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    Investigating the health benefits of Monty’s Surprise apple phytochemicals : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatū, New Zealand. EMBARGOED until 18 November 2027.
    (Massey University, 2024) Nezbedová, Linda
    Cancer is a major health concern worldwide, requiring effective prevention strategies to reduce its burden. There is evidence suggesting that diet plays an important role in disease prevention. Apples are an example of a commonly available fruit that can be easily incorporated into a diet to reduce the risk of chronic disease and some cancers. Apples are rich in phytochemicals with chemo-preventative properties. However, assessing their benefits in humans is complex due to various factors affecting phytochemical composition and low bioavailability. Understanding and controlling for these factors is crucial for maximising their potential in preventing cancer and promoting human health. The PhD project presented in this thesis uses a multidisciplinary ‘from orchard to fork to function’ approach to evaluate the health benefits of phytochemicals in a New Zealand heritage apple cultivar known as Monty’s Surprise, with an emphasis on cancer prevention. This thesis particularly focuses on apple’s most abundant phytochemical group known as phenolic compounds. After controlling fruit harvest and maturity, Monty’s Surprise apple was found to contain high concentrations of health beneficial phenolics, especially procyanidins, with apple skin having higher total phenolic content compared to its flesh. Pureeing was identified as a suitable processing technique with minimal impact on apple’s phenolics, allowing long-term preservation and creating uniform material for use in intervention and feeding trials. Moreover, Monty’s Surprise apple extract depleted of sugars inhibited the proliferation of lung, breast, and colorectal cancer cells in a dose-dependent manner in vitro. Lastly, in a human acute feeding trial, Monty’s Surprise apple puree increased plasma antioxidant capacity and decreased postprandial glucose response compared to sugar-matched placebo. The feeding trial also showed that procyanidins were not detected in the participant’s blood, while epicatechin and chlorogenic acid, which are prevalent phenolics in Monty’s Surprise apple puree, were absorbed into the bloodstream. Therefore, these compounds were available to contribute to the improved health parameters measured in the study. This study offers practical recommendations for optimising harvesting, storage, and processing practices to preserve and more clearly elucidate the health-promoting properties of phenolics in apples. Overall, this research demonstrated the potential health benefits of Monty’s Surprise apple and its phenolics, suggesting its value as a functional food for improving human health and well-being.
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    Unravelling the molecular basis of subcuticular host-colonization by the apple scab fungus, Venturia inaequalis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, School of Agriculture and Environment, Massey University
    (Massey University, 2023) Rocafort Ferrer, Mercedes
    Scab, caused by the fungal pathogen V. inaequalis (Vi), is the most economically important apple disease. During infection, Vi occupies the subcuticular environment, where it develops specialized infection structures, called stromata and runner hyphae. These structures are thought to be important for fungal nutrition and the delivery of proteins, with many of these anticipated to function as virulence factors (effectors) in promoting host infection or avirulence factors (Avr effectors) in triggering host resistance. To date, nothing is known about how these structures are differentiated and protected from recognition by the host immune system. Likewise, little is known about the identity and function of Vi effector proteins. To better control scab, a greater understanding of the molecular mechanisms underpinning infection structure differentiation and protection, as well as Vi virulence and avirulence, is first needed. In Chapter 2, a comprehensive review of apoplastic effector proteins from plant-associated fungi (and oomycetes) was provided. Given that Vi is an extracellular pathogen, this review provided insights into the potential types of effector proteins secreted by Vi into the subcuticular environment. Then, in Chapter 3, a multidisciplinary approach based on bioinformatics, transcriptomics, and structural biology was used to identify and characterize Vi effector candidates (ECs). This revealed that ECs were predominantly expressed in two temporal waves, and that many belonged to expanded protein families with predicted structural similarity to virulence and avirulence effectors from other plant-pathogenic fungi. This analysis helped to generate a list of ECs for further study and contributed to a better understanding of effector biology and evolution. Next, in Chapter 4, a multidisciplinary approach based on transcriptomics, proteomics, glycomics, and confocal microscopy was used to study Vi cell wall carbohydrate composition during the differentiation of infection structures. This iii revealed that Vi down-regulates genes putatively associated with the biosynthesis of immunogenic carbohydrates, and deacetylates surface-exposed chitin to the less immunogenic carbohydrate, chitosan. Finally, in Chapter 5, CRISPR-Cas9 technology was applied to Vi for the first time, which will enable genes identified in this study to be functionally characterized. Altogether, this thesis has furthered our understanding of the Vi –apple pathosystem and has provided novel data that can be used to inform the development of new scab control strategies against Vi.
