Adaptation of Grass Pea (Lathyrus Sativus Cv

Grass pea (Lathyrus sativus cv. Ceora) − adaptation to water deficit and benefit in crop rotation

MARCAL GUSMAO M.Sc. School of Earth and Environmental Sciences Faculty of Sciences The University of Adelaide

This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia Faculty of Natural and Agricultural Sciences School of Plant Biology and The UWA Institute of Agriculture

December 2010

i Abstract

Grass pea (Lathyrus sativus cv. Ceora) is a multipurpose grain legume with an indeterminate growth habit. Adaptation of grass pea to water deficits and its potential rotational benefits in the Mediterranean-type environment of southern Australia are not well understood.

The first objective of the thesis was to identify adaptation mechanisms of grass pea to water deficits. This was done by imposing water deficit during the reproductive period on plants grown in pots in a glasshouse. In the first experiment, a moderate water deficit was imposed on Ceora and a well-adapted field pea (Pisum sativum cv. Kaspa), by reducing soil water content from 80 to 50% field capacity (FC) during seed filling. Water deficit decreased pre-dawn leaf water potential (Ψ) of Ceora and Kaspa, as well as stomatal conductance (gs) of Ceora, but no reduction in photosynthesis occurred. Water deficit reduced green leaf area of Ceora resulting in 30 and 24% reduction in plant dry mass and seed yield at maturity, respectively. Seed size and harvest indices (HI) of Ceora did not differ between the treatments. Ceora produced more dry matter than Kaspa in both treatments, but produced 22 (control) and 33% (water deficit) lower seed yields. Kaspa had higher HI and water use efficiency for grain than Ceora.

In the second experiment, severe water deficit was imposed on Ceora plants by withholding water from first flowering until Ψ fell to -3.12 MPa, when the plants were rewatered. At maturity, dry matter, seed yield and harvest index decreased by 60%, 87% and 67%, respectively, compared with the control. Flower production stopped at Ψ -1.8 MPa. At Ψ=-1.5 MPa, only 25% of the total flowers produced filled pods (compared with 95% in the control) and the rest aborted as flowers (48%) and pods (27%). Filled pods had more aborted ovules resulting in 29% less seeds per pod than the control. Water deficit reduced pollen viability (from 88 to 75%) and germination (from 53 to 28%) compared with the control. Of the germinated pollen, pollen tubes reaching the ovary were reduced by water deficit from 70 to 39% compared with the control. Seed size did not differ between the treatments.

A second objective of the thesis was to assess the effect of a grass pea crop on soil N and P availabilities, and the persistence of this effect over the summer period. This was done by comparing growth, dry matter and N and P uptake of wheat plants in soil collected from plots previously grown to grass pea or wheat at Cunderdin in the

ii Western Australian grain belt. Soil from each plot was sampled monthly from Nov 2008 to Feb 2009. Wheat was grown in pots containing these soils with no fertiliser (control), with N or P, or with N and P combined. After four weeks in a controlled environment room, with pots well-watered at 75% FC, wheat after grass pea produced greater dry mass (12%) and green area (16%) than wheat after wheat. The addition of N or combined N and P fertilisers reduced the beneficial effect of grass pea to the subsequent wheat crop. Wheat shoot N content was higher after grass pea than after wheat. Wheat shoot P content was higher after grass pea than wheat at the first sample date, but the opposite occurred at the last sampling.

This study showed that under moderate water deficit, grass pea avoids dehydration through a reduction in green leaf area and stomatal conductance. This enables plants to maintain the water status and photosynthesis of the remaining green leaves to support seed yield. High seed loss under severe water deficit was due to the cessation of flower production, reduced pollen fertility and high flower, pod and ovule abortion. Nonetheless, grass pea was able to produce some seed by concentrating limited resources to a smaller number of viable pods. The plants also matured early to escape drought. These adaptation strategies are important in southern Australia where rainfall in the growing season is variable and terminal drought is a common feature. Growing wheat after grass pea resulted in increased growth and N uptake compared with wheat after wheat. Thus, use of grass pea in the rotation could enhance soil N availability and growth and yield of subsequent cereal crops.

iii Table of Contents

Content Page

Abstract ………………………………………………………………………….. ii

Acknowledgements ……………………………………………………………... ix

Statement of candidate contribution ………………………………………...... xi

Chapter 1. General introduction ………………………………………………. 1

Chapter 2. Literature review 2.1 Introduction ………………………………………………………………...... 5 2.2 Taxonomy and common names of grass pea (Lathyrus sativus) …………...... 5 2.3 Description of L. sativus ……………………………………………………... 5 2.4 Origin and distribution of L. sativus …………………………………………. 6 2.5 Uses, production, toxicity and detoxification of L. sativus ………………...... 7 2.6 Environmental effect on ODAP concentration and breeding for low ODAP concentration of L. sativus …………………………………………………… 11 2.7 Achievement and recommendations for new cultivar of grass pea developed in Western Australia ………………………………………………………….. 12 2.8 Plant-soil water relations …………………………………………………….. 12 2.9 Crop adaptation to water deficits …………………………………………...... 14 2.10 Morphological adaptation mechanisms …………………………………...... 15 2.11 Physiological adaptation mechanisms to water deficits …………………..... 18 2.12 The effect of water deficit on yield, yield components and harvest indices… 22 2.13 Water use and water use efficiency (WUE) ………………………………… 24 2.14 The effect of legumes on growth, N and P uptake and yield of subsequent cereal crops ………………………………………………………………….. 26 2.15 Conclusion ………………………………………………………………...... 28

Chapter 3. Grass pea avoids dehydration and escapes drought under moderate water deficit during reproductive period Abstract …………………………………………………………..………………. 29 3.1 Introduction ………………………………………………………………...... 30 3.2 Materials and methods ……………………………………………………...... 33 3.3 Results ……………………………………………………………………...... 37 3.4 Discussion ……………………………………………………………………. 47 3.5 Conclusion …………………………………………………………………... 53

Chapter 4. Grass pea tolerates severe water deficit and sets normal sized seed when the deficit is relieved Abstract ……………………………………………….………..………………… 54 4.1 Introduction ………………………………………………………………...... 54 4.2 Materials and methods ……………………………………………………..... 56 4.3 Results ……………………………………………………………………...... 62 4.4 Discussion …………………………………………………………………… 77 4.5 Conclusion …………………………………………………………………... 85

Table of Contents (Continued)

Content Page

Chapter 5. Grass pea enhances growth and N uptake of a subsequent wheat crop Abstract ……………………………………………………………..……………. 86 5.1 Introduction ………………………………………………………………...... 86 5.2 Materials and Methods ……………………………………………………...... 89 5.3 Results ……………………….……………………………………………...... 92 5.4 Discussion ……………………………………………………………………. 98 5.5 Conclusion …………………………………………………………………… 102

Chapter 6. General discussion 6.1 Introduction ………………………………………………………………...... 103 6.2 Adaptation of grass pea to water deficits …………………………………….. 103 6.3 Soil N and P availability under grass pea crop ………………………………. 108 6.4 General conclusion …………………………………………...……………… 109

References ……………………………………………………………………...... 110

Appendices ………………………………………………………………………. 124

v List of Figures

Figure Page

Figure 3.1 Daily maximum and minimum growing season glasshouse temperatures (oC) ………………………………………………………………… 37

Figure 3.2 Cumulative transpiration of Ceora (LS) and Kaspa (FP) ……………. 39

Figure 3.4 Stomatal conductance and net photosynthesis for Ceora (a, c) and for Kaspa (b, d) ………………………………………………………...... 41

Figure 3.5 Osmotic potential for Ceora (a) and for Kaspa (b) ……………...... 42

Figure 3.6 Plant height and node number for Ceora (a, c) and for Kaspa (b, d) ……………………………………………………………………………………. 42

Figure 3.7 Green leaf area development and dry matter production for Ceora (a, c) and for Kaspa (b, d) …………………………………………………………… 44

Figure 3.8 Root dry matter for Ceora (a) and Kaspa (b) …………………...... 44

Figure 3.9 Dry matter partitioning (g plant-1) of Ceora and Kaspa ………...... 45

Figure 4.1 Daily maximum and minimum growing season glasshouse temperatures (oC) ...... 62

Figure 4.2 Pot weight during the water deficit treatment ……………………….. 63

Figure 4.3 Pre-dawn leaf water potential (Ψ) ……………………………...... 64

Figure 4.4 Relationship between pre-dawn Ψ and gravimetric soil water content during the treatment period (82 to 100 DAS) ……………………...... 64

Figure 4.5 Growth of grass pea: a) at first flowering (82 DAS) when water stress was imposed, b) difference between control and water deficient plants at the lowest Ψ (-3.12 MPa) (100 DAS) just prior to rewatering, and c) appearance 6 days after rewatering (Ψ = -1.4 MPa) (106 DAS) when regular watering (to 80% FC) was applied ………………………………………………………………….. 65

Figure 4.6 The effect of withholding water from 82 to 100 DAS, and then rewatering, on plant height and number of nodes measured on main stem and tillers ……………………………….………………………….…………………. 66

Figure 4.7 Dry matter production and partitioning ………………………...... 67

vi List of Figures (Continued)

Figure Page

Figure 4.8 Cumulative numbers of (a) flowers, (b) aborted flowers, (c) filled pods and (d) aborted pods per plant between water deficient and control plants ... 69

Figure 4.9 Cumulative numbers of (a) aborted ovules, (b) aborted seeds and (c) seeds per plant between water deficient and control plants ………...... 70

Figure 4.10 Mature pods from (a) control and (b) water deficient plants, and an example of a pod with (c) normal and aborted seeds and an aborted ovule ……... 71

Figure 4.11 Weight of mature seed as a function of flowering time ……...... 71

Figure 4.12 Percentage of flower abortion (FA), pod abortion (PA), and pod set (PS) for (a) control and (b) water deficient plants expressed as a percentage of total flower number, and within the retained flowers, the percentage of ovule abortion (OA), seed abortion (SA) and seed set (SS) for (c) control and (d) water stressed plants expressed as a percentage of total ovule number ………………... 72

Figure 4.13 Flower development at 92 DAS (pre-dawn Ψ =-1.8 MPa) ………… 74

Figure 4.14 Pollen viability and germination in vitro observed at 92 DAS for (a) control and (b) water deficient plants ……………………………………………. 75

Figure 4.15 Percentage of (a) viable pollen and (b) germinated pollen in in vitro assessments ………………………………………………………………………. 76

Figure 4.16 Pollen tube present (a) number of pollen tubes in the style and (b) percentage of pollen tubes in the ovary for (FC–MC) female and male control plants and (FC–MS) female control and male water deficient plants ……………. 76

Figure 4.17 Percentage of ovule and seed abortion and seed set for (a) FC–MC and (b) FC–MS ………………………………………………………...... 76

Figure 4.18 Cumulative emergence of seeds derived from water deficient (WD) and control (WW) plants …………………………………………………………. 77

Figure 5.1 Wheat dry mass (a) and green area (b) (per plant) following wheat (W) or grass pea (G) as the previous crop, without N (0N) or P (0P) or with N (1N) or P (1P) fertiliser …………………………………………………………... 93

Figure 5.2 Wheat shoot N (a) and P (b) concentrations of wheat grown in soils from plots with wheat (W) or grass pea (G) in the previous crop, and without N (0N) or P (0P) or with N (1N) or P (1P) fertiliser ………………………………... 95

Figure 5.3 Wheat shoot N (a) and P (b) contents of wheat grown in soils from plots with wheat (W) or grass pea (G) in the previous crop, and without N (0N) or P (0P) or with N (1N) or P (1P) fertiliser ……………………………………... 96

vii

List of Tables

Table Page

Table 2.1 Common names of L. sativus from several countries around the world 6

Table 2.2 Physiological and morphological adaptation mechanisms to water deficits; adapted from Begg and Turner (1976) and Turner and Begg (1981) …... 15

Table 2.3 Variation in harvest indices of grain legumes observed in some growing areas of Western Australia and in glasshouse studies ………………….. 25

-1 -1 -1 Table 2.4 Water use efficiency (kg ha mm ) for dry matter (WUEdm) and grain (WUEgr) of various prostrate grain legumes at two contrasting growing sites, Merredin and Mullewa, Western Australia ………………………………... 26

Table 3.1 Key stages of phenological development (days after sowing) and seed filling duration (days) of Ceora and Kaspa ………………………………………. 38

Table 3.2 Yield, yield components and harvest index of Ceora and Kaspa at maturity …………………………………………………………………………... 46

Table 4.1 Key stages of phenological development (days after sowing) and seed filling duration (days) ……………………………………………………………. 63

Table 4.2 Yield, yield components, harvest index, water use efficiency and ODAP concentration of seeds .…………………………………………………... 67

Table 5.1 Soil chemical properties measured near experimental plots ………….. 90

viii Acknowledgements

I gratefully acknowledge the Australian Centre for International Agricultural Research (ACIAR) for providing the John Allwright Fellowship to undertake my PhD studies at the School of Plant Biology, Faculty of Natural and Agricultural Sciences and The UWA Institute of Agriculture, The University of Western Australia. Thanks to Dr. Reinhardt Howeler at the International Centre for Tropical Agriculture (CIAT) who suggested me to apply for this Fellowship. I thank to the National University of East Timor (Universidade Nacional de Timor Leste - UNTL), East Timor for granting me a study leave. I also acknowledge the UWA Convocation Postgraduate Research Travel Award that allowed me to visit the International Centre for Agricultural Research in the Dry Areas (ICARDA), Syria and to present my study results at the 5th International Food Legume Research Conference (IFLRC) and 7th European Conference on Grain Legumes (AEP) in Antalya, Turkey in April 2010. I thank Centre for Legumes in Mediterranean Agriculture (CLIMA), UWA, for providing me with operating funds to undertake the experiments reported in the thesis. I also acknowledge The Australian Government’s Overseas Aid Program (AusAID) for providing me a grant for proof reading of my thesis.

I would like to express my heartfelt gratitude to my supervisors Prof. Erik J. Veneklaas, Winthrop Prof. Kadambot H. M. Siddique, Dr. Ken Flower, and Adjunct Prof. Harry Nesbitt. They provided significant contribution to my PhD through project formulation, experimental design, monitoring research activities, formulating results and thesis writing. Prof. Erik J. Veneklaas gave me in depth comments on both my English and thesis content that allowed me to think more critically in developing my thesis. I consider Prof Kadambot Siddique as a motivator and mediator of my PhD study. He always encouraged me to take every opportunity to discuss, develop my research, and progress on the thesis writing. Dr. Ken Flower helped me to set up a field trial which was also a part of my PhD research project. He also assisted and gave valuable statistical advice that helped me to interpret my experimental results. Adjunct Prof Harry Nesbitt helped me a great deal with English and general aspects of the thesis. Together, they gave an excellent support and guidance to my PhD research and university life. I also thank to Assoc/Prof David Turner for his valuable suggestions and comments on my thesis.

ix The work provided in this thesis would have been impossible without assistance of staffs, friends and colleagues, particularly at the School of Plant Biology, in carrying out the research. I would like to thank to Dr. Perry Swanborough for his assistance particularly on using Li-Cor-6400 for photosynthesis measurement. I also thank Dr. Tsun-Thai Chai for his assistance in pollen studies and Dr. Takami Saito and Mr. Raphael Bailly for their assistances in collecting soil from the field site. Ms. Mariana Cruz Campos and Ana Luiza Muler for their assistance in plant P measurement, Mr. Michael Smirk for his assistance in plant N measurement using CHN combustion analyzer and several friends and colleagues who also assisted me during my PhD are acknowledged. Thanks to Mr. John Murphy at the Centre for Microscopy, Characterisation and Analysis, UWA, who allowed me to use a microscope in his laboratory.

Last but not least, my heartfelt gratitude goes to my wife, Ms Lamdor T. Sitorus, my son Andre Gusmao and my daughter Eunike Gusmao who was born when I was very busy with my experiment. Even though I spent less time with them during my PhD, my wife always encouraged and motivated me to continue my work. She also assisted me in some glasshouse and laboratory work. I also thank to all my family members back in East Timor who I never visited during my PhD, but who always supported me to finish my PhD.

Statement of candidate contribution

The work presented in this thesis does not contain any material that has been accepted for award of any other degree or diploma at any other University.

I declare that this thesis is my own work and contains no material previously published or written by another person except where acknowledgement is made in the text.

Marcal Gusmao

xii Chapter 1

General introduction

The low 3- (-N-oxalyl)-L-2,3-diaminopropionic acid (ODAP) Ceora cultivar of grass pea (Lathyrus sativus) is a new multipurpose grain legume bred at the Centre for Legumes in Mediterranean Agriculture (CLIMA) at The University of Western Australia. It is a cross between a low ODAP (0.05%) male parent 8604 from Bangladesh and a high ODAP (0.43) female parent K33 from Pakistan (Hanbury et al. 2005; Siddique et al. 2006) for cultivation in the Mediterranean-type environment of southern Australia. Field trials show that grass pea is well-adapted to this region and has potential as a grain feed, forage, hay and green manure (Hanbury et al. 2005; Hanbury et al. 2000). Adaptation mechanisms of Ceora to water deficits and its potential benefits to following cereal crops are not fully understood. The general objective of this study is to investigate Ceora’s response to water deficits during reproduction and, to a lesser extent, its rotational benefit to a subsequent wheat crop.

The Mediterranean-type environment in Southtern Australia is characterised by a short, cool and wet winter from May to August and hot and dry conditions for the rest of the year. Annual rainfall ranges from 250 to 600 mm. Cultivation in this region relies on winter rainfall with seasonal crop production from May to October (Turner 1992). Most annual cereal and grain legume crops are in the reproductive stage in September (spring) when terminal drought occurs (Turner 1992). Increasing soil water deficits in spring due to little or no rainfall, accompanied by higher air temperatures and evaporation, affect growth, reproductive development and yields of drought-sensitive annual crops. Terminal drought has been shown to decrease seed yield in other crops. For example, in chickpea, grain yield was reduced by up to 80% compared with the irrigated control (Leport et al. 1999), due to high flower and pod abortion (Leport et al. 2006) in response to water deficit. High flower and pod abortion has been associated with reduced pollen and ovule fertility (Fang et al. 2010). Moreover, plants surviving a period of water deficit have poor seed set, less seeds per pod and smaller seed size which all contribute to low grain yield (Turner et al. 2005).

An important adaptation to increasing water deficit during reproduction is the plant's ability to escape drought. Drought escape enables plants to complete their reproductive cycle before severe water deficit (Begg and Turner 1976; Chaves et al. 2003) and is

1 particularly important in the Mediterranean-type environment of southern Australia. Therefore, it is essential to grow species or genotypes which are fast growing, flower early, pod and mature before the onset of water deficit (Ludlow and Muchow 1990). Flower development in some plants, chickpea for example, is sensitive to low temperatures early in the season, reducing early growth potential (Croser et al. 2003); although some progress has been made towards better adaptation of this crop to low temperature (Clarke et al. 2004) to ensure drought escape. The importance of drought escape has been demonstrated in, for example, wheat in the Mediterranean-type environment of southern Australia (Siddique et al. 1990), chickpea in the drought-prone environment of India (Berger et al. 2006) and in common beans (Rosales-Serna et al. 2004). Certainly, early species that produce more leaf area when soil water is still adequate intercept more radiation (but experience less evaporation) leading to high dry matter production at anthesis. Dry matter partitioning (remobilisation of assimilated reserves) is important for seed filling under water deficit. This has been demonstrated in cereals (Yang and Zhang 2006) such as wheat cultivars (Yang et al. 2001) and barley genotypes (Austin et al. 1980) and in grain legumes such as chickpea (Davies et al. 2000) where remobilisation enhanced seed filling and harvest index (Yang and Zhang 2006). Early flowering and seed set also enables the plant to use water more efficiently post-anthesis resulting in high harvest indices (Siddique et al. 1990).

There are also physiological and morphological strategies of adaptation that enable plants to maintain high water status (dehydration avoidance) under water deficit (Begg and Turner 1976; Blum 2005; Turner and Begg 1981). There are two major physiological adaptations: reduced stomatal conductance and osmotic adjustment (Turner and Begg 1981). A reduction in stomatal conductance in response to water deficit is the first step in the adaptation of a plant to maintain high water status (Stoddard et al. 2006), and has been observed in all species investigated to date, such as soybean (Glycine max) (Lawn 1982b), white lupin (Lupinus albus L.), chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), field pea (Pisum sativum L.), grass pea (Lathyrus sativus L.) and lentil (Lens culinaris Med.) (Leport et al. 1999; Leport et al. 1998). For example, Leport et al. (1998) observed a reduction in stomatal conductance of grain legumes when leaf water potential (Ψ) dropped below -0.9 MPa. This reduction was associated with stomatal closure which was linked to increased concentrations of abscisic acid (ABA) in leaves (Comstock 2002; Davies and Zhang 1991; Turner et al. 2001). There is also evidence that water deficit increases osmotic adjustment associated

2 with increased solutes in plant tissues (Chaves and Oliveira 2004; Lambers et al. 2008). It is reported that plants with higher osmotic adjustment have the ability to maintain higher plant water status (Blum 2005).

Two morphological adaptations that enable plants to maintain high plant water status in response to water deficits are reductions in green leaf area and the extraction of soil water from deeper in the soil. Reduction in leaf expansion in response to water deficit (Boyer 1970; Hsiao 1973; Wery 2005) is the first contributor to a reduction in green leaf area. Two other contributors are reduced rates of leaf production as observed in bean (Boutraa and Sanders 2001) and accelerated leaf senescence (Morgan 1984; Turner and Begg 1981). Plants with less green leaf area use less water and therefore maintain high water status. It is also recognised that plants under water deficit extending their roots deeper into the soil profile to obtain more soil water (Begg and Turner 1976) to avoid dehydration. As a result, root dry matter and root/shoot ratio increase (Blum 1996; Rodrigues et al. 1995; Stoddard et al. 2006).

Ceora appears to possess both drought escape and dehydration avoidance strategies in response to water deficit. Earlier lines of L. sativus, introduced directly from different countries of origin (Siddique et al. 1996), flowered late in the season. Siddique et al. (1996) reported that although these lines had similar dry matter production near flowering compared with other grain legumes in a favourable environment in Western Australia, they had the lowest grain yield (1.6 t ha-1) compared with L. cicera (2.6 t ha- 1), L. ochrus (1.7 t ha-1) and field pea (3.1 t ha-1). It was suggested that the low grain yield and harvest index may have been due to water deficit late in the season. Ceora is reported to reach flowering four weeks earlier than previous lines (Siddique et al. 1996) suggesting that it is better adapted to the low to medium rainfall environments of Western Australia. Field experiments at Merredin and Mullewa, Western Australia, reported that Ceora yielded higher than previous lines (Siddique et al. 2001). Just as importantly, Ceora maintains its low concentrations of the toxin β-N-oxalyl-L-α-β- diaminopropionic acid known as ODAP which is a problem for some cultivars of Lathyrus in other countries. Grass pea has also been shown to avoid drought by delaying leaf senescence and maturity (Leport et al. 1998; Thomson and Siddique 1997). This was possibly associated with the extraction of soil water from deeper in the profile through root extension (Begg and Turner 1976; Blum 1996; Sinclair and Muchow 2001).

The benefits of rotating grain legumes and cereals in low to medium rainfall environments has been known for some time (Siddique and Sykes 1997; Turner 2004). However, some legume species are hindered by the presence of duplex or contrasting soil textures in agricultural areas in southern Australia (Turner 1992). For example, soils with low hydraulic conductivity tend to waterlog during winter, thereby impeding growth and development of lupins (Belford et al. 1992). Narrow-leafed lupin is poorly adapted to such conditions (Siddique et al. 1993). The introduction of new grain legume cultivars adapted to this adverse environment will provide more options for farmers to improve productivity (Siddique et al. 1996). A range of grain legumes is required in these cropping rotations to reduce disease build-up. Moreover, grain legumes can improve the soil by increasing soil organic matter, enhancing biological activity, enhancing soil fertility with biological nitrogen (N) (Adu and Oades 1978; Evans et al. 2001; Peoples et al. 1995a; Rochester et al. 1998; Siddique and Sykes 1997) and remobilisation of residual phosphorus (P) (Ae et al. 1990; Neumann and Römheld 2000; Nuruzzaman et al. 2005b; Nuruzzaman et al. 2006; Veneklaas et al. 2003). Such soil conditions enhance the growth and productivity of subsequent cereal crops. Grass pea is one good option as a legume break crop because it is adapted to both waterlogging and drought (Campbell 1997). The cultivar Ceora is particularly suitable as it is already regarded as being adapted to the Mediterranean-type environment of southern Australia (Hanbury et al. 2005; Siddique et al. 1996; Siddique et al. 1999).

This study aimed to (i) identify adaptation mechanisms of Ceora to water deficits during the reproductive period and (ii) assess N and/or P benefits from Ceora crops to subsequent wheat growth, and whether these benefits increased or decreased over summer between growing seasons.

4 Chapter 2 Literature review

2.1. Introduction This review on grass pea (Lathyrus sativus) provides background information on its taxonomy, description, origin and distribution, uses and production, toxicity associated with 3-(N-oxyalyl)-L-2,3-diaminopropionic acid (ODAP) and environmental effects on ODAP concentration. As a basis for this study, more detailed information is provided on plant-soil water relations, crop adaptation to water deficits, the effect of water deficits on yield and yield components, water use and water use efficiency, and the role of L. sativus in N fixation and P mobilisation on the growth and yield of a subsequent wheat crop. There is little published information on the effect of water deficit on growth and development of L. sativus and its potential benefits in a rotation with cereal crops; hence relevant information from other grain legumes and cereals is included as background.

2.2. Taxonomy and common names of L. sativus The Lathyrus genus is large with 187 species and subspecies, and belongs to (Campbell 1997): Family : Leguminosae (=Fabaceae) Subfamily: Papilionoideae Tribe : Vicieae Among Lathyrus species, L. sativus is grown worldwide—it is adapted to adverse environments and has high nutritional value for humans and animals (Campbell 1997; Erskine et al. 2001; Sarker et al. 2001). As it has been used as a crop for thousands of years in many different parts of the world, L. sativus has many local names (Table 2.1).

2.3. Description of L. sativus L. sativus is a herbaceous indeterminate annual plant with a branched, weak-stemmed, prostrate growth habit (Campbell 1997; Hanbury et al. 2005). Early in development, seedlings of L. sativus form a number of branches from the base of the plant, and later, also during reproductive growth, further branches are formed from the main stem. Once flowering starts, one flower is formed for every new node on the stem. Time to first flowering varies between grass pea germplasm ranging from 47 to 94 days after sowing (DAS) (Campbell 1997). In a field experiment in southern Australia, L. sativus started flowering between 90 and 110 days after sowing and ended between 120 and 145 DAS

5 (Siddique et al. 1999). Podding started between 10 and 16 days after flower bud appearance, indicating that L. sativus has a long period of flowering, podding and seed filling. Therefore, water deficit at early flowering, pod set or seed set may potentially affect growth and reproductive development. The plant has a well-developed tap root system; rootlets are usually covered with small, cylindrical, branched nodules, which often cluster in dense groups (for a more detail description see Campbell (1997)).

Table 2.1 Common names of L. sativus from several countries around the world. Country Local name Bangladesh Khesari Burma Pe-kyin-baung, pe-sa-li, muter pea China San lee dow Cyprus Foveta, pharetta, dog-toothed pea Ethiopia Sabberi, guaya France Lentille d’Espagne, pois carré, gesse blanche, gesse chichi, gesse commune, gessette Germany Saatplatterbse Nepal Khesari India Kesare, khesari, karas, karil, kasar, khesari dhal, khesra, lang, chural, latri, lakhori, lakhodi, chattra matur, santal, teora, tiuri, batura, chickling vetch, chickling pea Italy Cicerchia coltivata, pisello, bretonne, pisello cicerchia Pakistan Matri, mattra Spain, Portugal, Chicharo Latin-American countries Sudan Gilban(eh) Source: Adapted from Campbell (1997)

2.4. Origin and distribution of L. sativus Grass pea has a long history of cultivation for human consumption and forage in a range of geographical and environmental conditions, and it remains popular today. Despite its popularity, the origin of grass pea (Lathyrus species) remains ambiguous. Archeological records of eastern Mediterranean pulses indicate that Lathyrus sativus was found in the Balkan Peninsula (Campbell 1997; Kislev and Bar-Yosef 1988) and L. cicera was found in eastern Europe (Kislev and Bar-Yosef 1988). Recent archaeobotany of Indian pulses suggests that L. sativus originated in south-west Asia (Colledge et al. 2005; Fuller and Harvey 2006; Smartt 1990) along with other pulses such as pea (Pisum sativum), lentil

6 (Lens culinaris), bitter vetch (Vicia ervilia) and chickpea (Cicer arietinum). L. sativus then dispersed to the eastern Mediterranean region, SE Europe, reaching Britain and Scandinavia (Colledge et al. 2005). The distribution was assumed to be related to domestication and farming practices in the Near East (Campbell 1997; Kislev and Bar- Yosef 1988). Kislev and Bar-Yosef (1988) argued that the distribution of pulses including L. sativus occurred because: ...their nutritional value, timing of availability, and accessibility would have made them ideal candidates for gathering and later for cultivation by the latest Levantine hunter-gatherers or earliest farmers so called the legumes: the earliest domesticated plants in the near east.

It is also argued that L. sativus in India was related to the diffusion of crops from West Asia (Campbell 1997; Jackson and Yunus 1984). Some wild types of L. sativus species were reported from Iraq and Iran in a different time period (Campbell 1997; Fuller and Harvey 2006; Jackson and Yunus 1984), but it remains unclear whether they were wild or escaped from cultivation (Campbell 1997).

Robertson and Abd-El-Moneim (1999) showed the distribution of Lathyrus germplasm accessions held at the International Centre for Agricultural Research in Dry Areas (ICARDA), being L. sativus, L. cicera and L. ochrus. Of these, L. sativus is the most widely grown. The most L. sativus accessions were recorded in Bangladesh (1,115) followed by 178 accessions in Ethiopia, 83 in Palestine, 76 in Pakistan and a small number recorded elsewhere (Robertson and Abd-El-Moneim 1999).

2.5. Uses, production, toxicity and detoxification of L. sativus 2.5.1 Uses L. sativus is a multipurpose grain legume crop. It can grow in both drought- and flooding-prone environments and has high nutritional value (Erskine et al. 2001; Patto et al. 2006; Sarker et al. 2001). L. sativus seeds are high in protein (25–30%) which is important for humans and animals. Cultivar Ceora has a protein content of approximately 30% (Siddique et al. 2006). L. sativus is used as feed grain, forage, green manure and for human consumption (Abd-El-Moneim et al. 1999; Abd-El Moneim et al. 2001; Campbell 1997; Chowdhury et al. 2005; Cocks et al. 2000; Erskine et al. 2001; Gizachew and Smit 2005; Hanbury et al. 2005; Hanbury et al. 2000; Malek et al. 1996; Peña-Chocarro and Peña 1999; Sarker et al. 2001; White et al. 2002; Yang and Zhang 2005). In addition, L. sativus can be used to sustain production of other grain crops through intercropping, rotations and relay cropping systems (Section 2.14).

In Western Australia, L. sativus is regarded as a potential source for green feed, fodder, hay and green manure as it is well adapted to the low-to-medium winter rainfall zone (250–500 mm per year) of southern Australia (Hanbury et al. 2005; White et al. 2002). Similar potential uses of L. sativus exist in Europe and USA. Meanwhile, in other parts of the world such as the Indian sub-continent, China and Ethiopia, L. sativus is grown mainly for human consumption in addition to green feed, fodder, hay and green manure (Campbell 1997; Erskine et al. 2001; Malek et al. 1996). Campbell (1997) reported that in Pakistan, especially in the province of Sind, 60% of seed was used for forage and 40% for human consumption. Meanwhile, in the Indian sub-continent and Ethiopia, L. sativus can sometimes be considered as a survival crop when others fail to produce a harvestable yield due to either drought or flooding (Smartt 1990). This crop is now considered one of the important food legumes cultivated in the highlands of Ethiopia (Butler et al. 1999) and in Bangladesh (Abd-El-Moneim et al. 1999; Abd-El Moneim et al. 2001; Malek et al. 1996).

2.5.2 Cultivation and production The area devoted to grass pea cultivation varies in different parts of the world. In Ethiopia, where L. sativus is an important food legume in the highland areas, the area sown to grass pea was about 9% of the total area used for grain legume production up to 1999 (Butler et al. 1999; Teklehaimanot et al. 1993) and may have increased more recently (Patto et al. 2006). In Bangladesh, this crop is even more important. Malek et al. (1996) estimated that, in this country, during the late 1980s and early 1990s, grass pea occupied 36% of total area under grain legumes.

Grass pea grain yields are variable depending on variety, ultimate crop use and cropping system. In Ethiopia, where the crop is grown as a food crop, under favourable conditions, L. sativus grain yields for local cultivars were 3.7 t ha-1 and for improved cultivars, 4.2–5.2 t ha-1 (Tsegaye et al. 2005). A similar seed yield was also reported for a new large-seeded cultivar Luanco-INIA in Chile (Mera et al. 2003). In Bangladesh, the average grain yield observed before 1996 was 0.73 t ha-1 and contributed 34% of grain produced from legumes (Malek et al. 1996). This yield was still noted in 2000 (Rahman et al. 2001). Grain yield of 17 lines of L. sativus from ICARDA grown in three agro-climatically different sites in the Mediterranean-type environments of southern Australia ranged from 0.72 to 1.56 t ha-1 at Northam, 0.24 to 0.68 t ha-1 at

8 Merredin and 0.24 to 0.75 t ha-1 at Mullewa (Siddique et al. 1996). Yield of improved grass pea lines observed at Tel Hadya, Aleppo in Syria, ranged from 0.8 to 1.6 t ha-1 (Kumar et al. 2010). Kumar et al. (2010) also showed a high variation in seed size of improved grass pea varieties ranging from 0.06 g per seed for variety Bari in Bangladesh to 0.19 g per seed for variety Krab in Poland. Interestingly, the higher seed size of 0.20 g per seed was found from plants that had experienced severe water deficit (Herwig 2001).

