La elaboración del plan estratégico 2016-2018 tiene como objetivo determinar estrategias que permitan aprovechar las oportunidades del mercado y que mantengan el liderazgo que la compañía Apple Inc. tiene en la industria. Esto se logrará a través del incremento en ventas de los productos ya existentes, con funcionalidades mejoradas; así como con el lanzamiento de un nuevo producto que revolucione el mercado.

The influence of different concentrations of a film- forming antitranspirant (Wilt-Pruf ) on the transpiration rate of young apple trees (Ml06) was studied. One-year-old Mailing 106 trees were potted in 10.2 em (4 inch) metal pots. The plants were placed in a controlled environmental chamber during the study period. Sufficient reductions in water loss resulted with all concentrations of Wilt- Pruf with a 63 percent reduction at the highest concentration. Phytotoxicity was not observed though the higher concentrations imparted a sticky film to plants and they appeared to be lighter green at the end of the test. The effect of Wilt-Pruf on four-year old apple trees (Golden Delicious) was conducted in August 1977 at Utah State University Experimental Farm i n Farmington, Utah. The resistance to water vapor diffusion from the leaves was increased on both Wilt-Pruf treated irrigated and unirrigated trees, and water balance was improved, increasing leaf and stern water potential. The improved water balance of treated apple trees resulted in fruit size increase . The higher concentration of antitranspirant in both irrigated and unirrigated apple trees resulted in a higher leaf temperature than the irrigated control.

A comparative study of phytoseiid populations was made of two well- cultivated and regularly sprayed apple orchards with two unsprayed orchards in Cache Valley, northern Utah. Two phytoseiid species, Typhlodromus mcgregori Chant and T. occidentalis Nesbitt were observed on the apple leaves, under the bark, and occasionally in the litter and soil. Amblyseius cucumeris (Oudemans) occurred in the soil and litter and occasionally under bark. T. mcgregori was dominant in the unsprayed orchards, and T. occidentalis in the sprayed. Of the phytophagous mites, which served as food for the phytoseiids, the two-spotted mite, Tetranychus urticae Koch was dominant. Other phytophagous mites were the brown mite, Bryobia rubrioculus (Scheuten), the European red mite, Panonychus ulmi (Koch), and the McDaniel mite, Tetranychus mcdanieli McGregor. The study suggests T. mcgregori to be a non-specific, facultative predator of phytophagous mites. T. mcgregori was adversely affected by standard pesticide practices, but T. occiden talis survived in larger numbers. The phytoseiids seem well adjusted to the environment of the unsprayed orchards and to contribute to the low phytophagous mite populations in those orchards. In the sprayed orchards, the phytoseiids failed to control the high populations of phytophagous mites which developed. No statistical differences were found in the efficiencies of the mite brushing machine and Berlese funnels in removing either phytoseiid or phytophagous mites from apple leaves.

In the spring, apple orchards are susceptible to freeze damage. Various approaches to orchard protection have been used in the past. Overhead sprinkling for bloom delay has been effective in extending to a later period in the spring the freeze hardiness of apple buds. Thus, protection against a late spring freeze is obtained. Previous research has not been conducted to determine optimum sprinkling times in the spring, the most effective threshold temperature, the amount of water needed to provide adequate bud protection, and the daily length of sprinkling time. A 2-year investigation was conducted with the objective of obtaining basic information to help in system design and the operational aspects of overhead sprinkling. The experiment was designed to determine the effectiveness of a system sprinkling beginning at the end of rest and at Celsius growing degree hour accumulations relating to stages bud development. The evaluation of limited and unlimited water use and the relation to bloom delay was obtained. The two sprinkler types studied were umbrella and impact. On the umbrella sprinklers, four cycling times (1/2, 1/3, 1/4, and 1/6 time) and three threshold temperatures (7, 10, and 13 C) were tested. On the impact sprinkler, three nozzle sizes (4.0, 3.6, and 2.8 mm) were evaluated. The investigation answered many questions, the most important of which were: (1) when only limited bloom delay is needed, more freeze protection can be obtained by sprinkling in the early spring than in the late spring; (2) bloom delay can be regulated by terminating sprinkling at different stages of bud development; (3) to obtain maximum delay, sprinkling should begin at the end of rest and a low threshold temperature for sprinkling should be used for sprinkler initiation; (4) when limited delay is required, greater efficiency of water use can be obtained by lowering the threshold temperature and lowering the application rate.

