Orange pectin mediated growth and stability of aqueous gold and silver nanocolloids

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Survival of Mycobacterium bovis on Feedstuffs Commonly Used as Supplemental Feed for White-tailed Deer (Odocoileus virginianus)

Mycobacterium bovis, the causative agent of bovine tuberculosis, has become established in free-ranging white-tailed deer Odocoileus virginianus in northeastern Michigan. The practice of supplemental feeding of white-tailed deer during the winter is believed to contribute to transmission of M. bovis between deer. The current study was conducted to determine the ability of M. bovis to survive on various feedstuffs commonly used as supplemental feed for deer in northeast Michigan (i.e., apples, corn, carrots, sugar beets, potatoes, and hay) and the effect of maintenance at 220 C, 8 C, and 23 C on survival. Mycobacterium bovis survived on all feedstuffs at all temperatures tested for at least 7 days. At 23 C, M. bovis could still be isolated from samples of apples, corn and potatoes at 112 days. This study suggests that contamination of feedstuffs by M. bovis-infected deer could act as a source of indirect transmission between deer because M. bovis is able to survive in temperatures similar to those recorded during winter months in northeastern Michigan. Current efforts to ban or control supplemental feeding of deer should have a positive effect on decreasing transmission of M. bovis among deer.

Genotypic variation in Actinidia deliciosa fruit size and carbohydrate content

In a global and increasingly competitive fresh produce market, more attention is being given to fruit quality traits and consumer satisfaction. Kiwifruit occupies a niche position in the worldwide market, when compared to apples, oranges or bananas. It is a fruit with extraordinarily good nutritional traits, and its benefits to human health have been widely described. Until recently, international trade in kiwifruit was restricted to a single cultivar, but different types of kiwifruit are now becoming available in the market. Effective programmes of kiwifruit improvement start by considering the requirements of consumers, and recent surveys indicate that sweeter fruit with better flavour are generally preferred. There is a strong correlation between at-harvest dry matter and starch content, and soluble solid concentration and flavour when fruit are eating ripe. This suggests that carbon accumulation strongly influences the development of kiwifruit taste. The overall aim of the present study was to determine what factors affect carbon accumulation during Actinidia deliciosa berry development. One way of doing this is by comparing kiwifruit genotypes that differ greatly in their ability to accumulate dry matter in their fruit. Starch is the major component of dry matter content. It was hypothesized that genotypes were different in sink strength. Sink strength, by definition, is the effect of sink size and sink activity. Chapter 1 reviews fruit growth, kiwifruit growth and development and carbon metabolism. Chapter 2 describes the materials and methods used. Chapter 3, 4, 5 and 6 describes different types of experimental work. Chapter 7 contains the final discussions and the conclusions Three Actinidia deliciosa breeding populations were analysed in detail to confirm that observed differences in dry matter content were genetically determined. Fruit of the different genotypes differed in dry matter content mainly because of differences in starch concentrations and dry weight accumulation rates, irrespective of fruit size. More detailed experiments were therefore carried out on genotypes which varied most in fruit starch concentrations to determine why sink strengths were so different. The kiwifruit berry comprises three tissues which differ in dry matter content. It was initially hypothesised that observed differences in starch content could be due to a larger proportion of one or other of these tissues, for example, of the central core which is highest in dry matter content. The study results showed that this was not the case. Sink size, intended as cell number or cell size, was then investigated. The outer pericarp makes up about 60% of berry weight in ‘Hayward’ kiwifruit. The outer pericarp contains two types of parenchyma cells: large cells with low starch concentration, and small cells with high starch concentration. Large cell, small cell and total cell densities in the outer pericarp