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Item Screening of effector proteins from the Kauri dieback pathogen Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Biological Sciences at Massey University, Manawatū, New Zealand(Massey University, 2024) Heslop, Scott JohnPhytophthora agathidicida is the causative agent of kauri dieback, a destructive disease threatening kauri that are endemic to the Northernmost regions of New Zealand. Relatively little is understood of this pathogen and its interaction with kauri at the molecular level. However, advances in the understanding of other Phytophthora pathogens, and the completion of a chromosome-level P. agathidicida genome sequence, now allow for this interaction to be studied in greater detail. Core components of any pathogen-plant pathosystem are effectors, which are proteins released by the pathogen during infection that promote virulence. A well characterised type of effector from Phytophthora are the glycoside hydrolases (GHs), a broad group of extracellular effectors that include XEG1 from the soybean pathogen Phytophthora sojae. In the model host Nicotiana benthamiana, XEG1 is recognised by an extracellular, membrane-bound receptor-like protein (RLP) that associates with the co-receptor SOBIR1 to begin the signalling cascade needed for plant immunity. In line with this, the presence of SOBIR1 strongly impedes the ability of P. agathidicida to grow on N. benthamiana. However, it is unclear what role RLPs may have in the immunity of kauri against kauri dieback disease via recognition of the XEG1 homolog from P. agathidicida. In this study, we aimed to characterise the role of XEG1 and a novel kauri dieback effector, Pa8011, to determine if the immunity observed in N. benthamiana could also be found in kauri and how this influences the host-pathogen interaction. Kauri from families with differing levels of tolerance to kauri dieback were kindly provided by Te Roroa, with the requirement that XEG1 testing in kauri leaves was performed using purified protein in place of transient expression used in N. benthamiana. Experiments were first conducted in N. benthamiana to confirm that the P. agathidicida XEG1 protein elicits a plant defence response in the form of localised cell death and that this response depends on the SOBIR1 co-receptor. The purified proteins were then infiltrated into kauri leaves where neither XEG1 or Pa8011 caused localised cell death, despite the response seen with XEG1 in N. benthamiana. To assess how the effectors influence the ability of P. agathidicida to infect kauri, protein-infiltrated leaf tissue was then inoculated with the pathogen, but neither effector caused a significant increase or decrease in the size of pathogen lesions formed. The results obtained indicate a lack of XEG1/Pa8011-specific RLP immune receptors in kauri and opens up new questions about what form this system takes in kauri and how comparable it is to the model host.Item Ally, adversary or something else : do co-occurring Phytophthora pathogens influence each other in culture? : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Biological Science at Massey University, Manawatu, New Zealand(Massey University, 2023) Dagg, Taylah JosieKauri (Agathis australis), a New Zealand native and taonga tree species, is currently under threat from the devasting disease, kauri dieback, caused by the oomycete Phytophthora agathidicida. Recent research highlighted that, along with P. agathidicida, two other Phytophthora species, Phytophthora cinnamomi and Phytophthora multivora, can co-occur in the soil surrounding kauri trees, and that both P. cinnamomi and P. multivora cause lesions on kauri seedlings in glasshouse trials. Whether all three Phytophthora species interact, whether cooperatively or antagonistically, remains unknown. Such information is needed because it may mean that kauri dieback disease control strategies need to target all three species. In an attempt to gain such information, macroscopic inter- and intra-species colony interaction assays were first carried out on solid growth media; however all colonies formed a zone of growth inhibition around themselves irrespective of the media type or species tested. However, it was anticipated that interactions between the three species could occur at the molecular level through secreted proteins, a proteomic analysis was then carried out to determine how the repertoire of secreted proteins changed when multiple species were grown together in liquid culture, relative to grown alone. In total, 20 putatively secreted proteins, termed effector candidates (most of which had predicted enzymatic functions), were identified that were only produced, or that significantly increased in abundance, during co-culturing. All but one of these were from P. agathidicida and most were encoded by genes that were upregulated during infection of kauri leaves and/or roots, suggesting that they may play an important role during host colonisation. As a starting point for future experiments aiming to investigate