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Common valerian (Valeriana officinalis L.) is an important medicinal plant revealing sedative, hypotensive, anti-spasmodic and anxiolytic activity. The purpose of the study was to determine the intraspecific variability of the common valerian growing wild in Poland and the ‘Lubelski’ landrace, as to their developmental traits, chemical composition and selected genetic parameters. Both wild-growing populations (19) and the landrace (1) were evaluated under ex situ conditions. Observations of the underground organs parameters, both developmental and chemical (according to the European Pharmacopoeia) were carried out in the first year of the plant’s development, while the characteristics of the aboveground organs, followed by the sowing value of seeds (according to the International Seed Testing Association)—in the second year. The genetic analyses were performed using the NGS-DArT-seq method. Results indicate the presence of five different gene pools covering the regions of population’s origin, with a gene flow within and between them. A high level of developmental and chemical variabilities among the wild-growing populations was noticed, however without a clear relation to the region of the origin. The mass of underground organs ranged from 107.4 to 403.6 g FW × plant−1 with the content of sesquiterpenic acids at the level of 0.004–0.094%. Population no 18 was distinguished by the highest content of sesquiterpenic acids and the relatively high mass of underground organs, followed by the admixture of the gene pool, typical for the ‘Lubelski’ landrace. Unlike the ‘Lubelski’ landrace, the wild-growing populations were characterized by a high amount of an essential oils (3.90 to 10.04 mL/kg), which may be promising from the perspective of their potential use. In turn, the sowing value of the seeds obtained from the populations, expressed as the germinability, was rather low (25.25–62.25%).

Grapevine fanleaf virus (GFLV) is one of the main causes of grapevine fanleaf degeneration disease (GFDD) and is present in almost all areas where grapevine is cultivated. In this work, we ascertained the presence and spread of GFLV in different commercial vineyards in four Sicilian provinces (Italy), and its genetic structure and molecular variability were studied. In detail, a total of 617 grapevine samples of 11 autochthonous grapevine cultivars were collected in 20 commercial vineyards. Preliminary screening by serological (DAS-ELISA) and molecular (RT-PCR) analyses for ArMV (arabis mosaic virus) and GFLV detection were conducted. Results obtained showed the absence of ArMV in all the samples analyzed, while 48 out of 617 samples gave positive results to GFLV, for a total of 9 out of 11 cultivars analyzed. Phylogenetic analyses carried out on the GFLV-CP gene of 18 Sicilian GFLV sequences selected in this study showed a certain degree of variability among the Sicilian isolates, suggesting a different origin, probably as a consequence of the continuous interchange of GFLV-infected propagating material with other Italian regions or viticultural areas located in other countries.

[ES] El virus de las venas amarillas del pimiento 5 (PeVYV-5), un virus con genoma de RNA de simple cadena y polaridad positiva perteneciente al género Polerovirus (familia Luteoviridae), fue detectado por primera y única vez en nuestro país en 2013. Este virus está estrechamente relacionado con al menos diez especies virales (englobadas bajo el acrónimo PeVYV seguido por un número) descritas en distintas partes del mundo, una diversificación que parece haber sido el resultado de mutaciones y/o recombinaciones, fenómenos frecuentes en virus de RNA y, de forma remarcable, en polerovirus. A pesar de que cualquiera de estos virus constituye una amenaza importante para el cultivo de Capsicum spp., desde la detección del PeVYV-5 y posterior secuenciación de su genoma en 2018, no hay más datos publicados sobre la presencia y/o variabilidad de este virus (o de cualquier otro PeVYV) en España. Recientemente, se han vuelto a observar los síntomas asociados al PeVYV en plantas de pimiento crecidas en invernaderos de Almería. En este trabajo se ha pretendido determinar si dichas plantas estaban, efectivamente, infectadas por PeVYV y, en tal caso, por qué especie concreta. Mediante hibridación molecular con sondas no radioactivas y RT-PCR se ha podido constatar la presencia del PeVYV-5 en algunas de ellas. Un análisis de la distribución y acumulación de este virus en plantas infectadas ha mostrado que los frutos presentan una carga viral superior a la de los peciolos, los pedúnculos de frutos y/o las hojas. La comparación de secuencias derivadas de dos regiones del genoma de los aislados del PeVYV-5 identificados en este trabajo, ha mostrado una heterogeneidad molecular considerable tanto a nivel nucleotídico como aminoacídico, lo que subraya la gran capacidad evolutiva de este patógeno., [EN] The Pepper vein yellows virus 5 (PeVYV-5), a virus with a single-stranded RNA genome and positive polarity belonging to the genus Polerovirus (Luteoviridae family), was detected for the first and only time in our country in 2013. This virus is closely related to at least ten viral species (included under the acronym PeVYV followed by a number) described in different parts of the world, a diversification that seems to have been the result of mutations and/or recombinations, frequent phenomena in RNA viruses and, remarkably, in polerovirus. Despite the fact that any of these viruses constitutes an important threat to the cultivation of Capsicum spp., since the detection of PeVYV-5 and subsequent sequencing of its genome in 2018, there are no more published data on its presence and/or variation of this virus (or any other PeVYV) in Spain. Recently, the symptoms associated with PeVYV have been observed again in pepper plants grown in greenhouses in Almería. In this work we have tried to determine if these plants were, indeed, infected by PeVYV and, if so, by which specific species. By means of molecular hybridation with non-radioactive probes and RT-PCR it has been possible to verify the presence of PeVYV-5 in some of them. An analysis of the distribution and accumulation of this virus in infected plants has shown that the fruits present a viral load higher tan that of the petioles, fruit peduncles and/or leaves. The comparison of sequences derived from two regions of the genome of the PeVYV-5 isolates identified in this work has shown considerable molecular heterogeneity both at the nucleotide and amino acid levels, which underlines the great evolutionary capacity of this pathogen.

