Anàlisi polifàsica de soques de "Pseudomonas fluorescens" potencials agents de biocontrol de malalties de fruiters

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases. L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP. Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn. S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes. Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies. El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempelts La caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri. Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP. Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades.

Genetic variation and population structuring in two brooding coral species (Siderastrea stellata and Siderastrea radians) from Brazil

Siderastrea stellata and S. radians are scleractinian coral species that present a remarkable overlap of diagnostic characteristics and sympatric distribution. Moreover, both are viviparous with similar reproductive strategies and with a gregarious larval behavior. Samples of both species from the Brazilian coast were analyzed using 18 isozymic loci to quantify their genetic variability and populational structure. Results confirmed species identity, high intrapopulational variability and revealed moderate genetic structuring among all samples (S. stellata: F(ST) = 0.070; S. radians: F(ST) = 0.092). Based on genotypic diversity analysis, there was evidence that local recruitment may have a minor role in the populations (mean, G(o) :G(e) = 1.00 +/- 0.0003 SD for S. stellata and 0.99 +/- 0.0023 SD for S. radians). Deviations towards heterozygote deficiencies found in both Siderastrea species could be explained by the Wahlund effect, since there was evidence that populations might be composed of colonies of different ages. In S. radians it is also likely that there is some inbreeding occurring in the studied populations. Despite the brooding pattern and the gregarious larval behavior, our data suggest the occurrence of gene flow along the Brazilian coast. This is the first study on population genetics of Brazilian reef corals.

Collagenase production and hemolytic activity related to 16S rRNA variability among Parvimonas micra oral isolates

Parvimonas micra are gram positive anaerobic cocci isolated from the oral cavity and frequently related to polymicrobial infections in humans. Despite reports about phenotypic differences, the genotypic variation of P. micra and its role in virulence are still not elucidated. The aim of this study was to determine the genotypic diversity of P. micra isolates obtained from the subgingival biofilm of subjects with different periodontal conditions and to correlate these findings with phenotypic traits. Three reference strains and 35 isolates of P. micro were genotyped by 16S rRNA PCR-RFLP and phenotypic traits such as collagenase production, elastolytic and hemolytic activities were evaluated. 16S rRNA PCR-RFLP showed that P. micra could be grouped into two main clusters: C1 and C2; cluster C1 harbored three genotypes (HG1259-like, HG1467-like and ICBM0583-like) while cluster C2 harbored two genotypes (ATC03270-like and ICBM036). A wide variability in collagenolytic activity intensities was observed among all isolates, while elastolytic activity was detected in only two isolates. There was an association between hemolytic activity in rabbit erythrocytes and cluster C2. There was an association between hemolytic activity in rabbit erythrocytes and cluster C1. Although these data suggest a possible association between P. micra genetic diversity and their pathogenic potential, further investigations are needed to confirm this hypothesis. (C) 2009 Elsevier Ltd. All rights reserved.

Intraspecific variation in 16S rRNA gene of Mycoplasma synoviae determined by DNA sequencing

Mycoplasma synoviae (MS) is an important avian pathogen may cause both respiratory disease and joint inflammation synovitis in poultry, causing economic losses to the Brazilian poultry industry. The genotypic variation in 16S rRNA gene is unknown. Partial sequences of 16S rRNA gene of 19 strains of M. synoviae were sequenced and analyzed in order to obtain molecular characterization and evaluation of the genetic variability of strains from distinct Brazilian areas of poultry production. Different polymorphic patterns were observed. The number of polymorphic alterations in the studied strains ranged from 0 to 6. The nucleotide variations, including deletion, insertion and substitutions, ranged from 3 to 5. The genotypic diversity observed in this study may be explained by spontaneous mutations that may occur when a lineage remains in the same flock for long periods. The culling and reposition in poultry flocks may be responsible for the entry of new strains in different areas. (C) 2008 Elsevier Ltd. All rights reserved.

