Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

On-line MRI sequences for the evaluation of apple internal quality.

On-line dynamic MRI, which is oriented to industrial grading lines, requires high-speed sequences with motion correction artefacts. In this study two different types of motion correction sequences have been used and have been implemented in real-time (FLASH and UFLARE). They are based on T2* and T2 respectively and their selection depends on the expected contrast effect of the disorder: while watercore enhances bright areas due to higher fluid mobility, internal breakdown potentiates low signal due to texture degradation. For watercore study, five different apple cultivars were used (Normanda-18-, Fuji-35-, Helada-36-, Verde Doncella-54-, Esperiega-75-) along two seasons (2011 and 2012). In total 218 fruits were measured under both, static conditions (20 slices per fruit) and under dynamic conditions (3 repetitions without slice selection). For internal breakdown, Braeburn cultivar has been studied (in total 106 fruits) under both static (20 slices per fruit) and dynamic conditions (3 replicates with slice selection). Metrological aspects such as repeatability of dynamic images and subsequent histogram feature stability become of major interest for further industrial application. Segregation ability among varying degrees of disorder is also analyzed.

The fruiting habit of the apple tree.

Thesis (M. S. in Agr.) - Cornell Univ., June 1918.

Touches on agriculture : including a treatise on the preservation of the apple tree. Together with family recipes, experiments on insects, &c. /

Signatures: [1]⁴ 2-8⁴

Development and productivity of apple tree cv. MaxiGala on different planting depth.

2016

Evaluation of the productive and vegetative aspects of the cultivar 'Imperial Gala' apple tree with EM-9 interstem in different lengths.

The obtaining of a compact plant, with less vigor and high productivity, equivalent to a conventional plant, constitutes a strong tendency in the current horticulture, aiming at a raising of the fruit production at the same planted area. One of the techniques that have had success nowadays is the interstem use. This study was developed in a commercial orchard of Randon Agro Silvo Pastoril S.A. (RASIP), located in the Rio Grande do Sul state, Brazil. The purpose of this work was to evaluate the vegetative and productive development of apple trees of 'Imperial Gala' with different lengths of EM-9 interstem. The treatments consisted of five interstem lengths: 10, 15, 20, 25, 30 cm. In the seventh year of implantation the following parameters were evaluated: the height of the plant, the diameter of the 'Imperial Gala' 5 cm above the second graft point, the volume of the tree-head (height, width and length), the number of bud per branch, and the number of fruits per lineal centimeter of branch. Through this study it could be concluded that the greater interstem (30 cm) presented better indices with relation of vigor control. However, the number of fruits per lineal centimeter of branch with the interstem of 10 cm offered only significant superiority, when compared with the interstem of 30 cm. Using interstem technique allows to gather the benefits of the rootstock 'Marubakaido' and to control excessive vigour with the interstem EM-9.

Avaluació de la pèrdua de qualitat de la poma “Fuji” deguda al dany produït pel cop de sol

Estudi de l’afectació del cop de sol a les pomes de varietat fuji, sobretot tenint en compte que l’aspecte visual té una repercussió econòmica molt important

Sensory and instrumental profiling of apples: a new tool for quality assessment

Food suppliers currently measure apple quality considering basic pomological descriptors. Sensory analysis is expensive, does not permit to analyse many samples, and cannot be implemented for measuring quality properties in real time. However, sensory analysis is the best way to precisely describe food eating quality, since it is able to define, measure, and explain what is really perceivable by human senses and using a language that closely reflects the consumers’ perception. On the basis of such observations, we developed a detailed protocol for apple sensory profiling by descriptive sensory analysis and instrumental measurements. The collected sensory data were validated by applying rigorous scientific criteria for sensory analysis. The method was then applied for studying sensory properties of apples and their changes in relation to different pre- and post-harvest factors affecting fruit quality, and demonstrated to be able to discriminate fruit varieties and to highlight differences in terms of sensory properties. The instrumental measurements confirmed such results. Moreover, the correlation between sensory and instrumental data was studied, and a new effective approach was defined for the reliable prediction of sensory properties by instrumental characterisation. It is therefore possible to propose the application of this sensory-instrumental tool to all the stakeholders involved in apple production and marketing, to have a reliable description of apple fruit quality.

