus from the soil to their host plant. These organisms are also sometimes involved
in mediation of allelopathic interference as plant-produced allelochemicals, herbicides
and other metabolites present in the soil rhizosphere are released into the rhizosphere and
later taken up and transported to other plants by way of the endophyte’s network of
underground hyphal networks, sometimes referred to as the below-ground
“superhighway” facilitating interplant transport of allelochemicals (Delaux et al., 2013;
Souto et al., 2000).
1.2.1. Allelochemical Localization and Release into the Environment
Secondary metabolites including allelochemicals are ubiquitous in nature and can
be released over time from all plant tissues including leaves, stem, roots, flowers, seed,
rhizomes, pollen, bark and buds (Weston & Duke, 2003). A number of mechanisms
including volatilization or leaching from aerial parts, exudation from roots and
decomposition of plant residues in soil (Figure 1) have been well characterized with
respect to the release of allelochemicals over time in the environment (Cheng & Cheng,
2015; Putnam & Tang, 1986). Direct volatilization and precipitation, including rain, fog
and dew, also play an important role in the solubilisation and release of allelochemicals
from foliar parts of the plants, particularly those that possess glandular trichomes
containing allelochemicals (Bais et al., 2006; Inderjit & Duke, 2003). Trichomes are hair-
48
like appendages on the surface of the plant leaf, stem or inflorescence. They provide a
physical defense against mammalian herbivores upon ingestion, due to unpleasant
sensorial experience associated with the allelochemical release. In addition to a physical
barrier, glandular trichomes have specialized cells, which can synthesize allelochemicals
as part of a constitutive chemical defense system (Champagne & Boutry, 2016).
Figure 1. Chemical (leaching, volatilization) and biological (herbivory, microbial
degradation, competition) mechanisms of release of allelochemicals from various plant
tissues.
1.3 Classification of Secondary Metabolites
Secondary metabolites including allelochemicals can logically be classified according
to their carbon skeletal structure and type of functional groups; however, the most useful
classification system is based on the biogenetic origin of metabolites (Figure 2). As
presented by Walton and Brown (1999) and Rice (1974) plant metabolites and
allelochemicals can be further divided into 3 major groups: phenolics, terpenoids, and
alkaloids.
49
Figure 2. Major families of allelochemicals and their common biosynthetic pathways
originating from numerous key precursors (Rice, 1974). Amino acids and polypeptides
as well as purines and nucleosides are often categorized as primary metabolites due to
their roles in primary biosynthetic pathways of proteins and DNA. However, some of
these molecules are also bioactive in their own right and can serve as allelochemicals,
thereby impacting higher plant growth.
1. 3.1 Phenolic Compounds and their Derivatives
Plant phenolics (Figure 3) represent an extremely diverse group of organic
compounds, which share a common structure; they consist of an aromatic ring possessing
at least one hydroxyl group and possibly other substituents (Stalikas, 2007). They are
generally categorized as phenolic acids, flavonoids, stilbenes, coumarins, lignins and
tannins (Cheynier et al., 2013; Dinelli et al., 2009). The term phenolic is descriptive as
phenolics are secondary metabolites derived from the phenylpropanoid-acetate
biosynthetic pathway (Croteau et al., 2000; Haig, 2008; Quideau et al., 2011).
Approximately 8000 naturally occurring secondary metabolites belonging to this group
50
have been identified so far. They perform a broad array of structural and physiochemical
roles in planta (Croteau et al., 2000). Phenolics are the most commonly reported
metabolites known to play a role in defense mechanisms in higher plants; with variable
toxicity, they target cellular functions at multiple sites (Haig, 2008). They also represent
40% of the structural matrix of plants in form of lignins and tannins, and through these
molecules, which degrade over time in the soil rhizosphere, maintain their abundance
through the important ecological cycle of synthesis, transformation, release and
decomposition (Dalton, 1999; Hattenschwiler & Vitousek, 2000).
The mechanism of allelopathy associated with phenolic compounds has been studied
extensively and reports indicate that phenolics interfere with several key plant enzymes
and physiological processes. For example, cinnamic and benzoic acids (Figure 3)
interfere with hormone activity, membrane permeability, photosynthesis, respiration and
synthesis of organic compounds. However, there has been no evidence to date for
phenolics impacting cell division or gene translation (Einhellig, 2004).
Perennial legumes including alfalfa (Medicago sativa L.) and clovers (Trifolium spp.) are
known for their allelopathic properties following pasture establishment (Weston &
Mathesius, 2013). Some species including alfalfa and clover can also prove autotoxic
once they are established. These perennial plant stands tend to inhibit the growth of plants
of the same species over time. Carlsen et al. (2012) studied the phenomenon of
phytotoxicity following clover establishment and reported that release of several
secondary metabolites, including flavonoids and their glycosides, causes weed
suppression and negative plant-plant interactions associated with replanting following
legume establishment.
51
Figure. 3. Some examples of diverse plant phenolics with known allelopathic properties.
1.3.2 Terpenoids
Terpenoids are specific allelochemicals that are biosynthetically derived from the
mevalonic acid and isopentenyl pyrophosphate pathways (Haig, 2008). The term
‘’terpene’’ or the preferred term ‘’terpenoid’’ comes from the German word ‘’terpentin’’
52
(terpentine) due to the fact that first reported terpenoid was isolated from turpentine
(Herz, 1963). Naturally occurring, approximately 24,000 terpenoids are comprised of five
carbon isoprene subunits (Figure 4) linked together through the common head-to-head or
less common head-to-tail linkages but some are also characterized by head-to-middle ring
closure (Croteau et al., 2000). Based on the relative number of isoprene subunits
possessed, they are classified into hemiterpenes (single five carbon isoprene unit)
monoterpenes (two isoprene units), sesquiterpenes, (three isoprene units), diterpenes
(four isoprene units), triterpenes (six isoprene units), tetraterpenes (eight isoprene units)
and polyterpenes with more than eight isoprene subunits. Terpenoids are typically volatile
compounds having multiple biological activities in plants as signaling molecules,
photoprotective agents, reproductive hormones but also allelochemicals (Croteau et al.
2000).
Figure 4. The structure of an isoprene unit, the backbone of terpenoids.
Monoterpenes, the main constituents of plant essential oils, are widely known for their
strong inhibitory effects on plant growth and seedling germination (Haig, 2008). For
example, the monoterpenes 1,4-cineole and 1,8-cineole (Figure 5) are well studied as
growth inhibitors and potential candidates for herbicides (Duke et al., 2004). Despite
structural similarities, these monoterpenes have very different modes of action where 1,
8-cineole affects all stages of mitosis and 1, 4-cineole causes growth abnormalities in
shoots.
53
Figure 5. Some examples of plant monoterpenes with known allelopathic properties.
Sesquiterpenes and their associated metabolites along with the monoterpenes in essential
oils have been well characterized with respect to their phytotoxic effects on plants. The
sesquiterpene β-caryophyllene (Figure 6) is present in numerous plant volatiles and
inhibits germination and seedling growth of Brassica napus L. and Raphanus sativus L.
at very low concentrations (Wang et al., 2009). Geometric isomers of xanthoxin (Figure
6) present in leaves of Pueraria thunbergiana L. (Sieb. and Zucc.) Benth., also known as
the invasive kudzu vine, inhibited seedling root growth of numerous small-seeded annual
broadleafs and grasses at very low concentrations (Kato-Noguchi, 2003; Rashid et. al.,
2010).
54
Figure 6. Some examples of plant sesquiterpenes with known allelopathic properties.
1.3.3 Alkaloids
Alkaloids (Figure 7) are heterocyclic nitrogen-containing basic compounds of
plant origin and are named accordingly, due to their alkaline chemical nature. Plant
alkaloids predominate in four families of plants including the Asteraceae, Apocynaceae,
Boraginaceae, and Fabaceae (Haig, 2008).They have been important since antiquity due
to their pharmacological properties and are among the largest group of secondary
metabolites with approximately 20,000 compounds identified to date representing great
structural biosynthetic diversity (Yang & Stöckigt, 2010). Based on their biosynthetic
origin, alkaloids are classified into different classes; e.g. indole alkaloids are derived from
tryptophan, pyrrolizidine alkaloids are derived from ornithine or arginine, and
quinolizidine alkaloids are derived from lysine (Seigler, 1998).
Alkaloids are widely distributed across the plant kingdom and frequently reported to play
key defensive roles in plant interactions against herbivores, microorganisms, fungi, and
in some cases, neighboring plants. Several naturally occurring pyrrolizidine alkaloids
55
have been evaluated for hepatotoxicity to humans and grazing livestock (Quinn et al.,
2014), and also deter herbivory as part of the plant’s above-ground defense system.
However, other alkaloids have in some cases shown activity as inhibitors of plant growth.
Nicotine production in wild tobacco is up-regulated after grazing or damage by
herbivores; interestingly nicotine also possesses allelopathic activity against common
annual weeds. Alkaloids are thought to inhibit plant growth by several mechanisms
including interference with DNA, enzyme activity, protein biosynthesis, and membrane
integrity in developing plants (Wink, 2004). Lovett and Hoult (1995) studied defense
mechanisms in barley (Hordeum spp.) through the release of the plant-produced alkaloids
gramine and hordenine (Figure 7) from living roots; these were shown to have allelopathic
effects in seedling bioassays. The quinolizidine alkaloids studied by Wink (2004),
including lupanine and sparteine, are produced by legumes and are thought to cause
potent inhibition through interference with membrane permeability and protein synthesis.
Figure 7. Some examples of plant alkaloids with known allelopathic properties.
56
1.3.4 Hydroxamic Acids of Benzoxazinoids: A Case Study from the Agronomy
Hydroxamic acids are phenol derivatives, known as cyclic 2-hydroxy-2H-1,4-
benzoxazin-3-(4H)-ones, which are a basic skeleton of a broader family of bioactive plant
secondary metabolites referred to as benzoxazinoids (Figure 8). They have been well
characterized by numerous scientists and have antimicrobial, antifungal, antifeedant and
phytotoxic activities (Chiapusio et al., 2005).
Figure 8. The structure of 2-hydroxy-2H-1, 4-benzoxazin-3-(4H)-ones, also known as
hydroxamic acids of the benzoxazinoid family of allelochemicals.
Hydroxamic acids of benzoxazinoids present in cultivated cereals of the Graminaceae
including maize, rye and wheat have been widely studied for their allelopathic potential
and their abundance has been highly correlated with resistance to insects and other
pathogens (Elek et al., 2013; Niemeyer, 2009). DIMBOA (2,4-dihydroxy-7-methoxy-2H-
1,4-benzoxazin-3-one) and DIBOA (2,4-dihydroxy-2H-1,4-benzoxazin-3-one) and their
associated microbial conversion products (Figure 9) are prominent hydroxamic acids with
potent allelopathic effects on numerous broadleaf weeds and crops including lettuce
(Lactuca sativa L.) and wheat (Triticum aestivum L.) (Macías et al., 2003). They are
typically released into soil from injured plant tissues or by degradation of crop residues
after harvest. The glucosides of DIBOA and DIMBOA, together with their respective
aglycones and degradation products released from rye (Secale cereal L.) root residues,
are mainly responsible for allelopathic properties on developing broadleaf crops and
weeds and several annual grasses (Schulz et al., 2013).
57
Figure 9. Common examples of benzoxazinoids with known phytotoxic properties.
1.4 Allelochemical Mode of Action
One of the current challenges in allelopathy is to determine the specific mode(s) of
action of allelochemicals in association with their diverse chemical nature and multiple
target sites in higher plants. Various bioassays have been designed to carefully study the
direct effects of allelochemicals on higher plant growth as well as evaluate their
underlying mechanism of action. This can be a particularly difficult task, as bioassays for
specific enzymes that may be potential target sites of allelochemicals are often
challenging to devise, and require prior knowledge of the structure of the the catalytic or
target site. In some cases, the actual target site of allelochemical action is not evident by
visual observation of affected plant growth and/or morphology. In addition, the relevance
of some in vitro bioassays with respect to the provision of conclusive evidence related to
allelochemical mode of action has come under scrutiny, and the relationship of in vitro
effects to those seen in a natural setting has also been questioned (Inderjit and Weston,
2000).
58
It has also been suggested that the relative concentration of allelochemicals typically
released into the environment is often quite low and transient; therefore, the concentration
of an allelochemical or complex mixtures of allelochemicals encountered in the soil or in
the environment is often difficult to estimate or measure (Weir et al., 2004). Therefore, it
is particularly important to use appropriate bioassays at relevant concentrations to assess
the potential inhibitory effects of allelochemicals on higher plant growth. In many cases,
it may be critical to use more than one bioassay to assess impacts on plant growth. For
example, assays utilizing local soils, plant species of importance in natural systems and
those providing uniform, repeatable results are critical for the development of useful data
sets. Other investigators, including those assessing mode of action of synthetic and natural
products such as allelochemicals in the agrochemical industry have developed robust
models to assess plant growth using very sensitive assays. One can typically use several
assay conditions to generate data including the use of an aqueous plant known as
pondweed or duckweed (Lemna spp.) that is rapidly growing and uniformly reproduces
additional plantlets over a one week period. The duckweed assay allows the testing of
very low concentrations of allelochemicals as well as mixtures of metabolites, provided
the compounds can be solubilized in an aqueous solution. In addition, various small pot
assays using local soils and/or agar have been adopted to assess impacts both above and
below ground on shoots and roots, respectively (Blum, 2014). These small pot or
container assays typically require a longer period to generate plant growth for assessment.
Finally, a bioassay to assess direct impacts of decomposing plant residues has also been
utilized to assess phytotoxicity associated with plant residues following a crop or a weed
infestation using recovered plant tissue (Weston et al., 1989). By use of appropriate assays
for screening, a number of different mechanisms of action have been postulated, and later
confirmed using very specific assessment at the site of action; these include membrane
59
permeability, water and nutrient uptake, respiration, photosynthesis, protein and nucleic
acid synthesis, and growth regulation in susceptible plants (Einhellig, 1995).
1.4.1 Membrane Permeability
Many secondary metabolites including allelochemicals alter cell permeability and
membrane function with exposure at adequate concentrations. Exposure to such
metabolites may result in leakage of cellular contents and consequently cell death through
apoptosis and necrosis (Li et al., 2010). This process eventually leads to tissue death and
loss of specific function. For example, several phenolic compounds are reported to exhibit
allelopathic effects by altering plasma membrane permeability. The compounds are easily
able to cross cellular membranes either by diffusion or assisted transport mechanisms.
Once membranes have experienced altered permeability, potassium channels are
impacted and decreased permeability to chloride ions is often encountered. Yoon et al.
(2000) and Singh et al. (2006) reported that the monoterpenoid, α- pinene, caused
oxidative stress to the plasma membrane and disruption of membrane structural integrity,
which led to cell death. Chai et al. (2013) also found that concentrations of phenolic
compounds greater than 1mM and mimosine (a non-protein amino acid) in Leucaena
leucocephala (Lam.) leachates caused increased membrane permeability in leaf tissue of
hyacinth (Hyacinthus orientalis L.). Membrane injury is typically assessed in vitro by
measuring relative electrolyte leakage in treated plant culture bioassays in contrast to an
untreated control.
1.4.2 Water and Nutrient Uptake
To further investigate the potential mode of action of allelochemicals released into
the environment, it is important to focus upon the relative health and function of the
plant’s root system. In the living roots, allelochemicals can frequently affect the activity
of Na+/K+ pumps involved in the absorption of ions across the plasma membrane. For
60
example, Abenavoli et al. (2010) found that nitrate uptake and membrane H+-ATPase
activity in Zea mays L. (corn) roots were inhibited by cinnamic acid, ferulic acid, and p-
coumaric acid. Franche et al. (2009) also reported that the phenolic compounds cinnamic
and ferulic acid reduced the nutrient uptake of P and Fe and thereby inhibited the growth
of target plants. Phytotoxins produced by Beta vulgaris subspp. vulgaris (sugar beet)
make zinc more available for zinc-sensitive crops such as beans (Phaseolis vulgaris L.)
and corn when planted following sugar beet (Boawn 1965).
1.4.3 Respiration
Allelochemicals released from donor plants can also seriously affect the growth
of plants by impacting the process of respiration including electron transfer in the
mitochondria, oxygen uptake, CO2 generation, and oxidative phosphorylation for ATP
generation (Cheng & Cheng, 2015). For example, Abrahim et al. (2003) studied the
monoterpenes camphor, α-pinene and limonene, and determined that each influences
respiratory activity in hypocotyl mitochondria of various donor plants through different
mechanisms i.e., mitochondrial uncoupling, electron flow in the cytochrome pathways
and oxygen uptake, respectively. Czarnota et al. (2001) found that the inhibitor
sorgoleone, exuded by living sorghum roots in copious quantities, is both an inhibitor of
electron transport in photosystem II at the D1 binding protein and also inhibits
mitochondrial electron transport by uncoupling electron flow in all plant species
evaluated, including weeds and crops. Allelochemicals typically exhibit a stronger effect
on seedling growth and germination in contrast to the growth of mature plants, and it has
been suggested that one of the underlying mechanisms of germination and seedling
growth inhibition is through frequent disruption of mitochondrial ability to carry out
respiration (Weir et al., 2004).
61
1.4.4 Photosynthesis
Allelochemicals can significantly influence the process of photosynthesis in
several ways including disruption of electron flow in PS I and II, impacting the synthesis
of photosynthetic pigments or stimulation of the decomposition of photosynthetic
pigments. Consequently, decreased concentrations of photosynthetic pigments in plant
shoots can lead to reduced synthesis of ATP, primarily in photosystem II (Cheng &
Cheng, 2015; Fengzhi et al., 2004; Weir et al., 2004). For example, sorgoleone reacts at
the D1 binding protein site in photosystem II and has shown strong competitive
interference with electron transport (Einhellig, 1995; Gonzalez et al., 1997). Weeds
(Amaranthus retroflexus L.) that were resistant to PS II herbicide inhibitors by mutational
changes at the D1 binding site were also resistant to the impact of sorgoleone at this site,
providing further evidence for activity at the D1 binding protein in PS II (Nimbal et al.,
1996). Poonpaiboonpipat et al. (2013) observed that essential oil from Cymbopogon
citratus (DC.) Stapf. (lemongrass) significantly decreased the concentration of
chlorophyll a and b in the seedling barnyard grass, thereby reducing photosynthesis and
subsequent plant growth. These findings suggest that essential oils have a direct impact
on chlorophyll biosynthesis. Studies performed with the allelochemical citral, a volatile
monoterpene found in many plants, has shown that that citral is effective not only on
inhibition of seedling metabolism through respiration and reduced photosynthesis but also
on adult plants by inhibiting growth and altering the plant oxidative status (Graña et al.,
2013). This suggests that mode of action of certain allelochemicals is age and tissue
dependant and also dependant on the site of uptake in the plant.
1.4.5 Protein and Nucleic Acid Synthesis and Growth Regulation
Allelochemicals can impact the growth of neighboring plants by targeting key
regulatory mechanisms affecting plant growth. These mechanisms include intercalation
of DNA, inhibition of DNA polymerase I and inhibition of protein biosynthesis, and have
62
been associated with reduction in plant growth due to allelochemical exposure (Wink &
Latz-Bruning, 1995). Phenolic allelochemicals can also interfere with the formation of
nucleic acids, critical in cellular metabolism and gene expression. Li et al. (2010)
determined that the phenolic compounds ferulic and cinnamic acid adversely affected
total protein synthesis. Allelochemicals from donor plants have also been reported to
regulate the expression of other defense genes in the target plant. For example, synthesis
of enzymes, which are involved in the biosynthesis of phenolic compounds in rice are
upregulated in the presence of barnyard grass (Echinochloa crus-galli L.), a common
weed in cultivated rice (He et al., 2012). In the case of seedling rice, the growth of rice
with the weed competitor barnyard grass results in an increase in production of the
allelochemical momilolactone in rice (Kato-Noguchi, 2011). This suggests that stress
caused by plant competition or interference due to allelopathy alters a target plant’s ability
to produce defense compounds and response may occur in a matter of hours to days as
measured by enhanced allelochemical production. Similar phenolics have also been
shown to oxidize indole-3-acetic acid, a hormone required for cell elongation in higher
plants (Yang et al., 2005).
1.5. Localization and Transport of Allelochemicals in Donor Plants
Plants have specialized tissues for synthesis and release of secondary metabolites
in the environment including stomata and glandular trichomes in plant shoots, and root
hairs, border cells, epidermis and periderm in roots. The root hair is a single cell extension
of the root epidermis and plays an important role in complex interactions at root-soil
interface (Weston et al., 2012). Root hairs have the ability to exude both low and high
molecular weight organic molecules including ions, amino acids, growth regulators and
allelochemicals (Table 1). Some compounds are synthesized within the root hair and
directly exuded into the environment, such as sorgoleone (Weston et. al., 2012) and
shikonins, which are directly exuded by Echium plantagineum L. seedlings (Zhu et al.,
63
2016). Recently, the localization of red-colored bioactive naphthoquinones (also known
as shikonins) from the outer periderm of living roots of the highly invasive weed Echium
plantagineum L. has also been studied; this plant accumulates these antimicrobial and
phytotoxic naphthoquinones in the periderm of the primary taproot over time and also
releases these molecules from seedling root hairs as red-colored droplets. These
localization studies were facilitated through the use of confocal microspectrofluorometry
(Zhu et al., 2016).
64
In the shoot, allelochemicals can be directly exuded by glandular trichomes on the leaf
surface, while in other cases they are translocated to the trichomes from other parts of the
Table 1. Diverse organic metabolites known to be released in root exudates of living
plants (Bertin et al., 2003)
Class of Compounds Metabolites Functions
Carbohydrates arabinose, glucose, raffinose,
rhamnose, ribose, fructose,
galactose, maltose, sucrose, and
xylose
Provide favorable
environment for the
growth of
microorganisms
Amino acids and
amides
all 20 proteinogenic amino
acids, aminobutyric acid,
homoserine, cystathionine,
mugineic acid
Inhibit nematodes and
root growth of different
plant species
Aliphatic acids formic, acetic, butyric, isocitric,
oxalic, fumaric, propionic,
maleic, citric, tartaric,
oxaloacetic, pyruvic,
oxaloglutaric, glycolic,
shikimic, acetonic, valeric,
gluconic
Plant growth regulation
and inhibition
Aromatic acids p-hydroxybenzoic, caffeic, p-
coumaric, ferulic, gallic,
gentisic, protocatechuic,
salicylic, sinapic, syringic
Stimulation depending
on concentration
Miscellaneous
phenolics
flavanol, flavones, flavanones,
anthocyanins, isoflavonoids
Plant growth inhibition
or stimulation
depending upon
concentration
Fatty acids linoleic, linolenic, oleic,
palmitic, stearic
Plant growth regulation
Sterols campestrol, cholesterol,
sitosterol, stigmasterol
Plant growth regulation
Enzymes and
miscellaneous
Unknown
65
plant (Eom et al., 2005). In the case of catmint, Nepeta x faassenii, 21 volatile constituents
were noted in the glandular exudates of the plant and proved to potently inhibit seed
germination and seedling plant growth, when assays were conducted with foliage
suspended in the proximity of germinating seedlings, but not in contact with seedlings.
This suggested that volatilization is important for dispersal and inhibition of plant growth
processes. Three components, 2-(2-ethoxyethoxy)-ethanol, alloaromadendrene, and chi-
cadinene, were not only detected in both the volatile mixture and the methanolic extract,
but also in an aqueous foliar extract that exhibited potential allelopathic activity on curly
cress (Lepidium sativum L.) growth. Eom et al. (2006) studied the allelopathic potential
of perennial groundcovers and found that foliar volatile compounds from Nepta x
faassenii (catmint) significantly decreased the growth of Lepidium sativum (curly cress)
and also common roadside weeds under field conditions. Lipophilic or volatile
compounds such as those produced by catmint can directly diffuse through the lipid
bilayer while more polar compounds require specialized membrane-bound transport
proteins to assist the transport process (Weston et al., 2012).
Once synthesized and accumulated in plant tissues, most allelochemicals are excreted
from the plant to prevent autotoxicity or stored in vacuoles and small membranous
vesicles or storage organs, such as the periderm, waxes and glands on the leaf surface or
in extracellular spaces or compartments in plant tissues. They can also be transported to
other plant tissues using diffusion or more likely specific transport proteins, which are
embedded in the plasma membrane and actively assist in the transport of a particular class
of compounds, including allelochemicals. Figure 10 illustrates ABC (ATP-binding
cassette), MATE (multidrug and toxic compound extrusion), MFS (major facilitator
superfamily) and ALMT (aluminium-activate malate transport) transport protein families
potentially operational in plant donor tissues producing allelochemicals for their eventual
transport in the plant (Weston et al., 2012).
66
Figure 10. Transport of organic compounds in root cells through membrane-bound
proteins including ABC (ATP binding cassette family), MATE (multidrug and toxic
compound extrusion family), MFS (the major facilitator superfamily), and the ALMT
(aluminium-activated malate transport family) (Weston et al., 2012).
1.5.1 Root Exudation of Allelochemicals
Root exudation is a common mechanism of release of allelochemicals from living
plant roots, specifically through root hairs, which are individual cells associated with
synthesis and excretion of bioactive metabolites, or by the actively growing tips of
primary and secondary roots (Table 1) (Weston et al. 2013; Zhu et al., 2016). The soil-
root interface or rhizosphere is considered as the site of greatest activity in the soil matrix
as a myriad of organic compounds in varying concentrations are released from living
roots, decomposing plant material, and by associated microorganisms. The rhizosphere is
an extremely complex environment to chemically survey due to actively changing the
concentration of secondary metabolites and relative difficulty in extraction from soil
matrix. Most recently, the development of silicone microprobe tubing and solid phase
67
root zone extraction techniques have provided an opportunity to precisely profile non-
polar to moderately polar allelochemicals released from living plant roots in the soil (Zhu
et al., 2016). In this case, a silicon tube is placed in the soil around the root surface and
less polar molecules adhere to the silicone exterior of the tube, and can be selectively
removed by later solvent extraction (Weidenhamer et al. 2009)
The composition of root exudates is complex and generally consists of carbon-containing
compounds including polysaccharides (for example arabinose, glucose, fructose,
maltose), amino acids (for example arginine asparagine, cysteine, glutamine), organic
acids (for example acetic, ascorbic, benzoic, ferulic acids), phenolic compounds,
alkaloids, tannins, terpenoids, flavonoids, growth regulators, vitamins and nutrients and
to a lesser extent, non-carbon compounds including ions, water and electrolytes (Table 1)
(Baetz & Martinoia, 2014; Bertin et al., 2003). Once released, allelochemicals undergo
physical, chemical and biological changes in the soil. Therefore, the biological activity of
an allelochemical might be altered before reaching the potential target due to microbial
degradation, oxidation or immobilization by irreversible binding to soil particles (Cheng,
1995).
Neumann and Römheld (2007) and Weston et al. (2013) described various mechanisms
of root exudation including diffusion, vesicle transport and ion channels. A brief
description of these mechanisms is given below.
1.5.2 Diffusion
Low molecular weight organic compounds such as sugars, amino acids,
carboxylic acids, and phenolics are released passively through a gradient of concentration
between the cytoplasm of living root cells and the surrounding soil. This particular type
of excretion depends on the physiological state of the root cells and the polarity of the
68
organic compounds. The hydrophobic nature of plasma bilayer generally facilitates the
transport of lipophilic compounds by diffusion.
1.5.3 Vesicle Transport
Transport of high molecular weight organic compounds (e.g. mucilage
polysaccharides) is mediated through the formation of Golgi vesicles from secretory cells
of the root cap. These cells are subsequently degenerated or sloughed off from the surface
of the roots (Bertin et al., 2003). Enzymes such as acid phosphatase and peroxidase,
synthesized at the level of membrane-bound polysomes, enter the lumen of endoplasmic
reticulum using vectorial segregation (Neumann & Römheld, 2007). This exocytosis of
vesicles depends on intracellular and extracellular calcium level.
1.5.4 Ion Channels
Exudation of some compounds (e.g., citrate, malate, oxalate) at higher
concentrations, particularly under stressful conditions, cannot take place though simple
diffusion in certain plant species (Neumann & Römheld, 2007). The use of patch clamp
approaches and ion channel antagonist studies indicated the presence of ion channels on
the plasma membrane of epidermal root cells involved in the release of malate and citrate
in the Triticum spp (wheat) and corn (Weston et al., 2013).
1.6. Factors Influencing the Release of Allelochemicals from the Plant
The nature and concentration of allelochemicals released by the plant into the
environment, including the rhizosphere, is dependent on the plant itself and several other
biotic and abiotic factors as shown in Figure 11. Plant factors include plant species and
cultivar, plant age and the type of tissue under consideration for allelochemical
production. Environmental factors regulating allelochemical production and release
include pathogen infestation or physical injury or abiotic factors including drought,
temperature, soil characteristics, rainfall, nutrient deficiency, irradiation, competitors and
69
exposure to ultra-violet radiation (Brimecombe et al., 2001; Cseke & Kaufman, 1999;
Einhellig, 1996; Iannucci et al., 2013; Mahmood et al., 2013). A changing climate may
also influence allelochemical production and this has been an area of particular interest
in recent years (Jassey et al., 2013; Putten et al., 2013; Weston et al., 2013).
Taxonomically related species do not always release similar allelochemicals or
concentrations of metabolites into the environment (Imatomi et al., 2013). The release
and dispersal of allelochemicals into the external environment are influenced by the
chemical nature of allelochemicals including their respective molecular weight, polarity,
and concentration in the plant. Allelochemicals are most often released in mixtures in
conjunction with other closely related metabolites. It is important to note that these
metabolites can serve more than one biological role in the plant, and one compound can
also defend its host from several unrelated competitors or predators (Macías et al., 2007).
For example, phenolic compounds are known to stimulate and/or inhibit fungi in
association with higher plants; certain fungi utilize phenyloxidase activities to metabolize
and thus mitigate the toxicity of phenolic compounds produced by higher plants (Jassey
et al., 2011; Sinsabaugh, 2010).
The selection and use of bioassays to investigate the allelopathic potential of related
metabolites must be carefully planned in any experimentation performed. Allelopathic
activity may be seriously impacted by the mixture of metabolites present in complex
mixtures, and therefore activity might be associated with complex molecular interactions
including synergy, antagonism and enhanced effects in the presence of other metabolites
(Albuquerque et al., 2011; Einhellig, 1996; Neilson et al., 2013). To estimate the
bioactivity of allelochemicals, a dose/response study is essential to establish their
potential effects on the environment. When studies utilize a dose significantly higher than
the concentration(s) naturally present in the soil, the results are generally difficult to
interpret from an allelopathic perspective. Moreover, the mode of action of
70
allelochemicals can differ when applied at a dose well beyond that encountered in nature
(Fujii & Hiradate, 2007).
Figure 11. The release of allelochemicals in the environment under the influence
of various factors (Albuquerque et al., 2011).
1.7. Role (s) of Allelochemicals in the Rhizosphere, in Neighboring Plants and
Other Organisms
The rhizosphere is a narrow zone of soil around living plant roots that is also
inhabited by diverse groups of microorganisms; here plant roots compete with root
systems of neighboring plants and with other soil-born organisms, including bacteria and
fungi (Weston et al., 2012). The rhizosphere itself is not easily defined by size but rather
by its physical and biochemical properties, with a decreasing chemical gradient away
from living plant roots. Newman et al. (1985) estimated that the roots of a typical higher
plant can release 10-40 % of photosynthetically fixed carbon in the form of organic and
71
inorganic compounds into the rhizosphere. These root-produced products are collectively
called rhizodeposits and include exudate, mucilage, sloughed off border cells and root
cap cells (McNear Jr, 2013). As presented in Table 1, the majority of the root exudate
consists of diverse low molecular weight organic compounds (Baetz & Martinoia, 2014).
Allelopathic interference in the rhizosphere is well documented under controlled
conditions. However, allelopathy remains difficult to separate from competition among
plants of different species for growth limiting nutrient resources under field conditions
(Weston & Duke, 2003). However, recent advances have increased our knowledge about
physiochemical properties of roots. Below-ground interactions between roots and
microbes are continuously occurring in the rhizosphere and are often difficult to study
due to their presence in a complex soil matrix (Cipollini et al., 2012; Inderjit et al., 2005;
McCully, 1999). There has been increasing evidence which suggests that root exudates
provide a system of simultaneous communication with neighboring plants, as well as with
symbiotic and pathogenic organisms through biological and physical interactions (Figure
11) (Bais et al., 2004). Root exudates have been shown to regulate symbiotic relationships
with microorganisms in the rhizosphere, protect against herbivory, alter the chemical and
physiochemical properties of the soil and affect the growth of other plants in the
rhizosphere (Mathesius & Watt, 2010; Nardi et al., 2000). Recently, secondary
metabolites produced by plants have been shown to inhibit microbially-mediated
denitrification in the rhizosphere of invasive plants, thereby impacting growth and
available N for the invasive community (Bardon et al., 2014). Effective symbioses
between plants and microorganisms are generally facilitated by electrostatic and
chemotactic responses created by the root and its exudates (Van West et al., 2002; Zheng
& Sinclair, 1996).
Root exudates can also be associated with stimulation of plant growth, through the
attraction of useful microbial communities to the plant rhizosphere having subsequent
72
positive effects on plant growth or by direct effects of the plant-produced metabolites
(Bais et al., 2006). The impact of allelochemicals in root exudates on neighboring plant
growth is dependent on the concentration of allelochemicals in the exudate, their relative
rate of release, the vegetative stage of the target plant, as well as on the biotic and abiotic
environmental conditions (Gniazdowska & Bogatek, 2005; Hill et al., 2006; Putnam,
1988). It is important to consider that allelochemicals can only mediate negative
interference if they are released in sufficient quantities and persist long enough in the
environment to remain active. Weidenhamer (1996) has demonstrated in controlled
conditions that growth stage, biomass and density of target plants are important factors
that alter the availability of allelochemicals released into the rhizosphere and should be
considered when performing studies with allelochemicals. A greater density of receiver
plants will thus require higher concentrations of allelochemicals provided by the donor to
generate similar results in contrast to a reduced density. Therefore, interactions that are
documented in one location may not be easily replicated in another natural setting due to
varying conditions, including plant densities, encountered.
After release from plant tissue, other processes including leaching, oxidation,
biodegradation and uptake by neighboring plants can influence interference of
allelochemicals upon target plants (Inderjit, 2001). Root exudates produced under the
influence of chemical and biological elicitors may also act as antimicrobial agents against
pathogenic bacteria in the rhizosphere (Zhu et al., 2016). Plant roots typically defend
themselves against bacteria and fungi through the action of detachable border cells; these
cells located at the tips of actively growing roots produce a vast assortment of bioactive
secondary metabolites including allelochemicals. As an example, a newly germinating
plant uses products generated by border cells to aid in plant defense, particularly during
this sensitive period before the root system is well-established in a field setting. Root
border cells also produce extracellular DNA and associated proteins, which can act as a
73
trap for soil-borne pathogens, which are later negatively impacted by associated
allelochemical production (Hawes et al., 2016). Oats produce avenicin, an important
allelochemical and metabolite with potent activity against plant pathogenic organisms.