Grass pea is also grown as a fodder crop. A high yield of fodder of 7–10 t ha-1 was reported for L. sativus when intercropped with maize (Campbell 1997). A 6-year study on legumes as green manure in dryland cropping systems found that L. sativus yielded 2.2 t ha-1 vegetative growth and concluded that this crop was one of the most suited to green manure in semi-arid climates (Biederbeck et al. 1993).

Production systems that include grass pea vary in different parts of the world. In Bangladesh, grass pea is grown as a relay crop with rice. Seeds of grass pea are simply broadcast into standing rice one or two weeks before harvesting. Herwig (2001) reported a similar cultivation method used in India, Nepal and Pakistan. In Spain, both L. sativus and L. cicera are grown in winter, but only L sativus is grown in summer due to its potential for human consumption (Peña-Chocarro and Peña 1999). L. sativus and L. cicera are also considered multipurpose grain legumes that adapt well to a range of environments in the grain belt of Western Australia (Hanbury and Siddique 1998; Hanbury et al. 2005; Hanbury et al. 2000; Leport et al. 1998; Siddique et al. 1996; Siddique et al. 2001). In this region, L. sativus has potential for rotation with cereal crops (Hanbury et al. 2005). Hanbury et al. (2005) reported that L. sativus in rotation will act as a break crop for root- and stubble-borne diseases of cereals, weed control and nitrogen fixation, and also produce a good yield.

2.5.3 Toxicity L. sativus contains the toxin 3-(N-oxyalyl)-L-2,3-diaminopropionic acid (ODAP) in the grain. This toxin causes lathyrism in humans and livestock. Lathyrism is a neurological disorder causing irreversible paralysis when humans consume grain in large quantities over prolonged periods (Hanbury et al. 2005; Yan et al. 2006). The disorder is endemic in countries of the Indian-subcontinent and Ethiopia where L. sativus is commonly grown (Hanbury et al. 2000).

2.5.4 Human consumption and detoxification The presence of ODAP at toxic concentrations in the grains of L. sativus has caused some governments like India (in parts of the country) to discourage human consumption of this crop by prohibiting its trade (Hanbury et al. 2005; Smartt 1990). Smartt (1990) claimed that preventing this crop from being cultivated and consumed was detrimental in some cases where flood or drought caused starvation due to a lack of alternative food sources.

There are a number of ways to detoxify pods and grain of L. sativus for consumption. One method is to steep grain in a double quantity of water for 8 hours at 60–70oC, changing the water seven times, followed by sun drying. This method reduces neurotoxins by up to 93% (Salim ur et al. 2008). Some consumers also dilute the toxin by mixing water-soaked grass pea with non-toxic cereals (Dr Shiv Kumar, pers comm. 2010). This method is being used in some parts of Spain (Peña-Chocarro and Peña 1999).

Campbell (1997) described some common practices in Asia for processing L. sativus before consumption. In India, grains are occasionally boiled whole, but more often processed through a dal mill to obtain a split dal, a soup-like dish. Recently, grass pea dal has been used to adulterate pigeon pea dal and chickpea dal at a proportion of 1:4 which can reduce ODAP concentration in the consumed product by 80% (Kumar et al. 2010). In many parts of Bangladesh, roti (unleavened bread), made from grass pea and flour, is a staple for landless labourers. Dal is also made in Nepal and eaten with rice, while flour made from grains is used for badi or pakoda (pancake-like). Kitta, a kind of unleavened bread made from grass pea, is mostly consumed when there are food shortages. Roasted or boiled grass pea is consumed as a snack in most areas. Young pods are also boiled, salted and sold or consumed as a snack in many parts of South Asia.

Recently, a study based on household individual and dietary risk factors for neurolathyrism found that soaking grass pea seeds before cooking reduced the risk of neurolathyrism by 50% or more (Getahun et al. 2005). In contrast, the risk of neurolathyrism increased 10-fold or more when green-boiled pods or seeds of L. sativus were consumed. This observation revealed that some traditional (cooking) methods

10 described earlier are inappropriate and may lead to neurolathyrism. The findings also suggest the need for breeding and release of low and stable toxin cultivars to minimise toxicity risks (Kumar et al. 2010).

2.6. Environmental effect on ODAP concentration and breeding for low ODAP concentration of L. sativus 2.6.1 Environmental effect on ODAP concentration ODAP concentration of Lathyrus spp. especially L. sativus, varies widely across growth environment, genotype and species (Erskine et al. 2001; Kumar et al. 2010). For example, ODAP concentrations of L. sativus studied at three contrasting agro-climatic environments of Western Australia ranged from 0.12% to 0.54% for two grass pea accessions grown at three locations (Siddique et al. 1996). The ODAP concentrations of grass pea lines (bred for high yield and low ODAP concentration) at Tel Hadya, Syria, ranged from 0.09% to 0.27% (Kumar et al. 2010). A study of genotype-environment interactions for seed yield and ODAP concentration of L. sativus and L. cicera in the Mediterranean-type environment of southern Australia demonstrated that the strongest determinant of ODAP concentration was genotype, and that it is less likely to be affected by environment (Hanbury et al. 1999). Nonetheless, a glasshouse study observing the effect of water deficit on ODAP concentration of grass pea lines 526 and 80392 from Ethiopia and 455 from Germany indicated that severe water deficit increased ODAP concentration by 33−52% compared with the control which was associated with reduced number of pods per plant (Herwig 2001). This relationship was also observed in a field study conducted in Lanzhou, China where water deficit increased seed ODAP concentration by 12 to 20% (Yang et al. 2004). Increased ODAP concentrations in grain may be associated with increased abscisic acid concentrations in response to water deficit (Xiong et al. 2006).

2.6.2 Breeding programs Breeders at different institutions have investigated the development of new varieties with low ODAP concentration. Erskine et al. (2001) suggested that this could be achieved using a combination of biotechnology and interspecific hybridisation. Some Lathyrus genotypes and cultivars with low concentrations of ODAP have been bred and are commercially available for cultivation and marketing for consumption. Kumar et al. (2010) summarised improved varieties of grass pea for cultivation in different countries.

11 2.7. Achievement and recommendations for new cultivar of grass pea developed in Western Australia 2.7.1 Achievements After confirming in field trials that grass pea was well-adapted and produced good seed yields in the Mediterranean-type environments of southern Australia (Siddique et al. 1996; Siddique et al. 1999), the cultivar Ceora of L. sativus, was developed for low ODAP concentration at the Centre for Legumes in Mediterranean Agriculture (CLIMA), The University of Western Australia (UWA). Low ODAP concentration was obtained by cross-breeding a low ODAP (0.05%) accession from Bangladesh and a high ODAP (0.43%) accession from Pakistan (Hanbury et al. 2005). Testing for ODAP concentration and yield over three years found that grain ODAP concentration remained low at 0.04%, 0.09%, and 0.05% in 1997, 1998 and 1999, respectively and produced good seed yield. The Ceora cultivar was registered and released (Hanbury et al. 2005; Siddique et al. 2006) and was considered as a new multipurpose grain legume in this environment.

2.7.2 Recommendations Adaptation mechanisms of L. sativus cv. Ceora to water deficits during reproduction and its rotational benefits to subsequent cereal crops have not been documented. This thesis aimed to fill this gap.

2.8. Plant-soil water relations Plants are living media that absorb water from soil, store and release it to the atmosphere as water vapour. The storage and release of water by plants enhance biochemical and physiological activities leading to growth and development through enlargement of plant cells (Lambers et al. 2008; Vaadia et al. 1961). Evaporation of water from plant tissues, known as transpiration, exists when vapour pressure of air is lower than vapour pressure of plant tissues (Begg and Turner 1976; Lambers et al. 2008). This usually occurs during the daytime. The stomata of plant tissues are open allowing water to evaporate, but at the same time letting CO2 enter the tissues for photosynthesis. In photosynthesis, CO2 is synthesised into compounds that enhance growth and development. Maintaining plant water status at full turgor is therefore important to accommodate transpiration and photosynthesis.

12 It is well-known that plant water status depends on soil water availability (Kramer and Boyer 1995). The maximum plant available soil water is at field capacity. In non-saline soils, soil water potential at field capacity is around -0.01 or -0.03 MPa (Lambers et al. 2008). However, when the amount of available soil water decreases as a result of no rainfall or irrigation, it affects plant water status and therefore restricts the opening of stomata for transpiration. As a consequence, photosynthesis is limited along with growth, development and yield. Photosynthetic sensitivity to water deficit has been recently reported (Ghannoum 2009; Souza et al. 2004) (Section 2.11.1). Therefore, in water-limited arid and semi-arid climates, where both rainfall and irrigation are in limited supply, crops always experience drought which results in reduced yield (Section 2.12).

The amount of water retained in plant tissues indicates its water content. Plant tissue water content can be expressed as relative water content (RWC) or based on potential energy. RWC is defined as the ratio of current water content to water content at full saturation (RWC is 100% at full turgor). Plant water potential is defined as the pressure or energy applied to release water from tissues. At full turgor or saturation, total plant water potential is 0 MPa. Under equilibrium conditions, leaf and soil water potential is equal. However when transpiration exists, leaf water potential (Ψ) falls below soil water potential. This condition creates a water potential gradient between plant and soil. As water always moves from higher to lower potential, there is an upward movement of soil water into the plant. The rate of movement is determined by transpiration demand (Aston and Lawlor 1979; Lambers et al. 2008; Turner and Begg 1981; Vaadia et al. 1961). However, due to the liquid flow resistance in the pathway of plants tissues, it may not meet transpiration demand at its maximum thus water deficit in plant tissues occurs (Turner and Begg 1981) even though there is no deficit in soil water content. Turgidity recovers at night if there is no deficit in soil water content. Plants' ability to maintain tissue water content varies between species and genotypes depending on their adaptation to water deficit (Section 2.9), but most species may lose turgidity at soil water potentials between -1.5 and -2.0 MPa (or approximately 10 to 15% soil water content) when permanent wilting point occurs (Serraj and Sinclair 2002).

13 2.9. Crop adaptation to water deficits Crops that perform satisfactorily in growth and development in water-limited environments and hence produce comparatively high biomass and/or grain yield are defined as drought-resistant species. Drought resistance is generally recognised to occur in plants that are resistant to water deficits because of their ability to escape the development of severe deficits or to tolerate severe deficits (Begg and Turner 1976; Chaves et al. 2003; Levit 1972).

2.9.1 Drought escape Drought escape is defined as the ability of a crop to complete its growth cycle before onset of severe water deficit (Begg and Turner 1976; Chaves et al. 2003). The drought escape strategy involves rapid growth, development, increased assimilate reserves, early flowering, early pod and seed set, and earlier maturity. This growth habit may also provide good ground cover, thereby reducing the direct impact of radiation and evaporation, resulting in more soil water retention for transpiration and photosynthesis (Ludlow and Muchow 1990). For example, a comparative study on old and modern cultivars of wheat at Merredin, Western Australia, found that modern wheat cultivars grew faster, with more rapid leaf development which reduced soil evaporation compared with old cultivars (Siddique et al. 1990). Similar observations were reported for field pea and faba bean (Siddique et al. 2001), two grain legumes adapted to the Mediterranean-type environment of southern Australia. Plant species with rapid development characteristics have improved light interception and photosynthesis (Ludlow and Muchow 1990; Siddique et al. 1990) and radiation use efficiency (De Costa et al. 1999). Pre-anthesis assimilate reserves are particularly important for their contribution to grain filling when water deficit develops during the reproductive period as observed in barley genotypes (Austin et al. 1980) (Section 2.11.4).

The benefit of drought escape has been demonstrated for some grain legume species in southern Australia (Leport et al. 1998; Siddique et al. 2001; Thomson et al. 1997) and in drought-prone environments of India (Berger et al. 2006). Siddique et al. (2001) reported that crops with rapid growth and development, and early flowering, podding and seed set would have more chance of maturing before the onset of severe water deficit. To demonstrate this, Lawn (1982b) conducted a comparative study of four grain legumes—soybean (Glycine max CPI 26671), black gram (Vigna mungo cv. Regur), mung bean (V. radiata cv. Berken) and cowpea (V. unguiculata CPI 28215). He found

14 that mung bean (determinate species) escaped drought by flowering and maturing earlier (by one and 4 weeks, respectively) compared with other indeterminate species. Water deficit also accelerated seed filling and maturity of indeterminate species earlier than irrigated plants (Lawn 1982b). Similarly, by maturing earlier, upland rice gained a yield advantage in a water-deficit environment (Lafitte and Courtois 2002). Faba bean and field pea are considered well-adapted to cultivation in southern Australia because they can escape severe water deficit late in the season (Leport et al. 1998; Siddique et al. 1999; Siddique et al. 2001).

2.9.2 Drought tolerance Drought tolerance is defined as the ability of a crop to sustain its water status to avoid dehydration during water deficit or the ability of a crop to recover after desiccation at a certain water potential (Begg and Turner 1976; Blum 2005; Turner and Begg 1981). Plants tolerate drought using either physiological and/or morphological mechanisms (Table 2.2).

Table 2.2 Physiological and morphological adaptation mechanisms to water deficits; adapted from Begg and Turner (1976) and Turner and Begg (1981) Physiological mechanisms Morphological mechanisms

reduce stomatal conductance (reduce reduce leaf expansion and water use) production maintain photosynthesis accelerate leaf senescence osmotic adjustment leaf movement and rolling pollen and ovule fertility and sensitivity root development assimilate reserves and remobilisation

2.10. Morphological adaptation mechanisms Morphological mechanisms of adaptation are changes in parts of a plant to reduce water use and thus maintain plant water status. This often occurs in response to increases in soil water deficit. Responses to water deficit include reduced leaf expansion and production, and increased root growth into deep soil to explore soil moisture (Table 2.2).

2.10.1 Shoot adaptive mechanisms to water deficits Reduced green leaf area is an important adaptation to water deficit that enables plants to maintain their water status (avoid dehydration). There are at least three major shoot

15 adaptive mechanisms that reduce green leaf area in response to water deficit: reduced stem growth resulting in less leaf production, reduced leaf expansion, and increased leaf senescence. Sensitivity of leaf development and senescence in response to water deficit has been reported for indeterminate crops (Turner and Begg 1981; Wery 2005). In faba bean (genotype Icarus), leaf expansion decreased by approximately 4 mm, three days after imposition of water deficit (Mwanamwenge et al. 1999). Leaf expansion stopped when leaf water potential dropped below -0.4 MPa in sunflower and -1.2 MPa in soybean (Boyer 1970; Hsiao 1973; Hsiao and Xu 2000; Wery 2005). Moreover, water deficit also decreases the rate of leaf production through reduction in number of nodes (Blum 1996). For example, in bean water deficit imposed at flowering decreased node number by 4 compared with the control (Boutraa and Sanders 2001). On the other hand, water deficit increases leaf senescence as reported by Morgan (1984) and Turner and Begg (1981). Consequently, green leaf area is reduced, e.g. water deficit reduced green leaf area of soybean by 30% compared with the irrigated control (Neyshabouri and Hatfield 1986). Similar observations were also reported for soybean, black gram, mung bean and cowpea when plants were exposed to rainfed conditions (Lawn 1982a). In lupin, green leaf area decreased from 43 to 52% two weeks after withholding water compared with the controls (Rodrigues et al. 1995).

2.10.2 Leaf movement and rolling in response to water deficits There are two major leaf movement responses: solar tracking (diaheliotropism) and light avoidance (paraheliotropism). Under water deficit, leaves were more diaheliotropic in the afternoon and paraheliotropic around midday compared with well-watered control plants (Kao and Forseth 1992; Ludlow and Muchow 1990). Paraheliotropic leaf movement has been investigated in some species and cultivars and may be associated with a passive wilting response due to a general decline in leaf water status (Turner and Begg 1981). A comparative study of four grain legumes of soybean (Glycine max CPI 26671), black gram (V. mungo cv. Regur), mung bean (V. radiata cv. Berken), and cowpea (V. unguiculata CPI 28215) in response to water deficit found that paraheliotropic leaf movement was strongest in Vigna spp. (Lawn 1982b). Lawn (1982b) reported that a reduction in leaf stomatal conductance in soybean was related to high leaf temperature where there was some paraheliotropy. Kao and Forseth (1992) demonstrated that paraheliotropic leaf movement of soybean occurred earlier and longer in plants grown under water deficit and with low nitrogen input compared with well- watered plants receiving high nitrogen input.

Plant leaf rolling in response to water deficit is another strategy to avoid dehydration. This has been observed in tomato (Wudiri and Henderson 1985) and rice (Dingkuhn et al. 1989; O'Toole and Cruz 1980). O’Toole and Cruz (1980) reported that leaves of rice cultivars started to roll when midday leaf water potential (Ψ) was -0.8 MPa 10 days after treatment imposition. Leaves of IR28 completely rolled when midday Ψ was close to -2.4 MPa. Despite this, there was no clear evidence suggesting that the higher leaf rolling cultivar (IR28) maintained higher plant water status than Kinandang Patong cultivar, because both pre-dawn and midday Ψ were also lower in IR28. However, in tomato, studies carried out in both the greenhouse and field reported that the drought- resistant cultivar Saladete maintained higher leaf water potential under severe water deficit through leaf rolling during high evaporative demand allowing more fruit set (50%) than a semi-indeterminate cultivar VF145B-7879 (Wudiri and Henderson 1985). It appears that leaf rolling may be important in some species to enable plants to maintain high water status. The degree of leaf rolling may also be modified by root development under water deficit.

2.10.3 Root adaptive mechanisms in response to water deficit In contrast to reduced shoot growth in response to drought, roots tend to explore deeper into the soil profile for soil moisture (Begg and Turner 1976). Root development varies between and within species. Deeper root systems are better able to use water stored deeper in the soil profile and thus achieve greater yield compared with shallow-rooting species. Yield advantages have been reported for wheat and tomato cultivars with deeper root systems (Begg and Turner 1976). A comparative study of six field pea (Pisum sativum L.) genotypes investigated patterns of water use and root development in both glasshouse and field in southern Australia (Armstrong et al. 1994). The research found that cultivar Wirrega extended its roots deeper into the soil (40 cm deeper than others) and thus extracted more soil moisture, making Wirrega one of the superior genotypes observed. Similar field investigations were also reported for soybean in comparison with Vigna spp. (Lawn 1982a), chickpea (Serraj et al. 2004) and wheat (Morgan and Condon 1986). It was observed that L. sativus could extract water from deeper into the soil profile through its root development during water deficit at Mullewa, Western Australia, that enhanced harvest indices and water use efficiency (Siddique et al. 2001). Serraj et al. (2004) reported that high yields of recombinant inbred lines of chickpea were associated with deeper root growth and greater water

17 extraction during terminal drought. Increased root dry weights and root/shoot ratios suggest root adaptation to water deficit conditions (Blum 1996; Stoddard et al. 2006).

2.11. Physiological adaptation mechanisms to water deficits There is a range of physiological mechanisms that enable plants to adapt to water deficits. Turner and Begg (1981) proposed four adaptive mechanisms: seed priming, stomatal control of water loss, osmotic adjustment and cellular tolerance of dehydration. Here, the focus is on the effect of water deficit on gas exchange and the response in leaf water potential (Ψ) across species and genotypes. Moreover, the role of osmotic adjustment in maintaining leaf water status, reproductive physiological adaptation and the role of assimilate reserves to grain yield is reviewed (Table 2.2).

2.11.1 Changes in gas exchange in response to water deficits Reduced stomatal conductance is an important adaptive mechanism to maintain plant water status in response to soil water deficit (Stoddard et al. 2006), and has been observed in sorghum cultivars (Premachandra et al. 1992), soybean (Lawn 1982b), white lupin, chickpea, faba bean, field pea, grass pea and lentil (Leport et al. 1998). This reduction is associated with stomatal closure and is linked to increased abscisic acid (ABA) in leaves (Comstock 2002; Davies and Zhang 1991). It is reported that ABA is synthesised in roots exposed to water deficit (Davies and Zhang 1991) due to changes in gene expression through transduction mechanisms that first sense water deficit conditions (Bray 2002; Nambara and Marion-Poll 2005). ABA is transported to shoots via xylem where it then modifies stomatal opening (Davies and Zhang 1991). In lupin, it was reported that increased ABA in leaves decreased stomatal conductance (Turner et al. 2001). In wheat, stomatal closure initially occurred at a Ψ of -1.1 MPa (Seaton et al. 1977). Stomatal conductance of several grain legumes including grass pea decreased markedly when Ψ fell below -0.9 MPa and close to zero when Ψ was between -1.0 and 2.0 MPa (Leport et al. 1998). In a pot experiment, water deficit reduced stomatal conductance of cowpea genotypes by 71% on average compared with the controls (Anyia and Herzog 2004). This physiological adaptation enables plants under water deficit to maintain high water status, at least in the short term.

Reduced stomatal conductance due to water deficit generally reduces photosynthesis, but the degree of reduction varies between species and genotypes and water deficit itself. Under moderate water deficit, reduced stomatal conductance increases water use

18 efficiency (CO2 uptake per unit H2O lost) (Chaves and Oliveira 2004; Hetherington and Woodward 2003), but had no effect on photosynthetic capacity (Chaves and Oliveira

2004). Therefore, high water use efficiency of, for example, C3 plants occurred when stomatal conductance fell between 50 and 200 mmol m-2 s-1 (Hetherington and Woodward 2003). In grass pea, water deficit decreased photosynthesis by 3 to 6 μmol m-2 s-1, but increased WUE (mmol mol-1 by 33 to 100% compared with control (Yang et al. 2004). However, when water deficit is severe, photosynthesis is limited. For example, in chickpea, photosynthesis dropped to one-tenth of its maximum when Ψ dropped to -3 MPa (Leport et al. 1999). Davies et al. (1999) found that leaf photosynthesis dropped below 10 μmol m-2 s-1 as leaf water potential dropped to -1.2 MPa and less than 5 μmol m-2 s-1 (e.g. cultivar ICCV88201) when Ψ was approximately -3.0 MPa. Between cultivars, photosynthesis dropped to 2 μmol m-2 s-1 when Ψ decreased to -1.7 MPa in M129 and -2.4 MPa in Tyson cultivars (Basu et al. 2007). Continued photosynthesis under moderate water deficit enables plants to support seed filling. Seed filling relies on remobilisation of assimilated reserves when water deficit is severe (Section 2.11.4).

2.11.2 Osmotic adjustment Osmotic adjustment (OA) refers to an adjustment of living cells by net accumulation of osmotically-active compounds (solutes) that reduce osmotic potential in response to water deficit (Lambers et al. 2008). This enables plants to continue water uptake and therefore maintain turgor at low leaf water potential (Chaves and Oliveira 2004; Lambers et al. 2008; Ludlow and Muchow 1990; Morgan 1984). Species with high OA maintain higher leaf turgor than low OA species in response to water deficits (Lecoeur et al. 1992; Premachandra et al. 1992; Turner and Begg 1981). Premachandra et al. (1992), for example, observed that OA of sorghum ranged from 0.25 to 0.48 MPa for cultivar Zairai Tokin (ZT) and Snow Brand-Hybrid HI-Sugar (HS) when leaf water potential (Ψ) was -1.65 MPa for ZT and -1.50 MPa for HS. OA increased by 0.24 in ZT and 0.31 in HS when Ψ decreased by 0.25 MPa in ZT and 0.36 MPa in HS. The authors concluded that HS had greater OA than ZT allowing plants to withstand low leaf water potential by maintaining leaf turgor and increasing protoplasmic tolerance (Premachandra et al. 1992). A high variation of OA in various grain legumes from different studies was summarised by Turner et al. (2001); ranging from 0 in chickpea to 1.6 MPa in peanut. There was also high variation between genotypes; in chickpea it ranged from 0 to 1.3 MPa, and in peanut it ranged from 0.3 to 1.6 MPa (Turner et al.

19 2001). These authors showed that OA exists in certain crop plants and is a major response of tissues that enables the plant to avoid dehydration through maintaining high leaf water status (Blum 2005; Flower and Ludlow 1986).

Recent reviews and studies have shown that the role of OA may be limited in certain species (Blum 2005; Rodrigues et al. 1995; Turner et al. 2007). In a review on drought resistance, water use efficiency and yield potential, OA was a major cellular response to drought in some crop plants, enhancing dehydration avoidance and supporting seed yield (Blum 2005). Flower and Ludlow (1986) concluded that increased tolerance of dehydration in pigeon pea due to a high OA allowed many leaves to survive water deficit, contributing to improved plant productivity after the water deficit. High OA genotypes of wheat and sorghum yielded up to 60% more than low OA genotypes (Turner et al. 2001). It was suggested that OA enhanced carbon partitioning contributing to pod set and yield of pigeon pea genotypes (Subbarao et al. 2000a; Subbarao et al. 2000b). A linear relationship (r2 = 70 and r2 = 90) between OA and yield was observed for various pea cultivars and breeding lines under severe water deficit (Rodriguez-Maribona et al. 1992). A greater capacity of OA in mustard contributed to improved water extraction and thus radiation use efficiency compared with canola (Gunasekera et al. 2009). This may have been due to induced root growth as demonstrated in peas (Andersen and Aremu 1991) and sunflower (Chimenti et al. 2002). Recent studies observed no relationship between OA and yield of chickpeas (Basu et al. 2007; Turner et al. 2007). Turner et al. (2007) found that, in a study conducted in Australia and India, OA from terminal drought varied from year to year, did not benefit yields in Australia and had an inconsistent effect on yield in India. This suggests that OA does exist and that it enhances yield of some but not all genotypes and species.

2.11.3 Reproductive adaptation to water deficits Water deficit during flowering and podding affects reproductive output. It is common for water deficit during reproduction to increase flower and pod abortion resulting in less grain yield (Section 2.12). Leport et al (1999), for example, showed that water deficit during reproduction of chickpea genotypes decreased seed yield by 50–80% compared with the irrigated control. This resulted from 27–47% less filled pods suggesting significant flower and pod abortion. Similar flower and pod abortion rates (56–73%) were observed in other studies (Fang et al. 2010; Leport et al. 2006).

20 Moreover, Leport et al. (2006) demonstrated that pods on secondary branches were more sensitive to water deficit than the main stem when water deficit was applied at early podding. Abortion rates of 68% for desi and 93% for kabuli types were observed during the study. In addition, water deficit decreased seed size by 19–25% (Leport et al. 1999).

High flower and pod abortion, poor pod and seed set are the consequence of pollen and ovule sensitivity to water deficit (Turner et al. 2005). A study on soybean suggested that ovule sensitivity contributed more to flower abortion under water deficit than pollen fertility (Kokubun et al. 2001). Similarly, in white clover, water deficit reduced the number of ovules per floret (Turner 1993). In rice, however, water deficit deformed and reduced the size of pollen resulting in 80% floret failure (Sheoran and Saini 1996). More recently, Fang et al. (2010) demonstrated that in chickpeas, high flower and pod abortion was due to reduced pollen viability (by 50%) and germination (by 80%) compared with the well-watered control. In addition, water deficit decreases pistil function which may retard pollen tube growth into the ovary as observed in chickpeas (Fang et al. 2010). The yield of grass pea (L. sativus) grown under rainfed conditions in southern Australia decreased by 46% compared with the irrigated control (Leport et al. 1998) suggesting significant flower and pod abortion or a reduction in the number of fruiting sites. However, little is known about the reproductive response of grass pea to water deficits.

The above reviews suggest that the response of pollen fertility, ovule sensitivity and pistil function to water deficit contribute to the abortion of flowers, pods and seeds of many crops. Water deficit may also reduce seed size. However, these responses vary between species and genotypes and the degree of water deficit.

2.11.4 Role of assimilate reserves in adaptation to water deficits Turner and Begg (1981) distinguished two sources of assimilates for grain production of cereals and legumes: photosynthesis after anthesis, and assimilated reserves from photosynthesis before anthesis that is translocated into the grain. Pre-anthesis assimilated reserves are temporarily stored in leaves, stems and roots. These become important resources for seed filling when current photosynthesis is limited by water deficit (Yang et al. 2001) (Section 2.11.1). This has been observed in wheat (Asseng and van Herwaarden 2003; Austin et al. 1980; Bidinger et al. 1977; Gebbing and

21 Schnyder 1999; Xue et al. 2006; Yang et al. 2000; Yang et al. 2001), barley (Austin et al. 1980; Bidinger et al. 1977), pigeon pea (Subbarao et al. 2000a) and chickpea (Davies et al. 2000; Turner et al. 2005). Xue et al. (2006) estimated that the contribution of pre-anthesis stem reserves to grain yield was 88.3% in rainfed compared with 38.3% in irrigated plants. In a separate study, water deficit during reproduction resulted in 75 to 95% of pre-anthesis straw reserves being converted into wheat grain (Yang et al. 2001). A field experiment on barley genotypes found that 44% of pre- anthesis reserves remobilised into grain dry matter in a dry and hot year compared with <11% in a wetter and cooler year (Austin et al. 1980). Similar observations were also reported for pigeon pea (Subbarao et al. 2000b) and wheat and barley (Asseng and van Herwaarden 2003); when there was less extractable soil moisture, a greater proportion of pre-anthesis assimilates was remobilised into grain. In chickpea, average pre-podding C and N translocated into grain was 9 and 55%, respectively, in controls and 13 and 93% in water deficit treatments. It was concluded that lower C remobilisation was due to the limited number of pods (sink strength) (Davies et al. 2000). In pearl millet (Pennisetum glaucum L.) under water deficit, 25% of pre-anthesis assimilate was translocated to grain at harvest (Mahalakshmi et al. 1993). Yang et al. (2001) suggested that water deficit during reproduction enhanced leaf senescence leading to remobilisation of pre-anthesis reserves into grain. Species or genotypes with high dry matter production at anthesis are therefore important in southern Australia where water deficit during reproductive development is common (Siddique et al. 1999; Siddique et al. 2001).

2.12. The effect of water deficit on yield, yield components and harvest indices Terminal drought is the most yield-limiting factor for most cereal and grain legume crops in southern Australia. A study comparing commercial grain legumes at Merredin, Western Australia, found that rainfed water deficit reduced grain yield by 28% in Vicia narbonensis, 66% in V. faba, 85% for both Lupinus angustifolius and L. pilosus, and 89% in Cicer arietinum, compared with irrigated controls (Thomson et al. 1997). In a pot experiment, yield of grass pea was reduced by 40% in mild water deficit and 82% under severe water deficit during the reproductive period compared with the controls (Herwig 2001). At the same location, water deficit reduced chickpea seed yield by 27% and 45% for Kaniva and Tyson cultivars, respectively, compared with the irrigated controls (Davies et al. 1999). Similar observations were also reported for chickpea in another study (Leport et al. 1999). High variation of seed yield between species and

22 genotypes reflects variation in adaptation to water deficits during reproduction. Siddique et al. (2001) argued that high sub-soil compaction in this growing area also restricted root penetration of deep-rooted species contributing to low grain yields. Recently, low seed yields under terminal drought were reported to be due to reproductive sensitivity to water deficits (Fang et al. 2010; Leport et al. 2006) (Section 2.11.3).

In some situations in southern Australia, rainfall occurs during the reproductive period such that terminal drought is delayed or does not occur. For example, Gunasekera et al. (2009) reported up to 20 mm of rainfall at Merredin, Western Australia, during September which contributed to pod filling and thus enhanced grain yield. Many studies have addressed the contribution of such rainfall to yield by manipulating irrigation or watering systems during the reproductive period (Gunasekera et al. 2009; Mwanamwenge et al. 1999). Leport et al. (2006) applied water deficit to chickpea genotypes at three different stages—early podding, mid and then at late podding—to observe the effect of timing of water deficit on pod abortion, seed yield and seed size. Although there was significant variation in pod abortion and thus seed yield between treatments, water deficit applied at late podding caused the least reduction in seed yield. Similar results were also observed in a three-year study on lupins where water deficit applied at early podding resulted in lower seed yields than late podding (Palta et al. 2007). In faba bean (Vicia faba L.) genotypes (ACC286, Fiord and Icarus), water deficit applied at early podding for approximately 8 days reduced seed yield by 50% in all genotypes, but no reduction was observed when water deficit was applied at floral initiation or at 50% flowering (Mwanamwenge et al. 1999). Plants recovered quicker when the water deficit was applied before podding rather than at podding.

Genotype and species variation in growth, development and seed yield under water deficits are reflected in harvest indices (HI) (Table 2.3). HI at Merredin, WA, ranged from 0.04 to 0.55 for rainfed and irrigated treatments, and was associated with adaptation of the species to water deficits (Siddique et al. 2001). These authors compared growth and development of various grain legumes at Merredin and Mullewa. At both sites, species with high HIs (P. sativum and V. faba) were able to escape or tolerate end-season drought and better partition dry matter into grain (Sections 2.9.1, 2.9.2). L. sativus had a maximum HI of 0.25 at Merredin and 0.31 at Mullewa in the two-year study (Siddique et al. 2001). This late-flowering species was likely to be

23 affected by drought late in the season particularly at Merredin where subsoil conditions are less hostile than at Mullewa (Siddique et al. 2001). At Mullewa, L. sativus possibly avoided dehydration through greater root development to extract water from deeper in the soil profile (Section 2.10.3), hence the higher HI at this site. Similar observations were reported in earlier field studies by Siddique et al. (1996).