Pasteurization of Apple Cider with UV Irradiation

In a period of increasing concern about food safety, food poisoning outbreaks where unpasterurized apple cider or apple juice was found contaminated with Escherichia coli 0157:H7 reinforces the need for using the best technologies in apple cider production. Most apple cider is sold as an unpasteurized raw product. Because of their acidity, it was believed that juice products do not usually contain microorganisms such as E. coli 0157:H7, Salmonella, and Crytosporidium. Yet all of these foodborne pathogens are capable of being transmitted in unpasteurized juices. It is known that these pathogens can survive for several weeks in a variety of acidic juices. Although heat pasteurization is probably the best method to eliminate these pathogens, it is not the most desirable method as it changes sensory properties and also is very costly for small to mid-sized apple cider processors. Pasteurization of apple cider with Ultraviolet Irradiation (UV) is a potential alternative to heat pasteurization. Germicidal W irradiation is effective in inactivating microorganisms without producing undesirable by-products and changing sensory properties. Unpasteurized raw apple cider from a small local processor was purchased for this study. The effects of physical parameters, exposure time and dosage on the W treatment efficacy were examined as well as the effects of the UV light on apple cider quality. W light with principal energy at a wavelength of 254.7 nm, was effective in reducing bacteria (E .coli, ATCC 25922) inoculated apple cider. The W dosage absorbed by the apple cider was mathematically calculated. A radiation dose of 8,777 μW-s/cm2 reduced bacteria an average of 2.20 logs and in multiple passes, the FDA mandated 5-log reduction was achieved. Sensory analysis showed there was no significant difference between the W treated and non-treated cider. Experiments with W treated apple cider indicated a significant (p < 0.01) extension of product shelf life through inhibition of yeast and mold growth. The extension of the researched performed is applicable to other fruit juice processing operations.

Application of NMR techniques for the evaluation of fruit internal quality and physical properties of food

