Diversidade de maçãs (Malus Domestica Borkh.) nas feiras de produtores de Nova Iorque

Visitando feiras de produtores locais na cidade de Nova Iorque por um ano, entre junho de 2008 e maio de 2009, verificou-se significativa amplitude varietal das maçãs, demonstrando-se um aspecto particular da diversidade vegetal. Evidenciadas no verão pela presença volumosa, é no outono e inverno que se nota a diversidade intraespecífica, atravessando todos os períodos sazonais. Verificou-se a existência de mais de quatro centenas de espécies vegetais disponíveis no mercado, escolhendo-se a maçã como um dos tipos mais evidentes para a análise da diversidade, uma vez que se oferecem em 65 cultivares nas feiras do estado ao qual emprestam o apelido. Representam um tipo de diversidade que extrapola ao conceito de especialização, usualmente atribuído à agricultura dos países desenvolvidos, uma vez que convivem com outras espécies nos espaços em que são cultivadas. O objetivo deste artigo é demonstrar a diversidade genética e de usos das maçãs que se expressa na atividade comercial exercida nas ruas e praças nova iorquinas.

Estudo químico e das atividades citotóxica, antioxidante e antifúngica de Prunus myrtifolia L. (Urban.) (ROSACEAE)

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Diferença entre as espécies de Timbó (Derris Spp., Fabaceae) de diferentes regiões da Amazônia no controle da Musca domestica L.

The roots' powder of timbo species collected in different regions of Amazonia that were tested in larvae groupings, didn't produce differential significative effects in two strains of Musca domestica. The two species with the greater number of plants used in the trial were Derris urucu and Derris nicou; the individuals from the species came from regions considered as "forestal refugies" during the Amazonian pleistocene. Among each species the plants varied since that inefficient to control, until plants lethals to the fies. This differential capacity for larvae control among plants of the same species, originated from different regions, suggests that both species had their populations isolated, during the quaternary epoch. In regions or "forestal refugies", where both species were represented, D. urucu was superior to D. nicou in the capacity to control larvae. While among plants from F region (Peruvian-East refuge) of the State of Acre, the two species had convergence in the values of damage to larvae groupings. Among the another species, Derris sp. (yellow timbo or watermelon timbo) didn't show differences in larvae control between samples from the two regions; while the species that was introduced in the Amazonia Denis elleptica showed damage in the larvae groupings similar to the most effective plants of D. nicou and D. urucu.

Teores de rotenona em Clones de Timbó (Derris Spp. Fabaceae) de diferentes regiões da Amazônia e os seus efeitos na emergência de Imagos em Musca Domestica L

The rotenone contents analysis in clones of three timbó species permited the classification of these plants based on their efficiency to control Musca domestica larvae. The results showed significant correlations between the rotenone contents in the Derris urucu and D. nicou clones with the capacity to control the larvae. The plants with high rotenone contents were more effective. The rotenone contents, the effect of Derris species clones on the flics and the localization of the plants origin, showed that among the plants could have happened a populational isolation in the Amazonia during the pleistocene epoch. In Derris sp., that presented lower rotenone contents and was ineffective to control of the larvae, these differences were not observed.

Musca domestica as a host for mass rearing of parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae)

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Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 degrees C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila. (C) 2012 Elsevier Ltd. All rights reserved.

Role of reactive oxygen and nitrogen species in the response of Prunus spp. to bicarbonate

In calcareous soils, which are a large share of agricultural soils worldwide, iron availability is limited. Consequently, the whole plant physiology is affected, because of the key role of iron in redox metabolism, resulting in reduced crop yield and quality. Peach cultivation is economically important in northern Italy, and is easily subjected to iron chlorosis. The management of iron nutrition in peach includes grafting on bicarbonate-tolerant rootstocks; other forms of management may be expensive and environmentally impacting. Four genotypes, used as rootstocks for peach and characterized by different degrees of tolerance to chlorosis, were tested in vitro on optimal and bicarbonate-enriched medium. Their redox status and antioxidant responses were assayed; the production and possible roles of nitric oxide (NO) and related compounds were also studied. The most sensitive genotypes show a stronger reduction of the antioxidant enzymatic activities and an increased oxidative stress. A high production of NO was found to be associated to resistant genotypes, whereas sensitive genotypes reacted to stress by downregulating nitrosoglutathione reductase activity. Therefore, NO is proposed to improve the internal iron availability, or to stimulate iron intake.

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.