Among the wild relatives of wheat, Triticum urartu Tumanian ex Gandilyan is of interest to wheat breeders. Numerous works have focussed on its utilization for enrichment of the genetic variation of cultivated genotypes. In the present study, the genetic diversity and population structure in 85 accessions of T. urartu were investigated using start codon targeted (SCoT) polymorphism and CAAT-box derived polymorphism (CBDP) markers. Nineteen SCoT primers and fifteen CBDP primers amplified 185 and 141 polymorphic fragments with an average of 9.74 and 9.40 fragments per primer, respectively. The polymorphic information content (PIC) for the SCoT and CBDP primers ranged from 0.41 to 0.50 and 0.40 to 0.49, with the resolving power (Rp) ranging from 21.61 to 3.97 and 13.08 to 28.02, respectively. Neighbour-joining (NJ) based clustering grouped 72 accessions into two main clusters based on the two sets of markers and the combined data. Genetic relationships inferred from STRUCTURE analysis was matched with cluster analysis and principal coordinate analysis (PCoA), indicating that the accessions were grouped into two major clades and the grouping patterns were not correlated with the geographic origins. The results from this study revealed that Iranian T. urartu, especially Kerend-e-Gharb and Sisakht-Pataveh populations, can be interesting for wheat improvement. Hence, conservation of this region is recommended.

In order to study the current situation of Citrus psorosis virus (CPsV) in Tunisia, field surveys were conducted in commercial orchards in Cap Bon region and 575 citrus trees were sampled. As determined by DAS-ELISA, the CPsV incidence in Tunisia was high, and ranged between 20 and 47%, according to geographical location, citrus species and varieties. cDNA encompassing the full-length RNA3 from Tunisian isolates was synthesized by RT-PCR using specific primers and two isolates were cloned and sequenced. Full length sequences of the coat protein (CP) gene were obtained and deposited in GenBank. Based on CP gene sequences, phylogenetic clustering of isolates revealed two populations, one includes isolates from Spain, Morocco and New Zealand and the other includes those from Tunisia, Spain, Italy, Mexico and Egypt. Genetic haplotype network provided evidence of the existence of two distinct clusters with an ambiguous origin. Selective neutrality test was significantly positive, suggesting a balancing selection of CPsV CP gene.

Genetic diversity in safflower blight pathogen (Alternaria carthami) was analyzed using 20 isolates collected from major safflower growing regions of Maharashtra state in India. Experiment was conducted at Department of Agricultural Bioatechnology, College of Agriculture, Latur, VNMKV, Parbhani (M.S.). Genomic DNA extracted from each isolates of Alternaria carthami was subjected to polymerase chain reaction using 20 random decamer primers from OPA series. Only 08 of the 20 RAPD primers were selected based on repeatability. Result revealed that 100 per cent polymorphism were found. The dendrogram analysis revealed two clusters, Cluster I comprised twelve isolates viz., Ac-1, Ac-8, Ac-4, Ac-5, Ac-7, Ac-11, Ac-18, Ac-15, Ac-17, Ac-9, Ac-20 and Ac-19 with similarity coefficient of 77 per cent. Cluster II comprised of eight isolates viz., Ac-2, Ac-3, Ac-6, Ac-13, Ac-12, Ac-14, Ac-10 and Ac-16 with similarity coefficient of 76 per cent.