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    Water stress and apple fruit quality : a thesis presented in partial fulfilment of the requirements for the degree of Master of Horticultural Science at Massey University
    (Massey University, 1992) Moremong, Mapaseka
    Regulated Deficit Initiation (RDI) has been used successfully in dry climates to control vegetative growth of fruit trees during the early part of the growing season without seriously disadvantaging fruit growth or quality. This project was undertaken as part of a study to investigate the feasibility and practicality of using the RDI concept in a humid fruit growing environment using Royal Gala apple trees. Treatments consisted of a lucerne cover crop, black polyethylene undertree covers and a within-row herbicide strip which is the normal commercial practice in New Zealand orchards. A full irrigation treatment (FI) was used on half the experimental trees and an RDI treatment was used on the other half of the trees. The RDI treatment consisted of withholding water until 105 days after full bloom, then using a full irrigation for the remainder of the season. Integration with depth of the soil moisture content (θ) (measured with a neutron probe) at the commencement of the experiment revealed 230 mm of water was stored in the top 900 mm of soil. Full irrigation resulted in θ increasing, with storage of about 250 mm 83 days after bloom and remaining at this level for the remainder of the season. The storage in the RDI treatments decreased in a linear manner until 58 days after bloom, after which it remained constant until initiation was started 105 days after bloom. The lucerne RDI (LRDI) treatment had a lower storage (105 mm) during this constant period than plastic RDI (PRDI), or herbicide (HRDI) treatments which both had a storage of 130 mm. The amount of water in the soil at this time for LRDI and for PRDI/HRDI was 42% and 52% of the total available capacity. Immediately after irrigation commenced on the RDI treatments, profile water storage returned to the values of the FI treatments remaining at these values for the rest of the season. Removal of water from the soil profile was not uniform. It appeared that lucerne removed moisture from the upper horizons first, before extracting it from the zone below 500 mm. Leaf water potentials (ψₑ) were lower in RDI treatments, when measured at midday (but not pre-dawn), than in FI treatments. Leaves from LRDI trees had lower ψₑ values than did leaves from PRDI and HRDI trees. Rate of fruit growth was reduced in all RDI treatments during the early part of the season, but returned to the same value as FI fruit once irrigation was resumed, except for LRDI fruit which did not attain the same growth rate of FI fruit. There were less large fruit and more small fruit from LRDI treatments than from other treatments where no significant effects on fruit size were measured. Fruit from RDI treatments were firmer, less mature and contained more soluble solids at harvest than FI fruit; some of these differences were maintained through 12 weeks storage at 4°C. There was no consistent effect of irrigation or cover treatment on fruit colour, mineral content or disease incidence at harvest or after storage. Vegetative growth, measured as pruning weights and the increment in trunk diameter, was significantly reduced by RDI treatments with LRDI causing the greatest reduction in pruning weight and PRDI inducing the smallest trunk diameter increase. The combined lucerne cover crop and RDI treatment was the most successful method found for reducing soil moisture in a humid climate. It also resulted in the greatest amount of stress being induced in these trees, reducing both vegetative growth - a desirable effect, but also fruit growth, a commercially undesirable effect. It is suggested that different methods of managing lucerne, or the use of less successful water extracting plants will need to be evaluated before recommending a successful, yet practical method of using the RDI concept in apple orchards growing in humid environments.
