Stability of Citrus tristeza virus protective isolate 'Pêra IAC' according to SSCP analysis of old and new lines of three sweet orange varieties

Clonal cleaning, followed by pre-immunization with protective complexes of Citrus tristeza virus(CTV), allowed the commercial cultivation of Pêra sweet orange, a variety that has great importance for Brazilian citriculture but is sensitive to the virus. The use of mild protective isolates in other citrus varieties, even those more tolerant to CTV, can also be of interest to prevent the spread of severe isolates. The aim of this study was to characterize, by means of SSCP (Single Strand Conformational Polymorphism) analysis of the coat protein gene, CTV isolates present in plants of the sweet orange cultivars Pêra, Hamlin and Valencia propagated from four budwood sources: 1) old lines, 2) nucellar lines, 3) shoot-tip-grafted lines, and 4) shoot-tip-grafted lines pre-immunized with the mild CTV protective isolate 'PIAC'. We also evaluated the correlation of the obtained SSCP patterns to stem pitting intensity, tree vigor and fruit yield. SSCP results showed low genetic diversity among the isolates present in different trees of the same variety and same budwood source and, in some cases, in different budwood sources and varieties. Considering tristeza symptoms, lower intensity was noted for plants of new, shoot-tip-grafted and pre-immunized shoot-tip-grafted lines, compared to old lines of the three varieties. The observed SSCP patterns and symptomatology suggested that more severe CTV complexes infect the plants of old lines of all three varieties. The protective complex stability was observed in the SSCP patterns of CTV isolates of some shoot-tip-grafted and pre-immunized clones. It was concluded that the changes detected in other electrophoretic profiles of this treatment did not cause loss of the protective capacity of CTV isolate 'PIAC' inoculated in the pre-immunization.

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

A molecular epidemiological study was performed with Babesia bigemina isolates from five geographical regions of Brazil. The genetic analysis was done with random amplification of polymorphic DNA (RAPD), repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) and enterobacterial repetitive intergenic consensus sequences-polymerase chain reaction (ERIC-PCR) that showed genetic polymorphism between these isolates and generated fingerprinting. In RAPD, ILO872 and ILO876 primers were able to detect at least one fingerprinting for each B. bigemina isolate. The amplification of B. bigemina DNA fragments by REP-PCR and ERIC-PCR gave evidence for the presence in this haemoprotozoan of the sequences described previously in microorganisms of the bacterial kingdom. For the first time it was demonstrated that both techniques can be used for genetic analysis of a protozoan parasite, although the ERIC-PCR was more discriminatory than REP-PCR. The dendogram with similarity coefficient among isolates showed two clusters and one subcluster. The Northeastern and Mid-Western isolates showed the greatest genetic diversity, while the Southeastern and Southern isolates were the closest. The antigenic analysis was done through indirect fluorescent antibody technique and Western blotting using a panel of monoclonal antibodies directed against epitopes on the merozoite membrane surface, rhoptries and membrane of infected erythrocytes. As expected, the merozoite variable surface antigens, major surface antigen (MSA)-1 and MSA-2 showed antigenic diversity. However, B cell epitopes on rhoptries and infected erythrocytes were conserved among all isolates studied. In this study it was possible to identify variable and conserved antigens, which had already been described as potential immunogens. Considering that an attenuated Babesia clone used as immunogen selected populations capable of evading the immunity induced by this vaccine, it is necessary to evaluate more deeply the cross-protection conferred by genetically more distant Brazilian B. bigemina isolates and make an evaluation of the polymorphism degree of variable antigens such as MSA-1 and MSA-2.

Genetic variation of and environmental effects on inducibility of resistance in tomatoes (Solanum lycopersicum L.) to Phytophthora infestans (Mont) de Bary

Many plant strengtheners are promoted for their supposed effects on nutrient uptake and/or resistance induction (IR). In addition, many organic fertilizers are supposed to enhance plant health and several studies have shown that tomatoes grown organically are more resistant to late blight, caused by Phytophthora infestans to tomatoes grown conventionally. Much is known about the mechanisms underlying IR. In contrast, there is no systematic knowledge about genetic variation for IR. Therefore, the following questions were addressed in the presented dissertation: (i) Is there genetic variation among tomato genotypes for inducibility of resistance to P. infestans? (ii) How do different PS compare with the chemical inducer BABA in their ability to IR? (iii) Does IR interact with the inducer used and different organic fertilizers? A varietal screening showed that contrary to the commonly held belief IR in tomatoes is genotype and isolate specific. These results indicate that it should be possible to select for inducibility of resistance in tomato breeding. However, isolate specificity also suggests that there could be pathogen adaptation. The three tested PS as well as two of the three tested organic fertilisers all induced resistance in the tomatoes. Depending on PS or BABA variety and isolate effects varied. In contrast, there were no variety and isolate specific effects of the fertilisers and no interactions with the PS and fertilisers. This suggests that the different PS should work independent of the soil substrate used. In contrast the results were markedly different when isolate mixtures were used for challenge inoculations. Plants were generally less susceptible to isolate mixtures than to single isolates. In addition, the effectiveness of the PS was greater and more similar to BABA when isolate mixtures were used. The fact that the different PS and BABA differed in their ability to induce resistance in different host genotype -pathogen isolate combinations puts the usefulness of IR as a breeding goal in question. This would result in varieties depending on specific inducers. The results with the isolate mixtures are highly relevant. On the one hand they increase the effectiveness of the resistance inducers. On the other hand, measures that increase the pathogen diversity such as the use of diversified host populations will also increase the overall resistance of the hosts. For organic tomato production the results indicate that it is possible to enhance the tomato growing system with respect to plant health management by using optimal fertilisers, plant strengtheners and any measures that increase system diversity.