Rodents are a very diverse group with large chromosome variability. One of the most species rich linage in the Neotropics is the Sigmodontinae. Among them, the tribe Abrotrichini was recently defined and its taxonomy and phylogeny were mostly elucidated through molecular and morphological evidence. Meanwhile, chromosome data were only secondarily used because of fragmentary information. In this contribution, we conduct a chromosome characterization of Abrothrix hirta, A. olivacea, A. andina, and Paynomys macronyx, review the cytogenetic background of the tribe, and contrast it with molecular data. Chromosomes were analyzed by conventional and differential techniques. All Abrothrix species presented 2n = 52/FNa = 56, with a high similarity in the banding patterns reflecting a conserved karyotype, which does not coincide with its high molecular variability. In turn, P. macronyx have 2n = 54/FNa = 58–59, varying due to a heteromorphic pair of autosomes. In addition, in this last species, different morphologies of the X chromosome and the presence of B chromosomes were detected. Heterochromatin was involved in these variants. The telomeric probe in P. macronyx marks terminal regions of all chromosomes. B chromosomes generated strong telomeric signals. The Ag-NORs banding revealed the same patterns in Abrothrix and Paynomys. Cytogenetic data support phylogenetic relationships previously proposed and suggest that the specious genus Abrothrix could have retained the ancestral karyotype of the subfamily. In the tribe, the relatively conserved chromosome complement contrasts with its high molecular variability. This indicates decoupling between the rates of chromosomal and molecular divergence, as observed in other rodent lineages. In abrotrichines, chromosome evolution was slower., Facultad de Ciencias Naturales y Museo

One of the hypotheses about the causes of invasive transmission of Phalaris arundinacea in the US is, that this species have repeatively introducted from Europe or by hybridization of the introduced genotypes with the native species. This is the same situation as Phragmites australis. Part of this intercontinental gene flow is enhanced by the production of Phalaris cultivars in Minnesota. The consequences of these exchanges have a significant impact on the management of invasive crops from both continents. Previous studies confirm only minimal morphological heterogeneity between cultivated and native (wild) forms of Phalaris arundinacea. In Phalaris arundinacea, we analyzed genetic similarities and differences between the US populations (Minnesota) and population of the Czech Republic. The extent of genetic variation in native and invasive populations was compared using ISSR markers. The occurence of commercial genotypes was observed across the full spectrum of invasive genotypes, which suggests a relatively frequent exchange of genes among forage, ornamental, and native US populations. The genus Myriophyllum is mainly significant because of its invasive species, which have always been in a special position as the course of ecological and evolutionary changes may be observed in real time. In Central Europe it is represented by three native species (M. spicatum L., M. alterniflorum and M. verticillatum) and two non-native species of North American M. heterophyllum and South American M. aquaticum. Species M. spicatum, together with M. heterophyllum and M. aquaticum, is one of the most important invasive plants that, due to their aggressive growth, cause many problems, especially in North America. The main theme of this Ph.D. is the evaluation of the role of polyploidization in processes leading to invasive character of some species of the genus Myriophyllum in the USA, cytological and population-genetic comparison of native (Eurasia) and invasive populations (US) of Myriophyllum spicatum. Moreover, possible "Pools" of genetic and cytotype variability of invasive populations and evaluation of usability of simple genome size were determinated in taxonomic determination by classical morphological procedure of unclassifiable plants, which might reduce the cost and efficiency of invasive stool biomonitoring and thus the management of its invasive populations

Cucurbits are widely grown vegetables in different provinces of Turkey. However their production is restricted by pests and pathogens including different plant viruses, among which Zucchini yellow mosaic virus (ZYMV) is particularly damaging. Infections by this virus during the early stages of plant development causes deformation of fruits and significant yield losses. During 2009-2014, 221 cucurbits samples were collected from fields and greenhouses from different provinces of Turkey. Serological testing was performed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for presence of Watermelon mosaic virus (WMV), ZYMV, Cucumber mosaic virus (CMV), Papaya ring spot virus (PRSV), cucumber green mottle mosaic virus (CGMMV) and Squash mosaic virus (SqMV). For molecular analaysis of ZYMV, full-length CP nucleotide sequences were obtained for 45 ZYMV isolates and compared with sequences from worldwide isolates available in databases. The CP coding regions of Turkish ZYMV were 837 nt long and encoded 279 amino acids (aa). Forty two isolates from different regions were classified in the molecular subgroup Al, the most frequent in the Mediterranean Basin, Europe and Africa. Three isolates collected from Antalya region were classified in subgroup A4 and showed 99 % sequence identity with isolates from the same subgroup that have emerged recently in France.