Asexual reproduction does not produce clonal populations of the brooding coral Pocillopora damicornis on the Great Barrier Reef, Australia

We have investigated the relationship between genotypic diversity, the mode of production of brooded larvae and disturbance in a range of reef habitats, in order to resolve the disparity between the reproductive mode and population structure reported for the brooding coral Pocillopora damicornis. Within 14 sites across six habitats, the ratio of the observed (G o) to the expected (G e) genotypic diversity ranged from 69 to 100% of that expected for random mating. At three other sites in two habitats the G o /G e ranged from 35 to 53%. Two of these sites were recently bleached, suggesting that asexual recruitment may be favoured after disturbance. Nevertheless, our data suggest that brooded larvae, from each of five habitats surveyed, were asexually produced. While clonal recruitment may be important in disturbed habitats, the lack of clonality detected, both in this and earlier surveys of 40 other sites, implies that a disturbance is normally insufficient to explain this species’ continued investment in clonal reproduction.

Do reproductive tactics vary with habitat heterogeneity in the intertidal sea anemone Actinia tenebrosa?

Cnidarians display a diverse range of reproductive tactics including sexual and asexual modes of reproduction. Although few studies have looked for intraspecific variation in reproductive tactics, flexible expression of such life-history traits may be favoured in species that occupy a range of habitats. We tested this in the sea anemone Actinia tenebrosa by comparing cycles of reproductive activity and the mode of production of brooded larvae in local populations occupying boulder fields and stable rock platforms. We determined the mode of production of broods from eight rock platforms (separated by up to 1600 km) and two boulder shores on the south east coast of Australia using a combination of allozyme data and four newly characterised microsatellite markers.

We determined seasonal patterns of brooding and gonad development by monthly dissection of 15–30 adults from each of two boulder fields and two stable platforms. Previous genetic studies have shown that populations of A. tenebrosa on rock platforms can be highly clonal, whereas anemones on more heterogeneous boulder habitats display levels of genotypic diversity similar to that expected for sexual reproduction. We genotyped a total of 221 juveniles from 37 brooding adults including 11 broods and 80 juveniles from boulder shores. We did not detect any evidence of sexual production of broods. All brooded juveniles displayed identical multi-locus genotypes to their brood parent irrespective of habitat of origin or location, including 28 broods (200 juveniles) that were heterozygous at one or more locus. Similarly, we found that temporal patterns of gonad formation and brooding were consistent across habitats and locations. We detected 346 mature males, 234 non-reproductive or immature individuals, and no mature females within a total of 580 dissected individuals. These data suggest that the reproductive tactics of A. tenebrosa are essentially fixed and that variation in the genotypic diversity of populations may reflect variation in factors such as the input of sexually derived planktonically dispersed recruits or post-settlement processes. However, the apparent lack of females paradoxically implies that sexual reproduction, and hence recruitment, must be rare or no longer possible within some populations, and highlights the need for long-term studies of these populations.

Fine-scale patterns of genetic variation in a widespread clonal seagrass species

Seagrasses are ecosystem engineers that offer important habitat for a large number of species and provide a range of ecosystem services. Many seagrass ecosystems are dominated by a single species, with research showing that genotypic diversity at fine spatial scales plays an important role in maintaining a range of ecosystem functions. However, for most seagrass species, information on fine-scale patterns of genetic variation in natural populations is lacking. In this study, we use a hierarchical sampling design to determine the levels of genetic and genotypic diversity at different spatial scales (centimeters, meters, kilometers) in the Australian seagrass Zostera muelleri. Our analysis shows that at fine spatial scales (<1 m), levels of genotypic diversity are relatively low (R(Plots) = 0.37 ± 0.06 SE), although there is some intermingling of genotypes. At the site (10’s m) and meadow location (km) scale, we found higher levels of genotypic diversity (R(sites) = 0.79 ± 0.04 SE; R(Locations) = 0.78 ± 0.04 SE). We found some sharing of genotypes between sites within meadows, but no sharing of genotypes between meadow locations. We also detected a high level of genetic structuring between meadow locations (FST = 0.278). Taken together, our results indicate that both sexual and asexual reproductions are important in maintaining meadows of Z. muelleri. The dominant mechanism of asexual reproduction appears to occur via localized rhizome extension, although the sharing of a limited number of genotypes over the scale of 10’s of meters could also result from the localized dispersal and recruitment of fragments. The large number of unique genotypes at the meadow scale indicates that sexual reproduction is important in maintaining these populations, while the high level of genetic structuring suggests little gene flow and connectivity between our study sites. These results imply that recovery from disturbances will occur through both sexual and asexual regeneration, but the limited connectivity at the landscape scale implies that recovery at meadow-scale losses is likely to be limited.

