Caracterização molecular e virulência de isolados de Beauveria spp. contra a lagarta-do-cartucho

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.Beauveria es un hongo deuteromiceto de gran espectro entomopatogénico. Existe mucho interés en el uso de ese hongo en programas integrados de manejo de plagas, como una alternativa a los defensivos químicos. La caracterización molecular de los aislados altamente virulentos es esencial en la selección de potenciales candidatos para utilización en control biológico. Se han evaluado en este trabajo 24 aislados de Beauveria, en bioensayos contra gusanos de Spodoptera frugiperda y molecularmente caracterizados por secuenciamiento de la región ITS del rDNA y marcadores RAPD. El secuenciamiento de fragmentos de 570 pares de bases de la región ITS del rDNA posibilitó la identificación de los aislados como B. bassiana y B. brongniarti, por la comparación con secuencias depositadas en el GenBank, pero no fue suficiente para discriminar los aislados dentro de cada especie. Sin embargo, 67 fragmentos polimórficos de RAPD fueron capaces de diferenciar casi todos los aislados de Beauveria, agrupándolos de acuerdo con el insecto hospedero y la virulencia contra el gusano cogollero del maíz. Los marcadores RAPD fueron altamente asociados a la virulencia contra S. frugiperda, explicando hasta el 67% de la variación fenotípica. Por lo tanto, además de la identificación y caracterización molecular de aislados de Beauveria, el secuenciamiento de la región ITS y los marcadores RAPD fueron útiles en la selección de aislados potencialmente eficientes contra gusanos de S. frugiperda e en el monitoramiento de liberaciones de esos microrganismos, en programas de biocontrolO objetivo deste trabalho foi avaliar a patogenicidade de 24 isolados de Beauveria contra larvas de Spodoptera frugiperda e caracterizá-los molecularmente, por meio do seqüenciamento da região ITS do rDNA e de marcadores RAPD. O seqüenciamento de fragmentos de 570 pares de bases, da região ITS do rDNA, possibilitou a identificação dos isolados como B. bassiana e B. brongniarti, pela comparação com seqüências depositadas no GenBank. Sessenta e sete fragmentos polimórficos de RAPD foram capazes de diferenciar 20 entre 24 isolados de Beauveria, e agrupá-los de acordo com o inseto hospedeiro e com a patogenicidade contra a lagarta-do-cartucho do milho. Os marcadores RAPD foram altamente associados à patogenicidade contra S. frugiperda e explicaram até 67% da variação fenotípica. Além da identificação e caracterização molecular de isolados de Beauveria, o seqüenciamento da região ITS, aliado aos marcadores RAPD, é útil na seleção de isolados potencialmente eficazes contra larvas de S. frugiperda e no monitoramento de liberações desses microrganismos em programas de biocontrole

Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines

O objetivo deste trabalho foi desenvolver um sistema de transformação genética para genótipos tropicais de milho pelo bombardeamento de embriões imaturos com micropartículas. O bombardeamento compartículas foi realizado utilizando-se uma construção gênica contendo os genes bar e uidA sob o controle do promotor CaMV35S. As melhores condições para transformação das linhagens de milho tropical L3 e L1345foram obtidas após o cultivo de embriões imaturos durante 4 horas, antes do bombardeamento, em alta osmolaridade, e bombardeados com a pressão de 1.100 psi de gás hélio, dois tiros por placa e uma distância de vôo da micropartícula de 6,6 cm. As freqüências de transformação obtidas variaram entre 0,9 e 2,31%. A integração dos genes heterólogos no genoma das plantas transgênicas de milho foi confirmada por análises de Southern blot e expressão dos genes bar e uidA. O protocolo de transformação genética de milho desenvolvido neste estudo irá aumentar, provavelmente, a eficiência de produção de novas linhagens tropicais de milho expressando características agronômicas desejáveis.The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditionsranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics

Molecular caracterization of a corn core collection–landraces subgroup

The efficient use of genetic resources- stored in germplasm collections can be maximized if morphoagronomic and molecular information on the accessions is made available. To achieve this, a collection that is well-structured, well-curated and easily accessible (the core collection) is required. Consequently, the objective of the current study was to characterize 80 landrace accessions from the maize core collection of the Federal University of Viçosa (UFV), and assay thenngenetic diversity of the various landraces, considering grain type and ecogeographic origin. For this, AFLP analysis was performed using 12 primer combinations. Genetic diversity of the collection was quantified with the UPGMA method, using the Jaccard Index to quantify dissimilarity. The core collection was divided into four sub-populations by grain type, and into six sub-populations based on ecogeographic origin. Genetic diversity analysis was performed both within and between sub-populations. A high level of genetic variability was found among the landrace accessions of UFV Core Collection, principally among those accessions with dentate type grains.Classification by grain type andecogeographic origin allowed genetically divergent groups to be distinguished.The efficient use of genetic resources- stored in germplasm collections can be maximized if morphoagronomic and molecular information on the accessions is made available. To achieve this, a collection that is well-structured, well-curated and easily accessible (the core collection) is required. Consequently, the objective of the current study was to characterize 80 landrace accessions from the maize core collection of the Federal University of Viçosa (UFV), and assay thenngenetic diversity of the various landraces, considering grain type and ecogeographic origin. For this, AFLP analysis was performed using 12 primer combinations. Genetic diversity of the collection was quantified with the UPGMA method, using the Jaccard Index to quantify dissimilarity. The core collection was divided into four sub-populations by grain type, and into six sub-populations based on ecogeographic origin. Genetic diversity analysis was performed both within and between sub-populations. A high level of genetic variability was found among the landrace accessions of UFV Core Collection, principally among those accessions with dentate type grains.Classification by grain type and ecogeographic origin allowed genetically divergent groups to be distinguished