Genetic variability of locally adapted Serbian and Austrian winter wheat varieties was evaluated in order to assess their potential as a genetic material that can be exploited in crosses between two different European breeding pools. A field trial with 20 elite wheat varieties from each country was set at the Institute of Field and Vegetable Crops, Novi Sad, Serbia in a row-column design with three replications during the 2016/2017 season. The genotypes were phenotyped for tillering, heading and flowering time, plant height, ear length, number of spikelets per spike, number of grains per spike, thousand-kernel weight, chlorophyll content and resistance to prevalent wheat diseases. Additionally, the varieties were genotyped with 30 microsatellites. The varieties from two geographic regions and different release periods were clearly differentiated with population structure obtained from marker data. A significant phenotypic variation was found for most of the traits. Coefficients of variation were the largest for chlorophyll content (16.5%) and plant height (10.1%). Generally, the early genotypes were more susceptible to leaf rust (r = -0.6), while the late maturing genotypes produced more grains per spike (r = 0.4). The Serbian varieties had earlier tillering (p < 0.03), heading (p < 0.00) and flowering (p < 0.00) dates, shorter plant stems (p < 0.00), higher chlorophyll content (p < 0.00) and were more susceptible to leaf rust (p < 0.00) than the Austrian ones. The principal component analysis indicated general properties of the groups that would facilitate the choice of parent combinations for crossings.