Molecular characterization of group A bovine rotavirus in southeastern and central-western Brazil, 2009-2010

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

Genetic structure of populations of Rhizoctonia solani anastomosis group-1 IA from soybean in Brazil

The Basidiomycete fungus Rhizoctonia solani anastomosis group (AG)-1 IA is a major pathogen of soybean in Brazil, where the average yield losses have reached 30 to 60% in some states in Northern Brazil. No information is currently available concerning levels of genetic diversity and population structure for this pathogen in Brazil. A total of 232 isolates of R. solani AG1 IA were collected from five soybean fields in the most important soybean production areas in central-western, northern, and northeastern Brazil. These isolates were genotyped using 10 microsatellite loci. Most of the multilocus genotypes (MLGTs) were site-specific, with few MLGTs shared among populations. Significant population subdivision was evident. High levels of admixture were observed for populations from Mato Grosso and Tocantins. After removing admixed genotypes, three out of five field populations (Maranhao, Mato Grosso, and Tocantins), were in Hardy-Weinberg (HW) equilibrium, consistent with sexual recombination. HW and gametic disequilibrium were found for the remaining soybean-infecting populations. The findings of low genotypic diversity, departures from HW equilibrium, gametic disequilibrium, and high degree of population subdivision in these R. solani AG-1 IA populations from Brazil are consistent with predominantly asexual reproduction, short-distance dispersal of vegetative propagules (mycelium or sclerotia), and limited long-distance dispersal, possibly via contaminated seed. None of the soybean-infecting populations showed a reduction in population size (bottleneck effect). We detected asymmetric historical migration among the soybean-infecting populations, which could explain the observed levels of subdivision.

Biopatologia do Helicobacter pylori

A infecção pelo Helicobacter pylori (H. pylori) induz inflamação persistente na mucosa gástrica com diferentes lesões orgânicas em humanos, tais como gastrite crônica, úlcera péptica e câncer gástrico. Os fatores determinantes desses diferentes resultados incluem a intensidade e a distribuição da inflamação induzida pelo H. pylori na mucosa gástrica. Evidências recentes demonstram que cepas do H. pylori apresentam diversidade genotípica, cujos produtos acionam o processo inflamatório por meio de mediadores e citocinas, que podem levar a diferentes graus de resposta inflamatória do hospedeiro, resultando em diferentes destinos patológicos. Cepas H. pylori com a ilha de patogenicidade cag induzem resposta inflamatória mais grave, através da ativação da transcrição de genes, aumentando o risco para desenvolvimento de úlcera péptica e câncer gástrico. O estresse oxidativo e nitrosativo induzido pela inflamação desempenha importante papel na carcinogênese gástrica como mediador da formação ou ativação de cancerígenos, danos no DNA, bem como de alterações da proliferação celular e da apoptose.

Genetic structure of populations of Rhizoctonia solani AG-3 on potato in eastern North Carolina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solani anastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 PT on potato that includes both recombination and clonality.

Avaliação da expressão da talassemia Beta no Brasil pela coherança com defeitos de hemocromatose

Pós-graduação em Genética - IBILCE

Diversidade genética e estrutura de populações de Rhizoctonia solani AG-1 IA no Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estrutura genética de populações de Crinipellis perniciosa e Moniliophthora roreri utilizando marcadores RAPD e SSR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)