This interaction has been well characterized by the Osbourn laboratory, which has shown
that triterpenes produced by oats play critical roles in defense against soil microbes and
plant pathogens as well as abiotic stressors (Owatworakit et al., 2013). Interestingly, oats
have diverged chemically from other cereal grasses, which do not produce this family of
compounds.
1.7.1 Tolerance to Allelochemicals
Some plant species are intrinsically insensitive to specific phytotoxins or have
evolved mechanisms of defense from allelochemicals produced by the plant itself or from
other species (Shitan, 2016). Plants producing allelochemicals as well as those absorbing
them can resist toxicity by actively sequestering toxins in membrane-bound structures,
vacuoles or vesicles, secreting the compounds immediately after absorption, or altering
their chemical structure by primary and secondary metabolism (Bais et al., 2006). For
example, Polygonella myriophylla (Small) Horton releases hydroquinone and
benzoquinone allelochemicals in the form of arbutin, a glycoside of hydroquinone, which
prevents autotoxicity (Weidenhamer & Romeo, 2004). Likewise, corn (Zea mays L.)
produces inactive N--glycosylated forms of DIMBOA, DIBOA and BOA (Figure 9),
characteristic benzoxazolinones produced by several Poaceae spp; (Schulz et al., 2016).
Over millions of years, evolution has endowed selected plant species with the capacity to
tolerate or metabolize commonly encountered phytotoxins. However, those species which
do not encounter phytotoxins frequently are predicted to be less likely to develop
resistance (Bais et al., 2006). A recent hypothesis suggests that greater success of invasive
weeds is associated with their allelopathic potential to negatively affect native species by
the production of unique allelochemicals or ‘’novel weapons’’ which can successfully
74
interfere with the growth of native plant communities that are not well-adapted to the
presence of these bioactive metabolites (Callaway & Aschehoug, 2000).
1.8. Metabolic Profiling of Allelochemicals in Complex Plant or Soil Extracts or
Mixtures
The biochemical interactions occurring in the rhizosphere are the least well-
characterized in all of the biotic zones studied in terrestrial ecology. Despite these
challenges, new technological advancements in metabolite detection and identification
are proving useful when studying complex systems and interactions, and solving
ecological questions about regulation of allelochemicals and their roles in ecosystem
function. Metabolic profiling is proving to be an important tool when studying these
complex plant interactions and is utilized to study the plant metabolome (the total
collection of primary and secondary products produced by a plant). By performing an
extraction of plant tissue and evaluating the metabolome, one can assess the functional
state of a biological system at a particular point in time (Roessner & Bacic, 2009).
Weston et al. (2015) utilized a type of metabolomics referred to as metabolic profiling
that is a set of analytical procedures designed to study targeted compounds of interest,
including specific allelochemicals, in a biological system in response to a particular
treatment. Metabolic profiling can provide a strong insight on the biochemical status of
an organism so one might further understand complex interactions following the release
of allelochemicals in the rhizosphere, or in some cases their intermediate metabolites
(Putnam, 1988; Rice, 1974). It can also be used in conjunction with other omics
technologies (proteomics and transcriptomics) to fully construct the biosynthetic
pathways of targeted compounds of interest. Metabolic profiling of specific
allelochemicals may also be employed to study possible mechanisms associated with
invasion of weeds. Skoneczny et al. (2015) described significant upregulation of
pyrrolizidine alkaloids and their N-oxides in the highly invasive species, Echium
75
plantagineum L., in contrast to the less successful invader, Echium vulgare L., in both
field and laboratory settings, through the use of liquid chromatography coupled to mass
spectrometry (LC-MS) for evaluation of a series of related pyrrolizidine alkaloids,
including the toxins echiumine, leptanthine and echimidine.
Metabolomics is a high throughput approach which produces results in a relatively short
period of time when analyzing large sample sets (Scognamiglio et al., 2015). Progress in
this field has been associated with the development of a diverse range of analytical
platforms including gas and liquid column chromatography coupled with high-resolution
mass spectrometry for targeted and non-targeted metabolic profiling of secondary
metabolites and allelochemicals (Kim et al., 2010; Weston et al., 2015). More recently,
the development of triple quadrupole MS or sensitive ion trap MS has facilitated the
precise and accurate profiling for putative annotation of thousands of bioactive
compounds present in very low quantity in complex matrices (Weston et al., 2015). A
thorough description of how allelochemicals may be profiled by using metabolomics
approaches is discussed in the review prepared by Weston et al., (2015). Most recently,
metabolic profiling has also been applied to studies of the plant rhizosphere. One study
was performed to evaluate the release of diverse flavonoids from clover (Trifolium spp.)
in the soil rhizosphere when clovers have been utilized as cover crops, pointing to the role
of the allelochemical kaempferol, which persisted in the soil rhizosphere and was likely
to be associated with consequent allelopathy in perennial stands of white clover (Weston
& Mathesius, 2013). Additional studies to evaluate the role of a complex group of over
15 bioactive naphthoquinones, the shikonins, in plant defense and allelopathy in the
rhizosphere, have also been facilitated by the use of sensitive metabolic profiling
performed by UPLC QToF-MS (Zhu et al., 2016). Recovery of some of the more
persistent allelochemicals including acetyl shikonin and shikonin in ppm levels in
infested soils was also successful using LC/MS technologies. The increasing use of
76
metabolomics to study metabolite dynamics and biochemical pathways in plants will no
doubt result in an improved understanding of the presence and the role of allelochemicals
in the soil environment.
After analysis by metabolomics, processing of substantial amounts of mass spectral data
and subsequent characterization of metabolites are attempted (Smith et al., 2005).
Although it is never possible to identify all plant metabolites in a complex mixture, and
many remain currently unidentified or are undescribed. Such challenges are dealt with
using efficient throughput computational and analysis tools, which often involve data
conversion, features detection, normalization and recently developed quality control of
data processing (Sugimoto et al., 2012). The molecular features of the compounds present
are normalized for baseline variation and aligned with available structural libraries. The
METLIN library is one of the largest databases used for identification of natural products
(Weston et al., 2015). Greater flexibility across its catalogs and multipurpose search
options make it very convenient and widely used database (Smith et al., 2005).
1.9 Conclusions
Allelopathy, the study of complex plant–plant and plant-microbial interactions, is a
very active field of research in the plant sciences. In the past 20 years, the number of
researchers and research papers in the field has increased exponentially. Enhanced
collaboration among soil scientists, chemists, ecologists, geneticists and molecular
biologists will undoubtedly lead to new insights as to the factors regulating the production
of allelochemicals in higher plants and how these metabolites impact invasion ecology,
plant competition, plant interference, vegetation dynamics and crop production. The use
of new analytical techniques associated with metabolomics in concert with other omics
technologies has led to new advances in the identification of unique allelochemicals, the
biosynthetic pathways associated with their production, their complex role(s) in the soil
rhizosphere, and their production as impacted by a changing climate. A better
77
understanding of allelochemical production with respect to plant defense strategy may
also allow us to better protect and manage developing crops, limit the spread of invasive
weeds, preserve native plant stands, and create strategies for allelochemical development
and application as novel pesticides.
Currently, increasing public awareness regarding safety and environmental issues
associated with the use of herbicides, as well as profitable markets for organic
commodities has resulted in greater emphasis on the development of natural product-
based pesticide discovery programs (Duke et al., 2002; Weston & Duke, 2003).
Identification of novel plant metabolites, including allelochemicals, may result in a source
for future development of biologically based pesticides (Chiapusio et al., 2005), through
the provision of complementary structures for synthetic compounds (Ghisalberti, 2007)
and as an aid in the development of new molecular target sites (Duke et al., 2002).
1.10 References
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Chapter. 2
Weed Suppressive Potential of Selected Pasture Legumes against
Annual Weeds in Southeastern Australia
Chapter 2 presents results from preliminary field studies conducted in 2016 and
2017 in Wagga Wagga to assess the weed suppressive potential of eight selected annual
pasture legumes sown either as pure or mixed stands, both in the year of their
establishment and the subsequent year following regeneration. The experiment further
evaluated the effect of early sowing of pasture species in autumn to produce critical early
ground cover to compete against early emerging winter weeds. Results suggested that
species mixtures containing either two annual pasture legumes or an annual pasture
legume mixed with a grass species did not differ significantly with respect to relative
weed infestation when compared to monocultures of the same pasture legume species.
Arrowleaf clover and biserrula cv. Casbah produced significantly greater pasture biomass
resulting in reduced weed biomass. Results from this field study provided the basis for
further exploration of the fundamental mechanisms associated with weed suppression in
Chapter 3 and 4 in selected pasture legumes.
The data presented in this Chapter was published as a peer-reviewed manuscript
in the Proceedings of the 21st Australasian Weeds Conference, 2018.
Latif, S., Weston, P., Piltz, J., Gurusinghe, S., Brown, W., Quinn, J., & Weston,
L. A. (2018). Weed suppressive potential of selected pasture legumes against annual
weeds in southeastern Australia. In proceeding of the 21st Australasian Weeds
Conference 2018 in Sydney, Australia (pp. 378-382).
As first author, I, Sajid Latif, wrote the first and final drafts and led manuscript
development. All named authors contributed to the writing or editing of this conference
92
proceeding, Specifically, Drs. Leslie A. Weston, William Brown and Mr. John Piltz were
involved in establishing and monitoring the field trials while also providing technical
support and assistance with the collection of field data. Others co-author edited the
manuscript and provided suggestions related to data presentation. Other contributing to
this study are acknowledged in the manuscript.
93
Weed Suppressive Potential of Selected Pasture Legumes against
Annual Weeds in Southeastern Australia
Sajid Latif 1,2*, Paul A. Weston1,3, John W. Piltz1,4, Saliya Gurusinghe1, William B. Brown1,3,
Jane C. Quinn1,2, and Leslie A Weston1,3
1 Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga NSW
2678
2 School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga NSW
2678
3 School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga NSW 2678
4 NSW Department of Primary Industries, Agriculture Institute, Wagga Wagga NSW 2650
*Corresponding author: ([email protected])
Summary: Poor adaptation of numerous annual and perennial legumes across south
eastern Australia due to acidic soils, insufficient rainfall resulting in false breaks, high
input costs, soil acidification and the emergence of herbicide resistant weeds have resulted
in limited options for producers seeking to establish productive pastures in the Riverina
region of NSW. To overcome these challenges, numerous novel pasture species have been
selected and recently released for establishment. However, limited knowledge exists
regarding their ability to suppress weeds during establishment and in subsequent years
when they regenerate. Field trials were conducted at Wagga Wagga NSW over a two year
period to evaluate: (a) the suppressive potential of eight selected pasture legumes against
annual weeds; and (b) the impact of autumn (March) and winter (June) sowing dates on
stand establishment. Weed and crop cover and biomass were assessed each year in
94
replicated trials in 2016–2017. Results suggested that species mixtures with more than
one pasture species were not significantly different in terms of (P<0.05) establishment
and subsequent weed infestation. However, autumn sowing of arrowleaf clover, yellow
serradella cv. Avila and French serradella/bladder clover generally resulted in increased
(P<0.05) pasture crop cover over two growing seasons. Arrowleaf clover and biserrula cv.
Casbah were strong performers and produced significantly greater crop biomass and also
less weed biomass. However, weed suppression and subsequent biomass was not always
related to competition for resources and production of total crop biomass. This was the
case for yellow serradella cv. Santorini in both 2016 and 2017 where weed biomass was
significantly reduced but total crop biomass produced was limited. This suggested that
factors other than resource competition, such as allelopathy, were associated with weed
suppression. Arrowleaf clover, biserrula cv. Casbah and yellow serradella cv. Santorini
were generally the best and most reliable annual legumes with respect to yearly
regeneration and pasture weed suppression in the Riverina.
Keywords pasture, hard-seeded legume, weed suppression, competition, establishment.
2.1 Introduction
Pasture legumes have been an integral part of mixed farming systems in
southeastern Australia as they provide high-quality nutrition for livestock and improve
soil fertility due to their ability to fix atmospheric nitrogen. The selective use of pasture
legumes has also been instrumental in effectively managing weeds in such rotational
cropping systems (Mengel et al. 2001, Loi et al. 2005a, Loi et al. 2005b). Traditionally,
subterranean clover has been the most widely used pasture in southern Australia but
unpredictable weather patterns resulting in lower and inconsistent spring rainfall, high
cost of legume establishment and low persistence mainly due to low hardseed levels have
impacted the suitability of subterranean clover in the region (Loi et al. 2001, Loi et al.
2005a).
95
To overcome these challenges, numerous novel self-regenerating annual pasture
legumes including Biserrula pelecinus L. (biserrula), Ornithopus sativus Brot. (French
serradella), Ornithopus compressus L. (yellow serradella), Trifolium glanduliferum Boiss.
(gland clover), Trifolium spumosum L. (bladder clover) and Trifolium vesiculosum Savi.
(arrowleaf clover) have been recently released in New South Wales (NSW). These pasture
legumes are well adapted to the south eastern region, due to their successful establishment
in diverse soils, drought tolerance, higher herbage production and nutritional quality
relative to traditional pasture species. However, limited knowledge exists regarding their
establishment following initial seeding and in subsequent years of regeneration. To
maximize the adoption of introduced species/cultivars under suitable soil types and
rainfall zones, further research is required in NSW to assess the ability of selected pasture
legumes to establish and produce improved yields while suppressing annual weeds to
determine their suitability within sustainable cropping systems. Therefore, the objectives
of this study were to evaluate: (a) the suppressive potential of selected annual pasture
legumes against dominant annual weeds; and (b) the impact of sowing season; autumn
(March) or winter (June) on establishment and regeneration of annual legumes in
southeastern Australia.
2.2 Materials and Methods
Field experiments were established in 2016 and 2017 at the Graham Centre field
site and the Agricultural Research Institute field site in Wagga Wagga NSW (35°S, 147
E). All experiments were arranged in a randomised complete block design with five
replicates per treatment. The soil type was characterised as a fine red sodosol at both trial
sites. Treatments were planted at standard sowing rates using a precision cone planter in
20 x 3 metre with 15 cm row spacing. All pasture treatments were sown on two different
sowing dates either in the first week of March or first week of June and thereafter referred
to as autumn and winter sowing dates respectively, except in the case of those not suitable
96
agronomically for autumn sowing. All plots were fertilised with 100 kg ha-1 of fertiliser
(Croplift® 12, 1:2:1, Incitec Pivot, Victoria) at the time of sowing. The second trial site
was established similarly in winter 2017 at the Wagga Wagga Agricultural Research
Institute using only monoculture pasture treatments in 9 x 2 m plots with similar row
spacing and seeding rates.
Percent stand establishment of pasture crops and weeds was recorded using visual
ratings on a scale of 0 to 100 performed at physiological maturity of the pasture crops
when crops were at the flowering stage. In addition to qualitative data, two representative
pasture samples were collected from each plot at a height of approximately 2 cm the soil
surface (to simulate grazing by sheep) using 0.25 m2 quadrants from monoculture plots to
determine the total biomass of crop and weeds in 2016 and 2017. Plant matter was sorted
into pasture legume and weed species before being dried at 90°C for 96 hours and
weighed. All weeds were collected and bulked irrespective of species and dry weights
were recorded. Above ground crop competitive traits including normalised vegetative
index (NDVI), leaf area index (LAI), and light interception (LI) were assessed for each
pasture legume species at physiological maturity in 2017 to determine the relative effect
of each trait on total weed suppression. An analysis of variance (ANOVA) was performed
to determine the effect of sowing season and pasture crop on ground cover and biomass
of crops and weeds using the GenStat statistical software package (18th Edition, VSN
International Ltd, Hertfordshire, United Kingdom). The fixed model included year,
sowing date and pasture crops while the random effect was block number. A multivariate
analysis was performed using IBM SPSS statistical package (IBM SPSS® Inc. 2001,
version 24.0 for Windows, United States of America) using a partial least square
regression (PLSR) analysis model to determine the relative effect of competitive traits of
weed suppression (Reiss et al. 2018). PLSR is linear regression model that examines
97
relationships between latent variables (predicted variables and observed variable) in the z
axis related to structural covariance (Abdi & Williams, 2013).
2.3 Results
Results of visual ratings performed for pasture crops at physiological maturity
demonstrated that percentage ground cover of crops and weeds varied significantly
(P<0.05) across pasture species. Arrowleaf clover produced the highest ground cover of
all pasture species followed by biserrula cv. Casbah and gland clover, while cultivars of
yellow serradella produced the least pasture ground cover. Arrowleaf clover initially
exhibited the lowest percentage of weed infestation followed by biserrula cv. Casbah and
yellow serradella cv. Santorini (Figure 1).
Generally, mean percentage crop cover was slightly to moderately higher for the
autumn sowing date compared to winter sowing but the overall effect of sowing date was
not significant (P>0.05) except for arrowleaf clover and both cultivars of yellow
serradella.
Biomass of pasture crops and weed species was also obtained in at crop maturity
in 2016 and 2017 and these varied significantly (P<0.05) across pasture species. Overall,
crop biomass of all treatments ranged from 44 to 90 g m-2 dry weight (DW) while weed
biomass ranged from 5 to 30 g m-2 DW (Figure 1). Arrowleaf clover produced the highest
crop biomass followed by biserrula cv. Casbah and gland clover. In contrast, yellow
serradella cv. Santorini and subterranean clover produced the lowest crop biomass.
Interestingly, arrowleaf clover had the lowest weed biomass followed by yellow serradella
cv. Santorini. Remarkably, yellow serradella cv. Santorini produced the lowest biomass
but also maintained low weed infestation.
A PLSR analysis was conducted to determine the relative effect of competitive
traits on weed biomass. Competitive traits explained up to 30% of the variance in two
98
latent factors when dataset from all species was analysed in one model suggesting a
separate analysis for each species separately is desired. Overall, total weed biomass at
physiological maturity was strongly correlated with crop biomass followed by LI.
Figure 1. Visual ratings performed on a scale of 0 to 100 where zero represents no crop
cover and 100 represents complete and uniform ground cover established in Wagga
Wagga, NSW. Ratings were taken at physiological maturity of the pasture crops which
was achieved in the third week of October and November, respectively, and averaged
over both years. Error bars indicate the standard error of means.
0 10 20 30 40 50 60 70 80 90 100
Yellow serradella cv. Santorini
Yellow serradella cv. Avila
Subterranean clover
Gland clover
French serradella
Bladder clover
Biserrula cv. Casbah
Arrowleaf clover
Crop cover Weed cover
99
Figure 2. Biomass of crop and weeds (g m-2 DW) assessed at physiological maturity
of the pasture crops averaged across 2016 and 2017 Wagga Wagga NSW. Weed
biomass represents the composite biomass of all weeds. Error bars indicate the
standard error of means.
Crop biomass appeared to have the strongest inverse effect on total weed
biomass in the case of biserrula cv. Casbah, bladder clover, French serradella,
subterranean clover and yellow serradella cv. Avila. In addition, LAI was observed to
have a strong inverse effect on total weed biomass in the case of arrowleaf clover and
gland clover. Clearly, crop biomass, NDVI and LAI were not associated with weed
suppression in case of yellow serradella cv. Santorini.
2.4 Discussion
Results of two years of several field trials in the Riverina region of NSW clearly
demonstrated significant variation in the competitive ability of individual pasture legumes
species and cultivars, resulting in variable weed suppression against annual weeds.
Arrowleaf clover, biserrula and yellow serradella were generally the most reliable and
competitive annual legumes with respect to stand establishment, yearly regeneration and
0 10 20 30 40 50 60 70 80 90 100
Yellow serradella cv. Santorini
Yellow serradella cv. Avila
Subterranean clover
Gland clover
French serradella
Bladder clover
Biserrula cv. Casbah
Arrowleaf clover
Crop cover Weed cover
100
consistent suppression of pasture weed species in two years of experiments performed at
multiple field sites in Wagga Wagga. Sowing of pasture legumes in autumn or winter is
feasible but crops performed more consistently when sown in June. In this study autumn
sowing did not offer significant advantages compared to winter sowing in suppressing annual
weeds. Arrowleaf clover and biserrula have been reported to have greater ability to
successfully regenerate in Mediterranean-like climatic conditions. In general, these two
legumes produced significantly greater crop biomass, and as a result, considerably less weed
biomass due to low light penetration of the dense crop canopy and competition for resources.
Results of PLSR analysis demonstrated that, in general, crop biomass had the strongest
inverse effect on total weed biomass for many pasture species and likely also impacted weed
seed germination and seedling growth. Several mechanisms could be associated with the
ability of these pasture legumes to suppress weeds including their ability to rapidly develop
dense canopies limiting light interception at the soil surface. In addition, yellow serradella
may exhibit the ability to suppress weeds through allelopathic interference. Additional
information related to the mechanisms involved in weed suppression, including the potential
for some species to release allelochemicals over time, will be useful in the selection of new
pasture species/cultivars for sustainable weed management solutions, particularly when
considering the increasing importance of herbicide resistance in pasture weeds.
2.5 Acknowledgments
The authors acknowledge funding support for this project from Meat and Livestock
Australia (MLA) Project B. WEE 0146 and the Australian Centre for International Agricultural
Research (ACIAR) for providing a PhD scholarship for SL. The author also acknowledges
technical support provided by Graeme Heath and Simon Flynn in collecting field data.
101
2.6 References
Abdi, H., & Williams, L. J. (2013). Partial least squares methods: partial least squares
correlation and partial least square regression Computational Toxicology (pp. 549-
579): Springer.
Loi, A., Howieson, J.G., Nutt, B.J. and Carr, S.J. (2005a). A second generation of annual pasture
legumes and their potential for inclusion in Mediterranean-type farming systems.
Australian Journal of Experimental Agriculture 45, 289–99.
Loi, A., Howieson, J.H. and Carr, S.J. (2001). Register of Australian herbage plant cultivars.
Biserrula pelecinus L. (biserrula) cv. Casbah. Australian Journal of Experimental
Agriculture 41, 841–2.
Loi, A., Revell, C, Nutt, B.J. (2005b). Domestication of new forage legumes improves the
productivity and persistence of pastures in Mediterranean environments. Proceedings of
the 1st COST 852 Workshop, Eds B.E. Frankow-Lindberg, R.P. Collins, A Luscher, M.T.
Sebasria, A. Helgadottir, pp 165-175(wedish University of Agricultural Sciences,
Sweden).
Mengel, K., Kirkby, E.A., Kosegarten, H. and Appel, T. (2001) 'Principles of Plant
Nutrition'.(Springer, Dordrecht).
Reiss, A., Fomsgaard, I.S., Mathiassen, S.K. and Kudsk, P. (2018). Weed suppressive traits of
winter cereals: Allelopathy and competition. Biochemical Systematics and Ecology 76,
35–41.
102
Chapter 3.
Performance and Mechanism of Weed Suppression in Selected Pasture
Legumes against Annual Weeds in Southeastern Australia
Chapter 3, in contrast to Chapter 2 which presents preliminary data, presents the
compiled results of a three year replicated field experiment performed at two different
locations in southern NSW evaluating the weed suppression potential of pasture legumes
as monocultures and mixed stands against dominant annual weed species through critical
evaluation of various weed competitive crop traits. This study also successfully attempted
to evaluate underlying mechanisms associated with weed suppression in the case of each
individual pasture species. Data collected included qualitative analysis of the pasture crop
and weed ground cover through repeated visual assessment. Quantitative assessments
included normalised vegetative index (NDVI), light inception (LI), leaf area index (LAI),
interception of photosynthetically active radiation by the pasture crop and accumulation
of above-ground crop biomass. Partial least squares analyses were performed to estimate
the relative effect of crop competitive traits on weed suppression.
Outcomes of this research were published as a research article in the journal Crop
and Pasture Science.
Latif, S., Gurusinghe, S., Weston, P. A., Brown, W. B., Quinn, J. C., Piltz, J. W.,
& Weston, L. A. (2019). Performance and weed-suppressive potential of selected pasture
legumes against annual weeds in southeastern Australia. Crop and Pasture Science 70(2),
147-158.
Contributions to this work were as follows:
I, Sajid Latif, undertook the main body of work presented in this chapter including
field data collection, statistical analysis and writing first draft of the manuscript. Prof.
103
Leslie A. Weston assisted in experimental design of all experiments, performing visual
assessments in the field, statistical analysis and presentation of all data in tables and
figures. In addition, Dr. William Brown and Mr. John W Piltz were involved in
experimental design, establishment and monitoring of the field trials and providing
technical support for sample collection while Dr. Paul Weston provided technical support
for statistical analysis of the data. Drs. Gurusinghe and Quinn provided assistance in
editing the manuscript, presentation of data and Dr. Gurusinghe in model development.
As primary author, I led the manuscript preparation of this journal article by writing the
first and final drafts and performing all data analyses. All authors contributed to the
preparation and editing of the manuscript during the process of acceptance for
publication. Other contributors to this study are included in the acknowledgements.
104
CSIRO PUBLISHING
Performance and weed suppressive potential of selected pasture
legumes against annual weeds in southeastern Australia
Sajid Latif A,B, Saliya Gurusinghe A, Paul A. Weston A,C, William B. Brown A,C,
Jane C. Quinn A,B, John W. Piltz A, D and Leslie A Weston A,C
A Graham Centre for Agricultural Innovation (NSW Department of Primary Industry),
Locked Bag 588, Wagga Wagga, NSW 2678
B School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678
C School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678
D NSW Department of Primary Industries, Agricultural Institute, Wagga Wagga, NSW
2650
Abstract
Mixed farming systems have traditionally incorporated Trifolium subterraneum L.
(subterranean clover) and Medicago sativa L. (lucerne) as key components of the pasture
phase across southeastern Australia. However, poor adaptation of subterranean clover to
acidic soils, insufficient and inconsistent rainfall, high input costs, soil acidification and
the emergence of herbicide-resistant weeds have reduced efficacy of some traditional
clover species in recent years. To overcome these challenges, numerous novel pasture
species have been selectively improved and released recently for establishment in
Australia. Despite their suitability to Australian climate and soils, limited knowledge
exists regarding their weed suppressive ability in relation to establishment and
regeneration. Field trials were therefore conducted over three years in New South Wales
to evaluate the suppressive potential of selected pasture legumes as monocultures and in
mixed stands against dominant annual pasture weeds. Pasture and weed biomass varied
105
significantly between pasture species when sown as monocultures, but mixtures of several
species did not differ with regard to establishment and subsequent weed infestation.
Arrowleaf clover and biserrula cv. Casbah showed improved stand establishment, with
higher biomass and reduced weed infestation in comparison to other pasture species. In
most cases, weed suppression was positively correlated with pasture biomass; however,
yellow serradella cv. Santorini exhibited greater weed suppression compared to other
pasture legumes while producing lower biomass, thereby suggesting a mechanism other
than competition for resources impacting weed suppressive ability. Over the period of
2015-2017, arrowleaf clover and biserrula cv. Casbah were generally the most consistent
annual pasture legumes with respect to yearly regeneration and suppression of annual
pasture weed species.
Additional keywords: hard-seeded legume, establishment, competition and weed
suppression
3.1 Introduction
Pasture legumes are an integral part of mixed farming systems in southeastern
Australia, as they provide high-quality pasture biomass for livestock production and if
effectively nodulated with compatible rhizobia, improve soil fertility through fixation of
atmospheric nitrogen. Many pasture legume species provide additional ecosystem
benefits through interference with pest life cycles and often improve soil tilth when
included in cropping rotations (Howieson et al. 2000; Nichols et al. 2010; Jeger et al.
2014). Pasture legumes have also been selectively utilised as a weed management tool in
rotational cropping systems (Mengel et al. 2001; Loi et al. 2005a; Loi et al. 2005b).
However, the choice of appropriate pasture legume species or cultivar for depends upon
climate, soil type and farming system.
106
The Riverina region of southeastern Australia has a temperate climate, characterised by
winter dominant rainfall (350-500 mm/year) pattern and predominantly mixed cropping
systems (Dear and Ewing 2008). In such moderate to high rainfall zones, Trifolium
subterraneum L. (subterranean clover) has been a key component of pasture systems
across southeastern Australia, while Medicago sativa L. (lucerne) and several other
species of annual medics (Medicago spp.) have been utilised in low to moderate rainfall
zones (Hackney et al. 2008). Reliance on subterranean clover has, however, resulted in a
lack of pasture legume diversity (Howieson et al. 2000; Nichols et al. 2007). Moreover,
unpredictable weather patterns resulting in lower and/ or inconsistent rainfall, the
relatively high cost of legume establishment and cases of low pasture persistence have
impacted the suitability of subterranean clover in the region (Loi et al. 2001; Loi et al.
2005a; Hackney et al. 2015). In addition, subterranean clover also contributes to soil
acidification through short- and long-term imbalances in the carbon and nitrogen cycles
(Williams 1980; Carr et al. 1999). Finally, the emergence and spread of herbicide-
resistant weeds have also limited the use of subterranean clover in southeastern Australia
in mixed cropping rotations (Powles and Yu 2010).
It has been shown that increasing legume diversity may stabilise and enhance long-term
pasture production in the face of variable seasonal conditions and soil types and may also
impede pest and disease spread and persistence (Smith and Allcock 1985; Gould et al.
2016). Improved selection and breeding for potentially adaptable and robust self-
regenerating annual pasture legumes has been the focus of recent research efforts in
southern Australia. These efforts have led to the introduction of novel annual pasture
legumes including Biserrula pelecinus L. (biserrula), Ornithopus sativus Brot. (French
serradella), Ornithopus compressus L. (yellow serradella), Trifolium glanduliferum
Boiss. (gland clover), Trifolium spumosum L. (bladder clover) and Trifolium vesiculosum
Savi. (arrowleaf clover). Most of these species originated in temperate to Mediterranean
107
regions of Europe and Northern Africa with bioclimatic environments similar to that of
southeastern Australia, including seasonal rainfall distribution and soil types (Dear and
Ewing 2008). Many of the newly introduced annual pasture species exhibit the capacity
to resist late germination events in late summer and early autumn arising from
inconsequential rainfall events due to higher hard seed levels, which can elicit high
mortality in softer-seeded species such as subterranean clover. They can produce large
quantities of high quality livestock feed while tolerating moisture stress throughout the
growing season, generating ample seed that can be harvested using a conventional
harvester and fixing appreciable quantities of atmospheric nitrogen (Carr et al. 1999; Dear
et al. 2003; Hackney et al. 2008; Banik et al. 2013).
Following evaluation in recent field demonstration trials, producers and agronomists have
generally embraced the introduction of annual pasture legumes (Hackney et al. 2015).
However, a survey conducted in 2008 indicated adoption by producers was hampered by
lack of technical knowledge in their establishment and management, and a shortfall of
information on long-term performance in low to moderate rainfall zones have led to
limited adoption (Nichols et al. 2007; Hackney et al. 2008). Many producers prefer
establishing self-regenerating annual pasture legumes as monoculture stands for ease of
management, while others established mixtures, either with grasses or other legumes,
with an aim to prolong the duration of production, enhance stress tolerance and improve
nutrition, while reducing the annual weed burden and establishment costs (Loi et al
2005a).
Although pasture weed management has relied heavily on herbicide application in recent
decades since the application of minimum tillage practices, the availability of labeled
herbicide options for newly developed annual pasture legume species is limited. The
development of herbicide resistance by an increasing number of pasture weed species has
further contributed to weed management challenges faced by producers in mixed farming
108
systems (Broster et al. 2014). According to the Cooperative Research Centre for
Australian Weed Management (Richards 1957; Department of the Environment and
Heritage 2004), pasture weeds incur an estimated cost of AUD 2.2 billion per year in
management expenses and yield loss to Australian livestock producers. Recent shifts in
weed populations and widespread emergence of herbicide resistance in major weed
species including Raphanus raphanistrum L. (wild radish) (Walsh et al. 2007), Lolium
rigidum Gaudin. (annual ryegrass) (Owen et al. 2007) and barley grass (Hordeum
leporinum L.) (Owen et al. 2012) suggest an urgent need for alternative or complementary
weed management strategies. However, limited information is currently available
regarding competitiveness of many of the newly introduced annual pasture legumes
against pasture weeds.
Crop competition has been widely accepted as an important tactic in managing pasture
weeds (Bajwa et al. 2017). Agronomic choices and cultural practices including choice of
crop species and cultivars, row spacing and orientation and crop density are shown to
impact the outcomes of crop-weed interactions and the relative competitiveness of pasture
legumes. Annual pasture legumes sown as forage crops can provide an important non-
chemical weed management option for sustainable crop production (Dear et al. 2006).
Highly competitive pasture species and/or cultivars generally have the ability to better
access resources including light, soil moisture and nutrients, thus suppressing the growth
of weed species in close proximity (Lemerle et al. 1995; Saito et al. 2010; Worthington
et al. 2015; Sardana et al. 2017). Establishment and performance of many species/
cultivars may also be influenced by modifications to agronomic practices including
planting time and pasture composition.
Pasture legumes with the ability to produce higher biomass with early vigorous growth
and dense canopy architecture have been shown to be highly competitive against annual
weeds (Dear et al. 2006; Saito et al. 2010; Sardana et al. 2017). The selection of highly
109
competitive species and cultivars could potentially provide cost-effective weed
management strategies by reducing the reliance on herbicides and also reduce the risk of
developing herbicide resistance. In addition to selecting competitive species, early sowing
strategies could further exploit the benefits provided by competition through improved
suppression of germinating weeds (Bertholdsson 2005) and extension of the seasonal
availability of quality feed for grazing livestock. To maximize the adoption of introduced
species / or cultivars suited to a variety of soil types and rainfall zones, further research
is required to assess their performance and weed suppression potential for sustainable
pasture cropping mixed system. Therefore, the objectives of this study were to evaluate
(a) the establishment and biomass production of selected annual pasture legumes grown
either as a monoculture or in binary mixtures with another legume or temperate grass
species, (b) the impact of sowing season; March (autumn) vs June (winter) on
establishment of annual legumes, and (c) the weed suppressive potential of pasture
species against dominant annual pasture weeds of southeastern Australia.
3.2 Materials and Methods
3.2.1 Pasture Establishment
Field experiments were established in 2015 and 2017 at the Graham Centre field
site (field site 1) and the Wagga Wagga Agricultural Institute (field site 2), at Wagga
Wagga, NSW (35.04°S, 147.36 E and 35.02 S, 147.33 E, respectively). All experiments
were arranged in a randomised complete block design with five replicates per treatment.
The soil type was characterised as a fine red sodosol (Isbell 1996) at both trial sites with
pH of 6.2 (Mwendwa et al. 2018). The pasture species and/or cultivars used in this study
and their respective cultivars, seasonal maturity, morphological growth habits and
inoculants used are listed in Table 1. All pasture species either in monoculture or mixtures
were sown at commercially recommended sowing rates in NSW regardless of seed weight
(Lattimore and McCormick 2012) (Table 1).
110
Plots were managed without any grazing or mowing throughout the growing season.
Daily rainfall was recorded on site while other climate including number of frost days
and temperature range was acquired from nearby weather station. The first experimental
site was established in 2015, with 17 treatments including monocultures and binary
mixtures of pasture legumes with other legumes or temperate perennial grasses sown at
two times (Table 2). Seeds of legumes species were coated with the appropriate inoculant
for each species (as recommended by inoculant producers) by application of a slurry to
seed, followed by a period of drying (overnight). Glyphosate was applied at a rate of 1.6
L/ha prior to sowing in all plots in February to remove residual weed infestation. All
pasture treatments were then sown at two different dates, either in the first week of March
or first week of June. These were subsequently referred to as autumn and winter sowing,
respectively, except for those not agronomically suitable for autumn sowing, including
Dactylis glomerata L. (cocksfoot), Medicago sativa L. (lucerne) and Phalaris aquatic L.