were shown to be not correlated with either dry matter content or fruit size but further investigation of volume proportion among cell types seemed justified. It was then shown that genotypes with fruit having higher dry matter contents also had a higher proportion of small cells. However, the higher proportion of small cell volume could only explain half of the observed differences in starch content. So, sink activity, intended as sucrose to starch metabolism, was investigated. In transiently starch storing sinks, such as tomato fruit and potato tubers, a pivotal role in carbon metabolism has been attributed to sucrose cleaving enzymes (mainly sucrose synthase and cell wall invertase) and to ADP-glucose pyrophosphorylase (the committed step in starch synthesis). Studies on tomato and potato genotypes differing in starch content or in final fruit soluble solid concentrations have demonstrated a strong link with either sucrose synthase or ADP-glucose pyrophosphorylase, at both enzyme activity and gene expression levels, depending on the case. Little is known about sucrose cleaving enzyme and ADP-glucose pyrophosphorylase isoforms. The HortResearch Actinidia EST database was then screened to identify sequences putatively encoding for sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoforms and specific primers were designed. Sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoform transcript levels were anlayzed throughout fruit development of a selection of four genotypes (two high dry matter and two low dry matter). High dry matter genotypes showed higher amounts of sucrose synthase transcripts (SUS1, SUS2 or both) and higher ADP-glucose pyrophosphorylase (AGPL4, large subunit 4) gene expression, mainly early in fruit development. SUS1- like gene expression has been linked with starch biosynthesis in several crop (tomato, potato and maize). An enhancement of its transcript level early in fruit development of high dry matter genotypes means that more activated glucose (UDP-glucose) is available for starch synthesis. This can be then correlated to the higher starch observed since soon after the onset of net starch accumulation. The higher expression level of AGPL4 observed in high dry matter genotypes suggests an involvement of this subunit in drive carbon flux into starch. Changes in both enzymes (SUSY and AGPse) are then responsible of higher starch concentrations. Low dry matter genotypes showed generally higher vacuolar invertase gene expression (and also enzyme activity), early in fruit development. This alternative cleavage strategy can possibly contribute to energy loss, in that invertases’ products are not adenylated, and further reactions and transport are needed to convert carbon into starch. Although these elements match well with observed differences in starch contents, other factors could be involved in carbon metabolism control. From the microarray experiment, in fact, several kinases and transcription factors have been found to be differentially expressed. Sink strength is known to be modified by application of regulators. In ‘Hayward’ kiwifruit, the synthetic cytokinin CPPU (N-(2-Chloro-4-Pyridyl)-N-Phenylurea) promotes a dramatic increase in fruit size, whereas dry matter content decreases. The behaviour of CPPU-treated ‘Hayward’ kiwifruit was similar to that of fruit from low dry matter genotypes: dry matter and starch concentrations were lower. However, the CPPU effect was strongly source limited, whereas in genotype variation it was not. Moreover, CPPU-treated fruit gene expression (at sucrose cleavage and AGPase levels) was similar to that in high dry matter genotypes. It was therefore concluded that CPPU promotes both sink size and sink activity, but at different “speeds” and this ends in the observed decrease in dry matter content and starch concentration. The lower “speed” in sink activity is probably due to a differential partitioning of activated glucose between starch storage and cell wall synthesis to sustain cell expansion. Starch is the main carbohydrate accumulated in growing Actinidia deliciosa fruit. Results obtained in the present study suggest that sucrose synthase and AGPase enzymes contribute to sucrose to starch conversion, and differences in their gene expression levels, mainly early in fruit development, strongly affect the rate at which starch is therefore accumulated. This results are interesting in that starch and Actinidia deliciosa fruit quality are tightly connected.