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    Temperature and atmosphere composition influence on colour change of apples : a dissertation presented in partial fulfilment of the requirements for a Masterate of Horticultural Science, Massey University, Palmerston North, New Zealand
    (Massey University, 1993) Dixon, Jonathan
    In apples colour is a major quality parameter used by consumers to determine apple maturity. A full understanding of the nature of the relationship between storage conditions and apple fruit colour change would be of advantage in formulating models to predict how changes to handling systems would affect fruit colour. While much is known in a general way about how environmental conditions affect colour change, little information is available to characterise the nature of the relationships between temperature, oxygen and carbon dioxide. The postharvest change in colour was measured for two export apple cultivars; Cox's Orange Pippin and Granny Smith. Previous research on these and other apple cultivars has determined that colour change is from green to yellow. The colour of Cox's Orange Pippin and Granny Smith apples were measured by subjective and objective methods during experiments to investigate the effect of temperature and atmosphere composition on colour change. The objective methods used were: chlorophyll extraction and colour using a Minolta chromameter. The subjective method was colour matching for Granny Smith using the NZAPMB maturity colour charts. When related to changes in chlorophyll, the principal skin pigment, the colour parameters used had non-linear relationships. Lightness, hue angle and colour chart score all reflect pigment changes occurring as apples change colour from green to yellow. Lightness values were the least variable followed by hue angle then colour chart score. All methods used showed more sensitivity to changes in chlorophyll content when chlorophyll content was low compared to when chlorophyll content was high. The objective measurements were highly correlated with the subjective measurements and the conclusion was that the use of hue angle or lightness to follow colour change in the skin of Granny Smith and Cox's Orange Pippin apples is an accurate indirect measure of chlorophyll and other pigments. The rate constant of colour change (k), measured using a declining exponential function, from green to yellow, at eleven temperatures over two seasons, two harvests per season and several growers was investigated in order to characterise the relationship between yellowing and temperature. All the methods of colour measurement used had the same relationship with temperature which was described by a modified form of the Arrenhius equation. Re-worked published data also fitted the modified Arrenhius equation. The modified Arrenhius equation was used to generate k for the various colour parameters measured (chlorophyll, hue angle, lightness and colour charts score). The value of k, as a function of temperature, increases slowly between 0°C and 6°C (the lag phase), increases exponentially between 6°C and 20°c and reaches a maximum at 25.3°C for Cox's Orange Pippin and 23.5°C for Granny Smith before declining. Pattern of response to temperature was the same for each cultivar although Granny Smith yellowed more slowly than Cox's Orange Pippin. For Cox's Orange Pippin apples more variation was accounted for by differences between growers than years or harvests within a year. For Granny Smith fruit most variation was accounted for by differences between years. Sixteen atmospheres were used each year for Cox's Orange Pippin and Granny Smith apples from one harvest in order to characterise the relationship between yellowing and oxygen or carbon dioxide. Cox's Orange Pippin and Granny Smith apples differ in their response to oxygen. For Cox's Orange Pippin the value of k as a function of oxygen level increased slowly from 0% to 6% and thereafter increased exponentially from 6% to 19%. This function may be sigmoidal as the k values increase slows above 17% oxygen. The relationship for Granny Smith was poorly defined by this function, k values increased slowly as the oxygen level rose. This could be due to a fundamental physiological or biochemical difference between these two cultivars. Each cultivar had a similar response to carbon dioxide, described by a declining exponential function, with the relationship for Granny Smith being better defined than for Cox's Orange Pippin. The relationship of carbon dioxide with colour change was poorly defined as the effects of oxygen on colour change were not removed from the analysis. Oxygen appears to have a greater influence on colour change than carbon dioxide. Atmospheres for Cox's Orange Pippin apples were not scrubbed for carbon dioxide in 1989 but were in 1990. The pattern of response to oxygen in the absence of levels of carbon dioxide above 1% in the atmosphere did not alter the sigmoidal relationship found. This may be evidence that the effect on yellowing by oxygen and carbon dioxide is by separate processes. Ethylene levels in the atmosphere appeared to have little effect on the rate of yellowing in all the atmospheres studied. The carbon dioxide and oxygen functions were combined into a single equation for use as a predictive model. The temperature function, the modified Arrenhius equation, and the atmosphere functions were combined into one equation to which different environmental values were added. The use of such a model and other practical applications for the information gathered for this thesis are discussed and a chart drawn comparing the hue angle, lightness and colour chart score to chlorophyll level.