(phalaris) mixtures with subterranean clover.
111
Table 1. Pasture species, cultivars, seeding rate, seasonal maturity, growth habit, and inoculants used in the field trials at field sites 1 and 2 in Wagga
Wagga, NSW.
Scientific name Common name Cultivar Seasonal
maturity
Growth habit Seeding
rate*
(kg/ha)
Seed
weight**
(mg)
Inoculant**
*
Trifolium vesiculosum Savi. Arrowleaf
clover
Cefalu Early Erect to semi-
erect
8 1.2 C
(WSM1325)
Biserrula pelecinus L.
Biserrula
Casbah
Early-mid
Erect to semi-
erect
8
1.2
WSM 1497
Mauro Mid-late Erect to semi-
erect
8 1.2 WSM 1497
Trifolium spumosum L. Bladder clover Bartolo Early-mid Semi-erect 10 2.6 C
Ornithopus sativus Brot. French
serradella
Margarita Mid Erect to semi-
erect
10 0.9 S (WSM471)
Trifolium glanduliferum
Boiss.
Gland clover Prima Early Erect to semi-
erect
8 0.7 C
Trifolium subterraneum L. Subterranean
clover
Seaton Park Early-mid Prostrate 10 6.7 C
Ornithopus compressus L. Yellow
serradella
Avila Mid-late Semi-erect 10 1.4 S
Santorini Early-mid Semi-erect 10 1.2 S
Medicago sativa L. Lucerne Aurora Perennial Erect 4 2.1 AL (RRI128)
Phalaris aquatic L. Phalaris Holdfast Perennial Prostrate 2 0.6 -
Dactylis glomerata L. Cocksfoot Uplands Perennial Semi-erect 2 0.55 -
*Seeding rate here refers to seed not pods.
**Seed weight referred here is based on published literature.
*** Letters C, S are commercial inoculants; WSM refers to the specific strain of inoculant (supplementary Table 1).
112
Treatments were planted using a precision cone planter in 20 × 3 metre plots with 15 cm
row spacing. All plots were fertilised with 100 kg/ha of fertiliser (Croplift® 12, 12:18:6
NPS Incitec Pivot, VIC) at the time of sowing. In 2016, pasture crops regenerated
naturally following 2015 seeding and subsequent seed dispersal. A second trial site was
established similarly in winter 2017 at field site 2 using only monoculture pasture
treatments in 9 × 2 m plots with similar row spacing and seeding rates (Table 2).
Table 2. Species/cultivars and mixtures of pasture species sown in autumn and winter
in 2015 and monoculture pasture species sown in 2017 at field sites 1 and 2,
respectively.
Pasture species Autumn Winter Winter
2015 2015 2017
Arrowleaf clover ✓ ✓ ✓
Biserrula cv. Casbah ✓ ✓ ✓
Biserrula cv. Mauro ✓ ✓ ˗
Biserrula cv. Casbah + French serradella ✓ ✓ ˗
Biserrula cv. Casbah + Gland clover ✓ ✓ ˗
Bladder clover ✓ ✓ ✓
Bladder clover + Gland clover ✓ ✓ ˗
Cocksfoot + Biserrula cv. Casbah ˗ ✓ ˗
French serradella ✓ ✓ ✓
French serradella + Bladder clover ✓ ✓ ˗
Gland clover ✓ ✓ ✓
Lucerne + Subterranean clover ˗ ✓ ˗
Phalaris + Subterranean clover ˗ ✓ ˗
Subterranean clover ˗ ✓ ✓
Yellow serradella cv. Avila ✓ ✓ ✓
Yellow serradella cv. Avila + Gland clover ✓ ✓ ˗
Yellow serradella cv. Santorini ✓ ✓ ✓
113
3.2.2 Data Collection
Percentage cover of pasture species and weeds was recorded separately using
visual ratings of ground cover, on a scale of 0 to 100 performed when pasture species
were at approximately 50% flowering stage (People and Craswell 1992; Bell and Fischer
1994). This occurred in the third week of October in 2015 and 2016 at field site 1 and in
the third week of November in 2017 at field site 2. A rating of 100 represented complete
and uniform ground cover while 0 represented no establishment (Weston 1990). In 2015,
site 1 was hand weeded for Papaver spp. (poppy). Visual ratings for dominant annual
weed species were conducted only in 2016 and 2017. In 2016, predominant weed species
included barley grass, Sonchus spp. (sowthistle) and poppy spp.; and other common
species including annual ryegrass, Fumaria spp. (fumitory), Artotheca spp. (capeweed)
and Echium plantagineum L. (Paterson’s curse). In 2017, visual ratings were conducted
for barley grass, fumitory, annual ryegrass and wild radish. Two representative pasture
samples were collected from each plot at a height of approximately 2 centimetres above
the soil surface (to simulate grazing by sheep) using 0.25 m2 quadrants only from
monoculture plots to determine the total biomass of both pasture and weed species in
2016 and 2017. Plant matter was then sorted into pasture crops or weeds before drying at
60 °C for 96 h. Bulked pasture species and weed biomass were then obtained by
assessment of dry weights.
3.2.3 Measurement of Ecophysiological Traits Associated with Competitive Ability
Aboveground competitive traits including canopy architecture as measured by
leaf area index, light interception and biomass (Bajwa et al. 2017, Sardana et al. 2017)
accumulation were assessed for each pasture legume species at approximately 50 %
flowering in 2017 at field site 2 to determine the relative effect of each trait on total weed
biomass in each of five replicates. Soil coverage of green plant material at approximately
50 % flowering was recorded by measuring the reflectance of red light (R) (650 nm) and
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near infra-red (NIR) (810 nm) light with a handheld sensor (GreenSeeker 505®,
Trimble®) to determine irradiation at the top of the canopy in each treatment at mid-day
on a cloudless day. For each measurement, the normalised difference of vegetative index
(NDVI) was calculated (Tucker and Sellers 1986) for each treatment as follows:
𝐍𝐃𝐕𝐈 =𝐍𝐈𝐑 − 𝐑
𝐑 + 𝐍𝐈𝐑
Canopy cover was also assessed by measuring the leaf area index (LAI) and light
interception (LI) as measured by percent interception of photosynthetically active
radiation (PAR) at the base of the pasture using a light ceptometer (AccuPAR LP-80
Ceptometer, Decagon Devices®) on a cloudless day. The intercepted PAR is calculated
as PAR at the base of the canopy subtracted from the PAR above the level of the canopy.
3.2.4 Statistical Analysis
Statistical analyses were conducted to determine the effect of sowing date, pasture
species and their mixtures on pasture establishment and weed suppression using the
GenStat statistical software package (18th Edition, VSN International Ltd, Hertfordshire,
UK). A linear mixed model was fitted to the data due to an unequal number of treatments
by calculating restricted maximum likelihood (REML) estimation. The fixed model
included year, sowing date and pasture treatments while the random effect was block
number. Since REML analysis identified a significant interaction between years, an
independent analysis was carried out for each year using REML analysis. Least
significant difference (LSD) was used to evaluate the difference between treatment
means. Significance was declared at P = 0.05. A multivariate analysis was performed
with SPSS (IBM SPSS® Inc. 2001, version 24.0 for Windows, USA) using a partial least
square regression (PLSR) model to determine the relative effect of competitive traits on
weed suppression in individual species/cultivars (Reiss et al. 2018). The model was fitted
on a scaled and centered dataset. Percent variance was calculated based on the first two
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latent factors. An angle of > 90° between dependent variable and independent variables
indicates an inverse relationship while an angle of < 90° indicates a positive correlation,
while an angle close to 90° indicated no impact.
3.3 Results
3.3.1 Climatic Conditions
In 2015 and 2016, weather conditions including growing-season (from April to
October) rainfall (332 and 624 mm respectively) and temperature in Wagga Wagga region
were favourable for the establishment of pasture legumes. However, the 2017 growing
season, experienced low rainfall with only 181 mm rainfall received, which was markedly
below the long-term average (1948 to 2017) rainfall of 373 mm during a typical growing
season. Therefore, the total annual mean precipitation over the three years represented a
range of below, within and above average growing-season rainfall (Table 3). The mean
temperature in the 2017 cropping season was also lower compared to 2015 and 2016
(Table 3). In addition to a cold and dry growing season, an above average number of frost
days (37 days) were noted in 2017 when compared to the long-term average (21 days).
Table 3. Meteorological data for the years 2015, 2016, 2017 and long-term average
(1948 to 2017), including the average minimum and maximum temperature, total
precipitation, and number of frost days in each cropping season (April-October)
recorded at field site 1 in Wagga Wagga, NSW.
Meteorological data 2015 2016 2017 Long term average
Average maximum temperature (°C) 16.3 17.4 18.1 16.9
Average minimum temperature (°C) 5.1 7.1 3.9 6.17
Total rainfall (mm) 333 625 181 374
No. of frost days 16 7 37 9
3.3.2 Effect of Sowing Date on Pasture and Weed Cover
Weed infestation as assessed by percentage weed cover varied significantly
depending on sowing date (P < 0.05) in the year of pasture establishment (2015).
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However, the different sowing dates had no significant impact on mean percentage
pasture cover (P > 0.05). Average percentage pasture cover ranged from 18 to 95 % for
the autumn sowing date and from 39 to 80 % for the winter sowing date (Table 4).
Arrowleaf clover produced the highest proportion of ground cover (95 %) of all pasture
species followed by biserrula cv. Mauro (81 %) for autumn sowing, while gland clover
(80 %) and biserrula treatments (73-74 %) produced the highest ground cover for winter
sowing when evaluated at approximately 50 % flowering. In contrast, weed infestation
was highest in yellow serradella cv. Santorini at autumn and bladder clover at winter
sowing dates, when evaluated at flowering (Table 4).
3.3.3 Impact of Pasture Mixtures on Weed Infestation
A separate analysis was performed on visual ratings to compare weed infestation
between pasture mixtures and corresponding monocultures. Visual ratings of weed
infestation as a percentage of ground cover were not significantly (P > 0.05) impacted by
seeding in mixtures and subsequent establishment in mixed stands in 2015 when
compared to monocultures of the same cultivars (data not presented). Of note was the
observation that mixtures generally exhibited comparable weed infestation to
monocultures. A similar effect was also noted in 2016, with several exceptions. Phalaris
/ subterranean clover and cocksfoot / biserrula cv. Casbah mixed stands showed the
highest ratings for all pasture covers while having the lowest weed infestation levels (see
supplementary data Figure 1 and 2).
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Table 4. Visual ratings performed on both monocultures and mixtures of pasture legumes established in autumn or winter a in 2015 including
percentage pasture and weed cover b at field site 1 in Wagga Wagga, NSW. Ratings were taken after approximately 50 % floweringc of the
pasture legumes.
Pasture species Pasture cover (%) Weed cover (%)
autumn winter autumn winter
Arrowleaf clover 95 66 3 13
Biserrula cv. Casbah / Gland clover 76 74 13 10
Biserrula cv. Casbah 75 73 16 8
Biserrula cv. Mauro 81 74 18 11
Biserrula cv. Casbah / French serradella 74 73 15 8
Bladder clover 54 39 29 37
Bladder/ Gland clover 68 70 17 8
Cocksfoot/ Biserrula cv. Casbahd - 62 - 19
French serradella 73 70 13 6
French serradella/ Bladder clover 70 60 17 15
Gland clover 75 80 9 16
Lucerne/ Subterranean cloverd - 56 - 26
Phalaris/ Subterranean cloverd - 53 - 13
Subterranean cloverd - 64 - 20
Yellow serradella cv. Avila 80 42 10 32
Yellow serradella cv. Avila / Gland clover 71 67 15 18
Yellow serradella cv. Santorini 18 45 57 33
LSD* 27 17.4 20.6 14.1
LSD** 22.2 17.5 a Autumn sowing was performed in the first week of March and winter sowing in the first week of June 2015.
b Percentage pasture and weed cover were rated on a scale of 0 to 100 where zero represents no pasture cover and 100 represents complete and uniform
ground cover. c Approximately 50% of the pasture flowering was observed in the third week of October in both 2015 and 2016. d Treatments were only sown in winter
* Least significant difference between pasture species at each sowing date
** Least significant difference between autumn and winter sowing dates.
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3.3.4 Impact of Pasture Monoculture Treatment on Weed Cover
Estimated cover of pasture legumes and weeds measured by visual ratings, also
varied significantly (P < 0.05) across pasture species in 2016 and 2017 (Table 5).
Table 5. Visual ratings of pasture and weed cover a performed on monoculture of
pasture legumes established in 2016 or 2017 at field sites 1 and 2 in Wagga Wagga,
NSW, respectively. Ratings were taken at 50% flowering b of the pasture species.
Pasture species Pasture cover (%) Weed cover (%)
2016 2017 2016 2017
Arrowleaf clover 91 87 1 8
Biserrula cv. Casbah 81 53 9 13
Biserrula cv. Mauro 63 - 26 -
Bladder clover 57 47 30 32
French serradella 50 70 37 23
Gland clover 71 70 20 17
Subterranean clover 65 45 27 29
Yellow serradella cv. Avila 41 49 36 22
Yellow serradella cv.
Santorini 24
63 15 11
P value < 0.001 < 0.01 < 0.01 < 0.01
LSD 15.8 19.6 13.8 14.0
a Percentage pasture and weed cover were assessed on a scale of 0 to 100 where zero represents
no pasture cover and 100 represents complete and uniform ground cover. b Approximately 50% of the pasture flowering was observed third week of the October and third
week of November in 2016 and 2017, respectively.
(-) Biserrula cv. Mauro was not included in 2017 field trial.
Arrowleaf clover (91%) produced the highest ground cover, followed by biserrula cv.
Casbah (81 %) and gland clover (71 %) in 2016. Similarly, arrowleaf (87%) produced the
highest ground cover in 2017. In contrast, yellow serradella cv. Santorini (24 %) produced
the lowest cover in 2016 and bladder clover (47 %) in 2017. Further, arrowleaf clover
exhibited the lowest level of weed infestation (1 and 8 %) in both years, respectively
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followed by biserrula cv. Casbah (9 %) in 2016 and yellow serradella cv. Santorini (11
%) in 2017 (Table 5).
3.3.5 Effect of Pasture Treatment on Dominant Weed Species
Percentage cover of dominant weed species was recorded in 2016 and 2017 and
differed significantly (P < 0.05) depending on pasture species and composition for each
weed species (Table 6 and 7). Sowing date significantly impacted the percentage cover
of dominant weed species between treatments in 2015 (P < 0.05) (Table 6). Yellow
serradella cv. Avila/ gland and yellow serradella cv. Santorini exhibited significantly (P
< 0.05) higher infestation of barley grass (20 %) when compared to yellow serradella cv.
Avila, gland clover, biserrula cv. Casbah/ French serradella, biserrula/gland clover and
arrowleaf clover in autumn-sown species. French serradella/ bladder clover (19 %)
exhibited significantly (P < 0.05) higher infestation of barley grass when compared to
arrowleaf clover, biserrula cv. Casbah/ gland clover, biserrula cv. Casbah, biserrula cv.
Casbah/ French serradella, cocksfoot/ biserrula cv. Casbah and phalaris/ subterranean
clover in winter sown treatments.
The establishment of sowthistle was not affected by the sowing date of most pasture
species with the exception of gland clover (16 %) exhibiting significantly (P < 0.05)
higher infestation of sowthistle compared with arrowleaf clover, biserrula cv. Casbah,
biserrula cv. Mauro, bladder clover, French serradella, French serradella/ bladder clover,
yellow serradella cv. Avila in autumn sown treatments. Poppy infestation was
significantly (P < 0.05) higher in bladder clover/ gland clover mixture compared to
arrowleaf clover, biserrula cv. Casbah, yellow serradella cv. Avila in autumn-sowing
treatments. Bladder clover and a mixture of bladder clover/ gland clover exhibited
significantly (P < 0.05) higher infestations of poppy compared with arrowleaf clover,
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biserrula cv. Mauro, French serradella/ bladder clover, lucerne/ subterranean clover and
phalaris/ subterranean clover in winter-sown treatments.
Barley grass, fumitory, annual ryegrass and wild radish were the most prevalent annual
weed species at field site 2, when evaluated at approximately 50 % flowering stage in
winter 2017. Average weed cover differed significantly (P < 0.05) depending on pasture
species in 2017 (Table 7) with the exception of barley grass. Arrowleaf clover displayed
significantly (P < 0.05) higher suppression against fumitory when compared to yellow
serradella cv. Avila and yellow serradella cv. Santorini. Arrowleaf significantly
suppressed annual ryegrass establishment when compared to bladder clover, yellow
serradella cv. Avila, yellow serradella cv. Santorini (P < 0.05). Arrowleaf clover also
offered significantly (P < 0.05) higher suppression against wild radish when compared
to biserrula cv. Mauro and yellow serradella cv. Santorini.
Table 7. Visual ratingsa of dominant annual weeds established in monoculture pasture
monocultures in 2017 at field site 2 in Wagga Wagga, NSW. Ratings were taken at 50%
flowering b of the pasture species.
Pasture species Barley
grass
Fumitory Annual ryegrass Wild
radish
Arrowleaf clover 6 9 6 2
Biserrula cv. Casbah 9 12 12 13
Bladder clover 10 13 20 6
French serradella 6 15 7 4
Gland clover 5 12 11 4
Subterranean clover 4 16 11 6
Yellow serradella cv. Avila 10 23 16 4
Yellow serradella cv. Santorini 7 21 16 8
P value < 0.05 < 0.05 < 0.05 < 0.05
LSD 11.2 10.6 10.5 4.01 a Percentage weed cover was measured on a scale of 0 to 100 where zero represents no pasture
cover and 100 represents complete and uniform ground cover. b Approximately 50% of the pasture flowering was observed in the third week of November
in 2017.
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Table 6. Visual ratings a of dominant annual weeds established in autumn and winter sown b pasture species in 2016 at field site 1 in
Wagga Wagga, NSW. Ratings were taken at 50% flowering c of the pasture species.
Pasture species Barley grass Sowthistle spp. Poppy spp.
autumn winter autumn winter autumn winter
Arrowleaf clover 0 1 6 4 1 1
Biserrula / Gland clover 6 7 14 10 9 9
Biserrula cv. Casbah 10 6 7 5 4 5
Biserrula cv. Mauro 16 16 8 7 7 12
Biserrula cv. Casbah / French serradella 4 2 11 9 11 10
Bladder clover 8 12 8 11 9 14
Bladder/ Gland clover 7 10 15 8 13 14
Cocksfoot/ Biserrula cv. Casbah - 4 - 0 - 10
French serradella 17 16 7 8 7 10
French serradella/ Bladder clover 10 19 8 9 11 6
Gland clover 6 10 16 9 9 8
Lucerne/ Subterranean clover - 17 - 5 - 3
Phalaris/ Subterranean clover - 2 - 0 - 0
Subterranean clover - 13 - 8 - 8
Yellow serradella cv. Avila 3 17 6 9 6 12
Yellow serradella cv. Avila / Gland clover 20 14 9 7 9 7
Yellow serradella cv. Santorini 19 11 12 6 11 13
P value < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
LSD* 12.4 8.9 7.5 7.1 6.9 7.3
LSD** 4.6 4.4 3.3 a Percentage weed cover was assessed on a scale of 0 to 100 where zero represents no pasture cover and 100 represents complete and uniform
ground cover. b Autumn sowing was carried out in the first week of March and winter sowing in the first week of June in 2015. c Approximately 50% of the pasture flowering was observed in the third week of October in 2016.
(-) Indicates the pasture species not suitable for autumn sowing. * Least significant difference between pasture species in each sowing date. ** Least significant difference between autumn and winter sowing dates.
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3.3.6 Effect of Pasture Biomass on Weed Suppression
Aboveground biomass of both pasture and weed species was significantly impacted by
year at both experimental sites (P < 0.05). Among pasture species, arrowleaf clover
produced the highest biomass (1940 kg DM/ha) (Figure 1A). In contrast, yellow
serradella cv. Santorini (270 kg DM/ha) and subterranean clover (340 kg DM/ha)
produced the lowest pasture biomass in 2016 and 2017, respectively (Figure 1A and 1B).
Weed biomass ranged from 210 - 930 kg DM/ha in 2016 and from 140 - 680 kg DM/ha
in 2017 (Figure 1A and1B). Arrowleaf clover had the lowest weed biomass in both years.
Interestingly, yellow serradella cv. Santorini produced limited pasture biomass in both
2016 and 2017 but also maintained significantly low weed infestation in contrast to most
other species.
Table 8. Competitive traits of pasture species as measured by normalised vegetative index
(NDVI), percentage light interception (% LI) and leaf area index (LAI) at approximately 50
% flowering of the pasture species in 2017 at field site 2 in Wagga Wagga, NSW.
Pasture species NDVI LI (%) LAI
Arrowleaf clover 0.52 10.5 1.02
Biserrula cv. Casbah 0.49 31.6 0.68
Bladder clover 0.20 40.7 0.54
French serradella 0.36 17.2 0.92
Gland clover 0.29 18.4 0.93
Subterranean clover 0.22 45.6 0.68
Yellow serradella cv. Avila 0.24 38.3 0.56
Yellow serradella cv. Santorini 0.22 23.9 0.58
P value < 0.05 < 0.05 > 0.05
LSD 0.19 20.4 0.6
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Figure 1. Mean pasture and weed biomass in various pasture species treatments assessed
at~50%flowering at Wagga Wagga, NSW: (a) dried biomass in 2016 at field site 1; (b)
total biomass in 2017 at field site 2. Weed biomass represents the composite biomass of
all weeds regardless of species. Error bars indicate the standard error of means.
3.3.7 Effect of Competitive Traits on Weed Suppression
Among pasture species, NDVI and % LI varied significantly while LAI did not differ
statistically among the pasture treatments (P > 0.05) (Table 8). Partial least squares
regression (PLSR) analysis revealed that competitive traits explained up to 30% of the
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variance associated with weed infestation in all pasture species when datasets from all
species were analysed in one model. Therefore, a separate PLSR analyses were performed
for each pasture species and proved more revealing in terms of those factors associated
with weed infestation in pasture species (Figure 2). In individual loading plots (Figures
2), of latent factors, independent variables located orthogonally near the origin
contributed little to the model, while variables located further from the origin contributed
more to weed suppression. The angle to the dependent variable determines the effect of
each independent variable. A separate PLSR was performed to examine the effect of
independent variables on weed suppression for individual species/cultivars (Figure 2),
pasture biomass had the greatest inverse effect on total weed biomass in the case of
biserrula cv. Casbah, bladder clover, French serradella, subterranean clover and yellow
serradella cv. Avila. LAI was observed to have a strong inverse effect on total weed
biomass in the case of arrowleaf clover and gland clover (Figure 2). Interestingly, pasture
biomass, NDVI and LAI had no impact on weed biomass in yellow serradella cv. Santorini
(Figure 2).
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Figure 2. Individual loading plots of PLSR analysis of pasture species crops based on a
correlation matrix of four competitive traits including pasture species biomass, percent light
interception (LI), leaf area index (LAI) and normalised vegetative index (NVDI) of pasture
legumes on weed biomass using pairwise comparison of first two weights of latent factors.
Arrowleaf clover (A), biserrula cv. Casbah (B), bladder clover (C), French serradella (D),
gland clover (E), subterranean clover (F), yellow serradella cv. Santorini (G), yellow serradella
cv. Avila (H).
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3.4 Discussion
This study attempted to quantify the potential effect of crop competitive traits on
the establishment and performance of annual pasture legumes as assessed by crop cover
and biomass production and subsequent impact on weed cover and biomass in the absence
of soil-applied pre-emergent herbicides in southeastern Australia. The results presented
here from three years of field experimentation, which included monocultures and
mixtures of a variety of annual pasture legumes in Wagga Wagga, NSW demonstrated
significant variation in the competitiveness of individual pasture legumes, resulting in
variable weed suppression among pasture species and cultivars. Arrowleaf clover and
biserrula were the most consistent and competitive annual legumes with respect to stand
establishment and consistent suppression of pasture weed species.
Both arrowleaf clover and biserrula significantly reduced light interception at the base of
the canopy and had higher leaf area indices when compared to other pasture species,
potentially contributing to a reduction in weed seed germination. This observation is
complementary to findings from a previous study that indicated both light interception
and crop biomass contributed to the weed suppressive potential of various forage legumes
against annual ryegrass (Dear et al. 2006). Therefore, competition for resources and
biomass accumulation are key mechanisms associated with weed suppression in both
arrowleaf clover and biserrula. Early biomass accumulation, high relative growth rate and
root system development compared to weeds in pasture legumes is reported to have
significant negative impact on weed establishment (Evans et al. 2002; Saito et al. 2010).
However, our field results demonstrated that weed suppression was independent of total
pasture biomass in the case of yellow serradella cv. Santorini, which produced limited
biomass over all experimental sites and years, while significantly inhibiting weed
infestation (Table 5). The reduction of weed infestation in subsequent years of
establishment in this particular case may also be attributed to the overall reduction of N
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fixation by the crop, given its low biomass, affecting the plant available N for weeds. It
is plausible that a reduction in N levels may have affected weed growth. However, as N
levels were not monitored at treatment level, further experimentation is required to
determine the effect of nutrient deficiency.
Lower weed infestations in arrowleaf clover and biserrula treatments may also be due to
the high planting densities achieved by higher number of seeds at recommended sowing
rates when compared to pasture species planted less densely (eg. subterranean clover)
contributing to higher biomass accumulation. In support of this observation previous
studies have shown that small-seeded annual clovers adversely impact weed infestation
due to higher plant densities achieved per unit area, resulting in accumulation of greater
biomass (Evans et al. 2002; Dear et al. 2006). Multivariate PLSR analysis demonstrated
that of the physical parameters measured, pasture species biomass had the greatest effect
on total weed biomass for the majority of pasture species. Laboratory bioassays
evaluating the impact of pasture crop residues on weed seedling germination and growth
have recently indicated that both yellow serradella cv. Santorini and biserrula may
suppress weeds by mechanisms other than competition for resources (unpublished data).
PLSR analysis further supported the hypothesis that weed biomass was not necessarily
correlated with pasture biomass in the case of yellow serradella cv. Santorini. Pasture
legume breeders in WA also obtained similar results with biserrula and yellow serradella
(Revell and Thomas 2004; Loi et al. 2008). Laboratory experimentation performed with
dried residues and extracts of fresh pasture legumes has indicated that phytotoxins
produced by leachates from selected hard-seeded legume residues collected from field
experimentation subsequently suppressed weed establishment when released into field
soil, in the presence of soil microbiota (Gurusinghe et al. 2018). The underlying
mechanism of weed suppression is thus potentially associated with the plant- or
microbially- produced metabolites (Weston and Mathesius 2013). Further laboratory and
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additional field experiments are underway to evaluate the prevalence of key phytotoxic
metabolites associated with pasture legumes and their residues, including those produced
by both cultivars of yellow serradella, biserrula, and arrowleaf clover. In addition, the
direct impact of soil microbial communities present in the soil rhizosphere is also under
evaluation.
Various cereal or food legume and grasses have been reported to selectively inhibit weed
species in field and greenhouse experiments (Weston 1996). Visual ratings performed to
evaluate the impact of pasture species establishment on establishment and growth of weed
species suggested that dominant species differed with the exception of barley grass but
no specific selectivity was found in terms of weed suppression (Table 6 & 7).
This study also evaluated the impact of planting date on the potential of annual of annual
pasture legumes to suppress weeds. However, field observations do not support this
hypothesis (Table 4 & 5). The effect of early establishment of pasture species by autumn
sowing, either in mixtures or in monoculture, was not significant when compared to
winter sowing, with the exception of yellow serradella cv. Santorini in 2015 and 2016.
Generally, autumn sowing of monocultures and mixed swards produced somewhat
improved pasture cover in 2015 but this effect was not significant in 2016 since
successive stands occurred through self-regeneration and weather condition might have
impact the establishment in autumn. This could also be attributed to climatic conditions
in the growing season in 2016. Previous studies have shown that early pasture biomass
and root system establishment contributed to weed suppression by competition for
resources and/or the release of secondary metabolites by the pasture species in the
rhizosphere (López-Castañeda et al. 1996; Bertholdsson 2005; Navas and Moreau-
Richard 2005). The disparity between our results and those of others may potentially be
associated with the limited rainfall recently experienced in the autumn months in the
Riverina, negatively impacting pasture establishment. As such, the potential advantage of
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early growth and ground cover offered by early sowing of some rapidly maturing species
may have been masked by limited soil moisture.
Relative hard-seededness or the rate of hard seed breakdown has been shown to differ
regionally for various legumes based on weather patterns (Loi et al. 1999). Arrowleaf
clover, biserrula cv. Casbah and bladder clover have previously demonstrated satisfactory
regeneration ability in southeastern climatic conditions in contrast to Western Australian
conditions (Hackney et al. 2012). These species require moderate temperature and soil
moisture in early spring for optimum establishment and herbage production. Overall, we
noted a significant reduction in pasture establishment and biomass production in all
pasture species in 2017, in contrast to 2015 and 2016, most likely as a consequence of
prolonged cool and dry weather conditions coupled with a higher number of frost days
early in the growing season in 2017. Poor germination of some annual legumes,
particularly hardseeded species including yellow serradella, has previously been
associated with low soil moisture availability (Taylor et al. 1991; Norman et al. 1998). It
is possible that the poor regeneration of biserrula cv. Mauro, French serradella and gland
clover over consecutive growing seasons may be due to hardseededness, thereby
contributing to poor subsequent establishment. The presence of grazing livestock in
established pastures may enhance seed breakdown and consequently, improve pasture
establishment through mechanical breakdown of seed pods (Phelan et al. 2015). It is also
likely that high relative humidity and a prolonged spring growing season could hasten the
breakdown of hardseeded annual legumes in Wagga Wagga region. Understanding the
seed breakdown patterns of various pasture legumes through additional future research
may facilitate their improved establishment and persistence, thereby contributing to
enhanced weed management over time.
Our results indicated that mixtures of pastures at two sowing dates provided no particular
advantage over the same species established as monocultures with respect to pasture
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establishment or weed suppression. These findings could potentially be attributed to
competition between co-existing species in mixed stands for similar ecological niches and
resources, including soil moisture and nutrients, thereby impacting overall establishment.
Our findings are in agreement with those of Conning et al. (2011), who observed that
increasing densities of subterranean clover had an adverse impact on biomass
accumulation in the co-culture species biserrula, potentially through occupation of the
same ecological niche as subterranean clover. Conning et al. further recommended a
seeding rate of 7:1 (biserrula to subterranean clover) for a balanced or equivalent stand.
Previous reports also highlighted species incompatibilities when establishing mixtures of
temperate grasses and legumes, due to poor early growth vigour and low persistence of
legumes under southeastern climatic conditions (Lodge 2010). This may also be a
consequence of different temperature and soil moisture optima for the growth of pasture
species utilised in mixtures (Haynes 1980). Pasture monocultures and mixtures sown in
autumn and winter of 2015 while established successfully, failed to regenerate in 2017.
The poor regeneration was likely due to the dry conditions experienced during the 2017
growing season (Table 3), delaying hard seed breakdown and reducing plant available
moisture (Loi et al. 2005). As the 2015 and 2016 growing seasons provided clear
indications on comparable weed suppressive potential between pasture mixtures and their
respective monocultures, the 2017 sowing only included monocultures of annual pasture
legumes.
Physiological and morphological growth differences were observed between pasture
species sown in binary mixtures, which may have contributed to poor suppression of
weeds over time. As an example, yellow serradella cv. Avila, a mid- to late-maturing
species, produced limited ground cover, while gland clover, an early-maturing species,
established a dense ground cover early in the growing season. Differences in a pasture’s
seasonal growth patterns, time of emergence and establishment strongly impact the
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competitive ability of certain species utilised in mixed swards. Therefore, pasture
compatibility based on establishment, persistence and sowing rate must be carefully
evaluated before selection of species for use in mixed swards (Conning et al. 2011).
Pasture mixtures evaluated in this study were selected based on common species and
cultivar choices available to livestock producers in southern NSW, and crop-weed
interactions were evaluated at approximately 50 % flowering of the crop.
Additional knowledge of both weed and pasture species emergence, phenology and
ecological niche occupied over several growth seasons are required to better exploit the
competitiveness of individual pasture species for optimal suppression of local weed
populations.
3.5 Conclusions
Results of three years of field experimentation performed at two field sites in
Wagga Wagga, NSW with a variety of annual pasture legumes demonstrated that
arrowleaf clover and biserrula were the most consistent and competitive annual legumes
with respect to stand establishment, yearly regeneration and consistent suppression of
common pasture weed species. Recent laboratory results also point to the conclusion that
weed suppressive potential of yellow serradella cv. Santorini may be associated with the
production of phytotoxic secondary metabolites, either de novo or through subsequent
microbial transformation. Annual pasture legumes may offer a cost-effective opportunity
to improve existing weed management strategies through selection of competitive pasture
species and cultivars along with modifications to agronomic practices such as planting
date, thereby providing improved management of annual pasture weeds. While
competition for resources was identified as a key factor contributing to weed suppression,
additional research is now underway to confirm the mechanisms driving enhanced weed
suppression by pasture legumes. In general, the rapid establishment of pasture species, as
well as optimal production of biomass, contributes to weed suppression. In addition,
132
phytochemicals produced by legumes and their residues in the presence of a viable soil
microbial community are likely drivers of weed suppression by such legumes as yellow
serradella. Future research will address the role of canopy architecture, competition for
resources and allelopathic tendencies in the selection of Australian pasture species for
sustainable weed management.
3.6 Acknowledgments
The authors declare no conflict of interest in submitting the manuscript. The
authors also acknowledge Belinda Hackney for reviewing the manuscript and funding
support from Meat and Livestock Australia (MLA) (Grant: B.WEE.0146) for establishing
and managing research trials and the Australian Centre for International Agricultural
Research (ACIAR) for sponsoring a student fellowship for the PhD project. The authors
also acknowledge the technical support provided by Graeme Heath and Simon Flinn in
collecting data at the field sites.
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140
3.8 Supplementary Materials
Figure 1. Mean pasture and weed cover in various pasture mixtures assessed at physiological
maturity in 2015 at the Graham Centre field site Wagga Wagga NSW. Error bars indicate the
standard error of mean.
-20
0
20
40
60
80
100
% P
astu
re c
over
Pasture species
141
Figure 2. Mean pasture and weed cover in various pasture mixtures assessed at physiological
maturity in 2016 at the Graham Centre field site Wagga Wagga NSW. Error bars indicate the
standard error of mean.