Análise do N-terminal da proteína capsidial de SCMV infectando milho e sorgo no Brasil

Os objetivos do presente estudo foram (i) identificar por meio do sequenciamento da proteína capsidial a espécie de potyvírus causando sintomas de mosaico em milho e sorgo no Brasil, e (ii) analisar a sequência de aminoácidos (aa) do N-terminal do gene da proteína capsidial.Fil: Souza, Isabel Regina Prazeres de. Embrapa Milho e Sorgo, Sete Lagoas; BrasilFil: Carneiro, Newton Portilho. Embrapa Milho e Sorgo, Sete Lagoas; BrasilFil: Giolitti, Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Lenardon, Sergio Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Oliveira Sabato, Elizabeth de. Embrapa Milho e Sorgo, Sete Lagoas; BrasilFil: Gomes, Eliane Aparecida. Embrapa Milho e Sorgo, Sete Lagoas; BrasilFil: Noda, Roberto Willians. Embrapa Milho e Sorgo, Sete Lagoas; BrasilFil: Souza, Francisco Adriano de. Embrapa Milho e Sorgo, Sete Lagoas; Brasi

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

Patterns of gene expression in maize endosperm: Characterization of the eEF1A gene family

One of the major challenges for maize improvement is to enhance the protein quality of the endosperm. We do not know what genes encode the proteins that contribute most to the protein quality, but lysine is the most limiting amino acid. One way to determine the proteins made in the endosperm is by isolating the genes expressed in this tissue and identifying their function. The results of the maize genome project with etiolated seedling and endosperm cDNA libraries support the basis of this strategy, in that the putative functions of a large number of cDNA sequences were identified through sequence similarity comparison and genomic Southern analyses. One of the genes I selected for further analyses is the elongation factor 1 alpha (eEF1A) gene family. eEF1A interacts with many cellular components and is therefore classified as a multifunction protein. In addition, its level is highly correlated with the amount of protein-bound lysine in maize endosperm. Even though many proteins are known to interact with eEF1A, the basis for the relationship between eEF1A and endosperm lysine content is not known. Transcript level and sequence of different members of the maize eEF1A gene family were analyzed in this work. All the eEF1A maize genes examined had GTP, aminoacyl tRNA, eEF1B and actin binding domains. The substitution of glutamic acid for aspartic acid in a region that has been shown to be important for actin binding in eEF1Aa, eEF1Ae and eEF1Af suggest that there might be two groups of eEF1A genes that bind differently to actin. Analysis of transcript levels demonstrated that different members of the eEF1A gene family are expressed at different levels in the tissues examined. The results from the analyses of transcript levels demonstrated that most of the maize eEF1A gene family members are expressed and their mRNAs vary in different tissues and in developing endosperm. Physiological differences were also determined for the two most abundant eEF1As members by the yeast two-hybrid system. This research provides a significant step toward understanding eEF1A functions in maize and why this protein correlates with the lysine content of the endosperm

Molecular characterization and pathogenicity of isolates of Beauveria spp. to fall armyworm

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs

Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines Otimização dos parâmetros de bombardeamento de partículas para a transformação genética de linhagens brasileiras de milho

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.O objetivo deste trabalho foi desenvolver um sistema de transformação genética para genótipos tropicais de milho pelo bombardeamento de embriões imaturos com micropartículas. O bombardeamento com partículas foi realizado utilizando-se uma construção gênica contendo os genes bar e uidA sob o controle do promotor CaMV35S. As melhores condições para transformação das linhagens de milho tropical L3 e L1345 foram obtidas após o cultivo de embriões imaturos durante 4 horas, antes do bombardeamento, em alta osmolaridade, e bombardeados com a pressão de 1.100 psi de gás hélio, dois tiros por placa e uma distância de vôo da micropartícula de 6,6 cm. As freqüências de transformação obtidas variaram entre 0,9 e 2,31%. A integração dos genes heterólogos no genoma das plantas transgênicas de milho foi confirmada por análises de Southern blot e expressão dos genes bar e uidA. O protocolo de transformação genética de milho desenvolvido neste estudo irá aumentar, provavelmente, a eficiência de produção de novas linhagens tropicais de milho expressando características agronômicas desejáveis