Innovative treatments for shelf-life extension of whole and fresh-cut fruit

Maintaining the postharvest quality of whole and fresh-cut fruit during storage and distribution is the major challenge facing fruit industry. For this purpose, industry adopt a wide range of technologies to enable extended shelf-life. Many factors can lead to loss of quality in fresh product, hence the common description of these products as ‘perishable’. As a consequence normal factors such as transpiration and respiration lead ultimately to water loss and senescence of the product. Fruits and vegetables are living commodities and their rate of respiration is of key importance to maintenance of quality. It has been commonly observed that the greater the respiration rate of a product, the shorter the shelf-life. The principal problem for fresh-cut fruit industries is the relative shorter shelf-life of minimally processed fruit (MPF) compared to intact product. This fact is strictly connected with the higher ethylene production of fruit tissue stimulated during fresh-cut processing (peeling, cutting, dipping). 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene action and several researches have shown its effectiveness on the inhibition of ripening and senescence incidence for intact fruit and consequently on their shelf-life extension. More recently 1-MCP treatment has been tested also for shelf-life extension of MPF but discordant results have been obtained. Considering that in some countries 1-MCP is already a commercial product registered for the use on a number of horticultural products, the main aim of this actual study was to enhance our understanding on the effects of 1-MCP treatment on the quality maintenance of whole and fresh-cut climacteric and non-climacteric fruit (apple, kiwifruit and pineapple). Concerning the effects of 1-MCP on whole fruit, was investigated the effects of a semi-commercial postharvest treatment with 1-MCP on the quality of Pink Lady apples as functions of fruit ripening stage, 1-MCP dose, storage time and also in combination with controlled atmospheres storage in order to better understand what is the relationship among these parameters and if is possible to maximize the 1-MCP treatment to meet the market/consumer needs and then in order to put in the market excellent fruit. To achieve this purpose an incomplete three-level three-factor design was adopted. During the storage were monitored several quality parameters: firmness, ripening index, ethylene and carbon dioxide production and were also performed a sensory evaluations after 6 month of storage. In this study the higher retention of firmness (at the end of storage) was achieved by applying the greatest 1-MCP concentration to fruits with the lowest maturity stage. This finding means that in these semi-commercial conditions we may considerate completely blocked the fruit softening. 1-MCP was able to delay also the ethylene and CO2 production and the maturity parameters (soluble solids content and total acidity). Only in some cases 1-MCP generate a synergistic effect with the CA storage. The results of sensory analyses indicated that, the 1-MCP treatment did not affect the sweetness and whole fruit flavour while had a little effect on the decreasing cut fruit flavour. On the contrary the treated apple was more sour, crisp, firm and juicy. The effects of some treatment (dipping and MAP) on the nutrient stability were also investigated showing that in this case study the adopted treatments did not have drastic effects on the antioxidant compounds on the contrary the dipping may enhance the total antioxidant activity by the accumulation of ascorbic acid on the apple cut surface. Results concerning the effects of 1-MCP in combination with MAP on the quality parameters behaviour of the kiwifruit were not always consistent and clear: in terms of colour maintenance, it seemed to have a synergistic effect with N2O MAP; as far as ripening index is concerned, 1-MCP had a preservative effect, but just for sample packed in air.

Genomic and transcriptional analysis of allergen genes in apple (Malus x domestica)

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Quali-quantitative study of the phenolic and polyphenolic compounds and their antioxidant capacity in vegetal matrix processed with different technologies