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Apple is one of the most valuable fruit consumed worldwide due to its excellent quality features and high degree of acceptation by consumers. However, apples are susceptible to various postharvest disorders, leading to significant economic losses. To slow down food decay and provide constant fresh food to the market, postharvest strategies including decreasing the temperature can slows down the metabolism of fruits, but in parallel can also trigger the occurrence of typical chilling injury related disorders. Among all the physiological disorders, superficial scald is one of the most challenging, causing discoloration and skin browning triggering therefore consumers unacceptance. The development of superficial scald in apple fruit is a complex process influenced by various factors, including genetic background, storage conditions and preharvest management practices. Recent research partially disclosed the molecular mechanisms underlying the development of superficial scald in apple fruit. It has been shown that this disorder is associated with the accumulation of reactive oxygen species (ROS) and the oxidation of alfa-farnesene, a sesquiterpene largely considered as a major actor in the onset of this disorder. Various postharvest treatments have been used to control the development of superficial scald in apple fruit, such as including the application of the ethylene competitor 1-methylcyclopropene (1-MCP) or controlled atmosphere storage. This thesis aimed to identify and characterize the genetic factors involved in symptoms development and to disclose the regulation of this phenomenon. To this end, a comparative multidisciplinary study integrating transcriptomics and metabolomics analysis have been performed in a wide range of storage conditions and across different genetic background. The mechanism of action and effectiveness of two preventing strategies, such as the exogenous application of 1-MCP and storage at low oxygen concentration were assessed in two apple cultivars, ‘Granny Smith’ and ‘Ladina’. The metabolite and transcriptomic profiles revealed that 1-MCP treatment actively stimulated the production of unsaturated fatty acids such as oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3), thereby successfully preventing the development of superficial scald in ‘Granny Smith’. ‘Ladina’, on the other hand, was less responsive to these strategies and was characterized by a higher accumulation of very long chain fatty acids (VLCFAs), together with an increased level of fatty acid saturation. Additionally, the integration of the transcriptomic survey and VOCs assessment underlined that storage at low oxygen concentration stimulated the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase, and pyruvate decarboxylase, leading to the accumulation of ethanol and acetaldehyde in both cultivars. The expression of three members of the VII subgroups of ERF genes was also observed in ‘Granny Smith’, which coordinate the acclimation process to hypoxia in plants. The effect of controlled atmosphere in the prevention of superficial scald was furthermore investigated in fruit of ‘Granny Smith’ stored for different time and employing both a static and dynamic controlled atmosphere approach. The transcriptome analysis found that the duration of storage had an important effect on the coordination of gene expression, and the expression patterns of DEGs identified a distinct networks and types of transcriptomic hubs. Samples that developed superficial scald were characterized by higher concentration of chlorogenic acid and a time-specific increase in the expression of PAL and PPO genes. Prevention of superficial scald was related to a peculiar realigning episodes that involved the accumulation of specific antioxidant metabolites, very long chain fatty acids, and the expression of genes that coordinate the hypoxia acclimation process, such as RAP2-like and PCO. A complementary research was conducted to investigate the connection between the application of 1-MCP, low oxygen, and superficial scald. The study highlighted the possible application of genes involved in key fruit ripening pathways, including ethylene-related pathways and sugar/fermentation metabolism, as potential biomarkers for molecular characterization of the major changes that occur during fruit storage. The specific expression of polyphenol oxidase, along with sorbitol-6-phosphate dehydrogenase, were also used to investigate the onset of superficial scald in apples and correlated with the accumulation of the sugar alcohol sorbitol, which plays a protective role against chilling injuries. Assessing the transcriptional signature of these biomarkers can contribute to the development of new tools for a more informed understanding of the physiological progression of postharvest ripening in apples. Overall, these studies provide insights into the genetic and metabolic mechanisms underlying the development and prevention of superficial scald in apple cultivars during storage. The results obtained here paved the way for the identification of a core set of genes to be used as functional marker suitable to better comprehend this phenomenon and to select new accession characterized by a superior fruit quality and postharvest performance.