Impact of forest fragmentation and disturbance on the endangered tropical tree Prunus africana (Rosaceae)

Anthropogene Fragmentierung und Störung von Wäldern beeinflussen ökologische Prozesse. Darüber hinaus werden genetische Drift und Inzucht verstärkt und die Fitness von Populationen beeinträchtigt. Um die Einflüsse von Fragmentierung und Störung auf die Biodiversität und Prozesse in tropischen Wäldern zu ermitteln, habe ich im „Kakamega Forest“, West-Kenia, die Baumart Prunus africana genauer untersucht. Dabei lag der Fokus auf (i) der Frugivorengemeinschaft und Samenausbreitung, (ii) der Kleinsäugergemeinschaft im Kontext der Samenprädation und (iii) der genetische Populationsstruktur von Keimlingen und adulten Bäumen. Der Vergleich von Keimlingen mit adulten Bäumen ermöglicht es, Veränderungen im Genfluss zwischen Generationen festzustellen. Die Ergebnisse zeigten, dass im untersuchten Waldgebiet insgesamt 49 frugivore Arten (Affen und Vögel) vorkommen. Dabei lag die Gesamtartenzahl im zusammenhängenden Wald höher als in den isoliert liegenden Fragmenten. An den Früchten von P. africana konnten insgesamt 36 Arten fressend beobachtet werden. Hier jedoch wurden in Fragmenten eine leicht erhöhte Frugivorenzahl sowie marginal signifikant erhöhte Samenausbreitungsraten nachgewiesen. Der Vergleich von stark gestörten mit weniger gestörten Flächen zeigte eine höhere Gesamtartenzahl sowie eine signifikant höhere Frugivorenzahl in P. africana in stark gestörten Flächen. Entsprechend war die Samenausbreitungsrate in stark gestörten Flächen marginal signifikant erhöht. Diese Ergebnisse deuten darauf hin, dass die quantitative Samenausbreitung in fragmentierten und gestörten Flächen etwas erhöht ist und somit eine gewisse Artenredundanz besteht, die den Verlust einzelner Arten ausgleichen könnte. Prunus africana Samen, die auf dem Boden lagen, wurden hauptsächlich von einer Nagerart (Praomys cf. jacksonii) erbeutet. Dabei war in gestörten Waldbereichen eine tendenziell höhere Prädatoraktivität zu beobachten als in weniger gestörten. Zudem waren einzelne Samen im Gegensatz zu Samengruppen in gestörten Flächen signifikant höherem Prädationsdruck ausgesetzt. Diese Ergebnisse zeigen, dass Fragmentierung sowie anthropogene Störungen auf unterschiedliche Prozesse im Lebenszyklus eines tropischen Baumes gegensätzliche Effekte haben können. Eine Extrapolation von einem auf einen anderen Prozess kann somit nicht erfolgen. Die genetische Differenzierung der adulten Baumpopulationen war gering (FST = 0.026). Der Großteil ihrer Variation (~ 97 %) lag innerhalb der Populationen, was intensiven Genfluss in der Vergangenheit widerspiegelt. Die genetische Differenzierung der Keimlinge war etwas erhöht (FST = 0.086) und ~ 91 % ihrer Variation lag innerhalb der Populationen. Im Gegensatz zu den adulten Bäumen konnte ich für Keimlinge ein „Isolation-by-distance“-Muster feststellen. Somit sind erste Hinweise auf begrenzten Genfluss im Keimlingsstadium infolge von Fragmentierung gegeben. Obwohl die Momentaufnahmen im Freiland keine Abnahme in der Frugivorenzahl und Samenausbreitung von P. africana als Folge von Fragmentierung beobachten ließen, weisen die Ergebnisse der genetischen Studie auf einen bereits reduzierten Genaustausch zwischen den Populationen hin. Somit lässt sich feststellen, dass die Faktoren Fragmentierung und Störung genetische Diversität, ökologische Prozesse und Artendiversität in Wäldern jeweils auf unterschiedliche Weise beeinflussen. Um Konsequenzen derartiger Einflüsse folgerichtig abschätzen zu können, sind Studien auf unterschiedlichen Diversitätsebenen unabdingbar.