Phytophthora–Phytophthora as well as Phytophthora–candidate effector interactions in planta, each Phytophthora species was tested for its ability to infect the model host plant, Nicotiana benthamiana. This test revealed large differences in the consistency and rate of infection between the Phytophthora species, meaning that interaction assays in this host would likely be difficult. But interestingly, P. agathidicida and P. multivora more readily infected plants lacking SOBIR1, a major component of extracellular immunity-related signalling, suggesting that extracellular immunity plays a significant role in slowing infection of N. benthamiana by these species. Taken together, the results of this study have considerably advanced our understanding of how Phytophthora species interact at the molecular level and provide a solid foundation for determining whether Phytophthora species work together to cause kauri dieback disease.Item Molecular breeding for resistance to biotic threats in kiwifruit (Actinidia chinensis) : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, Manawatu, New Zealand(Massey University, 2023-12-08) Flay, Casey DamianThis thesis takes a genomic approach to assist kiwifruit breeding programs by identifying genomic loci that contribute to resistance to biotic threats. Three different biotic threats were investigated that represent some of the most destructive to the kiwifruit industry. These were made up of two pests including latania scale (Hemiberlesia lataniae) and Brown headed leafroller (Ctenopseustis obliquana) and the pathogen Psa (Pseudomonas syringae pv. actinidiae). Together these pests and pathogen represent the predominant feeding mechanisms of, sucking from H. lataniae, chewing from C. obliquana, and infection from the necrotrophic pathogen Psa. The inclusion of these pests and pathogen was thought to provide a well-rounded knowledge of the molecular mechanisms that influence both monogenic and polygenic resistance traits in kiwifruit. Moreover, because these experiments were conducted on multiple kiwifruit families at different sites over time with multiple interactions occurring between organisms, an appreciation was gained for the sources of error that affect experiments, and the effects of those errors on the interpretation of the resulting genomic data. The projects that make up this thesis first analyse kiwifruit (Actinidia chinensis var. chinensis) families of different sizes to identify the molecular architecture for resistance to H. lataniae. Once the monogenic region for resistance to H. lataniae was identified, existing datasets were interrogated for individuals with the loci associated with resistance. Furthermore, due to an absence of H. lataniae resistance in the elite tetraploid gold fleshed A. chinensis var. chinensis breeding program, a diploid individual resistant to H. lataniae that produced unreduced gametes was crossed with tetraploid males and screened for tetraploid individuals that contained markers for H. lataniae resistance. Unfortunately, the experiments investigating the molecular architecture for resistance of A. chinensis var. chinensis to C. obliquana using genotyping by sequencing (GBS) data was unsuccessful due to multiple factors discussed in chapter 4. The last project broke away from the traditional association mapping approach to identify quantitative trait loci (QTL) for Psa resistance from several A. chinensis families that had lost individuals due to Psa. This fast, inexpensive, and powerful approach used a modified bulked segregant analysis (BSA) with bulking methods similar to that of selection mapping. The methodology was validated using pools created with a variable number of males and the sensitivity of detection determined by identifying QTL at the known sex locus. The full experiment then analysed whole genome sequence (WGS) data from eight pools containing multiple families to identify QTL associated with survival to Psa. Together these experiments will advance the kiwifruit breeding program by providing the molecular architecture for two of the most prevalent biotic threats affecting kiwifruit production. Utilising the markers for resistance to H. lataniae and developing markers to target the QTL for resistance to Psa will enable breeding programs to use these markers as selection criteria in commercial breeding programs, increasing the rate at which cultivars resistant to these biotic threats can be developed.Item A population genetics investigation of the New Zealand endemic Lophomyrtus bullata (Myrtaceae), a species of conservation concern due to the threat of myrtle rust : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand(Massey University, 2023) Macdonald, Amy LeighLophomyrtus bullata Burret (Myrtaceae) is a shrub or small tree species endemic to New Zealand. The species is a member of the Myrtaceae family and is one of two species in the Lophomyrtus genus. The conservation status of L. bullata was raised to “Threatened – Nationally Critical” following the establishment of the fungal pathogen Austropuccinia psidii (G. Winter) Beenken 2017 in New Zealand in 2017. Repeated infections of the fungal disease can lead to extensive loss of foliage, premature dropping of fruits and plant death. Infected L. bullata exhibit all these symptoms, and rapidly declining regional populations indicate a high potential for localised extinction. The regional disappearance of L. bullata could isolate remaining populations, decreasing gene flow and connectivity. Additionally, rapidly decreasing population sizes could increase the potentially harmful effects of inbreeding and genetic drift, reducing the species’ fitness and increasing its risk of extinction. An understanding of the genetic variation and structure of L. bullata populations could help determine its vulnerability to such genetic effects. To this end, Illumina sequencing was used to develop microsatellite markers for L. bullata. From the 1,351,112 successfully paired and merged sequences, 55 microsatellite loci were isolated, and primer pairs were designed. After an initial screening of the 55 primer pairs, 12 were identified as polymorphic and amplified consistently. These 12 microsatellite markers were genotyped across 452 samples representing 18 populations of L. bullata, one population of L. obcordata, and two populations of L. bullata x L. obcordata putative hybrids. The 18 populations of L. bullata showed low genetic differentiation, low expected heterozygosity and very few private alleles. Lophomyrtus bullata populations in the upper North Island region had the highest expected heterozygosity, and there is evidence of a correlation between expected heterozygosity and decreasing latitudinal distance. High genetic differentiation was observed between the populations of L. bullata and L. obcordata and between the two hybrid populations and the L. bullata populations. The population structure results suggest the presence of two to five genetic clusters within L. bullata. The clusters reflect the geographic location of the populations, in addition to a potential North versus South cline. These patterns could have formed due to past climatic events such as glacial cycling and volcanic activity, isolation by distance, or some combination of these. These results suggest that L. bullata is potentially vulnerable to the negative effects of genetic stochasticity. The continued spread and infection of Austropuccinia psidii could significantly exacerbate these effects. However, the higher genetic diversity and differentiation of the putative hybrid populations suggest a possible avenue for the species to acquire new adaptive variation, though this would heavily depend on the hybridisation mechanics of the species, for which there is little information. Additionally, the significant population structure of L. bullata identified in this study can be used as a guide for conservation practices, including seed banking, population management and re-vegetation projects.Item Identification and characterization of effector proteins from pine needle pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2022) Massoco Tarallo, MarianaCollectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.Item The mode of action of sodium bicarbonate / additive mixtures against cucumber powdery mildew disease : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Applied Science in Plant Pathology, Massey University(Massey University, 1997) Bell, Wendy JacqueleneThe host-parasite-complex of cucumber (Cucumis sativus L.) and Sphaerotheca fulginea (Schlechtendal ex Fr.) Pollacci, the causal agent of cucumber powdery mildew, was studied to determine whether stimulation of host defences was a mode of action of sodium bicarbonate/additive mixtures. Additives included shellspray mineral oil and sunlight dishwashing liquid. The roles in cucumber deface of phenolic compounds, enzymes: peroxidase (PO) and phenylalanine ammonia lyase (PAL), and induced structural barriers, papillae, were investigated. Comparisons were made between the effects of bicarbonate/'additive mixtures and the plant extract Milsana on these host defences, as related to three levels (cultivars) of host resistance to disease: susceptible, disease-tolerant and resistant. Long and short duration glasshouse studies found disease incidence highest on less resistant cultivars and lowest on the most resistant cultivar, 'Slice King' ("SK'). Applied alone at 2 g/litre, sodium bicarbonate failed as a protectant. When combined with either additive, the fungicidal activity of bicarbonate was enhanced. Shellspray oil and bicarbonate-oil mixtures were the most effective treatments for less resistant cultivars, 'Lebanese' ('Leb') and 'Slicemaster' ('SM'). These treatments and Milsana provided comparable control against disease affecting 'SK'. The role of phenolic compounds was investigated in thin-layer chromatogram bioassays for antifungal phenolics in leaf extracts. This revealed much variability in presence and absence of disease. Glycosidically-bound phenolics hydrolysed into their free form, aglycones, were more abundant than free