Various glasshouse studies on other grain legumes confirmed that terminal drought starting at flowering decreased HI severely as reported by Leport et al. (1999) (Table 2.3). Delaying terminal drought would improve HI, as shown in chickpeas (Leport et al. 2006). In faba bean, water deficit imposed at podding for approximately 8 days reduced HI more than water deficit applied at floral initiation (Mwanamwenge et al. 1999). This suggests that pod development was more sensitive to water deficit. Interestingly, in lupin, there was no difference in HI when water deficit was imposed at first pod on the main stem, first pod on apical branches, or 13 days later; but HI was 6% less than the control.

2.13. Water use and water use efficiency (WUE) On a crop basis, WUE can be defined as the ratio of total biomass or grain yield produced to the input of water (Siddique et al. 2001; Sinclair et al. 1984). Siddique et al. (2001) reported that WUE can be improved by changing the cropping pattern. In southern Australia, the strategy to improve WUE is to grow crops that develop rapidly

(Section 2.9.1). Water use efficiency for dry matter (WUEdm) and for grain (WUEgr) vary between species, genotypes and location (Leport et al. 1999; Siddique et al. 2001) (Table 2.4). In a two-year study, Siddique et al. (2001) observed much lower and more variable WUEdm and WUEgr at Merredin than at Mullewa. WUEdm for L. sativus ranged from approximately 11 kg-1 ha-1 mm-1 at Merredin to 27 kg-1 ha-1 mm-1 at Mullewa

(Siddique et al. 2001) (Table 2.4). Similarly, a higher WUEgr was observed at Mullewa than at Merredin. This trend reflects the grain yield and HI of L. sativus shown in Table

2.3. WUEgr of L. sativus was comparable with other grain legumes observed at Mullewa under favourable rainfall and soil growing conditions (Table 2.4).

24 Table 2.3 Variation in harvest indices of grain legumes observed in some growing areas of Western Australia and in glasshouse studies. Values in parentheses are means, excluding species affected by thunderstorms, disease or insects. Species/Genotypes Growth condition HI (range) Year References Various grain legumes Merredin (rainfed) 0.25–0.43 (0.33) 1993–94 (Siddique et al. 2001) Mullewa (rainfed) 0.24–0.52 (0.40) 1993–94 Various grain legumes Merredin (rainfed) 0.16–0.43 (0.30) 1993–94 (Thomson et al. 1997) Merredin (irrigated) 0.21–0.55 (0.41) 1993–94 Chickpeas Merredin (rainfed) 0.25–0.39 (0.33) 1995 (Leport et al. 1999) Merredin (irrigated) 0.31–0.43 (0.38) 1995 Chickpeas Floreat Park, Perth (rainout shelter from 0.09–0.31 (0.20) 1997 (Leport et al. 1999) flowering to maturity) Floreat Park (irrigated) 0.14–0.47 (0.30) 1997 Faba bean (ACC286, Fiord, Icarus) Glasshouse, UWA (floral initiation water deficit) 0.31–0.43 (0.37) 1995 (Mwanamwenge et al. 1999) Glasshouse, UWA (flowering water deficit) 0.16–0.44 (0.33) Glasshouse, UWA (early podding water deficit; 0.10–0.25 (0.17) on average WD was 8 days then relieved) Glasshouse, UWA (control) 0.33–0.48 (0.40) Lupins (cv Merrit) East Beverley (water stress at first pod on 0.32 ? (Palta and Plaut 1999) main stem; 5 days WD) East Beverley (water stress at first pod on 0.32 apical branches; 5 days WD) East Beverley (control) 0.34 L. sativus Northam (rainfed) 0.15–0.27 (0.22) 1993 (Siddique et al. 1996) Merredin (rainfed) 0.04–0.16 (0.10) 1993 Mullewa (rainfed) 0.23–0.41 (0.33) 1993

25 -1 -1 -1 Table 2.4 Water use efficiency (kg ha mm ) for dry matter (WUEdm) and grain (WUEgr) of various prostrate grain legumes at two contrasting growing sites, Merredin and Mullewa, Western Australia. Values in parentheses are means. Experiments were under rainfed conditions. Species WUEdm WUEgr Year Reference Merredin Mullewa Merredin Mullewa Prostrate 13.7–27.4 19.4–27.3 5.6–8.1 7.0–12.6 1993 (Siddique et al. grain (21.1) (23.9) (5.9) (9.3) 2001) legumes L. sativus 13.7 27.3 - 9.0 Prostrate 11.7–17.6 20.6–38.7 2.8–6.0 4.9–15.9 1994 grain (13.4) (25.8) (4.7) (9.3) legumes L. sativus 11.7 25.1 2.8 7.1 1994 Chickpea 19.5–25.2 6.22–8.22 1995 (Leport et al. (23.0) (7.54) 1999)

2.14. The effect of legumes on growth, N and P uptake and yield of subsequent cereal crops Grain legumes, including grass pea, are important in agriculture for their ability to fix

N2 and to sustain production of cereal crops using rotational farming systems (Adu and Oades 1978; Evans et al. 2001; Herridge et al. 1995; Ladd 1981; Ladd and Amato 1986; Ladd et al. 1994; Peoples et al. 2009; Peoples et al. 1995a; Peoples et al. 1995b; Rochester et al. 1998; Siddique and Sykes 1997). Rochester et al. (1998) reported that the amount of N fixed by legumes ranged from 20 kg ha-1 for adzuki bean and droughted lablab (Lablab purpureus) to >450 kg ha-1 for irrigated soybean. Recent reviews indicated that grass pea fixes N between 67 and 125 kg ha-1 (Kumar et al. 2010; McCutchan 2003). Another study reported that shoot N concentration in grass pea was up to 55 mg g-1 dry mass (Rao et al. 2005). Tissue N content of grass pea cultivar Ceora is about 45% (Siddique et al. 2006).

Much of the N fixed by legumes is removed from the field as harvested grain (Peoples et al. 1995a), but Rochester et al. (1998) found that this does not account for all fixed N. In drought-prone environments, it is common for both grain and stubble of multipurpose legume crops (e.g. grass pea) to be removed for human consumption and animal feed (Cuttle et al. 2003). Therefore, in these crops, shoots provide little N for recycling, but a significant amount of N may still be released from belowground sources through leaching from and decomposition of roots.

26 Evans et al. (2001), in their review, concluded that N mineralised from lupin and pea residues contributed 40% and 15–30%, respectively, of N in grain yield in the following wheat crop. It was observed that 40% of total N fixed by winter- and summer-irrigated faba bean in the cotton-growing regions of northern New South Wales was in the belowground portion of the plant at peak biomass production (Rochester et al. 1998). Similar observations were reported for chickpea, albus lupin and alfalfa (Medicago sativa) (Unkovich and Pate 2000). In a glasshouse study, it was reported that the proportion of belowground N determined using isotopic and mass N balance methods ranged from 28% in mungbean to 48% in chickpea (Khan et al. 2002). In another study, it was estimated that between 20 and 25% of lupin-N mineralised belowground during summer fallow was available for the subsequent wheat crop (McNeill and Fillery 2008). Herridge et al. (1995) reported that although chickpea (sown in May and harvested in November) decreased soil nitrate-N during vegetative growth, soil nitrate-N observed at 1.2 m depth consistently increased again from approximately 50 kg ha-1 in November to 100 kg ha-1 in May. This increase was associated with a rapid decomposition of root and nodule material over the summer and autumn fallow (Rochester et al. 1998; Unkovich and Pate 2000). The effect of chickpea on N uptake and the yield of subsequent wheat was observed on two different soil types, and it was found that wheat vegetative biomass and N uptake after chickpea increased by 64% and 69%, respectively, on black soil and 52% and 50%, respectively, on red clayey soil compared with wheat after wheat (Holford and Crocker 1997). These differences were due, firstly, to poor wheat growth and N uptake after wheat and, secondly, that chickpea increased N availability for the following wheat.

To a limited extent, legumes have also been shown to enhance P availability to subsequent crops through the development of specialised cluster roots (Lambers et al. 2006), which are associated with low soil P (Dakora and Phillips 2002). Cluster roots exude carboxylates into the rhizosphere that often reduce pH and chelate sparingly soluble P (Dakora and Phillips 2002; Lambers et al. 2006). Increased P availability under legumes has been demonstrated in white lupin, field pea and faba bean (Nuruzzaman et al. 2005a; Nuruzzaman et al. 2005b; Veneklaas et al. 2003), pigeon pea (Ae et al. 1990), and chickpea (Li et al. 2004; Veneklaas et al. 2003). In a pot experiment, Nuruzzaman et al (2005b) reported that wheat P content increased by 30– 50% following legumes compared with wheat after wheat. The persistence of carboxylates may be short-lived in the soil, but mineralisation of legume residues

27 increases over time (Nuruzzaman et al. 2005b) as demonstrated in pea and clover (Lupwayi et al. 2007). It is not known whether grass pea increases soil N and P availability for a subsequent cereal crop.

2.15. Conclusion Grass pea (Lathyrus sativus cv Ceora) is a new multipurpose grain legume that was bred for low ODAP concentration at CLIMA, UWA. Testing this cultivar in a range of agro- climatic environments of Western Australia found that ODAP concentration remained low, with good yields. Despite this, little is known about the adaptation mechanisms of this cultivar to water deficits during reproduction and its potential benefit for cereals in a crop rotation. This review suggests that Ceora may escape or tolerate drought in response to water deficits during reproduction thereby enabling plants to produce good seed yield in the field. Ceora has an indeterminate growth habit that may disadvantage plant growth over water use and drought escape. This growth habit slows reproductive development causing high flower and pod abortion. However, evidence suggests that water deficit can accelerate seed filling of surviving pods and mature earlier than controls (drought escape). Ceora may also tolerate drought through physiological and morphological adaptations that enhance seed production. Reduction in stomatal conductance and osmotic adjustment may help plants to avoid dehydration under water deficits. Moreover, Ceora appears to explore soil water deeper in the soil profile through root development in response to water deficits. These adaptive mechanisms may help plants to remobilise pre-anthesis reserves into seed filling that improves harvest index and water use efficiency for grain. As a grain legume, Ceora may increase soil N and P availability through atmospheric nitrogen fixation and remobilisation of residual P for a subsequent cereal crop.

28 Chapter 3

Grass pea avoids dehydration and escapes drought under moderate water deficit during reproductive period

Background and aims Grass pea (Lathyrus sativus L.) grows well and produces good yield in the Mediterranean-type environment of southern Australia where water deficits during the reproductive phase are common. Mechanisms by which grass pea adapts to water deficits are not well understood. This study aimed to identify these mechanisms at the whole plant and tissue level. Methods Ceora grass pea of indeterminate growth habit and Kaspa, a semi- indeterminate and drought-adapted field pea (Pisum sativum) were grown in pots containing 10 kg of a mixture of clayey loam soil and sand in a glasshouse. Plants were well-watered at 80% field capacity (FC) from sowing to seed filling when moderate water deficit (50% FC) was imposed until maturity. Plant-water relations, leaf area development, dry matter production, yield components and water use efficiency were measured. Results Water deficit had a greater effect on Ceora than Kaspa which was due to the more indeterminate growth habit than Kaspa. In Ceora, water deficit significantly decreased pre-dawn leaf water potential (Ψ) and stomatal conductance, but did not significantly reduce photosynthesis. Water deficit significantly reduced green leaf area by 29% at maximum growth of the control leading to a 30 and 24% reduction in plant dry mass and seed yield, respectively, at maturity compared with the control. In Kaspa, water deficit decreased pre-dawn Ψ, but there was no reduction in stomatal conductance or photosynthesis, green leaf area and yield between the treatments. Within the species, there was no significant treatment differences for seed size or water use efficiency but harvest index (HI) was reduced in Kaspa by 8% compared with the control. Between the species, Ceora produced 41% and 19% more dry matter for well-watered control and water deficient plants, respectively, but produced 22% (control) and 33% (water deficit) less seed yield compared with Kaspa. Harvest indices and water use efficiency for grain were 53% and 50% less in Ceora than Kaspa in the control and 44% and 46% less under water deficit. Conclusion This study confirmed the adaptation of Ceora to water deficit. Ceora was able to avoid dehydration by maintaining high plant water status through a reduction in green leaf area, stomatal conductance and possibly osmotic adjustment under water deficit. These strategies allowed maintenance of a high photosynthetic rate enabling seed filling even under water deficit. This resulted in similar harvest index and water use efficiency between treatments. Ceora also demonstrated the ability to escape drought through early maturity. Thus, it is concluded, that Ceora displayed dehydration avoidance and drought escape adaptation mechanisms in response to moderate water deficit during the reproductive period.

29 3.1. Introduction Grass pea (Lathyrus sativus cv. Ceora), a new multipurpose grain legume with indeterminate growth habit (Hanbury et al. 2005), adapts well to the Mediterranean-type environment of southern Australia (Siddique et al. 1996), where water deficit during reproduction is common (Turner 1992). Mechanisms by which grass pea adapts to water deficit during reproduction are not well understood. Water deficits often develop during crop reproduction stages as a consequence of little or no rainfall in spring. This period is accompanied by increasing air temperatures and high evaporation which also affect growth, reproductive development and yields of drought-sensitive annual crops. The sensitivity of crop growth and development to water deficit has long been recognised (Boyer 1970; Chaves et al. 2003; Desclaux et al. 2000; Farooq et al. 2009; Hsiao 1973; Wery 2005). For example, Desclaux et al. (2000) observed that water deficit applied at flowering decreased plant height of indeterminate soybean (Weber) through reduction of internode length by 3 and 8 mm in moderate and severe water deficit compared with the well-watered control. Moreover, in bean (Phaseolus vulgaris L), water deficit applied at flowering decreased node number of the drought tolerant genotype Carioca by 4 nodes (at 83 days after planting) compared with the control (Boutraa and Sanders 2001). As a consequence, plant height of Carioca was approximately 20 cm shorter than the control. Water deficit imposed at the later stage of podding, however had no effect on node number or plant height of Carioca.

The growth process most sensitive to water deficit is leaf expansion. For example, leaf expansion of sunflower, soybean and maize started to decrease at leaf water potential (Ψ) less than -0.2 MPa and completely stopped at Ψ -0.4 MPa in sunflower, -0.7 MPa in maize and -1.2 MPa in soybean (Boyer 1970; Hsiao 1973; Hsiao and Xu 2000; Wery 2005). Water deficit also decreased the rate of leaf production (Blum 1996) as demonstrated in bean genotype Carioca (Boutraa and Sanders 2001), and increased leaf senescence (Morgan 1984; Turner and Begg 1981). As a result, there was an overall reduction in green leaf area. Since plants lose water mostly through their leaves, a reduction in leaf area minimises water use and potentially enables plants to maintain a high plant water status (dehydration avoidance). Moreover, water deficit has been shown to increase leaf rolling, and decrease stomatal conductance, thus further reducing water loss per unit leaf area in the remaining green leaves, and contributing to dehydration avoidance (Morgan 1984; Stoddard et al. 2006). For example, stomatal conductance of white lupin, chickpea, faba bean, field pea, grass pea and lentil

30 decreased when Ψ dropped below -0.9 MPa (Leport et al. 1998). Stomatal closure is often observed during midday when evaporative demand is high (Turner and Begg 1981), for example in lupin (Rodrigues et al. 1995), grass pea (Yang et al. 2004) and other grain legumes (Lawn 1982b).

Reduction in stomatal conductance in response to water deficits decreases photosynthesis, however the decrease in photosynthesis is not proportional to the decrease in conductance under moderate water deficit (Farquhar and Sharkey 1982; Hetherington and Woodward 2003; Wery 2005; Yang et al. 2004). Hetherington and

Woodward (2003) reported that in C3 plants, high water use efficiency (CO2 uptake per unit increase in stomatal conductance) occurred when stomatal conductance was between 50 and 200 mmol m-2 s-1, respectively. This would enable water deficient plants to continue their reproductive development and seed yield, despite the fact that many plants sacrifice some of their reproductive parts such as flowers, pods and seeds through abortion as observed in chickpeas (Davies et al. 2000; Fang et al. 2010; Leport et al. 2006; Leport et al. 1999). Davies et al. (2000) demonstrated that seed yield of chickpea cv Tyson was decreased by 74% in rainfed conditions compared with irrigated controls. This was associated with a 60 and 16% reduction in number of pods per plant and seeds per pod, respectively, compared with the control.

In the Mediterranean-type environment of southern Australia, crop species are needed that have an improved ability to escape drought through early vigour, flowering, podding and seed set. It is preferable for the crops to mature before they experience severe water deficit (Leport et al. 1998; Ludlow and Muchow 1990; Siddique et al. 2001; Siddique and Sedgley 1986; Siddique et al. 1990; Thomson and Siddique 1997; Thomson et al. 1997). Siddique, et al. (1990) demonstrated that higher water use efficiency (grain yield per unit area per mm rainfall) of modern wheat cultivars was associated with faster growth and development, earlier flowering resulting in higher harvest indices compared with older cultivars. Similar observations were made with chickpea in drought-prone environments of India (Berger et al. 2006). Rapid leaf development decreased soil evaporation and increased light interception resulting in high dry matter production at anthesis (Siddique and Sedgley 1986; Siddique et al. 1990). In fact, dry matter partitioning into seeds as water deficit develops during reproduction is important to increase harvest indices (Yang and Zhang 2006). For example, in wheat cultivars, it was observed that water deficit during reproduction

31 enhanced translocation of pre-anthesis assimilated carbon into grain which ranged from 50 to 80% higher than the control (Yang et al. 2001). In barley, it was estimated that pre-anthesis carbon assimilation contribution to the grain was 44% in a very dry and hot year and 11% in a wetter year (Austin et al. 1980). In chickpea, average pre-podding C and N translocated into grain was 9 and 55% in controls and 13 and 93% in water deficient plants (Davies et al. 2000). Leport et al. (1998) commented that a strong correlation existed between yield, early vigour, pod development and dry matter production in cool-season grain legumes. The Ceora cultivar of grass pea (L. sativus cv Ceora) has been reported as being an early flowering line (Siddique et al. 1996) that enables it to escape severe water deficit.

Under rainfed conditions, grass pea (L. sativus), has been shown to avoid and/or tolerate drought by delaying leaf senescence and maturity (Leport et al. 1998; Thomson and Siddique 1997). This adaptation was possibly due to the exploration of more soil water from a deeper soil profile. It is well noted that under water deficit, drought tolerant plants increase root surface area by extending root growth deeper into the soil to explore for more water (Begg and Turner 1976; Manavalan et al. 2009; Sinclair and Muchow 2001). Blum (1984) concluded that water deficits increased the root/shoot ratio of sorghum along with a larger root length density compared with leaf area. This adaptation enabled the plant to sustain its water status and therefore enhanced seed filling and yield as observed in chickpea genotypes under terminal drought (Kashiwagi et al. 2006; Serraj et al. 2004). In addition, osmotic adjustment in plant tissues also contributed to the ability to sustain water uptake (Blum 1996).

This study aimed to identify mechanisms by which grass pea (L. sativus L.) adapts to water deficits by imposing on plants to a moderate water deficit during seed filling. Field pea (Pisum sativum L.), a drought-adapted grain legume species, was included for comparison. Measurements included growth, development and water relations. The hypotheses were that (i) Ceora would grow and develop sufficiently rapidly that pre- anthesis CO2 assimilation would enable seed filling during the imposition of a water deficit. (ii) moderate drought would reduce green leaf area and stomatal conductance in order to maintain plant water status (drought avoidance). (iii) Ceora would escape water deficit by maturing earlier than in well-watered conditions. (iv) seed filling and yield would be less affected than growth and water use.

32 3.2. Materials and methods 3.2.1 Study location, experimental design and experimental details The experiment was conducted in a glasshouse at the Faculty of Natural and Agricultural Sciences, the University of Western Australia, from May to October 2008. Growth of L. sativus cv. Ceora and field pea (Pisum sativum L. cv. Kaspa) were compared. Two different watering regimes, well-watered and water deficient (section 3.2.2), were applied to pots arranged on benches in a completely randomised design (CRD) with four replications.

Pots were filled with approximately 10 kg of a mixture of sieved air-dried reddish- brown clay loam soil (with a pHCaCl2 of 7.0) from Merredin, Western Australia and yellow sand at a proportion of 4:1. The mixture had a water holding capacity of 1.7 L per pot at 80% FC. Pots were made from PVC pipes, 15 cm in diameter and 45 cm in depth (with a bottom cap having four 12 mm diameter drainage holes). A piece of polyester cloth was placed over the holes before pot-filling to prevent soil loss. Superphosphate (1.2 g) and potassium sulphate (0.8 g) (obtained from Richgro Garden Products, WA) were incorporated into the top 3 to 4 cm of each pot before watering to 80% FC. After watering, a full teaspoon of a commercial version of Group F Rhizobium (Australian Pulse) was also incorporated into the top 3 to 4 cm of soil in each pot. Immediately following fertiliser and rhizobium application, four seeds of Ceora or Kaspa were sown 3–4 cm deep, slightly less than the recommended field sowing depth for Ceora of 4–6 cm (Hanbury and Siddique 1998; Hanbury et al. 2005). A preliminary study observed that Ceora flowered a week earlier than Kaspa, therefore it was decided to synchronize flowering time by sowing Kaspa earlier than Ceora. Seeds of Kaspa and Ceora were sown on 20 and 28 May 2008, respectively, and thinned to two plants per pot after two weeks. The soil was then covered with plastic beads (150 g per pot) to minimise soil evaporation. A wire support of known weight was set around the plants in each pot to encourage an upright growth habit. Pots were regularly rearranged within and between the benches during the experiment to minimise variation caused by position in the glasshouse.

3.2.2 Watering details Pot weight at field capacity (FC) was determined after over-saturating three spare pots and allowing them to drain freely (the top of the pot was sealed to prevent evaporation) for 48 hours. Mean pot weight was calculated for 80% FC to be used as the well-

33 watered treatment. All pots were watered to 80% FC before sowing and maintained at this water content. Several pots were weighed daily to estimate evapotranspiration (Et) and to calculate the water requirement for the next watering. The pots were watered at 2 to 3 day intervals. All pots were weighed once a week for fine adjustments to the watering regime. Moderate drought was applied by reducing soil water content from 80 to 50% FC when Ceora and Kaspa were at first podding and seed set. Treatments occurred at the same date for the two species from 27 August to 14 September for Kaspa and until 21 September 2008 for Ceora. Daily crop transpiration (Ct) was determined as the difference between daily Et and evaporation (E) from unplanted pots. Daily Ct was summed for cumulative transpiration.

3.2.3 Glasshouse temperature Daily maximum and minimum glasshouse temperatures were recorded by the climate control system using shielded aspirated thermistors at the height of the plants, respectively, during the experiment.

3.2.4 Phenology Time to emergence was recorded when 90%of the seeds in all pots had broken through the soil surface. Times to first flowering, podding, seed set and physical maturity were recorded when 50% of plants had started flowering (one plant in each pot had at least one fully open flower), 50% had started podding (one plant in each pot had at least one visible pod), 50% had started setting seed (when one pod in each pot had set seed) and 90% of the vegetation in each pot had turned golden brown. The time difference between seed set and maturity was taken as the seed filling period (Egli 2004).

3.2.5 Growth, development and yield components At each harvest, number of nodes was counted and plant height measured on the main stem and the tillers of Ceora and on the main stem of Kaspa. Green leaf area was determined by scanning all green leaves (stipules of Kaspa were included, but tendrils were excluded) from each pot using a Li-Cor area meter (Model LI 3100 Li-Cor Inc, Lincoln, Nebraska, USA). Other parts of the plant such as petioles, tendrils, stems, pod wall (including young pods, aborted pods and flowers), senesced leaves, seeds and roots were separated before oven-drying at 70 C for 48 h and then weighed. Roots were separated from the soil by washing the soil through a 2 mm mesh. Soil samples for soil water content were taken immediately after removal from the pot.

Yield, yield components and harvest index were determined at final harvest. Seed and pod numbers, seeds per pod, and aborted pods and flowers were recorded and separated before oven drying at 70 C for 48 h. Total seed dry matter was recorded for grain yield, while mean seed weight was determined by weighing 50 randomly selected seeds per pot. Aborted flowers and pods were determined from dead flowers that were still attached to the plants and pods that had no seeds. All plant parts and components were summed for total dry matter production. Total dry matter production and components in each pot were divided by two to calculate total dry matter and components per plant. The harvest index was determined as the ratio of grain yield to total dry mass (including roots).

3.2.6 Soil-plant water relations At each harvest (excluding final harvest), soil water content was determined by taking six soil samples (two from top, middle and bottom parts of the pot) in each pot before washing out the root system. Samples of soil were wrapped in aluminum foil, immediately weighed, oven dried at 105oC for 48 h and re-weighed. Soil water content was determined as the ratio of water lost to total dry soil (g g-1).

Pre-dawn leaf water potential (Ψ), leaf relative water content, osmotic potential, photosynthesis and leaf stomatal conductance were measured the day before each harvest (except at maturity). Pre-dawn leaf water potential (MPa) was measured between 4.30 am and 7.00 am (before sunrise) using a pressure chamber (Scholander et al. 1964) with appropriate precautions (Turner 1988). A petiole with fully expanded leaflets for Ceora or a tendril for Kaspa was wrapped in a plastic bag, excised and immediately sealed in the chamber with the cut end of the petiole or tendril exposed to allow sap to exude as pressure was applied.

For relative water content (RWC) at pre-dawn, two fully expanded leaflets (between second and fourth) were excised, covered with aluminum foil and wrapped into a polythene bag to prevent water loss. The leaves were immediately re-cut and weighed to determine their fresh weight (FW) and placed in a vial filled with DI water for rehydration. Samples were stored overnight in a dark room at 4oC to become fully turgid and then weighed for turgid weight (TW). Leaves were scanned for their green area, oven dried at 70oC for 48 h and then weighed for dry weight (DW) (Turner and

35 Begg 1981). Relative water content was calculated using the following equation: RWC (%) = (FW–DW)/(TW–DW)*100.

Leaf osmotic potential (OP) at pre-dawn was determined from four fully expanded leaflets for well-watered plants and up to eight fully expanded leaflets for water deficient plants. The leaves were excised, covered with aluminum foil and wrapped into a polythene bag to prevent water loss. The samples were immediately stored in a freezer at –16°C until measured. Osmolality (milliosmols) of expressed sap was determined using a Fiske® Micro-Osmometer Model 210 (Norwood, Massachusetts 02062, USA), after which OP was calculated as OP (MPa) = (2.447 osmolarity)/1000. OP at full turgor (OP100) was calculated using the following equation: (OP RWC)/100 and the difference between OP100 (well-watered) and OP100 (water deficit) was assumed to indicate osmotic adjustment.

Photosynthesis and stomatal conductance of fully expanded leaves (between second and fourth) were measured in the glasshouse using a portable open gas-exchange system Li- Cor-6400 (LI-COR Bioscience, Inc, Lincoln, Nebraska 68504 USA), with a blue/red light source (Li-Cor-6400-02(02B)) providing 1500 μmol m-2 s-1 of photosynthetically active radiation (PAR). Measurements were made on the day before harvesting (except measurements made at 100 for Ceora or 108 DAS for Kaspa which were in between harvests) under sunny conditions between 9.30 am and 3.00 pm. After measurement, the leaf in the cuvette was carefully marked, excised at harvesting, covered with aluminum foil and wrapped into a sealed polythene bag and immediately scanned to determine its area. This leaf area was required to recalculate photosynthesis and stomatal conductance obtained from the Li-Cor-6400.

3.2.7 Water use and water use efficiency Crop water use (Ct) from sowing to the start of water treatment was recorded as pre- podding or pre-seed set water use. Water use from the start of water treatment to maturity was recorded as post-podding or post-seed set water use for Ceora and Kaspa, respectively. The ratio of pre-to-post water treatment crop water use (hereafter expressed as pre-to-post podding water use (Ctp/Ctpp)) was determined to compare the pattern of water use between species and treatment. Water use efficiency (WUE) was calculated as the ratio of total dry mass including roots (WUEdm) or grain yield (WUEgr) to total Ct.

3.2.8 Statistical analysis The statistical package GenStat 10.2 was used to analyse the experimental data. A one- way analysis of variance (ANOVA) was calculated to compare the data for the well- watered and water deficit treatments. Analysis was performed separately for each species due to different sowing times and phenology. At final harvest, a two-way ANOVA was calculated to compare yield and yield components, water use and water use efficiency between species and treatments.

3.3. Results 3.3.1 Seasonal glasshouse temperature The average maximum and minimum glasshouse temperatures during the experiment were 21oC and 10oC, respectively (Figure 3.1). At sowing, the maximum glasshouse temperature was 20oC for Kaspa and 25oC for Ceora with a similar minimum temperature of around 15oC. The maximum and minimum temperature reached their lowest point of below 15oC and 5oC, respectively, at 70 (for Ceora) or 78 (for Kaspa) days after sowing (DAS). A moderate increase in maximum and minimum glasshouse temperatures occurred during the reproductive period reaching over 30oC and 17oC, respectively, at maturity.

Figure 3.1 Daily maximum and minimum growing season glasshouse temperatures (oC). Arrows indicate first flowering of Kaspa (first arrow) and Ceora (second arrow). Gaps indicate missing data.

3.3.2 Crop phenology There were no differences between species in the time to germination, first flowering and podding (Table 3.1) although Kaspa flowered and podded earlier (due to earlier

37 sowing) than Ceora. Water deficit did not affect flowering period in Kaspa and seed set and flowering period in Ceora. Ceora was more indeterminate than Kaspa and took longer to finish flowering and reach 50% seed set. Imposition of water deficit accelerated seed filling of both species which matured earlier than well-watered treatments. Kaspa matured in approximately 14 and 18 days earlier than Ceora in water deficit and well-watered plants, respectively. In Ceora, water deficient plants matured a week earlier than well-watered plants.

Table 3.1 Key stages of phenological development (days after sowing) and seed filling duration (days) of Ceora and Kaspa. Symbols are well-watered (WW) and water deficit (WD). Species Treatment Phenology

90% 50% End of 50% 50% Physical Seed emergen flowering flowering podding seed set maturity filling ce duration

Ceora WW 7 80 106 90 96 141 45 WD 7 80 106 90 96 133 37 Kaspa WW 7 80 98 88 90 122 32 WD 7 80 98 88 90 119 29

3.3.2 Cumulative water use The well-watered plants of Ceora used more water (9.6 L plant-1) than those of Kaspa (6.2 L plant-1). Water deficit reduced water use in both species, but Ceora still used more water (7.0 L plant-1) than Kaspa (5.6 L plant-1) (Figure 3.2). Cumulative plant water use of Kaspa was slightly higher than Ceora from around 40 DAS until first flowering (at 80 DAS). At the imposition of water deficit, cumulative plant water use was 4.3 L plant-1 for Kaspa and 4.4 L plant-1 for Ceora. Under well-watered controls, plant water use of Kaspa declined, but it was maintained in Ceora reaching a peak of approximately 9.4 L plant-1 at 127 DAS resulting in 3.3 L plant-1 more than Kaspa at maturity. In Ceora, well-watered plants used approximately 2.6 L plant-1 more than water deficient plants. In Kaspa, well-watered plants used approximately 0.7 L plant-1 more water than water deficient plants (but well below water deficient plants of Ceora).

Figure 3.2 Cumulative transpiration of Ceora (LS) and Kaspa (FP) for well-watered (WW) and water deficit (WD) treatments. Arrows indicate first flowering (first arrow) and start of water deficit (second arrow).

3.3.3 Soil-plant water relations There was considerable variation in soil-plant water relations caused by differences between species and treatments (Figure. 3.3). Soil water content for water deficient plants was significantly less (by 0.05 g g-1) at 104 and 110 DAS for Ceora and 112 and 119 DAS for Kaspa, respectively, compared with well-watered plants. This led to lowered pre-dawn leaf water potential by 0.4 MPa (P=0.007) and 0.2 MPa (P=0.001) at 104 and 110 DAS for Ceora and 0.5 (P=0.045) and 0.2 MPa (P=0.004) at 112 and 119 DAS for Kaspa compared with well-watered plants, respectively. At 112 DAS, water deficit significantly reduced leaf relative water content by 4% (P=0.024) in Ceora but not in Kaspa.

Water deficit reduced stomatal conductance by 128 mmol m-2 s-1 (P=0.029) and 144 mmol m-2 s-1 (P=0.02) at 104 and 110 DAS for Ceora and by 114 and 160 mmol m-2 s-1 (P>0.05) at 112 and 119 DAS for Kaspa compared to well-watered plants, respectively (Figure 3.4). Despite a significant reduction in stomatal conductance in Ceora, there was no effect on net photosynthesis. In Kaspa, net photosynthesis decreased in both water deficit and well-watered plants (except from 108 to 112 DAS when net photosynthesis for well-watered plants was maintained (P>0.05)).

Figure 3.3 Soil-plant water relations: gravimetric soil water content for Ceora (a) and for Kaspa (b), pre-dawn leaf water potential for Ceora (c) and for Kaspa (d), and leaf relative water content for Ceora (e) and for Kaspa (f). Symbols are well-watered plants (closed symbols) and water deficient plants (open symbols). Bars indicate standard error of means (n = 4). There were insufficient green leaves of water deficient plants of Kaspa available for RWC measurement at 119 DAS.

Leaf osmotic potential (OP) varied between treatments and species (Figure 3.5). OP of Ceora significantly decreased by 0.39 (P=0.031) and 0.53 MPa (P=0.027) at 104 and 110 DAS, respectively. In Kaspa, no significant reduction was observed at either date. Osmotic adjustment, calculated as the difference in OP between well-watered and water deficient plants adjusted to full turgor, was 0.26 MPa at 104 DAS for Ceora and 0.19 MPa at 112 DAS for Kaspa. Six days later, OA for Ceora increased to 0.4 MPa, but in Kaspa it could not be measured accurately anymore as most plants had senesced.

