WRINKLED 1(WRI1), a member of AP2/EREBP class of transcription factors regulates carbon allocation between glycolytic and fatty acid biosynthetic pathway. Additionally, among the four WRI1 paralogs in arabidopsis, WRI3 and 4 but not WRI2, are also able to increase fatty acid content in different tissues. While the role of WRI1 is well established in seeds, the potential or WRI1 or its paralogs as master regulators in oil-rich nonseed tissues is poorly understood. Recent transcriptome studies of avocado (Persea americana) mesocarp revealed that the ortholog of WRI2, along with WRI1 and WRI3 was highly expressed during oil accumulation. Through transient expression assays, we further demonstrated that both PaWRI1 and PaWRI2 can accumulate oil in tobacco leaves. We conducted a comprehensive and comparative in silico analysis of WRI paralogs from a dicot, monocot and a basal angiosperm to identify distinct features associated with function. These data provide insights into the possible evolutionary changes in WRI1 homologs and allow for identification of new targets to enhance oil biosynthesis in diverse tissues.

Plants synthesize and store oil, mostly as triacylglycerols (TAG), in seeds that is transcriptionally controlled by WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Additionally, two rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid diacylglycerol acyltransferase (PDAT) catalyze the terminal step in TAG assembly and contribute to oil accumulation. Avocado (Persea americana) mesocarp, a non-seed tissue, accumulates significant amounts of TAG (~70% by dry weight) enriched with heart-healthy oleic acid. The oil accumulation stages in mesocarp coincide with the high expression of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional implication of these genes is not explored. Using structural analyses, we showed that PaWRI2 is a relatively stable protein, has a single intact AP2 DNA-binding domain, and has different C-terminal properties compared to its ortholog in Arabidopsis. Through transient expression, we demonstrated that PaWRI2 is functional and drives TAG accumulation in Nicotiana benthamiana leaves, unlike Arabidopsis WRI2. Additionally, co-infiltration of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1 further increased the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of >46 fatty acid biosynthetic pathway genes revealed that several were distinctly up- or down-regulated by the expression of PaWRI2 and PaWRI1. Further yeast-one-hybrid assay showed a unique characteristic of PaWRI2 being autoregulated and by PaWRI1. Also, both the proteins could bind to AW-box promoter elements in specific avocado genes. Deletion of the C-terminally-located ordered region in both the proteins further improved the lipid content with an altered composition in the leaf tissue. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in non-seed tissues. This study provides a functional role for WRI2 in a basal angiosperm species that is likely lost in modern angiosperms and thus provides basis for mechanistic differences in the transcriptional regulation of lipid biosynthesis among different plant species and between seed and non-seed tissues.