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    An economic analysis of a robotic harvest technology in New Zealand fresh apple industry : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Agribusiness, Massey University School of Agriculture and Environment, Manawatu, New Zealand
    (Massey University, 2020) Ghahremani, Morteza
    The New Zealand apple industry is predominately an export-oriented industry relying on manual labour throughout the year. In recent years, however, labour shortages for harvesting have been jeopardising its competitiveness and profitability. Temporary immigration labour programs, such as the Recognised Seasonal Employer (RSE) program have not been able to solve the labour shortages, urging the industry to consider use of harvesting automation, i.e. robotic technology, as a solution. Harvesting robots are still in commercial trial stage and no studies have assessed the economic feasibility of such technology. The present study for the first time develops a bio-economic model to analyse the investment decision for adopting harvesting robots compared to available alternatives, i.e. platform and manual harvesting systems, using net present value (NPV) as the method of analysis; for newly established single-, bi-, and multi-varietal orchards across different orchard sizes, and three apple varieties (Envy, Jazz, and Royal Gala); and implications of orchard canopy transition and associated sensitivities are considered. The results of the analysis identified fruit value and yield as the key drivers for the adoption of harvesting automation. For relatively low value and or yielding varieties such as Jazz or Royal Gala, robots are less profitable in single-varietal orchard compared to bi-varietal orchard planted with relatively low value and yielding varieties. In a multi-varietal orchard, a relatively high value and high yield variety, such as Envy, is crucial to compensate for the costs incurred for harvesting other varieties using robots or platforms. The greatest potential benefit of utilising harvesting robots was reducing pickers required by an average of 54% for Envy and 48% for each of Jazz and Royal Gala across all orchard sizes compared to manual harvesting; and 7% in average for each of Envy, Jazz, and Royal Gala across all orchard sizes compared to platform harvesting system. This study also identified the break-even price for a robotic harvester in a single-varietal orchard, showed that the break-even prices exceeded the assumed price of the robot, and are highly variable depending on the varietal value and yield, where Envy as a relatively higher value and yielding variety returns a break-even price of $2.92 million compared to relatively lower value and yielding varieties, Jazz with $674,895, and Royal Gala with $689,608. Sensitivity analyses showed that both harvesting speed and efficiency are key parameters in the modelled orchard and positively affected the net returns of the investment and must be considered by researchers and manufacturers. However, for developers and potential adopters of robots, it should be more important that robots operate faster, but not necessarily as more efficient in order to generate a high return while substituting the highest number of pickers and leaving less unharvested fruit on trees in the limited harvesting window. Reducing robot price by 12% and 42% can generate an equivalent level of profit similar to manual or platform harvesting, respectively. Increases in labour wages, and decreases in labour availability and efficiency adversely affected the NPV and profitability outlook of the investment, but NPV was more affected by the decreases in labour efficiency and availability than wage increases. This research has important science and policy implications for policy makers, academics, growers, engineers, and manufacturers. From an economic perspective, for late adopters or those growers who may not be financially able to invest in robots or may be uncertain about their performance, platform harvesting system can be utilised as an alternative solution that is commercially available until robotic harvesting technology improves or becomes more affordable, and commercially available. Alternatively, it may be possible for these orchardists to benefit from utilising the robotic harvester in the form of a co-operative or contract-harvesting business model to avoid the capital costs associated with purchasing and operating the robots. Besides the economic factors, robotic harvesters have the potential to be considered as a solution for non-economic factors such as food safety problems. This is more apparent in the post-Covid-19 pandemic era, which has not only made it more difficult for growers to source their required workers due to border closures, but also has led consumers to be more cautious about food safety when they make purchase decisions and prefer to have their fresh fruit touchless from farm to plate. This may not be a problem for packhouses as most are automated, but it may be an issue for harvesting operations, because pickers have to pick apples by hand. Even though robots cannot be the only option for growers to rely on for the foreseeable future as they are not commercially available, in the current situation robot harvesting may be the most ideal solution.