Los alimentos son sistemas complejos, formados por diversas estructuras a diferentes escalas: macroscópica y microscópica. Muchas propiedades de los alimentos, que son importantes para su procesamiento, calidad y tratamiento postcosecha, están relacionados con su microestructura. La presente tesis doctoral propone una metodología completa para la determinación de la estructura de alimentos desde un punto de vista multi-escala, basándose en métodos de Resonancia Magnética Nuclear (NMR). Las técnicas de NMR son no invasivas y no destructivas y permiten el estudio tanto de macro- como de microestructura. Se han utilizado distintos procedimientos de NMR dependiendo del nivel que se desea estudiar. Para el nivel macroestructural, la Imagen de Resonancia Magnética (MRI) ha resultado ser muy útil para la caracterización de alimentos. Para el estudio microestructural, la MRI requiere altos tiempos de adquisición, lo que hace muy difícil la transferencia de esta técnica a aplicaciones en industria. Por tanto, la optimización de procedimientos de NMR basados en secuencias relaxometría 2D T1/T2 ha resultado ser una estrategia primordial en esta tesis. Estos protocolos de NMR se han implementado satisfactoriamente por primera vez en alto campo magnético. Se ha caracterizado la microestructura de productos alimentarios enteros por primera vez utilizando este tipo de protocolos. Como muestras, se han utilizado dos tipos de productos: modelos de alimentos y alimentos reales (manzanas). Además, como primer paso para su posterior implementación en la industria agroalimentaria, se ha mejorado una línea transportadora, especialmente diseñada para trabajar bajo condiciones de NMR en trabajos anteriores del grupo LPF-TAGRALIA. Se han estudiado y seleccionado las secuencias más rápidas y óptimas para la detección de dos tipos de desórdenes internos en manzanas: vitrescencia y roturas internas. La corrección de las imágenes en movimiento se realiza en tiempo real. Asimismo, se han utilizado protocolos de visión artificial para la clasificación automática de manzanas potencialmente afectadas por vitrescencia. El presente documento está dividido en diferentes capítulos: el Capítulo 2 explica los antecedentes de la presente tesis y el marco del proyecto en el que se ha desarrollado. El Capítulo 3 recoge el estado del arte. El Capítulo 4 establece los objetivos de esta tesis doctoral. Los resultados se dividen en cinco sub-secciones (dentro del Capítulo 5) que corresponden con los trabajos publicados bien en revistas revisadas por pares, bien en congresos internacionales o bien como capítulos de libros revisados por pares. La Sección 5.1. es un estudio del desarrollo de la vitrescencia en manzanas mediante MRI y lo relaciona con la posición de la fruta dentro de la copa del árbol. La Sección 5.2 presenta un trabajo sobre macro- y microestructura en modelos de alimentos. La Sección 5.3 es un artículo en revisión en una revista revisada por pares, en el que se hace un estudio microestrcutural no destructivo mediante relaxometría 2D T1/T2. la Sección 5.4, hace una comparación entre manzanas afectadas por vitrescencia mediante dos técnicas: tomografía de rayos X e MRI, en manzana. Por último, en la Sección 5.5 se muestra un trabajo en el que se hace un estudio de secuencias de MRI en línea para la evaluación de calidad interna en manzanas. Los siguientes capítulos ofrecen una discusión y conclusiones (Capítulo 6 y 7 respectivamente) de todos los capítulos de esta tesis doctoral. Finalmente, se han añadido tres apéndices: el primero con una introducción de los principios básicos de resonancia magnética nuclear (NMR) y en los otros dos, se presentan sendos estudios sobre el efecto de las fibras en la rehidratación de cereales de desayuno extrusionados, mediante diversas técnicas. Ambos trabajos se presentaron en un congreso internacional. Los resultados más relevantes de la presente tesis doctoral, se pueden dividir en tres grandes bloques: resultados sobre macroestructura, resultados sobre microestructura y resultados sobre MRI en línea. Resultados sobre macroestructura: - La imagen de resonancia magnética (MRI) se aplicó satisfactoriamente para la caracterización de macroestructura. En particular, la reconstrucción 3D de imágenes de resonancia magnética permitió identificar y caracterizar dos tipos distintos de vitrescencia en manzanas: central y radial, que se caracterizan por el porcentaje de daño y la conectividad (número de Euler). - La MRI proveía un mejor contraste para manzanas afectadas por vitrescencia que las imágenes de tomografía de rayos X (X-Ray CT), como se pudo verificar en muestras idénticas de manzana. Además, el tiempo de adquisición de la tomografía de rayos X fue alrededor de 12 veces mayor (25 minutos) que la adquisición de las imágenes de resonancia magnética (2 minutos 2 segundos). Resultados sobre microestructura: - Para el estudio de microestructura (nivel subcelular) se utilizaron con éxito secuencias de relaxometría 2D T1/T2. Estas secuencias se usaron por primera vez en alto campo y sobre piezas de alimento completo, convirtiéndose en una forma no destructiva de llevar a cabo estudios de microestructura. - El uso de MRI junto con relaxometría 2D T1/T2 permite realizar estudios multiescala en alimentos de forma no destructiva. Resultados sobre MRI en línea: - El uso de imagen de resonancia magnética en línea fue factible para la identificación de dos tipos de desórdenes internos en manzanas: vitrescencia y podredumbre interna. Las secuencias de imagen tipo FLASH resultaron adecuadas para la identificación en línea de vitrescencia en manzanas. Se realizó sin selección de corte, debido a que la vitrescencia puede desarrollarse en