phenolics. Because of their frequency and arbitrary concentrations, it was unclear whether stimulation of aglycone production had occurred in infected leaves, as a result of any specific treatment or level of host resistance. The presence of these fungitoxic substances could not solely account tor differences in disease severity. Hence, data did not support the concept of stimulation of host materials by either mildew infection or the treatments applied. The responses of PO and PAL to treatment and infection were assessed by spectrophotometric measurements of activity in leaf samples. Higher enzymic activities in healthy leaves were coupled with increasing levels of host resistance to disease. During the 14 days post-treatment, significant increases were detected in healthy and inoculated leaves treated with bicarbonate and Milsana, levels varied between cultivars. General enzymic-activity was higher in inoculated than noninoculated 'Leb' and 'SM', and of a comparable level in 'SK' controls and leaves treated with materials other than shellspray oil and bicarbonate-oil mixtures. For these apparent "normal" increases in plants treated with other materials occurred during this time. Fluorescence and bright-field microscopy revealed similar numbers of conidia germinated 24 h after inoculation, irrespective of treatment and cultivar, although significantly fewer conidia germinated on leaves treated with oil and bicarbonate-oil mixtures. With increasing host resistance the number of multiple germ tubes produced 72 h post-infection was less, papillae deposited in epidermal cells had increased, and the number of haustoria less, 120 h post-infection. Shellspray oil and bicarbonate-oil mixtures provided the most significant reduction. in multiple germ tubes and haustoria and the least apparent stimulation of papillae production.Item Identification and functional characterisation of glycoside hydrolases from the kauri dieback pathogen, Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science at Massey University, Manawatū, New Zealand(Massey University, 2022) Bradley, Ellie LynnThe survival of kauri, an ancient conifer species endemic to New Zealand, is currently threatened by kauri dieback disease, caused by the oomycete plant pathogen Phytophthora agathidicida. As P. agathidicida continues to spread throughout kauri forests in the northern North Island of New Zealand, encouraging research has indicated there may be natural tolerance to the disease within the kauri population. This resistance is likely governed, in part, by the plant immune system, which is activated upon recognition of pathogen invasion patterns such as microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and effectors (virulence factors required for host colonisation), which are recognised at the plant cell surface by plant immune receptors. To better understand how P. agathidicida interacts with its plant host on a molecular level, pathogenproduced proteinaceous invasion patterns need to be identified and characterized to aid in the identification of cognate immune receptors in the plant host, which may be involved in activation of the plant immune system. As the role of glycoside hydrolase (GH) proteins in virulence and pathogenicity of fungal and oomycete plant pathogens is well established (Chapter two), an effectoromics approach was used to identify six P. agathidicida GH12 proteins that appear to act as MAMPs in both Nicotiana benthamiana and Nicotiana tabacum (Chapter three). Furthermore, nuclear magnetic resonance was used to identify considerable changes in kauri leaf apoplastic wash fluid of approximately 17 metabolites, including sucrose and glucose, in response to P. agathidicida inoculation (Chapter four), thus suggesting a role for GH proteins in the hydrolysis of some of these metabolites. Finally, proteomic analysis of P. agathidicida culture filtrates via liquid chromatography-mass spectrometry (LC–MS) was used to validate the expression of predicted P. agathidicida proteins and to investigate the capacity of this method to identify candidate invasion patterns for future analysis. Chapter five established that LC–MS analysis of Phytophthora culture filtrate was an effective method for the identification of putative apoplastic invasion pattern candidates and confirmed the production of all six P. agathidicida GH12 cell death elicitors in culture. Collectively, this thesis has advanced our understanding of the molecular mechanisms underpinning the interaction of P. agathidicida with its host and has contributed to the identification of candidate apoplastic effectors.Item Identification of novel avirulence effectors in the Dothideomycete plant pathogens, Venturia inaequalis and Cladosporium fulvum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Sciences at Massey University, Manawatū, New Zealand(Massey University, 2022) de la Rosa, SilviaVenturia inaequalis and Cladosporium fulvum are important fungal pathogens of crop species, causing scab and leaf mould disease of apple and tomato, respectively. Resistance to