Figure 3.4 Stomatal conductance and net photosynthesis for Ceora (a, c) and for Kaspa (b, d). Symbols are well-watered plants (closed symbols) and water deficit plants (open symbols), respectively. Bars indicate standard error of means (n = 4).

3.3.4 Effect of water deficit on growth, green leaf area development, dry matter production and partitioning Plant growth Plant height measured for main stem and tillers (average) for Ceora and main stem for Kaspa varied significantly between species and treatments (Figure 3.6). Plant height at first harvest was 132 cm for Ceora (91 DAS) and 139 cm for Kaspa (99 DAS). In Ceora, water deficit reduced plant height by 9.0 and 9.9 cm (P>0.05) at 104 and 110 DAS, respectively, and by 16.7 cm (P<0.001) at maturity compared with well-watered plants. In Kaspa, there were no differences between treatments, except at 119 DAS when it was 10.8 cm (P=0.016) shorter in water deficient plants resulting in one less node per plant (P=0.029) compared with well-watered plants. In Ceora, water deficit reduced plant node numbers (average of the main stem and tillers) by 2.7 (P=0.036) at 110 DAS and 5.2 (P<0.001) at maturity compared with well-watered plants (Figure 3.6).

Figure 3.5 Osmotic potential for Ceora (a) and for Kaspa (b). Bars indicate standard error of means (n=4). Symbols are well-watered (WW) and water deficit (WD) plants. There were insufficient green leaves of water deficit plants of Kaspa available for osmotic potential measurement at 118 DAS.

Figure 3.6 Plant height and node number for Ceora (a, c) and for Kaspa (b, d). Symbols are well-watered plants (closed symbols) and water deficit plants (open symbols). Bars indicate standard error of means (n=4).

42 Green leaf area development and dry matter production Mean green leaf area at first harvest was 1264 cm2 plant-1 for Ceora (91 DAS) and 536 cm2 plant-1 for Kaspa (99 DAS) (Figure 3.7). Water deficit reduced green leaf area by 540 cm2 (P=0.012) and 336 cm2 plant-1 (P=0.033) at 104 and 110 DAS for Ceora and 121 cm2 (P>0.05) and 100 cm2 plant-1 (P=0.013) at 112 and 119 DAS for Kaspa compared with well-watered plants, respectively. In Ceora, green leaf area continued to increase in well-watered plants reaching its maximum at 104 DAS; meanwhile there was no change in green leaf area in water deficient plants for the same period. In Kaspa, green leaf area was at its maximum at 99 DAS and thus in the post-water treatment, green leaf area in both well-watered and water deficient plants continued to decline with a more rapid decrease in water deficient compared with well-watered plants towards maturity.

Dry matter production (including root dry mass) reflected cumulative water use and green leaf area development of both species (Figure 3.7). Average whole plant dry mass at first harvest was 14.4 g plant-1 for Ceora and 15.9 g plant-1 for Kaspa. As green leaf area of Ceora continued to increase in well-watered plants, whole plant dry mass was 3.4 (P=0.053) and 10.5 g plant-1 (P<0.001) higher than water deficient plants at 110 DAS and at maturity, respectively. In contrast, whole plant dry mass of Kaspa was slightly lower in water deficient compared with well-watered plants, but no significant difference was observed at either harvest.

Root dry mass was significantly higher in Ceora than Kaspa in all four harvests (P<0.001) (Figure 3.8). In Kaspa, root dry mass consistently decreased in subsequent harvests with no differences between treatments. In Ceora, however, root dry mass consistently increased with no differences between treatments until maturity when root dry matter of water deficient plants was significantly lower than well-watered plants (P<0.001)

Figure 3.7 Green leaf area development and dry matter production for Ceora (a, c) and for Kaspa (b, d). Symbols are well-watered plants (closed symbols) and water deficit plants (open symbols), respectively. Bars indicate standard error of means (n = 4).

Figure 3.8 Root dry matter for Ceora (a) and Kaspa (b). Symbols are well-watered plants (closed symbols) and water deficit plants (open symbols), respectively. Bars indicate standard error of means (n = 4). Note that scales are different between graph a and b.

Dry matter partitioning Dry matter partitioning varied between species and treatments (Figure 3.9). At first harvest in Ceora (91 DAS), stems accounted for 46% followed by green leaves (25%), while the pod wall was the smallest dry matter component (2%). There were no seeds or dead leaves at this stage. In Kaspa, the largest dry matter component at 99 DAS was tendrils (38%) followed by stems (23%). Seed dry matter accounted for 9% of total dry

44 matter and no dead leaves were observed. Dry matter components changed following the imposition of water deficit.

In Ceora, at 104 DAS, water deficit reduced dry matter components of stems and green leaves by 3% and 2%, respectively, while dead leaves and pod walls increased by 3% and 2%, respectively, compared with well-watered plants. In Kaspa, at 112 DAS, tendrils were reduced the most (by 2%), while dead leaves and seeds increased by 2% compared with well-watered plants. Six days later, dead leaf dry matter of Ceora continued to increase due to increasing leaf senescence accompanied by a rapid decline in green leaf dry mass. Water deficit decreased pod wall dry matter by 3% compared with well-watered plants, despite the proportion of seed dry matter in water deficit plants being 0.6% of the total higher than well-watered plants. In Kaspa, dry matter components continued to decline in both well-watered and water deficit treatments apart from dead leaves and seed weight. At 118 DAS, seed weight accounted for 39 and 36% of total dry matter in well-watered and water deficit plants, respectively.

Figure 3.9 Dry matter partitioning (g plant-1) of Ceora and Kaspa. Well-watered plants are (a) for Ceora and (b) for Kaspa and water deficit plants are (c) for Ceora and (d) for Kaspa. Note that tendril and petiole refer to Ceora and Kaspa, respectively. Note that the time scale is not linear.

At maturity, leaves, stems, petioles and root dry mass in Ceora were similar for both treatments. In contrast, pod wall yield was 4% lower in water deficient plants compared with well-watered plants. Seed yield accounted for 25% of total dry matter production in water deficient plants which was 2% higher than well-watered plants. In Kaspa, grain

45 yield accounted for almost 45 and 50% of plant dry mass in water deficient and well- watered plants, respectively.

3.3.5 Yield components Yield and yield components varied between species and treatments (Table 3.2). At maturity, Ceora had significantly higher total dry matter production (P<0.001) than Kaspa and there was a significant interaction (P=0.002) which was due to water deficit having a small effect on growth and dry mass of Kaspa and a relatively large effect on Ceora. Seed yield differed significantly between species (P<0.001) and water deficit treatments (P=0.013), but there was no significant interaction (at P≤0.05) between species and treatments. Although Kaspa produced less pods (P<0.001) and seeds (P<0.001) compared with Ceora, it had more seeds per pod (P<0.001) and larger mean seed size (P<0.001) that contributed to higher total seed yield. This resulted in higher harvest indices in Kaspa than in Ceora (P<0.001).

Water deficit affected Ceora more than Kaspa. Water deficit reduced seed yield of Ceora by 1.9 g plant-1 (P=0.034) compared with well-watered plants. Water deficient plants had 18 (38%) fewer pods (P<0.001) than well-watered plants, which was the main cause of the 24% lower seed yield. Reduced pod number was somewhat compensated for by increased seed numbers per pod (13%) and mean seed size (11%) compared with well-watered plants (P>0.05). Water deficient plants had slightly higher harvest indices (P>0.05) than well-watered plants. In Kaspa, water deficit significantly reduced harvest index by 0.04 (P<0.033).

Table 3.2 Yield, yield components and harvest index of Ceora and Kaspa at maturity. LSD refers to interaction between species and treatment. Well-watered Water deficit LSD Components Ceora Kaspa Ceora Kaspa P<0.05 DM (g plant-1) 34.81 20.72 24.34 19.63 3.77*** Seed yield (g plant-1) 7.87 10.13 5.99 8.88 1.66ns Harvest index 0.23 0.49 0.25 0.45 0.03** Pod number (plant-1) 47.25 9.13 29.25 8.88 3.54** Seed number (plant-1) 99.38 51.13 68.75 47.13 16.41** Seed number (pod-1) 2.09 5.61 2.36 5.32 0.41ns Weight of 50 seeds (g plant-1) 4.24 10.50 4.69 9.75 1.03ns *** significant at P≤0.001 ** significant at P≤0.01 * significant at P≤0.05 ns not significant

46 a 3.3.6 Water use and water use efficiency be d c Total crop water use (Ct) differed between species and water deficit treatment (P<0.001) with a strong interaction (P<0.001) (Table 3.3). This was due to the small difference in Ct between well-watered and water deficit for Kaspa and a large difference for Ceora plants. A significant interaction also occurred for the ratio of pre-to-post podding crop water use (Ctp/Ctpp) (P=0.031). Kaspa had higher Ctp/Ctpp than Ceora (P<0.001) indicating Kaspa used proportionately less water than Ceora during seed filling. Due to its more indeterminate growth habit, Ceora maintained its high rate of water use during the reproductive period. Water use efficiency for dry matter (WUEdm) and grain (WUEgr) did not differ between species or treatments (P>0.05), except WUEgr when species were the main effect (P<0.001). In Ceora, WUE did not differ between treatments.

Table 3.3 Total crop transpiration (Ct, L), ratio of pre-to-post podding water use (Ctp/Ctpp), and water use efficiency for dry matter production (WUEdm, g L-1) and grain yield (WUEgr, g L-1). Symbols are well-watered (WD) and water deficit (WD). Ct Ctp/Ctpp WUEdm WUEgr Treatment Ceora Kaspa Ceora Kaspa Ceora Kaspa Ceora Kaspa WW 9.68 6.26 0.87 2.32 3.60 3.31 0.81 1.62 WD 6.82 5.52 1.66 3.62 3.56 3.54 0.87 1.60

LSD- Treatment 0.54*** 0.23*** 0.18ns 0.10ns LSD- Species 0.54*** 0.23*** 0.18ns 0.10*** LSD- Interaction 0.76*** 0.33* 0.26ns 0.14ns *** significant at P≤0.001 ** significant at P≤0.01 * significant at P≤0.05 ns not significant

3.4. Discussion The findings in this experiment support the hypothesis that Ceora grass pea grows rapidly, and produces an amount of dry matter that should be sufficient to support some filling of grain in case of water deficit. Ceora is a more indeterminate crop; therefore growth and yield were more affected by water deficit compared with Kaspa. Water deficient plants of Ceora avoided dehydration through reduction in green leaf area and stomatal conductance without a significant effect on photosynthesis. This enabled seed filling of water deficient plants that resulted in a similar harvest indices and water use efficiency compared with the control plants. Water deficient plants escaped drought by maturing earlier than control plants.

3.4.1 Crop phenology and its importance for adaptation to water deficits This study confirmed earlier studies with grass pea that partly attributed its adaptation to water deficit environments of southern Australia to the crop’s phenology. The time from sowing to 90% emergence and 50% flowering of Ceora was similar to Kaspa, a species that is well-adapted to water limited environments of southern Australia (Thomson and Siddique 1997; Thomson et al. 1997) (Table 3.1). This result agreed with field experiments in a range of environments of Western Australia which found that Ceora was well-adapted to this environment (Siddique et al. 1996; Siddique et al. 2001; Thomson and Siddique 1997; Thomson et al. 1997). These studies found that Ceora was fast growing, early flowering and early podding. This early phenology is important in southern Australia where terminal drought during the reproduction phase is common (Ludlow and Muchow 1990; Siddique et al. 1999; Siddique et al. 2001; Turner et al. 2001). In the present study, the imposition of water deficit at early pod development did not affect the time to reach 50% pod set, seed set and end of flowering compared with the control. This was consistent with a review that indeterminate crops are less sensitive to moderate drought in various stages of reproductive development (Wery 2005).

Ceora escaped the full impact of drought through early maturity. It was observed that in both species, plants under water deficit accelerated seed filling and matured earlier (by 8 days in Ceora and 3 days in Kaspa) than control plants. This demonstrated the ability of Ceora and Kaspa to escape drought by completing the reproductive cycle before the onset of severe drought and this was consistent with reports on chickpea (Berger et al. 2006; Turner et al. 2005), field pea (Siddique et al. 2001; Thomson et al. 1997), wheat (Siddique et al. 1990) and upland rice (Lafitte and Courtois 2002) . The present study also observed that due to a more indeterminate growth habit, Ceora took longer (by 19 days in well-watered and 14 days in water deficient plants) to reach maturity compared with semi-indeterminate Kaspa (Table 3.1). This was due to a continued flowering that slowed seed filling and thus delayed maturity compared with semi-indeterminate Kaspa. Similar observations were reported for Lathyrus sativus accessions (Thomson et al. 1997) and chickpea (Turner et al. 2005).

48 3.4.2 Physiological and morphological adaptation mechanisms in response to moderate water deficit Under moderate water deficit, Ceora in the present study avoided dehydration through a reduction in stomatal conductance without having a significant effect on photosynthesis. The study was consistent with a field study observing that stomatal conductance of grain legumes markedly decreased when leaf water potential dropped below -0.9 MPa (Leport et al. 1998). This reduction is a primary response to water deficits to avoid dehydration by minimising water loss (Stoddard et al. 2006). This effect has also been demonstrated in lupin (Rodrigues et al. 1995). Normally, a moderate reduction in stomatal conductance causes a less-than-proportional decrease in photosynthesis (Farquhar and Sharkey 1982). It has been reported that photosynthesis of indeterminate crops is less sensitive to moderate drought (Hetherington and Woodward 2003; Wery

2005). Hetherington and Woodward (2003), for example, reported that, in C3 plants, high photosynthetic water use efficiency (CO2 uptake per unit increase in stomatal conductance) occurred when stomatal conductance fell between 50 and 200 mmol m-2 s- 1. Ceora, in the present study, responded similarly, as there was no significant reduction in photosynthesis when stomatal conductance fell between 50 and 200 mmol m-2 s-1. This was possibly associated with stomatal opening strategies (Turner and Begg 1981); where stomata open in the morning or late in the afternoon (and thus photosynthesis continues) when evaporative demand is low, and close during midday when evaporative demand is high. This physiological adaptation enables plants exposed to drying soil to avoid dehydration, while continuing photosynthesis.

Osmotic adjustment (OA) may contribute to dehydration avoidance of Ceora. It was observed that moderate water deficits significantly decreased leaf relative water content (RWC) at 104 DAS in Ceora and 118 DAS in Kaspa (Figure 3.3 e, f). This reduction gradually decreased osmotic potential of water deficient plants (Figure 3.5) leading to a significant reduction in pre-dawn leaf water potential (Figure 3.5 c, d). This physiological change was possibly due to increased soluble sugar contents in leaves, as reported by Chaves and Oliveira (2004). Some plants and genotypes have the capacity to adjust osmotically as plant tissue osmotic potential decreases, but the degree of OA varies between species and genotypes; being 0.4 MPa in soybean and 0.7 MPa in sorghum (Morgan 1984). In chickpea, it was reported that OA varied from 0.0 to 1.2 MPa depending on genotypes, level of water deficit, location and physiological stage of the plant (Turner et al. 2007). OA of Ceora in the present study was around 0.3 MPa at

49 104 DAS and increased to 0.4 MPa at 110 DAS. Increased OA enabled plants subject to drying soil to maintain high plant water status (Blum 2005) which was consistent with the observation in pigeon pea (Flower and Ludlow 1986). Flower and Ludlow (1986) stated that OA is particularly important to maintain turgor and high leaf relative water content when plants are exposed to severe water deficit.

Reduction in green leaf area (by 29% at maximum growth of the control) of Ceora in response to moderate water deficit (Figure 3.7a) contributed to dehydration avoidance. There were three major changes in leaf development that caused a reduction in green leaf area of water deficient plants compared with the control. First, visual observation suggested that there was less leaf expansion of water deficient plants compared with well-watered plants which was in agreement with reports by Boyer (1970), Hsiao (1973) and Wery (2005). Second, the number of nodes which could potentially develop leaves was less under water deficit (Figure 3.6c). Boutraa and Sanders (2001) also observed fewer nodes under water deficit in bean. Last, water deficit increased leaf senescence of older leaves (based on reduction in leaf dry mass, Figure 3.9) which was consistent with reports by Morgan (1984) and Turner and Begg (1981). Reduction in green leaf area reduced water loss. Moreover, visual observation also indicated that remaining green leaves rolled during midday when there was high atmospheric demand at low leaf water potential as reported by Ludlow and Muschow (1990) and Rodrigues et al. (1995). This morphological adaptation may also have contributed to dehydration avoidance through reduced transpiration rates.

Ceora maintained high root dry matter which was an adaptive characteristic in response to water deficit that contributed to dehydration avoidance. Ceora not only produced much more root dry mass compared with Kaspa in the well-watered treatment, but also maintained root dry mass under water deficit (Figure 3.8), resulting in a markedly higher root/shoot ratio (P>0.005) compared with the control. High root dry mass under water deficit potentially increased Ceora’s ability to tolerate drought. This may be even more important in the field, where roots may grow deeper into the soil profile and extract more soil water. This may contribute to drought tolerance as reported by Siddique et al. (2001), Leport et al. (1998), Thomson and Siddique (1997), Begg and Turner (1976) and Sinclair and Muchow (2001).

50 3.4.3 Yield and yield components High dry matter production in Ceora in the present study was consistent with that observed in field experiments in southern Australia (Siddique et al. 1996; Siddique et al. 2001; Thomson and Siddique 1997). Dry matter production in Kaspa did not differ between treatments in any harvest (Figure 3.7). In Ceora, water deficit decreased total dry matter by 30% compared with the control which corresponded with a 29% reduced green leaf area at the time of maximum growth of control plants. Between species, Ceora produced more dry matter than Kaspa in both treatments. High dry matter production of Ceora at anthesis and its partitioning into seed as water deficit developed during seed filling was important to improve harvest indices (see below).

Water deficit accelerated seed filling with better dry matter partitioning into seeds compared with controls in both species. At 110 DAS, the proportion of dry matter partitioned into seed mass of Ceora was 0.6% higher in water deficient than control plants, which corresponded with the increased leaf senescence. At 118 DAS (a week later compared with Ceora), seed mass of Kaspa was already 39% of total dry mass in control and 36% of total in water deficient plants. This demonstrated faster seed filling of Kaspa than Ceora, explaining better adaptation to terminal drought in southern Australia (Thomson et al. 1997). The proportion of plant parts, such as leaf, stem, petiole and root, in Ceora at maturity was similar between treatments, but pod wall was 13% of total dry mass in water deficient plants and 17% in control plants, suggesting that assimilates of water deficient plants were preferentially allocated to seeds, and/or reallocated from pod walls into seeds. This resulted in a 25% (of total) seed dry mass in water deficient plants and that was 2% higher than control which was consistent with the evidence in lupin (Rodrigues et al. 1995), chickpea (Davies et al. 2000; Turner et al. 2005) and in rice and wheat (Yang and Zhang 2006).

At maturity, although Ceora produced more dry matter than Kaspa, Ceora had less seed yield than Kaspa in both treatments. Kaspa produced less pods than Ceora, but its seed number per pod and seed size were double than that of Ceora resulting in more seed dry mass than Ceora. This led to higher harvest indices of Kaspa than Ceora. This result confirmed greater grain yield stability of Kaspa that demonstrates its superior adaptation to the drought prone environments of southern Australia (Ludlow and Muchow 1990; Thomson and Siddique 1997; Thomson et al. 1997).

51 In Ceora, water deficit significantly decreased grain yield by 19% compared with the control. This was due to the lower number of pods and thus seeds per plant compared with the control (Table 3.2). This result was consistent with similar studies in chickpea (Davies et al. 2000; Leport et al. 2006) and narrow-leafed lupin (Palta and Plaut 1999). Leport et al. (2006) reported that water deficit applied at early podding affected pod abortion more than later water deficit. This was probably associated with a reduction of pollen fertility in response to water deficit (Fang et al. 2010) (Chapter 4) contributing to a lower seed number per pod as observed in narrow-leafed lupin (Palta and Plaut 1999). In the present study, there was no reduction in seed size compared with the control which was consistent with other reports on grass pea (Herwig 2001; Polignano et al. 2009). Seed size was comparable with other improved grass pea lines observed at Tel Hadya (Kumar et al. 2010). Seed number per pod was slightly higher under water deficit compared with control plants which somewhat compensated for the lower number of seeds per plant. As a result, there was no difference in harvest index between water treatments which is consistent with studies on lupin (Palta and Plaut 1999) and grass pea (Thomson et al. 1997). Consistency in harvest indices of Ceora confirmed its good adaptation to moderate water deficit, agreeing with an earlier report on the growth and yield of grass pea in high rainfall environments of southern Australia (Siddique et al. 1996; Siddique et al. 2001). Harvest indices may be decreased when Ceora is exposed to a severe water deficit during reproduction as reported for other species (Ludlow and Muchow 1990; Passioura 1977) (Chapter 4).

3.4.4 Water use and water use efficiency Water use influenced growth, development, dry matter production and yield of Ceora and Kaspa. Cumulative water use was similar between species at the start of soil drying (Figure 3.2). However, due to a more indeterminate growth habit, Ceora maintained a high rate of water use for a longer period compared with Kaspa which started to decrease the rate of water use from around seed set. Therefore, Ctp/Ctpp of Ceora was significantly lower than that of Kaspa (Table 3.3). The relatively low water use of Kaspa during the reproductive period without grain yield penalties contributes to its good adaptation to water limited environments (Siddique et al. 1999). This was possibly associated with the much more advanced seed filling of Kaspa resulting in higher water use efficiency for grain compared with Ceora. There were no differences in water use efficiency for dry matter between species. In Ceora, there were no significant differences in WUEs between treatments suggesting its good adaptation to moderate

52 water deficit which was consistent with reports based on field studies (Leport et al. 1998; Thomson et al. 1997). The present study distinguished the pattern of water use between Ceora and Kaspa that was due to the more indeterminate growth habit of the former; consequently, Ceora plants used more water during reproduction than Kaspa.

3.5. Conclusion Ceora and Kaspa have similar growth patterns for being fast growing, early flowering and having high dry matter production at flowering. Ceora’s disadvantages compared with Kaspa were due to its more indeterminate growth habit resulting in a longer reproductive period. Water deficit, therefore, had a greater effect on Ceora than Kaspa. It reduced the number of pods per plant in Ceora leading to decreased seed yield compared with the well-watered control. However, this study confirmed the adaptation of Ceora to water deficit. Ceora was able to avoid dehydration through a reduction in green leaf area, stomatal conductance and possibly osmotic adjustment under water deficit. These strategies allowed maintenance of a high photosynthetic rate enabling seed filling of water deficient plants. This resulted in similar harvest index and water use efficiency for grain between treatments. Ceora also demonstrated an ability to escape drought through early maturity. Thus, it is concluded, that Ceora displayed mechanisms of dehydration avoidance and drought escape in response to moderate water deficit during the reproductive period.

53 Chapter 4

Grass pea tolerates severe water deficit and sets normal sized seed when the deficit is relieved

Background and aims This study investigated the effect of severe water deficit on yield and yield components, reproductive development, seed 3-(N-oxyalyl)- L-2,3-diaminopropionic acid (ODAP) concentration and seed quality (in terms of emergence and seedling growth) of grass pea (L. sativus cv Ceora). Methods Plants were grown in PVC pots filled with 10 kg of a 4:1 mixture of clay loam soil and sand in a glasshouse at UWA. Watering was stopped at first flowering, leading to severe water deficit. When pre-dawn leaf water potential (Ψ) was -3.12 MPa, the plants were rewatered. Key results At maturity, water deficit reduced dry matter by 60%, seed yield by 87%, harvest index by 67% and 75% water use efficiency for grain, compared with the control. Flower production stopped when Ψ fell to -1.8 MPa. At Ψ=-1.5 MPa, only 25% of the total flowers filled pods (compared with 95% filled pods in the control) and the rest aborted as flowers (48%) or pods (27%). Filled pods had more aborted ovules resulting in 29% less seeds per pod than the control. Water deficit reduced pollen viability (from 88 to 75%) and germination (from 53 to 28%) compared with the control. Of the germinated pollen, pollen tubes reaching the ovary were reduced from 70 to 39% compared with the control. Seed size was slightly higher under water deficit than control, but it did not differ significantly between the treatments. Seed ODAP concentration was below the detection limit (<0.05%) in both treatments. Seeds produced from water deficient mother plants had 21% less emergence than those from well- watered plants, but the days from sowing to emergence did not differ between treatments. Seedling shoot dry mass was significantly higher (18%) in water deficient than control plants which matched a 19% greater seed mass. Conclusion Under severe water deficit, Ceora was able to tolerate desiccation and produce some seed yield by concentrating its limited resources to a smaller number of viable surviving pods. Low seed yield in water deficient plants was due to the cessation of flower production and high flower, pod and ovule abortion which were associated with reduced pollen fertility. Seeds produced from water deficient plants were not smaller than those from well-watered control plants and did not have high ODAP concentrations. Seedlings from these seeds had somewhat lower emergence rates but similar growth as those from control plants.

4.1. Introduction In the experiments of Chapter 3 (Sections 3.3, 3.5 and 3.6), moderate drought reduced yield, but it did not affect photosynthesis, harvest indices or water use efficiency at the whole plant level in grass pea. It was concluded that maintenance of photosynthesis from remaining green leaves enabled seed filling in plants that had water deficit imposed at podding. However, there is evidence of suppressed photosynthetic rate, yield, harvest index and water use efficiency when plants are exposed to severe water deficit during reproduction. In chickpea genotypes, seed yield decreased by 50–80% when terminal drought was imposed (Leport et al. 1999) due to reduced seed numbers 54 and size. Low seed numbers resulted from increased flower and pod abortion when plants experienced water deficit. Flower and pod abortion in chickpea (Almaz) exposed to terminal drought at flowering increased by up to 56 and 73% compared with 15 and 67% for controls, respectively (Fang et al. 2010). The sensitivity of flowers and pods to water deficit has been investigated in various grain legume and cereal crops. In soybean, flower abortion in response to water deficit was reportedly due to ovule sensitivity rather than reduced pollen fertility (Kokubun et al. 2001), but generally, male reproductive development is more sensitive to water deficit than female (Saini 1997; Saini and Westgate 2000). In chickpea, pollen viability decreased by 50% and germination by 80%, compared with control plants when pre-dawn Ψ dropped below - 1.0 MPa (Fang et al. 2010). A high reduction in pollen germination due to water deficit has been reported before (Saini 1997; Saini and Westgate 2000). In rice, water deficit deformed and reduced the size of pollen leading to 80% floret failure (Sheoran and Saini 1996). However, in white clover (Trifolium repens L.), water deficit reduced the number of ovules (Turner 1993).

Little is known about reproductive development of Ceora in response to water deficits and its consequences for grain yield. In a field study, it was reported that yield of grass pea (L. sativus) decreased by up to 46% in rainfed conditions compared with the irrigated control (Leport et al. 1998). In a glasshouse experiment, water deficits during the reproductive period reduced seed yield of grass pea by 40−82% compared with the controls (Herwig 2001). Under moderate water deficit, yield of Ceora was reduced by 24% compared with the control (Chapter 3). This suggests that water deficit during the reproductive period causes Ceora to abort flowers and pods, possibly associated with reduced pollen fertility.

The relationship between seed ODAP concentration of grass pea and environmental factors is not fully resolved. For example, average seed ODAP concentration of L. sativus (among 17 accessions) obtained from ICARDA observed in three contrasting agro-climatic environments of Western Australia was highly variable ranging from 0.17% at Merredin, 0.38% at Northam to 0.44% at Mullewa (Siddique et al. 1996). This study found that low seed ODAP concentration observed at Merredin was positively correlated with low grain yield. Contradictory results have been shown on the effect of planting time on seed ODAP concentration. In a field study at The University of Western Australia, it was found that seed ODAP concentration of L. sativus line SEL

55 455 decreased from 0.38% in seeds sown in early winter to 0.26% for seeds sown in late winter (Herwig 2001). In contrast, in a recent study at Tel Hadya, Syria, it was found that early planting had lower seed ODAP concentration than later planting (Kumar et al. 2010). Above reports suggest that there may be several factors that contribute to an increase in seed ODAP concentration. Water deficit seems to be a clear factor. For example, in a glasshouse study, Herwig (2001) showed that severe water deficit increased ODAP concentration of L. sativus from 0.15% to 0.31% in the line SEL455, from 0.25% to 0.38% in the line 80092 and 0.18% to 0.26% in the line SEL 526 (Herwig 2001). In a field study, water deficit (40% FC) increased ODAP concentration of L. sativus from 0.5% to 0.6% in Xide variety and 0.8% to 0.9% in Yongshou compared with the irrigated controls (80% FC) (Yang et al. 2004). Previous reports indicated that seed ODAP concentration of the new cultivar Ceora was low (0.04 − 0.09%) (Hanbury et al. 2005; Siddique et al. 2006), but no study has been conducted on the effect of water deficit on ODAP concentration of this cultivar.

This study examined grass pea cv. Ceora under severe water deficit imposed from first flowering to (i) compare growth and development, (ii) compare yield, yield components, seed ODAP concentration and abortion of flowers, pods, seeds and ovules, (iii) investigate pollen viability, germination, pollen tube growth and number of ovules per flower, and (iv) seed quality in terms of emergence and seedling shoot dry mass between water deficient and well-watered parent plants. The hypothesis of this study was that severe water deficit (i) suppresses growth and development, (ii) reduces harvest index and water use efficiency (iii) increases seed ODAP concentration, (iv) increases flower, pod, seed and ovule abortion resulting in low seed yield, (v) reduces pollen fertility and number of ovules per flower, and (vi) does not affect the size and quality of seeds produced.

4.2. Materials and methods 4.2.1 Study location, experimental design and experimental details The experiment was conducted in the same glasshouse as that used for the study described in Chapter 3 from May to October 2009. Pots were arranged on benches in a completely randomised design (CRD) with five replications for leaf water potential, growth, development and yield studies, six replications for crosses and eight replications for pollen viability and germination studies. Two different watering regimes (well-watered control and water deficient) were used.

Pots were filled with approximately 10 kg of a 4:1 mixture of sieved air-dried reddish- brown clay loam soil (pH 7.8) collected from a farm site at Bindi Bindi, Piawanning, WA and river sand. The mixture had a water holding capacity of 2.5 L pot-1. Pot dimensions, pot filling and seed sowing were the same as described in Chapter 3. Fertilisers were applied before watering to field capacity followed by application of Group F rhizobium (recommended for faba bean, lentil, vetch and field pea) and seeding. Four seeds were sown in each pot on 23 May 2009 and thinned to one plant per pot 10 days after sowing (DAS) followed by the application of plastic beads (details described in Chapter 3). Wire frames with known weight were set on each pot to encourage upright growth. Bamboo sticks with known weight were additionally set on the wires when plants, particularly control plants, grew over the wire.

4.2.2 Watering details Field capacity (FC) of the soil was measured using the same techniques as described in Chapter 3. Pots were watered to 80% FC before sowing and maintained at this water content by regular watering. Pots were weighed every two to three days to estimate crop water use and determine next watering requirement until first flowering at 82 DAS, when the water deficit treatment began. Control plants were watered as usual, but water deficient plants had watering withheld for 19 days before slowly being relieved through rewatering to normal at 100 DAS. During the treatment, pots were weighed in the afternoon, followed by watering of the control plants (between 4−6 pm). Watering (for both control and restored plants) was stopped at 125 DAS. Total crop transpiration was determined as the difference between evapotranspiration (Et) and evaporation (E). Et was determined by summing daily Et from sowing to maturity.

4.2.3 Phenology Crop phenology methods are described in Chapter 3.

4.2.4 Soil-plant water relations Pot weight from water deficient plants was used to calculate gravimetric soil water content (SWC) and presented against pre-dawn Ψ during the treatment period. Ψ is commonly used as an indicator of plant water status (Davies and Zhang 1991). Pre- dawn Ψ was regularly measured from initiation of the treatment (83 DAS) until Ψ

57 between water deficient and control plants was similar (114 DAS) after rewatering of water deficient plants. Measurement details for pre-dawn Ψ are described in Chapter 3.

4.2.5 Growth, harvest index and water use efficiency Growth and development were determined by counting the number of nodes and measuring the height of main stem and tillers at each harvest. There were four harvest times: at treatment imposition (82 DAS), rewatering of water deficient plants (100 DAS), full recovery of water deficient plants (114 DAS) and maturity (145 for control plants and 149 DAS for water deficient plants). Different parts of the plant such as petioles, stems, pod walls (including young pods, aborted pods and flowers), senesced leaves, green leaves, seeds and roots were separated before oven-drying at 70 C for 48 h and weighed. Roots were washed from the soil as described in Chapter 3.

Yield, yield components, harvest index and water use efficiency were determined from tagged plants (section 4.2.6) at maturity. Pods with the same flowering date (based on tagged date on pod) were combined and number of filled pods, aborted pods and flowers were noted for these groups. Empty pods were counted as aborted pods, while dead flowers and/or stalks with no flower (fall-off) were counted as aborted flowers. In addition, number of seeds, seeds per pod, aborted seeds and ovules (definitions in next paragraph) were also noted (Figure 4.10c). Plant components such as seeds (seeds were kept separate according to flowering date), pod walls, stems, petioles and dead leaves were separated before being oven-dried at 70 C for 48 h and weighed. Seeds were further dried in the same oven-drier for about a week and then weighed. Seed dry weights from different flowering dates were summed for cumulative dry weight, and calculation of dry matter partitioning, harvest index and water use efficiency (details provided in Chapter 3).