cualquier punto del volumen de la manzana. Se consiguió reducir el tiempo de adquisición, de modo que se llegaron a adquirir 1.3 frutos por segundos (758 ms por fruto). Las secuencias de imagen tipo UFLARE fueron adecuadas para la detección en línea de la podredumbre interna en manzanas. En este caso, se utilizó selección de corte, ya que se trata de un desorden que se suele localizar en la parte central del volumen de la manzana. Se consiguió reducir el tiempo de adquisicón hasta 0.67 frutos por segundo (1475 ms por fruto). En ambos casos (FLASH y UFLARE) fueron necesarios algoritmos para la corrección del movimiento de las imágenes en tiempo real. ABSTRACT Food is a complex system formed by several structures at different scales: macroscopic and microscopic. Many properties of foods that are relevant to process engineering or quality and postharvest treatments are related to their microstructure. This Ph.D Thesis proposes a complete methodology for food structure determination, in a multiscale way, based on the Nuclear Magnetic Resonance (NMR) phenomenon since NMR techniques are non-invasive and non-destructive, and allow both, macro- and micro-structure study. Different NMR procedures are used depending on the structure level under study. For the macrostructure level, Magnetic Resonance Imaging (MRI) revealed its usefulness for food characterization. For microstructure insight, MRI required high acquisition times, which is a hindrance for transference to industry applications. Therefore, optimization of NMR procedures based on T1/T2 relaxometry sequences was a key strategy in this Thesis. These NMR relaxometry protocols, are successfully implemented in high magnetic field. Microstructure of entire food products have been characterized for the first time using these protocols. Two different types of food products have been studied: food models and actual food (apples). Furthermore, as a first step for the food industry implementation, a grading line system, specially designed for working under NMR conditions in previous works of the LPF-TAGRALIA group, is improved. The study and selection of the most suitable rapid sequence to detect two different types of disorders in apples (watercore and internal breakdown) is performed and the real time image motion correction is applied. In addition, artificial vision protocols for the automatic classification of apples potentially affected by watercore are applied. This document is divided into seven different chapters: Chapter 2 explains the thesis background and the framework of the project in which it has been worked. Chapter 3 comprises the state of the art. Chapter 4 establishes de objectives of this Ph.D thesis. The results are divided into five different sections (in Chapter 5) that correspond to published peered reviewed works. Section 5.1 assesses the watercore development in apples with MRI and studies the effect of fruit location in the canopy. Section 5.2 is an MRI and 2D relaxometry study for macro- and microstructure assessment in food models. Section 5.3 is a non-destructive microstructural study using 2D T1/T2 relaxometry on watercore affected apples. Section 5.4 makes a comparison of X-ray CT and MRI on watercore disorder of different apple cultivars. Section 5.5, that is a study of online MRI sequences for the evaluation of apple internal quality. The subsequent chapters offer a general discussion and conclusions (Chapter 6 and Chapter 7 respectively) of all the works performed in the frame of this Ph.D thesis (two peer reviewed journals, one book chapter and one international congress).Finally, three appendices are included in which an introduction to NMR principles is offered and two published proceedings regarding the effect of fiber on the rehydration of extruded breakfast cereal are displayed. The most relevant results can be summarized into three sections: results on macrostructure, results on microstructure and results on on-line MRI. Results on macrostructure: - MRI was successfully used for macrostructure characterization. Indeed, 3D reconstruction of MRI in apples allows to identify two different types of watercore (radial and block), which are characterized by the percentage of damage and the connectivity (Euler number). - MRI provides better contrast for watercore than X-Ray CT as verified on identical samples. Furthermore, X-Ray CT images acquisition time was around 12 times higher (25 minutes) than MRI acquisition time (2 minutes 2 seconds). Results on microstructure: - 2D T1/T2 relaxometry were successfully applied for microstructure (subcellular level) characterization. 2D T1/T2 relaxometry sequences have been applied for the first time on high field for entire food pieces, being a non-destructive way to achieve microstructure study. - The use of MRI together with 2D T1/T2 relaxometry sequences allows a non-destructive multiscale study of food. Results on on-line MRI: - The use of on-line MRI was successful for the identification of two different internal disorders in apples: watercore and internal breakdown. FLASH imaging was a suitable technique for the on-line detection of watercore disorder in apples, with no slice selection, since watercore is a physiological disorder that may be developed anywhere in the apple volume. 1.3 fruits were imaged per second (768 ms per fruit). UFLARE imaging is a suitable sequence for the on-line detection of internal breakdown disorder in apples. Slice selection was used, as internal breakdown is usually located in the central slice of the apple volume. 0.67 fruits were imaged per second (1475 ms per fruit). In both cases (FLASH and UFLARE) motion correction was performed in real time, during the acquisition of the images.