0
20
40
60
80
100
% P
astu
re c
over
Pasture species
Tables S1. Examples of legume inoculant groups used in Australian agriculture and their
respective rhizobia. Currently, 39 different legume inoculants are manufactured in Australia,
covering about 100 legume species (Adopted from GRDC webpage and modified). Rhizobia Commercial inoculant group Legumes nodulated Sinorhizobium spp. AL Lucerne, strand and disc
medic AM All other annual medics Rhizobium leguminosarum
bv. trifolii B Perennial clovers
C Most annual clovers Bradyrhizobium spp. G1 Lupin, serradella S1 Serradella, lupin Mesorhizobium ciceri N Chickpea Rhizobium leguminosarum
bv. viciae E2 Field pea and vetch
F2 Faba bean and lentil Bradyrhizobium japonicum H Soybean Bradyrhizobium spp. I Cowpea, mungbean
142
Chapter 4
Metabolomic Approaches for the Identification of Flavonoids
Associated with Weed Suppression in Selected Hardseeded Annual
Pasture Legumes
Results from the field study presented in Chapter 3 provided strong evidence for
the in-field weed suppressive potential of certain annual pasture legumes i. e. arrowleaf
clover and biserrula cultivars through competition for resources (Latif et al., 2019), and
suggested that weed suppression observed in selected pasture i.e. yellow serradella
species was likely due to chemical interference. To investigate this hypothesis,
comprehensive targeted and non-targeted metabolic profiling resulted in the identification
of key metabolites associated with weed suppression of selected annual pasture legumes.
The findings from these experiments are presented and discussed in this chapter. In
addition, the phytotoxic potential of methanolic extracts prepared from various plant
tissues as well as the dried residues of pasture legumes was demonstrated in a series of
laboratory bioassays. Non-targeted and targeted metabolic profiling of above and below
ground pasture tissue and rhizosphere soils revealed an abundance of bioactive flavonoids
and their precursors in those legumes demonstrating high levels of weed suppression in
the field, including yellow serradella and biserrula. Stepwise multiple linear regression
analyses performed with in-field assessment of weed suppression and weed biomass and
data from non-targeted metabolic profiling demonstrated the strong association of several
flavonoids, including quercetin, isoquercetin, kaempferol and kaempferol-7-O-glucoside,
with weed suppression.
Results from these experiments was published as a journal article in Plant and
Soil.
143
Latif, S., Gurusinghe, S., Weston, P. A., Quinn, J. C., Piltz, J. W., & Weston, L. A.
(2019). Metabolomic approaches for the identification of flavonoids associated with weed
suppression in selected hardseeded annual pasture legumes. Plant and Soil.
doi;10.1007/s11104-019-04225-4
I undertook the main body of work presented in this chapter which was discussed
by my committee members and I conducted all experimentation following on from
committee input. Prof. Leslie A Weston and Dr. Saliya Gurusinghe assisted in
experimental design and analyses for laboratory bioassays and metabolic profiling, and
were instrumental in data presentation and discussion of results. Dr Saliya Gurusinghe
also provided technical support in performing laboratory bioassays and assisted with data
analysis. Dr. Paul A. Weston assisted in data acquisition, data processing, and
bioinformatics. I performed all data analytics, and prepared the first and second drafts of
the manuscript. Drs. Saliya Gurusinghe and Leslie Weston edited these and subsequent
versions and final versions were edited by Drs. Paul Weston and Jane Quinn. All authors
contributed in preparation and editing of the final manuscript before publication. Other
contributors to this study are described in the acknowledgements.
144
Metabolomic Approaches for the Identification of Flavonoids
Associated with Weed Suppression in Selected Hardseeded
Annual Pasture Legumes
Sajid Latif A,B, Saliya Gurusinghe A, Paul A. Weston A,C, Jane C. Quinn A,B, John Piltz D,
Leslie A Weston A,C
A Graham Centre for Agricultural Innovation (NSW Department of Primary Industries),
Locked Bag 588, Wagga Wagga, NSW 2678
B School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678
C School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678
D NSW Department of Primary Industries, Agricultural Institute, Wagga Wagga, NSW
2650
Abstract
Background and aims
Incorporation of competitive pasture legumes in conservation agricultural systems
provides a non-chemical alternative towards integrated and sustainable weed
management strategies. While the in-field weed suppressive potential of annual pasture
legumes has been previously described, the mechanism of interference with weeds has
not been clearly elucidated. We, therefore, aimed to delineate the role of secondary
metabolites synthesized and released by pasture legumes through a series of studies to: 1)
characterize key metabolites present in plant tissues, residue and the rhizosphere and 2)
correlate their presence with weed suppressive properties.
145
Methods
Extensive field analysis of competitive pasture legumes was performed from 2015 to
2017 in southern Australia. Based on these results, in vitro experimentation was
performed to assess the phytotoxic potential of selected annual pasture legumes through
targeted and non-targeted metabolic profiling to evaluate the abundance of key
metabolites using UHPLC QTOF-MS. Further, those metabolites strongly correlated with
weed suppression and phytotoxicity were predicted by chemometric analyses and their
concentration evaluated in field soils collected from the same legume site.
Results
Field experimentation demonstrated consistent weed suppressive potential of certain
annual pasture legumes, particularly arrowleaf clover (Trifolium vesiculosum Savi.),
biserrula (Biserrula pelecinus L.) and yellow serradella (Ornithopus compressus L.).
Methanolic extracts and dried residues of biserrula and yellow serradella, but not
arrowleaf clover, exhibited marked phytotoxicity in a series of laboratory experiments.
Metabolic profiling revealed that both foliar tissues and rhizosphere soils of annual
pasture legumes possessed a high abundance of various flavonoids and their precursors.
Chemometric analyses suggested the strong and positive association of quercetin,
isoquercetin, kaempferol, and kaempferol-7-O-glucoside with phytotoxicity and weed
suppression under field conditions. Specifically, the abundance of quercetin and
kaempferol was significantly higher in soils collected from established stands of biserrula
and yellow serradella in contrast to arrowleaf, gland and subterranean clover.
Conclusion
Both field and laboratory experimentation provided evidence for the role of certain annual
legume-produced flavonoids in weed suppression in southern Australia and further
insight into their localization and release in the soil rhizosphere.
146
Keywords: annual hard-seeded pasture legumes, phytotoxicity, root exudation, weed
suppression, chemometric analysis, metabolic profiling, flavonoids
4.1 Introduction
Weeds are considered a global threat to primary production industries and cost
over $4 billion per year due to yield losses and costs associated with weed control in
Australia (Patel et al. 2016). Recently, an increased incidence of herbicide resistance has
highlighted the need for alternative weed management strategies for more sustainable
production systems (Powles et al. 2010). It has been suggested that bio-herbicidal or
phytotoxic plant secondary metabolites (PSMs) may offer supplemental weed
management opportunities (Bhadoria 2011; Dayan et al. 2014).
Annual pasture legumes are an integral part of mixed farming systems as they provide
quality nutrition for grazing livestock, prevent erosion, and can improve soil physical
properties and fertility (Carlsen et al. 2008b; Keating 1999). As a component of non-
chemical weed control strategies, pasture legumes have also been shown to effectively
suppress weeds when used in rotational cropping systems (Clayton et al. 1997;
Macfarlane et al. 1982). Recent introductions of various annual pasture legumes in
Australia include Biserrula pelecinus L. (biserrula), Ornithopus sativus Brot. (French
serradella), Ornithopus compressus L. (yellow serradella), Trifolium glanduliferum
Boiss. (gland clover), Trifolium spumosum L. (bladder clover) and Trifolium vesiculosum
Savi. (arrowleaf clover), all of which originated in temperate to Mediterranean Europe
and Northern Africa (Loi et al. 2005). Australian research has identified significant weed
suppressive potential of several newly introduced annual pasture legumes (Latif et al.
2019; Nichols et al. 2012; Howieson et al. 2000). However, the exact mechanisms of
crop-weed interference have not yet been clearly elucidated, but were suggested to
include competition for resources, suppression of light at the soil surface due to closed
147
canopy formation and chemical inference through the release of secondary metabolites in
soil.
Previous research suggests that phytotoxicity elicited by crops and their residues
could play an important role in crop-weed interference, and supplement existing
integrated weed management practices (Kim and Shin, 2003). Selection for species and/or
cultivars possessing strong phytotoxic potential resulting from the release of
allelochemicals into the rhizosphere, either through the degradation of crop residues
(Jabran 2017; Mwangi et al. 2015; Teasdale et al. 2012) or through root exudation of
living plants (Carlsen et al. 2012; Uddin et al. 2010) has presented an alternative tool to
growers for managing newly establishing weeds. However, the clear demonstration of
biologically relevant concentrations of such bio-herbicides or allelochemicals in soil and
their subsequent interactions with soil microorganisms requires additional evaluation
(Duke 2015; Hiradate et al. 2010).
Phytotoxic plant secondary products are typically synthesized and/or localized in
diverse plant parts and/or organelles (Weston et al. 2003). Determination of the specific
mode of action of plant-produced phytotoxins can be challenging due to their structural
diversity and multiple target sites. In addition, the relative concentration of phytotoxins
after their release into the soil is impacted by both biotic and abiotic factors including soil
moisture availability, plant phenology, soil nutrient dynamics and microbial interactions
(Einhellig 1996; Mahmood et al. 2013; Nakabayashi et al. 2015). Numerous studies have
shown that suppression observed under controlled conditions may potentially be masked
or modified under field conditions due to the complexity of rhizosphere interactions
(Duke 2015; Latif et al. 2017).
Clearly, it has been difficult to separate plant growth suppression caused by
allelopathy or chemical interference from competition for resources under field
148
conditions. Laboratory and glasshouse assays have been successfully designed to
differentiate resource competition from chemical interference (Hoffman et al. 1996) but
generally, do not attempt to mimic field conditions (Seal et al. 2008). The use of in vitro
seedling growth assays with dried crop residues or fresh plant extracts offer several
advantages for evaluation of biological activity including demonstrated sensitivity under
controlled conditions, scalability and the ability to screen for bioactive molecules in a
high throughput setting (Munzuroglu et al. 2002; Wang et al. 2001; Weston et al. 1989).
However, the comprehensive evaluation of weed suppression under both field and
laboratory conditions is thus essential from a mechanistic standpoint to differentiate
phytotoxicity from other unrelated interactions (Inderjit et al. 2003; Inderjit et al. 2000).
One family of secondary plant products, the flavonoids, is frequently noted in
reference to regulation of plant defences against competing organisms, particularly in
legumes (Mathesius 2018; Weston and Mathesius 2013). Flavonoids are ubiquitous
across the plant kingdom and are known to perform a range of functions, which include
the regulation of symbiotic or pathogenic plant-microbial signaling as well as associated
plant-plant interactions. Some pasture legumes have been noted to produce an abundance
of flavonoids and several in white clover and other legume crops have been identified as
potential phytotoxins (Carlsen et al. 2008a; Carlsen et al. 2012; Gomaa et al. 2015a).
Recent research on the biosynthetic pathway of flavonoids has been performed,
particularly with respect to their use in breeding for strategic incorporation for enhanced
resistance to pests and pathogens (Mathesius 2018).
Traditionally, bioassay-guided fractionation techniques have been employed for the
isolation and identification of such bioactive metabolites, but this approach can be time-
consuming, costly, and typically generates low yields of purified compounds for
structural elucidation (Duke 2015; Weston et al. 1987). More recently, metabolomics has
been employed, not only as a systematic approach for studying plants’ chemical
149
fingerprints (Breitling et al. 2013), but also for identification of plant secondary
metabolites (PSMs) associated with plant defence or stress (Kim et al. 2010; Weston et
al. 2015). Recent advances in high precision analytical instrumentation, particularly mass
spectrometry, have enabled accurate and robust methodologies for screening, annotation
and identification of bioactive PSMs. Liquid chromatography systems interfaced with
time-of-flight (TOF) mass spectrometers have enabled determination of mass values to ±
5 ppm, allowing more precise prediction of molecular formulae (Deng et al. 2013; Liu et
al. 2017; Zhu et al. 2013).
In Australia, recent field experimentation on a diverse collection of annual pasture
legumes revealed strong weed suppressive potential in several unrelated species (Latif et
al. 2019) and suggested that serradella and biserrula pastures, in particular, exhibited
weed suppression through mechanisms other than competition for resources, as weed
suppression was not correlated with crop biomass (Latif et al. 2019). Currently, the
putative allelochemicals associated with weed suppression in these annual pasture
legumes are unknown. Therefore, the aims of our subsequent experimentation with this
diverse collection of pasture legumes were to (a) evaluate the phytotoxic potential of soil-
incorporated dried plant residues and fresh tissue extracts of selected annual pasture
legumes, (b) characterize the relative abundance of plant-produced metabolites present in
fresh tissue extracts through non-targeted metabolic profiling, (c) identify key
metabolites, including flavonoids, associated with weed suppression through correlative
chemometric analyses and (d) quantify identified metabolites associated with weed
suppression in both above- and below-ground plant tissues and associated rhizosphere
soils.
150
4.2 Materials and Methods
4.2.1 Field Experimentation
Field experimentation with monocultures of selected pasture legumes was
performed from 2015-2017 in Wagga Wagga, NSW, Australia at the Charles Sturt
University research farm (35.04°S, 147.36 °E). Experiments were arranged in a series of
randomized complete block designs with five replicates per treatment. Individual pasture
legumes were established in bare previously harrowed plots by direct-seeding with a drill
adapted for small-seeded legumes in late May-early June of each year, with plots
measuring approximately 4 m in width × 20 m in length (Table 1). The soil type was
characterized as a fine, red, sodosol (Isbell 1996) with a pH of 6.2. Glyphosate was
applied at a rate of 1.6 L/ha before sowing to remove any residual weed infestation and
plots were managed without any grazing or mowing management throughout the grazing
season (Latif et al. 2019b). Above- and below-ground plant tissue samples (shoots and
roots) along with rhizosphere soils at a 5-10 cm depth were collected from established
plots in the third week of October in each year when pasture plots exhibited > 50%
flowering of all pasture species. In late October, two representative sub-samples of above-
ground biomass (leaf and stem tissues) were also collected from each plot (n=2 × 5
replicates) in all years using 0.25 m2 quadrants to estimate total weed biomass as direct
measure of weed suppression (Jannink et al. 2000). Plant matter was subsequently sorted
into pasture crops and weeds before drying at 60 °C for 96 hours. Fresh plant material
was also collected at this time from each replicate in a similar manner and separated into
the various tissues including leaf, stem, inflorescence and root tissues. All plant material
and rhizosphere soils were immediately transported to the laboratory where soil samples
were screened to remove remaining root tissue or plant residue and stored at -20 °C until
subsequent processing.
151
Table 1. Pasture species, common name, cultivars, seasonal maturity, growth habit, seeding rates, seed weights and inoculant used in field trials in
2015 to 2017 in Wagga Wagga, NSW, Australia (Adapted from Latif et al. (2019).
Scientific name Common name Cultivar Seasonal
maturity
Growth habit Seeding
rate*
(kg ha-1)
Seed
weight**
(mg)
Inoculant**
*
Trifolium vesiculosum Savi. Arrowleaf clover Cefalu Early Erect to semi-
erect
8 1.2 C
(WSM1325)
Biserrula pelecinus L. Biserrula Casbah Early-mid Erect to semi-
erect
8 1.2 WSM 1497
Mauro Mid-late Erect to semi-
erect
8 1.2 WSM 1497
Trifolium spumosum L. Bladder clover Bartolo Early-mid Semi-erect 10 2.6 C
Ornithopus sativus Brot. French serradella Margarita Mid Erect to semi-
erect
10 0.9 S (WSM471)
Trifolium glanduliferum
Boiss.
Gland clover Prima Early Erect to semi-
erect
8 0.7 C
Trifolium subterraneum L. Subterranean
clover
Seaton Park Early-mid Prostrate 10 6.7 C
Ornithopus compressus L. Yellow serradella Santorini Early-mid Semi-erect 10 1.2 S
*Seeding rate refers to seed not pods.
**Seed weight is based on published suggested rates.
** Letters C, S and WSM refer to the specific group or strain of rhizobia used in inoculants for various legumes.
152
4.2.2 A Modified Parker Assay for Assessment of Plant Residue Phytotoxicity in
Annual Pasture Legumes
Above-ground plant material collected from field trials was dehydrated at 40 °C
for 96 hours in a drying oven. The dried residues were cut into pieces of approximately 1
cm2 in size and stored at 4 °C until further use. A modified Parker assay was performed
similar to Weston et al. (1989) using 125 g of non-cropped field soil collected from the
same field site (fine, red sodosol, pH 5.8) and combined with washed river sand (1:1 v/v)
for improved moisture permeation, and placed in sterile square Petri dishes (100 × 100 ×
15 mm; Interpath, VIC). Crop residues were subsequently and uniformly incorporated
into the soil: sand mixture at a rate of either 5 or 10 mg∙cm-3. The control treatment
consisted of an equivalent mass of inert cellulose fibres, which were incorporated into the
soil media. Sterile distilled water (50 mL) was added to the soil to moisten the entire
soil/residue mixture. The soil surface was then covered with one layer of sterile filter
paper (Whatman No.1), and 15 seeds of each of two weed indicator species, Lepidium
sativum L. and Lolium rigidum Guad., were placed in two horizontal rows, which were
two cm apart, on the dish. These species were found to be suitable indicators after testing
various other weed species, primarily due to their sensitivity, rapid germination and
uniform growth patterns following germination. The Petri dish was covered and secured
with adhesive tape sealed with Parafilm®, and placed vertically in an incubator for 96
hours at 25 °C in dark conditions to encourage gravitropic rooting. Each treatment was
replicated four times and the experiment was duplicated over time. Following incubation,
the length of each radicle was recorded. The percentage inhibition of radicle length was
calculated relative to the control using the following formula;
% 𝐈𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 = [𝟏 − (𝐫𝐚𝐝𝐢𝐜𝐥𝐞 𝐥𝐞𝐧𝐠𝐭𝐡 𝐨𝐟 𝐭𝐫𝐞𝐚𝐭𝐦𝐞𝐧𝐭
𝐫𝐚𝐝𝐢𝐜𝐥𝐞 𝐥𝐞𝐧𝐠𝐭𝐡 𝐨𝐟 𝐜𝐨𝐧𝐭𝐫𝐨𝐥)] × 𝟏𝟎𝟎
153
4.2.3 Extraction Procedure
Freshly collected plant tissue was extracted using a pressurized solvent extraction
system (E-916 Büchi, Switzerland) in HPLC- grade methanol (Honeywell Burdick &
Jackson, Muskegon, USA) as solvent (Dayan et al. 2010). This system operated with
nitrogen gas at high pressure to facilitate rapid infiltration of the solvent into plant cells
while limiting oxidation of secondary metabolites. Plant material (5 g) was mixed
thoroughly with quartz sand, (particle size, 0.3-0.9 mm) (Büchi, Switzerland, 034925)
and placed in 10 mL extraction cells. The extraction was performed in two consecutive
cycles conducted under the following conditions: solvent: 100% methanol; temperature:
35 °C; pressure: 1400 psi. Samples were dried using a rotary evaporator (Multivapour P-
6, Büchi, Switzerland) at 35 °C and reconstituted to 20 mL in methanol and stored in the
dark at -20 °C for use in bioassays and UHPLC-QTOF-MS without any further dilution.
Rhizosphere soils (10 g) were extracted by placing them in 250 mL conical flasks with
50 mL methanol in a rotary shaker at 125 rpm for 16 hours at room temperature. The soil
suspension was filtered through Whatman No 1. filter paper. All extracts including those
of rhizosphere soils were filtered through 0.20 µm polytetrafluoroethylene (PTFE)
syringe filters (Captiva Econofilter, Agilent Technologies, Australia) and stored at -20°C
until UHPLC-QTOF-MS analysis without any further dilution. Recovery of key
phytotoxic flavonoids after spiking tissue and soil samples with appropriate flavonoid
standards suggested recovery to be generally greater than 80%.
4.2.4 Bioassay of Pasture Legume Extracts for Phytotoxicity
The phytotoxic potential of methanolic extracts prepared from both fresh leaf and
root tissues of pasture species were evaluated against both a monocot and a dicot weed
seed indicator, L. rigidum. (annual ryegrass) and L. sativum (garden cress) as described
by Weston et al. (1987) with minor modifications. Methanolic extracts (0.5 mL) of
various plant parts and dilutions were evenly spread over filter paper (Whatman No. 1)
154
in glass Petri dishes (30 mm diameter) and methanol was subsequently allowed to
evaporate and replaced with 0.5 mL water resulting in final extract concentrations ranging
from 0.0625 to 2 ppm in six doubling dilutions. Ten seeds of each indicator species were
placed on moistened filter paper. Petri dishes were placed in a humidified germination
box in completely randomized design for incubation at 25°C in dark conditions. The
radicle and hypocotyl lengths were measured at 72 hours for L. sativum seedlings and 96
hours for L. rigidum seedlings. All experiments were duplicated with three replicates each
time. The percentage inhibition of radicle and hypocotyl length for each treatment was
calculated relative to control using the following formula (Pandya et al. 1978);
% 𝐈𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 = [𝟏 − (𝐫𝐞𝐬𝐩𝐨𝐧𝐬𝐞 𝐨𝐟 𝐭𝐫𝐞𝐚𝐭𝐦𝐞𝐧𝐭
𝐫𝐞𝐬𝐩𝐨𝐧𝐬𝐞 𝐨𝐟 𝐜𝐨𝐧𝐭𝐫𝐨𝐥)] × 𝟏𝟎𝟎
Linear quadratic regression (first order) was fitted separately for all extracts to
characterize the trends between concentration and percentage inhibition. Inhibitory
concentrations of 50% seedling hypocotyl or radicle length (IC50) were then calculated
by linear regression analysis. Commercial standards of bioactive flavonoids were also
subjected to similar bioassay for confirmation of phytotoxicity. Photos of treated and
untreated seedlings were obtained using a M205 FA stereomicroscope (Leica
Microsystem).
4.2.5 UHPLC-QTOF-MS Analysis
Non-targeted and targeted metabolic profiling of plant tissues and rhizosphere
soils were performed using an Agilent 1290 Infinity UHPLC system equipped with a
quaternary pump, diode array detector (DAD), degasser, temperature controlled column
(25 °C) and cooled auto-sampler compartments (4 °C) which were coupled to an Agilent
6530 quadrupole time-of-flight (QTOF) mass spectrometer (MS) with an Agilent Dual
Jet Stream ionisation source (Agilent Technologies, Australia). Full scan mass spectra
were acquired over a range of 100-1700 mz-1 at a rate of two spectra/second using both
155
positive and negative ion modes. Chromatographic separation was performed in two
experimental runs using a reverse-phase C18 Poroshell column (2.1 × 100 mm, 2.7 μm
particle size) (Agilent Technologies, Santa Clara, CA, USA) preceded by a C18 guard
column (2.1 × 12.5 mm, 5 μm particle size) (Agilent Technologies, CA, USA) and
subsequently to a Kinetex HILIC (2.1 × 50 mm, 2.6 µm particle size) (Phenomenex,
Torrance, CA USA) column preceded by a HILIC guard column (2.1 × 10 mm, 2.6 µm
particle size ). The flow rate of the mobile phase was 0.3 mL min-1. The columns were
equilibrated for 40 min prior to analysis. Reverse-phase separation was obtained using a
gradient of solvent A [water (Milli-Q, TKA-GenPure) + 0.1% formic acid (LC–MS grade,
LiChropur®, 98–100%, Sigma-Aldrich, USA) and solvent B [95 % HPLC-grade
acetonitrile (RCI Labscan, Thailand) + 0.1% formic acid]. The solvent gradient was as
follows: 5-100% B over 0-17 min, 100-5 % B from 17-29 minutes for C18
chromatography. The DAD monitored absorbance across a range of wavelengths from
210 nm to 635 nm. Separation with the HILIC column was obtained using a gradient of
solvent A (100 % water with 10 mM ammonium acetate and 0.2 % formic acid) and
solvent B (100 % acetonitrile). The solvent gradient was as follows: 95 % B from 0-0.5
min, 95-35% B over 0.5-12.5 min, 35 % B for 12.5-13 min, 35-95% over 13-14 min and
re-equilibration at 95% B from14-24 min. The DAD monitored absorbance across a range
of wavelengths from 190 nm to 640 nm The QTOF-MS was calibrated in positive and
subsequently, in negative ion mode with nebulizer gas set at 35 psi, capillary voltage at
3500 V and fragmentor voltage at 135 V. Injection volume was 10 µL for each sample.
Nitrogen was used as the drying gas at 250 °C at a flow rate of 9 L min-1. MS-MS spectra
were obtained using Auto MS/MS mode at three collision energies (10, 20 and 40 eV).
Quantification of associated compounds was also performed using UHPLC-QTOF-MS.
Analytical standards of quercetin, isoquercetin, kaempferol and kaempferol-7-O-
glucoside were purchased from Sigma Aldrich (St. Louis, Missouri, USA).
156
4.2.6 Data Analysis
An analysis of variance (ANOVA) was performed to determine the effect of year
and pasture crop on ground cover and biomass of crops and weeds using the GenStat
statistical software package (18th Edition, VSN International Ltd, Hertfordshire, United
Kingdom). The fixed model included year and pasture species while the random effect
was block number. A matrix of molecular features characterized by mass to charge ratio
(m/z) and retention time (RT) was generated using MassHunter Workstation Qualitative
software version B07.00, MassHunter Profinder (version B.08.00), Mass Profiler
Professional (MPP version 14.5) and Personal Compound Database Library (PCDL)
(Agilent Technologies, CA, USA). The parameters for molecular feature extraction and
peak binning /alignment using Profinder were as follows: peak height ≥ 10000 counts,
compound ion count threshold two or more ions, compound alignment tolerances were
0.00% + 0.15 minutes for RT and 20.00 ppm + 2.00 mDa for mass. The extracted ion
chromatogram was smoothed with Gaussian smoothing before integration. The data files
were converted to compound exchange file (.cef) format and visualized and analyzed in
MPP using multivariate analysis including principal component analysis. Molecular
features were required to be present in a minimum of three out of five replicates to be
included in the analysis. Identification was performed comparing molecular entities with
the METLIN metabolomics database (version B 07.00, Agilent Technologies, CA, USA)
and, when possible, confirmed using available standards based on accurate mass, RT and
mass spectra.
Chemometric analyses were performed to detect associations between relative
abundance of each molecular feature observed in the positive ion mass spectra of both
C18 and HILIC chromatography of leaf tissue samples with weed suppression, as
described by Weston et al. (2019). Stepwise linear regression was performed using SPSS
(IBM SPSS Statistics v. 20, 2015, IBM Corporation, Sydney, Australia) with molecular
157
features as the independent variable and weed biomass (measured in field plots) as the
dependent variable. Redundant accurate masses and database identifiers were removed
before analysis. Significance levels for molecular features (compounds) entering and
leaving the model were P ≤ 0.5 and P ≤ 0.1, respectively. A series of models with an
increasing number of molecular features as independent variables were generated until a
substantial increase in variance was explained from the observed data. A total of six
models were generated explaining up to 60 % of the variance in the data set with six
predictor variables associated with activity or phytotoxicity in this case.
4.3 Results
4.3.1 Field Experimentation for Assessment of Weed Suppression by Annual
Pasture Legumes
Weed suppressive potential of annual pasture legumes was assessed based on the
accumulated weed biomass obtained at physiological maturity (> 50% flowering) in field
trials in 2015-2016. Weed biomass ranged from 19.5 to 181.1 g∙m-2 and varied
significantly across pasture species (P < 0.05) (Table 2). Arrowleaf clover recorded the
lowest weed biomass (19.5 g∙m-2) while gland clover exhibited the highest at 181.1 g∙m-
2. Weed biomass in arrowleaf clover, French serradella, yellow serradella and both
cultivars of biserrula was significantly reduced compared to that in bladder clover,
subterranean clover and gland clover (P < 0.05). Weed biomass, along with other
competitive traits assessed in our trials such as leaf area index, photosynthetic efficiency,
light interception by canopy and at the soil surface, were included in the multivariate
analysis and revealed that weed suppression was not always dependent on characteristics
associated with competition for resources in pasture legumes (Latif et al. 2019a),
suggesting that other mechanisms of suppression might be in play for some pasture
legumes, such as yellow serradella and biserrula.
158
4.3.2 A Modified Parker Assay for Assessment of Phytotoxicity of Dried Residues
of Annual Pasture Legumes
Germination of both L. sativum and L. rigidum seeds was not impacted at the two
residue rates tested (5 and 10 mg∙cm-3) in comparison to the inert cellulose control, with
the exception of L. sativum indicator seeds exposed to biserrula cv. Mauro and
subterranean clover residues at 10 mg∙cm-3 (P < 0.05) (data not shown). In contrast,
radicle elongation was consistently and negatively impacted by the presence of legume
crop residues, with greater inhibition observed at the higher residue rate (Table 3).
Generally, the inhibition of radicle elongation in L. sativum was more pronounced than
in L. rigidum. The greatest inhibition against both indicator species was observed with
dried residues from both biserrula cultivars, French and yellow serradella and
subterranean clover in decreasing order of activity, followed by arrowleaf clover, gland
clover and bladder clover (Table 3).
Table 2. Averaged weed biomass collected in 2016-2017 from five replicates of
legume pasture species in Wagga Wagga, NSW, Australia field site. The values
followed by the same superscript in each column were not significantly different
(P < 0.05)
Pasture species Weed Biomass (g∙m-2)
Arrowleaf clover 19.5 ± 11.2a
Biserrula cv. Casbah 28.8 ± 21.2ab
Yellow serradella 29.7 ± 66.9ab
French serradella 33.5 ± 9.7ab
Biserrula cv. Mauro 38.9 ± 21.2ab
Bladder clover 112.1 ± 69.6c
Subterranean clover 136.5 ± 53.5c
Gland clover 181.1 ± 48.4d
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4.3.3 Bioassay of Pasture Legume Extracts for Phytotoxicity
Exposure to the methanolic extracts of foliar tissues of pasture legumes had a
significant impact on radicle and hypocotyl elongation of both indicator species.
Generally, radicle elongation was more negatively impacted than hypocotyl growth in the
dicotyledonous indicator species L. sativum, while hypocotyl growth was inhibited more
severely in the monocotyledonous indicator species L. rigidum (Table 4). Relative
inhibition of radicle and hypocotyl elongation increased with increasing concentration of
the extracts. The IC50 for radicle elongation ranged from 0.78 to 2.10 mg∙mL-1 against L.
sativum and 0.62 to 3.61 mg∙mL-1 against L. rigidum seedlings when considered over all
species extracts. Extracts of biserrula cv. Casbah exhibited greatest inhibitory activity on
radicle elongation of L. sativum seedlings followed by subterranean clover. In contrast,
yellow serradella showed the greatest inhibitory activity on radicle elongation of L.
rigidum followed by subterranean clover. Interestingly, biserrula extracts were selective
Table 3. Impact of soil incorporated dried residues of annual pasture legumes at two
rates of incorporation on radicle elongation of L. sativum and L. rigidum. Mean percent
inhibition of radicle length relative to the inert control are presented with standard error
of the mean. Values followed by the same superscript in each column are not
significantly different (P < 0.05).
Pasture species L. sativum (%) L. rigidum (%)
5 mg∙cm-3 10 mg∙cm-3 5 mg∙cm-3 10 mg∙cm-3
Biserrula cv.
Casbah
76.3 ± 4.0a 96.4 ± 0.5a 68.3 ± 5.1a 75.6 ± 7.2a
Biserrula cv. Mauro 76.0 ± 4.4a 89.5 ± 0.9a 71.1 ± 6.8a 74.8 ± 6.5a
French serradella 58.8 ± 7.3a 72.5 ± 3.7b 21.3 ± 4.3b 51.9 ± 8.8ab
Subterranean
clover
57.9 ± 9.6a 89.1 ± 3.9a 47.6 ± 4.7ab 45.9 ± 18.3ab
Yellow serradella 48.3 ± 7.0ab 84.1 ± 4.3b 26.6 ± 8.8b 48.1 ± 7.29ab
Arrowleaf clover 42.2 ± 17.6ab 82.3 ± 4.7b 22.8 ± 11.3b 58.9 ± 11.3ab
Gland clover 36.0 ± 12.2b 35.7 ± 7.6b 37.9 ± 2.9ab 35.7 ± 8.6ab
Bladder clover 23.1 ± 7.4b 57.6 ± 1.9b 26.2 ± 13.6b 27.5 ± 9.2b
160
in their enhanced ability to inhibit the growth of dicotyledonous species while yellow
serradella extracts were more active against monocotyledonous species. Similarly, the
inhibitory effect of foliar extracts on hypocotyl elongation followed a similar trend, with
biserrula cv. Casbah displaying improved activity against L. sativum seedlings and yellow
serradella exhibiting greatest phytotoxicity against L. rigidum seedlings. Seedlings of L.
sativum exposed to crude extracts of phytotoxic pasture legumes typically displayed
browning at the radicle terminal, with stunted growth eventually leading to significant
and generalized necrosis of the radicle (Fig. 1).
Figure 1. A) Representative images of L. sativum seedlings treated with biserrula cv.
Casbah crude extract (A and D) and untreated control (B and C) at 48 hours post-
germination. E) Image of radicle treated with crude extract of biserrula cv. Casbah
using a M205 FA stereo microscope (Leica microsystem); scale bar: 5 mm.
161
Table 4. IC50 of crude leaf and root tissue extracts (mg∙mL-1) of selected pasture
species for radicle and hypocotyl elongation.
Tissue Pasture species L. sativum (mg∙mL-1) L. rigidum (mg∙mL-1)
radicle hypocotyl radicle hypocotyl
Leaf
Arrowleaf clover 2.10 2.01 0.89 0.81
Biserrula cv. Casbah 0.78 0.85 1.18 0.81
Biserrula cv. Mauro 1.15 1.23 1.93 0.65
Bladder clover 1.73 1.59 1.05 1.96
French serradella 1.12 0.93 3.61 2.41
Gland clover 1.13 1.35 2.30 1.58
Subterranean clover 0.83 1.16 0.84 0.66
Yellow serradella 1.74 2.01 0.77 0.62
Root
Arrowleaf clover 1.94 2.29 1.21 1.29
Biserrula cv. Casbah 1.31 1.95 3.65 3.12
Biserrula cv. Mauro 1.37 1.73 2.87 3.45
Bladder clover 3.45 3.98 3.54 3.92
French serradella 2.70 3.47 1.45 2.92
Gland clover 4.41 3.19 2.63 3.96
Subterranean clover 2.47 2.29 3.01 3.54
Yellow serradella 4.21 3.93 1.37 2.69
Methanolic extracts of root tissues of annual pasture legumes followed the same
trend in phytotoxic activity as leaf tissues, however, their corresponding IC50s were higher
and ranged from 1.31 to 4.41 mg∙mL-1 for L. sativum and 1.21 and 3.92 mg∙mL-1 for the
L. rigidum seedlings (Table 4). Root extracts of both cultivars of biserrula exhibited
marked inhibitory activity against the dicotyledonous indicator while yellow serradella
root extracts were most active against monocotyledonous seedlings.
162
4.3.4 Non-Targeted Metabolic Profiling
The molecular features extracted after data normalization and noise removal were
subjected to unsupervised principal component analysis (PCA) as described in figure 2.
The total molecular features (2827) included in the analysis demonstrated clear
differences among pasture species from different genera by spatially separated clustering
of entities while molecular features from both cultivars of biserrula and the two species
of serradella were closely grouped (Fig. 2). At QTOF-MS spectral signal of threshold
15,000, the number of features contributing to differentiation was in the range of 1000 to
2500 depending upon the pasture species.