Reproductive development of water deficient and control plants was compared between 78 and 90 DAS when water deficient plants produced viable flowers and pods (or before cessation of flowering). Total flower production, filled pods, aborted pods and flowers, aborted seeds and ovules were compared according to time of flowering (DAS). An aborted ovule is defined here as one that does not develop either with or without fertilisation and an aborted seed is an abnormally small seed which aborts after fertilisation. Aborted seeds are darker and larger than aborted ovules which are pale in colour.

4.2.6 Monitoring of flowers Flowers from water deficient and control plants allocated for final harvesting at maturity were tagged as they formed from the beginning to the end of flowering to quantify cumulative flower number, pod set and abortion, seed set, seed and ovule abortion. All newly-developed flower buds appearing within three-day intervals were tagged with the same date (date of tagging). In water deficient plants, new shoots that developed after rewatering were tagged to separate flowers and pods developed during water deficit with those post water deficit. All flowers and pods developed in the post water deficit period were totalled at maturity.

4.2.7 Pollen viability and germination in vitro and pollen tube growth in vivo Methods and solutions used in these studies were optimised in a preliminary study.

Pollen viability and germination studies in vitro This study was done once every three days from 83 DAS (a day after treatment imposition) to 95 DAS (when flowering ceased in water deficient plants), with eight replications. Sixteen independent fully developed flowers (at least 7 days after flower bud appeared, Figure 4.13d, h) were excised and immediately placed in closed Petri dishes with moist Whatman filter paper 1001-085 (Whatman International Ltd), and were prepared in the laboratory using the sucrose method (Shivana and Rangaswamy 1992). A drop of sucrose solution (containing 10 g sucrose, 0.01 g boric acid, 0.03 g calcium nitrate, 0.02 g magnesium sulphate and 0.01 g potassium nitrate in 100 mL deionised water) was placed on a labelled microscope slide followed by squeezing anthers with pollen grains (after carefully removing sepal and corolla) into the solution. Pollen grains in the solution were gently mixed using forceps for a uniform distribution. The slides were put in a humid container in a dark room (at about 20oC) for about 1 h. A drop of Alexander solution (Alexander 1969) was added to the sucrose solution for staining and sealed with a microscope cover slide. Slides were gently warmed (using a light flame) for about 8 seconds for staining and immediately observed using a fluorescence microscope (Zeiss, Oberkochen, Germany). Pollen germination was recorded when the pollen tube exceeded the diameter of the pollen grain. Pollen grains that stained and/or germinated were regarded as viable. An average of 250 pollen grains were counted from 4 to 6 photos (depending on the density of pollen) taken from a

59 randomly selected area under the microscope. The number of viable, germinated and non-viable pollens (unstained pollen) was counted as a proportion of the total.

Hand crossings for in vivo pollen germination and pollen tube growth Grass pea is a self-pollinated species (see Figure 4.13 for the development stages of Ceora’s flower). Before making crosses, pots were designated as female control (FC), male control (MC), female water deficit (FS) and male water deficit (MS), where control is well-watered and deficit is water withheld. Crosses were made on four occasions (86, 89, 92 and 95 DAS) with 8 replications by collecting pollen from the male flower and pollinating the emasculated female flower of the second plant, following the order FC–MC, FC–MS, FS–MC and FS–MS. Emasculation occurred in the afternoon (between 3 and 5 pm) and pollination the following day (between 7 and 11 am). A flower was emasculated when it had approximately the same length sepal and corolla. A mature flower (between 5 and 6 days after flower bud appearance) (Figure 4.13) from a male plant was excised and half of the sepal and corolla were removed. Pollen grains were collected using forceps and carefully set on the stigma of the emasculated female flower (at least 1 emasculated flower in each plant per cross was not pollinated as a check). In this study, pollen from one male flower was applied to one emasculated female flower. The number of crosses in each plant varied due to variation in numbers of newly-developed flowers. In each cross, some pollinated flowers were removed for in vivo pollen germination and examination of pollen tube growth, while the rest were retained on the host plant for pod and seed set studies. Observations were limited to FC–MC and FC–MS due to the large proportion of emasculated flowers that died in water deficient plants (FS–MC and FS–MS). This was possibly due to the combined effect of water deficit and exposure of the pistil to high temperature (mean maximum temperature during the crossings was around 25oC) (Figure 4.1).

For crosses made at 86 and 89 DAS, three pollinated flowers (from three independent plants) were sampled for in vivo pollen tube growth after two days. Flower parts such as anthers, standard and wings were removed from the pistil. Pistils (tissues) were fixed in a labelled tube containing 10 to 15 ml acetic acid:ethanol (1:3 v/v) solution and stored in a cool room for a minimum of 72 h. The solution was changed to sodium hydroxide (4N NaOH) for about 48 h to soften the tissues, which were then rinsed by soaking in regularly changed (every 5 min) deionised water for 30 min. The tissues were stained with aniline blue solution at pH 11 (Shivana and Rangaswamy 1992) and put in a cool

60 room for 72 h prior to observation. The tissue was then set on a labelled microscope slide and mounted with two drops of glycerin and covered with a microscope cover slide. The number of pollen tubes present in the style and ovary were counted under a fluorescence microscope (Zeiss, Oberkochen, Germany). The percent of pollen tubes reaching the ovary was calculated as the ratio of the number of tubes in the ovary to the total number in the style. Pistils used for the pollen tube growth study were also used to determine the number of ovules per flower.

4.2.8 ODAP concentration Matured seeds from each of five replicates were randomly selected for ODAP determination. ODAP concentration was analysed using Capillary Zone Electrophoresis (CZE) (Arentoft and Greirson 1995) at the Chemistry Centre Western Australia.

4.2.9 Glasshouse temperature Daily maximum and minimum glasshouse temperatures were recorded during the experiment.

4.2.10 Seed emergence and seedling dry mass following drought A total of 210 seeds (105 for water deficient and 105 for control plants) were randomly selected and weighed individually. Each seed was sown in a labelled small square pot filled with river sand at 1 cm deep, in the same glasshouse as the previous experiments on 22 June 2010, followed by regular watering using deionised water until harvest at 30 days after sowing. Seedling emergence was counted daily from the start to the end of emergence. At harvest, shoots were cut at the soil surface, oven-dried at 70 C for 48 h and weighed.

4.2.11 Statistical analyses The statistical package GenStat 10.2 was used to analyse experimental data. A one-way analysis of variance (ANOVA) compared control and water deficit treatments. Two- sample (unpaired) t-test was used to compare days to emergence, emerged seed size and seedling shoot dry mass between treatments. In addition, emerged and non-emerged seeds within the treatment were also compared.

61 4.3. Results 4.3.1 Seasonal glasshouse temperature The average maximum and minimum glasshouse temperature during the experiment were 25oC and 12oC, respectively (Figure 4.1). Although the maximum glasshouse temperature dropped below 20oC at 34 and 89 DAS, in general, the maximum glasshouse temperature was maintained at around 25oC from sowing to physiological maturity before rising to over 30oC at harvest. The minimum glasshouse temperature fluctuated over the growing season, ranging from 6oC to 15oC from sowing to physiological maturity before increasing to around 19oC at harvest.

Figure 4.1 Daily maximum and minimum growing season glasshouse temperatures (oC). Arrows indicate 90% flowering (first arrow), rewatering of water deficient plants (second arrow) and maturity (third arrow).

4.3.2 Crop phenology Water deficit imposed at first flowering (82 DAS) reduced the flowering period by 3 weeks compared with control plants, disregarding flowers developed in the post water deficit period (Table 4.1). There were no differences in time to 50% podding or seed set between water deficient and control plants. Control plants reached maturity 4 days earlier than water deficient plants. Later maturity in water deficient plants resulted from extended vegetative and reproductive growth after the water deficit was relieved at 100 DAS. Rewatering enabled seed filling in pods that survived water deficit and also encouraged new shoot development that produced flowers and pods, despite the fact that most flowers and pods developed in the recovery period were aborted.

62 Table 4.1 Key stages of phenological development (days after sowing) and seed filling duration (days). Water deficit and control plants are represented by WD and WW, respectively. Phenology Treatment 90% 50% End of 50% 50% Physical Seed filling emergence flowering flowering Podding seed set maturity duration WW 7 80 111 92 98 145 47 WD 7 80 90* 92 98 149 51 * Refers to the cessation of flowering before water deficit was relieved at 100 DAS.

4.3.3 Soil-plant water relations Average daily water use (based on changes in pot weight) in control plants was approximately 400 ml day-1 plant-1 (Figure 4.2). In contrast, the rate of daily water use in water deficient plants decreased gradually following imposition of the deficit to 45 ml day-1 plant-1 at 93 DAS. Plant water use declined further, reaching less than 9 ml day-1 plant-1 at 100 DAS just before the water deficit was relieved. Water deficient plants slowly recovered and the rate of daily water use gradually increased, but was not to the level of control plants.

Figure 4.2 Pot weight during the water deficit treatment. Control plants were watered regularly to 80% FC (2–3 days intervals) and water deficient plants were not watered for 19 days before the deficit was relieved gradually between day 100−106 DAS when regular watering to 80% FC was applied. Values are means ± SE (n = 5).

Plant pre-dawn leaf water potential (Ψ) for control plants was constant around -0.5 MPa (Figure 4.3). In water deficient plants, pre-dawn Ψ gradually decreased from -0.5 MPa at 83 DAS to below -3.0 MPa at 100 DAS when the stress was relieved. Pre-dawn Ψ gradually increased after rewatering, reaching the control value of approximately -0.5 MPa at 114 DAS. 63

Figure 4.3 Pre-dawn leaf water potential (Ψ). Pre-dawn Ψ was measured once every two or three days starting from the first day after treatment imposition at 83 DAS (first arrow) to 100 DAS (second arrow) when water deficit was relieved and continuing until 114 DAS (third arrow) when pre-dawn Ψ was similar between treated and control plants. Values are means ± SE (n = 5).

The relationship between pre-dawn Ψ and gravimetric soil water content (SWC) for water deficient plants is shown in Figure 4.4. This curve confirms that drying of the soil and imposition of stress on the plant happened gradually. Gravimetric SWC and pre- dawn Ψ were approximately 30% and -0.5 MPa, respectively, at 80% FC (83 DAS). Pre-dawn Ψ changed little even though SWC decreased to 20%. A further reduction in gravimetric SWC gradually decreased pre-dawn Ψ, reaching below -3.0 MPa when SWC was approximately 10% at 100 DAS.

Figure 4. 4 Relationship between pre-dawn Ψ and gravimetric soil water content during the treatment period (82 to 100 DAS). Values are means ± SE (n = 5).

64 a)

b) c)

Figure 4.5 Growth of grass pea: a) at first flowering (82 DAS) when water deficit was imposed, b) difference between control and water deficient plants at the lowest Ψ (- 3.12 MPa) (100 DAS) just before rewatering, and c) appearance 6 days after rewatering (Ψ = -1.4 MPa) (106 DAS) when regular watering (to 80% FC) was applied. White labels marking flowers and pods can be seen.

4.3.3 Growth, development and dry matter partitioning At 82 DAS, plant height and number of nodes measured on main stem and tillers were 115 cm and 20 nodes, respectively (Figures 4.5a and 4.6). At 100 DAS, water deficient plants had significantly lower plant height by 20.5 cm (P = 0.06) compared with the control plants (Figures 4.5b and 4.6a). Nodes of water deficient plants were still being

65 produced during water deficit, but at a reduced rate resulting in a 2.5 nodes less (P=0.015) than control plants (Figure 4.6b) at 100 DAS when rewatering was initiated. Rewatering (at 100 DAS) water deficient plants had no effect on plant height and so these plants were 46 cm shorter (P<0.001) than the controls at 114 DAS and maturity (Figures 4.5c and 4.6a). Water deficient plants had seven less nodes (P<0.001) at 104 DAS and six less nodes (P=0.011) at maturity, compared with the control plants (Figure 4.6b).

Figure 4.6 The effect of withholding water from 82 to 100 DAS, and then rewatering, on plant height and number of nodes measured on main stem and tillers. Values are means ± SE (n = 5).

Total dry matter production when drying began (82 DAS) was 20 g plant-1 (Figure 4.7). Most dry matter was in the stems (48%) followed by green leaves (32%), petioles (13%) and roots (8%). Water deficit reduced total dry matter production by 47 g plant-1 at 114 DAS and 61 g plant-1 at maturity (P<0.001) compared with the controls. At maturity, in control plants, the proportion of total dry matter as stems, leaves, petioles, pod walls and roots decreased, corresponding with an increase in seed dry matter (37%). In contrast, water deficient plants had 12% seed dry matter leaving 88% in stems, dead leaves, petioles and roots.

Figure 4.7 Dry matter production and partitioning for (a) well-watered and (b) water deficient plants. Values are means (n = 5). Note that the time scale is not linear.

Table 4.2 Yield, yield components, harvest index, water use efficiency at maturity and ODAP concentration of seeds. Values are means (n = 5). Components Treatment LSD WW WD P<0.05 DM (g plant-1) 102.0 41.0 11.44*** Seed yield (g plant-1) 37.6 5.0 5.22*** Pod number (plant-1) 133.0 20.0 9.71*** Seed number (plant-1) 353.0 37.8 27.33*** Seed number (pod-1) 2.66 1.89 0.36*** Seed size (g seed-1) 0.11 0.13 0.03ns Harvest index 0.37 0.12 0.05*** WUEdm (g L-1) 4.22 3.28 0.46*** WUEgr (g L-1) 1.56 0.40 0.25*** ODAP concentration (%) <0.05 <0.05 na *** significant at P≤0.001 ** significant at P≤0.01 * significant at P≤0.05 ns not significant, na not applicable

4.3.4 Yield and yield components Water deficit reduced seed yield by 33 g plant-1 (P<0.001) compared with control plants (Table 4.2). Low grain yield in water deficient plants was due to fewer pods per plant (113 pods; P<0.001) and fewer seeds per pod (0.8 seeds; P<0.001) resulting in 315 fewer seeds (P<0.001) than control plants. In contrast, seed size was the only component not affected by water deficit compared with control plants (P>0.05) (Figure 4.11 provides details on weight of mature seed as a function of flowering time). Water deficit significantly (P<0.001) decreased harvest index and water use efficiency for dry matter and grain yield compared with control plants. ODAP concentration was below the detection limit (<0.05%) in both treatments, thus no comparisons were made.

4.3.5 Cumulative flower, pod and seed production and abortion Grass pea required at least 18 days to set seed after the appearance of a flower bud. Flowers needed at least 6 days from the appearance of flower buds before fertilisation occurred (Figure 4.13 indicates some stages of the development) and about 4 to 6 days for pod set—a pod that is visible after petal senescence. After this, the pods needed a further 6 days to set seed.

Control plants produced 174 flowers and water deficient plants produced 96 flowers (Figure 4.8a). Flower production in water deficient plants was affected soon after treatment began. As a result, the cumulative number of flowers increased slowly from 81 to 90 DAS reaching a maximum of 45 flowers, 38 flowers less than control plants at 93 DAS. After 93 DAS, young flowers aborted in the very early stages. Pre-dawn leaf water potential (Ψ) was -1.8 MPa (Figure 4.3) at this stage. Flower production in control plants increased rapidly until 99 DAS (126 flowers) before slowing to reach a maximum of 174 flowers at 114 DAS. Water deficient plants started flowering again (from newly developed-shoots) at least 10 days after the water deficit was relieved, resulting in total flower production of 96 flowers in treated plants.

Water deficient plants aborted 22 more flowers than control plants (Figure 4.8b). Water deficit imposed at 82 DAS increased flower abortion—16 aborted flowers at 93 DAS. At this stage, water deficient plants aborted 5 flowers more than control plants. Flower production in water deficient plants stopped from 93 DAS (Ψ was -1.8 MPa) to 100 DAS (Ψ was -3.12 MPa) when rewatering was initiated. Rewatering encouraged development of new flowers from newly-developed shoots, but most of these flowers aborted, resulting in 54 aborted flowers. Water deficit rapidly increased pod abortion to 10 aborted pods which was almost six times more than control plants at 93 DAS (Figure 4.8d). The number of aborted pods increased by 50% post-water deficit, when most of the pods developed during this period aborted. As a consequence of high flower and pod abortion in water deficient plants, there were 51.4 less filled pods at 93 DAS and 113 less at 114 DAS compared with control plants (Figure 4.8c). Abortion of newly-formed pods after relief of the water deficit meant that hardly any new pods developed in this treatment. Section 4.3.6 compares treatments between 78 and 90 DAS.

Figure 4.8 Cumulative numbers of (a) flowers, (b) aborted flowers, (c) filled pods and (d) aborted pods per plant between water deficient and control plants. Values are means (n = 5). Values at a given DAS refer to flowers developed in the three days prior to that date.

Of all the filled pods in water deficient plants, 23 ovules and 6 seeds aborted (Figure 4.9a, b) suggesting that in each pod there was at least one aborted ovule in addition to 0.3 aborted seeds. In control plants, although cumulative ovule and seed abortion was higher than in water deficient plants, ovule abortion per pod was 0.3 less than water deficient plants. However, seed abortion per pod was 0.3 more in control plants than in water deficient plants (see also Figure 4.12c, d). Increased ovule abortion in water deficient plants resulted in less seeds per pod (section 4.3.6 provides details). Figure 4.10b shows a deformed pod affected by water deficit compared with a pod from control plants (Figure 4.10a).

Figure 4.9 Cumulative numbers of (a) aborted ovules, (b) aborted seeds and (c) seeds per plant between water deficient and control plants. Values are means (n = 5).

Production of less flowers and abortion of more flowers, pods (Figure 4.8b, d) and ovules (Figure 4.9a) in water deficient plants resulted in 152 less seeds at 93 DAS and 315 less seeds at 114 DAS than control plants (Figure 4.9c). This resulted in reduced seed yield (by 33 g plant-1) in water deficient compared with control plants (Table 4.2). Despite this, the weight of each seed of water deficient plants was the same as those

70 from control plants (Table 4.2 and Figure 4.11). Figure 4.11 shows the reduction in seed size for later-formed flowers.

Normal seeds Aborted seed

Aborted ovule

Figure 4.10 Mature pods from (a) control and (b) water deficient plants, and an example of a pod with (c) normal and aborted seeds and an aborted ovule.

Figure 4.11 Weight of mature seed as a function of flowering time. Arrows indicate the time when the water deficit was imposed at 82 DAS (first arrow) and the time when plants were rewatered at 100 DAS (second arrow). Values are means ± SE (n = 5). Values at a given DAS refer to flowers developed in the three days before that date.

4.3.6 Percentage of flower abortion, pod set and abortion, ovule and seed abortion and seed set This section compares percentages of flower, pod, seed and ovule abortion, and pod and seed set for water deficient and control plants between 78 and 90 DAS. Flowers that developed up to 78 DAS aborted more in control than water deficient plants (Figure 4.12a, b). In control plants, the proportion of flower abortion decreased steeply to around 5% at 90 DAS resulting in a dramatic increase in pod production. In contrast, in

71 water deficient plants, the proportion of flower and pod abortion increased by 16% and 15%, respectively, at 90 DAS resulting in a significant reduction in pod production from approximately 60% at 75 DAS to 25% at 90 DAS.

For flowers developed up to 78 DAS, ovule and seed abortion accounted for 45% (55% set seed) of total ovule numbers in control plants and around 26% (74% set seed) in water deficient plants (Figure 4.12c, d). In control plants, ovule and seed abortion decreased, corresponding with an increase in seed set to 68% at 90 DAS. In contrast, in water deficient plants, seed set decreased from approximately 74% at 78 DAS to 55% at 90 DAS in response to a steady increase in ovule abortion to 45% at 90 DAS. It is clear that water deficient plants proportionally increased flower and pod abortion as water deficit progressed. Similarly, water deficit progressively increased ovule abortion from surviving pods leading to less seeds per pod in these plants. Observation from tissues used for pollen tube growth studies revealed no significant reduction in ovule numbers per flower in water deficient plants compared with controls (data not shown).

Figure 4.12 Percentage of flower abortion (FA), pod abortion (PA), and pod set (PS) for (a) control and (b) water deficient plants expressed as a percentage of total flower number, and, within the retained flowers, the percentage of ovule abortion (OA), seed abortion (SA) and seed set (SS) for (c) control and (d) water deficient plants expressed as a percentage of total ovule number. Values are means ± SE (n = 5). Values at a given DAS refer to flowers developed in the three days before that date (date of tagging).

4.3.7 Flower development, pollen viability and germination, and pollen tube growth At a pre-dawn Ψ of -1.8 MPa (92 DAS), flower stalks were shorter in water deficient plants compared with control plants (picture not shown). However, whole flower buds looked similar (Figure 4.13). There were no differences in anthers of very young flowers between water deficient and control plants (Figure 4.13a, e). However, more pollen either dropped onto the base or stuck to the anthers of more mature flowers in water deficient plants (Figure 4.13f, g, h) compared with control plants (Figure 4.13b, c, d).

In vitro pollen viability of water deficient plants was less than control plants by 15% (P=0.053) at 92 DAS and 13% (P<0.001) at 95 DAS (Figure 4.15a). The proportion of germinated pollen in vitro decreased in both control and water deficient plants from 86 DAS, but there was much less germinated pollen in water deficient plants (Figure 4.15b, Figure 4.14). Pollen germination of water deficient plants was less than control plants by 19% (P=0.062) at 92 DAS and 26% (P<0.001) at 95 DAS.

Pollen tube growth in vivo was studied to determine if pollen quality was affected by water deficit thereby reducing the number and rate of growth into the style and ovary. There were no differences in the number of pollen tubes present in the style or ovary between FC–MC and FC–MS crosses (P>0.05) at 86 DAS (Figure 4.16). At 89 DAS, even though there was no significant reduction in number of pollen tubes present in the style, the percentage of pollen tubes reaching the ovary was 31% (P=0.014) less in FC– MS than FC–MC (Figure 4.16b).

Observations of ovule and seed abortion and seed set from cross-pollination (Figure 4.17) revealed a similar trend to non-cross-pollination between water deficient and control plants (Figure 4.12c, d). At 89 DAS, FC–MS aborted 27% ovules (P = 0.018) less than FC–MC. Conversely, FC–MS aborted 14% more seeds (P = 0.028) than FC– MC. Seed set was maintained at around 60% in FC–MC from 89 to 95 DAS, but for the same period in FC–MS, there was a decrease from 74 to 54% in FC–MS, corresponding with increased ovule abortion. There was no significant difference in weight of seeds from FC–MC and FC–MS crosses (data not shown), in agreement with the observation that seeds of non-crossed water deficient plants had normal seed weight (Table 4.2 and Figure 4.11).

73 a b c d a a a

e f g h a a a a

Figure 4.13 Flower development at 92 DAS (pre-dawn Ψ=-1.8 MPa). Photos a, b, c and d represent control plants and e, f, g and h represent water deficient plants. From left to right, (a and e) very young flowers (3–4 days after flower bud appeared [DAFBA]), (b and f) young flowers (5 DAFBA), (c and g) slightly open flowers (6 DAFBA) and (d and h) fully developed flowers (approximately 7 DAFBA). Scale bar = 1 mm. Photographer: Dr. Tsun- Thai Chai.

74 a b a

Figure 4.14 Pollen viability and germination in vitro observed at 92 DAS for (a) control and (b) water deficient plants. Pre-dawn leaf water potential (Ψ) for control was -0.5 MPa and -1.8 MPa for water deficient plants. Arrows indicate viable and germinated pollen (black arrows), viable but ungerminated pollen (blue arrows) and non-viable pollen (red arrows).

Figure 4.15 Percentage of (a) viable pollen and (b) germinated pollen in in vitro assessments. Values are means ± SE (n = 8). Pollen taken from flowers formed in the approximately 6 days before DAS indicated.

Figure 4.17 Percentage of ovule and seed abortion and seed set for (a) FC–MC and (b) FC–MS. Values are means ± SE (n = 6). DAS refers to the time when crosses occurred; the assessment occurred at maturity.

4.3.8 Seed emergence and shoot dry mass following water deficit Overall, weight of seeds used in this experiment was larger in water deficient than control parent plants. Most seed emerged 5-10 DAS for both water deficient and control

76 plants (Figure 4.18). Peak emergence occurred on the eighth day in both treatments. A t- test revealed that the percentage emergence for seed produced from water deficient plants was 21% (P=0.033) less than control plants. There was no significant difference in days to emergence between treatments (Table 4.3). Seedling shoot dry mass was 18% (P=0.001) more in water deficient than control plants which was in agreement with a 19% difference in seed mass in the batch of seeds used for this experiment. Size (dry weight) of seeds that did not produce seedlings was not significantly different from size of seeds that did produce seedlings in both treatments (P>0.05; data not shown).

Figure 4.18 Cumulative emergence of seeds derived from water deficient (WD) and control (WW) plants.

Table 4.3 Summary of number of days from sowing to emergence, seedling shoot dry mass, and size of seeds producing seedlings for previously water deficient (WD) and control (WW) plants. Parameters Mean t-test WD WW Days to emergence (DAS) 8.6 8.4 P>0.05 Seedling shoot dry mass (mg plant-1) 74.0 62.8 P=0.001 Emerged seed size (mg seed-1) 168.9 141.5 P<0.001

4.4. Discussion The findings in this experiment support the hypothesis that growth and development of Ceora was suppressed by the severe water deficit. Severe water deficit reduced seed yield, harvest index and water use efficiency for grain which were consequences of the cessation of flower production at pre-dawn Ψ=-1.8 MPa and high flower, pod and ovule abortion. These abortions were due to reduced pollen fertility. Rewatering at pre-dawn Ψ=-3.12 MPa enabled plants to fill seed of the pods that had survived the severe water deficit. Seed size was slightly higher in water deficient plants, but no significant

77 differences occurred. Seed ODAP concentrations remained low, however treatment effects could not be tested as the concentrations were below detection limit in both treatments. Seeds produced from water deficient parent plants had somewhat lower emergence but similar growth compared to controls.

4.4.1 Crop phenology This study observed similar crop phenology to that of in Chapter 3, such as time to 90% emergence and 50% flowering, confirming that Ceora is an early vigour and early flowering cultivar suited to the Mediterranean-type environment of southern Australia. In the present study, water deficit imposed at first flowering had no effect on time to first pod or seed set. However, due to the severe water deficit, the period of flower production was shortened by 3 weeks (disregarding flowers developed during the recovery phase) compared with control plants. Rewatering of water deficient plants, at a pre-dawn leaf water potential (Ψ)=-3.12 MPa at 100 DAS, encouraged development of new shoots which are important adaptive features of indeterminate crops to severe water stress (Blum 1996), leading to later maturity than control plants. Later maturity in water deficient plants was due to extended growth and development during recovery compared with control plants that were already at maximum growth and development when watering was stopped (in both treatments).

4.4.2 Dehydration tolerance of Ceora in response to severe soil water deficits Daily plant water use was approximately 400 ml day-1 plant-1 at the beginning of treatment. This high plant water use rapidly decreased soil water content (SWC) (Figures 4.2, 4.4), which led to a decline in pre-dawn leaf water potential Ψ (Figures 4.3, 4.4). The current study observed that predawn Ψ of Ceora was around -3.12 MPa at which point plants appeared severely affected, suggesting that the lethal water potential (Turner et al. 2001) of grass pea is between -3 and -4 MPa.

4.4.3 Growth and development of Ceora in response to severe soil water deficits Water deficit imposed at 82 DAS significantly decreased the rate of increase in plant height and the rate of node production, compared with the control at 100 DAS (Figure 4.6). There were no assessments between 82 and 100 DAS when SWC and plant Ψ progressively decreased (Figures 4.2, 4.3, 4.4). Desclaux et al. (2000) reported the sensitivity of plant height of an indeterminate soybean, through reduced internode length in response to water deficit. Rewatering at 100 DAS did not increase plant height

78 despite a slight increase in node number. Rewatering encouraged development of new branches which are an important component of desiccation tolerance (Blum 1996).

Plant dry mass at first flowering when water deficit was imposed was 20 g (Figure 4.7), with stems contributing the largest proportion. The imposition of water deficit significantly decreased total dry matter production by 39% at 100 DAS. Growth and development were slower post water deficit, while control plants continued with rapid growth and development, leading to a 60% reduction in dry matter production in water deficient plants at 114 DAS, compared with controls. After this, growth and development in water deficient plants slowly recovered producing an additional 24% of dry matter at maturity, but overall, this was 60% lower than control plants at maturity. The seed dry mass at final harvest accounted for 37% and 12% in control and water deficient plants, respectively. This study demonstrated that the limited number of surviving pods and thus, seeds per plant, in water deficient plants was responsible for the lower dry matter partitioning into seed, which was consistent with reports on chickpea (Davies et al. 2000) and wheat (Asseng and van Herwaarden 2003).

4.4.4 Yield and yield components of Ceora in response to severe soil water deficits It is recognised that plants under severe water deficit, particularly during reproduction, have less dry matter and grain yield, as observed in pearl millet (Bidinger et al. 1987), chickpea (Davies et al. 1999; Fang et al. 2010; Leport et al. 2006; Leport et al. 1999), faba bean (Mwanamwenge et al. 1999), rice (Boonjung and Fukai 1996) and corn (Çakir 2004); but the degree of reduction varies between species and genotypes. For example, Leport et al. (1996) demonstrated that yield reduction due to terminal drought observed in the Mediterranean-type environment of southern Australia varied from 50 to 80% for kabuli and desi chickpeas compared with an irrigated control.

Maintenance of normal seed size of Ceora under water deficits is an adaptive trait for water limited environments. In the current study, severe water deficit significantly decreased total dry matter production and grain yield by 60% (as described in section 4.4.3) and 87%, respectively, compared with control plants (Table 4.2). Low grain yield in water deficient plants was due to significantly less pods (85%), seeds per pod (29%) and thus seeds per plant (89%) compared with the control, which corresponded with less flower production (the most important factor) and increased flower, pod and ovule abortion (Section 4.4.5). This result is consistent with recent studies on chickpea (Fang

79 et al. 2010; Leport et al. 2006; Leport et al. 1999). Leport et al. (1999) reported reduced seed size in chickpea under terminal drought. In a review on soybean, it was reported that terminal drought reduced seed size by up to 32% compared with the control (Manavalan et al. 2009). In the present study, severe water deficit had no effect on seed size of Ceora and similar results were also observed from cross–pollinated plants (P>0.05; data not shown). Similar results were also reported for other genotypes of L. sativus observed in a glasshouse and field study at UWA (Herwig 2001) and in a Mediterranean environment of the Basilicata region in South Italy during a three-year study (Polignano et al. 2009). It is not known if seed size of larger seeded genotypes of grass pea is also maintained under water deficit conditions. Thus, a further investigation is required to clarify this. Consistency in seed size of Ceora in response to water deficit provides some evidence of the adaptation of Ceora to the Mediterranean-type environment of southern Australia. This is consistent with reports by Blum (1996) and Ludlow and Muchow (1990). In the present study, water deficit decreased harvest index by 67% compared with control plants, which was much more severe compared with a less than 20% reduction in chickpea under rainfed terminal drought (Leport et al. 1999). In the present study, HI of 0.12 in water deficient plants was comparable to L. sativus (0.10 on average) in a field study at Merredin, Western Australia where subsoil compaction restricted the development of roots to extract soil moisture during reproduction (Siddique et al. 1996; Siddique et al. 2001). In the present study, reduced harvest index in water deficient plants was probably due to the limited number of seeds per plant that could be filled (Section 4.4.3). Low harvest index resulted in a 75% reduction in water use efficiency for grain compared with the control.

4.4.5 Flower and pod production and abortion of Ceora in response to severe soil water deficits In the Mediterranean-type environment of southern Australia, flower and pod production and abortion are key factors in determining final grain yield, as demonstrated in chickpeas (Fang et al. 2010; Leport et al. 2006). However, the sensitivity of flowers and pods to water deficit varies between species and genotypes as well as growth environment (Turner et al. 2005). For example, in a glasshouse study, Fang et al. (2010) showed that flower and pod abortion due to terminal drought ranged from 37 to 56% and from 54 to 73%, respectively, for desi (cv Rupali) and kabuli (cv Almaz) chickpeas. In a similar controlled environment study, pod abortion ranged from

80 50 to 75% for desi and kabuli cultivars (Leport et al. 2006). These studies concluded that both flower and pod abortions are determining factors for seed yield of chickpeas.

In the present study, severe water deficit decreased total flower production by 45% compared with the control. In plants of the water deficit treatment, 47% of total flower production occurred after water was withheld (but before re-watering), and 53% after rewatering. Almost all flowers produced post water deficit aborted as either flowers or pods (Figure 4.8b, d). Rewatering may have been too slow or too late in development to allow those flowers or pods to set more pods or filling seeds. This is a good mimic of the field conditions in southern Australia where there is little rainfall during the late reproductive phase. Therefore, discussion is limited to flowers that developed during water deficit.

Ceora produced flowers until predawn Ψ declined to -1.8 MPa at 90 DAS and stopped flowering until the water deficit was relieved. Some flowers produced at a predawn Ψ of -1.8 MPa were able to grow pods at Ψ of below -2.5 MPa (Figure 4.3). These observations indicate that water potentials in pods were below -2.5 MPa at pre-dawn, and lower still at midday. It is important to note that Ψ during the day was considerably lower than pre-dawn as observed in lupin (Rodrigues et al. 1995). In chickpea, however, flower production ceased at predawn Ψ of -1.0 MPa in a greenhouse experiment (Fang et al. 2010). This suggests that Ceora can produce flowers and pods at a lower Ψ than chickpea.