Food technologies today mean reducing agricultural food waste, improvement of food security, enhancement of food sensory properties, enlargement of food market and food economies. Food technologists must be high-skilled technicians with good scientific knowledge of food hygiene, food chemistry, industrial technologies and food engineering, sensory evaluation experience and analytical chemistry. Their role is to apply the modern vision of science in the field of human nutrition, rising up knowledge in food science. The present PhD project starts with the aim of studying and improving frozen fruits quality. Freezing process in very powerful in preserve initial raw material characteristics, but pre-treatment before the freezing process are necessary to improve quality, in particular to improve texture and enzymatic activity of frozen foods. Osmotic Dehydration (OD) and Vacuum Impregnation (VI), are useful techniques to modify fruits and vegetables composition and prepare them to freezing process. These techniques permit to introduce cryo-protective agent into the food matrices, without significant changes of the original structure, but cause a slight leaching of important intrinsic compounds. Phenolic and polyphenolic compounds for example in apples and nectarines treated with hypertonic solutions are slightly decreased, but the effect of concentration due to water removal driven out from the osmotic gradient, cause a final content of phenolic compounds similar to that of the raw material. In many experiment, a very important change in fruit composition regard the aroma profile. This occur in strawberries osmo-dehydrated under vacuum condition or under atmospheric pressure condition. The increment of some volatiles, probably due to fermentative metabolism induced by the osmotic stress of hypertonic treatment, induce a sensory profile modification of frozen fruits, that in some way result in a better acceptability of consumer, that prefer treated frozen fruits to untreated frozen fruits. Among different processes used, a very interesting result was obtained with the application of a osmotic pre-treatment driven out at refrigerated temperature for long time. The final quality of frozen strawberries was very high and a peculiar increment of phenolic profile was detected. This interesting phenomenon was probably due to induction of phenolic biological synthesis (for example as reaction to osmotic stress), or to hydrolysis of polymeric phenolic compounds. Aside this investigation in the cryo-stabilization and dehydrofreezing of fruits, deeper investigation in VI techniques were carried out, as studies of changes in vacuum impregnated prickly pear texture, and in use of VI and ultrasound (US) in aroma enrichment of fruit pieces. Moreover, to develop sensory evaluation tools and analytical chemistry determination (of volatiles and phenolic compounds), some researches were bring off and published in these fields. Specifically dealing with off-flavour development during storage of boiled potato, and capillary zonal electrophoresis (CZE) and high performance liquid chromatography (HPLC) determination of phenolic compounds.

Morphological and physiological responses of apple trees under photoselective colored nets

In recent years and thanks to innovative technological advances in supplemental lighting sources and photo-selective filters, light quality manipulation (i.e. spectral composition of sunlight) have demonstrated positive effects on plant performance in ornamentals and vegetable crops. However, this aspect has been much less studied in fruit trees due to the difficulty of conditioning the light environment of orchards. The aim of the present PhD research was to study the use of different colored nets with selective light transmission in the blue (400 – 500 nm), red (600 – 700 nm) and near infrared (700 – 1100 nm) wavelengths as a tool to the light quality management and its morphological and physiological effects in field-grown apple trees. Chapter I provides a review the current status on physiological and technological advances on light quality management in fruit trees. Chapter II shows the main effect of colored nets on morpho-anatomical (stomata density, mesophyll structure and leaf mass area index) characteristics in apple leaves. Chapter III provides an analysis about the effect of micro-environmental conditions under colored nets on leaf stomatal conductance and leaf photosynthetic capacity. Chapter IV describes a study approach to evaluate the impact of colored nets on fruit growth potential in apples. Summing up results obtained in the present PhD dissertation clearly demonstrate that light quality management through photo-selective colored nets presents an interesting potential for the manipulation of plant morphological and physiological traits in apple trees. Cover orchards with colored nets might be and alternative technology to address many of the most important challenges of modern fruit growing, such as: the need for the efficient use of natural resources (water, soil and nutrients) the reduction of environmental impacts and the mitigation of possible negative effects of global climate change.