Flavonoids, anthocyanins and their glycosides accounted for the majority of
constituents among all identified major classes of secondary metabolites through
metabolic profiling, based on METLIN database comparisons and verification with
analytical standards. Differences in relative abundance of various flavonoids, their
glycosides and anthocyanin are presented in Fig. 5.
4.3.5 Chemometric Analysis
Stepwise multiple linear regression analyses yielded a model (model 6), which
included six predictor variables that explained over 60 % of the variability associated with
weed suppression (Table 5).
Table 5. Stepwise multiple linear regression models performed with weed suppression
as dependent variable and metabolic profiles acquired from foliar tissue extracts using
positive ion mode C18 and HILIC chromatography interfaced with QTOF-MS as
independent variables.
Model Summary
Model R R Square
Adjusted R
Square
Std. Error of the
Estimate
1 .338a .114 .093 2.7567
2 .492b .242 .205 2.5811
3 .585c .342 .293 2.4345
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4 .676d .456 .401 2.2408
5 .729e .531 .469 2.1091
6 .770f .593 .526 1.9919
In this model, four metabolites (quercetin, kaempferol, isoquercetin and
kaempferol-7-O-glucoside) were identified in the model; these compounds were
annotated by comparing accurate mass, RT and mass spectra with available libraries and
known standards while two additional metabolites associated with weed suppression
currently are unknown with accurate masses of 790.5411 and 459.4685, eluting at 25.22
min and 12.17 min RT, respectively (Table 6).
The phytotoxicity elicited by the four flavonoids correlated with bioactivity in the
regression model was assessed by evaluating commercial standards of those metabolites
separately in a similar in vitro phytotoxicity assay. Using an array of serial dilutions of
the commercial standards, phytotoxic activity was observed in both indicator species with
each of the standards.
Table 6. Name, empirical formula, accurate mass, m/z, RT and chromatographic
condition of compounds associated with phytotoxicity in stepwise multiple linear
regression model 6 presented in Figure 5. Identification was performed by comparing
accurate mass and RT with analytical standards using UHPLC-QTOF-MS.
Sr.
No
.
Predictor Name Formula Mass m/z Appro
x. RT
(min)
Chromat
ography
1 22 unknown C42H60O16 790.541 791.078 25.217 C18
2 33 isoquercetin C21H20O12 464.379 465.102 8.16 C18
3 49 unknown C22H34O10 458.216 459.468 12.17 HILIC
4 73 kaempferol C15H10O7 286.239 287.056 10.99 C18
5 103 quercetin C15H10O7 302.238 303.05 10.04 C18
6
139 kaempferol-7-
O-glucoside
C21H20O11 448.38 449.108 8.70 C18
164
Similarly to experimentation with methanolic extracts of foliar tissue, elongation
of radicles was impacted more than that of hypocotyls following exposure to authentic
commercial standards (Table 7). However, no selectivity in phytotoxicity was observed
for monocotyledonous and dicotyledonous indicator species. Kaempferol exhibited the
greatest activity in contrast to other analytical standards, with an IC50 of 189 µM and 223
µM for radicle elongation against L. sativum and L. rigidum, respectively. Kaempferol-
7-O-glucoside exhibited similar phytotoxicity when compared to its aglycone (Table 7).
4.3.6 Quantification of Phytotoxic Flavonoids in Plant Tissue and Rhizosphere Soil
The generation of standard curves for each commercial analyte allowed the
flavonoids identified in leaf, stem, flower, root and rhizosphere soil samples from various
pasture legumes to be successfully quantified (Fig. 3 and 4). With respect to leaf tissues,
quercetin content was significantly higher in biserrula cultivar (968 and 808 ppb)
compared to all other species except arrowleaf clover (P < 0.05) (Supplementary Table
S1). Quercetin in stem tissues was highest in biserrula cv. Casbah (135.0 ppb) and was
significantly different when compared to levels found in yellow serradella, biserrula cv.
Mauro, French serradella, gland clover and subterranean clover (P < 0.05). Interestingly,
gland clover contained greater amounts of quercetin in inflorescence tissues (566.7 ppb)
compared to all other species (P < 0.05) (Supplementary Table S1).
165
Figure 2. Three dimensional visual representation of principal component analysis
(PCA) of molecular features characterized in positive ion mode of leaf tissue collected
in 2016 using UHPLC-QTOF-MS with five replicates for each pasture species.
Components 1, 2 and 3 contributed to separation by 25.87%, 19.09% and 14.05%
respectively.
166
Figure 3. Concentration of key phytotoxic flavonoids in above ground tissues (leaf,
stem and flower) of annual pasture legumes at physiological maturity averaged over
two years (2015 and 2016). Values represent the mean of five replicates. Error bars
indicate standard error of means.
167
Table 7. Phytotoxic response of commercial standards of flavonoids quercetin, kaempferol, isoquercetin and kaempferol-7-O-glucoside for radicle
growth inhibition of various indicator species utilized in in vitro bioassay including our results on L. sativa and L. rigidum as well others reported in the
current literature.
Compound Test species Phytotoxicity References Structure
quercetin Lepidium sativum L. IC50 450-550 µM *
Lolium rigidum Gaud. IC50 400-450 µM *
Lolium perenne L. IC50 270 µM (Kalinova et al. 2009)
Lactuca sativa L. IC50 150-200 µM (Nasir et al. 2005)
Lactuca sativa L. IC50 300-650 µM (Kuang et al. 2017)
Lepidium sativum L. IC50 700-850 µM (De Martino et al. 2012)
Lepidium sativum L. 10% inhibition of growth at
300 µM
(Okada et al. 2018)
isoquercetin Lepidium sativum L. IC50 250-350 µM *
Lolium rigidum Gaud. IC50 250-350 µM (Okada et al. 2018)(Okada et al.
2018)(Okada et al. 2017)
kaempferol Lepidium sativum L. IC50 150-250 µM *
Lolium rigidum Gaud. IC50 150-300 µM *
Arabidopsis thaliana (L.)
Heynh.
65% inhibition relative to
control at 875 µM
(Bais et al. 2003)
Lycopersicon esculentum
L.
60% inhibition relative to
control at 875 µM
(Bais et al. 2003)
kaempferol-7-
O-glucoside
Lepidium sativum L. IC50 150-200 µM *
Lolium rigidum Gaud. IC50 200-300 µM *
*Refers to results from current study
168
Quantification of isoquercetin revealed that subterranean clover (79.8 ppb) and
arrowleaf clover (74.7 ppb) produced greater levels in foliar tissue in contrast to other
species, while stem tissue of subterranean (18.9 ppb) and arrowleaf clover (10.7 ppb)
contained the greatest amount of isoquercetin. Arrowleaf clover (82.9) possessed higher
isoquercetin content in its flowers compared to those of all other species (Supplementary
Table S1).
Kaempferol content in leaves was greatest in biserrula cv. Casbah (189.33) as
compared to bladder clover, French serradella, gland clover, subterranean clover and
yellow serradella (P < 0.05). In stem tissue, gland clover contained higher kaempferol
content (106.33 ppb) in contrast to other species (P < 0.05). Kaempferol content in
flowers was significantly greater in both biserrula cultivars than in all other species (P <
0.05). Kaempferol-7-O-glucoside was present in significantly higher concentration in
gland clover (1015.39 ppb), French serradella (996.8 ppb), and subterranean clover
(950.88 ppb) foliar tissues (P < 0.05). Stem tissue of subterranean clover (185.33 ppb)
contained a significantly higher concentration of kaempferol compared to the same tissue
in all other species (P < 0.05). Interestingly, the inflorescence of both species of serradella
contained significantly higher concentrations of this flavonoid when compared to that of
other species (P < 0.05).
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Figure 4. Concentration of key phytotoxic flavonoids in roots and rhizosphere soils of
annual pasture legumes at physiological maturity averaged over two years (2015 and
2016). Each value represents the mean of five replicates. Error bars indicate standard
error of means.
Quantification of these phytotoxic flavonoids in root and rhizosphere soil
collected from the field experimental site revealed that arrowleaf clover roots exhibited a
significantly higher concentration of isoquercetin, kaempferol and kaempferol-7-O-
glucoside at physiological maturity (> 50% flowering had occurred in the stand) while
roots of yellow serradella contained the highest concentration of quercetin (Fig. 4 ) in
contrast to other pasture legumes. Rhizosphere soil collected from yellow serradella sites
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contained significantly higher concentrations of quercetin, isoquercetin and kaempferol
than other pasture legumes, while rhizosphere soil from biserrula cv. Casbah contained
the highest kaempferol-7-O-glucoside content compared to all other species
(Supplementary Table S2).
Figure 5. Variation in average relative abundance of 18 flavonoids, their glycosides
and anthocyanin in leaf of annual pasture legumes collected from field trials in 2015
and 2016. Hierarchical clustering algorithm and Euclidean distance metric were
generated using logarithmic normalisation of relative abundance (analysed with Mass
Profiler Professional ver. 14.5, Agilent Santa Clara, USA). The scale presented above
represents relative abundance of each compound, transformed to log scale.
171
4.4 Discussion
Experimental modelling developed as a result of field experimentation
investigating the weed suppressive potential of annual pasture legumes in three
consecutive growing seasons (2015-2017) showed that above-ground competition for
resources was likely to have only partially accounted for the reduction in weed biomass
in plots of biserrula cv. Casbah and yellow serradella. Factors other than competitive
interference were thus likely to have been associated with reduction in weed biomass
accumulation over time in these two species of pasture legumes (Latif et al. 2019a).
Consequently, a series of studies was developed to assess the potential role of plant-
produced secondary metabolites present either in plant tissues or rhizosphere soils
beneath pasture legumes in eliciting a phytotoxic response towards the establishment of
annual weeds, as observed previously in other annual pasture legumes (Carlsen et al.
2012; Clayton et al. 1997; Hale et al. 1979).
Currently, the phytotoxic potential of hardseeded annual pasture legumes, the
identity of PSMs associated with weed suppression, and the quantity of phytotoxic
metabolites present in various plant tissues and rhizosphere soil have remained largely
unexplored. Moreover, release mechanisms of phytotoxic metabolites either through
leachates from plant residue or root exudation, their accumulation and potential
transformation/degradation in soil, and their respective mode(s) of action are unknown in
these annual pasture species (Albuquerque et al. 2011; Duke 2015; Latif et al. 2017). In
this study, through in vitro phytotoxic assessment against weed seed indicators exposed
to both leachates from dried crop residues and methanolic foliar extracts, we provide
evidence of the phytotoxic potential of certain commonly produced annual pasture
legumes in Australia. Through non-targeted metabolic profiling of plant tissues and
rhizosphere soil followed by chemometric analysis incorporating field observations, we
also predicted and quantified key phytotoxic metabolites present in pasture legumes, and
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for the first time demonstrated the presence of these metabolites in biologically relevant
concentrations in rhizosphere soil beneath established stands. Our results strongly suggest
a role for key flavonoids, particularly kaempferol and quercetin and their respective
glycosides, in weed interference.
Although in vitro seedling bioassays have been widely used to test the phytotoxic
potential of terrestrial plants and offer several advantages including high sensitivity,
reproducibility and high throughput formats for screening large numbers of plant
secondary products under controlled conditions (Bertin et al. 2007; Leather et al. 1988;
Wang et al. 2011), it is important to correlate laboratory findings with those obtained
from replicated field experimentation for proof of concept (Inderjit and Weston 2000;
Duke 2015). Based on our initial field results demonstrating significantly higher weed
suppression in some annual pasture species compared to the traditional pasture species
such as subterranean clover, we postulated that mechanisms of weed suppression of some
of the pasture legumes, including yellow serradella and biserrula, may be through
chemical interference rather than resource-based competition, including competition for
light through dense canopy formation (Latif et al. 2019a). To further evaluate this
hypothesis, we performed a series of assays using either methanolic extracts or dried plant
residues of selected pasture legumes to assess their phytotoxic potential. The dominant
mechanism of phytotoxicity could potentially be associated with membrane disruption in
sensitive weed indicator species, followed by necrosis of actively growing tissue resulting
in discolouration of the tips of primary roots, but at this time the mode of action of such
extracts remains unknown (Figure 1). To date, this is the first study reporting on the
phytotoxic potential of dried residue and extracts from leaf and root tissues of annual
hardseeded pasture legumes. Initially, we employed a modified Parker assay to assess the
phytotoxic potential of dried crop residues and their leachates in the presence of field soil,
thereby mimicking field conditions in which crop residues degrade and release secondary
173
metabolites into the rhizosphere. Results suggested the production of phytotoxic
metabolites or allelochemicals from incorporation of legumes residues at relatively low
incorporation rates (1 to 2 MT ha-1 = 5 to 10 g dish-1), and moreover the subsequent
generation of potential phytotoxins at concentrations adequate to elicit growth inhibition
in both seedling indicators (Table 3).
This use of a Parker bioassay facilitates rapid observation of crop residue-soil and
crop residue-microbial interactions, in contrast to simple in vitro seed germination assays
commonly used for bioassay-directed isolation of potential metabolites. Previous studies
have also successfully utilized this assay to assess the uptake and impact of soil-
incorporated herbicides on growth of sorghum seedlings (Parker 1966) as well as for the
evaluation of weed suppressive potential of dried crop residues (Barnes et al., 1987;
Weston et al., 1989). Methanolic extracts of various tissues of pasture legumes were
subsequently utilized to confirm phytotoxicity against similar monocot and dicot seedling
indicators (Table 4). Overall, radicle and hypocotyl growth of both seedling indicators
were inhibited with increasing concentration compared to the untreated control, similar
to findings of others who have assessed crop residue phytotoxicity (Einhellig 1996;
Siddiqui et al. 2009). Methanolic extracts of foliar tissues of both cultivars of biserrula
were active and selective against the dicotyledonous weed seed indicator, while yellow
serradella exhibited selective activity against the monocotyledonous test species, with
radicle elongation most sensitive in contrast to hypocotyl growth as observed by others
(Bertin et al. 2007; Inderjit 1996; Weston et al. 1987). In contrast, the response of the
monocot indicator showed greater hypocotyl sensitivity, suggesting potential differences
in the site or mode of action of such phytotoxic extracts between dicots and monocots.
We also observed that the presence of bioactive metabolites with phytotoxic
potential was not limited to foliar plant tissues, but also extended to the roots (Table 4).
The relatively higher IC50 values of root extracts in some annual pasture species in
174
contrast to foliar extracts suggested that the concentration of active PSMs was lower in
roots compared to aerial tissues, and this was subsequently confirmed by quantification
of PSMs (quercetin, kaempferol, isoquercetin and kaempferol-7-O-glucoside) in tissue
extracts. However, the subsequent detection and quantification of the same active
metabolites in rhizosphere soils collected from established replicated field sites of annual
pasture legumes post-flowering is a critical finding emanating from this research. In
particular, our samples were collected from rhizosphere soils in active rooting zones of
each living legume (n=10). Although considerable variation is expected in rooting
patterns and residue decomposition rates under field conditions, our results were highly
consistent and showed that exudation of PSMs directly from living roots or by
degradation of the above-ground plant residues led to their accumulation in the
rhizosphere at levels that have previously shown phytotoxicity to sensitive indicators.
These findings are in agreement with previous studies that identified phytotoxic
flavonoids, including kaempferol, in the rhizosphere following extended growth and/or
soil incorporation of Trifolium spp. (white clover) (Carlsen et al., 2012; Weston and
Mathesius 2013).
Non-targeted metabolic profiling utilizing LC-MS provides a unique advantage in
the detection and identification of biologically active metabolites at parts per billion level
in complex mixtures such as plant extracts (Griffiths et al. 2009; Weston et al. 2015) and
was instrumental in the identification of PSMs associated with pasture legume
phytotoxicity in this study. The data was acquired using electrospray ionization operated
in positive and negative ion mode using both C18 and HILIC columns to include a
maximal number of known and unknown entities for further multivariate analyses.
Positive ion mode mass spectrometry generated a more intense ion current, hence a higher
number of molecular features were observed when compared to negative ion mode mass
spectrometry. Data generated in positive ion mode was utilized for chemometric analysis
175
after eliminating redundancies based on accurate mass. The clustering of molecular
features showed that molecular entities varied between species but were similar within
species or those within the same genus (Fig. 2).
Of particular note is the fact that the majority of the most abundant compounds identified
in leaf extracts were flavonoids, followed by metabolites associated with chlorophyll
biosynthesis and photosynthesis, including pheophorbide a and pheophytin a. At a species
level, the relative abundance of flavonoids was highest in both cultivars of biserrula,
followed by French and yellow serradella and lowest in gland clover (Supplementary Fig.
S1). The differential abundance of flavonoids between species and the relative similarity
of chemical profiles within closely related species are strongly suggestive of the degree
of conservation of the flavonoid biosynthesis pathway and its regulation among the
various forage legumes under study. Plants are dependent on the enzymes F3H (flavanone
3-hydroxylase) and FLS (flavonol synthase) for the synthesis of phytotoxic flavonoids
such as kaempferol and quercetin directly from naringenin (Gholami et al. 2014). It is
therefore likely that the expression of these enzymes is regulated at either transcriptional
or post-translational level, resulting in the synthesis of various secondary metabolites
derived from the same precursor. Based on the relative differential abundance of
secondary metabolites it may also be possible to predict genetic similarities between
species using a reverse genetics approach (Weston et al. 2015).
The variance explained by models produced by stepwise linear regression
between molecular features and in-field weed suppression was not appreciably improved
by any additional modelling, so model 6 was chosen, and discriminated six predictors
(molecular features) of strong association. The level of variation (~60%) accounted by
model development is reasonably high for complex field/laboratory analyses and is not
logically easily exceeded due to the complexity in the data matrix employed in non-
targeted metabolomics analysis, as described previously (Dixon et al. 2003; Grata et al.
176
2008). The most significant features were assigned to compound numbers 33, 73, 103,
and 109, and together along with two unidentified metabolites (22, 49), explained the
majority of the variance (Table 6). The identity of the predicted compounds (kaempferol,
quercetin, isoquercetin, and kaempferol-7-O-glucoside) was further confirmed by
comparing accurate mass, RT and MS/MS spectra using analytical standards.
These particular flavonoids and their glycosides have previously been frequently
characterized in various legumes (Carlsen et al. 2008b; Carlsen et al. 2012; Weston et al.
2013) and their biological activity as potential allelochemicals has also been reported
(Carlsen et al. 2012; Gomaa et al. 2015b). While our model predicted six compounds
associated with phytotoxicity, of which four were identified flavonoids, the two unknown
molecules were not successfully matched by further comparisons with known
metabolites. In this case, complex sugar glycosylation leading to difficulty in
identification was possible but the existence of other classes of secondary metabolites
such as saponins or condensed polyphenols has not been ruled out.
While several flavonoids identified in plant tissues and rhizosphere soils in this
study have previously been reported to inhibit the growth of Raphanus sativus L. and
Lactuca sativa L. (Basile et al. 2000; Kalinova et al. 2009), their specific activity against
L. sativum and L. rigidum have not yet been reported. Therefore, the phytotoxic potential
of kaempferol, quercetin and their glycosyl intermediates as predicted by the regression
model were examined using a similar in vitro bioassay with authentic standards from
which their respective IC50 values were determined. Not surprisingly, the methanolic
dilutions prepared with authentic standards were comparatively more phytotoxic than the
crude plant extracts previously evaluated, with kaempferol exhibiting the lowest IC50
value (and thus the greatest toxicity) in range of 150-250 µM for L. sativa and 150-300
µM for L. rigidum, respectively, while that of quercetin was 450-550 µM for L. sativa
and 400-450 µM for L. rigidum, respectively. The increased specific activity in the
177
standards with respect to extracts could likely be attributed to the lower concentration of
phytotoxic flavonoids in crude extracts and/ or antagonistic effect of other metabolites
present in crude extracts (Einhellig 2018; Razzaq et al. 2012; Shah et al. 2014).
Isoquercetin was less active as a plant growth inhibitor, with IC50s of 200 and 800 µM for
L. sativa and L. rigidum, respectively, in contrast to its parent compound. In comparison,
kaempferol-7-O-glucoside had similar specific activity to its parent compound
kaempferol. Our findings are in agreement in most cases with the results of previous
studies showing IC50 values for quercetin and kaempferol of 300-800 µM and 150-300
µM, respectively, in in vitro phytotoxicity bioassays using various indicator species (Bais
et al. 2003; De Martino et al. 2012; Kuang et al. 2017; Okada et al. 2018).
Following biosynthesis in the cytosol, flavonoids are generally stored in plants in
their glycosylated form, resulting in increased solubility in water, thereby facilitating their
potential entry into and transport across rhizosphere soils. In most cases, flavonoid
glycosides are rapidly degraded through soil microbial transformation processes and the
activity of plant exo-enzymes, resulting in relatively more stable but hydrophobic
aglycones (Cao et al. 2015; Okada et al. 2018; Wang et al. 2013). Certain soil microbes
catabolize flavonoids and subsequently use them as a carbon source, for example through
the action of intracellular glycosidases (Ciegler et al. 1971; Hartwig et al. 1991; Hassan
et al. 2012). Carlsen et al. (2012) have shown that numerous flavonoid glycosides exuded
by white clover (Trifolium repens L.) were active in the rhizosphere in their aglycone
forms following potential deglycosylation. Bais et al. (2003) observed the conversion of
kaempferol into naringenin and (±) - catechin when incubated with root total protein
extract of Centaurea maculosa L., suggesting that transformation of kaempferol took
place after translocation into the root system, but these results have not been substantiated
in pasture legume systems.
178
Kaempferol is reported to be particularly persistent in temperate field soils under
cool temperatures, with a longer half - life than other flavonoids and potential to remain
at detectable levels for many months following clover establishment and degradation
(Carlsen et al. 2012). Together, results on different continents and under different climatic
conditions provide evidence of potential weed suppression provided by pasture legumes
and associated phytotoxicity resulting from the bioaccumulation of specific flavonoids in
the soil rhizosphere. Although the mode of action of kaempferol and quercetin remains
unclear, various flavonoids have been suggested to act as phytotoxins through cell
membrane disruption or through inhibition of cell division, photosynthesis, specific
enzymes, plant hormones or protein synthesis (Li et al. 2010; Singh et al. 2006).
Quercetin, isoquercetin, kaempferol and kaempferol-7-O-glucoside
concentrations in legume foliage varied between stem and leaf, and also with species.
Flavonoid concentrations remained similar among related species and cultivars in contrast
to unrelated species, suggesting that related species have conserved pathways for
flavonoid biosynthesis, which was not unexpected. Plant phenology and plant sampling
techniques may also play a role in the observed differential abundance of these
compounds among plant populations. In a previous study, the concentration of quercetin
and isoquercetin was highest in leaf tissue at physiological maturity in Fagopyrum
exculentum Moench. when compared to stem and flower (Kalinova et al. 2009). Carlsen
et al. (2008) reported a higher concentration of aglycone products in flowers while
glycosides in leaf tissues of white clover were abundant. Our findings were consistent
with Carlsen et al. (2008) with respect to levels of glycosides and aglycones in serradella
cultivars, gland clover and subterranean clover. However, arrowleaf clover, biserrula
cultivars and bladder clover all exhibited higher concentrations of aglycone in leaves and
glycosides in flowers. This could be attributed to both inherent genetic variation and also
differences in pasture phenology at the time of sampling. Serradella, gland clover and
179
subterranean clover are all early maturing species while arrowleaf clover, biserrula and
bladder clover are later maturing (Table 1). Our results suggest variation exists in the
biosynthesis and distribution of flavonoids in plant tissues depending on maturity, life
cycle and plant part, and that these processes are tightly regulated through highly
conserved defence systems, but some flavonoid expression may also be inducible based
upon weed, insect and/or pathogen pressure. Further investigation on pathway regulation
in annual pasture legumes is required to verify this.
Significantly higher concentrations of quercetin, isoquercetin and kaempferol were
detected in rhizosphere soils of yellow serradella and of kaempferol-7-O-glucoside in
biserrula cv. Casbah, and suggested that the weed suppressive potential displayed by these
species may occur through the release of these PSMs into the rhizosphere, either through
plant residue degradation in soil or directly through root exudation by living roots and
bioaccumulation over time. The concentration of kaempferol-7-O-glucoside in the
rhizospheres of biserrula cv. Casbah and French serradella was within the biologically
active range against L. sativum and L. rigidum, judging for radicle elongation determined
inhibition in our bioassays. Further investigations are currently underway to characterize
the dynamics of phytotoxic metabolites in annual pasture legumes and rhizosphere soils
over time to gain a better understanding of their biosynthesis, and environmental triggers
that may facilitate their production, and influence both the emergence and persistence of
pasture weeds. In addition, the impact of soil microbial communities on the
biotransformation of phytotoxic metabolites present in soils and residues of annual
pasture legumes is currently under evaluation.
4.5 Conclusions
Field experimentation revealed that the weed-suppressive potential of certain
annual pasture legumes, including biserrula cv. Casbah and yellow serradella, were
associated with mechanisms other than competition for resources. Both dried plant
180
residues and crude extracts limited weed seedling growth and metabolic profiles of
various tissue extracts pointed to a high abundance of flavonoids along with anthocyanins
and chlorophyll derivatives. Chemometric analysis predicted in-field and laboratory weed
suppression was associated with the presence of several flavonoids and their glycosides,
specifically quercetin, kaempferol, isoquercetin, and kaempferol-7-O-glucoside. The
abundance of the phytotoxic flavonoids quercetin and kaempferol were significantly
higher in plant tissues and soils collected from established stands of biserrula and yellow
serradella in contrast to other pasture legumes. This study provides strong preliminary
evidence for the role of key flavonoids produced in abundance by annual pasture legumes
with weed suppression and phytotoxicity.
4.6 Acknowledgments
The authors acknowledge funding support from Meat and Livestock Australia
(MLA) (Grant: B.WEE.0146 awarded to L.A. Weston and J. W Piltz) for field trial
research and the Australian Centre for International Agricultural Research (ACIAR) for
sponsoring the PhD fellowship awarded to S. Latif for his studies associated with the
project. The authors also wish to acknowledge Ulrike Mathesius of The Australian
National University (Acton, ACT, Australia) for providing analytical standards of
numerous flavonoids, Douglas Rutledge from AgroParis Tech (Paris, France) for his
support in chemometric analysis, Dr. Russell Barrow for reviewing the manuscript and
Dr. Willian Brown, Vincent West, Graeme Heath, Simon Flinn and Agasthya
Thotagamuwa for their support in data collection and bioassay performance.
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4.8 Supplementary Material
Table S1. Concentration of key phytotoxic flavonoids in foliar tissues (leaf, stem and
flower) of annual pasture legumes at physiological maturity. Values represent the mean
of five replicates. Values followed by the same superscript in each column are not
significantly different (P < 0.05).
Tissue
Pasture species
quercetin
(ppb)
isoquercetin
(ppb)
kaempferol
(ppb)
kaempferol-
7-O-glucoside
(ppb)
Leaf
Arrowleaf clover 835.04ab 74.68ab 184.35ab 530.72d
Biserrula cv. Casbah 968.32a 0.00e 189.33a 16.00f
Biserrula cv. Mauro 808.38ab 0.00e 185.25ab 29.00f
Bladder clover 147.15c 2.62e 30.85e 481.20e
French serradella 97.23c 44.87c 109.13cd 996.80ab
Gland clover 421.42c 23.69d 69.25d 1015.39a
Subterranean clover 42.48c 79.81a 115.01c 950.88b
Yellow serradella 150.27c 54.19b 95.23c 702.75c
Stem
Arrowleaf clover 111.55ab 10.71b 44.17bc 130.72b
Biserrula cv. Casbah 135.04ab 0.32c 22.88b 7.53d
Biserrula cv. Mauro 56.37c 4.23bc 29.01c 7.07d
Bladder clover 110.32ab 1.96c 0.00d 16.35d
French serradella 141.00a 0.87c 2.42d 7.57d
Gland clover 6.91c 1.23c 106.33a 9.09d
Subterranean clover 3.38c 18.85a 61.11b 185.33a
Yellow serradella 79.48c 1.22c 59.17b 9.01d
Flower
Arrowleaf clover 159.65bcd 82.85a 57.72e 9.76c
Biserrula cv. Casbah 223.76bc 0.96c 450.54a 35.25c
Biserrula cv. Mauro 89.12cd 1.71c 297.20b 39.25c
Bladder clover 29.71d 11.85c 0.00f 32.19c
French serradella 244.35b 39.83b 131.69cd 115.33b
Gland clover 566.67a 5.76c 129.40d 51.33c
Subterranean clover 136.53bcd 13.27c 143.00c 152.37b
Yellow serradella 210.40bc 19.64c 105.17e 1186.67a
Table S2. Concentration of key phytotoxic flavonoids in roots and rhizosphere soils of annual
pasture legumes at physiological maturity. Each value represents the mean of five replicates.
190
Values followed by the similar superscript in a column for each of four compounds in each
tissue type are not significantly different (P < 0.05).
Tissue Pasture species quercetin isoquercetin kaempferol
kaempferol-
7-O-
glucoside
(ppb) (ppb) (ppb) (ppb)
Arrowleaf clover 41.02e 2.45a 28.05a 102.76a
Biserrula cv. Casbah 91.97b 1.00c 15.16c 6.65e
Biserrula cv. Mauro 55.92c 1.71b 18.36b 11.83d
Root Bladder clover 16.57g 0.00d 0.00f 0.00g
French serradella 49.57d 1.03c 4.04d 12.34c
Gland clover 31.86f 0.00d 0.43ef 0.13g
Subterranean clover 48.78d 0.00d 1.55e 26.46b
Yellow serradella 113.90a 0.06d 3.76d 3.19f
Rhizosphere
Arrowleaf clover 37.37c 2.14b 32.40b 59.22d
Biserrula cv. Casbah 88.33b 2.08b 22.60c 149.93a
Biserrula cv. Mauro 52.27c 0.65c 11.59d 117.94b
Bladder clover 12.92d 0.41c 3.33ef 19.41e
French serradella 45.93c 1.65c 31.26b 153.10a
Gland clover 28.22d 0.93c 2.18f 97.19c
Subterranean clover 45.14c 0.63c 3.87e 75.03d
Yellow serradella 110.26a 2.65a 35.17a 95.64c
The concentration of kaempferol-7-O-glucoside in the rhizospheres of biserrula cv. Casbah and
French serradella was within the biologically active range against L. sativum and L .rigidum as
calculated for inhibition of radicle elongation.
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Table S3. Concentration of key phytotoxic flavonoids (µM) in above ground tissues (leaf,
stem and flower) of annual pasture legumes at physiological maturity averaged over two years
(2015 and 2016). Values represent the mean of five replicates. Error bars indicate standard
error of means.
Pasture species quercetin isoquercetin kaempferol
kaempferol-7-O-
glucoside
µM µM µM µM
Leaf
arrowleaf clover 276.3 16.1 64.4 118.4
biserrula cv. Casbah 320.4 0.0 66.2 3.6
biserrula cv. Mauro 267.5 0.0 64.7 6.5
bladder clover 48.7 0.6 10.8 107.3
French serradella 32.2 9.7 38.1 222.3
gland clover 139.4 5.1 24.2 226.4
subterranean clover 14.1 17.2 40.2 212.0
yellow serradella 49.7 11.7 33.3 156.7
Stem
arrowleaf clover 36.9 2.3 15.4 29.2
biserrula cv. Casbah 44.7 0.1 8.0 1.7
biserrula cv. Mauro 18.7 0.9 10.1 1.6
bladder clover 36.5 0.4 0.0 3.6
French serradella 46.7 0.2 0.8 1.7
gland clover 2.3 0.3 37.2 2.0
subterranean clover 1.1 4.1 21.4 41.3
yellow serradella 26.3 0.3 20.7 2.0
Flower
arrowleaf clover 52.8 17.8 20.2 2.2
biserrula cv. Casbah 74.0 0.2 157.4 7.9
biserrula cv. Mauro 29.5 0.4 103.8 8.8
bladder clover 9.8 2.6 0.0 7.2
French serradella 80.9 8.6 46.0 25.7
gland clover 187.5 1.2 45.2 11.4
subterranean clover 45.2 2.9 50.0 34.0
yellow serradella 69.6 4.2 36.7 264.6
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Table S4. Concentration of key phytotoxic flavonoids (µM) in roots and rhizosphere soils of
annual pasture legumes at physiological maturity averaged over two years (2015 and 2016).
Each value represents the mean of five replicates. Error bars indicate standard error of
means.
Pasture species
quercetin isoquercetin kaempferol
kaempferol-7-
O-glucoside
µM µM µM µM
Root
arrowleaf clover 13.6 0.5 9.8 22.9
biserrula cv.
Casbah 30.4 0.2 5.3 1.5
biserrula cv.
Mauro 18.5 0.4 6.4 2.6
bladder clover 5.5 0.0 0.0 0.0
French serradella 16.4 0.2 1.4 2.8
gland clover 10.5 0.0 0.2 0.0
subterranean
clover 16.1 0.0 0.5 5.9
yellow serradella 37.7 0.0 1.3 0.7
Rhizosphere
arrowleaf clover 12.4 0.5 11.3 13.2
biserrula cv.
Casbah 29.2 0.4 7.9 33.4
biserrula cv.
Mauro 17.3 0.1 4.0 26.3
bladder clover 4.3 0.1 1.2 4.3
French serradella 15.2 0.4 10.9 34.1
gland clover 9.3 0.2 0.8 21.7
subterranean
clover 14.9 0.1 1.4 16.7
yellow serradella 36.5 0.6 12.3 21.3
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Chapter 5
Analysis of Coumestans, Flavonoids and Proanthocyanidins in Selected
Annual Hardseeded Pasture Legumes of Mediterranean Origin
Chapter 5 presents a comprehensive study of metabolic profiling of coumestans,
isoflavonoids and proanthocyanidins in annual pasture legumes grown in replicated field
trials performed at two different locations in 2016 and 2017 in southern NSW.
Coumestans (coumestrol and 4’-meythoxycoumestrol) and isoflavonoids (formononetin,
daidzein and genistein) were detected and quantified in aerial tissues of pasture legumes
at physiological maturity due to their known impact on livestock health and reproductive
performance , of which I was very interested, given my background as a veterinarian.
Total polyphenol and proanthocyanidin content were also quantified due to their potential
role in protein digestibility and palatability for grazing livestock. Metabolic profiling
provided unique insights into phytoestrogen and flavonoid biosynthesis and accumulation
in selected annual pasture legumes under the climatic conditions experienced in
southeastern Australia.
I led the task of writing this manuscript by designing and performing all
experimentation including all data analytics. I also prepared the first and second draft of
this manuscript, with direction and assistance provided mainly by Drs Russell A. Barrow,
Saliya Gurusinghe and Leslie A. Weston, with additional bioinformatics input from Dr.
Paul A. Weston. All authors contributed in preparation and editing of the final manuscript
before publication. Prof Leslie A Weston assisted in designing all the experiments
including field trials and metabolic profiling, and was instrumental in data presentation
and crafting of the introduction and discussion, along with Dr. Russell Barrow. Dr Paul
A. Weston assisted in data acquisition, data processing, and bioinformatics. Other
194
contributors to this study are described in the acknowledgements. Chapter 5 was
submitted for publication as a research article in the Journal Agriculture and Food
Chemistry.