In the present study, in control plants, a high proportion of flowers that developed in the early stage of flowering were aborted and this was possibly due to competition between reproductive development and vegetative growth. This proportion decreased dramatically to around 5% within a week which corresponded with a dramatic increase in pod set to 95% with no further pods aborting (Figure 4.12). In contrast, in water deficient plants, more than half of flowers developed at the early stage formed pods with seeds (filled pods), but filled pods decreased to around 25% at 90 DAS due to increased flower and pod abortion as the water deficit developed. The high proportion of pod set in the early stage as water deficit progressed demonstrated the ability of Ceora to allocate limited resources to reproduction processes, rather than vegetative growth. Moreover, high flower abortion in the later stage resulted in lower competition

81 between pods. This strategy allows water deficient plants to accelerate seed filling if sufficient water is available.

Water deficient plants had fewer seeds per pod than control plants (Table 4.2 and Figure 4.12). In the current study, on average filled pods of control plants aborted about 40% of seed and ovules (or 60% set seed) between 78 and 90 DAS. Similar percentages of abortion occurred to flowers developed before the imposition of water deficit (at 78 DAS). Water deficit significantly increased ovule abortion (Figure 4.12d) from 20% (74% seed set) at 78 DAS to 45% (or 55% set seed) at 90 DAS. Increased ovule, flower, and pod abortion may be as a result of reduced pollen germination and pollen tube growth (Section 4.4.6) or related to a direct effect of water deficit on ovule fertility. It was attempted to observe ovule fertility by crossing pollen from control or water deficient plants to emasculated female water deficient plants. However, a high proportion of these crosses failed to set pods, which was probably due to a combination of water deficit and exposure to high temperature.

Slow reproductive development of Ceora (Section 4.3.5) may have contributed to high flower and pod abortion under water deficits. The development of a flower, from the time of flower bud initiation to setting pods and seed, is the most critical stage of reproductive development in grain crops (Saini and Westgate 2000). A Ceora flower requires at least 16 to 18 days to set seed after the appearance of a flower bud (Section 4.3.7) and this was consistent with grass pea (L. sativus) in a field experiment in southern Australia (Siddique et al. 1999). The time between flower bud initiation and pod set is similar (10−12 days) to field pea (Kaspa) (Chapter 3); however, Kaspa sets seed much more rapidly than Ceora. In chickpea, pod set occurs about 6 days after the appearance of flower bud (Turner et al. 2005), a time where Ceora is still in the fertilization stage. Slower reproductive development of Ceora compared with field pea is probably related to its indeterminate growth habit compared with the semi- indeterminate field pea (Chapter 3). Chickpea is also an indeterminate growth habit, but it sets pod much earlier than grass pea (Turner et al. 2005) suggesting that there are other factors regulating growth and reproductive development.

82 4.4.6 Pollen viability, germination and pollen tube growth of Ceora in response to severe soil water deficits Male reproductive development is more sensitive to water deficit than female (Saini 1997; Saini and Westgate 2000). The present study demonstrated that flower, pod and ovule abortion in water deficient plants (Section 4.4.5) was linked to decreased pollen viability and germination in vitro and pollen tube growth in vivo (Figures 4.15 and 4.16) and this was consistent with a study on chickpea (Fang et al. 2010). In the present study, pollen viability decreased by 15% compared with the control at pre-dawn Ψ -1.8 MPa. However, most viable grains did not germinate, resulting in 48% less pollen germination in vitro compared with the control. In chickpea, at pre-dawn Ψ below -1.0 MPa, pollen viability and germination in vitro decreased from 80% to 40% for pollen viability and to 10% for germination compared with the well-watered control, resulting in high flower and pod abortion. Thus, pollen viability and germination of Ceora were less sensitive to severe water deficit than in chickpea which was in agreement with flower and pod production described in Section 4.4.5. Reduced fertility of pollen grains at low pre-dawn Ψ is a consequence of photosynthetic inhibition under water deficit that decreases sugar delivery to reproductive tissue leading to disturbances in carbohydrate metabolism and distribution within anthers (Saini 1997; Saini and Westgate 2000).

The high proportion of non-germinated pollen grains may interfere with germinated pollen on the receptive stigma resulting in less pollen tubes (Fang et al. 2010; Turner 1993) in the pistil. Moreover, it has been demonstrated that water deficit decreased pistil function leading to retarded pollen tube growth resulting in a low number of pollen tubes reaching the ovary (Fang et al. 2010). The present study was consistent with this finding as the proportion of pollen tubes present in the ovary significantly decreased, despite no reduction in the number of pollen tubes in the style (near stigma). It is also likely that the quality of pollen grains decreased (Lee 1988) due to water deficit, irrespective of their germination, leading to decreased pollen tube growth down the style, into the ovary and penetrating the ovule. Turner (1993) demonstrated that pollen grains of plants exposed to water deficit may start to germinate but pollen tubes failed to reach the ovary due to their shorter life span. It is clear that in the present study, reduced germination of viable pollen and pollen tube growth were responsible for high flower, pod and ovule abortion in response to severe water stress. However, there is still the possibility for fertilisation as the proportion of pollen germination was relatively high at a Ψ of about -2.5 MPa. For example, hand-crossed pollination between treatments using

83 pollen grains from water deficient plants set on the stigma of the emasculated female control at Ψ -2.5 MPa (Figure 4.17) confirmed that seed set occurred, despite the high proportion of ovule aborted which was consistent with non-crossed pollination (Section 4.4.5).

4.4.7 Seed ODAP concentration of Ceora in response to severe soil water deficit Treatment effect on the Ceora seed ODAP concentration in the present study cannot be compared because the level of ODAP concentration was below detection limit in both treatments (Table 4.2). However, results from the present study showed that the level of ODAP remained low which was consistent with earlier reports (Hanbury et al. 2005; Siddique et al. 2006). Indeed, ODAP concentration in Ceora seeds (<0.05%) in the present study is much less than other improved grass pea lines (for high yield and low ODAP concentration) observed at Tel Hadya, Syria which ranged from 0.09 to 0.41% (Kumar et al. 2010). Such levels are safe for human and animal consumption (Hanbury et al. 2005; Siddique et al. 2006).

4.4.8 Seed emergence and seedling shoot dry mass following water stressed treatment Many studies have addressed reproductive development in cereal and grain legume crops in response to water deficits because of its consequences for seed yield (Section 4.4.6), however viability of seed (developed during and/ or after water deficit) in terms of its ability to germinate and emerge, and seedling growth, are barely understood. This is probably due to increasing availability of high-quality seeds that have a better ability to germinate (Ellis 1992) than seeds produced from water deficient plants. As reported earlier, seed size of Ceora was not reduced by water deficits. Thus, it was of interest to observe if this seed was of equal quality in terms of emergence and early seedling growth, compared with seed produced under well-watered conditions.

Interestingly, percentage emergence of seeds from water deficient plants was less than from control plants (Figure 4.18), indicating that seed quality is somewhat compromised by the water deficit. The present study observed that there was no difference in time from sowing to emergence between the somewhat larger seeds of the water deficit treatment and the somewhat smaller seeds of the control treatment. This is consistent with a report on wild radish that seed size did not affect emergence time (Stanton 1984). Despite this, the percentage emergence for seed produced from water deficient plants was only 66% compared with 87% from control plants. Un-emerged seeds rotted, but

84 the reason behind this was unclear. However, this may be associated with fungal infection after testa rupture. In conclusion, differences in emergence of seeds from water-stressed or control plants may be due to structural and/or chemical factors. In the post emergence phase, seedling vigour depends on food reserves it contains that are available to sustain the young plant in the early stages before it becomes independent to use other resources e.g. light energy (Bewley 1997; Bewley and Black 1994). In the present study, the apex of some seedlings was necrotic resulting in no further growth or increased branching, but it was not clear if it was also related to root infection, or to insect damage. There was no correlation between time to emergence and seed size or shoot dry mass in either treatment. Seedling shoot dry mass was 18% more in water deficient than control plants but this can most likely be attributed to the 19% larger seed size (Table 4.3).

4.5. Conclusion Ceora was able to tolerate desiccation and produce some seed yield by concentrating its limited resources to a smaller number of viable surviving pods. Low seed yield in water deficient plants was due to: first, the cessation of growth and flowering when pre-dawn Ψ was below -1.8 MPa and secondly, increased flower, pod and ovule abortion which were associated with reduced in pollen fertility. Rewatering at pre-dawn leaf water potential Ψ of -3.12 MPa was important to allow seed filling of surviving pods, nevertheless, seed yield was severely reduced by water deficit through a reduced seed number. This led to a severe reduction in HI and WUE for grain, despite a slightly greater seed size of water deficient plants. Seed ODAP concentration remained low under severe water deficit. Seeds produced from water deficient plants had somewhat reduced emergence but seedlings had similar growth to that of controls, indicating that seeds from drought-affected crops can be used for subsequent crops.

This study’s results indicate opportunities for future studies. Maintenance of seed size under various water deficits can be used as an adaptive trait for the improvement of drought resistance crops. The study on pollen tube growth was limited to its presence in the ovary, thus a further investigation on its entrance into the ovule would enable a better understanding on this subject.

85 Chapter 5 Grass pea enhances growth and N uptake of a subsequent wheat crop

Back ground and aims Potential benefits of rotating grass pea (Lathyrus sativus cv Ceora) with cereal crops are not known. This study assessed whether or not a grass pea crop has a positive effect on soil N and P and whether that effect persists over the summer. Methods In adjacent plots seeded with grass pea and wheat (Triticum aestivum) soils were sampled (0–10 cm depth) over the summer period: 25 Nov 2008 (D1, during seed filling, early summer), 18 Dec 2008 (D2, after harvest of wheat), and 18 Feb 2009 (D4, late summer). Three uniform pre-germinated seedlings of wheat (cv Bonnie Rock) were grown in in square pot filled with 1 kg sieved soil in a controlled environment room. Plants received either no fertiliser (control), or N, or P, or N and P and they were well-watered at 75% field capacity until harvest at 4 weeks. Growth, N and P uptake were measured and compared. Key results Wheat dry mass (12%) and green area (16%) were greater after grass pea than after wheat. The addition of N fertiliser or combined N and P reduced the beneficial effect of grass pea to subsequent wheat. There was a small P treatment effect on the growth of wheat on both soils. Wheat shoot N content was higher after grass pea than after wheat in both dates. Wheat shoot N content was greater at D4 than D1 in all treatments. Wheat shoot P content was higher after grass pea than wheat in all treatments at D1, but the opposite was true at D4. Conclusion Soil N availability was increased after a grass pea crop and therefore enhanced growth and shoot N content of the following wheat crop. There was no evidence of increased P availability under grass pea due to a relatively high P availability in soils used. Further research may reveal an effect on P availability.

5.1. Introduction

Chapters 3 and 4 demonstrated the adaptation of Ceora to water deficit during reproduction. Besides yield benefits, another advantage of introducing a new crop in a farming system can be the potential positive effect on other crops in the rotation. This chapter examines the benefits of Ceora to subsequent crops.

It is well known that legumes, including grass pea, are important in agriculture for their ability to fix N2 and to sustain production of cereal crops in rotational farming systems (Adu and Oades 1978; Evans et al. 1991; Evans et al. 2001; Ladd 1981; Ladd and Amato 1986; Ladd et al. 1994; Peoples et al. 2009; Peoples et al. 1995a; Peoples et al. 1995b; Rochester et al. 1998; Siddique and Sykes 1997). Rochester et al (1998), for example, reported that total N-fixed by irrigated soybean was >450 kg N ha-1 and up to 350 kg N ha-1 in winter faba bean. In a recent review, it was reported that grass pea fixed shoot-N at approximately 67 kg ha-1 (McCutchan 2003). Another study reported that shoot N concentration of grass pea was up to 55 mg g-1 dry mass (Rao et al. 2005).

86 High N concentration in grass pea residues would contribute a significant amount to soil N and therefore subsequent crops. Mineralized N from lupin and pea residues was reported to contribute 40% and 15-30% respectively of N in grain yield in the following wheat crop (Evans et al. 2001). The effect of chickpea on N uptake and the yield of subsequent wheat was observed on two different soil types and found that wheat vegetative dry matter and N uptake after chickpea increased by 64 and 69% respectively on black soil and 52 and 50% respectively on red clayey soil compared with wheat after wheat (Holford and Crocker 1997).

Much of the N fixed by legumes is removed from the field as harvested grain (Peoples et al. 1995a), but Rochester et al. (1998) found that this does not account for all the fixed N. In drought prone environments it is common for both grain and stubble of multipurpose legume crops (e.g. grass pea) to be removed for human consumption and animal feed (Cuttle et al. 2003). Therefore, in these crops, shoots provide little N for recycling. Nevertheless, a significant amount of N may still be released from belowground sources through decomposition and leaching from roots, as reported by

Unkovich and Pate (2000). It was observed that 40% of total N fixed (derived from N2 fixation) by winter and summer irrigated faba bean in the cotton growing-regions of northern New South Wales was in the below-ground portion of the plant at peak dry matter production (Rochester et al. 1998) and a similar proportion was found in root and nodules of chickpea, albus lupin and alfalfa (Unkovich and Pate 2000). In a series of glasshouse studies, it was observed that of total plant N derived from the atmosphere, the proportion remaining in roots ranged from 28% for mungbean to 48% for chickpea (Khan et al. 2002). It is estimated that between 20-25% of lupin N, found below ground, was mineralized during summer fallow and was available for the subsequent wheat crop (McNeill and Fillery 2008). Herridge et al. (1995) reported that although chickpea (sown in May and harvested in November in northern New South Wales cereal belt) decreased soil nitrate-N during vegetative growth, soil nitrate-N observed at 1.2 m depth consistently increased again from approximately 50 kg ha-1 in November to 100 kg ha-1 in May. This increase was associated with a rapid decomposition of root and nodule material over the summer and autumn fallow (Rochester et al. 1998; Unkovich and Pate 2000). In addition, legume crops also use less soil-derived N compared with cereal crops as demonstrated for the use of soil nitrate-N between chickpea and wheat (Herridge et al. 1995). Therefore, legumes have multiple beneficial effects on soil N which are an important resource for the following cereal crops.

87 Certain legumes have also been shown to enhance P availability to subsequent crops. Unlike the case of N, enhanced P availability is not due to additional P inputs, but to changes in the rhizosphere or uptake and subsequent release of P from legume tissues. Legumes tend to have a good ability to take up P from soils, including P forms that require specialised root functions such as exudation of carboxylates and phosphatases, and specialised root morphology such as cluster roots (Ae et al. 1990; Lambers et al. 2006; Tarafdar et al. 2001; Vance 2001). Increased uptake of poorly soluble P has been demonstrated in white lupin (Neumann et al. 2000), field pea and faba bean (Nuruzzaman et al. 2005a; Nuruzzaman et al. 2005b), pigeon pea (Ae et al. 1990) and chickpea (Li et al. 2004; Veneklaas et al. 2003). The ability to take up poorly soluble P depends on the production of carboxylates (mainly citrate, oxalate and malate) that chelate sparingly soluble P from Ca-, Fe-, Al/Fe-P complexes in the rhizosphere (Langlade 2002; Neumann and Martinoia 2002), often accompanied by a decrease in pH. Carboxylates are probably too short-lived in the soil to have a positive effect on subsequent crops, but decomposition and mineralization of legume residues continue over time as demonstrated in pea and clover (Lupwayi et al. 2007). This would increase N and P availability for the subsequent crops. In a pot experiment, it was reported that wheat P content was increased by 30-50% following legumes compared with wheat after wheat (Nuruzzaman et al. 2005b). It is not known if grass pea, at maturity, increases soil N and P availability, and if these effects persist over the summer period before a subsequent wheat crop. Further increases of N and P could point to decomposition of grass pea residues over time, whereas decreases of N and P may indicate immobilization by microbes or, in the case of P, reconversion to sparingly soluble forms of P.

Two adjacent field plots were grown with grass pea and wheat during the 2008 growing season followed by the collection of soil samples at monthly intervals during the summer period for wheat bioassay studies in a controlled environment. This study determined if there was N and/or P benefit from growing Ceora grass pea and if this benefit increased or decreased between growing seasons by comparing (i) shoot N and P concentration and content and (ii) growth of the following wheat. The hypotheses were that (i) wheat grows better on soil collected from plots previously sown with grass pea than wheat, (ii) wheat takes up more N and P from soil collected from plots previously sown with grass pea than wheat and (iii) there will be no effect of previous crop if soil N and P status is increased using fertiliser.

88 5.2. Materials and Methods 5.2.1 Study location, experimental design and experimental details The influence of grass pea on N and P availability for wheat was tested using soils collected in the field. Grass pea (Lathyrus sativus cv Ceora) and wheat (Triticum aestivum cv Bonnie Rock) were grown in the 2008 crop season in two adjacent zero- tillage plots, each approximately 6.6 m wide and 80 m long, at the College of Agriculture, Cunderdin, Western Australia. Plots were seeded at a rate of 90 and 80 kg ha-1 for Ceora and Bonnie Rock, respectively, on 5 June 2008. Ceora was inoculated with a Group F ALOSCA, a clay based inoculants of Rhizobium, at a rate of 10 kg ha-1. The wheat plants were supplied with 100 kg ha-1Agstar Zn (containing 13.9% N [or 13.9 kg N ha-1], 14.1% P [or 14.1 kg P ha-1], 9.0% S, 1.1% Cu and 0.82% Zn) and the grass pea plants were supplied with 80 kg ha-1 Double Phos (containing 17.7% P [or 14.2 kg P ha-1], 3.6% S, 16.2% Ca, 0.08% Cu and 0.08% Zn) (obtained from Cuming Smith British Petroleum [CSBP] Ltd., Kwinana, WA). The wheat growth was average with approximately 2 t ha-1 yield. The grass pea had close to average growth, but was affected by Rhizoctonia in the later stage of the crop. The soil was a red sandy clay loam (Table 5.1). Soil was collected four times during summer for the pot experiments: 25 Nov 2008 (D1, during seed filling or early summer), 18 Dec 2008 (D2, a week after harvest of wheat, grass pea was not harvested), 15 Jan 2009 (D3) and 18 Feb 2009 (D4). Top soil (0–10 cm depth) from each plot was sampled (after removing standing crop residues) at six random locations within the plot. Soil collected from each plot was sieved (2 mm) and mixed before sowing wheat in a controlled environment room (CER) with 12:12 day:night hours and 20/15oC day/night temperatures at the Faculty of Natural and Agricultural Sciences, University of Western Australia. Light was provided by metal halide lamps (EYE Hortilux). Due to a problem with the lighting during the D3 experiment, results from this experiment were excluded from the Results section (Section 5.3).

Square pots (top 8x8 cm, bottom 6.5x6.5 cm, height 18 cm) were filled with 1 kg fresh sieved soil. Gravel and polyester cloth were put over the drainage holes in the bottom of the pot before filling to prevent soil loss. Wheat seeds were selected for uniform size and pre-germinated in a Petri dish with moist Whatman paper 1001-085 (Whatman International Ltd) for 48 h. Five seedlings were transplanted into each pot and later thinned to three plants per pot after fertiliser application (section 5.2.2). Pots were

89 arranged in a completely randomised design (CRD) with 6 replications and were regularly repositioned.

Table 5.1 Soil chemical properties measured near experimental plots. Soil sampled March 2007. The analysis was done by CSBP Ltd. Analytical laboratories at Bibra Lake, WA. Soil was extracted with a 1 M potassium chloride solution for nitrate and ammonium, 0.5 M sodium bicarbonate solution for phosphorus and potassium, a 0.1 M NH4Cl/0.1 M BaCl2 for exchangeable Ca, Mg, Na and K, dichromate solution for organic carbon and with a calcium chloride (CaCl2) solution for pH determinations. Depth (cm) Soil properties 0-10 10-20 Nitrate (mg kg-1) 17 11 Ammonium (mg kg-1) 7 2 Phosphorus (mg kg-1) 32 9 Potassium (mg kg-1) 684 525 Exc-Ca (meq 100g-1) 16.2 23.1 Exc-Mg (meq 100g-1) 1.86 3.23 Exc-Na (meq 100g-1) 0.19 0.26 Exc-K (meq 100g-1) 1.79 1.36 Organic Carbon (%) 1.02 0.95 pH 7.7 7.6

5.2.2 Treatments There were four fertiliser treatments: plants without N or P (0N0P) (control), with added N (1N0P), with added P (0N1P) and with added N and P (1N1P). Di-potassium orthophosphate (K2HPO4) and ammonium nitrate (NH4NO3) were applied to pots with 1N and/or 1P treatment/s, while the rest were sown without fertiliser (control). For P, a total of 0.01 g P (dissolved in 10 ml water) was applied in a single dose before transplanting seedlings. This equates to 15.63 kg P ha-1. For N, 0.02 g N was applied, half (0.01) before transplanting seedlings, and again 0.01 g after two weeks. The total of 0.02 g N equates to 31.25 kg N ha-1. No other nutrients were added because their levels are considered adequate for wheat production.

5.2.3 Watering Field capacity (FC) was determined by saturating six pots (three for soil from the grass pea plot and three for soil from the wheat plot) for 24 h and weighing (details are described in Chapter 3). Before and during the experiments, all pots were watered to 75% FC with DI-water based on their weight at 2-3 days intervals.

90 5.2.4 Measurement and harvesting After four weeks of growth, plants were harvested by cutting wheat tops at the soil surface. Immediately before harvest, plant height was measured from the soil surface to the end of the longest leaf using a ruler followed by counting leaf and tiller numbers. Leaf blades were separated from stems and both were scanned for green area using a Li- Cor area meter (Model LI 3100 Li-Cor Inc, Lincoln, Nebraska, USA) before being oven-dried at 70oC for 48 h and weighed. Roots were separated from soil by washing over a 2-mm mesh, then oven dried at 70oC for 48 h and weighed.

5.2.6 Determination of plant N and P Shoot dry material (for D1 and D4) was finely ground in a steel ball-mill to determine shoot N and P concentration and content. An average of 80 mg powder was combusted using a CHN combustion analyzer (Model VARIO Macro, Elementar, Hanau, Germany) for N determination. A similar amount of powder was digested in nitric/perchloric acid to determine total P using colorimetry by the molybdovanadophosphate method (Kitson and Mellon 1944). Total P was determined by extracting digested samples (30 µl) with vanadate (150 µl) and molybdate (120 µl) solutions in a Greiner plate. The Greiner plate (with samples) was inserted into a microplate spectrophotometer (Multiskan Spectrum, Thermo Scientific Co., Victoria, Australia) and samples were measured at 460 nm absorbance wavelength. Shoot N and P concentration (mg g-1 dry weight) and shoot N and P content (mg plant-1) was calculated by multiplying concentration and shoot dry mass.

5.2.7 Statistical analysis An analysis of variance (ANOVA), with the factors of date, previous crop, fertiliser N and fertiliser P, was applied to the data using the statistical package Genstat 10.2. A split-plot design was used where the previous crop, fertiliser N and fertiliser P factors were nested within date of sampling.

91 5.3. Results The results provided in the table and figures refer to wheat and its growth, N and P uptake in response to the previous crops (grass pea and wheat).

5.3.1 Effect of date of sampling on growth, shoot N and P concentration and content There was a significant effect of date of sampling on dry mass, green area and N and P uptake (Appendixes 1, 2, 3, 4, 5 and 6). However, this effect was not consistent across treatments (Figures 5.1, 5.2 and 5.3), except wheat shoot N concentration and content, which was higher at D4 than D1 in all treatments. Overall, wheat grown in soil from the grass pea plot produced greater dry matter, green area and shoot N concentration and content compared with soil from the wheat plot, but not greater shoot P concentration or content (Table 5.2).

Table 5.2 Summary of average values over three sampling dates for wheat dry matter (g plant-1), green area (cm2 plant-1), shoot N and P concentration (mg g-1 dry weight) and shoot N and P content (mg plant-1). Symbols are without N (0N) or P (0P) or with N (1N) or P (1P) fertiliser. Details for individual dates are shown in Figures 5.1, 2 and 3, with statistical analyses in Appendices 1, 2, 3, 4, 5 and 6. Previous crop

Wheat Grass pea Parameters measured Treatment

0N0P 1N0P 0N1P 1N1P Mean 0N0P 1N0P 0N1P 1N1P Mean

Dry matter 0.296 0.365 0.322 0.435 0.355 0.351 0.360 0.413 0.486 0.403

Green area 25.7 36.7 27.1 40.7 32.6 33.4 38.6 36.5 47.0 38.9

N conc. 18.3 27.8 17.4 24.7 22.0 23.9 33.8 21.6 28.9 27.1

P conc. 2.35 2.80 2.50 2.83 2.62 2.20 2.63 2.45 2.85 2.53

N content 3.57 6.72 3.72 6.89 5.22 5.15 7.71 5.39 8.56 6.70

P content 0.459 0.680 0.534 0.790 0.616 0.497 0.606 0.608 0.856 0.642

Figure 5.1 Wheat dry mass (a) and green area (b) (per plant) following wheat (W) or grass pea (G) as the previous crop, without N (0N) or P (0P) or with N (1N) or P (1P) fertiliser. Values are means ± SE (n = 6); D is date (D1 was at seed filling, D2 was approximately a week after harvest of wheat and D4 was approximately 2 months after harvest of wheat).

5.3.2 Dry matter production, green area, shoot N and P concentration and content Wheat dry mass after four weeks of growth reflected its green area in all treatments and dates. There were no treatments effects (or interactions between factors) on the ratio of green area to total dry mass.

Wheat after grass pea In the control (0N0P), wheat dry mass was 0.36 g per plant at D1, 0.33 g at D2 and 0.37 g per plant at D4 (Figure 5.1a) and this corresponded with a green area of 33, 32 and 35 cm2 per plant, respectively (Figure 5.1b). The addition of N fertiliser (1N0P) increased wheat dry mass by 12% and green area by 21% at D1, compared with the controls. Total wheat dry mass was slightly below the control at D2 and D4, despite this,

93 however, there was no reduction in green area at either date. Wheat dry mass increased by 12% at D1, 24% at D2 and 7% at D4 when P fertiliser (0N1P) was added. This corresponded with an increase of wheat green area by 4% at D1, 15% at D2 and 6% at D4. The largest increase in wheat dry mass occurred when both N and P (1N1P) were supplied and this was 34% greater than the control at D1, 32% greater at D2 and 15% greater at D4. This corresponded with an increase in wheat green area of 33% at D1 and D2 and 20% at D4 compared with the control.

Wheat shoot N concentration and N content were higher at D4 than D1 in all treatments (Figures 5.2a and 5.3a). In the controls, wheat shoot N concentration was 19.7 mg per g dry weight at D1 and 28.2 mg per g dry weight at D4. Shoot N content was 3.90 mg per plant at D1 and 6.40 mg per plant at D4. The addition of N fertiliser (1N0P) increased wheat shoot N concentration by 32% at D1 and 27% at D4 and shoot N content by 44% at D1 and 24% at D4, compared with the controls. Added P fertiliser (0N1P) reduced shoot N concentration by 18% at D1 and 6% at D4, despite there being a slight increase in shoot N content compared with the controls. The addition of N and P (1N1P) increased wheat shoot N concentration by 13% at D1 and 20% at D4 and increased shoot N content by 46% at D1 and 36% at D4.

Wheat shoot P concentration and P content varied between treatments and dates (Figures 5.2b and 5.3b). In the controls, wheat shoot P concentration was 2.11 mg per g dry weight at D1 and 2.28 mg per g dry weight at D4. Shoot P content was 0.47 mg per plant at D1 and 0.52 mg per plant at D4. The addition of N (1N0P) increased shoot P concentration by 24% at D1 and 8% at D4 and shoot P content by 29% at D1 and 4% at D4 compared with the controls. Added P (0N1P) increased wheat shoot concentration by 11% at D1 and 10% at D4 and shoot P content by 18% at D1 and 19% at D4. The addition of N and P (1N1P) increased wheat shoot N concentration by 25% at D1 and 21% at D4 and shoot P content by 47% at D1 and 37% at D4.

Wheat after wheat In the control (0N0P), wheat dry mass was 0.31 g per plant at D1, 0.23 g per plant at D2 and 0.35 g per plant at D4 (Figure 5.1a) corresponding with a green area of 26.0 cm2 per plant at D1, 20.8 cm2 per plant at D2 and 30.2 cm2 per plant at D4 (Figure 5.1b). Compared with the control, the addition of N (1N0P) increased dry mass by 20% at D1, 23% at D2 and 16% at D4 and this corresponded with an increase in green area of 31%,

94 36% and 25%, respectively. There was only a slight increase in wheat dry mass and green area when P (0N1P) was added. When both N and P were combined (1N1P), wheat dry mass increased by 31% at D1, 43% at D2 and 22% at D4 which corresponded with an increase in green area of 37%, 48% and 26%, respectively, compared with the control.

Wheat shoot N concentration and shoot N content were higher at D4 than D1 in all treatments (Figures 5.2a and 5.3a). In the controls, wheat shoot N concentration was 14.9 mg per g dry weight at D1 and 21.6 mg per g dry weight at D4. Shoot N content was 2.66 mg per plant at D1 increasing to 4.48 mg per plant at D4. The addition of N (1N0P) increased wheat shoot N concentration by 41% at D1 and 29% at D4 and shoot N content by 52% at D1 and 43% at D4 compared with the controls. Added P (0N1P) reduced shoot N concentration by 9% at D1 and 3% at D4, but there was a slight increase in shoot N content linked to the higher amount of dry matter produced. Added

95 N and P (1N0P) increased shoot N concentration by 30% at D1 and 23% at D4 and shoot N content by 54% at D1 and 44% at D4, compared with the controls.

Wheat shoot P concentration and shoot P content were higher at D4 than D1 in all treatments (Figure 5.2b and 5.3b). In the controls, wheat shoot P concentration was 1.96 mg per g dry weight at D1 and 2.75 mg per g dry weight at D4. Shoot P content was 0.35 mg per plant at D1 and 0.57 mg per plant at D4. The addition of N (1N0P) increased shoot P concentration by 22% at D1 and 11% at D4 and shoot P content by 38% at D1 and 29% at D4, compared with the controls. Added P (0N1P) increased shoot P concentration by 10% at D1 and 3% at D4 and shoot N content by 19% at D1 and 11% at D4. The addition of N and P (1N1P) increased shoot P concentration by 21% at D1 and 14% at D4 and shoot P content by 48% at D1 and 37% at D4, compared with the controls.

96 Comparison of the effects of previous crop and fertiliser treatments Across all fertiliser treatments and dates, total wheat dry matter production was 12% higher after grass pea than after wheat (Figure 5.1a, Table 5.2) which corresponded with a greater green area (16%) after grass pea than after wheat (Figure 5.1b). Larger wheat green area after grass pea corresponded with a greater (P<0.001) number of tillers (7%), leaves (8%) and increased plant height (3%) compared with wheat after wheat (data not shown). In both dry matter production and green area, there were two-way interactions between date and previous crop, between date and N and between date and P, as well as three-way interactions between date, previous crop and N (Appendices 1, 2). In the controls, total wheat dry mass was 16% higher after grass pea than after wheat which corresponded with a greater green area (24%) after grass pea than after wheat (Figure 5.1a, b and Table 5.2). The addition of N fertiliser (1N0P) increased wheat dry mass and green area following both previous crops at D1. However, the proportion of the increase in dry mass (compared with their controls) was 8% less after grass pea than after wheat. This corresponded with 10% less increase in wheat green area after grass pea than after wheat at D1. Although the addition of P fertiliser (0N1P) did not greatly increase wheat dry mass and green area, compared with the controls, the response after grass pea was more than double that after wheat. The addition of both N and P (1N1P) increased wheat dry mass following both previous crops, but the increase was 13% higher after grass pea than after wheat. However, the proportion of the increase in dry mass (compared with their controls) was 5% less after grass pea than after wheat.

On average, wheat shoot N concentration and shoot N content were 19 and 22% higher after grass pea than after wheat (Figures 5.2a, 5.3a and Table 5.2). For the wheat shoot N concentration, there were two-way interactions between date and previous crop (P<0.001) and N and P (P=0.023) (Appendix 3). In the controls, wheat shoot N concentration was 24% higher after grass pea than after wheat at D1 and 23% higher at D4. The addition of N (1N0P) increased wheat shoot N concentration for both previous crops, but the proportion of increase (compared with their controls) was 8% less after grass pea than wheat at D1 and 2% at D4. Similarly, when N and P were added, the proportional increase, compared with the controls, was 17% less shoot N concentration after grass pea than wheat at D1 and 3% less at D4. For the wheat shoot N content, there were significant interactions between factors (except date by N) including four-way interactions between date, previous crop, N and P (P<0.001) (Appendix 4). In the controls, wheat shoot N content was 32% higher after grass pea than after wheat at D1

97 and 30% at D4. Although wheat shoot N content was higher after grass pea than after wheat in response to the addition of N (1N0P), the proportion of increase (compared with their controls) was less (8% at D1 and 19% at D4) than after wheat. Wheat shoot N was 4% higher after grass pea than wheat at D1, but it was 2% less than after wheat at D4 when P (0N1P) was added. When N and P (1N1P) were combined, the proportional increase in shoot N content after grass pea than after wheat, compared with the controls, was 8% lower at D1 and 9% lower at D4.