Molecular variability was assessed for 18 isolates of cowpea mild mottle virus (CPMMV, genus Carlavirus, family Betaflexiviridae) found infecting soybean in various Brazilian states (Bahia, Goias, Maranhao, ́ ) in 2001 and 2010. A Mato Grosso, Minas Gerais, Para variety of symptoms was expressed in soybean cv. CD206, ranging from mild (crinkle/blistering leaves, mosaic and vein clearing) to severe (bud blight, dwarfing, leaf and stem necrosis). Recombination analysis revealed only one CPMMV isolate to be recombinant. Pairwise comparisons and phylogenetic analysis were performed for partial genomes (ORF 2 to the 3’ terminus) and for each ORF individually (ORFs 2 to 6), showing the isolates to be distinct. The topology of the phylogenetic tree could be related to symptoms, but not to the year of collection or geographical origin. Additionally, the phylogenetic analysis supported the existence of two distinct strains of the virus, designated CPMMV-BR1 and CPMMV-BR2, with molecular variations between them.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Soybeans are one of the crops with the highest expression in Brazil, both in area planted as in sales volume. The culture has as main pests defoliating caterpillars. Among these, stands out Chrysodeixis includens, pest that went from of secondary status in the 90s to a major defoliating caterpillar soybeans. Studies on genetic variation among populations and genetic structure of this species have not yet been carried out, and can help to indicate management practices. Molecular markers are tools indicated to genetically characterize insect populations. In order to use molecular tools, first, it is necessary a method for DNA extraction in quantity and quality that enables the practice in the laboratory. The goals of the study were to compare three DNA extraction methods for soybean caterpillars to applicate in PCR techniques, and analyze the molecular variability and the genetic structure of populations of C. includens in soybeans. Caterpillars of C. includens and S. eridania were collected from different sites in center soybean regions of Brazil, in 2011/12. The confirmation of the species was based on morphological characteristics of caterpillars according to identification keys. Samples of C. includes and S. eridania coming from Goiás were used for DNA extraction comparison test. The methods used were based on cell lysis by Sarcosyl, CTAB and SDS. Each method was compared for quantity, quality, economy and performance in PCR. The best DNA extraction method was chosen for extraction of all the caterpillars samples for molecular characterization work. Thirty populations of C. includens from nine Brazilian states were subjected to analysis of molecular variability and genetic structure with ISSR markers. The observed DNA extraction method with the best performance in the variables o was the DNA extraction method by Sarcosyl. ISSR generated 247 loci in 262 specimens analyzed. The estimated genetic diversity (HE) in populations ranged between 0.072 and 0.120, while the average was 0.094. The analysis of molecular variance indicates that 94% of the variability between individuals was expressed in 6% of the population and among populations (FST = 0.056, p = 0.001). The high level of gene flow and low genetic structure are indicatives of genetic information exchange between different sampling locations. The analysis of the genetic structure suggests the presence of two major groups which are not correlated to their sampling locations in Brazil. These results may indicate the recent colonization of C. includens in Brazil or the pattern of migration of moths following the cropping system in Brazil. Furthermore, the presence of two groups of C. includens suggest that the studies of development of resistance need to be further assessed for them both. A soja é uma das culturas de maior expressão no Brasil, tanto em área plantada como em volume comercializado, e tem como principais pragas desfolhadoras as lagartas. Dentre essas, se destaca Chrysodeixis includens, praga que passou de secundária na década de 90 para uma das principais lagartas desfolhadoras da soja. Estudos sobre variações genéticas entre populações e estruturação genética dessa espécie ainda não foram realizados, e podem auxiliar na indicação práticas de manejo. Marcadores moleculares são ferramentas indicadas para caracterizar geneticamente populações de insetos. Mas para utilizar ferramentas moleculares, primeiramente, se faz necessário a extração de DNA em quantidade e qualidade que possibilita a prática no laboratório. Dessa forma, os objetivos do trabalho foram comparar três métodos de extração de DNA para lagartas da soja visando a aplicação com técnicas que utilizem a PCR. E analisar a variabilidade molecular e a estruturação genética de populações de C. includens na cultura da soja. Espécimes de lagartas de C. includens e Spodoptera eridania foram coletados de diferentes sítios de coleta nas principais áreas produtoras de soja do Brasil, safra de 2011/12. A confirmação da espécie foi baseada em características morfológicas das lagartas de acordo com chaves de identificação. As amostras de C. includens e S. eridania procedentes de Goiás foram utilizadas para o teste de comparação de extração de DNA. Os métodos utilizados eram cada um baseados na lise celular por Sarcosyl, CTAB e SDS. Cada método foi comparado quanto à quantidade, qualidade, economia e desempenho na PCR. O melhor método de extração de DNA foi utilizado para a extração de todas as amostras de lagartas para o trabalho de caracterização molecular. Trinta populações de C. includens de nove estados brasileiros foram submetidas a análise da variabilidade molecular e estrutura genética com marcadores ISSR. O método de extração de DNA que apresentou melhor desempenho nas variáveis observadas foi o método de extração de DNA por Sarcosyl. Os marcadores ISSR geraram 247 locos em 262 espécimes analisados. A diversidade genética estimada (HE) nas populações variaram entre 0,072 e 0,120, enquanto a média foi 0,094. A análise da variância molecular indica que 94% da variabilidade foi expressa entre indivíduos dentro das populações e 6% entre populações (FST = 0,056, p = 0,001). O alto nível de fluxo gênico e baixa estrutura genética são indicativos de troca de informação genética entre as diferentes locais de amostra. A análise da estrutura genética sugere a presença de dois grupos maiores que não se correlacionam com seus locais de amostragem no Brasil. Esses resultados podem indicar a recente colonização de C. includens no Brasil ou o padrão de migração das mariposas seguindo o sistema de cultivo no Brasil. Além disso, a presença de dois grupos de C. includens sugere que estudos sobre o desenvolvimento de resistência precisa ser avaliado sob outros ângulos para ambos os grupos.

The genes encoding 13 classes of pathogenesis-related (PR) proteins were examined for positive selection using maximum-likelihood (ML) models of codon substitution. The study involved 194 sequences from 54 species belonging to 37 genera. Although the sizes of the sequences examined varied from 237 bp for PR12 to 1,110 bp for PR7, most classes (9 out of 13) contained sequences made up of more than 400 nucleotides. Signs of positive selection were obtained for sites in PR proteins 4, 6, 8, 9 and 15 using an ML-based Bayesian method and likelihood ratio tests. These results confirm the importance of positive selection in proteins related to defense mechanisms already observed in a wide array of organisms.