Populationsdynamik des Cydia pomonella Granulovirus

Das Cydia pomonella Granulovirus (CpGV, Fam. Baculoviridae) ist ein sehr virulentes und hoch spezifisches Pathogen des Apfelwicklers (Cydia pomonella), das seit mehreren Jahren in der Bundesrepublik Deutschland und anderen Ländern der EU als Insektizid zugelassen ist. Wie andere Baculoviren auch befällt es die Larven der Insekten und ist aufgrund seiner Selektivität für Nicht-Zielorganismen unbedenklich. In der Vergangenheit konzentrierte sich die Erforschung des CpGV auf Bereiche, die für die Anwendung im Pflanzenschutz relevant waren, wobei nach fast 20 Jahren nach der ersten Zulassung noch immer nicht bekannt ist, ob und wie sich das CpGV in der Umwelt etablieren kann. Im Rahmen der vorliegenden Arbeit wurden verschiedene Parameter, mit deren Hilfe die Populationsdynamik des CpGV beschrieben werden kann, analysiert und quantitativ bestimmt. Neben den biologischen Eigenschaften wie Virulenz, DNA-Charakterisierung und Quantifizierung der Virusnachkommenschaft wurden insbesondere die horizontale sowie die vertikale Transmission, die Inaktivierung und die Infektion später Larvenstadien untersucht. Letztlich wurden die ermittelten Parameter zusammen mit Daten aus der Literatur in ein mathematisches Modell integriert. Um die Wahrscheinlichkeit der horizontalen Transmission zu quantifizieren, wurde ein Modellsystem mit losen Äpfeln etabliert, in dem verschiedene Szenarien möglicher horizontaler Transmission unter definierten Laborbedingungen getestet wurden. In Versuchsserien, in denen ein Virusfleck, entsprechend der produzierten Virusmenge einer Eilarve, auf einen Apfel appliziert worden war, war unter den aufgesetzten Apfelwicklerlarven lediglich eine sehr geringe Mortalität von 3 - 6% zu beobachten. Wurde jedoch ein an einer Virusinfektion gestorbener Larvenkadaver als Inokulum verwendet, lag die Mortalitätsrate aufgesetzter Larven bei über 40%. Diese beobachtete hohe horizontale Transmissionsrate konnte mit dem Verhalten der Larven erklärt werden. Die Larven zeigten eine deutliche Einbohrpräferenz für den Stielansatz bzw. den Kelch, wodurch die Wahrscheinlichkeit des Zusammentreffens einer an der Infektion verendeten Larve mit einer gesunden Larve um ein Vielfaches zunahm. In einem ähnlich angelegten Freilandversuch konnte eine horizontale Transmission nicht belegt werden. Der Unterschied zur Kontrollgruppe fiel aufgrund einer hohen natürlichen Mortalität und einer damit einhergehenden niedrigen Dichte der Larven zu gering aus. Parallel hierzu wurde außerdem eine Halbwertszeit von 52 Sonnenstunden für das CpGV ermittelt. Weiterhin konnte festgestellt werden, dass die Mortalität von späteren Larvenstadien, die 14 Tage Zeit hatten sich in die Äpfel einzubohren, bevor eine CpGV-Applikation durchgeführt wurde, ebenso hoch war wie bei Larven, die sich im L1-Stadium auf der Apfeloberfläche infizierten. Aufgrund des höheren Alters jener Larven war der Fraßschaden an befallenen Äpfeln jedoch wesentlich größer und vergleichbar mit dem Fraßschaden einer unbehandelten Kontrolle. Der Versuch zur vertikalen Transmission zeigte dass, obwohl die verwendete Apfelwicklerzucht nicht frei von CpGV war, die Mortalitätsrate der Nachkommen subletal infizierter Weibchen (44%) jedoch deutlich höher war als die der Nachkommen subletal infizierter Männchen (28%) und der unbehandelten Kontrolle (27%). Auch in den PCR-Analysen konnte eine größere Menge an CpGV-Trägern bei den Nachkommen subletal infizierter Weibchen (67%) als bei den Nachkommen subletal infizierter Männchen (49%) und bei der Kontrolle (42%) nachgewiesen werden. Die Ergebnisse deuten darauf hin, dass eine Infektion durch subletal infizierte Weibchen vertikal in die nächste Generation übertragen werden kann. Dies lässt erkennen, dass in der Folgegeneration des Apfelwicklers eine zusätzliche Wirkung des CpGV durch vertikale Transmission auftreten kann. Hierin wäre auch ein potentieller Mechanismus für eine dauerhafte Etablierung des Virus zu sehen. Letztlich wurden alle Parameter, die die CpGV-Apfelwickler-Beziehung beschreiben, in ein mathematisches Modell GRANULO integriert. Nach einer Sensitivitätsanalyse wurde GRANULO teilweise mit Daten aus den Freilandversuchen verifiziert. Durch Modifikation der Virusparameter im Modell konnte anschließend der Einfluss veränderter biologischer Eigenschaften (UV-Stabilität und Transmissionsraten) der Viren in Simulationen theoretisch erprobt werden. Das beschriebene Modell, das allerdings noch einer weitergehenden Verifizierung und Validierung bedarf, ist eine erste Annäherung an die quantitative Erfassung und Modellierung der Populationsdynamik des Systems CpGV-Apfelwickler. Die im Zusammenhang mit der Populationsdynamik des Apfelwicklers erhobenen Daten können einen wertvollen Beitrag zur Optimierung von Kontrollstrategien des Apfelwicklers mittels CpGV leisten. Außerdem geben sie Aufschluss über die Etablierungsmöglichkeiten dieses Bioinsektizids.