Optical genetic mapping defines regions of chromosomal variation in serovars of S. enterica subsp enterica of concern for human and animal health

Infections involving Salmonella enterica subsp. enterica serovars have serious animal and human health implications; causing gastroenteritis in humans and clinical symptoms, such as diarrhoea and abortion, in livestock. In this study an optical genetic mapping technique was used to screen 20 field isolate strains from four serovars implicated in disease outbreaks. The technique was able to distinguish between the serovars and the available sequenced strains and group them in agreement with similar data from microarrays and PFGE. The optical maps revealed variation in genome maps associated with antimicrobial resistance and prophage content in S. Typhimurium, and separated the S. Newport strains into two clear geographical lineages defined by the presence of prophage sequences. The technique was also able to detect novel insertions that may have had effects on the central metabolism of some strains. Overall optical mapping allowed a greater level of differentiation of genomic content and spatial information than more traditional typing methods.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

Background. The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. Results. Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. Conclusions. The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI. while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Molecular and biological characterization of Lettuce mosaic virus (LMV) isolates reveals a distinct and widespread type of resistance-breaking isolate: LMV-Most

Lettuce mosaic virus (LMV) causes an economically important seedborne and aphid-transmitted disease of lettuce and ornamental crops worldwide. The genetic diversity among 73 LMV isolates was examined based on a 216-nucleotide sequence at the variable region encoding the NIb-coat protein junction, Three clusters of LMV isolates were distinguished: LMV-Yar, LMV-Greck, and LMV-RoW. In the latter cluster, two subgroups of isolates, LMV-Common and LMV-Most, accounted for a large proportion of the LMV isolates analyzed. These two subgroups included the seedborne isolates, consistent with this property contributing a selective advantage and resulting in widespread distribution. In addition to being seedborne, LMV-Most isolates overcome the two resistance genes commonly used in lettuce, mol(1) and mol(2), and thus represent a potential threat to lettuce cultivation. The complete sequence of an LMV-Most isolate (LMV-AF199) was determined, allowing a better definition of the genetic relationships among LMV-Most, LMV-Common, and an additional isolate of the LMV-RoW cluster.

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment 1), we analyzed two antigen lots of two human isolates (Bt1 and Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh and Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment 2), we compared the antigenic profile of three antigen hatches from three human isolates (Bt1, Bt2 and Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment 1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment 2, the reference system for P brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.

Genetic diversity of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina

Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cryl genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.

Comparison of three molecular typing methods to assess genetic diversity for Mycobacterium tuberculosis

This study describes the comparison of three methods for genotyping of Mycobacterium tuberculosis, namely MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats), spoligotyping and, for the first time, MLST (Multilocus Sequence Typing). In order to evaluate the discriminatory power of these methods, a total of 44 M. tuberculosis isolates obtained from sputum specimens of patients from Brazil were genotyped. Among the three methods, MLST showed the lowest discriminatory power compared to the other two techniques. MIRU-VNTR showed better discriminatory power when compared to spoligotyping, however, the combination of both methods provides the greatest level of discrimination and therefore this combination is the most useful genotyping tool to be applied to M. tuberculosis isolates. © 2013 Elsevier B.V.

Frequency and genetic diversity of the MAT1 locus of Histoplasma capsulatum isolates in Mexico and Brazil

The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+mating type) and G-186AR (-mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective+ and-mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources. © 2013, American Society for Microbiology. All Rights Reserved.

Genetic characterization of Toxoplasma gondii isolates from eared doves (Zenaida auriculata) in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular strategies for genetic diversity analysis and development of markers linked to resistance traits in apple

The goal of many plant scientists’ research is to explain natural phenotypic variation in term of simple changes in DNA sequence. DNA-based molecular markers are extensively used for the construction of genome-wide molecular maps and to perform genetic analysis for simple and complex traits. The PhD thesis was divided into two main research lines according to the different approaches adopted. The first research line is to analyze the genetic diversity in an Italian apple germplasm collection for the identification of markers tightly linked to targeted genes by an association genetic method. This made it possible to identify synomym and homonym accessions and triploids. The fruit red skin color trait has been used to test the reliability of the genetic approaches in this species. The second line is related to the development of molecular markers closely linked to the Rvi13 and Rvi5 scab resistance genes, previously mapped on apple’s chromosome 10 and 17 respectively by using the traditional linkage mapping method. Both region have been fine-mapped with various type of markers that could be used for marker-assisted selection in future breeding programs and to isolate the two resistance genes.

Funduscopy in adult zebrafish and its application to isolate mutant strains with ocular defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals.A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out.Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas.In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.