Recientemente, el papel de los factores ecológicos en la especiación ha comenzado aevaluarse críticamente. En insectos mariposas y moscas de la fruta, la evidenciadisponible indica que los cambios de hábitat/dieta están correlacionados con elaislamiento reproductivo apuntando a que la adquisición de nuevas plantas hospedadoraspodría ser clave en la sorprendente diversidad de los insectos fitófagos. Las especiescactófilas del género Drosophila son modelos ideales para estudiar el papel de loscambios de planta hospedadora en la adaptación y la especiación. D. buzzatii y D.koepferae se crían en los tejidos necróticos de cactáceas de los géneros Opuntia (tunas) y Trichocereus (cardones) y exhiben un cierto grado de especificidad de nicho. Se hapropuesto que los cactus columnares y las tunas podrían representar dos tipos de recursosclaramente diferenciados, ya que difieren en varios aspectos relevantes para el ciclo devida de las moscas, como por ejemplo en su composición química. Los primerospresentan una mayor concentración de compuestos tóxicos, como alcaloides, y por lotanto representan un ambiente estresante durante el desarrollo larval; en tanto que lastunas ofrecen un ambiente químico más benigno y rico desde el punto de vista nutricional. Asimismo, cardones y tunas se diferencian en su predecibilidad espacial y temporal y suspatrones de distribución geográfica. Una predicción basada en la relación entre el uso deplantas hospedadoras diferentes y la estructuración poblacional es que el grado decompartimentalización del recurso impondría diferentes grados de restricción al flujogénico. En este trabajo se estudió la variación molecular y la estructura poblacional deestas dos especies utilizando dos marcadores moleculares, COI y un grupo demicrosatélites específicos para estas especies. Se encontró que no hay estructuracióngenético-poblacional en D. buzzatii como cabría esperar para una especie que utiliza unrecurso con una distribución espacialmente homogénea (más que los cardones) yespacialmente predecible; en cambio en D. koepferae, asociada a un recurso con unadistribución más compartimentalizada y menos predecible espacialmente, se encontró unaestructuración poblacional. The role of ecological factors in speciation has recently began to be critically evaluated. In insects, the available evidence indicates that changes in habitat/diet are correlated withreproductive isolation, particularly, in butterflies and fruit flies, suggesting that theacquisition of new host plants could be a key factor in the amazing diversity ofphytophagous insects. Cactophilic species of the genus Drosophila are ideal models forstudying the role of changes in host plant adaptation and speciation. D. buzzatii and D.koepferae are two cactophilic species that inhabit the arid and semiarid lands of Argentinaand Bolivia. These species exhibit a certain degree of niche specificity since the formerbreeds mainly in the necrotic tissues of cacti of the genus Opuntia (prickly pears) and thelatter on the necroses of plants of the genera Trichocereus (cardón) and Cereus, howeverthe available evidence points to a certain degree of niche overlap. Previous studies haveproposed that columnar cacti and prickly pears represent two distinct types of resourcessince they differ in many aspects such as geographic distribution, chemical compositionand spatial and temporal predictability that are relevant to the life cycle of flies. On onehand, columnar cacti represent stressful environments for larval development since theyhave a high concentration of toxic compounds such as alkaloids and triterpenes. On theother hand, unas offer a more benign chemical environment due to the absence of toxiccompounds and greater nutritional richness. A prediction based on the relationshipbetween the use of different host plants and population structure is that the degree ofcompartmentalization of the specific resource imposes varying constraints to gene flow. In this tesis, we studied molecular variation and population structure in D. buzzatii and D. koepferae using two types of molecular markers, the mitochondrial gene COI and aset of species-specific microsatellites. Our main findings are that natural populations of D. buzzatii are not genetically structured as expected for a species that uses a resourcewith a spatially homogeneous distribution, while significant population structure wasdetected in D. koepferae, a species associated with resources exhibiting morecompartmentalized and spatially less predictable breeding sites. Fil: Lipko, Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The genetic variability of the Brazilian physic nut (Jatropha curcas) germplasm bank (117 accessions) was assessed using a combination of phenotypic and molecular data. The joint dissimilarity matrix showed moderate correlation with the original matrices of phenotypic and molecular data. However, the correlation between the phenotypic dissimilarity matrix and the genotypic dissimilarity matrix was low. This finding indicated that molecular markers (RAPD and SSR) did not adequately sample the genomic regions that were relevant for phenotypic differentiation of the accessions. The dissimilarity values of the joint dissimilarity matrix were used to measure phenotypic + molecular diversity. This diversity varied from 0 to 1.29 among the 117 accessions, with an average dissimilarity among genotypes of 0.51. Joint analysis of phenotypic and molecular diversity indicated that the genetic diversity of the physic nut germplasm was 156% and 64% higher than the diversity estimated from phenotypic and molecular data, respectively. These results show that Jatropha genetic variability in Brazil is not as limited as previously thought.

Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.

“Flavescence dorée” (FD) is a disease still of relevant importance considering the ability of phytoplasmas associated with it to differentiate new strains in short periods of time. The first finding of FD in Tuscany dates back to 2003 and concerns a vineyard sited at Montignoso, in Massa Carrara province. In the last decade the monitoring of phytoplasma has highlighted a fairly widespread presence of the disease, only in the wine-growing areas of the provinces of Massa Carrara and Lucca. In the context of the program EUPHRESCO GRAFDEPI, in 2012 surveys were carried out mainly in vineyards in the province of Massa Carrara. The vineyard reported was monitored in 2009 as well, it is made of six varieties, four native (‘Albarola’, ‘Ciliegiolo’, ‘Pollera Pontremolese’ and ‘Vermentino’) plus ‘Chardonnay’ and ‘Merlot’, all grafted on SO4 rootstock. The flora of the border is made up by chestnut plants and also several shrubs including, blackberries, hawthorn, plum, elderberry and especially greybeard. A total of 30 samples of leaves and branches were collected from symptomatic vines (nine in 2009 and 21 in 2012) and a total of six samples of Clematis vitalba were collected from border flora in 2012. All grapevine samples positive to phytoplasmas (7/9 in 2009 and 14/21 in 2012) resulted infected by FD-C. Regarding to the greybeard a sample resulted positive and infected by 16SrXII-A (“stolbur”) phytoplasmas. Molecular characterization was carried out on FD strains identify that all the samples were infected by FD-D phytoplasmas, however the fine characterization on the SecY gene by RFLP analyses showed that all strains but one have a profile indistinguishable from the one typically described in FD-C strains. One sample showed profile mixed between typical FD-C and FD-D strains. This result indicates that also in Tuscany FD-D phytoplasma is producing strains as already reported in other Italian FD-D epidemic as well as in one strain from France. This finding is quite important since the possibility to early identify new FD strains allows to better manage the disease before these strains become epidemically relevant.

Therefore, species are expected to move towards the poles in response to shifting climate ones. This warming trend also includes the establishment of tropical algae in temperate systems of the Northern Hemisphere. Numerous tropical and subtropical species of microalgae have been reported in the last decades in the continental waters of Europe. The aims of the Bachelor thesis are to summarize articles concerning changes in diversity and dynamics focused on algae and blue-green algae in temperate water bodies. Several hypotheses about the origin, modes of distribution and the ability of microorganisms to overcome stressful condition during the transportation are also discussed in this work.

Phytoplasmas within single plants can be considered as populations of individuals and as phytoplasmas are obligate parasites of plants, single clones cannot be obtained. For identification of phytoplasmas, PCR followed by RFLP, sequencing PCR products directly or sequencing cloned PCR products are standard procedures. These procedures will, however, not show the diversity of populations within single plants, as only the most frequent genotypes will be detected and identified. A number of grapevine samples, in which mixed phytoplasma infections were detected by a nested PCR, were used for deep amplicon sequencing on the Roche Genome Sequencer FLX system. 50,926 phytoplasma sequences from the 5’end of the 16Sr DNA gene were obtained from 16 phytoplasma-infected grapevine samples. After clustering and alignment to phytoplasma reference sequences it was shown that the phytoplasmas in the grapevine plants belonged to diverse 16Sr groups, mainly the same groups that were detected using nested/PCR/RFLP. Furthermore, a high number of single nucleotide polymorphisms were present in individual samples.

The Polimedia describes the use of the PHYLIP package to develop cluster analysis from molecular data. Concretely, an example using data form RAPD markers is explained., Visualización y desarrollo del ejemplo utilizando los programas