Architectural development and dry matter production in a multisite trial on single and multiaxis apple trees (Malus domestica Borkh) grafted on different rootstocks

In two Italian sites, multiaxis trees slightly reduced primary axis length and secondary axis length of newly grafted trees, and increased the number of secondary shoots. The total length, node production, and total dry matter gain were proportional to the number of axis. Growth of both primary and secondary shoots, and dry matter accumulation, have been found to be also well related to rootstock vigour. A great variability in axillary shoot production was recorded among different environments. Grafted trees had higher primary growth, secondary axis growth, and dry matter gain than chip budded trees. Stem water potential measured in the second year after grafting was not affected by rootstocks or number of leaders. Measurements performed in New Zealand (Hawke’s Bay) during the second year after grafting revealed that both final length and growth rate of primary and secondary axis were related to the rootstock rather than to the training system. Dwarfing rootstocks reduced the number of long vegetative shoots and increased the proportion of less vigorous shoots.

Genetic dynamics across early life stages in the tropical tree Prunus africana (Rosaceae)

In many plant species, the genetic template of early life-stages is formed by animal-mediated pollination and seed dispersal and has profound impact on further recruitment and population dynamics. Understanding the impact of pollination and seed dispersal on genetic patterns is a central issue in plant population biology. In my thesis, I investigated (i) contemporary dispersal and gene flow distances as well as (ii) genetic diversity and spatial genetic structure (SGS) across subsequent recruitment stages in a population of the animal-pollinated and dispersed tree Prunus africana in Kakamega Forest, West Kenya. Using microsatellite markers and parentage analyses, I inferred distances of pollen dispersal (father-to-mother), seed dispersal/maternal gene flow (mother-to-offspring) as well as paternal gene flow (father-to-offspring) for four early life stages of the species (seeds and fruits, current year seedlings, seedlings ≤ 3yr, seedlings > 3yr). Distances of pollen and seed dispersal as well as paternal gene flow were significantly shorter than expected from the spatial arrangement of trees and sampling plots. They were not affected by the density of conspecific trees in the surrounding. At the propagule stage, mean pollen dispersal distances were considerably (23-fold) longer than seed dispersal distances, and paternal gene flow distances exceeded maternal gene flow by a factor of 25. Seed dispersal distances were remarkably restricted, potentially leading to a strong initial SGS. The initial genetic template created by pollination and seed dispersal was extensively altered during later recruitment stages. Potential Janzen-Connell effects led to markedly increasing distances between offspring and both parental trees in older life stages. This showed that distance and density-dependent mortality factors are not exclusively related to the mother tree, but also to the father. Across subsequent recruitment stages, the pollen to seed dispersal ratio and the paternal to maternal gene flow ratio dropped to 2.1 and 3.4, respectively, in seedlings > 3yr. The relative changes in effective pollen dispersal, seed dispersal, and paternal gene flow distances across recruitment stages elucidate the mechanisms affecting the contribution of the two processes pollen and seed dispersal to overall gene flow. Using the same six microsatellite loci, I analyzed genetic diversity and SGS across five life stages, from seed rain to adults. Levels of genetic diversity within the studied P. africana population were comparable to other Prunus species and did not vary across life stages. In congruence with the short seed dispersal distances, I found significant SGS in all life stages. SGS decreased from seed and early seedling stages to older juvenile stages, and it was higher in adults than in late juveniles of the next generation. A comparison of the data with direct assessments of contemporary gene flow patterns indicate that distance- or density-dependent mortality, potentially due to Janzen-Connell effects, led to the initial decrease in SGS. Intergeneration variation in SGS could have been driven by variation in demographic processes, the effect of overlapping generations, and local selection processes. Overall, my study showed that complex sequential processes during recruitment contribute to the spatial genetic structure of tree populations. It highlights the importance of a multistage perspective for a comprehensive understanding of the impact of animal-mediated pollen and seed dispersal on spatial population dynamics and genetic patterns of trees.

Molekulare Analyse des Kolumnar-Gens beim Apfel (Malus x domestica)