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    Process for recovery of smooth fibre ingredient from pomace : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Food Technology at Massey University, Manawatu, New Zealand
    (Massey University, 2020) Eblaghi, Marzieh
    This research aimed to develop a process to convert apple pomace into a food ingredient which can provide functional properties such as water-binding in baked goods, stability in aqueous suspension and smooth mouthfeel. This was achieved by modifying the apple pomace through three main steps: heating, shearing and enzymatic hydrolysis. Firstly, the effect of sample preparation (addition of water to fresh pomace, temperature and shearing apple pomace) on the solubility of pectin was investigated. Secondly, kinetics of main reactions involved in pomace while heating at temperatures between 90-140 °C (10 °C intervals) and incubation times between 0-360 min, were studied at bench scale. These reactions were: solubilisation and depolymerisation reactions of pectin, degradation of sugars and production of secondary products such as organic acids and 5-hydroxymethylfurfural (5-HMF). After that, the kinetics of these reactions were modelled for determining the rate constants and activation energies. The kinetic models of pectin solubilisation and 5-HMF formation were then used for scaling up the hydrothermal process in a more complex heat transfer situation, using a pilot scale retort. Finally, the effect of particle size distribution and molecular weight of solubilised components (mainly pectin) on physicochemical and sensorial properties of pomace material was investigated. Solubilisation of pectin at room temperature was independent of addition of water and shearing treatment of pomace. However, heating at temperatures > 100 °C, combined with increasing the amounts of water added to pomace (from 0 to 8 mL water/ g pomace) resulted in increasing the pectin solubility up to a pomace-water ratio of 1:2. The maximum amount of solubilised pectin (~ 605 μmol galacturonic acid/ g dry pomace) was determined when heating pomace at 130 and 140 °C for 15 and 7 min, respectively. Hydrothermal depolymerisation of pectin through acid hydrolysis and β-elimination reactions also showed temperature-dependent behaviour. Depolymerisation reactions resulted in degradation of pectin polymers into ethanol-soluble forms (galacturonic acid). Depolymerisation seemed more likely to happen from non-esterified sites of pectin polymers, as suggested by the high degree of esterification of the remaining insoluble pectin. Increasing amounts of glucose and fructose were observed in the serum phase of pomace to about five times their initial values when pomace was heated at temperatures >120 °C. This was accompanied by a reduction in sucrose content, suggesting hydrothermal hydrolysis of sucrose to its subunits. A complete conversion of sucrose was recorded at temperatures > 120 °C and times > ~30 min. Glucose, fructose and galacturonic acid underwent further transformation at temperatures > 100 °C forming secondary products of organic acids, (such as acetic acid, formic acid and lactic acid), furfural and 5-HMF. Modelling the kinetics of pectin solubilisation and 5-HMF production resulted in activation energies of 81 and 105 kJ/mol, respectively. The effects of hydrothermal treatment were modelled using COMSOL for heating a slab of pomace in a pilot scale retort with a maximum steam temperature of 125 °C. In this model, heat transfer through the pomace and chemical reactions of pectin solubilisation and 5-HMF production were predicted. The aim for this model was to identify conditions permitting solubilisation enough to double the amount of pectin from the amount initially present at room temperature while limiting the production of 5-HMF to the range permitted in food standards (bulk averaged). The validity of the model was confirmed in the pilot plant condition. Another objective of this research study was to investigate the effects of particle size distributions and molecular weight of pectin on sensory properties and physical stability of pomace samples. A controlled modification of particle size and molecular weight of solubilised pectin was achieved by fractionation of heat-treated pomace into insoluble solid and serum parts. Shearing insoluble particles for 5 min showed significant particle size reduction from 496 μm (initial shearing for 2 min) to 165 μm. Further shearing did not affect the average size of the particles. Microscopy revealed the effect of shearing in separating cell aggregations, resulting in individual cells. Shearing also fractured some individual cells, the oval shape of most cells was still visible. The pectin in the serum phase of pomace before and after heat treatment was analysed for molecular weight distribution. Results confirmed the effectiveness of heat treatment on solubilising high molecular weight pectins into the serum phase. Two ranges of high (between 4000-73 kDa) and low (< 73 kDa) pectin molecular weights were analysed in heat-treated pomace. These two ranges were separated from each other by ultrafiltration. High molecular weight of fraction (with the average size of 380 kDa) was enzymatically hydrolysed into two pectin components with average molecular weights of 30 and 150 kDa. Finally, six pomace ingredients were produced from the pomace fractions with two insoluble particle size distributions (with the average size ranges of 500 and 160 μm) and three ranges of molecular weights (380, 150 and 30 kDa) being blended. Particle size reduction had a significant effect on the physical stability of pomace suspensions. Samples containing particles >496 μm showed phase separation during storage (5 days at 4 °C), while samples with smaller particle size did not show any phase separation.