Thesis (M. Sc. (Horticulture)) -- University of Limpopo, 2018 / ‘Hass’ avocado fruit changes skin colour from green to purple and then black during ripening. However, markets importing South African avocado fruit have been complaining about the ‘Hass’ skin colour not changing to purple/black during ripening. Thus, the study aimed to investigate the effect of tree girdling, harvest time and ripening temperature on ‘Hass’ avocado fruit skin colour development during ripening. The mature ‘Hass’ avocado fruit were harvested from girdled and non-girdled trees during early (April), mid- (May) and late (June) harvest times. Upon arrival, in the laboratory fruit were cold stored at 5.5°C for 28 days. After storage, fruit were ripened at 25, 21 and 16°C for 8, 6 and 4 days, respectively. After withdrawal from clod storage fruit were evaluated for skin colour development, ripening and physiological disorders (chilling injury). Fruit from girdled trees showed high maturity (low moisture content) when compared with fruit from non-girdled trees during early and mid-harvest. With respect to skin colour development, the results indicate that skin eye colour development of fruit from girdled and non-girdled trees minimally increased from emerald green (1) to olive green (3) across all harvest times, ripening temperature and ripening duration. However, late season fruit from non-girdled trees improved to purple (4) when ripened at 21°C when compared with fruit from girdled trees. In terms of objective colour, lightness, hue angle and chroma decreased for fruit from girdled and non-girdled trees, across all harvest times, ripening temperature and ripening duration. Lightness and hue angle of fruit from girdled trees were slightly reduced when compared with fruit from non-girdled trees, throughout all harvest times, ripening temperature and duration. Early and mid-season fruit harvested from girdled trees showed rapid decrease of chroma when compared with fruit from non-girdled trees, throughout ripening temperature and x duration. In terms of softening, fruit from girdled trees showed higher firmness loss and ripening percentage within 6 (16°C) and 4 (21 and 25°C) days when compared with fruit from non-girdled trees during early and mid-harvest, whereas, late harvest fruit from girdled trees reached higher ripening percentage and firmness loss within 4 days throughout ripening temperatures. With respect to cold damage, late harvested fruit from girdled trees showed higher external chilling injury when compared with non-girdled trees, throughout ripening temperature. In general, girdling treatment improved fruit maturity, ripening rate and firmness loss. However, the incidence of variable skin colouring of ‘Hass’ avocado fruit during ripening was also prevalent in early harvested fruit from girdled tree, irrespective of ripening temperature. Keywords: girdling, harvest time, physiological disorder, ripening temperature, variable colouring

Thesis (M.Sc. (Horticulture)) -- University of Limpopo, 2022 / The South African Avocado Industry has recently announced plans to expand exports into new markets, such as the United States (US). As a requirement for these markets, fruit of high quality must be stored at ultra-low temperature to mitigate phytosanitary risks. However, ‘Hass’ avocado fruit are susceptible to chilling injury when stored at temperatures below 3°C. Moreover, CI development resulted in uneven ripening and disease infestation due to damaged cell membranes. Therefore, the objective of this study was to evaluate the potential of methyl jasmonate (MeJA) and salicylic acid (SA) on quality maintenance of 'Hass' avocado fruit during ultra-low cold storage. Matured ‘Hass’ avocado fruit were harvested at commercial dry matter (22%). The experiment was conducted using a completely randomized design (CRD) with eight replications per treatment. Treatment concentrations for methyl jasmonate (MeJA) were 0 (control), 10 and 100 μmol•L−1 , while those for salicylic acid (SA) were 0 (control), 1.0, 2.0 and 3.0 mM. After treatments, fruit were stored at 2°C for 31 days and thereafter, ripened at ambient temperature (±25°C) until fully ripe. During ripening, fruit were evaluated for weight loss, exocarp colour, firmness, chilling injury, as well as physiological (vascular browning) and pathological disorders (fruit rot). In this study, dipping fruit in MeJA solution significantly (P < 0.05) reduced ‘Hass’ avocado fruit firmness loss. Moreover, MeJA showed a significant effect (P < 0.05) on hue angle (h°) but did not significantly affect (P > 0.05) visual colour rating, chroma (C*), lightness (L*) and weight loss. The results showed that ‘Hass’ avocado fruit treated with 10 μmol•L−1 MeJA reduced weight loss when compared with 100 μmol•L−1 MeJA from day 2 to day 8 of ripening. Overall results showed a visual change in ‘Hass’ avocado fruit exocarp colour, with eye colour changing from rating 1 (emerald-green) to 3 (olive- green) for control and fruit treated with MeJA throughout the ripening days. Furthermore, MeJA reduced ‘Hass’ avocado fruit external chilling injury, physiological and pathological disorders. With respect to SA treatments, the result showed that dipping fruit at 1.0 and 2.0 mM SA had a significant effect (P < 0.05) on reducing firmness loss during ripening. Salicylic acid (1.0 mM) reduced and alleviated ‘Hass’ avocado fruit external chilling injury during ultra-low cold storage. Furthermore, result showed that 1.0 and 2.0 mM SA treatments had significant affect (P < 0.05) on firmness loss. Moreover, a significant effect was observed on visual colour and C* but did not affect (P > 0.05) L* and h°. Fruit treated with SA showed poor exocarp colour development with extended exposure to ultra-low cold storage, as a result, developed chilling symptoms. The treatment of ‘Hass’ avocado fruit with 1.0 mM SA inhibited the incidence of fruit rot and vascular browning when compared with control and fruit treated with 2.0 and 3.0 mM SA. In conclusion, 10 and 100 μmol•L−1 MeJA and 1.0, 2.0 and 3.0 mM SA effectively preserved ‘Hass’ avocado fruit quality during storage at ultra-low temperature. / Agricultural Sector Education Training Authority (AgriSeta) and National Research Foundation (NRF)