Wheat shoot P concentration was 3% lower after grass pea than after wheat, but shoot N content was 4% higher than after wheat (Figures 5.2b, 3b and Table 5.2). For the wheat shoot P concentration, there were two-way interactions between date and previous crop (P<0.01) and date and N (P=0.054) (Appendix 5). In the controls, wheat shoot P concentration was 7% higher after grass pea than wheat at D1, but it was 21% less than after wheat at D4. The addition of N fertiliser (1N0P) increased wheat shoot P concentration for both previous crops, but proportion of increase (compared with their controls) was 2% higher after grass pea than after wheat at D1, but it was 3% less than after wheat at D4. For the wheat shoot P content, there were two-way interactions between factors (except date by P), as well as three-way interactions between previous crop, N and P (P=0.014) (Appendix 6). In the controls, wheat shoot P content was 26% higher after grass pea than after wheat at D1, but it was 10% less than after wheat at D4. Wheat shoot P contents following both previous crops were improved with the addition of N (1N0P). However, the proportion of increase (compared with their controls) was 9% less after grass pea than after wheat at D1 and 25% less at D4. When P (0N1P) was added, shoot P content was 1% less after grass pea than after wheat at D1, but it was 8% higher than after wheat at D4. The proportion of increase compared with the controls was 1% less after grass pea than after wheat at both dates when N and P (1N1P) were added.

5.4. Discussion The findings in this experiment support the hypothesis that wheat grew better on soil collected from plots previously sown with grass pea than wheat. This was mainly due to the improvement of soil N that enhanced growth, N and P uptake of the following wheat crop. There was no evidence that grass pea increased P availability for the following wheat crop which was due to a relatively high P status of soils used.

98 5.4.1 Wheat growth following grass pea or wheat The addition of N or combined N and P fertilisers removed the beneficial effect of grass pea to the subsequent wheat crop, which suggests that the beneficial effect of grass pea, in this experiment, was mainly due to N. Total wheat dry matter production was higher after grass pea than after wheat in all treatments which was due to the greater green area after grass pea than after wheat (Figures 5.1a, b and Table 5.2). Increased plant dry matter after grass pea, particularly in the control, was consistent with a study on wheat dry matter after lupin (38%) and pea (33%) compared with wheat after wheat (Evans et al. 1991). Positive vegetative yield responses of wheat to various legumes under different rotational systems in two different soil types were also observed by Holford and Crocker (1997). They found that wheat vegetative dry matter produced after chickpea increased by 64% on black soil and 52% on red clayey soil in northern New South Wales compared with wheat after wheat. The above studies showed that the greater wheat dry matter after legumes was associated with improved wheat N content which was consistent with the present study (Section 5.4.2). The present study demonstrated that the addition of N fertiliser to soil collected from grass pea plot had similar dry mass to that of the control. However, when N fertiliser was added to soil collected from the wheat plot, there was a large increase in dry mass and this was comparable to that of control in grass pea. This treatment effect suggests that the beneficial effect of grass pea was due to N. While there was little effect on growth of adding P only, greater growth in the treatment where N and P were given suggests that P may become co-limiting at higher soil N availability.

In a pot experiment, legumes in the preceding crop increased P availability and therefore enhanced growth of the following wheat crop (Nuruzzaman et al. 2005a). This study found that wheat dry mass, after 5 weeks of growth in pots previously grown with legumes, increased by approximately 50% after faba bean and 25% after field pea without P treatment or more than double with added P in the previous crops, compared with wheat after wheat. In the present study, there was only a small P treatment effect on growth of wheat on soils from grass pea and wheat plots which was due to the high P status of soils used. In the controls, wheat grown on soils collected from grass pea and wheat plots produced similar dry mass, except at D2 when it was higher after grass pea than after wheat (Figure 5.1a and Table 5.2). The addition of P increased wheat dry mass by an average of 15% after grass pea and 9% after wheat, compared with the controls. Greater wheat dry mass and P content after grass pea was associated with

99 better growth which was probably due to the positive effect of grass pea on N availability, compared with wheat after wheat (Section 5.4.2).

In the present study, greater growth and N content of wheat after grass pea suggested that grass pea improved the N status of the soil. Addition of fertiliser N had the same effect on growth and reduced or removed the positive effect of the previous grass pea crop compared with the previous wheat crop. Consistently higher N content from soil collected later in summer suggests that more N becomes available through decomposition of plant residues which is in agreement with a report by Herridge et al. (1995), but this effect was not greater in the soil from the grass pea plots than from the wheat plots, and did not lead to better growth, possibly because P availability was not simultaneously enhanced (Section 5.4.2).

5.4.2 Wheat shoot N and P concentration and content following grass pea or wheat There was no evidence showing that grass pea increased soil P availability for the subsequent wheat which was probably due to high P status of the soils used in this experiment. The addition of P fertiliser did not affect wheat shoot P concentration grown on soils collected from grass pea and wheat plots in the early summer (D1) (Figure 5.2b), however wheat shoot P content was higher after grass pea than wheat in all treatments (Figure 5.3b). In the controls, at D1, wheat shoot P concentrations were similar between both previous crops and this was comparable with wheat shoot P concentration following faba bean with P treatment as reported by Nuruzzaman et al. (2005a). This suggests that early in the summer there was no P limitation in soils previously grown with grass pea or wheat crops. Total P uptake depends on root surface area as reported in wheat and barley cultivars (Gahoonia et al. 1997). In the present study, the greater wheat shoot P content after grass pea was mostly the result of a better overall growth due to the increased N availability. The larger root system (P=0.001, data not shown) probably allowed greater P uptake. Similarly increased P content and growth was evident in the N fertiliser treatment. In the late summer (D4), P availability of the soil collected from the grass pea plot became less available compared with that of wheat. This resulted in lower shoot P concentration and shoot P content compared with wheat after wheat (Figures 5.2b, 5.3b).

Wheat shoot N concentration and shoot N content were higher after grass pea than after wheat, which was consistent with the greater dry matter production after grass pea than

100 after wheat (Figures 5.2a, 5.1 and Table 5.2). Wheat shoot N concentration and shoot N content were higher when growing in soil collected in late summer (D4) than early summer (D1) in all treatments suggesting an increase in N availability over time. Increased N concentration at D4 compared with D1 did not lead to better growth, despite N concentrations being somewhat below levels considered adequate (Weir and Cresswell 1994), possibly due to reduced P availability. The possibility that one or more other mineral element was at suboptimal level cannot be fully excluded. A total of 44.6 mm rainfall occurred between November 2008 and February 2009, which may have enhanced mineralization and decomposition of grass pea residues that increased N availability in late summer. An increase in soil nitrate-N was observed during a fallow summer and autumn after chickpea (Herridge et al. 1995) and at wheat seeding in the following season after lupin (McNeill and Fillery 2008). The contribution of belowground N derived from lupin to total wheat shoot-N of the following wheat was 40% with no N fertiliser and 15-20% with N fertiliser (30 kg N ha-1) (Russell and Fillery 1996). This study was comparable with the results in the present study that wheat shoot N content was 31% (control) and 13% (with added N at a rate of 31.25 kg N ha-1) higher after grass pea than after wheat. The present study also showed a higher shoot N concentration of 24% (control) and 18% (with added N) after grass pea than wheat. This result demonstrated that grass pea in the previous crop improved soil N that benefited a subsequent wheat crop. N availability increased towards the end of the summer and this trend is important for the following season’s cereal crops. Since these results depend on a single pot experiment, it would be important to undertake field experiments to confirm the amounts available from grass pea.

In addition to the soil nitrogen improvement, grass pea may be an important disease break crop. It was demonstrated that yield of wheat increased by 43% following pea compared with wheat after wheat (Rovira 1990). This was due to both soil nitrogen improvement by the pea crop, and reduction of the take-all fungus (Gaeumannomyces graminis). Take-all is a wheat disease worldwide (Kirkegaard et al. 2008) and is one of the most important root diseases in southern Australia (Rovira 1990). Similar combined advantages of nitrogen improvement and disease break were also reported for wheat after lupin (Reeves et al. 1984). A further study is needed to provide a better understanding of the potential of grass pea as a disease break. Such a role would contribute to the potential of this species as a rotational legume species in southern

101 Australia, in addition to its good adaptation to water deficits (Chapter 3 and 4) and potential role in soil N improvement.

5.5. Conclusion There was no evidence that grass pea (cv. Ceora) increased P availability for the subsequent wheat crop, due to a relatively high P status of the soils used in the experiment. By contrast, grass pea increased the availability of soil N and therefore enhanced growth, dry matter production and shoot N content of the following wheat crop. N availability was higher in the soil collected in late summer than in early summer. Wheat growth was less responsive to the origin of the soil (previously cropped with grass pea or wheat) after the addition of N or combined N and P, suggesting that the benefit of grass pea was due to improved N availability. It is concluded that Ceora is a species that can potentially be used to sustain soil N for subsequent cereal crops through rotational farming systems in the Mediterranean-type environments of southern Australia. Further studies are needed to assess potential P benefits, and to identify the role of grass pea as a disease break crop.

102 Chapter 6

General discussion

6.1. Introduction Grass pea (L. sativus) is a multipurpose grain legume that adapts well to the Mediterranean-type environment of southern Australia where grain legumes are grown in rotation with cereal crops (Siddique and Sykes 1997). Grain legumes benefit farmers through grain production, forage, reduced disease build-up, increased soil N and increased P availability from residual P. However, transient waterlogging during the wet winter and terminal drought during the reproductive period in spring limits adaptation of some species in southern Australia. For example, the most-used legume, narrow- leafed lupin, has been reported to be poorly adapted to this adverse environment (Belford et al. 1992; Dracup et al. 1992; Dracup et al. 1998). Lupin also poorly tolerates alkaline soil (Tang et al. 1995). Grass pea adapts to both waterlogging and drought environments (Campbell 1997) and is regarded as a potential species for southern Australia (Hanbury et al. 2005; Siddique et al. 1999). Grass pea adaptation to waterlogging was demonstrated in a pot experiment (Solaiman et al. 2007) and to alkaline soil in southern Australia was reported (Siddique et al. 1999), but its adaptation mechanisms to water deficits were poorly known until the present studies where key adaptive mechanisms were identified (Section 6.2). Similarly, no studies had been conducted on the changes in soil N and P under a grass pea crop. Wheat bio-assays following a grass pea crop yielded results indicating grass pea increases soil N (Section 6.3). Adaptation mechanisms of grass pea to water deficit during the reproductive period and its potential role in soil N and P availability are discussed.

6.2. Adaptation of grass pea to water deficits Water deficit during the reproductive phase is common in southern Australia with rainfall of 250−600 mm per year. The studies reported in this thesis aimed to identify adaptation mechanisms of grass pea by imposing moderate (Chapter 3) and severe (Chapter 4) water deficits on Ceora plants during the reproductive period. These studies identified two mechanisms of adaptation: (1) drought escape, and (2) dehydration avoidance and tolerance, through physiological and morphological adaptations. The effect of severe water deficit on reproductive development was also observed (Section 6.2.3).

103 6.2.1 Drought escape Drought escape refers to the ability of a plant to complete its lifecycle before a severe water deficit occurs (Ludlow and Muchow 1990). This involves fast growth, early flowering and pod set, high dry matter production at anthesis and dry matter partitioning into seed. The present study revealed that grass pea is fast growing, producing high dry matter at anthesis and it flowers early compared with field pea, a species well-adapted to terminal drought in the Mediterranean-type environment of southern Australia. High dry matter production at anthesis is important as it enables seed filling and maturity before the water deficit becomes severe. Potentially less favourable is the fact that grass pea is an indeterminate species that continues its vegetative growth after the initiation of flowering. This growth pattern slows reproductive development; one can assume that seed filling may be altered by severe water deficit. However, when water deficit develops, grass pea limits its vegetative growth which enables plants to accelerate seed filling and maturity before severe water deficit (Chapter 3). Grass pea can tolerate a severe water deficit that might occur during reproduction and responds well to a later rainfall. For example, in southern Australia, rainfall is quite variable and unpredictable, often with a long period of drought during reproduction. Grass pea is able to withstand dehydration and restart growth and seed filling when there is a sufficient rainfall later in the season (Chapter 4). Another strategy of grass pea appears to be an ability to explore soil water from deep in the soil profile (Section 6.2.2) enabling seed filling and maturity before soil water content is exhausted. In conclusion, grass pea is able to escape drought by early maturity, completing flowering and seed production when there is sufficient extractable soil water.

6.2.2 Dehydration avoidance and tolerance Dehydration avoidance refers to the changes in parts of a plant to reduce plant water use and/or obtain water from deeper in the soil profile enabling the plant to maintain water status. Dehydration tolerance is the ability of a plant to tolerate a severe soil water deficit or low leaf water potential (desiccation tolerance) (Ludlow and Muchow 1990). Grass pea avoided and tolerated dehydration through physiological and morphological adaptations in response to water deficits in the experiments described in this thesis. Since plant water use is mostly through green leaves, a reduction in green leaf area can reduce transpiration and therefore maintain plant water status (dehydration avoidance). Reduction in green leaf area was a fast response of grass pea to water deficit (Chapter 3). Growth components that contributed to a reduction in green leaf area were a

104 reduction in the production of stem nodes, and an increase in leaf senescence (Chapter 3). Moreover, it was also observed that grass pea limited leaf expansion when water deficit developed (Chapters 3 and 4). The extent to which reduction of leaf area reduces plant water use in the field needs to be investigated further. Grass pea was able to withstand dehydration and recover after rewatering, indicating that grass pea tolerates dehydration (Chapter 4). Osmotic adjustment (OA) as observed in the experiment in Chapter 3 may play an important role in dehydration tolerance. This physiological adaptation enables plants to maintain turgor at low Ψ (Blum 2005; Morgan 1984; Serraj and Sinclair 2002). This is consistent with the observation in the present study that there were small differences in leaf relative water content between treatments, despite a significant reduction in pre-dawn leaf water potential (Ψ) (Chapter 3). These findings suggest that, in the field, grass pea may respond well to rainfall after a period of severe water deficit, which could be an important advantage of a crop in southern Australia where rainfall is unpredictable during the reproductive phase. It is claimed that OA allows greater soil water uptake through better root development (Serraj and Sinclair 2002). As demonstrated in Chapter 3, water deficit did not reduce root dry mass of grass pea compared with control plant. In fact, roots of grass pea were already at the bottom of the pot (45 cm in depth) during the treatment suggesting that, in the field, grass pea roots grow deep into the soil profile to explore more soil moisture. Unless other factors exist that alter the development of grass pea roots, e.g. subsoil compaction, this would enable grass pea plants to avoid dehydration and maintain physiological processes that enhance grain yield, as reported in chickpea (Kashiwagi et al. 2006; Serraj et al. 2004). Reduced stomatal conductance is another physiological process that contributes to dehydration avoidance. It is assumed that reduction in stomatal conductance of grass pea is the primary response to water deficit to maintain high plant water status. Although reduction in stomatal conductance decreases photosynthesis in most crops, in grass pea, the reduction in photosynthesis per unit leaf area was smaller and was not statistically significant compared with the control (Chapter 3). Photosynthetic capacity of the few leaves that remained after many of the older leaves were shed, was possibly increased by remobilisation of nitrogen into these leaves. Photosynthesis of the remaining leaves supported filling of the relatively small number of seeds (Section 6.2.4). In conclusion, grass pea responds to water deficits not only by avoiding dehydration, but also by tolerating dehydration. This enables grass pea to be well- adapted to the Mediterranean-type environment of southern Australia.

105 6.2.3 Reproductive adaptation/responses to water deficits It has been reported that male reproductive function is more sensitive to water deficits than female reproductive function (Nguyen et al. 2009; Saini and Westgate 2000) and therefore pollen fertility of grass pea was the main focus in the present study (Chapter 4). As expected, male reproduction of grass pea was highly sensitive to water deficit (Chapter 4). Water deficit decreased pollen fertility resulting in less germinated pollen and high flower, pod and ovule abortion. Reduction in pollen fertility was assumed to be a result of the decreased photosynthesis in response to severe water deficit that reduced carbohydrate supply for microspore development. Interestingly, there was still a relatively high proportion of pollen germination (30%) at pre-dawn Ψ of around -2.5 MPa. It was found that the quality of the germinated pollen was also affected by water deficit and therefore fewer pollen tubes reached the ovary. Despite this, some ovules were fertilised and set pods with normal seed size, even though the number of seeds per pod was reduced (Section 6.2.4). While ovule number per flower was not reduced by water deficit, a decrease in ovule fertility may have contributed to the abortion of flowers, pods and ovules. A further study is needed to clarify this. In conclusion, although severe water deficit reduced pollen fertility of grass pea resulting in high flower, pod and ovule abortion, some ovules were fertilised by pollen and produced some seed yield by concentrating its limited resources to a smaller number of viable surviving pods (Section 6.2.4).

6.2.4 Yield and yield components, ODAP concentration and seed quality Yield of crops is reduced less in a moderate water deficit than severe water deficit. For example, in the present study, yield of Ceora grass pea was reduced by 24% in moderate water deficit (Chapter 3) and by 87% in severe water deficit (Chapter 4) compared with the controls. In southern Australia, grass pea was thought to be more suitable for the higher rainfall areas (Siddique et al. 2001), but limited information was available. The present study provided detailed information on the physiological, morphological and reproductive development processes (Section 6.2.2) and how they enhance or reduce seed yield in response to water deficits. Under moderate water deficit, grass pea avoided dehydration by reducing green leaf area and stomatal conductance without a significant reduction in photosynthesis. This adaptation enabled plants to fill more pods and seeds as well as more seeds per pod compared with a severe water deficit. Severe water deficit caused plants to stop flowering or abort many flowers, pods and ovules, mainly due to the reduction in pollen fertility (Section 6.2.3) (Chapter 4). This led to a reduced

106 number of filled pods and seeds and therefore lower seed yield compared with a moderate water deficit. Interestingly, grass pea (carrying some viable pods) had the ability to withstand severe water deficit at a pre-dawn Ψ below -3.0 MPa and to recover well when the water deficit was relieved. This suggests a good adaptation of grass pea to the Mediterranean-type environment of southern Australia. In the field, soil water may decrease more slowly than in a pot experiment allowing roots to develop deeper into the soil profile to extract more water (Section 6.2.2). This allows plants to continue photosynthesis and therefore fill more pods and seeds and thus minimise seed losses, as demonstrated in Chapter 3. However, when there is a severe drought during the reproductive period, grass pea is able to survive and respond well to later rainfall and produce some viable seeds from pods that were set before the water deficit became too severe (Chapter 4).

A particular interesting observation was that water deficit did not reduce seed size. In the present studies (Chapters 3 and 4), seed size of water deficient plants was even slightly higher than controls (number of seeds per pod was also slightly higher than the control in Chapter 3). Similar results were reported for grass pea by Herwig (2001) and Polignano et al. (2009), and agree with a report on sorghum that, under water deficit, kernel weight usually increased to compensate for decreased panicle numbers (Blum 1996). In contrast, in grain legumes such as chickpea and soybean water deficit reduced seed size (Leport et al. 1999; Manavalan et al. 2009). In the present study, in well- watered plants, seeds that filled early in the reproductive phase were smaller than those filled later on (Chapter 4). All pods on water deficient plants had been formed early in development, and any flowers formed later aborted. The smaller number of seeds on these plants therefore experienced less competition for assimilates. However, the amount of assimilates available for seed filling was undoubtedly reduced as well. The net result in grass pea, due to its tendency to abort flowers, pods and seeds, was that remaining seeds were filled to reach a greater dry weight than the seeds on well-watered plants. Grass pea’s physiological and developmental responses seem particularly suitable where seed size is important for economic reasons or where these seeds are used for sowing the next crop. In contrast, these responses may be less advantageous where periods of soil water deficits are too long to ensure sufficient numbers of pods. Further experiments into the effect of timing and duration of drought on seed number and seed size would help target the ideal agro-climatic conditions for grass pea. In the present study, HI and WUE for grain did not differ between treatments in a moderate

107 water deficit (Chapter 3), but were greatly reduced by severe water deficit (Chapter 4). Maintenance of seed size in grass pea under extreme water deficit is a trait that is fairly unique in terms producing a few seeds that are viable for producing the next generation. HI and WUE can be used as traits for crop improvement in areas where only moderate drought during reproduction exists. Although proportion of seedling emergence was 21% less in seeds from water deficient plants, compared with control plants, time to seedling emergence and rate of seedling growth were not negatively affected by drought, suggesting that seeds from water deficient plants can be used for subsequent crops. The present study also confirmed that seeds produced from water deficient plants (Chapter 4) had low ODAP concentration (Hanbury et al. 2005; Siddique et al. 2006).

6.3. Soil N and P availability under grass pea crop It is well known that legumes, including grass pea, fix atmospheric nitrogen and therefore improve soil nitrogen through mineralisation and decomposition of plant material. Similarly, grass pea may be able to remobilise residual P through root exudates (Lambers et al. 2006). Limited studies have been conducted on the contribution of grass pea to the subsequent availability of soil N and P. The present study assessed whether a grass pea crop increased the availability of soil N and P and whether that effect increased or decreased over the summer period. This was done by comparing wheat growth and N and P uptake on soils collected from plots previously sown with grass pea and wheat during the winter of 2008 (Chapter 5). This study yielded no evidence that grass pea increased soil P availability for the subsequent wheat crop, due to a relatively high soil P status of the soils used in the experiment. A further study using low and high soil P status is needed to provide a better understanding on the role of grass pea in P remobilisation. In contrast, this study found that grass pea increased soil N availability and therefore enhanced growth and N and P uptake of subsequent wheat plants. Higher shoot N content from soil collected later in the summer suggests that more N becomes available through the decomposition of plant residues. This is important for the following winter crop in the Mediterranean-type environment of southern Australia. This experiment observed the contribution of the belowground benefits from grass pea to subsequent wheat by excluding aboveground biomass at soil sampling. A high proportion of N may have remained in shoot residuals that further contribute to soil N through mineralisation and decomposition. Thus, in the field, retaining plant material in the soil or on the soil surface could potentially increase soil N availability for following cereal crops.

108 6.4. General conclusion This study confirmed that grass pea (L. sativus cv Ceora) is a multipurpose grain legumes that adapts well to the Mediterranean-type environment of southern Australia. The study showed that grass pea avoided dehydration through reduction in green leaf area, stomatal conductance and possibly osmotic adjustment. This enabled plants to maintain water status and photosynthesis of the remaining green leaves to support seed yield. Under moderate water deficit, grass pea escaped drought through early maturity. Grass pea also tolerated severe water deficit and responded well to later rainfall. These adaptations are important in southern Australia which has variable rainfall during the winter/spring growing season. Grass pea displayed the ability to maintain its seed size under extreme water deficits. Whether this ability is also present when drought stress occurs at different times, and in genotypes with larger seed than Ceora, remains to be investigated. Despite maintenance of seed size, seed yield was reduced severely under severe water deficit, due to cessation of flower production and reduced pollen fertility. Grass pea also improves soil nitrogen that potentially benefits other cereal crops through rotational systems. The role of grass pea in P remobilisation requires further study.

109 References Abd-El-Moneim AM, van Dorrestein B, Baum M, Mulugeta W (1999) Role of ICARDA in improving the nutritional quality and yield potential of grass pea (Lathyrus sativus L.) for subsistence farmers in developing countries. In 'Improving human nutrition through agriculture: the role of international agricultural research'. (International Food Policy Research Institute: International Rice Research Institute, Los Banos, Philippines organized ).

Abd-El Moneim AM, Van Dorrestein B, Baum M, Ryan J, Bejiga G (2001) Role of ICARDA in improving the nutritional quality and yield potential of grasspea (Lathyrus sativus L.), for subsistence farmers in dry areas. Lathyrus Latyrism Newsletter 2, 55–58.

Adu JK, Oades JM (1978) Physical factors influencing decomposition of organic materials in soil aggregates. Soil Biology and Biochemistry 10, 109–115.

Ae N, Arihara J, Okada K, Yoshihara T, Johansen C (1990) Phosphorus uptake by pigeon pea and its role in cropping systems of the Indian subcontinent. Science 248, 477–480.

Alexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technology 44, 117–122.

Andersen MN, Aremu JA (1991) Drought sensitivity, root development and osmotic adjustment in field grown peas. Irrigation Science 12, 45–51.

Anyia AO, Herzog H (2004) Water-use efficiency, leaf area and leaf gas exchange of cowpeas under mid-season drought. European Journal of Agronomy 20, 327–339.

Arentoft AMK, Greirson BN (1995) Analysis of 3-(N-Oxalyl)-L-2,3-diaminopropanoic acid and Its alpha-isomer in grass pea (Lathyrus-sativus) by capillary zone electrophoresis. Journal of Agricultural and Food Chemistry 43, 942–945.

Armstrong EL, Pate JS, Tennant D (1994) The field pea crop in south Western Australia – patterns of water use and root growth in genotypes of contrasting morphology and growth habit. Australian Journal of Plant Physiology 21, 517–532.

Asseng S, van Herwaarden AF (2003) Analysis of the benefits to wheat yield from assimilates stored prior to grain filling in a range of environments. Plant and Soil 256, 217–229.

Aston MJ, Lawlor DW (1979) The relationship between transpiration, root water uptake, and leaf water potential. Journal of Experimental Botany 30, 169–181.

Austin RB, Morgan CL, Ford MA, Blackwell RD (1980) Contributions to grain yield from pre-anthesis assimilation in tall and dwarf barley phenotypes in two contrasting seasons. Annals of Botany 45, 309–319.

Basu PS, Berger JD, Turner NC, Chaturvedi SK, Ali M, Siddique KHM (2007) Osmotic adjustment of chickpea (Cicer arietinum) is not associated with changes in carbohydrate composition or leaf gas exchange under drought. Annals of Applied Biology 150, 217– 225.

110 Begg JE, Turner NC (1976) Crop water deficits. Advances in Agronomy 28, 161–215.

Belford RK, Dracup M, Tennant D (1992) Limitations to growth and yield of cereal and lupin crops on duplex soils. Australian Journal of Experimental Agriculture 32, 929– 945.

Berger JD, Ali M, et al. (2006) Genotype by environment studies demonstrate the critical role of phenology in adaptation of chickpea (Cicer arietinum L.) to high and low yielding environments of India. Field Crops Research 98, 230–244.

Bewley JD (1997) Seed germination and dormancy. Plant Cell 9, 1055–1066.

Bewley JD, Black M (1994) 'Seeds: physiology of development and emergence.' (Plenum Press: New York).

Bidinger F, Musgrave RB, Fischer RA (1977) Contribution of stored pre-anthesis assimilate to grain yield in wheat and barley. Nature 270, 431–433.

Bidinger FR, Mahalakshmi V, Rao GDP (1987) Assessment of drought resistance in pearl millet [Pennisetum americanum (L.) Leeke]. I. Factors affecting yields under stress. Australian Journal of Agricultural Research 38, 37–48.

Biederbeck VO, Bouman OT, Looman J, Slinkard AE, Bailey LD, Rice WA, Janzen HH (1993) Productivity of four annual legumes as green manure in dryland cropping systems. Journal of Agronomy 85, 1035–1043.

Blum A (1996) Crop responses to drought and the interpretation of adaptation. Plant Growth Regulation 20, 135–148.

Blum A (2005) Drought resistance, water-use efficiency, and yield potential—are they compatible, dissonant, or mutually exclusive? Australian Journal of Agricultural Research 56, 1159–1168.

Boonjung H, Fukai S (1996) Effects of soil water deficit at different growth stages on rice growth and yield under upland conditions. 2. Phenology, biomass production and yield. Field Crops Research 48, 47–55.

Boutraa T, Sanders FE (2001) Influence of water stress on grain yield and vegetative growth of two cultivars of bean (Phaseolus vulgaris L.). Journal of Agronomy and Crop Science 187, 251–257.

Boyer JS (1970) Leaf enlargement and metabolic rates in corn, soybean, and sunflower at various leaf water potentials. Plant Physiology 46, 233–235.

Bray EA (2002) Abscisic acid regulation of gene expression during water-deficit stress in the era of the Arabidopsis genome. Plant Cell and Environment 25, 153–161.

Butler A, Tesfay Z, D'Andrea C, Lyons D (1999) 'The Ethnobotany of Lathyrus sativus L. in the Highlands of Ethiopia ' (Cluiver Academic/Plenum Publishers: New York).

Çakir R (2004) Effect of water stress at different development stages on vegetative and reproductive growth of corn. Field Crops Research 89, 1–16.

111

Campbell CG (1997) Grasspea. Lathyrus sativus L. Promoting the conservation and use of underutilized and neglected crops. 18. Institute of Plant Genetics and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute, Rome, Italy.

Chaves MM, Maroco JP, Pereira JS (2003) Understanding plant responses to drought – from genes to the whole plant. Functional Plant Biology 30, 239–264.

Chaves MM, Oliveira MM (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. Journal of Experimental Botany 55, 2365–2384.

Chimenti CA, Pearson J, Hall AJ (2002) Osmotic adjustment and yield maintenance under drought in sunflower. Field Crops Research 75, 235–246.

Clarke HJ, Khan TN, Siddique KHM (2004) Pollen selection for chilling tolerance at hybridisation leads to improved chickpea cultivars. Euphytica 139, 65–74.

Colledge S, Conolly J, Shennan S (2005) The evolution of Neolithic farming from SW Asian origins to NW European limits. European Journal of Archaeology 8, 137–156.

Comstock JP (2002) Hydraulic and chemical signalling in the control of stomatal conductance and transpiration. Journal of Experimental Botany 53, 195–200.

Croser JS, Clarke HJ, Siddique KHM, Khan TN (2003) Low-temperature stress: Implications for chickpea (Cicer arietinum L.) improvement. Critical Reviews in Plant Sciences 22, 185–219.

Cuttle S, Shepherd M, Goodlass G (2003) A review of leguminous fertility-building crops, with particular refence to nitrogen fixation and utilisation. In 'The development of improved guidance on the use of fertility-building crops in organic farming'.

Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant and Soil 245, 35–47.

Davies SL, Turner NC, Palta JA, Siddique KHM, Plummer JA (2000) Remobilisation of carbon and nitrogen supports seed filling in chickpea subjected to water deficit. Australian Journal of Agricultural Research 51, 855–866.

Davies SL, Turner NC, Siddique KHM, Plummer JA, Leport L (1999) Seed growth of desi and kabuli chickpea (Cicer arietinum L.) in a short-season Mediterranean-type environment. Australian Journal of Experimental Agriculture 39, 181–188.

Davies WJ, Zhang J (1991) Root signals and the regulation of growth and development of plants in drying soil. Annual Review of Plant Physiology and Plant Molecular Biology 42, 55–76.

De Costa W, Shanmugathasan KN, Joseph K (1999) Physiology of yield determination of mung bean (Vigna radiata (L.) Wilczek) under various irrigation regimes in the dry and intermediate zones of Sri Lanka. Field Crops Research 61, 1–12.

112 Desclaux D, Huynh TT, Roumet P (2000) Identification of soybean plant characteristics that indicate the timing of drought stress. Crop Science 40, 716–722.

Dingkuhn M, Cruz RT, O'Toole JC, Dorffling K (1989) Net photosynthesis, water use efficiency, leaf water potential and leaf rolling as affected by water deficit in tropical upland rice. Australian Journal of Agricultural Research 40, 1171–1181.

Dracup M, Belford RK, Gregory PJ (1992) Constraints to root-growth of wheat and lupin crops in duplex soils. Australian Journal of Experimental Agriculture 32, 947– 961.

Dracup M, Reader MA, Palta JA (1998) Variation in yield of narrow-leafed lupin caused by terminal drought. Australian Journal of Agricultural Research 49, 799–810.

Egli DB (2004) Seed-fill duration and yield of grain crops. Advances in Agronomy 80, 243–276.

Ellis RH (1992) Seed and seedling vigor in relation to crop growth and yield. Plant Growth Regulation 11, 249–255.

Erskine W, Siddique KHM, Khan T, Cowling W (2001) Utilisation of grain legume diversity. In 'Plant Genetic Resources of Legumes in the Mediterranean'. (Eds N Maxted, SJ Bennett). (Kluwer Academic Publishers: Netherlands).

Evans J, Fettell NA, Coventry DR, Oconnor GE, Walsgott DN, Mahoney J, Armstrong EL (1991) Wheat response after temperate crop legumes in South-Eastern Australia. Australian Journal of Agricultural Research 42, 31–43.

Evans J, McNeill AM, Unkovich MJ, Fettell NA, Heenan DP (2001) Net nitrogen balances for cool-season grain legume crops and contributions to wheat nitrogen uptake: a review. Australian Journal of Experimental Agriculture 41, 347–359.

Fang XW, Turner NC, Yan GJ, Li FM, Siddique KHM (2010) Flower numbers, pod production, pollen viability, and pistil function are reduced and flower and pod abortion increased in chickpea (Cicer arietinum L.) under terminal drought. Journal of Experimental Botany 61, 335–345.

Farooq M, Wahid A, Kobayashi N, Fujita D, Basra SMA (2009) Plant drought stress: effects, mechanisms and management. Agronomy for Sustainable Development 29, 185–212.

Farquhar GD, Sharkey TD (1982) Stomatal conductance and photosynthesis. Annual Review of Plant Physiology 33, 317–345.

Flower DJ, Ludlow MM (1986) Contribution of osmotic adjustment to the dehydration tolerance of water-stressed pigeonpea (Cajanus cajan (L.) millsp.) leaves. Plant, Cell and Environment 9, 33–40.

Fuller DQ, Harvey EL (2006) The archaeobotany of Indian pulses: identification, processing and evidence for cultivation. Environmental Archaeology 11, 219–246.

113 Gahoonia TS, Care D, Nielsen NE (1997) Root hairs and phosphorus acquisition of wheat and barley cultivars. Plant and Soil 191, 181–188.

Gebbing T, Schnyder H (1999) Pre-anthesis reserve utilization for protein and carbohydrate synthesis in grains of wheat. Plant Physiology 121, 871–878.

Getahun H, Lambein F, Vanhoorne M, Van der Stuyft P (2005) Neurolathyrism risk depends on type of grass pea preparation and on mixing with cereals and antioxidants. Tropical Medicine and International Health 10, 169–178.