195
Analysis of Coumestans, Flavonoids and Proanthocyanidins in Selected Annual
Hardseeded Pasture Legumes of Mediterranean Origin
Sajid Latif A,B, Paul A. Weston A,C, Russell A. Barrow A,D,
Saliya Gurusinghe A, and Leslie A. Weston A,C
A Graham Center for Agricultural Innovation, Locked Bag 588, Wagga Wagga, NSW 2678
B School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW 2678
C School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW 2678
D Plus 3 Australia Pty Ltd, PO Box 4345, Hawker, ACT 2614
Abstract
The presence and abundance of key secondary metabolites in selected field-grown annual
pasture legumes (lucerne, biserrula, French and yellow serradella, and gland, bladder,
arrowleaf and subterranean clover) were quantified using standard colorimetric assays
and novel metabolic profiling techniques. Marked differences in the abundance of
coumestans, flavonoids, polyphenols and proanthocyanidins were observed: 1) gland
clover, bladder clover, subterranean clover and lucerne contained phytoestrogen levels
that were above reported tolerable concentrations for grazing livestock; 2) total
polyphenol content was highest in gland clover, and 3) biserrula cultivars and
subterranean clover accumulated more proanthocyanidins than other species. Genetically
related annual pasture legumes segregated similarly from a chemotaxonomic perspective;
concentration of phytoestrogens was associated with upregulation of the flavonoid
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biosynthetic pathway, and variable abundance of isoflavones suggest evolutionary
differences in metabolic dynamics and biosynthetic regulation in related pasture legumes.
Metabolic profiling revealed that several Trifolium spp. and lucerne upregulate
coumestans and other flavonoid phytoestrogens when produced under the climatic
conditions experienced in southeastern Australia. These differences in metabolomic
profiles likely contribute to differences in fodder quality and palatability, and may
contribute to reproductive problems in grazing livestock.
5.1 Introduction
Mixed farming is practised throughout southeastern Australia, with the pasture phase of
crop rotations sustaining both lamb and cattle enterprises 1. Lamb and beef production
account for the majority of livestock-related income in southeastern Australia (AU$22
billion in 2017) and demand is projected to increase over the next decade
(Australian Bureau of Statistics, 2018). Establishment of pasture species that are non-
toxic, persistent and high in nutritional quality is therefore critical for continued
improvement of livestock productivity.
Currently, subterranean clover (Trifolium subterraneum L.) and lucerne
(Medicago sativa L.) are the most widely utilized pasture species in prime lamb, wool
and cattle producing regions across southeastern Australia. Subterranean clover is widely
used due to its compatibility across various soil types and tolerance of pH extremes 2, 3.
Lucerne is a deep-rooted perennial species that is established across low, medium and
high rainfall regions; it is also suitable for neutral or mildly alkaline soils. However, due
to the high protein content in this pasture species, it undergoes rapid fermentation in the
rumen, resulting in increased incidence of bloat and potential nitrogen loss due to
excretion into the grazing environment 4. In addition, ingestion of these pasture legumes
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may result in acute inflammation of both the small and large intestine (red gut), sodium
deficiency and pregnancy-related toxaemia 5-7.
The high cost associated with establishment of standard legume pastures coupled
with their low persistence due to inconsistent rainfall patterns 8, 9 led to the introduction
to Australia of novel annual pasture legume species originating from temperate regions
including Mediterranean Europe and Northern Africa 3. These included Biserrula
pelecinus L. (biserrula), Ornithopus sativus Brot. (French serradella), Ornithopus
compressus L. (yellow serradella), Trifolium glanduliferum Boiss. (gland clover),
Trifolium spumosum L. (bladder clover) and Trifolium vesiculosum Savi. (arrowleaf
clover), which are characterized by their adaptation to deep, acid and sandy soils, drought
tolerance, weed suppressive potential and prolonged feed availability for livestock 10, 11.
While the nutritive value of these pasture legumes has been studied in vivo in South and
Western Australia, detailed investigation of the phenolic chemistry of these species has
not been performed with respect to livestock production 8.
Flavonoids represent a very distinct class of secondary products with both positive
and negative impacts on plant-microbial and plant-livestock interactions 12, 13. Over 5000
naturally occurring flavonoids have been characterised in plant tissues. In general, these
compounds are classified by chemical structure and are subdivided into subgroups
including anthocyanidins, anthoxanthins, flavanones, flavans, and flavanonols.
Isoflavonoids are derived from 3-phenylchromen-4-one (3-phenyl-1, 4-benzopyrone) and
are important in regulating interactions in higher plants. The isoflavone biosynthetic
pathway is one of the well-elaborated in plant secondary metabolism due to the
importance of these compounds as chemoattractants (endosymbiotic bacteria responsible
for nitrogen fixation in legumes) and their involvement in plant defence. Isoflavones,
while mainly derived from the phenylpropanoid pathway, are generated through multiple
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branch pathways in many plant species. Legumes possess a unique enzyme, isoflavone
synthase (IFS), a cytochrome P450 monooxygenase that catalyzes the 2, 3 migration of
the B-ring of naringenin or liquiritigenin, resulting in the production of various
biologically active isoflavonoids 14. The genes encoding enzymes in this pathway are
tissue-specific and are regulated both spatially and temporally in legumes. Synthesis of
catalytic enzymes is induced by various stress factors influencing plant condition,
including climate, temperature, soil moisture availability, nutrient deficiency and predator
attack 15.
Phytoestrogens are polyphenolic compounds produced in legumes and other
plants that may exert estrogenic actions in mammalian systems. Elevated concentrations
of phytoestrogens, including isoflavones, coumestrol and related metabolites, have been
implicated in estrogenic clinical signs in livestock with edematous vaginal and cervical
tissue, hypertrophy of mammary glands, and milky secretions from elongated teats from
ingestion of fodder or feedstock. Elevated phytoestrogen concentrations in forage may
also cause adverse effects in ovarian function, resulting in loss of fecundity or early
embryonic death 16. Significant levels of phytoestrogens are produced in pasture legumes
including lucerne as well as various clover species, and when present at significant levels,
can seriously reduce reproductive efficiency and livestock fertility 13, 17, 18.
Legume-produced phytoestrogens include coumestrol and several key
isoflavones, namely daidzein, formononetin, and genistein. (Figure 1). Such metabolites
constitute a subset of plant defence metabolites targeting herbivory and are highly
species-specific 19. Coumestans and isoflavone phytoestrogens are stable, non-steroidal
secondary metabolites that mimic mammalian estrogen, an endogenous female sex
hormone 13, 20. The affinity of these phytoestrogens in binding estrogen receptor β can
result in reproductive abnormalities during embryonic development and infertility in both
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male and female grazing livestock 21. Isoflavone phytoestrogens are typically stored either
as glycosides or aglycones in pasture legumes 13, 22.
Figure 1: Phytoestrogens found in abundance in lucerne (M. sativa) and subterranean
clover (T. subterraneum).
Polyphenolic compounds including condensed tannins (proanthocyanidins) are
another large group of metabolites also evaluated for a range of biological and
nutritional properties in grazing livestock, based on chemical structure, plant source
and target animal species 23. Monomeric phenolic acids in forages are associated with
enhanced milk production and acid-base imbalance in the rumen following microbial
degradation 24. Traditionally, the proanthocyanidins, oligomeric polyphenols, were
considered as anti-nutritional factors leading to lack of palatability in forage species,
but recent research has shown several potential advantages to grazing livestock,
including reduced risk of bloat at moderate intake of 3-4% of dry matter (DM) 25,
increased weight gain, reduced greenhouse gas emissions 26-28 and reduced parasite
burden 29, 30. However, the effect of plant secondary metabolites on livestock
performance can vary with concentration, chemical structure, feed composition, age
and physiological status of the animal 31, 32.
Currently, there is a marked lack of information on the phytochemical profiles
of aerial tissues of annual pasture legumes, particularly those recently introduced to the
200
southern hemisphere from the Mediterranean. Therefore, the aim of this study was to:
a) investigate the distribution of secondary metabolites that may impact livestock
performance including flavonoids and phytoestrogens (coumestrol, 4’-
methoxycoumestrol, formononetin, genistein and daidzein) in above-ground tissues of
selected traditional and newly introduced annual pasture legumes grown under field
conditions in southern Australia (Wagga Wagga NSW), b) estimate the total
polyphenol and proanthocyanidin content to better understand their prevalence in
pasture legumes grown in southern Australia and c) elucidate variation in expression
of biosynthetic pathways associated with the production of flavonoids and certain
phytoestrogens in selected annual pasture legumes through metabolomics analyses.
5.2. Materials and Methods
5.2.1 Chemicals. Analytical grade acetone, acetic acid, anhydrous sodium acetate,
ethanol, hexane, hydrochloric acid, potassium chloride, sodium carbonate and sulfuric
acid were sourced from Chem Supply (Port Adelaide, South Australia, Australia).
HPLC grade methanol, acetonitrile, formic acid and ammonium acetate were purchased
from Sigma Aldrich (Australia). Analytical standards (Folin-Ciocalteu reagent,
vanillin, gallic acid, cyanidin-3-O-glucoside, (+)-catechin, coumestrol, formononetin,
genistein and naringenin) were purchased from Sigma Aldrich (Australia).
5.2.2 Plant Material. Monocultures of selected pasture legumes listed in Table 1 were
established in 2016 and 2017 (Experiment 1) at the Charles Sturt University research
farm in Wagga Wagga, NSW, Australia (35.04° S, 147.36° E) on a red sodosol soil 11.
Each planting was arranged as a randomized complete block design with five
replications. Individual pasture legumes were established in plots by direct-seeding
with a drill adapted for small-seeded legumes in late May to early June of each year,
with plots measuring approximately 4 × 20 m. Following on from experimentation to
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assess establishment 11, above-ground plant tissue samples (leaf, stem and flower) were
collected from established plots in the third week of October in each year after > 50%
flowering was achieved in each species, which corresponded to the growth stage at
which peak concentrations of phytoestrogens in vegetative tissues have been reported
33. An additional trial was established at Charles Sturt University research farm in 2016
and 2017 and focused on two cultivars of biserrula (cv. Casbah and Mauro) to evaluate
cultivar differences in chemical composition of foliar tissues with five replicates per
cultivar (Experiment 2). Composite samples were collected from each plot (two sub-
samples per cultivar treatment) at pre-bud, pre-bloom, 50% bloom, full bloom and
senescence between mid-July to mid-November in 2016 and 2017, approximately
every three weeks. All plant material was placed on ice immediately after collections
and stored at -20 °C until subsequent extraction.
Table 1. Scientific, common and cultivar names of annual pasture legumes
evaluated in this study.
Scientific name common name Cultivar
Trifolium vesiculosum Savi. arrowleaf clover Cefalu
Biserrula pelecinus L. biserrula 1-Casbah
2-Mauro
Trifolium spumosum L. bladder clover Bartolo
Ornithopus sativus Brot. French serradella Margarita
Trifolium glanduliferum Boiss. gland clover Prima
Trifolium subterraneum L. subterranean clover Seaton Park
Ornithopus compressus L. yellow serradella Santorini
Medicago sativa L. lucerne Aurora
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5.2.3 UHPLC-QTOF-MS Analysis. Metabolic profiling of plant tissues was
performed using an Agilent 1290 Infinity UHPLC system equipped with a quaternary
pump, diode array detector (DAD), degasser, temperature-controlled column (25 °C)
and cooled auto-sampler compartments (4 °C), which was coupled to an Agilent 6530
quadrupole time-of-flight (QTOF) mass spectrometer (MS) with an Agilent Dual Jet
Stream electrospray ionisation source (Agilent Technologies, Australia). Full scan
mass spectra were acquired over an m/z range of 100-1700 Da at a rate of two
spectra/second in both positive and negative ion modes. Chromatographic separation
was performed using a reverse-phase C18 Poroshell column (2.1 × 100 mm, 2.7 μm
particle size) (Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 guard
column (2.1 × 12.5 mm, 5 μm particle size) (Agilent Technologies, CA, USA) using a
flow rate of 0.3 mL min-1. The column was equilibrated for 40 min prior to analysis.
Reverse-phase separation was obtained with a gradient of solvent A, [water (Milli-Q,
TKA-GenPure) + 0.1% formic acid (LC-MS grade, LiChropur®, 98–100%, Sigma-
Aldrich, USA)] and solvent B [95 % HPLC-grade acetonitrile (RCI Labscan, Thailand)
+ 0.1% formic acid]. The solvent gradient was as follows: 5% B (0-1 min) ramping
linearly from 5 to 100% B from 1-17 min, followed by a hold of 100% B for 8 min,
then returning to 5% B for 4 min to re-equilibrate the column. The DAD monitored
absorbance across a range of wavelengths from 210 nm to 635 nm. Injection volume
was 10 µL for each sample. Nitrogen was used as the drying gas at 250 °C at a flow
rate of 9 L min-1. Five biological replicates for each treatment were employed for the
analyses. Phytoestrogens (isoflavones and coumestans) identified in annual pasture
legumes in the current study are summarized in Table 2.
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5.2.4 Extraction for Polyphenols. Foliar and inflorescence samples (1g) from each
pasture legume were freeze-dried and ground manually to a fine powder ( 1-2 mm
size) using a mortar and pestle. Extraction of samples was performed as described
previously 34 with minor modifications. Briefly, ground foliar tissue was defatted with
n-hexane. Residues were extracted three times in 20 mL of solvent
(acetone:water:acetic acid 70:29.5:0.5 v/v/v). Solvent was removed using a rotary
evaporator and the residue lyophilized before it was reconstituted in 50% aqueous methanol
to a final concentration of 1 g mL-1 for storage at -20 °C until required.
5.2.5 Quantification of Total Polyphenol Content (TPC). Total free phenolic content
was determined as described previously 35 with minor alterations. Briefly, 125 µL of
the extract was mixed with 125 µL of Folin-Ciocalteu reagent and 500 µL of deionized
water and incubated in the dark for 6 min. The solution was neutralized by adding 1.5
mL of 7% aqueous sodium carbonate solution and further incubated in the dark for 90
min after which absorbance was measured at 725 nm on a 200 µL aliquot using a
UV/Vis spectrophotometer (FLUOstar Omega, BMG Labtech, Offenburg, Germany)
against a control containing methanol. Total phenolic content was expressed as mg 100-
Table 2. Phytoestrogens from pasture legume species identified in this study by
UHPLC-QTOF-MS in positive ionisation mode.
Name Molecular
formula M+H
Basis for
identificationa
Isoflavones
daidzein C15H10O4 255.0652 STD
formononetin C16H12O4 269.0808 STD
genistein C15H10O5 271.0601 STD
coumestans
coumestrol C15H8O5 269.0444 STD
4’-methoxycoumestrol C16H10O5 283.0579 AM
a Basis for identification codes: AM – match to accurate mass/molecular formula;
STD – match to accurate mass and retention time of analytical standards.
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1 g of gallic acid equivalent (GAE). The experiment was repeated twice with three
technical replicates.
5.2.6 Quantification of Total Proanthocyanidin Content (TPAC). TPAC was
quantified using a vanillin assay as described previously 36, 37. Briefly, 0.2 mL of extract
was added to 0.5 mL of 1% (w/v) vanillin in methanol and 0.5 mL of 25% sulfuric acid
in methanol. The extract was thoroughly blended using a vortex mixer and placed in a
water bath at 37 °C for 15 min. Absorbance of a 200 µL aliquot was measured at room
temperature at a wavelength of 500 nm. The total proanthocyanidin content was
determined as mg100-1 g of (+)-catechin equivalent (CE). The experiment was repeated
twice with three technical replicates.
5.2.7 Statistical Analysis. A matrix of molecular features characterized by mass to
charge ratio (m/z) and retention time (RT) was generated using MassHunter
Workstation Qualitative software version B07.00, MassHunter Profinder (version
B.08.00), Mass Profiler Professional (MPP version 14.5) and Personal Compound
Database Library (PCDL) (Agilent Technologies, CA, USA). Molecular features were
extracted and binned/aligned using parameters as follows: peak height ≥ 10000 counts,
compound ion count threshold two or more ions, compound alignment tolerances were
0.00% + 0.15 minutes for RT and 20 ± 2.00 m Da for mass using Profinder. Molecular
features which were present in three samples out of five were included in the analysis.
Identification was performed comparing molecular entities with entries in a library of
compounds of interest using PCDL, with matches based on a high degree of similarity
(>95%) of monoisotopic mass, RT and mass spectra generated from analytical
standards where possible and METLIN metabolomics database (version B 07.00,
Agilent Technologies, CA, USA) otherwise. All descriptive statistical analyses were
performed using Statistix version 10 (Analytical Software, Tallahassee, FL, USA) and
205
R (R core Team, 2015) and standard deviations were calculated and reported where
possible.
5.3. Results
Metabolomic experimentation was performed using UHPLC- QTOF-MS to assess the
abundance of major defence metabolites in selected annual pasture legumes, with an
emphasis on those potentially impacting livestock health and performance. In addition,
standard assays for the quantification of total polyphenols and TPAC were also
performed to assess potential for impacts on legume palatability and nutritive quality.
5.3.1 Quantification of Phytoestrogens. The phytoestrogenic compounds,
coumestrol, 4’-methoxycoumestrol (coumestans), daidzein, formononetin and
genistein (isoflavones), were detected and quantified at physiological maturity in all
pasture legumes (Tables 3 and 4). Concentration of coumestans ranged between 0.13
and 48.4 mg kg-1 and varied significantly across pasture species (Table 3). Both
coumestrol and 4’-methoxycoumestrol accumulated at higher concentrations in leaf
and stem compared to inflorescence tissues. Bladder clover possessed significantly
higher concentrations of both coumestrol and 4’-methoxycoumestrol in leaf tissue
compared to other pasture legumes (48.4 and 24.8 mg kg-1 respectively), while in stem
tissue, bladder clover had the highest concentration of coumestrol (39.6 mg kg-1).
Lucerne had the highest concentration of 4’-methoxycoumestrol (27.7 mg kg-1) when
compared across similar tissues of other pasture species in this study and had higher
concentrations of both coumestans (0.5 and 0.2 mg kg-1 respectively) in inflorescence
tissue compared to other annual pasture species.
Three isoflavones; daidzein, formononetin and genistein, are commonly found
in pasture legumes and were profiled in this study (Table 4). Isoflavone content in leaf
(113.3- 443.9 mg kg-1), stem (37.5-968.1 mg kg-1) and inflorescence (29.8- 614.1 mg
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kg-1) varied significantly by both pasture species and tissue type (Table 4). Gland
clover exhibited the highest concentration of total isoflavones in leaf tissue (443.9 mg
kg-1) and bladder clover the greatest concentration in stem and inflorescence tissue
(968.1 and 604.1 mg kg-1 respectively). Of the phytoestrogenic isoflavones in aerial
tissues, formononetin was observed at the highest concentration followed by genistein
at physiological maturity (Table 4). The clover species dominated in the production of
phytoestrogenic isoflavones; specifically, daidzein concentration was greatest in gland
clover leaf tissue at 120.2 mg kg-1, while in stem tissue was maximal in bladder clover
and subterranean clover (112.0 and 107.8 mg kg-1 respectively) compared to other
pasture species. Inflorescence tissue of subterranean clover contained the highest levels
of daidzein (128.9 mg kg-1) when compared to the inflorescence of other species
evaluated.
Formononetin concentration in leaf tissue was significantly higher in lucerne
(329.4 mg kg-1) when compared to other species in this study; while the concentration
in stem tissue was greatest in bladder clover (829.8 mg kg-1). Interestingly, genistein
levels were greatest in all three tissues types in bladder clover compared to other species
assessed.
Table 3. The concentration of coumestrol and 4’-methoxycoumestrol (mg kg-1) at
physiological maturity in selected annual pasture legume species.
Tissue type
Pasture Species coumestrol
4’-
methoxycoumestrol
Total
leaf
arrowleaf clover 14.7±2.2 5.9±1.5 20.6±5.1
biserrula cv.
Casbah 13.1±2.7 5.2±1.7 18.2±6.5
biserrula cv.
Mauro 14.6±2.4 7.4±5.5 22.0±4.2
bladder clover 48.4±10.6 24.8±17.7 73.2±13.6
French serradella ND ND ND
gland clover 27.1±11.1 21.1±5.6 48.2±3.5
lucerne 31.8±14.4 20.2±7.5 52.0±6.7
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Table 4. The concentration of daidzein, formononetin and genistein (mg kg-1) in annual pasture legume
species at physiological maturity, post-flowering.
Tissue type Pasture species daidzein formononetin genistein Total
leaf
arrowleaf clover 55.1±26.4 222.9±84.4 28.3±6.8 306.3±94.3
biserrula cv. Casbah 11.5±3.5 73.6±51.3 116.8±2.8 192.5±18.7
biserrula cv. Mauro 6.1±0.7 58.9±17.3 124.7±12.5 189.7±24.0
bladder clover 95.0±2.1 152.3±18.9 184.1±26.3 431.4±32.8
French serradella 47.7±12.5 35.5±48.6 130.1±7.6 113.3±8.1
gland clover 120.3±16.1 226.1±20.5 97.5±10.8 443.9±61.4
lucerne 48.2±13.3 329.4±192.7 61.5±10.1 439.1±141.9
subterranean clover 109.1±14.8 157.6±47.3 72.1±244.3 318.8±40.5
yellow serradella 89.0±13. 91.6±16.6 118.4±5.4 219.1±43.0
subterranean
clover
25.1±8.6 12.5±4.5
37.6±7.3
yellow serradella ND ND ND
LSD (P<0.05) 9.3 5.4 13.1
stem
arrowleaf clover 24.2±5.3 6.1±4.6 30.3±10.5
biserrula cv.
Casbah
ND ND
ND
biserrula cv.
Mauro
ND ND
ND
bladder clover 39.6±7.7 18.7±3.6 58.3±12.1
French serradella ND ND ND
gland clover 26.2±14.4 3.6±1.9 29.8±13.0
lucerne 36.6±16.5 27.7±12.8 64.3±5.1
subterranean
clover
25.4±7.5 7.1±1.6
32.5±10.6
yellow serradella ND ND ND
LSD (P < 0.05) 9.4 14.5 17.5
inflorescence
arrowleaf clover ND 0.1±0.1 <0.1
biserrula cv.
Casbah
ND
ND ND
biserrula cv.
Mauro
ND
0.2±0.1 <0.1
bladder clover 0.2±0.2 <0.1 <0.1
French serradella ND ND ND
gland clover 0.1 ND <0.1
Lucerne 0.5±0.1 0.2±0.1 0.7±0.1
subterranean
clover
0.3±0.1 ND
0.3±
yellow serradella ND ND ND
LSD (P < 0.05) 0.2 0.1 0.2
ND denotes when compound was not detected. Least significant difference LSD
differentiated between concentration means. Each value represents the arithmetic mean of
five replicates with associated standard deviation. Results are presented as means ±
standard deviations.
208
LSD (P < 0.05) 24.9 37.5 34.8 67.8
stem
arrowleaf clover 9.8±3.2 147.7±58.2 27.6±5.2 185.1±67.1
biserrula cv. Casbah 7.5±0.9 23.9±12.2 6.1±1.9 37.5±8.9
biserrula cv. Mauro 5.1±1.4 60.4±18.4 9.9±3.7 75.4±27.4
bladder clover 112.0±33.5 829.8±144.9 126.1±5.4 968.1±394.7
French serradella 75.2±23.3 96.7±34.4 104.1±18.9 276.0±13.4
gland clover 3.4±0.5 239.5±18.4 88.1±37.4 331.0±107.0
lucerne 7.5±2.3 120.6±35.3 15.9±5.1 144.0±56.4
subterranean clover 107.8±18.9 36.2±6.4 18.2±8.3 162.2±42.4
yellow serradella 42.5±7.2 77.7±14.2 20.3±6.9 140.5±25.9
LSD (P < 0.05) 20.8 47.9 19.8 139.4
inflorescence
arrowleaf clover 1.3±0.3 98.2±24.5 18.4±4.6 117.9±45.3
biserrula cv. Casbah 2.2±0.5 39.6±9.9 10.2±2.5 52.0±17.6
biserrula cv. Mauro 1.7±0.4 18.5±4.6 9.6±4.1 29.8±7.5
bladder clover 19.3±4.8 435.8±108.9 158.9±37.2 614.1±190.6
French serradella 10.5±2.6 98.8±24.7 19.9±4.9 129.2±43.4
gland clover 68.3±17.1 184.8±46.2 68.3±17.1 321.0±60.1
lucerne 25.5±6.4 182.6±45.6 25.7±6.4 233.8±81.1
subterranean clover 128.9±32.2 187.6±46.9 148.7±39.8 465.2±26.3
yellow serradella 14.6±3.6 17.6±4.4 25.8±6.6 58.00±5.2
LSD (P < 0.05) 16.8 39.2 24.5 124.8
Least significant difference (LSD) was used to differentiate between concentration means.
Each value represents the arithmetic mean of five replicates with associated standard deviation.
Results are presented as means ± standard deviations,
5.3.2 Effect of Cultivars and Growth Stage on Phytoestrogen Levels. Analysis of
phytoestrogen by in aerial tissues of biserrula cv. Casbah and biserrula cv. Mauro at
five different growth stages showed clear differences between the cultivars (Figures
2a and 2b). The phytoestrogens profiled in this study reached their maxima either at
the 50% bloom or full bloom growth stage while the lowest concentrations were
observed at crop senescence. Significant differences in the concentration of coumestans
between biserrula cultivars was restricted to coumestrol at pre-bloom and 50% bloom
stages of legume growth; in this case cv. Mauro were observed to produce greater levels
of coumestans than cv. Casbah. In the case of phytoestrogenic isoflavones, biserrula
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cv. Casbah possessed greater concentrations of formononetin and daidzein compared
than biserrula cv. Mauro while the opposite was true for genistein.
Figure 2a. The concentration of coumestrol and 4’-methoxycoumestrol at five
vegetative stages i.e., pre bud, pre bloom, 50% bloom, full bloom and senescence of
biserrula cv. Casbah and biserrula cv. Mauro averaged over two years. Error bars
indicate standard deviations.
210
Figure 2b. The concentration of selected isoflavones at five growth stages i.e., pre
bud, pre bloom, 50% bloom, full bloom and senescence in biserrula cv. Casbah and
biserrula cv. Mauro samples averaged over two years. Error bars indicate standard
deviation.
5.3.3 Quantification of Total Polyphenols and Proanthocyanidins. Total polyphenol
and proanthocyanidin content was also assessed in physiologically mature annual
legume pastures. Extractable TPC (total polyphenol content) and TPAC (total
proanthocyanidins) ranged from 4.40 to 13.84 mg 100-1 g of gallic acid and 1.73 to 6.49
cyanidin-3-glucoside equivalents, respectively (Table 5). Gland clover contained
significantly higher extractable TPC levels (13.84 mg 100-1 g) than all other pasture
species examined. Extractable and bound TPAC was significantly higher in biserrula
cv. Casbah compared to other species, while bound TPAC was only detected in both
biserrula cultivars (Casbah and Mauro) and the perennial legume lucerne.
a Values represent arithmetic mean of five replicates. Values followed by the same superscript
in each column are not significantly different (P < 0.05).
211
5.3.4 Abundance of Flavonoids and their Glycosides. The clustering of molecular
features profiled through non-targeted metabolic profiling revealed that molecular
entities varied between species but were similar in those species within the same genus
(supplementary data S1). Over 5000 molecular features in total were profiled in legume
leaf tissues, a smaller number in stem (3258) and 1854 in inflorescence tissues
(supplementary data S2). Flavonoids and their glycosides accounted for the majority of
constituents among all identified major classes of secondary metabolites, based on
verification with analytical standards as well as METLIN database comparisons
(supplementary data T1). The relative abundance of various flavonoids and their
glycosides in selected pasture legumes is presented in Figure 3. Interestingly, the total
number of molecular features characterized in this study was highest in biserrula
followed by French and yellow serradella, while gland clover exhibited the fewest total
molecular features (supplementary data S1). However, gland clover, a recent
introduction to Australia sourced from native pastures in the Mediterranean with
Tables 5. Concentration of extractable and bound total polyphenol and total
proanthocyanidins in foliar tissues of pasture legumes.
Pasture species TPCa TPAC
Extractable Bound Extractable Bound
arrowleaf clover 10.50b 0.24de 3.71c ND
biserrula cv. Casbah 7.69cde 0.43cd 6.49a 8.87a
biserrula cv. Mauro 6.24def 0.11e 4.29b 4.06b
bladder clover 6.67def 0.45bc 1.73c ND
French serradella 8.29bcd 0.35cd 1.94c ND
gland clover 13.84a 0.57bc 2.12c ND
lucerne 4.40f 0.11de 2.05c 0.33c
subterranean clover 9.39bc 0.62bc 4.52ab ND
yellow serradella 5.17ef 0.72b 1.94c ND
LSD 2.62 0.28 2.16 1.95
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relatively limited selection, exhibited a considerably higher abundance of flavonoids
and their glycosides as characterized in the heatmaps presented (Figure 3 &
supplementary Figure S3 and S4). The two biserrula cultivars (Casbah and Mauro)
exhibited similar profiles, and had relatively low levels of numerous flavonoids and
their glycosides, in contrast to subterranean and gland clover. The clustering of the
pasture species according to the similarity in their metabolomic profiles (as indicated
by the dendrogram above the heatmap in Fig. 3) was consistent with the taxonomic
relatedness of the species (e.g. the two serradella cultivars and the two biserrula
cultivars formed separate clades which were distinct from the clovers and lucerne,
which formed another clade).
213
Figure 3. Hierarchal clustering of relative abundance of flavonoids, their glycosides
and coumestrol in leaf tissue in pasture legumes collected in 2016. Hierarchical
clustering algorithm and Euclidean distance metric were used on normalized
abundance using R (R core Team 2015). Compound classes (see legend) for each
compound is indicated by coloured boxes to the left of the heat map.
214
5.4. Discussion
Plants, particularly legumes, have evolved chemical defence mechanisms mediated
by secondary metabolites, including phytoestrogens, to deter herbivores and plant
pests 38, 39. To our knowledge, the study presented herein is the first report
investigating the detection, quantification and distribution of secondary products,
including flavonoids, phenolics, proanthocyanidins and phytoestrogens, in aerial
tissues of several newly introduced annual pasture species in southeastern Australia.
The metabolic profiling and metabolomics analyses employed in this study provided
a comprehensive overview of the specific metabolites in annual pasture legumes
species pertaining to the biosynthesis and regulation of flavonoids and related
metabolites, including those with adverse impacts on grazing livestock in Australia,
the phytoestrogens 40, 41. The accurate identification of secondary metabolites was
facilitated by use of quantitative liquid chromatography time-of-flight mass
spectrometry (LC-QTOF-MS), allowing their detection and quantification at low
concentrations (ng kg-1). In this study, identification of putative compounds was
confirmed by comparison with authentic standards or the METLIN library of
secondary metabolites. In recent years, time-of-flight (TOF) instruments have
increasingly been used as quantitation tools as well as for verification of empirical
formulae because of their high resolving power. Historically, TOF instruments have
had limited capacity for quantification due to a relatively narrow dynamic range, but
recent advances have resolved these limitations to a degree 41, 42.
Along with various isoflavonoids, coumestans are produced in large
quantities by members of the Fabaceae family, commonly known as legumes, and
many of these metabolites contribute to plant defence. Coumestans, mainly
215
coumestrol and 4’-methoxycoumestol (Figure 1), are polycyclic aromatic
metabolites and are closely related biosynthetically to flavonoids.
Plant-produced coumestans are known for a large number of biological
activities, many of which can be attributed to their action as phytoestrogens and
polyphenols 43. Livestock grazing various Trifolium species exhibit varying tolerance
to coumestans, which typically range from 25 to 200 mg kg -1 DM in grazing sheep
and cattle (Reed, 2016). The presence of coumestrol at concentrations greater than
~40 mg kg-1 DM in plant tissues is associated with reproductive inefficiency in
grazing livestock through disruption of several endocrine mechanisms 44, 45. Our
results suggest that the greatest concentration of coumestans occurs in leaf and stem
tissues, opposed to floral tissues, which exhibited only trace quantities of coumestan
metabolites. This is in agreement with previous studies of traditional pasture legumes
46. In addition, the results also indicate that consumption of pure stands of novel
annual pasture legumes such as biserrula, French serradella, yellow serradella and
arrowleaf clover at physiological maturity would likely pose no threat to herd
fertility. In contrast, gland and bladder clover possessed concentrations of total
coumestans at levels above the suggested tolerance limit of ~40 mg kg-1 (ca. 48 and
70 mg kg-1, respectively).
Interestingly, traditional pasture legumes in Australia, i.e. lucerne and
subterranean clover, have consistently been reported to accumulate higher
concentrations of coumestans at flowering in both leaf and stem tissues, but recurrent
selection by plant breeders has resulted in reduced levels observed in many
commercial cultivars in recent years. Our results indicate that both the lucerne and
subterranean clover cultivars included in this study produced substantial
concentrations of both metabolites, reaching concentrations of ca.52 and 38 mg kg-1,
216
respectively which also exceed tolerance limits. The considerable accumulation of
coumestan metabolites in leaf and stem tissues of gland and bladder clover, which
have experienced only limited genetic improvement since their introduction to
Australia from the Mediterranean in the last decades, provides further support for
their potential role in plant defence when established in natural settings 47. These
findings unfortunately has implications for livestock health if these clovers are
utilised as a sole source of feed.
Naturally occurring isoflavones found predominately in the Fabaceae family
have been well described, largely based on their pharmacological activities in
humans and animals, which also includes estrogenic activities. The isoflavonoids
profiled in the present study demonstrated clear intra-species variation as well as
variation associated with tissue type. Others have also noted similar variance within
the genus Trifolium 48. The plant’s ability to regulate allelochemical metabolism in
response to biotic and abiotic stressors, including climate, has been recently
described 49-52, and also with respect to flavonoid production (Mathesius, 2018;
Weston and Mathesius, 2013). Qualitative and quantitative variation in flavonoids
and associated phytoestrogens profiled in our study is an indicator of both species
and tissue-specific adaptation to the environment, resulting in modulation in the
expression of associated genes 53. Although recent pasture studies have suggested
that isoflavones are highly abundant in Medicago and Trifolium spp. and are
frequently highest before 100% flowering 54, considerable variation in total
concentration has been observed in mature Trifolium pratense pastures, with those
surveyed ranging between 9000 and 27000 mg kg-1 DM 52, 55, 56. Such variation may
be due to environmentally mediated response, time of sampling, extraction efficiency
and recovery or previous analytical workflow employed to profile individual
217
isoflavones 22. We specifically attempted to address these issues experienced in other
published works by conducting experimentation in uniform and replicated field sites,
creating composite samples for each pasture plot, and performing sampling at similar
stages of physiological maturity in both 2016 and 2017. In addition, we utilised an
automated high-pressure extraction device to rapidly and uniformly extract all
samples at 35°C, thereby providing uniform extraction across treatments and
replicates, and reducing the possibility of plant to plant variation or that associated
with manual extraction protocols.