Surveys carried out to identify ‘bois noir’ (BN) phytoplasmas in grapevine yellows outbreaks in vineyards located in Serbia and in several Italian regions were carried out from 2008 to 2010. The presence of BN phytoplasmas was preliminarily demonstrated by RFLP analyses with TruI restriction enzyme on R16F2/R2 amplicons. About 60 samples were selected for further molecular characterization. Reference strains maintained in periwinkle were STOL (from Serbia), STOLC and STOL-PO (from France). RFLP analyses on R16F2/R2 amplicons with Hpy188I, Hpy8I, MboI, MboII, TruI, RsaI, BstUI, AluI, and Tsp509I restriction enzymes were carried out. Tsp509I was only differentiating one samples from Veneto region in Italy, while different profiles were observed in several of the examined samples when MboII, Hpy188I, and AluI were used. In particular, three different profiles were observed with AluI in samples from Veneto and Tuscany (Italy), and Serbia. Using MboII four different profiles (a-b-c-d) were observed in samples from Veneto, Tuscany and Emilia, while only one of them (profile a) was observed in the samples from Serbia. RFLP profiles of reference strains were a for STOL, b for STOL-PO and c for STOLC. Considering that the profile c is referring to an amplicons longer than the expected one, the presence of interoperon heterogeneity and/or of mixed BN strain infection could be hypnotized for this profile. Some of the samples having profile c when digested with Hpy188I showed further polymorphism. The reference strains plus 7 field collected BN strains chosen among those showing the above described polymorphisms were sequenced on the full 16S gene. The sequences were assembled using DNA STAR software, and compared with selected sequences of phytoplasmas in GenBank database using Blast N 2.2.18. Obtained aligned sequences ranged from 1,300 to 1,500 bp, all showing 99% homology among them self and with several of the 16SrXII-related strains deposited in Genbank. Virtual RFLP analyses on R16F2/R2 amplicons were carried out, using pDRAW32 program (AcaClone Software) and it was possible to confirm the variability detected in real RFLP analyses in the majority of BN sequenced strains. The comparison between real and virtual RFLP analyses showed in some cases different profiles when digested with MboII. Strain STOLC showed in this case a b profile confirming that profile c is formed by mixed infection or interoperon heterogeneity. On the other hand the presence of a d profile was detected in one of the sequenced strains from Serbia showing an a profile in RFLP analyses. BN strains differentiation on 16S rDNA on field collected uncloned amplicons indicates that the detected variability could be related with different epidemiological behaviours.

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In previous studies, Aster Yellows (AY) phytoplasmas were identified in Populus nigra L. ‘ Italica’ (Lombardy poplar) trees from the urban area of Zagreb. This pathogen was classified as a member of a newly described ribosomal subgroup 16SrI-P and ribosomal protein (rp) subgroup rp-O. The aim of this research was to examine the incidence and molecular variability of phytoplasmas in Lombardy poplar tree population of wider Zagreb urban area. Total nucleic acid was extracted from leaf and branch samples of 34 symptomatic and asymptomatic trees by using 3% CTAB extraction procedure. Phytoplasma 16S rDNA was amplified in direct PCR by using R16F1/R0 and/or P1/P7 universal phytoplasma primer pairs. Nested PCR reactions were primed by R16F2n/R2, 16R738f/R1232r, as well as group-specific R16(I)F1/R1 oligonucleotides. A major portion of the elongation factor EF-Tu gene, ribosomal protein L22 gene, and the region encoding putative aa kinase and ribosomal recycling factor were also amplified by using primers fTufu/rTufu, rpF1/R1, and BB88F1/R1, respectively, and followed by RFLP analyses. Phytoplasmas with restriction profiles referable to AY were detected in 12 samples. PCR/RFLP analyses of 16S rRNA and of other conserved phytoplasma gene regions revealed that amplicons from 10 positive samples had profiles identical to the phytoplasma belonging to the ribosomal 16SrI-P and rp-O subgroup. In the remaining 2 positive samples, amplicons obtained with rpF1/rpR1, fTufu/rTufu and BB88/F1/R1 primers, had unique, previously unrecorded, RFLP profiles. In order to analyze these two phytoplasmas in more detail and to infer phylogenetic relationships, their 16S rRNA and spacer regions were amplified, analyzed by RFLP, cloned and sequenced. Virtual RFLP analyses were also performed on the obtained sequences. Comparisons with the 16SrI-P phytoplasma profiles revealed differential restriction sites confirming the presence of molecular variability within their 16S and the spacer regions. Positions of these sequences in the phylogenetic tree reflected the molecular variability found in the 16S region.

The genes encoding 13 classes of pathogenesis-related (PR) proteins were examined for positive selection using maximum-likelihood (ML) models of codon substitution. The study involved 194 sequences from 54 species belonging to 37 genera. Although the sizes of the sequences examined varied from 237 bp for PR12 to 1,110 bp for PR7, most classes (9 out of 13) contained sequences made up of more than 400 nucleotides. Signs of positive selection were obtained for sites in PR proteins 4, 6, 8, 9 and 15 using an ML-based Bayesian method and likelihood ratio tests. These results confirm the importance of positive selection in proteins related to defense mechanisms already observed in a wide array of organisms.