Phylogenetic analyses of symbiotic genes and characterization of functional traits of Mesorhizobium spp. strains associated with the promiscuous species Acacia seyal Del.

Aims: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro‐ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. Methods and Results: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal‐nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. Conclusions: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal‐nodulating rhizobia could enhance plant growth. Significance and Impact of the Study: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.     

DNA fingerprinting reveals polygyny in the subterranean rodent Ctenomys talarum

DNA fingerprinting was used to characterize patterns of paternity in two populations of Ctenomys talarum from Buenos Aires Province, Argentina. The multilocus probe PV47-2 was used to detect variation in genomic DNA extracted from 12 females, their 32 offspring, and 14 putative sires. For 11 out of 12 litters examined, a single male capable of providing all nonmaternal bands was identified. Within each study population, individual males sired more than one litter, suggesting that C. talarum is polygynous. No evidence of multiple paternity of litters was found. High band-sharing values among females suggest that further research is needed to assess the population genetic structure of this species.     

Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated “core” sequences (GACA, GATA, and GCAC). The principal objectives of this investigation were to determine whether: (1) the previously reported almost complete lack of polymorphism at allozyme loci in this species was indicative of a reduced amount of genetic diversity at VNTR loci as well; (2) VNTR markers were informative about possible clonal propagation; and (3) significant differences in genetic structure of sampling sites were associated with differences in environmental levels of pollutants at those sites. Previously, widespread sampling across the eastern United States, surveying across ten allozyme loci, has detected only two genotypes, involving a difference at a single locus, among 104 populations. In this study, the amount of genetic diversity detected at VNTR loci: (1) among ramets (N = 40; 40 genotypes detected) collected at ∼8-km intervals along a 320-km transect; (2) among ramets (N = 220; 117 genotypes detected) from five study sites separated by 50–3000 m; and (3) even among ramets within each study site [N = 44 per site; from 13 to 34 genotypes detected per site (270 m²)] exceeds that previously found in those more geographically widespread allozyme surveys. Among the 260 ramets analyzed here, the mean number of bands scored per individual was 48.61 (SD = 2.80). Mean genetic similarity among ramets collected along the 320-km transect was 0.91, which was within the range of mean genetic similarity within the five study sites (range: 0.89–0.95). Among the five study sites, 61% of the samples analyzed appeared to be clonal ramets, with up to 12 clones detected for 44 ramets sampled within a site. Clones grew intermingled and ranged up to 39 m in extent. Permutation tests of genetic similarity revealed significant genetic differentiation between each of the five study sites. Consistent with the previous allozyme studies, T. latifolia was characterized by extremely low genetic variation relative to levels of polymorphism detected at VNTR loci in other plant species. Estimated heterozygosity among ramets along the 320-km transect ranged from 0.11 to 0.13, while that within the five study sites ranged from 0.05 to 0.12. Estimates of F_st (0.32–0.41) also indicated considerable genetic subdivision among these stands. Significantly higher genetic diversity was detected at the two study sites that chemistry and toxicity data indicate to be the most severely impacted by pollutants. Although this correlation does not establish cause and effect, the results of this study indicate that the analysis of genetic diversity at VNTR loci may be a useful tool for monitoring anthropogenic-induced changes in the genetic structure of natural populations of plants.     

Epidemiology and molecular typing of Candida isolates from burn patients

This study, spread over a span of 2 years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45) followed by Candida tropicalis(33), Candida glabrata (13.5), C. parapsilosis (4), C. krusei (2.75) and C. kefyr (1.75). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe. Fingerprinting analyses of all the C. albicans strains revealed that strains collected from different patients were different. It is noteworthy that patients with disseminated candidiasis had a similar, but unique strain isolated from all body locations, suggesting a possibility that commensal isolates might be turning pathogenic. Taken together, this is probably the first ever detailed survey of Candidainfections in burn patients in India and is expected to lead to better clinical management of this group of patients.     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.     

High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.     