Dihydrochalcones (DHCs) are specialised metabolites with a limited natural distribution, found in significant amounts in Malus x domestica Borkh. (cultivated apple) and wild Malus species. Among them, M. x domestica accumulates significant amounts of phloridzin, whilst trilobatin and sieboldin are abundant in some wild relatives. DHCs have demonstrated a wide range of bioactive properties in biomedical models. Some DHCs have also been reported to act as flavour enhancers. Phloridzin may act as an anti-diabetic compound by blocking sodium-linked glucose transport and renal reabsorption of glucose in kidneys. Despite the protective effects reported in mammal models, little is known about how these metabolites are biosynthesised and what is their function in planta, where it has been hypothesised a role for phloridzin in plant growth. The biosynthetic pathway leading to DHC formation has been proposed in apple, and some steps have been characterised recently. DHC pathway diverts from the main phenylpropanoid pathway most probably from 4-coumaroyl-CoA by the action of a yet unknown reductase that would produce 4-dihydrocoumaroyl-CoA. Then, chalcone synthase (CHS) catalyses its condensation to form phloretin. Phloretin can be directly glycosylated at position 2′- or 4′ by the previously characterised 2′- and 4′-O-UDP-glycosyltransferases PGT1 and PGT2, to produce phloridzin or trilobatin, respectively. However, sieboldin has been postulated to derive from hydroxylation in position 3 of phloretin before been glycosylated, and the key responsible enzyme producing 3-hydroxyphloretin has not been yet discovered. The main aim of this PhD proposal was to provide a better understanding of the physiological functions of DHCs in apple, as well as to contribute to the elucidation of the biosynthetic pathway as the molecular basis for future genetic engineering in apple. Towards this aim, functional characterisation was conducted in MdPGT1 knockdown apple lines by RNAi silencing and CRISPR/Cas9 genome editing to assess the physiological effect of targeting a key biosynthetic gene involved in phloridzin biosynthesis. In addition, molecular, transcriptomic and metabolomic analyses were integrated to evaluate candidate genes accounting for 3-hydroxylase activity involved in DHC biosynthesis in wild Malus species accumulating sieboldin. Moreover, a de novo transcriptome assembly was carried out in an intergeneric hybrid between M. x domestica and Pyrus communis L. known to accumulate intermediate levels of DHCs compared to apple, in order to identify additional genes potentially involved in DHC pathway. We compared the physiological effect of reducing phloridzin through PGT1 knockdown by RNAi silencing and CRISPR/Cas9 genome editing. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology as reported in literature, whereas genome edited lines exhibited normal growth despite reduced foliar phloridzin. Bioactive brassinosteroids and gibberellins were found to be key players involved in the contrasting effects on growth observed following phloridzin reduction. Moreover, a cytochrome P450 from wild M. toringo (K. Koch) Carriere syn. sieboldii Rehder, and M. micromalus Makino was identified as dihydrochalcone 3-hydroxylase (DHCH), proving to produce 3-hydroxyphloretin and sieboldin in yeast. Different DHCH allele isoforms found in domesticated apple and M. toringo and M. micromalus correlated with sieboldin accumulation in a Malus germplasm collection. Finally, the assembled de novo transcriptome of the intergeneric apple/pear hybrid integrated to functional annotation and metabolomic analysis resulted in the identification of genes potentially involved in DHC biosynthesis, providing the basis for future biochemical characterisation. Altogether these results contribute to get insight into the roles of DHCs in apple and to illustrate how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple. Furthermore, the present PhD thesis contributes to the state-of-the-art by elucidating key missing steps in the biosynthesis of DHCs, which could be relevant for future establishment of genetic engineered lines that contribute to assess physiological effects of altering DHCs content, as well as to establish heterologous expression systems to produce de novo DHCs.

A 2 year study was conducted to investigate the influence of Atar on young apple trees. In 1996 concentrations of 0, 500, 1000, 2000, 4000, and 8000 ppm of Alar were applied to 'Golden Delicious', 'York Imperial' and 'Delicious' cultivars growing outside in gallon containers. / Ph. D.