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    Genetic dissection of the dwarfing effect of the apple rootstock 'M.9' : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2007) Celton, Jean-Marc
    The dwarfing phenomenon in apple is mainly characterized by the ability of a rootstock to reduce the vegetative growth and ultimate size of the scion grafted onto it. Many hypotheses have been proposed to explain the dwarfing effect of rootstocks, from the production and translocation of hormones to the altered anatomy of the graft union. However, despite the numerous studies undertaken, none of the studies support a single hypothesis. This study focussed on identifying genetic markers for genomic regions influencing the dwarfing effect of 'Malling 9' ('M.9'). Two rootstock populations derived from crosses between 'M.9' and the vigorous rootstock 'Robusta 5' ('R5') were propagated and phenotyped at the HortResearch Havelock North Research Centre (New Zealand). Bulked segregant analysis (BSA) and QTL analysis were performed using phenotypic data collected from scions grafted onto the rootstock populations. Several genomic regions in 'M.9' and 'R5' were found to have a significant influence on the dwarfing phenotype and other related traits. The percentage of variation explained by these QTLs ranged from 4.2% to 57.2%. A large number of traits had significant variation associated with the major dwarfing QTL DWJ (identified previously by BSA), confirming the influence of this gene on tree architecture. To identify the genes responsible for the dwarfing effect, a microarray analysis on RNA extracted from bark tissues was performed to detect genes differentially expressed among dwarfing and vigorous rootstocks derived from the 'M.9' x 'R5' family. Following the mapping of 16 markers developed from 12 candidate genes, their position was compared with those of the QTLs identified previously and co­ localisations among genes and QTLs were identified. Results to date indicate that none of these particular CGs co-segregate with DW1. In order to estimate the number of different genetic sources of dwarfing present in commercial rootstocks, two SSR markers mapping about 0.5 cM away from the dwarfing QTL DWJ were screened over 58 rootstock accessions. The majority of the dwarf and semi-dwarf accessions screened carried the locus DWl, indicating that there may be only a single genetic source of dwarfing in apple rootstocks. The identification of markers for dwarfing will have a major impact on apple rootstock breeding, which currently relies on laborious phenotyping of individuals in breeding populations that presently takes at least 5-7 years to adequately perform. The identification of the genetic function of DWJ would provide an opportunity to develop dwarfing rootstocks for other members of the Rosaceae family for which such rootstocks have not yet been developed.
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    An investigation into the mode of action of alar (succinic acid, 2, 2-dimethyl hydrazide) on apples : a thesis ... for the degree of Master of Agricultural Science.
    (Massey University, 1968) Hopping, Murray Edwin
    Application of the growth retardant succinic acid 2,2-dimethyl hydrazide as a foliar spray to seven year old Gravenstein apple trees at full bloom and eleven days after, reduced extension growth in comparison to that on untreated trees. This retardation was characterized by a reduction in internode length and node number without formative effects on leaf area or observable leaf chlorophyll. Shoot and petiole dry weight was decreased with Alar treatment; leaf dry weight increased. Fruit size and weight was increased at 1000 and 2000 ppm Alar treatment; the converse relationship occurred at 4000 ppm Alar. Alar treatment improved apple keeping quality and enhanced fruit skin colouration. Acidic gibberellin-like substances extracted from shoot apices decreased with Alar treatment and this reduction was accompanied by an increase in 'abnormal' gibberellin-like substances. Acidic, neutral, and basic auxins extracted from shoot apices also decreased with Alar treatment although evidence was not as conclusive as that shown by gibberellin-like substances. A study of the interaction between Alar, auxin (IAA) and gibberellin (GA 3) on Avena 1st internode sections was used as the basis for a suggested mode of action of Alar on apple extension growth.