Advances on apple production under semiarid climate: N fertigation

The fertilizing management for apple tree is essential, especially for nitrogen, one of the most important nutrients affecting fruit yield. Thus, an experiment was conducted in 2012 and 2013 to evaluate the fruit production, yield and leaf chlorophyll of ?Princesa? and ?Eva? apples as a function of nitrogen fertigation under Brazilian semiarid conditions. The experimental design consisted of randomised blocks, with treatments distributed in a factorial arrangement 2 x 4, corresponding to apple cultivars (Eva and Princesa); and nitrogen doses (160, 120, 80 and 40 kg of N ha-1), with four replications and three plants. Calcium nitrate was used as nitrogen source (15.5% of N) with applications twice a week during 40 days, reaching 12 fertilizing performances through irrigation water. The following variables were evaluated: i) fruit production per plant (kg plant-1); ii) fruit yield (t ha-1); iii) number of fruits per plant; iv) leaf chlorophyll meter readings (index); and v) leaf nitrogen concentration (g kg-1). Princesa apple cultivar if compared to ?Eva? presents a better fruit production performance under Brazilian semiarid. Furthermore, nitrogen doses fertilized through irrigation water have no effect on fruit production of Eva and Princesa apple cultivars during the first production cycle.

The effect of different training systems on vegetative and productive performance of apple trees cultivar 'maxigala' - in southern Brazil.

2016

Nuorten männiköiden kehitys - taimikonhoito ja ensiharvennus

The aim of this study was to investigate the effects of location, site type, regeneration method and precommercial thinning on the characteristics and development of young, even-aged, pure Scots pine stands. In addition, the effects of timing and intensity of first commercial thinning on the yield and profitability during the rotation period were also studied. The stand characteristics and external quality of young Scots pine stands and stand-level growth models were based on extensive inventory data of the Finnish Forest Research Institute for young Scots pine stands (3 measurement times, 192 stands). The effect of precommercial thinning on stand development was examined on the basis of long-term experiments (13 stands, 169 plots). The effect of timing and intensity of the first commercial thinning on yield and profitability were based on measurements made in first commercial thinnings (27 stands of Metsähallitus), and the further stand development was modeled using the MOTTI simulator. The thesis was based on four articles and a summary. Stand level growth models were developed for young, even-aged Scots pine stands. The models reliably predicted the development up until the first commercial thinning stage. The stand density of young Scots pine stands in Finland was moderately low compared to the target values. In addition, the external quality of pines was low on average. The low stand density and poor external quality will result in the need for quality tree selection in thinnings, if high quality sawn timber is required. In Northern Finland, only 20% of the dominant trees were classified as normal. This will lead to the situation where external quality will remain relatively poor up until the end of rotation. Early and light precommercial thinning (Hdom 3 m, to a density of 3000 trees per hectare) increased the thinning removal by 40% compared to late and more intensive precommercial thinning (at 7 meters to a density of 2000 trees per hectare). A model for the effect of precommercial thinning on merchantable thinning removal at the first commercial thinning was developed for forest management planning purposes. When the recommended time of first commercial thinning was delayed from a dominant height of 12 m to 16 m, or by ten years, the yield of merchantable wood was doubled. Simultaneously, the current value of the stumpage revenues (with 4% interest rate) was increased on the average by 65% (330 € per hectare). Variation in stumpage prices or interest rates did not have any effect on the final results. Without exception, delaying the first commercial thinning by ten years seemed to be the most profitable method. This presupposes that precommercial thinning has been carried out at the right time and that tree quality aspects do not be specially considered. Furthermore, the wood yield and economic outcome from the entire rotation were similar regardless of whether the first thinning was performed at the time currently recommended or ten years later.

黄土高原沟壑区苹果园土壤重金属含量特征研究

以黄土高原沟壑区的苹果园为研究对象,对6~36 a苹果园土壤重金属含量状况进行研究,结果发现,该区苹果园的高投入种植管理模式,能够影响重金属在土壤中的迁移与富集,使土壤重金属含量发生明显变化。土壤Cu含量随树龄增加而增加,20 a以上的土壤-果树系统对土壤Cu的输入与输出趋于平衡,Cu含量变化不大,且耕层土壤Cu含量较高。Cr含量随树龄线性递增,36 a果园0~20 cm,20~40 cm和40~60 cm土层Cr含量分别比6 a果园增加27.14%,17.09%和19.17%。Cd含量随树龄增加先增加后减少,长期大量施用磷肥是土壤Cd的主要来源,果园生态系统深层土壤Cd含量的峰值比耕层提前出现。Pb含量以15~26 a果园含量最高,树龄<15 a和>26 a时Pb含量较低。Hg含量则以15 a为转折点,在不同土层上呈现出不同的变化趋势。As含量在树龄<15 a时逐渐降低,15~20 a时逐渐增加,20 a以后果园土壤As含量趋于不变,且各土层之间差异不显著。

Gwaith Madog Dwygraig

Edwards, Huw, Gwaith Madog Dwygraig (Canolfan Uwchefrydiau Cymreig a Cheltaidd Prifysgol Cymru, 2007)