The effect of controlled atmosphere (CA) storage, and antioxidant treatment on polyphenoloxidase (EC 1. 1418.1:PPO), peroxidase (EC11.11.1.7:POD), and superoxide dismutase (EC 1.15.1.1:S0D) activities and superficial scald and soft scald development in 'Virginiagold','Stayman', and 'Rome' apples (Malus domestica Borkh.) was investigated. 'Virginiagold' apples treated postharvest with an aqueous solution of diphenylamine (DPA) + 1,2, dihydro -6- ethoxy -2,2,4-trimethylquinoline (ethoxyquin) and stored in CA exhibited lower soft scald incidence and higher firmness than comparable fruits stored at O°C in air storage. Fruit firmness and titratable acids (TA) decreased continuously, while ethylene increased in storage. Titratable acids and firmness were positively correlated, while TA and ethylene and firmness and ethylene were negatively correlated. 'Stayman' and 'Rome' apples analyzed during storage showed increase in levels of λ±-famesene and its oxidation products conjugated trienes. During storage fruits showed an increase in the activities of polyphenoloxidase (PPO), peroxidase (POD), and superoxide dismutase (SOD), which reached a maximum when scald symptoms in 'Stayman' were observed. Our results indicate that 'Stayman' apples exhibited scald and 'Rome' did not, because 'Stayman' apples had a ten fold higher PPO activity than 'Rome.' / Master of Science

With post-bloom chemical fruit thinning responses being heavily influenced by tree carbohydrate reserves and weather conditions, there is a need for alternative thinning practices such as chemical blossom thinning in the Mid-Atlantic apple growing region. This project sought to 1) evaluate timing strategies for lime sulfur + stylet-oil blossom thinning sprays and 2) screen chemical agents for their suitability and efficacy as blossom thinners. In the first study, two 'Gala'/'M.9' blocks in different states (North Carolina and Virginia) were utilized in 2019 to compare between pollen tube growth model-guided blossom thinning sprays and those that are based on fixed time intervals between the initial application and subsequent thinning sprays. It was generally found that model-guided blossom thinning sprays and sprays applied at 20% open bloom and 48 hours after reduced fruit set, crop load, and improved fruit weight. In the second study, a 'Honeycrisp'/'B.9' and 'Cripps Pink'/'M.9' apple block in Virginia were used in 2018 and 2019 to evaluate multiple chemical agents with and without stylet-oil. Most of the treatments under-thinned compared to untreated control trees. However, it was determined that ammonium thiosulfate with and without stylet-oil was the most effective thinning agent. Potassium bicarbonate + stylet-oil was observed to cause excessive fruit russeting and phytotoxicity. This project demonstrated that optimum apple crop loads can be obtained if lime sulfur + stylet-oil blossom thinning sprays are applied at the proper time, and that multiple chemical agents offer potential use for chemical blossom thinning in the Mid-Atlantic region / Master of Science in Life Sciences / For apple trees to produce high quality fruit, a proportion of the flowers and/or fruit must be removed in a practice known as "thinning". Chemical blossom thinning is a relatively new method of thinning in the Mid-Atlantic apple growing region. This project sought to 1) evaluate spray timing of blossom thinning sprays and 2) evaluate different chemicals for their suitability in blossom thinning. In the first study, two 'Gala' apple orchards in two states (North Carolina and Virginia) were utilized in 2019 to compare a computer model-guided spray timing to structured spray timings based on the amount of time between the first and second sprays. It was found that the model-guided sprays, and sprays occurring once 20% of the blossoms had opened and reapplied 48 hours after, were the most effective in thinning the flowers. In the second part of the study, a 'Honeycrisp' orchard and a 'Cripps Pink' orchard in Virginia were used in 2018 and 2019 to evaluate different chemicals for their potential as blossom thinners. Unfortunately, most of the treatments did not achieve sufficient thinning results compared to untreated trees. However, it was determined that ammonium thiosulfate with and without stylet-oil was the most effective thinning chemical. Potassium bicarbonate with stylet-oil was found to cause excessive injury to the fruit and foliage. This project demonstrated that blossom thinning can be effective when the sprays are applied at the correct time and that multiple chemical agents offer potential for blossom thinning in the Mid-Atlantic.