Thesis (M.Sc. Agriculture (Horticulture)) -- University of Limpopo, 2017 / Avocado ‘Hass’ fruit characteristically change skin colour from green to deep purple or black during ripening. However, there is an ongoing debate about the use of ‘Hass’ avocado fruit skin colour change as an indicator for ripening and whether pre- and post-harvest factors can alter this relationship. Thus, the aim of the study was to investigate the effect of harvest season, harvest time, ripening temperature and ripening days on ‘Hass’ avocado fruit skin colour change during ripening. The experiment was carried out as 2 x 3 x 3 x 5 factorial with three replicates. The experiment consisted of four treatment factors: 2 x harvest season (2014 and 2015), 3 x harvest time (May-early, June-mid and July-late), 3 x ripening temperature (16, 21 and 25°C) and 5 x ripening days (0, 2, 4, 6 and 8). Fruit were stored at industry recommended temperature of 5.5°C. After storage, fruit were ripened at 16, 21 and 25°C, therefore, evaluated at 0, 2, 4, 6 and 8 days for subjective and objective skin colour, fruit firmness, ripening percentage and chilling injury. Harvest season, harvest time, ripening temperature and ripening days had a significant effect (P<0.001) on ‘Hass’ avocado fruit skin subjective colour development during ripening. However, amongst the treatment factors; harvest time, ripening temperature and ripening days were the predominant factors in skin subjective colour development variation. Hence, late harvest fruit showed an improved skin colour development at higher temperature (25°C) at day 4 to ripening when compared with early and mid-harvest fruit. Moreover, skin lightness showed a decreasing trend during all harvest time and ripening temperature throughout days to ripening during 2014 and 2015 harvest season. Furthermore, ripening at higher temperature (25°C) resulted in rapid decrease on fruit firmness when compared with lower temperature (16°C), irrespective of harvest season and harvest time. In addition, mid-season fruit showed significantly higher chilling damage during the 2014 harvest season. In conclusion, the study showed that harvest season, harvest time, ripening temperature and ripening days factors had a significant influence on ‘Hass’ avocado fruit skin colour development, firmness and susceptibility to chilling injury. / Agricultural Sector Education Training Authority (AgriSeta) and Postharvest Innovation Programme (PHI)

Hábitos de vida saudáveis e uma dieta balanceada aliados ao alto consumo de frutas e vegetais, estão associados a redução do risco de doenças e à manutenção da saúde. O óleo de abacate possui em sua composição substâncias bioativas capazes de prevenir e controlar as dislipidemias. Como existem poucas pesquisas científicas avaliando o potencial deste óleo para consumo humano, o presente trabalho estudou os processos de extração e refino do óleo de abacate, bem como suas propriedades funcionais. Os resultados mostraram que os processos de extração e refino do óleo a partir da variedade Margarida são tecnicamente viáveis, o que o torna excelente matéria-prima para a indústria alimentícia. Além disso, possui um perfil de ácidos graxos muito semelhantes ao azeite de oliva, predominando em ambos o ácido oléico, que em conjunto com os esteróis vegetais e a vitamina E presentes, é capaz de influenciar positivamente o controle metabólico do colesterol, prevenindo ou retardando as doenças cardiovasculares. / Healthy life habits and an equilibrate diet, associated with a high fruit and vegetable intake, are joined with the prevention of diseases and health maintenance. The avocado oil has in these composition bioactives substances that can help in the prevention and control of hyperlipidemia. As there are a few scientific researches evaluating the oil potential for human consumption, the present proposal studied the extraction and refining process of avocado oil and its functional properties. The results have been showed that extraction and refining process of Margarida's variety are technically possible, became it an excellent raw material for food industry. Besides, the fatty acid composition of avocado oil are similar to olive oil, predominating in both, the oleic acid, that together of sterols and vitamin E presents, can to influence to metabolic control of cholesterol, preventing or delaying the cardiovascular disease.