(De)colonization through topophilia: Marjorie Kinnan Rawlings’s life and work in Florida

(De)colonization Through Topophilia: Marjorie Kinnan Rawlings’s Life and Work in Florida attempts to reveal the author’s intimate connection to and mental growth through her place, namely the Cross Creek environs, and its subsequent effect on her writing. In 1928, Marjorie Kinnan Rawlings and her first husband Charles Rawlings came to Cross Creek, Florida. They bought the shabby farmhouse on Cross Creek Road, trying to be both, writers and farmers. However, while Charles Rawlings was unable to write in the backwoods of the Florida Interior, Rawlings found her literary voice and entered a symbiotic, reciprocal relationship with the natural world of the Cracker frontier. Her biographical preconditions – a childhood spent in the rural area of Rock Creek, outside of Washington D. C. - and a father who had instilled in her a sense of place or topophilia, enabled her to overcome severe marriage tensions and the hostile climate women writers faced during the Depression era. Nature as a helping ally and as an “undomesticated”(1) space/place is a recurrent motif throughout most of Rawlings’s Florida literature. At a time when writing the American landscape/documentary and the extraction of the self from texts was the prevalent literary genre, Marjorie Kinnan Rawlings inscribed herself into her texts. However, she knew that the American public was not yet ready for a ‘feminist revolt’, but was receptive of the longtime ‘inaudible’ voices from America’s regions, especially with regard to urban poverty and a homeward yearning during the Depression years. Fusing with the dynamic eco-consciousness of her Cracker friends and neighbors, Rawlings wrote in the literary category of regionalism enabling her to pursue three of her major aims: an individuated self, a self that assimilated with the ‘master narratives’ of her time and the recognition of the Florida Cracker and Scrub region. The first part of this dissertation briefly introduces the largely unknown and underestimated writer Marjorie Kinnan Rawlings, providing background information on her younger years, the relationship toward her family and other influential persons in her life. Furthermore, it takes a closer look at the literary category of regionalism and Rawlings’s use of ‘place’ in her writings. The second part is concerned with the ‘region’ itself, the state of Florida. It focuses on the natural peculiarities of the state’s Interior, the scrub and hammock land around her Cracker hamlet as well as the unique culture of the Florida Cracker. Part IV is concerned with the analysis of her four Florida books. The author is still widely related to the ever-popular novel The Yearling (1938). South Moon Under (1933) and Golden Apples (1935), her first two novels, have not been frequently republished and have subsequently fallen into oblivion. Cross Creek (1942), Rawlings’s last Florida book, however, has recently gained renewed popularity through its use in classes on nature writers and the non-fiction essay but it requires and is here re-evaluated as the author’s (relational) autobiography. The analysis through place is brought to completion in this work and seems to intentionally close the circle of Rawlings’s Florida writings. It exemplifies once more that detachment from place is impossible for Rawlings and that the intermingling of life and place in literature, is essential for the (re)creation of her identity. Cross Creek is therefore not only one of Rawlings’s greatest achievements; it is more importantly the key to understanding the author’s self and her fiction. Through the ‘natural’ interrelationship of place and self and by looking “mutually outward and inward,”(2) Marjorie Kinnan Rawlings finds her literary voice, a home and ‘a room of her own’ in which to write and come to consciousness. Her Florida literature is not only product but also medium and process in her assessment of her identity and self. _____________ (1) Alaimo, Stacy. Undomesticated Ground: Recasting Nature as Feminist Space (Ithaca: Cornell UP, 2000) 23. (2) Libby, Brooke. “Nature Writing as Refuge: Autobiography in the Natural World” Reading Under the Sign of Nature. New Essays in Ecocriticism. Ed. John Tallmadge and Henry Harrington. (Salt Lake City: The U of Utah P, 2000) 200.