Several phytoestrogenic flavonoids were prevalent at physiological maturity
(50% flowering stage) in all pasture legumes, specifically formononetin, the most
abundant isoflavone detected, and genistein. These observations were in agreement
with selected previous reports in various Trifolium species 52, 57-59. Recent findings
also suggested a greater abundance of phytoestrogens and isoflavonoids in leaf
compared to stem tissue and inflorescence tissue 54. Biosynthesis and subsequent
distribution of isoflavones between leaf, stem and floral tissues appears to be
impacted by cultivar, physiological growth stage and the climatic conditions under
which plants were maintained 60. In Trifolium pratense (perennial red clover),
isoflavone concentrations were also impacted by growth stage, with inflorescence
tissue containing equivalent concentrations of isoflavones to those in leaf tissue in
initial bloom stages, with subsequent declines in floral tissue as the crop entered full
bloom stage 52. The disproportionately high levels of formononetin in bladder clover
observed in this study are unexplained but suggest that modulation of the
phenylpropanoid pathway may, in fact, be species-specific and thus determine
terminal isoflavone accumulation in the pathway.
218
Only gland clover, bladder clover and subterranean clover exhibited total
isoflavone concentrations above the livestock-safe threshold level of ~280 mg kg-1
DM (Table 4) and therefore, all of the Trifolium spp. profiled, with the exception of
arrowleaf clover, could theoretically adversely impact the reproductive performance
of livestock. Hashem et al. (2016) similarly reported that cattle fed solely on
Trifolium alexandrinum (berseem clover) containing isoflavones at high
concentrations of ~280 kg-1 DM exhibited hormonal disruption and reduced fertility
compared to similar cattle grazing other species. Interestingly, lambs grazing
Trifolium pratense with high levels of isoflavones exhibited weight gain compared
to cultivars low in isoflavones, but macromolecular interactions impacting plant
nutrition and subsequent weight gain were not fully explored in this study 61. Thus,
future studies to investigate the production of phytoestrogens including coumestans
and isoflavones at various growth stages will be important to optimize site selective
seasonal grazing.
Isoflavones are synthesized as part of the phenylpropanoid pathway (Figure
4), which has multiple branches common to both legumes and non-legumes. The
phenylpropanoid pathway is capable of generating lignins, anthocyanins,
phytoalexins and flavonoids, including isoflavones, as a means of plant protection
against stress or predation (Dixon et al., 1995). Gene sets encoding enzymes in this
pathway are both developmentally and tissue-specifically regulated, and such
environmental stressors as exposure to UV light, drought, prolonged cold, pathogen
attack, and nutrient deficiency may impact expression. Isoflavone synthase (IFS) is
a key enzyme involved in the production of an array of isoflavones from naringenin,
a common phenylpropanoid pathway intermediate (Yu et al., 2000). Legumes can
produce both daidzein from the intermediate compound liquiritigenin, and genistein
219
from the intermediate naringenin chalcone in alternative branches of the
phenylpropanoid pathway (Figure 4). The presence of multiple copies of enzymes
such as chalcone synthase and IFS in species within the Leguminosae allows for the
differential regulation of isoflavone biosynthesis in response to both developmental
and environmental stimuli 62, 63.
The flavonoid precursors and various isoflavones profiled in this study varied
qualitatively and quantitatively among the annual pasture species investigated,
suggesting differences in their metabolic dynamics and ability to regulate flavonoid
biosynthesis. Daidzein and formononetin are produced from one branch of the
phenylpropanoid pathway while genistein is produced from another branch point.
Both branches originate from p-coumaroyl-CoA, but the propensity of the pathway
bias towards dual endpoints is typically determined by the equilibrium between the
enzymes chalcone synthase (CS) and chalcone reductase (CR) (Yu et al., 2000).
Greater production of formononetin as evidenced by higher concentrations observed
in the pasture legume extracts profiled in this study, in contrast to the other
isoflavones measured in this study (daidzein and genistein) (Table 4) suggests that
certain pasture legumes have greater ability to convert formononetin from daidzein
(in contrast to genistein from naringenin) through modulation of the isoflavone
biosynthetic pathway. These findings are also consistent with previous observations
14, 64. Of note, close metabolite clustering and a higher concentration of genistein in
both cultivars of biserrula and serradella species (as opposed to the more typical
daidzein and formononetin) suggested potential overexpression of the gene encoding
CS for the conversion of coumaroyl-CoA to naringenin chalcone in biserrula and
serradella (Figure 4). This observation is consistent with recent results of a study
investigating isoflavone profiles in food-related species in the Fabaceae family
220
where higher genistein concentrations discriminated the genera Biserrula and
Ornithopus from other members 53.
Isoflavones. tend to accumulate at highest concentrations at physiological
maturity in Trifolium spp. as they commence flowering 65. To assess the impact of
growth stage on the abundance of key phytoestrogens in selected novel annual
legume species for livestock production, we further profiled for the abundance of
phytoestrogens in two common biserrula cultivars, first commercialized in Australia
9, at five different growth stages. Interestingly, we observed no significant
differences in coumestan concentration between the two cultivars, despite the fact
that the cultivars were initially isolated from geographically separate locations in the
Mediterranean. This suggests that the bias of the phenylproponoid pathway was
highly conserved in this species, particularly in early growth stages, and was not
affected by genotypic differences. However, the production of phytoestrogenic
isoflavones varied significantly at 50 % bloom and full bloom stages (Figures 2a
and 2b). A previous study also described a significant difference in the accumulation
of isoflavonoids (formononetin and biochanin A) in different cultivars of red clover,
peaking at 50% bloom and full bloom stage 52.
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Figure 4. Biosynthetic pathway leading to the formation of various isoflavones
including phytoestrogenic isoflavones, coumestans and proanthocyanidins (boxed)
(Dixon et al., 1995, 2013).
Our results in biserrula demonstrate that concentrations of all phytoestrogens
assessed were lowest at senescence, which is in agreement with earlier observations
in red clover 65. As noted previously, the genes encoding enzymes for the
biosynthesis of isoflavones are developmentally regulated and are frequently
influenced by environmental stressors and various biotic factors 15, 51, 63, some of
222
which are likely experienced in southeastern Australia during a typical growing
season. Breeding programs in annual pasture legumes can therefore potentially
exploit the bias of the phenylpropanoid pathway by targeted manipulation of genes
involved in the biosynthesis of genistein and daidzein through expression of chimeric
transcription factors over various growth stages 66.
Pasture legume samples collected from multi-year field trials were also
subjected to quantification of extractable and bound TPC and TPAC (Table 5). The
role of TPAC in pasture legumes in reducing herbivory by impacting palatability
attributes has been previously elucidated 47. This is the first study to report on both
TPC and TPAC in annual self-regenerating pasture legumes at physiological
maturity and our findings support previous reports suggesting a similar range for
other related pasture legumes 26, 67, 68. Aerts et al. (1999) demonstrated that the range
of TPAC in Ornithopus was between 2 and 2.5 g 100 g-1, which is also in agreement
with the current study. However, proanthocyanidins in Onobrychis viciifolia
(sainfoin) were reported to be up to ten times higher than measured in this study and
this discrepancy could be associated with species, methodology or growth stage of
sampling 69. Of note is that previous studies using similar assays did not detect
measurable TPAC in lucerne 70, 71. This could be due to differences in cultivar
genetics and growth conditions; however, we also observed that lucerne exhibited
the lowest extractable TP and TPAC levels of all legumes surveyed. A high
concentration of extractable TPAC was noted in biserrula cv. Casbah; both cultivars
also exhibited high levels of bound TPAC in their cell walls. High TPAC levels
suggest the potential for biserrula to limit pathogen and herbivore attack or reduced
palatability to grazing livestock 72, 73 but may potentially offer a cost-effective
opportunity for this species to be integrated in multi-species mixtures to reduce
223
parasite burden. At this time specific bioactive proanthocyanidins in biserrula remain
unidentified.
This study used metabolic profiling to advantage to identify and quantitate a
number of more common secondary plant metabolites, particularly flavonoids. Our
results clearly demonstrate that 1) this collection of annual pasture legumes produce
a diverse array of flavonoids and other phytochemicals associated with plant defence,
and in some cases less desirable phytoestrogens or proanthocyanidins and 2) the
regulation of flavonoid and related metabolites such as coumestans through the
phenyl propanoid pathway occurs at multiple branch sites and as reported, could be
impacted by elicitation in response to various biotic factors including predation and
herbivory. Plants have thus developed various evolutionary adaptations for making
regulatory decisions to produce an array of secondary metabolites, which can provide
them with multiple ecological benefits. In some cases, a single compound or related
family of compounds can exhibit multiple biological functions in plants 74, 75. Despite
significant differences in biological functions of various plant metabolites, which are
frequently concentration-dependent, related compounds often share common
biosynthetic pathways while others, including flavonoids, can originate from diverse
biosynthetic pathways and precursors 49, 76. Evolutionarily, this chemical diversity
provides higher plants with a cost-effective strategy for resource allocation. As an
example, the study results suggest that both lucerne and gland clover have the ability
to upregulate the synthesis of formononetin from daidzein while bladder clover
evolved to upregulate biosynthesis of genistein from 2’, 4’, 5, 7 tetrahydroxy-
isoflavone (Figure 4).
Our study results also support the hypothesis that high concentrations of key
phytoestrogens, TP and TPAC in annual pasture legumes are associated with
224
flavonoid abundance. Interestingly, the relative abundance of total molecular features
profiled through non-targeted metabolic profiling was highest in biserrula leaf tissues
followed by serradella, while gland clover leaf tissue exhibited the fewest molecular
features. In contrast, gland clover exhibited a higher concentration of flavonoids and
their glycosides, suggesting that trade-offs in metabolite production and regulation
occur in pasture legume species; i.e. if flavonoids are high, then expression of TPAC,
TP and other polyphenols might potentially be reduced.
In conclusion, this study has extended our understanding of secondary
metabolism associated with the biosynthesis of phytoestrogens and
proanthocyanidins. Metabolic profiling performed with a variety of hardseeded
annual pasture legumes demonstrated that phytoestrogens quantified in this study
were at concentrations insufficient to pose negative impact on livestock production
with the exception of gland clover, bladder clover and lucerne. Results of the study
suggest that annual hardseeded pasture legumes of Mediterranean origin offer viable
alternative pasture options for more sustainable mixed farming systems of
southeastern New South Wales. In the future, the use of an integrated experimental
approach including multi-omics platforms could also potentially provide deeper
insights into pathway dynamics and regulation of associated genes important in
production of secondary metabolites in pasture legumes. Our findings along with
those of Butkute et al. (2018) suggest great potential to improve legume-based forage
quality through recurrent selection or engineering for species- or cultivar-specific
phytochemicals. In addition, optimisation of livestock management to reduce health
and reproductive issues by selective grazing through timing of animal movement,
manipulation of plant growth stage at harvest and appropriate selection of pasture
species mixtures is also warranted.
225
5.5. Acknowledgements
The authors acknowledge funding support from Meat and Livestock Australia
(MLA) (Grant: B.WEE.0146 awarded to L.A. Weston and J. Piltz) for field trials and
the Australian Center for International Agricultural Research (ACIAR) for
sponsoring the PhD fellowship awarded to S. Latif for studies associated with this
project. The authors also wish to acknowledge Ulrike Mathesius of The Australian
National University (Acton, ACT, Australia) for providing analytical standards of
flavonoids used in this study, and Agasthya Thotagamuwa, Dr. Willian Brown,
Graeme Heath, and Simon Flinn for their technical support in data collection.
5.6. Conflict of Interest
Authors declare no conflict of interest.
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5.8. Supplementary data
Figure S1. Hierarchal clustering of molecular features in leaf tissue of annual
pasture legumes acquired using UHPLC-QTOF-MS in positive and negative
mode. Hierarchical clustering algorithm and Euclidean distance metric were used
on normalized abundance using MPP (ver. 14.5 Agilent Santa Clara, CA, USA).
236
Figure S2 Three dimensional visual representation of principal component
analysis (PCA) of molecular features characterized in positive ion mode of leaf
stem and inflorescence tissues collected in 2016 using UHPLC-QTOF-MS with
five replicates for each pasture species. Components 1, 2 and 3 contributed to
separation by 22.17%, 18.66% and 16.76% respectively.
237
Figure S3. Hierarchal clustering of relative abundance of flavonoids, their
glycosides and coumestrol in stem tissue in pasture legumes collected in 2016.
Hierarchical clustering algorithm and Euclidean distance metric were used on
normalized abundance using R (R core Team 2015). Compound classes (see
legend) for each compound is indicated by coloured boxes to the left of the heat
map.
238
Figure S4. Hierarchal clustering of relative abundance of flavonoids, their
glycosides and coumestrol in inflorescence tissue in pasture legumes collected in
2016. Hierarchical clustering algorithm and Euclidean distance metric were used
on normalized abundance using R (R core Team 2015). Compound classes (see
legend) for each compound is indicated by coloured boxes to the left of the heat
map.
239
Table T1. Mass spectrometry and chemical properties of flavonoids and their
glycosides profiled in this study. Molecular weight, accurate mass of molecular
ion that are unique for a compound and retention time was listed. Identification
was performed either using the Personal Compound Data Base Library (PCDL)
or match with Metlin library.
Sr.
No
Compound Formula MW M+H RT PCD
L
1 apigenin C15H10O6 270.24
271.0605 10.9 Yes
2 apigenin 7-
rhamnoside
C21H20O9 416.4 417.2548 9.0 No
3 apigenin 7-
galactoside
C21H20O10 432.4 433.5781 7.5 No
4 astragalin C21H20O11 448.4 449.107 8.6 Yes
5 biochanin A C16H12O5 284.3 285.0757 13.1 Yes
6 coumarin C9H6O2 146.1 147.1051 8.0 Yes
7 coumestrol C15H8O5 268.2 269.0444 10.9 Yes
8 coumestrol
diacetate
C19H12O7 352.3 353.1821 7.8 No
9 cyanidin C15H11O6 287.2 288.158 12.7 No
10 cyanidin 3-
arabinoside
C20H19O10 419.4 420.251 12.6 No
11 cyanidin 3-
glucoside-7-
rhamnoside
C27H31O15 595.5 596.174 6.4 No
12 apigenin-7-O-
neohesperidoside
C27H30O14 578.5 579.1698 8.6 No
13 daidzein C15H10O4 254.2 255.0652 9.7 Yes
14 daidzin C21H20O9 416.4 417.2458 7.9 No
15 formononetin C16H12O4 268.3 269.0808 11.8 Yes
16 formononetin 7-O-
glucoside-6''-O-
malonate
C25H24O12 516.4 517.318 14.7 No
17 formononetin 7-O-
glucuronide
C22H20O10 444.4 445.102 8.1 No
18 formononetin 7-O-
rutinoside
C28H32O13
576.5 577.312 14.4 No
19 genistein C15H10O5 270.2 271.0601 10.9 Yes
20 isoliquiritigenin C15H12O4 256.3 257.0808 11.6 Yes
21 isoliquiritigenin 2-
glucoside
C32H40O18 712.6 713.543 23.1 No
22 isomollupentin 7-O-
glucoside
C26H28O14 564.5 565.5051 14.6 No
23 isovitexin C21H20O10 432.4 433.5481 13.3 No
24 kaempferol C15H10O6 286.2 287.0556 11.0 Yes
25 kaempferol-7-O-
glucoside
C21H20O11 448.4 449.1078 8.7 Yes
26 luteolin 3'-glucoside C21H20O11 448.4 449.133 10.9 No
27 luteolin 3'-
rhamnoside
C21H20O10 432.4 433.196 7.4 No
240
28 luteolin 5-
galactoside
C22H22O10 446.4 465.257 9.6 No
29 luteolin 5-O-
rutinoside
C27H30O15 594.5 595.304 14.3 No
30 luteolin-3 ,7-di-O-
glucoside
C27H30O16 610.5 611.156 22.6 No
31 7,3',4'-
trihydroxyisoflavon
e
C15H10O5 270.2 271.06 8.9 No
32 6,7,4-
trihydroxyisoflavon
e
C15H10O5 270.1 271.0596 9.0 No
33 luteone 7-glucoside C26H28O11 516.5 517.318 14.7 No
34 medicarpin C16H14O4 270.3 271.214 12.5 No
35 medicarpin 3-O-
glucoside
C22H24O9 432.4 433.196 7.4 No
36 naringenin C15H12O5 272.3 273.0757 10.8 Yes
37 naringenin-7-O-
glucoside
C21H22O10 434.4 435.1286 8.7 Yes
38 p-coumaric acid C9H8O3 164.0 165.0547 7.6 Yes
39 Taxifolin C15H12O7 304.4 305.0657 8.2 No
40 naringenin-7-O-
rhamnoglucoside
C27H32O14 580.5 581.1865 8.6 No
41 puerarin C21H20O9 416.4 417.118 6.8 Yes
42 quercetin C21H20O12 464.4 465.1024 8.2 Yes
43 quercetin 3-
rutinoside-7-
galactoside
C33H40O21 772.7 773.494 17.1 No
44 quercetin-3-O-β-D-
galactoside
C21H20O12 464.4 465.257 9.6 No
45 quercetin-Xyl-Gal C21H20O12 596.5 596.389 18.2 No
46 quercitrin C21H20O11 448.4 449.169 10.0 No
48 isoquercetin C21H20O12 464.4 465.1024 8.2 Yes
49 rhamnetin C16H12O7 316.3 317.184 13.5 No
50 rhamnetin 3-
galactoside
C22H22O12 478.4 479.218 6.8 No
51 rhamnetin 3-
rhamninoside
C34H42O20 770.7 771.513 12.3 No
52 rutin C27H30O16 610.5 611.162 8.0 Yes
54 scopoletin C10H8O4 192.2 193.11 13.4 No
55 scutellarein C15H10O6 286.2 287.137 5.8 No
56 scutellarein 7-
glucoside
C21H20O11 448.4 449.169 10.0 No
57 umbelliferone C9H6O3 162.1 163.0391 7.9 Yes
241
Chapter 6
General Discussion
Conventional food and fibre-based cropping practices in the developed world
have frequently resulted in unsustainable agroecosystems due to high energy inputs
leading to high production costs, and creation of challenged soils due to continual erosion,
compaction, low organic matter content, nutrient and water retention, less diverse soil
microbiomes and pest management problems. Currently, a recent review on weed impacts
by Grains Research and Development Corporation (GRDC) has suggested that one major
limitation to broadacre and pasture crop productivity in Australia is the lack of suitable
legumes for rotational diversity (GRDC 2018). Therefore, the introduction of annual self-
regenerating pasture legumes for use in rotational pastures or mixed cropping systems
could provide a particularly useful option for improved sustainability for several
important reasons. The Mediterranean-type climatic conditions with winter dominant
rainfall in southeastern Australia is well-suited to a range of introduced annual pasture
legumes. Mixed farmers in this region continue to search for pasture crops that meet
multiple demands including the production of quality feed or fodder for a prolonged
period, provision of enhanced nitrogen to the agricultural system through nitrogen
fixation, ability to tolerate temperature extremes and drought, and potentially reduce the
weed burden for the following cropping season.
The research encompassed in this thesis performed both field and laboratory
studies with the objective of 1) assessing in-field weed suppressive potential of several
recently introduced annual pasture legume species originating from the Mediterranean
(Chapter 2) 2) evaluating fundamental mechanisms associated with weed suppression in
these species through competition for resources or chemical interference (Chapter 3) 3)
242
assessment of phytotoxicity exhibited by soil-incorporated crop residues and plant
extracts followed by correlation of their biological activity with the phytotoxic secondary
metabolites present in shoots, roots and rhizosphere soils through targeted and non-
targeted metabolic profiling techniques (Chapter 4) and 4) using devlopment of novel
metabolomics approaches for the evaluation of the chemical composition of coumestans,
proanthocyanidins and flavonoids present in the afore-mentioned annual pasture legumes
that potentially impact the health or fecundity of grazing livestock (Chapter 5).
This project has performed the first detailed study published on the weed
suppressive ability of a select group of newly introduced pasture legumes (arrowleaf
clover, bladder clover, gland clover, biserrula, French serradella, yellow serradella) in
contrast to the more commonly established pasture legumes lucerne and subterranean
clover by evaluation of crop competitive ability and potential for weed suppression due
to allelopathy or phytotoxicity of residues. To accomplish this, both targeted and non-
targeted metabolic profiling was performed in all pasture legume shoot, inflorescence and
root tissues as well as rhizosphere soil. In addition, the same pasture legumes were
evaluated for their ability to produce phytoestrogens and chemistry derived from the
phenylpropanoid biosynthetic pathway, with a focus on flavonoids, coumestans and
proanthocyanidins due to their potential to adversely impact livestock health and
reproduction.
Field research findings demonstrated that the choice of annual pasture species
impacted stand establishment, yearly regeneration and consistent suppression of common
annual pasture weed species when nutrient and water supply were not restricting pasture
growth, with arrowleaf clover and biserrula being the most competitive annual legumes
aboveground, exhibiting the ability to rapidly produce biomass and develop a closed
canopy leading to weed suppression at the soil surface (Chapter 2). Multivariate partial
243
least square regression (PLSR) analysis identified that pasture species biomass had the
greatest impact on reducing weed biomass for the majority of pasture species followed
by light interception (LI) at the base of canopy (Chapter 3). This observation is
complementary to findings from a previous study that indicated both crop biomass and
LI contributed to the weed-suppressive potential of various forage legumes against annual
ryegrass (Dear et al., 2006). Early biomass accumulation, root system development and
high relative growth rate compared with weeds are reported to have a significantly
negative impact on weed establishment in pasture legumes. Field results demonstrated
that yellow serradella cv. Santorini, consistently produced limited crop biomass overall
experimental sites and years, while significantly reducing the accumulation of weed
biomass, indicating that the mechanism of weed suppression of this pasture species was
independent of competition for resources (Chapter 3). It is generally considered that the
weed-suppressive ability is determined by a combination of the competitive and
allelopathic properties of a crop (Worthington & Reberg-Horton, 2013). Observations
from the field study were suggestive of chemical interference as a potential mode of
action for weed suppression in some annual pasture legume species, prompting the design
of several in vitro bioassays to identify and characterise phytotoxic plant secondary
metabolites released to the soil rhizosphere through degradation of crop residues or root
exudation.
Laboratory bioassays revealed that both decomposing crop residues and
methanolic extracts prepared from foliar tissues of annual pasture legumes strongly
inhibited the growth of monocot and dicot weed seedling indicators. In particular, yellow
serradella cv. Santorini and biserrula suppressed weed growth through mechanisms other
than, or in addition to, competition for resources, in the presence of soil rhizosphere
microbiota. The underlying mechanism of weed suppression in certain annual pasture
legumes may thus potentially be associated with the bioactive secondary metabolites
244
produced by plants and associated microbiota. It was concluded from this multi-year and
multi-location study that weed suppression could be effectively achieved by establishing
pasture legumes which demonstrated early vigour and growth, accumulated biomass
leading to dense canopy architecture and also released several flavonoids and their
glycosides through root exudation or degradation of dried residue. These traits are
associated with crop competition and interference with neighbouring plants, and are likely
to be the key mechanisms associated with weed suppression in pasture legumes.
Results of our studies on weed suppressive annual pasture legumes will better
inform growers wishing to manage weeds in this region through use of an integrated
management approach, instead of relying solely on herbicides. This non-chemical
approach is frequently the approach most practiced by local livestock producers who must
cost-effectively manage large well-stocked acreage. It has been demonstrated in
broadacre crops that the choice of a weed suppressive cultivar can cost-effectively prevent
future weed propagules from entering the weed seedbank. However, consistent
performance in weed suppression of newly introduced annual pasture species although
desirable, is strongly impacted by the environment, given the complex suite of factors
associated with local climate change, including temperature extremes, limited and
unpredictable rainfall patterns resulting in drought events and greenhouse gas emissions
by grazing animals. Annual pasture legumes of Mediterranean origin therefore provide
an opportunity to enhance legume diversity in southern Australian mixed farming regions
due to their adaptation to acid soils and similar climatic conditions. However, results of
this PhD study have also pointed out that further breeding efforts are potentially required
to produce pasture legumes better adapted to increasingly droughty conditions while
supporting the profitability and sustainability of livestock production in southeastern
Australia.
245
The ability of a plant to produce and release phytotoxic metabolites into the
environment and/or to tolerate the presence of allelochemicals released by neighbouring
plants including weeds can be crucial to the ability of a species to survive and reproduce
(Bertholdsson et al. 2012, Worthington et al. 2015). This project has developed a
comprehensive protocol for non-targeted metabolic profiling leading to the identification
of key secondary metabolites associated with weed suppression, in this case of annual
pasture legumes. Non-targeted metabolic profiling developed in the course of this
research using UHPLC QTOF-MS provided a unique advantage in the annotation and
subsequent identification of biologically active metabolites in complex mixtures such as
plant extracts and field-collected rhizosphere soils of pasture legumes (Griffiths & Wang,
2009; Weston et al., 2015). Chemometric analysis performed in this body of research
predicted that both in-field weed suppression and inhibition of seedling development
under laboratory conditions were associated with the presence of several flavonoids and
their glycosides, specifically quercetin, kaempferol, isoquercetin, and kaempferol-7-O-
glucoside (Chapter 4). The phytotoxic potential of these specific metabolites was further
confirmed by assessing the impact of their authentic standards on the germination and
growth of weed seedling indicators in vitro. It was further established that the abundance
of such phytotoxic secondary metabolites occurred at biologically relevant concentration
in rhizosphere soils, strongly supporting the hypothesis that exudation and accumulation
of phytotoxins in the rhizosphere of some annual pasture legumes promote suppression
of weed seedling growth and development.
Our results suggest that the biosynthesis and distribution of flavonoids in plant
tissues within the genera of the Fabaceae family vary depending on species, growth stage,
life cycle and plant part, and that these processes are tightly regulated through highly
conserved defence mechanisms, while the expression of some flavonoids may also be
induced by weed, insect and/or pathogen pressure. This work is also the first to
246
demonstrate that in Australia the accumulation of quercetin, kaempferol, isoquercetin,
and kaempferol-7-O-glucoside from annual pasture legumes can occur at ecologically
relevant concentrations, such that weed suppression may be clearly impacted under field
conditions (Chapter 4). Comprehensive metabolic profiling of allelochemicals in both the
plant and the soil rhizosphere, using sensitive analytical instrumentation (Agilent 6530
LC-QTOF-MS) also provided strong insights into the dynamics of the release of bioactive
metabolites following incorporation of plant material or living root exudates into the soil
as previously suggested (Krogh et al., 2006; Weston et al., 2015).
Legume-produced phytoestrogens including coumestrol and several key
isoflavones, (mainly daidzein, formononetin, and genistein) have been consistently
reported to reduce reproductive efficiency and livestock fertility (Hashem et al., 2016;
Reed, 2016; Wocławek-Potocka et al., 2013). While the nutritive value of annual pasture
legumes has been studied previously, there was a need for additionalinformation on
pasture legume phytochemical profiles in aerial tissues, particularly with respect to
implications for livestock production and health (Chapter 5).
The flavonoid precursors and various isoflavones profiled in this study varied both
qualitatively and quantitatively among the annual pasture species investigated, suggesting
differences in their metabolic dynamics and ability to regulate flavonoid biosynthesis.
The variation in the concentrations of isoflavonoids observed across pasture legume
species was not limited to inter-species/cultivar differences but also associated with tissue
type within a species, as reported previously in other Trifolium species (Oleszek et al.,
2007). This further suggested that annual pasture legumes have adapted to regulate their
secondary metabolism through modulation of genes encoding enzymes that catalyse the
synthesis of specific isoflavones, in response to biotic and abiotic stressors (Einhellig,
2018; Iannucci et al., 2013; Visnevschi-Necrasov et al., 2012). Of note, close metabolite
247
clustering and a higher concentration of genistein in both cultivars of biserrula and
serradella species (as opposed to the more typical daidzein and formononetin) suggested
potential overexpression of the gene encoding CS for the conversion of coumaroyl-CoA
to naringenin chalcone in biserrula and serradella (Chapter 5).
In conclusion, field and laboratory experimentations revealed the marked weed
suppressive potential of biserrula cv. Casbah and yellow serradella cv. Santorini,
potentially due to phytochemical interference, while field studies supported the
competitive edge of both arrowleaf clover and biserrula over annual pasture weeds due to
the rapid formation of closed canopies under optimal growth conditions. Non-targeted
and targeted metabolic profiling and subsequent chemometric analysis revealed an
abundance of flavonoids released from annual legumes to the rhizosphere, either through
decomposition of dried residue or exudation of living roots by weed suppressive annual
legumes. In addition, phytoestrogens observed in numerous hardseeded annual pasture
legumes occurred at concentrations were not sufficient to pose adverse effects on
livestock production, with the exception of two newly introduced Trifolium spp., gland
clover and bladder clover and also the more traditional pasture legumes subterranean
clover and lucerne.
This study has also extended our current understanding of the secondary
metabolism associated with the biosynthesis of phytoestrogens and proanthocyanidins.
Results clearly suggest that certain annual hardseeded pasture legumes of Mediterranean
origin such as arrowleaf clover, and in some cases serradella and biserrula, offer viable
alternative pasture options for biodiverse and sustainable mixed farming systems of
southeastern New South Wales, provided sufficient soil moisture is present for successful
establishment.
248
6.1.1 Recommendations for Future Studies
The research presented in this PhD study has contributed to the extension of
current knowledge on performance and weed suppressive potential of annual pasture
legumes under southeastern Australian climatic conditions. Importantly, the study
identified for the first time the possible underlying mechanisms of weed suppression
associated with several annual pasture legumes. I also further characterised the
metabolomes of several novel annual pasture species with an emphasis on polyphenolic
chemistry, including several intensive investigation of the flavonoid biosynthetic pathway
and production of related phytoestrogens. Additional knowledge about the optimal timing
of both weed and pasture species emergence from the standpoint of success of the pasture
crop, crop phenology and the ecological niche occupied over multiple growing seasons
and additional locations is required for better exploitation of the competitiveness of
individual pasture species for optimal suppression of local weed populations in this
region.
The next phase of experimentation to complement this work will entail further
characterisation of phytotoxic flavonoids during various growth stages of annual pasture
legumes to better understand the impact of plant phenology and environment on
biosynthesis and release of phytotoxins under controlled conditions. Knowledge of
phytotoxin production will, therefore, enable growers to manage grazing practices for
optimum productivity, while also managing weed emergence. It will also better inform
growers on ideal rates of crop residue retention for the purpose of weed suppression as a
cover crop, particularly between growing seasons. The impact of accumulated crop
residue on the germination and growth of crops in the subsequent growing season also
requires investigation to ascertain any negative impacts on yield. Furthermore, it will be
interesting to assess phytotoxicity of annual pasture legumes posed by phytoestrogen
content due to their abundance in annual legume crops. In addition, the selectivity of
249
pasture crop residues and leachates may influence subsequent weed establishment, and
therefore additional studies evaluating crop specific selectivity on emerging weed species
are desirable.
There is a need for a model annual pasture legume species to assess the potential
for exploitation of biosynthetic pathways and pathway flux dynamics associated with
flavonoid and isoflavone synthesis, which we observed were differentially regulated
between legume species. This is particularly important with respect to developing
pastures with capacity to produce high biomass and also demonstrated phytotoxicity
towards annual weeds. Complex evolutionary changes may potentially be driven by long-
term exposure to environmental stimuli. While Arabidopsis is frequently considered a
universal and often ubiquitous model, legume secondary chemistries, particularly with
respect to flavonoid synthesis are unique to this family and are not fully represented in an
Arabidopsis model. Advancements in gene editing technologies may further enable the
development of robust, yet cost-effective pasture legume model species, such as
Medicago trunculata, similar to soybean as a model species for the pulses, in terms of
biotechnological manipulation.
Given the accumulation of phytotoxic flavonoids in annual pasture legumes and
their release into the soil rhizosphere as revealed in this work, further research is required
to evaluate their capacity to create disease suppressive soils, which would greatly benefit
rotational mixed farming enterprises by reducing pathogen levels in the soil (Carrión et
al., 2019). Profiling of soil microbial communities over multiple growing seasons are thus
also required to further understand the impact of plant phenology and rhizosphere
chemistry on modification of soil rhizosphere microbial communities under field and
controlled conditions, and how these communities may impact annual legume residues
250
and/or decomposition rates of residues, and signalling processes stimulating root
exudation and defense metabolite production.
6.1.2 References
Carrión, V. J., Perez-Jaramillo, J., Cordovez, V., Tracanna, V., De Hollander, M., Ruiz-
Buck, D., Mendes, L. W., van Ijcken, W. F., Gomez-Exposito, R., & Elsayed, S.
S. (2019). Pathogen-induced activation of disease-suppressive functions in the
endophytic root microbiome. Science, 366 (6465), 606-612.
Dear, B., Hodge, A., Lemerle, D., Pratley, J., Orchard, B., & Kaiser, A. (2006). Influence
of forage legume species, seeding rate and seed size on competitiveness with
annual ryegrass (Lolium rigidum) seedlings. Australian Journal of Experimental
Agriculture, 46(5), 627-636.
Einhellig, F. A. (2018). Allelopathy—a natural protection, allelochemicals Handbook of
Natural Pesticides: Methods (pp. 161-200): CRC Press.
Grains Research Development Corporation (GRDC), 2018. Choosing rotational crops.
Available from
https://grdc.com.au/data/assets/pdffile/0024/223683/grdcfsbreakcrops north.pdf
Griffiths, W. J., & Wang, Y. (2009). Mass spectrometry: from proteomics to
metabolomics and lipidomics. Chemical Society Reviews, 38(7), 1882-1896.
Hashem, N., El-Azrak, K., & Sallam, S. (2016). Hormonal concentrations and
reproductive performance of Holstein heifers fed Trifolium alexandrinum as a
phytoestrogenic roughage. Animal Reproduction Science, 170, 121-127.
Iannucci, A., Fragasso, M., Platani, C., & Papa, R. (2013). Plant growth and phenolic
compounds in the rhizosphere soil of wild oat (Avena fatua L.). Frontiers in Plant
Science, 4(509).
Krogh, S. S., Mensz, S. J., Nielsen, S. T., Mortensen, A. G., Christophersen, C., &
Fomsgaard, I. S. (2006). Fate of benzoxazinone allelochemicals in soil after
251
incorporation of wheat and rye sprouts. Journal of Agricultural and Food
Chemistry, 54(4), 1064-1074.
Oleszek, W., Stochmal, A., & Janda, B. (2007). Concentration of isoflavones and other
phenolics in the aerial parts of Trifolium species. Journal of Agricultural and
Food Chemistry, 55(20), 8095-8100.
Reed, K. F. M. (2016). Fertility of herbivores consuming phytoestrogen-containing
Medicago and Trifolium species. Agriculture, 6(3), 35.
Visnevschi-Necrasov, T., Harris, D., Faria, M., Pereira, G., & Nunes, E. (2012). Short
communication. Phylogeny and genetic diversity within Iberian populations of
Ornithopus L. and Biserrula L. estimated using ITS DNA sequences T. Spanish
Journal of Agricultural Research, 10(1), 149-154.
Weston, L. A., Skoneczny, D., Weston, P. A., & Weidenhamer, J. D. (2015). Metabolic
profiling: An overview—New approaches for the detection and functional
analysis of biologically active secondary plant products. Journal of
Allelochemical Interactions, 1, 15-27.
Wocławek-Potocka, I., Mannelli, C., Boruszewska, D., Kowalczyk-Zieba, I.,
Waśniewski, T., & Skarżyński, D. J. (2013). Diverse effects of phytoestrogens on
the reproductive performance: cow as a model. International Journal of
Endocrinology, 2013. 15.