The anthracnose pathogen, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., is a major cause of disease in the avocado industry, causing significant economic losses, and infects all cultivars. In South Africa, Fuerte and Hass varieties are the most widely grown. Identification of genes differentially expressed in avocado during infection with the fungus represents an important step towards understanding the plant’s defence responses and would assist in designing appropriate intervention strategies. In this study, 454 sequencing and analysis of the transcriptome of infected Fuerte avocado fruits were performed using the Roche 454 GS FLX Titanium platform. cDNA libraries enriched for differentially expressed genes were constructed from unharvested and harvested avocado fruit tissues collected after 1, 4 and 24 h post-infection and after 3, 4, 5 and 7 day post-infection, then sequenced.The expression profiles of the genes expressed were measured by a hierarchical clustering algorithm.Subsequently, quantitative real-time PCR was employed to measure the expression of some candidate resistance genes to anthracnose disease and to validate the sequencing results. The single sequencing run produced 215 781 reads from the transcriptome. A total of 70.6 MB of sequence data was generated and subjected to BLAST searches of which about 1500 genes encoding proteins predicted to function in signal transduction, transcriptional control, metabolism, defence, stress response, transportation processes and some genes with unknown functions were identified. The expression profiles studies showed that many expressed genes were either up or down regulated after infection in avocado fruits when compared to the uninfected sample. Salicylic acid and ethylene were identified to be involved in the signalling networks activated in avocado fruit during C. gloeosporioides infection. This study showed that avocado is able to respond to C. gloeosporioides infection by exhibiting a sophisticated molecular system for pathogen recognition and by activating structural and biochemical defence mechanisms.

Este proyecto de investigación contiene un diseño de un proceso de Gestión de Seguridad y Salud Ocupacional basado en la ISO 45001, propuesto para las MYPES productoras de palta Hass, con el objetivo de disminuir los riesgos presentes en sus actividades, lo cual es un problema relevante debido a los accidentes y enfermedades comunes que presentan. El primer capítulo está compuesto por un estado del arte en base a artículos científicos que se encuentran en un periodo de 5 años antes, además, cuenta con un marco teórico con temas relacionados a las MYPES en el Perú, asociatividad, gestión por procesos, productividad, y seguridad y salud ocupacional. En el segundo capítulo, se efectúa un diagnóstico de la situación actual de las MYPES productoras de palta hass, en donde se identificaron distintos problemas que originan una baja productividad en la producción de la palta hass. En el tercer capítulo, se plantea un diseño de un sistema de gestión de seguridad y salud ocupacional basada en la gestión por procesos, con la finalidad de fomentar una cultura de preventiva, la cual reduce los riesgos y mejora la productividad en el sector agrícola de palta Hass. En el capítulo cuatro, se presenta la validación tanto de los expertos como de los agricultores, con la finalidad de obtener recomendaciones y poder mejorar el diseño del proceso que se propone. Por último, en el capítulo 5 se presenta las conclusiones, recomendaciones y las futuras líneas de investigación acerca del modelo propuesto. / This research project contains a design of an Occupational Health and Safety Management process based on ISO 45001, proposed for the MYPES producing Hass avocado, with the objective of reducing the risks present in their activities, which is a relevant problem due to the accidents and common diseases they present. The first chapter is composed of a state of the art based on scientific articles that are found in a period of 5 years before, in addition, it has a theoretical framework with issues related to MYPES in Peru, associativity, process management, productivity , and occupational health and safety. In the second chapter, a diagnosis is made of the current situation of the MYPES producing hass avocado, where different problems were identified that cause low productivity in the production of hass avocado. In the third chapter, a design of an occupational health and safety management system based on process management is proposed, in order to foster a culture of prevention, which reduces risks and improves productivity in the agricultural sector of Hass avocado. In the fourth chapter, the validation of both experts and farmers is presented, in order to obtain recommendations and to improve the design of the proposed process. Finally, chapter 5 presents the conclusions, recommendations and future lines of research on the proposed model. / Tesis