Carbon fluxes and allocation pattern in an apple orchard

Carbon fluxes and allocation pattern, and their relationship with the main environmental and physiological parameters, were studied in an apple orchard for one year (2010). I combined three widely used methods: eddy covariance, soil respiration and biometric measurements, and I applied a measurement protocol allowing a cross-check between C fluxes estimated using different methods. I attributed NPP components to standing biomass increment, detritus cycle and lateral export. The influence of environmental and physiological parameters on NEE, GPP and Reco was analyzed with a multiple regression model approach. I found that both NEP and GPP of the apple orchard were of similar magnitude to those of forests growing in similar climate conditions, while large differences occurred in the allocation pattern and in the fate of produced biomass. Apple production accounted for 49% of annual NPP, organic material (leaves, fine root litter, pruned wood and early fruit drop) contributing to detritus cycle was 46%, and only 5% went to standing biomass increment. The carbon use efficiency (CUE), with an annual average of 0.68 ± 0.10, was higher than the previously suggested constant values of 0.47-0.50. Light and leaf area index had the strongest influence on both NEE and GPP. On a diurnal basis, NEE and GPP reached their peak approximately at noon, while they appeared to be limited by high values of VPD and air temperature in the afternoon. The proposed models can be used to explain and simulate current relations between carbon fluxes and environmental parameters at daily and yearly time scale. On average, the annual NEP balanced the carbon annually exported with the harvested apples. These data support the hypothesis of a minimal or null impact of the apple orchard ecosystem on net C emission to the atmosphere.

Phenotype and genotype characterization of Monilinia spp. isolates and preformed antifungal compounds in peach peel fruit at different developmental stages

The brown rot fungi belong to a group of fungal pathogens that causes considerable damage to cultivated fruits trees, particularly stone fruits and apples in the temperate regions of the World and during the postharvest with an important economic impact. In particular in Italy, it is important to monitor the Monilinia population to control economic losses associated to the peach and nectarine market. This motivates the research steps presented in this dissertation on Monilinia Italian isolates. The Monilinia species collected from stone fruits have been identified using molecular analysis based on specific primers. The relevant role of M. fructicola was confirmed and, for the first time, it was found also on apple fruits. To avoid the development of resistant strains and implement valid treatment strategies, the understanding of the fruit natural resistance during different developmental stages and the assessment of the Monilinia sensitivity/resistance to fungicides are required. The relationship between the inhibition spots and the phenolic compounds in peach fruit peel was highlighted in this research. Three methods were used to assess isolate resistance/sensitivity, the amended medium, the Spiral Gradient Endpoint Method (SGD) and the Alamar Blue method. The PCR was used to find possible mutation points in the b-tubulin gene that is responsible for fungicide resistance. Interestingly, no mutation points were observed in resistant M. laxa isolates, suggesting that the resistance could be stimulated by environmental factors. This lead to the study of the effect of the temperature on the resistance and the preliminary results of in vitro tests showed that maximum inhibition was observed at 30°C.