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    Molecular analysis of plant innate immunity triggered by secreted effectors from bacterial and fungal pathogens of apple : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, Institute of Agriculture and Environment, Massey University, New Zealand
    (Massey University, 2017) Prokchorchik, Maxim
    In comparison to animals, plants do not have a dedicated immune system with mobile immune cells to protect themselves. Instead they rely on the innate immunity of each cell. Plant immunity branches into two classical layers: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI detects the conserved molecular patterns (PAMPs) associated with pathogens and often can be overcome by pathogens translocating effector molecules into plant cells to inhibit the PTI. ETI, in turn, relies on intracellular receptors that can specifically recognize effectors or their activity and activate a rapid and robust response. The research presented in this thesis is focused on two pathogens of apple plants: the bacterial pathogen Erwinia amylovora (the causal agent of fire blight) and fungal pathogen Venturia inaequalis (the causal agent of apple scab disease). As both bacterial and fungal pathogens deliver effector molecules in order to promote their virulence, ETI engineering is a promising universal strategy to control these pathogens. In Chapter 3, the main aim was to elucidate the requirements and precise mechanism of how an important effector of E. amylovora, AvrRpt2, is recognized by the MR5 disease resistance (R) protein, derived from a hybrid apple Malus x robusta 5. I identified that a fragment of the guardee apple protein RIN4 was required and sufficient and required for MR5 activation. I further identified crucial amino acid residues responsible for this activation. Interestingly, cognate residues in RIN4 guardee homolog from Arabidopsis thaliana are responsible for suppression of the autoactivity of R protein RPS2. These findings led to the proposal of a novel hypothesis for evolutionary guardee adaption to the pool of R proteins present in plants. In Chapter 4, the main focus was to apply newly acquired whole-genome sequencing data of V. inaequalis for identifying the previously mapped AvrRvi8 effector, as well as several novel effectors predicted in silico. The sequences of these effectors were validated by amplification and resequencing of candidate genes from V. inaequalis cDNA. Further functional analysis of the selected gene candidates was performed. In addition, a library of constructs for generating V. inaequalis knock-out strains was prepared for future work. The findings from this thesis expected to be useful for breeders of apple to battle two economically important pathogens devastating the industry. Deployment of the MR5 system in apples should facilitate fire blight resistance in pipfruit and offers the opportunity for further engineering of MR5 to detect other pathogens. Furthermore, the effector library developed for V. inaequalis offers a novel tool for studying both virulence and avirulence mechanisms present in the applescab pathosystem. It is envisaged that further effector research will elucidate authentic targets critical for resistance development in apple.
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    Differentiating apple sports by pollen ultrastructure : a thesis presented in partial fulfilment of the requirements for the degree of Master of Horticultural Science at Massey University
    (Massey University, 1995) Currie, Alastair John
    Cultivars are plants that form distinct, uniform and stable phenotypes. New cultivars can be protected by Plant Variety Rights (PVR) which allow the owner exclusive rights to the propagation and sale of the plant material. Current PVR identification methods for apple cultivars require detailed records of tree, flower and fruit characteristics to differentiate the new cultivars from known cultivars. This method is slow, expensive and unable to cope with the increasing numbers of sports. Biochemical identification methods such as isozymes, restriction fragment length polymerisation (RFLP), random amplified polymorphism DNAs (RAPD), and minisatellite probes, can quickly and objectively differentiate cultivars, but cannot differentiate apple sports. Previous research suggested that pollen ultrastructure could be an alternative method for plant identification. This thesis is concerned with the development of a technique to differentiate apple sports using pollen exine patterns. Scanning electron microscopy was used to capture images of the apple pollen grain and the exine surface. A digital image analysis algorithm was developed to extract quantitative data from the pollen grain dimensions and pore characteristics, and a Fast Fourier transform extracted quantitative data from the ridge patterns. Statistical methods were applied to the data to differentiate the sports. Pollen harvested from apple flowers in the spring were wider than pollen harvested from flowers forced out of season under artificial conditions. Significant differences between trees were found for pollen grain length:width ratio, percent pore coverage, pore area and pore length but further research is required. However, apple cultivars types 'Red Delicious' and 'Gala' were successfully differentiated by pore and pollen grain variables, and 'Aversang' and 'Ultrared' sports of 'Red Delicious', and 'Splenola' and 'Galalea' sports Of 'Gala' were successfully differentiated by exine ridge patterns and pollen grain measurements. Differentiation of apple sports by pollen requires further development but may be one of the only quick, objective identification methods that can differentiate sports. Sport differentiation would greatly aid PVR establishment and enforcement.