A proteomic approach to studying plant response to crenate broomrape (Orobanche crenata) in pea (Pisum sativum)

Crenate broomrape (Orobanche crenata) is a parasitic plant that threatens legume production in Mediterranean areas. Pea (Pisum sativum) is severely affected, and only moderate levels of genetic resistance have so far been identified. In the present work we selected the most resistant accession available (Ps 624) and compared it with a susceptible (Messire) cultivar. Experiments were performed by using pot and Petri dish bioassays, showing little differences in the percentage of broomrape seed germination induced by both genotypes, but a significant hamper in the number of successfully installed tubercles and their developmental stage in the Ps 624 compared to Messire. The protein profile of healthy and infected P. sativum root tissue were analysed by two-dimensional electrophoresis. Approximately 500 individual protein spots could be detected on silver stained gels. At least 22 different protein spots differentiated control, non-infected, Messire and Ps 624 accessions. Some of them were identified by MALDI-TOF mass spectrometry and database searching as cysteine proteinase, beta-1,3-glucanase, endochitinase, profucosidase, and ABA-responsive protein. Both qualitative and quantitative differences have been found among infected and non-infected root extracts. Thus, in the infected susceptible Messire genotype 34 spots were decreased, one increased and three newly detected, while in Ps 624, 15 spots were increased, three decreased and one newly detected. In response to the inoculation, proteins that correspond to enzymes of the carbohydrate metabolism (fructokinase, fructose-bisphosphate aldolase), nitrogen metabolism (ferredoxin-NADP reductase) and mitochondrial electronic chain transport (alternative oxidase 2) decreased in the susceptible check, while proteins that correspond to enzymes of the nitrogen assimilation pathway (glutamine synthetase) or typical pathogen defence, PR proteins, including beta-1,3-glucanase and peroxidases, increased in Ps 624. Results are discussed in terms of changes in the carbohydrate and nitrogen metabolism an induction of defence proteins in response to broomrape parasitism.     

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.     

Performance of phaseolus bean rhizobia in soils from the major production sites in the Nile Delta

The symbiotic and competitive performances of two highly effective rhizobia nodulating French bean P. vulgaris were studied in silty loam and clayey soils. The experiments were carried out to address the performance of two rhizobia strains (CE3 and Ph. 163] and the mixture thereof with the two major cultivated bean cultivars in two soil types from major growing French bean areas in Egypt. Clay and silty loam soils from Menoufia and Ismailia respectively were planted with Bronco and Giza 6 phaseolus bean cultivars. The data obtained from this study indicated that rhizobial inoculation of Giza 6 cultivar in clayey soil showed a positive response to inoculation in terms of nodule numbers and dry weight. This response was also positive in dry matter and biomass accumulation by the plants. The inoculant of strain CE3 enhanced plant growth and N-uptake relative to Ph. 163. However, the mixed inoculant strains were not always as good as single strain inoculants. The competition for nodulation was assessed using two techniques namely fluorescent antibody testing (FA) and REP-PCR fingerprinting. The nodule occupancy by inoculant strain Ph. 163 in both soils occupied 30-40% and 38-50 of nodules of cultivar Bronco. The mixed inocula resulted in higher proportions of nodules containing CE3 in silty loam soil and Ph. 163 in clayey soil. The native rhizobia occupied at least 50% of the nodules on the Bronco cultivar. For cultivar Giza 6, the native rhizobia were more competitive with the inoculant strains. Therefore, we suggest using the studied strains as commercial inocula for phaseolus bean.     

Utility of Amplified Fragment Length Polymorphisms (AFLP) to analyse genetic structures within the Alexandrium tamarense species complex

Phylogenetic analyses of the Alexandrium tamarense species complex using ribosomal RNA sequences show a differentiation of ribotypes/clades into geographic areas and not into the three morphotypes/species A. tamarense, A. fundyense and A. catenella. Different parts of the rRNA operon have proven informative in revealing the existence and the relationships of these geographic clades, whereas even internal transcribed spacer (ITS) regions lack the resolution required to gain a deeper insight into the population structure of the species complex. Here, the utility of the DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) as a possible tool for such purposes was tested. A mixed sampling strategy was used in order to assess the amount of variation of AFLP banding patterns at the level of populations and geographic clades. We also describe optimized methods to achieve a good reproducibility. Our results suggest that AFLPs can provide useful information at the population level using clonal samples from a certain bloom, whereas the amount of variation that we found is too high to allow for meaningful comparisons of a few strains collected from different localities at different time points even though they belong to one geographic clade.