Molecular strategies for genetic diversity analysis and development of markers linked to resistance traits in apple

The goal of many plant scientists’ research is to explain natural phenotypic variation in term of simple changes in DNA sequence. DNA-based molecular markers are extensively used for the construction of genome-wide molecular maps and to perform genetic analysis for simple and complex traits. The PhD thesis was divided into two main research lines according to the different approaches adopted. The first research line is to analyze the genetic diversity in an Italian apple germplasm collection for the identification of markers tightly linked to targeted genes by an association genetic method. This made it possible to identify synomym and homonym accessions and triploids. The fruit red skin color trait has been used to test the reliability of the genetic approaches in this species. The second line is related to the development of molecular markers closely linked to the Rvi13 and Rvi5 scab resistance genes, previously mapped on apple’s chromosome 10 and 17 respectively by using the traditional linkage mapping method. Both region have been fine-mapped with various type of markers that could be used for marker-assisted selection in future breeding programs and to isolate the two resistance genes.

Use of essential oils and biocontrol cultures for the improvement of shelf-life of fresh cut products

The demand of minimally processed fruits and vegetables has increased in the last years. However, their intrinsic characteristics may favor the growth of pathogens and spoilage microbiota. The negative effects on human health reported for some traditional chemical sanitizers have justified the search for substitutes to guarantee food safety and quality. In this work we have evaluate the potential of some essential oils and their components to improve the safety and the shelf life of Lamb’s lettuce (Valerianella locusta) and apples (Golden delicious). Moreover, the effects of selected lactic acid bacteria alone or in combination with essential oils or their components, on the shelf-life and safety as well as organoleptic properties of minimally processed products, were evaluated. Since the lack of knowledge of microbial cell targets of essential oils represent one of the most important limit to the use of these molecules at industrial level, another aim of this thesis was the study of the action mechanisms of essential oils and their components. The results obtained showed the beneficial effects of the natural antimicrobials as well as the selected lactic acid bacteria on minimally processed fruit and vegetable safety and shelf-life, without detrimental effects on the quality parameters. The beneficial effects obtained by the use of the selected biocontrol agents were further increased combining them with selected natural antimicrobials. The natural antimicrobial employed induced noticeable modifications of membrane fatty acid profiles and volatile compounds produced by microbial cells during the growth. The modification of the expression in genes involved in fatty acid biosynthesis suggesting that the cytoplasmic membrane of microbial cells is one of the major cellular target of essential oils and their components. The comprehension of microbial stress response mechanisms can contribute to the scaling up of natural antimicrobials and bio-control agents at industrial level.

Atmospheric plasma processes for microbial inactivation: food applications and stress response in Listeria monocytogenes

This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes.

The examination and identification of bite marks in foods using 3D scanning and 3D comparison methods

Bite mark analysis offers the opportunity to identify the biter based on the individual characteristics of the dentitions. Normally, the main focus is on analysing bite mark injuries on human bodies, but also, bite marks in food may play an important role in the forensic investigation of a crime. This study presents a comparison of simulated bite marks in different kinds of food with the dentitions of the presumed biter. Bite marks were produced by six adults in slices of buttered bread, apples, different kinds of Swiss chocolate and Swiss cheese. The time-lapse influence of the bite mark in food, under room temperature conditions, was also examined. For the documentation of the bite marks and the dentitions of the biters, 3D optical surface scanning technology was used. The comparison was performed using two different software packages: the ATOS modelling and analysing software and the 3D studio max animation software. The ATOS software enables an automatic computation of the deviation between the two meshes. In the present study, the bite marks and the dentitions were compared, as well as the meshes of each bite mark which were recorded in the different stages of time lapse. In the 3D studio max software, the act of biting was animated to compare the dentitions with the bite mark. The examined food recorded the individual characteristics of the dentitions very well. In all cases, the biter could be identified, and the dentitions of the other presumed biters could be excluded. The influence of the time lapse on the food depends on the kind of food and is shown on the diagrams. However, the identification of the biter could still be performed after a period of time, based on the recorded individual characteristics of the dentitions.