Ghannoum O (2009) Photosynthesis and water stress. Annals of Botany 103, 635–644.

Gunasekera CP, French RJ, Martin LD, Siddique KHM (2009) Comparison of the responses of two Indian mustard (Brassica juncea L.) genotypes to post-flowering soil water deficit with the response of canola (B. napus L.) cv. Monty. Crop and Pasture Science 60, 251–261.

Hanbury C, Siddique KHM (1998) Growing dwarf chickling (Lathyrus cicera). (Ed. SP Department of Agriculture Western Australia and Centre for Legumes in Mediterranean Agriculture (CLIMA) and Agriculture Western Australia). (Farmnote, Department of Agriculture, Western Australia.

Hanbury C, Siddique KHM, Seymour M, Jones R, MacLeod B (2005) Growing Ceora grass pea (Lathyrus sativus) in Western Australia. (Ed. SP Department of Agriculture Western Australia and Centre for Legumes in Mediterranean Agriculture (CLIMA)). (Farmnote, Government of Western Australia.

Hanbury CD, Siddique KHM, Galwey NW, Cocks PS (1999) Genotype-environment interaction for seed yield and ODAP concentration of Lathyrus sativus L. and L. cicera L. in Mediterranean-type environments. Euphytica 110, 45–60.

Hanbury CD, White CL, Mullan BP, Siddique KHM (2000) A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Animal Feed Science and Technology 87, 1–27.

Herridge DF, Marcellos H, Felton WL, Turner GL, Peoples MB (1995) Chickpea increases soil-N fertility in cereal systems through nitrate sparing and N2 fixation. Soil Biology and Biochemistry 27, 545–551.

Herwig S (2001) The effect of genotype and enviroment [sic] on β-N-oxalyl-L-A-B- diaminopropionic acid (ODAP) concentration in Lathyrus sativus (L.) seeds (PhD thesis). The University of Western Australia.

Hetherington AM, Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature 424, 901–908.

Holford ICR, Crocker GJ (1997) A comparison of chickpeas and pasture legumes for sustaining yields and nitrogen status of subsequent wheat. Australian Journal of Agricultural Research 48, 305–315.

Hsiao TC (1973) Plant responses to water stress. Annual Review of Plant Physiology 24, 519–570.

114

Hsiao TC, Xu LK (2000) Sensitivity of growth of roots versus leaves to water stress: biophysical analysis and relation to water transport. Journal of Experimental Botany 51, 1595–1616.

Jackson MT, Yunus AG (1984) Variation in the grass pea (Lathyrus sativus L.) and wild species. Euphytica 33, 549–559.

Kao WY, Forseth IN (1992) Diurnal leaf movement, chlorophyll fluorescence and carbon assimilation in soybean grown under different nitrogen and water availabilities. Plant, Cell and Environment 15, 703–710.

Kashiwagi J, Krishnamurthy L, Crouch JH, Serraj R (2006) Variability of root length density and its contributions to seed yield in chickpea (Cicer arietinum L.) under terminal drought stress. Field Crops Research 95, 171–181.

Khan DF, Peoples MB, Chalk PM, Herridge DF (2002) Quantifying below-ground nitrogen of legumes. 2. A comparison of 15N and non isotopic methods. Plant and Soil 239, 277–289.

Kirkegaard J, Christen O, Krupinsky J, Layzell D (2008) Break crop benefits in temperate wheat production. Field Crops Research 107, 185–195.

Kislev ME, Bar-Yosef O (1988) The legumes: the earliest domesticated plants in the Near East? Current Anthropology 29, 175–179.

Kitson RE, Mellon MG (1944) 'Colorimetric determination of phosphorus as molybdivanadophosphoric acid.' (Purdue University, Lafayette, Ind.).

Kokubun M, Shimada S, Takahashi M (2001) Flower abortion caused by preanthesis water deficit is not attributed to impairment of pollen in soybean. Crop Science 41, 1517–1521.

Kramer PJ, Boyer JS (1995) Water relations of plants and soils. Academic Press, Inc. New York.

Kumar S, Bejiga G, Ahmed S, Nakkoul H, Sarker A (2010) Genetic improvement of grass pea for low neurotoxin ([beta]-ODAP) content. Food and Chemical Toxicology In Press, Corrected Proof, doi: DOI: 10.1016/j.fct.2010.1006.1051.

Ladd JN (1981) The use of 15N in following organic-matter turnover, with specific reference to rotation systems. Plant and Soil 58, 401–411.

Ladd JN, Amato M (1986) The fate of nitrogen from legume and fertilizer sources in soils successively cropped with wheat under field conditions. Soil Biology and Biochemistry 18, 417–425.

Ladd JN, Amato M, Zhou LK, Schultz JE (1994) Differential effects of rotation, plant residue and nitrogen fertilizer on microbial biomass and organic matter in an Australian alfisol. Soil Biology and Biochemistry 26, 821–831.

115 Lafitte HR, Courtois B (2002) Interpreting cultivar x environment interactions for yield in upland rice: Assigning value to drought-adaptive traits. Crop Science 42, 1409–1420.

Lambers H, Chapin III FS, Pons TL (2008) 'Plant Phisiological Ecology.' (Springer: The Netherlands).

Lambers H, Shane MW, Cramer MD, Pearse SJ, Veneklaas EJ (2006) Root structure and functioning for efficient acquisition of phosphorus: matching morphological and physiological traits. Annals of Botany 98, 693–713.

Langlade NB (2002) A physiological and molecular approach to study organic acid exudation and development of cluster roots in Lupinus albus L. (PhD thesis). University of Neuchatel.

Lawn RJ (1982a) Response of 4 grain legumes to water-stress in southeastern Queensland. 2. Plant-growth and soil-water extraction patterns. Australian Journal of Agricultural Research 33, 497–509.

Lawn RJ (1982b) Response of four grain legumes to water-stress in southeastern Queensland. 1. Physiological-response mechanisms. Australian Journal of Agricultural Research 33, 481–496.

Lecoeur J, Wery J, Turc O (1992) Osmotic adjustment as a mechanism of dehydration postponement in chickpea (Cicer arietinum L.) leaves. Plant and Soil 144, 177–189.

Lee TD (1988) 'Pattern of fruit and seed production.' (Oxford University Press: New York).

Leport L, Turner NC, Davies SL, Siddique KHM (2006) Variation in pod production and abortion among chickpea cultivars under terminal drought. European Journal of Agronomy 24, 236–246.

Leport L, Turner NC, French RJ, Barr MD, Duda R, Davies SL, Tennant D, Siddique KHM (1999) Physiological responses of chickpea genotypes to terminal drought in a Mediterranean-type environment. European Journal of Agronomy 11, 279–291.

Leport L, Turner NC, French RJ, Tennant D, Thomson BD, Siddique KHM (1998) Water relations, gas exchange and growth of cool-season grain legumes in a Mediterranean-type environment. European Journal of Agronomy 9, 295–303.

Levit J (1972) 'Responses of plants to environmental stresses.' (Academic Press: New York).

Li SM, Li L, Zhang FS, Tang C (2004) Acid phosphatase role in chickpea/maize intercropping. Annals of Botany 94, 297–303.

Ludlow MM, Muchow RC (1990) A critical evaluation of traits for improving crop yields in water-limited environments. Advances in Agronomy 43, 107–153.

Lupwayi NZ, Clayton GW, O'Donovan JT, Harker KN, Turkington TK, Soon YK (2007) Phosphorus release during decomposition of crop residues under conventional and zero tillage. Soil and Tillage Research 95, 231–239.

116

Mahalakshmi V, Sivaramakrishnan S, Bidinger FR (1993) Contribution of pre-and post- anthesis photosynthates to grains in pearl millet under water deficit. Journal of Agronomy and Crop Science 170, 91–96.

Malek MA, Sarwar CDM, Sarker A, Hassan MS (1996) Status of grasspea research and future strategy in Bangladesh. In 'Proceedings of a Regional Workshop, 27–29 December 1995'. New Delhi, India. (Eds RK Arora, PN Mathur, KW Riley, Y Adham) pp. 7–12. ( International Plant Genetic Resources Institute).

Manavalan LP, Guttikonda SK, Tran LSP, Nguyen HT (2009) Physiological and molecular approaches to improve drought resistance in soybean. Plant and Cell Physiology 50, 1260–1276.

McCutchan JS (2003) A brief history of grass pea and its use in crop improvement. Lathyrus Lathyrism Newsletter 3, 18–23.

McNeill AM, Fillery IRP (2008) Field measurement of lupin belowground nitrogen accumulation and recovery in the subsequent cereal-soil system in a semi-arid Mediterranean-type climate. Plant and Soil 302, 297–316.

Mera M, Tay J, France A, Montenegro A, Espinoza N, Gaete N (2003) Luanco-INIA, a large-seeded cultivar of Lathyrus sativus released in Chile. Lathyrus Lathyrism Newsletter 3, 26.

Morgan JM (1984) Osmoregulation and water stress in higher plants. Annual Review of Plant Physiology 35, 299–319.

Morgan JM, Condon AG (1986) Water use, grain yield, and osmoregulation in wheat. Australian Journal of Plant Physiology 13, 523–532.

Mwanamwenge J, Loss SP, Siddique KHM, Cocks PS (1999) Effect of water stress during floral initiation, flowering and podding on the growth and yield of faba bean (Vicia faba L.). European Journal of Agronomy 11, 1–11.

Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annual Review of Plant Biology 56, 165–185.

Neumann G, Martinoia E (2002) Cluster roots - an underground adaptation for survival in extreme environments. Trends in Plant Science 7, 162–167.

Neumann G, Massonneau A, Langlade N, Dinkelaker B, Hengeler C, Römheld V, Martinoia E (2000) Physiological aspects of cluster root function and development in phosphorus-deficient white lupin (Lupinus albus L.). Annals of Botany 85, 909–919.

Neumann G, Römheld V (2000) 'The release of root exudates as affected by the plant physiological status.' (Marcel Deker (in Press): Basel, Switzerland).

Neyshabouri MR, Hatfield JL (1986) Soil water deficit effects on semi-determinate and indeterminate soybean growth and yield. Field Crops Research 15, 73–84.

117 Nguyen GN, Hailstones DL, Wilkes M, Sutton BG (2009) Drought-induced oxidative conditions in rice anthers leading to a programmed cell death and pollen abortion. Journal of Agronomy and Crop Science 195, 157–164.

Nuruzzaman M, Lambers H, Bolland MDA, Veneklaas EJ (2005a) Phosphorus benefits of different legume crops to subsequent wheat grown in different soils of Western Australia. Plant and Soil 271, 175–187.

Nuruzzaman M, Lambers H, Bolland MDA, Veneklaas EJ (2005b) Phosphorus uptake by grain legumes and subsequently grown wheat at different levels of residual phosphorus fertiliser. Australian Journal of Agricultural Research 56, 1041–1047.

Nuruzzaman M, Lambers H, Bolland MDA, Veneklaas EJ (2006) Distribution of carboxylates and acid phosphatase and depletion of different phosphorus fractions in the rhizosphere of a cereal and three grain legumes. Plant and Soil 281, 109–120.

O'Toole JC, Cruz RT (1980) Response of leaf water potential, stomatal resistance, and leaf rolling to water stress. Plant Physiology 65, 428–432.

Palta JA, Plaut Z (1999) Yield and components of seed yield of indeterminate narrow- leafed lupin (Lupinus angustifolius L.) subjected to transient water deficit. Australian Journal of Agricultural Research 50, 1225–1232.

Palta JA, Turner NC, French RJ, Buirchell BJ (2007) Physiological responses of lupin genotypes to terminal drought in a Mediterranean-type environment. Annals of Applied Biology 150, 269–279.

Passioura JB (1977) Grain yield, harvest index, and water use of wheat. Journal of the Australia Institute of Agricultural Science 43, 117–120.

Patto MCV, Skiba B, Pang ECK, Ochatt SJ, Lambein F, Rubiales D (2006) Lathyrus improvement for resistance against biotic and abiotic stresses: From classical breeding to marker assisted selection. Euphytica 147, 133–147.

Peña-Chocarro L, Peña LZ (1999) History and traditional cultivation of Lathyrus sativus L. and Lathyrus cicera L. in the Iberian peninsula. Vegetation History and Archaeobotany 8, 49–52.

Peoples MB, Brockwell J, et al. (2009) The contributions of nitrogen-fixing crop legumes to the productivity of agricultural systems. Symbiosis 48, 1–17.

Peoples MB, Herridge DF, Ladha JK (1995a) Biological nitrogen-fixation: An efficient source of nitrogen for sustainable agricultural production? Plant and Soil 174, 3–28.

Peoples MB, Ladha JK, Herridge DF (1995b) Enhancing legume N2 fixation through plant and soil management. Plant and Soil 174, 83–101.

Polignano GB, Bisignano V, Tomaselli V, Uggenti P, Alba V, Gatta CD (2009) Genotype x environment interaction in grass pea (Lathyrus sativus L.) lines. International Journal of Agronomy doi:10.1155/2009/898396, 1–7.

118 Premachandra GS, Saneoka H, Fujita K, Ogata S (1992) Leaf water relations, osmotic adjustment, cell membrane stability, epicuticular wax load and growth as affected by increasing water deficits in sorghum. Journal of Experimental Botany 43, 1569–1576.

Rahman MA, Rahman MM, Sarkar MA (2001) Progress in isolation and purification of Lathyrus sativus breeding lines. Lathyrus Lathyrism Newsletter 2, 39–40.

Rao SC, Northup BK, Mayeux HS (2005) Candidate cool-season legumes for filling forage deficit periods in the southern Great Plains. Crop Science 45, 2068–2074.

Reeves TG, Ellington A, Brooke HD (1984) Effects of lupin-wheat rotations on soil fertility, crop disease and crop yields. Australian Journal of Experimental Agriculture 24, 595–600.

Robertson LD, Abd-El-Moneim AM (1999) Status of Lathyrus germplasm held at ICARDA and its use in breeding programmes. In 'Lathyrus Genetic Resources Network'. Aleppo, Syria. (International Centre for Agricultural Research in Dry Areas).

Rochester IJ, Peoples MB, Constable GA, Gault RR (1998) Faba beans and other legumes add nitrogen to irrigated cotton cropping systems. Australian Journal of Experimental Agriculture 38, 253–260.

Rodrigues ML, Pacheco CMA, Chaves MM (1995) Soil-plant water relations, root distribution and biomass partitioning in Lupinus albus L. under drought conditions. Journal of Experimental Botany 46, 947–956.

Rodriguez-Maribona B, Tenorio JL, Conde JR, Ayerbe L (1992) Correlation between yield and osmotic adjustment of peas (Pisum-sativum L.) under drought stress. Field Crops Research 29, 15–22.

Rosales-Serna R, Kohashi-Shibata J, Acosta-Gallegos JA, Trejo-Lopez C, Ortiz- Cereceres J, Kelly JD (2004) Biomass distribution, maturity acceleration and yield in drought-stressed common bean cultivars. Field Crops Research 85, 203–211.

Rovira AD (1990) The impact of soil and crop management-practices on soil-borne root diseases and wheat yields. Soil Use and Management 6, 195–200.

Russell CA, Fillery IRP (1996) Estimates of lupin below-ground biomass nitrogen, dry matter, and nitrogen turnover to wheat. Australian Journal of Agricultural Research 47, 1047–1059.

Saini HS (1997) Effects of water stress on male gametophyte development in plants. Sexual Plant Reproduction 10, 67–73.

Saini HS, Westgate ME (2000) Reproductive development in grain crops during drought. Advances in Agronomy 68, 59–96.

Salim ur R, Piggott JR, Ahmad MM, Hussain S, Ahmad N, Owusu-Darko P (2008) Preparation and evaluation of pizza cheese made from blend of vetch-bovine milk. International Journal of Food Science and Technology 43, 770–778.

119 Sarker A, El Moneim AMA, Maxted N (2001) 'Grass pea and chicklings (Lathyrus L.).' (Kluwer Academic Publishers: The Netherlands).

Scholander PF, Hemmingsen EA, Hammel HT, Bradstreet EDP (1964) Hydrostatic pressure and osmotic potential in leaves of mangroves and some other plants. Proceedings of the National Academy of Sciences of the United States of America 52, 119–124.

Seaton KA, Landsberg JJ, Sedgley RH (1977) Transpiration and leaf water potentials of wheat in relation to changing soil-water potential. Australian Journal of Agricultural Research 28, 355–367.

Serraj R, Krishnamurthy L, Kashiwagi J, Kumar J, Chandra S, Crouch JH (2004) Variation in root traits of chickpea (Cicer arietinum L.) grown under terminal drought. Field Crops Research 88, 115–127.

Serraj R, Sinclair TR (2002) Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant, Cell and Environment 25, 333–341.

Sheoran IS, Saini HS (1996) Drought-induced male sterility in rice: Changes in carbohydrate levels and enzyme activities associated with the inhibition of starch accumulation in pollen. Sexual Plant Reproduction 9, 161–169.

Shivana KR, Rangaswamy NS (1992) 'Pollen biology: A laboratory manual.' (Springer- Verlag: Berlin).

Siddique KHM, Hanbury CL, Sarker A (2006) Registration of 'Ceora' grass pea. Crop Science 46, 986.

Siddique KHM, Loss SP, Herwig SP, Wilson JM (1996) Growth, yield and neurotoxin (ODAP) concentration of three Lathyrus species in Mediterranean-type environments of Western Australia. Australian Journal of Experimental Agriculture 36, 209–218.

Siddique KHM, Loss SP, Regan KL, Jettner RL (1999) Adaptation and seed yield of cool season grain legumes in Mediterranean environments of south-western Australia. Australian Journal of Agricultural Research 50, 375–387.

Siddique KHM, Regan KL, Tennant D, Thomson BD (2001) Water use and water use efficiency of cool season grain legumes in low rainfall Mediterranean-type environments. European Journal of Agronomy 15, 267–280.

Siddique KHM, Sedgley RH (1986) Canopy development modifies the water economy of chickpea (Cicer arietinum L.) in Southwestern Australia. Australian Journal of Agricultural Research 37, 599–610.

Siddique KHM, Sykes J (1997) Pulse production in Australia past, present and future. Australian Journal of Experimental Agriculture 37, 103–111.

Siddique KHM, Tennant D, Perry MW, Belford RK (1990) Water use and water use efficiency of old and modern wheat cultivars in a Mediterranean-type environment. Australian Journal of Agricultural Research 41, 431– 447.

120 Siddique KHM, Walton GH, Seymour M (1993) A comparison of seed yields of winter grain legumes in Western Australia. Australian Journal of Experimental Agriculture 33, 915–922.

Sinclair TR, Muchow RC (2001) System analysis of plant traits to increase grain yield on limited water supplies. Agronomy Journal 93, 263–270.

Sinclair TR, Tanner CB, Bennett JM (1984) Water-use efficiency in crop production. BioScience 34, 36–40.

Smartt J (1990) 'Pulses of the clasical world: the grass pea (Lathyrus sativus L.).' (Cambridge University Press: New York).

Solaiman Z, Colmer TD, Loss SP, Thomson BD, Siddique KHM (2007) Growth responses of cool-season grain legumes to transient waterlogging. Australian Journal of Agricultural Research 58, 406–412.

Souza RP, Machado EC, Silva JAB, Lagoa A, Silveira JAG (2004) Photosynthetic gas exchange, chlorophyll fluorescence and some associated metabolic changes in cowpea (Vigna unguiculata) during water stress and recovery. Environmental and Experimental Botany 51, 45–56.

Stanton ML (1984) Seed variation in wild radish: effect of seed size on components of seedling and adult fitness. Ecology 65, 1105–1112.

Stoddard FL, Balko C, Erskine W, Khan HR, Link W, Sarker A (2006) Screening techniques and sources of resistance to abiotic stresses in cool-season food legumes. Euphytica 147, 167–186.

Subbarao GV, Chauhan YS, Johansen C (2000a) Patterns of osmotic adjustment in pigeonpea — its importance as a mechanism of drought resistance. European Journal of Agronomy 12 239–249.

Subbarao GV, Nam NH, Chauhan YS, Chris J (2000b) Osmotic adjustment, water relations and carbohydrate remobilization in pigeonpea under water deficits. Journal of Plant Physiology 157, 651.

Tang C, Robson AD, Longnecker NE, Buirchell BJ (1995) The growth of lupinus species on alkaline soils. Australian Journal of Agricultural Research 46, 255–268.

Tarafdar JC, Yadav RS, Meena SC (2001) Comparative efficiency of acid phosphatase originated from plant and fungal sources. Journal of Plant Nutrition and Soil Science- Zeitschrift Fur Pflanzenernahrung Und Bodenkunde 164, 279–282.

Teklehaimanot R, Abegaz BM, Wuhib E, Kassina A, Kidane Y, Kebede N, Alemu T, Spencer PS (1993) Pattern of Lathyrus-sativus (Grass pea) consumption and beta-N- Oxalyl-alpha-beta-diaminoproprionic acid (Beta-ODAP) content of food samples in the lathyrism endemic region of Northwest Ethiopia. Nutrition Research 13, 1113–1126.

Thomson BD, Siddique KHM (1997) Grain legume species in low rainfall Mediterranean-type environments. 2. Canopy development, radiation interception, and dry-matter production. Field Crops Research 54, 189–199.

121

Thomson BD, Siddique KHM, Barr MD, Wilson JM (1997) Grain legume species in low rainfall Mediterranean-type environments. 1. Phenology and seed yield. Field Crops Research 54, 173–187.

Tsegaye D, Tadesse W, Bayable M (2005) Performance of grass pea (Lathyrus sativus L.) somaclones at Adet, northwest Ethiopia. Lathyrus Lathyrism Newsletter 4, 5–6.

Turner LB (1993) The effect of water stress on floral characters, pollination and seed set in white clover (Trifolium repens L.). Journal of Experimental Botany 44, 1155–1160.

Turner NC (1988) Measurement of plant water status by the pressure chamber technique. Irrigation Science 9, 289–308.

Turner NC (1992) Crop production on duplex soils: An introduction. Australian Journal of Experimental Agriculture 32, 797−800.

Turner NC (2004) Sustainable production of crops and pastures under drought in a Mediterranean environment. Annals of Applied Biology 144, 139–147.

Turner NC, Abbo S, Berger JD, Chaturvedi SK, French RJ, Ludwig C, Mannur DM, Singh SJ, Yadava HS (2007) Osmotic adjustment in chickpea (Cicer arietinum L.) results in no yield benefit under terminal drought. Journal of Experimental Botany 58, 187–194.

Turner NC, Begg JE (1981) Plant-water relations and adaptation to stress. Plant and Soil 58, 97–131.

Turner NC, Davies SL, Plummer JA, Siddique KHM (2005) Seed filling in grain legumes under water deficits, with emphasis on chickpeas. Advances in Agronomy 87, 211–250.

Turner NC, Wright GC, Siddique KHM (2001) Adaptation of grain legumes (pulses) to water-limited environments. Advances in Agronomy 71, 193–231.

Unkovich MJ, Pate JS (2000) An appraisal of recent field measurements of symbiotic N2 fixation by annual legumes. Field Crops Research 65, 211–228.

Vaadia Y, Raney FC, Hagan RM (1961) Plant water deficits and physiological processes. Annual Review of Plant Physiology 12, 265–292.

Vance CP (2001) Symbiotic nitrogen fixation and phosphorus acquisition. Plant nutrition in a world of declining renewable resources. Plant Physiology 127, 390–397.

Veneklaas EJ, Stevens J, Cawthray GR, Turner S, Grigg AM, Lambers H (2003) Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake. Plant and Soil 248, 187–197.

Weir RG, Cresswell GC (1994) 'Plant nutrient disorders 4: Pastures and field crops ' (Inkata Press: Melbourne).

122 Wery J (2005) Differential effects of soil water deficit on the basic plant functions and their significance to analyse crop responses to water deficit in indeterminate plants. Australian Journal of Agricultural Research 56, 1201–1209.

White CL, Hanbury CD, Young P, Phillips N, Wiese SC, Milton JB, Davidson RH, Siddique KHM, Harris D (2002) The nutritional value of Lathyrus cicera and Lupinus angustifolius grain for sheep. Animal Feed Science and Technology 99, 45–64.

Wudiri BB, Henderson DW (1985) Effects of water-stress on flowering and fruit-set in processing-tomatoes. Scientia Horticulturae 27, 189–198.

Xiong YC, Xing GM, Li FM, Wang SM, Fan XW, Li ZX, Wang YF (2006) Abscisic acid promotes accumulation of toxin ODAP in relation to free spermine level in grass pea seedlings (Lathyrus sativus L.). Plant Physiology and Biochemistry 44, 161–169.

Xue QW, Zhu ZX, Musick JT, Stewart BA, Dusek DA (2006) Physiological mechanisms contributing to the increased water-use efficiency in winter wheat under deficit irrigation. Journal of Plant Physiology 163, 154–164.

Yan ZY, Spencer PS, Li ZX, Liang YM, Wang YF, Wang CY, Li FM (2006) Lathyrus sativus (grass pea) and its neurotoxin ODAP. Phytochemistry 67, 107–121.

Yang HM, Zhang XY, Wang GX (2004) Relationships between stomatal character, photosynthetic character and seed chemical composition in grass pea at different water availabilities. Journal of Agricultural Science 142, 675–681.

Yang JC, Zhang JH (2006) Grain filling of cereals under soil drying. New Phytologist 169, 223–236.

Yang JC, Zhang JH, Huang ZL, Zhu QS, Wang L (2000) Remobilization of carbon reserves is improved by controlled soil-drying during grain filling of wheat. Crop Science 40, 1645–1655.

Yang JC, Zhang JH, Wang ZQ, Zhu QS, Liu LJ (2001) Water deficit-induced senescence and its relationship to the remobilization of pre-stored carbon in wheat during grain filling. Agronomy Journal 93, 196–206.

123 Appendices

Appendix 1. Table of ANOVA for total plant dry mass (g plant-1).

Source of variation d.f. s.s. m.s. v.r. F pr.

Date.Reps stratum Date 2 0.075229 0.037615 40.14 <.001 Residual 15 0.014058 0.000937 0.69

Date.Reps.*Units* stratum Prev_Crop 1 0.083328 0.083328 61.16 <.001 N 1 0.158183 0.158183 116.10 <.001 P 1 0.179823 0.179823 131.98 <.001 Date.Prev_Crop 2 0.048869 0.024434 17.93 <.001 Date.N 2 0.015700 0.007850 5.76 0.004 Prev_Crop.N 1 0.022234 0.022234 16.32 <.001 Date.P 2 0.021933 0.010967 8.05 <.001 Prev_Crop.P 1 0.018799 0.018799 13.80 <.001 N.P 1 0.026515 0.026515 19.46 <.001 Date.Prev_Crop.N 2 0.010898 0.005449 4.00 0.021 Date.Prev_Crop.P 2 0.001119 0.000560 0.41 0.664 Date.N.P 2 0.001009 0.000505 0.37 0.691 Prev_Crop.N.P 1 0.001003 0.001003 0.74 0.393 Date.Prev_Crop.N.P 2 0.002289 0.001145 0.84 0.435 Residual 105 0.143059 0.001362

Total 143 0.824049

Appendix 2. Table of ANOVA for wheat green area (cm2 plant-1).

Source of variation d.f. s.s. m.s. v.r. F pr.

Date.Reps stratum Date 2 222.562 111.281 33.91 <.001 Residual 15 49.221 3.281 0.64

Date.Reps.*Units* stratum Prev_Crop 1 1430.519 1430.519 280.84 <.001 N 1 3647.687 3647.687 716.11 <.001 P 1 641.211 641.211 125.88 <.001 Date.Prev_Crop 2 358.515 179.258 35.19 <.001 Date.N 2 205.596 102.798 20.18 <.001 Prev_Crop.N 1 179.331 179.331 35.21 <.001 Date.P 2 103.151 51.575 10.13 <.001 Prev_Crop.P 1 82.509 82.509 16.20 <.001 N.P 1 134.416 134.416 26.39 <.001 Date.Prev_Crop.N 2 59.042 29.521 5.80 0.004 Date.Prev_Crop.P 2 6.828 3.414 0.67 0.514 Date.N.P 2 12.716 6.358 1.25 0.291 Prev_Crop.N.P 1 18.162 18.162 3.57 0.062 Date.Prev_Crop.N.P 2 15.934 7.967 1.56 0.214 Residual 105 534.844 5.094

Total 143 7702.244

124 Appendix 3. Table of ANOVA for wheat shoot N concentration (mg g-1 dry weight).

Source of variation d.f. s.s. m.s. v.r. F pr.

Date.Reps stratum Date 1 1684.107 1684.107 206.93 <.001 Residual 10 81.386 8.139 1.27

Date.Reps.*Units* stratum Prev_Crop 1 605.919 605.919 94.22 <.001 N 1 1741.811 1741.811 270.85 <.001 P 1 189.317 189.317 29.44 <.001 Date.Prev_Crop 1 71.924 71.924 11.18 0.001 Date.N 1 1.409 1.409 0.22 0.641 Prev_Crop.N 1 0.070 0.070 0.01 0.917 Date.P 1 15.692 15.692 2.44 0.123 Prev_Crop.P 1 16.209 16.209 2.52 0.117 N.P 1 34.865 34.865 5.42 0.023 Date.Prev_Crop.N 1 11.776 11.776 1.83 0.180 Date.Prev_Crop.P 1 2.288 2.288 0.36 0.553 Date.N.P 1 1.936 1.936 0.30 0.585 Prev_Crop.N.P 1 0.382 0.382 0.06 0.808 Date.Prev_Crop.N.P 1 0.448 0.448 0.07 0.793 Residual 70 450.160 6.431

Total 95 4909.699

Appendix 4. Table of ANOVA for wheat shoot N content (mg plant-1).

Source of variation d.f. s.s. m.s. v.r. F pr.

Date.Reps stratum Date 1 120.37236 120.37236 3675.36 <.001 Residual 10 0.32751 0.03275 0.65

Date.Reps.*Units* stratum Prev_Crop 1 52.36883 52.36883 1044.00 <.001 N 1 217.47093 217.47093 4335.41 <.001 P 1 2.94827 2.94827 58.78 <.001 Date.Prev_Crop 1 0.25660 0.25660 5.12 0.027 Date.N 1 0.00012 0.00012 0.00 0.962 Prev_Crop.N 1 0.50959 0.50959 10.16 0.002 Date.P 1 0.89333 0.89333 17.81 <.001 Prev_Crop.P 1 0.88648 0.88648 17.67 <.001 N.P 1 0.60093 0.60093 11.98 <.001 Date.Prev_Crop.N 1 0.89904 0.89904 17.92 <.001 Date.Prev_Crop.P 1 0.55869 0.55869 11.14 0.001 Date.N.P 1 0.32566 0.32566 6.49 0.013 Prev_Crop.N.P 1 0.54262 0.54262 10.82 0.002 Date.Prev_Crop.N.P 1 0.91189 0.91189 18.18 <.001 Residual 70 3.51131 0.05016

Total 95 403.38416

125 Appendix 5. Table of ANOVA for wheat shoot P concentration (mg g-1 dw).

Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr.

Date.Reps stratum Date 1 3.05891 3.05891 34.64 <.001 Residual 10 0.88317 0.08832 1.56

Date.Reps.*Units* stratum Prev_Crop 1 0.19610 0.19610 3.47 0.067 N 1 3.90717 3.90717 69.23 <.001 P 1 0.64116 0.64116 11.36 0.001 Date.Prev_Crop 1 2.55135 2.55135 45.20 <.001 Date.N 1 0.21656 0.21656 3.84 0.054 Prev_Crop.N 1 0.00428 0.00428 0.08 0.784 Date.P 1 0.05645 0.05645 1.00 0.321 Prev_Crop.P 1 0.12890 0.12890 2.28 0.135 N.P 1 0.03066 0.03066 0.54 0.464 Date.Prev_Crop.N 1 0.05976 0.05976 1.06 0.307 Date.Prev_Crop.P 1 0.06346 0.06346 1.12 0.293 Date.N.P 1 0.14808 0.14808 2.62 0.110 Prev_Crop.N.P 1 0.01323 0.01323 0.23 0.630 Date.Prev_Crop.N.P 1 0.00474 0.00474 0.08 0.773 Residual 69 (1) 3.89437 0.05644

Total 94 (1) 15.66745

Appendix 6. Table of ANOVA for wheat shoot P content (mg plant-1).

Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr.

Date.Reps stratum Date 1 0.249594 0.249594 99.77 <.001 Residual 10 0.025018 0.002502 0.97

Date.Reps.*Units* stratum Prev_Crop 1 0.016099 0.016099 6.26 0.015 N 1 1.042654 1.042654 405.61 <.001 P 1 0.446940 0.446940 173.87 <.001 Date.Prev_Crop 1 0.370116 0.370116 143.98 <.001 Date.N 1 0.025823 0.025823 10.05 0.002 Prev_Crop.N 1 0.021365 0.021365 8.31 0.005 Date.P 1 0.001356 0.001356 0.53 0.470 Prev_Crop.P 1 0.047527 0.047527 18.49 <.001 N.P 1 0.045377 0.045377 17.65 <.001 Date.Prev_Crop.N 1 0.047028 0.047028 18.29 <.001 Date.Prev_Crop.P 1 0.002729 0.002729 1.06 0.306 Date.N.P 1 0.000816 0.000816 0.32 0.575 Prev_Crop.N.P 1 0.016183 0.016183 6.30 0.014 Date.Prev_Crop.N.P 1 0.000313 0.000313 0.12 0.728 Residual 69 (1) 0.177370 0.002571

Total 94 (1) 2.511809

126