Das Kolumnarwachstum beim Apfel (Malus x domestica) geht auf eine in den frühen 1960er Jahren entdeckte Zufallsmutation zurück. Die daraus resultierende Sprossmutante ist von großem wirtschaftlichem Interesse, da diese sehr kompakte Wuchsform unter anderem zu einer enormen Ertragssteigerung durch eine hohe Pflanzdichte der Bäume führt. Das Ziel der Arbeit ist die Entschlüsselung der molekularen Ursache dieser Mutation, die bisher weitgehend ungeklärt ist. Die Analyse wurde durch die Erstellung einer Referenzsequenz der Co-Zielregion einer kolumnaren Apfelsorte sowie durch die Konstruktion eng gekoppelter molekularer Marker realisiert. Durch die Konstruktion von genomischen Apfel-BAC-Bibliotheken mit mehrfacher Genomabdeckung und die Erstellung geeigneter Sonden wurde die Co-Region kloniert und deren Sequenz bestimmt. In Kombination zu dieser klassischen positionellen Klonierungsstrategie wurden genomische Illumina „mate pair“-Bibliotheken erstellt, sequenziert und bioinformatisch analysiert, um die genomische Region vollständig zu annotieren. Somit wurde eine vollständige genomische Referenz der Co-Region einer kolumnaren Apfelsorte erstellt, die die Grundlage für weitere Analysen bildet. Auf Basis dieser Referenz konnte die Co-Mutation in Form der Integration des LTR-Retrotransposons Gypsy-44 im kolumnaren Chromosom an Position 18,79 Mbp auf Chromosom 10 lokalisiert werden. Darüber hinaus konnten Transposon-basierende molekulare Marker erstellt werden, die eine verlässliche Genotypisierung von Apfelbäumen in Bezug auf das Kolumnarwachstum ermöglichen und dies unabhängig von der verwendeten Apfelsorte. Der genaue Wirkmechanismus von Gypsy-44, der zur Ausprägung dieses extremen Phänotyps führt, ist bislang unklar. Zusammenfassend lässt sich sagen, dass die molekulare Ursache für das kolumnare Wachstum aufgeklärt werden konnte und zudem die ersten molekularen Marker erstellt wurden, die eine sortenunabhängige Differenzierung zwischen kolumnaren und nicht kolumnaren Apfelbäumen ermöglichen.

Molecular genetic causes of columnar growth in apple (Malus x domestica)

The columnar growth habit of apple is interesting from an economic point of view as the pillar-like trees require little space and labor. Genetic engineering could be used to speed up breeding for columnar trees with high fruit quality and disease resistance. For this purpose, this study dealt with the molecular causes of this interesting phenotype. The original bud sport mutation that led to the columnar growth habit was found to be a novel nested insertion of a Gypsy-44 LTR retrotransposon on chromosome 10 at 18.79 Mb. This subsequently causes tissue-specific differential expression of nearby downstream genes, particularly of a gene encoding a 2OG-Fe(II) oxygenase of unknown function (dmr6-like) that is strongly upregulated in developing aerial tissues of columnar trees. The tissue-specificity of the differential expression suggests involvement of cis-regulatory regions and/or tissue-specific epigenetic markers whose influence on gene expression is altered due to the retrotransposon insertion. This eventually leads to changes in genes associated with stress and defense reactions, cell wall and cell membrane metabolism as well as phytohormone biosynthesis and signaling, which act together to cause the typical phenotype characteristics of columnar trees such as short internodes and the absence of long lateral branches. In future, transformation experiments introducing Gypsy-44 into non-columnar varieties or excising Gypsy-44 from columnar varieties would provide proof for our hypotheses. However, since site-specific transformation of a nested retrotransposon is a (too) ambitious objective, silencing of the Gypsy-44 transcripts or the nearby genes would also provide helpful clues.

Valutazione del contributo emissivo in condizioni non-stazionarie alle emissioni totali di una stufa a pellet domestica

The aim of this work is to evaluate the emissions of the main pollutants of a pellet stove, by trying to simulate the real use in domestic operations. All the operating phases of this system were considered: ignition, partial load, increase in power, and nominal load. In each phase, quantity and type of some pollutants in emissions were determined: the main pollutant gases (CO, NOx, SO2, H2S and volatile organic compounds (VOCs)), total dust (PM) and its content of polycyclic aromatic hydrocarbons (PAHs), regulated heavy metals (Ni, Cd, As and Pb), main soluble ions and Total Carbon (TC). Results show that emission factors of TSP, CO, and of the main determined pollutants (TC, Cd and PAHs) are higher during ignition phase. In particular, this phase prevalently contributes to PAHs emissions. During increase in power phase, gas and particulate emissions do not appreciably differ from nominal load ones; nevertheless, PAH emission factors are higher than steady state ones, but lower than ignition phase. Moreover, during not-steady state phases, PAH mixture is more toxic than during steady state phases. In conclusion, this study allowed to go deeper in pellet stove environmental impact, by pointing out how the different operating conditions can modify the emissions. These are different from certificated data, which are based exclusively on measurements in steady state conditions.