Worthington, M., & Reberg-Horton, C. (2013). Breeding cereal crops for enhanced weed
suppression: optimizing allelopathy and competitive ability. Journal of Chemical
Ecology, 39(2), 213-231.
252
Appendix A –Book Chapter
Role of Allelopathy in the Persistence of Weeds
Sajid Latif 1,3, Saliya Gurusinghe 3 and Leslie A. Weston 2,3
1School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678, Australia
2School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678, Australia
3Graham Center for Agricultural Innovation, Charles Sturt University Locked Bag 588,
Wagga Wagga, NSW 2678, Australia
7.1 Introduction
Environmental and crop weeds display a number of physiochemical,
physiological, agronomic and reproductive characteristics which enable them to
successfully establish and persist (Cobb & Reade, 2011). Weeds interact with other
plants in close proximity through competition for resources and chemical
interference through release of bioactive secondary metabolites. Secondary
metabolites produced by plants, fungi, algae and associated microflora frequently
serve as natural defenses against potential predators. This ecological phenomenon is
referred as allelopathy, a term first developed by Molisch (1937) and broadly defined
as direct or indirect biochemical interactions among plants and microorganisms
living in a certain ecosystem, mediated through the release of allelochemicals (Rice,
1974; Weston, 1996). While their capacity to outcompete other plants through
competition for finite resources such as light, water, nutrients and space (Bajwa,
Walsh, & Chauhan, 2017; Niklas & Hammond, 2013) have been studied extensively
in the past, there is an increasing body of evidence suggesting that allelopathic
interference may impact plant growth as well as interference caused by direct
competition for resources (Callaway & Aschehoug, 2000; Fernandez et al., 2013).
253
Allelopathy has been reported in the literature for over 2000 years, through ancient
manuscripts, where certain plants were frequently reported to “sicken the soil’’,
thereby adversely affecting the growth and development of other plants (Weston &
Duke, 2003). It is evident through several historical references in the literature that
the inhibitory effect of target species was due to release allelochemicals from donor
plants (Weir, Park, & Vivanco, 2004). The Greek botanist Theophrastus (300 BC)
first observed that chickpeas cultivated over several years inhibited weeds by
exhausting the soil. Toxicity of the walnut tree to neighboring plants was later
described by the Roman scholar Pliny (1 AD). De Candolle (1832) performed the
first well-described experiment to study toxicity associated with root exudates of
allelopathic species particularly noxious weeds. Following on from these early
studies, recent research into the mechanisms associated with allelopathic interactions
has increased exponentially in the past 25 years, mainly through recent advancements
in analytical detection systems providing high accuracy and precision and enhanced
detection.
Allelopathy has also been suggested as one of the potential mechanisms of weed
invasion and persistence in agricultural production systems (Hierro & Callaway,
2003). The latest studies on allelopathy have been critical in advancing our
understanding of plant ecosystems and their drivers, and have focused on the
following: the impact of allelochemicals on successful invasion by plant invaders in
the non-native range; the role of allelochemicals in plant succession in dynamic
ecosystems; and the role of root exudates in driving rhizosphere interactions with
plants and/or microbial symbionts. This chapter aims to present allelopathic
interactions as defence strategies of invasive weeds against native plants and crops,
thereby enhancing their persistence in the ecosystem over time.
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7.2 Classification of Allelochemicals
Allelochemicals generally consist of secondary metabolites that do not have apparent
roles in primary metabolism, and are numerous. More than 100,000 plant-produced
secondary metabolites of low molecular weight have been characterized and were
initially categorized into classes including phenolics, terpenoids and alkaloids based on
their chemical structure and functional groups (Table 1) (Ahuja, Kissen, & Bones, 2012;
Rice, 1980; Walton & Brown, 1999). Some allelochemicals are expressed ubiquitously
in higher plants while others are synthesized or activated from precursor molecules in
response to specific biotic or abiotic stressors (Bartwal, Mall, Lohani, Guru, & Arora,
2013).
Phenolic compounds and their derivatives are the largest and most diverse group of
secondary metabolites performing a vast array of biological functions in plants (Croteau,
Kutchan, & Lewis, 2000). They originate from the phenylpropanoid-acetate biosynthetic
pathway and contain a hydroxylated aromatic hydrocarbon ring with various
substitutions. Phenolics include benzoic acids, aldehydes, cinnamic acids, coumarins,
tannins and flavonoids (Zeng, Mallik, & Luo, 2008) making up to 40% of the structural
matrix of plants (Dalton, 1999). Simple phenolics have been the largest group of
secondary metabolites extensively reported in studies investigating allelopathy in the last
decade, followed by flavonoids (Macías, Mejías, & Molinillo, 2019). Consequently
phenolics and their derivatives represent the most common class of secondary metabolites
known to play a role in defense mechanisms in higher plants, with varying degrees of
phytotoxicity and multiple cellular target sites (Haig, 2008). Several in vitro and in vivo
studies have attempted to explain the allelopathic effect of Centaurea maculosa Lam.
(spotted knapweed) and Centaurea diffusa Lam. (diffuse knapweed) in North American
grasslands through the root exudation of (+) – catechin. Other studies have described
phytotoxic effects of Stellaria media (chickweed) (Inderjit, Muramatsu, & Nishimura,
255
1997), Parthenium hysterophorus L., Asphodelus tenuifolius Cavase. and Pluchea
lanceolata Oliver & Hiern. on seed germination and seedling growth in wheat (Triticum
aestivum L.) through the release of water-soluble phenolics (Yadav & Chauhan, 1998).
Terpenoids, a term adopted from the German word ‘turpentine’ due to the isolation
of earliest terpenoids with turpentine (Herz, 1963), are derived from both the mevalonic
acid and isopentyl pyrophosphate pathways (Haig, 2008). Terpenoids are comprised of
five carbon isoprene subunits linked together to constitute hemiterpenes, monoterpenes,
sesquiterpenes, diterpenes, triterpenes, tetraterpenes and polyterpenes depending upon
the number isoprene subunits. More than 36,000 terpenoids have been characterized
(Buckingham, Cooper, & Purchase, 2016) to date, many of which are volatiles and are
known to perform a variety of biological functions in plants including signaling, photo-
protection, production of reproductive hormones and allelopathy (Croteau et al., 2000).
For example, monoterpenes found in many essential oils are reported to exhibit
phytotoxic effects on seed germination and seedling growth (Haig, 2008). Terpenoids
were shown to be the major chemical constituents of Echinochloa crus-galli L.
(barnyardgrass) in allelopathic interactions with Oryza sativa L. (rice) in several
greenhouse and field experiments (Khanh, Trung, Anh, & Xuan, 2018).
Alkaloids are nitrogen-containing heterocyclic compounds of plant origin, named
due to their alkaline nature. Historically, they are known for their biological and
pharmacological properties, including plant defense, and represent great structural
diversity, with over 21,000 compounds characterized in plants (Wink, 2011; Yang &
Stöckigt, 2010). Biosynthetically, they can typically be categorized into different classes
based on the original amino acid from which they were derived, while others are derived
from terpenoids (i.e. steroidal alkaloids) or polyketide pathways (Macías et al., 2019). As
an example, indole alkaloids originate from tryptophan, pyrrolizidine alkaloids from
ornithine or arginine and quinolizidine alkaloids from lysine (Seigler, 1998).
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Pyrrolizidine alkaloids produced from weed species Senecio angustifolius
Thunb., commonly known as ‘bitter bush’, were found to be the main source of soil
contamination in Aspalathus linearis L. (Rooibos tea) crop causing yield loss and toxicity
(Van Wyk, Stander, & Long, 2016).
Benzoxazinoids are an interesting group of secondary metabolites studied for their
potential use as natural herbicides and insect deterrents, or as lead molecules for the
development of new herbicide modes of action (Macías et al., 2019; Rice, Cai, &
Teasdale, 2012). They are degradation products of hydroxamic acids, mainly synthesized
in the Poaceae family (M. Schulz, A. Marocco, V. Tabaglio, F. A. Macias, & J. M.
Molinillo, 2013). Numerous studies have described the role of these metabolites in
suppressing weeds, their release, microbial degradation, mode of action, and
quantification in above- and below-ground parts of the donor and target plants.
Table 1. Major classes of allelopathic phytochemicals synthesized by invasive plant
species
Class of
compound
Weed species Structure
Phenolic
compounds
Bidens pilosa, (Campbell, Lambert,
Arnason, & Towers, 1982).
Imperata cylindrica (Eussen, 1978)
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Terpenoids Parthenium hysterophorus
(Reinhardt, Belz, & Hurle, 2009)
Pueraria thunbergiana L. Sieb. and
Zucc.
Lantana camara (Ghisalberti,
2000)
Benzoxazinoid
s
Secale cereale L. (Copaja,
Villarroel, Bravo, Pizarro, &
Argandoñ, 2006)
Quinone Sorghum bicolor L. Moench.
as well as in soil (Macías et al., 2014). 2-Aminophenoxazine-3-one (APO), a degradation
product from rye, wheat and corn exudation, is regarded as the most potent and present a
lead molecule for a new class of herbicides due to unique modes of action (Macías et al.,
2019).
7.3 Modes of Action of Allelochemicals
The diverse nature of allelochemicals reported in literature presents a challenge to
those who study their mode(s) pf actions and specific target sites as these are also diverse
258
and frequently unknown (Latif, Chiapusio, & Weston, 2017). Bioassays designed to
study specific mode of actions of allelochemicals often do not provide sufficient evidence
to determine mode of action, as prior knowledge of enzymes, catalysts and target sites
involved in the activity are frequently required for confirmation. This becomes even
more challenging when an allelochemical acts at multiple sites and impacts multiple
biochemical processes in the target plant.
Characterising the mode of action of potential allelochemicals may be highly
beneficial for development of novel herbicides with new modes of action. A number of
mechanisms of action have been confirmed using specific bioassays for the identification
of specific site of action, and include target sites that limit functions of fundamental
physiological processes including membrane permeability, water and nutrient uptake,
respiration, photosynthesis, protein and nucleic acid synthesis, and growth regulation in
susceptible plants (Einhellig, 2004). However, the use of omics and transcriptomics
technologies is now common to point out key genes and processes involved in treated
plant tissues, and can frequently point to pathways that may be impacted by specific
herbicides
Table 2. Modes of action of various allelochemicals associated with invasive weeds
Mode of action Allelochemicals Reference
Membrane
permeability
a-pinene, mimosine, benzoic
acid, cimnamic acid, sarmentine,
limonene, camphor,
(Yoon et al., 2000), (Chai,
Ooh, Ooi, Chue, & Wong,
2013), (Baziramakenga,
Leroux, & Simard, 1995;
Baziramakenga, Leroux,
Simard, & Nadeau, 1997),
(Huazhang Huang, Morgan,
Asolkar, Koivunen, &
Marrone, 2010), (Young &
Bush, 2009)
Nutrient and water
intake
p-coumaric, ferullic, vanillic,
salicylic, and caffeic acids, 2,3,
benzoxazolinones, p-
hydroxybenzoic acid
(Barkosky & Einhellig, 1993),
(Sánchez-Moreiras & Reigosa,
2005), (Barkosky & Einhellig,
2003), (Abenavoli, Lupini,
Oliva, & Sorgonà, 2010)
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Respiration quercetin, kaempferol, camphor,
a-pinene and limonene,
sorgoleone, juglone, lawsone,
naphthotectone and
anthratectone
(Latif et al., 2017) (Abrahim,
Takahashi, Kelmer-Bracht, &
Ishii-Iwamoto, 2003),
(Czarnota, Paul, Dayan,
Nimbal, & Weston, 2001),
(Weir et al., 2004),
(Soderquist, 1973), (Durán,
Chinchilla, Molinillo, &
Macías, 2018)
Photosynthesis p-hydroxy phenyl acetic acid, p-
coumaric, ferulic acids,
sorgoleone, citral, sarmentine,
(Aslam et al., 2017), (Graña,
Sotelo, Díaz-Tielas, Reigosa,
& Sánchez-Moreiras, 2013),
(Dayan, Owens, Watson,
Asolkar, & Boddy, 2015)
Protein and nucleic
acid synthesis
m-tyrosine, vanillic, p-coumaric,
ferulic acid, momilactones,
camptothecin, alangiside,
palmatine, parthenin
(Bertin et al., 2007), (Mersie &
Singh, 1993), (Li, Wang, Ruan,
Pan, & Jiang, 2010), (Kato-
Noguchi, 2011), (Kumar &
Barthwal, 2018), (Belz, 2016)
It is important to note that phytotoxicity of any allelochemical is a function of its
concentration, flux rate, physiological stage of the target plant, climatic conditions and
microbial interactions in the rhizosphere (Einhellig, 2004; Rice, 2012; Weidenhamer,
1996; Weston, 1996). Further standardization of research efforts in allelopathy are
required with evidence of relative concentrations, degradation and/or transformation and
their possible antagonistic or synergistic effect in the soil (Duke, 2015).
7.4 Synthesis, Localization and Release of Allelochemcials from Donor Plants
Secondary metabolites are synthesized and localized in almost all plant parts and
tissues including stem, leaves, inflorescence, roots, pollen grains, bark, fruits, seeds and
rhizomes (Putnam & Tang, 1986; Rice, 2012). However, many studies have noted that
the diversity and concentration of allelochemicals is higher in the shoots instead of the
roots. Some studies have also reported high concentrations of allelochemicals in seeds,
particularly seed coats, that might contribute to the longevity of certain weed seeds in soil
from decay by microorganisms (Qasem & Foy, 2001).
260
However, for an optimum effect, allelochemicals must concentrate in leaves, stem
and roots for degradation or exudation in response to environmental. Allelochemicals are
generally synthesized in the cytoplasm or in cell organelles, such as the chloroplast, and
are frequently localized in vacuoles away from synthetic machinery, or deposited in
specialized storage structures such as glandular trichomes to prevent autotoxicity or
immediate release into the environment (Eom, Senesac, Tsontakis-Bradley, & Weston,
2005). Water soluble allelochemicals are often stored in vacuoles (Kutchan, 2005) while
lipophilic compounds are localized in resin ducts, laticifers, glandular trichomes or cuticle
members (Yazaki, 2006). Relatively high concentrations of allelochemicals are required
to elicit defense or signaling against potential threats. Significant allocations of carbon
and energy resources in term of adenosine triphosphate (ATP) and equivalent, i.e.
nicotinamide adenine dinucleotide phosphate (NADPH2) are required for the synthesis
of secondary metabolites, storage, transport and exudation (Wink, 2011). The
concentration and diversity of allelochemicals synthesized in plants varies with
physiological stage and is greatly impacted by various intrinsic and extrinsic factors
including genetics, tissue type and environment, and their interactions. Frequently the
abundance of allelochemicals is reported to be enhanced in response to exposure to
various stimuli including insects, pathogens, ultra-violet light, ionizing radiations,
temperature and moisture stress, and herbivory (Albuquerque et al., 2011; Wink, 2011).
Complicating these interactions even further is the fact that secondary metabolites are
often released in mixtures with other related metabolites, with one secondary metabolite
often performing more than one biological role, including defending the plant against
several, but often unrelated threats (Macias, Molinillo, Varela, & Galindo, 2007).
Plants have often developed specialized tissues for the release of allelochemicals in
to the environment including stomata, glandular trichomes in above-ground tissue and
root hairs, border cells, epidermis, and periderms in below-ground tissues. In the shoot,
261
glandular trichomes are often the primary tissues for the exudation of secondary volatile
metabolites, synthesized either within trichomes or translocated from other plant parts.
Volatile compounds can diffuse directly from cell walls on the plant surface, while non-
volatile polar compounds require specialized protein transporters (Weston, Ryan, & Watt,
2012).
Volatilization and leaching from foliar plant parts, decomposition of dead plant parts and
root exudation have been characterized with respect to release of allelochemicals over
time in the environment in numerous plant species. Root exudation is the most common
mechanism of release of allelochemicals into the below-ground environment or
rhizosphere, though exudation by primary and/or secondary roots and root hairs. Root
hairs, a unicellular extension of the epidermis, are frequently the interface for chemical
interactions in the soil rhizosphere as they exude the majority of secondary metabolites
at varying concentrations, from both living and decomposing roots. Some allelochemicals
are synthesized within root hairs and directly released into the soil; examples include
sorgoleone and certain naphthoquinones (shikonins) while others are translocated to root
hairs prior to exudation (Weston et al., 2012). Mechanisms of root exudation including
simple diffusion, vesicle transport and specific ion channels have been previously
described in greater detail (Weston et al., 2012). Extraction of plant-produced
allelochemicals from rhizosphere soil is challenging due to complex chemical nature of
soil matrices, but relatively recent developments in solid phase extraction and specialized
silicone microprobe tubing have facilitated the recovery of allelochemicals including a
range of both nonpolar to moderately polar metabolites (Weston et al., 2012).
7.5 Factors Affecting Biosynthesis of Allelochemicals
The exudation of allelochemicals from donor plants into the environment is one of
the basic phenomena one tries to evidence when conducting allelopathy studies in the
262
field or laboratory. Allelochemicals are released in response to several intrinsic and
extrinsic factors as summarized in figure 1. Intrinsic factors include plant species,
cultivar, age, physiological stage and tissue type. Extrinsic factors include pathogen
infestation, physical injury, nutrient stress and higher temperature and irradiation
exposure, soil characteristic and competitors.
Figure 1. Interactions of invasive plants and challenges to overcome with respect to
availability and competition for resources, environmental stessors and herbivory
Several studies also suggest that several physio-chemical and biotic characteristics
of soil affect allelochemical production and hence the phenomenon of allelopathy (Blum,
2011; Cheng, 1995; Einhellig, 1996). Soil texture can affect the degree of sorption of
allelochemicals, with loose and sandy soil generally exhibiting more rapid inhibitory
effects than heavy clays or loamy soils (Oleszek & Jurzysta, 1987). Other soil
characteristics including soil pH, organic matter, available carbon and nitrogen affecting
allelopathic activity by uptake, immobilization, microbial activity and accumulation of
inorganic ions and nutrients (Dalton, Blum, & Weed, 1983; Facelli & Pickett, 1991). For
example, nutrient stress can result in the up-regulation of allelochemicals particularly
263
phenolic acids which have the ability to interfere further with mineral uptake (Einhellig,
1996).
7.6 The Role of Microorganisms in the Release and Transformation of
Allelochemicals
Soil microbiota are important modulators of plant community structure. They can
either limit or promote the success of plant species through maintaining a balance between
antagonistic and mutualistic interactions. This factor is particularly vital with respect to
invasions, where soil microbiota have been shown to contribute to their dominance. While
certain invasive species would induce niche construction of microbial communities for
nutrient enrichment (Dassonville, Guillaumaud, Piola, Meerts, & Poly, 2011), others are
recruited to facilitate the bioactivation and release of defense-related secondary
metabolites. As the appreciation for soil microbiota and their ecological functions
improves, their impact on promoting the persistence of certain weeds through microbially
mediated bio-activation processes of secondary products is gaining traction in the field of
allelopathy.
One such biosynthetic pathway involved in microbial transformation is that of the
benzoxazinoids, produced by plants in the Poaceae family. The defensive properties of
benzoxazinoids were first identified in corn DIMBOA and DIBOA (resistance to
insects)and also cereal rye, where the crop was noted to display increased resistance to
fungal pathogens (M. Schulz, A. Marocco, V. Tabaglio, F. A. Macias, & J. M. G.
Molinillo, 2013). The benzoxazinoids released by plants through decomposition or root
exudation, 2(3H)-benzoxazolinone (BOA) and 6-methoxy-benzoxazolin-2-one (MBOA)
are further converted to their corresponding aminophenols. They are subsequently
converted to aminophenoxazinones, acetamides, and malonamic acids through bacterial
and fungal degradation (Friebe, Wieland, & Schulz, 1996). The end-products
aminophenoxazinone (APO and AMPO produced from BOA and MBOA, respectively)
264
are transformed by oxidation of aminophenols and form exceptionally active microbial
metabolites with the ability to persist for extended periods in soil.
7.7 Metabolic Profiling of Allelochemicals
The soil rhizosphere presents a complex medium for biochemical interactions
between roots, soil and microbiota and remains the least characterized in terrestrial
ecology. Advances in high precision analytical instrumentation, including high-pressure
liquid chromatography interfaced with mass spectrometry, has enabled accurate and
robust methodologies for screening, annotation and identifications of allelochemicals.
Due to the chemical diversity of secondary metabolites, multiple, often complementary
analytical platforms are employed to capture the entirety of metabolome of a biological
system. Weston et al., (2015) discussed a detailed description of metabolic profiling of
allelochemicals using metabolomics approaches. By performing efficient extractions
followed by metabolic profiling of complex matrices including rhizosphere, one can
describe the functional state of a biological system at a particular point in time.
Metabolomics in conjunction with other proteomics and transcriptomics has been
employed as a systematic approach for studying a plants' chemical fingerprint (Breitling,
Ceniceros, Jankevics, & Takano, 2013). After metabolic profiling, processing of a
substantial amount of spectral data is performed through computational and analysis tools
often including quality control, binning and alignment of molecular feature across
samples, feature extractions, conversion, normalization of data (Smith et al., 2005;
Sugimoto, Kawakami, Robert, Soga, & Tomita, 2012). However, as it is nearly
impossible to characterize all plant metabolites in a complex mixture, many remain
unidentified.
265
7.8 Case Studies of Invasive Plant Species Exhibiting Allelopathic Interactions
Several weed species, have escaped from their native ranges and established as invasive
species in various geographically distinct locations. The case studies presented here are
examples of several invasive species that have persisted over numerous continents. They
represent both gramminaceous and broadleaf species and are known to compete with
native flora through the production of plant secondary defense compounds. Furthermore,
the species presented here; Echium plantagineum L., Centaurea solstitialis L., Sorghum
halepense (L.) Pers., Fallopia japonica (Houtt.) Dcne. and Parthenium hysterophorus L.
represent excellent model species to study adaptations in plant secondary product
synthesis and exudation under different climatic conditions experienced in invaded
territories.
A
B
C
D
266
E
Figure 2: Representative images of invasive plant species presented as case studies.
Echium plantagineum L. growing in New South Wales, Australia (image courtesy of Dr
Xiaocheng Zhu,Charles Sturt University, Wagga Wagga, NSW, Australia) (A); Natural grassland
invaded by Centaurea solstitialis L. in California, USA (image courtesy of Dr Daniel Montesinos,
Australian Tropical Herbarium, Cairns, QLD, Australia) (B), Parthenium hysterophorus
L.infestation in Queensland, Australia (image courtesy of Dr Ali Bajwa, Department of Primary
Industries, NSW, Australia) (C); Fallopia japonica (Houtt.) Dcne. established in Nova scotia,
Canada (image courtesy of Dr David Clements, Trinity Western University, British Columbia,
Canada) (D); Sorghum halepense (L.) Pers. in its invaded territory, New South Wales, Australia
(image courtesy of Dr Xiaocheng Zhu,Charles Sturt University, Wagga Wagga, NSW, Australia)
(E).
7.8.1 Echium plantagineum L.
Commonly known as Patterson’s curse, Echium plantagineum is a weed introduced to
Australia in the 1800s. It is considered a highly invasive plant species in Australia,
whereas in the Iberian Peninsula, the native range of E. plantagineum, infestations are
limited. Recent research has strongly indicated that several factors contribute to the
invasion and persistence of the weed in Australia, including greater genetic diversity,
tolerance to environmental stressors and their capacity to produce defensive plant
267
secondary metabolites (Zhu et al., 2017). To protect the plant against herbivory above-
ground, foliar tissues of the plant were shown to produce significantly high concentrations
of pyrrolizidine alkaloids and their N-oxides when compared to its co-generic exotic
counterpart Echium vulgare, which although present in Australia is not considered
invasive (Skoneczny, 2017). E. plantagineum also produces and releases
naphthoquinones from its roots, including Acetylshikonin and acetylalkannin. In
laboratory bioassays root extracts prepared from roots of plantagineum collected from
Australia were shown to be highly inhibitory against wheat coleoptile development, in
comparison to roots collected in Spain (Durán et al., 2017). Overall, there is evidence that
plant secondary chemistry present in highly invasive E. plantagineum plants in Australia
evolved to adapt to environmental conditions in the island country. As shikonins are
known to function as microbial signaling molecules and that shikonin expression is
elevated in E. plantagineum, there may be potential impacts on the rhizosphere
microbiome which may enhance the persistence of the plant through disease suppression,
a mechanism of invasion previously discussed in other exotic plant introductions (Hierro
& Callaway, 2003).
7.8.2 Centaurea solstitialis L.
Native to Eurasia, Centaurea solstitialis L. (Asteraceae) commonly known as yellow
starthistle has established as a highly invasive ecologically impactful plant species in
north and south America and Australia (Eriksen et al., 2014; Montesinos & Callaway,
2018). Multiple introduction events have been shown to contribute to increased genetic
diversity of C. solstitialis in the introduced regions when compared to the eastern
Mediterranean region (Montesinos & Callaway, 2018). Contrasting herbivory patterns by
a generalist herbivore, the common garden snail against various Centaurea spp. has
shown that when fed with Centaurea solstitialis L. foliar tissues, the weight-gain was
differential between plants sourced from the native and invaded regions (Filipe et al.,
268
2016). While requiring further experimentation to identify the differentially expressed
plant defense secondary metabolites, it provides compelling evidence that defense
strategies towards generic and specific herbivores are altered by invasive plants.
7.8.3 Sorghum halepense (L.) Pers.
Commonly referred to as Johnsongrass, the perennial grass species is native to the
Mediterranean region, but has distributed globally (Holm, Plucknett, Pancho, &
Herberger, 1977). The weed is troublesome to control in broadacre cropping systems due
to its persistence in cereal grain crops, limiting herbicide options for control, and
exacerbated due to acquisition of resistance to acetolactate synthase inhibiting herbicides
(Powles & Yu, 2010; Werle, Jhala, Yerka, Anita Dille, & Lindquist, 2016). In addition
to developing herbicide resistance, Johnsongrass has been shown to synthesise and exude
the well-characterized photosystem II inhibitor, sorgoleone from roots (Czarnota et al.,
2001). More recent studies have identified that the production of phenolic compounds
by S. halepense differed based on the growth stage of plants (Hongjuan Huang, Liu,
Wei, Wang, & Zhang, 2015), with one dominant phenolic compound p-hydroxybenzoic
acid in the seedling stage; three phenolic compounds, p-hydroxybenzaldehyde, p-
hydroxybenzoic acid and ethyl p-hydroxybenzoate in the jointing stage and three phenolic
compounds, p-hydroxybenzaldehyde, p-hydroxybenzoic acid and ethyl p-
hydroxybenzoate dominant in the reproductive stage. The results strongly suggest that the
exudation profiles of these known allelochemicals were recruited to enhance the
competitiveness against other plants at the early growth stage, at which point other
physical competitive traits associated with leaf area index and light interception have not
been developed.
7.8.4 Fallopia japonica (Houtt.) Dcne.
Fallopia japonica, also commonly known as Japanese knotweed originating from Asia,
mainly Japan, Taiwan and China was introduced to north America, Canada and Europe
269
over 100 years ago (L. A. Weston, Barney, & DiTommaso, 2005). Being able to
regenerate vegetative and producing high levels of biomass, Japanese knotweed has
established as a successful invasive plant (Barney, Tharayil, DiTommaso, & Bhowmik,
2006). Importantly it was shown previously that allelopathic interactions with native
plants was not through the inhibition of germination in native plant seedlings by soil
incorporated plant litter, but through interference in the life-history stages (most notably,
time to flowering) (Parepa, Schaffner, & Bossdorf, 2012). The results indicate that F.
japonica potentially induces changes in pollination timing between native host plants and
their pollinators, impacting reproductive success through target plant specific root
exudates. Further studies have also pointed to the impact of soil microbiota on F. japonica
invasion success where a greater abundance of soil microbial presence was shown to
favour growth and development of F. japonica, whereas no significant impact on the
growth of native plants was observed through enrichment of soil microbiota (Parepa,
Schaffner, & Bossdorf, 2013). Interestingly it was observed that only microorganisms
between 20-200m were beneficial to the enhancement of F. japonica growth. In support
of this argument, Dasonville et al. (2011) showed that Asian knotweeds significantly alter
the rhizosphere microbial community structure of denitrifies and nitrifiers, thereby
constructing a niche microbiome and enhancing the persistence of the weed through
competition for resources. As a majority of rhizosphere microorganism recruitment and
enrichment occurs through root exudation, it is plausible that the chemical cues
modulating microbial communities in this examples also occur through synthesis and
exudation of specific plant secondary metabolites.
7.8.5 Parthenium hysterophorus L.
The invasive herbaceous plant Parthenium hysterophorus L., commonly known as
parthenium weed is native to tropical and subtropical regions of America (Dale, 1981),
but has since been introduced to over 40 countries worldwide including India, Pakistan,
270
Nepal, Bangladesh, Australia, Ethiopia, Tanzania and South Africa (Shi, Aslam, &
Adkins, 2015). Interestingly this highly invasive plant has many adverse effects on the
biodiversity of natural communities, agricultural crop production, and human and animal
health (Adkins & Shabbir, 2014; Allan, Shi, & Adkins, 2018). Apart from competition
for resources it is often proposed that allelopathic interactions play a major role in its
invasiveness. It has been established that both above- and below-ground litter of P.
hysterophorus inhibit germination and growth of various weed seedling indicators in a
dose-responsive manner, with litter collected from the invasive regions being exhibiting
a greater level of phytotoxicity when compared to litter from the native range (Shi &
Adkins, 2018). The plant secondary chemistry in P. hysterophorus is quite unique, in that
there is a high abundance of a sesquterpene lactone, parthenin in aerial parts of the plant
(Allan et al., 2018; Shi et al., 2015), which has been shown to inhibit germination and
growth of a number of seedlings of test species; Ageratum conyzoides L., Lactuca sativa
L. var. capitate, Eragrostis curvula (Schrad.) Nees, Eragrostis tef (Zucc.) Trotter and
Echinochloa crus-galli (L.) P. Beauv (Belz, Reinhardt, Foxcroft, & Hurle, 2007). Several
other phytotoxic sesquiterpene lactones are also known to be produced by P.
hysterophorus including Ambrosin, Hysterin, Coronopilin, Damsin and Hymenin (de la
Fuente, Uriburu, Burton, & Sosa, 2000; Rodríguez, Epstein, & Mitchell, 1977).
Furthermore, the presence of various phytotoxic flavonoids including Kaempferol and
Quercetin 3-o-glycoside have been established in foliar tissues of the plant (Mears, 1980).
7.9 Conclusion
Invasive weeds employ various strategies to persist in invaded territories, of which
allelopathic interactions is being appreciated to play a significant role. The synthesis,
translocation and activation of allelochemicals is highly dynamic and varies significantly
with respect to the class of compounds produced, the target species and intermediate
interactions with soil microbiota involved with the degradation of plant residues and/or
271
activation of root exudates. The specificity of allelochemicals towards target weed species
are affected by their mode of action, as they directly influence the uptake and
translocation of the phytotoxic metabolites in the tissues of the target species. New
methods for the identification of phytotoxic plant secondary products in complex matrices
such as soil and plant extracts through advanced mass-spectrometry based metabolomics
profiling strategies have enabled rapid advancements in the field of phytochemistry. The
study of model invasive species and their adaptations with respect to the production of
defense compounds, show clear trends in the enhancement of biosynthesis and release of
phytotoxins in invaded territories, in direct contrast to their native habitats. Results from
case studies show clear differences in defense mechanisms targeting specialised
herbivores, while enhancing the production of generic phytochemicals to suppress the
growth of native plant species, to ensure persistence in invaded territories. The study of
model invasive species has also highlighted that climatic adaptations by invasive plants
include enhanced production of plant defense compounds, and that there may be increased
persistence of invasive species under elevated temperatures in the future.
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Appedndix B: Media Article
Smother Winter Weeds with Annual Legumes
Recent research conducted over three years (2015-17) at two sites in the south
west slopes of Wagga Wagga district, noted arrowleaf clover and biserrula were the most
consistent and competitive annual legumes with respect to stand establishment and
consistent suppression of winter pasture weed species.
Biserrula, a newer annual legume, capable of being more suppressive of pasture weeds.
Other legumes assessed in the research included sub clover (Seaton Park), bladder clover,
gland clover, yellow serradella (Santorini and Avila) and French serradella.
Mixtures of pasture species were also assessed in combination with lucerne, phalaris and
cocksfoot.
282
Combinations did not perform as well or any better than best single species, with
production and competitiveness no better when annual legumes were in combination with
perennials.
Arrowleaf clover has also been shown to be very competitive against pasture weeds.
Sajid Latif (School of Animal and Veterinary Sciences, Charles Sturt University Wagga
Wagga), and colleagues Saliya Gurusinghe, Paul Weston, William B. Brown, Jane C.
Quinn, John Piltz and Leslie A. Weston, undertook the research.
Past research has generally indicated that the more productive a winter legume pasture,
the better able it is to compete against winter weeds.
Aspects associated with pasture competitiveness, such as correcting soil deficiencies and
selection of varieties and species suited to a given soil and environment are important.
This study supports these findings, plus confirms an important role for some newer
pasture species.
283
Sajid Latif and colleagues' research, has shown some pasture legumes, such as
arrowleaf clover and biserrula, were more competitive than more traditional species.
An important outcome from the research was that Santorini serradella did not perform
that well from a productivity aspect on this soil type (red Sodosol at both trial sites with
pH of 6.2), but was close to the more productive species of arrowleaf clover and biserrula
for suppressing weeds.
The researchers suggest serradella varieties like Santorini have mechanisms other than
competition for resources (water nutrients, sunlight) affecting weed suppressive ability.
Other research indicates species like yellow serradella, and biserrula may possess
phytotoxins produced by leachates from their residues, which suppress weed
establishment in the presence of specific soil microbiota.
The underlying mechanism of suppression is likely associated with metabolites produced
by plants or microbes.
Generally, the three years of research across the two trial sites showed weed suppression
was positively correlated with pasture biomass (except in the case of Santorini).
284
Arrowleaf and biserrula Casbah were generally the most consistent annual pasture
legumes with respect to productivity, yearly regeneration and suppression of annual
pasture weed species. Both arrowleaf clover and biserrula significantly reduced light
interception at the base of the canopy and had higher leaf area index than other pasture
species, potentially contributing to reduced weed-seed germination. Other research
supports that both leaf area index and pasture biomass contribute to weed suppression.
Previous studies have shown, that early pasture biomass and root-system establishment
contributed to weed suppression by competition for resources and/or the release of
secondary metabolites by the pasture species in the rhizosphere. However, because of
seasonal factors in the three years of this research, early autumn verses early winter
pasture establishment had little impact on pasture species weed suppression. Additional
research is now under way to confirm the mechanisms driving enhanced weed
suppression by pasture legumes.
In general, the rapid establishment of pasture species, as well as optimal production of
biomass, contributes to weed suppression the authors conclude.
Weed species assessed in the research included barley grass, sow thistle, poppy, ryegrass,
fumitory, capeweed and Paterson's curse.
Pasture weeds have been estimated to cost the Australian pastoral industry $2.2 billion
annually in management expenses and yield loss.
Winter pasture weeds are an important part of this annual loss and hence the importance
of this research.
285