Standoff detection of targets using volatiles is essential when considering substances that are hazardous or dangerous, or for which the presence or location is unknown. For many invasive biological threats, their presence is often not realized until they have begun visibly affecting and spreading through crops or forests. The fungus Raffaelea lauricola is a biothreat vectored by the invasive beetle Xyleborus glabratus, or redbay ambrosia beetle (RAB), whose presence in avocado groves is currently detectable by visual inspection. Once visually identified, the affected trees must be removed and destroyed to protect those remaining trees. However, if the fungus is identified via standoff volatile detection, there is anecdotal evidence that it can be treated with propiconazole and saved from progression to the fatal laurel wilt disease. As a result of the rapid spread of R. lauricola and the quick death of trees, early detection through standoff methods is essential. The only current method of pre-symptomatic identification is canine detection. Canines are sensitive and selective biological detectors that can trace odors to their source, despite the presence of a variety of background odors. The present research evaluated the volatile organic compounds (VOCs) of the laurel wilt disease and R. lauricola using headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Additionally, a new method for odor collection and presentation to trained detection canines was developed. Knowledge of the disease and standoff volatile detection capabilities are improved using this information.

The rural poor of Hazaribag suffer from vitamin A and iron deficiencies from meals containing rice (200-400g) and limited (25-50g) slow-cooked vegetables with potatoes. This causes undernourishment in 50% of children and 40% of women. The two main causes are lack of access to nutritious foods, and lack of knowledge about nutrition. Through extension, 24 women were taught and quantitatively assessed about growing avocado and drumstick trees, and proper nutrition. Participants were monitored for four months using persistent observation to qualitatively evaluate the adoption process of four behaviors (eating and feeding avocados to children; increasing consumption of drumstick leaves/pods; caring for grafted avocado trees, and planting avocado seeds) and their consequences. From the assessment of knowledge gained and retained, results indicate a statistically significant difference among all test scores, T1 and T2 (p = .001, t = -5.58), T1 and T3 (p = .001, t = -6.98), T2 and T3 (p = .038, t = -2.20), and T4 perceptions of knowledge before/after training (p = .001, t = -16.32). Qualitative studies show the women did adopt a majority of the behaviors, with 100% eating or feeding avocados to children; 60% eating more drumstick leaves/pods; 90% caring for avocado trees and 50% planting seeds. A number of consequences were associated with the adoption of the behaviors. Many of the women reported perceptions of improved health and expressed that children were falling sick less often due to the avocados and drumstick leaves/pods. The MUAC test showed that children who ate avocados weekly had increases in their mid-upper-arm circumference. And, people outside the study became interested in avocados because the participants shared their new knowledge. Most extension programs result in knowledge gains, but this study showed that with continued engagement and interaction, the participants were able to retain and gain additional knowledge about agriculture and proper nutrition. From the data collected, it is clear that the women learned and adopted the behaviors to improve their nutrition resulting in positive changes. Ultimately, there is a connection between extension, knowledge gains, and behavior adoption. This could be a powerful tool to address malnutrition in Hazaribag.