these pathogens is governed by Rvi (apple) and Cf (tomato) resistance (R) genes. These R genes encode immune receptors that recognize specific pathogen virulence factors, termed avirulence (Avr) effectors, to activate plant defenses. Notably, isolates or strains of V. inaequalis and C. fulvum have emerged that can overcome resistance mediated by specific R genes in their respective hosts. To better understand how these pathogens cause disease or overcome resistance, and to monitor the occurrence of resistance-breaking isolates or strains in the field, Avr effectors from V. inaequalis and C. fulvum must be identified and functionally characterized. Using a combined comparative genomics and phenotyping approach based on progeny from a sexual cross between V. inaequalis isolates that differ in their ability to overcome Rvi4 resistance in apple, a strong candidate for the corresponding AvrRvi4 effector gene was identified (Chapter 2). Similarly, using a comparative genomics approach based on in planta-expressed effector candidates from C. fulvum strains that differ in their ability to overcome Cf-9B resistance in tomato, combined with functional assays, the corresponding Avr9B effector gene was identified (Chapter 4). In the resistance-breaking isolates or strains studied, the candidate AvrRvi4 gene was disrupted, while the Avr9B gene had been deleted. Consistent with most fungal Avr effectors and their genes, both the AvrRvi4 candidate and Avr9B are highly expressed in planta, and encode small, secreted cysteine-rich proteins. The AvrRvi4 candidate forms part of an expanded protein family in V. inaequalis, with members predicted to adopt a β sandwich fold similar to structurally characterized fungal effectors. Avr9B, however, is predicted to adopt a novel protein fold. Finally, using a heterologous expression approach, three in planta-expressed candidate effectors from V. inaequalis were found to trigger defense responses in non-host plants (Nicotiana spp.), suggesting they are recognized by R proteins in these species (Chapter 3). Taken together, this thesis has increased our understanding of the molecular mechanisms responsible for the activation and circumvention of resistance by V. inaequalis and C. fulvum, which will in turn direct host cultivar deployment and disease control strategies in the field.Item Reactive oxygen species play a dual role in the resistance and susceptibility of Camellia to flower blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatū, New Zealand(Massey University, 2020) Fulton, CadeReactive oxygen species (ROS), a group of highly reactive biomolecules, are known to rapidly accumulate in plant tissue as an early defence response to pathogen invasion. However, ROS can also contribute to pathogen virulence. Currently, little is known about the activity of these compounds during the interaction between Camellia and the Camellia flower blight (CFB) necrotrophic fungal pathogen, Ciborinia camelliae L. M. Kohn (Sclerotiniaceae). It has been shown that there is a spectrum of resistance and susceptibility to the disease within the Camellia genus. This study aimed to elucidate the role that ROS play during C. camelliae interactions with Camellia on this spectrum of resistance. To achieve this, hydrogen peroxide accumulation was first visualised and compared between the CFB resistant Camellia lutchuensis and the susceptible Camellia ‘Nicky Crisp’ in response to C. camelliae. Following the inoculation of flower petals with C. camelliae ascospores, widespread apoplastic hydrogen peroxide accumulation and upregulation of genes encoding NADPH oxidase and cell wall peroxidase began 12 hours earlier in the resistant C. lutchuensis species than the susceptible C. ‘Nicky Crisp’, which showed very little observable accumulation. In addition, the quantity of hydrogen peroxide significantly increased in the resistant C. lutchuensis petals, but no change was observed in the susceptible C. ‘Nicky Crisp’ within the same timeframe. The application of exogenous antioxidants to scavenge the hydrogen peroxide accumulation resulted in disease development in the normally resistant C. lutchuensis, while the incidence of disease was significantly reduced in the susceptible C. ‘Nicky Crisp’. Therefore, it was hypothesised that early ROS accumulation contributes to CFB resistance and that late ROS accumulation contributes to CFB susceptibility. This work further expands knowledge of plant interactions with necrotrophic fungal pathogens from the Sclerotiniaceae family by demonstrating that ROS both positively and negatively regulates CFB development based on temporal accumulation, thereby discovering a dual role for ROS accumulation during this interaction.Item Water-use efficiency in perennial ryegrass (Lolium perenne L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand(Massey University, 2021) Weerarathne, Lekamalage Visna YahaniKnowledge of genetic and physiological bases of drought responses and stress tolerance properties of pasture plants is an integral part of designing efficient pasture improvement programs to combat the consequences of climate change. However, experimental evidence or theoretical analyses on that aspect is sparse in the literature. Therefore, considering wider applications, high economic importance, and acknowledged poor tolerance of (Lolium perenne L.; PRG) to drought, the main aim of this research was to evaluate morpho-physiological trait responses that are linked to water-use efficiency (WUE) in different PRG populations from different sources in simulated summer drought cycles under a controlled environment. This study also included a quantitative genetic analysis conducted on the key traits to ascertain which traits are under genetic control for future breeding purposes. Experiment 1 screened single potted PRG genotypes from three commercial cultivars with industry reputation for persistence for natural differences in morpho-physiological traits related to water use (WU) under simulated drought. Large within-population variation was observed for the measured traits which included, among others, WUE (g WU/g plant dry matter); shoot dry weight, SDW; leaf osmotic potential, OP; leaf relative water content; predawn leaf water potential; root dry weight at 20–50 cm depth, RDWD; gravimetric soil moisture at 30–40 cm depth, SMCD; post-cutting regrowth. Principal component analysis (PCA) identified important trait associations contributing to high WUE (i.e. WUE-OP-RDWD trait association) and one related to higher SDW together with ‘SMCD conservation’ indicative of ‘true WUE’ was used to make a divergent selection of 20 high- and 15 low-WUE genotypes (HWUE and LWUE, respectively). Experiments 2 and 3 were conducted simultaneously using the same methodology as Experiment 1 and inter-randomised in the same glasshouse space. Experiment 2 retested clones of HWUE and LWUE plants selected in Experiment 1 for consistency of trait expression across the two consecutive growing seasons, and also collected data for additional traits. Results confirmed that the key trait associations identified in Experiment 1 were almost identically expressed in Experiment 2. From data on additional traits, it was established that the accumulation of high molecular weight sugars in the shoots significantly contribute to ‘true WUE’ of a subset of PRG genotypes but, with the less involvement of gas exchange data under the conditions tested. It is speculated that enhanced mesophyll conductance of CO2 might underlie this important trait association. However, the large majority of genotypes exhibited a ‘SMCD-depleting’ trait association of WUE with improved gas exchange and maximum quantum efficiency of PSII, demonstrating late A.R. Blum’s theory of WUE. Thus, selection of PRG for drought tolerance should consider yield and soil moisture data together to establish the most appropriate category of WUE trait association in improved cultivars. Experiment 3 investigated drought response trait associations in two further populations: a group of elite plants from a commercial plant breeder’s breeding program (CBL) and a PRG germplasm line derived from crossing Mediterranean and Middle Eastern seed accessions (MMEL) compared with those from the HWUE selection. PCA results showed that the major trait associations found in the yield and water relations data of elite subsets of CBL closely followed those of the HWUE selection, but elite MMEL plants exhibited typical summer dormancy characteristics where the average SDW of MMEL was 40% of lower than that of the CBL plants. Results further suggested that the company field evaluation system could benefit from the consideration of water relations traits, including WUE and associated traits like OP, as externally-measured selection traits for PRG drought tolerance. Experiment 4 evaluated quantitative genetic parameters of the key traits using the same methodology from Experiment 1 in a breeding population of 36 ‘half-sib (HS) families’ under both stressed and non-stressed conditions. There were significant estimates of among- and within-HS family genetic variances, narrow-sense heritability, and predicted genetic gain estimates for the key traits, indicating high genetic potential of each trait for breeding purposes under the conditions tested. However, the correlated response to selection of each trait pair comprising highly genetically correlated morpho-physiological traits with WUE was lower than that gain from the single-trait selection, accommodating further research questions on the efficacy of indirect and multi-trait selection of key traits. Based on the current results, it was found that the direct selection of PRG for WUE or concurrent selection for OP, RDWD, SMCD, and SDW or RGS traits under drought is advisable. Furthermore, significant quantitative genetic parameters estimated for WUE under non-stressed conditions together with high genetic correlation observed for WUE between stressed and non-stressed conditions suggested that a PRG population can potentially be selected for this trait at early growth stages or before imposition of water deficit.